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Microarray Analysis Reveals Overexpression of CD163 and HO-1 in Symptomatic Carotid Plaques

【摘要】  Objective- We studied by microarray analysis whether symptomatic and asymptomatic carotid plaques from the same patient differ in gene expression and whether the same changes are present in an independent sample set.

Methods and Results- Carotid plaques from four patients with bilateral high-grade stenosis, one being symptomatic and the other asymptomatic, were analyzed on Affymetrix U95Av2 arrays. 1.5-fold change between symptomatic and asymptomatic plaques in an intraindividual comparison with FDR ranging from 0.28 to 0.40. Three genes involved in iron-heme homeostasis, CD163, HO-1, and transferrin receptor, were further analyzed in 40 independent plaques. HO-1 (fold-change 1.93, 95%CI 1.04 to 3.94, P =0.040) and CD163 (1.58, 1.11 to 2.40, P =0.013) mRNAs were again induced, and also HO-1 protein was overexpressed in symptomatic plaques (4.38, 1.54 to 12.20, P =0.024). The expression of HO-1 and CD163 correlated with tissue iron content but iron itself was not associated with the symptom status.

Conclusions- Symptomatic plaques show overexpression of CD163 and HO-1 both in intraindividual and interindividual comparison. Their expression correlates with iron deposits but asymptomatic and symptomatic plaques from isolated patients do not differ in macroscopic hemorrhages or iron deposits. We suggest that symptomatic plaques show a more pronounced induction of CD163 and HO-1 in response to plaque hemorrhages.

We showed that HO-1 and CD163 were overexpressed in symptomatic carotid plaques. Their expression correlated with iron deposition but iron itself was not associated with symptom status. Symptomatic plaques might show a more pronounced induction of CD163 and HO-1 in response to plaque hemorrhages.

【关键词】  atherosclerosis carotid arteries gene expression microarray stroke


Introduction


Severe atherosclerotic narrowing of the internal carotid artery was found in 20% to 30% of patients with ischemic stroke in its supply territory. 1 Features associated with a symptomatic plaque include the degree of vessel stenosis, prior symptoms, and plaque characteristics, such as ulceration, inflammatory cell infiltration, and a thin fibrous cap. 2-4 However, these characteristics are poor predictors of the risk of thromboembolism and, as a result, 80% of patients undergoing carotid endarterectomy are needlessly exposed to surgical risks. 1 Thus better markers for the symptom causing carotid disease are needed. See cover


Microarray technology provides a rapid means to screen gene expression in the tissues of interest. Several efforts have been made to study large-scale gene expression in human atherosclerosis, for example by comparing gene expression in normal and atherosclerotic arteries. 5,6 Changes involved in destabilization of the atherosclerotic plaque have been less in focus. 7-9


Whereas symptomatic high-grade carotid plaque remains highly susceptible to recurrent ipsilateral symptoms, the risk of stroke from contralateral asymptomatic plaque is low, comparable to that of asymptomatic carotid stenosis in general. 10 Thus intraindividual differences in the carotid stenoses exist in the same patient causing one plaque to become symptomatic and the other to remain silent. The causes of these differences are unknown. This led us to a microarray study on patients operated due to bilateral significant carotid stenosis, of which one is symptomatic and the other asymptomatic. These rare cases offer a valuable natural experiment, in which the symptom-causing unstable plaque can be compared with the stable one from the same patient, minimizing noise caused by interindividual differences in gene expression and giving insight into plaque-specific characteristics important in the destabilization. One main gene expression profile that appeared differentially regulated between symptomatic and asymptomatic plaques was genes involved in the metabolism of iron and heme. Three of these, CD163, HO-1, and transferrin receptor (TRFC), were further analyzed at mRNA and protein level in 40 independent carotid plaques.


Materials and Methods


Patients


Patients were selected from a larger study population, the He lsinki C arotid E ndarterectomy S tudy, HeCES, described elsewhere in detail. 11-13 Briefly, the study included 97 consecutive patients subjected to endarterectomy because of high-grade carotid stenosis. Division into asymptomatic or symptomatic plaque phenotypes was based on prior clinical symptoms; carotid stenosis was considered symptomatic if a patient had suffered an ipsilateral stroke, transient ischemic attack of the carotid territory, or amaurosis fugax within 120 days before endarterectomy. All the patients gave informed consent, and the appropriate departmental Ethics Committees of Helsinki University Central Hospital approved the study protocol.


The present microarray study includes the bilateral cases, ie, the patients (n=4) who had significant carotid stenosis operated bilaterally, one side being asymptomatic and the other symptomatic. Three patients had an acute infarction in the supply territory of the symptomatic carotid artery and the fourth patient had a clinical ischemic stroke (left hemiparesis). None of the patients had ever experienced symptoms from the contralateral, asymptomatic side, and the brain imaging of the contralateral hemisphere was normal. The characteristics of the bilateral cases are detailed in supplemental Table I (available online at http://atvb.ahajournals.org).


For follow-up study by quantitative real-time RT-PCR we included carotid plaques from all HeCES patients with ipsilateral stroke symptoms (symptomatic specimen, n=22) and all patients without cerebrovascular symptoms (asymptomatic specimen, n=18), who in contrast to bilateral patients underwent endarterectomy for only one carotid artery stenosis, either symptomatic or asymptomatic. From these, carotid specimen from patients with radiologically confirmed ipsilateral ischemic stroke (n=13) and symptom-free patients with normal brain imaging (n=9) were used for the Western blotting analysis and immunohistochemistry. Clinical characteristics of all 40 patients are given in the Table.


Characteristics of Patients and Carotid Plaques Used For The Follow-up Study


Tissue Sampling


Carotid plaques were removed en bloc in longitudinal endarterectomy, drained with saline and graded macroscopically for smoothness, ulceration, hemorrhage, loose atheroma, calcification, and intramural thrombus by the same vascular surgeon (ES, Table 1 and supplemental Table I ). All plaques were complicated lesions, ie, the AHA-class VI. 14 Specimen were divided into longitudinal slices used each for specific purposes such as RNA-extraction, biochemical analyses, detection of infectious agents, standard histological examination, and immunohistochemical stainings.


RNA Extraction and Microarray Analysis


Total cellular RNA was extracted from each specimen with Trizol-reagent (Invitrogen Life Technologies) and purified with the RNeasy Total RNA Isolation Kit (Qiagen) according to the manufacturers? recommendations.


Microarray experiments were performed using Affymetrix GeneChip U95Av2 arrays according to the manufacturer?s recommendations and the MIAME guidelines. 15 Detailed descriptions of all data and protocols were submitted to a public repository, ArrayExpress (http://www.ebi.ac.uk/miamexpress/login.htm, the accession number: E-MEXP-268). Each RNA sample was hybridized to its own microarray resulting in eight arrays from four patients. Hybridization data were analyzed using the BioC 1.8 Release of the Bioconductor packages. 16 First, signal intensities were calculated and hybridization data normalized across arrays using Robust Multi-array Average method. 17 Genes expressed at a reliable level and showing differential expression were identified by filtering, using the following 100 in either plaque from three patients and (2) 1.5-fold difference in the mean signal between symptomatic and asymptomatic plaques. Differential expression was tested by paired t test and Benjamin-Hoechberg multiple testing correction was applied to obtain the false discovery rates. Hierarchical clustering was performed using the GeneSpring v. 7.1 software (Silicon Genetics) and gene-class testing for functional pathways and Gene Ontology (GO) categories by using DAVID software applying gene enrichment analysis. 18


Quantitative Real-Time RT-PCR for CD163, HO-1, and TRFC


Quantitative real-time RT-PCR was performed using Assays-on-Demand Gene Expression Products and ABI PRISM 7000 Sequence Detection System (Applied Biosystems) according to the manufacturer?s recommendations. Gene expression was determined by the comparative Ct method, normalizing expression to ß-actin.


Protein Isolation and Western Blotting


Total cellular proteins were isolated from the phenol-chloroform phase left over from the RNA extraction with Trizol-reagent (Invitrogen Life Technologies). Proteins were quantitated by the Bradford method and 10 µg was separated on 10% SDS-polyacrylamide gel electrophoresis followed by electroblotting to PVFD membranes (Hybond P, Amersham Biosciences). Membranes were blocked in 5% skimmed milk and 0.1% Tween 20 in tris-buffered saline. HO-1 was detected using rabbit polyclonal antibody (Stressgen, 1:2000) and ß-actin using mouse monoclonal antibody (Sigma-Aldrich, 1:4800). Secondary antibodies were peroxidase-conjugated goat anti-rabbit IgG and anti-mouse IgG (Molecular Probes). Proteins were visualized using ECL Plus Western Blotting Detection Reagents and Typhoon Variable Mode Imager according to manufacturer?s recommendations, and HO-1 and ß-actin bands were quantitated using ImageQuant TL 1D Gel Analysis v2003.1 software (all from Amersham Biosciences). The amount of HO-1 protein is given as the volume of HO-1 band divided by the volume of ß-actin band.


Immunohistochemistry and Microscopy


Tissue slices embedded in paraffin were cut longitudinally into 5-µm sections, hence containing the region of highest stenosis. Sections were immunostained with primary antibodies: rabbit polyclonal antibody against HO-1 (Stressgen, 1:1500) and mouse monoclonal antibody against CD163 (Novocastra, 10D6, 1:100). Ferric iron was detected by Perls? Prussian blue staining. One investigator (KN), blinded to the data, performed light microscopy (Axioplan 2, Carl Zeiss) and scored semiquantitatively the quantity of protein expression or iron staining in the whole section (0=none, 1=weak, 2=moderate, 3=strong).


Statistical Analysis


Statistical analyses, apart from microarray analyses, were performed using SPSS 10.0.7 for Windows (SPSS Inc). Pearson chi-square or Fisher exact test was used to evaluate the differences in noncontinuous and the independent-samples t test for continuous clinical characteristics shown in Table 1. Results from quantitative real-time RT-PCR and Western blotting were evaluated by independent-samples t test ( Figures 1 to 3 ). Differences in protein and iron levels determined by immunohistochemistry were tested by the Mann-Whitney U test ( Figure 3 ). Correlations were analyzed by the Spearman rank correlation. Results are given as an expression ratio and 95% confidence interval (CI). A two-tailed P <0.05 was considered significant.


Figure 1. Gene expression of CD163, HO-1, and TRFC in asymptomatic and symptomatic plaques determined by quantitative real-time RT-PCR. Bars represent means±SEM. Asterisks indicate P <0.05.


Figure 2. Quantitation of HO-1 protein in asymptomatic and symptomatic plaques by Western blotting (A). Bars represent means±SEM. Asterisks indicate P <0.05. A representative immunoblot is shown in B.


Figure 3. Relationships between CD163 and HO-1 mRNA/protein expression and iron deposits in carotid plaques. B, Results from HO-1 quantitation by Western blotting were transformed to rank orders for the purpose of presentation together with immunohistochemistry results. D, Results from the immunohistochemical quantitation of CD163 and HO-1. Bars represent means±SEM. Spearman correlation coefficients and corresponding probability values are given below each graph. IHC indicates immunohistochemistry; WB, western blotting.


Results


Microarray Analysis in Patients With Bilateral Carotid Stenoses


Forty probes (33 genes) showed more than 1.5-fold change in expression in comparison between asymptomatic and symptomatic plaques (summarized in supplemental Table II). The expression of lysyl oxidase-like 1, was significantly different between symptomatic and asymptomatic samples and the expression of transferring receptor was borderline significant. However, if multiple testing corrections were applied, the false discovery rates were between 0.28 and 0.40, ie, the expected percent of false predictions in these 40 probes was 11 to 16.


The list of differentially regulated genes showed significant enrichment ( P <0.05) of 19 GO terms (please see http://atvb.ahajournals.org) with two main motifs: (1) a large immunoglobulin gene cluster down-regulated in the symptomatic samples and (2) a protease gene cluster (cathepsin L, matrix metalloproteinase 7, 9, and 12) upregulated in the symptomatic plaques. There was no enrichment in pathways in the publicly available repositories (KEGG, Biocharta) but one of the most prominent functional clusters was genes involved in the tissue homeostasis of iron and heme, namely HO-1, hemoglobin scavenger receptor CD163, hemoglobins beta and gamma, and transferrin receptor (TFRC). Therefore we decided to pursue further the expression of genes known to be induced in tissue response to hemorrhages, namely CD163, HO-1, and TFRC, in an independent set of 40 carotid plaques.


Overexpression of CD163 and HO-1 mRNAs in an Independent Set of Carotid Plaques


CD163, HO-1, and TRFC were analyzed by quantitative real-time RT-PCR in the bilateral plaques as well as in the larger set of 40 carotid plaques. Microarray and quantitative real-time RT-PCR results of bilateral plaques showed a good correlation (data not shown). When analyzed in the larger set of carotid plaques, the mRNA expression of both CD163 and HO-1 was significantly higher in symptomatic compared with asymptomatic plaques, revealing 1.58-fold induction for CD163 (95% CI 1.11 to 2.40, P =0.013) and 1.93-fold induction for HO-1 (95% CI 1.04 to 3.94, P =0.040) ( Figure 1 ). CD163 and HO-1 mRNA expressions were positively correlated with each other (r s =0.72, P <0.001). TRFC also showed a tendency toward higher expression in symptomatic than in asymptomatic plaques but this was not statistically significant. TRFC expression correlated with CD163 (r s =0.62, P <0.001) but not with HO-1 expression.


Quantitation of HO-1 Protein Expression by Western Blotting


HO-1 protein was quantitated by Western blotting in the set of 22 carotid plaques (asymptomatic n=9 and symptomatic n=13). The expression of HO-1 protein relative to ß-actin was significantly higher in the symptomatic than in the asymptomatic plaques (fold-change 4.38, 95% CI 1.54 to 12.20, P =0.024; Figure 2 ). HO-1 protein levels correlated with the HO-1 mRNA levels (r S =0.73, P <0.001, see Figure 3 B). CD163 protein was not quantitated because of incompatibilities related to the protein extraction method and available antibodies.


Immunohistochemistry of HO-1 and CD163


To determine the localization of CD163 and HO-1 expression, the carotid plaques from 22 cases were studied by immunohistochemistry. Representative stainings of CD163 and HO-1 are presented in Figure 4. CD163 expression was abundant in the shoulder and cap regions of the atheroma ( Figure 4A, 4D, 4G, and 4 J). Morphologically, the CD163 antibody stained a subpopulation of macrophages, which were either scattered or assembled into infiltrates ( Figure 4D, 4G, and 4 J). Two different staining patterns were observed: cytoplasmic and membranous patterns, the first being more abundant (not shown). HO-1 expression was often bordering the lipid core, mainly in macrophages but in some vascular smooth muscle cells (VSMCs) as well ( Figure 4B, 4E, 4H, and 4 K).


Figure 4. Representative staining patterns for CD163, HO-1, and iron in adjacent sections. CD163 on the left hand column (A, D, G, J), HO-1 in the middle (B, E, H, K), and Perls blue (iron) on the right (C, F, I, L). The upper row (A through C) visualizes a longitudinal cut of the whole plaque the endothelial side on top. The second row (D through F) shows in higher magnification the shoulder area of the plaque (indicated by the rectangles in panels A through C) revealing an infiltrate of inflammatory cells and some microvessels. Panels G through I show an area of disintegrated intima (luminal side on top) with inflammatory cells, lipids, cholesterol crystals, and a few red blood cells. In panels G through I, the rectangles indicate the areas enlarged in panels J through L.


Protein staining of CD163 and HO-1 was scored semiquantitatively. The amount of HO-1 and CD163 staining were correlated with each other (r s =0.61, P =0.002), and also partially colocalized ( Figure 4; compare the left and middle columns). Both CD163 and HO-1 tended to be expressed more intensively in symptomatic specimen in correlation with the corresponding mRNA levels ( Figure 3A and 3 B).


Correlations of CD163 and HO-1 Expression to Clinical and Macroscopic Plaque Characteristics


CD163 and HO-1 mRNA and protein levels showed association to morphological features of symptom-causing carotid disease, namely the degree of carotid stenosis (CD163 mRNA: r S =0.60, P =0.004; CD163 protein (immunohistochemistry) r S =0.42, P =0.042; HO-1 mRNA: r S =0.59, P =0.005; HO-1 protein(Western blotting): r S =0.37, P =0.098) and plaque ulceration (CD163 mRNA: r S =0.70, P =0.001; HO-1 mRNA: r S =0.47, P =0.035; HO-1 protein: r S =0.49, P =0.028). Both had a tendency to associate directly to total serum total cholesterol and LDL levels and indirectly to serum HDL and sensitized CRP.


Staining of Iron Deposits


Asymptomatic and symptomatic plaques did not differ in the frequency of macroscopic hemorrhages (data not shown) but HO-1 and CD163 mRNA as well as HO-1 protein level were correlated to plaque hemorrhages (CD163 mRNA: r S =0.44, P =0.005, HO-1 mRNA: r S =0.54, P <0.001, HO-1 protein: r S =0.52, P =0.016). To reveal prior microhemorrhages, iron deposits were stained ( Figure 4C, 4F, 4I and 4 L) with two emerging patterns: both extracellular and cytoplasmic granular staining ( Figure 4; panels I and L) and, on the other hand, large confluent homogeneously stained areas (not shown). Iron deposits were present in several locations, eg, near the luminal surface, around the lipid core and in the intima-media, but, interestingly, not always in the areas of past rupture or erosion. With respect to the bilateral cases, each symptomatic plaque contained more iron than the asymptomatic plaque from the same patient. However, the 22 independent specimen showed no differences in the amount of iron between symptomatic and asymptomatic plaques. Iron deposits correlated significantly with CD163 and HO-1 expression both at mRNA (CD163: r s =0.47, P =0.029, HO-1: r s =0.73, P <0.001) and protein levels (CD163 immunohistochemistry: r s =0.54, P =0.006, HO-1 immunohistochemistry: r s =0.54, P =0.02, HO-1 Western blotting: r S =0.71, P <0.001) (see Figure 3C and 3 D). Although iron deposits were mainly accompanied by CD163 and HO-1 expression ( Figure 4 G through 4L), not all the CD163- or HO-1-positive areas contained iron ( Figure 4A through 4 F).


Discussion


The present study started from a large-scale microarray analysis in four bilateral cases of carotid stenoses to screen genes that are differentially expressed between asymptomatic and symptomatic plaques within the same individual. One of the largest functional clusters included genes involved in the homeostasis of iron and heme and we could show that two of these, HO-1 and CD163, were induced both at the mRNA and protein level also in a larger set of carotid plaques (n=40). In line, their expression correlated with traditional markers of unstable carotid disease, the degree of carotid stenosis and plaque ulcerations. Finally, we could show that both mRNA and protein levels of CD163 and HO-1 correlated strongly with iron deposits.


To our knowledge this is the first report comparing gene expression between symptomatic and asymptomatic carotid plaques from the same bilaterally operated patients, which are very rare. Our cohort of 98 patients including all consecutive patients admitted to our hospital for carotid endarterectomy during 1995 to 2000 had only four such patients. Consequently, the microarray study has not enough power to provide significant results at the genome-wide level without independent replication, for which we used a larger set of carotid specimen from isolated patients. The two sample sets and their analysis differ in some respects. In the microarray analysis of the bilateral cases the genetic background is identical and thus "the unique environment" of each plaque, which might be, eg, distinct anatomy, infections, hemorrhages, must underlie the differences in the expression profiles. In the case of the replication material, where we compared the groups of isolated asymptomatic and symptomatic plaques, similar kind of factors should also play a role but individual differences (eg, genetic factors, comorbidities, smoking, medication) may confound the effect. Thus even though replication in isolated single endarterectomies can be criticized it can also be used to confirm expression profiles that have such a large impact on plaque stability that their effect can also be detected at the population level.


Intuitively, the induction of CD163 and HO-1 would fit with more frequent intraplaque hemorrhages in the symptomatic plaques. This proved true in the bilateral cases where each symptomatic plaque contained more iron in comparison to the asymptomatic one from the same patient. This suggests that intraplaque hemorrhages might be one important incidental factor causing plaque destabilization. However, considering the whole material the situation was more complex since we did not find differences in macroscopic hemorrhages or long-term iron deposition between asymptomatic and symptomatic plaques. This might also explain why the differential expression of TRFC seen in the bilateral cases did not recur in the isolated cases; the expression of TRFC is more directly regulated by iron through the iron-responsive elements. 19


Our findings are in line with previous pathological studies showing that intraplaque hemorrhages are correlated with the degree of carotid stenosis rather than with symptom-producing plaques. 3 Because symptomatic plaques did not show more iron deposition but showed higher expression of HO-1, this could imply that a stronger activation response to microhemorrhages and free iron takes place in symptomatic plaques, eg, attributable to differences in cellular composition or genetic background. In fact, regarding endothelial cells it has been shown that atherosclerosis-susceptible mouse strains demonstrate higher induction of HO-1 than atherosclerosis-resistant strains in response to modified LDL. 20 We suggest that intraplaque hemorrhages have a deleterious effect on plaque stability, but this is strongly modulated by individual environmental and genetic factors. This might explain why pathological studies on the role of hemorrhages in plaque destabilization have remained inconclusive.


Protein staining of CD163 and HO-1 was strongest in areas of active inflammation characterized by various inflammatory cells, foam cells, extracellular lipids, cholesterol crystals, and red blood cells. In these areas, their expression also mainly colocalized ( Figure 4 ), although not always in the same cells. CD163 is specifically expressed by monocyte-macrophage lineage and is commonly used as a macrophage marker. It is also regarded to mark "alternatively activated macrophages" that have a special regulatory role in immune responses. 21 It could be argued that overexpression of CD163 in symptomatic plaques reflects differences in the cellular composition between plaques. We have previously studied macrophage density in this same set of carotid plaques using HAM56-staining 12 and did not find increased density in symptomatic plaques. Still, it is possible that overexpression of CD163 indicates over-representation of a certain macrophage subtype in symptomatic plaques. HO-1, on the other hand, stained macrophages that had endocytosed red blood cells or hemosiderin and were surrounded by cholesterol crystals, which is in line with the results by others. 22,23


Especially HO-1 is regarded as an antiatherogenic agent during the early phases of atherosclerosis, 24 and it is possible that the induction of HO-1 and CD163 purely reflects immune activation in response to intraplaque hemorrhages without being destabilizing phenomena per se. Yet, both HO-1 and CD163 have characteristics that might paradoxically turn harmful in advanced atheromas. HO-1 is known to prevent proliferation of VSMCs and endothelial cells after vascular injury, 25 which can be important to sustain plaque integrity and stability. CD163, again, is linked to an increased risk of vascular complications via its different efficiencies to remove hemoglobin, depending on haptoglobin genotypes. 26 Only one study has previously compared HO-1 or CD163 expression in asymptomatic and symptomatic atherosclerotic plaques. 27 Ameriso and colleagues investigated HO-1 expression by immunohistochemistry in relation to Helicobacter pylori infection and concluded that HO-1 expression was more frequent in infected and asymptomatic carotid plaques. 27 Unfortunately, many methodological differences, eg, in the patient selection and immunohistochemical methods, hinder direct comparison between their study and the present one.


It could be claimed that the induction of CD163 and HO-1 occurred "post-hoc" in response to thromboembolic events and associated surface bleeding into the plaque. We cannot entirely preclude this possibility, but several facts argue against it. First, asymptomatic and symptomatic plaques did not show differences in macroscopic hemorrhages or iron deposits, which suggests that symptomatic plaques have not suffered more abundant or more recent hemorrhages than asymptomatic ones. The majority of the HO-1 and CD163 expression occurred in the deeper layers of the plaques ( Figure 4 ) rather than around the surface ulcerations/hemorrhages. Second, no correlation was found between CD163/HO-1 expression and time after onset of symptoms. Thus we suggest that the coinduction of CD163 and HO-1 does not merely reflect a surface hemorrhage associated with the latest thromboembolism but a more chronic process throughout the evolution of symptomatic plaques that could mark an increased risk for plaque destabilization and thromboembolic symptoms.


In conclusion, our study revealed several genes that are potentially important in the destabilization of atherosclerotic plaques and could all represent novel targets for development of plaque-stabilizing drugs. Especially, CD163 and HO-1 involved in the degradation of hemoglobin after intraplaque hemorrhage were induced in symptomatic plaques, warranting further studies on these signaling cascades in unstable atherosclerotic plaques.


Acknowledgments


Nuno Raimundo is thanked for technical advice and Tanja Eriksson, Saija Eirola and Riitta Kärkkäinen for technical assistance.


Sources of Funding


The study was funded by Helsinki University Central Hospital research grants, Academy of Finland, and Sigrid Juselius, Lundbeck, Aarne Koskelo, and Päivikki and Sakari Sohlberg Foundations. Wihuri Research Institute is maintained by Jenny and Antti Wihuri Foundation.


Disclosures


None.

【参考文献】
  Kappelle LJ. Symptomatic carotid artery stenosis. J Neurol. 2002; 249: 254-259.

Beneficial effect of carotid endarterectomy in symptomatic patients with high-grade carotid stenosis. North American Symptomatic Carotid Endarterectomy Trial Collaborators. N Engl J Med. 1991; 325: 445-453.

Golledge J, Greenhalgh RM, Davies AH. The symptomatic carotid plaque. Stroke. 2000; 3: 774-781.

Takaya N, Yuan C, Chu B, Saam T, Underhill H, Cai J, Tran N, Polissar NL, Isaac C, Ferguson MS, Garden GA, Cramer SC, Maravilla KR, Hashimoto B, Hatsukami TS. Association between carotid plaque characteristics and subsequent ischemic cerebrovascular events: a prospective assessment with MRI-initial results. Stroke. 2006; 37: 818-823.

Martinet W, Schrijvers DM, De Meyer GRY, Thielemans J, Knaapen MWM, Herman AG, Kockx MM. Gene Expression profiling of apoptosis-related genes in human atherosclerosis: upregulation of death-associated protein kinase. Arterioscler Thromb Vasc Biol. 2002; 22: 2023-2029.

McCaffrey TA, Fu C, Du B, Eksinar S, Kent KC, Bush H, Jr., Kreiger K, Rosengart T, Cybulsky MI, Silverman ES, Collins T. High-level expression of Egr-1 and Egr-1-inducible genes in mouse and human atherosclerosis. J Clin Invest. 2000; 105: 653-662.

Faber BCG, Cleutjens KBJM, Niessen RLJ, Aarts PLJW, Boon W, Greenberg AS, Kitslaar PJEHM, Tordoir JHM, Daemen MJAP. Identification of genes potentially involved in rupture of human atherosclerotic plaques. Circ Res. 2001; 89: 547-554.

Vemuganti R, Dempsey RJ. Carotid atherosclerotic plaques from symptomatic stroke patients share the molecular fingerprints to develop in a neoplastic fashion: A microarray analysis study. Neuroscience. 2005; 131: 359-374.

Papaspyridonos M, Smith A, Burnand KG, Taylor P, Padayachee S, Suckling KE, James CH, Greaves DR, Patel L. Novel Candidate Genes in unstable areas of human atherosclerotic plaques. Arterioscler Thromb Vasc Biol. 2006; 26: 1837-1844.

Inzitari D, Eliasziw M, Gates P, Sharpe BL, Chan RKT, Meldrum HE, Barnett HJM, The North Am Symptomatic Carotid Endarterectomy Trial Collaborators. The causes and risk of stroke in patients with asymptomatic internal-carotid-artery stenosis. N Engl J Med. 2000; 342: 1693-1701.

Nuotio K, Lindsberg PJ, Carpen O, Soinne L, Lehtonen-Smeds EMP, Saimanen E, Lassila R, Sairanen T, Sarna S, Salonen O, Kovanen PT, Kaste M. Adhesion molecule expression in symptomatic and asymptomatic carotid stenosis. Neurology. 2003; 60: 1890-1899.

Lehtonen-Smeds EMP, Mäyränpää M, Lindsberg PJ, Soinne L, Saimanen E, Järvinen AAJ, Salonen O, Carpen O, Lassila R, Sarna S, Kaste M, Kovanen PT. Carotid plaque mast cells associate with atherogenic serum lipids, high grade carotid stenosis and symptomatic carotid artery disease. Cerebrovasc Dis. 2005; 19: 291-301.

Soinne L, Saimanen E, Malmberg-Céder K, Kovanen P, Lindsberg PJ, Kaste M, Lassila R. Association of the fibrinolytic system and hemorheology with symptoms in patients with carotid occlusive disease. Cerebrovasc Dis. 2005; 20: 172-179.

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, Am Heart Association. Circulation. 1995; 92: 1355-1374.

Brazma A, Hingamp P, Quackenbush J, Sherlock G, Spellman P, Stoeckert C, Aach J, Ansorge W, Ball CA, Causton HC, Gaasterland T, Glenisson P, Holstege FC, Kim IF, Markowitz V, Matese JC, Parkinson H, Robinson A, Sarkans U, Schulze-Kremer S, Stewart J, Taylor R, Vilo J, Vingron M. Minimum information about a microarray experiment (MIAME)-toward standards for microarray data. Nat Genet. 2001; 29: 365-371.

