主题:Calcification

+ 关注 ≡ 收起全部文章
336*280_ads

Role of Osteoprotegerin in Arterial Calcification

【摘要】  Objectives— Enhanced osteoclastogenesis, increased bone resorption, and osteoporosis have been reported in osteoprotegerin-deficient (OPG (–/–)) mice. OPG (–/–) mice available in Japan usually do not show vascular calcification. We have found that arterial calcification can be quickly induced by a simple procedure in OPG (–/–) mice.

Methods and Results— Male OPG (–/–), OPG (+/–), and OPG (+/+) mice were fed a high phosphate diet from 6 to 10 weeks after birth, and then 1,25-dihydroxyvitamin D3 (calcitriol) was injected for 3 days. We found that severe calcification developed in the media of the aorta in OPG (–/–) mice. Under electron microscopy, calcium deposits were observed in the cytoplasm and extracellular matrix of vascular smooth muscle cells (VSMCs). Neither apoptosis of VSMCs nor infiltration of macrophages was observed. Alkaline phosphatase (ALP) activity of aortic tissue correlated with the calcified lesion area. Mouse aorta and bone extracts revealed an identical pattern by ALP electrophoresis.

Conclusions— Our results demonstrated that OPG had anticalcification activity in the aorta, probably through the downregulation of ALP activity. Because the time course of arterial calcification after the injection of calcitriol is accurate and reproducible, this mouse model will be useful for further investigation of vascular calcification.

Using osteoprotegerin-deficient mice, we established a mouse model in which arterial calcification can be quickly induced by treatment with a high-phosphate diet plus calcitriol injection. This model will allow us to perform detailed pathological and biochemical examinations at desired time points.

【关键词】  osteoprotegerin alkaline phosphate vascular smooth muscle cells calcium deposits


Introduction


Vascular calcification, which is frequently observed in patients with end-stage renal disease, diabetes, aging, and osteoporosis, can also lead to cardiovascular diseases and even sudden death. 1–3 Until recently, vascular calcification was considered to be a passive process that occurred as a nonspecific response to tissue injury or necrosis. Now it is becoming increasingly clear that vascular calcification is an actively regulated process that resembles bone metabolism and involves alkaline phosphatase (ALP) and other bone-related proteins. 4–7


Osteoprotegerin (OPG) is abundantly produced by osteoblasts at the bone surface and inhibits osteoclast activity, working as a key regulator of bone homeostasis. 8,9 Since it has been reported that OPG (–/–) mice exhibit severe osteoporosis attributable to enhanced osteoclastogenesis, OPG is considered to be a protective factor in bone metabolism. 10,11 In the vasculature, the function of OPG is unknown because it is unclear whether vascular calcification takes place in OPG (–/–) mice or not. 10,11 Moreover, it was reported that the serum OPG level is associated with the presence and severity of coronary artery disease (CAD). 12 It remains to be clarified whether OPG is involved in the progression of CAD or whether the upregulation of serum OPG concentration is a compensatory mechanism. ALP is a crucial enzyme for initiating mineralization in bone and is present in systemic arteries, arterioles, and some capillaries. 13 It is possible that this enzyme plays a role in arterial calcification by the same mechanism of action as that in bone. 14 Activation of ALP in the arterial wall may result in enhanced vascular calcification.


It is well known that either an elevated serum phosphate level or treatment with high doses of vitamin D induces vascular calcification in animal models as well as in humans. 15,16 In the present study, using OPG (–/–) mice, we established a mouse model in which arterial calcification can be quickly induced by treatment with a high phosphate diet plus 1,25-dihydroxyvitamin D3 (calcitriol) injection, and this model allowed us to perform detailed pathological and biochemical examinations at desired time points.


Materials and Methods


Male OPG (–/–), OPG (+/–), and OPG (+/+) mice, 6 weeks of age were used in this study. We divided the mice with 3 different genotypes into 3 different load groups; standard diet plus saline injection, high phosphate diet plus saline injection, and high phosphate diet plus calcitriol injection. The 9 groups of mice were fed a standard diet until 6 weeks. The mice were fed either a standard or high (1.5%) phosphate diet from 6 weeks to 10 weeks and water ad libitum throughout the study. At 9 weeks of age, a subcutaneous injection of saline or 5 µg/kg body weight of calcitriol was given for 3 days. The mice were euthanized at 10 weeks of age, and histopathologic and histochemical analyses were performed. For details, please see supplemental data, available online at http://atvb.ahajournals.org.


Results


Establishment of Vascular Calcification Model


We found that a combination of a lower dose of a high phosphate diet, containing 1.5% phosphorus, and injection of calcitriol (5 µg/kg, for 3 days) resulted in significant calcification in the arterial wall of OPG (–/–) mice ( Figure 1 ). The mortality rate of mice treated with a high phosphate diet plus calcitriol was 0%, 17%, and 25% in OPG (+/+), OPG (+/–), and OPG (–/–) mice, respectively. Most of the deaths took place on day 4 or 5, and hemorrhage into the thoracic cavity was frequently observed, probably attributable to aortic dissection that occurred during vascular calcification. After this critical time point, most of the mice could survive.


Figure 1. Representative serial sections of aortic sinus from mice given high-phosphate diet plus calcitriol injection. Sections were obtained from OPG (+/+) mice (A, D, G), OPG (+/–) mice (B, E, H), and OPG (–/–) mice (C, F, I). Hematoxylin and eosin stain (A-C), Azan stain (D-F), and von Kossa stain (G-I) were performed. Calcification of the aortic sinus is more obvious in OPG (–/–) mice (I) than in OPG (+/+) (G) and OPG (+/–) mice (H). Arrow, calcified lesion. Arrowhead, hemosiderin deposit.


Light Microscopic Analysis


Sections of the aortic sinus in OPG (+/+), OPG (+/–), and OPG (–/–) mice fed a high-phosphate diet plus calcitriol injection were observed by light microscopy ( Figure 1 ). In OPG (+/+) mice, there was no visible calcification ( Figure 1 G). On the other hand, in OPG (–/–) mice, calcification was detected by von Kossa staining, shown by dark brown in the arterial media ( Figure 1 I, arrow). Azan staining showed reduced blue staining in the same lesion, indicating decreased elastic fibers ( Figure 1 F, arrow). These lesions were associated with a reduction in the thickness of the vascular smooth muscle layer ( Figure 1 C). In immunohistochemical analysis, expression of -SM actin in these mice was not decreased in the calcified arterial lesions. In the arterial wall from all groups, there were no F4/80-positive cells (data not shown).


Measurement of Calcified Lesion Area


The calcified lesion area in the aortic sinus was carefully determined in 72 mice, and individual data points are plotted by genotype, diet, and calcitriol injection in Figure 2. In OPG (–/–) mice, aortic sinus calcification was significantly augmented by a high-phosphate diet plus calcitriol injection. Among the 3 genotypes with this treatment, there was a significant difference in the calcified lesion area, which was approximately 2.5 times higher in OPG (+/–) mice, and 17.7 times higher in OPG (–/–) mice than in OPG (+/+) mice ( Figure 3 ).


Figure 2. Effect of high-phosphate diet plus calcitriol injection on calcified area of aortic sinus in 3 genotypes. Calcified area was calculated in OPG (+/+) (white bar), OPG (+/–) (gray bar), and OPG (–/–) mice (black bar). OPG (–/–) mice given a high-phosphate diet plus calcitriol injection showed significant arterial calcification. Values are expressed as mean±SEM (n=8). * P <0.0001.


Figure 3. Electron micrographs of ascending aorta 4 days after initiation of saline or calcitriol injection. Samples were obtained from OPG (+/+) (A, B) and OPG (–/–) mice (C-F). A and C, Ascending aortic wall of mice treated with standard diet plus saline injection showed normal vascular smooth muscle cells, regardless of genotype. B, In OPG (+/+) mice treated with a high-phosphate diet plus calcitriol injection, dense deposits in the extracellular matrix and granular deposits in the cytoplasm of VSMC were occasionally seen. D through F, In OPG (–/–) mice treated with a high phosphate diet plus calcitriol injection, extensive diffuse calcification was observed in the cytoplasm and extracellular matrix of VSMC. E, In the panel, dense deposits in the extracellular matrix and needle-like crystals in the cytoplasm of VSMCs were observed. F, Higher magnification of panel E. In A-E, bar=2 µm; F, bar=500 nm.


Electron Microscopy


To clarify the time course of aortic calcification induced by a high phosphate diet plus calcitriol treatment, we obtained the ascending aortas from the 3 genotypes at 2, 4, and 7 days after the initiation of saline or calcitriol injection. On day 2, there were no abnormal findings in the 3 genotypes (data not shown). However, on day 4, treatment with a high phosphate diet plus calcitriol injection induced calcification, ranging from minimal to severe depending on the genotype. In OPG (+/+) mice treated with a high phosphate diet plus calcitriol, localized dense deposits in the extracellular matrix of vascular smooth muscle cells (VSMCs) and granular deposits in the cytoplasm of VSMCs were occasionally seen ( Figure 3 B). On the other hand, in OPG (–/–) mice treated with a high phosphate diet plus calcitriol, extensive diffuse calcification in the cytoplasm and extracellular matrix of VSMC was observed ( Figure 3D through 3 F). On day 7, the same results as those on day 4 were observed (data not shown). On intensive examination of specimens from the three genotypes (day 2, 4, and 7), we could not detect any apoptotic smooth muscle cells or infiltrating cells, such as macrophages.


Serum and Aortic Tissue ALP Activity


In the standard diet plus saline injection group at 10 weeks of age, OPG (–/–) mice showed significantly elevated serum ALP activity compared with that of OPG (+/+) mice and OPG (+/–) mice ( Figure 4 A). In the high phosphate diet plus saline injection groups, there was no difference in serum ALP activity compared with the standard diet plus saline injection groups in each genotype. On the other hand, in each genotype, calcitriol injection increased the serum ALP activity. In the high phosphate diet plus calcitriol injection groups, OPG (–/–) mice showed significantly elevated aortic tissue ALP activity compared with that of OPG (+/+) and OPG (+/–) mice ( Figure 4 B).


Figure 4. A, Effect of high-phosphate diet plus calcitriol injection on serum ALP activity in 3 genotypes. Values are expressed as mean±SEM (n=14). * P <0.05, ** P <0.01. B, Effect of high-phosphate diet plus calcitriol injection on aortic tissue ALP activity in 3 genotypes. Aortic tissue ALP activity was measured and normalized by tissue protein content and expressed as mean±SEM (n=9). Aortic tissue ALP activity in OPG (–/–) mice was significantly upregulated by administration of calcitriol compared with the other genotypes. * P <0.01, ** P <0.001.


ALP Isozymes


Representative ALP electrophoretic membranes of serum and organs from OPG (+/+) and OPG (–/–) mice given a standard diet plus saline injection or a high-phosphate diet plus calcitriol injection are shown in Figure 5. As mentioned in the methods, the running patterns of mouse samples are totally different from those of bovine and human samples. In OPG (–/–) mice given a high phosphate diet plus calcitriol injection, aortic and bone extracts revealed an identical pattern; however, mouse serum showed a completely different mobility pattern. Aortic extract in other groups did not show any distinct bands, probably because of weak ALP activity ( Figure 4 B).


Figure 5. Representative ALP electrophoretic membranes of serum and organs from OPG (+/+) and OPG (–/–) mice given standard diet plus saline injection or high phosphate diet plus calcitriol injection. Samples were run from the application point (–) to the front line (+). For the aortic extract, 2 aortas were combined. Serum was diluted 5-fold with saline. Bone extract was a homogenate of femur containing bone marrow. For loading, roughly 0.2, 0.2, 1.0, 5.0, and 0.4 mg protein was applied in lanes 2, 3, 4, 5, and 6, respectively.


PTHrP Level in Aortic Tissue


In the standard diet plus saline injection groups, aortic PTHrP level in OPG (–/–) mice was lower than that in OPG (+/+) mice (supplemental Figure I). Administration of a high phosphate diet plus calcitriol injection did not change PTHrP level in each genotype. There were no significant differences in the plasma level of PTHrP among the experimental groups (data not shown).


Histological Analysis of Femur


In the standard diet plus saline injection groups, the ratio of porous area/cortical area in OPG (–/–) mice was higher than that in OPG (+/+) mice (supplemental Figure II). Administration of a high-phosphate diet plus calcitriol injection did not change the ratio of porous area/cortical area in each genotype.


Serum Calcium and Inorganic Phosphate (P i ) Levels


Serum calcium level was significantly elevated by subcutaneous administration of calcitriol in OPG (–/–) mice (supplemental Figure IIIA). There was no significant difference in P i level among the experimental models (supplemental Figure III B).


Blood Pressure


While receiving a standard diet, systolic blood pressure was similar in the 3 genotypes (OPG (–/–) 109±7 mm Hg, OPG (+/–) 120±3 mm Hg, OPG (+/+) 118±4 mm Hg). A high-phosphate diet for 3 weeks before injection did not change the systolic blood pressure in any group. Under a high phosphate diet, 7 days after the initiation of calcitriol injection, systolic blood pressure in OPG (–/–) mice was significantly lower than that in OPG (+/–) and OPG (+/+) mice (95±15, 112±4, and 110±6 mm Hg, respectively, P <0.05).


Discussion


The present study shows that we have established a mouse model in which arterial calcification can be quickly induced by a simple procedure. This model provides both an accurate time course and high reproducibility for the development of vascular calcification, and will be useful to clarify the mechanism of arterial calcification. There are 2 well-known knockout mice—matrix Gla protein (MGP)-deficient mice and Klotho-deficient mice, which are reported to develop extensive vascular calcification and other organ disorders from a few weeks after birth. 17,18 Their average lifespan is about 2 months, therefore these mice are not suitable for vascular calcification research. For these reasons, we used OPG (–/–) mice to establish a mouse model to clarify the mechanism of arterial calcification. We could produce arterial calcification by a combination of a high (1.5%)-phosphate diet and calcitriol administration.


Vascular calcification is an actively regulated process that is associated with bone-related proteins bone morphogenetic protein-2, MGP, osteopontin (OPN), and osteocalcin (OC), as well as several transcription factors, including osteoblast transcription factors Runx2/Cbfa1, Msx2, and chondrocyte transcription factors, such as Sox9. 4–6,19,20 Various vascular cells in the arterial wall participate in the process of calcification. Especially, it is reported that VSMCs derived from the aorta have calcifying capacity and express MGP, OPN, and OC. 4,7,21,22 OPG is produced in various tissues including main components of the human vasculature, such as VSMCs and endothelial cells. 8,23


OPG has been isolated by 2 laboratories independently. 8,9 It is a secreted protein of the tumor necrosis factor (TNF) family that regulates bone mass by inhibiting osteoclast differentiation and activation. OPG exerts its inhibitory effects on osteoclasts by binding to receptor activator of nuclear factor B (RANK) ligand, thereby inhibiting the interaction between RANK and RANK ligand on osteoclasts and their precursors. 24 In mice, targeted deletion of the OPG gene resulted in an overall decrease in total bone density and a high incidence of bone fractures. 10,11 The osteoporosis with early onset observed in these mice was characterized by an increased number and activity of osteoclasts. In human, osteoporotic patients have a higher prevalence of arterial calcification. 25,26 Osteoporosis and vascular calcification frequently occur together and share many of the same risk factors. 3,25–27


OPG (–/–) mice reported by Mizuno et al are available in Japan. 10 Mizuno et al did not report whether or not vascular calcification occurred in their OPG (–/–) mice. 10 Bucay et al reported that approximately two-thirds of OPG (–/–) mice developed arterial calcification in the first several weeks after birth. 11 However, it was reported that the calcified lesions were extremely limited. 28 Unfortunately, we could not obtain the OPG (–/–) mice that Bucay et al developed and used in their study. According to the papers, the genetic backgrounds of the 2 mice might be very similar. 10,11 This discrepancy in phenotype between these reports and our results might be attributable to differences in environment such as diet and drinking water, especially in their mineral content.


Here, we focused on 2 molecules, 1,25-dihydroxyvitamin D3 (calcitriol) and phosphate, which have been implicated in the induction of vascular calcification. Vitamin D3 is critically important for the development, growth, and maintenance of a healthy skeleton from birth to death. However, its function is very complex. A physiological dose of vitamin D3 has the effect of promoting both bone resorption and formation. On the other hand, high-dose vitamin D3 increases bone resorption by activating osteoclastogenesis and inhibiting the expression of OPG. 29 In our experiments, the bone morphology in OPG (–/–) mice showed an osteoporotic phenotype including increased porous area and decreased bone volume of femoral cortex, consistent with earlier reports (supplemental Figure II). Administration of a high-phosphate diet plus calcitriol injection did not influence the bone morphology in both OPG (–/–) and OPG (+/+) mice. One possibility is that the effect of OPG deficiency was far more potent than the effects of a high phosphate diet plus calcitriol injection. The other possibility is that the time course after calcitriol treatment was only 1 week, which may be too short to affect bone density.


In the vasculature, it has been reported that high-dose vitamin D3 induces vascular calcification in animal studies, but the precise mechanism is not clearly understood. 15,30 It was reported that matrix metalloproteinase (MMP) is involved in aortic calcification, and inhibiting MMP activity could reduce calcium accumulation in the arterial wall. 31 It was also reported that excess vitamin D3 can increase calcium uptake into smooth muscle cells. 32,33 In VSMCs, vitamin D3 increases the expression of bone-related proteins such as OPN and ALP, which may be responsible for vascular calcification. 7,34 In our experiments, administration of calcitriol in OPG (–/–) mice given a high phosphate diet increased their aortic tissue ALP activity, and serum ALP and calcium level. It is thought that active osteoclastogenesis in OPG (–/–) mice was further enhanced by treatment with calcitriol, and resulted in elevation of serum calcium level.


Calcium-regulating hormones such as PTHrP may modulate atherosclerotic calcification. It was reported that PTHrP inhibits BVSMC calcification through depression of ALP activity, and that PTHrP secreted from BVSMCs acts as an endogenous inhibitor of vascular calcification, suggesting that VSMCs may be equipped with an autocrine or paracrine system that regulates calcium metabolism. 35 In our experiment, deficiency of the OPG gene decreased the PTHrP level in aortic tissue, suggesting that vascular calcification might be partly PTHrP-dependent. However, the load of a high phosphate diet plus calcitriol injection did not significantly change the PTHrP level in aortic tissue, but strongly enhanced vascular calcification. It is suggested that the enhancement may be PTHrP-independent.


Hyperphosphatemia is a frequent complication in patients with end-stage renal failure, who have severe calcification of vessel walls and high mortality from cardiovascular disease. In vitro, elevation of the phosphate level stimulates VSMC phenotypic transition and mineralization via the activity of a sodium-dependent phosphate cotransporter. 36–38 In spite of a high (1.5%)-phosphate diet, serum phosphate level was not changed in all genotypes, probably as a result of renal compensation. However, the serum calcium level of OPG (–/–) mice with high-phosphate diet plus calcitriol injection was relatively high. Interestingly, it was reported that in human VSMCs, calcium was a more potent inducer of vesicle-mediated calcification than phosphate. 39


In our light microscopic analysis, increased calcium deposition was seen in the medial layer of the aortic sinus in OPG (–/–) mice with a high-phosphate diet plus calcitriol treatment. However, OPG (–/–) mice did not show atherosclerotic lesions with lipid accumulation and inflammation, but these calcified lesions were confined to the media of arterial wall. In electron microscopic analysis, we observed a needle-like calcium matrix in the cytoplasm of VSMCs and calcium deposits around VSMCs in OPG (–/–) mice with a high-phosphate diet and calcitriol treatment. However, we could not detect any apoptotic cells or infiltrating macrophages in the arterial wall showing calcific changes. Endothelial cell morphology was normal in all groups. Our findings suggest that the mechanism of calcification in this mouse model is not related to apoptosis, but rather is derived from the cellular activity of VSMCs.


ALP are highly ubiquitous enzymes present in most species from bacteria to man. Although their wide distribution in nature indicates that these enzymes perform important biological functions, their detailed roles and natural substrate are not known. In humans, there are at least 4 ALP genes: liver/bone/kidney (non-tissue–specific), intestinal, placental, and placental-like. The non–tissue-specific form is located on chromosome 1, whereas the latter 3 are located together on chromosome 2. In human serum, there are 2 major circulating ALP isozymes—bone-type and liver-type. They are derived from a single gene and differ only in posttranslational glycosylation. Bone-type ALP is crucial for initiating mineralization of bone. Min et al reported that serum ALP activity in OPG (–/–) mice was elevated compared with that in wild-type mice, probably because of increased osteoclast activity in OPG (–/–) mice. 40 After administration of recombinant OPG in OPG (–/–) mice, serum ALP activity decreased to that in wild-type mice, probably because of normalization of osteoclast activity. However, they did not examine ALP activity in the aorta. In the present study, ALP activity was measured in both serum and aorta. Serum ALP activity was elevated regardless of a high phosphate diet plus calcitriol treatment; however, aortic tissue ALP activity was significantly upregulated only in OPG (–/–) mice treated with a high phosphate diet plus calcitriol. Shioi et al reported that cultured human VSMCs could express bone-type ALP derived from the non–tissue-specific ALP gene under certain circumstances. 7 Our electrophoretic data showed that the pattern in aortic extract resembled that in bone extract and did not resemble that in serum, liver extract, or intestinal extract. These results suggest that in OPG (–/–) mice treated with a high-phosphate diet and calcitriol, bone-type ALP activity in the aorta is increased and may contribute to aortic calcification.


Limitation


As is the case with animal experiments with serious histological changes in the arterial system, the mortality rate of our model is relatively high. We could not clarify the cause of death in OPG (–/–) mice when there was no hemorrhage into thoracic cavity. In such cases, renal failure may be responsible for a cause of death.


In conclusion, we have established an arterial calcification model with high reproducibility using OPG-deficient mice with a high (1.5%)-phosphate diet plus calcitriol treatment. In one sense, treatment with a high-phosphate diet plus calcitriol injection highlighted the role of OPG in the pathogenesis of vascular calcification. This OPG (–/–) mouse model will be useful for further investigation of vascular calcification.


Acknowledgments


The authors thank Katsuko Kataoka, MD, PhD, and Etsuko Suzaki, PhD, (Department of Histology and Cell Biology, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan) for technical guidance.


Sources of Funding


This study was supported in part by a Grants-in Aid for Scientific Research from Ministry of Education, Culture, Sports, Science, and Technology of Japan (18590814).


Disclosures


None.

【参考文献】
  Parfitt AM. Soft-tissue calcification in uremia. Arch Intern Med. 1969; 124: 544–556.

Blumenthal HT, Lansing AI, Wheeler PA. Calcification of the media of the human aorta and its relationship to intimal arteriosclerosis, aging and disease. Am J Pathol. 1944; 20: 665–687.

Hak AE, Pols HA, van Hemert AM, Hofman A, Witteman JC. Progression of aortic calcification is associated with metacarpal bone loss during menopause: a population-based longitudinal study. Arterioscler Thromb Vasc Biol. 2000; 20: 1926–1931.

Bostrom K, Watson KE, Horn S, Wortham C, Herman IM, Demer LL. Bone morphogenetic protein expression in human atherosclerotic lesions. J Clin Invest. 1993; 91: 1800–1809.

Giachelli CM, Bae N, Almeida M, Denhardt DT, Alpers CE, Schwartz SM. Osteopontin is elevated during neointima formation in rat arteries and is a novel component of human atherosclerotic plaques. J Clin Invest. 1993; 92: 1686–1696.

Shanahan CM, Cary NR, Metcalfe JC, Weissberg PL. High expression of genes for calcification-regulating proteins in human atherosclerotic plaques. J Clin Invest. 1994; 93: 2393–2402.

Shioi A, Katagi M, Okuno Y, Mori K, Jono S, Koyama H, Nishizawa Y. Induction of bone-type alkaline phosphatase in human vascular smooth muscle cells: roles of tumor necrosis factor-alpha and oncostatin M derived from macrophages. Circ Res. 2002; 91: 9–16.

Simonet WS, Lacey DL, Dunstan CR, Kelley M, Chang MS, Luthy R, Nguyen HQ, Wooden S, Bennett L, Boone T, Shimamoto G, DeRose M, Elliott R, Colombero A, Tan HL, Trail G, Sullivan J, Davy E, Bucay N, Renshaw-Gegg L, Hughes TM, Hill D, Pattison W, Campbell P, Sander S, Van G, Tarpley J, Derby P, Lee R, Boyle WJ. Osteoprotegerin: a novel secreted protein involved in the regulation of bone density. Cell. 1997; 89: 309–319.

Tsuda E, Goto M, Mochizuki S, Yano K, Kobayashi F, Morinaga T, Higashio K. Isolation of a novel cytokine from human fibroblasts that specifically inhibits osteoclastogenesis. Biochem Biophys Res Commun. 1997; 234: 137–142.

Mizuno A, Amizuka N, Irie K, Murakami A, Fujise N, Kanno T, Sato Y, Nakagawa N, Yasuda H, Mochizuki S, Gomibuchi T, Yano K, Shima N, Washida N, Tsuda E, Morinaga T, Higashio K, Ozawa H. Severe osteoporosis in mice lacking osteoclastogenesis inhibitory factor/osteoprotegerin. Biochem Biophys Res Commun. 1998; 247: 610–615.

Bucay N, Sarosi I, Dunstan CR, Morony S, Tarpley J, Capparelli C, Scully S, Tan HL, Xu W, Lacey DL, Boyle WJ, Simonet WS. osteoprotegerin-deficient mice develop early onset osteoporosis and arterial calcification. Genes Dev. 1998; 12: 1260–1268.

Jono S, Ikari Y, Shioi A, Mori K, Miki T, Hara K, Nishizawa Y. Serum osteoprotegerin levels are associated with the presence and severity of coronary artery disease. Circulation. 2002; 106: 1192–1194.

Ushiki T, Abe K. Identification of arterial and venous segments of blood vessels using alkaline phosphatase staining of ink/gelatin injected tissues. Arch Histol Cytol. 1998; 61: 215–219.

Hui M, Tenenbaum HC. New face of an old enzyme: alkaline phosphatase may contribute to human tissue aging by inducing tissue hardening and calcification. Anat Rec. 1998; 253: 91–94. <a href="/cgi/external_ref?access_num=10.1002/(SICI)1097-0185(199806)253:3

Price PA, Williamson MK, Nguyen TM, Than TN. Serum levels of the fetuin-mineral complex correlate with artery calcification in the rat. J Biol Chem. 2004; 279: 1594–1600.

Block GA, Hulbert-Shearon TE, Levin NW, Port FK. Association of serum phosphorus and calcium x phosphate product with mortality risk in chronic hemodialysis patients: a national study. Am J Kidney Dis. 1998; 31: 607–617.

Luo G, Ducy P, McKee MD, Pinero GJ, Loyer E, Behringer RR, Karsenty G. Spontaneous calcification of arteries and cartilage in mice lacking matrix GLA protein. Nature. 1997; 386: 78–81.

Kuro-o M, Matsumura Y, Aizawa H, Kawaguchi H, Suga T, Utsugi T, Ohyama Y, Kurabayashi M, Kaname T, Kume E, Iwasaki H, Iida A, Shiraki-Iida T, Nishikawa S, Nagai R, Nabeshima YI. Mutation of the mouse klotho gene leads to a syndrome resembling ageing. Nature. 1997; 390: 45–51.

Jono S, Peinado C, Giachelli CM. Phosphorylation of osteopontin is required for inhibition of vascular smooth muscle cell calcification. J Biol Chem. 2000; 275: 20197–20203.

Tyson KL, Reynolds JL, McNair R, Zhang Q, Weissberg PL, Shanahan CM. Osteo/chondrocytic transcription factors and their target genes exhibit distinct patterns of expression in human arterial calcification. Arterioscler Thromb Vasc Biol. 2003; 23: 489–494.

Shioi A, Nishizawa Y, Jono S, Koyama H, Hosoi M, Morii H. Beta-glycerophosphate accelerates calcification in cultured bovine vascular smooth muscle cells. Arterioscler Thromb Vasc Biol. 1995; 15: 2003–2009.

Doherty MJ, Ashton BA, Walsh S, Beresford JN, Grant ME, Canfield AE. Vascular pericytes express osteogenic potential in vitro and in vivo. J Bone Miner Res. 1998; 13: 828–838.

Schoppet M, Preissner KT, Hofbauer LC. RANK ligand and osteoprotegerin: paracrine regulators of bone metabolism and vascular function. Arterioscler Thromb Vasc Biol. 2002; 22: 549–553.

Lacey DL, Timms E, Tan HL, Kelley MJ, Dunstan CR, Burgess T, Elliott R, Colombero A, Elliott G, Scully S, Hsu H, Sullivan J, Hawkins N, Davy E, Capparelli C, Eli A, Qian YX, Kaufman S, Sarosi I, Shalhoub V, Senaldi G, Guo J, Delaney J, Boyle WJ. Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell. 1998; 93: 165–176.

Hofbauer LC, Schoppet M. Osteoprotegerin: a link between osteoporosis and arterial calcification? Lancet. 2001; 358: 257–259.

Kiel DP, Kauppila LI, Cupples LA, Hannan MT, O?Donnell CJ, Wilson PW. Bone loss and the progression of abdominal aortic calcification over a 25 year period: the Framingham Heart Study. Calcif Tissue Int. 2001; 68: 271–276.

Sattler AM, Schoppet M, Schaefer JR, Hofbauer LC. Novel aspects on RANK ligand and osteoprotegerin in osteoporosis and vascular disease. Calcif Tissue Int. 2004; 74: 103–106.

Wallin R, Wajih N, Greenwood GT, Sane DC. Arterial calcification: a review of mechanisms, animal models, and the prospects for therapy. Med Res Rev. 2001; 21: 274–301.

Kondo T, Kitazawa R, Maeda S, Kitazawa S. 1 alpha,25 dihydroxyvitamin D3 rapidly regulates the mouse osteoprotegerin gene through dual pathways. J Bone Miner Res. 2004; 19: 1411–1419.

Takeo S, Anan M, Fujioka K, Kajihara T, Hiraga S, Miyake K, Tanonaka K, Minematsu R, Mori H, Taniguchi Y. Functional changes of aorta with massive accumulation of calcium. Atherosclerosis. 1989; 77: 175–181.

Qin X, Corriere MA, Matrisian LM, Guzman RJ. Matrix metalloproteinase inhibition attenuates aortic calcification. Arterioscler Thromb Vasc Biol. 2006; 26: 1510–1516.

Inoue T, Kawashima H. 1,25-Dihydroxyvitamin D3 stimulates 45Ca2+-uptake by cultured vascular smooth muscle cells derived from rat aorta. Biochem Biophys Res Commun. 1988; 152: 1388–1394.

Rajasree S, Umashankar PR, Lal AV, Sarma PS, Kartha CC. 1,25-dihydroxyvitamin D3 receptor is upregulated in aortic smooth muscle cells during hypervitaminosis D. Life Sci. 2002; 70: 1777–1788.

Jono S, Nishizawa Y, Shioi A, Morii H. 1,25-Dihydroxyvitamin D3 increases in vitro vascular calcification by modulating secretion of endogenous parathyroid hormone-related peptide. Circulation. 1998; 98: 1302–1306.

Jono S, Nishizawa Y, Shioi A, Morii H. Parathyroid hormone-related peptide as a local regulator of vascular calcification. Its inhibitory action on in vitro calcification by bovine vascular smooth muscle cells. Arterioscler Thromb Vasc Biol. 1997; 17: 1135–1142.

Jono S, McKee MD, Murry CE, Shioi A, Nishizawa Y, Mori K, Morii H, Giachelli CM. Phosphate regulation of vascular smooth muscle cell calcification. Circ Res. 2000; 87: e10–e17.

Yang H, Curinga G, Giachelli CM. Elevated extracellular calcium levels induce smooth muscle cell matrix mineralization in vitro. Kidney Int. 2004; 66: 2293–2299.

Giachelli CM, Speer MY, Li X, Rajachar RM, Yang H. Regulation of vascular calcification: roles of phosphate and osteopontin. Circ Res. 2005; 96: 717–722.

Reynolds JL, Joannides AJ, Skepper JN, McNair R, Schurgers LJ, Proudfoot D, Jahnen-Dechent W, Weissberg PL, Shanahan CM. Human vascular smooth muscle cells undergo vesicle-mediated calcification in response to changes in extracellular calcium and phosphate concentrations: a potential mechanism for accelerated vascular calcification in ESRD. J Am Soc Nephrol. 2004; 15: 2857–2867.

Min H, Morony S, Sarosi I, Dunstan CR, Capparelli C, Scully S, Van G, Kaufman S, Kostenuik PJ, Lacey DL, Boyle WJ, Simonet WS. Osteoprotegerin reverses osteoporosis by inhibiting endosteal osteoclasts and prevents vascular calcification by blocking a process resembling osteoclastogenesis. J Exp Med. 2000; 192: 463–474.


作者单位:Yuichi Orita; Hideya Yamamoto; Nobuoki Kohno; Masaaki Sugihara; Hiroaki Honda; Seiichi Kawamata; Shinji Mito; Nwe Nwe Soe; Masao YoshizumiFrom the Department of Molecular and Internal Medicine (Y.O., H.Y., N.K., M.S.), Graduate School of Biomedical Sciences, the Department of Developmental Biology (

日期:2008年12月28日 - 来自[2007年第27卷第9期]栏目
循环ads

Aortic Valve Calcification

【摘要】  Background- Aortic valve calcification (AVC) is considered degenerative. Recent data suggested links to atherosclerosis or coronary disease (CAD).

Methods and Results- AVC and coronary artery calcifications (CAC) were prospectively assessed by Electron-Beam-Computed-Tomography in 262 population-based research participants 60 years. AVC was frequent (27%) with aging ( P <0.01) and in men ( P <0.05). AVC was associated with diabetes, hypertension, higher body-mass-index, and serum glucose (all P <0.05). AVC was a marker of higher prevalence ( P <0.01) and severity of CAD (CAC score: 441±802 versus 265±566, P <0.05) independently of age. After follow-up of 3.8±0.9 years, AVC score increased (94±271 versus 54±173, P <0.01, +11±32 U/year), faster with higher baseline AVC score ( P <0.01). Compared with participants remaining free of AVC, de novo acquisition of AVC was associated with higher LDL-cholesterol (141±31 versus 121±27 mg/dL, P <0.05) and faster CAC progression (+78±87 versus +28±47 U/year, P <0.05). In multivariate analysis, LDL-cholesterol independently determined AVC acquisition while higher baseline AVC scores determined faster progression of existing AVC.

Conclusion- In the population, AVC is frequent with aging and atherosclerotic risk factors. AVC is a marker of subclinical CAD. AVC is progressive, appearing de novo with progressive atherosclerosis whereas established AVC progresses independently of atherosclerotic risk factors and faster with increasing initial AVC loads.

Aortic valve calcification (AVC) in 262 population-based participants 60 years was frequent (27%) with aging, with diabetes, and with coronary calcification. After 3.8±0.9 years, AVC score and prevalence increased. New AVC acquisition occurs with high LDL-cholesterol and progressive atherosclerosis. Larger established AVC determines faster AVC progression independently of atherosclerotic risk factors.

【关键词】  aortic valve computed tomography calcification atherosclerosis epidemiology


Introduction


With the striking decrease in the prevalence of rheumatic disease, calcification of the aortic valve (AVC) is the most frequent cause of aortic stenosis (AS) in Western countries (Monckeberg disease). 1 Calcific AS has long been considered as a passive and degenerative process but recent data challenged this concept, showing that AVC is an active and highly regulated process, 2-4 with histological similarities to atherosclerosis. 5,6 The interpretation of AVC as an atherosclerotic phenomenon is supported by the 50% increased risk of cardiovascular death and myocardial infarction in subjects with aortic sclerosis (aortic valve thickening without obstruction to left ventricular outflow) and no known cardiovascular disease, 7 which is presumed to be attributable to associated coronary artery disease (CAD). This interpretation was extended to all AVC grades by observational studies reporting diffuse atherosclerosis with aortic sclerosis, 8-10 and suggesting that statin treatment reduced AS progression. 11-13 Thus, AS and AVC have been mostly considered atherosclerotic processes potentially preventable by lipid-lowering treatment using statins. 14


However, important discordant data have surfaced. Patients with the most extreme form of AVC, ie, AS, often have normal coronary angiograms, 15 and the proof of association of AVC with coronary atherosclerosis remains circumstantial 16 and uncertain. The link of hyperlipidemia to AS progression 17 appears weak 18 or even insignificant. 12 Most importantly, the only available clinical trial of high-dose statin treatment for AS, recently published, demonstrated no association between lipids and AS progression and showed no effect of statin therapy on AS or AVC progression. 19 Thus, to date, the determinants of AS and AVC progression remain unclear.