Gentleman RC, Carey VJ, Bates DM, Bolstad B, Dettling M, Dudoit S, Ellis B, Gautier L, Ge Y, Gentry J, Hornik K, Hothorn T, Huber W, Iacus S, Irizarry R, Leisch F, Li C, Maechler M, Rossini AJ, Sawitzki G, Smith C, Smyth G, Tierney L, Yang JY, Zhang J. Bioconductor: open software development for computational biology and bioinformatics. Genome Biol. 2004; 5: R80.

Irizarry RA, Hobbs B, Collin F, Beazer-Barclay YD, Antonellis KJ, Scherf U, Speed TP. Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics. 2003; 4: 249-264.

Dennis G, Jr, Sherman BT, Hosack DA, Yang J, Gao W, Lane HC, Lempicki RA. DAVID: Database for Annotation, Visualization, and Integrated Discovery. Genome Biol. 2003; 4: P3.

Haile DJ. Regulation of genes of iron metabolism by the iron-response proteins. Am J Med Sci. 1999; 318: 230-240.

Shi W, Haberland ME, Jien ML, Shih DM, Lusis AJ. Endothelial responses to oxidized lipoproteins determine genetic susceptibility to atherosclerosis in mice. Circulation. 2000; 102: 75-81.

Gordon S. Alternative activation of macrophages. Nature Rev Immunol. 2003; 3: 23-35.

Kolodgie FD, Gold HK, Burke AP, Fowler DR, Kruth HS, Weber DK, Farb A, Guerrero LJ, Hayase M, Kutys R, Narula J, Finn AV, Virmani R. Intraplaque hemorrhage and progression of coronary atheroma. N Engl J Med. 2003; 349: 2316-2325.

Kockx MM, Cromheeke KM, Knaapen MW, Bosmans JM, De Meyer GR, Herman AG, Bult H. Phagocytosis and macrophage activation associated with hemorrhagic microvessels in human atherosclerosis. Arterioscler Thromb Vasc Biol. 2003; 23: 440-446.

Siow RC, Sato H, Mann GE. Heme oxygenase-carbon monoxide signalling pathway in atherosclerosis: anti-atherogenic actions of bilirubin and carbon monoxide? Cardiovasc Res. 1999; 41: 385-394.

Duckers HJ, Boehm M, True AL, Yet SF, San H, Park JL, Clinton Webb R, Lee ME, Nabel GJ, Nabel EG. Heme oxygenase-1 protects against vascular constriction and proliferation. Nat Med. 2001; 7: 693-698.

Schaer DJ. The macrophage hemoglobin scavenger receptor (CD163) as a genetically determined disease modifying pathway in atherosclerosis. Atherosclerosis. 2002; 163: 199-201.

Ameriso SF, Villamil AR, Zedda C, Parodi JC, Garrido S, Sarchi MI, Schultz M, Boczkowski J, Sevlever GE. Heme oxygenase-1 is expressed in carotid atherosclerotic plaques infected by Helicobacter pylori and is more prevalent in asymptomatic subjects. Stroke. 2005; 36: 1896-1900.


作者单位:Departments of Neurology (P.I., K.N., L.S., M.K., P.J.L.), Surgery (E.S.), Pathology (M.-L.K.-L.), and Radiology (O.S.), Helsinki University Central Hospital; the Neuroscience Program (P.I., K.N., J.S., P.J.L.), Biomedicum Helsinki; South Karelia Central Hospital (E.S.), Lappeenranta; the Department

日期:2008年12月28日 - 来自[2007年第27卷第1期]栏目

IL-20 Is Expressed in Atherosclerosis Plaques and Promotes Atherosclerosis in Apolipoprotein E-Deficient Mice

【摘要】  Objective- Atherosclerosis is a chronic inflammatory disease with immune cell infiltration. Various cytokines and chemokines have been characterized as pro- or antiatherogenic factors. Interleukin-20 (IL-20) belongs to the IL-10 family and is a proinflammatory cytokine involved in the pathogenesis of psoriasis. However, the association between IL-20 and atherosclerosis is undetermined. Therefore, we sought to investigate whether IL-20 is associated with atherosclerosis.

Methods and Results- We examined the expression of IL-20 and its receptor complex IL-20R1/IL-20R2 in atherosclerotic lesions of humans and mice using immunohistochemical staining. IL-20 was expressed in macrophage-rich areas. Both IL-20 and IL-20R1/IL-20R2 were expressed by endothelial cells lining the intimal microvessels, vasa vasorum, but rarely in nonatherosclerotic arteries. We used reverse-transcription polymerase chain reaction to analyze gene expression. IL-20 transcripts increased in hypoxic monocytes and monocytes treated with oxidized low-density lipoprotein. The expression of IL-20R1 and IL-20R2 was also upregulated by human umbilical vein endothelial cells in response to hypoxic treatment. Incubating IL-20 with human umbilical vein endothelial cells upregulated CXCL9 and CXCL11 transcripts. Furthermore, in vivo administration of IL-20 expression vector using intramuscular electroporation promoted atherosclerosis in apolipoprotein E-deficient mice.

Conclusions- Our data suggest that IL-20 is a proatherogenic cytokine that contributes to the progression of atherosclerosis.

We investigated the association between IL-20 and atherosclerosis. IL-20 and its receptors are expressed in the atherosclerosis plaques of human and mice. In vitro, oxidized LDL and hypoxia induced IL-20. In vivo, IL-20 promoted atherosclerosis in apoE -/- mice. Thus, we postulate that IL-20 is a proatherogenic cytokine.

【关键词】  atherosclerosis cytokines hypoxia IL oxidized lipids


Introduction


Atherosclerosis is a chronic inflammatory disease of the arterial wall characterized by the progressive accumulation of lipids, extracellular matrix, and cells, including macrophages, T lymphocytes, and smooth muscle cells. 1 Inflammation plays a major role in atherosclerotic plaque disruption and thrombosis; therefore, it greatly influences the occurrence of coronary syndromes and mortality. 2-4 Foam cell macrophages are generally thought to play a major role in the pathology of the disease. 2 Activated macrophages secrete cytokines and chemokines that direct and amplify the local immune response. Inflammatory stimuli include lipoproteins trapped within lesions in which protein and lipid moieties have been chemically modified. Lipid-engorged macrophages, or foam cells, comprise the major volume of the early lesions and are enriched in late-stage lesions as well. 3,5 In situ studies of human and animal atherosclerotic lesions have identified the increased expression of several chemokines, cytokines, and tissue-remodeling and lipid metabolism genes. 6 Antibodies to oxidized low-density lipoprotein (OxLDL) epitopes and increases in circulating proinflammatory cytokines correlate with vascular disease. 7 In addition, exposure to OxLDL alters the transcription response of macrophages to inflammatory stimuli. 8 These studies implicate activated macrophages and other inflammatory cells in the development and progression of atherosclerosis.


Angiogenesis is also crucial in the progression of atherosclerosis plaque formation. The clinical importance of the neovascularization of plaque has been demonstrated by a higher prevalence of neovascularization in lesions with plaque rupture, mural hemorrhage, or unstable angina. 9 Therefore, factors that stimulate plaque angiogenesis also contribute to plaque disruption, which is responsible for myocardial infarction and ischemic stroke. Angiogenesis inhibitors such as endostatin reduce intimal neovascularization and plaque growth in apolipoprotein E-deficient (apoE -/- ) mice. 10 This evidence further supports the importance of angiogenesis in the pathogenesis of atherosclerosis. See page 1929


An imbalance between the demand and supply of oxygen is another key factor for the development of atherosclerotic lesions. In vivo, zones of hypoxia occur within the atherosclerotic plaque through the mechanism of impaired oxygen diffusion capacity caused by the thickness of the lesion, together with high oxygen consumption by foam cells. 11 This observation supports the anoxemia theory of atherosclerosis. 12


IL-10, important in atherosclerotic lesion formation and stability, is a protective factor against the effect of environmental pathogens on atherosclerosis. 13 IL-10 was originally described as a cytokine-synthesis inhibitory factor because of its inhibitory effect on cytokine production. 14 Several new members of the IL-10 family, including IL-19, IL-20, IL-22, MDA-7 (IL-24), and AK155 (IL-26), have recently been discovered. 15 Overexpression of IL-20 in transgenic mice causes neonatal death as well as skin abnormalities, including aberrant epidermal differentiation. 16 IL-20 selectively enhances multipotential hematopoietic progenitors in vitro and in vivo. 17 IL-20 is preferentially expressed in monocytes 18 and induces STAT 3 activation through binding to 2 types of IL-20 receptor (R) complexes, either IL-20R1/IL-20R2 or IL-22R1/IL-20R2. 19 Our recent study 20 demonstrated that IL-20 induced endothelial cell proliferation, migration, and vascular tube formation in vitro and tumor angiogenesis in vivo. IL-20 stimulates angiogenic activity either directly or indirectly through inducing vascular endothelial growth factor (VEGF), bFGF, and IL-8 production. Angiogenesis is one of the characteristics of atherosclerosis. We therefore wanted to investigate the molecular mechanism of IL-20 to determine whether it, like IL-10, had any association with atherosclerosis. ApoE -/- mice reveal the phenotype of atherosclerosis. 21 Thus, we investigated the expression pattern of IL-20 and its receptors on atherosclerotic lesions of humans and apoE -/- mice using immunohistochemical staining. We also examined the regulation of IL-20 and its receptors under hypoxia or OxLDL stimulation and the effect of IL-20 on chemokine regulation in endothelial cells. To study the in vivo activity of IL-20, using intramuscular electroporation, we injected IL-20 expression vector into apoE -/- mice and monitored the progression of atherosclerosis plaques.


Materials and Methods


Please see http://atvb.ahajournals.org for detailed Materials and Methods


Results


Upregulation of IL-20 and Its Receptors in Atherosclerotic Lesions of apoE -/- Mice


Chronic inflammation plays a pivotal role in the progression of atherosclerosis. IL-10 exerts important protective effects against the development of atherosclerotic lesions in experimental animals. IL-20 promotes angiogenesis in vitro and in vivo. Therefore, we wanted to see whether IL-20 was also associated with atherosclerosis. ApoE -/- mice demonstrate the atherosclerosis phenotype. 21 Thus, we performed immunostaining to analyze the expression of IL-20 and its receptors in the atherosclerotic lesions in apoE -/- mice and normal C57BL/6 mice. We found that IL-20 was upregulated in the atherosclerosis plaque of apoE -/- mice compared with normal C57BL/6 mice (supplemental Figure I Ai to Aj, available online at http://atvb.ahajournals.org). Strong immunoreactivity was detected for both IL-20R1 (supplemental Figure I Aa) and IL-20R2 (supplemental Figure I Ae) in atherosclerosis plaque and the endothelium of the aortic arches of apoE -/- mice. By contrast, low levels of IL-20R1 and IL-20R2 were detected in a portion of the endothelial cells in the aortic arches of normal C57BL/6 mice (supplemental Figure IAb, f). Immunoreactivity was detected in the adventitia of the aortic arches of C57BL/6 and apoE -/- mice. In the aortic root lesions, IL-20 was also detected in Mac-3-rich (foam cells/macrophages) area (supplemental Figure I Ba-b). These results indicated that both IL-20 and IL-20 receptors were induced in atherosclerosis plaque and were markedly upregulated in the endothelium of atherosclerotic aortas. It is likely that foam cells/macrophages are the major producers of IL-20 in the plaques.


Expression of IL-20, IL-20R1, and IL-20R2 in Human Atherosclerotic Artery


The expression of IL-20, IL-20R1, and IL-20R2 was also examined in paraffined sections of surgical femoral arterial samples from peripheral arterial occlusion patients. In these sections, pronounced thickening occurred in the intima and media, both of which exhibited extensive angiogenesis. IL-20 was detected in endothelial cells lining the microvessels and macrophage-derived foam cells that exhibited positive staining for CD68 ( Figure 1 Aa, f). Both IL-20R1 and IL-20R2 were detected primarily in endothelial cells of the vasa vasorum ( Figure 1 Ab, d). By contrast, IL-20R1 and IL-20R2 were expressed at lower levels in the endothelium of normal aorta sections whereas IL-20 was not detected in normal aorta sections ( Figure 1 Ag, h, j). In addition, none of these proteins was significantly expressed in the smooth muscle cells of atherosclerotic lesions. A high-power field (200 x ) magnification of an atherosclerotic lesion is shown in Figure 1 B.


Figure 1. Immunohistochemical staining of IL-20 and receptor subunits in the human atherosclerotic lesions. Paraffin sections of human femoral artery were obtained from patients with peripheral artery occlusion disease after bypass surgery. A, IL-20 was detected by an anti-IL-20 monoclonal antibody 7E (a,g). IL-20R1 expression was detected by the mouse anti-hIL-20R1 monoclonal antibody (b,h). IL-20R2 expression was detected with a rabbit anti-hIL-20R2 polyclonal antibody (d,j). Staining with mouse IgG 1 isotype was used as the negative control for IL-20R1 and IL-20(c,i). Staining with rabbit preimmune serum alone was used as the negative control for IL-20R2 (e,k). Monocytes/macrophages were detected using an anti-CD68 monoclonal antibody (f,l). Reactions were detected by AEC (red, a-e, g-k) or DAB (brown, f,l), and nuclei were counterstained with hematoxylin (blue). B, 200-fold magnification. Macrophages ( ) and endothelial cells ( ) were indicated. Bar represents 100 µm. Sections represent similar patterns in 3 individual specimens.


OxLDL Induced the Expression of IL-20 in Monocytes


IL-20 and its receptors were upregulated both in human and mice atherosclerotic lesions (supplemental Figure I and Figure 1 ). We speculated that IL-20 was induced by existing inflammatory stimuli in the atherosclerotic lesions. OxLDL has been shown to promote foam cell formation and compromise endothelial function by triggering the secretion of chemokines and increasing the expression of leukocyte adhesion molecules. 5 To examine whether OxLDL stimulated IL-20 expression, we used reverse-transcription polymerase chain reaction (RT-PCR) to determine IL-20 mRNA level in OxLDL-treated human peripheral monocytes. Our results revealed that OxLDL, like the positive control lipopolysaccharide (LPS), induced IL-20 expression in human peripheral monocytes ( Figure 2 A) and mouse RAW264.7 macrophages ( Figure 2 B). We further confirmed the result using real-time PCR ( Figure 2 C). These results could explain the co-localization of foam cells and IL-20 in immunohistochemical staining ( Figure 1 ).


Figure 2. OxLDL induced IL-20 in monocytes. Human peripheral monocytes (A) and mouse RAW264.7 macrophages (B) were exposed to PBS, native LDL (100 µg/mL), OxLDL (1, 20, 100 µg/mL), or LPS (10 ng/mL) for 6 hours. Total RNA was then isolated for RT-PCR analyses, and ß-actin was the internal control. C, Total RNA underwent real-time PCR analysis to confirm the result of OxLDL-treated human monocytes. Fold increases of the transcripts are represented as 2 - Ct Ct: retention time.


IL-20 Did Not Affect OxLDL Uptake by Macrophages


We further analyzed whether IL-20 affected OxLDL uptake by macrophages in vitro. Mouse peritoneal macrophages (supplemental Figure II A-B) or human THP-1 macrophages (supplemental Figure II C-D) were incubated with IL-20, phorbol 12-myristate 13-acetate (PMA), or IL-10 together with OxLDL. Unlike IL-10-enhanced OxLDL uptake, 22 IL-20 had no effect on OxLDL uptake by macrophages. Thus, OxLDL induced IL-20 production by monocytes, but it did not enhance OxLDL-induced foam cells formation.


Hypoxia Induced the Expression of IL-20 and Its Receptors


Hypoxia induces various angiogenesis factors and results in neovascularization in ischemic tissues. Neovascularization in atherosclerotic plaque was associated with its hypoxic zones. IL-20 and its receptors were upregulated in atherosclerotic lesions in a mouse model and human samples. Therefore, we speculated that IL-20 and its receptors might be induced under hypoxic conditions. CoCl 2 is known to elicit hypoxia-like responses by activating hypoxia-inducible factor-1 (HIF-1 ) in vitro. 23,24 To evaluate whether IL-20 is upregulated under hypoxic conditions, we used RT-PCR to determine IL-20 mRNA level in CoCl 2 -treated human peripheral monocytes. Our results demonstrated that CoCl 2 induced IL-20 in monocytes ( Figure 3A, 3 B). We further examined the effect of hypoxia on the expression of IL-20 and its receptors in endothelial cells. CoCl 2 treatment upregulated the transcripts of IL-20, IL-20R1, and IL-20R2, but it did not significantly upregulate the transcripts of IL-22R1, in human umbilical vein endothelial cells (HUVECs) ( Figure 3 C). Furthermore, we incubated HUVECs in the chamber supplied with only 1% O 2 to mimic a hypoxia condition for 12 hours and analyzed the transcripts of IL-20 and its receptors. The similar results of upregulation of IL-20 and its receptors were observed ( Figure 3 D). Thus, hypoxia stimulated the expression of IL-20 on monocytes and HUVECs and its receptors on HUVECs. These data are consistent with observations in immunohistochemical staining of atherosclerotic lesions.


Figure 3. Hypoxia regulated the expression of IL-20 and its receptors. Human peripheral monocytes (A, B) and HUVECs (C) were exposed to normoxia (PBS), 100 µmol/L CoCl 2, 500 µmol/L CoCl 2, or 100 ng/mL LPS for 6 hours. In another experiment, HUVECs were incubated in chambers under normoxia (21% O 2, 5% CO 2 ) or hypoxia (1% O 2, 5% CO 2 ) condition for 12 hours (D). RNA was then isolated for RT-PCR analysis, and HPRT or ß-actin was used as an internal control. Equal amounts of cDNA and primers specific for IL-20, IL-20R1, IL-20R2, and IL-22R1 were used in PCR to amplify the transcripts. HUVECs (E) or HMECs (F) were incubated with PBS, hIL-20 (200 ng/mL), or IFN- (10 ng/mL) for 6 hours. Total RNA was then isolated for RT-PCR analysis as described. The relative quantity of PCR products was analyzed using the BIO-PROFIL program and expressed as a fold-increase relative to untreated control cells. The experiment was repeated three times with similar results.


IL-20 Induced Chemokines, Mig/CXCL9, and I-TAC/CXCL11, in Endothelial Cells


Recruitment of T lymphocyte into the atherosclerotic lesions is a prominent process during progression of atherosclerosis. 2 Three CXCR3 ligands, chemokines induced by IFN-, Mig/CXCL9, IP-10/CXCL10, and I-TAC/CXCL11, mediate the process of T lymphocyte recruitment and are present in atheroma-associated cells. 25 To investigate whether IL-20 induced chemokine production and thus, recruited T lymphocyte, we analyzed the effect of IL-20 on chemokines production in endothelial cells. HUVECs were incubated with IL-20 and analyzed using RT-PCR. The transcripts of CXCL9 and CXCL11, but not CXCL10, were upregulated by IL-20 in HUVECs ( Figure 3 E) as well as in human microvessel endothelial cells (HMECs) ( Figure 3 F).


Mouse IL-20 Promoted Angiogenesis In Vivo


We have shown human IL-20 promotes HUVECs proliferation, migration in vitro, and tumor angiogenesis in vivo. 20 To further examination whether mouse (m) IL-20 also enhanced angiogenesis in vivo, mouse hepatoma cells, ML-1, were dorsally co-injected with Matrigel-containing saline, mIL-20, mIL-20 plus anti-mIL-20R1 antibody, or VEGF into Balb/c mice (Laboratory Animal Center, NCKU; Tainan, Taiwan). After 7 days, tumors were excised and immunohistochemical stained with CD31 for microvessel density analysis (supplemental Figure III). mIL-20, similar to VEGF, enhanced vascularization around the solid tumors compared with saline-treated control (supplemental Figure IIIA). CD31 staining also showed higher microvessel density in mIL-20 treated tumors than that in saline-treated tumors (supplemental Figure IIIB-C). In addition, antibody against mouse IL-20R1 inhibited mIL-20-induced angiogenesis, indicating IL-20R1 was involved in the signaling of IL-20-induced angiogenesis. These results provided evidences of the angiogenic activity of mIL-20 in vivo.


IL-20 Promoted Atherosclerosis in ApoE -/- Mice


To further investigate the role of IL-20 in atherosclerosis in vivo, we performed intramuscular electroporation to deliver the mIL-20 expression construct into apoE -/- mice. The serum levels of mIL-20 after electroporation were measured using enzyme-linked immunosorbent assay ( Figure 4 A). The mIL-20 protein level peaked at the first week after electroporation and gradually leveled off in the mice treated with mIL-20 plasmid DNA. Administration of mIL-20 led to enhanced en face lesion areas of aortas ( Figure 4B, 4 C), lesion area of aortic sinus, and an increased area of Mac-3-positive (macrophages) in the aortic sinus. However, no significant difference in the percentage of Mac-3-positive area (Mac-3-positive area/lesion area) was observed between the groups (supplemental Table II). Additionally, in consistent with the in vitro result, the expression of Mig and I-TAC were also higher in aortas form pcDNA3.1-mIL-20-treated mice than that from control mice. Furthermore, proinflammatory cytokines tumor necrosis factor- and IL-6 were also upregulated in the aortas ( Figure 4 D). These results suggest that IL-20 is a proatherogenic factor and may contribute to the pathogenesis of atherosclerosis.


Figure 4. IL-20 promoted atherosclerosis in apoE -/- mice. Fourteen-week-old apoE -/- mice were fed by the atherogenic diet. 50 µg per mouse of pcDNA3.1 or pcDNA3.1-mIL-20 were delivered to the muscles of hind leg using intramuscular electroporation (n=7 in each group). Electroporation was performed once each week for a total of ten times per mouse. A, Serum levels of mIL-20 in apoE -/- mice were analyzed using enzyme-linked immunosorbent assay. B, The aortas were oil red O-stained and showed red lipid-rich atherosclerotic lesions. Figure represents 7 mice from each group. C, Quantitative analysis showed that the en face lesion area of aortas were higher in pcDNA3.1-mIL-20 treated mice than that in pcDNA3.1 treated mice. Bar represents 1 mm (A). * P <0.05. D, RT-PCR analyses of the expression of Mig and I-TAC in the aortas from mice treated with pcDNA3.1-mIL-20 or control vector.


Discussion


More characteristics of the biological activities of IL-20 were demonstrated by the expression profiles of IL-20 and its receptors. 16,18,26,27 Our current work provides evidence to support the notion that expression of IL-20 and its receptors is associated with the development of atherosclerosis, though much remains to be elucidated about the physiological and pathogenic roles of IL-20 and its receptors. Results from immunohistochemical staining demonstrated that IL-20 and its receptor complex IL-20R1/IL-20R2 were indeed expressed in atherosclerotic lesions from human patients as well as apoE -/- mice (supplemental Figure I and Figure 1 ). Because macrophages are known to secrete IL-20, co-distribution of IL-20 and the macrophage marker Mac-3 (mouse) and CD68 (human) in atherosclerotic lesions suggests that macrophages are the major source of IL-20 in the atherosclerotic lesions (supplemental Figure I and Figure 1 ). IL-20 and its receptor complex IL-20R1/IL-20R2 were expressed at minimum to undetectable level in the aorta of C57BL/6 normal mice and normal human aorta, whereas their expression increased in endothelial cells lining the intimal microvessel, vasa vasorum. These results indicate that IL-20 may involve in the progression of atherosclerosis plaque by regulating the intimal neovascularization.


The formation of new microvessels in human atherosclerotic plaque is associated with high cellular proliferation activity in the plaque. 28 Neovascularization is required for atherosclerotic plaque development. 12 Administration of the antiangiogenic drugs endostatin and TNP-470 to apoE -/- mice reduces both plaque growth and intimal neovascularization. 10 Recently, our study showed IL-20 induced the proliferation and migration of endothelial cells in vitro, and promoted tumor angiogenesis in vivo. In addition, IL-20 also induced matrix metalloproteinase-2, VEGF, and IL-8 expression by HUVECs. 20 In the present study, we also showed mouse IL-20 promoted angiogenesis in vivo through IL-20R1 in a mouse model (supplemental Figure III). Therefore, it can be postulated that an increased secretion of IL-20 by macrophages and endothelial cells may serve as paracrine or autocrine factors for endothelial cells. The increased IL-20 secretion in combination with upregulation of IL-20 receptors on endothelial cells will promote the migration and proliferation of endothelial cells, thereby stimulating angiogenesis in atherosclerotic lesions. In another report, 29 IL-20 inhibited PMA-induced angiogenesis by inhibiting COX-2. It is possible that IL-20 regulates angiogenesis via proangiogenic and antiangiogenic activities in different tissue types according to the microenvironment and different receptor complex used.


In the animal model, we did not detect significant CD31-positive staining on the intimal vasa vasorum in the aortic arches sections (data not shown). This may be attributed to the angiogenesis-mediated neovascularization occurred at the late stage of plaque development. 30 Thus, it was not clear whether IL-20 regulated angiogenesis during atherosclerotic plaque development in the apoE -/- model. Nevertheless, we did not exclude the possibility that IL-20 regulates angiogenesis in human atherosclerotic lesions. There may be some difference in the molecular mechanism of atherosclerotic plaque development between animal model and human. The effect of IL-20 on the regulation of angiogenesis during atherosclerotic plaque development awaits further investigation.


Initiation and progression of atherosclerosis are dependent on an inflammatory environment. Proinflammatory cytokines play a pivotal role in perpetuating this environment. However, mechanisms regulating the expression of IL-20 and its receptors remain poorly understood. Our results demonstrated that LPS, OxLDL, and hypoxic condition could contribute to increase of IL-20 production by monocytes. Furthermore, hypoxic condition also increased the expression of IL-20, IL-20R1, and IL-20R2 by HUVECs ( Figure 3 ).


A hypoxic zone occurs at an atherosclerotic lesion 11 and is associated with the angiogenesis process and inflammation. 31 In response to hypoxia, HIF-1 induces gene expression by binding to the hypoxia response element on the promoter of hypoxia-inducible genes. Treatment with CoCl 2 induced IL-20 transcription by human monocytes. IL-20 and the receptors, IL-20R1 and IL-20R2, were expressed on HUVECs under normoxia. Induction of hypoxia by CoCl 2 or 1% O 2 treatment significantly increased the expression of IL-20 and its receptors in HUVECs ( Figure 3 ). We have found some potential hypoxia response elements upstream of IL-20 gene. These HIF-1 binding motifs may be responsible for upregulation of IL-20 under hypoxia condition (unpublished data). This provides a mechanism for upregulation of IL-20 in atherosclerotic lesions by macrophages and endothelial cells, and upregulation of the IL-20 receptors by endothelial cells.


The 3 CXCR3 ligands (Mig/CXCL9, IP-10/CXCL10 and I-TAC/CXCL11) were differentially expressed by human atheroma-associated cells and mediate the recruitment of T lymphocytes into the inflammation sites. 25,32 Our result demonstrated that IL-20 induced Mig/CXCL9 and I-TAC/CXCL11 but not IP-10/CXCL10 in endothelial cells ( Figure 3 E, F). In consistent with the in vitro data, the mRNA levels of Mig and I-TAC were also elevated in the aortas from IL-20 plasmid DNA treated mice ( Figure 4 D). Thus, IL-20 induced Mig and I-TAC in vitro and in vivo, indicating IL-20 may play an important role to recruit T lymphocytes to accumulate in the atheroma during the plaque formation.


Systemic delivery of IL-20 by intramuscular electroporation resulted in increased atherosclerotic lesion areas in apoE -/- mice ( Figure 4 and supplemental Table II). These results suggest that IL-20 acts as a proatherogenic cytokine. We found that IL-20, unlike IL-10, did not enhance OxLDL uptake and foam cells formation in vitro (supplemental Figure II). The proatherogenic effect of IL-20 might be mediated through persistent higher serum levels of IL-20 by acting on vascular endothelial cells directly or indirectly through other mediators (Mig, I-TAC, VEGF) in inflamed lesions. The upregulation of IL-6 and tumor necrosis factor- also revealed the role of IL-20 in enhancing intraplaque inflammation ( Figure 4 D). Thus, neutralization of IL-20 may constitute an attractive new strategy for the treatment of atherosclerosis.


Transgenic mice expressing IL-20 controlled by various tissue promoters reveal similar neonatal death effect. 16 However, injection of IL-20 protein into normal Balb/c mice has no effect on skin phenotype or any lethal effect. 17 In this study, we used the intramuscular electroporation to systemically deliver the IL-20 gene construct to the apoE -/- mice without any lethal effect or significant pathological change. Therefore this approach could be useful for further exploring the biological function of IL-20 in vivo. However, repeated electroporation may induce muscle necrosis or tolerance that could explain mIL-20 protein gradually leveled off after the first week.


In summary, we found that IL-20 is associated with atherosclerosis because both IL-20 and its receptor subunits are expressed in atherosclerotic lesions of apoE -/- mice and in human atherosclerotic arteries. In vitro, the expression of IL-20 was upregulated in monocytes with OxLDL and hypoxic stimulation. IL-20 also induced chemokines expression in HUVECs and systemic delivery of IL-20 in apoE -/- mice led to increased atherosclerotic lesion areas, suggesting that IL-20 is a novel proatherogenic cytokine. Because the biological functions and clinical implications of IL-20 remain to be explored, our findings provide evidence of the association between IL-20 and atherosclerosis.