Another source of uncertainty relates to AVC assessment by standard Fluoroscopy or Echocardiography, both subjective and qualitative. Thus, Echocardiographic data, although pioneering, 7 warrant population assessment of reliably measured AVC to assess their determinants and progression which are uncertain. Electron-Beam Computed Tomography (EBCT) is a reliable, objective, and quantitative method to measure simultaneously AVC 20 and coronary arteries calcifications (CAC) 21,22 an established marker of CAD. 23,24


The Epidemiology of Coronary Artery Calcification study is a population-based study in which randomly selected Olmsted County residents are prospectively followed with sequential evaluation of risk factors and EBCT. 25,26 This epidemiological study offers the unique opportunity to evaluate AVC prevalence, progression and link to CAD.


Methods


Study Population


In the Epidemiology of Coronary Artery Calcification (ECAC) study, 25,26 1376 randomly selected adult Olmsted County residents of all ages gave written informed consent and were prospectively and repeatedly examined by EBCT for heart calcifications along with comprehensive clinical and cardiac risk factors assessment. Participants were excluded from the study if they were pregnant or lactating at enrollment or had previous cardiac surgery. The present substudy limited enrollment to participants 60 years of age who had baseline and follow-up EBCT performed after 1995 (date after which scans were electronically saved). The Aortic Valve Calcification substudy was approved by our Institutional Review Board. At baseline and follow-up, cardiovascular risk factors were prospectively determined by participants? interview, physical examination, and fasting blood sampling. Fibrinogen levels and Creatinine clearance were measured at follow-up. Follow-up to assess AS development was obtained clinically with physical examination in all patients and with clinically indicated Doppler-Echocardiography in 129 participants.


Measures and Definitions


Participants reported current medication use, education, history of smoking, and physician-diagnosed hypertension, myocardial infarction, stroke, or diabetes. Resting systolic blood pressure (SBP) and diastolic blood pressure (DBP) levels were measured in the right arm with a random-zero sphygmomanometer (Hawksley and Sons). Three measures at least 2 minutes apart were taken and the average of the second and third measurements was used. Participants were considered hypertensive if the average SBP was 140 mm Hg and/or the average DBP was 90 mm Hg, or if they reported a prior diagnosis of and treatment for hypertension and were currently using antihypertensive medications. Participants were considered diabetic if they were using insulin or oral hypoglycemic agents. Body mass index was calculated as weight/height 2 (kg/m 2 ).


Standard enzymatic methods were used to measure total cholesterol, HDL cholesterol, and triglycerides after overnight fasting.15 LDL cholesterol was calculated with the Friedwald equation. Plasma glucose was measured by the glucose oxidase method after overnight fasting. Creatinine clearance was calculated using the Cockroft formula and the fibrinogen measured by immunoturbidimetric assay.


Electron-Beam-Computed-Tomography


We and others have demonstrated the accuracy and reproducibility of EBCT for AVC 20 and CAC quantification. 23,24 Acquisition with Imatron C-100 or C-150 was triggered at 80% of RR interval for two chest scan-runs 27 of 30 to 40 contiguous transverses slices (3-mm thickness, 100 ms/slice). Calcification was defined as 130 Hounsfield units. Integrative scores were calculated (accounting for pixel density) 21 in Agatston units (AU) separately for aortic valve and coronary artery calcifications, using a dedicated software 28,29 by observers blinded to all other data. Calcium scoring was semi-automated: areas of calcification are highlighted and those corresponding to the aortic valve or the coronary arteries are selected by the operator. Two runs were scored separately and averaged and calcification was considered present on the basis of this final score. Annualized progression rates of AVC and CAC were calculated as the difference between baseline and follow-up score divided by the follow-up duration. Progression, stability, or regression were also defined categorically based on a previously validated regression approach, 25 which avoid misdiagnosis of change in regard to inter-run variability. Follow-up score (average of the 2 runs) is compared with the 95% confidence limits of the baseline score (based on inter-run variability specific to the specific baseline score). This 95% confidence interval increases with the baseline score (eg, [78 to 123] for a score of 100 and [440 to 580] for a score of 500). Progression was 95% CI upper limit, regression by a follow-up score <95% CI lower limit and stability by a follow-up score between the 95% CI lower and upper limits.


Statistical Analysis


Data are presented as mean±SD or percent. Comparisons between groups used Student t test or 2 test as appropriate and the Wilcoxon rank sum test for non-normally distributed variables. Bivariate logistic regressions assessed the association between AVC presence and each cardiovascular risk factor after adjustment for age and gender. Correlations between AVC and CAC scores or annualized progression rates used Spearman correlations. Determinants of AVC progression overall and in patients with no AVC at baseline were assessed by logistic regression. Stepwise multiple linear regression analysis using logarithmic transformation of baseline AVC score (which was close to normally distributed) identified determinants of AVC progression with established AVC at baseline. To assess whether CAC score was higher in patients with AVC after adjustment for age and gender, CAC score was divided into quartiles and a proportional odds model was performed. A proportional odds model was also used to compare annualized CAC progression rate after adjustment for baseline CAC quartiles. For both analyses, proportional odds assumptions were verified and not violated. P <0.05 was considered significant.


Results


Baseline Characteristics


Two hundred sixty two participants aged 60 or older were enrolled, and their characteristics are summarized in Table 1. History of smoking was observed in 124 participants (47%), hypertension in 179 (68%), and diabetes in 25 (10%). Fourteen participants (5%) had clinical history of CAD and 8 (3%) of cerebrovascular disease. AVC and CAC scores varied widely, respectively: 54±173 (range 0 to 1944) and 312±640 (range 0 to 5711) AU.


TABLE 1. Baseline Clinical Characteristics, Laboratory Measurements, Coronary Artery, and Aortic Valve Calcification Scores in the Overall Population and in Subgroups Defined According to the Presence or not of Aortic Valve Calcification


Determinants of AVC and Association With Cardiac Risk Factors


Baseline AVC prevalence was 27% and increased with age: 19% between 60 and 69 years (n=171) and 42% after 70 years (n=91) (Odds ratio 1.12 [1.06 to 1.18] per year, P <0.01). AVC prevalence was higher in men than in women (33 versus 22%, P =0.05) despite similar age (68±5 versus 68±5 years, P =0.94), but AVC prevalence increased with age both in men and women (23% and 16% between 60 and 69 years and 50% and 35% after 70 years, respectively). Higher prevalence of AVC in men persisted after adjustment for age (OR=1.79 [1.01 to 3.19], P <0.05). Participants with AVC had higher prevalence of cardiovascular risk factors ( Table 1 ), with more frequent diabetes, higher body mass index, systolic blood pressure, serum glucose, and marginally more frequent hypertension ( P =0.06). No difference in tobacco-pack-years consumed or cholesterol levels was observed. After adjustment for age and gender, diabetes (OR=2.9 [1.2 to 6.9], P <0.05), hypertension (OR=2.0 [1.1 to 3.9], P <0.05), serum glucose (OR=4.5 per mg/dL increment [1.2 to 16.9], P <0.05), and body mass index (OR=1.12 per kg/m 2 increment [1.06 to 1.19], P <0.01) remained independently associated with the presence of AVC. Diabetes, serum glucose, and body mass index were also significantly associated with AVC score (used as a continuous variable) (all P <0.05).


Progression of Aortic Valve Calcification


Overall


After 3.8±0.9 years (range 1.8 to 5.8) AVC prevalence and score increased (respectively, 34 versus 27% and 94±271 versus 54±173 AU, P <0.01) with an annualized score progression rate of +11±32 AU/year. Based of the regression approach described in methods, progression was noted in 74 participants (28%). No regression was observed. Progression was observed in 55/70 participants with baseline AVC and in 19/192 participants with no AVC at baseline (acquisition of AVC or de novo AVC) (79 versus 10%, P <0.01, OR=33,[16-72] P <0.01). Participants with AVC at baseline had also higher annualized aortic progression rate (+39±53 versus +1±4 U/year, P <0.01) ( Figure, left panel). No atherosclerotic factor showed linear association to AVC progression. Using a logistic regression, presence of baseline AVC ( P <0.001) and LDL ( P =0.01) predicted AVC progression but with a significant interaction AVC-LDL ( P =0.04). Thus, determinants of AVC progression were analyzed separately in participants with and without AVC at baseline.


Effect of baseline aortic valve calcification (AVC) on subsequent calcification progression: In panel A (left), AVC score (mean±SE) progression from baseline to follow-up is displayed stratified according to presence or absence of AVC at baseline, showing that AVC score progression is higher in participants with baseline AVC. In panel B (right), displaying only participants with established baseline AVC, the AVC score (mean±SE) progression from baseline to follow-up is stratified according to baseline AVC score tercile showing faster progression with higher AVC load.


Progression in Participants With no AVC at Baseline


Among the 192 participants with no AVC at baseline, 19 developed de novo AVC at follow-up (10%). Total- and LDL-cholesterol were higher in participants with de novo AVC than in those who remained free of AVC at follow-up (235±39 versus 209±33 mg/dL, P <0.001 and 141±31 versus 121±27 mg/dL, P =0.003 respectively). Triglycerides was of borderline statistical significance (209±101 versus 175±77 mg/dL, P =0.07) while age (67±4 versus 67±5 years, P =0.85) and male gender (37% versus 40%, P =0.80) were not different. These participants showed also at follow-up a trend for higher fibrinogen level (351±65 versus 318±75 mg/dL, P =0.08) but no difference in creatinine clearance (51±17 versus 55±17 mL/min, P =0.34). In multivariate analysis, the only independent baseline predictor of AVC acquisition was LDL-cholesterol (RR=1.29 per 10 mg/dL increment [1.08 to 1.55], P <0.01).


Progression in Participants With Baseline AVC


In participants with baseline AVC, aortic score increased at follow-up (337±441 versus 197±291, P <0.01) and faster with baseline aortic score tercile (+11±12, +20±17, and +86±71/year, respectively in the lowest, mid, and highest AVC score tercile, P <0.01) ( Figure, right panel). AVC progression rate was not different according to age, gender, or cardiovascular risk factors ( Table 2 ). There was no association with follow-up Fibrinogen ( P =0.53) and creatinine clearance ( P =0.16). In multivariate analysis, the only independent determinant of faster annualized AVC progression was higher baseline AVC score ( P <0.01).


TABLE 2. Annualized Progression Rate of Aortic Valve Calcification in Participants With Established Baseline Aortic Calcification According to Baseline Characteristics (Presence for Categorical Variables and Median for Continuous Variables)


Clinical Correlates


Progression to AS (mean gradient 10 mm Hg) was observed in 10 participants at last follow-up. These 10 participants reached a lower aortic valve area (AVA) (1.66±0.48 versus 2.28±0.56 cm2, P <0.01) and had faster AVC progression rates than the remaining population (96±80 versus 8±24 AU/year, P <0.01). Participants with baseline AVC compared with those with no AVC at baseline developed AS more frequently during follow-up (13% versus 0.5%, P <0.0001). Five participants developed moderate or severe AS (mean gradient 20 mm Hg or AVA <1.5 cm2) and were only noted among those with AVC at baseline (7% versus 0%, P <0.001).


Association With Subclinical Coronary Atherosclerosis (CAC)


Baseline


Although correlation between AVC and CAC scores was weak (Rho=0.28, P <0.01), AVC was associated with higher prevalence of CAC (94 versus 72%, P <0.01; OR=6.3 [2.4 to 21.4], P <0.01), higher prevalence of CAC score 200 23 (47 versus 27%, OR=2.5 [1.4 to 4.4], P <0.01), and with higher CAC score (441±802 versus 265±566, P <0.01). CAC score increased with age ( r =0.26, P <0.01), but prevalence of CAC score 200 was higher in participants with AVC irrespective of age (41 versus 23% under 70 years and 53 versus 36% after 70 years, P =0.01). Adjusting for age and gender, AVC was independently associated with high odds of CAC presence (Odds-Ratio 4.7[1.7 to 16.6], P <0.01), high odds of CAC score 200 (Odds-Ratio 1.9[1.0 to 3.5], P =0.05), and with higher CAC score ( P =0.01). The independent association of AVC with high odds of CAC presence persisted unchanged (Odds-Ratio 4.4[1.5 to 15.9], P <0.01) after further adjustment for cardiovascular risk factors (blood pressure, hypertension, smoking, diabetes, cholesterol) and persisted ( P =0.01) even after exclusion of diabetic participants.


CAC and AVC Progression


Coronary score also increased (422±705 versus 312±640, P <0.01) with an annualized progression rate of +38±55 AU/year. There was a modest but significant correlation between AVC and CAC progression rates (Rho=0.35, P <0.01).


Compared with participants remaining free of AVC, participants with de novo AVC had higher CAC score at baseline (643±901 versus 223±503 AU, P =0.04) and at follow-up (967±1214 versus 328±637 AU, P =0.008), and most importantly had the faster CAC annualized progression rate (78±87 versus 29±47 AU per year, P =0.003) even after adjustment for baseline coronary score ( P <0.05). In contrast, adjusting for baseline CAC, annualized CAC progression rate was not different in participants with established AVC and in those remaining free of AVC ( P =0.41).


Discussion


This first population-based study with prospective EBCT performance allows determination of AVC, CAC, and atherosclerotic risk factors, and allows analysis of the determinants of AVC progression in the population. After the age of 60 years, AVC prevalence is high (27%), increases with age, and is higher in men than women. AVC presence is associated with cardiovascular risk factors, is a marker of subclinical CAD revealed by CAC independently of age and diabetes, and is associated with a higher risk of developing AS. AVC is progressive, with two distinctive patterns. Acquisition of AVC (de novo AVC) is characterized by high lipid and fibrinogen levels and fast CAC progression suggestive of rapidly progressive atherosclerosis. Conversely, progression of established AVC is fastest with highest baseline calcification score and unrelated to cardiovascular risk factors. These data emphasize the importance of diagnosing AVC as a marker of subclinical CAD, which may lead to more intense risk factor management. It is also essential to monitor AVC progression to AS and to devise new strategies of prevention of AVC progression.


Prevalence and Determinants of AVC


In our population based study, AVC measured by EBCT was common, present in 27% of subjects 60 or older, similar to the Cardiovascular Health Study aortic valve sclerosis prevalence after 65 (29%), 7 showing that this observation is not an echocardiographic artifact. AVC is an active valve lesion and not simply a consequence of aging. Indeed, prevalence of AVC increases with age and is higher in men than women, but shows strong association to cardiovascular risk factors, 30 even after adjustment for age and gender. Our study provides novel insights in demonstrating the association of AVC to diabetes, body mass index, and elevated serum glucose. Vascular calcification is a hallmark of diabetes and insulin resistance, 31 and our study extends this observation to aortic valve calcification. The mechanistic link between glycemic dysregulation and AVC is yet undefined and appears unrelated to diabetic nephropathy.


Progression of AVC


AVC progression is the mechanism by which the intrinsic valvular complication of AVC, ie, progression to AS, occurs. Our study shows that the observation of AVC is not benign and that progression to AS occurs frequently. Although it is known that AS hemodynamically progresses over time, with valve area declining approximately by 0.1 cm 2 /year, 12 the processes and determinants leading to this progression are poorly known. The concept of "degenerative" passive calcific valvular deposition is no longer accepted, and AS is considered an active atherosclerotic disease. 14 AVC progression was even found in pilot data, 17 directly linked to hyperlipidemia. However, the link AS-atherosclerosis was challenged by the lack of association between cholesterol levels and AS progression, 12,13,18 and most importantly, by the lack of effect of the strongest antiatherosclerotic treatment available, high-dose statins, on AS and AVC progression. 19 Our study provides insights dissociating two different phases of AVC progression and allows reconciling apparently discordant data.


The early phase fits well the atherosclerotic concepts of aortic calcification with hyperlipidemia, inflammation, and rapid coronary calcification. This phase is consistent with previous studies on early aortic lesion pathology, 6 characterized by inflammation and oxidized lipoproteins deposition 5,32 colocalized with early calcium deposit, 6,32 and with recent experimental data. 33 The secondary phase of calcium accumulation with ultimately ossification, 4 is unrelated to vascular risk factors and AVC grows faster with calcification load. 34 This phase fits the biological concept of centripetal expansion of calcific nodules, which are surrounded by osteoblast-like cells, 35 and fits the observed independence of AS progression from lipid profile. 12,19 This concept of two different phases of AVC progression is essential in comprehending statin effect and properly targeting a clinical trial. Observational retrospective studies suggested that statins may reduce AS progression, 12,13,36,37 but a recent clinical trial showed that, at an advanced stage, statins prevented neither AS progression nor calcium deposition. 19 Thus, when AVC grows independently of lipid levels as shown by our study, statins do not prevent calcium accumulation. Conversely, at an early stage of aortic sclerosis, when de novo calcification is lipid-dependent and inflammatory, statins may prevent AVC progression, as shown experimentally, 33,38 and may prevent the frequent progression to AS. 39 Thus our study, as well as the most recent data, provide essential support for a clinical trial of statins in patients with early rather than advanced valve lesions, sclerosis or early AVC, to prevent progression to AS.


Clinically, calcified valves accumulate calcium faster but the relationship between calcification load and hemodynamic AS severity is non-linear. 20 The same calcium accumulation may result in smaller valve area reduction in patients with more severe AS. Combinations of these various factors explain the large variability of AS progression between individuals. 12 However, faster calcific growth with AVC load noted in the present study, suggests that for any given AS severity, a larger baseline calcific load may lead to faster calcific and hemodynamic progression and worse prognosis. 20,40 Therefore, monitoring AVC load in patients with aortic valve disease is an important part of the clinical monitoring.


Recent ACC/AHA guidelines underline the importance of risk stratification by calcium measurements, especially in asymptomatic patients with severe AS or in patients with less severe AS undergoing coronary artery bypass graft. 41 The method for AVC assessment is not specifically mentioned. Echocardiography versus CT is simple but qualitative, subjective, depends on gain settings, and is not specific (reflects both fibrosis and calcification), resulting in more severe estimation of AVC grade. 20


AVC and Subclinical Coronary Artery Atherosclerosis


Recently, the Cardiovascular Health Study reported increased cardiac mortality in participants with aortic valve sclerosis, 7 a finding quite surprising with nonobstructive valve lesions. Whether echocardiographic underestimation of the valve stenosis or true linkage of minor AVC with subclinical CAD explained the excess mortality remained mysterious as most studies attempting to address this issue were subject to referral and selection bias. 16,42,43 Our study provides novel population-based information on the association independent of age between CAD and AVC. The association of AVC to cardiovascular risk factors 30 led to the suspicion of a CAD-AVC association, 7 but our study proves the excess CAD burden with AVC in the general population even accounting for atherosclerotic risk factors. CAC assessed by EBCT is a sensitive and reliable marker of presence and extent of CAD, 23,24,44 and a high score predicts high rate of subsequent coronary events. 45-47 Thus, by demonstrating that AVC is a marker of higher prevalence and severity of CAC in the population independently of age and gender, the present study links formally AVC to the CAD that leads to poor outcome. Also, the rapidly progressive CAD accompanying AVC acquisition emphasizes the link of AVC presence to subsequent risk. Future prospective studies should evaluate whether AVC presence and/or acquisition identify groups at high risk for coronary events and whether active treatment prevents these events.


However, our study also provides important insights regarding association between CAD and AVC. Indeed, in a purely atherosclerotic hypothesis of calcification progression, 14 the fact that most patients with severe AS attributable to massive valve calcification present without obstructive CAD, 15,48,49 would be incomprehensible. AVC, after the initial atherosclerotic phase, grows independently of most risk factors. 4 Thus, AVC presence is a marker of presence and extend of CAD but massive AVC is not a marker for extensive CAD.


Limitations


The present study is, with the Cardiovascular Health Study (CHS), 7,30 the only population-based study of early aortic valve lesions. Nevertheless biases cannot be ruled out, either because of the methods (echocardiography for CHS) or the requirement of a second EBCT (excluding patients who died in the interim). However, both studies are consistent regarding the high prevalence of aortic valve sclerosis or calcification, and the association to cardiovascular risk factors. Thus, this consistency underscores the public health problem detected. Our study is smaller in size and the association of risk factors of lesser significance to AVC may not be detected. However, our sample provides 84% power to detect correlations of Rho=0.18, so that the major determinants of AVC presence and progression can be detected. Only patients 60 years old were considered in the present study. Whether our findings can be extended to younger patients, especially those with bicuspid aortic valve, needs further evaluation. Importantly, although we cannot assess prognosis because participants with both baseline and follow-up CT were enrolled, we can demonstrate AVC progression leading to aortic stenosis, its determinants, and association to CAD, which represent novel information of particular value because population-based.


We did not perform repeated coronary angiography to assess coronary disease, but CAC burden is a reliable surrogate of subclinical coronary atherosclerosis. 24 Coronary and aortic calcifications appear with aging, but their association is not incidental and is independent of age, gender, and diabetes, and as such may be a marker of future coronary events. 47,50 Also, to assess for AS development, Doppler-Echocardiography was performed when clinically indicated in only 129 participants. In addition, anatomy of the aortic valve (bi- or tricuspide) was not available. Finally, for AVC and CAC quantification, we used the previously validated Agatston score. Similar results were obtained using a volumetric method.


Conclusion


In the population, AVC prevalence is high, increases with age, and is higher in men than women. However, AVC is not a passive, degenerative phenomenon. AVC is associated with cardiovascular risk factors, particularly diabetes, and is a marker of subclinical CAD. AVC is progressive leading frequently to AS, with two different patterns. De novo AVC occurs in a context of hyperlipidemia and progressive atherosclerosis. Progression of established AVC is unrelated to cardio-vascular risk factors and is faster with high AVC load suggesting exponential calcification growth. These new epidemiologic data emphasize the importance of detecting and of testing new approaches for preventing AVC.


Acknowledgments


We appreciate the help of Professor Richard Robb, Ron Karwoski, and Mahlon Stacy from Mayo Biomedical Imaging Resource with Analyze software and image processing.


Sources of Funding


Dr Messika-Zeitoun was supported by a grant from the Fédération Française de Cardiologie.


This study was supported in part by National Institutes of Health grants HL46292 and HL64928.


Disclosures


None.

【参考文献】
  Iung B, Baron G, Butchart EG, Delahaye F, Gohlke-Barwolf C, Levang OW, Tornos P, Vanoverschelde JL, Vermeer F, Boersma E, Ravaud P, Vahanian A. A prospective survey of patients with valvular heart disease in Europe: The Euro Heart Survey on Valvular Heart Disease. Eur Heart J. 2003; 24: 1231-1243.

Rajamannan NM, Subramaniam M, Rickard D, Stock SR, Donovan J, Springett M, Orszulak T, Fullerton DA, Tajik AJ, Bonow RO, Spelsberg T. Human aortic valve calcification is associated with an osteoblast phenotype. Circulation. 2003; 107: 2181-2184.

O?Brien KD, Kuusisto J, Reichenbach DD, Ferguson M, Giachelli C, Alpers CE, Otto CM. Osteopontin is expressed in human aortic valvular lesions. Circulation. 1995; 92: 2163-2168.

Mohler ER, 3rd, Gannon F, Reynolds C, Zimmerman R, Keane MG, Kaplan FS. Bone formation and inflammation in cardiac valves. Circulation. 2001; 103: 1522-1528.

O?Brien KD, Reichenbach DD, Marcovina SM, Kuusisto J, Alpers CE, Otto CM. Apolipoproteins B, (a), and E accumulate in the morphologically early lesion of ?degenerative? valvular aortic stenosis. Arterioscler Thromb Vasc Biol. 1996; 16: 523-532.

Otto CM, Kuusisto J, Reichenbach DD, Gown AM, O?Brien KD. Characterization of the early lesion of ?degenerative? valvular aortic stenosis. Histological and immunohistochemical studies. Circulation. 1994; 90: 844-853.

Otto CM, Lind BK, Kitzman DW, Gersh BJ, Siscovick DS. Association of aortic-valve sclerosis with cardiovascular mortality and morbidity in the elderly. N Engl J Med. 1999; 341: 142-147.

Agmon Y, Khandheria BK, Meissner I, Sicks JR, O?Fallon WM, Wiebers DO, Whisnant JP, Seward JB, Tajik AJ. Aortic valve sclerosis and aortic atherosclerosis: different manifestations of the same disease? Insights from a population-based study. J Am Coll Cardiol. 2001; 38: 827-834.

Adler Y, Vaturi M, Wiser I, Shapira Y, Herz I, Weisenberg D, Sela N, Battler A, Sagie A. Nonobstructive aortic valve calcium as a window to atherosclerosis of the aorta. Am J Cardiol. 2000; 86: 68-71.

Aronow WS, Ahn C, Kronzon I. Association of valvular aortic stenosis with symptomatic peripheral arterial disease in older persons. Am J Cardiol. 2001; 88: 1046-1047.

Novaro GM, Tiong IY, Pearce GL, Lauer MS, Sprecher DL, Griffin BP. Effect of hydroxymethylglutaryl coenzyme a reductase inhibitors on the progression of calcific aortic stenosis. Circulation. 2001; 104: 2205-2209.

Bellamy MF, Pellikka PA, Klarich KW, Tajik AJ, Enriquez-Sarano M. Association of cholesterol levels, hydroxymethylglutaryl coenzyme-A reductase inhibitor treatment, and progression of aortic stenosis in the community. J Am Coll Cardiol. 2002; 40: 1723-1730.

Rosenhek R, Rader F, Loho N, Gabriel H, Heger M, Klaar U, Schemper M, Binder T, Maurer G, Baumgartner H. Statins but not angiotensin-converting enzyme inhibitors delay progression of aortic stenosis. Circulation. 2004; 110: 1291-1295.

Chan KL. Is aortic stenosis a preventable disease? J Am Coll Cardiol. 2003; 42: 593-599.

Rapp AH, Hillis LD, Lange RA, Cigarroa JE. Prevalence of coronary artery disease in patients with aortic stenosis with and without angina pectoris. Am J Cardiol. 2001; 87: 1216-1217; A7.

Adler Y, Vaturi M, Herz I, Iakobishvili Z, Toaf J, Fink N, Battler A, Sagie A. Nonobstructive aortic valve calcification: a window to significant coronary artery disease. Atherosclerosis. 2002; 161: 193-197.

Pohle K, Maffert R, Ropers D, Moshage W, Stilianakis N, Daniel WG, Achenbach S. Progression of aortic valve calcification: association with coronary atherosclerosis and cardiovascular risk factors. Circulation. 2001; 104: 1927-1932.

Palta S, Pai AM, Gill KS, Pai RG. New insights into the progression of aortic stenosis: implications for secondary prevention. Circulation. 2000; 101: 2497-2502.

Cowell SJ, Newby DE, Prescott RJ, Bloomfield P, Reid J, Northridge DB, Boon NA. A randomized trial of intensive lipid-lowering therapy in calcific aortic stenosis. N Engl J Med. 2005; 352: 2389-2397.

Messika-Zeitoun D, Aubry MC, Detaint D, Bielak LF, Peyser PA, Sheedy PF, Turner ST, Breen JF, Scott C, Tajik AJ, Enriquez-Sarano M. Evaluation and clinical implications of aortic valve calcification measured by electron-beam computed tomography. Circulation. 2004; 110: 356-362.

Agatston AS, Janowitz WR, Hildner FJ, Zusmer NR, Viamonte M Jr, Detrano R. Quantification of coronary artery calcium using ultrafast computed tomography. J Am Coll Cardiol. 1990; 15: 827-832.

O?Rourke RA, Brundage BH, Froelicher VF, Greenland P, Grundy SM, Hachamovitch R, Pohost GM, Shaw LJ, Weintraub WS, Winters WL Jr, Forrester JS, Douglas PS, Faxon DP, Fisher JD, Gregoratos G, Hochman JS, Hutter AM Jr, Kaul S, Wolk MJ. Am College of Cardiology/Am Heart Association Expert Consensus document on electron-beam computed tomography for the diagnosis and prognosis of coronary artery disease. Circulation. 2000; 102: 126-140.

Rumberger JA, Simons DB, Fitzpatrick LA, Sheedy PF, Schwartz RS. Coronary artery calcium area by electron-beam computed tomography and coronary atherosclerotic plaque area. A histopathologic correlative study. Circulation. 1995; 92: 2157-2162.

Sangiorgi G, Rumberger JA, Severson A, Edwards WD, Gregoire J, Fitzpatrick LA, Schwartz RS. Arterial calcification and not lumen stenosis is highly correlated with atherosclerotic plaque burden in humans: a histologic study of 723 coronary artery segments using nondecalcifying methodology. J Am Coll Cardiol. 1998; 31: 126-133.

Bielak LF, Sheedy PF, 2nd, Peyser PA. Coronary artery calcification measured at electron-beam CT: agreement in dual scan runs and change over time. Radiology. 2001; 218: 224-229.

Peyser PA, Bielak LF, Chu JS, Turner ST, Ellsworth DL, Boerwinkle E, Sheedy PF, 2nd. Heritability of coronary artery calcium quantity measured by electron beam computed tomography in asymptomatic adults. Circulation. 2002; 106: 304-308.

Maher JE, Raz JA, Bielak LF, Sheedy PF, 2nd, Schwartz RS, Peyser PA. Potential of quantity of coronary artery calcification to identify new risk factors for asymptomatic atherosclerosis. Am J Epidemiol. 1996; 144: 943-953.

Reed JE, Rumberger JA, Davitt PJ, Kaufmann RB, Sheedy PF, 3rd. System for quantitative analysis of coronary calcification via electron beam computed tomography. In: Hoffman EA, Archarya R.S., ed. Medical imaging, 1994: physiology and function from multidimensional images. Bellingham Wash: International Society for Optical Engineereing; 1994: 43-53.

Robb RA. The biomedical imaging resource at Mayo Clinic. IEEE Trans Med Imaging. 2001; 20: 854-867.

Stewart BF, Siscovick D, Lind BK, Gardin JM, Gottdiener JS, Smith VE, Kitzman DW, Otto CM. Clinical factors associated with calcific aortic valve disease. Cardiovascular Health Study. J Am Coll Cardiol. 1997; 29: 630-634.

Meigs JB, Larson MG, D?Agostino RB, Levy D, Clouse ME, Nathan DM, Wilson PW, O?Donnell CJ. Coronary artery calcification in type 2 diabetes and insulin resistance: the framingham offspring study. Diabetes Care. 2002; 25: 1313-1319.

Olsson M, Thyberg J, Nilsson J. Presence of oxidized low density lipoprotein in nonrheumatic stenotic aortic valves. Arterioscler Thromb Vasc Biol. 1999; 19: 1218-1222.

Rajamannan NM, Subramaniam M, Springett M, Sebo TC, Niekrasz M, McConnell JP, Singh RJ, Stone NJ, Bonow RO, Spelsberg TC. Atorvastatin inhibits hypercholesterolemia-induced cellular proliferation and bone matrix production in the rabbit aortic valve. Circulation. 2002; 105: 2660-2665.

Yoon HC, Emerick AM, Hill JA, Gjertson DW, Goldin JG. Calcium begets calcium: progression of coronary artery calcification in asymptomatic subjects. Radiology. 2002; 224: 236-241.

Mohler ER, 3rd, Chawla MK, Chang AW, Vyavahare N, Levy RJ, Graham L, Gannon FH. Identification and characterization of calcifying valve cells from human and canine aortic valves. J Heart Valve Dis. 1999; 8: 254-260.

Novaro GM, Sachar R, Pearce GL, Sprecher DL, Griffin BP. Association between apolipoprotein E alleles and calcific valvular heart disease. Circulation. 2003; 108: 1804-1808.

Shavelle DM, Takasu J, Budoff MJ, Mao S, Zhao XQ, O?Brien KD. HMG CoA reductase inhibitor (statin) and aortic valve calcium. Lancet. 2002; 359: 1125-1126.

Rajamannan NM, Subramaniam M, Stock SR, Stone NJ, Springett M, Ignatiev KI, McConnell JP, Singh RJ, Bonow RO, Spelsberg TC. Atorvastatin inhibits calcification and enhances nitric oxide synthase production in the hypercholesterolaemic aortic valve. Heart. 2005; 91: 806-810.

Cosmi JE, Kort S, Tunick PA, Rosenzweig BP, Freedberg RS, Katz ES, Applebaum RM, Kronzon I. The risk of the development of aortic stenosis in patients with "benign" aortic valve thickening. Arch Intern Med. 2002; 162: 2345-2347.

Rosenhek R, Binder T, Porenta G, Lang I, Christ G, Schemper M, Maurer G, Baumgartner H. Predictors of outcome in severe, asymptomatic aortic stenosis. N Engl J Med. 2000; 343: 611-617.

Bonow RO, Carabello BA, Kanu C, de Leon AC Jr, Faxon DP, Freed MD, Gaasch WH, Lytle BW, Nishimura RA, O?Gara PT, O?Rourke RA, Otto CM, Shah PM, Shanewise JS, Smith SC Jr, Jacobs AK, Adams CD, Anderson JL, Antman EM, Fuster V, Halperin JL, Hiratzka LF, Hunt SA, Nishimura R, Page RL, Riegel B. ACC/AHA 2006 guidelines for the management of patients with valvular heart disease: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines (writing committee to revise the 1998 Guidelines for the Management of Patients With Valvular Heart Disease): developed in collaboration with the Society of Cardiovascular Anesthesiologists: endorsed by the Society for Cardiovascular Angiography and Interventions and the Society of Thoracic Surgeons. Circulation. 2006; 114: e84-e231.

Chandra HR, Goldstein JA, Choudhary N, O?Neill CS, George PB, Gangasani SR, Cronin L, Marcovitz PA, Hauser AM, O?Neill WW. Adverse outcome in aortic sclerosis is associated with coronary artery disease and inflammation. J Am Coll Cardiol. 2004; 43: 169-175.

Aronow WS, Ahn C, Shirani J, Kronzon I. Comparison of frequency of new coronary events in older subjects with and without valvular aortic sclerosis. Am J Cardiol. 1999; 83: 599-600, A8.

Rumberger JA, Sheedy PF, 3rd, Breen JF, Schwartz RS. Coronary calcium, as determined by electron beam computed tomography, and coronary disease on arteriogram. Effect of patient?s sex on diagnosis. Circulation. 1995; 91: 1363-1367.

Secci A, Wong N, Tang W, Wang S, Doherty T, Detrano R. Electron beam computed tomographic coronary calcium as a predictor of coronary events: comparison of two protocols. Circulation. 1997; 96: 1122-1129.

Arad Y, Spadaro LA, Goodman K, Lledo-Perez A, Sherman S, Lerner G, Guerci AD. Predictive value of electron beam computed tomography of the coronary arteries. 19-month follow-up of 1173 asymptomatic subjects. Circulation. 1996; 93: 1951-1953.

Detrano R, Hsiai T, Wang S, Puentes G, Fallavollita J, Shields P, Stanford W, Wolfkiel C, Georgiou D, Budoff M, Reed J. Prognostic value of coronary calcification and angiographic stenoses in patients undergoing coronary angiography. J Am Coll Cardiol. 1996; 27: 285-290.

Peltier M, Trojette F, Sarano ME, Grigioni F, Slama MA, Tribouilloy CM. Relation between cardiovascular risk factors and nonrheumatic severe calcific aortic stenosis among patients with a three-cuspid aortic valve. Am J Cardiol. 2003; 91: 97-99.

Mautner GC, Roberts WC. Reported frequency of coronary arterial narrowing by angiogram in patients with valvular aortic stenosis. Am J Cardiol. 1992; 70: 539-540.

Greenland P, LaBree L, Azen SP, Doherty TM, Detrano RC. Coronary artery calcium score combined with Framingham score for risk prediction in asymptomatic individuals. Jama. 2004; 291: 210-215.


作者单位:David Messika-Zeitoun; Lawrence F. Bielak; Patricia A. Peyser; Patrick F. Sheedy; Stephen T. Turner; Vuyisile T. Nkomo; Jerome F. Breen; Joseph Maalouf; Christopher Scott; A. Jamil Tajik; Maurice Enriquez-SaranoFrom the Divisions of Cardiovascular Diseases and Internal Medicine (D.M.-Z., V.T.N., J.M

日期:2008年12月28日 - 来自[2007年第27卷第3期]栏目

Molecular Mechanisms of Vascular Calcification

【摘要】  Vascular calcification increasingly afflicts our aging and dysmetabolic population. Once considered a passive process, it has emerged as an actively regulated form of calcified tissue metabolism, resembling the mineralization of endochondral and membranous bone. Executive cell types familiar to bone biologists, osteoblasts, chondrocytes, and osteoclasts, are seen in calcifying macrovascular specimens. Lipidaceous matrix vesicles, with biochemical and ultrastructural "signatures" of skeletal matrix vesicles, nucleate vascular mineralization in diabetes, dyslipidemia, and uremia. Skeletal morphogens (bone morphogenetic protein-2 (BMP) and BMP4 and Wnts) divert aortic mesoangioblasts, mural pericytes (calcifying vascular cells), or valve myofibroblasts to osteogenic fates. Paracrine signals provided by these molecules mimic the epithelial-mesenchymal interactions that induce skeletal development. Vascular expression of pro-osteogenic morphogens is entrained to physiological stimuli that promote calcification. Inflammation, shear, oxidative stress, hyperphosphatemia, and elastinolysis provide stimuli that: (1) promote vascular BMP2/4 signaling and matrix remodeling; and (2) compromise vascular defenses that limit calcium deposition, inhibit osteo/chondrogenic trans-differentiation, and enhance matrix vesicle clearance. In this review, we discuss the biology of vascular calcification. We highlight how aortic fibrofatty tissue expansion (adventitia, valve interstitium), the adventitial-medial vasa, vascular matrix, and matrix vesicle metabolism contribute to the regulation of aortic calcium deposition, with greatest emphasis placed on diabetic vascular disease.