Acknowledgments


We thank Dr Chauying J. Jen and S.L. Kao (Department of Physiology, NCKU) for their help in preparing the OxLDL, and Dr Pin Ling (Department of Microbiology & Immunology, NCKU) for his assistance in preparing the manuscript.


Sources of Funding


This study was supported by grants CMFHT-9303 from the Chi-Mei Medical Center and 94-2320-B-006-093 from the National Science Council, Taiwan.


Disclosures


None.

【参考文献】
  Ross R. Atherosclerosis-an inflammatory disease. N Engl J Med. 1999; 340: 115-126.

Glass CK, Witztum JL. Atherosclerosis. the road ahead. Cell. 2001; 104: 503-516.

Lusis AJ. Atherosclerosis. Nature. 2000; 407: 233-241.

Lee RT, Libby P. The unstable atheroma. Arterioscler Thromb Vasc Biol. 1997; 17: 1859-1867.

Steinberg D. Atherogenesis in perspective: hypercholesterolemia and inflammation as partners in crime. Nat Med. 2002; 8: 1211-1217.

Lawn RM, Wade DP, Couse TL, Wilcox JN. Localization of human ATP-binding cassette transporter 1 (ABC1) in normal and atherosclerotic tissues. Arterioscler Thromb Vasc Biol. 2001; 21: 378-385.

Hansson GK. Cell-mediated immunity in atherosclerosis. Curr Opin Lipidol. 1997; 8: 301-311.

Mikita T, Porter G, Lawn RM, Shiffman D. Oxidized low density lipoprotein exposure alters the transcriptional response of macrophages to inflammatory stimulus. J Biol Chem. 2001; 276: 45729-45739.

Tenaglia AN, Peters KG, Sketch MH, Jr., Annex BH. Neovascularization in atherectomy specimens from patients with unstable angina: implications for pathogenesis of unstable angina. Am Heart J. 1998; 135: 10-14.

Moulton KS, Heller E, Konerding MA, Flynn E, Palinski W, Folkman J. Angiogenesis inhibitors endostatin or TNP-470 reduce intimal neovascularization and plaque growth in apolipoprotein E-deficient mice. Circulation. 1999; 99: 1726-1732.

Bjornheden T, Levin M, Evaldsson M, Wiklund O. Evidence of hypoxic areas within the arterial wall in vivo. Arterioscler Thromb Vasc Biol. 1999; 19: 870-876.

Barger AC, Beeuwkes R, 3rd, Lainey LL, Silverman KJ. Hypothesis: vasa vasorum and neovascularization of human coronary arteries. A possible role in the pathophysiology of atherosclerosis. N Engl J Med. 1984; 310: 175-177.

Mallat Z, Besnard S, Duriez M, Deleuze V, Emmanuel F, Bureau MF, Soubrier F, Esposito B, Duez H, Fievet C, Staels B, Duverger N, Scherman D, Tedgui A. Protective role of interleukin-10 in atherosclerosis. Circ Res. 1999; 85: e17-24.

Gesser B, Leffers H, Jinquan T, Vestergaard C, Kirstein N, Sindet-Pedersen S, Jensen SL, Thestrup-Pedersen K, Larsen CG. Identification of functional domains on human interleukin 10. Proc Natl Acad Sci U S A. 1997; 94: 14620-14625.

Pestka S, Krause CD, Sarkar D, Walter MR, Shi Y, Fisher PB. Interleukin-10 and related cytokines and receptors. Annu Rev Immunol. 2004; 22: 929-979.

Blumberg H, Conklin D, Xu WF, Grossmann A, Brender T, Carollo S, Eagan M, Foster D, Haldeman BA, Hammond A, Haugen H, Jelinek L, Kelly JD, Madden K, Maurer MF, Parrish-Novak J, Prunkard D, Sexson S, Sprecher C, Waggie K, West J, Whitmore TE, Yao L, Kuechle MK, Dale BA, Chandrasekher YA. Interleukin 20: discovery, receptor identification, and role in epidermal function. Cell. 2001; 104: 9-19.

Liu L, Ding C, Zeng W, Heuer JG, Tetreault JW, Noblitt TW, Hangoc G, Cooper S, Brune KA, Sharma G, Fox N, Rowlinson SW, Rogers DP, Witcher DR, Lambooy PK, Wroblewski VJ, Miller JR, Broxmeyer HE. Selective enhancement of multipotential hematopoietic progenitors in vitro and in vivo by IL-20. Blood. 2003; 102: 3206-3209.

Wolk K, Kunz S, Asadullah K, Sabat R. Cutting edge: immune cells as sources and targets of the IL-10 family members? J Immunol. 2002; 168: 5397-5402.

Dumoutier L, Leemans C, Lejeune D, Kotenko SV, Renauld JC. Cutting edge: STAT activation by IL-19, IL-20 and mda-7 through IL-20 receptor complexes of two types. J Immunol. 2001; 167: 3545-3549.

Hsieh MY, Chen WY, Jiang MJ, Cheng BC, Huang TY, Chang MS. Interleukin-20 promotes angiogenesis in a direct and indirect manner. Genes Immun. 2006; 7: 234-242.

Zhang SH, Reddick RL, Piedrahita JA, Maeda N. Spontaneous hypercholesterolemia and arterial lesions in mice lacking apolipoprotein E. Science. 1992; 258: 468-471.

Halvorsen B, Waehre T, Scholz H, Clausen OP, von der Thusen JH, Muller F, Heimli H, Tonstad S, Hall C, Froland SS, Biessen EA, Damas JK, Aukrust P. Interleukin-10 enhances the oxidized LDL-induced foam cell formation of macrophages by antiapoptotic mechanisms. J Lipid Res. 2005; 46: 211-219.

Yuan Y, Hilliard G, Ferguson T, Millhorn DE. Cobalt inhibits the interaction between hypoxia-inducible factor-alpha and von Hippel-Lindau protein by direct binding to hypoxia-inducible factor-alpha. J Biol Chem. 2003; 278: 15911-15916.

Semenza GL. Regulation of mammalian O2 homeostasis by hypoxia-inducible factor 1. Annu Rev Cell Dev Biol. 1999; 15: 551-578.

Mach F, Sauty A, Iarossi AS, Sukhova GK, Neote K, Libby P, Luster AD. Differential expression of three T lymphocyte-activating CXC chemokines by human atheroma-associated cells. J Clin Invest. 1999; 104: 1041-1050.

Parrish-Novak J, Xu W, Brender T, Yao L, Jones C, West J, Brandt C, Jelinek L, Madden K, McKernan PA, Foster DC, Jaspers S, Chandrasekher YA. Interleukins 19, 20, and 24 signal through two distinct receptor complexes. Differences in receptor-ligand interactions mediate unique biological functions. J Biol Chem. 2002; 277: 47517-47523.

Nagalakshmi ML, Murphy E, McClanahan T, de Waal Malefyt R. Expression patterns of IL-10 ligand and receptor gene families provide leads for biological characterization. Int Immunopharmacol. 2004; 4: 577-592.

O?Brien ER, Garvin MR, Dev R, Stewart DK, Hinohara T, Simpson JB, Schwartz SM. Angiogenesis in human coronary atherosclerotic plaques. Am J Pathol. 1994; 145: 883-894.

Heuze-Vourc?h N, Liu M, Dalwadi H, Baratelli FE, Zhu L, Goodglick L, Pold M, Sharma S, Ramirez RD, Shay JW, Minna JD, Strieter RM, Dubinett SM. IL-20, an anti-angiogenic cytokine that inhibits COX-2 expression. Biochem Biophys Res Commun. 2005; 333: 470-475.

Khurana R, Simons M, Martin JF, Zachary IC. Role of angiogenesis in cardiovascular disease: a critical appraisal. Circulation. 2005; 112: 1813-1824.

Murdoch C, Giannoudis A, Lewis CE. Mechanisms regulating the recruitment of macrophages into hypoxic areas of tumors and other ischemic tissues. Blood. 2004; 104: 2224-2234.

Piali L, Weber C, LaRosa G, Mackay CR, Springer TA, Clark-Lewis I, Moser B. The chemokine receptor CXCR3 mediates rapid and shear-resistant adhesion-induction of effector T lymphocytes by the chemokines IP10 and Mig. Eur J Immunol. 1998; 28: 961-972. <a href="/cgi/external_ref?access_num=10.1002/(SICI)1521-4141(199803)28:03


作者单位:Institute of Basic Medical Sciences (W.Y.C., B.C.C., M.S.C.), Department of Biochemistry and Molecular Biology (W.Y.C., M.Y.H., M.S.C.), Department of Cell Biology and Anatomy (M.J.J.), College of Medicine, National Cheng Kung University, and Chi-Mei Medical Center (W.Y.C., B.C.C., M.S.C.), Tainan,

日期:2008年12月28日 - 来自[2006年第26卷第9期]栏目

Identifying Inflamed Carotid Plaques Using In Vivo USPIO-Enhanced MR Imaging to Label Plaque Macrophages

【摘要】  Background- Inflammation within atherosclerotic lesions contributes to plaque instability and vulnerability to rupture. We set out to evaluate the use of a macrophage labeling agent to identify carotid plaque inflammation by in vivo magnetic resonance imaging (MRI).

Methods and Results- Thirty patients with symptomatic severe carotid stenosis scheduled for carotid endarterectomy underwent multi-sequence MRI of the carotid bifurcation before and after injection of ultrasmall superparamagnetic particles of iron oxide (USPIOs). USPIO particles accumulated in macrophages in 24 of 30 plaques (80%). Areas of signal intensity reduction, corresponding to USPIO/macrophage-positive histological sections, were visualized in 24 of 27 (89%) patients, with an average reduction in signal intensity induced by the USPIO particles of 24% (range, 3.1% to 60.8%).

Conclusions- USPIO-enhanced MRI can identify plaque inflammation in vivo by accumulation of USPIO within macrophages in carotid plaques.

The in vivo detection of plaque inflammation was evaluated using a macrophage labeling MRI contrast agent. Areas of focal signal loss on MRI corresponded to macrophage populations, thereby identifying inflamed plaques in 24 of 27 symptomatic individuals with severe ICA stenosis were detected and confirmed histologically.

【关键词】  carotid inflammation MRI USPIO vulnerable plaque


Introduction


Although conventional angiographic measurements of luminal stenosis do not reflect disease burden in carotid atherosclerosis, 1 they are still used as the primary criteria for definitive surgical therapy. Histological studies have identified features that may better predict rupture in "high-risk" plaques; these plaques have thin/eroded fibrous caps that overly large necrotic lipid cores and have an abundance of inflammatory cells (macrophages). 2 Inflammation within atherosclerotic plaques increases vulnerability to rupture and subsequent thromboembolism and presents itself as a target for plaque stabilization therapies.


Animal studies of atherosclerosis have shown that superparamagnetic iron oxide (SPIO) particles are taken up by inflamed plaques rich in macrophages as intracellular deposits 3 that induce areas of signal loss on T 2 *-weighted magnetic resonance imaging (MRI) within the vessel wall. 4 More recently, in vivo human studies using the ultrasmall SPIOs (USPIO) agent, Sinerem, have confirmed these findings and also refined optimal MRI parameters to detect inflamed plaques. 5,6 These pilot studies, while demonstrating the potential of USPIO enhanced MRI to visualize plaque macrophages, are limited by sample size and methodology issues. This report describes the findings of the largest in vivo human study evaluating Sinerem-enhanced MRI to identify inflammation within atherosclerotic plaques with histological correlation.


Materials and Methods


Patients


The carotid arteries of 30 nonconsecutive (patients with contraindications to MRI or for whom MRI slots could not be obtained were excluded) symptomatic patients (22 males, 8 females; median age, 70; range, 48 to 83 years) with severe internal carotid artery (ICA) stenosis 1 (mean (±SD) ICA stenosis 77% (±7%), measured by digital subtraction angiography), recruited from a specialist neurovascular clinic, scheduled for carotid endarterectomy were imaged. The overall median time from symptom onset to surgery was 3.5 months (range, 0.5 to 7 months). Approval for the study was obtained from the Local Research Ethics Committee. All patients gave informed consent.


MR Contrast Agent


The USPIO contrast agent, Sinerem (Guerbet, Roissy, France) consisting of ferromagnetic iron oxide particles with an overall size of &30 nm, was suspended in normal saline and given as an intravenous infusion (2.6 mg/kg) over 30 minutes. 5


All the imaging studies were conducted on a 1.5-Tesla system (CV/I; GE Medical Systems, USA) using a customized 4-channel phased array coil (Flick Engineering Solutions, the Netherlands) wrapped around the neck. Images were acquired through the carotid bifurcation using the following EKG-gated, fat-suppressed pulse sequence using double inversion blood suppression before and 36 hours after USPIO injection 5 : 2-dimensional T 2 *-weighted spiral acquisitions using spectral-spatial excitation pulses (echo time/repetition time 5.6/1 R-R). The multi-shot spiral sequence involved the acquisition of 22 spiral interleaves each of 4096 data points, resulting in an effective in-plane pixel size of 0.42 x 0.42 mm, 2 signal averages were performed. The field of view was 12-cm x 12-cm and slice thickness was 3 mm for all sequences. Typically between 4 to 6 plaques containing images were generated for each vessel, covering the length of the plaque. The time from USPIO infusion to endarterectomy ranged from 40 hours (1 patient) to 18 days (1 patient) with a mean (±SD) interval of 6.9 (±4.8) days.


Histological Staining


Histology sections underwent H&E, Elastin van Gieson, and immunostaining for macrophages CD68 (mature macrophages), MAC387 (immature macrophages), smooth muscle cells ( -SMA), and endothelial cells (CD31). Perls reagent was used to identify the contrast agent. 7 Double immunostaining and Perls staining was performed on serial sections from several plaques that had evidence of strong Perls positivity to determine the distribution and localization of Sinerem.


MRI Analysis


Images were viewed on a standard computer workstation attached to a high-resolution display screen using an image analysis software package (CMRTools, Imperial College, UK). Images were viewed at 200% magnification and pre-infusion and post-infusion images from any individual were adjusted to ensure identical window/level settings. Coregistration of histological sections and MR images was performed in a similar manner to that previously described. 5,6 In brief, the carotid bifurcation was used as reference marker for both MR and histology section localization and corresponding images were re-orientated according to gross morphological features, such as lumen position.


Qualitative Image Analysis


Images were deemed acceptable for analysis if the entire border of the carotid vessel wall was visible and the lumen free of flow artifacts. The presence of USPIO within the plaque was confirmed by noting whether the matched post-infusion image contained a new region(s) of low signal intensity (SI) within the vessel wall (plaque). The nature of any new area of SI reduction was noted as "focal," if the region of signal change was localized to 1 well-circumscribed area, as "multi-focal" if there was 1 such nonconfluent area or as "diffuse," for any other pattern of signal change. The location (quadrant) of the region of signal change was also noted, which was determined by constructing an imaginary set of perpendicular axes with their inter-section at the center of the vessel lumen. This approach was taken for pre-infusion and post-infusion images. Then each pair of matched quadrants (right upper, left upper, right lower, left lower) was viewed in turn and the presence or absence (and nature where present) of USPIO effect noted.


Quantitative Image Analysis


The maximum SI change within a matched region of interest (ROI) was determined, because this was thought to better reflect the Sinerem load within plaques. The following method was used to define the ROI from which the SI was measured. On the post-infusion image, the ROI was defined to include and circumscribe the region of SI change (focal or diffuse). The delineated ROI was then copied and transposed to the appropriate location on the pre-infusion image to provide a "mirror" location for comparative analysis. The SI of these ROIs was then measured. The SI within the ROI (SI plaque ) was then normalized to that of a similar sized ROI in the adjacent sternocleidomastoid muscle (SI muscle ) (we had previously observed that the SI of such a ROI varied minimally in relation to its position). The relative signal intensity (rSI) was calculated as follows: SI plaque / SI muscle. The magnitude of SI change was quantified separately for the "diffuse" and "focal" effect groups; images that were classified as "multi-focal" had the area with the most pronounced signal effect used as the ROI. If there was no such area, then each "focus" was used as a separate ROI and the mean value used to describe the magnitude of signal change in this image.


Histological Image Analysis


Determination of USPIO Accumulation Within Plaques


Perls reagent uptake was used as a surrogate marker for the presence of USPIO. 6,7 Previous analysis of Perls reagent stained sections from subjects not given USPIO injections revealed only sporadic Perls positivity in a few plaque sections. Using low-power magnification ( x 1.6 lens), which allows the majority of the vessel cross-sections to be viewed in one field of view, the overall distribution pattern of Perls positive cells was determined in a similar manner to that used for the USPIO-induced MR signal effect (focal, multi-focal, diffuse, absent). At higher magnification ( x 40 lens) cell counts were performed on sections stained for USPIO (Perls) and macrophages (CD68/MAC 387). For each section, the total from 10 randomly selected high-power fields (hpf) was determined. Only positively stained material with the morphological appearance of cells (nucleus, cytoplasm) was counted as cells.


Colocalization of USPIO Particles


USPIO particles were deemed to have colocalized with a particular cell type (macrophage, smooth muscle cell, endothelial cell) if both the typical blue appearance of the Perls reagent and the brown appearance of the antibody revealing chromogen were present in the same cell.


Plaque Characterization


Plaque sections were classified by an independent histopathologist as either vulnerable/ruptured or stable by a grading system loosely based on the American Heart Association criteria 2 : plaques that had evidence of plaque erosion, fissure, or rupture or had a thin fibrous cap and/or had large necrotic lipid cores were considered "vulnerable/ruptured" and other morphological types were considered "stable." When there was uncertainty, the plaques sections were considered "unclassifiable."


Statistical Analysis


The magnitude of change in signal intensity induced by Sinerem was quantified as the percent change in SI within the defined ROI. For statistical comparisons of rSI between pre-infusion and post-infusion images, the data were analyzed separately for those images where the signal effect was diffuse and where it was focal (where the effect was multi-focal, the mean value was used). Significance in any differences in pre- and post-infusion rSI was measured using the Wilcoxan-Rank test, with P <0.05 indicating statistical significance. The Mann-Whitney U test was used to determine whether there were any significant differences in SI change between the "focal" and "diffuse" effect groups. The relationship between % change in SI and the number of Perls positive and CD68/MAC387 cells was determined by calculating Pearson?s correlation coefficient. Agreement between location of signal loss on MR and location of Perls positive cells on histology was measured by computing Cohen?s kappa. The relationship between the number of Perls positive cells and macrophages in individual slices was determined by calculating Spearman?s rho coefficient. A 2 test was performed to determine whether Sinerem positivity was associated with vulnerable/ruptured plaques.


Results


Clinical Details


All patients were either current or former smokers and had 1 other risk factors for cerebrovascular disease (22 had hypertension, 11 had diabetes, 20 had hypercholesterolemia). Seven patients had ischemic heart disease; no patients had other systemic inflammatory conditions. All subjects had been in sinus rhythm for the previous 6 months and were taking anti-platelet therapy; half were taking cholesterol-lowering medication; 12 were taking an angiotensin-converting enzyme inhibitor. No patients had evidence of aortic atherosclerotic disease on digital subtraction angiography.


Image Analysis


There were 210 matched MR and histology image pairs generated following the coregistration process from 27 patients; 3 patients had excessive movement artifacts during the post-infusion MR study resulting in incomplete image acquisition, and these were therefore excluded. After review of the coregistered image pairs, 54 were excluded from further analyses because MRI revealed no vessel wall thickening (and consequently no signal change within the wall was detectable) and histological sections revealed vessels with normal intima with no Perls staining (concordant absence). Because the endarterectomy specimen always included the region of the carotid bifurcation, to allow ex vivo coregistration, if the atheromatous plaque was distant to this reference landmark, then it was foreseen that there would be disease-free vessel cross-sections among the histology-MRI pairs. This left a total of 156 image pairs available for further comparative analyses.


Histological Analysis


Distribution of Perls Stain


There were 97 (62%) histological sections from 23 patients that demonstrated Perls positivity. At low magnification, the distribution of the Perls stain in 45 (46%) of these was thought to be diffuse, with another 45 (46%) slices showing a focal accumulation and the remainder a multi-focal distribution. At high magnification, Perls staining appeared to be both intracellular and extracellular in location. In those plaques with focal Perls staining, this was almost always in the subendothelial fibrous cap region. In particular, Perls staining appeared to colocalize to macrophages in the shoulder regions of the plaque ( Figure 1 ). In those sections where Perls staining was diffuse, this was visualized at every depth of the atheromatous plaque; in the peri-luminal region of the fibrous cap, within the thickened intima in close proximity to the necrotic lipid core, at the intima/media border (region of the internal elastic lamina), and in a few sections in the adventitial region of the vessel wall. In all of these latter sections, there was also staining in the fibrous cap region ( Figure 2 ).


Figure 1. Localization of macrophages to fibrous cap. CD68+ macrophages (a) accumulating in shoulder regions of the plaque ( x 4) and high-power view ( x 80) (b) showing both intracellular accumulation of (white arrowheads) and extracellular location of USPIO particles (black arrowheads).


Figure 2. Location of Perls stain within plaques with diffuse staining. Subendothelial fibrous cap region ( x 4) (a), fibrous cap/lipid core border ( x 4) (b), intima/media border ( x 1.5) (c), and adventitial region ( x 4) (d). FC indicates fibrous cap; LC, lipid core.


Frequently, Perls staining was observed in close proximity to areas of neovascularization within deep portions of the intima and distant from the peri-luminal region. In the individual in whom there was a short interval between USPIO infusion and surgery, there appeared to be a greater concentration of Perls staining in areas of neovascularization than in other sites ( Figure 3 ).


Figure 3. Proximity of Perls stain to areas of neovascularization (CD31/Perls double stain, x 30). Dense Perls positive staining (a) (blue) near vascular channels (white arrowheads) compared with scant Perls positivity (b) (black) in other regions in the same cross-section.


Colocalization of USPIO Particles


In the majority of the 97 Perls positive sections, there were macrophage accumulations located in similar regions of the plaque. There were areas in a few plaques where macrophages were not identified, but where Perls staining was evident; serial sections, however, stained for smooth muscle or endothelial cells also revealed an absence of these cell types in these locations. Conversely, there were locations within plaque sections, where there were abundant macrophages, but where Perls staining was absent, including several sections from the 4 subjects in whose plaques no Perls staining was seen whatsoever. These later 4 subjects had plaques harvested within 5 days of the USPIO infusion.


Double immunostaining/Perls staining performed on serial sections of randomly selected Perls positive sections confirmed that there was colocalization of the Perls stain with macrophages ( Figure 1 ) but not with either smooth muscle or endothelial cells (not shown).


Relationship of Perls Stain to Macrophage Content


A poor but significant correlation between the total number of Perls positive cells and MAC 387-stained macrophages (Spearman rho=0.24, P <0.001) and a slightly stronger correlation with CD68-stained macrophages (Spearman rho=0.29, P <0.001) was found, suggesting that either not all plaque macrophages were internalizing Sinerem or that the Perls stain was an inadequate method for detecting Sinerem.


MRI Analyses


Detection of Sinerem-Induced Signal Effect


Comparison between the pre- and post-Sinerem infusion image pairs resulted in 128 (82%) post-infusion images being deemed positive for USPIO-induced signal effect, from a total of 26 individuals. In 56 (44%) of these images, the induced signal loss was described as focal, whereas it was seen as multi-focal in nature in 12 (9%) and diffuse in 60 (47%) slices. There was no USPIO effect observed in the remainder images (28, 18%).


Location of Sinerem Signal Within Plaque


In the "focal" effect images the induced signal effect was consistently in the peri-luminal region corresponding to the fibrous cap region ( Figure 4 ). In the "diffuse" effect images the extent of the induced signal loss extended between the peri-adventitial and peri-luminal regions ( Figure 5 ).


Figure 4. Pattern of USPIO induced signal loss. Axial MR images (T 2 *W [TE=5.6 ms]) through the common carotid artery. Diffuse USPIO signal effect (a,b). Focal USPIO signal effect (c,d). Pre-USPIO infusion (a,c). 36 hours after infusion (b,d) (*lumen).


Figure 5. USPIO induced signal loss in subendothelial fibrous cap region. Pre-USPIO axial MR image (STIR sequence) (a) through common carotid artery showing high-signal fibrous cap region. Post-infusion T 2 *W image (b) showing evidence of new area of signal loss in peri-luminal region (arrowhead) corresponding to fibrous cap region on multi-sequence review. Matched Perls-stained (c) cross-section showing corresponding location of USPIOs within fibrous cap region (arrowhead).


Quantification of Sinerem-Induced Signal Effect


Comparison of the rSI between pre- and post-infusion images of the "focal" effect group revealed a significant difference between the 2, attributable to Sinerem (median change in rSI=-24.1%; range, -3.1% to -60.8%; P <0.0001). In the "diffuse" effect group, the overall reduction in rSI was not statistically significant (median change in rSI=-3.5%; range, -65.1% to 58.7%; P =0.06), with an increase in rSI being measured between some image pairs. There was a significant difference in the magnitude of the USPIO-induced signal change between the "focal" and "diffuse" effect groups (median difference in signal change between "focal" and "diffuse" groups=-17.7%; range, -10.8% to -27.6%; P <0.0001).


Comparative Analyses Between MR and Histology


Qualitative MRI analysis was highly sensitive (92.5%) and moderately specific (64%) for detection of Sinerem particles within atheromatous plaques. In addition, good agreement between location of Perls stain and Sinerem signal effect was observed (Cohen kappa=0.60); however, the agreement between MRI and histology for characterizing the nature of the Sinerem signal effect and Perls stain was only moderate (Cohen kappa=0.48).


There was a significant correlation between magnitude of Sinerem effect and Perls staining in the "focal" effect group (Spearman rho=-0.63, P <0.001) but not in the "diffuse" effect group (Spearman rho=0.17, P 0.05). There was a significant correlation between the magnitude of Sinerem effect and macrophage count in the "focal" effect group (Spearman rho=-0.49 [CD68] and -0.60 [MAC 387], P <0.001 for both). There was no statistically significant correlation in the "diffuse" effect group.


There was a stronger association between the "focal" signal effect group and histologically "unstable/vulnerable" plaques than with the "diffuse" signal effect group (72.4% versus 27.6%, 2 =20.7, 2 degrees of freedom, P <0.001).


Discussion


It has been difficult to visualize atheromatous plaque inflammation in vivo because of limitations in the resolution obtainable with standard imaging technology. Contrast-enhanced MRI with USPIO particles has been shown in animal models to overcome these issues by exploiting the physico-chemical properties of this new genre of MR contrast agent. A recent study of such a contrast agent, Sinerem, prompted exploration of whether this might prove to be a useful in vivo marker of inflammation in human atherosclerosis. 6 The present report suggests that Sinerem-enhanced MRI of human carotid atherosclerotic plaques is a sensitive way of identifying inflamed plaques in symptomatic individuals.


Interpretation of Sinerem-Induced Signal Effect


The finding of a significant correlation between SI reduction and number of macrophages/Perls-positive cells within plaques would suggest that a linear relationship exists between Sinerem accumulation and macrophage burden. However, whereas a linear relationship appears to exist between magnitude of SI reduction and macrophage or Perls positive cells independently, there was no strong correlation between the Perls stain and macrophage counts. Because the size of the area of SI reduction observes a nonlinear relationship with Sinerem particle content, and because the SI change induced by these particles is heavily dependent on dense packing within intracellular organelles, it is difficult to know how to interpret the magnitude of SI change in terms of macrophage burden. 8 Furthermore, this linear relationship did not hold true for the ?diffuse" group where occasionally SI increases were measured. This group is likely to represent mainly extra-cellular Sinerem, and the increase in SI may be explained by the known T 1 effect of Sinerem. 5,7 From the data presented here, it is probable that only a "focal" area of signal loss represents USPIO accumulating in macrophages.


Suitability of Perls Stain for Detecting Sinerem Uptake


The qualitative and quantitative histological analyses suggest no consistent pattern of Perls stain and macrophage accumulation. There are several possible explanations for this. First, it is possible that there is a heterogeneous population of macrophages with differing phagocytic capacities for USPIO. Second, labeling of macrophages by Sinerem is a dynamic process, 5 dependent on individual cell kinetics and, as has been recently suggested, the kinetics of Sinerem differs to those of other USPIO. 9 It is possible that Sinerem may have slower kinetics in inflammatory tissue than in other tissue types. Thus, performing a detection stain at any given time (time form imaging to surgery) may result in a significant number of cells being classified as negative, either because the iron moiety has not yet become detached from the dextran coating or because it has become sequestered by intracellular proteins such as ferritin. A more likely explanation, we feel, is that the Perls stain may not be a very sensitive marker for USPIO, in keeping with others who have used an enhanced Perls stain to increase their detection at the expense of specificity. 10,11 The individual in whom no Sinerem effect/Perls staining was observed had tandem carotid stenosis. The failure to detect Sinerem or induced signal loss could be explained by the fact that the more proximal lesion (acute plaque) was just out of range of the imaging coil and could not be visualized and was not the lesion that was excised. The more distal lesion appeared heavily calcified (plaque characterization sequences) and made any smaller accumulation of Sinerem more difficult to visualize.