Vascular calcification increasingly afflicts our aging and dysmetabolic population. Once considered a passive process, it has emerged as an actively regulated form of calcified tissue metabolism, resembling the mineralization of endochondral and membranous bone. In this review, we discuss the biology of vascular calcification. We highlight how aortic fibrofatty tissue expansion (adventitia, valve interstitium), the adventitial-medial vasa, vascular matrix, and matrix vesicle metabolism contribute to the regulation of aortic calcium deposition, with greatest emphasis placed on diabetic vascular disease.

【关键词】  diabetes vascular calcification Wnt signaling bone morphogenetic proteins oxylipids


Introduction


With advanced age, vascular inflammation, hypertension, and certain metabolic disorders, calcium accumulates in the arterial macrovasculature. 1 Calcification of aortic valve leaflets and atherosclerotic plaques have long been recognized as clinically important. 2 However, medial artery calcification (MAC) also portends mortality and amputation risk. 1,3-5 Studies of vascular calcified tissue metabolism significantly lag behind those of skeletal metabolism. Executive cell types familiar to bone biologists are seen in calcifying aortic specimens. 1,6 As in bone, endothelial, mesenchymal, and hematopoietic cell lineages control vascular mineral accumulation, with cellular activities entrained to morphogenetic, metabolic, inflammatory, and mechanical demands placed on each vascular segment. 1


We provide a brief overview of vascular calcification, emphasizing how paracrine osteogenic signals recruited by dysmetabolic insults promote aortic calcium deposition in diabetic vascular disease. We point to emerging evidence that inflammation, mechanical, and metabolic oxidative stresses not only provide stimuli that induce vascular osteogenic morphogens but also compromise defense mechanisms that limit vascular calcium deposition. 1


Aortic MAC


MAC is a highly characteristic feature of diabetes and chronic kidney disease (CKD). 3,4 Although diabetes is the major cause of CKD, hyperglycemia conveys independent risk for vascular calcification. 5,7 Aortic calcium scores, but not coronary calcium scores, are linearly related to fasting blood glucose. 8 MAC has emerged as an exceptionally strong predictor of lower extremity amputation and mortality in patients with type II diabetes; 4,9 mechanisms are still unclear but may relate to abnormal aortofemoral Windkessel physiology that generates systolic hypertension, increases myocardial workload, and perturbs normal microvascular tissue perfusion. Aortic pulse wave velocity, an index of vascular stiffness, is highly correlated with the prevalence of aortic calcification and diabetes in CKD5. 10 Increases in aortic stiffness convey the impact of diabetes-enhanced cardiovascular mortality. 11 Thus, a better understanding of the mechanisms controlling aortic MAC is required to address the burgeoning unmet clinical needs of diabetic vascular disease and CKD.


In diabetes and CKD, MAC proceeds via matrix vesicle-nucleated mineralization, 12-14 with apatitic calcium phosphate deposition in the tunica media occurring in the absence of atheroma and neointima. (Of note, this differs from calcific uremic arteriolopathy, an uncommon disorder in which fibroproliferative occlusion and medial calcification of arterioles cause skin and sometimes intestinal necrosis 15 ). The concentric nature of MAC stands in stark contrast to the eccentric, calcified atherosclerotic plaque. 1,16 At least 2 types of lipid vesicles have been identified to date that nucleate vascular calcification: (1) the apoptotic bodies (250-nm diameter) of dead and dying cells; and (2) mineralizing matrix vesicles (100 nm diameter) actively extruded by viable vascular smooth muscle cells (VSMCs) and calcifying vascular cells (CVCs). 12-14,17 The latter resembles the mineralization of membranous bone, 13,17 is intensely procalcific, 14 and appears predominate in aortic calcification. 12,14,17


Mechanisms controlling MAC in type II diabetes are beginning to be understood. High-fat diets that induce obesity, insulin-resistant diabetes, and dyslipidemia promote aortic MAC and valve calcification in male low-density lipoprotein receptor (LDLR)-deficient mice. 18,19 An aortic bone morphogenetic protein-2 (BMP2)-muscle segment homeobox homolog (Msx2) signaling cascade is activated by mural oxidative stress and inflammatory cytokines 18,19 ( Figure 1 ). Because Msx2-dependent gene expression is critical for craniofacial bone formation, 20 this suggested that similar signals participate in diabetic MAC. Intriguingly, a subset of myofibroblasts in the fibrofatty aortic adventitia and aortic valve interstitium, but not the tunica media, elaborated this early BMP2-Msx2 response. 18,19


Figure 1. Evolving model of diabetic MAC. Diabetes and dyslipidemia induce oxidative stress, low-grade inflammation, and angiogenesis in the adventitia of diabetic arteries. Glucose, reactive oxygen species, and TNF- upregulate BMP2/4 production by pericytes and endothelial cells in the vessel wall; this promotes adventitial Msx2-Wnt signaling. Subsequently, enhanced adventitial Wnt production (increased Wnt3a and Wnt7a, decreased Dkk1) augments medial nuclear ß-catenin accumulation, ALP activity, and osteogenic differentiation. The mural CVC, a macrovascular myofibroblast related to the microvascular pericyte, is thought to be the resident osteoprogenitor. Adventitial Sca1+ mesenchymal progenitors are present in murine aortas, contribute to medial and intimal disease processes, and can undergo osteogenic differentiation in response to BMP2-Wnt signaling. However, the lineage relationship between Sca1+ progenitors and the CVC is currently unknown; speculation based on studies of aortic mesoangioblast development suggests that CVCs arise from Sca1+ cells. 35 Hyperphosphatemia, a common feature of diabetes in the setting of CKD, promotes osteo/chondrogenic "trans-differentiation" of aortic VSMCs via Runx2. 37 The relative extent to which CVC recruitment vs VSMC "trans-differentiation" mechanisms contribute to the osteogenic calcification in diabetic MAC has yet to be determined.


Emerging evidence indicates that vascular osteogenic signals, initiated by adventitial BMP2-Msx2 actions, are concentrically conveyed to the calcifying tunica media via the vasa vasorum 18,19,21,22 ( Figure 1 ). Diabetes causes low-grade adventitial and medial inflammation, with adipocyte-laden expansion and associated mural neoangiogenesis 23,24 ( Figure 1 ). Primary vasa, arising from overt branch points in the arterial tree, sprout and meander through the adventitia, then ramify to form secondary vasa that circumferentially penetrate and percolate the aortic tunica media 25 ( Figure 1 ). The vasa vasorum is most evident in larger mammals 25 and becomes grossly manifest in dyslipidic mice. 26,27


What molecules convey vascular osteogenic signals? Recent data from our laboratory 19 and the Rajamannan laboratory 28 have shown that Wnts are important. Wnts are secreted polypeptides that bind specific LDLR-related protein (LRP)/frizzled heterodimers, activate LRP5- and LRP6- signaling cascades, and augment gene expression via nuclear ß-catenin in the canonical pathway. 19,28 Cultured Msx2-expressing mesenchymal cells secrete an osteogenic activity that is antagonized by Dickkopf homolog (Dkk1), an inhibitory ligand of LRP5/6 and paracrine Wnt signaling. 19 These results were confirmed in vivo using cytomegalovirus promoter/immediate early enhancer-Msx2 transgenic mice, 19 a model validated previously in studies of ectopic calvarial bone formation. Whereas Msx2 accumulates in the aortic adventitia, alkaline phosphatase (ALP) induction occurs in the tunica media with concomitant MAC. 19 Importantly, the Msx2 transgene selectively upregulated galactosidase (LacZ) in the tunica media of TOPGAL mice (T-cell factor/lympoid enhancer binding factor optimal promoter-galactosidase reporter mouse; demarcates canonical Wnt actions in vivo). 19 The vector of mural microvascular flow is from adventitia to media 25 ( Figure 1 ); therefore, we posited that paracrine Wnt signals were elaborated by Msx2-expressing cells of the adventitia, and that these Wnt signals programmed concentric mineralization via the CVCs 29 of the tunica media. 19 Surgical stripping of the adventitia significantly reduces MAC in rats fed high-fat diets, consistent with this notion. 21


How does induction of vascular BMP contribute to activation of this osteogenic signal? In craniofacial osteoblasts, BMP2 is a key stimulus for Msx2 expression and enhances Wnt signaling. 30 Aortic Msx2-Wnt signaling is also stimulated by BMP2 ( Figure 2 ). Intraperitoneal BMP2 administration upregulates aortic Msx2 and LacZ mRNAs in TOPGAL mice ( Figure 2 A). LacZ histochemistry localizes enhanced canonical Wnt signaling to the aortic tunica media ( Figure 2 B). Moreover, thrice-weekly BMP2 treatment for 4 weeks augments aortic calcium 2-fold in LDLR-/- mice fed high-fat diabetogenic diets ( Figure 2 C). Calcium accumulation (alizarin red stain) again localizes to the aortic tunica media ( Figure 2 D). Thus, BMP2 can activate an aortic Wnt signaling cascade that drives osteogenic mineralization of vascular progenitors via processes that resemble craniofacial membranous bone formation. 19,31 The concentric medial calcification of diabetes is proposed to arise in part from the vasa-dominated relationship between: (1) BMP2-stimulated cells of the periaortic adventitia that express Msx2 and elaborate Wnts; 19 and (2) the CVCs in the tunica media 6,29 ( Figure 1 ). It remains possible that the osteogenic potential of vascular progenitors is programmed within the adventitia but is elaborated only when these progenitors migrate with the vasa into the tunica media. 22,32


Figure 2. BMP2 activation of aortic Msx2-Wnt signaling and MAC in vivo. A and B, TOPGAL+;LDLR+/- mice were intraperitoneally injected with either vehicle (VEH) or 50 ng/gm recombinant human BMP2 (R&D Systems) for 3 days. A, Aortic tissues were harvested for analysis of mRNA accumulation by fluorescence RT-qPCR normalized to 18S signal. Data are presented as percentage of vehicle-treated controls. BMP2 upregulated aortic Msx2 and canonical Wnt signaling, the latter indicated by the accumulation of LacZ mRNA. B, Histochemical staining for LacZ (blue, nuclear red counterstain) in thoracic aortas of BMP2-treated animals confirmed that Wnt signaling is enhanced in the tunica media. C, LDLR-/- mice fed high-fat Western diets (HFD) were treated 3 days per week with either vehicle (HFD+VEH) or 50 ng/gm BMP2 (HFD + BMP2) for 4 weeks. At death, aortic calcium content was measured. BMP2 significantly increased aortic calcification (Student 2-sided t test). D, Alizarin red staining revealed that calcification occurred within the aortic tunica media. A diet-induced factor, potentially an oxylipid-containing matrix vesicle, 29,64,65,85 is required to robustly elaborate MAC in BMP2-treated, Msx2 transgenic, and uremic murine disease models. 19,84 Methods have been detailed previously. 19


What are the origins of aortic osteogenic cells and Msx2-expressing adventitial cells? At least 2 aortic mesenchymal cell types can contribute to the ectopic osteogenic programs of vascular calcification: (1) multipotent vascular mesenchymal progenitors that are recruited to form the mural CVCs; and (2) VSMCs that can undergo osteo/chondrogenic trans-differentiation in response to hyperphosphatemia. Demer first described the aortic CVCs. 6,29 The CVC is a macrovascular myofibroblast subtype related to the microvascular pericyte. 33 Of note, pericytes from multiple vascular beds function as osteoprogenitors in vitro. 33 It is highly probable that CVCs arise from local mesenchymal progenitors recruited during vascular injury responses. 22,29,32,33 Markers for pericytes and CVCs are few but include 3G5, smooth muscle -actin, and Stro1 (human). 33 Importantly, an abundant Sca1+ (stem cell antigen) cell population resides within the aortic adventitia in dyslipidemic apolipoprotein E-/- 22 and LDLR-/- (our unpublished data, 2005) mice that contributes to medial and intimal injury responses. During vertebrate development, a Sca1+ CD34+ mesenchymal progenitor, the mesoangioblast, 34,35 resides in the dorsal aorta that is programmed by Msx2. 36 In response to BMP2, mesoangioblasts upregulate ALP and differentiate into mineralizing osteoblasts. 34 Because neoangiogenesis generates bipotential endothelial cell, VSMC progenitors resembling the mesoangioblast, 34,36 our working model posits that dysmetabolic signals that expand the adventitial vasa simultaneously expand the mural pool of Sca1+ mesenchymal progenitors. In addition, many laboratories have demonstrated that aortic VSMCs can undergo a type of phenotypic modulation: "trans-differentiating" into mineralizing VSMCs that elaborate markers of the osteo/chondrogenic lineage. 1,37 The hyperphosphatemia of CKD is an important stimulus for this process, 38 signaling through cell surface Na/phosphate cotransporter Pit1/Glvr1. 37 Elevated extracellular phosphate upregulates VSMC expression of Runx2/Cbaf1, the prototypic osteo/chondrogenic transcription factor. 37 Moreover, hyperphosphatemia, a common metabolic insult in patients with CKD5, 38 enhances production of apoptotic bodies and matrix vesicles that nucleate vascular mineral deposition. 14 Intriguingly, apoptotic bodies simultaneously upregulate the expression of stromal cell-derived factor (SDF)-1 /CXCL12; 32 because SDF-1 mediates vascular homing of Sca1+ progenitors, medial VSMC vesiculation 14 could help recruit adventitial osteoprogenitors. Whether Sca1+ aortic adventitial cells differentially express Msx2, elaborate canonical Wnts, or contribute to the CVC lineage has yet to be determined. Moreover, the relative contribution of Sca1+ progenitor recruitment 22,29,32 versus VSMC "trans-differentiation" 14,37 to the birth of vascular osteogenic cells has yet to be examined in diabetic MAC and may change if CKD ensues. 5,7


What signals recruit vascular BMP2 signaling in diabetes? High glucose concentrations upregulate BMP2 production in pericytes and mesangial myofibroblasts. 18,39 In response to tumor necrosis factor- (TNF- ), peroxides, and shear stress, the endothelial cell also produces BMP2 40 and BMP4. 41 Adipose tissue itself is an endocrine gland that produces TNF-, interleukin-6, and adipokines. 24 The inflammatory fibrofatty adipose tissue expansion in the periaortic adventitia 18,21,22,24 and aortic valve interstitium 42 before vascular calcification is likely to play an important role in disease initiation ( Figure 1 ). The oxidative stress, inflammation, fatty connective tissue expansion, and neovascularization of the diabetic adventitia 24 can all serve to stimulate aortic production of BMPs 31,43 that exert paracrine influence on regional mesenchymal progenitors ( Figure 1 ).


Why don?t all vascular beds calcify in response to metabolic insult and BMP signaling? The answer lies in the number of defense mechanisms that prevent tissue mineralization. Inorganic pyrophosphate (PPi), matrix Gla protein (MGP), and fetuin are chief among these. PPi is a VSMC-generated organic anion that inhibits mineralization and is a physiological substrate that matrix vesicle-associated ALP must hydrolyze to promote calcium deposition. 12 Extracellular PPi is generated by 2 mechanisms. First, the membrane transporter ankylosis (ank) directs secretion of PPi. 44 Second, the enzyme ectonucleotide pyrophosphatase/phosphodiesterase I (NPP1) cleaves extracellular NTPs to generate PPi. Loss of extracellular PPi from either NPP1 or ank deficiency predisposes to massive aortic calcification. 44 PPi is required to stabilize the VSMC phenotype; VSMCs that cannot generate a PPi-replete extracellular milieu undergo osteo/chondrogenic trans-differentiation. 44 MGP is a calcium-binding matrix protein that binds and inhibits BMP2 induction of ALP. 45 In addition, carboxylated MGP produced by VSMCs binds matrix elastin and inhibits calcification. 46,47 Mice lacking MGP develop profound panarterial vascular calcification (endochondral ossification) and die from aortic rupture. 48 The diverse roles of BMP2 and BMP4 during vasculogenesis and development are regulated by a diverse cadre of vascular BMP inhibitors. 45,49 Intracellular defenses to osteogenic vascular BMP signaling also exist; Smad6, an inhibitory vascular Smad, attenuates BMP2 activation of receptor Smad trans-activators. 49 Mice lacking Smad6 develop aortic valve and outflow tract ossification. 49 Fetuin is an important humoral inhibitor of soft tissue mineralization that controls the metabolism of vascular matrix vesicles 50 (vide infra). Deficiencies in serum fetuin arising from genetic, inflammatory, or metabolic insult promote widespread tissue calcium deposition (eg, heart and lung) that curiously spares the aorta in mouse models. 51 Other molecules such as osteopontin have more complex roles, inhibiting calcification but also promoting calcium egress via extracellular matrix acidification. 52 Procalcific vascular cytokine signaling is held in check by osteoprotegerin, most probably via inhibition of RANKL. 1,53


Thus, in addition to the upregulation of pro-osteogenic signals, inhibitors of mineral accumulation must be inactivated to permit robust aortic calcification. Both regulatory arms are profoundly perturbed in CKD. The mechanisms controlling aortic MAC in CKD overlap those of diabetes; however, the hyperphosphatemia, reduced serum PPi and fetuin, and secondary hyperparathyroidism of CKD accentuate aortic calcium accumulation. 38 Hyperphosphatemia promotes VSMC matrix vesicle formation. 14 Intriguingly, matrix vesicles may either promote or inhibit calcium deposition, dependent on whether fetuin is recruited. 50 Moreover, fetuin promotes "phagocytotic clearance" of pro-osteogenic matrix vesicles. 50 Thus, in the setting of CKD, reduced serum fetuin levels contribute to the vascular procalcific milieu. 50


Electron microscopy studies of human postmortem specimens demonstrated early on that aortic calcium deposition in both MAC and atherosclerosis initiates at lipid vesicles located along and between elastic laminae but not within the elastin fibers. 12,13,17 However, aortic calcification in association with primary alterations in elastin matrix metabolism represents a unique entry point in a feed-forward cycle of MAC. The most aggressive drug-induced animal models of MAC combine either nicotine, a stimulus for elastinolysis, 54 or warfarin, a mechanism for inhibiting MGP-elastin interaction, 46,47 with excessive vitamin D. Ex vivo, devitalized aortic valves and aortas depleted of VSMCs and myofibroblasts, the sources of matrix vesicles, can calcify by elastin-mediated nucleation. 55-57 However, matrix-bound lipids still play a role because ethanolic delipidation inhibits ex vivo calcium deposition of devitalized aortic valves. 55 In vivo, aberrant elastin organization and metabolism is characterized by aortic root dilatation and MAC, as evident in Marfan syndrome. Primary fibrillin 1 insufficiency causes abnormal adventitial microfibrillar matrix organization; 58 secondary changes in elastin metabolism impair medial VSMC terminal differentiation 59 and promote elastin-nucleated medial calcification. 58 Direct elastin-nucleated calcification also occurs in pseudoxanthoma elasticum; 60 electron microscopy confirms calcium deposition along elastin fibers in the absence of matrix vesicle formation. 60 Thus, although mechanisms are still being elucidated, altered elastin matrix metabolism enhances aortic calcium deposition and VSMC phenotypic drift. 58,59 Because calcium phosphate mineral deposition suppresses VSMC production of tropoelastin, 61 elastin matrix metabolism no doubt contributes to the progression of vascular calcium load in all forms of MAC.


Atherosclerotic Aortic Calcification


Mechanisms of aortic atherosclerotic calcification are overlapping yet distinct from those of MAC. This form of aortic calcium deposition, the type Vb atherosclerotic plaque, 62 has been described excellently 1 and will be considered only briefly. Atherosclerotic calcification is intimally oriented, eccentric, initiating at the base of necrotic fibrofatty plaques via apoptotic vesicles arising from dead and dying VSMCs. 1,62 Adjacent chondrogenic and osteogenic processes are recruited by CVC activation and contribute to procalcific matrix remodeling. 63 As in endochondral bone formation, ALP induction, Cbfa1/Runx2 and Msx2 expression, type II and type I collagen deposition, and angiogenic invasion are salient components. 63 The initiating stimuli are inflammatory and redox dependent, 64 and bone morphogens are recruited with disease progression. 6 Indeed, Demer first identified vascular BMP2 expression within calcifying atherosclerotic plaques. 6 Oxidation of cholesterol-laden lipoprotein deposits generate bioactive oxysterols that synergize with vascular BMP2 to promote ectopic osteogenic gene regulatory programs. 65


Major features of atherosclerotic calcification that differ from diabetic MAC include abundant fibrosis, extensive cellular necrosis, apoptotic body formation, and cholesterol crystal accumulation that can support some epitaxial calcium phosphate deposition. 1,14,50,66 By histology, endochondral bone formation very commonly ensues; in advanced disease, this ectopic bone can support hematopoietic marrow elements. 6 The high level of Cbfa1/Runx2 observed in calcifying atherosclerotic plaques is particularly important. 38,48,63 In addition to promoting ALP expression, Runx2: (1) strongly promotes expression of type I collagen, and (2) upregulates the expression of vascular endothelial growth factor, the prototypic osteogenic-angiogenic coupling factor. 67 Karsenty demonstrated that sustained ectopic expression of dermal ALP with the regional type I collagen deposition was sufficient to drive heterotopic dermal mineralization. 68 However, this type of ectopic dermal mineralization was not associated with matrix vesicles or apoptotic bodies that characterize MAC or atherosclerotic calcification. 12-14,17,50 Thus, drawing on lessons learned from skeletal development, synergistic interactions between paracrine BMP and vascular endothelial growth factor signaling with neoangiogenesis, robust aortic expression of Runx2, matricrine cues provided by type I collagen accumulation, and vascular lipidaceous matrix vesicle production likely combine to drive ectopic bone formation in advanced type Vb plaques. 1


Aortic Valve Calcification


Approximately 30% of patients 65 years of age have echocardographic evidence of aortic sclerosis, with 2% overall exhibiting aortic stenosis. 69 Calcium deposition is a particularly ominous feature of aortic valve disease; in patients with asymptomatic aortic stenosis, moderate to severe valve calcification is the single most significant determinant of clinical disease progression. 70 In a cross-sectional study, Otto described the histopathologic progression of aortic valve calcification. 42 Degenerative lipid accumulation, fatty expansion of the valve fibrosus, neoangiogenesis, and stippled interstitial calcium deposition are accompanied by macrophage and T-cell infiltrates at the earliest stages of disease. 42 Neoangiogenesis is also a key concomitant of valve calcification. 71,72 In many ways, the early changes of the aortic valve interstitium 42 are reminiscent of those described in the tunica adventitia with diabetes and dyslipidemia (vide supra). Similar inflammatory histology occurs in calcifying bicuspid aortic valves in the complete absence of atherosclerosis. 73


Thus, aortic valve calcification occurs in response to mechanical stressors, inflammation, and the metabolic challenges of diabetes, dyslipidemia, and uremia. 1,38,74,75 During disease progression, histological and molecular analyses clearly demonstrate that a phase of active osteogenic mineral deposition contributes to vascular calcium accumulation. By histology, this appears to occur principally via nonendochondral processes in aortic valves, 75 although the chondrogenic transcription factor Sox9 is upregulated in both calcifying and noncalcifying diseased valves. 76 In advanced disease, woven bone formation is histologically evident in 13% of cases. 77 At the molecular level, active BMP-Msx2-Wnt signaling is detectable in virtually all calcifying aortic valves 76,77 (D.A. Towler, unpublished data, 2002). However, by histology, massive concretions of acellular amorphous calcium phosphate are also seen, suggesting that profound epitaxial mineral deposition occurs once cell-based mineralization has initiated. 77 The disappointing effects of statins on the progression of established aortic valve calcification may reflect this fact. 78


Mechanisms controlling initiation and progression of aortic valve calcification are poorly understood, largely because of the limitations of current animal models. Rajamannan first demonstrated that aortic myofibroblasts undergo osteogenic trans-differentiation. 76,79 Pharmacological doses of statins prevented osteogenic trans-differentiation of aortic valve myofibroblasts in culture. 79 Recently, she has identified the mechanistic underpinnings; Wnt/LRP5/ß-catenin signaling, a signaling cascade absolutely required for osteoblast differentiation in the skeleton, 80 is activated by oxidized LDL cholesterol in valve myofibroblasts and is inhibited by statin administration. 28 This elegant work provides robust evidence that mineral deposition directed by aortic valve myofibroblasts is osteogenic in nature and is potentially preventable via pharmacological intervention.


Summary


Over the past 25 years, we have learned much concerning the biology of aortic calcification. Active mineralization mechanisms clearly resembling those of skeletal endochondral and membranous ossification participate in vascular calcium accumulation. However, the endocrine physiology of vascular calcium deposition and its turnover is poorly understood and will depend on the histoanatomic, mechanical, developmental, matricrine, and metabolic features of the diseased vascular segment. Many fundamental questions remain to be addressed. The field of vascular matrix vesicle metabolism is in its infancy; mechanisms whereby cells shed and endocytose vascular matrix vesicles 14,50 and how osteogenic morphogens and matrix control these processes are poorly characterized. The origins of vascular osteoprogenitors must be clearly delineated. Although circulating progenitors may contribute, 32 most mineralizing osteoprogenitors appear locally recruited in response to paracrine osteogenic cues 19,22,34 or hyperphosphatemic stimulation. 14,37,38 The "paracrinology" of vascular adventitial-medial signaling and its metabolic regulation is poorly characterized. Although canonical Wnt signals are capable of mediating these interactions, 19,28 the specific LRP5 and LRP6 ligands that participate in vascular Wnt signaling cascades have yet to be determined.


Novel therapeutics are needed. 78 Dependent on disease stage and setting, the metabolic, endocrine, inflammatory, elastinolytic, and mechanical insults will differ in relative contribution to the extent of aortic calcification. Stage-specific mechanisms contributing to calcific aortic disease must be carefully considered as clinical studies are designed and therapeutic strategies crafted. Given the benefits of sevelamer on aortic calcification in dialysis patients, 81 this phosphate- and sterol-binding resin also holds promise for diabetic patients with declining renal function. 38 Although statins cannot treat established aortic valve calcification, 78 early treatment with statins may prevent valve mineralization, 28,79 particularly in high-risk patients after bioprosthetic valve implantation. 1 Small studies suggest that bisphosphonates can inhibit aortic mineral deposition in extant disease; 82 mechanisms are unknown, and the potential role of Pit1 37 as a pyrophosphate/bisphosphonate receptor 44 has yet to be explored. Whether soluble inhibitors of the BMP2-Msx2-Wnt signaling cascade 19 alter the progression of aortic calcium accumulation will soon be tested. As the mechanisms that initiate and propagate vascular pro-osteogenic morphogen signaling are better understood, other potential therapeutic strategies will emerge. 1 Antagonism of the endothelin A receptor, a receptor that enhances vascular BMP2 signaling, 43 has been shown to promote regression of medial calcium deposition. 83 BMP7, a unique BMP that stabilizes VSMC phenotype and inhibits vascular BMP2 signaling, holds promise for ameliorating uremic vasculopathy. 84 The absence of a robust murine model of calcific aortic stenosis remains a major scientific shortcoming. This limits our ability to examine how strategies targeting inflammation, oxidative stress, morphogen signaling, angiogenesis, matrix metabolism, and epitaxial mineral deposition may differentially influence initiation versus progression phases of aortic stenosis. Nevertheless, given the blossoming field of cardiovascular endocrinology, the near future holds tremendous promise that new pharmacotherapies will emerge to help address the unmet clinical need in calcific aortic disease.


Acknowledgments


Sources of Funding


This work was supported by the National Heart, Lung, and Blood Institute and the Barnes-Jewish Hospital Foundation.


Disclosures


None.

【参考文献】
  Abedin M, Tintut Y, Demer LL. Vascular calcification: mechanisms and clinical ramifications. Arterioscler Thromb Vasc Biol. 2004; 24: 1161-1170.

Virchow R. Cellular Pathology as Based Upon Physiological and Pathological Histology. New York, NY: Dover Publications; 1863.

London GM, Marchais SJ, Guerin AP, Metivier F. Arteriosclerosis, vascular calcifications and cardiovascular disease in uremia. Curr Opin Nephrol Hypertens. 2005; 14: 525-531.

Lehto S, Niskanen L, Suhonen M, Ronnemaa T, Laakso M. Medial artery calcification. A neglected harbinger of cardiovascular complications in non-insulin-dependent diabetes mellitus. Arterioscler Thromb Vasc Biol. 1996; 16: 978-983.

Ishimura E, Okuno S, Kitatani K, Kim M, Shoji T, Nakatani T, Inaba M, Nishizawa Y. Different risk factors for peripheral vascular calcification between diabetic and non-diabetic haemodialysis patients-importance of glycaemic control. Diabetologia. 2002; 45: 1446-1448.

Bostrom K, Watson KE, Horn S, Wortham C, Herman IM, Demer LL. Bone morphogenetic protein expression in human atherosclerotic lesions. J Clin Invest. 1993; 91: 1800-1809.

Reaven PD, Sacks J. Coronary artery and abdominal aortic calcification are associated with cardiovascular disease in type 2 diabetes. Diabetologia. 2005; 48: 379-385.

Kuller LH, Matthews KA, Sutton-Tyrrell K, Edmundowicz D, Bunker CH. Coronary and aortic calcification among women 8 years after menopause and their premenopausal risk factors: the healthy women study. Arterioscler Thromb Vasc Biol. 1999; 19: 2189-2198.

Nelson RG, Gohdes DM, Everhart JE, Hartner JA, Zwemer FL, Pettitt DJ, Knowler WC. Lower-extremity amputations in NIDDM. 12-yr follow-up study in Pima Indians. Diabetes Care. 1988; 11: 8-16.

Blacher J, Demuth K, Guerin AP, Safar ME, Moatti N, London GM. Influence of biochemical alterations on arterial stiffness in patients with end-stage renal disease. Arterioscler Thromb Vasc Biol. 1998; 18: 535-541.

Shoji T, Emoto M, Shinohara K, Kakiya R, Tsujimoto Y, Kishimoto H, Ishimura E, Tabata T, Nishizawa Y. Diabetes mellitus, aortic stiffness, and cardiovascular mortality in end-stage renal disease. J Am Soc Nephrol. 2001; 12: 2117-2124.

Tanimura A, McGregor DH, Anderson HC. Calcification in atherosclerosis. I. Human studies. J Exp Pathol. 1986; 2: 261-273.

Tanimura A, McGregor DH, Anderson HC. Matrix vesicles in atherosclerotic calcification. Proc Soc Exp Biol Med. 1983; 172: 173-177.

Reynolds JL, Joannides AJ, Skepper JN, McNair R, Schurgers LJ, Proudfoot D, Jahnen-Dechent W, Weissberg PL, Shanahan CM. Human vascular smooth muscle cells undergo vesicle-mediated calcification in response to changes in extracellular calcium and phosphate concentrations: a potential mechanism for accelerated vascular calcification in ESRD. J Am Soc Nephrol. 2004; 15: 2857-2867.

Coates T, Kirkland GS, Dymock RB, Murphy BF, Brealey JK, Mathew TH, Disney AP. Cutaneous necrosis from calcific uremic arteriolopathy. Am J Kidney Dis. 1998; 32: 384-391.

Vattikuti R, Towler DA. Osteogenic regulation of vascular calcification: an early perspective. Am J Physiol Endocrinol Metab. 2004; 286: E686-E696.

Kim KM. Calcification of matrix vesicles in human aortic valve and aortic media. Fed Proc. 1976; 35: 156-162.

Towler DA, Bidder M, Latifi T, Coleman T, Semenkovich CF. Diet-induced diabetes activates an osteogenic gene regulatory program in the aortas of low density lipoprotein receptor-deficient mice. J Biol Chem. 1998; 273: 30427-30434.

Shao JS, Cheng SL, Pingsterhaus JM, Charlton-Kachigian N, Loewy AP, Towler DA. Msx2 promotes cardiovascular calcification by activating paracrine Wnt signals. J Clin Invest. 2005; 115: 1210-1220.

Satokata I, Ma L, Ohshima H, Bei M, Woo I, Nishizawa K, Maeda T, Takano Y, Uchiyama M, Heaney S, Peters H, Tang Z, Maxson R, Maas R. Msx2 deficiency in mice causes pleiotropic defects in bone growth and ectodermal organ formation. Nat Genet. 2000; 24: 391-395.

Bujan J, Bellon JM, Sabater C, Jurado F, Garcia-Honduvilla N, Dominguez B, Jorge E. Modifications induced by atherogenic diet in the capacity of the arterial wall in rats to respond to surgical insult. Atherosclerosis. 1996; 122: 141-152.

Hu Y, Zhang Z, Torsney E, Afzal AR, Davison F, Metzler B, Xu Q. Abundant progenitor cells in the adventitia contribute to atherosclerosis of vein grafts in ApoE-deficient mice. J Clin Invest. 2004; 113: 1258-1265.

Moreno PR, Fuster V. New aspects in the pathogenesis of diabetic atherothrombosis. J Am Coll Cardiol. 2004; 44: 2293-2300.

Zhang L, Zalewski A, Liu Y, Mazurek T, Cowan S, Martin JL, Hofmann SM, Vlassara H, Shi Y. Diabetes-induced oxidative stress and low-grade inflammation in porcine coronary arteries. Circulation. 2003; 108: 472-478.

Heistad DD, Marcus ML, Law EG, Armstrong ML, Ehrhardt JC, Abboud FM. Regulation of blood flow to the aortic media in dogs. J Clin Invest. 1978; 62: 133-140.

Moulton KS, Olsen BR, Sonn S, Fukai N, Zurakowski D, Zeng X. Loss of collagen XVIII enhances neovascularization and vascular permeability in atherosclerosis. Circulation. 2004; 110: 1330-1336.

Moulton KS, Vakili K, Zurakowski D, Soliman M, Butterfield C, Sylvin E, Lo KM, Gillies S, Javaherian K, Folkman J. Inhibition of plaque neovascularization reduces macrophage accumulation and progression of advanced atherosclerosis. Proc Natl Acad Sci U S A. 2003; 100: 4736-4741.

Rajamannan NM, Subramaniam M, Caira F, Stock SR, Spelsberg TC. Atorvastatin inhibits hypercholesterolemia-induced calcification in the aortic valves via the Lrp5 receptor pathway. Circulation. 2005; 112: I229-234.

Tintut Y, Alfonso Z, Saini T, Radcliff K, Watson K, Bostrom K, Demer LL. Multilineage potential of cells from the artery wall. Circulation. 2003; 108: 2505-2510.

Rawadi G, Vayssiere B, Dunn F, Baron R, Roman-Roman S. BMP-2 controls alkaline phosphatase expression and osteoblast mineralization by a Wnt autocrine loop. J Bone Miner Res. 2003; 18: 1842-1853.

Cheng SL, Shao JS, Charlton-Kachigian N, Loewy AP, Towler DA. MSX2 promotes osteogenesis and suppresses adipogenic differentiation of multipotent mesenchymal progenitors. J Biol Chem. 2003; 278: 45969-45977.

Zernecke A, Schober A, Bot I, von Hundelshausen P, Liehn EA, Mopps B, Mericskay M, Gierschik P, Biessen EA, Weber C. SDF-1alpha/CXCR4 axis is instrumental in neointimal hyperplasia and recruitment of smooth muscle progenitor cells. Circ Res. 2005; 96: 784-791.

Collett GD, Canfield AE. Angiogenesis and pericytes in the initiation of ectopic calcification. Circ Res. 2005; 96: 930-938.

Tagliafico E, Brunelli S, Bergamaschi A, De Angelis L, Scardigli R, Galli D, Battini R, Bianco P, Ferrari S, Cossu G. TGFbeta/BMP activate the smooth muscle/bone differentiation programs in mesoangioblasts. J Cell Sci. 2004; 117: 4377-4388.

Esner M, Meilhac SM, Relaix F, Nicolas JF, Cossu G, Buckingham ME. Smooth muscle of the dorsal aorta shares a common clonal origin with skeletal muscle of the myotome. Development. 2006; 133: 737-749.

Brunelli S, Cossu G. A role for MSX2 and necdin in smooth muscle differentiation of mesoangioblasts and other mesoderm progenitor cells. Trends Cardiovasc Med. 2005; 15: 96-100.

Li X, Yang HY, Giachelli CM. Role of the sodium-dependent phosphate cotransporter, Pit-1, in vascular smooth muscle cell calcification. Circ Res. 2006; .