Specificity of Sinerem for Macrophage Labeling


There are some who have described Sinerem uptake in smooth muscle and/or endothelial cells, although no colocalization with either of these cell types was found in this study. This might be because of a sampling bias, however, smooth muscle cells are not naturally phagocytic and it is possible that pinocytosis alone might not be sufficient to allow detectable amounts of Sinerem to be internalized and subsequently visualized within these cells.


It is more surprising that there was no colocalization with endothelial cells, because Sinerem entry within plaques may be mediated by entry across dysfunctional endothelium. 6 Explanations for this lack of uptake come from knowledge of the endothelial cell physiology. Endothelial cells have a known capacity to transcytoses particles across from the luminal surface. This process is likely to be rapid, if endothelial cell contraction occurs, because this will result in disruption, albeit transient, to the integrity of the endoluminal barrier. Also, plaque endothelium is not fenestrated, unlike in glands, and this therefore limits internalization.


Safety of Sinerem MRI


It has been speculated that iron-borne free radicals might contribute to atherosclerotic plaque instability, 12 thereby raising some concerns about the use of an iron-based contrast agent to visualize inflammatory cells. Sinerem has been extensively evaluated in both the preclinical and clinical setting, with only mild adverse events being reported with the slow intravenous infusion method, as was used in this study. In this larger study of carotid atherosclerosis, 1 subject reported a transient alteration to taste, which resolved within a few seconds, and did not require cessation of the USPIO infusion. There were no other adverse events reported in the period up to the 1 year postoperative visit, confirming the safety of this compound.


In summary, this report suggests that high-risk individuals with inflamed plaques may be identified on the basis of a "focal" area of signal loss visualized on MRI after Sinerem infusion.


Acknowledgments


None.


Sources of Funding


This work was supported by a grant from Stroke Association UK.


Disclosures


None.

【参考文献】
  Beneficial effect of carotid endarterectomy in symptomatic patients with high-grade carotid stenosis. North American Symptomatic Carotid Endarterectomy Trial Collaborators. N Engl J Med. 1991; 325: 445-453.

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. Circulation. 1995; 92: 1355-1374.

Ruehm SG, Corot C, Vogt P, Kolb S, Debatin JF. Magnetic resonance imaging of atherosclerotic plaque with ultrasmall superparamagnetic particles of iron oxide in hyperlipidemic rabbits. Circulation. 2001; 103: 415-422.

Schmitz SA, Coupland SE, Gust R, Winterhalter S, Wagner S, Kresse M, Semmler W, Wolf KJ. Superparamagnetic iron oxide-enhanced MRI of atherosclerotic plaques in Watanabe hereditable hyperlipidemic rabbits. Invest Radiol. 2000; 35: 460-471.

Trivedi RA, U-King-Im JM, Graves MJ, Cross JJ, Horsley J, Goddard MJ, Skepper JN, Quartey G, Warburton E, Joubert I, Wang L, Kirkpatrick PJ, Brown J, Gillard JH. In vivo detection of macrophages in human carotid atheroma. Temporal dependence of ultrasmall superparamagnetic particles of iron oxide-enhanced MRI. Stroke. 2004; 35: 1631-1635.

Kooi ME, Cappendijk VC, Cleutjens KB, Kessels AG, Kitslaar PJ, Borgers M, Frederik PM, Daemen MJ, van Engelshoven JM. Accumulation of ultrasmall superparamagnetic particles of iron oxide in human atherosclerotic plaques can be detected by in vivo magnetic resonance imaging. Circulation. 2003; 107: 2453-2458.

Corot C, Petry KG, Trivedi R, Saleh A, Jonkmanns C, Le Bas JF, Blezer E, Rausch M, Brochet B, Foster-Gareau P, Baleriaux D, Gaillard S, Dousset V. Macrophage imaging in central nervous system and in carotid atherosclerotic plaque using ultrasmall superparamagnetic iron oxide in magnetic resonance imaging. Invest Radiol. 2004; 39: 619-625.

Billotey C, Wilhelm C, Devaud M, Bacri JC, Bittoun J, Gazeau F. Cell internalization of anionic maghemite nanoparticles: quantitative effect on magnetic resonance imaging. Magn Reson Med. 2003; 49: 646-654.

Penno E, Johnsson C, Johansson L, Ahlstrom H. Comparison of ultrasmall superparamagnetic iron oxide particles and low molecular weight contrast agents to detect rejecting transplanted hearts with magnetic resonance imaging. Invest Radiol. 2005; 40: 648-654.

Bulte JW, Duncan ID, Frank JA. In vivo magnetic resonance tracking of magnetically labeled cells after transplantation. J Cereb Blood Flow Metab. 2002; 22: 899-907.

Dousset V, Ballarino L, Delalande C, Coussemacq M, Canioni P, Petry KG, Caille JM. Imaging macrophage activity in the brain by using ultrasmall particles of iron oxide. (AJNR) Am J Radiol. 2000; 21: 1768.

Stadler N, Lindner RA, Davies MJ. Direct detection and quantification of transition metal ions in human atherosclerotic plaques: evidence for the presence of elevated levels of iron and copper. Arterioscler Thromb Vasc Biol. 2004; 24: 949-954.


作者单位:University Department of Radiology (R.A.T., C.M., J.-M.U., M.J.G., J.H.G.), Addenbrooke?s Hospital, Cambridge, UK; the Department of Pathology (J.H., M.J.G.), Papworth Hospital, Cambridge, UK; GlaxoSmithKline (A.B., L.W., J.B.), Translational Medicine and Technology, Addenbrooke?s Centre for Clinica

日期:2008年12月28日 - 来自[2006年第26卷第7期]栏目

Microparticles of Human Atherosclerotic Plaques Enhance the Shedding of the Tumor Necrosis Factor- Converting Enzyme/ADAM Substrates Tumor Necrosis Factor and

【摘要】  Human atherosclerotic plaques express the metalloprotease tumor necrosis factor (TNF)- converting enzyme (TACE/ADAM-17), which cleaves several transmembrane proteins including TNF and its receptors (TNFR-1 and TNFR-2). Plaques also harbor submicron vesicles (microparticles, MPs) released from plasma membranes after cell activation or apoptosis. We sought to examine whether TACE/ADAM17 is present on human plaque MPs and whether these MPs would affect TNF and TNFR-1 cellular shedding. Flow cytometry analysis detected 12,867 ?? 2007 TACE/ADAM17+ MPs/mg of plaques isolated from 25 patients undergoing endarterectomy but none in healthy human internal mammary arteries. Plaque MPs harbored mainly mature active TACE/ADAM17 and dose dependently cleaved a pro-TNF mimetic peptide, whereas a preferential TACE/ADAM17 inhibitor (TMI-2) and recombinant TIMP-3 prevented this cleavage. Plaque MPs increased TNF shedding from the human cell line ECV-304 overexpressing TNF (ECV-304TNF), as well as TNFR-1 shedding from activated human umbilical vein endothelial cells or ECV-304TNF cells, without affecting TNF or TNFR-1 synthesis. MPs also activated the shedding of the endothelial protein C receptor from human umbilical vein endothelial cells. All these effects were inhibited by TMI-2. The present study shows that human plaque MPs carry catalytically active TACE/ADAM17 and significantly enhance the cell surface processing of the TACE/ADAM17 substrates TNF, TNFR-1, and endothelial protein C receptor, suggesting that TACE/ADAM17+ MPs could regulate the inflammatory balance in the culprit lesion.
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Atherosclerosis is a chronic inflammatory disease of the vessel wall resulting from the interactions between modified lipoproteins, monocytes/macrophages, lymphocytes, and vascular cells.1 The development and the progression of atherosclerotic plaques are associated with apoptotic cell death and accumulation of microparticles (MPs) within the lesion.1-3 MPs are submicron plasma membrane vesicles released during cell activation or apoptosis and harbor at their surface transmembrane proteins initially present at the parent cell surface, conferring to MPs a dynamic storage pool of bioactive molecules.4-6 MPs have been isolated from human atherosclerotic plaque but are absent in healthy blood vessels.7 Human plaque MPs originate mainly from leukocytes, red blood cells, endothelial cells, and smooth muscle cells.7 They also express a procoagulant activity associated with the presence of phosphatidylserine and tissue factor at their surface, which could lead to thrombus formation at the time of plaque rupture.7-9
Inflammatory processes are regulated by the balance between pro- and anti-inflammatory mediators or cytokines. Sheddases also modulate this equilibrium by cleaving transmembrane proteins (cytokines, receptors, adhesion molecules, and so forth) at the cell surface, releasing soluble ectodomains with altered function.10 The typical example is the tumor necrosis factor (TNF)- converting enzyme (TACE). Initially discovered as the protease that cleaves the 26-kDa proform of TNF (pro-TNF) to yield the TNF soluble form (sTNF),11,12 TACE also cleaves ectodomains of several other transmembrane proteins13 such as TNFR-1 and TNFR-2.14,15 TACE belongs to the ADAM family (ADAM17) and is synthesized as an inactive proform that is further cleaved into an active form by proprotein convertases, such as furin.16,17
We recently reported that TACE/ADAM17 is expressed in both cellular and acellular areas of lesions from apoEC/C mice and in human atherosclerotic plaques.18 We therefore hypothesized that MPs present in the plaque are potential carriers of TACE/ADAM17. MPs were isolated from human atherosclerotic plaques and analyzed for their content in TACE/ADAM17 protein and activity. Results showed that MPs carry TACE/ADAM17, mainly in its mature active form, catalyze in vitro hydrolysis of a mimetic peptide containing the cleavage site of pro-TNF, and activate the shedding of TACE/ADAM17 substrates such as TNF, TNFR-1, and endothelial protein C receptor (EPCR).

【关键词】  microparticles atherosclerotic shedding necrosis converting enzyme/adam substrates necrosis necrosis receptor-

Materials and Methods

Isolation of MPs from Human Atherosclerotic Plaque and Human Umbilical Vein Endothelial Cells

MPs were isolated from human atherosclerotic plaques removed from 25 patients undergoing carotid endarterectomy (73 ?? 2 years of age; 79% male), as recently reported.7 Plaques were obtained either from symptomatic patients (70% with ischemic attacks and 30% with stroke, n = 10) or from asymptomatic patients (n = 15) with critical asymptomatic stenosis of the carotid artery (>75% narrowing). As control experiments, healthy human internal mammary arteries (n = 3, obtained as surgical waste) were submitted to the same isolation protocol. All patients gave their informed consent to the study, which was approved by our local ethical committee. Surgical samples obtained within 90 minutes after excision were rapidly rinsed in ice-cold sterile phosphate-buffered saline (PBS) solution supplemented with streptomycin and penicillin (100 U/ml each). Atherosclerotic lesions were then mechanically separated from the apparently healthy vessel wall. Plaques were thoroughly minced for 15 minutes into 1-mm3 tissue fragments using fine scissors in a volume of Dulbecco??s modified Eagle??s medium (supplemented with 10 µg/ml polymyxin B, streptomycin, and penicillin, and filtered through a 0.22-µm membrane) corresponding to the respective weight of each lesion. The resulting preparations were centrifuged first at 400 x g (15 minutes) and then at 12,500 x g (5 minutes) to remove cells and cell debris. The resulting supernatants referred to as plaque homogenates were subsequently used for flow cytometry analysis of plaque microparticle cellular origins.7 The remaining plaque homogenate was further centrifuged at 20,500 x g for 150 minutes at 4??C to pellet MPs. The supernatant was discarded, and MP pellets were gently suspended in fresh Dulbecco??s modified Eagle??s medium (1/10 of volume corresponding to the respective weight of each lesion) and were used for in vitro purposes (activity and cell stimulation). Endothelial MPs were obtained from human umbilical vein endothelial cells (HUVECs) (fourth passage) maintained in serum-deprived medium for 72 hours. The medium was first centrifuged at 300 x g for 10 minutes to eliminate cell debris and then at 20,500 x g for 150 minutes at 4??C to pellet MPs that were subsequently resuspended in fresh Dulbecco??s modified Eagle??s medium.

Flow Cytometry Analysis of MPs

All analyses were performed on homogenates prepared from atherosclerotic plaques or normal arteries and were performed on a Coulter EPICS XL flow cytometer (Beckman Coulter, Villepinte, France) as recently reported.7 MP gate was defined as events with a 0.1- to 1-µm diameter and then plotted on a FL/FSC fluorescence dot plot to determinate positively labeled MPs by specific antibodies. MP concentration was assessed by comparison to Flowcount calibrator beads. MPs bearing phosphatidylserine were labeled using fluorescein isothiocyanate (FITC)-conjugated Annexin V (Roche Diagnostics, Meylan, France) in the presence or in the absence of CaCl2 (5 mmol/L). The cellular origin of human plaque MPs was determined as follows. We incubated 10 µl of plaque homogenates with different fluorochrome-labeled antibodies or their corresponding isotype-matched IgG controls at room temperature for 30 minutes. Anti-CD4-phycoerythrin (PE) was provided by BD Biosciences Pharmingen (Le Pont-de-Claise, France); anti-CD41-PE-cyanin5 (PC5), anti-CD66b-FITC, anti-CD144-PE, and anti-CD235a-FITC were obtained from Beckman Coulter; anti-CD14-PE was from Caltag Laboratories (Burlingame, CA). MPs derived from endothelial cells, monocytes/macrophages, lymphocytes, erythrocytes, and granulocytes were identified as CD144+, CD14+, CD4+, CD235a+, and CD66b+, respectively. The presence of intracellular smooth muscle cell actin was assayed after MP fixation in paraformaldehyde (2%) and permeabilization by saponin (0.1%). Anti-smooth muscle cell actin antibodies (rabbit IgG, dilution 1:2; LabVision, Runcorn, UK), or rabbit IgG (as a negative control), were incubated for 1 hour at room temperature. MP samples were washed once in PBS, and Alexa Fluor 555 donkey anti-rabbit IgGs (Invitrogen, Cergy-Pontaise, France) were then added for 30 minutes at room temperature. To investigate the presence of TACE/ADAM17 at the surface of plaque MPs, we incubated 10 µl of homogenate with 5 µl of monoclonal anti-human TACE/ADAM17-PE (clone no. 111633) or its corresponding isotype-matched IgG control (R&D Systems, Lille, France) at room temperature for 20 minutes in the dark. The same anti-TACE-PE antibody (5 µl) was used in co-labeling with the anti-CD66b-FITC (20 µl) and the anti-235a-FITC (20 µl). For the co-labeling experiments with PE-conjugated antibodies (20 µl each of anti-CD4, -CD14, and -CD144), we used the monoclonal antibody anti-human TACE/ADAM17 conjugated to fluorescein (clone no. 111633) (5 µl). All subsequent in vitro experiments were performed using the 20,500 x g-pelleted plaque MPs. In these assays, plaque MPs reached a final concentration of 14,250 ?? 4990 Annexin V+ MPs/µl, which corresponded to 11% of the average MP concentration in the plaque.

Fluorogenic Assays of Protease Activity of MPs

Plaque MPs (n = 4) were incubated with the classical pro-TNF mimetic fluorogenic peptide (peptide III; R&D Systems) harboring the consensus sequence A-V (Mca-PLAQAV-Dpa-RSSSR-NH2) cleaved by TACE/ADAM17.19 MPs (10 µl) were suspended in 100 µl of the final activity buffer (25 mmol/L Tris/HCl, pH 8.0, containing 2.5 µmol/L ZnCl2). Fluorogenic peptide I (substrate of MMP-1, -2, -7, -8, -9, -12, -13, -14, -15, and -16), peptide II (substrate of MMP-3 and -10) (R&D Systems), and peptide III were diluted in the activity buffer at the final concentration of 10 µmol/L. Recombinant ectodomain of human TACE/ADAM17 (R&D Systems), used as the positive control, or MPs were extemporaneously mixed with the substrate in a final volume of 100 µl at room temperature to initiate the reaction. TACE/ADAM17 inhibitor was premixed with MPs or recombinant TACE/ADAM17 at 4??C for 15 minutes. Mixtures were immediately delivered in a 96-well black plate and read in a microplate fluorescence reader (Chameleon; Hidex, Turku, Finland). For all substrates, fluorescence-related enzymatic cleavage was monitored at 320-nm excitation and 405-nm emission wavelength for 2 to 3 hours. Blank (buffer and MPs and substrate, separately) was subtracted from sample measurements for calculations.

Cell Culture

HUVECs were isolated and cultured as previously described.20 They were used at the third passage for MP stimulation experiments. The human endothelial cell line ECV-304 and ECV-304 cells stably overexpressing TNF (ECV-304TNF) were cultured as described.21 Monocytic mouse cell line homozygous for TACE/ADAM17 mutation, which deletes the Zn2+ binding domain (TACE/ADAM17Zn/Zn cells), and monocytic mouse TACE/ADAM17 cells expressing active TACE/ADAM17 were kindly provided by Dr. J. Peschon (Amgen Inc., Thousand Oaks, CA).11

Incubation of Cells with MPs

In HUVECs, the constitutive release of TNF or TNFR-1 was less than the detection limit in the present experimental conditions. Thus, HUVECs were first exposed to phorbol ester (PMA; 20 nmol/L, 4 hours), washed with serum-free medium (Dulbecco??s modified Eagle??s medium, 0.2% bovine serum albumin, 0.1 µmol ZnCl2, 1% penicillin/streptomycin, and L-Glu) and incubated for 2 hours in this medium with plaque MPs with or without TMI-2 (1.0 µmol/L) (VF 0.4 ml). ECV-304TNF cells were exposed for 2 hours to plaque-pelleted MPs in the presence or the absence of TMI-2 (1.0 µmol/L) in the above serum-free medium (VF 0.4 ml). Conditioned medium was collected and centrifuged at 20,500 x g to remove the MPs. Cells were lysed with a lysis buffer (PBS, 0.2% Triton X-100, and 1 µg/ml Pefabloc) for subsequent cellular protein assay (enzyme-linked immunosorbent assay and total proteins). In some cases, the 20,500 x g supernatant cleared of the MPs was divided in two parts: one was stored at 4??C, and the other one was centrifuged at 170,000 x g for 16 hours at 4??C (Beckman Optima TLX ultracentrifuge, TLA-100.2 rotor) to sediment exosome-like particles that could be potentially released by cells.22 TNF or TNFR-1 shedding was expressed as the ratio of TNF or TNFR-1 in the culture medium over cellular TNF or TNFR-1, respectively, to take into account variations in cellular protein level.

Western Blotting

MP pellets were lysed with the above lysis buffer containing TMI-2 to prevent the autocleavage of TACE/ADAM17. MP proteins were first submitted to concanavalin A column separation and then submitted to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12% acrylamide or NuPAGE 10% acrylamide; Invitrogen) followed by immunoblotting as previously described.21 TACE/ADAM17 and ADAM10 were revealed with the rabbit polyclonal anti-human TACE/ADAM17 (R&D Systems) and the rabbit polyclonal anti-human ADAM10 (eBioscience, Montrouge, France), respectively. The potential presence of exosomes in MP preparations was analyzed using the anti-TSG-101 antibody (Sigma, L??Isles d??Abeau Chesnes, France) and the anti-lactadherin antibody.23 The MW was estimated with the BenchMark ladder (Invitrogen).

Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)

Total RNA from HUVECs was extracted with the RNeasy mini kit from Qiagen (Courtaboeuf, France). Primer pairs for human TNFR-1 and eEF1 and RT-PCR conditions were described previously.24

Protein Assay

Total protein content of cell lysates was assayed using the bicinchoninic acid protein assay kit from Sigma-Aldrich. Levels of human sTNF, sTNFR-1, sICAM-1, and MCP-1, and murine sTNFR-1 were determined by enzyme-linked immunosorbent assay according to the specifications of the supplier (R&D Systems). Soluble endothelial protein C receptor (sEPCR) was assayed by enzyme-linked immunosorbent assay using the specification of the Asserachrom sEPCR kit (Diagnostica Stago, Asnires, France).

Culture media and reagents were from Gibco BRL (Invitrogen). Bovine serum albumin and Pefabloc were from Sigma. TACE/ADAM17 inhibitor TMI-2 was kindly donated by Dr. J. Levin (Wyeth Research, Cambridge, MA). Recombinant active human TACE/ADAM17 was from R&D Systems.

Results were expressed as mean ?? SD or SEM where indicated. Differences of the means between two groups were evaluated by the Mann-Whitney U-test, with P < 0.05 considered as significant.

Presence of the Mature Form of TACE/ADAM17 on MPs Isolated from Human Atherosclerotic Plaques

Flow cytometry analysis of plaque homogenates (Figure 1A) detected the presence of 119,639 ?? 26,325 Annexin V+ MPs, and 12,867 ?? 2007 TACE/ADAM17+ MPs per mg of plaque (Figure 1B) (mean ?? SEM, n = 25). In contrast, isolation of MPs from healthy human internal mammary arteries did not yield detectable levels of TACE/ADAM17+ MPs (n = 3) (Figure 1B) . TACE+ MP abundance was not different between asymptomatic and symptomatic plaques (13,487 ?? 2529 versus 11,975 ?? 3397 TACE+ MPs/mg plaque, respectively; P = 0.48).

Figure 1. MPs isolated from atherosclerotic human plaque, which contain the mature form of TACE/ADAM17, are of diverse cellular origin and do not contain exosomes: Analysis of TACE/ADAM17 on MPs isolated from human atherosclerotic plaque and human internal mammary arteries. A: Expression of TACE/ADAM17 on MPs from plaque homogenates. This graph is representative of the different plaque preparations. The shaded peak corresponds to negative isotype control. B: Levels of TACE/ADAM17+ MPs in human internal mammary arteries (M.Art., n = 3) and atherosclerotic plaque (plaque, n = 25); values are mean ?? SEM. C: Co-labeling of TACE/ADAM17+ MPs with various cellular markers (from left to right: lymphocytes, monocytes, granulocytes, endothelial cells, and erythrocytes) (n = 12). Results are expressed as percentage of total TACE/ADAM17+ MPs (mean ?? SEM). D: Immunoblotting of the exosomal marker TSG-101 in the post 20,500 x g pellet (left) and corresponding supernatant further centrifuged at 170,000 x g (right). Because protein profiles were different in both fractions (see Ponceau red staining), two times more MP materials (20 µg) than exosomal-like material were loaded. Representative of three different samples analyzed. E: Immunoblotting of TACE/ADAM17 from two different MP preparations containing 2.8 x 106 and 0.1 x 106 Annexin V+ MPs/µl on the middle and right lanes, respectively. The TACE/ADAM17 of MPs in the middle lane is to illustrate that only a highly MP-enriched plaque allows the detection of TACE proform. The right lane is representative of the five MP preparations tested. mTACE and pTACE indicate the positions of the mature and proform of TACE/ADAM17, respectively, validated by the migration of these forms present in COS-7 cells.

The cellular origin of TACE/ADAM17+ MPs was determined by positive co-labeling of TACE/ADAM17 and cellular markers (n = 12, Figure 1C ). TACE/ADAM17+ MPs mostly originated from leukocytes (lymphocytes, granulocytes, and monocytes/macrophages) and also from erythrocytes and endothelial cells. None seems to be of smooth muscle cell origin because TACE/ADAM17+ MPs did not co-label with the smooth muscle cell actin antibody. There was no difference in the cellular origin of TACE/ADAM17+ MPs between symptomatic and asymptomatic plaques (data not shown).

Co-pelleting of plaque MPs with exosomes that sediment at much higher centrifugation speeds than MPs was excluded in the 20,500 x g pellet because there was no significant labeling for the exosomal markers TSG-10125,26 (Figure 1D) or lactadherin (data not shown).22,27,28 In plaque MP pellets, the mature form of TACE/ADAM17 was primarily predominant over the proform. Only a minor band of the proform was detected when a preparation exceptionally rich in MPs was examined (Figure 1E) .

The Mature Form of TACE/ADAM17 Present on Plaque MPs Is Active in Vitro

The presence of the mature form of TACE/ADAM17 on MPs prompted us to investigate whether MPs could be catalytically active in vitro. The human recombinant TACE/ADAM17 ectodomain (10 ng/assay), which contains the active catalytic site, time dependently cleaved the pro-TNF mimetic peptide (peptide III). TMI-2, a preferential inhibitor of TACE/ADAM17,29 inhibited this cleavage by 87 and 100% at 5 and 50 nmol/L, respectively (Figure 2A) . The natural endogenous TACE/ADAM17 inhibitor TIMP-330 (100 nmol/L) inhibited by 85% the TACE/ADAM17-dependent cleavage of the peptide.

Figure 2. MPs isolated from human atherosclerotic plaque are active on fluorogenic peptides that are substrates of TACE/ADAM17 or MMPs. Proteolytic activity of recombinant human TACE/ADAM17 and MPs were measured on various fluorogenic substrates. Details of assay conditions are indicated in Materials and Methods. A: Time-dependent cleavage by recombinant TACE/ADAM17 of the fluorogenic peptide III, mimetic of the cleavage zone of pro-TNF (10 ng) in the presence or not of TMI-2 at 5 and 50 nmol/L or TIMP-3 (100 nmol/L), mean ?? SD of two separate measurements. B: Time-dependent cleavage by MPs (10 µl) of the fluorogenic peptide III in the presence of various concentrations of TMI-2, and TIMP-3. For the sake of clarity, data are presented only as the dose of 100 nmol/L TIMP-3 because 200 nmol/L gave similar inhibition, and SEM in place of SD to avoid overlapping of error bars. n = 4. C: Dose-dependent effect of MPs on the cleavage of the fluorogenic peptide III. For clarity of the graph, only two time points are presented. Values are mean ?? SD of two separate MP preparations. D: Time-dependent cleavage by MPs of fluorogenic peptide I and peptide II in the presence or not of TMI-2 (1 µmol/L). Values are mean ?? SD of four separate MP preparations identical to those used in B.

Plaque MPs pelleted from homogenates cleaved the fluorogenic peptide in a time-dependent manner (Figure 2B) . The MP-induced increase in fluorescence was strongly reduced by TMI-2, in a concentration-dependent manner. Inhibition by TMI-2 averaged 63% at 5 nmol/L and was optimal (87%) at 500 nmol/L, after 120 minutes of incubation (Figure 2B) . TIMP-3 (100 nmol/L) inhibited the MP-induced increase in fluorescence to the same extent as TMI-2 (5 nmol/L) did. The TACE/ADAM17-dependent hydrolysis of the peptide augmented with the increasing concentrations of MPs (Figure 2C) . No TACE/ADAM17 activity could be detected in the supernatant resulting from MP pelleting (45 minutes; 20,500 x g), indicating that the activity is carried by MPs and not by smaller vesicular structures.

We also investigated whether plaque MPs hydrolyze two other fluorogenic peptides that are substrates of several MMPs. Peptide I is cleaved by a large panel of MMPs (MMP-1, -2, -7, -8, -9, -12, -13, -14, -15, and -16), whereas peptide II is preferentially cleaved by MMP-3 and MMP-10. Peptides I and II were not hydrolyzed by recombinant TACE/ADAM17 because the increase in fluorescence after 120 minutes resulting from their cleavage was less than 10% of the initial fluorescence value (data not shown). However, peptides I and II were cleaved by plaque MPs (Figure 2D) , but TMI-2 (1 µmol/L) inhibited their cleavage by only 28 and 20%, respectively, indicating that plaque MPs carry other active protease(s)31 in addition to TACE/ADAM17.

We observed that plaque MPs also contain ADAM10 (Supplemental Figure 1, see http://ajp.amjpathol.org), a protease able to cleave in vitro pro-TNF,32 although its physiological relevance as a TNF convertase is considered to be of minor importance.33,34 When testing the cleavage of the pro-TNF mimetic peptide, we observed that the increase in fluorescence induced by recombinant ADAM10 (100 ng) was only 5% of that recorded for recombinant TACE/ADAM17 (100 ng), indicating that, in TACE/ADAM17 assay conditions, ADAM10 activity on pro-TNF was minimal. Furthermore, ADAM10 activity was inhibited by 10 and 40% in the presence of TMI-2 concentrations (5 and 50 nmol/L, respectively) that inhibited TACE/ADAM17 activity by 87 and 100%. We then investigated if plaque MPs were able to stimulate the cleavage of TACE/ADAM17 substrates present on the cell surface.