Moe SM, Chen NX. Pathophysiology of vascular calcification in chronic kidney disease. Circ Res. 2004; 95: 560-567.

McMahon R, Murphy M, Clarkson M, Taal M, Mackenzie HS, Godson C, Martin F, Brady HR. IHG-2, a mesangial cell gene induced by high glucose, is human gremlin. Regulation by extracellular glucose concentration, cyclic mechanical strain, and transforming growth factor-beta1. J Biol Chem. 2000; 275: 9901-9904.

Csiszar A, Smith KE, Koller A, Kaley G, Edwards JG, Ungvari Z. Regulation of bone morphogenetic protein-2 expression in endothelial cells: role of nuclear factor-kappaB activation by tumor necrosis factor-alpha, H2O2, and high intravascular pressure. Circulation. 2005; 111: 2364-2372.

Sorescu GP, Song H, Tressel SL, Hwang J, Dikalov S, Smith DA, Boyd NL, Platt MO, Lassegue B, Griendling KK, Jo H. Bone morphogenic protein 4 produced in endothelial cells by oscillatory shear stress induces monocyte adhesion by stimulating reactive oxygen species production from a nox1-based NADPH oxidase. Circ Res. 2004; 95: 773-779.

Otto CM, Kuusisto J, Reichenbach DD, Gown AM, O?Brien KD. Characterization of the early lesion of ?degenerative? valvular aortic stenosis. Histological and immunohistochemical studies. Circulation. 1994; 90: 844-853.

Nett PC, Ortmann J, Celeiro J, Haas E, Hofmann-Lehmann R, Tornillo L, Terraciano LM, Barton M. Transcriptional regulation of vascular bone morphogenetic protein by endothelin receptors in early autoimmune diabetes mellitus. Life Sci. 2005; 78: 2213-2218.

Johnson K, Polewski M, van Etten D, Terkeltaub R. Chondrogenesis mediated by PPi depletion promotes spontaneous aortic calcification in NPP1-/- mice. Arterioscler Thromb Vasc Biol. 2005; 25: 686-691.

Zebboudj AF, Imura M, Bostrom K. Matrix GLA protein, a regulatory protein for bone morphogenetic protein-2. J Biol Chem. 2002; 277: 4388-4394.

Schurgers LJ, Teunissen KJ, Knapen MH, Kwaijtaal M, van Diest R, Appels A, Reutelingsperger CP, Cleutjens JP, Vermeer C. Novel conformation-specific antibodies against matrix gamma-carboxyglutamic acid (Gla) protein: undercarboxylated matrix Gla protein as marker for vascular calcification. Arterioscler Thromb Vasc Biol. 2005; 25: 1629-1633.

Price PA, Faus SA, Williamson MK. Warfarin causes rapid calcification of the elastic lamellae in rat arteries and heart valves. Arterioscler Thromb Vasc Biol. 1998; 18: 1400-1407.

Steitz SA, Speer MY, Curinga G, Yang HY, Haynes P, Aebersold R, Schinke T, Karsenty G, Giachelli CM. Smooth muscle cell phenotypic transition associated with calcification: upregulation of Cbfa1 and downregulation of smooth muscle lineage markers. Circ Res. 2001; 89: 1147-1154.

Galvin KM, Donovan MJ, Lynch CA, Meyer RI, Paul RJ, Lorenz JN, Fairchild-Huntress V, Dixon KL, Dunmore JH, Gimbrone MA Jr, Falb D, Huszar D. A role for smad6 in development and homeostasis of the cardiovascular system. Nat Genet. 2000; 24: 171-174.

Reynolds JL, Skepper JN, McNair R, Kasama T, Gupta K, Weissberg PL, Jahnen-Dechent W, Shanahan CM. Multifunctional roles for serum protein fetuin-a in inhibition of human vascular smooth muscle cell calcification. J Am Soc Nephrol. 2005; 16: 2920-2930.

Merx MW, Schafer C, Westenfeld R, Brandenburg V, Hidajat S, Weber C, Ketteler M, Jahnen-Dechent W. Myocardial stiffness, cardiac remodeling, and diastolic dysfunction in calcification-prone fetuin-A-deficient mice. J Am Soc Nephrol. 2005; 16: 3357-3364.

Steitz SA, Speer MY, McKee MD, Liaw L, Almeida M, Yang H, Giachelli CM. Osteopontin inhibits mineral deposition and promotes regression of ectopic calcification. Am J Pathol. 2002; 161: 2035-2046.

Kaden JJ, Bickelhaupt S, Grobholz R, Haase KK, Sarikoc A, Kilic R, Brueckmann M, Lang S, Zahn I, Vahl C, Hagl S, Dempfle CE, Borggrefe M. Receptor activator of nuclear factor kappaB ligand and osteoprotegerin regulate aortic valve calcification. J Mol Cell Cardiol. 2004; 36: 57-66.

Niederhoffer N, Bobryshev YV, Lartaud-Idjouadiene I, Giummelly P, Atkinson J. Aortic calcification produced by vitamin D3 plus nicotine. J Vasc Res. 1997; 34: 386-398.

Vyavahare N, Hirsch D, Lerner E, Baskin JZ, Schoen FJ, Bianco R, Kruth HS, Zand R, Levy RJ. Prevention of bioprosthetic heart valve calcification by ethanol preincubation. Efficacy and mechanisms. Circulation. 1997; 95: 479-488.

Price PA, Si Chan W, Jolson DM, Williamson MK. The elastic lamellae of devitalized arteries calcify when incubated in serum. Evidence for a serum calcification factor. Arterioscler Thromb Vasc Biol. 2006;.

Sakata N, Noma A, Yamamoto Y, Okamoto K, Meng J, Takebayashi S, Nagai R, Horiuchi S. Modification of elastin by pentosidine is associated with the calcification of aortic media in patients with end-stage renal disease. Nephrol Dial Transplant. 2003; 18: 1601-1609.

Pereira L, Andrikopoulos K, Tian J, Lee SY, Keene DR, Ono R, Reinhardt DP, Sakai LY, Biery NJ, Bunton T, Dietz HC, Ramirez F. Targeting of the gene encoding fibrillin-1 recapitulates the vascular aspect of Marfan syndrome. Nat Genet. 1997; 17: 218-222.

Bunton TE, Biery NJ, Myers L, Gayraud B, Ramirez F, Dietz HC. Phenotypic alteration of vascular smooth muscle cells precedes elastolysis in a mouse model of Marfan syndrome. Circ Res. 2001; 88: 37-43.

Klement JF, Matsuzaki Y, Jiang QJ, Terlizzi J, Choi HY, Fujimoto N, Li K, Pulkkinen L, Birk DE, Sundberg JP, Uitto J. Targeted ablation of the abcc6 gene results in ectopic mineralization of connective tissues. Mol Cell Biol. 2005; 25: 8299-8310.

Sugitani H, Wachi H, Mecham RP, Seyama Y. Accelerated calcification represses the expression of elastic fiber components and lysyl oxidase in cultured bovine aortic smooth muscle cells. J Atheroscler Thromb. 2002; 9: 292-298.

Stary HC, Chandler AB, Dinsmore RE, Fuster V, Glagov S, Insull W, Jr., Rosenfeld ME, Schwartz CJ, Wagner WD, Wissler RW. A definition of advanced types of atherosclerotic lesions and a histological classification of atherosclerosis. A report from the Committee on Vascular Lesions of the Council on Arteriosclerosis, American Heart Association. Arterioscler Thromb Vasc Biol. 1995; 15: 1512-1531.

Tyson KL, Reynolds JL, McNair R, Zhang Q, Weissberg PL, Shanahan CM. Osteo/chondrocytic transcription factors and their target genes exhibit distinct patterns of expression in human arterial calcification. Arterioscler Thromb Vasc Biol. 2003; 23: 489-494.

Proudfoot D, Davies JD, Skepper JN, Weissberg PL, Shanahan CM. Acetylated low-density lipoprotein stimulates human vascular smooth muscle cell calcification by promoting osteoblastic differentiation and inhibiting phagocytosis. Circulation. 2002; 106: 3044-3050.

Parhami F, Morrow AD, Balucan J, Leitinger N, Watson AD, Tintut Y, Berliner JA, Demer LL. Lipid oxidation products have opposite effects on calcifying vascular cell and bone cell differentiation. A possible explanation for the paradox of arterial calcification in osteoporotic patients. Arterioscler Thromb Vasc Biol. 1997; 17: 680-687.

Laird DF, Mucalo MR, Yokogawa Y. Growth of calcium hydroxyapatite (Ca-HAp) on cholesterol and cholestanol crystals from a simulated body fluid: A possible insight into the pathological calcifications associated with atherosclerosis. J Colloid Interface Sci. 2006; 295: 348-363.

Stein GS, Lian JB, van Wijnen AJ, Stein JL, Montecino M, Javed A, Zaidi SK, Young DW, Choi JY, Pockwinse SM. Runx2 control of organization, assembly and activity of the regulatory machinery for skeletal gene expression. Oncogene. 2004; 23: 4315-4329.

Murshed M, Harmey D, Millan JL, McKee MD, Karsenty G. Unique coexpression in osteoblasts of broadly expressed genes accounts for the spatial restriction of ECM mineralization to bone. Genes Dev. 2005; 19: 1093-1104.

Otto CM, Lind BK, Kitzman DW, Gersh BJ, Siscovick DS. Association of aortic-valve sclerosis with cardiovascular mortality and morbidity in the elderly. N Engl J Med. 1999; 341: 142-147.

Rosenhek R, Binder T, Porenta G, Lang I, Christ G, Schemper M, Maurer G, Baumgartner H. Predictors of outcome in severe, asymptomatic aortic stenosis. N Engl J Med. 2000; 343: 611-617.

Chalajour F, Treede H, Ebrahimnejad A, Lauke H, Reichenspurner H, Ergun S. Angiogenic activation of valvular endothelial cells in aortic valve stenosis. Exp Cell Res. 2004; 298: 455-464.

Soini Y, Salo T, Satta J. Angiogenesis is involved in the pathogenesis of nonrheumatic aortic valve stenosis. Hum Pathol. 2003; 34: 756-763.

Wallby L, Janerot-Sjoberg B, Steffensen T, Broqvist M. T lymphocyte infiltration in non-rheumatic aortic stenosis: a comparative descriptive study between tricuspid and bicuspid aortic valves. Heart. 2002; 88: 348-351.

Pohle K, Otte M, Maffert R, Ropers D, Schmid M, Daniel WG, Achenbach S. Association of cardiovascular risk factors to aortic valve calcification as quantified by electron beam computed tomography. Mayo Clin Proc. 2004; 79: 1242-1246.

Rajamannan NM, Subramaniam M, Rickard D, Stock SR, Donovan J, Springett M, Orszulak T, Fullerton DA, Tajik AJ, Bonow RO, Spelsberg T. Human aortic valve calcification is associated with an osteoblast phenotype. Circulation. 2003; 107: 2181-2184.

Caira FC, Stock SR, Gleason TG, McGee EC, Huang J, Bonow RO, Spelsberg TC, McCarthy PM, Rahimtoola SH, Rajamannan NM. Human degenerative valve disease is associated with up-regulation of low-density lipoprotein receptor-related protein 5 receptor-mediated bone formation. JACC. 2006; 47: 1707-1712.

Mohler ER III, Gannon F, Reynolds C, Zimmerman R, Keane MG, Kaplan FS. Bone formation and inflammation in cardiac valves. Circulation. 2001; 103: 1522-1528.

Cowell SJ, Newby DE, Prescott RJ, Bloomfield P, Reid J, Northridge DB, Boon NA. A randomized trial of intensive lipid-lowering therapy in calcific aortic stenosis. N Engl J Med. 2005; 352: 2389-2397.

Rajamannan NM, Subramaniam M, Springett M, Sebo TC, Niekrasz M, McConnell JP, Singh RJ, Stone NJ, Bonow RO, Spelsberg TC. Atorvastatin inhibits hypercholesterolemia-induced cellular proliferation and bone matrix production in the rabbit aortic valve. Circulation. 2002; 105: 2660-2665.

Hu H, Hilton MJ, Tu X, Yu K, Ornitz DM, Long F. Sequential roles of Hedgehog and Wnt signaling in osteoblast development. Development. 2005; 132: 49-60.

Chertow GM, Burke SK, Raggi P. Sevelamer attenuates the progression of coronary and aortic calcification in hemodialysis patients. Kidney Int. 2002; 62: 245-252.

Hashiba H, Aizawa S, Tamura K, Shigematsu T, Kogo H. Inhibitory effects of etidronate on the progression of vascular calcification in hemodialysis patients. Ther Apher Dial. 2004; 8: 241-247.

Essalihi R, Dao HH, Gilbert LA, Bouvet C, Semerjian Y, McKee MD, Moreau P. Regression of medial elastocalcinosis in rat aorta: a new vascular function for carbonic anhydrase. Circulation. 2005; 112: 1628-1635.

Davies MR, Lund RJ, Hruska KA. BMP-7 is an efficacious treatment of vascular calcification in a murine model of atherosclerosis and chronic renal failure. J Am Soc Nephrol. 2003; 14: 1559-1567.

Hsu HH, Camacho NP, Sun F, Tawfik O, Aono H. Isolation of calcifiable vesicles from aortas of rabbits fed with high cholesterol diets. Atherosclerosis. 2000; 153: 337-348.


作者单位:Jian-Su Shao; Jun Cai; Dwight A. TowlerFrom the Washington University School of Medicine, Department of Medicine, St. Louis, Mo.

日期:2008年12月28日 - 来自[2006年第26卷第7期]栏目
循环ads

Elastin Calcification in the Rat Subdermal Model Is Accompanied by Up-Regulation of Degradative and Osteogenic Cellular Responses

【摘要】  Calcification of vascular elastin occurs in patients with arteriosclerosis, renal failure, diabetes, and vascular graft implants. We hypothesized that pathological elastin calcification is related to degenerative and osteogenic mechanisms. To test this hypothesis, the temporal expression of genes and proteins associated with elastin degradation and osteogenesis was examined in the rat subdermal calcification model by quantitative real-time reverse transcription-polymerase chain reaction and specific protein assays. Purified elastin implanted subdermally in juvenile rats exhibited progressive calcification in a time-dependent manner along with fibroblast and macrophage infiltration. Reverse transcription-polymerase chain reaction analysis showed that relative gene expression levels of matrix metalloproteinases (MMP-2and MMP-9) and transforming growth factor-ß1 were increased in parallel with calcification. Gelatin zymography showed strong MMP activities at early time points, which were associated with high levels of soluble elastin peptides. Gene expression of core binding factor-1, an osteoblast-specific transcription factor, increased in parallel with elastin calcification and attained 9.5-fold higher expression at 21 days compared to 3 days after implantation. Similarly, mRNA levels of the bone markers osteopontinand alkaline phosphatasealso increased progressively, but osteocalcinlevels remained unchanged. We conclude that degenerative and osteogenic processes may be involved in elastin calcification.
--------------------------------------------------------------------------------
Vascular calcification occurs at two distinct sites in the blood vessels. In atherosclerosis, calcification occurs mainly in the intima associated with lipid deposition, macrophages, and activated vascular smooth muscle cells, whereas medial calcification, also known as Möncke-berg??s medial sclerosis, occurs in the media and is typically associated with elastin.1 Vascular calcification is known to cause decreased elasticity, which is partially responsible for disorders related to elastic fibers.2 In the early stages of medial calcification, morphology differs distinctly from intimal calcification, appearing as linear deposits along elastic lamina throughout most of the medial width, and in advanced lesions, in which the media is filled with circumferential rings of mineral deposits. At later stages of the disease, osteocytes are seen within bone trabeculae with apparent bone marrow formation.3 Despite its clinical significance, the molecular mechanisms regulating calcification are still unclear. Several recent studies indicate that ectopic mineralization is a highly regulated active process exhibiting many characteristics of bone formation.4,5 In this study, we focused on evaluating the cellular and molecular mechanisms of elastin-specific calcification in an animal model.
Elastin is a major component of the extracellular matrix in cardiovascular connective tissues. Matrix metalloproteinases (MMPs) are expressed in normal physiological processes such as wound healing and angiogenesis, but increased levels of MMPs have also been identified in many cardiovascular pathologies.6 MMP-9 and MMP-2 bind and degrade insoluble elastin to generate soluble peptides.7 These elastin peptides can interact with a 67-kd transmembrane protein, the elastin laminin receptor (ELR),8 which is present on the surface of most cells. Activation of ELR by elastin peptides triggers diverse biological activities in various cell types including synthesis and release of elastase, liberation of free radicals, increased Ca2+ influx in endothelial cells, NO-dependent vasorelaxation, proliferation of arterial smooth muscle cells, chemotaxis of monocytes and fibroblasts, and apoptosis.9-13 Taken together, these data suggest a possible correlation between elastin degradation, activation of ELR, and elastin calcification, but the mechanisms that link these processes are as yet unknown. We have reported that MMP-mediated elastin degradation is the initial step in elastin calcification in the rat subdermal implantation model and that inhibition of MMPs leads to significant reduction in calcification.14,15 Moreover, we have recently shown in an abdominal aorta injury model in rats and MMP-knockout mice that MMP-mediated elastin degradation precedes elastin calcification and that the presence of active MMP-2 and MMP-9 are required for elastin calcification.16
We hypothesized that pathological elastin calcification is governed by two processes: enzyme-mediated elastin degeneration and cell-facilitated ectopic osteogenesis. As a result of elastin degradation, soluble peptides are released locally, where they activate quiescent cells, resulting in ELR and MMP up-regulation and enhanced matrix degradation. The degraded elastin-rich extracellular matrix becomes a calcification-prone substrate. We further hypothesized that elastin degradation also induces differentiation of nonbone cells into osteoblast-like cells, which secrete bone proteins that promote elastin calcification.17 To evaluate this hypothesis, we examined the expression of genes implicated in elastin degeneration such as MMP-2, MMP-9, ELR, and transforming growth factor(TGF-ß1) as well as osteogenesis-associated genes such as core binding factor--1(CBFA-1), osteocalcin(OCN), alkaline phosphatase(ALP), and osteopontin(OPN) using real-time reverse transcription-polymerase chain reaction (RT-PCR) in a time-course study in a rat subdermal elastin calcification model. We also evaluated soluble elastin peptide production and MMP activity, as markers of elastin degradation, as well as ALP enzyme activity and OCN secretion, as markers of osteoblastic differentiation. In the present study, we report that elastin remodeling and osteogenesis may be involved in elastin calcification.

【关键词】  calcification subdermal accompanied up-regulation degradative osteogenic cellular responses

Materials and Methods

Preparation of Pure Porcine Aortic Elastin

Porcine hearts were obtained from a local slaughterhouse and transported to the laboratory on ice. A supravalvular aortic segment (3 to 4 cm) was dissected, cleaned to remove fat and adherent tissues, and rinsed in cold saline. Aortic elastin was purified by autoclaving, as previously described by Partridge and Keeley18 Briefly, each aorta was cut into 2-mm strips, rinsed with distilled water, and shredded using a blender. The shredded aorta was washed with cold saline to remove soluble proteins until no protein was detected by BCA assay (Pierce, Rockford, IL). The washed aorta was autoclaved four times in distilled water (1 hour per cycle) and washed with distilled water until no protein was detected in the solution by BCA assay. This was then followed by defatting the aorta with ethanol and diethyl ether and lyophilization. This procedure extracts all cellular materials, collagenous and noncollagenous components, including elastin-associated glycoproteins, leaving pure elastin intact.

Subdermal Implantation of Elastin

Juvenile male Sprague-Dawley rats (21 days old, 35 to 40 g; Harlan, Indianapolis, IN) were anesthetized with acepromazine (0.5 mg/kg; Ayerst Laboratories, Inc., Rouse Point, NJ) and maintained on isoflurane gas (2 to 2.5%) throughout surgery. A small incision was made on the back of the rats, and three subdermal pouches were formed by blunt dissection. Each rat received three 30- to 40-mg elastin implants (one per pouch), which were rehydrated in sterile saline 1 to 2 hours before implantation. Four rats were sacrificed by CO2 asphyxiation at each time point (3, 7, 14, and 21 days after implantation), and the implants were retrieved along with the surrounding fibrous capsule. One explant from each rat was frozen in OCT (Sakura Finetek, Zoeterwoude, The Netherlands) on dry ice and stored at C80??C for immunohistochemistry; another explant was frozen on dry ice for protein analysis. The third explant from each rat was stored in RNAlater (Ambion, Austin, TX) for quantitative real-time RT-PCR. Small segments from each explant were frozen on dry ice for calcium and phosphorous analysis. As a control, uninjured subdermal tissue samples were collected from four rats and divided into two segments. One from each rat was stored in RNAlater for quantitative real-time RT-PCR and the other was frozen on dry ice for zymography. As a control medical grade polyester fabric (Dacron) samples (30 to 40 mg each, 4 x 4 mm pieces) were implanted subdermally in juvenile rats (n = 4) and explanted at 21 days as described above. Animal experiments were conducted according to National Institutes of Health guidelines for the care and use of laboratory animal (NIH publication no. 86-23, revised 1996). The animal protocol was approved by the Animal Research Committee at Clemson University.

Calcium and Phosphorous Determination

Analysis of calcium and phosphorous content in rat subdermal explants was performed by previously described procedures.19 Briefly, lyophilized elastin explants (16 to 23 mg) were placed in 1 ml of 6 N HCl and hydrolyzed in a boiling water bath for 8 hours. Samples were evaporated under a continuous stream of nitrogen gas and residual material dissolved in 1 ml of 0.01 N HCl. Calcium content in explants was determined (n = 8 per time point) with an atomic absorption spectrophotometer (model 3030; Perkin-Elmer, Norwalk, CT). Phosphorous content (n = 8 per time point) was measured on the same acid hydrolysates using the molybdate complexation assay.20

Immunohistochemical (IHC) Staining for Characterization of Cell Infiltrates

Explants embedded in OCT were cryosectioned (6 µm) and mounted onto poly-L-lysine-coated glass slides. Sections were fixed in cold acetone for 5 minutes and blocked with 0.3% hydrogen peroxide in 0.3% normal sera in Tris-buffered saline for 5 minutes to neutralize endogenous peroxidase activity. Sections were incubated with mouse-derived primary antisera directed against rat monocytes/macrophages (1:200 dilution, MAB1435; Chemicon, Temecula, CA), vimentin (1:5000 dilution; Sigma, St. Louis, MO) diluted in TNB blocking buffer (Perkin Elmer Life Sciences, Boston, MA), and -smooth muscle actin (Sigma) for 1 hour at room temperature. TNB buffer alone was used as negative control. Staining was visualized using rat-adsorbed biotinylated anti-mouse IgG secondary antibody (5 µg/ml; Vector Laboratories, Burlingame, CA), avidin-biotin-horseradish peroxidase complex (Vectastain Elite kit, Vector Laboratories), and diaminobenzidine substrate (Vector Laboratories). Sections were counterstained with hematoxylin and mounted. IHC for macrophages, fibroblasts, and pericytes was also performed on paraffin-embedded sections of uninjured rat skin as a control.

Alizarin Red Staining for Mineralization

Mineralization in elastin implants and Dacron implants was examined by Alizarin red staining.14 Frozen sections were warmed to room temperature, fixed in 95% ethanol for 30 seconds, stained for 3 minutes at room temperature with 1% Alizarin red solution, and rinsed with distilled water. Sections were counterstained with 1% light green solution for 8 to 10 seconds and rinsed with distilled water and mounted.

Gelatin Zymography for MMP Detection

Proteins from elastin explants with associated capsules and control rat subdermal tissue were extracted in a guanidine buffer, dialyzed, and analyzed in triplicates by gelatin zymography using 10 µg of protein per lane (as per BCA assay), as described before.21 Intensity of MMP bands (white on dark background) were evaluated by densitometry using LabImage software (Labsoft Diagnostics AG, Halle, Germany) and expressed as relative density units (RDUs) normalized to protein content.

Enzyme-Linked Immunosorbent Assay (ELISA) for Elastin Peptides

Quantitation of soluble elastin peptides was performed in the same extracts that were used for zymography by a competitive ELISA as described by Wei and colleagues22 with some modifications. Wells of microtiter plates were coated with 2 µg/ml of elastin peptides (CB573; Elastin Products Company Inc., Owensville, MO). Simultaneously, in a separate plate (precoated with 0.5% bovine serum albumin), samples were mixed 1:1 (v/v) with a 1:500 dilution of rabbit anti-bovine neck elastin antibody (PR403, Elastin Products Company Inc.). This primary antibody is specific for bovine and porcine elastin but does not cross-react with rat elastin.

After overnight incubation of both plates at 4??C, contents of each sample-antibody mixture well were transferred to corresponding wells in the elastin peptide-coated plate and allowed to react for 30 minutes at 37??C, followed by secondary antibody (anti-rabbit IgG peroxidase conjugate, diluted 1:2000). After 60 minutes at 37??C, peroxidase activity was detected using o-phenylene diamine hydrochloride as a substrate at 490 nm. All washes between steps and dilutions of samples and antibodies were performed in phosphate-buffered saline containing 0.05% Tween 20 and 0.5% bovine serum albumin. Extracts obtained from unimplanted elastin served as controls. All assays were done in triplicates and concentrations of soluble elastin peptides were calculated from a standard curve obtained with 0 to 2 µg/ml of competing elastin peptides and expressed as µg per mg protein.

Assays for ALP, OCN, and CBFA Protein Expression

Elastin explants with associated capsules were homogenized in RIPA extraction buffer (50 mmol/L Tris, 150 mmol/L NaCl, 1 mmol/L ethylenediaminetetraacetic acid, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, pH 7.4) including protease inhibitor cocktail (Sigma). Homogenized explants were centrifuged at 13,000 rpm for 15 minutes, supernatants were collected, and protein concentrations were determined using a BCA assay kit (Pierce). ALP activities were examined with p-nitrophenyl phosphate as a substrate (procedure no. 104; Sigma Diagnostic Inc., St. Louis, MO). Enzyme activity was calculated using a p-nitrophenol standard curve and expressed as Sigma U per mg protein. The OCN ELISA was performed using a rat OCN EIA kit (Biomedical Technologies Inc., Stoughton, MA) and following the manufacturer??s instructions. Rat OCN antibody-precoated well strips were incubated with samples at 4??C overnight and reacted with OCN antiserum at 37??C for 1 hour. Donkey anti-goat IgG peroxidase conjugate was used as secondary antibody and 3,3',5,5'-tetra-methyl benzidine was used as the peroxidase substrate. Levels of OCN were calculated using a rat OCN standard curve and normalized to total protein concentration. CBFA-1 was assayed in elastin explants using the TransAM AML-3/Runx2 transcription factor assay kit (Active Motif, Carlsbad, CA) according to the manufacturer??s instructions. Levels of CBFA-1 were calculated using an osteoblast nuclear extract standard curve and normalized to total protein.

RNA Isolation and Characterization

Total RNA was isolated at predetermined time points from elastin-explanted and uninjured rat subdermal tissue using RNeasy fibrous tissue kit (Qiagen, Valencia, CA). RNA quality and quantity were evaluated for each sample using the RNA 6000 Nano Assay, Agilent Bioanalyzer 2100 (Agilent Technologies, Inc., Germantown, MD). The purity of RNA was determined by RIN (RNA integrity number) software algorithm (Agilent Technologies, Inc.), which allows for the classification of eukaryotic total RNA based on a numbering system from 1 to 10, with 1 being the most degraded RNA and 10 being the most intact RNA. The RINs of all RNAs extracted were greater than 9 (data not shown).

Real-Time RT-PCR Analysis

Reverse transcription reactions were performed with 1 µg of total RNA for each 20-µl RT reaction using Moloney murine leukemia virus reverse transcriptase with oligo (dT) primers (RetroScript Kit; Ambion). Target-specific PCR primers were designed using Primer3 software and synthesized by Integrated DNA Technologies Inc. (Coralville, IA). Primer sets, gene accession numbers, and PCR product sizes are listed in Table 1 . Real-time PCR amplifications were performed using SYBR Green PCR kit (Qiagen) in a Rotorgene 3000 thermal cycler (Corbett Research, Mortlake, NSW, Australia), and the cycle number at which the amplification plot crosses the threshold was calculated (CT). The 2CCT method was used to analyze the relative changes in gene expression from real-time quantitative PCR using ß2-microglobulin (ß2-MG) as a housekeeping gene. Minus RT reactions performed on a representative subset of samples demonstrated that genomic DNA contamination was insignificant (data not shown). Reaction specificities were routinely verified by product purification, agarose gel electrophoresis, as well as routine melting curve analysis. The identities of PCR products were determined by automated sequencing at Arizona State University DNA Analysis Facility. The levels of gene expression were presented as the change in expression of a given time point (X) relative to 3 days as time 0 in a time-course study using the 2CCT method23 as follows: CT = (CT target gene C CT ß2-MG)Time X C (CT target gene C CT ß2-MG)Time 0.

Table 1. Primers for Real-Time RT-PCR

Statistical Analysis

Data are reported as mean ?? SEM. Statistical significance of value at a given time point as compared to the 3-day time point was determined by Student??s t-test with statistical significance defined as P < 0.05.

Assessment of Calcification

Calcium content in elastin explants increased progressively with time (P < 0.05 for all time points) and reached 133.65 ?? 5.82 µg Ca/mg dry explant at 21 days. Phosphorous content also increased in parallel with calcium content (Figure 1) . The molar ratios of Ca/P were 2.5, 1.6, 1.5, and 1.4 at 3, 7, 14, and 21 days, respectively, suggesting the presence of poorly crystalline hydroxyapatite deposition. We have previously analyzed the mineral associated with elastin in this animal model by X-ray diffraction and have shown that it resembles hydroxyapatite.24 Control Dacron implants did not show any calcification in this model (0.8 ?? 0.09 µg Ca/mg dry explants at 21 days).

Figure 1. Time course of elastin calcification in the rat subdermal model. The calcium and phosphorous contents are expressed as µg/mg dry explant and are presented as means ?? SEM (n = 8).

Characterization of Cellular Infiltration and Mineralization

To evaluate the incidence of infiltrating cells in the implants, IHC was performed to identify macrophages, fibroblasts, and pericytes/activated myofibroblasts. Vimentin-positive fibroblasts were observed throughout the capsule and within the elastin implants and they were apparently the dominant cells alongside with infiltrating macrophages (Figure 2, A and B) . These cells appeared to make intimate contact with the elastin fibers. -Smooth muscle cell actin staining showed the presence of pericytes in small blood vessels forming within the capsule (Figure 2C) . Normal skin samples of rats showed strong staining for fibroblasts throughout and for pericytes around the vasculature but weak staining for few scattered macrophages (Figure 2, DCF) . Alizarin red staining showed that elastin fibers started to calcify at the edge of elastin implants as early as 3 days after implantation (data not shown), and significant calcium deposition was noted throughout the entire elastin implant at 21 days after implantation (Figure 2G) whereas no calcification was observed in Dacron implant at 21 days after implantation (Figure 2H) .

Figure 2. Cellular infiltration of elastin implants. At 21 days after implantation, IHC staining showed that the elastin implant (EL) was invaded by numerous macrophage/monocytes (A), fibroblast-like cells (B), and pericytes/activated fibroblasts outlined by arrows (C). IHC staining for macrophages/monocytes (D), fibroblasts (E), pericytes/activated fibroblasts (F) is also shown for normal rat skin. Alizarin red staining showed mineralization of elastin fibers (G) at 21 days after implantation compared to no calcification in Dacron implant (DC) as a control (H). Insets show corresponding negative controls for IHC staining. Original magnifications, x200.

Characterization of Elastin Degradation

Relative gene expression levels for MMP-2, MMP-9, and TGF-ß1were up-regulated in elastin subdermal implants with increasing elastin calcification (Figure 3) . mRNA levels of TGF-ß1and MMP-9significantly increased at 7 days and remained at high levels at 2 and 3 weeks, respectively (P < 0.05), in parallel with increasing elastin calcification. In the case of MMP-2and ELR, mRNA levels were significantly increased (P < 0.05) at 7 and 14 days as compared to 3 days after implantation and then decreased at 21 days; overall the levels were lower than control rat subdermal tissue. To evaluate MMP activities, gelatin zymography was performed on protein extracts obtained at various time points from elastin explants and control rat subdermal tissue (Figure 4A) . At 3 days, MMP-9 activity was the strongest and then decreased slightly with time. MMP-2 activity was also increased at earlier time points and then decreased with time. Overall MMP levels were significantly higher in elastin implants as compared to control subdermal tissue. The content of soluble elastin peptides as a consequence of elastin degradation in elastin explants was determined by ELISA assay and showed the highest levels at 7 days, in parallel with the MMP activities in elastin explants (Figure 4B) .

Figure 3. Time course of gene expression associated with elastin degradation and remodeling in elastin implants. Isolated RNA was analyzed by real-time RT-PCR for MMP-2, MMP-9, ELR, TGF-ß1, and ß2-microglobulin. Gene expression levels are presented as the change in expression of a given time point relative to 3 days in a time course study using the 2CCT method and normalized to ß2-MG. Data points represent means ?? SEM (n = 4). *P < 0.05.

Figure 4. Protein expression associated with elastin degradation. A: MMP activities from elastin explants were assayed by gelatin zymography (top) and expressed as RDU (relative density units)/mg protein (bottom). Data points represent means ?? SEM (n = 3). B: Content of soluble elastin peptides was determined by ELISA and expressed as µg elastin peptides/mg protein. Data points represent means ?? SEM (n = 3).

Characterization of Osteogenic Signals

Real-time RT-PCR was performed on the isolated RNA from elastin explants at different time points to evaluate osteogenic gene expression in cells associated with elastin implants. Relative gene expression of CBFA-1, OPN, and ALPincreased in elastin subdermal implants with increasing elastin calcification, but OCNlevels, a late marker of calcification in osteogenesis, remained unchanged (Figure 5) . In particular, mRNA levels of CBFA-1, an osteoblast-specific transcription factor, increased significantly at all time points compared to 3 days and attained 9.5-fold higher expression at 21 days than that at 3 days (P < 0.05). ELISA quantitation detected CBFA-1 protein levels in elastin explants at 14 and 21 days after implantation (12.62 ?? 0.66 and 6.88 ?? 3.27 µg nuclear extract equivalent of osteoblasts/mg protein, respectively).

Figure 5. Time course of osteoblast-specific gene expression in elastin implants. Isolated RNA was analyzed by RT-PCR for CBFA-1, OCN, OPN, and ALP, and ß2-microglobulin. The levels of mRNA expression are presented as the change in expression relative to 3 days in a time course study using the 2CCT method and normalized to ß2-microglobulin. Data points represent means ?? SEM (n = 4). *P < 0.05.

mRNA levels of OPNand ALPwere higher at all time points relative to those at 3 days. ALP activity and OCN protein production in elastin explants as markers of osteogenic remodeling were also evaluated. ALP activity increased 1.32-, 5.44-, and 4.07-fold at 7, 14, and 21 days, respectively, compared to 3 days (Figure 6A) . OCN levels were, however, not significantly changed (P > 0.05) in parallel with mineralization (Figure 6B) . Overall, protein secretion of ALP and OCN matched with mRNA levels at various time points.

Figure 6. Protein expression associated with osteoblast-like cell differentiation in elastin implants. A: ALP activity in protein extracts of elastin explants were assayed and expressed as Sigma units/mg protein. Data points represent means ?? SEM (n = 3). B: OCN was determined by ELISA assay and expressed as ng OCN/mg protein. Data points represent means ?? SEM (n = 3). *P < 0.05.

Pathological vascular elastin-specific calcification is seen in a variety of diseases including patients with arteriosclerosis, renal failure and diabetes, implanted vascular grafts, and aging aortic stenosis.25-27 We have previously reported that purified porcine aortic elastin, when implanted subdermally in rats, exhibited progressive calcification.15 The present work is an extension of previous studies to evaluate the cellular and molecular mechanisms involved in elastin calcification. We hypothesized that pathological calcification of medial arterial calcification is associated with two processes: elastin degeneration and ectopic osteogenesis. To evaluate this hypothesis, we subdermally implanted purified porcine aortic elastin in rats and evaluated gene expression accompanying elastin calcification.

MMPs have been identified in pathological conditions in many cardiovascular diseases.6 Longo and colleagues28 reported that macrophage-derived MMP-9 and MMP-2 derived from mesenchymal cells (smooth muscle cells and fibroblasts) work in concert to produce abdominal aortic aneurysms, which involves elastic lamina degeneration. MMP-9 and MMP-2 degrade insoluble elastin to soluble peptides. Elastin peptides have been shown to activate the ELR.8 The ELR was identified on lymphocytes, macrophages, granulocytes, smooth muscle cells, and endothelial cells.29,30 Activation of this receptor by binding of elastin peptides triggers diverse biological activities. These peptides stimulate fibroblast adhesion to elastin fibers,31 regulate cellular proliferation,32 and are chemotactic for several cell types, such as monocytes33 and fibroblasts.9 Substantial infiltration of elastin implants by macrophages/monocytes and fibroblasts, observed by IHC staining (Figure 2, A and B) , resulted in higher cell densities than those observed in normal rat skin (Figure 2, D and E) . Increased mRNA expression of MMP-9and MMP-2was observed at all time points compared to 3 days as an earliest time point of calcification (Figure 3) . Both MMP-9 and MMP-2 showed high enzyme activities at early time points in calcification (Figure 4A) , and the level of degraded elastin peptides in elastin explants was also increased in parallel with MMP expression (Figure 4B) . The mRNA level of ELRwas also increased significantly at 7 and 14 days compared to 3 days although the expression levels in implants were lower than those found in normal skin. Thus, it is possible that MMP-mediated degradation of elastin after implantation leads to binding of soluble elastin peptides to ELR on both fibroblasts and macrophages in our implants. The mRNA level of TGF-ß1was also increased in parallel with MMP-2and MMP-9gene expression. Several studies have demonstrated multiple interactions between MMPs and TGF-ß1 that suggest simultaneous expression of these molecules may promote elastin calcification through a positive-feedback mechanism. MMP-2 and MMP-9 can activate TGF-ß1 by cleaving its latent form,34 and TGF-ß1 has been shown to induce an elevated level of MMPgene expression.35,36 TGF-ß1 promotes osteoprogenitor cell proliferation and osteogenesis. TGF-ß1 also stimulates ALP activity in cells.37 Thus, increased expression of TGF-ß1 in our implants may promote osteogenesis.

The occurrence of ectopic osteogenesis process was confirmed by the up-regulated expression of bone-specific genes such as CBFA-1, OCN, OPN, and ALPin elastin implants. Of particular interest, progressive and sustained increases in the expression of the osteoblast-specific transcription factor CBFA-1were observed, attaining 9.5-fold higher expression at 21 days compared to 3 days. Moreover, CBFA-1gene expression was observed 2.3-, 5.7-, 8.7-, and 20.2-fold higher expression at 3, 7, 14, and 21 days, respectively, than that of rat subdermal tissue. Detectable levels of CBFA-1 protein were found at 14 days and 21 days. CBFA-1is the earliest and most specific marker of developmental osteogenesis. CBFA-1acts as an activator of transcription and can induce osteoblast-specific gene expression in fibroblasts and myoblasts.38,39 Significantly increased OPNgene expression was also observed at 7 days and 21 days compared to 3 days and control rat subdermal tissue. OPN is a phosphorylated protein of wide tissue distribution that is found in calcified vascular tissues.40 Giachelli and colleagues41 reported that OPNexpression is increased under injury and disease in many tissues and it is closely related to calcified deposits found in numerous pathologies. We also demonstrated that production of bone marker proteins such as ALP was increased in parallel with calcification on days 14 and 21. OCN content did not significantly change when normalized to total protein.

In the present studies, IHC analysis showed that infiltration by pericytes/activated myofibroblasts was increased in a time-dependent manner along with fibroblast and macrophage invasion. Moreover, RT-PCR and protein analysis clearly indicated the presence of osteoblast-like cells in the vicinity of calcifying elastin. Although the origin of osteoblast-like cells in calcified elastin is not clearly defined in our study, we speculate that activated fibroblasts may undergo cell differentiation and express an osteoblast-like phenotype. In support of this speculation, we have recently observed increased gene and protein expression of CBFA-1 in primary rat skin fibroblast cell cultures exposed to elastin peptides and TGF-ß1 for several days (data not shown). However, more research is needed to identify what specific types of cells are involved in osteogenesis in the rat subdermal implantation model.

The present work involved study of elastin calcification in the rat subdermal implantation model, which may provide some insights for elastin-specific medial calcification seen in vasculature. Elastin in native arteries is surrounded by acidic glycoproteins, including fibrillin, fibulin, and latent TGF-ß1-binding protein among others.42 These glycoproteins may protect elastin from calcification. For example, fibrillin mutant mice develop severe elastin-specific vascular calcification.43 Glycoprotein levels in the arteries decrease with age, in parallel with the propensity for vascular calcification.44 Physical (plaque formation) or biochemical (inflammatory reactions) injury may expose elastin to cells. Cells may then produce MMPs and other serine elastases to degrade elastin. Elastin peptides would then activate ELR on a variety of cells, which in turn up-regulates MMP expression by positive feedback mechanisms. MMPs are also known to degrade glycoproteins surrounding elastin, including fibrillin,45 thus exposing more elastin for degradation. Several stimuli induce up-regulation of MMPgene expression and increase the degradation of matrix. The elastin degradation may release matrix-bound cytokines such as TGF-ß1, which are chemotactic for several cells and induce inflammatory reactions.46,47 These released cytokines may also induce the differentiation of smooth muscle cells or fibroblasts into osteoblast-like cells, which would produce bone-specific proteins and aid the calcification process.35 Vascular smooth muscle cells and pericytes, considered resting mesenchymal stem cells, can assume osteoblastic/chondrocytic phenotypes,3,48,49 implying a phenotypic modulation of resident vascular cells in a permissive matrix environment. TGF-ß1 has also been demonstrated to have osteogenic activity50,51 and to promote the calcification of aortic smooth muscle cells in culture.52

There is also a possibility of cross-talk between the ELR and TGF-ß/BMP-Smad intracellular pathways. The signal transduction pathway of ELR involves activation of phospholipase C by a pertussis toxin-sensitive G-protein. Phospholipase C induces the production of inositol triphosphate (IP3) leading to the increase in the intracellular free calcium.53 Free calcium would then bind to calmodulin. Calmodulin has been shown to bind Smads 1 to 4 in a calcium-dependent manner.54 The Smads were shown to play an important role in transducing specific TGF-ß1 signaling pathways. Recently, Smads were also shown to have links to CBFA-1.55 Thus, interrelations between ELR activation and osteogenic differentiation may exist. However, more research is needed to confirm these interactions.

In conclusion, initiation and progression of elastin calcification in the rat subdermal model involves MMP-mediated elastin degeneration and localized ectopic osteogenesis. The present study may shed some light on the mechanisms of elastin calcification in medial pathological vascular calcification. Ongoing gene silencing studies will extend our investigations of these mechanisms by using delivery of MMP siRNA and CBFA-1 siRNA to block elastin degradation and osteogenesis processes in the rat subdermal model and circulatory aortic injury models.

We thank Dr. Ken Webb for his assistance with real-time RT-PCR.

【参考文献】
  Proudfoot D, Shanahan CM: Biology of calcification in vascular cells: intima versus media. Herz 2001, 26:245-251

Niederhoffer N, Lartaud-Idjouadiene I, Giummelly P, Duvivier C, Peslin R, Atkinson J: Calcification of medial elastic fibers and aortic elasticity. Hypertension 1997, 29:999-1006

Shanahan CM, Cary NR, Salisbury JR, Proudfoot D, Weissberg PL, Edmonds ME: Medial localization of mineralization-regulating proteins in association with Monckeberg??s sclerosis: evidence for smooth muscle cell-mediated vascular calcification. Circulation 1999, 100:2168-2176

Rajamannan NM, Subramaniam M, Rickard D, Stock SR, Donovan J, Springett M, Orszulak T, Fullerton DA, Tajik AJ, Bonow RO, Spelsberg T: Human aortic valve calcification is associated with an osteoblast phenotype. Circulation 2003, 107:2181-2184

Bostrom K: Insights into the mechanism of vascular calcification. Am J Cardiol 2001, 88:20E-22E

Nagase H, Woessner JF, Jr: Matrix metalloproteinases. J Biol Chem 1999, 274:21491-21494

Mecham RP, Broekelmann TJ, Fliszar CJ, Shapiro SD, Welgus HG, Senior RM: Elastin degradation by matrix metalloproteinases. Cleavage site specificity and mechanisms of elastolysis. J Biol Chem 1992, 272:18071-18076

Hinek A, Wrenn DS, Mecham RP, Barondes SH: The elastin receptor: a galactoside-binding protein. Science 1988, 239:1539-1541

Mecham RP, Griffin GL, Madaras JG, Senior RM: Appearance of chemotactic responsiveness to elastin peptides by developing fetal bovine ligament fibroblasts parallels the onset of elastin production. J Cell Biol 1984, 98:1813-1816

Faury G, Garnier S, Weiss AS, Wallach J, Fulop T, Jr, Jacob MP, Mecham RP, Robert L, Verdetti J: Action of tropoelastin and synthetic elastin sequences on vascular tone and on free Ca2+ level in human vascular endothelial cells. Circ Res 1998, 82:328-336

Hinek A: Biological roles of the non-integrin elastin/laminin receptor. Biol Chem 1996, 377:471-480

Mochizuki S, Brassart B, Hinek A: Signaling pathways transduced through the elastin receptor facilitate proliferation of arterial smooth muscle cells. J Biol Chem 2002, 277:44854-44863

Hance KA, Tataria M, Ziporin SJ, Lee JK, Thompson RW: Monocyte chemotactic activity in human abdominal aortic aneurysms: role of elastin degradation peptides and the 67-kD cell surface elastin receptor. J Vasc Surg 2002, 35:254-261

Vyavahare N, Jones PL, Tallapragada S, Levy RJ: Inhibition of matrix metalloproteinase activity attenuates tenascin-C production and calcification of implanted purified elastin in rats. Am J Pathol 2000, 157:885-893

Bailey MT, Pillarisetti S, Xiao H, Vyavahare NR: Role of elastin in pathologic calcification of xenograft heart valves. J Biomed Mater Res A 2003, 66:93-102

Basalyga DM, Simionescu DT, Xiong W, Baxter BT, Starcher BC, Vyavahare NR: Elastin degradation and calcification in an abdominal aorta injury model: role of matrix metalloproteinases. Circulation 2004, 110:3480-3487

Simionescu A, Philips K, Vyavahare N: Elastin-derived peptides and TGF-beta1 induce osteogenic responses in smooth muscle cells. Biochem Biophys Res Commun 2005, 334:524-532

Partridge SM, Keeley FW: Age related and atherosclerotic changes in aortic elastin. Adv Exp Med Biol 1974, 43:173-191

Schoen FJ, Levy RJ: Pathology of substitute heart valves: new concepts and developments. J Card Surg 1994, 9:222-227

Chen P, Toribara T, Warner H: Microdetermination of phosphorous. Anal Chem 1956, 28:1756-1758

Bailey M, Pillarisetti S, Jones P, Xiao H, Simionescu D, Vyavahare N: Involvement of matrix metalloproteinases and tenascin-C in elastin calcification. Cardiovasc Pathol 2004, 13:146-155

Wei SM, Erdei J, Fulop T, Jr, Robert L, Jacob MP: Elastin peptide concentration in human serum: variation with antibodies and elastin peptides used for the enzyme-linked immunosorbent assay. J Immunol Methods 1993, 164:175-187

Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(CDelta Delta C(T)) method. Methods 2001, 25:402-408

Vyavahare N, Ogle M, Schoen FJ, Levy RJ: Elastin calcification and its prevention with aluminum chloride pretreatment. Am J Pathol 1999, 155:973-982

Bestetti-Bosisio M, Cotelli F, Schiaffino E, Sorgato G, Schmid C: Lung calcification in long-term dialysed patients: a light and electronmicroscopic study. Histopathology 1984, 8:69-79

Bobryshev YV, Lord RS, Warren BA: Calcified deposit formation in intimal thickenings of the human aorta. Atherosclerosis 1995, 118:9-21

David TE, Bos J: Aortic valve replacement with stentless porcine aortic valve: a pioneer series. Semin Thorac Cardiovasc Surg 1999, 11:9-11

Longo GM, Xiong W, Greiner TC, Zhao Y, Fiotti N, Baxter BT: Matrix metalloproteinases 2 and 9 work in concert to produce aortic aneurysms. J Clin Invest 2002, 110:625-632

Fulop T, Jr, Jacob MP, Khalil A, Wallach J, Robert L: Biological effects of elastin peptides. Pathol Biol (Paris) 1998, 46:497-506

Peterszegi G, Texier S, Robert L: Human helper and memory lymphocytes exhibit an inducible elastin-laminin receptor. Int Arch Allergy Immunol 1997, 114:218-223

Groult V, Hornebeck W, Ferrari P, Tixier JM, Robert L, Jacob MP: Mechanisms of interaction between human skin fibroblasts and elastin: differences between elastin fibres and derived peptides. Cell Biochem Funct 1991, 9:171-182

Ghuysen-Itard AF, Robert L, Jacob MP: Effect of elastin peptides on cell proliferation. C R Acad Sci III 1992, 315:473-478

Senior RM, Griffin GL, Mecham RP: Chemotactic activity of elastin-derived peptides. J Clin Invest 1980, 66:859-862

Yu Q, Stamenkovic I: Cell surface-localized matrix metalloproteinase-9 proteolytically activates TGF-beta and promotes tumor invasion and angiogenesis. Genes Dev 2000, 14:163-176

Jeziorska M: Transforming growth factor-betas and CD105 expression in calcification and bone formation in human atherosclerotic lesions. Z Kardiol 2001, 90(Suppl 3):23-26

Sternlicht MD, Werb Z: How matrix metalloproteinases regulate cell behavior. Annu Rev Cell Dev Biol 2001, 17:463-516

Yeung HY, Lee KM, Fung KP, Leung KS: Sustained expression of transforming growth factor-beta1 by distraction during distraction osteogenesis. Life Sci 2002, 71:67-79

Ducy P, Zhang R, Geoffroy V, Ridall AL, Karsenty G: Osf2/Cbfa1: a transcriptional activator of osteoblast differentiation. Cell 1997, 89:747-754

Harada H, Tagashira S, Fujiwara M, Ogawa S, Katsumata T, Yamaguchi A, Komori T, Nakatsuka M: Cbfa1 isoforms exert functional differences in osteoblast differentiation. J Biol Chem 1999, 274:6972-6978

Giachelli CM: Ectopic calcification: gathering hard facts about soft tissue mineralization. Am J Pathol 1999, 154:671-675

Giachelli CM, Liaw L, Murry CE, Schwartz SM, Almeida M: Osteopontin expression in cardiovascular diseases. Ann NY Acad Sci 1995, 760:109-126

Rosenbloom J, Abrams WR, Mecham R: Extracellular matrix 4: the elastic fiber. FASEB J 1993, 7:1208-1218

Pereira L, Lee SY, Gayraud B, Andrikopoulos K, Shapiro SD, Bunton T, Biery NJ, Dietz HC, Sakai LY, Ramirez F: Pathogenetic sequence for aneurysm revealed in mice underexpressing fibrillin-1. Proc Natl Acad Sci USA 1999, 96:3819-3823

Godfrey M, Nejezchleb PA, Schaefer GB, Minion DJ, Wang Y, Baxter BT: Elastin and fibrillin mRNA and protein levels in the ontogeny of normal human aorta. Connect Tissue Res 1993, 29:61-69

Ashworth JL, Murphy G, Rock MJ, Sherratt MJ, Shapiro SD, Shuttleworth CA, Kielty CM: Fibrillin degradation by matrix metalloproteinases: implications for connective tissue remodelling. Biochem J 1999, 340:171-181

Gregory AK, Yin NX, Capella J, Xia S, Newman KM, Tilson MD: Features of autoimmunity in the abdominal aortic aneurysm. Arch Surg 1996, 131:85-88

Shapiro SD: Matrix metalloproteinase degradation of extracellular matrix: biological consequences. Curr Opin Cell Biol 1998, 10:602-608

Diefenderfer DL, Brighton CT: Microvascular pericytes express aggrecan message which is regulated by BMP-2. Biochem Biophys Res Commun 2000, 269:172-178

Doherty MJ, Ashton BA, Walsh S, Beresford JN, Grant ME, Canfield AE: Vascular pericytes express osteogenic potential in vitro and in vivo. J Bone Miner Res 1998, 13:828-838

Miyazono K, Kusanagi K, Inoue H: Divergence and convergence of TGF-beta/BMP signaling. J Cell Physiol 2001, 187:265-276

Bonewald LF, Dallas SL: Role of active and latent transforming growth factor beta in bone formation. J Cell Biochem 1994, 55:350-357

Jian B, Narula N, Li QY, Mohler ER, III, Levy RJ: Progression of aortic valve stenosis: TGF-beta1 is present in calcified aortic valve cusps and promotes aortic valve interstitial cell calcification via apoptosis. Ann Thorac Surg 2003, 75:456-465

Fulop T, Jr, Douziech N, Jacob MP, Hauck M, Wallach J, Robert L: Age-related alterations in the signal transduction pathways of the elastin-laminin receptor. Pathol Biol (Paris) 2001, 49:339-348

von Bubnoff A, Cho KWY: Intracellular BMP signalling regulation in vertebrates: pathway or network? Dev Biol 2001, 239:1-14

Ito Y, Zhang YW: A RUNX2/PEBP2alphaA/CBFA1 mutation in cleidocranial dysplasia revealing the link between the gene and Smad. J Bone Miner Metab 2001, 19:188-194


作者单位:From the Department of Bioengineering, Clemson University, Clemson, South Carolina

日期:2008年5月29日 - 来自[2006年第168卷第2期]栏目

Regulation of Vascular Calcification

    the Bioengineering Department, University of Washington, Seattle, Wash.

    Abstract

    Vascular calcification is prevalent in aging as well as a number of pathological conditions, and it is now recognized as a strong predictor of cardiovascular events in the general population as well as diabetic and end-stage renal disease patients. Vascular calcification is a highly regulated process involving inductive and inhibitory mechanisms. This article focuses on two molecules, phosphate and osteopontin, that have been implicated in the induction or inhibition of vascular calcification, respectively. Elevated phosphate is of interest because hyperphosphatemia is recognized as a major nonconventional risk factor for cardiovascular disease mortality in end-stage renal disease patients. Studies to date suggest that elevated phosphate stimulates smooth muscle cell phenotypic transition and mineralization via the activity of a sodium-dependent phosphate cotransporter. Osteopontin, however, appears to block vascular calcification most likely by preventing calcium phosphate crystal growth and inducing cellular mineral resorption.

    Key Words: cardiovascular disease  osteopontin  phosphate  pyrophosphate  smooth muscle cell  vascular calcification

    Introduction

    Calcification of the cardiovascular system is associated with a number of diseases, including end-stage renal disease (ESRD) and cardiovascular disease. Calcium phosphate deposition is the hallmark of vascular calcification and can occur in the blood vessels, myocardium, and cardiac valves. Calcium phosphate deposits are found in distinct layers of the blood vessel and are associated with specific pathologies. Intimal calcification is observed in atherosclerotic lesions,1,2 whereas medial calcification is common to the arteriosclerosis observed with age, diabetes, and ESRD.3,4 Intimal and medial calcification may occur independently of each other. In ESRD patients, intimal and medial calcification have been observed in affected vessels,5,6 although the etiological and clinical significance of this finding is not yet clear.

    Vascular calcification can lead to life-threatening organ dysfunction depending on its extent and the organ affected. For example, calcification of cardiac valve leaflets is recognized as a major mode of failure of native and bioprosthetic valves.7,8 Furthermore, vascular calcification is responsible for calcific uremic arteriolopathy, a necrotizing skin condition associated with extremely high mortality rates.9 Finally, idiopathic infantile arterial calcification, a genetic disease characterized by arterial calcification, fibrosis, and stenosis, leads to premature death in afflicted neonates.10

    In contrast, age and vascular disease-related vascular calcifications were previously considered benign. However, recent clinical studies have challenged this dogma. Calcification has been positively correlated with coronary atherosclerotic plaque burden,11,12 increased risk of myocardial infarction,13eC15 and plaque instability.2,16 Furthermore, in the Rotterdam Coronary Calcification Study, a large population-based study, graded associations between coronary calcification score and stroke were identified.17 Similarly, medial arterial calcification is strongly correlated with coronary artery disease and future cardiovascular events in type I diabetic subjects,18,19 and is a strong prognostic marker of cardiovascular disease mortality in ESRD patients.20 These findings may be explained by growing evidence that vascular medial calcification in large arteries leads to increased stiffness and therefore decreased compliance of these vessels. These mechanical changes are associated with increased arterial pulse wave velocity and pulse pressure, and lead to impaired arterial distensibility, increased afterload favoring left ventricular hypertrophy, and compromised coronary perfusion.21 Thus, intimal and medial calcifications may contribute to the morbidity and mortality associated with cardiovascular disease.

    It is becoming increasingly clear that vascular calcification is an actively regulated process that may be initiated by a number of different, nonmutually exclusive mechanisms. These mechanisms have been extensively reviewed elsewhere22 and include: (1) loss of mineral inhibiting factors; (2) induction of bone formation; (3) cell death; and (4) circulating nucleational complexes (ie, aggregates of calcium phosphate and proteins released from remodeling bone that may initiate ectopic mineralization). Abnormalities in mineral metabolism that enhance the calcium x phosphate product (Ca x P) may further exacerbate vascular calcification initiated by any of these mechanisms. This article focuses on recent evidence implicating elevated phosphate as a major inductive factor for vascular calcification and osteopontin as an inducible inhibitor of vascular calcification, and our current understanding of their mechanisms of action.

    Role of Phosphate

    Hyperphosphatemia is commonly observed in renal disease, especially in ESRD patients.23 Elevated serum phosphorus (in the form of phosphate) is a major risk factor for vascular calcification and cardiovascular mortality in these patients.23,24 Although elevations in the Ca x P may thermodynamically drive calcification, growing evidence indicates that direct effects of elevated phosphate on vessel wall cells may be more important in regulating the propensity of the vessel to calcify. We and others have found that heterogeneous, uncloned populations of vascular smooth muscle cells (VSMCs) do not spontaneously mineralize in culture, but can be induced to mineralize by elevating phosphate levels in the culture medium to those typically observed in hyperphosphatemic individuals (>2 mmol/L).25eC29 Under these conditions, the extracellular matrix surrounding the VSMCs undergoes calcification with features similar to that observed in bone and in pathological vascular calcification in vivo, including the presence of calcifying collagen fibers, matrix vesicles, and bioapatite.30 In VSMC cultures, calcification does not appear to require apoptosis or to be associated with apoptotic cells30 (Giachelli and Li unpublished observations), although induction of apoptosis may accelerate calcification.31

    Concomitant with induction of VSMC mineralization, treatment with elevated phosphate induces cultured VSMCs to undergo a profound phenotypic transition. Under normal phosphate conditions, VSMCs express smooth muscle lineage markers representative of the contractile phenotype, including smooth muscle (SM) -actin and SM22. After treatment with elevated phosphate, there is a dramatic loss of these smooth muscle cell (SMC) lineage markers, and simultaneous gain of osteochondrogenic markers such as osteopontin, Cbfa-1/Runx2, alkaline phosphatase, and osteocalcin.27,32 Importantly, almost identical changes in smooth muscle gene expression are observed in biopsy specimens from ESRD patients with calciphylaxis32 and with calcified inferior epigastric arteries.33 Finally, in an experimental mouse model of vascular calcification, the matrix Gla protein null (MGPeC/eC) mice, spontaneous vascular calcification was found in mice older than 2 weeks of age.34 Before mineralization, the vessels appear normal and medial SMCs express abundant SM lineage genes. However, as mineralization of the elastic lamellae ensues, medial cells lose SM -actin and SM22 expression and gain expression of osteopontin, alkaline phosphatase, and Cbfa1/Runx230 (Speer and Giachelli, unpublished observation). Furthermore, analysis of older MGPeC/eC mice with advanced vascular calcification showed clusters of cells with chondrocytic features including type II collagen expression.34

    Based on these findings, we have hypothesized that VSMCs have the capacity to undergo modulation from a contractile to an osteochondrogenic phenotypic state that is controlled by local environmental cues such as elevated phosphate levels (Figure). According to gene expression patterns, this phenotypic state appears to be distinct from the previously characterized synthetic/dedifferentiated state seen in arteries injured by chemicals, disease, or trauma.35 The osteochondrogenic state may be exquisitely designed to repair and/or adapt to a mineralizing microenvironment, with enhanced expression of a number of mineral regulating molecules including the mineralization inhibitor, osteopontin, discussed later. Recent studies suggest that other molecules that promote or inhibit vascular calcification may also act, in part, by regulating VSMC phenotypic change, including elevated calcium29 and BMP-7.36

    SMC phenotypic modulation associated with vascular calcification. It is hypothesized that VSMCs have the capacity to undergo modulation from a contractile to an osteochondrogenic phenotypic state that is controlled by local environmental cues such as elevated phosphate (P) or calcium (Ca) levels. The osteochondrogenic state is proposed to regulate matrix mineralization and to participate in the formation of bone and cartilage-like structures observed in calcified vascular lesions. SM indicates smooth muscle; alk phos, alkaline phosphatase; OPN, osteopontin; MHC, myosin heavy chain. The (+) indicates expression and (eC) indicates lack of expression.

    Mechanisms controlling this phenotypic transition in response to elevated phosphate in VSMCs are currently undergoing active investigation. We have determined that mineralization and VSMC phenotypic modulation in response to elevated phosphate are dependent on the activity of sodium-dependent phosphate cotransporters in the cells. Sodium-dependent phosphate cotransporters use the sodium gradient to actively transport phosphate into the cell. Three types of sodium dependent phosphate cotransporters have been identified based on structure, tissue expression, and regulation, and each family contains several members identified from various species. The type I and type II sodium-dependent phosphate cotransporters are predominantly expressed in intestine and kidney, and function to control phosphate reabsorption by these tissues.37,38 The type III sodium-dependent phosphate cotransporters are represented by Pit-1 (also named Glvr-1 and SLC20A1) and Pit-2 (also named Ram-1 and SLC20A2).39 These proteins are more ubiquitously expressed in tissues including kidney, heart, lung, brain, liver, and bone.40 Although the physiological functions of type III sodium-dependent phosphate cotransporters have not yet been identified, they may serve a more generalized function to allow phosphate movement into cells in support of oxidative phosphorylation. Although type III sodium-dependent phosphate cotransporters appear to be constitutively expressed in many tissues, phosphate deficiency39 and certain cytokines like insulin-like growth factor, transforming growth factor-, and platelet-derived growth factor41eC43 induce expression, whereas PTH reduces expression.44 We have found that Pit-1 and Pit-2 are the only sodium-dependent phosphate cotransporters expressed in human VSMCs29 (Li and Giachelli, unpublished data). Inhibition of sodium-dependent phosphate transport by phosphonoformic acid, a generic sodium-dependent phosphate cotransporter inhibitor, blocked elevated phosphate-induced SMC mineralization.26,29,45 Furthermore, elevated phosphate-induced Cbfa-1 and osteocalcin expression were also inhibited by phosphonoformic acid.26,45 Most recently, we have found that suppression of endogenous Pit-1 expression by small interfering RNAs inhibits SMC mineralization in response to elevated phosphate, and overexpression of either Pit-1 or Pit-2 is able to rescue the phosphate-induced mineralization in Pit-1eCdeficient cells.46 In addition, Suzuki et al showed that vasopressin induces SMC mineralization through the enhancement of the phosphate transport activity of Pit-1.47 These findings suggest that Pit-1 and phosphate transport play a crucial role in SMC mineralization and phenotypic modulation by elevated phosphate.

    Growing evidence suggests that type III sodium-dependent phosphate cotransporters are likely to be important mediators of cell-mediated matrix mineralization in general. In osteoblasts, Pit-1 mRNA levels increase and correlate with differentiation and mineralization.48 Furthermore, inhibition of sodium-dependent phosphate cotransporters by phosphonoformic acid also blocked phosphate-induced mineralization and osteopontin (OPN) expression in this cell type.49,50 Finally, phosphate uptake as well as mineralization of chondrocyte-derived matrix vesicles have been shown to depend on the activity of phosphate transporters, including type III sodium-dependent phosphate cotransporters.51,52 Matrix vesicles are thought to be key nucleating structures during endochondral ossification, and matrix vesicles have been described in calcified vascular lesions53 and cultured SMCs.28 Thus, these studies suggest that phosphate must be transported through mineral-forming cells, perhaps via matrix vesicles, to participate in matrix mineralization, and additionally that phosphate may have a unique signaling role(s) in these cells.

    Although this review has focused on potential roles of VSMCs, it is important to stress that other cell types may also contribute to the osteochondrogenic processes observed in vascular calcification. In a series of elegant experiments, Demer et al identified and cloned a population of bovine arterial medial cells, termed calcifying vascular cells, that spontaneously form nodules that mineralize in vitro under long-term culture.54,55 These cells lack characteristic VSMC markers and display pericyte-like properties early in culture, and develop osteoblastic features, including expression of alkaline phosphatase, osteocalcin, and mineralization, with time in culture. Nodulation and mineralization of calcifying vascular cells are modulated by a large number of proatherogenic factors, and have more recently been shown to undergo additional developmental fates, including leiomyogenesis, depending on the culture conditions.55 Thus, these cells behave like pericytes that have long been postulated as a reservoir of multipotent stem cells in adults and can be induced to differentiate into multiple lineages, including osteoblasts.56 Likewise, Towler et al have described vascular myofibroblasts expressing Msx2 associated with mineralization in diabetic low-density lipoprotein receptor-null mice.57eC59 These data support the presence of non-SMeCderived, pluripotent stem cell-like populations within the artery wall capable of osteogenic differentiation that might be involved in vascular calcification under pathological conditions. Thus, lineage studies are critically needed to determine conditions and disease states under which pluripotent stem cells, phenotypic modulation of SMCs, or both contribute to vascular calcification.

    Role of Osteopontin

    It has long been known that blood and body fluids are at or near saturation with respect to calcium and phosphate levels, suggesting that mechanisms must exist to prevent ectopic calcification. Human and mouse genetic findings have now determined that most tissues, including blood vessels, normally express inhibitors of mineralization, and lack of these molecules ("loss of inhibition") leads to calcification. In humans, a dramatic example of the importance of this mechanism is genetic deficiency of the small molecule, pyrophosphate, that leads to idiopathic infantile arterial calcification.10,60 In this condition, calcification of the arteries leads to a severe fibroproliferative vascular disease that culminates in heart failure soon after birth. Thus, pyrophosphate has emerged as a major regulator of vascular calcification during human development. A model mimicking the human disease, the tip-toe walking mouse, has a naturally occurring mutation in nucleotide pyrophosphate/phosphodiesterase I, which leads to pyrophosphate deficiency. In addition to articular cartilage calcification, ankylosis, and increased cementum, these animals display vascular calcification.61eC65 A growing number of other putative calcification inhibitory molecules have been identified using mouse mutational analyses, including MGP, -glucosidase, carbonic anhydrase II, fetuin, osteoprotegerin, desmin, and Smad 6.34,66eC73 Mutant mice deficient in these molecules present with enhanced cardiovascular calcification as part of their phenotype and demonstrate that specific proteins and small molecules are normally important in suppressing ectopic calcification, including vascular calcification.

    Another important molecule is OPN. OPN is an acidic phosphoprotein normally found in mineralized tissues such as bones and teeth, and it is involved in regulation of mineralization by acting as an inhibitor of apatite crystal growth, as well as promoting osteoclast function through the v3 integrin.74 Although OPN is not found in normal arteries, we75eC77 and others78eC82 have reported that OPN is abundant at sites of calcification in human atherosclerotic plaques and in calcified aortic valves. In addition, OPN levels are greatly elevated in the spontaneously mineralizing arteries of MGPeC/eC mice.27 These findings suggest that OPN may be an important regulator of arterial mineral deposition under conditions of injury and disease.

    To examine the role of OPN in vascular calcification, we bred OPN-null mice (OPNeC/eC) that have no overt vascular phenotype to MGPeC/eC mice in which vascular calcification spontaneously develops. Mice deficient in both MGP and OPN (MGPeC/eCOPNeC/eC) showed accelerated and enhanced vascular calcification compared with mice deficient in MGP alone (MGPeC/eCOPN+/+).71 These studies indicate that OPN is an inducible inhibitor of vascular calcification in vivo and may play an important role in the adaptive response of the body to injury and disease. In light of our previous in vitro findings, part of the inhibition of arterial calcification in MGPeC/eC mice may be accounted for by the potent apatite inhibitory activity of phosphorylated OPN.30,83 Furthermore, our most recent studies point to a novel role for OPN in promoting ectopic mineral resorption as well.

    In a subcutaneous implantation model, a 5- to 10-fold greater calcification was observed in glutaraldehyde-fixed porcine aortic valve leaflets explanted from OPNeC/eC mice versus OPN wild-types (OPN+/+), verifying again the inhibitory effect of OPN in calcification in vivo. More importantly, heterozygous mice showed early calcification of implants at 14 days, with subsequent regression at 30 days. The regression was found to correlate with the accumulation of OPN and carbonic anhydrase II expressing monocyte-derived cells, including macrophages and foreign body giant cells, and with subsequent acidification of the implants.70 Rescue of the calcification phenotype in the OPNeC/eC subcutaneous implantation model could be achieved by the administration of exogenous OPN. Significant inhibition of calcification in glutaraldehyde-fixed bovine pericardial implants was achieved compared with controls by delivering soluble phosphorylated rat recombinant OPN via injection at the implant site (72% inhibition) or adsorption to the implant surface (91% inhibition). Reduced phosphorylation and inactivation of the arginine-glycine-aspartate motif in adsorbed OPN resulted in significant loss of inhibition, indicating that the optimal anti-calcific effect required sufficient phosphorylation (10 to 14 phosphate groups/molecule of OPN) and a functional RGD motif. More importantly, quantitative immunostaining showed a strong positive correlation between carbonic anhydrase II expression localized to glutaraldehyde-fixed bovine pericardial implants and adsorbed OPN having sufficient phosphorylation and a functional RGD motif.84 These studies suggest that OPN acts as an inducible inhibitor of calcification not only by inhibiting crystal growth but also by promoting active regression.

    Conclusions

    Vascular calcification is highly correlated with cardiovascular disease mortality, especially in ESRD and diabetic patients. In addition to the devastating effects of inappropriate biomineralization seen in cardiac valvulopathies, calciphylaxis, and idiopathic infantile arterial calcification, vascular calcification is now recognized as a marker of atherosclerotic plaque burden as well as a major contributor to loss of arterial compliance and increased pulse pressure seen with age, diabetes, and renal insufficiency. The presence of inducers, such as phosphate, and inhibitors, such as pyrophosphate and osteopontin, are likely to control whether calcification occurs under pathological conditions. Furthermore, arterial wall cells appear to play a particularly important role in mediating vascular calcification. Understanding the origins of the cells participating in osteochondral tissue formation, and mechanisms controlling their differentiation may aid in the development of novel therapeutic strategies to prevent and potentially reverse vascular calcification, which is an urgent need in the ESRD population.

    Acknowledgments

    This work was supported by National Institutes of Health grant AR 48798 and R01 HL62329, and National Science Foundation grant EEC-9529161 to C.M.G.

    References

    Hunt JL, Fairman R, Mitchell ME, Carpenter JP, Golden M, Khalapyan T, Wolfe M, Neschis D, Milner R, Scoll B, Cusack A, Mohler ER III. Bone formation in carotid plaques: a clinicopathological study. Stroke. 2002; 33: 1214eC1219.

    Burke AP, Taylor A, Farb A, Malcom GT, Virmani R. Coronary calcification: insights from sudden coronary death victims. Z Kardiol. 2000; 89: 49eC53.

    Monckeberg JG. Uber die reine mediaverkalkung der extremitatenarterien und ihr verhalten zur arteriosklerose. Virchows Arch Pathol Anat. 1902; 171: 141eC167.

    Edmonds ME, Morrison N, Laws JW, Watkins PJ. Medial arterial calcification and diabetic neuropathy. BMJ (Clin Res Ed). 1982; 284: 6928eC6930.

    Schwarz U, Buzello M, Ritz E, Stein G, Raabe G, Wiest G, Mall G, Amann K. Morphology of coronary atherosclerotic lesions in patients with end-stage renal failure. Nephrol Dial Transplant. 2000; 15: 218eC223.

    Ibels LS, Alfrey AC, Huffer WE, Craswell PW, Anderson JT, Weil R III. Arterial calcification and pathology in uremic patients undergoing dialysis. Am J Med. 1979; 66: 790eC796.

    Schoen FJ, Levy RJ. Tissue heart valves: current challenges and future research perspectives. J Biomed Mater Res. 1999; 47: 439eC465.

    O’Keefe JH Jr, Lavie CJ, Nishimura RA, Edwards WD. Degenerative aortic stenosis. One effect of the graying of America. Postgrad Med. 1991; 89: 143eC144.

    Coates T, Kirkland GS, Dymock RB, Murphy BF, Brealey JK, Mathew TH, Disney AP. Cutaneous necrosis from calcific uremic arteriolopathy. Am J Kidney Dis. 1998; 32: 384eC391.