Effects of TMI-2 on the Release of TACE/ADAM17 Substrates in Cell-Based Conditions

We first examined TMI-2 inhibitory activity on TNF cleavage, the prototypical substrate of TACE/ADAM17, in ECV-304TNF cells that constitutively express TACE/ADAM17 and produce and release TNF.21 ECV-304TNF cells were stimulated for 1 hour by PMA (200 nmol/L), which preferentially activates TACE/ADAM17 over ADAM10.35,36 As shown in Figure 3A , significant inhibition of TNF release occurred for concentrations of TMI-2 at 0.5 µmol/L and reached a plateau at 1.0 µmol/L (63% inhibition). We investigated the effect of TMI-2 also in a murine monocytic cell line deficient in TACE/ADAM17 activity (TACE/ADAM17Zn/Zn).11 After PMA stimulation, TACE/ADAM17Zn/Zn cells did not significantly release TNF as expected, but presented a residual significant release of TNFR-1 (Figure 3B) , which was much lower than that released by TACE/ADAM17+/+ wild-type cells, as previously described.37 TMI-2 (0.1 to 1.0 µmol/L) dose dependently decreased up to 50% the TNFR-1 release from wild-type cells, without affecting the residual release in TACE/ADAM17Zn/Zn cells. These data indicate that in these monocytic cells, the TACE/ADAM17-independent TNFR-1 shedding is insensitive to TMI-2, even at 1.0 µmol/L. A concentration of 1.0 µmol/L TMI-2 was selected for further studies.

Figure 3. Effects of TMI-2 on TNF and TNFR-1 release in cell-based conditions. A: ECV-304TNF cells were preincubated for 10 minutes with TMI-2 at concentrations ranging from 0 to 2 µmol/L and then stimulated with PMA (200 nmol/L) for 1 hour. Culture medium was collected for TNF assay. Values are mean ?? SD of two separate experiments each performed in duplicate. B: Murine TACE/ADAM17+/+ and TACE/ADAM17Zn/Zn monocytic cells were preincubated with TMI-2 at concentrations ranging from 0 to 1.0 µmol/L and then stimulated with PMA (200 nmol/L) for 1 hour. Culture medium was collected for TNFR-1 assay. Values are mean ?? SD of two separate experiments each performed in triplicate.

MPs Isolated from Human Atherosclerotic Plaques Stimulate the Release of the TACE/ADAM17 Substrates, TNFR-1 and EPCR, from HUVECs

HUVECs, even after PMA stimulation, did not release detectable amounts of TNF, in agreement with previous studies showing that HUVECs did not release TNF.38 We therefore focused on TNFR-1, which was measurable in the culture medium of PMA-treated HUVECs. Exposure of PMA-treated HUVECs to plaque MPs significantly augmented by 76% the amount of TNFR-1 in the culture medium, without affecting TNFR-1 mRNA expression (Figure 4A) . The increased TNFR-1 levels in the culture medium probably did not result from MPs themselves because TNFR-1 was undetectable in plaque MPs. TMI-2 (1.0 µmol/L) significantly reduced by 83 and 70% the amount of TNFR-1 released from unstimulated and MP-stimulated HUVECs, respectively.

Figure 4. MPs isolated from human atherosclerotic plaque activate the release of TNFR-1, ICAM-1, and EPCR from HUVECs. HUVECs were incubated with MPs as described in Materials and Methods. Release in the culture medium of TNFR-1 (A) (values are mean ?? SD, n = 12, each in duplicate), ICAM-1 (B) (values are mean ?? SD, n = 3, each in duplicate), and EPCR (C) (values are mean ?? SD, n = 8, each in duplicate). Significance of MP and TMI-2 effects was calculated by t-test (Mann-Whitney U-test).

We also investigated MP effects on the release of ICAM-1 and EPCR and on the secretion of MCP-1. MPs significantly increased the amount of ICAM-1 by twofold in the culture medium (Figure 4B) , but both basal and MP-induced release of ICAM-1 were unaffected by TMI-2. Plaque MPs significantly augmented the release of EPCR by 62% (Figure 4C) . Both basal and MP-stimulated releases of EPCR were inhibited by TMI-2 by 82 and 85%, respectively. MCP-1 secretion was analyzed as a marker of cell activation. Exposure of HUVECs to plaque MPs did not affect the secretion of MCP-1 (control, 43 ?? 3; with MPs, 41 ?? 7 pg/µg cell proteins; mean ?? SD, n = 6; P = 0.7), and this secretion was unaffected by TMI-2 (1.0 µmol/L), both in control and MP-treated cells. These results suggest that MPs did not alter the cell secretory activity within the 2 hours of incubation. Exposure of PMA-treated HUVECs to the recombinant active soluble ectodomain of TACE/ADAM/17 for 2 hours did not alter the amount of TNFR-1 in the culture medium (data not shown).

MPs Isolated from Human Atherosclerotic Plaques Stimulate the Release of the TACE/ADAM17 Substrates, TNF and TNFR-1, from ECV-304TNF Cells

ECV-304 cells did not release significant measurable amounts of TNF, either under basal conditions or in the presence of plaque MPs (data not shown). However, in ECV-304TNF cells,21 plaque MPs significantly increased the release of TNF by 47% (Figure 5A) . TMI-2 (1.0 µmol/L) inhibited both basal and MP-stimulated release of TNF by 59 and 54%, respectively. Plaque MP-induced TNF release increased with the amount of MPs (Figure 5B) . In the culture medium of ECV-304TNF cells, the amount of TNFR-1 was significantly increased by approximately twofold in the presence of plaque MPs. TMI-2 (1.0 µmol/L) inhibited both basal and MP-stimulated TNFR-1 release by 66 and 70%, respectively (Figure 5C) . The addition of the active soluble ectodomain of TACE/ADAM/17 did not enhance TNF release (data not shown).

Figure 5. MPs isolated from human atherosclerotic plaque activate the release of TNF and TNFR-1 from the human cell line ECV-304TNF. ECV-304TNF cells were incubated with MPs as described in Materials and Methods. A: Release of TNF. Values are mean ?? SD (n = 6, each performed in duplicate). Significance of MP and TMI-2 effects was calculated by t-test (Mann-Whitney U-test). B: Dose-dependent effect of MPs (expressed as Annexin V+/µl) on TNF release. C: Release of TNFR-1 measured on the same samples as in A.

MPs Isolated from Human Atherosclerotic Plaques Did Not Induce the Release of Exosome-Associated Full-Length TNFR-1 and TNF from Endothelial Cells

It has been shown that under certain conditions, full-length TNFR-122 and full-length TNF39 can be found on exosomes. We therefore examined if this process might account for the increase in TNFR-1 or TNF in culture medium of HUVECs and ECV-304TNF cells stimulated by plaque MPs. The conditioned medium was sequentially centrifuged to pellet exosome-like vesicles, possibly released during incubation (see Materials and Methods). In HUVECs (Figure 6A) , tiny amounts of TNFR-1 sedimented at 170,000 x g, whereas the amounts of TNFR-1 present in the supernatants before and after the 170,000 x g centrifugation were not significantly changed whether MPs were added or not. Similar results were obtained with ECV-304TNF cells releasing TNFR-1 (data not shown) or TNF (Figure 6B) . The low amount of TNFR-1 released by HUVECs or ECV-304TNF cells did not allow a reliable detection of the cleaved form by immunoblotting even with enhanced detection. However, the higher amount of TNF released from ECV-304TNF cells exposed to plaque MPs allowed detection of only the cleaved TNF soluble form (Figure 6C) .

Figure 6. Plaque MPs do not induce the release of exosome-associated full-length TNFR-1 and TNF from endothelial cells. HUVECs (A) or ECV-304TNF cells (B) were incubated with or without plaque MPs as indicated in Figures 4 and 5 , respectively. The culture medium was centrifuged at 20,500 x g (45 minutes at 4??C) to pellet MPs, and half of the resulting supernatant (Sn 20,500 x g) was further centrifuged at 170,000 x g (16 hours at 4??C) to pellet exosomes and the other half left for the same time at 4??C. The two supernatants (Sn 20,500 x g; Sn 170,000 x g) and the 170,000 x g pellet were assayed for TNFR-1 (expressed as total amount, pg). For both cellular types, at least 98% of TNFR-1 or TNF was recovered in the 170,000 x g supernatant (Sn). Values are mean ?? SD; n = 3. C: The culture medium of ECV-304TNF cells exposed to plaque MPs (Sn) contain only the cleaved form of TNF compared with 10 µg of recombinant human TNF (rTNF) on the left lane. Representative of two different samples analyzed.

MPs Devoid of TACE/ADAM17 Did Not Enhance TNFR-1 Release from HUVECs

To examine the contribution of TACE/ADAM17 carried by MPs in the observed effects, we took advantage of MPs emitted by apoptotic HUVECs. These MPs did not express detectable levels of TACE/ADAM17 as judged by Western blot and flow cytometry analyses. PMA-stimulated HUVECs were exposed to apoptotic HUVEC-derived MPs (40,260 Annexin V+ MPs/µl) for 2 hours. These MPs did not enhance significant TNFR-1 release, whereas endothelial ICAM-1 release was concomitantly augmented after HUVEC-derived MP exposure by a factor of 5.3 (P < 0.001) (data not shown).

The present study demonstrates that the mature active form of TACE/ADAM17 is present at the surface of MPs isolated from human atherosclerotic lesions and that plaque MPs stimulate the shedding of the TACE/ADAM17 substrates TNF, TNFR-1, and EPCR. The present data also confirm that MPs, which have been previously identified in human plaques, can be isolated from human carotid atherosclerotic lesions but not from healthy arteries.2,7,8 The large preponderance of the mature form of TACE/ADAM17 on MPs raises the question on how MPs are particularly enriched with this form. We recently reported that lipid rafts of THP-1 cell membranes contain specifically the mature form of TACE/ADAM17 and that THP-1-derived MPs are enriched in lipid rafts and mature active form of TACE/ADAM17.40 MPs generated from human blood-derived monocytes are also enriched in lipid rafts.41 Similarly, exosomes in which the mature form of TACE/ADAM17 was found,28 also contain large amounts of lipid rafts.42 Thus, the exclusive sequestration of the mature form of TACE/ADAM17 in lipid rafts may account for the preferential recovery of this form in lipid raft-enriched membrane vesicular structures, herein plaque MPs.

The mature form of TACE/ADAM17 detected on plaque MPs is active as judged by their ability to cleave in vitro a mimetic peptide of the cleavage site of pro-TNF. The pro-TNF cleavage induced by MPs was strongly inhibited by TMI-2, an inhibitor reported to preferentially (500-fold) inhibit TACE/ADAM17 over ADAM10,29 a property confirmed herein, and by TIMP-3, the unique known endogenous TACE/ADAM17 inhibitor.30 Human plaque MPs mainly derive from activated leukocytes and also from erythrocytes.7,8 Interestingly, the present study shows that TACE/ADAM17 labeling is preferentially associated with plaque MPs originating from leukocytes or erythrocytes, but also with MPs of endothelial origin, consistent with previous findings.11,43

Our results demonstrate that plaque MPs stimulate the release of TNF, TNFR-1, and EPCR from endothelial cells. The contribution of the sheddase activity of TACE/ADAM17 carried by plaque MPs to these ectodomain cleavages is supported by several findings. First, TNFR-1 or TNF released by target cells did not pellet at 170,000 x g, excluding a possible release of the full-length molecule associated to exosome-like vesicles.22 Second, TACE/ADAM17-negative-like MPs prepared from apoptotic HUVECs, although not completely assimilated to plaque MPs, did not significantly enhance the shedding of TNFR-1, which supports the role of TACE/ADAM17 carried by plaque MPs in substrate ectodomain cleavage. Third, a global increase in protein synthesis caused by MPs within the 2 hours of incubation might have accounted for higher TNFR-1 or TNF secretion. This was, however, not the case because in HUVECs MPs altered neither TNFR-1 mRNA levels nor MCP-1 secretion, and in ECV-304TNF cells TNF synthesis is under a strong but poorly regulated viral promoter. This is consistent with previous studies examining MP effects on endothelial cell cytokine secretion, for which a significant effect could not be seen before 6 or 12 hours of incubation.44,45 Finally, the contribution of TACE/ADAM17 to MP-induced TNF and TNFR-1 shedding is supported by the TMI-2-dependent inhibition of MP effect on TNF, TNFR-1, and EPCR release. Although TACE/ADAM17 is more efficient in TNF shedding when compared with other ADAMs,33,34 we do not exclude the possibility that these proteases, such as ADAM10, which was detected on plaque MPs, may also contribute to TNF shedding. However, TNFR-1 and EPCR shedding is, to the best of our knowledge, recognized to be processed only by TACE/ADAM17. We also observed that plaque MPs stimulated the shedding of ICAM-1, but neither basal nor MP-induced shedding of ICAM-1 was affected by TMI-2. These findings suggest that TACE/ADAM17 was not involved in ICAM-1 shedding, in agreement with previous studies21,46 but not with another one.47

The cellular mechanisms at the basis of MP-induced TACE/ADAM17 substrate shedding from the cell surface remain to be clarified. These may include i) a direct trans-cleavage, ii) a fusion of MPs with cells and delivery of their active TACE/ADAM17 to the cell plasma membrane subsequently allowing cis-cleavage, and iii) a specific activation of the endothelial sheddase at the cell surface by the vesicle itself through an unknown mechanism. The third hypothesis seems unlikely because there was no enhancement of TNFR-1 release on exposure to TACE/ADAM17-negative-like MPs. Our data also indicated that the recombinant soluble active ectodomain of TACE/ADAM17 is unable to cleave TNFR-1 or TNF at the cell surface. This suggests that the integration of TACE/ADAM17 in a cell-derived structure like MPs, may favor its subsequent delivery at the cell surface through fusion of MPs with membrane and then allowing the cis-cleavage of transmembrane substrates. Indeed, a membrane-to-membrane protein transfer has already been reported after MP interaction with target cells.41,48,49 This process may also account for the drastic enhancement of endothelial sICAM-1 release induced by MPs.

The present data also demonstrate that plaque MPs cause the release of endothelial EPCR, a receptor involved in protein C activation at the cell surface. Higher plasma levels of sEPCR, which are associated with an increased risk of thrombosis, are linked to EPCR gene polymorphisms responsible for increased EPCR ectodomain shedding.50 This results in an elevated procoagulant state at the endothelial surface when compared with the common genotype.51 We show herein that the constitutive and MP-stimulated release of EPCR from HUVECs is strongly inhibited by TMI-2, pointing out TACE/ADAM17 as a likely candidate on plaque MPs to cleave EPCR, consistent with the recent data demonstrating that TACE/ADAM17 cleaves EPCR ectodomain.52 Thus, our data raise the possibility that an enhanced TACE/ADAM17-dependent cleavage of EPCR by MPs could add to their already known prothombogenic effect in human atherosclerotic plaques.8 Taken altogether, these results warrant further study to examine the potential role of TACE/ADAM17+ MPs in atherosclerotic plaque development and rupture.

In conclusion, the present study demonstrates that MPs isolated from human carotid atherosclerotic plaques harbor active TACE/ADAM17 and significantly enhance the shedding of TACE/ADAM17 substrates such as TNF, TNFR-1, and EPCR from endothelial cells. This process could contribute to MP-induced alteration and propagation of inflammatory signals in human atherosclerotic lesions.

【参考文献】
  Hansson GK: Inflammation, atherosclerosis, and coronary artery disease. N Engl J Med 2005, 352:1685-1695

Kockx MM, De Meyer GRY, Muhring J, Jacob W, Bult H, Herman AG: Apoptosis and related proteins in different stages of human atherosclerotic plaques. Circulation 1998, 97:2307-2315

Mallat Z, Tedgui A: Current perspective on the role of apoptosis in atherothrombotic disease. Circ Res 2001, 88:998-1003

Morel O, Toti F, Hugel B, Freyssinet JM: Cellular microparticles: a disseminated storage pool of bioactive vascular effectors. Curr Opin Hematol 2004, 11:156-164

Distler JH, Pisetsky DS, Huber LC, Kalden JR, Gay S, Distler O: Microparticles as regulators of inflammation: novel players of cellular crosstalk in the rheumatic diseases. Arthritis Rheum 2005, 52:3337-3348

Boulanger CM, Amabile N, Tedgui A: Circulating microparticles: a potential prognostic marker for atherosclerotic vascular disease. Hypertension 2006, 48:180-186

Leroyer AS, Isobe H, Leseche G, Castier Y, Wassef M, Mallat Z, Binder BR, Tedgui A, Boulanger CM: Cellular origins and thrombogenic activity of microparticles isolated from human atherosclerotic plaques. J Am Coll Cardiol 2007, 49:772-777

Mallat Z, Hugel B, Ohan J, Leseche G, Freyssinet J-M, Tedgui A: Shed membrane microparticles with procoagulant potential in human atherosclerotic plaques: a role for apoptosis in plaque thrombogenicity. Circulation 1999, 99:348-353

Bonderman D, Teml A, Jakowitsch J, Adlbrecht C, Gyongyosi M, Sperker W, Lass H, Mosgoeller W, Glogar DH, Probst P, Maurer G, Nemerson Y, Lang IM: Coronary no-reflow is caused by shedding of active tissue factor from dissected atherosclerotic plaque. Blood 2002, 99:2794-2800

Garton KJ, Gough PJ, Raines EW: Emerging roles for ectodomain shedding in the regulation of inflammatory responses. J Leukoc Biol 2006, 79:1105-1116

Black RA, Rauch CT, Kozlosky CJ, Peschon JJ, Slack JL, Wolfson MF, Castner BJ, Stocking KL, Reddy P, Srinivasan S, Nelson N, Boiani N, Schooley KA, Gerhart M, Davis R, Fitzner JN, Johnson RS, Paxton RJ, March CJ, Cerretti DP: A metalloproteinase disintegrin that releases tumour-necrosis factor-alpha from cells. Nature 1997, 385:729-733

Moss ML, Jin SL, Milla ME, Bickett DM, Burkhart W, Carter HL, Chen WJ, Clay WC, Didsbury JR, Hassler D, Hoffman CR, Kost TA, Lambert MH, Leesnitzer MA, McCauley P, McGeehan G, Mitchell J, Moyer M, Pahel G, Rocque W, Overton LK, Schoenen F, Seaton T, Su JL, Warner J, Willard D, Becherer JD: Cloning of a disintegrin metalloproteinase that processes precursor tumour-necrosis factor-alpha. Nature 1997, 385:733-736

Smalley DM, Ley K: L-selectin: mechanisms and physiological significance of ectodomain cleavage. J Cell Mol Med 2005, 9:255-266

Reddy P, Slack JL, Davis R, Cerretti DP, Kozlosky CJ, Blanton RA, Shows D, Peschon JJ, Black RA: Functional analysis of the domain structure of tumor necrosis factor- converting enzyme. J Biol Chem 2000, 275:14608-14614

Solomon KA, Pesti N, Wu G, Newton RC: Cutting edge: a dominant negative form of TNF- converting enzyme inhibits ProTNF and TNFRII secretion. J Immunol 1999, 163:4105-4108

Peiretti F, Canault M, Deprez-Beauclair P, Berthet V, Bonardo B, Juhan-Vague I, Nalbone G: Intracellular maturation and transport of tumor necrosis factor alpha converting enzyme. Exp Cell Res 2003, 285:278-285

Srour N, Lebel A, McMahon S, Fournier I, Fugere M, Day R, Dubois CM: TACE/ADAM-17 maturation and activation of sheddase activity require proprotein convertase activity. FEBS Lett 2003, 554:275-283

Canault M, Peiretti F, Kopp F, Bonardo B, Bonzi M-F, Coudeyre J-C, Alessi M-C, Juhan-Vague I, Nalbone G: The TNF alpha converting enzyme (TACE/ADAM17) is expressed in the atherosclerotic lesions of apolipoprotein E-deficient mice: possible contribution to elevated plasma levels of soluble TNF alpha receptors. Atherosclerosis 2006, 187:82-91

Black RA, Doedens JR, Mahimkar R, Johnson R, Guo L, Wallace A, Virca D, Eisenman J, Slack J, Castner B, Sunnarborg SW, Lee DC, Cowling R, Jin G, Charrier K, Peschon JJ, Paxton R: Substrate specificity and inducibility of TACE (tumour necrosis factor -converting enzyme) revisited: the Ala-Val preference, and induced intrinsic activity. Biochem Soc Symp 2003, 70:39-52

Peiretti F, Alessi MC, Henry M, Anfosso F, Juhan-Vague I, Nalbone G: Intracellular calcium mobilization suppresses the TNF--stimulated synthesis of PAI-1 in human endothelial cells. Indications that calcium acts at a translational level. Arterioscler Thromb Vasc Biol 1997, 17:1550-1560

Peiretti F, Canault M, Bernot D, Bonardo B, Deprez-Beauclair P, Juhan-Vague I, Nalbone G: Proteasome inhibition activates the transport and the ectodomain shedding of TNF- receptors in human endothelial cells. J Cell Sci 2005, 118:1061-1070

Hawari FI, Rouhani FN, Cui X, Yu Z-X, Buckley C, Kaler M, Levine SJ: Release of full-length 55-kDa TNF receptor 1 in exosome-like vesicles: a mechanism for generation of soluble cytokine receptors. Proc Natl Acad Sci USA 2004, 101:1297-1302

Silvestre J-S, Thery C, Hamard G, Boddaert J, Aguilar B, Delcayre A, Houbron C, Tamarat R, Blanc-Brude O, Heeneman S, Clergue M, Duriez M, Merval R, Levy B, Tedgui A, Amigorena S, Mallat Z: Lactadherin promotes VEGF-dependent neovascularization. Nat Med 2005, 11:499-506

Lopez S, Peiretti F, Bonardo B, Juhan-Vague I, Nalbone G: Tumor necrosis factor alpha up-regulates in an autocrine manner the synthesis of plasminogen activator inhibitor type-1 during induction of monocytic differentiation of human HL-60 leukemia cells. J Biol Chem 2000, 275:3081-3087

Stoorvogel W, Kleijmeer MJ, Geuze HJ, Raposo G: The biogenesis and functions of exosomes. Traffic 2002, 3:321-330

Th?ry C, Zitvogel L, Amigorena S: Exosomes: composition, biogenesis and function. Nat Rev Immunol 2002, 2:569-579

Th?ry C, Regnault A, Garin J, Wolfers J, Zitvogel L, Ricciardi-Castagnoli P, Raposo G, Amigorena S: Molecular characterization of dendritic cell-derived exosomes: selective accumulation of the heat shock protein hsc73. J Cell Biol 1999, 147:599-610

Stoeck A, Keller S, Riedle S, Sanderson MP, Runz S, Le Naour F, Gutwein P, Ludwig A, Rubinstein E, Altevogt P: A role for exosomes in the constitutive and stimulus-induced ectodomain cleavage of L1 and CD44. Biochem J 2006, 393:609-618

Zhang Y, Hegen M, Xu J, Keith JC, Jin G, Du X, Cummons T, Sheppard BJ, Sun L, Zhu Y, Rao VR, Wang Q, Xu W, Cowling R, Nickerson-Nutter C, Gibbons J, Skotnicki J, Lin L-L, Levin J: Characterization of (2R,3S)-2-(

Amour A, Slocombe PM, Webster A, Butler M, Knight CG, Smith BJ, Stephens PE, Shelley C, Hutton M, Knauper V, Docherty AJ, Murphy G: TNF-alpha converting enzyme (TACE) is inhibited by TIMP-3. FEBS Lett 1998, 435:39-44

Taraboletti G, D??Ascenzo S, Borsotti P, Giavazzi R, Pavan A, Dolo V: Shedding of the matrix metalloproteinases MMP-2, MMP-9, and MT1-MMP as membrane vesicle-associated components by endothelial cells. Am J Pathol 2002, 160:673-680

Rosendahl MS, Ko SC, Long DL, Brewer MT, Rosenzweig B, Hedl E, Anderson L, Pyle SM, Moreland J, Meyers MA, Kohno T, Lyons D, Lichenstein HS: Identification and characterization of a pro-tumor necrosis factor--processing enzyme from the ADAM family of zinc metalloproteases. J Biol Chem 1997, 272:24588-24593

Killar L, White J, Black R, Peschon J: Adamalysins: a family of metzincins including TNF- converting enzyme (TACE). Ann NY Acad Sci 1999, 878:442-452

Zheng Y, Saftig P, Hartmann D, Blobel C: Evaluation of the contribution of different ADAMs to tumor necrosis factor (TNF) shedding and of the function of the TNF ectodomain in ensuring selective stimulated shedding by the TNF convertase (TACE/ADAM17). J Biol Chem 2004, 279:42898-42906

Blobel CP: ADAMS: key components in EGFR signalling and development. Nat Rev Mol Cell Biol 2005, 6:32-43

Ludwig A, Hundhausen C, Lambert MH, Broadway N, Andrews RC, Bickett DM, Leesnitzer MA, Becherer JD: Metalloproteinase inhibitors for the disintegrin-like metalloproteinases ADAM10 and ADAM17 that differentially block constitutive and phorbol ester-inducible shedding of cell surface molecules. Comb Chem High Throughput Screen 2005, 8:161-171

Peschon JJ, Slack JL, Reddy P, Stocking KL, Sunnarborg SW, Lee DC, Russell WE, Castner BJ, Johnson RS, Fitzner JN, Boyce RW, Nelson N, Kozlosky CJ, Wolfson MF, Rauch CT, Cerretti DP, Paxton RJ, March CJ, Black RA: An essential role for ectodomain shedding in mammalian development. Science 1998, 282:1281-1284

Imaizumi T, Itaya H, Fujita K, Kudoh D, Kudoh S, Mori K, Fujimoto K, Matsumiya T, Yoshida H, Satoh K: Expression of tumor necrosis factor- in cultured human endothelial cells stimulated with lipopolysaccharide or interleukin-1. Arterioscler Thromb Vasc Biol 2000, 20:410-415

Zhang H-G, Liu C, Su K, Yu S, Zhang L, Zhang S, Wang J, Cao X, Grizzle W, Kimberly RP: A membrane form of TNF- presented by exosomes delays T cell activation-induced cell death. J Immunol 2006, 176:7385-7393

Tellier E, Canault M, Rebsomen L, Bonardo B, Juhan-Vague I, Nalbone G, Peiretti F: The shedding activity of ADAM17 is sequestered in lipid rafts. Exp Cell Res 2006, 312:3969-3980

Del Conde I, Shrimpton CN, Thiagarajan P, Lopez JA: Tissue-factor-bearing microvesicles arise from lipid rafts and fuse with activated platelets to initiate coagulation. Blood 2005, 106:1604-1611

de Gassart A, Geminard C, Fevrier B, Raposo G, Vidal M: Lipid raft-associated protein sorting in exosomes. Blood 2003, 102:4336-4344

Kieseier BC, Pischel H, Neuen-Jacob E, Tourtellotte WW, Hartung HP: ADAM-10 and ADAM-17 in the inflamed human CNS. Glia 2003, 42:398-405

Nomura S, Tandon NN, Nakamura T, Cone J, Fukuhara S, Kambayashi J: High-shear-stress-induced activation of platelets and microparticles enhances expression of cell adhesion molecules in THP-1 and endothelial cells. Atherosclerosis 2001, 158:277-287

Mesri M, Altieri DC: Leukocyte microparticles stimulate endothelial cell cytokine release and tissue factor induction in a JNK1 signaling pathway. J Biol Chem 1999, 274:23111-23118

Garton KJ, Gough PJ, Philalay J, Wille PT, Blobel CP, Whitehead RH, Dempsey PJ, Raines EW: Stimulated shedding of vascular cell adhesion molecule 1 (VCAM-1) is mediated by tumor necrosis factor--converting enzyme (ADAM 17). J Biol Chem 2003, 278:37459-37464

Tsakadze NL, Sithu SD, Sen U, English WR, Murphy G, D??Souza SE: Tumor necrosis factor- converting enzyme (TACE/ADAM-17) mediates the ectodomain cleavage of intercellular adhesion molecule-1 (ICAM-1). J Biol Chem 2006, 281:3157-3164

Mack M, Kleinschmidt A, Bruhl H, Klier C, Nelson PJ, Cihak J, Plachy J, Stangassinger M, Erfle V, Schlondorff D: Transfer of the chemokine receptor CCR5 between cells by membrane-derived microparticles: a mechanism for cellular human immunodeficiency virus 1 infection. Nat Med 2000, 6:769-775

Mause SF, von Hundelshausen P, Zernecke A, Koenen RR, Weber C: Platelet microparticles: a transcellular delivery system for RANTES-promoting monocyte recruitment on endothelium. Arterioscler Thromb Vasc Biol 2005, 25:1512-1518

Saposnik B, Reny J-L, Gaussem P, Emmerich J, Aiach M, Gandrille S: A haplotype of the EPCR gene is associated with increased plasma levels of sEPCR and is a candidate risk factor for thrombosis. Blood 2004, 103:1311-1318

Qu D, Wang Y, Song Y, Esmon NL, Esmon CT: The Ser219Gly dimorphism of the endothelial protein C receptor contributes to the higher soluble protein levels observed in individuals with the A3 haplotype. J Thromb Haemost 2006, 4:229-235

Qu DWY, Esmon NL, Esmon CT: Regulated endothelial protein C receptor shedding is mediated by tumor necrosis factor- converting enzyme/ADAM17. J Thromb Haemost 2007, 5:395-402


作者单位:From INSERM, U626,* Marseille; the Universit? de la M?diterran?e, Marseille; INSERM, U689, Centre de Recherche Cardiovasculaire Lariboisire, Paris; and the Hôpital Beaujon, Clichy, France

日期:2008年5月29日 - 来自[2007年第169卷第11期]栏目

Finding Vulnerable Atherosclerotic Plaques

From the Department of Internal Medicine/Division of Cardiology (M.M., A.Z.) and Department of Pathology (S.L.), School of Medicine, University of Texas–Houston Health Science Center and Texas Heart Institute, Houston, Tex; the Department of Internal Medicine and President of the University of Texas Health Science Center at Houston (J.T.W.), Medical Director, Texas Heart Institute, and Chief of Cardiology at St. Luke’s Episcopal Hospital, Houston, Tex; the Department of Internal Medicine/Division of Cardiology and Public Health (W.C.), Vice President of Biotechnology, School of Medicine, University of Texas–Houston Health Science Center, and Associate Director of Cardiology Research Texas Heart Institute/St. Luke’s Episcopal Hospital, Houston, Tex, and Division of Cardiology/Department of Internal Medicine, Medical School, The University of Texas Health Science Center at Houston, The Texas Heart Institute at St. Luke’s Episcopal Hospital, and President Bush Center for Cardiovascular Health at Memorial Hermann Hospital, Houston, Tex.

Series Editor: William Haynes

ATVB in Focus

Noninvasive Assessment of Atherosclerosis: from Structure to Function

Previous Brief Review in this Series:

?Choudhury RP, Fuster V, Badimon JJ. Fisher EA, Fayad ZA. MRI and characterization of atherosclerotic plaque: emerging appliations and molecular imaging. 2002;22:1065–1074.

?Bonetti PO, Lerman LO, Lerman A. Endothelial dysfunction: a marker of atherosclerotic risk. 2003;23:168–175.

?Oliver JJ, Webb DJ. Noninvasive assessment of arterial stiffness and risk of atherosclerotic events. 2003;23:554–566.

ABSTRACT

Techniques to identify and treat vulnerable plaques are the focus of enormous research. Some have questioned the benefit of locating individual vulnerable plaque in a multifocal disease. On autopsy, it is found that most deaths are caused by thrombotic occlusion of a single plaque; simultaneous occurrence of 2 occlusive thrombi is rare, but a second vulnerable plaque is common, particularly in acute myocardial infarction (MI). Angiographic progression is poorly predicted by risk factors, and angiographic progression is a weak predictor of MI or death. Intravascular ultrasonography (intravascular ultrasound ) studies find plaque rupture in most MI patients and in approximately half with unstable angina, but in only a minority of patients with stable angina. IVUS identifies a second vulnerable plaque in many patients with unstable angina, and in most MI patients. Angioscopy reveals a very low incidence of a second vulnerable plaque compared with angiography and IVUS, but identifies additional yellow plaques in many patients with stable angina and in most patients with unstable angina or MI. Using thermography catheters and a temperature cutoff of 0.1°C, approximately half the patients with stable angina have >1 hot lesion; however, if the cutoff is 0.2°C, only 15% have a second hot lesion. New imaging techniques may detect additional characteristics of plaques and new predictive models may assess the risk of vulnerable plaques and patients. This approach enables physicians to "buy time" by application of local therapies until systemic therapies stabilize plaques. This may also reduce the risk in subjects in whom systemic therapies do not work.

Techniques to identify and treat vulnerable plaques are the focus of enormous research. Here, we discuss the potential benefit of locating individual vulnerable plaques. We review the multifocal nature of the disease in autopsy series and studies using angiography, IVUS, thermography, and angioscopy. The use of new imaging techniques and the development of predictive models may enable physicians to identify plaques that may benefit from local therapies.

Key Words: plaque rupture ? atherosclerosis ? stents ? coronary imaging

Introduction

Coronary heart disease is a leading cause of death in the United States, accounting for >500 000 lives each year.1 Atherosclerosis is the underlying mechanism for unstable angina, myocardial infarction (MI), and sudden cardiac death. Luminal narrowing of arteries caused by atherosclerotic plaque enlargement causes the chronic ischemic manifestations of coronary heart disease, whereas superimpositions of thrombi over the plaques lead to acute coronary syndromes. To date, angiography has been the method of choice for detecting these problematic arterial lesions. However, this diagnostic technique, which approximately compares the degree of stenosis of arterial lesions relative to the proximal segments of the artery, does not provide insight into the disease state within the artery, and often fails to detect those lesions prone to thrombosis.

A series of landmark angiographic studies in the mid 1980s demonstrated that nearly two-thirds of all MI originate at atherosclerotic lesions that lack hemodynamic significance.2–6 Unfortunately, these "culprit" lesions, which have been termed vulnerable plaques, are undetectable using routine clinical methods of disease evaluation (eg, electrocardiography, angiography, stress test). Of utmost importance is the need to develop new diagnostic techniques for detecting vulnerable plaques. Several studies have shown that some patients may have >1 vulnerable plaque. This has led to the debate on whether it is justified to identify vulnerable plaques.

Reported herein, we review the currently available evidence implicating vulnerable plaques in the development of coronary events and disease progression. For the purpose of this report, the term vulnerable plaque will be used to describe plaques prone to disruption and/or thrombosis. We review emerging techniques for lesion detection and consider the benefits of detecting individual vulnerable plaques and whether focal and/or systemic therapies will be of value.

Pathology of Plaque Rupture and Erosion

Atherosclerosis begins as fatty streaks and over time progresses toward more advances lesions. Rupture of atherosclerotic plaques accounts for nearly two-thirds of all coronary deaths, and plaque erosion accounts for the majority of the remaining cases. Their underlying pathology is markedly heterogeneous, but the ruptured plaques typically have a large core of free cholesterol, necrotic foam cells, cholesterol crystals, hyalinized hemorrhage, calcification, angiogenesis, and inflammation.7 The fibrous cap is thin (50 to 100 micrometers) and deficient in the matrix-synthesizing smooth muscle cells. Almost all ruptured plaques contain numerous macrophages whose matrix metalloproteinases can digest the cap. In contrast, the eroded plaques are denuded of endothelium and have varying degrees of inflammation and superficial ulceration, which promote thrombosis.8 Inflammatory cells play a major role in initiation and progression of the atherosclerosis, and also in the development of its acute complications, by releasing different pro-inflammatory and pro-thrombotic cytokines.

Most ruptured plaques have foci of hemorrhage of varying ages, at varying stages of organization and fibrosis, suggesting discrete episodes of rupture or erosion leading to thrombosis, followed by partial lysis, then re-endothelialization. Serial angiographic studies also suggest that episodic plaque growth is more common than continuous gradual growth, and that many such episodes are often asymptomatic.9 The fact that some acute thrombi remain mural, whereas others progress to complete occlusion, is probably attributable to differences in coagulability. Conditions that favor thrombosis include differences in smoking, hydration, hormones, catecholamines, fibrinogen, cholesterol, erythrocyte count, leukocyte count, platelet count, protein S, and protein C (including mutated protein C, such as factor V Leiden, etc), and local concentration of thromboxane A2, serotonin, ADP, and tissue factor.10 Another likely reason is low flow caused by upstream or downstream stenosis (whether fixed or vasospastic), which promotes thrombosis.11

Techniques for Identification of Vulnerable Plaque

The natural history of vulnerable plaques is obviously difficult to infer from autopsy studies, and angiography has its limitations. It correlates only modestly with other measures of ischemia, and its angiographic progression is poorly predicted by risk factors and angiographic variables.12 Moreover, angiographic progression is a weak predictor of MI or death (relative risk=2.3).13 Even a wide array of clinical and angiographic factors, taken together, have been disappointing in their ability to predict clinical events. Hence, there is intense interest in developing new ways to identify vulnerable plaques by means of new risk factors, such as C-reactive protein (CRP), myeloperoxidase (MPO), lipoprotein-associated phospholipase A2, and pregnancy-associated plasma protein A, and to identify vulnerable plaques by noninvasive means, such as magnetic resonance imaging (MRI), computed tomography (CT), and intravascular methods, such as ultrasound (and the related techniques of integrated backscatter and elastography), thermography, near-infrared spectroscopy, angioscopy, and optical coherence tomography (Figure).14

Different diagnostic methods for detection of vulnerable plaques. Counterclockwise: MRI, CT scan, angiography, IVUS, angioscopy, OCT, thermography, virtual histology, near-infrared (NIR).

Single Versus Multifocal Nature of the Disease

Autopsy Studies

Will it help to identify the individual vulnerable plaque in a disease that is often multifocal or even diffuse? Most deaths are caused by thrombotic occlusion of a single plaque. For example, Levin and Fallon described a careful postmortem angiographic and histological study of patients with fatal MI and did not describe multiple plaque ruptures or multiple ulcerations.15 In a dozen autopsy series of patients with sudden coronary atherosclerotic death from 1970 to 1990, Liberthson’s study was the only one that described any second thrombi. These were noted in 16% of the autopsies.16 Ridolfi and Hutchins studied 494 large fatal myocardial infarcts, almost all of which were caused by thrombotic occlusion of an atherosclerotic coronary artery.17 A second area of ulceration was seen "frequently," but no second thrombus was noted. Horie described 108 autopsies with an occlusive coronary thrombus found in 80%.18 Of these, 91% were complications of a ruptured plaque. Only 6% of patients had a second thrombus. Qiao and Fishbein studied patients with fresh coronary thrombosis and found a second thrombus in 14%.19 In a series of 47 sudden cardiac deaths in those who underwent autopsy Falk, 2 patients had a second occlusive thrombus, although there were 63 foci with rupture and no thrombosis.20

In recent years, the incidence of multiple thrombosis has been lower, but the incidence of plaque rupture or vulnerable plaque (defined as a large atheroma with a thin, inflamed cap, or an erosion) has been higher. This trend may be caused by the increasing use of heparin, aspirin, ticlopidine, and clopidogrel. For example, in a series of 168 sudden coronary deaths described by Davies, 73% of the victims had a mural or occlusive thrombus.21 Fissuring without thrombosis was seen in 7.7%. Among age-matched controls without clinical coronary disease, 8.7% exhibited fissuring without thrombosis, and fissuring was found in 17% of coronary patients who died of other causes. Five percent of these patients had a thrombus, which must have been clinically silent if the death was truly caused by noncoronary cause. In this series, however, no patient with a second thrombus was described.

In a series of sudden coronary deaths caused by plaque rupture and thrombosis reported by Farb, each victim had a second vulnerable plaque (defined as a thin cap overlying an atheroma; other high-risk features, such as inflammation, hemorrhage, calcification, angiogenesis, large core size, or fissuring, were not required).8 Burke studied 113 victims of sudden cardiac death, 59 of whom had coronary thrombosis.22 Rupture was found in 41 of these and 18 others were eroded. There were 79 plaques described as vulnerable because of a thin cap with macrophage infiltration, with or without rupture. These plaques were not thrombosed. Frink reported a series of 83 patients with sudden cardiac death in whom 211 disrupted plaques were found, 102 of which had luminal thrombosis.23 A study of 298 fatal MI by Arbustini found that 2% had no thrombus, 88% had a single thrombus, 9% had 2 thrombi, and 1% had 3 thrombi.24

In some of these series, a distinction was made between a luminal versus occlusive thrombus and between fresh versus organizing thrombus. Most of the second thrombi were mural or recent rather than occlusive or acute. In other words, simultaneous occurrence of 2 occlusive coronary thromboses is rare.

To summarize dozens of previous studies, most fatal infarcts and sudden deaths are caused by coronary thrombosis or by rupture or erosion of a single plaque, and most have an additional 1—and occasionally 2—vulnerable plaques. Many of the latter exhibit rupture, usually with mural rather than occlusive thrombus.25

Angiography Studies

Angiographic series likewise suggest that a second acute thrombus is rare, but a second vulnerable plaque is common, particularly in patients with acute MI.26 Studying angiographic progression in symptomatic patients, Shub noted progression over a 2-year period in only 22% of lesions, with an average of 1.1 progressing lesions per patient.12 This suggests that simultaneous lesion progression is uncommon. Ge et al found no instance of a second vulnerable plaque in an angiographic and ultrasound study of patients with stable and unstable angina pectoris.27 In a study of asymptomatic angiographic progression over an 8-month period in patients with stabilized angina awaiting surgery, Kaski et al reported that among the 24% of patients who showed progression, only 1 lesion progressed in each patient.28 Subsequently, the same group reported an average of 2.6 angiographically complex plaques per patient with unstable angina.29 In that study, a sensitive but nonspecific definition of angiographic complexity was used: either irregularity or angiographic thrombus. Angiographic findings in 350 patients with non–Q-wave MI were described by Kerensky et al.30 Fourteen percent had >1 culprit lesion. Goldstein et al reported a large series of patients with ST-segment elevation/Q-wave MI. Forty percent had a second vulnerable plaque, as defined by 2 or more of 4 criteria: slow flow, ulceration, irregular surface, or angiographic thrombosis.26 If those characteristics were evenly distributed, then 20% of the patients had a second thrombus.

Intravascular Ultrasound Studies

Intravascular ultrasound (IVUS) is better than angiography at measuring the lumen area and can also detect calcification in some areas of low density in the plaque. With recent advances in radiofrequency signal analysis, integrated backscatter may be able to distinguish the very-low-density fatty areas from areas of hemorrhage.31,32 Ultrasound is also able to detect evidence of plaque remodeling and can identify large ruptures and clots, although it is not as sensitive as angiography in the detection of mural thrombosis or fissures. IVUS studies find plaque rupture in most patients with MI and in approximately half the patients with unstable angina or MI but in only a minority of patients with stable angina. Patients with unstable angina or MI have been found to have, in many or most cases, a second vulnerable plaque that is detectable by IVUS.33 Unfortunately, IVUS cannot easily distinguish caps of 0.4 mm in thickness from those that are 0.1 mm or less in thickness. This limitation may be, in the near future, addressed indirectly by the technique of elastography (also known as palpography), which can detect systolic dimpling of the thin-capped soft plaque.34

Optical Coherence Tomography Studies

Like ultrasound, optical coherence tomography (OCT) shows an image from a reflected wave, but because it uses near-infrared (shorter wave lengths than ultrasound) and interferometry, OCT yields much finer spatial resolution (10 to 20 micrometers).35 Unfortunately, OCT requires inflation of a proximal balloon to obstruct blood flow (to flush the artery to obtain a clear field of view). This could cause ischemia and/or vessel injury, and the balloon precludes assessment of the proximal segments, as with angioscopy.

Angioscopy Studies

Intracoronary angioscopy is superior to angiography and ultrasound in detecting fissuring or thrombus, but it characterizes only the luminal surface and requires a proximal balloon. However, useful information has come from angioscopic clinical research. Eighty to 85% of plaques thought to be the culprit in MI are found by angioscopy to be thrombosed, versus half of those with unstable angina. In contrast, only 15% of those with stable angina have a thrombus by angioscopy.36–41 Most of the thrombosed plaques have a complex topography, and most complex plaques are yellow.42 One study suggests that a bright, glistening yellow color is a specific predictor of infarction, although the sensitivity was only 50%.43

With regard to the prevalence of a second complex or thrombosed lesion, Sherman et al described none, whereas Uchida et al found that 20% of patients had a second yellow plaque, and nearly 10% had a second ulcerated plaque.37,43 Asakura et al found a second thrombus in 2% of the patients with MI, but most studies do not mention a second complex or thrombosed lesion.36 All of the many angioscopic series describe a very low incidence of a second disrupted plaque compared with angiographic and IVUS studies, but angioscopy identified additional yellow plaques in many patients with stable angina and most patients with an unstable angina or MI.

Thermography Studies

Normal arteries are uniform in temperature, but in living atherosclerotic plaques, there are hot spots that overlie regions where inflammatory cells are dense or close to the lumen surface. Further evidence that it is the inflammatory cells that generate the heat is suggested by the diminution in temperature by indomethacin in organ culture and by statins in a clinical series.44,45 Moreover, thermal heterogeneity correlated with levels of CRP.46 However, this could be a spurious finding, probably because patients with little thermal heterogeneity in the coronaries can have an elevated CRP caused by inflamed plaques elsewhere, or caused by arthritis, infection, trauma, or malignancy. In contrast, some patients have hot spots in their coronary arteries despite undetectable low levels in high-sensitivity CRP assays.47 The greatest thermal heterogeneity is found in patients with acute MI.48 Those with unstable angina pectoris have less heterogeneity, and patients with stable angina have the least. Yet among patients undergoing a percutaneous coronary intervention, those who do have hot plaques have the highest rate of adverse clinical events, according to Stefanadis et al.49

The number of hot plaques depends on the definition (the arbitrary temperature cutoff) such that if the temperature cutoff is 0.1°C, approximately half the patients with stable angina have >1 hot lesion; whereas if the cutoff is 0.2°C, only 15% have a second hot lesion. Larger temperature differences are found in the Stefanadis study patients probably because—compared with the reports from New Zealand and Europe—most were not using aspirin and statins and had higher CRP levels. Also, Stefanadis used a large, insulated thermistor that occluded flow, minimizing the dilutional cooling by the flowing blood.

Value of Systemic Markers of Inflammation

Maseri et al and other groups have described activation of circulating T lymphocytes in patients with unstable angina or MI.50,51 In patients with fatal MI, Spagnoli et al found nearly as many macrophages in the nonculprit artery as in the culprit artery, although the latter had more activated T cells, and numerous studies have now documented that using a sensitive assay, the inflammatory serum marker, CRP, is elevated in patients with unstable angina and more so with MI.52,53 Moreover, the levels are predictive of risk of MI in every category of patients studied to date.54 The caveat is that patients with renal disease, infection, cancer, autoimmune diseases, liver and kidney disease, and trauma are excluded because these problems can elevate serum CRP levels.

Buffon et al found the gradient (from coronary ostium to sinus) in neutrophil MPO was similar across "culprit left anterior descending coronary artery (LAD)" and "nonculprit" coronary stenoses in other vessels.55 They concluded that plaque inflammation is diffuse. However, a few caveats should be noted. First, neutrophils (the main source of MPO) are rare even in inflamed plaques, moderately numerous in ruptured plaques, and omnipresent in MI plaques and reperfused microvessels.56 Buffon’s myeloperoxidase may have come mainly from micro-infarcts, which are expected, because the study included patients with angina at rest. Thus, their data may relate more to thrombosed or embolizing plaques than to vulnerable plaques. Second, the great cardiac vein does not receive blood exclusively from the LAD but from the entire left ventricle, particularly when there is a tight stenosis in the LAD that leads to the development of collaterals.57,58 Finally, noncoronary sites of inflammation may contribute to circulating levels of MPO.

If Most Patients Have Multiple Inflamed Plaques Detectable by CRP, Why Locate Vulnerable Plaques?

The lesions at higher risk have not only inflammation but also a thin and/or fissured cap. The risk is higher still if the plaque has a large lipid core and a history of rupture or remodeling. Moreover, by LaPlace’s law, the wall stress (and presumably the risk) is higher if the lumen is large. Furthermore, the cap can be vulnerable if the endothelium is prothrombotic ("activated") or absent ("denuded"), or if the surface is irregular, which promotes thrombosis by means of the reduced shear rates and increased stasis.

Thrombosis is also promoted if there is an upstream or downstream flow-limiting stenosis. Moreover, the lesion has a greater capacity to cause clinical ischemia if it is proximal, because of the larger territory it serves. In addition, if collateralization is absent or inadequate, as is usually the case in patients with MI, the risk of infarction is obviously greater.

The severity of inflammation is probably critical, as well. van der Wal et al, who described inflammation as being widespread, nevertheless found that only 2.5% of plaques had moderate or severe infiltration by both macrophages and T lymphocytes, which regulate macrophages.59 Because macrophages can be activated by T cells, and because markers of T-cell activation in the coronary circulation identify patients at high risk for MI or death, it is likely that the lesions at highest risk are those with activated T cells and activated macrophages.

These features are likely to be detectable in the near future by some combination of CT, MRI, angiography, angioscopy, IVUS (particularly with elastography and/or integrated backscatter), OCT, thermography, near-infrared spectroscopy, or molecular imaging.14 Using a more complete list of predictive criteria will increase the sensitivity and specificity of detecting vulnerable plaques. A gradient of risks will result and will permit a cutoff based on whether the risk of that plaque exceeds the risk of therapy. These criteria could provide a relative risk for a given plaque. The absolute risk could then be estimated by summing the number and vulnerability of each individual plaque and adding the patient’s history and symptoms, family history, or genetic information, together with data from the physical examination, electrocardiogram, exercise test, and laboratory findings, such as risk factors for thrombosis and inflammation (including genetic polymorphisms). Long-term outcome studies are needed to estimate and validate the absolute risk score.

Even if the Number of Vulnerable Plaques Adds Prognostic Data, How Will This Help the Patient?

The usefulness of locating vulnerable plaque is unproven, but it must be recognized that prognosis is valuable to the patient, who may defer travel, relocation, or elective surgery, to cite just a few examples. Prognosis may prompt a patient to delay a major purchase or new venture, but not other things, such as a family meeting, a reconciliation, or even a last will and testament. More importantly, information about plaque vulnerability may lead to a life-saving change in diet, activity (eg, initial rest, followed by a gradual exercise program), or goals (eg, better management of low-density lipoprotein, blood pressure, and weight). This may influence the number and dose of medications, the patient’s adherence to a medical regimen, and the frequency of monitoring.

Treatment

Most importantly, vulnerability may be reduced by broader uses of existing therapies, such as the combination of aspirin, clopidogrel, and/or warfarin, of angiotensin-converting enzyme inhibitors (or angiotensin-receptor blockers), plus beta blockers and nitrates. The choice of beta blockers may be particularly important, because these are likely to lower the rate-related risk of rupture and, should ischemia develop, the myocardial contractility, the loss of diastolic filling time, and the vulnerability to ventricular fibrillation.60 Other patients may be treated not only with statins but also with niacin, fibrates, and a resin (or ezetimibe) or newer agents such as cholesteryl ester transfer protein inhibitors and agents that raise high-density lipoprotein.61–64

Administration of high-density lipoprotein cholesterol or apolipoprotein (Apo) A-I Milano are other potentially promising approaches toward stabilizing vulnerable plaques. Infusion of recombinant ApoA-I Milano–phospholipid complexes produces rapid regression and stabilization of atherosclerotic plaques in animal models.65 A recent human clinical trial demonstrated significant regression of coronary atherosclerosis as measured by IVUS after infusion of a recombinant ApoA-I Milano–phospholipid complex.66 Low-density lipoprotein apheresis, already used in some patients with familial hypercholesterolemia, is yet another possibility.67–69

In addition to statins and angiotensin-converting enzyme inhibitors, other potential anti-inflammatory and antiproliferative treatments include corticosteroids, cyclosporin, antithymocyte globulin, and rapamycin.70 Short courses may be well-tolerated.

Numerous novel anti-inflammatory agents and local gene therapies are in development, targeting tumor necrosis factor-, interferon-, monocyte chemotactic protein-1, vascular cell adhesion molecule 1 (VCAM1), and NF-B. Other gene therapies are directed at enhancing local culprit lesions’ availability of prostacyclin or tissue factor pathway inhibitor (tissue factor pathology inhibitor), transforming growth factor-?1, or interleukin-10. Even a simple warm infusion may be helpful, because there is evidence that gentle heating broadly downregulates the inflammatory process.71

However, long-term anti-inflammatory therapies are likely to be contraindicated because of the risks of infection, hypertension, renal failure, impaired healing, etc.72,73 Thus, it may be important to try to eliminate the antigens, such as oxidized low-density lipoprotein cholesterol, and infection (such as influenza).74

Our experience is that many doctors and patients are unaware of the proven benefits of a Mediterranean-type diet and of cost-effective interventions, such as influenza vaccine, in reducing cardiovascular and all-cause mortality.74 However, even these approaches are not likely to stabilize all plaques quickly enough to eliminate the need for interventional therapies, and some patients cannot tolerate polypharmacy. Higher doses of available drugs or novel therapies may work faster in these circumstances.

Clinical trials are needed to test the hypothesis that some patients with vulnerable plaques benefit from stenting of a lesion that is only 50% stenosed and not flow-limiting, but which has vulnerable features. Or a longer stent may be chosen to dilate not only the ischemia-causing stenosis but also the adjacent vulnerable lesion. Because statins do not reduce mortality for several months, local interventional therapies are likely to be needed to "buy time" until the medical regimen confers significant protection. Even if occlusion of the 95% LAD stenosis rarely causes MI (because of extensive collaterals), opening that segment may ensure collateral support in case the right or circumflex coronary occludes. Numerous trials have found improved exercise tolerance but no survival benefit from elective stenting (mainly of flow-limiting lesions in stable patients).75 Yet stenting in acute MI (stenting of proven vulnerable plaques) saves more lives than does thrombolytic therapy.76 This suggests that stenting vulnerable plaques before MI could save even more lives—those now lost to cardiac arrest and heart failure caused by MI.

Finally, given the emerging technologies for local therapy and for gene therapy, it may be possible soon to evaluate peptides, such as fibroblast growth factor-2 or fibroblast growth factor-4, to accelerate endothelialization of eroded plaques, and to stimulate smooth muscle proliferation and matrix synthesis.77,78 Other angiogenic factors are also in clinical trials, although use of vascular endothelial growth factor, the best-studied, is hampered by pro-inflammatory properties.79,80

Necessary Clinical Trials

Determining whether localization of vulnerable plaques can save lives will require trials that determine: (1) who benefits from measuring serum CRP levels and related markers, such as pregnancy-associated plasma protein A, MPO, phospholipase A2, serum amyloid A, homocysteine, fibrinogen, lipoprotein (a), D-dimer, etc; (2) who should have a stress test, CT scan, or MRI scan; and (3) who should undergo coronary angiography, IVUS, thermography, etc. Natural history trials are needed to determine the natural history and long-term outcome of different types of lesions detected by each imaging method. These studies need to be followed by trials to determine what combination of diet, drugs, local therapies, and devices will optimally help a given patient.

Our prediction is that such trials will lead to a graded and patient-specific approach, rather than a one-size-fits-all approach. Because atherosclerosis is a systemic and multifocal disease, systemic and multifocal therapies (or a combination of them) are likely to be required. The effort and cost will be considerable, but the potential savings from prevention of MI and stroke may offset these costs, and the value of the lives saved will be incalculable.

Acknowledgments

Supported in part by Department of Defense grant DAMD 17-01-2-0047. The authors thank Deborah Vela, MD for her help in preparing this manuscript.

References

American Heart Association. 2002 Heart and Stroke Statistical Update. Dallas, Texas: American Heart Association; 2002.

Ambrose JA, Tannenbaum MA, Alexopoulos D, Hjemdahl-Monsen CE, Leavy J, Weiss M, Borrico S, Gorlin R, Fuster V. Angiographic progression of coronary artery disease and the development of myocardial infarction. J Am Coll Cardiol. 1988; 12: 56–62.

Little WC, Constantinescu M, Applegate RJ, Kutcher MA, Burrows MT, Kahl FR, Santamore WP. Can coronary angiography predict the site of a subsequent myocardial infarction in patients with mild-to-moderate coronary artery disease? Circulation. 1988; 78: 1157–1166.

Hackett D, Davies G, Maseri A. Pre-existing coronary stenoses in patients with first myocardial infarction are not necessarily severe. Eur Heart J. 1988; 9: 1317–1323.

Lichtlen PR, Nikutta P, Jost S, Deckers J, Wiese B, Rafflenbeul W. Anatomical progression of coronary artery disease in humans as seen by prospective, repeated, quantitated coronary angiography. Relation to clinical events and risk factors. The INTACT Study Group. Circulation. 1992; 86: 828–838.

Giroud D, Li JM, Urban P, Meier B, Rutishauer W. Relation of the site of acute myocardial infarction to the most severe coronary arterial stenosis at prior angiography. Am J Cardiol. 1992; 69: 729–732.

Virmani R, Kolodgie FD, Burke AP, Farb A, Schwartz SM. Lessons from sudden coronary death: a comprehensive morphological classification scheme for atherosclerotic lesions. Arterioscler Thromb Vasc Biol. 2000; 20: 1262–1275.

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: 1354–1363.

Singh RN. Progression of coronary atherosclerosis. Clues to pathogenesis from serial coronary arteriography. Br Heart J. 1984; 52: 451–461.

Willerson JT, Golino P, Eidt J, Yao S, Buja LM. Evidence that combined thromboxane A2 and serotonin receptor blockade might prevent coronary artery thrombosis and the conversion from chronic to acute coronary heart disease syndromes. Blood Coagul Fibrinolysis. 1990; 1: 211–218.

Feldman CL, Stone PH. Intravascular hemodynamic factors responsible for progression of coronary atherosclerosis and development of vulnerable plaque. Curr Opin Cardiol. 2000; 15: 430–440.

Shub C, Vlietstra RE, Smith HC, Fulton RE, Elveback LR. The unpredictable progression of symptomatic coronary artery disease: a serial clinical-angiographic analysis. Mayo Clin Proc. 1981; 56: 155–160.

Waters D, Craven TE, Lesperance J. Prognostic significance of progression of coronary atherosclerosis. Circulation. 1993; 87: 1067–1075.

Naghavi M, Madjid M, Khan MR, Mohammadi RM, Willerson JT, Casscells SW. New developments in the detection of vulnerable plaque. Curr Atheroscler Rep. 2001; 3: 125–135.

Levin DC, Fallon JT. Significance of the angiographic morphology of localized coronary stenoses: histopathologic correlations. Circulation. 1982; 66: 316–320.

Liberthson RR, Nagel EL, Hirschman JC, Nussenfeld SR, Blackbourne BD, Davis JH. Pathophysiologic observations in prehospital ventricular fibrillation and sudden cardiac death. Circulation. 1974; 49: 790–798.

Ridolfi RL, Hutchins GM. The relationship between coronary artery lesions and myocardial infarcts: ulceration of atherosclerotic plaques precipitating coronary thrombosis. Am Heart J. 1977; 93: 468–486.

Horie T, Sekiguchi M, Hirosawa K. Coronary thrombosis in pathogenesis of acute myocardial infarction. Histopathological study of coronary arteries in 108 necropsied cases using serial section. Br Heart J. 1978; 40: 153–161.

Qiao JH, Fishbein MC. The severity of coronary atherosclerosis at sites of plaque rupture with occlusive thrombosis. J Am Coll Cardiol. 1991; 17: 1138–1142.

Falk E. Plaque rupture with severe pre-existing stenosis precipitating coronary thrombosis. Characteristics of coronary atherosclerotic plaques underlying fatal occlusive thrombi. Br Heart J. 1983; 50: 127–134.

Davies MJ. Anatomic features in victims of sudden coronary death. Coronary artery pathology. Circulation. 1992; 85: I19–24.

Burke AP, Farb A, Malcom GT, Liang YH, Smialek J, Virmani R. Coronary risk factors and plaque morphology in men with coronary disease who died suddenly. N Engl J Med. 1997; 336: 1276–1282.

Frink RJ. Chronic ulcerated plaques: new insights into the pathogenesis of acute coronary disease. J Invasive Cardiol. 1994; 6: 173–185.

Arbustini E, Dal Bello B, Morbini P, Burke AP, Bocciarelli M, Specchia G, Virmani R. Plaque erosion is a major substrate for coronary thrombosis in acute myocardial infarction. Heart. 1999; 82: 269–272.

Mann J, Davies MJ. Mechanisms of progression in native coronary artery disease: role of healed plaque disruption. Heart. 1999; 82: 265–268.

Goldstein JA, Demetriou D, Grines CL, Pica M, Shoukfeh M, O’Neill WW. Multiple complex coronary plaques in patients with acute myocardial infarction. N Engl J Med. 2000; 343: 915–922.

Ge J, Chirillo F, Schwedtmann J, Gorge G, Haude M, Baumgart D, Shah V, von Birgelen C, Sack S, Boudoulas H, Erbel R. Screening of ruptured plaques in patients with coronary artery disease by intravascular ultrasound. Heart. 1999; 81: 621–627.

Kaski JC, Chester MR, Chen L, Katritsis D. Rapid angiographic progression of coronary artery disease in patients with angina pectoris. The role of complex stenosis morphology. Circulation. 1995; 92: 2058–2065.

Garcia-Moll X, Coccolo F, Cole D, Kaski JC. Serum neopterin and complex stenosis morphology in patients with unstable angina. J Am Coll Cardiol. 2000; 35: 956–962.

Kerensky RA, Wade M, Deedwania P, Boden WE, Pepine CJ. Revisiting the culprit lesion in non-Q-wave myocardial infarction. Results from the VANQWISH trial angiographic core laboratory. J Am Coll Cardiol. 2002; 39: 1456–1463.

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.

Nissen SE. Application of intravascular ultrasound to characterize coronary artery disease and assess the progression or regression of atherosclerosis. Am J Cardiol. 2002; 89: 24B–31B.

Rioufol G, Finet G, Ginon I, Andre-Fouet X, Rossi R, Vialle E, Desjoyaux E, Convert G, Huret JF, Tabib A. Multiple atherosclerotic plaque rupture in acute coronary syndrome: a three-vessel intravascular ultrasound study. Circulation. 2002; 106: 804–808.

de Korte CL, van der Steen AF. Intravascular ultrasound elastography: an overview. Ultrasonics. 2002; 40: 859–865.

Jang IK, Bouma BE, Kang DH, Park SJ, Park SW, Seung KB, Choi KB, Shishkov M, Schlendorf K, Pomerantsev E, Houser SL, Aretz HT, Tearney GJ. Visualization of coronary atherosclerotic plaques in patients using optical coherence tomography: comparison with intravascular ultrasound. J Am Coll Cardiol. 2002; 39: 604–609.

Asakura M, Ueda Y, Yamaguchi O, Adachi T, Hirayama A, Hori M, Kodama K. Extensive development of vulnerable plaques as a pan-coronary process in patients with myocardial infarction: an angioscopic study. J Am Coll Cardiol. 2001; 37: 1284–1288.

Sherman CT, Litvack F, Grundfest W, Lee M, Hickey A, Chaux A, Kass R, Blanche C, Matloff J, Morgenstern L, et al. Coronary angioscopy in patients with unstable angina pectoris. N Engl J Med. 1986; 315: 913–919.

Waxman S, Sassower MA, Mittleman MA, Zarich S, Miyamoto A, Manzo KS, Muller JE, Abela GS, Nesto RW. Angioscopic predictors of early adverse outcome after coronary angioplasty in patients with unstable angina and non-Q-wave myocardial infarction. Circulation. 1996; 93: 2106–2113.

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.

Van Belle E, Lablanche JM, Bauters C, Renaud N, McFadden EP, Bertrand ME. Coronary angioscopic findings in the infarct-related vessel within 1 month of acute myocardial infarction: natural history and the effect of thrombolysis. Circulation. 1998; 97: 26–33.

Mizuno K, Miyamoto A, Satomura K, Kurita A, Arai T, Sakurada M, Yanagida S, Nakamura H. Angioscopic coronary macromorphology in patients with acute coronary disorders. Lancet. 1991; 337: 809–812.

Alfonso F, Fernandez-Ortiz A, Goicolea J, Hernandez R, Segovia J, Phillips P, Banuelos C, Macaya C. Angioscopic evaluation of angiographically complex coronary lesions. Am Heart J. 1997; 134: 703–711.

Uchida Y, Nakamura F, Tomaru T, Morita T, Oshima T, Sasaki T, Morizuki S, Hirose J. Prediction of acute coronary syndromes by percutaneous coronary angioscopy in patients with stable angina. Am Heart J. 1995; 130: 195–203.

Madjid M, Naghavi M, Malik BA, Litovsky S, Willerson JT, Casscells W. Thermal detection of vulnerable plaque. Am J Cardiol. 2002; 90: 36L–39L.

Stefanadis C, Toutouzas K, Vavuranakis M, Tsiamis E, Tousoulis D, Panagiotakos DB, Vaina S, Pitsavos C, Toutouzas P. Statin treatment is associated with reduced thermal heterogeneity in human atherosclerotic plaques. Eur Heart J. 2002; 23: 1664–1669.

Stefanadis C, Diamantopoulos L, Dernellis J, Economou E, Tsiamis E, Toutouzas K, Vlachopoulos C, Toutouzas P. Heat production of atherosclerotic plaques and inflammation assessed by the acute phase proteins in acute coronary syndromes. J Mol Cell Cardiol. 2000; 32: 43–52.

Webster M, Stewart J, Ruygrok P, Ormiston J, Scott D, Gray B, Fraser A. Intracoronary thermogrophy with a multiple thermocouple catheter: Initial human experience (abstract). Am J Cardiol. 2002; 90: 24H.

Stefanadis C, Diamantopoulos L, Vlachopoulos C, Tsiamis E, Dernellis J, Toutouzas K, Stefanadi E, Toutouzas P. Thermal heterogeneity within human atherosclerotic coronary arteries detected in vivo: A new method of detection by application of a special thermography catheter. Circulation. 1999; 99: 1965–1971.

Stefanadis C, Toutouzas K, Tsiamis E, Stratos C, Vavuranakis M, Kallikazaros I, Panagiotakos D, Toutouzas P. Increased local temperature in human coronary atherosclerotic plaques: an independent predictor of clinical outcome in patients undergoing a percutaneous coronary intervention. J Am Coll Cardiol. 2001; 37: 1277–1283.

Neri Serneri GG, Prisco D, Martini F, Gori AM, Brunelli T, Poggesi L, Rostagno C, Gensini GF, Abbate R. Acute T-cell activation is detectable in unstable angina. Circulation. 1997; 95: 1806–1812.

Caligiuri G, Liuzzo G, Biasucci LM, Maseri A. Immune system activation follows inflammation in unstable angina: pathogenetic implications. J Am Coll Cardiol. 1998; 32: 1295–1304.

Liuzzo G, Kopecky SL, Frye RL, Fallon WMO, Maseri A, Goronzy JJ, Weyand CM. Perturbation of the T-cell repertoire in patients with unstable angina. Circulation. 1999; 100: 2135–2139.

de Beer FC, Hind CR, Fox KM, Allan RM, Maseri A, Pepys MB. Measurement of serum C-reactive protein concentration in myocardial ischaemia and infarction. Br Heart J. 1982; 47: 239–243.

Ridker PM, Hennekens CH, Buring JE, Rifai N. C-reactive protein and other markers of inflammation in the prediction of cardiovascular disease in women. N Engl J Med. 2000; 342: 836–843.

Buffon A, Biasucci LM, Liuzzo G, D’Onofrio G, Crea F, Maseri A. Widespread coronary inflammation in unstable angina. N Engl J Med. 2002; 347: 5–12.

Naruko T, Ueda M, Haze K, van der Wal AC, van der Loos CM, Itoh A, Komatsu R, Ikura Y, Ogami M, Shimada Y, Ehara S, Yoshiyama M, Takeuchi K, Yoshikawa J, Becker AE. Neutrophil infiltration of culprit lesions in acute coronary syndromes. Circulation. 2002; 106: 2894–2900.

Gabella G. Cardiac Veins. In: Williams PL, ed. Gray’s Anatomy. New York: Churchill Livingstone; 1995.

Russell CJ, Exley AR, Ritchie AJ. Widespread coronary inflammation in unstable angina. N Engl J Med. 2003; 348: 1931.

van der Wal AC, Becker AE, van der Loos CM, Das PK. Site of intimal rupture or erosion of thrombosed coronary atherosclerotic plaques is characterized by an inflammatory process irrespective of the dominant plaque morphology. Circulation. 1994; 89: 36–44.

Waeber B, Brunner HR, Burnier M, Cohn JN. Hypertention. In: Willerson JT, Cohn JN, eds. Cardiovascular Medicine. Churchill Livingstone; 2000.

Dujovne CA. New lipid lowering drugs and new effects of old drugs. Curr Opin Lipidol. 1997; 8: 362–368.

Wang T, Stafford R, Ausiello J, Chaisson C. Randomized clinical trials and recent patterns in the use of statins. Am Heart J. 2001; 141: 957–963.

Kerzner B, Corbelli J, Sharp S, Lipka LJ, Melani L, LeBeaut A, Suresh R, Mukhopadhyay P, Veltri EP. Efficacy and safety of ezetimibe coadministered with lovastatin in primary hypercholesterolemia. Am J Cardiol. 2003; 91: 418–424.

de Grooth GJ, Kuivenhoven JA, Stalenhoef AFH, de Graaf J, Zwinderman AH, Posma JL, van Tol A, Kastelein JJP. Efficacy and safety of a novel cholesteryl ester transfer protein inhibitor, JTT-705, in humans: A randomized phase II dose-response study. Circulation. 2002; 105: 2159–2165.

Shah PK, Yano J, Reyes O, Chyu KY, Kaul S, Bisgaier CL, Drake S, Cercek B. High-dose recombinant apolipoprotein A-I(Milano) mobilizes tissue cholesterol and rapidly reduces plaque lipid and macrophage content in apolipoprotein e-deficient mice. Potential implications for acute plaque stabilization. Circulation. 2001; 103: 3047–3050.

Nissen SE, Tsunoda T, Tuzcu EM, Schoenhagen P, Cooper CJ, Yasin M, Eaton GM, Lauer MA, Sheldon WS, Grines CL, Halpern S, Crowe T, Blankenship JC, Kerensky R. Effect of recombinant ApoA-I Milano on coronary atherosclerosis in patients with acute coronary syndromes: a randomized controlled trial. JAMA. 2003; 290: 2292–2300.

Barter PJ. Coronary plaque regression: role of low density lipoprotein-apheresis. J Am Coll Cardiol. 2002; 40: 228–230.

Nishimura S, Sekiguchi M, Kano T, Ishiwata S, Nagasaki F, Nishide T, Okimoto T, Kutsumi Y, Kuwabara Y, Takatsu F, Nishikawa H, Daida H, Yamaguchi H. Effects of intensive lipid lowering by low-density lipoprotein apheresis on regression of coronary atherosclerosis in patients with familial hypercholesterolemia: Japan Low-density Lipoprotein Apheresis Coronary Atherosclerosis Prospective Study (L-CAPS). Atherosclerosis. 1999; 144: 409–417.

Hoffmann U, Derfler K, Haas M, Stadler A, Brady TJ, Kostner K. Effects of combined low-density lipoprotein apheresis and aggressive statin therapy on coronary calcified plaque as measured by computed tomography. Am J Cardiol. 2003; 91: 461–464.

Moses JW, Leon MB, Popma JJ, Fitzgerald PJ, Holmes DR, O’Shaughnessy C, Caputo RP, Kereiakes DJ, Williams DO, Teirstein PS, Jaeger JL, Kuntz RE. Sirolimus-eluting stents versus standard stents in patients with stenosis in a native coronary artery. N Engl J Med. 2003; 349: 1315–1323.

Geng Y-J, Phillips JE, Mason RP, Casscells SW. Cholesterol crystallization and macrophage apoptosis: implication for atherosclerotic plaque instability and rupture. Biochem Pharmacol. 2003; 66: 1485–1492.

Keane J, Gershon S, Wise RP, Mirabile-Levens E, Kasznica J, Schwieterman WD, Siegel JN, Braun MM. Tuberculosis associated with Infliximab, a tumor necrosis factor {alpha}-neutralizing agent. N Engl J Med. 2001; 345: 1098–1104.

Chung ES, Packer M, Lo KH, Fasanmade AA, Willerson JT. Randomized, double-blind, placebo-controlled, pilot trial of Infliximab, a chimeric monoclonal antibody to tumor necrosis factor-{alpha}, in patients with moderate-to-severe heart failure: Results of the anti-TNF therapy against congestive heart failure (ATTACH) trial. Circulation. 2003; 107: 3133–3140.

Madjid M, Naghavi M, Litovsky S, Casscells SW. Influenza and cardiovascular disease: a new opportunity for prevention and the need for further studies. Circulation. 2003; 108: 2730–2736.

King I, Spencer B. Dilate or defer? view of a skeptic. J Am Coll Cardiol. 2003; 42: 1171–1172.

Grines CL, Serruys P, O’Neill WW. Fibrinolytic therapy: Is it a treatment of the past? Circulation. 2003; 107: 2538–2542.

Casscells W. Migration of smooth muscle and endothelial cells. Critical events in restenosis. Circulation. 1992; 86: 723–729.

Simons M, Annex BH, Laham RJ, Kleiman N, Henry T, Dauerman H, Udelson JE, Gervino EV, Pike M, Whitehouse MJ, Moon T, Chronos NA. Pharmacological treatment of coronary artery disease with recombinant fibroblast growth factor-2: Double-blind, randomized, controlled clinical trial. Circulation. 2002; 105: 788–793.

Banai S, Jaklitsch MT, Shou M, Lazarous DF, Scheinowitz M, Biro S, Epstein SE, Unger EF. Angiogenic-induced enhancement of collateral blood flow to ischemic myocardium by vascular endothelial growth factor in dogs. Circulation. 1994; 89: 2183–2189.

Celletti FL, Waugh JM, Amabile PG, Brendolan A, Hilfiker PR, Dake MD. Vascular endothelial growth factor enhances atherosclerotic plaque progression. Nat Med. 2001; 7: 425–429.

 

日期:2007年5月18日 - 来自[2004年第24卷第10期]栏目

Reduction of Atherosclerotic Plaques by Lysosomal Acid Lipase Supplementation

From the Division and Program in Human Genetics (H.D., G.A.G.) and Division of Pathology and Laboratory Medicine (D.P.W.), Children’s Hospital Research Foundation, Cincinnati, OH, 45229; and Genzyme Corporation (S.S., N.W., M.L.), Cambridge, MA.

Correspondence to Gregory A. Grabowski, MD, Professor and Director, Division and Program in Human Genetics, 3333 Burnet Ave, Cincinnati, OH 45229-3039. E-mail greg.grabowski@cchmc.org

    Abstract

Objective— Proof of principle is presented for targeted enzyme supplementation by using lysosomal acid lipase to decrease aortic and coronary wall lipid accumulation in a mouse model of atherosclerosis.

Methods and Results— Mice with LDL receptor deficiency were placed on an atherogenic diet and developed predictable aortic and coronary atheroma. -Mannosyl-terminated human lysosomal acid lipase (phLAL) was produced in Pichia pastoris, purified, and administered intravenously to such mice with either early or late lesions. phLAL injections reduced plasma, hepatic, and splenic cholesteryl esters and triglycerides in affected mice. phLAL was detected in hepatic Kupffer cells and in atheromatous foam cells. Repeated enzyme injections were well tolerated, with no obvious adverse effects. In addition, the coronary and aortic atheromatous lesions were (1) eliminated in their early stages and (2) quantitatively and qualitatively reduced in their advanced stages.

Conclusion— These results support the potential utility of lysosomal acid lipase supplementation for the treatment of atherosclerosis, a leading cause of mortality and morbidity in Westernized nations.

Key Words: lysosomal acid lipase ? atherosclerosis ? lesion ? mice

    Introduction

Atherosclerosis is the number 1 cause of mortality and morbidity in the developed countries. A number of interventions or preventions delay the consequences of atherosclerosis, eg, low cholesterol diet and exercise, HMG-CoA reductase inhibitors, and coronary artery bypass. However, they are not suitable for all patients, and few have been shown to promote regression of lesions. Therefore, new approaches are needed for the treatment and prevention of atherosclerosis. Several stages characterize the progression of atherosclerotic lesions.1,2 The earliest lesion is the "fatty streak," an aggregation of lipid-rich macrophages and T-lymphocytes within the intimae layer of an artery. The fatty streaks evolve into intermediate lesions that have a layer of foamy macrophages and smooth muscle cells. This develops into complex and occlusive lesions, as well as fibrous plaques. These plaques have a dense cap of connective tissue, with embedded smooth muscle cells overlaying a core of lipid and necrotic debris. Macrophages are present at all lesional stages with excessive cholesterol and cholesteryl esters in lysosomes. Continuing development of atherosclerotic plaques requires progressive macrophage processing of cholesteryl esters in and through the lysosomes. Perpetuation and maturation of the plaques depends on additional complex inflammatory and scarring processes. Lysosomal acid lipase (LAL) is the only hydrolase for cleavage of cholesteryl esters delivered to the lysosomes.3

The receptors that mediate cholesteryl ester uptake into macrophage include those for LDL and oxidized LDL (oxLDL), ie, LDL receptor (LDL-R), the scavenger receptors type AI and AII (SR-AI and SR-AII),4–6 the scavenger receptor type B1 (SR-BI),7–9 CD36,10 and the LDL receptor related protein (LRP).11 All of these, except SR-BI, direct associated lipids to the lysosome.12–15 Furthermore, modification of LDL by oxidation, aggregation, and glycation occurs in the atherosclerotic lesions by histochemical and biochemical studies.16–20 The delivery of oxidized LDL to lysosomes of macrophages decreases cholesteryl ester hydrolysis, apolipoprotein degradation, and cholesterol to acyl-CoA/cholesterol acyltransferase (ACAT) activity.21–25 Aggregated LDL induces cholesteryl ester accumulation in cultured macrophages.18,20,26,27 Also, LDL particles, modified by advanced glycation, promote cholesterol accumulation in cultured macrophages.19,28 Consequently, enhancement of LAL activity in macrophages could provide a means to decrease accumulated, pathologic cholesteryl esters and triglycerides (TGs) that are causally related to atherosclerosis.

LAL is an essential enzyme for the cleavage of cholesteryl esters and TGs delivered to the lysosomes. The biochemical phenotype of the LAL knockout mouse indicates that other lipases cannot compensate for the loss of LAL activity in the lysosomes.3 The endogenously synthesized mature, soluble glycoprotein has 372 amino acids, is trafficked to the lysosome by the mannose-6-phosphate receptor, and has active site properties similar to those of other lipases.29,30 LAL contributes to cholesterol and fatty acid homeostasis by several mechanisms. Once cholesteryl esters are cleaved by LAL, free cholesterol exits the lysosome and participates in the modulation of cholesterol and fatty acid metabolism via the sterol regulatory element-binding protein (SREBP) system.31–34 Free cholesterol, a product of LAL hydrolysis of CE, leaves the lysosome and leads to downregulation of endogenous cellular cholesterol synthesis and LDL receptor-mediated uptake through the cholesterol sensing mechanisms of the cell. The free fatty acids generated by LAL and their metabolites released from the lysosome could be ligands for the peroxisome proliferated-activator receptor- (PPAR), a master regulator of lipogenesis, macrophage maturation, anti-inflammation, and glucose homeostasis in the body.13,35–38 Deficiency of LAL via targeted gene disruption leads to a multisystemic disease that includes the phenotype of human Wolman disease, aberrant cholesterol and TG metabolism, and macrophage proliferation.39 These diverse effects support the important and central role of LAL in the control of cholesterol and fat metabolism and macrophage proliferation, as well as a potential role in the treatment of atherosclerosis.

Expression of the human LAL (hLAL) in Pichia pastoris (phLAL) systems produced active hLAL that was targeted to macrophage lysosomes.39 Intravenous injections of phLAL into LAL-deficient mice (lal-/-) corrected the lipid storage phenotype in multiple tissues.39 Here, phLAL was administered to the mouse model of atherosclerosis, the LDL-receptor knockout (ldlr-/-) mice on a high fat/high cholesterol diet (HFCD). Repeated doses of phLAL resulted in almost complete elimination of early stage lesions and significant improvement in the quality and quantity of advanced lesions.

    Methods

Animal Models

Homozygous ldlr-/- and apoE-/- mice in a C57BL/6 background were from The Jackson Laboratory (Bar Harbor, Maine). Homozygous lal-/- mice were created in this laboratory.3 Cross breeding of lal-/- and apoE-/- mice generated double knockout of lal-/-;apoE-/- mice. Mice were genotyped by PCR using tail DNA.3 The ldlr-/- mice were fed a high fat/high cholesterol diet (HFCD, 7.5% cocoa butter fat, 1.25% cholesterol, TD86257, Harlan Tekland) from the age of 1.5 months. All animals had ad libitum access to food and water. The animal experiments were performed according to National Institutes of Health guidelines and were approved by IACUC at Cincinnati Children’s Hospital Research Foundation. phLAL was produced in Pichia pastoris as previously described.39

Study Design

Experiments were designed to evaluate the efficacy of LAL enzyme supplementation therapy to treat the athero/arterial-sclerosis at the early foam cell stage (group A) and at the advanced lesional stages (group B). The group A mice received phLAL (1.5 U/injection, n=8) after 1 month of HFCD, ie, beginning at 2.5 months of age. The group B mice received phLAL (6 U/injection, n=7) after 2 months of HFCD, ie, beginning at 3.5 months of age. The control mice for each group were injected with PBS (n=4 to 5). Mice in both groups started HFCD at 1.5 months of age and continued HFCD for the entire study period. PBS or phLAL injections (100 μL) were done every third day for 10 injections. Mice were harvested 2 days after the 10th injection. Tissues were processed for histologic, immunohistochemical, and biochemical analyses. phLAL was 96% pure on silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Plasma Lipids, Lipoprotein Profile, and Tissue Lipids

Blood was collected from the inferior vena cava (IVC) of anesthetized mice (ketamine, acepromazine, and xylazine, 0.2 mL, intraperitoneal injection) after an overnight fast. Plasma was collected (5000g, 10 minutes, 4°C) and stored (4°C and -20°C). Plasma free cholesterol, cholesteryl esters, and TG concentrations were tested by using colorimetric assays (COD-PAP kit for free cholesterol, Wako Chemicals GmbH; TGs/GB kit and cholesterol/HP kit were from Boehringer, GmbH). Plasma from group B mice was pooled (200 μL) and subjected to fast protein liquid chromatography by using 2 Superose 6 HR columns (Pharmacia Biotech Inc.).39,40 Cholesterol concentrations were determined in each fraction (0.5 mL; cholesterol/HP kits). Total lipids were extracted from liver and spleen by the Folch method.3,41 TG concentrations were estimated as described.39,42,43 Total tissue cholesterol concentrations were estimated using O-phthalaldehyde.39,43,44

Tissue Preparation and Morphometric Analyses of Atherosclerotic Lesions

After blood collection under anesthesia, livers, spleens, and small intestines were removed and processed for histologic and lipid analyses. The hearts were perfused through the left ventricle with PBS followed by 10 mL of 4% paraformaldehyde. The aortas were isolated under microscopy and photographed. For qualitative analysis, hearts were split in half along the aortas, embedded in paraffin with the open aorta upward, and sectioned at 4-μm thickness from midsagittal to parasagittal regions of the heart. Aortic arches were cut to 5 fragments at same relative positions, embedded in paraffin with distal end upward, and cross-sectioned at 4-μm thickness from the distal end of each segment of the aorta. Samples were mounted on slides and stained with hematoxylin/eosin (H&E), elastic Van Gieson, or Gomori’s trichrome. For quantitative analyses, 40 to 60 sections (7 μm) were collected from each heart with same orientation and stained with H&E. Lesional area was defined by vessel wall involvement on H&E staining. Images of every fifth sections were captured with a digital camera and analyzed by blinded readers by using computerized morphometric (MetaMorph) software. Briefly, images at the same magnification were captured, and a grid (19x19 lines) divided the image into 400 U. The area covered by lesional macrophages, collagen positive extracellular matrix and necrotic core, and the vessel wall were numbered and manually counted. The areas were converted into μM2 by using an integrated size marker in the MetaMorph program. Staining by Gomori’s trichrome and elastic Van Gieson were used to identify the collagen positive areas, macrophage cell areas, and elastic vessel wall areas. The lesional area was defined from the first elastin positive vessel wall section. Extensive sections and H&E staining (160 to 210 sections, 7 μm) were also performed for one representative heart in control group and 2 representative heart in treated group A. The positive coronary arterial lesions were scored by a blinded reader. The severity of the coronary lesion was scored by a percentage of the sections that had atherosclerotic lesions in arterial vessels and by an estimation of vessel area that was blocked by the lesion as defined in the legend for Table I (available online at http://atvb.ahajournals.org).

Immunohistochemical Staining

Immunohistochemical analyses were performed with frozen or paraffin-embedded aortic valve sections by using rabbit anti-hLAL antibody (1:500) or rat anti-mouse Mac-3 monoclonal antibody (PharMingen). Biotinylated conjugated anti-rabbit or anti-rat IgG (Vector) were used as secondary antibody, respectively.45 The signal was detected by using VECTASTAIN ABC kit (Vector) and counter-stained with Nuclear Fast Red.

Statistical Analysis

All data are expressed as mean±SEM. A 2-tailed unpaired t test was used to compare tissue lipid concentration, lesion area between PBS and phLAL treated mice. A P<0.05 was considered significant.

    Results

Reduction of Plasma and Tissue Lipids by phLAL

Two groups were studied: group A received a lower dose (1.5 U phLAL /injection, 60 U/kg), beginning at 2.5 months, and group B received higher doses (6 U phLAL/injection, 240 U/kg), beginning at 3.5 months. phLAL was given every third day for a total of 10 injections as tail vein boluses. Age-matched control mice received PBS injections. Both groups and their controls received HFCD beginning at 1.5 months, and the diet was continued throughout the study. Compared with wild-type mice, the plasma free cholesterol and cholesteryl esters were increased 9.1-fold and 4.7-fold, respectively, in ldlr-/- mice on HFCD (Table I). In group A, phLAL reduced plasma free cholesterol (29.8%, P=0.089) and cholesteryl esters (52.1%, P=0.0025) relative to control mice. In group B, phLAL decreased plasma free cholesterol (27.6%, P=0.0135), cholesteryl esters (28.6%, P=0.0107), and plasma TGs (46.2%, P=0.0045) in ldlr-/- mice compared with their controls.

In control ldlr-/- mice receiving HFCD, cholesterol and (TGs) levels in liver and spleen were increased relative to those of wild-type mice receiving chow diet (Table I). Decreases in total hepatic (43.2%, P=0.002) or splenic (52.6%, P=0.0351) cholesteryl esters were observed in group B mice. Total hepatic TGs were reduced by 34.9% and 62.2% in groups A and B, respectively (P=0.002 and P=0.0218). Total splenic TGs were reduced by 46.6% and 70% in groups A and B, respectively (P=0.0183 and P=0.0589).

Effects of phLAL Injections on Lipoprotein Profiles

The plasma lipoprotein profiles were compared between control and phLAL treated mice in group B. A large amount of VLDL cholesterol accumulated in ldlr-/- mice receiving HFCD for 3 months. Treatment with phLAL did not alter the VLDL cholesterol, but there was a small consistent decrease in IDL/LDL cholesterol in lipoprotein profile.

Reduction in Atherosclerotic Lesions in the Aorta and Aortic Valves

To evaluate the gross effects of phLAL injections on the progression of atherosclerotic lesions, whole mounts of aortic arches were examined by transillumination. In group A, all (3/3) control mice showed extensive lesions of the arch and branch points for the major vessels, eg, brachiocephalic arteries. The lesions were particularly prominent at the lesser curvature of the arch and the subclavian branch points. Treatment of phLAL at lower dose (1.5 U/injection) had little effect on the extent of the lesions in aorta of ldlr-/- mice (data not shown).

With high dose-treated mice (group B), the lesional area was reduced in the lesser curvature of aortic arch. The lesions at the bifurcation points of major vessels were similar in the test and control mice (Figure 1). Lesional reductions in isolated aortas from 5 enzyme treated mice were somewhat variable (Figure 1B and 1C), but this variation did not affect the significance of lesional reduction by phLAL treatment.

   Figure 1. Effect of phLAL on aortic lesions: Representative aortic arches were isolated from typical controls after 10 injections of PBS (A) and 2 group B mice, T1 (B) and T2 (C), that are typical of this group after receiving 10 injections of phLAL These were then photographed under transillumination (bright field). The dark lesions in the arch and the branch points of the brachiocephalic trunk are atherosclerotic plaques. Magnification, 25x.

Histologic analyses of atherosclerotic lesions of group A mice are in Figure 2 (typical results). Five of 8 group A mice were processed for aortic valve foam cell accumulations and these showed major reductions [from ++++ (30% to 40% of aortic valve areas, AVAs), to ++ (10% to 19% of AVA); 2/5, or 0 (<10% of AVA, 2/5)] (Figure 2B). In control mice the aortic valves showed large atherosclerotic lesions with large numbers of foam cells (Figure 2A).

   Figure 2. Histology of typical groups A and B and control mice. Representative H&E (100x) aortic valve sections of group A mice—control (A) and phLAL-treated (B)—and group B mice—control (C) and phLAL-treated (D). The asterisks indicate necrotic zones next to disrupted medial layers. The arrow indicates a fibrous cap, and arrowheads highlight cholesterol crystals. Reduced foamy cells were evident in the lesions of the aortic valve lesions in group A (B) or in group B (D) mice.

In control mice for group B, the atherosclerotic lesions at 4.5 months were very complex with fibrous caps, necrotic cores, and cholesterol crystals (Figure 2C). In comparison, phLAL-treated mice showed reductions, but not elimination, of atherosclerotic lesions in the aortic valve (Figure 2D). The number of foam cells was greatly decreased, but the necrotic core and cholesterol crystals remained. Quantitative analyses of the lesional areas were conducted by using serial sections of the aortic valve from the control and group B (n=5 in each). Every fifth section (10 μm) was mounted and stained for a total of 15 sections from each heart. This covered 750 μm around the aortic valves. The lesional areas on the aortic valves were quantified by using computerized morphometric software (MetaMorph). Significant reductions (P=0.0115) in lesional area were found in phLAL-treated mice compared with controls. The mean lesional area in phLAL-treated mice was similar to the pretreatment values (Figure 3).

   Figure 3. Quantitative analyses of aortic valve lesional areas. Lesion areas of ldlr-- mice on HFCD before receiving (C0) either PBS (C) or phLAL (T) injections. The lesion area was defined from the first elastin positive area. C and T mice each received 10 injections of PBS or phLAL (6 U/injection) and were analyzed 2 days after injection 10. n=5 for each group, P=0.0115 for C vs T.

Effects of phLAL Treatment on Coronary Artery Lesions

Control mice for groups A and B had extensive multifocal lesions in the coronary arteries. All had heavy infiltration with foamy macrophages and plaques extending a considerable distance into the coronary arteries. In one case, the main branch of the left coronary artery was completely obliterated with an advanced lesion containing cholesterol crystals and inflammatory cells Figure I, available online at http://atvb.ahajournals.org). In comparison, 7/8 of the phLAL treated mice in group A had normal coronary vessels Figure I). One enzyme treated mouse in group A had foamy cells in a small intramuscular coronary vessel. The other coronary arteries in this mouse did not have lesions. These results were confirmed in a more quantitative assessment of the coronary arterial lesions as obtained by sequential H & E sections (total=210 sections; 10 μm thickness) of the hearts from a control and 2 treated group A mice. The control mouse had multiple severe plaques (filled 80% to 100% area of coronary artery lumen) in the coronary arteries in 48% of the sections. One treated mouse had completely normal coronary arteries, and another treated mouse had mild lesions (filled 7% to 24% area of artery lumen) in the coronary arteries in 10% of the sections.

Reduction in the Complexity of the Lesions in phLAL-Treated ldlr-/- Mice

The effects of phLAL on the complexity of atherosclerotic lesions in group B mice were evaluated by histological analyses of lesional elastin and collagen staining. The aortas from group B control and treated mice were sectioned into 5 fragments (3/5 segment positions) (Figure 1A). Sections from distal end of fragment 1 were stained for elastin and collagen. Compared with the control mice, the group B treated mice had the lesional areas that were much smaller and with less extracellular matrix content, ie, collagen, and less vessel wall dilation (Figure II, available online at http://atvb.ahajournals. org). The lesional areas and collagen contents in group B mice were similar to those isolated from the mice before the initiation of enzyme administration. The macrophage area and collagen positive area were quantitatively analyzed by MetaMorph software (Figure IIG). The phLAL-treated mice had less area of macrophages and collagen-rich extracellular matrix. The atherosclerotic lesions from control mice were highly complex, with fibrous caps (Figure 4 A and 4B, arrow), and large amounts of collagen (Figure 4D and 4E, blue). The clear areas within these advanced, complicated lesions had lipid or/and cholesterol crystal accumulation and necrotic cores (Figure 4A, 4B, 4D, and 4E, asterisk). Collagen-rich fibrosis was present in aortic valves of control mice (Figure 4B and 4E, arrowhead). In the group B treated mice the lesions were less complex, with lesser degrees of macrophage infiltration and fibrous caps were lacking (Figure 4C and 4F). The deposition of excess extracellular matrix also was much less in treated mice (Figure 4E versus 4F). The macrophage area, collagen positive area, and necrotic core were quantitatively analyzed by using MetaMorph software (Figure 5).

   Figure 4. Reduced the foam cells in atherosclerotic lesion in aortic valve of group B treated mice. Sections of aortic valvular lesions were stained for elastin (A through C, black) and collagen (D through F, blue). C0, ldlr-- mice prior to injection at age 3.5 months. C, PBS (10 injections) control mice. T indicates phLAL-treated (10 injections) mice. *Area of necrotic core. Fibrous caps are indicated with arrow, and thrombosis is indicated with arrowhead. Notice the reduction the number of foam macrophages in phLAL treated samples. Magnification, 100x.

   Figure 5. Quantitative analysis of areas in the lesion of aortic valve. The methods are detailed in Methods. The macrophage and collagen positive areas were defined by van Gieson and Gomori’s trichrome stains.

Macrophage Targeting of phLAL to the Atherosclerotic Lesions

Endogenous LAL protein in ldlr-/- mice could confound these analyses. Thus, ldlr-/-;lal-/- mice were developed and placed on a HFCD. However, lal-/-;ldlr-/- mice died within a few days of HFCD initiation (data not shown). The alternative of apoE-/-;lal-/- mice was developed and used. These mice had no detectible LAL protein and developed spontaneous atherosclerotic lesions without dietary challenge (Figure 6A). By immunohistochemical staining, phLAL protein was taken up into the foam cells of atherosclerotic lesions (Figure 6C). Serial sections were stained with the macrophage marker, Mac-3 (Figure 6D), or oil red O (Figure 6B). The results show uptake of phLAL protein into foamy macrophages and surface endothelial cells.

   Figure 6. Localization of phLAL in foam cells of atherosclerotic lesion. Sections of the lesions at the aortic lesions of lal--;apoE-- mice stained with H&E (A), oil red O (B, red droplets indicates lipids), anti-hLAL antibody (C, brown), or macrophage marker, anti-Mac-3 (D, brown). The tissues were harvested 4 hours after injection of phLAL (24 U). LAL-positive cells were positive for Mac-3 (arrows). Not all macrophages have uptake of hLAL enzyme. Magnification, 400x.

Survival and Antibody Development

All ldlr-/- mice in group A survived. Fifteen of 17 ldlr-/- mice in group B survived. In group B, 1 mouse died after 2 injections and another after 5 injections of phLAL. There was no obvious cause for the death. No other adverse effects were noted in either treated or untreated groups. All ldlr-/- mice injected with phLAL developed anti-phLAL antibodies as assessed by Western blot analyses. One developed a high titer (1:32 000) antibody (data not shown). The earliest development of anti-phLAL antibody was after 5 injections (15 days). These antibodies were not inhibitory toward activity. The antibodies were directed against the LAL protein because they reacted similarly with the deglycosylated and the glycosylated proteins.

    Discussion

This study demonstrates that enzyme supplementation with phLAL had significant effects on the atherosclerotic lesions in ldlr-/- mice receiving HFCD. The lesions were diminished or absent in treated mice compared with the extensive and very severe lesions in the untreated cohorts. This was most evident during early lesional development. For preexisting advanced lesions, treatment of LAL reduced the lesional area and the macrophage component and, thus, appeared to prevent progression of and stabilized the preexisting lesions. The majority of the enzyme was localized to the liver macrophages, Kupffer cells, by immunohistochemical staining after intravenous injection.39 Biological activity of phLAL was assessed previously in lal-/- mice.39 Such in vivo activity also was evident from the reductions in tissue CEs and TGs in lal-/- and HFCD-fed ldlr-/- mice. Injection of phLAL in the ldlr-/- mice also led to significant reductions of plasma CE in group A mice, and of plasma free cholesterol, CE, and TG in group B mice. This suggested the dose-dependent systemic effect of phLAL. The reduction of plasma lipids by phLAL might be mediated by a decrease of lipoprotein production or an increase of lipoprotein uptake in these ldlr-/- mice. Based on an effect of phLAL in reduction of tissue lipid in the liver, we speculate that plasma lipid reductions are mediated though a decrease of lipoprotein production in these ldlr-/- mice.

At least 3 mechanisms could account for the lesional reduction by phLAL administration. First, LAL enters the lesional foam cells and hydrolyzes the stored CEs and TGs.46 Electron microscopic analyses of lesions confirmed that much of the accumulated lipid in foam cells occurred within large, lipid-filled lysosome.47 Most of the cholesterol in atherosclerotic lesions is esterified with linoleate rather than oleate,48,49 which indicates a LDL source rather than a product of ACAT.50 Here macrophages in the atherosclerotic lesions of apoE-/-;lal-/- mice were positive for injected phLAL. By incubation of phLAL enzyme with cultured mouse macrophages, J-774E cells, hLAL is colocalized with the lysosomal marker, lysosomal associated membrane protein 1 (LAMP-1)39. This suggests an important protective effect of LAL by locally reducing the accumulated CEs and TGs in the foamy macrophages. Second, LAL promotes lysosomal egress of free cholesterol that modulates cellular lipid biosynthesis mediated by the SREBPs. This would lead to increased egress of free cholesterol from lysosomes that would inhibit the endogenous synthesis of cholesterol and fatty acid through the SREBP system.34 Most administered phLAL localized to macrophages of the liver and spleen where it hydrolyzed CEs and TGs and reduced tissue lipids in ldlr-/- mice. Third, LAL cleaves oxLDL and generates 9-hydroxyocatadecadienoic acid (9-HODE) and 13-HODE that are ligands for PPAR.13 The ligand activated PPAR could have beneficial effects on the reduction of atherosclerotic lesion by its anti-inflammatory activity.51–54 LAL activity is required to produce ligands for PPAR from oxLDL13 that can contribute to in vivo PPAR ligand generation. Because synthetic ligands for PPAR, eg, rosiglitazone, reduced dietary induced atherosclerotic lesions in male ldlr-/- mice.54 The natural ligands generated by LAL lipid hydrolysis might be expected to have similar effects on reducing atherosclerotic lesions by activation of PPAR and its anti-inflammatory function.54 Here, quantitative analyses showed that hLAL administration in group B mice reduced macrophages in the lesions, but also reduced the areas of extension of collagen positive advanced plaques, cholesterol crystal-rich necrotic cores, and the vessel wall dilation (Figure 5). This suggested that LAL also could have anti inflammatory effects. Alternatively, ligand activated PPAR could stimulate the gene expression of LXR, with downstream stimulation of ABCA1 expression.55,56 The outcome of this cascade of gene regulation is to promote cholesterol efflux from macrophages in the lesion. Peritoneal macrophages isolated from lal-/- mice have lower cholesterol efflux than those from lal+/+ mice (data not shown). These data suggest that LAL derived ligands could promote cholesterol efflux from the cells through the ABCA1 transporter. Finally, recent studies have shown the role of free cholesterol in promoting apoptosis in cultured macrophages.57,58 It is not clear whether the free cholesterol has same role in lesional macrophages in vivo. The remaining question here is the balance between the production of free cholesterol by LAL that potentially could be toxic and a cause of macrophage cell death and the production of free fatty acid by LAL that could be a ligand for PPAR to activate the cholesterol efflux through LXR and ABCAI pathway and anti-inflammatory activity of PPAR.

Why does supplemental LAL work? Alternatively, at what level of substrate presentation to the lysosome does endogenous LAL activity become insufficient to maintain normal CE and TG flux through this organelle? Studies in atherosclerotic rabbit model indicated that the density of lysosomes was decreased and lysosomal cholesteryl esters was increased in lesional smooth muscle cells and endothelial cells compared with that in normal vessel walls.47 These data suggested that LAL activity in lesional cells was/is relatively decreased compared with that in nonlesional vessel walls. Also, LAL activity tends to decrease with age.59 Additional factors include the acetylation and oxidation of LDL leading to modified LDL uptake by unregulated scavenger receptors. Because oxLDL can inactivate lysosomal proteases and cause poor degradation of oxLDL in mouse peritoneal macrophages,60 one might speculate that oxLDL could potentially inactivate lysosomal lipases as well. Indeed, oxLDL CE are resistant to lysosomal hydrolysis in human macrophage THP-1 cells.21 These data suggest that oxLDL could accentuate lysosomal lipid accumulation in foam cells due to direct effects on LAL. As a result, LAL supplementation could compensate for this loss of endogenous LAL activity and reestablish the lysosomal flux of lipids.

    Acknowledgments

 

We thank Jaya Mishra and Bradley B. Jarrold for their excellent technical assistance, Lisa McMillin for the histology analyses, and Chris Woods for the color photography. This work was partially supported by grants from Genzyme Corporation (G.A.G) and from NIH DK 54930 (H.D). Additional support was provided by the Cincinnati Children’s Hospital Research Foundation.

References

Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature. 1993; 362: 801–809.

Ross R. Atherosclerosis is an inflammatory disease. Am Heart J. 1999; 138: S419–420.

Du H, Duanmu M, Witte D, Grabowski GA. Targeted disruption of the mouse lysosomal acid lipase gene: long-term survival with massive cholesteryl ester and triglyceride storage. Hum Mol Genet. 1998; 7: 1347–1354.

Lougheed M, Lum CM, Ling W, Suzuki H, Kodama T, Steinbrecher U. High affinity saturable uptake of oxidized low density lipoprotein by macrophages from mice lacking the scavenger receptor class A type I/II. J Biol Chem. 1997; 272: 12938–12944.

Ling W, Lougheed M, Suzuki H, Buchan A, Kodama T, Steinbrecher UP. Oxidized or acetylated low density lipoproteins are rapidly cleared by the liver in mice with disruption of the scavenger receptor class A type I/II gene. J Clin Invest. 1997; 100: 244–252.

Lysko PG, Weinstock J, Webb CL, Brawner ME, Elshourbagy NA. Identification of a small-molecule, nonpeptide macrophage scavenger receptor antagonist. J Pharmacol Exp Ther. 1999; 289: 1277–1285.

Acton S, Rigotti A, Landschulz KT, Xu S, Hobbs HH, Krieger M. Identification of scavenger receptor SR-BI as a high density lipoprotein receptor. Science. 1996; 271: 518–520.

de Villiers WJ, Smart EJ. Macrophage scavenger receptors and foam cell formation. J Leukoc Biol. 1999; 66: 740–746.

Swarnakar S, Temel RE, Connelly MA, Azhar S, Williams DL. Scavenger receptor class B, type I, mediates selective uptake of low density lipoprotein cholesteryl ester. J Biol Chem. 1999; 274: 29733–29739.

Calvo D, Gomez-Coronado D, Suarez Y, Lasuncion MA, Vega MA. Human CD36 is a high affinity receptor for the native lipoproteins HDL, LDL, and VLDL. J Lipid Res. 1998; 39: 777–788.

Kounnas MZ, Morris RE, Thompson MR, FitzGerald DJ, Strickland DK, Saelinger CB. The á2-macroglobulin receptor/low density lipoprotein receptor-related protein binds and internalizes Pseudomonas exotoxin A. J Biol Chem. 1992; 267: 12420–12423.

Leppanen P, Luoma, J. S., Hofker, M. H., Havekes, L. M., Yla-Herttuala, S. Characterization of atherosclerotic lesion in apo E3-leiden transgenic mice. Atherosclerosis. 1998; 136: 147–152.

Nagy L, Tontonoz P, Alvarez JG, Chen H, Evans RM. Oxidized LDL regulates macrophage gene expression through ligand activation of PPAR?. Cell. 1998; 93: 229–240.

Godyna S, Liau G, Popa I, Stefansson S, Argraves WS. Identification of the low density lipoprotein receptor-related protein (LRP) as an endocytic receptor for thrombospondin-1. J Cell Biol. 1995; 129: 1403–1410.

Herz J, Kowal RC, Ho YK, Brown MS, Goldstein JL. Low density lipoprotein receptor-related protein mediates endocytosis of monoclonal antibodies in cultured cells and rabbit liver. J Biol Chem. 1990; 265: 21355–21362.

Rosenfeld ME, Khoo JC, Miller E, Parthasarathy S, Palinski W, Witztum JL. Macrophage-derived foam cells freshly isolated from rabbit atherosclerotic lesions degrade modified lipoproteins, promote oxidation of low-density lipoproteins, and contain oxidation-specific lipid-protein adducts. J Clin Invest. 1991; 87: 90–99.

Yla-Herttuala S, Palinski W, Rosenfeld ME, Parthasarathy S, Carew TE, Butler S, Witztum JL, Steinberg D. Evidence for the presence of oxidatively modified low density lipoprotein in atherosclerotic lesions of rabbit and man. J Clin Invest. 1989; 84: 1086–1095.

Khoo JC, Miller E, McLoughlin P, Steinberg D. Enhanced macrophage uptake of low density lipoprotein after self-aggregation. Arteriosclerosis. 1988; 8: 348–358.

Vlassara H. Advanced glycation end-products and atherosclerosis. Ann Med. 1996; 28: 419–426.

Suits AG, Chait A, Aviram M, Heinecke JW. Phagocytosis of aggregated lipoprotein by macrophages: low density lipoprotein receptor-dependent foam-cell formation. Proc Natl Acad Sci U S A. 1989; 86: 2713–2717.

Yancey PG, Jerome WG. Lysosomal cholesterol derived from mildly oxidized low density lipoprotein is resistant to efflux. J Lipid Res. 2001; 42: 317–327.

Jessup W, Mander EL, Dean RT. The intracellular storage and turnover of apolipoprotein B of oxidized LDL in macrophages. Biochim Biophys Acta. 1992; 1126: 167–177.

Jialal I, Chait A. Differences in the metabolism of oxidatively modified low density lipoprotein and acetylated low density lipoprotein by human endothelial cells: inhibition of cholesterol esterification by oxidatively modified low density lipoprotein. J Lipid Res. 1989; 30: 1561–1568.

Roma P, Catapano AL, Bertulli SM, Varesi L, Fumagalli R, Bernini F. Oxidized LDL increase free cholesterol and fail to stimulate cholesterol esterification in murine macrophages. Biochem Biophys Res Commun. 1990; 171: 123–131.

Lougheed M, Zhang HF, Steinbrecher UP. Oxidized low density lipoprotein is resistant to cathepsins and accumulates within macrophages. J Biol Chem. 1991; 266: 14519–14525.

Tertov VV, Orekhov AN, Sobenin IA, Gabbasov ZA, Popov EG, Yaroslavov AA, Smirnov VN. Three types of naturally occurring modified lipoproteins induce intracellular lipid accumulation due to lipoprotein aggregation. Circ Res. 1992; 71: 218–228.

Hoff HF, O’Neil J, Pepin JM, Cole TB. Macrophage uptake of cholesterol-containing particles derived from LDL and isolated from atherosclerotic lesions. Eur Heart J. 1990; 11 Suppl E: 105–115.

Lopes-Virella MF, Klein RL, Lyons TJ, Stevenson HC, Witztum JL. Glycosylation of low-density lipoprotein enhances cholesteryl ester synthesis in human monocyte-derived macrophages. Diabetes. 1988; 37: 550–557.

Lohse P, Chahrokh-Zadeh S, Seidel D. Human lysosomal acid lipase/cholesteryl ester hydrolase and human gastric lipase: identification of the catalytically active serine, aspartic acid, and histidine residues. J Lipid Res. 1997; 38: 892–903.

Assmann G, Seedorf U. Acid lipase deficiency: Wolman disease and cholesteryl eser storage disease. New York: McGraw-Hill; 2001.

Hua X, Yokoyama C, Wu J, Briggs MR, Brown MS, Goldstein JL, Wang X. SREBP-2, a second basic-helix-loop-helix-leucine zipper protein that stimulates transcription by binding to a sterol regulatory element. Proc Natl Acad Sci U S A. 1993; 90: 11603–11607.

Kawabe Y, Honda M, Wada Y, Yazaki Y, Suzuki T, Ohba Y, Nabata H, Endo A, Matsumoto A, Itakura H, et al. Sterol mediated regulation of SREBP-1a, 1b, 1c and SREBP-2 in cultured human cells. Biochem Biophys Res Commun. 1994; 202: 1460–1467.

Wang X, Briggs MR, Hua X, Yokoyama C, Goldstein JL, Brown MS. Nuclear protein that binds sterol regulatory element of low density lipoprotein receptor promoter, II: purification and characterization. J Biol Chem. 1993; 268: 14497–14504.

Wang X, Sato R, Brown MS, Hua X, Goldstein JL. SREBP-1, a membrane-bound transcription factor released by sterol-regulated proteolysis. Cell. 1994; 77: 53–62.

Tontonoz P, Nagy L, Alvarez JG, Thomazy VA, Evans RM. PPAR? promotes monocyte/macrophage differentiation and uptake of oxidized LDL. Cell. 1998; 93: 241–252.

Kubota N, Terauchi Y, Miki H, Tamemoto H, Yamauchi T, Komeda K, Satoh S, Nakano R, Ishii C, Sugiyama T, Eto K, Tsubamoto Y, Okuno A, Murakami K, Sekihara H, Hasegawa G, Naito M, Toyoshima Y, Tanaka S, Shiota K, Kitamura T, Fujita T, Ezaki O, Aizawa S, Kadowaki T, et al. PPAR? mediates high-fat diet-induced adipocyte hypertrophy and insulin resistance. Mol Cell. 1999; 4: 597–609.

Rosen ED, Sarraf P, Troy AE, Bradwin G, Moore K, Milstone DS, Spiegelman BM, Mortensen RM. PPAR? is required for the differentiation of adipose tissue in vivo and in vitro. Mol Cell. 1999; 4: 611–617.

Heur M. Lysosomal regulation of gene expression, PhD thesis. Cincinnati: University of Cincinnati; 2002.

Du H, Schiavi S, Levine M, Mishra J, Heur M, Grabowski GA. Enzyme therapy for lysosomal acid lipase deficiency in the mouse. Hum Mol Genet. 2001; 10: 1639–1648.

Gerdes LU, Gerdes, C., Klausen, I. C., and Faegeman, O. Generation of analytic plasma lipoprotein profiles using prepacked superose 6B columns. Clin. Chim. Acta. 1992; 205: 1–9.

Folch J, Lees M, Stanley GHS. A simple method for the isolation and purification of total lipids from animal tissues. J Biol. Chem. 1957; 125: 497–509.

Biggs HG, Erikson JM, Moorehead WR. A manual colorimetric assay of triglycerides in serium. Clin Chem. 1975; 21: 437–441.

Du H, Heur M, Duanmu M, Grabowski GA, Hui DY, Witte DP, Mishra J. Lysosomal acid lipase-deficient mice: depletion of white and brown fat, severe hepatosplenomegaly, and shortened life span. J Lipid Res. 2001; 42: 489–500.

Rudel LL, Morris MD. Determination of cholesterol usingO-phthalaldehyde. J lipid Res. 1973; 14: 164–366.

Du H, Sheriff S, Bezerra J, Leonova T, Grabowski GA. Molecular and enzymatic analyses of lysosomal acid lipase in cholesteryl ester storage disease. Mol Genet Metab. 1998; 64: 126–134.

Sheriff S, Du H, Grabowski GA. Characterization of lysosomal acid lipase by site-directed mutagenesis and heterologous expression. J Biol Chem. 1995; 270: 27766–27772.

Peters TJ, De Duve C. Lysosomes of the arterial wall, II: subcellular fractionation of aortic cells from rabbits with experimental atheroma. Exp Mol Pathol. 1974; 20: 228–256.

Guyton JR, Klemp KF. Development of the atherosclerotic core region: chemical and ultrastructural analysis of microdissected atherosclerotic lesions from human aorta. Arterioscler Thromb. 1994; 14: 1305–1314.

Smith EB, Slater RS, Chu PK. The lipids in raised fatty and fibrous lesions in human aorta: a comparison of the changes at different stages of development. J Atheroscler Res. 1968; 8: 399–419.

Smith EB, Slater RS. Relationship between plasma lipids and arerial tissue lipids. Nutr Metab. 1973; 15: 17–26.

Ricote M, Li AC, Willson TM, Kelly CJ, Glass CK. The peroxisome proliferator-activated receptor-? is a negative regulator of macrophage activation. Nature. 1998; 391: 79–82.

Ricote M, Huang JT, Welch JS, Glass CK. The peroxisome proliferator-activated receptor (PPAR ?) as a regulator of monocyte/macrophage function. J Leukoc Biol. 1999; 66: 733–739.

Marx N, Sukhova G, Murphy C, Libby P, Plutzky J. Macrophages in human atheroma contain PPAR?: differentiation-dependent peroxisomal proliferator-activated receptor ? (PPAR?) expression and reduction of MMP-9 activity through PPAR? activation in mononuclear phagocytes in vitro. Am J Pathol. 1998; 153: 17–23.

Li AC, Brown KK, Silvestre MJ, Willson TM, Palinski W, Glass CK. Peroxisome proliferator-activated receptor ? ligands inhibit development of atherosclerosis in LDL receptor-deficient mice. J Clin Invest. 2000; 106: 523–531.

Chawla A, Boisvert WA, Lee CH, Laffitte BA, Barak Y, Joseph SB, Liao D, Nagy L, Edwards PA, Curtiss LK, Evans RM, Tontonoz P. A PPAR ?-LXR-ABCA1 pathway in macrophages is involved in cholesterol efflux and atherogenesis. Mol Cell. 2001; 7: 161–171.

Chinetti G, Lestavel S, Bocher V, Remaley AT, Neve B, Torra IP, Teissier E, Minnich A, Jaye M, Duverger N, Brewer HB, Fruchart JC, Clavey V, Staels B. PPAR-á and PPAR-? activators induce cholesterol removal from human macrophage foam cells through stimulation of the ABCA1 pathway. Nat Med. 2001; 7: 53–58.

Yao PM, Tabas I. Free cholesterol loading of macrophages induces apoptosis involving the fas pathway. J Biol Chem. 2000; 275: 23807–23813.

Yao PM, Tabas I. Free cholesterol loading of macrophages is associated with widespread mitochondrial dysfunction and activation of the mitochondrial apoptosis pathway. J Biol Chem. 2001; 276: 42468–42476.

Maehira F, Harada K, Shimoji J, Miyagi I, Nakano M. Age-related changes in the activation of aortic cholesteryl ester hydrolases by protein kinase in rats. Bioch. Biophy. Acta. 1998; 1389: 197–205.

Hoppe G, O’Neil, J., Hoff, H. F. Inactivation of lysosomal protease by oxidized low density lipoprotein is partially responsible for its poor degradation by mouse peritoneal macrophages. J. Clin. Invest. 1994; 94: 1506–1512.

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