    Rutsch F, Ruf N, Vaingankar S, Toliat MR, Suk A, Hohne W, Schauer G, Lehmann M, Roscioli T, Schnabel D, Epplen JT, Knisely A, Superti-Furga A, McGill J, Filippone M, Sinaiko AR, Vallance H, Hinrichs B, Smith W, Ferre M, Terkeltaub R, Nurnberg P. Mutations in ENPP1 are associated with ‘idiopathic’ infantile arterial calcification. Nat Genet. 2003; 34: 379eC381.

    Rumberger JA, Simons DB, Fitzpatrick LA, Sheedy PF, Schwartz RS. Coronary artery calcium area by electron-beam computed tomography and coronary atherosclerotic plaque area. A histopathologic correlative study. Circulation. 1995; 92: 2157eC2162.

    Sangiorgi G, Rumberger JA, Severson A, Edwards WD, Gregoire J, Fitzpatrick LA, Schwartz RS. Arterial calcification and not lumen stenosis is highly correlated with atherosclerotic plaque burden in humans: a histologic study of 723 coronary artery segments using nondecalcifying methodology. J Am Coll Cardiol. 1998; 31: 126eC133.

    Beadenkopf WG, Daoud AS, Love BM. Calcification in the coronary arteries and its relationship to arterioscler and myocardial infarction. AJR Am J Roentgenel. 1964; 92: 865eC871.

    Locker TH, Schwartz RS, Cotta CW, Hickman JR. Fluoroscopic coronary artery calcificcation and associated coronary disease in asymptomatic young men. J Am Coll Cardiol. 1992; 19: 1167eC1192.

    Puentes G, Detrano R, Tang W, Wong N, French W, Narahara K, Burndage B, Baksheshi H. Estimation of coronary calcium mass using electron beam computed tomography: a promising approach for predicting coronary events Circulation. 1995; 92: I313.

    Fitzgerald PJ, Ports TA, Yock PG. Contribution of localized calcium deposits to dissection after angioplasty. An observational study using intravascular ultrasound. Circulation. 1992; 86: 64eC70.

    Vliegenthart R, Hollander M, Breteler MM, van der Kuip DA, Hofman A, Oudkerk M, Witteman JC. Stroke is associated with coronary calcification as detected by electron-beam CT: the Rotterdam Coronary Calcification Study. Stroke. 2002; 33: 462eC465.

    Olson JC, Edmundowicz D, Becker DJ, Kuller LH, Orchard TJ. Coronary calcium in adults with type 1 diabetes: a stronger correlate of clinical coronary artery disease in men than in women. Diabetes. 2000; 49: 1571eC1578.

    Lehto S, Niskanen L, Suhonen M, Ronnemaa T, Laakso M. Medial artery calcification. A neglected harbinger of cardiovascular complications in noneCinsulin-dependent diabetes mellitus. Arterioscler Thromb Vasc Biol. 1996; 16: 978eC983.

    London GM, Guerin AP, Marchais SJ, Metivier F, Pannier B, Adda H. Arterial media calcification in end-stage renal disease: impact on all-cause and cardiovascular mortality. Nephrol Dial Transplant. 2003; 18: 1731eC1740.

    Guerin AP, London GM, Marchais SJ, Metivier F. Arterial stiffening and vascular calcifications in end-stage renal disease. Nephrol Dial Transplant. 2000; 15: 1014eC1021.

    Speer MY, Giachelli CM. Regulation of cardiovascular calcification. Cardiovasc Pathol. 2004; 13: 63eC70.

    Block GA, Hulbert-Shearon TE, Levin NW, Port FK. Association of serum phosphorus and calcium x phosphate product with mortality risk in chronic hemodialysis patients: a national study. Am J Kidney Dis. 1998; 31: 607eC617.

    Raggi P. Detection and quantification of cardiovascular calcifications with electron beam tomography to estimate risk in hemodialysis patients. Clin Nephrol. 2000; 54: 325eC333.

    Shioi A, Nishizawa Y, Jono S, Koyama H, Hosoi M, Morii H. Beta-glycerophosphate accelerates calcification in cultured bovine vascular smooth muscle cells. Arterioscler Thromb Vasc Biol. 1995; 15: 2003eC2009.

    Jono S, McKee MD, Murry CE, Shioi A, Nishizawa Y, Mori K, Morii H, Giachelli CM. Phosphate regulation of vascular smooth muscle cell calcification. Circ Res. 2000; 87: e10eCe17.

    Steitz SA, Speer MY, Curinga G, Yang HY, Haynes P, Aebersold R, Schinke T, Karsenty G, Giachelli CM. Smooth muscle cell phenotypic transition associated with calcification - Upregulation of Cbfa1 and downregulation of smooth muscle lineage markers. Circ Res. 2001; 89: 1147eC1154.

    Reynolds JL, Joannides AJ, Skepper JN, McNair R, Schurgers LJ, Proudfoot D, Jahnen-Dechent W, Weissberg PL, Shanahan CM. Human vascular smooth muscle cells undergo vesicle-mediated calcification in response to changes in extracellular calcium and phosphate concentrations: a potential mechanism for accelerated vascular calcification in ESRD. J Am Soc Nephrol. 2004; 15: 2857eC2867.

    Yang H, Curinga G, Giachelli CM. Elevated extracellular calcium levels induce smooth muscle cell matrix mineralization in vitro. Kidney Int. 2004; 66: 2293eC2299.

    Wada T, McKee MD, Steitz S, Giachelli CM. Calcification of vascular smooth muscle cell cultures: inhibition by osteopontin. Circ Res. 1999; 84: 166eC178.

    Proudfoot D, Skepper JN, Hegyi L, Bennett MR, Shanahan CM, Weissberg PL. Apoptosis regulates human vascular calcification in vitro: evidence for initiation of vascular calcification by apoptotic bodies. Circ Res. 2000; 87: 1055eC1062.

    Moe SM, Duan D, Doehle BP, O’Neill KD, Chen NX. Uremia induces the osteoblast differentiation factor Cbfa1 in human blood vessels. Kidney Int. 2003; 63: 1003eC1011.

    Moe SM, O’Neill KD, Duan D, Ahmed S, Chen NX, Leapman SB, Fineberg N, Kopecky K. Medial artery calcification in ESRD patients is associated with deposition of bone matrix proteins. Kidney Int. 2002; 61: 638eC647.

    Luo G, Ducy P, McKee MD, Pinero GJ, Loyer E, Behringer RR, Karsenty G. Spontaneous calcification of arteries and cartilage in mice lacking matrix GLA protein. Nature. 1997; 386: 78eC81.

    Owens GK, Kumar MS, Wamhoff BR. Molecular regulation of vascular smooth muscle cell differentiation in development and disease. Physiol Rev. 2004; 84: 767eC801.

    Davies MR, Lund RJ, Hruska KA. BMP-7 Is an Efficacious Treatment of Vascular Calcification in a Murine Model of Atherosclerosis and Chronic Renal Failure. J Am Soc Nephrol. 2003; 14: 1559eC1567.

    Murer H, Forster I, Biber J. The sodium phosphate cotransporter family SLC34. Pflugers Arch. 2004; 447: 763eC767.

    Reimer RJ, Edwards RH. Organic anion transport is the primary function of the SLC17/type I phosphate transporter family. Pflugers Arch. 2004; 447: 629eC635.

    Collins JF, Bai L, Ghishan FK. The SLC20 family of proteins: dual functions as sodium-phosphate cotransporters and viral receptors. Pflugers Arch. 2004; 447: 647eC652.

    Kavanaugh MP, Kabat D. Identification and characterization of a widely expressed phosphate transporter/retrovirus receptor family. Kidney Int. 1996; 49: 959eC963.

    Palmer G, Bonjour JP, Caverzasio J. Expression of a newly identified phosphate transporter/retrovirus receptor in human SaOS-2 osteoblast-like cells and its regulation by insulin-like growth factor I. Endocrinology. 1997; 138: 5202eC5209.

    Palmer G, Guicheux J, Bonjour JP, Caverzasio J. Transforming growth factor-beta stimulates inorganic phosphate transport and expression of the type III phosphate transporter Glvr-1 in chondrogenic ATDC5 cells. Endocrinology. 2000; 141: 2236eC2243.

    Giachelli CM, Jono S, Shioi A, Nishizawa Y, Mori K, Morii H. Vascular calcification and inorganic phosphate. Am J Kidney Dis. 2001; 38: S34eCS37.

    Tatsumi S, Segawa H, Morita K, Haga H, Kouda T, Yamamoto H, Inoue Y, Nii T, Katai K, Taketani Y, Miyamoto KI, Takeda E. Molecular cloning and hormonal regulation of PiT-1, a sodium-dependent phosphate cotransporter from rat parathyroid glands. Endocrinology. 1998; 139: 1692eC1699.

    Chen NX, O’Neill KD, Duan D, Moe SM. Phosphorus and uremic serum up-regulate osteopontin expression in vascular smooth muscle cells. Kidney Int. 2002; 62: 1724eC1731.

    Li X, Giachelli CM. The role of type III sodium-dependent phosphate cotransporter Pit-1in smooth muscle cell calcification. Cardiovasc Pathol. 2004; 13 (Suppl 1): 185.

    Suzuki A, Nishiwaki-Yasuda K, Ono Y, Kakita A, Ishiwata Y, Matsumoto T, Imamura S, Kato T, Hayakawa N, Oda N, Oiso Y. Vasopressin enhanced Na-dependent Pi transport activity and the mineralization in vascular smooth muscle cells. J Bone Miner Res. 2004; 19 (Suppl 1): S114.

    Nielsen LB, Pedersen FS, Pedersen L. Expression of type III sodium-dependent phosphate transporters/retroviral receptors mRNAs during osteoblast differentiation. Bone. 2001; 28: 160eC166.

    Beck GR Jr, Moran E, Knecht N. Inorganic phosphate regulates multiple genes during osteoblast differentiation, including Nrf2. Exp Cell Res. 2003; 288: 288eC300.

    Beck GR Jr, Zerler B, Moran E. Phosphate is a specific signal for induction of osteopontin gene expression. Proc Natl Acad Sci U S A. 2000; 97: 8352eC8357.

    Guicheux J, Palmer G, Shukunami C, Hiraki Y, Bonjour JP, Caverzasio J. A novel in vitro culture system for analysis of functional role of phosphate transport in endochondral ossification. Bone. 2000; 27: 69eC74.

    Wu LN, Guo Y, Genge BR, Ishikawa Y, Wuthier RE. Transport of inorganic phosphate in primary cultures of chondrocytes isolated from the tibial growth plate of normal adolescent chickens. J Cell Biochem. 2002; 86: 475eC489.

    Kim KM. Calcification of matrix vesicles in human aortic valve and aortic media. Fed Proc. 1976; 35: 156eC162.

    Bostrom K, Watson KE, Horn S, Wortham C, Herman IM, Demer LL. Bone morphogenetic protein expression in human atherosclerotic lesions. J Clin Invest. 1993; 91: 1800eC1809.

    Abedin M, Tintut Y, Demer LL. Mesenchymal stem cells and the artery wall. Circ Res. 2004; 95: 671eC676.

    Brighton CT, Lorich DG, Kupcha R, Reilly TM, Jones AR, Woodbury RA. The pericyte as a possible osteoblast progenitor cell. Clin Orthop. 1992; 287eC299.

    Cheng SL, Shao JS, Charlton-Kachigian N, Loewy AP, Towler DA. MSX2 promotes osteogenesis and suppresses adipogenic differentiation of multipotent mesenchymal progenitors. J Biol Chem. 2003; 278: 45969eC45977.

    Towler DA, Bidder M, Latifi T, Coleman T, Semenkovich CF. Diet-induced diabetes activates an osteogenic gene regulatory program in the aortas of low density lipoprotein receptor-deficient mice. J Biol Chem. 1998; 273: 30427eC30434.

    Shao JS, Cheng SL, Charlton-Kachigian N, Loewy AP, Towler DA. Teriparatide (human parathyroid hormone (1eC34)) inhibits osteogenic vascular calcification in diabetic low density lipoprotein receptor-deficient mice. J Biol Chem. 2003; 278: 50195eC50202.

    Rutsch F, Vaingankar S, Johnson K, Goldfine I, Maddux B, Schauerte P, Kalhoff H, Sano K, Boisvert WA, Superti-Furga A, Terkeltaub R. PC-1 nucleoside triphosphate pyrophosphohydrolase deficiency in idiopathic infantile arterial calcification. Am J Pathol. 2001; 158: 543eC554.

    Okawa A, Nakamura I, Goto S, Moriya H, Nakamura Y, Ikegawa S. Mutation in Npps in a mouse model of ossification of the posterior longitudinal ligament of the spine. Nat Genet. 1998; 19: 271eC273.

    Sali A, Favaloro JM, Terkeltaub R, Goding JW. Germline deletion of the nucleoside triphosphosphate (NTPPPH) plasma cell membrane glycoprotein (PC-1) produces abnormal calcification of periarticular tissues. In: Vanduffel L, Lemmens R, eds. EctoATPase and related ectonucleotidases. Maastricht, The Netherlands: Shaker Publishing BV; 1999.

    Harmey D, Hessle L, Narisawa S, Johnson KA, Terkeltaub R, Millan JL. Concerted regulation of inorganic pyrophosphate and osteopontin by akp2, enpp1, and ank: an integrated model of the pathogenesis of mineralization disorders. Am J Pathol. 2004; 164: 1199eC1209.

    Johnson K, Goding J, Van Etten D, Sali A, Hu SI, Farley D, Krug H, Hessle L, Millan JL, Terkeltaub R. Linked deficiencies in extracellular PP(i) and osteopontin mediate pathologic calcification associated with defective PC-1 and ANK expression. J Bone Miner Res. 2003; 18: 994eC1004.

    Nociti FH, Jr Berry JE, Foster BL, Gurley KA, Kingsley DM, Takata T, Miyauchi M, Somerman MJ. Cementum: a phosphate-sensitive tissue. J Dent Res. 2002; 81: 817eC821.

    Spicer SS, Lewis SE, Tashian RE, Schulte BA. Mice carrying a CAR-2 null allele lack carbonic anhydrase II immunohistochemically and show vascular calcification. Am J Pathol. 1989; 134: 947eC954.

    Schafer C, Heiss A, Schwarz A, Westenfeld R, Ketteler M, Floege J, Muller-Esterl W, Schinke T, Jahnen-Dechent W. The serum protein alpha 2-Heremans-Schmid glycoprotein/fetuin-A is a systemically acting inhibitor of ectopic calcification. J Clin Invest. 2003; 112: 357eC366.

    Pereira L, Andrikopoulos K, Tian J, Lee SY, Keene DR, Ono R, Reinhardt DP, Sakai LY, Biery NJ, Bunton T, Dietz HC, Ramirez F. Targetting of the gene encoding fibrillin-1 recapitulates the vascular aspect of Marfan syndrome. Nat Genet. 1997; 17: 218eC222.

    Kuroo M, Matsumura Y, Aizawa H, Kawaguchi H, Suga T, Utsugi T, Ohyama Y, Kurabayashi M, Kaname T, Kume E, Iwasaki H, Iida A, ShirakiIida T, Nishikawa S, Nagai R, Nabeshima Y. Mutation of the mouse klotho gene leads to a syndrome resembling aging. Nature. 1997; 390: 45eC51.

    Steitz SA, Speer MY, McKee MD, Liaw L, Almeida M, Yang H, Giachelli CM. Osteopontin inhibits mineral deposition and promotes regression of ectopic calcification. Am J Pathol. 2002; 161: 2035eC2046.

    Speer MY, McKee MD, Guldberg RE, Liaw L, Yang HY, Tung E, Karsenty G, Giachelli CM. Inactivation of the Osteopontin Gene Enhances Vascular Calcification of Matrix Gla Protein-deficient Mice: Evidence for Osteopontin as an Inducible Inhibitor of Vascular Calcification In Vivo. J Exp Med. 2002; 196: 1047eC1055.

    Bucay N, Sarosi I, Dunstan CR, Morony S, Tarpley J, Capparelli C, Scully S, Tan HL, Xu W, Lacey DL, Boyle WJ, Simonet WS. osteoprotegerin-deficient mice develop early onset osteoporosis and arterial calcification. Genes Dev. 1998; 12: 1260eC1268.

    Galvin KM, Donovan MJ, Lynch CA, Meyer RI, Paul RJ, Lorenz JN, Fairchild-Huntress V, Dixon KL, Dunmore JH, Gimbrone MA Jr, Falb D, Huszar D. A role for smad6 in development and homeostasis of the cardiovascular system. Nat Genet. 2000; 24: 171eC174.

    Giachelli CM, Steitz S. Osteopontin: a versatile regulator of inflammation and biomineralization. Matrix Biology. 2000; 19: 622.

    Giachelli CM, Bae N, Almeida M, Denhardt DT, Alpers CE, Schwartz SM. Osteopontin is elevated during neointima formation in rat arteries and is a novel component of human atherosclerotic plaques. J Clin Invest. 1993; 92: 1686eC1696.

    O’Brien ER, Garvin MR, Stewart DK, Hinohara T, Simpson JB, Schwartz SM, Giachelli CM. Osteopontin is synthesized by macrophage, smooth muscle, and endothelial cells in primary and restenotic human coronary atherosclerotic plaques. Arterioscler Thromb. 1994; 14: 1648eC1656.

    O’Brien KD, Kuusisto J, Reichenbach DD, Ferguson M, Giachelli C, Alpers CE, Otto CM. Osteopontin is expressed in human aortic valvular lesions. Circulation. 1995; 92: 2163eC2168.

    Ikeda T, Shirasawa T, Esaki Y, Yoshiki S, Hirokawa K. Osteopontin mRNA is expressed by smooth muscle-derived foam cells in human atherosclerotic lesions of the aorta. J Clin Invest. 1993; 92: 2814eC2820.

    Hirota S, Imakita M, Kohri K, Ito A, Morii E, Adachi S, Kim HM, Kitamura Y, Yutani C, Nomura S. Expression of osteopontin messenger RNA by macrophages in atherosclerotic plaques. A possible association with calcification. Am J Pathol. 1993; 143: 1003eC1008.

    Fitzpatrick LA, Severson A, Edwards WD, Ingram RT. Diffuse calcification in human coronary arteries. Association of osteopontin with atherosclerosis. J Clin Invest. 1994; 94: 1597eC1604.

    Shen M, Marie P, Farge D, Carpentier S, De Pollak C, Hott M, Chen L, Martinet B, Carpentier A. Osteopontin is associated with bioprosthetic heart valve calcification in humans. C R Acad Sci III. 1997; 320: 49eC57.

    Srivatsa SS, Harrity PJ, Maercklein PB, Kleppe L, Veinot J, Edwards WD, Johnson CM, Fitzpatrick LA. Increased cellular expression of matrix proteins that regulate mineralization is associated with calcification of native human and porcine xenograft bioprosthetic heart valves. J Clin Invest. 1997; 99: 996eC1009.

    Jono S, Peinado C, Giachelli CM. Phosphorylation of osteopontin is required for inhibition of vascular smooth muscle cell calcification. J Biol Chem. 2000; 275: 20197eC20203.

    Ohri R, Tung E, Rajachar RM, Giachelli CM. Mitigation of ectopic calcification in osteopontin-null mice by exogenous osteopontin. Calcif Tissue Int. In press.

日期:2007年5月18日 - 来自[2005年第95卷第4期]栏目
循环ads

Quantification of Calcification in Atherosclerotic Lesions

From the Departments of Medicine and Biochemistry/Molecular Biology, Baylor College of Medicine, Houston, Tex.

Correspondence to Dr Joel D. Morrisett, The Methodist Hospital, A601, 6565 Fannin St, Houston, TX 77030. E-mail morriset@bcm.tmc.edu

    Abstract

Calcification can be deposited throughout the vasculature in several forms of calcium phosphate, including calcium hydroxyapatite (CHA). Calcium accumulation in arteries by mineralization and calcium loss from bone by osteoporosis often coexist, and vascular calcification may share common mechanisms with bone remodeling. Deposition of calcification in valves and arteries diminishes the valvular or arterial wall elasticity, a major cause of aneurysm and stenosis. Obstruction of arteries by calcification and other components can lead to heart attack and stroke. Mineralization in the femoral arteries can cause intermittent claudication in the legs, causing decreased mobility. Accurate measurement of calcification is essential for identifying other factors associated with this process and ultimately for elucidating the mechanism(s) of calcification. A wide range of methods for visualizing and measuring calcification for diagnosis and treatment in vivo and for studying the calcification process ex vivo are available. This review provides a critical comparison of older established methods and newer evolving technologies for quantifying calcification.

Key Words: calcification ? atherosclerosis ? MRI, micro-computed tomography ? ultrasound

    Introduction

Calcium and phosphorus are the most abundant minerals in the body and are involved in a wide range of biochemical pathways, but mostly in the formation of calcium hydroxyapatite (CHA; Ca10(PO4)6(OH)2). Although CHA is the major natural component of bones and teeth, it can also deposit along with other forms of calcium phosphate in the vasculature with adverse affects. This type of deposit, known as vascular calcification, can ultimately lead to blood vessel stenosis, ischemia, and death. Approximately 90% of patients with cardiovascular disease (CVD) have vascular calcification. Because CVDs are the leading cause of death in the United States, there is considerable interest in understanding the mechanism(s) of vascular calcification and the implications for CVD.

Healthy bone exists in a dynamic state of remodeling, requiring osteoblasts that build bone under alkaline conditions and osteoclasts that degrade it under acidic conditions.1 Cytokines, such as bone morphogenetic proteins (BMPs), interleukin-6, insulin-like growth factor-1, as well as various hormones, regulate bone remodeling.2 As a consequence of remodeling, bone calcium and phosphate turnover occurs. In this turnover process, resorbed minerals are used to regenerate bone. When the calcium lost in degradation exceeds the calcium deposited in remodeling, there is a net loss of bone mass. With increasing age, the rate of bone degradation exceeds the rate of formation, resulting in osteoporosis, particularly in postmenopausal women, with decreased estrogens, which inhibit cytokines.3 Some of the calcium and phosphate mobilized by remodeling may become deposited in the arterial wall, leading to atherosclerosis in 1 arterial bed.

CHA is the most stable form of insoluble calcium phosphate.4 Under biological conditions, formation of CHA proceeds through noncrystalline amorphous calcium phosphate (ACP), which is stable under alkaline conditions (pH 8). Formation of ACP and CHA can be inhibited by many ions and other factors at their normal tissue concentrations. Osteoblast activity regulates formation of CHA, and alkaline phosphatase, which operates under alkaline conditions, is a marker for formation of ACP and its conversion to CHA.4

Although the mechanism of nucleation of ACP and its conversion into CHA in bone mineralization is not entirely clear, some factors of the process are known. ACP is isothermally metastable compared with the more ordered CHA. Initial mineral deposits are associated with membrane vesicles and specific bone-associated proteins such as osteonectin, osteocalcin, and matrix -carboxy glutamate (Gla) protein.2,5 On nucleation, growth of CHA crystals requires increased concentrations of Ca2+ and PO43– ions.6 The transformation of ACP to CHA in vitro occurs at slightly alkaline pH (7.4 to 7.8) and is temperature and time dependent, suggesting that the process is autocatalytic.7

Presumably, decomposition of CHA in bone to its constituent ions follows the reverse process. Osteoclast activity, inhibited by increased Ca2+ concentrations, involves resorption and phagocytosis of calcium phosphate in an environment made acidic by carbonic anhydrase.8,9 Osteoclasts form tunnel capillaries into the bone.1 Interfacing with the bone surface is a ruffled border, one of the domains into which the osteoclast plasma membrane is divided.10 Formation of a sealed compartment between the ruffled border and bone surface results in an acidic compartment in which CHA crystals disintegrate.4 Subsequently, the crystals are fragmented, transported through the osteoclast by a transcytotic vesicle, and exported to the vasculature.10

Vascular calcification is a well-ordered, regulated process similar to mineralization of bone tissue.11 Modulation of this process includes apoptosis of vascular smooth muscle cells (VSMCs), cell–cell interactions (macrophages and VSMCs), lipids, and plasma inorganic phosphate (Pi) levels.2 Four types of vascular calcification have been identified: atherosclerotic (fibrotic), cardiac valve, medial artery, and vascular calciphylaxis.2 In the atherosclerotic type, calcification initially occurs in the necrotic core of the plaque, and these types of lesions typically occur in or near bifurcations of arteries.2 Mechanical stressors and inflammation exacerbate cardiac valve calcification. Medial artery calcification often occurs in the femorals and is characteristic of diabetes and end-stage renal disease.2 Vascular calciphylaxis, or soft tissue calcification, is associated with a serum calcium–phosphate solubility product (Ksp) >60 mg2/dL2.2 The extent of calcification is strongly associated with stroke, amputation, and cardiovascular mortality.2

Calcification in atherosclerotic lesions involves factors important for bone mineralization, including matrix vesicles, BMP-2, osteopontin, osteocalcin, and collagen I.2 However, a major difference between vascular calcification and bone mineralization is the presence of oxidized lipids in the former but not the latter.12 The accumulation of oxidized lipids in the subendothelial space of arteries promotes arterial calcification, whereas these lipids in skeletal bone inhibit bone formation, suggesting another link between osteoporosis and vascular calcification.13

Osteoprotegerin, which protects against osteoporosis, forms a perimeter around calcified lesions.14 This protein is also in equilibrium with receptor activator of nuclear factor kappa and receptor activator of nuclear factor kappa ligand, which regulate the transition of preosteoclasts to fully differentiated osteoclasts.15 Several current investigations are focused on elucidating the mechanism(s) of vascular calcification. An important aspect of the task is quantification of arterial calcification.

The major objective of this review is to critically evaluate the diversity of methods available for quantifying calcification by in vivo and ex vivo methods (Table). Whereas in vivo methods are used primarily for clinical assessment and treatment of CVD, ex vivo methods are critical to mechanistic studies. Among ex vivo analytical methods, cadaveric carotid arteries (CCAs) and carotid endarterectomy (CEA) tissues have been studied for measuring calcium content. The wide variety of approaches enables comprehensive study of calcification quantification.

   Comparison of Methodologies for Calcification Quantitation

    In Vivo Analysis

Detecting lesions in vivo has clinical importance for diagnosis and treatment as well as in research. These in vivo methods of quantifying calcification will be discussed with regard to their methodology, practice, capabilities, and limitations.

Ultrasonography

Ultrasonography (US) involves the transmission of high-frequency sound waves (2 to 10 MHz) through an anatomic site of interest followed by conversion of echoes into electrical impulses, producing 2D images. Different modes and types of US are used in echocardiography: brightness or B-mode, motion or M-mode, spectral Doppler, and color flow mapping.

B-mode US produces a gray-scale image with good anatomic detail of the ventricular septum, ventricular free walls, heart valves, papillary muscle, and chordae tendineae. Using B-mode US to evaluate carotid plaques, weak reflections (echolucent) have been associated with a higher risk of neurological events than plaques giving strong reflections (echorich).16 Echolucent plaques have higher content of lipid and hemorrhage than echorich plaques, which usually contain more calcification and fibrous tissue.16 Arterial ulceration is sometimes assigned incorrectly to pits in fibrotic plaque, 2D calcification with shadowing, atheromatous debris, or ulcerated plaque hemorrhage.17 B-mode US has been used extensively to determine carotid intimal-medial far-wall thickness18–20 and calcification. Plaque calcification can be identified by a bright, hyperechogenic area resulting in cone-shaped echo shadowing. Such qualitative imaging was used in a study of calcification in atherosclerotic plaques in association with polymorphisms of the human matrix Gla protein (MGP) gene, which codes for a protein that inhibits calcification by strongly binding calcium ions to its Gla residues.21 Significantly, calcification has been found to be prevalent in femoral atherosclerotic plaques of patients carrying a particular MGP allele, but this association does not apply for carotid plaques. This study demonstrates the use of B-mode US in locating calcification; however, this method does not allow for reliable quantitation of calcification because the image resolution does not allow for accurate delineation of plaque components (Table).

Intravascular ultrasound (IVUS) is an invasive method that details the relationship between plaque and vessel wall in real time throughout the coronary artery tree. The invasive nature of IVUS allows exact definition of not only the quantity but also the distribution of calcification within the vessel wall and the ability to classify different plaque substructures, helping to clarify ambiguous angiograms and delineate the exact nature of luminal encroachment. The central positioning of a high-frequency transducer within the target vessel facilitates high resolution of the arterial lumen-wall border, permitting a more precise definition of small ulcerations than is available by other diagnostic methods.22 However, this method cannot be used routinely until the pathologic significance of plaque ulceration is clearly defined, thereby avoiding possible disturbance of an unstable plaque.22 This method also enables identification of lesion subsets that may have an important natural history in development of atherosclerosis.23,24 Four types of atherosclerotic plaque components can be distinguished using IVUS: (1) lipid-rich (hypoechoic), (2) fibromuscular (soft echoes), (3) fibrous (bright echoic), and (4) calcific (bright echoes with shadowing behind the lesion).25 IVUS has been used to differentiate these components in several studies in vivo.26 The precision and accuracy of calcification quantitation by IVUS is excellent.27 In a study of calcium in culprit lesions after the placement of a stent, IVUS was used to assess the arc of calcium, which appears as a bright echogenic signal accompanied by an acoustic shadow in the arterial wall.28 Calcium was quantified by 2 methods: (1) as the widest arc of calcium found in the stented segment, and (2) as the average arc of calcium in the proximal, middle, and distal sections of the stented segments. The results showed that calcium is less abundant in plaques associated with culprit stenoses and more abundant in plaques associated with stable angina.

Similarly, the calcium arc was used to quantify calcium in a study of coronary artery remodeling.29 Overall, coronary arteries were observed to enlarge in most patients, with the exception of smokers. Also, no specific morphological features were found to be predictive of arterial remodeling. In another study focused on determining the relationship between smoking and calcification, the arc of calcium as determined by IVUS was applied to a regression equation that took into account age, gender, and smoking.30 The results indicated that among patients with coronary artery disease, previous or current smokers have plaque areas with similar dimensions but less calcification than nonsmokers.

The method of measuring calcium content from IVUS images involves a process of gathering cross-sectional images at a reference site and at a plaque site to measure the arc of calcium.31 These measurements take into account the amount of calcium versus the lumen surface size by converting the calcium arc to a percentage of the lumen surface, producing a more accurate measure of calcification.

US tissue characterization coupled with integrated backscatter (IB) analysis is effective in distinguishing lipidic, fibrotic, and calcific components in human atherosclerotic plaques.32 This technique is capable of producing 2D images and IB images, the latter of which details biochemical and structural components of atherosclerotic lesions.33 In conjunction with IVUS, IB data have been used to make color-coded maps of coronary arterial plaques according to 5 tissue components: lipid core with fibrous cap, intimal hyperplasia, fibrous tissue, calcification, and thrombus.33,34

The most commonly used method to identify or quantify calcification by US is IVUS (Table). However, combinations of US methods are also incorporated but are used primarily for visualization purposes, not quantifying calcification.35–38 To determine the role of calcium–phosphate metabolism in cardiac valve calcification of hemodialysis patients, B-mode and Doppler US were used to image the areas of calcification and to determine the severity score of valvular calcifications on the basis of thickness.39 These scores were found to correlate with the calcium–phosphate product ([Ca]x[PO4]) calculated from the serum concentrations of the individual atoms.

Electron Beam CT

CT is based on x-ray technology that computes axial images of the body. In standard CT scanning of the heart, multiple cross-sectional images are acquired from different angles. A 3D view of the heart is then created by compiling the axial images. Although CT renders high-resolution images of still objects, it is not fast enough to acquire such images of a beating heart; however, it does allow noninvasive detection and reproducible quantification of calcification in vivo (Table).

Electron beam CT (EBCT) has been used to determine the presence and amount of calcium accumulated in the coronary arteries. It is much faster than standard CT scanning, produces images in a fraction of a second, and acquires high-resolutions images of an artery even while the heart is beating. EBCT uses an electron beam in stationary tungsten targets, permitting very rapid scanning times.40 Prospective electrocardiographic triggering is required for acquisition of images by EBCT to reduce cardiac motion artifacts. As a result, arterial fat and calcium accumulation can be visualized clearly by EBCT. In 100 milliseconds, serial transaxial images are obtained every 3 to 6 mm for purposes of detecting coronary artery calcium. Thinner sections have been found to provide more accurate results.41,42 Current EBCT software permits quantification of calcium area and density. The images produced provide the basis for a patient’s "calcium score," representing the total amount of calcium present in the artery and calculated using the following equation: calcium score=[sum of (suprathreshold areaxN)]xT/3, in which N is a density index with a value of 1 to 4 based on a truncated peak CT number (a measure of density with a range of 130 to 499) and T is the slice thickness.43 A calcium score of 0 indicates virtually no risk for a cardiac event; scores between 1 and 100 correlate with low risk for a cardiac event over the next 5 years; scores from 100 to 400 infer moderate risk for cardiac events; and scores >400 indicate high risk for a heart attack.40 Although this cardiac risk classification is not scientifically validated, it is useful as a clinical method of diagnosis and prognosis of heart disease. Another system of quantifying calcification as analyzed by CT is the Agatston score, which can be calculated using the number, area, and peak Hounsfield numbers of the detected calcified lesions and is based on 3-mm slices acquired without overlap.44 Volume and mass calculations also provide reproducible results. Additionally, percentiles of risk stratification have been suggested.45

EBCT provides an accurate, reliable alternative to the more commonly used stress tests but is more expensive than many comparable tests. Results of EBCT have been compared with those of 2D echocardiography,46 Doppler US,47 IVUS,48 and angiography48–50 and have been found to be highly accurate in localizing and quantifying calcifications in the heart. EBCT is also very effective for detecting stable calcification in the arterial wall, a feature highly correlated to age.51

An alternative method to EBCT is multidetector row spiral CT (MDCT) with electrocardiography gating.52–56 MDCT allows image acquisition of thinner slices but requires higher radiation exposure. Quantification of calcification by MDCT is more accurate than by EBCT or IVUS. Noncalcified plaque can also be visualized by MDCT but requires the aid of injected contrast-enhancing dyes, and individual plaque components cannot be distinguished and quantified. Studies have been conducted to understand the differences in signal between stable and unstable angina57,58 and within plaques to differentiate composition,59 but CT is not yet a reliable source for quantifying calcification relative to other plaque components.

Magnetic Resonance Imaging

MRI is a powerful imaging technique that can produce images of anatomic structures and organs inside the body and often provides more spatial and contrast resolution than other imaging techniques such as CT, which requires x-ray images and the injection of a contrast dye. MRI is most effective at providing images of tissues or organs that contain water or lipid but is not as useful for imaging structures that contain rather low levels of these molecules, especially in the solid state. The sensitivity of in vivo images is enhanced by the use of phased array surface coils that can be placed near the anatomic site of interest.

MRI is capable of distinguishing various components of atherosclerotic plaques, such as fibrous tissue, lipids, calcification, and thrombus60–62 (Figure 1A). This capability enables determining lesion type and monitoring the progression and regression of atherosclerotic plaques (Table). Calcification typically appears dark in standard T1-, T2-, and proton density–weighted (PDW) images (Figure 1A) and, hence, can be difficult to distinguish from the lumen of a black blood image (Figure 1C).

   Figure 1. A, Three axial imaging sequences performed to generate different contrast weightings at each slice location: PDW, T1W, and T2W MR images of cadaveric arteries. Feature space analysis allows for quantitation of plaque components. TR indicates repetition time; TE, echo time; ETL, echo train length; FSE, fast spin-echo; FOV, field of view. B, Plotting intensities of pixels at the same x-y address in the T1W, T2W, and PDW images generates a feature space plot containing multiple clusters; each cluster corresponds to a component of the tissue: calcification, lumen, intima, lipid, necrotic core, and media. Each cluster was partially identified by imaging separate tissue components isolated by microdissection. Integration of the cluster volumes gives a quantitative measure of the corresponding component. (C. Karmonik and J. Morrisett, unpublished results, 2005). C, MRI axial slices (3 mm) of the left carotid artery of a 67-year-old woman presenting with >60% stenosis. The slices begin 9 mm below the bifurcation (C3) and extend 6 mm above it into the internal branch (I2). The compositional heterogeneity of the vessel wall and partially occluded lumen is evident. Slice C3 has a patent lumen, but disease of the wall is apparent from the darker area (calcification) at 7:00 (white arrow). This feature becomes more prominent in slice C2, extending from 6:00 to 12:00 (white arrows); concentric bright bands (fibrous cap and lipid core) are seen from 2:00 to 6:00 (red arrows). Slice C1 contains an indentation indicating the beginning of the flow divider (white arrow); the beginning of the orifice of the partially occluded internal branch is evident. Slice B cuts directly through the bifurcation, clearly showing a rather patent external carotid (white arrow) but a highly stenosed internal carotid (red arrow); the occluding plaque exhibits intermediate brightness typical of lipid rich lesions. Slice I1 shows complete separation of the 2 branches; the external carotid (white arrow) has virtually no disease, whereas the internal carotid shows significant wall thickening at 4:00 to 7:00 (red arrows). The spatial and contrast resolution of these representative images are essential for the separation and quantification of plaque components.

In a study of carotid artery calcification and white matter ischemia, calcification was graded using 2 methods: (1) extent of calcification based on degrees of the vessel circumference occupied by calcification, and (2) thickness of calcification.63 In a study of the effects of lipid-lowering drugs on atherosclerotic plaques, the investigators used MRI to determine the calcium cluster area and its percentage of the total plaque area.64 The other plaque components were quantified in the same way so that comparisons could be made. Patients treated with intensive lipid-altering therapy had significantly lower percentages of lipid and higher percentages of calcium than those who were untreated. However, changes in percentages of plaque components can be misleading because if the proportion of 1 component decreases (eg, lipid), then the proportion of the other components (eg, calcification) must necessarily increase if their masses do not change.

We are currently using MRI to quantify calcified atherosclerotic lesions in the superficial femoral artery. The resulting images are used to guide endovascular intervention such as stenting, bypass, or remote endarterectomy (J. Morrisett and A. Lumsden, unpublished results, 2005).

    Ex Vivo Analysis

Whereas in vivo analysis of atherosclerotic plaques is useful for diagnosis and treatment, ex vivo analysis is instructive for understanding mechanisms of plaque formation and establishing the identity of features of plaque images obtained in vivo. Ex vivo studies have been performed on CCA and CEA tissues. Carotid arteries from human cadavers provide anatomic information about the medial and adventitial layers, which are not usually present in CEA tissues. This advantage is diminished partly by the alteration in CCA tissue structure because of formalin fixation, in contrast to CEA tissue, which is obtained fresh or stored in a cryoprotecting buffer at –20°C without loss of structure

Micro-CT

Micro-CT (μCT) is similar to CT in that x-ray images are acquired at multiple angles around the object followed by computation of its tomogram. Currently available μCT instrumentation is typically a compact, desktop x-ray system for nondestructive reconstruction of 3D tissue microstructure with high spatial resolution. With μCT, it is possible to: (1) obtain transmission shadow images of tissue; (2) reconstruct any cross-section of the complete 3D object microstructure; (3) calculate distance, surface area, and volume; (4) analyze density and porosity of an object; and (5) achieve 3D rendering and realistic visualization through animation of the reconstructed images (Table).

With μCT, 3D radioscopic image data are acquired rapidly and noninvasively to capture thin cross-sections. Because of the low-dose radiation used, mice and rats can be imaged serially by this method. The resulting data, which have spatial resolution of 20 μm, are used in reconstruction calculations to generate realistic 3D images and to calculate morphological parameters. A major application of μCT has been to quantify the 3D microstructure of bone and to provide quantitative information about its functionality, porosity, and mineral density, making the technique useful for early detection of various bone pathologies, including osteoporosis.65–68

μCT can also be used to study calcification of the arterial wall68,69 and enables nondestructive visualization and localization of CHA in very thin tissue slices, although it does not allow for delineation of other plaque components (Figure 2). Two morphological distributions are observed: (1) calcification nodules localized mainly in the necrotic core or luminal surface, and (2) calcification plates localized more to the medial layer, often extending around a substantial fraction of its circumference. Calibrating the μCT system with known amounts of CHA is done by scanning a 96-well microtiter plate having wells filled with different amounts of hydrated CHA. The sum of electron dense areas in successive slices of CHA in each well enables construction of a standard calibration curve that can be used for calculation of unknown samples.

   Figure 2. μCT images of a CCA. Axial slice images (0.3 mm) reveal a large calcification plate (a and b) and small nodules (c) in the internal carotid but no calcification in the common carotid (d).

Magnetic Resonance Imaging

Ex vivo samples can be imaged by MRI using coils of different geometries. A solenoid coil (30-mm diameter) is convenient for imaging single samples at very high resolution. A phased array coil (6-cm width) is convenient for multiple samples at high resolution. MRI analysis of CEA specimens can be performed using the same sample holder but smaller sample tubes than used for CCA imaging.

Representative magnetic resonance (MR) images of CEA tissues are shown in Figure 1. Plaque components, including collagenous cap, necrotic core, hemorrhage, and calcification can be distinguished in MR images of plaques.70–72 These components can also be mapped according to their contrast weightings, which allow for integration of the components and quantification. Multicontrast-weighted MR images of carotid plaques have also been used to develop classification maps useful in distinguishing plaque components more effectively69 (Table).

In a study of left and right CCA, MRI and EBCT were used to determine arterial wall volume and calcification score.73 The results indicated that total wall volume and plaque calcification burden were similar for the left and right arteries, suggesting that atherosclerosis is a bilaterally symmetrical disease.

In PDW images of CEA tissues, regions of calcification appear dark (Figure 3b). When these tissues are embedded in paraffin, the calcified regions appear white (Figure 3a). Integration of the calcified regions visualized by each method provided 2 sets of integrated areas (MRI and embedded tissue imaging) that were highly correlated (R2=0.99; Figure 3c).

   Figure 3. a, A CEA specimen was microdissected into segments 1.0 cm in length. Selected segments were fixed and embedded in paraffin by conventional methods. From each block, excess paraffin was lifted with a microtome until a full artery cross-section was exposed. The fixed tissue cross-sections were digitally photographed and contrast-manually enhanced with the NIH image analysis program ImageJ 1.30v. b, Each digital image was matched using eFilm Workstation 1.8.3 with the MRI image of the closest morphology. For each image, the total artery area, total lumen area, and total calcified area were circumscribed independently using freehand area selection in ImageJ and measured by automated integration. Percent calcification was then calculated for each image. c, Comparison of percent calcification in CEA specimens detected by MRI (b) and embedded tissue imaging (ETI; a; S. Marvel and J. Morrisett, unpublished results, 2005).

Three-D images have been obtained of human CCA using multicontrast-weighted fast spin-echo imaging.74 A cluster analysis technique called spatially enhanced cluster analysis objectively classified and quantified multicontrast MR images. The cluster technique divides data into groups with strong associations by iteratively minimizing a characteristic of the cluster. Using this technique, plaque components such as calcification can be differentiated by color and then quantified.

31P MR Spectroscopy

MR spectroscopy (MRS) is useful for quantifying nuclei in specific magnetic environments and has been used extensively to study biomolecules in isotropic solutions. Magic angle spinning (MAS) extends the power of MRS to include determining chemical and structural properties of anisotropic liquid crystalline and solid samples. The mineral content of bone can be quantified using 31P MRS because the phosphate of CHA is distinguishable from Pi, from phosphorylated metabolites dissolved in the cytosol, and from the polar head groups of phospholipids in membranes.75,76 Accordingly, 31P MAS MRS can be used for rapid quantification of CHA in atherosclerotic plaques.77,78

In a study of lipid phases and CHA deposits in human atherosclerotic lesions, a plaque with low lipid content (weak 13C MAS MRS signals) and extensive calcification (strong 31P MAS MRS signals) has been used to determine CHA content.77 After delipidation of the plaque, the 31P MAS MRS signal intensity showed no change, indicating that the 31P signal resulted from nonlipid phosphorus. Based on the intensity of the 31P MAS MRS peak, the phosphorus content could be calculated, followed by stoichiometric conversion to CHA content.

The integrated 31P signal intensity in plaques can be calibrated using synthetic CHA and chicken bone powder as reference compounds.78 Comparisons of 31P peak intensities showed that the chicken bone powder provided the best calibration, presumably because the synthetic CHA has a more ordered crystalline structure than the biological samples. This technique is somewhat destructive because delipidation leaves the tissue in a non-native state. 31P MAS MRS is generally restricted to ex vivo samples <1 cm3 (Table).

Tissue Digital Photography

Digital photographs of CEA specimens are useful for documenting tissue features lost during processing (eg, thrombus and calcification) for microscopy and can capture subtle textural and morphological features not detected by other techniques (Figure 4). Although differences in photographic color are useful for qualitatively distinguishing between calcified and lipid-rich regions, the contrast is not sufficient to accurately quantify the 2 types of plaque components. However, estimating calcium in these tissues becomes feasible when they are embedded in paraffin and digitally photographed (Figure 3). The total artery area, total lumen area, and total calcified area can be integrated and these areas used to calculate the percent calcification for each image. Although paraffin-thin sections frequently lose some of the calcified component during cutting, the remaining block has a smooth surface that facilitates quantitation (Table).

   Figure 4. Digital photograph of a fresh CEA specimen (a) illustrating the presence of extensive calcification and thrombus in segment I3 (b). Thrombus appears as a dark red area, and calcification appears as a white–pink area. The shiny white areas are optical artifacts.

Histology

Conventional histology is used widely to analyze the structure of animal and plant tissues. Fixed and stained CEA specimens viewed by light microscopy are useful for identifying plaque components.79 For calcified tissues in paraffin blocks, cutting intact cross-sections can be difficult. Exemplary images from histological analysis are shown in Figure 5. Cutting calcification in frozen sections is even more challenging, usually requiring at least treatment with nitric acid or EDTA. However, these treatments cause calcium depletion and can reduce immunoreactivity of calcium-binding proteins. The percentages of calcification and other plaque components can be determined by area integration.80,81

   Figure 5. From the internal branch of the left carotid artery, fibro-fatty lesions have been segmented and fixed in paraffin blocks. Then 10-μm histology sections prepared from original blocks were stained with: a, Oil Red O, to stain lipid purple, and nuclei brown; b, Van Kossa, to stain calcium salts black, nuclei red, and cytoplasm light pink; and c, trichrome, to stain muscle fibers red, collagen blue, and nuclei blue to black. These sections illustrate distribution of small calcification nodules through the midintimal area (heavy arrow) and loss of calcification from a deep-intimal area near 2:00 (light arrow).

Embedding tissue in plastic helps prevent loss of calcification during sectioning while retaining stain and antibody reactivity. Representative plastic embedded thin sections are shown in Figure 6. Paraffin blocks and frozen sections are attended by significant loss of calcification; however, the time-consuming process of tissue embedding in plastic allows retention of calcification and its quantification (Table).

   Figure 6. X-ray image and corresponding thin-section light micrographs from right CCA fixed in 10% formalin. After fixation, the CEA samples are dehydrated in a graded series of ethanol washes and embedded in glycomethylmethacrylate. After polymerization, thin sections (10 to 30 um) are prepared using the Exakt System modified sawing microtome technique.84 Sections are stained with a Modified Goldner Tri-Chrome reagent for plastic sections for which calcification appears blue–green.

Chemical Analysis

Atomic absorption spectroscopy (AAS) is a sensitive analytical technique used to determine the concentration of metals in liquid samples. In their elemental form, metals absorb ultraviolet light when excited by heat, and each metal has a characteristic wavelength that will be absorbed. The amount of light absorbed is proportional to the concentration of the element in the solution. Measurements are made separately for each element of interest. The method is very sensitive and can measure trace elements down to the parts per million level. Because calcium is one of the metals detectable by AAS, this method is useful in determining calcium concentration in CEA samples. Stoichiometry can be used to calculate the quantity of CHA in the samples (Table).

Another method for chemically analyzing CHA in CEA samples uses the phosphomolybdate reagent, which quantifies the amount of elemental phosphorus in a sample.82 The sample is digested in sulfuric acid and oxidized with hydrogen peroxide to liberate elemental phosphorus. Ammonium molybdate and 1-amino-2-naphthol-4-sulfonic acid are added to produce a solution that absorbs at 830 nm. The concentration of phosphorus is determined from a calibration curve of various concentrations of a standard phosphate salt. The stoichiometry of CHA, Ca10(PO4)6(OH)2, can be used to convert the measured phosphorus concentration to CHA mass. A useful alternative method uses o-cresolphthalein for calcium determination.83 Fura and Indo ratiometric calcium indicators are excited with UV light, allowing for quantification of calcium content as well.84

    Concluding Comments

Selecting an appropriate method of calcification quantification depends on the nature of the specimen and the desired results (Table). Although the in vivo methods of US, EBCT, and MRI are all capable of differentiating plaque components, IVUS and MRI have the highest spatial resolution. In ex vivo analyses, μCT and MRI are the principal methods for visualizing calcified thin slices and creating 3D reconstruction of the specimen. Digital photography is useful for capturing a visual record of intact tissues and tissue fragments before processing by embedding or homogenization. Histological sections provide morphological information that is currently the gold reference standard for validating many imaging techniques. 31P MAS MRS and the microphosphorus chemical assay for calcification quantification rely on chemical stoichiometry to convert the concentration of phosphorus atoms to CHA. The chemical assay is more amenable to high throughput but requires sample destruction. AAS is probably the most sensitive method for calcium quantification; it has the same capabilities and limitations as those of the microphosphorus assay. These qualitative and quantitative methods will likely see increasing use in future investigations of vascular calcification.

    Acknowledgments

This work was supported in part by the National Institutes of Health grants HL63090 and HL07812. The authors thank Catherine Ambrose, PhD, and Tiffany Sheffield of the Bone Histomorphometry and Biomaterials Laboratory in the Department of Orthopaedic Surgery at the University of Texas-Houston Medical School for the plastic embedded histochemical preparations.

References

Alberts B. Molecular Biology of the Cell. 4th ed. New York, NY: Garland Science; 2002.

Vattikuti R, Towler DA. Osteogenic regulation of vascular calcification: an early perspective. Am J Physiol Endocrinol Metab. 2004; 286: E686–E696.

Heino TJ, Hentunen TA, Vaananen HK. Osteocytes inhibit osteoclastic bone resorption through transforming growth factor-beta: enhancement by estrogen. J Cell Biochem. 2002; 85: 185–197.

Becker GL. Calcification mechanisms: roles for cells and mineral. J Oral Pathol. 1977; 6: 307–315.

Trion A, van der Laarse A. Vascular smooth muscle cells and calcification in atherosclerosis. Am Heart J. 2004; 147: 808–814.

Wu LN, Ishikawa Y, Sauer GR, Genge BR, Mwale F, Mishima H, Wuthier RE. Morphological and biochemical characterization of mineralizing primary cultures of avian growth plate chondrocytes: evidence for cellular processing of Ca2+ and Pi prior to matrix mineralization. J Cell Biochem. 1995; 57: 218–237.

Eanes ED, Gillessen IH, Posner AS. Intermediate states in the precipitation of hydroxyapatite. Nature. 1965; 208: 365–367.

Seuwen K, Boddeke HG, Migliaccio S, Perez M, Taranta A, Teti A. A novel calcium sensor stimulating inositol phosphate formation and i signaling expressed by GCT23 osteoclast-like cells. Proc Assoc Am Physicians. 1999; 111: 70–81.

Wenisch S, Stahl JP, Horas U, Heiss C, Kilian O, Trinkaus K, Hild A, Schnettler R. In vivo mechanisms of hydroxyapatite ceramic degradation by osteoclasts: fine structural microscopy. J Biomed Mater Res A. 2003; 67: 713–718.

Mostov K, Werb Z. Journey across the osteoclast. Science. 1997; 276: 219–220.

Doherty TM, Asotra K, Fitzpatrick LA, Qiao JH, Wilkin DJ, Detrano RC, Dunstan CR, Shah PK, Rajavashisth TB. Calcification in atherosclerosis: bone biology and chronic inflammation at the arterial crossroads. Proc Natl Acad Sci U S A. 2003; 100: 11201–11206.

Tintut Y, Demer LL. Recent advances in multifactorial regulation of vascular calcification. Curr Opin Lipidol. 2001; 12: 555–560.

Parhami F, Morrow AD, Balucan J, Leitinger N, Watson AD, Tintut Y, Berliner JA, Demer LL. Lipid oxidation products have opposite effects on calcifying vascular cell and bone cell differentiation. A possible explanation for the paradox of arterial calcification in osteoporotic patients. Arterioscler Thromb Vasc Biol. 1997; 17: 680–687.

Kiechl S, Schett G, Wenning G, Redlich K, Oberhollenzer M, Mayr A, Santer P, Smolen J, Poewe W, Willeit J. Osteoprotegerin is a risk factor for progressive atherosclerosis and cardiovascular disease. Circulation. 2004; 109: 2175–2180.

Golledge J, McCann M, Mangan S, Lam A, Karan M. Osteoprotegerin and osteopontin are expressed at high concentrations within symptomatic carotid atherosclerosis. Stroke. 2004; 35: 1636–1641.

Gronholdt ML. Ultrasound and lipoproteins as predictors of lipid-rich, rupture-prone plaques in the carotid artery. Arterioscler Thromb Vasc Biol. 1999; 19: 2–13.

Barry R, Pienaar C, Nel CJ. Accuracy of B-mode ultrasonography in detecting carotid plaque hemorrhage and ulceration. Ann Vasc Surg. 1990; 4: 466–470.

Schreiner PJ, Heiss G, Tyroler HA, Morrisett JD, Davis CE, Smith R. Race and gender differences in the association of Lp(a) with carotid artery wall thickness. The Atherosclerosis Risk in Communities (ARIC) Study. Arterioscler Thromb Vasc Biol. 1996; 16: 471–478.

de Groot E, Jukema JW, Montauban van Swijndregt AD, Zwinderman AH, Ackerstaff RG, van der Steen AF, Bom N, Lie KI, Bruschke AV. B-mode ultrasound assessment of pravastatin treatment effect on carotid and femoral artery walls and its correlations with coronary arteriographic findings: a report of the Regression Growth Evaluation Statin Study (REGRESS). J Am Coll Cardiol. 1998; 31: 1561–1567.

Wagenknecht LE, Langefeld CD, Carr JJ, Riley W, Freedman BI, Moossavi S, Bowden DW. Race-specific relationships between coronary and carotid artery calcification and carotid intimal medial thickness. Stroke. 2004; 35: e97–e99.

Herrmann SM, Whatling C, Brand E, Nicaud V, Gariepy J, Simon A, Evans A, Ruidavets JB, Arveiler D, Luc G, Tiret L, Henney A, Cambien F. Polymorphisms of the human matrix Gla protein (MGP) gene, vascular calcification, myocardial infarction. Arterioscler Thromb Vasc Biol. 2000; 20: 2386–2393.

Miskolczi L, Guterman LR, Flaherty JD, Hopkins LN. Depiction of carotid plaque ulceration and other plaque-related disorders by intravascular sonography: a flow chamber study. Am J Neuroradiol. 1996; 17: 1881–1890.

Maehara A, Fitzgerald PJ. Coronary calcification: assessment by intravascular ultrasound imaging. Z Kardiol. 2000; 89 (suppl 2): 112–116.

Tobis JM, Mallery J, Mahon D, Lehmann K, Zalesky P, Griffith J, Gessert J, Moriuchi M, McRae M, Dwyer ML, et al. Intravascular ultrasound imaging of human coronary arteries in vivo. Analysis of tissue characterizations with comparison to in vitro histological specimens. Circulation. 1991; 83: 913–926.

Gussenhoven EJ, Essed CE, Frietman P, van Egmond F, Lancee CT, van Kappellen WH, Roelandt J, Serruys PW, Gerritsen GP, van Urk H, et al. Intravascular ultrasonic imaging: histologic and echographic correlation. Eur J Vasc Surg. 1989; 3: 571–576.

Chiesa G, Di Mario C, Colombo N, Vignati L, Marchesi M, Monteggia E, Parolini C, Lorenzon P, Laucello M, Lorusso V, Adamian M, Franceschini G, Newton R, Sirtori CR. Development of a lipid-rich, soft plaque in rabbits, monitored by histology and intravascular ultrasound. Atherosclerosis. 2001; 156: 277–287.

Palmer ND, Northridge D, Lessells A, McDicken WN, Fox KA. In vitro analysis of coronary atheromatous lesions by intravascular ultrasound; reproducibility and histological correlation of lesion morphology. Eur Heart J. 1999; 20: 1701–1706.

Beckman JA, Ganz J, Creager MA, Ganz P, Kinlay S. Relationship of clinical presentation and calcification of culprit coronary artery stenoses. Arterioscler Thromb Vasc Biol. 2001; 21: 1618–1622.

Weissman NJ, Sheris SJ, Chari R, Mendelsohn FO, Anderson WD, Breall JA, Tanguay JF, Diver DJ. Intravascular ultrasonic analysis of plaque characteristics associated with coronary artery remodeling. Am J Cardiol. 1999; 84: 37–40.

Kornowski R. Impact of smoking on coronary atherosclerosis and remodeling as determined by intravascular ultrasonic imaging. Am J Cardiol. 1999; 83: 443–445, A449.

Scott DS, Arora UK, Farb A, Virmani R, Weissman NJ. Pathologic validation of a new method to quantify coronary calcific deposits in vivo using intravascular ultrasound. Am J Cardiol. 2000; 85: 37–40.

Urbani MP, Picano E, Parenti G, Mazzarisi A, Fiori L, Paterni M, Pelosi G, Landini L. In vivo radiofrequency-based ultrasonic tissue characterization of the atherosclerotic plaque. Stroke. 1993; 24: 1507–1512.

Kawasaki M, Takatsu H, Noda T, Ito Y, Kunishima A, Arai M, Nishigaki K, Takemura G, Morita N, Minatoguchi S, Fujiwara H. Noninvasive quantitative tissue characterization and two-dimensional color-coded map of human atherosclerotic lesions using ultrasound integrated backscatter: comparison between histology and integrated backscatter images. J Am Coll Cardiol. 2001; 38: 486–492.

Kawasaki M, Takatsu H, Noda T, Sano K, Ito Y, Hayakawa K, Tsuchiya K, Arai M, Nishigaki K, Takemura G, Minatoguchi S, Fujiwara T, Fujiwara H. In vivo quantitative tissue characterization of human coronary arterial plaques by use of integrated backscatter intravascular ultrasound and comparison with angioscopic findings. Circulation. 2002; 105: 2487–2492.

Brandt T, Knauth M, Wildermuth S, Winter R, von Kummer R, Sartor K, Hacke W. CT angiography and Doppler sonography for emergency assessment in acute basilar artery ischemia. Stroke. 1999; 30: 606–612.

London GM, Guerin AP, Marchais SJ, Metivier F, Pannier B, Adda H. Arterial media calcification in end-stage renal disease: impact on all-cause and cardiovascular mortality. Nephrol Dial Transplant. 2003; 18: 1731–1740.

Pai SS, Bude RO. Sonographic appearance of extensive hepatic arterial calcification mimicking pneumobilia. J Clin Ultrasound. 2002; 30: 38–41.

Whitehall J, Smith M, Altamirano L. Idiopathic infantile arterial calcification: sonographic findings. J Clin Ultrasound. 2003; 31: 497–501.

Ribeiro S, Ramos A, Brandao A, Rebelo JR, Guerra A, Resina C, Vila-Lobos A, Carvalho F, Remedio F, Ribeiro F. Cardiac valve calcification in haemodialysis patients: role of calcium-phosphate metabolism. Nephrol Dial Transplant. 1998; 13: 2037–2040.

O’Rourke RA, Brundage BH, Froelicher VF, Greenland P, Grundy SM, Hachamovitch R, Pohost GM, Shaw LJ, Weintraub WS, Winters WL Jr, Forrester JS, Douglas PS, Faxon DP, Fisher JD, Gregoratos G, Hochman JS, Hutter AM Jr, Kaul S, Wolk MJ. American College of Cardiology/American Heart Association Expert Consensus document on electron-beam computed tomography for the diagnosis and prognosis of coronary artery disease. Circulation. 2000; 102: 126–140.

Vliegenthart R, Song B, Hofman A, Witteman JC, Oudkerk M. Coronary calcification at electron-beam CT: effect of section thickness on calcium scoring in vitro and in vivo. Radiology. 2003; 229: 520–525.

Callister T, Janowitz W, Raggi P. Sensitivity of two electron beam tomography protocols for the detection and quantification of coronary artery calcium. Am J Roentgenol. 2000; 175: 1743–1746.

Watson KE, Abrolat ML, Malone LL, Hoeg JM, Doherty T, Detrano R, Demer LL. Active serum vitamin D levels are inversely correlated with coronary calcification. Circulation. 1997; 96: 1755–1760.

Agatston AS, Janowitz WR, Hildner FJ, Zusmer NR, Viamonte M Jr, Detrano R. Quantification of coronary artery calcium using ultrafast computed tomography. J Am Coll Cardiol. 1990; 15: 827–832.

Raggi P, James G. Coronary calcium screening and coronary risk stratification. Curr Atheroscler Rep. 2004; 6: 107–111.

Walsh CR, Larson MG, Kupka MJ, Levy D, Vasan RS, Benjamin EJ, Manning WJ, Clouse ME, O’Donnell CJ. Association of aortic valve calcium detected by electron beam computed tomography with echocardiographic aortic valve disease and with calcium deposits in the coronary arteries and thoracic aorta. Am J Cardiol. 2004; 93: 421–425.

Messika-Zeitoun D, Aubry MC, Detaint D, Bielak LF, Peyser PA, Sheedy PF, Turner ST, Breen JF, Scott C, Tajik AJ, Enriquez-Sarano M. Evaluation and clinical implications of aortic valve calcification measured by electron-beam computed tomography. Circulation. 2004; 110: 356–362.

Baumgart D, Schmermund A, Goerge G, Haude M, Ge J, Adamzik M, Sehnert C, Altmaier K, Groenemeyer D, Seibel R, Erbel R. Comparison of electron beam computed tomography with intracoronary ultrasound and coronary angiography for detection of coronary atherosclerosis. J Am Coll Cardiol. 1997; 30: 57–64.

Woodcock RJ Jr, Goldstein JH, Kallmes DF, Cloft HJ, Phillips CD. Angiographic correlation of CT calcification in the carotid siphon. Am J Neuroradiol. 1999; 20: 495–499.

Budoff MJ, Diamond GA, Raggi P, Arad Y, Guerci AD, Callister TQ, Berman D. Continuous probabilistic prediction of angiographically significant coronary artery disease using electron beam tomography. Circulation. 2002; 105: 1791–1796.

Pirich C, Leber A, Knez A, Bengel FM, Nekolla SG, Haberl R, Schwaiger M. Relation of coronary vasoreactivity and coronary calcification in asymptomatic subjects with a family history of premature coronary artery disease. Eur J Nucl Med Mol Imaging. 2004; 31: 663–670.

Achenbach S, Ropers D, Hoffmann U, MacNeill B, Baum U, Pohle K, Brady TJ, Pomerantsev E, Ludwig J, Flachskampf FA, Wicky S, Jang IK, Daniel WG. Assessment of coronary remodeling in stenotic and nonstenotic coronary atherosclerotic lesions by multidetector spiral computed tomography. J Am Coll Cardiol. 2004; 43: 842–847.

Achenbach S, Ropers D, Pohle K, Anders K, Baum U, Hoffmann U, Moselewski F, Ferencik M, Brady TJ. Clinical results of minimally invasive coronary angiography using computed tomography. Cardiol Clin. 2003; 21: 549–559.

Achenbach S, Hoffmann U, Ferencik M, Wicky S, Brady TJ. Tomographic coronary angiography by EBCT and MDCT. Prog Cardiovasc Dis. 2003; 46: 185–195.

Achenbach S, Daniel WG. Imaging of coronary atherosclerosis using computed tomography: current status and future directions. Curr Atheroscler Rep. 2004; 6: 213–218.

Schroeder S, Kuettner A, Kopp AF, Heuschmidt M, Burgstahler C, Herdeg C, Claussen CD. Noninvasive evaluation of the prevalence of noncalcified atherosclerotic plaques by multi-slice detector computed tomography: results of a pilot study. Int J Cardiol. 2003; 92: 151–155.

Leber AW, Knez A, Mukherjee R, White C, Huber A, Becker A, Becker CR, Reiser M, Haberl R, Steinbeck G. Usefulness of calcium scoring using electron beam computed tomography and noninvasive coronary angiography in patients with suspected coronary artery disease. Am J Cardiol. 2001; 88: 219–223.

Leber AW, Knez A, White CW, Becker A, von Ziegler F, Muehling O, Becker C, Reiser M, Steinbeck G, Boekstegers P. Composition of coronary atherosclerotic plaques in patients with acute myocardial infarction and stable angina pectoris determined by contrast-enhanced multislice computed tomography. Am J Cardiol. 2003; 91: 714–718.

Leber AW, Knez A, Becker A, Becker C, von Ziegler F, Nikolaou K, Rist C, Reiser M, White C, Steinbeck G, Boekstegers P. Accuracy of multidetector spiral computed tomography in identifying and differentiating the composition of coronary atherosclerotic plaques: a comparative study with intracoronary ultrasound. J Am Coll Cardiol. 2004; 43: 1241–1247.

Cai JM, Hatsukami TS, Ferguson MS, Small R, Polissar NL, Yuan C. Classification of human carotid atherosclerotic lesions with in vivo multicontrast magnetic resonance imaging. Circulation. 2002; 106: 1368–1373.

Cappendijk VC, Cleutjens KB, Heeneman S, Schurink GW, Welten RJ, Kessels AG, van Suylen RJ, Daemen MJ, van Engelshoven JM, Kooi ME. In vivo detection of hemorrhage in human atherosclerotic plaques with magnetic resonance imaging. J Magn Reson Imaging. 2004; 20: 105–110.

Correia LC, Atalar E, Kelemen MD, Ocali O, Hutchins GM, Fleg JL, Gerstenblith G, Zerhouni EA, Lima JA. Intravascular magnetic resonance imaging of aortic atherosclerotic plaque composition. Arterioscler Thromb Vasc Biol. 1997; 17: 3626–3632.

Babiarz LS, Yousem DM, Wasserman BA, Wu C, Bilker W, Beauchamp NJ Jr. Cavernous carotid artery calcification and white matter ischemia. Am J Neuroradiol. 2003; 24: 872–877.

Zhao XQ, Yuan C, Hatsukami TS, Frechette EH, Kang XJ, Maravilla KR, Brown BG. Effects of prolonged intensive lipid-lowering therapy on the characteristics of carotid atherosclerotic plaques in vivo by MRI: a case-control study. Arterioscler Thromb Vasc Biol. 2001; 21: 1623–1629.

Anderson HC, Sipe JB, Hessle L, Dhanyamraju R, Atti E, Camacho NP, Millan JL. Impaired calcification around matrix vesicles of growth plate and bone in alkaline phosphatase-deficient mice. Am J Pathol. 2004; 164: 841–847.

Doschak MR, Cooper DM, Huculak CN, Matyas JR, Hart DA, Hallgrimsson B, Zernicke RF, Bray RC. Angiogenesis in the distal femoral chondroepiphysis of the rabbit during development of the secondary centre of ossification. J Anat. 2003; 203: 223–233.

Verna C, Dalstra M, Wikesjo UM, Trombelli L. Healing patterns in calvarial bone defects following guided bone regeneration in rats. A micro-CT scan analysis. J Clin Periodontol. 2002; 29: 865–870.

Ritman EL, Bolander ME, Fitzpatrick LA, Turner RT. Micro-CT imaging of structure-to-function relationship of bone microstructure and associated vascular involvement. Technol Health Care. 1998; 6: 403–412.

Clarke SE, Hammond RR, Mitchell JR, Rutt BK. Quantitative assessment of carotid plaque composition using multicontrast MRI and registered histology. Magn Reson Med. 2003; 50: 1199–1208.

Coombs BD, Rapp JH, Ursell PC, Reilly LM, Saloner D. Structure of plaque at carotid bifurcation: high-resolution MRI with histological correlation. Stroke. 2001; 32: 2516–2521.

Shinnar M, Fallon JT, Wehrli S, Levin M, Dalmacy D, Fayad ZA, Badimon JJ, Harrington M, Harrington E, Fuster V. The diagnostic accuracy of ex vivo MRI for human atherosclerotic plaque characterization. Arterioscler Thromb Vasc Biol. 1999; 19: 2756–2761.

Morrisett J, Vick W, Sharma R, Lawrie G, Reardon M, Ezell E, Schwartz J, Hunter G, Gorenstein D. Discrimination of components in atherosclerotic plaques from human carotid endarterectomy specimens by magnetic resonance imaging ex vivo. Magn Reson Imaging. 2003; 21: 465–474.

Adams GJ, Simoni DM, Bordelon CB Jr, Vick GW III, Kimball KT, Insull W Jr, Morrisett JD. Bilateral symmetry of human carotid artery atherosclerosis. Stroke. 2002; 33: 2575–2580.

Itskovich VV, Samber DD, Mani V, Aguinaldo JG, Fallon JT, Tang CY, Fuster V, Fayad ZA. Quantification of human atherosclerotic plaques using spatially enhanced cluster analysis of multicontrast-weighted magnetic resonance images. Magn Reson Med. 2004; 52: 515–523.

Brown CE, Allaway JR, Brown KL, Battocletti JH. Noninvasive evaluation of mineral content of bone without use of ionizing radiation. Clin Chem. 1987; 33: 227–236.

Hsieh MF, Perng LH, Chin TS, Perng HG. Phase purity of sol-gel-derived hydroxyapatite ceramic. Biomaterials. 2001; 22: 2601–2607.

Guo W, Morrisett JD, Lawrie GM, DeBakey ME, Hamilton JA. Identification of different lipid phases and calcium phosphate deposits in human carotid artery plaques by MAS NMR spectroscopy. Magn Reson Med. 1998; 39: 184–189.

Guo W, Morrisett JD, DeBakey ME, Lawrie GM, Hamilton JA. Quantification in situ of crystalline cholesterol and calcium phosphate hydroxyapatite in human atherosclerotic plaques by solid-state magic angle spinning NMR. Arterioscler Thromb Vasc Biol. 2000; 20: 1630–1636.

Denzel C, Lell M, Maak M, Hockl M, Balzer K, Muller KM, Fellner C, Fellner FA, Lang W. Carotid artery calcium: accuracy of a calcium score by computed tomography-an in vitro study with comparison to sonography and histology. Eur J Vasc Endovasc Surg. 2004; 28: 214–220.

Schulte-Altedorneburg G, Droste DW, Haas N, Kemeny V, Nabavi DG, Fuzesi L, Ringelstein EB. Preoperative B-mode ultrasound plaque appearance compared with carotid endarterectomy specimen histology. Acta Neurol Scand. 2000; 101: 188–194.

Bassiouny HS, Davis H, Massawa N, Gewertz BL, Glagov S, Zarins CK. Critical carotid stenoses: morphologic and chemical similarity between symptomatic and asymptomatic plaques. J Vasc Surg. 1989; 9: 202–212.

Bartlett GR. Phosphorus assay in column chromatography. J Biol Chem. 1959; 234: 466–468.

Fischer JW, Steitz SA, Johnson PY, Burke A, Kolodgie F, Virmani R, Giachelli C, Wight TN. Decorin promotes aortic smooth muscle cell calcification and colocalizes to calcified regions in human atherosclerotic lesions. Arterioscler Thromb Vasc Biol. 2004; 24: 2391–2396.

Kieffer P, Robert A, Capdeville-Atkinson C, Atkinson J, Lartaud-Idjouadiene I. Age-related arterial calcification in rats. Life Sci. 2000; 66: 2371–2381.

 


 

日期:2007年5月18日 - 来自[2005年第25卷第8期]栏目

Calcification of Advanced Atherosclerotic Lesions in the Innominate Arteries of ApoE-Deficient Mice

From the Department of Pathobiology (M.R., F.B., E.A.K., J.L.R., M.E.R.), the Interdisciplinary Graduate Program in Nutritional Sciences (B.J.B., E.A.K., M.E.R.), the Department of Bioengineering (M.S., C.M.G.), and the Department of Pathology (S.M.S., C.M.G., M.E.R.), University of Washington, Seattle.

Correspondence to Michael E. Rosenfeld, Department of Pathobiology, Box 353410, University of Washington, Seattle, WA 98195. E-mail ssmjm@u.washington.edu

    Abstract

Objective— Advanced atherosclerotic lesions in the innominate arteries of chow-fed apolipoprotein E–deficient mice become highly calcified with 100% frequency by 75 weeks of age. The time course, cell types, and mechanism(s) associated with calcification were investigated.

Methods and Results— The deposition of hydroxyapatite is preceded by the formation of fibro-fatty nodules that are populated by cells that morphologically resemble chondrocytes. These cells are spatially associated with small deposits of hydroxyapatite in animals between 45 and 60 weeks of age. Immunocytochemical analyses with antibodies recognizing known chondrocyte proteins show that these cells express the same proteins as chondrocytes within developing bone. Histological and electron microscopic analyses of lesions from animals between 45 and 60 weeks of age show that the chondrocyte-like cells are surrounded by dense connective tissue that stains positive for type II collagen. Nanocrystals of hydroxyapatite can be seen within matrix vesicles derived from the chondrocyte-like cells. In mice between 75 and 104 weeks of age, the lesions have significantly reduced cellularity and contain large calcium deposits. The few remaining chondrocyte-like cells are located adjacent to or within the large areas of calcification.

Conclusions— Calcification of advanced lesions in chow-fed apolipoprotein E–deficient mice occurs reproducibly in mice between 45 and 75 weeks of age. The deposition of hydroxyapatite is mediated by chondrocytes, which suggests that the mechanism of calcification may in part recapitulate the process of endochondral bone formation.

Advanced atherosclerotic lesions in the innominate arteries of chow-fed apolipoprotein E–deficient mice become highly calcified. The cell types associated with calcification were investigated. The cells mediating the calcification have a chondrocyte-like phenotype. The process of calcification within advanced lesions of apoE–/– mice may recapitulate endochondral bone formation.

Key Words: atherosclerosis ? calcification ? chondrocytes ? apolipoprotein E–deficient mice

    Introduction

Many advanced human atherosclerotic lesions contain deposits of calcium phosphate.1,2 The significance of calcium deposition with regard to clinical events is still a matter of debate.3 Currently, the mechanisms by which advanced lesions become calcified are unknown, and may include both dystrophic calcification as well as direct ossification.4 The presence of ossified bone within plaques and the expression of osteogenic cell makers has been previously reported.5–9 The cell-mediated process of bone formation involves both osteoblasts and chondrocytes. Recent evidence suggests that both of these cell types may play a similar role in mediating the calcification of atherosclerotic lesions in humans and mice.10 Chondrocyte metaplasia within blood vessels has been previously reported in humans11 and is frequently observed in mouse models of atherosclerosis.12–14 The designation of these cells as chondrocytes is based on the morphological similarity to chondrocytes in cartilage and on the presence of cartilage and bone extracellular matrix proteins.15

See page 1307

This study is a continuation of our characterization of advanced atherosclerotic lesions in the innominate arteries of chow-fed apolipoprotein E–deficient (apoE–/–) mice.16 We previously reported that unstable lesions in the innominate arteries initially contain large central necrotic cores covered by thin fibrous caps, and that these plaques are converted to more stable fibro-fatty nodules by 1 year of age. This conversion is associated with the presence of chondrocyte-like cells.14 In the current study, we have evaluated the time course of calcification and the temporal and spatial association between the chondrocyte-like cells and the deposition of hydroxyapatite. In addition, we have used immunocytochemistry and electron microscopy to further define both the phenotype of the chondrocyte-like cells and the mechanism of calcification. The data suggest that calcification is initiated in younger mice and becomes apparent in mice between 45 and 60 weeks of age, and that the chondrocyte-like cells are responsible for depositing hydroxyapatite through a process that may recapitulate the cellular and temporal aspects of endochondral ossification.

    Methods

Animals

Male and female apoE–/– mice on a C57BL/6J background (n=50) were fed normal chow and water ad libitum throughout the study. Forty mice were euthanized by lethal injection (Ketamine/Xylaject; 28 between 45 and 75 weeks of age, and 12 between 75 and 104 weeks of age) and perfused with PBS at physiological pressure followed by 10% buffered formalin through the left ventricle. An additional subset of mice (n=4) was perfused only with PBS for frozen tissue analysis. Tissues from 6 mice were used for electron microscopic analysis. The femoral and humeral bones from two newborn mice were also included in this study. All protocols were approved by the University of Washington Institutional Animal Care and Use Committee.

Preparation and Analysis of Tissue

The base of the right carotid artery (also called the brachiocephalic trunk or the innominate artery) was dissected out, embedded in paraffin, and serially sectioned (5 μm). Every twenty-fifth section was stained with a modified Movat pentachrome stain.17 To identify vascular calcification, adjacent sections were stained with the von Kossa stain for hydroxyapatite. The lesion area, total number of cells, total number of chondrocyte-like cells, and the area of calcium deposition were determined in each stained section using computer assisted morphometry (Image Pro, Media Cybernetics).

In a subset of mice between 45 and 60 weeks of age (n=4), the innominate artery was dissected out, embedded in OCT, and frozen in liquid nitrogen (LN2). An additional group of animals between 45 and 104 weeks of age (n=4 between 45 and 75 weeks of age and n=2 at 104 weeks of age) was euthanized for analysis of lesion composition by transmission electron microscopy. The animals were perfusion fixed with 4% paraformaldehyde and immersion fixed with modified Karnovsky fixative. The innominate arteries were embedded in plastic and processed for transmission electron microscopy using standard techniques.

The femoral and humeral bones were removed from 2 newborn apoE–/– mice, fixed in 10% formalin for 24 hours, decalcified for 48 hours in 0.5 mol/L EDTA, and embedded as described above.

Immunocytochemistry

Sections adjacent to those stained with the Movat and von Kossa stains were stained with an anti-mouse macrophage antibody (Mac-2; Accurate Chemical), anti–smooth muscle actin antibody (1A4; Dako), type II collagen antibody (Novacastra), anti-osteoprotegerin (OPG; Santa Cruz Biotechnology), anti–receptor activator of NF-B ligand (RANKL; Santa Cruz Biotechnology), anti–parathyroid hormone related peptide (PTHrP; Santa Cruz Biotechnology), and anti-osteopontin (OPN; R&D Systems). Collagen II staining was done after antigen retrieval with trypsin (Zymed). Control sections were incubated with rat, goat, or rabbit IgG (Zymed). The number of chondrocyte-like cells that stained positively for OPG, PTHrP, RANKL, and OPN was established in 28 animals between 45 and 75 weeks of age and expressed as a percentage of the total number of chondrocyte-like cells that were counted in the adjacent Movat stained section.

Alkaline Phosphatase Activity

Cryosections of the innominate artery (8 μm) from 4 mice between 45 and 60 weeks of age were directly incubated with the working solution used for detecting alkaline phosphatase activity (ALP) conjugated antibodies (Red Alkaline Phosphatase Substrate Kit I, Vector Labs, Burlingame, CA). Adjacent sections were stained with the von Kossa and Movat stains to identify calcified areas and cellular composition, respectively.

Statistical Analysis

All data were expressed as mean±SD. Significant differences between means were determined by the nonparametric Mann–Whitney U test (STATA, Intercooled version 8).

    Results

Histology

Fibro-fatty nodules in the advanced lesions in the innominate arteries are a nidus for calcification and become highly calcified in mice at 75 weeks of age. However, the process of calcification is initiated in much younger mice and becomes apparent in mice between 45 and 60 weeks of age where small deposits of hydroxyapatite are observed (Figure 1A). These small deposits range from 17% to 34% of the plaque area (Table 1). The lesions exhibit a high degree of cellularity, ranging between 800 and 1500 cells per mm2 of lesion area, with an average of 13% of the cells being chondrocyte-like cells (Table 1). The chondrocyte-like cells are located both adjacent to and within areas of calcification, as well as in areas devoid of calcium (Figure 1A; Figures I through III, available online at http://atvb.ahajournals.org). Many of the chondrocyte-like cells are surrounded by a dense ring of connective tissue analogous to the appearance of chondrocytes within growth cartilage (Figure 3A). This ring of connective tissue stains positively with an antibody recognizing type II collagen (Figure 1B).

[in this window]

[in a new window]

   Figure 1. Chondrocyte-like cells in advanced atherosclerotic lesions in apoE–/– mice are surrounded by type II collagen and are spatially associated with calcified areas and matrix vesicles. This figure shows chondrocyte-like cells within small areas of calcification in the innominate arteries from apoE–/– mice between 45 and 75 weeks of age. A, von Kossa stain for hydroxyapatite (400x magnification). B, Immunostain with anti–type II collagen antibody (1000x magnification). C, Electron micrograph showing the dense matrix surrounding chondrocyte-like cells and their proximity to foam cells and cholesterol clefts (bar=1 μm). D, Electron micrograph showing that the cells are closely apposed to the deposited calcium and within a matrix rich in collagen fibers and vesicular material (bar=1 μm). E and F, Higher magnification electron micrographs showing vesicles derived from the cells that contain nanocrytals of calcium-phosphate (E, bar=200 nanometers; F, bar=100 nanometers).

[in this window]

[in a new window]

   TABLE 1. Temporal Changes of Calcified Plaques

[in this window]

[in a new window]

   Figure 3. Expression of cell markers by chondrocytes in developing bone from a newborn apoE–/– mice. This figure shows the results of immunostaining of serial sections of the femur of a newborn mouse with antibodies specific for chondrocyte proteins. Areas bracketed by black boxes in lower magnification micrographs (100x) are shown in higher magnification inserts (400x). A, Movat pentachrome stain; B, type II collagen; C, OPG; D, PTHrP; E, OPN; F, RANKL.

In mice between 75 and 104 weeks of age, there is a 100% frequency of calcification and the deposits are much larger, reaching an average of 50% of lesion area (Figure 2 and Table 1). These extremely advanced plaques also contain significant amounts of connective tissue and cholesterol clefts but have very few remaining cells (Table 1). The remaining chondrocyte-like cells have condensed pycnotic nuclei and are situated adjacent to or within the areas of calcium deposition (Figure 2D). Starting at 60 weeks of age, there is also a high frequency of chondrocyte-like cells and calcification in the medial layer (observed in 60% of the lesions; Figure IV, available online at http://atvb.ahajournals.org). This medial calcification is often associated with breaks in the internal elastic lamina (Figure 2C) and is observed in >80% of the lesions by 75 weeks of age.

[in this window]

[in a new window]

   Figure 2. Calcification of advanced atherosclerotic lesions in the innominate arteries of 2-year-old apoE–/– mice. Diffuse calcification occupies the majority of the lesion (A) or can occur within a smaller fibro-fatty nodule (B, N indicates nodule). Calcification often disrupts the internal elastic lamina (C, arrow points to disrupted internal elastic lamina). Chondrocyte-like cells within large calcified areas have condensed pycnotic nuclei (D, arrows point to condensed nuclei). A and B, 100x magnification; C and D, 400x. Movat pentachrome stain.

Electron Microscopy

Electron microscopic analysis of the lesions from mice between 60 and 75 weeks of age revealed that the extracellular matrix between chondrocyte-like cells is rich in collagen fibrils and vesicular material. Many of the cells are associated with early stages of calcium deposition (Figure 1C through 1F), where the matrix surrounding these cells contains needle-like structures compatible with nanocrystals of hydroxyapatite (Figure 1D through 1F). There are also vesicular structures which, based on their shape and size, resemble matrix vesicles. Some of these vesicles contain electron dense needle-like structures that appear to be associated with the initial process of mineralization (Figure 1F).

Immunocytochemical Analyses

Immunocytochemistry with antibodies specific for chondrocyte proteins was used to further characterize the phenotype of the chondrocyte-like cells. In addition, as a direct basis of comparison we generated sections of the growth plates of bones obtained from newborn apoE–/– mice. The Movat pentachrome staining of these sections of bone showed the characteristic zones containing proliferative and hypertrophic chondrocytes (Figure 3A). Staining patterns in the bone were compared with the expression patterns by chondrocyte-like cells within the advanced atherosclerotic lesions of mice between 45 and 60 weeks of age (Figures 3 and 4 and Table 2). As shown in Figure 3, resting/proliferative chondrocytes in the developing bone stain with antibodies for OPG and PTHrP (Figure 3C and 3D) and are surrounded by a matrix rich in type II collagen (Figure 4B). The resting/proliferative chondrocytes (that are not actively involved in calcification) do not stain for OPN and sporadically stain with anti-RANKL (Figure 4E and 4F). In contrast, hypertrophic chondrocytes that are actively engaged in the ossification process express all of the markers.

[in this window]

[in a new window]

   Figure 4. Expression of chondrocyte proteins and markers of smooth muscle cells and macrophages in serial sections of an advanced atherosclerotic lesion in the innominate artery of a 60-week-old apoE–/– mouse. This figure shows the results of immunostaining serial sections with antibodies recognizing known chondrocyte proteins. A, Movat pentachrome stain; B, Mac-2 (macrophage-specific); C,  actin (smooth muscle cell specific); D, OPG; E, RANKL; F, PTHrP; G, OPN; H, von Kossa stain (hydroxyapatite); I, Control IgG. Magnification=200x for all micrographs.

[in this window]

[in a new window]

   TABLE 2. Comparison of the Expression Pattern of Chondrocyte Markers by Chondrocyte-Like Cells in Advanced Atherosclerotic Lesions With Chondrocytes in Developing Bone

As shown in Figure 4, chondrocyte-like cells in the lesions do not stain with antibodies recognizing -actin or Mac-2. The staining for -actin is restricted to the medial layer and the fibrous cap of the lesions (Figures 4C and IB). Mac-2 predominately stains lateral xanthomas, aggregates of lipid loaded macrophage-derived foam cells (Figures 4B and IC). In contrast, in noncalcified lesions, only chondrocyte-like cells stain with antibodies specific for OPG, RANKL, and PTHrP (Figures 4D through 4F and IIF through IIH). Very few chondrocyte-like cells express OPN (<20% of the chondrocyte-like cells) (Figures 4G, ID, and IIE; Table 2). OPN staining colocalizes primarily with the cells that are positive for Mac-2 (Figures 4B and IC). In both calcified and noncalcified plaques, 80% of the chondrocyte-like cells stain positive for OPG, 70% for PThrP, and 50% of them express RANKL (Figures 4C, 4D, 4F, II, and III; Table 2). In calcified plaques there is increased staining for OPN by chondrocyte-like cells (>30% of cells, Figure III). Medial chondrocyte-like cells do not express -actin or mac-2 but are von Kossa and ALP positive (Figure 5) and surrounded by type II collagen (Figure IV).

[in this window]

[in a new window]

   Figure 5. Chondrocyte-like cells within areas of calcification in advanced atherosclerotic lesions of older apoE–/– mice contain active alkaline phosphatase. This figure shows serial sections of advanced lesions in the innominate arteries from 3 different mice between 60 and 75 weeks of age. A, D, and G, Movat pentachrome stain; D, E, and H, von Kossa stain; C, F, and I, alkaline phosphatase activity. A through F, 400x magnification; G through I, 100x magnification.

Alkaline Phosphatase Activity

Alkaline phosphatase is a key enzyme expressed by osteogenic cells during bone formation. As expected, there is ALP activity within the growth plate of the bones adjacent to the calcification zone (data not shown). There is also ALP activity in chondrocyte-like cells situated predominantly in areas surrounding the zone of mineralization in the advanced atherosclerotic lesions as demonstrated by Von Kossa staining (Figure 5). Areas without chondrocyte-like cells or not surrounding mineralization are devoid of ALP activity.

    Discussion

This study is a continuation of our characterization of advanced atherosclerotic lesions in the innominate arteries of chow-fed apoE–/– mice and focuses on plaque calcification, a process that also occurs with high frequency in older humans. We observed that small areas of calcification are apparent in many of the mice between 45 and 60 weeks of age and that by 75 weeks of age there is a 100% frequency of calcification. Furthermore, it appears that the chondrocyte-like cells are responsible for depositing the hydroxyapatite through a process that may recapitulate the cellular and temporal aspects of endochondral ossification. This conclusion is supported by the temporal and spatial association between the chondrocyte-like cells and hydroxyapatite deposits, the concordance in the patterns of expression of the chondrocyte markers by cells within both the developing bone and the advanced atherosclerotic lesions (Table 2), the presence of active alkaline phosphatase in the chondrocyte-like cells within and adjacent to areas of calcification, and by electron microscopic data showing matrix vesicles derived from the chondrocyte-like cells containing electron dense crystalline material consistent with hydroxyapatite.

The detection of OPG and RANKL in the mouse plaques is consistent with previous reports of RANKL and OPG expression in human plaques.7 It is currently unclear what role these molecules play within the plaques. However, recent epidemiological studies show that serum OPG levels are associated with the extent of atherosclerosis and calcification in humans18,19 and may predict the progression of atherosclerosis and cardiovascular mortality.20 The OPG–/– mouse is characterized by an osteoporotic phenotype and development of extensive medial calcification of the major blood vessels.21 To determine whether OPG regulates intimal calcification and atherosclerosis progression in the apoE–/– mouse lesions, we are currently generating OPG–/– x apoE–/– double knockout mice.

Osteopontin is normally expressed in bone and plays a role in regulating the process of mineralization.22 OPN is expressed by smooth muscle cells, endothelial cells, and macrophages in human atherosclerotic lesions especially in calcified areas.8,23 In the atherosclerotic lesions of the apoE–/– mice the chondrocyte-like cells show a similar pattern of OPN expression, staining positive for OPN more so when associated with areas of calcification. Both in vivo24,25 and vitro26,27 studies suggest that OPN may act locally to inhibit calcification by binding hydroxyapatite and reducing crystal growth. PTHrP is a critical mediator of chondrocyte growth and maturation as mice deficient in PTHrP have severe abnormalities in bone development.28 PTHrP is present in human atherosclerotic lesions,29 but whether it plays a role in the atherogenic process has not been determined.

ALP is crucial for initiating mineralization in bone. ALP activity has been shown inside matrix vesicles shed from chondrocytes and most likely works to increase the availability of inorganic phosphate (Pi) needed for hydroxyapatite crystal growth.30 Increases in ALP activity is a marker of the transition to an osteogenic phenotype by smooth muscle cells in vitro. ALP activity is increased after exposure of smooth muscle cells to inflammatory factors.31–33 In the present studies, ALP activity was documented only in areas of active calcification and in areas rich in chondrocyte-like cells and suggests that increases in ALP activity plays an active role in the deposition of hydroxyapaptite within the apoE–/– mouse lesions.

Matrix vesicles are believed to play a crucial role in initiating the process of mineralization by enabling the formation of the seed crystals of hydroxyapatite within the lumen of the vesicles.34 The presence of matrix vesicles associated with the chondrocyte-like cells in the lesions of the apoE–/– mice (Figure 1) is consistent with previous observations in calcified human lesions.35 However, it is also possible that these vesicles are apoptotic bodies rather than matrix vesicles, because apoptotic bodies have also been implicated in vascular calcification.36

The source of the osteogenic cells in the advanced lesions of the apoE–/– mice is unknown. However, in vitro studies have shown that vascular smooth muscle cells can undergo a phenotypic switch characterized by the loss of expression of smooth muscle cell markers and the gain of expression of molecules characteristic of osteoblasts and chondrocytes.37 In the advanced lesions of the apoE–/– mice, the staining of -actin was limited to the media or fibrous cap regions and was not expressed by the chondrocyte-like cells. This is consistent with the possibility that smooth muscle cells within the intima are converted to chondrocyte-like cells in response to signals such as elevated levels of Pi or factors such as tumor necrosis factor-, interleukin-6, and transforming growth factor-?, which can modulate the smooth muscle cell phenotype.31–33,38 However, it is also feasible that mesenchymal or hematopoietic stem cells are recruited into the plaques and are the source of the chondrocytes. For example, calcifying vascular cells (CVCs) retain a multi-lineage potential that is analogous to mesenchymal stem cells because these cells can be induced to express markers of chondrocytes, smooth muscle cells, and marrow stromal cells.39

Like humans, the arteries of older apoE–/– mice become calcified. The reproducibility of calcification in the lesions in the innominate arteries of the older apoE–/– mouse now provides a model in which the mechanisms that mediate plaque calcification can be investigated and in which interventions that target plaque calcification can be tested. Furthermore, coupled with studies on the modulation of cellular phenotypes and the role of stem cells, this model may enable us to determine the source of osteogenic cells and to elucidate the factors and signals that recruit or induce differentiation of osteogenic cells within the setting of atherosclerosis.

    Acknowledgments

These studies were supported by National Institutes of Health grants HL-01014 (to C.M.G.), HL-72262 (to S.M.S.), and HL-076748 (to M.E.R.) and funding provided by the Fondazione Cassamarca di Treviso (to M.R).

References

Eggen DA, Strong JP, McGill HC. Coronary calcification: relationship to clinically significant coronary lesions and race, sex and topographic distribution. Circulation. 1965; 32: 948–955.

Beadenkopf WG, Assaad DS, Love BM. Calcification in the coronary arteries and its relationship to arteriosclerosis and myocardial infarction. JAMA. 1964; 92: 865–871.

Abedin M, Tintut Y, Demer LL. Vascular calcification: mechanisms and clinical ramifications. Arterioscler Thromb Vasc Biol. 2004; 24: 1161–1170.

Speer MY, Giachelli CM. Regulation of cardiovascular calcification. Cardiovasc Pathol. 2004; 13: 63–70.

Tyson KL, Reynolds JL, McNair R, Zhang Q, Weissberg PL, Shanahan CM. Osteo/chondrocytic transcription factors and their target genes exhibit distinct patterns of expression in human arterial calcification. Arterioscler Thromb Vasc Biol. 2003; 23: 489–494.

Bostrom K, Watson KE, Horn S, Wortham C, Herman IM, Demer LL. Bone morphogenetic protein expression in human atherosclerotic lesions. J Clin Invest. 1993; 91: 1800–1809.

Dhore CR, Cleutjens JP, Lutgens E, Cleutjens KB, Geusens PP, Kitslaar PJ, Tordoir JH, Spronk HM, Vermeer C, Daemen MJ. Differential expression of bone matrix regulatory proteins in human atherosclerotic plaques. Arterioscler Thromb Vasc Biol. 2001; 21: 1998–2003.

Bini A, Mann KG, Kudryk BJ, Schoen FJ. Noncollagenous bone matrix proteins, calcification, and thrombosis in carotid artery atherosclerosis. Arterioscler Thromb Vasc Biol. 1999; 19: 1852–1861.

Giachelli CM, Bae N, Almeida M, Denhardt DT, Alpers CE, Schwartz SM. Osteopontin is elevated during neointima formation in rat arteries and is a novel component of human atherosclerotic plaques. J Clin Invest. 1993; 92: 1686–1696.

Rutsch F, Terkeltaub R. Parallels between arterial and cartilage calcification: what understanding artery calcification can teach us about chondrocalcinosis. Curr Opin Rheumatol. 2003; 15: 302–310.

Qiao JH, Mertens RB, Fishbein MC, Geller SA. Cartilaginous metaplasia in calcified diabetic peripheral vascular disease: morphologic evidence of enchondral ossification. Hum Pathol. 2003; 34: 402–407.

Qiao JH, Xie PZ, Fishbein MC, Kreuzer J, Drake TA, Demer LL, Lusis AJ. Pathology of atheromatous lesions in inbred and genetically engineered mice. Genetic determination of arterial calcification. Arterioscler Thromb. 1994; 14: 1480–1497.

Qiao JH, Fishbein MC, Demer LL, Lusis AJ. Genetic determination of cartilaginous metaplasia in mouse aorta. Arterioscler Thromb Vasc Biol. 1995; 15: 2265–2272.

Rosenfeld ME, Polinsky P, Virmani R, Kauser K, Rubanyi G, Schwartz SM. Advanced atherosclerotic lesions in the innominate artery of the ApoE knockout mouse. Arterioscler Thromb Vasc Biol. 2000; 20: 2587–2592.

Strom A, Ahlqvist E, Franzen A, Heinegard D, Hultgardh-Nilsson A. Extracellular matrix components in atherosclerotic arteries of ApoE/LDL receptor deficient mice: an immunohistochemical study. Histol Histopathol. 2004; 19: 337–347.

Seo HS, Lombardi DM, Polinsky P, Powell-Braxton L, Bunting S, Schwartz SM, Rosenfeld ME. Peripheral vascular stenosis in apolipoprotein E-deficient mice. Potential roles of lipid deposition, medial atrophy, and adventitial inflammation. Arterioscler Thromb Vasc Biol. 1997; 17: 3593–3601.

Bea F, Blessing E, Bennett B, Levitz M, Wallace EP, Rosenfeld ME. Simvastatin promotes atherosclerotic plaque stability in apoE-deficient mice independently of lipid lowering. Arterioscler Thromb Vasc Biol. 2002; 22: 1832–1837.

Jono S, Ikari Y, Shioi A, Mori K, Miki T, Hara K, Nishizawa Y. Serum osteoprotegerin levels are associated with the presence and severity of coronary artery disease. Circulation. 2002; 106: 1192–1194.

Nitta K, Akiba T, Uchida K, Otsubo S, Takei T, Yumura W, Kabaya T, Nihei H. Serum osteoprotegerin levels and the extent of vascular calcification in haemodialysis patients. Nephrol Dial Transplant. 2004; 19: 1886–1889.

Kiechl S, Schett G, Wenning G, Redlich K, Oberhollenzer M, Mayr A, Santer P, Smolen J, Poewe W, Willeit J. Osteoprotegerin is a risk factor for progressive atherosclerosis and cardiovascular disease. Circulation. 2004; 109: 2175–2180.

Bucay N, Sarosi I, Dunstan CR, Morony S, Tarpley J, Capparelli C, Scully S, Tan HL, Xu W, Lacey DL, Boyle WJ, Simonet WS. osteoprotegerin-deficient mice develop early onset osteoporosis and arterial calcification. Genes Dev. 1998; 12: 1260–1268.

Giachelli CM, Steitz S. Osteopontin: a versatile regulator of inflammation and biomineralization. Matrix Biol. 2000; 19: 615–622.

O’Brien ER, Garvin MR, Stewart DK, Hinohara T, Simpson JB, Schwartz SM, Giachelli CM. Osteopontin is synthesized by macrophage, smooth muscle, and endothelial cells in primary and restenotic human coronary atherosclerotic plaques. Arterioscler Thromb. 1994; 14: 1648–1656.

Speer MY, McKee MD, Guldberg RE, Liaw L, Yang HY, Tung E, Karsenty G, Giachelli CM. Inactivation of the osteopontin gene enhances vascular calcification of matrix Gla protein-deficient mice: evidence for osteopontin as an inducible inhibitor of vascular calcification in vivo. J Exp Med. 2002; 196: 1047–1055.

Matsui Y, Rittling SR, Okamoto H, Inobe M, Jia N, Shimizu T, Akino M, Sugawara T, Morimoto J, Kimura C, Kon S, Denhardt D, Kitabatake A, Uede T. Osteopontin deficiency attenuates atherosclerosis in female apolipoprotein e-deficient mice. Arterioscler Thromb Vasc Biol. 2003; 23: 1029–1034.

Jono S, Peinado C, Giachelli CM. Phosphorylation of osteopontin is required for inhibition of vascular smooth muscle cell calcification. J Biol Chem. 2000; 275: 20197–20203.

Wada T, McKee MD, Steitz S, Giachelli CM. Calcification of vascular smooth muscle cell cultures: inhibition by osteopontin. Circ Res. 1999; 84: 166–178.

Karaplis AC, Luz A, Glowacki J, Bronson RT, Tybulewicz VL, Kronenberg HM, Mulligan RC. Lethal skeletal dysplasia from targeted disruption of the parathyroid hormone-related peptide gene. Genes Dev. 1994; 8: 277–289.

Ishikawa M, Akishita M, Kozaki K, Toba K, Namiki A, Yamaguchi T, Orimo H, Ouchi Y. Expression of parathyroid hormone-related protein in human and experimental atherosclerotic lesions: functional role in arterial intimal thickening. Atherosclerosis. 2000; 152: 97–105.

Anderson HC, Sipe JB, Hessle L, Dhanyamraju R, Atti E, Camacho NP, Millan JL. Impaired calcification around matrix vesicles of growth plate and bone in alkaline phosphatase-deficient mice. Am J Pathol. 2004; 164: 841–847.

Parhami F, Basseri B, Hwang J, Tintut Y, Demer LL. High-density lipoprotein regulates calcification of vascular cells. Circ Res. 2002; 91: 570–576.

Proudfoot D, Davies JD, Skepper JN, Weissberg PL, Shanahan CM. Acetylated low-density lipoprotein stimulates human vascular smooth muscle cell calcification by promoting osteoblastic differentiation and inhibiting phagocytosis. Circulation. 2002; 106: 3044–3050.

Shioi A, Katagi M, Okuno Y, Mori K, Jono S, Koyama H, Nishizawa Y. Induction of bone-type alkaline phosphatase in human vascular smooth muscle cells: roles of tumor necrosis factor-alpha and oncostatin M derived from macrophages. Circ Res. 2002; 91: 9–16.

Anderson HC. Molecular biology of matrix vesicles. Clin Orthop. 1995; 266–280.

Hsu HH, Camacho NP. Isolation of calcifiable vesicles from human atherosclerotic aortas. Atherosclerosis. 1999; 143: 353–362.

Proudfoot D, Skepper JN, Hegyi L, Bennett MR, Shanahan CM, Weissberg PL. Apoptosis regulates human vascular calcification in vitro: evidence for initiation of vascular calcification by apoptotic bodies. Circ Res. 2000; 87: 1055–1062.

Steitz SA, Speer MY, Curinga G, Yang HY, Haynes P, Aebersold R, Schinke T, Karsenty G, Giachelli CM. Smooth muscle cell phenotypic transition associated with calcification: upregulation of Cbfa1 and downregulation of smooth muscle lineage markers. Circ Res. 2001; 89: 1147–1154.

Watson KE, Bostrom K, Ravindranath R, Lam T, Norton B, Demer LL. TGF-beta 1 and 25-hydroxycholesterol stimulate osteoblast-like vascular cells to calcify. J Clin Invest. 1994; 93: 2106–2113.

Tintut Y, Alfonso Z, Saini T, Radcliff K, Watson K, Bostrom K, Demer LL. Multilineage potential of cells from the artery wall. Circulation. 2003; 108: 2505–2510.

 


 

日期:2007年5月18日 - 来自[2005年第25卷第7期]栏目
循环ads

Clinical Significance of Coronary Calcification

Department of Cardiology, University Clinic Essen, Germany

To the Editor:

We read with interest the review by Abedin et al, who provide valuable insights into the mechanisms of vascular calcification and teleological concepts.1 However, regarding the clinical ramifications, they make diverse statements. We agree that, in patients selected by knowledgeable physicians, coronary artery calcification can be used "... for identifying patients at risk for adverse cardiac events." This position has been adopted in American and European guidelines.2,3 However, only 2 paragraphs below, the authors state that "a consensus has developed that coronary calcification is associated with chronic symptomatic coronary artery disease rather than with acute coronary events... " and that "these findings have been interpreted as evidence that vascular calcification is protective against acute events." We believe these statements are misleading. They are based on perceptions from cross-sectional data comparing patients who present with acute coronary syndromes and chronic stable angina pectoris. In many patients, the acute coronary syndrome is the first manifestation of coronary artery disease,4 whereas many patients with chronic disease have a long-standing diagnosis. When comparing findings in unstable and stable patients, the different medical history and time course of the disease needs to be taken into account. Intravascular ultrasound studies have produced contradictory results and have in part observed that calcification appeared neutral or even associated with acute coronary events.5,6

A series of histopathologic reports have been published by the Armed Forces Institute of Pathology group that characterize the relationship between plaque rupture and calcification in some detail.7–10 These reports demonstrate that calcification is a frequent feature of plaque rupture in victims of sudden coronary death, even in young adults. Among all types of histologically defined types of plaques, acute ruptures were calcified most frequently (80%), whereas healed ruptures were calcified most extensively.8 Plaque erosions, on the other hand, were associated with little calcium.7 Calcification was found preferentially in plaques with expansive ("positive") arterial remodeling,10 known to be associated with an increased risk of rupture.11

Most importantly, a number of prospective studies have consistently reported that the degree of coronary calcification is predictive of hard coronary events (for review, see Rumberger12). Abedin et al quote the only study that has ever reported a negative finding in that coronary calcification was not better than risk factors and ECG in predicting hard coronary events.13 However, follow-up reports from the same study have reported superior predictive ability of the coronary calcium score,14,15 in line with 4 large independent studies (see Rumberger12 for review) and 2 preliminary reports from the truly unselected general population.16,17 Measured in the clinical setting, coronary calcification clearly does not indicate protection but rather a relevant increase in coronary risk. It is our task as physicians to reduce that risk.

References

Abedin M, Tintut Y, Demer LL. Vascular Calcification. Mechanisms and Clinical Ramifications. Arterioscler Thromb Vasc Biol. 2004; 24: 1161–1170.

Third Report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III) Final Report. Circulation. 2002; 106: 3143–3421.

De Backer G, Ambrosioni E, Borch-Johnsen K, Brotons C, Cifkova R, Dallongeville J, Ebrahim S, Faergeman O, Graham I, Mancia G, Manger Cats V, Orth-Gomer K, Perk J, Pyorala K, Rodicio JL, Sans S, Sansoy V, Sechtem U, Silber S, Thomsen T, Wood D; Third Joint Task Force of European and Other Societies on Cardiovascular Disease Prevention in Clinical Practice. Executive Summary. European guidelines on cardiovascular disease prevention in clinical practice. Eur Heart J. 2003; 24: 1601–1610.

Am Heart Association Heart Disease and Stroke Statistics: 2004 Update. Available at: http://www.americanheart.org/downloadable/heart/1079736729696HDSStats2004UpdateREV3–19-04.pdf. Accessed July 19, 2004.

de Feyter PJ, Ozaki Y, Baptista J, Escaned J, Di Mario C, de Jaegere PP, Serruys PW, Roelandt JR. Ischemia-related lesion characteristics in patients with stable or unstable angina. A study with intracoronary angioscopy and ultrasound. Circulation. 1995; 92: 1408–1413.

Abizaid AS, Mintz GS, Abizaid A, Mehran R, Lansky AJ, Pichard AD, Satler LF, Wu H, Kent KM, Leon MB. One-year follow-up after intravascular-ultrasound assessment of moderate left main coronary artery disease in patients with ambiguous angiograms. J Am Coll Cardiol. 1999; 34: 707–715.

Farb A, Burke AP, Tang AL, Liang TY, Mannan P, Smialek J, Virmani R. Coronary plaque erosion without rupture into a lipid core. A frequent cause of coronary thrombosis in sudden coronary death. Circulation. 1996; 93: 1354–1364.

Burke AP, Taylor A, Farb A, Malcom GT, Virmani R. Coronary calcification: insights from sudden coronary death victims. Z Kardiol. 2000; 89 Suppl 2: 49–53.

Burke AP, Kolodgie FD, Farb A, Weber DK, Malcom GT, Smialek J, Virmani R. Healed plaque ruptures and sudden coronary death: evidence that subclinical rupture has a role in plaque progression. Circulation. 2001; 103: 934–940.

Burke AP, Kolodgie FD, Farb A, Weber D, Virmani R. Morphological predictors of arterial remodeling in coronary atherosclerosis. Circulation. 2002; 105: 297–303.

Ward MR, Pasterkamp G, Yeung AC, Borst C. Arterial remodeling. Mechanisms and clinical implications. Circulation. 2000; 102: 1186–1191.

Rumberger JA. Clinical use of coronary calcium scanning with computed tomography. Cardiol Clin. 2003; 21: 535–547.

Detrano RC, Wong ND, Doherty TM, Shavelle RM, Tang W, Ginzton LE, Budoff MJ, Narahara KA. Coronary calcium does not accurately predict near-term future coronary events in high-risk adults . Circulation. 1999; 99: 2633–2638.

Park R, Detrano R, Xiang M, Fu P, Ibrahim Y, LaBree L, Azen S. Combined use of computed tomography coronary calcium scores and C-reactive protein levels in predicting cardiovascular events in nondiabetic individuals. Circulation. 2002; 106: 2073–2077.

Greenland P, LaBree L, Azen SP, Doherty TM, Detrano RC. Coronary artery calcium score combined with Framingham score for risk prediction in asymptomatic individuals. JAMA. 2004; 291: 210–215.

Arad Y, Roth M, Newstein D, Guerci A. Coronary calcification, coronary disease risk factors, and atherosclerotic cardiovascular disease events: the St. Francis Heart Study. Hotline Session, ACC 2003.

Vliegenthart R. Coronary Calcification and the Risk of Cardiovascular Disease. An Epidemiologic Study. . Netherlands: Thoraxcentre Rotterdam; 2003.

Clinical Significance of Vascular Calcification

Moeen Abedin; Yin Tintut; Linda L. Demer

Departments of Medicine, Physiology, and Biomedical Engineering, University of California, Los Angeles

In response:

We were pleased to read the letter from Drs Schmermund and Erbel concerning our review on vascular calcification. In their letter, they point out the strong evidence that vascular calcification increases the risk of acute coronary events. We agree entirely with their view. Our reference to the concept that calcification is protective was merely intended to preface our analysis dispelling the notion. Our theoretical analysis showed that, as total calcification burden increases, the hard-soft interface area (hence rupture risk) peaks at intermediate levels of calcification due to coalescence. With progression, the interface area may decline but remains higher than in noncalcified plaque. Thus, both clinical and theoretical evidence support the concept that vascular calcification increases risk. We regret that our article was not clear on this point, and we greatly appreciate the important clarification provided by Drs Schmermund and Erbel.

 

日期:2007年5月18日 - 来自[2004年第24卷第10期]栏目
共 2 页,当前第 1 页 9 1 2 :

ads

关闭

网站地图 | RSS订阅 | 图文 | 版权说明 | 友情链接
Copyright © 2008 39kf.com All rights reserved. 医源世界 版权所有
医源世界所刊载之内容一般仅用于教育目的。您从医源世界获取的信息不得直接用于诊断、治疗疾病或应对您的健康问题。如果您怀疑自己有健康问题,请直接咨询您的保健医生。医源世界、作者、编辑都将不负任何责任和义务。
本站内容来源于网络,转载仅为传播信息促进医药行业发展,如果我们的行为侵犯了您的权益,请及时与我们联系我们将在收到通知后妥善处理该部分内容
联系Email: