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Mechanism of hypertensive nephropathy in the Dahl/Rapp rat: a primary disorder of vascular smooth muscle

【关键词】  muscle

    Nephrology Research and Training Center, Comprehensive Cancer Center, and Cell Adhesion and Matrix Research Center, Division of Nephrology, Departments of Medicine and of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham
    Department of Veterans Affairs Medical Center, Birmingham, Alabama


    The Dahl/Rapp salt-sensitive (S) rat is a model of salt-sensitive hypertension and hypertensive renal disease. This study explored the role of vascular remodeling in the development of renal failure in S rats. Groups of S and Sprague-Dawley rats were given 0.3 and 8.0% NaCl diets for up to 21 days and evidence of smooth muscle proliferation identified using immunohistochemistry that showed nuclear accumulation of proliferating cell nuclear antigen and 5-bromo-2'-deoxy-uridine. Compared with the other three groups, S rats on 8.0% NaCl diet showed increased nuclear labeling of cells of the aorta and arteries and arterioles of the kidney by the end of the first week of study. Progressive luminal narrowing of the interlobular arteries and preglomerular arterioles occurred in S rats over the 3 wk on the 8.0% NaCl diet. Accumulation of pimonidazole adducts and nuclear accumulation of hypoxia-inducible factor-1 (HIF-1) were used as markers of tissue hypoxia. By the end of the second week of study, pimonidazole levels increased in S rats on 8.0% NaCl diet and deposition was apparent in tubular cells in the cortex and medulla. At the completion of the experiment, HIF-1 levels were increased in nuclear extracts from the cortex and medulla of S rats on this diet, compared with the other three groups of rats. The data demonstrated a disorder of the vascular remodeling process with proliferation of vascular smooth muscle cells temporally followed by development of tissue hypoxia in the hypertensive nephropathy of S rats on 8.0% NaCl diet.

    salt-sensitive hypertension; kidney disease; hypoxia; hypoxia-inducible factor-1; pimonidazole

    RENAL FAILURE FROM HYPERTENSION is the second most common cause of end-stage kidney disease in the United States (25). However, hypertension is a very common medical problem, occurring in as many as 43 million individuals (3), so only about 1 in 2,500 hypertensive patients develops clinically important end-stage kidney failure from hypertension. While several interpretations are possible, this low frequency of end-organ renal damage suggests a potential genetic predisposition to this complication.

    The Dahl/Rapp salt-sensitive (S) rat is an inbred strain that serves as an excellent model of salt-sensitive hypertension. These animals also have a striking predisposition to develop progressive renal failure; within 3 wk following the development of hypertension, S rats uniformly demonstrated a severe reduction in glomerular filtration rate (6). Renal morphological abnormalities consisted of mesangial expansion and tubular atrophy with tubular epithelial cell dropout from apoptosis (22, 31, 36), but the striking changes were observed in the vessels. Small arteries and arterioles, which were indistinct in the kidneys of the Dahl/Rapp salt-resistant (R) rats and S rats treated to maintain normal blood pressures, were prominent in the kidneys of untreated, hypertensive S rats (6). Morphometric analysis demonstrated an increase in wall thickness of the interlobular arteries and preglomerular arterioles, whereas qualitative light microscopic and ultrastructural analyses suggested that the vascular remodeling that occurred in these hypertensive rats included excess matrix deposition and increased numbers of smooth muscle cells in the vessel wall (6).

    The intent of the present study was to characterize further the vascular remodeling process in S rats, focusing particularly on vascular smooth muscle proliferation. To determine if this vigorous remodeling contributed to the progressive renal failure in S rats, markers of tissue hypoxia were also examined over a 3-wk time frame.


    Animal preparation. The Institutional Animal Care and Use Committee at the University of Alabama at Birmingham approved the project. Studies were conducted using 86 male Sprague-Dawley (SD) rats and 86 Dahl/Rapp salt-sensitive (S) rats. The rats were obtained from Harlan Sprague Dawley (Indianapolis, IN) and were 28 days of age at the start of study. The protocol that was followed has been standardized in our laboratory (3234). The rats were housed under standard conditions and given formulated diets (AIN-76A, Dyets, Bethlehem, PA) that contained 0.3 or 8.0% NaCl. These diets were prepared specifically to be identical in protein composition and differed only in NaCl and sucrose content. The rats were studied at baseline and days 7, 14, and 21 of the study. Systolic blood pressures were determined in awake animals by tail-cuff sphygmomanometry (4). The rats were anesthetized by intraperitoneal injection of pentobarbital sodium injection (Abbott Laboratories, North Chicago, IL), 50 mg/kg body wt, and the kidneys were perfused in situ through the aorta for 2 min with 0.9% heparinized saline. Both kidneys and aorta were harvested and either placed in 4% paraformaldehyde or the cortex and medulla were dissected for protein analysis, as described below.

    Proliferating cell nuclear antigen staining and analysis. Proliferating cell nuclear antigen (PCNA) is a nuclear protein that is found in dividing cells and participates integrally in DNA replication (28). The appearance of PCNA provides supportive evidence of cell proliferation. Paraffin-embedded sections were deparaffinized by immersion twice into xylene for 5 min each, followed by immersion twice for 3 min each in 100% ethanol and then 95% ethanol. Slides were rinsed for 30 s using deionized water and then immersed twice in deionized water for 5 min. Slides were covered in 1% SDS in Tris-buffered saline (TBS), which contained 100 mM Tris?HCl, pH 7.4, 138 mM NaCl, and 27 mM KCl, for 5 min at room temperature, then rinsed in TBS. The slides were immersed in 0.1% H2O2 for 10 min at room temperature and then incubated for 1 h at room temperature in 50 μM Tris?HCl, pH 7.2, containing 10% goat serum and a mouse monoclonal antibody directed against PCNA (Dako, Carpinteria, CA), 1:1,600 dilution. Slides were rinsed with PBS and covered with 10% rat serum containing a peroxidase-labeled polymer conjugated to goat anti-mouse IgG (Dako Envision System, Dako) for 1 h at room temperature. Color was developed using 3,3'-diaminobenzidine (DAB) for 5 min. Cells were counterstained using hematoxylin and the slides were mounted in standard fashion. As a negative control, the primary antibody was omitted from the reaction.

    To quantify cellular proliferation, cells with nuclear staining for PCNA were counted manually in whole aortic cross-section and averaged for each of the four groups (n = 4 rats in each group) of rats at days 7 and 21 of the experiment. A total of 16 SD and 16 S rats were examined in this portion of the study. In the kidney, the entire trichrome-stained cortex was scanned for interlobular arteries and preglomerular arterioles that were completely cross-sectioned. Arteries and arterioles were identified by their anatomic location and branching pattern. Because of tapering of the interlobular artery, sections of interlobular artery located in the outer third of the cortex were excluded from analysis. Digitized photomicrographs obtained at the same magnification were projected on a monitor and the luminal diameter and outer diameter measured directly to calculate luminal area, wall area and the wall-to-lumen ratio (WTL). Nuclei present in the media were counted manually. An average of 5.9 interlobular arteries and 6.5 preglomerular arterioles were examined in each kidney. The mean diameters of the interlobular arteries and preglomerular arterioles were 49.8 ± 0.6 and 24.5 ± 0.2 μm, respectively.

    BrdU labeling and analysis. Thirty-two SD and 32 S rats were used in this study. To evaluate DNA synthesis in vascular cells, 5-Bromo-2'-deoxy-uridine (BrdU; Roche Molecular Biochemicals, Indianapolis, IN), 100 mg/kg body wt, was administered intraperitoneally 18 h before examination. The dose was similar to that published by other investigators (26). Kidney and aortic tissue were fixed in 4% paraformaldehyde and then transferred to 70% ethanol until paraffin embedding. The presence of BrdU in 5-μm sections was detected using an anti-BrdU antibody and a kit (BrdU Labeling and Detection Kit II, Roche Diagnostics, Indianapolis, IN). Briefly, antigen retrieval was performed by immersing the sections in 10 mM citrate buffer, pH 6.0, and warmed in a microwave oven at 700 W for 10 min. The slides were left in the hot citrate buffer for an additional 30 min and then were rinsed for 5 min in 50 mM TBS, pH 7.6. BrdU detection proceeded following the directions supplied by the manufacturer. The number of BrdU-labeled nuclei in a cross section of aorta was counted and averaged for each of the four groups (n = 4 rats in each group) of rats at days 7 and 21 of the experiment.

    In some experiments, double immunohistochemical staining was performed to demonstrate colocalization of cytoplasmic staining for smooth muscle -actin with nuclear labeling by BrdU. Deparaffinized tissue sections were dehydrated according to routine procedure. Monoclonal mouse anti-smooth muscle -actin (DakoCytomation, Carpinteria, CA), 1:200 dilution, was used initially as the primary antibody, followed by biotinylated secondary antibody and then peroxidase-conjugated streptavidin (LSAB2 Kit, DakoCytomation) and DAB, using the protocol provided by the manufacturer. Negative controls used mouse IgG2a (DakoCytomation). Following a 3-min incubation in the double-stain blocking solution, antigen retrieval proceeded as described in the preceding paragraph. The samples were then incubated in anti-BrdU monoclonal antibody (BrdU Labeling and Detection Kit II), 1:10 dilution, followed by alkaline phosphatase-conjugated anti-mouse IgG (EnVision Doublestain System, DakoCytomation), 1:10 dilution, for 30 min at 37°C and development using Fast Red Chromogen Solution (DakoCytomation).

    Protein-bound pimonidazole adduct analysis using immunohistochemistry and ELISA. Nitroimidazole compounds are activated at low-oxygen concentrations and form adducts with thiol groups of proteins (18); under hypoxic conditions, the rate of formation of adducts increases and can be quantified in a variety of tissues including the kidney (1, 2, 9, 19, 23, 30). At the initiation and on days 7, 14, and 21 of the study, SD and S rats received pimonidazole hydrochloride (Hypoxyprobe-1, Chemicon International, Temecula, CA), 120 mg/kg body wt ip, 2 h before death. Paraffin-embedded 5-μm kidney sections were deparaffinized, hydrated, and covered in 3% H2O2 for 5 min. Antigen retrieval consisted of incubation in 0.01% pronase at 40°C for 40 min. Slides were then washed with 0.2% Brij in PBS for 2 min at 0°C and then incubated in a blocking solution (DakoCytomation) for 5 min at room temperature. A mouse monoclonal IgG1 (Hypoxyprobe-1MAb1, Hypoxyprobe-1 Kit, Chemicon International), 1:100 dilution, was applied to each section for 40 min at room temperature. The sections were incubated for 10 min with a biotin-conjugated F(ab')2, 1:200 dilution. After being washed with 0.02% Brij in PBS buffer, the samples were incubated with peroxidase-conjugated streptavidin (DakoCytomation), followed by DAB (DakoCytomation). The sections were counterstained with hematoxylin and analyzed in standard fashion.

    For ELISA, 200 mg of kidney were homogenized and suspended in 10 volumes of PBS containing 0.05% Tween (PBS-Tween) solution. Protein concentration of the homogenates was determined using bicinchoninic acid reagent (Micro BCA Protein Assay Reagent Kit, Pierce, Rockford, IL). The homogenates were diluted 1:1 with PBS-Tween containing 1 mg/ml of proteinase K, 20 U/mg protein, and the incubated 37°C overnight in a shaking water bath. PMSF, 200 μM, was added and the homogenates were heated for 10 min at 95°C to completely inactivate the protease. The samples were centrifuged for 10 min at 9,300 g and the supernatant was used for the ELISA. To quantify pimonidazole-protein adducts, a competitive ELISA method that was described by Raleigh (2, 19) and successfully applied to kidney tissue (23, 30, 37) was followed. Hypoxyprobe-1 antigen and rabbit polyclonal anti-Hypoxyprobe primary antisera were generously provided by Dr. James A. Raleigh in the Department of Radiation Oncology at the University of North Carolina at Chapel Hill.

    Determination of nuclear levels of hypoxia-inducible factor-1 in the cortex and medulla. The protocol was similar to that published by Zou et al. (38). Briefly, dissected kidney cortical and medullary tissues from 8 S and 8 SD rats on the two diets for 21 days (n = 4 in each group) were minced and washed with PBS, then homogenized in ice-cold hypotonic buffer, which contained 10 mM HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol (DTT), 0.5 mM phenylmethylsulfonyl fluoride (PMSF), and 10% Nonidet P-40. The suspension was centrifuged at 10,000 g for 5 min at 4°C. The pellets were collected and incubated for 15 min in an ice-cold extraction buffer, which contained 5 mM HEPES (pH 7.9), 1.5 mM MgCl2, 300 mM NaCl, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF, and 26% glycerol. The nuclear extract was obtained by centrifugation at 32,140 g for 30 min. The nuclear extract was snap-frozen in liquid nitrogen and stored at 20°C until use. Protein concentrations were determined using bicinchoninic acid reagent (Micro BCA Protein Assay Reagent Kit, Pierce). Sixty-five micrograms of total protein were separated 8% SDS-PAGE and then transferred onto nitrocellulose membrane. After being blocked using 5% nonfat milk (Bio-Rad, Hercules, CA) in TBST (10 mM Tris?HCl, pH 8.0, 200 mM NaCl, 0.05% Tween 20) overnight at 4°C, the membranes were incubated for 4 h at room temperature with a rabbit polyclonal IgG antibody for hypoxia-inducible factor-1 (HIF-1; sc-10790, Santa Cruz Biotechnology, Santa Cruz, CA), 1:200 dilution in the blocking solution. After being washed with TBST, the blots were incubated 1 h with horseradish peroxidase-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology), 1:10,000 dilution in blocking solution, and the bound antibody was identified and quantified using chemiluminescence (SuperSignal Chemiluminescent reagents, Pierce) and densitometry.

    Statistical analysis. All data were presented as means ± SE. Statistical differences were determined using one-way analysis of variance with standard post hoc testing (Statview, version 5.0, SAS Institute, Cary, NC) for parametric data. Logarithmic transformation of the wall area, lumen area, and WTL permitted normal distribution of the data. Because the PCNA and BrdU analyses generated nonparametric data, the Kruskal-Wallis test was used to determine significance. A P value of <0.05 assigned significance.


    Although no significant changes in kidney morphology were observed in SD rats on either diet and S rats maintained on the 0.3% NaCl diet over the 21 days of the study, S rats on the 8.0% NaCl diet demonstrated marked thickening of the arteries and arterioles of the kidney by day 21 (Fig. 1A). Associated mesangial expansion and tubular atrophy with occasional cast formation were also present at that time. These findings reproduced those obtained from this laboratory previously (6, 22). Mean systolic blood pressures increased progressively in S rats on the 8.0% NaCl diet (109 ± 2 mmHg at day 0, 135 ± 2 mmHg at day 7, 132 ± 4 mmHg at day 14, 141 ± 8 mmHg at day 21). Morphometric analyses did not demonstrate differences in mean wall areas and luminal areas of interlobular arteries and preglomerular arterioles of SD and S rats at the start of the experiment (Table 1). At 1 wk on the 8.0% NaCl diet, the mean luminal areas of both interlobular arteries and preglomerular arterioles decreased and WTL ratios increased specifically in S rats and persisted through the duration of the experiment. At 3 wk on the 8.0% NaCl diet, mean wall thickness of interlobular arteries and preglomerular arterioles of S rats was greater than the other three groups of rats. A progressive decrease in luminal area and increase in the number of nuclei in the media of the interlobular arteries and preglomerular arterioles of S rats on 8.0% NaCl were apparent.

    View this table:

    Immunohistochemical detection of PCNA identified nuclear labeling of smooth muscle cells throughout the arterial tree of S rats maintained on the high-salt diet and were especially prominent in the small arteries and arterioles of the kidney by day 21 of the study (Fig. 1, BE). Nuclear labeling of cells of the tubular epithelium was also striking in S rats and was similar to that reported previously (36). To confirm these findings, BrdU labeling experiments were then performed. Incorporation of BrdU into nuclei of cells of the vascular tree of S rats on the 8.0% NaCl diet was observed by day 7 and progressed over the course of the study (Fig. 2). Double immunohistochemical staining experiments showed nuclear localization of BrdU in cells that also showed staining for cytoplasmic smooth muscle -actin (Fig. 2, D and E). Quantification of nuclear PCNA and BrdU labeling of aortic cross-sections demonstrated greater (P < 0.05) numbers of labeled nuclei in aortas from S rats on the 8.0% NaCl diet, compared with the other three groups at days 7 and 21 of study (Fig. 3).

    To determine if the blood supply to the kidney was compromised as a result of the vascular remodeling process, tissue hypoxia was determined using pimonidazole, which forms adducts with thiol groups at low-oxygen concentrations (18). While medullary localization of these adducts was shown in every specimen examined, staining of the tubules in the cortex was increased specifically in S rats on the 8.0% NaCl diet (Fig. 4). Protein-bound pimonidazole adducts in kidneys were quantified using an ELISA (Fig. 5). By day 14 of study, tissue pimonidazole levels were greater (P < 0.05) in S kidneys on the 8.0% NaCl diet, compared with the other three groups in the study.

    As an additional indicator of tissue hypoxia, nuclear accumulation of HIF-1 in cortical and medullary tissues from rats on the diets for 21 days was quantified using western analysis (Fig. 6). A single band that migrated to the same position as that described by Zou and associates (38) was identified. The mean relative intensity of HIF-1 in nuclei from medullary tissue of S rats on 8.0% NaCl (10,145 ± 825/mm2) was greater (P < 0.05) than mean intensities of bands from S rats on 0.3% NaCl (8,277 ± 277/mm2) and SD rats on 8.0% NaCl (3,997 ± 251/mm2) and 0.3% NaCl (2,814 ± 155/mm2). Using this technique, nuclear accumulation of HIF-1 in the cortex was not quantifiable in SD rats on either diet. Mean levels of HIF-1 in the cortex of S rats on 8.0% NaCl (56,797 ± 2,144/mm2) was greater (P < 0.05) than mean levels observed in S rats on 0.3% NaCl (42,279 ± 4,192/mm2).


    The Dahl/Rapp salt-sensitive (S) rat is a widely studied genetic model of salt-sensitive hypertension. Hypertensive S rats also rapidly and consistently develop end-organ kidney damage and have been used as a model of progressive renal injury. An interesting study by Cowley and associates (7) suggested that different genes confer salt-sensitive hypertension and the renal injury. A consomic strain in which chromosome 13 from the Brown-Norway strain was introgressed into the S genome demonstrated significantly less vascular reactivity and indices of tubulointerstitial damage compared with S rats on the same diet. Under the conditions of the present study, progressive increases in wall thickness and associated luminal narrowing of interlobular arteries and preglomerular arterioles occurred in S rats during the course of hypertension related to the 8.0% NaCl diet. In addition, two different methods (PCNA detection and BrdU incorporation) were used to demonstrate a role for hyperplasia of vascular smooth muscle in the exuberant vascular remodeling that emerged in the renal resistance vessels of S rats in the response to hypertension. Remodeling, however, promoted tissue hypoxia in the renal cortex and medulla. Dietary salt alone did not stimulate vascular smooth muscle proliferation, since the data did not differ between SD rats on either the low-salt or high-salt diet. The combined findings supported a primary role for dysregulated growth of vascular smooth muscle in the etiopathogenesis of hypertensive nephropathy in the Dahl/Rapp rat.

    Tissue hypoxia was demonstrated using two different methods. The first method used pimonidazole, which has been shown to form adducts with thiol groups under hypoxic conditions (18) and has been used to quantify tissue hypoxia in the kidney (23, 30, 37). As shown by other investigators (30, 37), medullary deposition of pimonidazole was routinely demonstrated in all the groups under study. Compared with the other three groups, however, after two wk on the high-salt diet, pimonidazole adducts in the kidney of S rats increased, particularly in the tubular epithelium of the kidney cortex. The findings correlated with a previous study that showed the reduction of the glomerular filtration rate in S rats was present by this time point; hypertension developed within the first week in S rats on 8.0% NaCl diet, but glomerular filtration rate was preserved until the end of the second week of study (6). Pimonidazole deposition in the renal cortex was therefore considered suggestive of tissue hypoxia.

    An important sensor of tissue levels of oxygen is HIF, which is a heterodimeric transcription factor that is composed of an -subunit bound to HIF-1; the biology of this system has been recently reviewed (21, 29). Hypoxia increases the cellular abundance of HIF-1 protein (10, 14, 27); the mechanism relates to oxygen-dependent proline hydroxylation of HIF-1 that in turn modulates the binding of an E3 ubiquitin ligase that contains the von Hippel-Lindau tumor suppressor protein (pVHL) (11, 12). Stabilization of HIF-1, which contains the transactivation domains, permits nuclear import by HIF-1, which possesses the nuclear transporter signal, and subsequent transcriptional activation of hypoxia-regulated genes (21, 29). HIF-1 is abundantly expressed in rodent kidney particularly in tubular epithelial cells and is regulated by changes in oxygen tension (20, 38). Because of the relative hypoxic conditions of the medulla, expression of HIF-1 is observed in normal rats (38). In the present study, using extracts of medullary tissue from SD rats on both diets, nuclear localization of HIF-1 was observed. Increased amounts of HIF-1 were seen in nuclear extracts from both the cortex and medulla of S rats on 8.0% NaCl diet for three wk, confirming a significant decrease in oxygen tension in these tissues.

    A recent study by Khan and associates (15) demonstrated the appearance of tissue hypoxia with an associated increase in apoptosis of tubular epithelial cells in a murine model of progressive renal failure, providing support for the link between hypoxia and apoptosis in the kidney. ATP-depleted Madin-Darby canine kidney cells exhibit apoptosis that is dependent upon upregulation and activation of Fas and Fas ligand (FasL) (8). A major mechanism of renal failure in S rats is tubular epithelial cell apoptosis, related to activation of Fas/FasL pathway and the intrinsic pathway involving mitochondrial release of cytochrome c (22, 31, 36); both pathways can be activated by hypoxia. While albuminuria and expansion of the glomerular mesangium are part of the features of hypertensive nephropathy in S rats (6), the present findings supported an important role for vascular smooth muscle hyperplasia with the subsequent development of tissue hypoxia as the proximate cause of progressive renal failure in this genetic model of hypertension and hypertensive renal disease. The underlying mechanism may be related to endothelial dysfunction, which is observed in S rats even prior to development of hypertension and may be related to an intrinsic disturbance of growth mechanisms of vascular smooth muscle cells, or to altered production of vasoactive agents (46, 35). The absence of augmented production of NO in response to an increase in dietary salt intake (46) may also unmask intrinsic renal vasoconstriction, as suggested to occur in other models of salt-sensitive hypertension (13). The imbalance between vasoconstrictor and vasodilator influences and subsequent vascular remodeling all contribute to the impaired myogenic responses of the renal resistance vessels of S rats (24). Progressive tissue hypoxia can also explain the paradoxical activation of the intrarenal renin-angiotensin axis in S rats on 8.0% NaCl diet (16) and facilitate the imbalance between vasoconstrictors and vasodilators. S rats demonstrate a progressive increase in blood pressure as renal function deteriorates (6); the increase in intrarenal angiotensin II promotes capillary rarefaction (17), which also contributes to salt-sensitive hypertension and tissue hypoxia. Thus a positive feedback loop is generated with the final result of end-stage kidney failure in this genetic model of salt-sensitive hypertension.


    National Institutes of Health Grant R01-DK-46199 and the Office of Research and Development, Medical Research Service, Department of Veterans Affairs supported this work.


    The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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日期:2013年9月26日 - 来自[2005年第288卷第1期]栏目

Inhibition of Vascular Endothelial Growth Factor Cotranslational Translocation by the Cyclopeptolide CAM741

【关键词】  Inhibition

    The cyclopeptolide CAM741 inhibits cotranslational translocation of vascular cell adhesion molecule 1 (VCAM1), which is dependent on its signal peptide. We now describe the identification of the signal peptide of vascular endothelial growth factor (VEGF) as the second target of CAM741. The mechanism by which the compound inhibits translocation of VEGF is very similar or identical to that of VCAM1, although the signal peptides share no obvious sequence similarities. By mutagenesis of the VEGF signal peptide, two important regions, located in the N-terminal and hydrophobic segments, were identified as critical for compound sensitivity. CAM741 alters positioning of the VEGF signal peptide at the translocon, and increasing hydrophobicity in the h-region reduces compound sensitivity and causes a different, possibly more efficient, interaction with the translocon. Although CAM741 is effective against translocation of both VEGF and VCAM1, the derivative NFI028 is able to inhibit only VCAM1, suggesting that chemical derivatization can alter not only potency, but also the specificity of the compounds.

    We have recently reported that the cyclopeptolide CAM741, a derivative of the naturally occurring substance Hun-7293, inhibits cotranslational translocation of vascular cell adhesion molecule 1 (VCAM1), which is dependent on its signal peptide (SP) and occurs at the level of VCAM1 SP insertion in the Sec61 translocon (Besemer et al., 2005; Harant et al., 2006). Very similar observations were reported by Garrison et al. (2005) using cotransin, a compound with related structure. These findings demonstrate for the first time that a compound can interfere with the process of cotranslational translocation in a SP-dependent manner.

    Amino-terminal, cleavable SPs, when emerging from the ribosome, are recognized by the signal recognition particle, which then directs the ribosome-nascent polypeptide chain complex to the heterotrimeric Sec61 (composed of the -, -, and  subunits) complex embedded in the membrane of the endoplasmic reticulum (ER) (for review, see Rapoport et al., 1996; Hegde and Lingappa, 1997; Matlack et al., 1998; Johnson and van Waes, 1999; Stroud and Walter, 1999; Osborne et al., 2005). SPs, usually of 20 to 30 amino acid residues in length, have a typical three-domain structure, representing a frequently positively charged n-region, a hydrophobic core region (h-region), and a more polar c-region containing the site for SP cleavage (Nielsen et al., 1997; Nielsen et al., 1999). It has been assumed that all SPs interact with the translocon in a similar manner, but there is now increasing evidence that there are remarkable differences between individual SPs (reviewed by Hegde and Bernstein, 2006). Some SPs have been shown to require interaction with accessory translocon components, such as translocating chain-associated membrane protein or translocon-associated protein, whereas others do not (High et al., 1993; Mothes et al., 1994; Voigt et al., 1996; Fons et al., 2003). SPs also contain information on the timing of SP cleavage and subsequent N-glycosylation of the translocated chains (Rutkowski et al., 2001, 2003). During our own studies on the VCAM1 SP, we observed that mutations of the CAM741-sensitive region of the VCAM1 SP caused different association with the translocon components Sec61 and -, indicating that these mutants are positioned differently within the translocon channel (Harant et al., 2006).

    Based on our findings that the cyclopeptolide CAM741 can inhibit the process of cotranslational translocation in an SP-dependent manner, we performed a search for other sensitive SPs to gain more insight into their functionality. We report here the identification of the SP of vascular endothelial growth factor-A (referred to as VEGF) as the second target of CAM741 action.

    VEGF, also termed vascular permeability factor, is one of the key factors in angiogenesis and induces endothelial cell proliferation, migration, and tube formation (Keck et al., 1989; Leung et al., 1989). Apart from its essential role in neovascularization, VEGF is also involved in several pathological conditions, such as tumor angiogenesis, diabetic retinopathy, age-related macular degeneration, and psoriasis, making it an attractive target for therapeutic intervention (for review, see Cardones and Banez, 2006; Eichler et al., 2006; Roy et al., 2006). There exist six isoforms of VEGF, differing in their lengths (121, 145, 165, 183, 189, and 206 amino acid residues). These isoforms are generated by alternative mRNA splicing, differ in sequences encoded by exons 6 and 7, and are differentially expressed between different cells types (reviewed by Robinson and Stringer, 2001). However, all six VEGF isoforms are controlled by the same 26 amino acid-residue signal peptide (SP).


    CAM741 and NFI028 were dissolved in dimethyl sulfoxide and stored at –20°C. Dulbecco's modified Eagle's medium (DMEM) was purchased from Invitrogen (Carlsbad, CA), and fetal calf serum (FCS) from Cambrex Bio Science Verviers S.p.r.l. (Verviers, Belgium). VEGF165 ELISA was purchased from R&D Systems (Minneapolis, MN). Superfect was purchased from QIAGEN (Hilden, Germany). AttoPhos reagent, rabbit reticulocyte lysate, canine pancreatic microsomal membranes, and RiboMAX Large-Scale RNA production System-T7 were purchased from Promega (Madison, WI). Polyclonal Sec61 or Sec61 antisera were purchased from Millipore (Billerica, MA). Excel Gel SDS 8–18%, Excel Gel SDS 12–14% gels and Redivue [35S]methionine were purchased from GE Healthcare (Chalfont St. Giles, Buckinghamshire, UK). Transforming growth factor- (TGF-) was purchased from BD Biosciences (San Jose, CA).


    ELISA. HaCaT keratinocytes were cultured in DMEM supplemented with 10% heat-inactivated FCS. Cells were seeded into 96-well plates at a density of 1.5 to 2 x 104 cells/well, grown to confluence, and then incubated with increasing concentrations of CAM741 for 16 h. VEGF production was stimulated by addition of 50 ng/ml TGF- for 24 h. Supernatants were collected and analyzed for VEGF165 by ELISA.

    Plasmid Constructions. The SP-secreted alkaline phosphatase (SEAP) fusion constructs, VEGF SP mutants fused to the SEAP mature domain, the construct encoding a fusion of the N-terminal tag to the VEGF SP, and the truncated VEGF cDNAs encoding either 81 or 131 amino acid residues were generated by polymerase chain reaction and subcloned into pcDNA3.1 (Invitrogen). All constructs were confirmed by sequencing. The numbering of amino acid residues refers to VEGF plus SP.

    Truncated cDNAs lacking a stop codon were generated by restriction digestion of the respective plasmid DNAs. In case of the SEAP fusion constructs, plasmids were linearized by digestion with either BamHI (encoding 54 amino acid residues, SEAP mature domain) or BstEII (encoding 146 amino acid residues, SEAP mature domain). Linearized plasmids were used as templates for creation of RNAs by the RiboMAX Large Scale RNA production System-T7.

    Transient Transfections of HEK293 Cells. HEK293 cells were cultivated in DMEM supplemented with 10% FCS and passaged twice a week. For transfection, 1.5 x 104 cells in a volume of 100 µl were seeded in each well of a 96-well plate, transfected with 0.2 µg of plasmid DNA and 0.5 µl of Superfect in each well and treated with increasing concentrations of CAM741 or NFI028. Supernatants were harvested after 24 h, and analyzed for SEAP secretion using the AttoPhos reagent. Fluorescence was recorded using the SPEKTRA-max GEMINI XS (Molecular Devices, Sunnyvale, CA).

    In Vitro Translocation Experiments. In vitro translation, targeting and translocation assays, and chemical cross-linking were performed with truncated RNAs using rabbit reticulocyte lysate, canine pancreatic microsomal membranes (Promega), and [35S]methionine (GE Healthcare), in the presence of CAM741 or dimethyl sulfoxide (vehicle control) as described previously (Besemer et al., 2005; Harant et al., 2006). Immunoprecipitations were performed with a polyclonal Sec61 or Sec61 antiserum. Proteins were separated on Excel Gel SDS 8–18% or, where stated, on high resolution Excel Gel SDS 12–14% gels. Fixed and dried gels were exposed to X-ray films.

    CAM741 Inhibited Cotranslational Translocation of VEGF, Which Was Dependent on Its SP. To search for SPs, which, apart from the known VCAM1 SP (Besemer et al., 2005), could be sensitive to inhibition of translocation by CAM741, a panel of 10 different SPs fused to the mature region of secreted alkaline phosphatase (SEAP) was tested as described in Table 1. The SPs were selected as representatives of different classes of secreted or membrane proteins involved in inflammation, immune regulation and angiogenesis. The chemokines CCL22 (macrophage-derived chemokine), CCL2 (monocyte chemoattractant protein), and CXCL8 (interleukin-8) were included. The inflammatory cytokines interferon-, interleukin-12p40 subunit, and interleukin-13 were also used, as was VEGF. From the membrane proteins, the SP of the chemokine C-C motif receptor 7, E-selectin, and intercellular adhesion molecule-1 were chosen. Although seven of the SP-SEAP constructs showed only partial inhibition of SEAP release at the highest concentration of CAM741 (10 µM), SEAP fusion constructs of CXCL8 and the ICAM1 SPs showed some sensitivity to inhibition by CAM741 (Table 1). However, of all SPs tested, that of VEGF was identified as most sensitive to inhibition by CAM741, being only 4-fold less sensitive than the VCAM1 SP (Harant et al., 2006) (Table 1).

    TABLE 1 Different sensitivity of SP-SEAP fusion constructs to inhibition by CAM741

    HEK293 cells were transfected with different SP-SEAP fusion constructs (in some cases SP + additional residues of the mature domain) and incubated with increasing concentrations of CAM741. Twenty-four hours after transfection, supernatants were harvested and analyzed for alkaline phosphatase activity. Results shown are IC50 values from at least three independent experiments performed in triplicates. The cleavage site is underlined; amino acid residues of the mature region are bold.

    To determine that inhibition of SEAP release of the transfected VEGF SP fusion construct by CAM741 also occurs at the level of cotranslational translocation, truncated RNAs encoding the VEGF SP fused to 146 amino acid residues of the SEAP mature domain were used for in vitro translocation experiments. The SEAP mature domain contains a glycosylation site at position 122, and glycosylation and protection from exogenous proteases therefore indicate translocation to the ER lumen after release of the nascent chains (NCs) from the ribosome by high salt/puromycin. In vitro translocation of truncated VEGF SP-SEAP NCs produced two glycosylated fragments, which were protected from protease digestion (Fig. 1A, left). Glycosylation was confirmed by treatment with endoglycosidase F (Fig. 1A, right). The slower migrating glycosylated fragment represents the VEGF SP-SEAP construct with the SP still attached to it, whereas the faster migrating glycosylated fragment represents the SEAP mature region after SP cleavage. The fastest migrating band, representing unprocessed NCs, was almost completely degraded by proteinase K treatment (Fig. 1A, left). However, in the presence of 1 µM CAM741, formation of both glycosylated fragments was inhibited, and again the remaining, unprocessed fragment was degraded by added protease (Fig. 1A, left).

    Fig. 1. CAM741 inhibits cotranslational translocation of VEGF SP-SEAP. A, sequence and schematic representation of the construct used. In vitro translocation of truncated VEGF SP-SEAP NCs (SP + 146 amino acid residues SEAP mature domain) in the absence or presence of CAM741 (1 µM), either untreated or treated with proteinase K (left); deglycosylation of sedimented VEGF SP-SEAP NCs with endoglycosidase F (Endo F; right). B, schematic representation of the construct used. In vitro translocation of truncated 131 amino acid residues VEGF NCs in the absence of microsomes (left), in the presence of microsomes (middle), or in the presence of microsomes and 1 µM CAM741 (right), either untreated or treated with proteinase K, or proteinase K and 1% Triton X-100. , glycosylated NCs with the SP attached; , glycosylated NCs without SP; , nonprocessed NCs; , NCs with SP cleaved off.

    To exclude any effect of the SEAP fusion partner on the VEGF SP, we also generated 131 amino acid residue translocation intermediates from wild-type (wt) VEGF165, which contains an N-glycosylation site at position 101 (position 75 of mature VEGF) (Fig. 1B, middle). In the control reaction, a slower migrating, glycosylated fragment was formed only in the presence of microsomal membranes, which was resistant to degradation by proteinase K. This fragment could be degraded only after permeabilization of the membranes with Triton X-100, whereas the residual unglycosylated product was almost completely degraded also in the absence of detergent. However, in the presence of CAM741, only the unglycosylated fragment formed which was largely degraded by proteinase K (Fig. 1B, right). Although CAM741 does not prevent formation of the tight, salt-resistant binding of VEGF NCs to the translocon, their translocation to the ER is prevented by the compound, witnessed by lack of SP cleavage and glycosylation and their sensitivity to degradation by exogenous protease.

    Finally, the effect of CAM741 on release of endogenously expressed VEGF was analyzed. HaCaT keratinocytes up-regulate the splice variant VEGF165 in response to treatment with TGF- (Gille et al., 1998). However, preincubation of HaCaT cells with increasing concentrations of CAM741 dose dependently inhibited TGF--induced VEGF165 release, as determined by ELISA (Fig. 2).

    Fig. 2. CAM741 inhibits release of endogenously expressed VEGF165. HaCaT cells were treated with TGF- in the presence of increasing concentrations of CAM741. The concentration of released VEGF165 (picograms per milliliter) was determined by ELISA.

    Mapping of the CAM741-Sensitive Region of the VEGF SP. Although the VEGF SP is sensitive to translocation inhibition by CAM741, it shares no similarities with the highly sensitive VCAM1 SP within the primary amino acid sequence apart from an identical cleavage site for the signal peptidase complex (Table 3). However, the cleavage site of the VCAM1 SP could be replaced without loss in sensitivity, indicating that it does not contain key residues required for the compound effect (Harant et al., 2006). To identify the region critical for inhibition by CAM741, mutagenesis of the VEGF SP was performed. Mutations at different positions of the VEGF SP caused changes in compound sensitivity; two regions, the n-region and parts of the h-region, were recognized as being essential for compound sensitivity (Table 2). The contribution of the n-region to compound sensitivity was identified by removal of the N-terminal amino acid residues 2 to 5 or by changing leucines at positions 4 and 5 into glutamine residues, because both mutants required higher concentrations of CAM741 for inhibition (Table 2). However, although these results strongly indicate an involvement of the n-region in compound sensitivity, it is dispensable for successful translocation, in that SEAP secretion was not affected in these less sensitive mutants.

    TABLE 3 Sensitivity of the VCAM1 (2-10) SP and VEGF SP mutants to inhibition by NFI028

    HEK293 cells were transfected with different SP-SEAP fusion constructs and incubated with increasing concentrations of NFI028. Twenty-four hours after transfection, supernatants were harvested and analyzed for alkaline phosphatase activity. Results shown are IC50 values from at least three independent experiments performed in triplicate. Mutations are indicated by double underlining, the cleavage site is single-underlined, and amino acid residues of the VCAM1 mature region are italic.

    TABLE 2 Modulation of the sensitivity to CAM741 by mutations in the VEGF SP

    HEK293 cells were transfected with different VEGF SP-SEAP fusion constructs and incubated with increasing concentrations of CAM741. Twenty-four hours after transfection, supernatants were harvested and analyzed for alkaline phosphatase activity. Results shown are IC50 values from at least three independent experiments performed in triplicates. Mutations are indicated by double underlining, the cleavage site is single-underlined, and amino-acid residues of the SEAP mature region are italic.

    Two residues within the central hydrophobic h-region were identified as critical for CAM741 sensitivity: leucine 12 and alanine 13. Substitutions of these residues by different aliphatic residues generated VEGF SP mutants with either increased or decreased compound sensitivities. Replacing leucine 12 with valine resulted in a mild decrease in sensitivity, whereas a more pronounced decrease was observed with isoleucine at this position. When alanine 13 was when changed to valine, leucine, or isoleucine, again a slight decrease was observed for valine, but a clear reduction in sensitivity for leucine or isoleucine at this position. However, when changing Leu12 and Ala13 to valines or isoleucines, sensitivity to CAM741 was further decreased (Table 2).

    Conversely, reducing hydrophobicity at these positions by replacing leucine 12 with glycine caused enhancement in sensitivity, as did the conversion into alanine. However, substitution of both Leu12 and Ala13 with glycine residues resulted in a hypersensitive VEGF SP variant that was inhibited by CAM741 at low nanomolar concentrations (Table 2). Taken together, these data indicate that the sensitivity to CAM741 can be modulated by increasing or decreasing hydrophobicity and/or size of the aliphatic residues at positions 12 and 13.

    Changing of other residues in the h-region, such as leucines 14 and 15, to either alanines or glycines did not affect sensitivity to CAM741. However, conversion of leucines at position 16 and 18 into valines also caused reduction in sensitivity (Table 2).

    Apart from hydrophobicity, a specific presentation of residues of the h-region may be required for the inhibitory effect of CAM741. Proline, a residue with known helix-breaking potential, was introduced at two different positions within the h-region. When alanine 13 was converted to proline, sensitivity to CAM741 markedly increased. In addition, changing tyrosine at position 17, a polar residue between leucines 16 and 18, into proline (Y17P) clearly increased sensitivity to CAM741. The results from both mutants would suggest that a specific optimal conformational presentation of residues of the h-region mediates sensitivity to CAM741, which additionally involves the n-region. We therefore tested the effect of removal of the N-terminal residues 2 to 5 in the highly sensitive VEGF SP mutant Y17P and show that in the absence of these residues, sensitivity was clearly reduced (Table 2). These data show that both the n- and h-regions mediate compound sensitivity, and maximal inhibition by CAM741 requires an interplay between these two segments.

    The response of selected VEGF SP mutants to inhibition by CAM741 at the level of cotranslational translocation was analyzed by in vitro translocation assays of truncated VEGF SP-SEAP NCs (all fused to 146 amino acid residues SEAP mature domain) in the presence of increasing concentrations of CAM741. The results from the transient transfections were also reflected by these experiments (Fig. 3).

    Fig. 3. Differential sensitivity of VEGF SP mutants to inhibition by CAM741. Schematic representation of the constructs used. In vitro translocation of fusion constructs of VEGF SP mutants and the 146 amino acid residues SEAP mature domain in the absence of microsomal membranes or in the presence of microsomes and increasing concentrations of CAM741. , glycosylated NCs; , unprocessed NCs.

    Altered Orientation of the VEGF NCs Relative to the Translocon Component Sec61 by CAM741. Targeting of the VCAM1 NCs to the translocon is not prevented by CAM741, but translocation inhibition occurs at the step of VCAM1 SP insertion into the translocon (Besemer et al., 2005; Harant et al., 2006). As the VEGF NCs could also be sedimented with the microsomal membranes after high salt/puromycin treatment in the presence of CAM741, we analyzed targeted VEGF NCs at the translocon by chemical cross-linking experiments.

    When using the heterobifunctional cross-linker m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), targeted VCAM1 NCs can be cross-linked to Sec61 in both the absence and the presence of CAM741. However, chemical cross-linking with the homobifunctional cysteine-reactive cross-linker bis-maleimidohexane (BMH) was observed only in the presence of compound a clearly enhanced cross-link to Sec61, suggesting an altered orientation of the VCAM1 NCs toward this translocon component (Besemer et al., 2005). This observation was also shared with Garrison et al. (2005) using the structurally related compound cotransin. We now employed the same chemical cross-linkers to study the translocon environment of targeted 81 amino acid residues VEGF NCs. When subjected to cross-linking with MBS followed by immunoprecipitation with a Sec61 antiserum, cross-links to Sec61 were observed in both the absence and the presence of CAM741 (Fig. 4A, left, labeled MBS). Chemical cross-linking was then performed with BMH followed by immunoprecipitation with a Sec61 antiserum. When using this cross-linker, visible cross-links can only form between the cysteine residues present in the mature region of VEGF (positions 52 and 77), and the single cysteine residue in the cytosolic tail of Sec61. However, only cysteine at position 52 is likely to be accessible to cross-linker, because cysteine at position 77 is too close to the peptidyl transferase site. Although cysteine 52 may also still be located within the ribosome, its distance from the peptidyl transferase site is sufficient for chemical cross-linking to the cytoplasmic tail of Sec61, as reported for opsin NCs (Laird and High, 1997). In the presence of CAM741, an enhanced cross-link between the VEGF NCs and Sec61 was observed, although basal cross-links to Sec61 were already seen in the absence of compound, and their enhancement by CAM741 was less pronounced (Fig. 4A, right, labeled BMH) compared with targeted VCAM1 NCs (Besemer et al., 2005). The identity of other cross-linked products that formed differently between control and CAM741-treated reactions is currently unknown (Fig. 4A, open arrowheads), with similarly sized products also formed with targeted VCAM1 NCs (Besemer et al., 2005).

    Fig. 4. CAM741 alters positioning of the VEGF NCs at the translocon. A, schematic representation of the construct used. In vitro targeting and chemical cross-linking with either MBS (left, labeled MBS) or BMH (right, labeled BMH) of truncated 81 amino acid residues VEGF NCs and immunoprecipitation with a Sec61 or Sec61 antiserum in the absence or presence of CAM741 (1 µM). , Sec61 cross-link; , Sec61 cross-link; , nonprocessed NCs; , additional unspecified cross-links; , residual peptidyl-tRNA-NCs. B, schematic representation of the constructs used. In vitro targeting and chemical cross-linking with BMH of truncated VEGF SP mutants fused to 54 amino acid residues SEAP mature domain containing a cysteine at position 30 (V30C; fourth position of the SEAP mature domain; left) and immunoprecipitation with a Sec61 antiserum (right). , Sec61 cross-link; , NCs.

    CAM741 Differentially Altered Positioning of VEGF SP Mutants at the Translocon. In the presence of CAM741, not only the VCAM1 mature region but also the VCAM1 SP has an altered orientation relative to Sec61 (Harant et al., 2006). We asked whether this is also true for VEGF. Valine 30 (fourth amino acid residue of the SEAP mature domain) was replaced by a cysteine residue in the wt VEGF SP-SEAP construct, the highly sensitive mutant VEGF (L12G, A13G) SP-SEAP, and in the constructs VEGF (L12I, A13I) SP-SEAP and VEGF (2–5) SP-SEAP, which have greatly reduced sensitivities to inhibition by CAM741. We chose position 30 for this substitution to exclude any effect of potential SP cleavage on cross-linking of targeted NCs, because it is located downstream of the cleavage site but near the SP. The constructs were then evaluated by transient transfections of HEK293 cells. As shown in Table 2, the constructs displayed a pattern of CAM741 sensitivity comparable with those containing valine at position 30, although overall sensitivity of the mutants was lower with cysteine at this position.

    Short translocation intermediates (VEGF SP + 54 amino acid residues SEAP mature domain) were generated and targeted nascent chains subjected to chemical cross-linking with BMH and immunoprecipitation with a Sec61 antiserum. At this length of the NCs, the only cysteine residue available for cross-linking was provided at position 30 (Fig. 4B). Similar to the VCAM1 SP (Harant et al., 2006), a dose-dependent increase in Sec61 cross-links of targeted VEGF (V30C) SP-SEAP NCs by CAM741 was observed. However, as also observed for wt VEGF NCs of 81 amino acid residues (Fig. 4A), some basal cross-links to Sec61 were already detected in the absence of compound (Fig. 4B). The highly sensitive construct VEGF (L12G, A13G, V30C) SP-SEAP showed only weak cross-links to Sec61 in the vehicle-treated reaction, but also formation of Sec61 cross-links with increasing concentrations of CAM741. In contrast, the construct VEGF (L12I, A13I, V30C) SP-SEAP with greatly reduced sensitivity to CAM741, formed basal cross-links to Sec61 in the absence of compound that were not enhanced by the presence of CAM741 (Fig. 4B). However, the VEGF SP mutant lacking the amino acid residues 2 to 5 [VEGF (2–5, V30C) SP-SEAP], although having strongly reduced sensitivity to CAM741, showed only weak basal cross-links with Sec61 and some enhanced formation of the Sec61 cross-link only at 1 µM CAM741 (Fig. 4B). These data indicate that substitution of Leu12 and Ala13 with isoleucine affects SP association with the translocon and could indicate a different and more efficient interaction with the translocon. In contrast, the presence of the glycines at positions 12 and 13 could cause inefficient association of the SP with the translocon, suggesting that the amino acid residues within the h-region may control the strength of translocon binding. The construct VEGF (2–5) SP-SEAP, which lacked the N-terminal amino acid residues 2 to 5 but contained no mutations within the h-region, showed only weak basal cross-links to Sec61, which further supports the idea that basal cross-linking to Sec61 was mediated through the h-region. However, the individual sensitivities of the VEGF SP mutants to CAM741 were mirrored by the different concentrations of compound required to induce Sec61 cross-links.

    CAM741 Inhibited N-Terminal Translocation of a Tag Fused to the VEGF SP. Translocation of a 17-amino acid residue tag (N-tag) fused to the VCAM1 SP was inhibited by CAM741, which additionally indicated incorrect SP insertion into the translocon channel caused by the presence of compound (Harant et al., 2006). We therefore performed targeting of 81 amino acid residue VEGF NCs fused to the 17 residues N-tag in the presence or absence of CAM741. This tag contains a diagnostic glycosylation site according to the constructs used by Heinrich et al. (2000), and N-terminal translocation of the tag is visualized by glycosylation (Fig. 5). Glycosylation can only occur at the N-tag, as at a chain length of 81 amino acid residues, the wt VEGF does not contain any glycosylation sites (Fig. 5, top left). Glycosylation of the N-terminal tag was further confirmed by treatment with endoglycosidase F (Fig. 5, top right). However, in the presence of the N-tag, efficient glycosylation was seen in the control reaction but was clearly reduced when CAM741 was present (Fig. 5, top panel). In addition, inhibition of N-terminal translocation of the tag by CAM741 was dose-dependent (Fig. 5, bottom panel). Together, these data indicate altered insertion of the VEGF SP in the presence of compound.

    Fig. 5. CAM741 inhibits N-terminal translocation of a 17 amino acid residue tag fused to the VEGF SP. Sequences and schematic representation of the constructs. The N-terminal tag is indicated in italic letters, the glycosylation site is underlined. In vitro targeting of truncated 81 amino acid residue VEGF NCs (top left), or 81 amino acid residues VEGF NCs containing the 17 amino acid residue N-terminal tag in the absence of microsomes, or in the presence of microsomes with or without 1 µM CAM741 (top middle). Deglycosylation with endoglycosidase F (Endo F; top right). In vitro targeting of 81 amino acid residues VEGF NCs containing the 17 amino acid residues N-terminal tag in the presence of increasing concentrations of CAM741 (bottom). , glycosylated NCs; , unprocessed NCs; , NCs with SP cleaved off.

    Differential Sensitivity of the VEGF and VCAM1 SPs to the Cyclopeptolide Derivative NFI028. In addition to side chain modification of HUN-7293, providing compounds such as CAM741, variation of the peptidic backbone was investigated. These efforts disclosed cyclic hexapeptides such as NFI028 as novel structural type of potent inhibitor of VCAM-1 expression (Schreiner et al., 2003; Fig. 6). To determine whether this fundamental structural difference has an impact on VCAM1 and VEGF SP-dependent translocation, HEK293 cells were transiently transfected with VCAM1 (2–10) SP-SEAP or VEGF SP-SEAP, and treated with increasing concentrations of NFI028. Although this compound was able to inhibit SEAP release from cells transfected with VCAM1 (2–10) SP-SEAP at low concentrations, it did not inhibit release of SEAP by cells transfected with VEGF SP-SEAP, demonstrating that although both SPs respond to CAM741, the VEGF SP shows no sensitivity to this derivative (Table 3). Based on the observations above, that the sensitivity of the VEGF SP to CAM741 could be enhanced by mutations within the h-region, we tested whether three of the highly sensitive mutants, VEGF (A13P) SP-SEAP, VEGF (Y17P) SP-SEAP, and VEGF (L12G, A13G) SP-SEAP could respond to inhibition by NFI028. However, only a partial response to NFI028 was observed with the mutants VEGF (A13P) SP-SEAP or VEGF (Y17P) SP-SEAP. The highly CAM741-sensitive mutant VEGF (L12G, A13G) SP-SEAP showed some increased sensitivity to NFI028, although it was markedly reduced compared with CAM741 (Table 3). These data indicate that, apart from general similarities between the VEGF and VCAM1 SPs that make them susceptible to inhibition by CAM741, differences in the composition of the SP may account for NFI028 selectivity.

    Fig. 6. Structures of CAM741 and NFI028

    Inhibition of cotranslational translocation through the SP was identified by us as a novel approach to interfere with the expression of proteins undergoing the secretory pathway. The proof of concept was provided by the discovery of the cyclopeptolide CAM741, which potently inhibits cotranslational translocation of VCAM1 (Besemer et al., 2005). At the same time, cotransin, a compound of similar structure and activity has been reported by Garrison et al. (2005). The mechanism by which this process is inhibited has been shown to be dependent on the VCAM1 SP at the level of its attachment to the Sec61 translocon (Besemer et al., 2005; Garrison et al., 2005; Harant et al., 2006). We were interested whether other SPs would also be able to respond to CAM741 to learn more about this mechanism. Garrison et al. reported some SPs with partial sensitivity to cotransin, which however lack any obvious consensus motif in their sequences (Garrison et al., 2005).

    From a panel of SPs analyzed, the most sensitive SP identified was that of VEGF; inhibition of VEGF SP-SEAP release required only 4-fold higher concentrations of CAM741 than inhibition of VCAM1 SP-SEAP (Besemer et al., 2005; Harant et al., 2006). By in vitro translocation experiments, we provide evidence that this inhibition also occurs at the level of cotranslational translocation. Moreover, the mechanism seems to be very similar or identical to that observed for VCAM1 translocation inhibition, although the two SPs share no obvious similarities within their primary sequences.

    These findings prompted us to analyze the features of the VEGF SP responsible for inhibition. In contrast to the VCAM1 SP, where the critical residues are located in the h-region and the polar c-region upstream of the cleavage site (Harant et al., 2006), residues of the VEGF SP required for translocation inhibition are located in the n-region and the h-region, where positions Leu12 and Ala13 were identified as most critical for sensitivity. Aliphatic residues with increased hydrophobicity and/or size at these positions resulted in a decrease in sensitivity; conversely, reduced hydrophobicity or tendency for -helix formation resulted in enhanced sensitivity to the compound. This indicates that these residues are located in or near a part of the h-region essential for translocon interaction. The h-region has been reported to be required for SP interaction with the translocon (Mothes et al., 1998), and CAM741 could interfere at this level. Amino acid changes within the h-region of the prolactin SP have been reported to alter its association with the translocon relative to Sec61 and Sec61, consequently affecting further processing such as SP cleavage and N-glycosylation of the mature domain (Rutkowski et al., 2003). We have observed that mutations in the VCAM1 SP, where hydrophobicity was increased in the CAM741-sensitive region, caused a different association with the translocon, witnessed by enhanced cross-links to Sec61 and Sec61 compared with the wt VCAM1 SP (Harant et al., 2006). Also with VEGF SP variants, a different association with the translocon was seen in chemical cross-linking experiments. Although wt VEGF showed some basal cross-links to Sec61, the less sensitive mutant VEGF (L12I, A13I, V30C) SP-SEAP already formed clearly visible basal cross-links with Sec61 in the absence of compound, possibly as a result of more efficient translocon interaction. This demonstrates individual association of the VEGF SP variants with the Sec61 translocon and could even involve different interaction sites.

    The strength or site of translocon binding may be one explanation for the individual compound sensitivities of the VEGF SP mutants. However, the less sensitive mutant VEGF (2–5, V30C) SP-SEAP, which lacks the N-terminal residues 2–5 but has no mutations in the h-region, gave only low basal cross-links to Sec61, and these only increased at the highest concentration of compound tested. This demonstrates that compound sensitivity can be reduced despite the presence of an intact h-region. Thus, this segment seems to mediate translocon interaction and determines proximity to Sec61, but compound sensitivity of the VEGF SP requires an interplay between the n- and h-regions, which could argue for a specific conformational requirement for optimal inhibition.

    During the translocation process, transmembrane domains can acquire a limited degree of protein folding (such as formation of an -helix) and, depending on the features of the transmembrane segment, folding can occur already inside the ribosome (Mingarro et al., 2000; Woolhead et al., 2004). Certain mutations in the h-region of the VEGF SP could support formation of a stabilized helix, which may enhance efficiency in translocon binding. In contrast, introduction of residues with helix-breaking potential, such as glycine or proline, could decrease helix formation propensities. Model SPs have been shown to initially insert with the N terminus facing toward the luminal side, followed by a reorientation with growing chain lengths. Such a dynamic reorientation has been suggested to occur more easily with unstable or kinked helices rather than stabilized helices (Rösch et al., 2000; Goder and Spiess, 2003). Alterations in helix formation propensity and thus enhanced flexibility of the VEGF SP within the translocon could be another reason for increased sensitivity to translocation inhibition by CAM741.

    Although some of the VEGF SP mutants showed higher sensitivity to inhibition by CAM741, they showed very little response to the derivative NFI028, which, however, is fully active against VCAM1. Only the mutant VEGF (L12G, A13G) SP-SEAP showed some sensitivity to NFI028, although a much higher concentration of the compound was required for inhibition compared with VCAM1, indicating that specific features of the VEGF SP account for the poor response to NFI028. Although currently a direct binding of compound to the SP cannot be fully excluded, from our data it seems more likely that there exists a competition between the compound and SP for binding to a specific site in the translocon required to initiate the translocation process. However, as shown previously, the compound does not prevent SP binding to the translocon but may rather force the SP into a position where luminal translocation cannot occur, resulting in synthesis of the growing polypeptide chains toward the cytosolic side (Besemer et al., 2005; Harant et al., 2006). According to this hypothesis, both CAM741 and NFI028 could interact with the translocon, although binding of NFI028 would be weaker. The compounds could compete with the VCAM1 SP, which interacts with the translocon inefficiently, whereas the VEGF SP binds more efficiently and cannot be competed by NFI028. This assumption is supported by the observation that mutants of the VCAM1 SP with only slightly reduced sensitivities to CAM741 had much lower sensitivities to NFI028 (H. Harant, unpublished observations). However, the similarity in CAM741 sensitivity coupled with the large difference in NFI028 sensitivity of the VCAM1 and VEGF (L12G, A13G) SP argues against this model and suggests that not only competition but also additional SP-dependent features contribute to this selectivity.

    The logical next step therefore will be the analysis of several SPs that show different degrees of sensitivity to CAM741 and the study of their association with the translocon. In addition, it would be interesting to evaluate whether insensitive SPs can be converted into sensitive ones by introduction of mutations based on our findings.


    We thank Roland Reuschel, Waltraud Mayer-Granitzer, and Eva-Marie Haupt for sequencing and Christiane Dascher-Nadel for generation of the VEGF constructs and SP-SEAP fusion constructs. We also thank Siegfried Höfinger, Piroska Devay, and Markus Jaritz for helpful discussions.

    ABBREVIATIONS: VCAM1, vascular cell adhesion molecule 1; SP, signal peptide; ER, endoplasmic reticulum; VEGF, vascular endothelial growth factor-A; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; ELISA, enzyme-linked immunosorbent assay; TGF-, transforming growth factor-; SEAP, secreted alkaline phosphatase; HEK, human embryonic kidney; NC, nascent chain; wt, wild-type; MBS, m-maleimidobenzoyl-N-hydroxysuccinimide ester; BMH, bis-maleimidohexane; HUN-7293, cyclo[N-methyl-L-alanyl-(2R)-4-cyano-2-hydroxybutanoyl-(2S,4R)-2-amino-4-methyloctanoyl-N-methyl-L-leucyl-L-leucyl-1-methoxy-N-methyl-L-tryptophyl-(2S,4R)-2-amino-4-methyloctanoyl].

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作者单位:Novartis Institutes for BioMedical Research, Vienna, Austria

日期:2009年8月25日 - 来自[2007年第69卷第6期]栏目

Neovasc与LeMaitre Vascular签署销售协议

    据悉,近日专业血管设备公司Neovasc与周围血管疾病治疗设备供应商LeMaitre Vascular签署了一项销售协议,销售Neovasc用于血管外科手术过程中的生物血管带。       此项协议期限为七年,根据协议,LeMaitre Vascular将享有在美国及欧洲销售Neovasc某些型号的组织补片产品的独家权利。       另外,根据协议,LeMaitre Vascular可以在五年后收购Neovasc生物血管带产品的技术。收购后,LeMaitre Vascular对于该技术的应用仅限于制造血管手术过程中的血管带产品,而Neovasc将保留其生物修补技术的其他一切应用的权利。两家公司未透露更多协议的有关细节。       Neovasc首席执行官Alexei Marko表示:“LeMaitre Vascular的技术创新、强大的销售及客户支持在血管外科医生中享有良好的声誉,我们很高兴其能销售我们的学管带产品。我们的组织产品的性能及特殊处理特性使得其适合应用于多种血管手术,其中包括颈动脉内膜切除术,而这将对LeMaitre Vascular的Pruitt-Inahara颈动脉分流管起到一定的补充。”(中国医药123网)        
日期:2009年2月6日 - 来自[环球]栏目

S-Adenosylhomocysteine—a better indicator of vascular disease than homocysteine?

Conrad Wagner and Mark J Koury

1 From the Departments of Biochemistry (CW) and Medicine (MJK), Vanderbilt University School of Medicine, Nashville, TN, and the Veterans Affairs Medical Center, Nashville, TN (CW and MJK)

2 Supported by grant no. DK15289 from the National Institutes of Health (to CW) and by a Merit Revue Award from the Department of Veterans Affairs (to MJK).

3 Reprints not available. Address correspondence to Conrad Wagner, Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232. E-mail: conrad.wagner{at}vanderbilt.edu.

It is widely accepted that elevated plasma total homocysteine is an independent risk factor for vascular disease. The relation is believed to be causal, but there is no generally accepted mechanism for the pathophysiology involved. The metabolic precursor of homocysteine in all tissues is S-adenosylhomocysteine (AdoHcy). AdoHcy is present in normal human plasma at concentrations approximately 1-500th of those of homocysteine, a fact that presents difficulties in measurement. The requirement for specialized equipment, complicated time-consuming methodology, or both is a reason that measurement of plasma AdoHcy has not generally been carried out in large studies. A recently published rapid immunoassay for AdoHcy in human plasma should make measurement of this important metabolite available for general use. Advantages of the measurement of plasma AdoHcy include 1) a smaller overlap of values between control subjects and patients, and thus the possibility of observing significant differences in fewer samples, 2) an accepted mechanism of metabolic activity as an inhibitor of all S-adenosylmethionine–mediated methyltransferases, and 3) evidence (from recent studies) that a higher plasma concentration of AdoHcy is a more sensitive indicator of vascular disease than is a higher plasma concentration of homocysteine.

Key Words: S-Adenosylhomocysteine • S-adenosylmethionine • homocysteine • vascular disease • methionine • risk factors • plasma

S-Adenosylhomocysteine (AdoHcy) is the immediate precursor of all of the homocysteine produced in the body. The reaction is catalyzed by S-adenosylhomocysteine hydrolase and is reversible with the equilibrium favoring formation of AdoHcy. In vivo, the reaction is driven in the direction of homocysteine formation by the action of the enzyme adenosine deaminase, which converts the second product of the S-adenosylhomocysteine hydrolase reaction, adenosine, to inosine (1). Homocysteine is a branch point in the metabolism of methionine. In one direction, it can be remethylated either by the vitamin B-12–dependent enzyme system, methionine synthase, or it can accept a methyl group from betaine to regenerate methionine. In a second direction, homocysteine can be degraded by the transsulfuration pathway by conversion to cystathionine with the use of the enzyme cystathionine-β-synthase (2).

The initial study by McCully (3) showed that homocystinuria resulted in massive thromboses and generalized vascular damage. The associated elevation in plasma homocysteine accompanying the homocystinuria in such cases can range from 150 to 500 µmol/L (normal: 10 µmol/L). However, it was not until Wilcken and Wilcken (4) examined patients with and without cardiovascular disease (CVD) that it was suggested that a moderate increase in plasma homocysteine was associated with vascular disease. Since that time, thousands of journal articles have been published on the relation between plasma or serum concentrations of homocysteine and vascular disease. Wilcken and Wilcken showed that 28% of patients with coronary heart disease had an abnormal methionine load test, which indicated a lower ability to metabolize methionine. An oral methionine load stresses the systems metabolizing methionine and results in a higher and more prolonged increase in plasma methionine in patients with CVD than in control subjects. An abnormal methionine load test was shown by Clarke et al (5) to be an independent risk factor for coronary, peripheral, and cerebral vascular disease. An elevation of plasma homocysteine in patients with vascular disease was also observed in those without a methionine load (6). Many subsequent studies have provided support for this conclusion. The meta-analysis of 27 studies by Boushey et al (7) concluded that there was a strong association of elevated plasma homocysteine with coronary, cerebrovascular, and peripheral vascular disease and that as much as 10% of the coronary artery disease in the United States could be attributed to high plasma concentrations of homocysteine. A survey of articles published between 1966 and 1998 analyzed results from 30 prospective or retrospective studies (8) and found a stronger association in the retrospective than in the prospective studies. The common polymorphism in the methylenetrahydrofolate reductase gene (677CT) found in 10% of the population is associated with higher plasma homocysteine concentrations in persons with below-normal folate concentrations (9). Another meta-analysis of 72 studies investigated the effect of mutations in this gene on homocysteine concentrations and vascular disease (10). The authors of the meta-analysis concluded from these genetic studies as well as from prospective studies showing a highly significant association between plasma homocysteine and a variety of vascular diseases that this association was causal, at least with respect to CVD (10).

There is also a dietary component. Many studies have provided evidence that, because folate, vitamin B-12, and vitamin B-6 are cofactors in the metabolic disposition of homocysteine, elevated plasma homocysteine is inversely correlated with plasma concentrations of these vitamins (11) (12). In the absence of vitamin B-12 deficiency, elevated plasma homocysteine concentrations are most responsive to folate supplementation and can be returned to normal (13) by that treatment.

There is no generally accepted mechanism for the pathophysiology of elevated plasma homocysteine as a cause of vascular disease. Various mechanisms for the toxic action of homocysteine include a change in the redox status of the tissues with production of reactive oxygen species (14); an inhibition of anticoagulation mechanisms mediated by the vascular endothelium (15); antiplatelet effects related to the reaction of elevated homocysteine with nitric oxide to form S-nitrosohomocysteine (16, 17); a direct effect of homocysteine on vascular endothelial (18) or smooth muscle cells (19); the formation of homocysteine thiolactone that modifies endothelial proteins (20); and the induction of programmed death of endothelial cells (21). In most cases, these actions have been indicated by effects caused by the addition of homocysteine to cells in culture. The principal problem with most of these studies has been the use of concentrations of homocysteine far higher (50–1000 µmol/L) than those present in plasma to show these effects. Rarely have any effects been shown with concentrations of homocysteine as low as 10 µmol/L. Homocysteine has a free sulfhydryl group and is oxidized with a second homocysteine molecule to form the disulfide, homocystine, and also with cysteine to form a mixed disulfide. In human plasma, most homocysteine exists in disulfide linkage to cysteine in albumin. For this reason, it has been the standard practice to measure total homocysteine (tHcy) that is produced after the reduction of the bound homocysteine. The normal concentration of tHcy in human males is 10 µmol/L. However, as was pointed out by Jacobsen (22), the amount of free homocysteine in human plasma is <1% (< 0.1 µmol/L). Therefore, although high plasma concentrations of homocysteine are associated with vascular disease, it has been difficult to show that they are the proximal cause of the damage.

An alternative possible cause of the pathophysiology associated with hyperhomocysteinemia is AdoHcy. This compound is the precursor of all of the homocysteine in tissues. Except for methyl transfer from betaine and from methylcobalamin in the methionine synthase reaction, AdoHcy is the product of all methylation reactions that involve S-adenosylmethionine (AdoMet) as the methyl donor. There are 50 reactions that carry out methyl transfer in cells. AdoHcy is well known as a potent inhibitor of most, if not all, methyltransferases (23). Increased concentrations of AdoHcy in tissues are usually accompanied by decreased concentrations of AdoMet. The use of the ratio of AdoMet to AdoHcy as an indicator of the methylating capacity of the cell was first suggested by Cantoni et al (24), and this ratio has been referred to as the "methylation index" (25). However, in certain situations, the elevation of AdoHcy appears to be a better indication of the inhibition of methylation than does the ratio of AdoMet to AdoHcy (26, 27). Methylation is significant in epigenetic regulation of protein expression via DNA and histone methylation. The inhibition of these AdoMet-mediated processes by AdoHcy is a proven mechanism for metabolic alteration. Because the conversion of AdoHcy to homocysteine is reversible, with the equilibrium favoring the formation of AdoHcy, increases in plasma homocysteine are accompanied by an elevation of AdoHcy in most cases. Measurement of plasma AdoHcy has not been carried out in most studies, mostly because the concentration of AdoHcy in plasma is 1-500th that of plasma tHcy, and complicated methods are needed to measure AdoHcy. Most of these methods are cumbersome and time-consuming, or they involve specialized equipment (28, 29).

Relatively few studies have directly compared plasma homocysteine and plasma AdoHcy as indicators of vascular disease, probably because of the complex methods involved in the measurement of plasma AdoHcy in large studies. Several small studies have shown that measurement of plasma AdoHcy is a better indicator of the risk of vascular disease than is measurement of plasma tHcy. Loehrer et al (30) first showed that, when compared with control subjects, patients with end-stage renal disease had 44-fold greater plasma AdoHcy but only 5-fold greater plasma homocysteine concentrations. Both measurements were significantly (P < 0.001) different, but the authors drew no conclusions about which measurement was more sensitive. In a study published in 2001 comparing patients with proven CVD and matched controls, there was a significant difference in the plasma AdoHcy concentrations between the patients and controls but no significant difference in the homocysteine concentrations (31). This inability to discriminate between patients with CVD and controls by using homocysteine concentrations was probably due to the small numbers of patients (n = 30) and control subjects (n = 29) in the study. This insensitivity illustrates one of the major problems of using plasma homocysteine as an indicator of the risk of vascular disease. Because there is a large overlap in values between patients and control subjects, large numbers of subjects are needed to show a relation between high homocysteine concentrations and vascular disease. This makes it impossible to predict that any one person with moderately elevated plasma homocysteine is at greater risk than any other person with the same plasma homocysteine concentration. An association of high homocysteine concentrations with low renal function has been noted many times. There is evidence for significant metabolism of methionine by the kidney (32). Plasma homocysteine is highly elevated in patients with renal disease—to concentrations that are generally higher than those in patients with CVD. Contrary to its effect in other patients with hyperhomocysteinemia, supplementation with folic acid in those with renal disease lowers, but does not normalize, plasma homocysteine (33, 34). A study comparing adult renal disease patients with control subjects showed that plasma AdoHcy was a significantly more sensitive test of renal insufficiency than was homocysteine (35). In the studies comparing plasma homocysteine and plasma AdoHcy in patients who were selected because they had renal disease (35) and in patients who were selected because they had CVD (31), the values for plasma AdoHcy in both patients and control subjects overlapped much less than those for homocysteine. Two studies have noted that both plasma AdoHcy and homocysteine are elevated in patients with kidney disease (30, 35). Adults with kidney disease generally are older and have other diseases that are known be associated with elevated plasma homocysteine (eg, hypertension, diabetes, and CVD), which makes it difficult to determine the primary reason for the elevated homocysteine. To determine whether decreased renal function was the reason for the elevated homocysteine, a group of children who had only renal disease were studied (36). In that group of patients, there was no statistical correlation between glomerular filtration rate and plasma tHcy, but there was a strong correlation with plasma AdoHcy. The study suggested that a reduction in the ability to metabolize or excrete AdoHcy (or both) is a primary event in renal disease. This change is probably a function of the fact that AdoHcy is readily excreted in the urine (37), whereas homocysteine is not (38). A significant correlation between elevated plasma homocysteine and serum creatinine has been noted in many previous studies of the association of homocysteine with CVD (39), which raises the question of whether decreased renal function and kidney disease due to the involvement of renal vessels with the vascular component of CVD may have been the underlying reason for the elevated plasma homocysteine in some of those earlier studies.

Although it may be expected that plasma AdoHcy and homocysteine values would tend to change in the same way, that is not always the case, as shown above for the children with renal disease only (36). In a particularly revealing study, Becker et al (40) showed that, in contrast to the plasma homocysteine concentration, the plasma AdoHcy concentration was not associated with folate concentrations. As pointed out by Becker et al, if AdoHcy is the actual toxic agent rather than homocysteine, then the use of folic acid supplementation to reduce plasma homocysteine concentrations will do nothing to reduce the incidence of vascular disease. This possibility is noteworthy in view of recent epidemiologic studies showing that folate supplementation did not reduce the risk of vascular disease, although plasma homocysteine was reduced (41, 42). It should be noted, however, that these studies were secondary prevention trials and that any effect of folate in reducing risk may have taken place at a time before supplementation was begun. Whether reduction of plasma homocysteine concentrations by B vitamin supplementation can reduce the incidence of vascular disease is under investigation in current clinical trials (41, 42). In a review of several large clinical trials, Clarke et al (43) carried out a meta-analysis of 4 trials that have been completed. It was concluded that there were no beneficial effects of B vitamin supplementation on either coronary heart disease or stroke. An additional 8 large studies are underway; together, these trials may have the statistical power to answer this question (43).

Many methods for the measurement of homocysteine in human plasma have been published. Because homocysteine contains a free thiol group and because it can form disulfide linkages with another molecule of homocysteine, with free cysteine, or with cysteine residues in proteins, only a small amount of the tHcy in plasma is free. Jacobsen has estimated that <1% is the free thiol (22). For this reason, plasma or serum must first be treated with a reducing agent to obtain the total amount of homocysteine. The normal concentration of tHcy is 10 µmol/L, and analytic methods usually involve a reduction step that is followed first by derivatization to a form more easily detected and then by separation with the use of HPLC (39). An immunoassay was developed to detect the homocysteine in plasma after reduction (44). Measurement of AdoHcy in plasma presents a greater challenge, because its concentrations are 1/500th of those of homocysteine in normal plasma—20 nmol/L. Indeed, the existence of AdoHcy in plasma was unexpected until Lohrer et al (45) devised the first sensitive method for its measurement. This method depended on the formation of the fluorescent 1,N6-etheno derivative of the adenosine portion of AdoHcy and then on separation by HPLC. This method measured AdoMet as well as AdoHcy in plasma; the reaction took a long time, although the results for measurement of AdoHcy compared favorably with those of other methods. More recently, Castro et al (46) were able to shorten the derivatization time from 8 to 4 h, and they could detect as little as 2.5 nmol AdoHcy/L in plasma. Their method uses a single HPLC column but requires the use of 1.0 mL plasma, an amount that may be difficult to obtain from small children. A method for measuring AdoHcy (and AdoMet) by using a very sensitive reaction with naphthalene dicarboxaldehyde to produce a fluorescent derivative was developed, but it too was cumbersome, requiring 2 HPLC separations in addition to the derivatization step (47).

Several other methods have used highly specialized equipment such as tandem mass spectrometry (29, 37, 48) and coulometric electrochemical detection (28) to obtain greater sensitivity. In hindsight, it would seem useful to have measured plasma AdoHcy as well as homocysteine and the relation of AdoHcy concentrations in response to folate and other B vitamins. The reason for not having done so is that the existing methods were not suitable for epidemiologic studies. Recently, a simple, rapid immunoassay developed for the measurement of AdoHcy in human plasma promises to be useful in such studies (49). No significant change was seen in concentrations of AdoHcy in plasma or serum samples that had been frozen at –80 °C and then thawed and kept for 2 h at 4 °C (37). We have seen no change in values for plasma AdoHcy kept for 4 y at –80 °C (C Wagner, unpublished data, 2007).

With regard to the stability of AdoHcy in freshly drawn plasma, we have noted, when using a method that measures both AdoMet and AdoHcy (47), that, when fresh plasma is kept at room temperature, there is little or no loss of either AdoMet or AdoHcy for 5 h. However, when frozen plasma is thawed and kept at room temperature for 5 h, there is a rapid loss of AdoMet but a very slight increase in AdoHcy. The amount of change varied from subject to subject. We ascribe these findings to some sort of activation of an enzyme in plasma, because it can be prevented by the addition of HgCL2 (C Wagner, unpublished data, 2005). We do not know whether the changes described above are due to the conversion of AdoMet to AdoHcy by a methyltransferase that is activated in frozen plasma. We believe that such changes are unlikely if the samples are kept on ice while being thawed and before analysis. The results described by Capdevila et al (49) for normal values obtained by this immunoassay are comparable to normal values published for several other, more complicated methods. If S-adenosylhomocysteine hydrolase is present and active in human plasma, there is a possibility that free AdoHcy in plasma could react with plasma adenosine to change AdoHcy concentrations; however, we are unaware of any reports of such activity in human plasma.

Elevated homocysteine has been implicated as a risk factor in numerous neurologic disorders (50-52), and a recent study showed that concentrations of homocysteine, AdoHcy, and AdoMet in plasma and cerebrospinal fluid are correlated (53). It would seem useful to determine whether cerebrospinal fluid measurements of AdoHcy are more informative than those of homocysteine in neurologic disorders such as dementia and Alzheimer disease.

The authors' responsibilities were as follows—CW and MJK: contributed equally to the writing of this article. Neither author had a personal or financial conflict of interest.


Received for publication March 23, 2007. Accepted for publication May 19, 2007.

日期:2008年12月28日 - 来自[2007年86卷第6期]栏目

Dietary fiber intake and retinal vascular caliber in the Atherosclerosis Risk in Communities Study

Haidong Kan, June Stevens, Gerardo Heiss, Ronald Klein, Kathryn M Rose and Stephanie J London

1 From the Epidemiology Branch, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, NC (HK and SJL); the Departments of Nutrition and Epidemiology (JS) and the Department of Epidemiology (GH and KMR), School of Public Health, the University of North Carolina at Chapel Hill, NC); and the Department of Ophthalmology and Visual Sciences, University of Wisconsin School of Medicine and Public Health, Madison, WI (RK)

2 Supported by grant Z01 ES043012 from the Intramural Research Program, National Institute of Environmental Health Sciences. The Atherosclerosis Risk in Communities Study is carried out as a collaborative study supported by National Heart, Lung, and Blood Institute contracts N01-HC-55015, N01-HC-55016, N01-HC-55018, N01-HC-55019, N01-HC-55020, N01-HC-55021, and N01-HC-55022.

3 Reprints not available. Address correspondence to SJ London, Epidemiology Branch, National Institute of Environmental Health Sciences, PO Box 12233, Mail Drop A3-05, Research Triangle Park, NC 27709. E-mail: london2{at}niehs.nih.gov.

Background: Dietary fiber appears to decrease the risk of cardiovascular morbidity and mortality. Microvascular abnormalities can be observed by retinal examination and contribute to the pathogenesis of various cardiovascular diseases. The impact of dietary fiber on the retinal microvasculature is not known.

Objective: We aimed to examine the association between dietary fiber intake and retinal vascular caliber.

Design: At the third visit (1993–1995) of the Atherosclerosis Risk in Communities (ARIC) Study, a population-based cohort of adults in 4 US communities, the retinal vascular caliber of 10 659 participants was measured and summarized from digital retinal photographs. Usual dietary intake during the same period was assessed with a 66-item food-frequency questionnaire.

Results: After control for potential confounders including hypertension, diabetes, lipids, demographic factors, cigarette smoking, total energy intake, micronutrients intake, and other cardiovascular disease risk factors, higher intake of fiber from all sources and from cereal were significantly associated with wider retinal arteriolar caliber and narrower venular caliber. Participants in the highest quintile of fiber intake from all sources had a 1.05-µm larger arteriolar caliber (P for trend = 0.012) and a 1.11-µm smaller venular caliber (P for trend = 0.029).

Conclusions: Dietary fiber was related to wider retinal arteriolar caliber and narrower venular caliber, which are associated with a lower risk of cardiovascular disease. These data add to the growing evidence of the benefits of fiber intake on various aspects of cardiovascular pathogenesis.

Key Words: Dietary fiber • cardiovascular diseases • microcirculation • retinal abnormalities • cereal

Dietary fiber intake is associated with a reduced risk of cardiovascular diseases, including ischemic heart disease (1-7), stroke (2, 7–9), peripheral arterial disease (10), hypertension (11), and atherosclerosis (12-14). The underlying mechanisms of the effect of dietary fiber on the cardiovascular system remain poorly understood, although previous studies have shown that fiber intake can affect blood pressure, systemic inflammation, serum lipid concentrations, postprandial absorption of carbohydrates, insulin sensitivity, fibrinolysis, coagulation, and endothelial cell function (15-21).

Microvascular dysfunction has long been implicated as a possible pathogenic factor in the development of various cardiovascular disorders (22). Observation of retinal vascular caliber may convey important information regarding the state of the microcirculation in the eyes and in other vascular beds (23). Several recent prospective studies have shown that a narrower retinal arteriolar diameter independently predicts incident severe hypertension (24), coronary heart disease (25, 26), and diabetes mellitus (27, 28). A wider retinal venular diameter has been associated with an increased risk of stroke, cerebral infarction (29), and cerebral small vessel disease (30).

We hypothesized that a higher intake of dietary fiber is associated with a wider retinal arteriolar and a narrower venular caliber. We examined this hypothesis in a population-based cohort of middle-aged men and women. We also examined potential modifying effects of cardiovascular disease risk factors, including sex, smoking status, diabetes status, hypertension, and physical activity.

The design and objectives of the Atherosclerosis Risk in Communities (ARIC) Study have been reported in detail (31). Briefly, the ARIC Study is a prospective epidemiologic study of new and established risk factors for atherosclerosis and community trends in coronary heart disease. The study population was selected as a probability sample of 15 792 men and women aged 45–64 y in Forsyth County, NC; Jackson, MS; selected suburbs of Minneapolis, MN; and Washington County, MD. Eligible participants were interviewed at home and then invited to a baseline clinical examination in 1987–1989. Three further examinations were carried out at approximately 3-y intervals, and participants were contacted annually by telephone between visits to the clinic.

Participants for the current analysis are limited to 12 887 who attended the third visit of the ARIC study cohort (1993–1995), at which time the retinal examinations occurred. This represented 86% of cohort survivors. We excluded persons who were of an ethnicity other than African American or white (n = 38) and who were missing data on retinal vascular caliber (n = 1849) or dietary fiber (n = 341). The final study sample consisted of 10 659 adults. The Institutional Review Board of the 4 participating centers approved the study.

Measurement of retinal vascular caliber
The procedures for retinal photography and the assessment of photographs were described in detail previously (32). Briefly, photographs of the retina were taken from a randomly selected eye after 5 min of dark adaptation. Trained graders, masked to all participant characteristics, used a standardized protocol to evaluate the photographs for microvascular signs.

Retinal arteriolar and venular calibers were measured with a computer-assisted technique, whereby photographs were digitized with a high-resolution scanner, and the diameters of all arterioles and venules in an area half to one disc diameter from the optic disc were measured. These diameters were summarized as the central retinal artery equivalent (CRAE) and the central retinal venular equivalent (CRVE), which represented average calibers of retinal arterioles and venules, respectively. A smaller CRAE value represents narrower retinal arterioles, and a higher CRVE value represents wider venular diameters. Quality control procedures were previously reported (32). For the retinal vascular caliber, reliability coefficients were 0.84 for within-grader and 0.79 for between-grader agreement.

Dietary assessment
The usual dietary intake of the participants at the third visit over the preceding year was assessed by using a 66-item semiquantitative food-frequency questionnaire. The questionnaire was a modified version of the 61-item instrument designed and validated by Willett et al for self-administration. The correlation coefficient of energy-adjusted crude fiber between the questionnaire and four 1-wk dietary records was 0.58 (33). To improve data quality and completeness, the questionnaire was administered by trained interviewers. Participants were asked to report the frequency of consumption of each food on the basis of 9 categories, which ranged from never or <1 time/mo to 6 times/d. Interviewers also obtained additional information, including the brand name of the breakfast cereal usually consumed. All dietary factors in our analysis were adjusted for total energy by using the residual method (34).

Other covariates
Blood pressure was measured with a random-zero sphygmomanometer according to a standardized protocol (35). We used the average values over the first 3 examinations (9-y mean blood pressure) to approximate the long-term blood pressure level. Hypertension was defined as a systolic blood pressure of 140 mm Hg, a diastolic blood pressure of 90 mm Hg, or the use of antihypertensive medication during the previous 2 wk. Diabetes mellitus was defined as a fasting glucose concentration of 126 mg/dL (7.0 mmol/L), a nonfasting glucose concentration of 200 mg/dL (11.1 mmol/L), or a self-reported history of or treatment for diabetes. Anthropometric measures (weight and height) were determined by trained, certified technicians who followed a detailed, standardized protocol (35). BMI was calculated as weight (kg)/[height squared (m)]. Blood collection and processing for concentrations of HDL cholesterol, LDL cholesterol, and triacylglycerol are described elsewhere (35). Trained and certified interviewers also collected information on age, ethnicity, sex, smoking, alcohol consumption status, medical history, occupation, education, and physical activity. We used the sports index, derived from the survey of Baecke et al (36), as a measure of physical activity. The index ranged from 1 (low) to 5 (high) for physical activity from sports during leisure time.

Statistical analysis
For this analysis, CRAE and CRVE were used in combination with fiber intake data from the same period in a cross-sectional analysis. SAS (version 9.1.2; SAS, Cary, NC) software was used for all statistical analyses. The distributions of CRAE and CRVE were continuous and relatively normally distributed in this population; therefore, we used linear regression models to examine the association of retinal vascular caliber with fiber intake. We analyzed energy-adjusted intake of fiber according to quintiles.

To assess for confounding, multivariate linear regression models were used. Our base model adjusted for age, sex, race and center. Several known and potential confounding factors were included in the multivariate models, either as indicator variables [sex, race, center, smoking status (never, former, and current smokers), occupation, education, alcohol intake, diabetes status, and hypertension] or continuous variables [age, smoking years, age at which smoking started, cigarettes smoked per day, BMI, physical activity, long-term systolic and diastolic blood pressure, serum lipids (HDL, LDL, and triacylglycerol), dietary factors from both food and supplements (total energy intake, glycemic index, carotenoids, folate, n–3 fatty acids, and vitamins B-6, B-12, C, and E), and other sources of fiber (total fiber intake not adjusted for the specific fiber types)]. Because CRAE and CRVE are correlated and might be confounders for each other (37), we included CRAE and CRVE in the models simultaneously (23, 38). Taking the lowest quintile of fiber intake as the reference, we estimated the difference of CRAE and CRVE with fiber intake after adjustment for the abovementioned covariates. In addition, we also conducted the stratified analysis by sex, smoking status, diabetes status, hypertension, and physical activity.

Given that the measurement error of dietary assessment may bias our findings, we repeated our analysis with the dietary data at the first visit (1987–1989). We also examined the association of fiber intake with frank retinal microvascular abnormalities such as arteriovenous nicking and retinopathy.

The descriptive characteristics of the ARIC participants at visit 3 stratified by quintiles of energy-adjusted total dietary fiber are shown in Table 1. Participants with a higher fiber intake generally had a higher intake of carotenoids (P < 0.001), folate (P < 0.001), n–3 fatty acids (P < 0.001), and vitamins B-6 (P < 0.001), B-12 (P = 0.009), C (P < 0.001), and E (P < 0.001). Subjects in the highest quintile of fiber intake were generally slightly older (P < 0.001), had lower BMI values (P < 0.001) and diastolic blood pressure (P < 0.001), were more likely to be female (P < 0.001), had more physical activity (P < 0.001), and were less likely to be current drinkers (P < 0.001), smokers (P < 0.001), or diabetes patients (P < 0.001).

View this table:
TABLE 1. Characteristics of participants of the Atherosclerosis Risk in Communities (ARIC) Study at the third visit (1993–1995) by quintiles of energy-adjusted total dietary fiber1

The mean (±SEM) retinal arteriolar caliber was 162.3 ± 0.2 µm, and the venular caliber was 193.1 ± 16.7 µm. Consistent with previous literature (36), we found that sex, age, BMI, alcohol drinking, smoking status, physical activity, blood pressure, and serum lipids (HDL and triacylglycerol) independently predicted retinal vascular caliber in our analysis (data not shown).

We found a statistically significant dose-response relation between CRAE and dietary fiber from all sources and from cereal (Table 2). After adjustment for CRVE, age, sex, race, center, BMI, smoking, alcohol drinking, occupation, education, physical activity, diabetes status, and other dietary factors (multivariate model 1), total fiber consumption was positively associated with arteriolar caliber (P for trend = 0.002); CRAE was 1.42 µm higher (95% CI: 0.42, 2.42 µm) in the highest quintile of intake than in the lowest quintile. Sports activity accounted for most of the difference between base model and multivariate model 1 (change in slope: –10%). After further adjustment for current hypertension, long-term systolic and diastolic blood pressure and lipids (HDL, LDL, and triacylglycerol) (multivariate model 2), the dose-response relation for total fiber attenuated (change in slope compared with base model: –30%) but remained significant (P for trend = 0.012); CRAE was 1.05 µm higher (95% CI: 0.09, 2.01 µm) in the highest quintile of intake than in the lowest quintile. A similar pattern of relation with CRAE was found for cereal fiber. The association of fruit fiber with CRAE was not significant in multivariate model 1 (P for trend = 0.114), although it became significant after further adjustment for current hypertension, long-term systolic and diastolic blood pressure, and lipids (multivariate model 2) (P for trend = 0.028). Vegetable fiber was not significantly associated with CRAE in either base model or after multivariate analyses (data not shown).

View this table:
TABLE 2. Differences in retinal arteriolar caliber (µm) across increasing quintiles of energy-adjusted fiber intake compared with the lowest quintile1

Similarly, we found significantly inverse dose-response associations between CRVE and fiber intake from all sources and from cereal, both before and after adjustment for covariates (Table 3). After adjustment for CRAE, age, sex, race, center, BMI, smoking, alcohol drinking, occupation, education, physical activity, diabetes, and dietary factors (multivariate model 1), the difference of CRVE in the highest quintile was –1.31 µm (95% CI: –2.27, –0.35 µm) relative to the lowest quintile of total fiber intake (P for trend = 0.011). Smoking and physical activity accounted for most of the difference between base model and multivariate model 1 (change in slope: –56%). The inverse association of total fiber with CRVE remained significant (change in slope compared with base model: –62%; P for trend = 0.029) after further adjustment for current hypertension, long-term systolic and diastolic blood pressure, and lipids (multivariate model 2); the CRVE was 1.11 µm lower (95% CI: 0.15, 2.08 µm) in the highest quintile of intake than in the lowest quintile. A similar pattern of relation with CRVE was found for cereal fiber. The association with CRVE was marginally significant for fruit fiber (P for trend = 0.066 in multivariate model 1) and was not significant for vegetable fiber.

View this table:
TABLE 3. Differences in retinal venular caliber (µm) across increasing quintiles of energy-adjusted fiber intake compared with the lowest quintile1

We examined whether sex, smoking status, diabetes, hypertension, and physical activity modified the associations of total fiber with arteriolar caliber (Table 4). We found no significant interaction terms. Similar patterns were found for venular caliber.

View this table:
TABLE 4. Adjusted differences in retinal arteriolar caliber (µm) between the highest and lowest quintiles of energy-adjusted intakes of total fiber, by sex, smoking status, diabetes status, hypertension, and physical activity1

Using the dietary data at the first visit (1987–1989), we found similar associations of dietary fiber with wider retinal arteriolar caliber and narrower venular caliber as we did with diet at visit 3 (1993–1995) when the retinal exams were done. We found no significant association of fiber intake with arteriovenous nicking or retinopathy.

In this cross-sectional analysis of a population-based cohort of middle-aged adults, we found significant associations of higher fiber intake from all sources and from cereal with wider retinal arteriolar caliber and narrower venular caliber. These associations were not explained by other dietary factors, including antioxidants, B vitamins, n–3 fatty acids, glycemic index, and fruit and vegetable fiber or by a large array of risk factors for this condition, including smoking, physical activity, hypertension, diabetes, and serum lipids.

Several mechanisms could underlie the associations we observed. Fiber intake may reduce known risk factors for smaller retinal arteriolar caliber and wider venular caliber. The primary risk factor for retinal arteriolar narrowing is hypertension (39). Several clinical trials and prospective studies suggest that fiber may protect against hypertension (40-42). In our analysis, the effect of fiber intake on CRAE or CRVE attenuated after adjustment for current hypertension and long-term blood pressure, which supports the hypothesis that the protective effect of fiber on arteriolar narrowing or venular widening may be mediated, in part, through its direct or indirect effects on blood pressure. Fiber intake may also reduce dyslipidemia, which is a risk factor for retinal microvascular abnormalities (39); attenuation of the association between dietary fiber and retinal vascular caliber when serum lipids were included in the regression model supports this potential mechanism. In addition, higher cereal fiber intake has been associated with reduced incident diabetes in the ARIC cohort (43), which is related with retinal venular widening (39). However, it should be noted that the significant associations between dietary fiber and retinal vascular caliber remained after we carefully controlled for hypertension, long-term blood pressure, lipids, and diabetes, which suggests that other mechanism may also play a role in the protective effect of dietary fiber. For example, fiber intake appears to reduce systemic inflammation, an important contributor to arteriolar narrowing and venular widening (19-21, 39); however, the markers of systemic inflammation, such as C-reactive protein, fibrinogen, and white blood cell count, were not available for most subjects at the third visit of the ARIC Study. Moreover, fiber intake was found to benefit endothelial cell function (18); several small clinical studies have suggested that endothelial dysfunction may influence retinal vascular caliber (44, 45). Fiber consumption may also replace intake of other foods with potentially detrimental effects on the microcirculation. Another possibility is that some constituents of dietary fiber, such as trace elements, may reduce cardiovascular disease risk (46).

As in most observational studies, residual confounding is possible. However, we found significant associations of dietary fiber with retinal vascular caliber after detailed adjustment for known and potential cardiovascular disease risk and protective factors (eg, hypertension, long-term blood pressure, lipids, diabetes, smoking, physical activity, alcohol intake, total energy intake, glycemic index, n–3 fatty acids, antioxidant vitamins, and specific sources of fiber), which suggests an independent role of dietary fiber in the etiology of retinal microvascular abnormalities. Although the concern may be raised that a diet high in fiber might be a marker of a healthy lifestyle, including less frequent smoking (Table 1), we carefully adjusted for smoking [smoking status (current, past, and never smokers), smoking years, age at which smoking started, and cigarettes smoked per day]. Although residual confounding by smoking could occur despite our careful control, we also found a protective effect of fiber in never smokers, which suggests that the benefits of fiber intake are not due to the correlation with smoking behavior (47).

In adjusted analyses, we found significant associations for total fiber and fiber from cereal, but not for vegetable fiber. The lack of an association of retinal vascular caliber and vegetable fiber is consistent with several prior reports on other cardiovascular outcomes (3-6, 9), which suggests that the effect of dietary fiber may vary depending on the food sources. However, the biological mechanisms for these differences are unclear.

We found no significant association of fiber with arteriovenous nicking and retinopathy, which suggests that fiber might be protective in the earlier stages of pathogenesis. The heterogeneity of these associations may reflect different pathophysiologic processes related with specific retinal microvascular signs (48).

On stratification by hypertension, subjects with hypertension were the smaller group. Although we did not observe a significant effect of fiber among subjects with hypertension (Table 4), there was no suggestion of interaction. This finding suggests limited power for this stratified analysis. However, it is possible that the effect of hypertension on retinal vascular caliber may dominate to such an extent that the additional exposure to fiber does not enhance effects in the same pathways.

The limitations of our analysis should be noted. We used a food-frequency questionnaire to characterize dietary fiber intake. Although fiber intake assessed by the food-frequency questionnaire was reasonably well correlated with intake measured by diet records, measurement error likely limited our ability to detect associations. In addition, caution must be made when interpreting the findings described herein that the current analyses were cross-sectional; thus, a temporal relation between fiber intake and retinal vascular caliber cannot be established. However, it should be noted that ARIC subjects would not have been aware of their retinal vascular caliber in advance and thus could not have changed their diet based on this result.

A major strength of our analysis was that it was based on carefully collected data on retinal abnormalities in a large cohort of the general population from 4 US communities. ARIC is also one of the largest studies of risk factors for these retinal microvascular signs. Confounding by hypertension, lipids, and diabetes was addressed by direct measurements made during the ARIC visit, and detailed data were available on other potential confounders.

In summary, in this cross-sectional analysis, a higher intake of fiber from all sources and from cereal was related to wider retinal arteriolar caliber and narrower venular caliber, both of which have been found to be associated with a lower risk of cardiovascular disease. These associations were independent of smoking, hypertension, diabetes, serum lipids, and other risk factors for cardiovascular disease. These data add to the evidence of a protective role for fiber in various aspects of the pathogenesis of cardiovascular disease.

The authors’ responsibilities were as follows—HK: contributed to the data analysis and manuscript preparation; JS, GH, RK, and KMR: contributed to the study design and manuscript preparation; and SJL: contributed to the study design, data analysis, and manuscript preparation. None of the authors had any financial or personal interest, except for JS, whose institution received unrestricted gifts from Sanofi-Aventis and the Gatorade Corporation.


Received for publication March 29, 2007. Accepted for publication July 27, 2007.

日期:2008年12月28日 - 来自[2007年86卷第6期]栏目

Homocysteine, vitamins, and vascular disease prevention

Kilmer S McCully

1 From the Pathology and Laboratory Medicine Service, Department of Veterans Affairs Medical Center, West Roxbury, MA

2 Presented at the Harvard College 50th Reunion, held in Cambridge, MA, June 6–9, 2005.

3 Address reprint requests to KS McCully, Pathology & Laboratory Service, Veterans Affairs Medical Center, 1400 Veterans of Foreign Wars Parkway, West Roxbury, MA 02132. E-mail: kilmer.mccully{at}med.va.gov.


In mid-20th century United States, deaths from vascular disease reached a peak incidence in 1955, but little was known about the underlying causes of this epidemic of disease. The significance of homocysteine in human disease was unknown until 1962, when cases of homocystinuria were first associated with vascular disease. Analysis of an archival case of homocystinuria from 1933 and a case of cobalamin C disease from 1968 led to the conclusion that homocysteine causes vascular disease by a direct effect of the amino acid on arterial cells and tissues. The homocysteine theory of arteriosclerosis attributes one of the underlying causes of vascular disease to elevation of blood homocysteine concentrations as the result of dietary, genetic, metabolic, hormonal, or toxic factors. Dietary deficiency of vitamin B-6 and folic acid and absorptive deficiency of vitamin B-12, which result from traditional food processing or abnormal absorption of B vitamins, are important factors in causing elevations in blood homocysteine. Numerous clinical and epidemiologic studies have established elevated blood homocysteine as a potent independent risk factor for vascular disease in the general population. Dietary improvement, providing abundant vitamin B-6, folic acid, and cobalamin, may prevent vascular disease by lowering blood homocysteine. The dramatic decline in cardiovascular mortality in the United States since 1950 may possibly be attributable in part to voluntary fortification of the food supply with vitamin B-6 and folic acid. Fortification of the US food supply with folic acid in 1998, as mandated by the US Food and Drug Administration, was associated with a further decline in mortality from vascular disease, presumably because of increased blood folate and decreased blood homocysteine in the population.

Key Words: Arteriosclerosis • cobalamin • folate • homocysteine • processed foods • vitamin B-6


In mid-20th century United States in 1955, deaths from heart attack and vascular disease reached a crescendo, becoming the leading cause of death and affecting many men and some women in the prime of their lives. Many physicians were appalled at the death toll from this epidemic and were baffled by the inability of medical science to understand the underlying cause of this disease. Scientists at the Framingham Heart Study were beginning to determine that smoking, high blood pressure, and blood cholesterol concentrations in middle-aged men were somehow related to this great increase in vascular disease. During this period, cholesterol chemistry and biosynthesis were studied by Louis Fieser and Konrad Bloch and their colleagues at Harvard. Also during this period, Frederick Stare and his colleagues produced vascular disease by cholesterol feeding in monkeys at the Harvard School of Public Health. Nevertheless, the way in which all of these "risk factors" contributed to the vascular disease problem seemed very difficult for physicians of the period to understand.

The American biochemist Vincent DuVigneaud won the Nobel Prize in Chemistry in 1955 for his pioneering studies of sulfur amino acid chemistry and for synthesizing a biologically active peptide hormone, oxytocin, from its constituent amino acids. In 1932 DuVigneaud discovered a new amino acid by treating methionine with sulfuric acid (1). Because this amino acid was similar in structure to cysteine and contained an extra carbon atom, he named it homocysteine. DuVigneaud investigated the role of homocysteine in metabolism and the ability of homocysteine and choline to replace methionine as an essential nutrient for growth of animals (2). However, little else was known about the importance of homocysteine in medicine or vascular disease in the 1950s. In 1953 Frederick Stare and his colleagues found that cholesterol concentrations and experimental atherogenesis in monkeys were inhibited by dietary methionine (3), which suggested a relation between arteriosclerosis and sulfur amino acid metabolism.


In 1962 children with mental retardation, dislocated ocular lenses, accelerated growth, osteoporosis, and a tendency to thrombosis of arteries and veins were discovered to excrete the amino acid homocystine in their urine (4–6). These children had a rare inherited enzymatic defect in homocysteine metabolism that was caused by deficiency of cystathionine synthase, an enzyme requiring pyridoxal phosphate (vitamin B-6) for normal activity (7).

Through some remarkable medical sleuth work, pediatricians at Massachusetts General Hospital discovered an archival case of homocystinuria published as a case report in 1933 (8). This 8-y-old boy was the uncle of a patient who was diagnosed with homocystinuria in 1965 (9). The boy was mentally retarded and had dislocated lenses and skeletal abnormalities. He expired with symptoms of a stroke in 1932. In discussing the pathological findings in this case, the pathologist Tracey Mallory found that the cause of death was thrombosis of the carotid artery with cerebral infarct and stroke. He remarked that the carotid arteries were narrowed by arteriosclerotic plaques caused by "a simple sclerotic process such as one sees in elderly people."

Because of my interest in amino acid metabolism and my experience in the laboratory of Giulio Cantoni and Harvey Mudd at the National Institutes of Health, I decided to restudy this interesting case of homocystinuria and arteriosclerosis. In 1968, the original protocol, 6 original slides, and several fragments of tissue imbedded in paraffin had survived since 1933 in the Pathology Department at Massachusetts General Hospital. In my review of this case, I found that arteriosclerotic plaques were scattered throughout the arteries in many organs. It was difficult to prove, however, that homocysteine was connected to the arteriosclerotic plaques and thrombosis that had caused the death of this child.

Later in 1968 I was fortunate to learn of another case of homocystinuria that had been studied at Massachusetts General Hospital, the National Institutes of Health, and Brandeis University. A 2-mo-old baby boy with growth failure and pneumonia was discovered to excrete homocysteine, cystathionine, and methylmalonic acid in the urine. Biochemical study disclosed deficiency of methionine synthase, an enzyme dependent on cobalamin (vitamin B-12) and methyltetrahydrofolate, and the case was reported as the index case of cobalamin C disease in the medical literature (10). In restudying the autopsy findings of this case, I discovered astonishingly advanced arteriosclerotic plaques scattered throughout the arteries in the major organs of the body. Because the accumulation of homocysteine was caused by a different enzyme abnormality from the earlier 1933 case, I concluded that homocysteine causes arteriosclerotic plaques by a direct effect of the amino acid on the cells and tissues of the arteries (11). In 1976, a child with the third major cause of homocystinuria, deficiency of methylenetetrahydrofolate reductase, was found to have similar arteriosclerotic plaques throughout the body, which independently corroborated my earlier conclusion (12).


In his classic monograph of 1923 entitled Inborn Errors of Metabolism, Sir Archibald Garrod pointed out that experiments of nature, consisting of inherited diseases of metabolism, help to illuminate the causes of disease processes (13). Investigation of cases of homocystinuria showed that 3 different inherited enzyme abnormalities cause elevation of blood homocysteine, producing arteriosclerotic changes in the arteries. This discovery suggested that elevation of blood homocysteine is likely to be a factor in the pathogenesis of arteriosclerosis in the general population (11, 14). Thus, dietary, genetic, metabolic, hormonal, or toxic factors cause arteriosclerotic plaques and thrombosis because of elevation of blood homocysteine, which affects the cells and tissues of the arteries (15).

This interpretation suggests that elevations in blood homocysteine may explain the experimental atherogenesis in animals caused by deficiency of vitamin B-6 in monkeys (16), deficiency of choline and other methyl donors in rats (17), methionine deficiency produced by feeding of cholic acid with thiouracil in rats (18, 19), and methionine deficiency produced by feeding soy protein and the goitrogenic isoflavones and saponins of soy in monkeys (3). Methionine deficiency elevates blood homocysteine concentrations because of decreased synthesis of adenosyl methionine and dysregulation of sulfur amino acid metabolism (20).

According to current concepts, homocysteine damages cells and tissues of arteries by inciting the release of cytokines, cyclins, and other mediators of inflammation and cell division (15). By affecting smooth muscle cells, homocysteine produces the connective tissue changes of arteriosclerotic plaques, causing fibrosis, calcification, proteoglycan deposition, and damage to elastic tissue layers. Homocysteine is a potent procoagulant that promotes the deposition of fibrin and mural thrombosis in artery walls. Homocysteine thiolactone is the reactive anhydride of homocysteine that interacts with LDL, causing aggregation, increased density, and uptake by vascular macrophages to form foam cells (21). Degradation of these aggregates leads to deposition of cholesterol and other fats in developing plaques. In addition, reaction of homocysteine thiolactone with serum proteins leads to the production of new protein antigens and autoimmune antibodies, facilitating the inflammatory response (22). Homocysteine causes oxidant stress by effects on cellular respiration, leading to oxidation of LDL and other constituents of plaques (23). Homocysteine also antagonizes the vasodilator properties of nitric oxide by the formation of S-nitrosohomocysteine, leading to endothelial dysfunction, the earliest stage in atherogenesis (24).

In the decades since the discovery and development of the homocysteine theory of arteriosclerosis, numerous clinical and epidemiologic studies have established elevation of blood homocysteine as a potent, independent risk factor for vascular disease (25). The results of the Physicians' Health Study, the Nurses' Health Study, the European Concerted Action Study, and the Hordaland Homocysteine Study all support the validity of the homocysteine theory of arteriosclerosis (26). Meta-analysis of published studies suggests that elevation of homocysteine is a causal factor in atherogenesis; such studies predicted that lowering homocysteine concentrations would be estimated to benefit 15–40% of the population by preventing vascular disease (27). This estimate is conservative, because it is based on an arbitrary definition of "normal" blood homocysteine concentrations in the population. In fact, many studies have shown that vascular disease risk is directly correlated with blood homocysteine over a wide range of values, which suggests that lowering blood homocysteine may benefit a large fraction of the population. Current trials have been designed to test this possibility.

The landmark report by the Framingham Heart Study in 1993 showed in a group of 1160 elderly participants between the ages of 67 and 96 y that blood homocysteine becomes elevated because of dietary deficiencies of vitamin B-6 and folic acid and decreased absorption of vitamin B-12 (28). Elevation of blood homocysteine is associated with increased prevalence of heart attack, as shown by the third National Health and Nutrition Examination Survey (29). These findings show that dietary deficiencies of vitamins B-6 and folic acid, and absorptive deficiency of vitamin B-12, lead to elevation in blood homocysteine concentrations, which produces vascular disease in the population. In addition, genetic factors are involved. A genetic variant of methylenetetrahydrofolate reductase, 677TT, that affects 12% of the population, leads to increased risk of vascular disease if dietary folic acid is marginal (27).

The amounts of dietary B vitamins needed to prevent elevations in blood homocysteine are 3 mg vitamin B-6 and 400 µg folic acid, as shown by the Framingham Heart Study (28). These figures agree well with the findings of the Nurses' Health Study, which showed that these amounts of dietary vitamin B-6 and folic acid are needed to prevent mortality and morbidity from heart disease (26). Before fortification of grain products with folic acid in 1998, intakes of vitamin B-6 and folic acid were well below these figures, amounting to 1.5 mg vitamin B-6 and 250 µg folic acid per day (30).

Vitamin B-12 intakes are generally adequate, except in vegans, who consume no meat, fish, or dairy foods. Vitamin B-12 is present only in foods of animal origin, so strict vegans may have insufficient intakes to prevent elevations in blood homocysteine (31). Vegans may obtain small amounts of vitamin B-12 from commercially baked goods, some of which contain lard or milk products. Vitamin B-12 absorption is inadequate in 15% of the elderly population aged >65 y because of lack of gastric acidity; decreased intrinsic factor synthesis by gastric mucosal cells, as originally discovered by William Castle at Harvard; and infection by Helicobacter pylori, the etiologic agent for gastric and duodenal peptic ulcers, as discovered by Barry Marshall and Robin Warren of Australia (32). For this reason, some elderly persons are susceptible to the subtle mental symptoms, neurological changes, weakness, and fatigue that are associated with deficiency of vitamin B-12.

In addition to vitamin B-6, folic acid, and vitamin B-12, vitamin B-2 (riboflavin) was recently shown to be a determinant of blood homocysteine (33–35). The requirement for riboflavin in preventing elevations in blood homocysteine is primarily found in persons with the common genetic variant of methylenetetrahydrofolate reductase, 677TT. These persons require adequate dietary folate and riboflavin for normal enzyme activity of methylenetetrahydrofolate reductase to prevent elevations in blood homocysteine. Other conditions that predispose to vascular disease, such as renal failure, hypothyroidism, and estrogen deficiency, are also characterized by elevated blood homocysteine concentrations (36). The hyperhomocysteinemia in hypothyroidism is likely related to the diminished conversion of dietary riboflavin to its coenzyme derivatives, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). FAD is the coenzyme required by methylenetetrahydrofolate reductase. Hypothyroidism in rodents depresses this conversion, which results in decreased hepatic concentrations of FMN and FAD (37, 38). These results have been confirmed and extended in human hypothyroidism. The metabolic defects of riboflavin metabolism in hypothyroid adults are completely corrected by treatment with thyroid hormones without increasing dietary riboflavin intake (39).

In many individuals, dietary vitamin B-6 and folic acid intakes are marginal because traditional methods of food processing partially destroy these sensitive B vitamins (40). Thus, milling of grains, canning, extraction of sugars and oils, and the addition of bleaching agents and other chemical additives account for losses of these B vitamins of 85% in highly processed foods. Countries such as Japan, France, and Spain with higher intakes of vitamin B-6 and folic acid have lower homocysteine concentrations, averaging 6–8 µmol/L, than do countries such as Finland, Scotland, and Northern Germany with lower B vitamin intakes and correspondingly higher homocysteine concentrations of 10–12 µmol/L. Because of these differences in vitamin B consumption, the mortality rates from coronary heart disease are related to homocysteine concentrations in a group of 30 countries on the basis of stored plasma samples collected in the 1970s (41).


Any "young elderly" person should have his or her blood homocysteine concentration monitored while in a fasting state every year. If the homocysteine concentration is in the range of 4–8 µmol/L, the risk of vascular disease from this etiology is low, and a healthful, nutritious diet, such as the Heart Revolution Diet, should be continued (42). Many authorities have advocated similar diets with abundant vitamin B-6, folic acid, and vitamin B-12 from fruit, vegetables, whole grains, fresh meats, and seafood. If the homocysteine concentration is in the range of 8–12 µmol/L, an effort should be made to improve the quality of the diet, providing sufficient vitamin B-6, folic acid, and vitamin B-12 to keep homocysteine concentrations low and to minimize disease risk. The aging process is associated with decreased ability to absorb these B vitamins, which results in a gradually rising homocysteine concentration with age, 1 µmol/L per decade. Over the age of 60 y, consideration should be given to consuming 3 mg vitamin B-6, 400 µg folic acid, and 100 µg vitamin B-12 as dietary supplements, most conveniently in a daily multivitamin pill, in addition to consuming the Heart Revolution Diet.

The following guidelines are personal recommendations based on clinical experience. If the elderly person is sedentary, obese, and a smoker consuming a poor diet, the homocysteine concentration may be in the range of 10–14 µmol/L. In addition to consuming an improved diet, supplements of 10 mg vitamin B-6, 1000 µg folic acid, and 100 µg vitamin B-12 should be considered to decrease disease risk. If there is a family history of heart disease, hypertension, and a low HDL concentration, the disease risk is high and the homocysteine concentration is likely to be in the range of 12–20 µmol/L. An improved diet and supplements of 50 mg vitamin B-6, 2000 µg folic acid, and 500 µg vitamin B-12 should be considered. If there is a history of angina, ischemic attacks, kidney failure, or diabetes and homocysteine concentrations are in the range of 16–30 µmol/L, disease risk is very high, and an improved diet with 100 mg vitamin B-6, 5000 µg folic acid, and 1000 µg vitamin B-12 should be considered. Another advisable supplement is fish oil, which decreases homocysteine concentrations when taken in doses of 12 g/d (43). Fish oil contains n–3 fatty acids that have a beneficial antiinflammatory effect.

The Heart Revolution Diet consists of fresh vegetables, fresh fruit, fresh meats and seafood, whole-grain foods, nuts, fresh eggs, yogurt, milk or cream, and occasional liver or liver pâté (42). Highly processed foods should be minimized, because they are partially depleted of vitamin B-6 and folic acid. Canned vegetables, fruit, meats, and seafoods contain only one-half or less the vitamin B-6 and folic acid that fresh foods do. Foods containing sugar, white flour, or white rice are seriously depleted of vitamin B-6 and folic acid, because these methods of food processing destroy 90% of these nutrients. Processed and packaged foods that are made with powdered eggs, powdered milk, and partially hydrogenated oils contain potentially damaging oxidized cholesterol and trans fats. Following the Heart Revolution Diet, combined with smoking cessation and moderate regular exercise, will help to control blood homocysteine concentrations and prevent vascular disease from this cause (42).

The Centers for Disease Control and Prevention issued a report on mortality from vascular disease in the 20th century (44). This report shows that mortality from vascular disease, in particular diseases of the heart, increased dramatically from 1900 to 1950, becoming the leading cause of death and reaching a peak in the late 1950s and early 1960s. The report stated, "Since 1950, age-adjusted death rates from cardiovascular disease have declined 60%, representing one of the most important public health achievements of the 20th century."

In 1978, almost 20 y after the dramatic decline in heart disease mortality became apparent, a nationwide conference at the National Institutes of Health concluded that none of the traditional risk factors, such as changes in dietary fats, blood cholesterol concentrations, smoking, hypertension, exercise, or coronary care units could explain this dramatic decline (45). In the 1950s and 1960s, synthetic vitamin B-6 was added to the US food supply in the form of fortification of cereals and supplements (15). In the 1960s, synthetic folic acid was also added to the food supply, and in 1998, the US Food and Drug Administration mandated the addition of folic acid to enriched flours and other refined-grain foods. Lowering of blood homocysteine concentrations by the addition of vitamin B-6 and folic acid to the US diet may explain in part the dramatic decline in vascular disease mortality in the United States to less than one-half the peak incidence. In recent years, additional factors such as smoking cessation; treatment of hypertension, hyperlipidemia, and diabetes; use of low-dose aspirin; and improved medical and surgical treatments (acute management of myocardial infarction, angioplasty, stenting, coronary bypass, etc) have also contributed to the decline in mortality.

Since 1998 folic acid fortification of refined grain foods has lowered the incidence of neural tube defects and other serious birth defects by as much as 78% in Newfoundland by lowering maternal blood homocysteine concentrations (46). A recent study by the Centers for Disease Control and Prevention found that the decline in stroke mortality in the United States and Canada from 1990 to 2002 accelerated from a 0.3% annual decline from 1990 to 1998 to a 2.9% annual decline beginning in 1998, accounting for 16 700 fewer deaths from stroke per year over a 6-y period (47). The accelerated rate of decline was attributed in part to lowering of blood homocysteine concentrations by folic acid fortification of refined grain foods, because other factors that might have accounted for this dramatic decline were unchanged. No change in stroke mortality was found during the same period in England and Wales, countries where there is no fortification of foods with folic acid. This study (47) and the Framingham Heart Study (48) showed that blood folate concentrations almost doubled and homocysteine concentrations declined 15% after folate fortification of enriched grains in the United States in 1998.

Current efforts to demonstrate reduced mortality and morbidity from vascular disease through interventional studies with dietary improvement and supplemental B vitamins to lower blood homocysteine are complicated by the large number of participants needed to power the studies, the length of the trials required, and the fortification of the North American food supply with folic acid (49). Recently, 3 large prospective trials of supplementation with B vitamins in patients with advanced vascular disease (VISP, NORVIT, and HOPE2) concluded that moderate doses of folic acid and vitamins B-6 and B-12 over a 3–5-y period have little effect on risk of recurrent heart attack or stroke (50–52). In the VISP trial of stroke survivors (50), a subgroup analysis concluded that those participants without renal impairment, without malabsorption of vitamin B-12, or who were not taking nonstudy vitamin B-12 supplements had a significant 21% reduction in adverse vascular events from B vitamin therapy (53). In the HOPE2 trial of patients with advanced vascular disease, there was a significant 24% reduction in stroke from B vitamin therapy, but the slight reductions in all-cause mortality, myocardial infarction, and cardiovascular death were not significant (52). Homocysteine concentrations were measured in only 19% of the HOPE2 participants after 5 y, and the lowering of homocysteine concentrations was not statistically significant (54). In the NORVIT trial of heart attack survivors (51), the placebo group had a significantly higher percentage of patients who were treated with cardiac bypass grafts or angioplasty (447/943 = 47.4%) than did the B vitamin group (395/937 = 42.2%), which may explain the decreased rate of late adverse vascular events in the placebo group (54).

In all of these trials, the participants had advanced disease that had been progressing for several decades, and the intervention with supplemental B vitamins was only for a 2–5-y period. Longer periods of intervention may be required. In addition, most of the participants were taking multiple drugs, including aspirin, statins, beta blockers, and other medications that may have obscured the potential beneficial effect of the B vitamin intervention. A review of 43 earlier studies of blood homocysteine concentrations and risk of cardiovascular disease concluded that most cross-sectional and case-control studies, with a few exceptions, supported elevation of blood homocysteine as a risk factor for coronary heart disease (55). Most of the prospective studies, however, did not support a relation between blood homocysteine and coronary heart disease, and the authors questioned whether blood homocysteine concentrations are a marker rather than a cause of the disease.

These findings, along with the generally negative results of the recent secondary prevention trials with B vitamin supplements (50–52), suggest that blood homocysteine, as measured by the present methods, likely reflects an underlying metabolic abnormality in the chronic disease process (25). In theory, the metabolic abnormality in advanced vascular disease is considered to involve depletion of the homocysteine derivative, thioretinaco ozonide, from cellular membranes (56). Thioretinaco, which is synthesized from homocysteine thiolactone, vitamin A, and vitamin B-12, prevents homocysteine-induced vascular disease in rats (57) and is anti-carcinogenic and anti-neoplastic in mice (58). Use of this compound in future human studies may be found to benefit advanced vascular disease by correcting this theoretical abnormality of homocysteine metabolism.

The most important role of B vitamin supplementation appears to be in primary prevention, as suggested by the reduction in stroke mortality after the institution of folic acid fortification (47). A recent study showed that folic acid supplementation suppresses the autoimmune response to homocysteinylated albumin and hemoglobin in hyperhomocysteinemic subjects without coronary artery disease but had no effect on the autoimmune response in subjects who already have coronary artery disease (59). A recent trial of B vitamin supplements in elderly patients with vascular disease showed no improvement in cognitive function despite a lowering of blood homocysteine concentrations (60). These results and the negative results from the secondary prevention trials of advanced vascular disease (50–52) do not support the use of B vitamin supplements to reverse the effects of advanced vascular disease.

My advice to keep the young elderly healthy is to eat an improved diet that is rich in nutrients, including vitamins, minerals, antioxidants, and phytochemicals (42). This simple strategy should help to prevent the life-long progression of vascular disease attributable to elevated blood homocysteine, which leads to life-threatening heart attack, stroke, amputations, and kidney failure. Additional helpful preventive measures are smoking cessation, stress reduction, moderate exercise, weight control, and treatment of malignant hypertension, dyslipidemia, and diabetes. Furthermore, in the young elderly who do not have advanced vascular disease, homocysteine reduction may have a role in disease prevention.


日期:2008年12月28日 - 来自[2007年86卷第5期]栏目

Randomized controlled trial of homocysteine-lowering vitamin treatment in elderly patients with vascular disease

David J Stott, Graham MacIntosh, Gordon DO Lowe, Ann Rumley, Alex D McMahon, Peter Langhorne, R Campbell Tait, Denis St J O’Reilly, Edward G Spilg, Jonathan B MacDonald, Peter W MacFarlane and Rudi GJ Westendorp

1 From the Division of Cardiovascular and Medical Sciences (DJS, GDOL, AR, PL, and PWM), the Nursing & Midwifery School (GM), and the Robertson Centre for Biostatistics (ADM), University of Glasgow, Glasgow, United Kingdom; the Departments of Haematology (RCT) and Pathological Biochemistry (DSJO), Glasgow Royal Infirmary, Glasgow, United Kingdom; the Department of Geriatric Medicine, Garnavel General Hospital, Glasgow, United Kingdom (EGS and JBM); and the Section of Gerontology and Geriatrics, Leiden University Medical Centre, Leiden, Netherlands (RGJW)

2 International Standard Randomized Controlled Trial Number (ISRCTN) 07337345.

3 Supported by a grant from the Healthcare Foundation (reference 112/57).

4 Reprints not available. Address reprint requests to DJ Stott, Academic Section of Geriatric Medicine, 3rd Floor Centre Block, Glasgow Royal Infirmary, Glasgow G4 0SF, United Kingdom. E-mail: d.j.stott{at}clinmed.gla.ac.uk.

Background: Homocysteine is an independent risk factor for vascular disease and is associated with dementia in older people. Potential mechanisms include altered endothelial and hemostatic function.

Objective: We aimed to determine the effects of folic acid plus vitamin B-12, riboflavin, and vitamin B-6 on homocysteine and cognitive function.

Design: This was a factorial 2 x 2 x 2, randomized, placebo-controlled, double-blind study with 3 active treatments: folic acid (2.5 mg) plus vitamin B-12 (500 µg), vitamin B-6 (25 mg), and riboflavin (25 mg). We studied 185 patients aged 65 y with ischemic vascular disease. Outcome measures included plasma homocysteine, fibrinogen, and von Willebrand factor at 3 mo and cognitive change (determined with the use of the Letter Digit Coding Test and on the basis of the Telephone Interview of Cognitive Status) after 1 y.

Results: The mean (±SD) baseline plasma homocysteine concentration was 16.5 ± 6.4 µmol/L. This value was 5.0 (95% CI: 3.8, 6.2) µmol/L lower in patients given folic acid plus vitamin B-12 than in patients not given folic acid plus vitamin B-12 but did not change significantly with vitamin B-6 or riboflavin treatment. Homocysteine lowering with folic acid plus vitamin B-12 had no significant effect, relative to the 2 other treatments, on fibrinogen, von Willebrand factor, or cognitive performance as measured by the Letter Digit Coding Test (mean change: –1; 95% CI: –2.3, 1.4) and the Telephone Interview of Cognitive Status (–0.7; 95% CI: –1.7, 0.4).

Conclusion: Oral folic acid plus vitamin B-12 decreased homocysteine concentrations in elderly patients with vascular disease but was not associated with statistically significant beneficial effects on cognitive function over the short or medium term.

Key Words: Elderly • homocysteine • folic acid • vitamin B-12 • riboflavin • vitamin B-6 • randomized controlled trial • cognitive function

In the developed world, vascular disease is the predominant cause of disability and death and is a major contributor to cognitive decline in elderly people. Serum total homocysteine (tHcy) is an independent risk factor for vascular disease, including myocardial infarction and stroke (1). An elevated tHcy concentration is also associated with dementia (2). Several plausible biological mechanisms have been proposed for these associations, including an enhanced tendency for thrombosis mediated via increased endothelial disturbance (3), platelet activation, reduced cell expression of thrombomodulin, and inhibition of activated protein C (4).

tHcy is a sulfhydryl amino acid. Its precursor, methionine, is an essential amino acid derived from dietary protein. The enzymes responsible for metabolizing tHcy are cystathionine synthase, methionine synthase, and 5,10 methylenetetrahydrofolate reductase. The activity of these enzymes is dependent on 4 micronutrients: folic acid, vitamin B-12, riboflavin (vitamin B-2), and vitamin B-6 (pyridoxal 6-phosphate), deficiencies of which cause elevations in plasma tHcy. Folic acid is the most important of these vitamins in treatment regimens designed to reduce tHcy (1, 5); folic acid supplementation reduces tHcy across a wide range of erythrocyte folate concentrations. However, in older patients, vitamin B-12 deficiency is often an important contributor to elevated tHcy concentrations (6). The importance of riboflavin and vitamin B-6 is less clear; however, both act as cofactors in the enzymatic breakdown of tHcy (7), and supplementation might play a role in ensuring maximal reductions in tHcy.

Aging is associated with elevated tHcy concentrations (7) and a reduced activity of cystathionine synthase (8), one of the key tHcy-metabolizing enzymes. Therefore, older patients may be at particular risk of tHcy-mediated disease. We aimed to determine whether vitamin supplementation with folic acid plus vitamin B-12, vitamin B-6, and riboflavin reduces plasma tHcy, alters hemostatic and endothelial function, and affects cognitive function in elderly patients with vascular disease.

A total of 185 patients was studied in this 2-center, hospital-based, randomized controlled trial (Figure 1). Inclusion criteria were age 65 y and ischemic vascular disease, defined as one or more of the following: history of angina pectoris, previous acute myocardial infarction, evidence of major ischemia or previous acute myocardial infarction on the basis of a 12-lead electrocardiogram, ischemic stroke, transient ischemic attack, intermittent claudication, or surgery for peripheral arterial disease. Exclusion criteria included an acute vascular event <1 wk previously; major surgery <1 mo previously; any other major acute illness <1 mo previously; severe renal impairment (serum creatinine > 400 µmol/L); severe hepatic impairment; malignancy within the previous year (excluding local skin cancer); severe congestive heart failure (New York Heart Association class IV); total anterior cerebral infarct with major residual disability; malabsorption; inability to give informed consent (eg, due to dementia or dysphasia); major cognitive impairment (Mini-Mental State Examination score <19); existing treatment with riboflavin, vitamin B-6, vitamin B-12, or folic acid preparations; hemoglobin concentration < 10 g/dL; and mean cell volume >100 fL plus either a low red blood cell folate concentration (<280 ng/mL) or a low serum vitamin B-12 concentration (<250 pg/mL). Baseline characteristics are presented in Table 1. Written informed consent was obtained from all eligible patients. The study was approved by the relevant local hospital ethical committees. The study was registered on the Cochrane Central Register of Controlled Trials (2001) and conducted and reported according to CONSORT (Consolidated Standards of Reporting Trials) guidelines (9).

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FIGURE 1.. Flow diagram of patient recruitment and progress throughout the study. F, folic acid; B12, vitamin B-12; B6, vitamin B-6; B2, riboflavin.


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TABLE 1. Baseline characteristics of all study groups1

The patients initially entered a single-blind, placebo, run-in (2 capsules/d as per the randomized phase of the trial) phase lasting between 2 and 4 wk. Patients who successfully completed the run-in phase were randomly allocated to receive folic acid (2.5 mg) plus vitamin B-12 (400 µg) or placebo, vitamin B-6 (25 mg) or placebo, or riboflavin (25 mg) or placebo for 12 wk in a factorial 2 x 2 x 2 design. The various combinations of vitamins and placebo were all packaged so that the daily dose was provided in a total of 2 capsules (1 red and 1 white), irrespective of patient group. The capsules were visually identical for the run-in phase and for all arms of the trial. Treatment allocation was concealed from the patients and the investigators (double-blind). Allocation was determined at a site remote from the clinical study (Robertson Centre for Biostatistics) in randomized permuted blocks of 8, stratified by hospital center.

Study measures
Patients were assessed at the beginning and end of the placebo run-in and at 3, 6, and 12 mo after randomization in the active phase of the trial. Baseline measures included height, weight, blood pressure (taken using a standard sphygmomanometer as the mean of 2 recordings after 5 min sitting, diastolic phase V), a 12-lead electrocardiogram, the Barthel index, and a short instrumental activities of daily living (IADL) scale (10).

A standard fasting venous blood sample was taken at both baseline visits and at 3 mo. Blood was anticoagulated with tripotassium citrate (0.109 mol/L; 9:1, by vol) and centrifuged at 2000 x g at 4 °C; citrated plasma aliquots were snap-frozen and stored at –50 °C until assayed for plasma total homocysteine (measured by HPLC), von Willebrand factor (measured by enzyme-linked immunosorbent assay; DAKO, High Wycombe, United Kingdom), and fibrinogen (Clauss assay, Coag-A-Mate X2; Organon Teknika, Cambridge, United Kingdom). Serum vitamin B-12 and red blood cell folate were measured with the chemiluminescence method (Centaur; Bayer, Newbury, United Kingdom). Plasma pyridoxal 5-phosphate was measured by using HPLC. Riboflavin status was assessed by measuring erythrocyte glutathione reductase activation coefficient (11).

General cognitive function was assessed by using the Telephone Interview for Cognitive status (TICSm; 12) at both baseline visits and at 6 and 12 mo; attention and speed of information processing were assessed by using the Letter Digit Coding Test (at both baseline visits and 12 mo) (13, 14). Baseline cognitive assessments were face-to-face, 6-mo reviews by telephone and 12-mo face-to-face reviews. The TICSm is composed of 21 items and has a maximum score of 39 (12); it was used in the Heart Protection Study (15). It correlates highly with the in-person Mini-Mental State Examination (MMSE) in healthy elderly subjects and in those with a diagnosis of Alzheimer disease (16). Serious adverse events, including incident vascular events, were recorded at each review.

Statistical analysis
Our target sample size was 200 individuals. The study was planned pragmatically, recognizing that it would not be powered to detect an effect on vascular endpoints but would provide valuable data in planning a large-scale intervention study. Data were analyzed by using the SAS version 8.02 software package (SAS Institute Inc, Cary, NC). All analyses were based on an intention-to-treat basis. Baseline summary statistics are presented as means ± SDs or as medians and interquartile ranges (IQRs) for continuous variables and as numbers and percentages for categorical variables. Baseline data are presented for all 8 groups of vitamin combinations; however, the analysis of each of the 3 active components of treatment was preplanned to be based on a factorial design, with a comparison of 1) all patients who received folic acid plus vitamin B-12 and those who did not receive folic acid plus vitamin B-12, 2) all patients who received riboflavin and those who did not receive riboflavin, and 3) all those who received vitamin B-6 and those who did not receive vitamin B-6.

The effects of the different vitamins on laboratory variables were analyzed by analysis of covariance (ANCOVA). The change from the mean of the 2 baseline measures was compared with the 3-mo measure, with adjustment for the baseline value of each variable. Statistical analysis of interactions of the different vitamin interventions on homocysteine concentrations was performed based on 3-factor ANCOVA (folic acid plus vitamin B-12, riboflavin, and vitamin B-6), seeking possible 3- and 2-factor interactions. An analysis of the changes in cognitive function was performed similarly to the laboratory analyses, but the change from baseline to 1 y of follow-up was examined. The effects of vitamins on incident vascular events were analyzed by calculating Mantel-Haenszel odds ratios and 95% CIs.

Baseline characteristics for all 8 different vitamin combination groups are presented in Table 1. Mean (±SD) baseline red blood cell folate and vitamin B-12 concentrations were 312 ± 127 ng/mL and 362 ± 136 pg/mL, respectively, in the group treated with folic acid plus vitamin B-12 and 294 ± 120 ng/mL and 371 ± 123 pg/mL, respectively, in those not treated with folic acid plus vitamin B-12. At baseline the median MMSE score for all patients was 28 (IQR: 26–29). Vitamin supplementation increased vitamin concentrations and improved vitamin status as expected (Table 2). Mean (±SD) baseline fasting plasma homocysteine was 16.5 ± 6.4 µmol/L. This was reduced significantly after 3 mo of treatment in all treatment groups that received folic acid plus vitamin B-12. Reductions in tHcy were seen across the entire range of baseline red blood cell folate concentrations; however, they were greatest in subjects in the lowest baseline quintiles of concentrations of this vitamin (P = 0.019, 4 df; interaction test) (Figure 2). There were no statistically significant effects of vitamin B-6 or riboflavin supplementation on homocysteine concentrations (Table 2). No statistically significant 2- or 3-factor interactions between the different components of vitamin treatment on homocysteine concentrations were seen (3-factor ANCOVA).

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TABLE 2. Changes in vitamin and homocysteine concentrations from baseline to 3 mo in the different vitamin-treatment groups1


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FIGURE 2.. Mean (±1 SE) change in total homocysteine concentrations with folic acid plus vitamin B-12 supplementation () compared with no folate and vitamin B-12 supplementation (•), by baseline concentration of red blood cell folate (quintile 1: 210 ng/mL; quintile 2: >210 to 252 ng/mL; quintile 3: >252 to 315 ng/mL; quintile 4: >315 to 387 ng/mL; quintile 5: >387 ng/mL). P values (analysis of covariance) for quintiles 1–5: <0.001, 0.004, 0.001, 0.004, and 0.039, respectively. P = 0.019 for the interaction between the baseline red blood cell folate concentration and the reduction in homocysteine concentrations with folic acid plus vitamin B-12.

No statistically significant 2- or 3-factor interactions between the different components of vitamin treatment on fibrinogen or von Willebrand factor were seen (3-factor ANCOVA) (Table 3). There was no significant difference in the 1-yr incidence of vascular events in subjects who received folic acid plus vitamin B-12 (17/92; 18%) compared with those who received no folic acid or vitamin B-12 (13/93; 14%); odds ratio 1.39 (95% CI: 0.63, 3.07).

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TABLE 3. Changes in hemostatic factors from baseline to 3 mo in the different vitamin-treatment groups1

There were no significant effects of any of the vitamins on change in cognitive function (as measured by TICSm and the Letter Digit Coding Test; Table 4). There was no evidence of beneficial effects on cognition from folic acid plus vitamin B-12, even in those in the lowest quintile of baseline red blood cell folate or of serum vitamin B-12 concentrations.

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TABLE 4. Effects of the different vitamin treatments on cognitive function1

The results of the preplanned marginal analyses are presented in Table 5. All patients who received folic acid plus vitamin B-12 were compared with all those who do not receive these vitamins, all who received riboflavin were compared with all those who did not receive riboflavin, and all those who received vitamin B-6 were compared with all those who did not receive vitamin B-6. Folic acid plus vitamin B-12 significantly decreased tHcy concentrations. However, there were no significant effects of any of the vitamins on fibrinogen or on the von Willebrand factor. No significant effects of any of the vitamins on cognition were seen; the between-group differences in change in cognition for all patients who received folic acid plus vitamin B-12 compared with all those who received no folic acid or vitamin B-12 were –0.7 (95% CI: –1.7, 0.4) for the TICSm score and –1.0 (–2.3, 0.4) for the Letter Digit Coding Test. Compared with the patients who did not receive riboflavin, the patients who received riboflavin had a change of –0.5 (95% CI: –1.5, 0.5) in the TICSm score and a change of 0.4 (–1.0, 1.7) in the Letter Digit Coding Test. Compared with those who received no vitamin B-6, the patients who received vitamin B-6 had a change of –0.1 (95% CI: –1.1, 0.9) in the TICSm score and a change of –0.6 (–2.0, 0.8) in the Letter Digit Coding Test.

View this table:
TABLE 5. Changes in homocysteine, fibrinogen, and the von Willebrand factor (baseline to 3 mo) and in cognitive function (1 y) in the different vitamin-treatment groups1


We found that folic acid plus vitamin B-12 had a large effect on tHcy in elderly patients with vascular disease; concentrations decreased by 33%. The effect was greatest in those with low baseline red blood cell folate and serum vitamin B-12 concentrations. However, folic acid plus vitamin B-12 supplementation reduced tHcy concentrations across the whole range of baseline folate concentrations, with no apparent ceiling effect, and in all except the top quintile of baseline vitamin B-12 concentrations.

We saw no significant effects of supplementation with vitamin B-6 or riboflavin, although there was a trend for vitamin B-6 to decrease tHcy. There were no statistically significant interactions between the different vitamins on changes in tHcy. Our results do not support previous suggestions that riboflavin interacts with folic acid to decrease tHcy by maximizing the catalytic activity of methylenetetrahydrofolate reductase (7). Riboflavin supplementation of older patients with biochemical deficiency of riboflavin does not affect tHcy (17). However, riboflavin status appears to be an important determinant of tHcy in homozygotes for the thermolabile (TT) variant of methylenetetrahydrofolate reductase, with high concentrations of tHcy in those with biochemical riboflavin deficiency (18). It is possible that riboflavin supplementation in this specific genetic subgroup might decrease tHcy.

The magnitude of reduction in tHcy that we observed with folic acid plus vitamin B-12 supplementation is larger than that seen in most other studies. The Vitamin Intervention for Stroke Prevention trial resulted in a reduction of 2 µmol tHcy/L with high-dose compared with low-dose folic acid plus vitamin B-6 and vitamin B-12 (7). High-dose folic acid decreased the tHcy concentration by 1.5 µmol/L in middle-aged patients with ischemic heart disease (19). The reductions seen after fortification of the diet with folic acid have ranged from 0.7 to 1.5 µmol/L (20, 21). The likely reasons for the large reduction in tHcy with folic acid plus vitamin B-12 supplementation seen in our study were the older age of our patients and the poor folate status at study entry. Both of these factors are associated with elevated tHcy concentrations.

We found no effect of folic acid plus vitamin B-12 supplementation on the von Willebrand factor, a marker of endothelial function. Previous studies of the effects of homocysteine-lowering vitamins (including folic acid) on flow-mediated (endothelium dependent) dilatation in the brachial artery have produced contradictory results (22, 23). Fibrinogen is the substrate for fibrin formation and a cofactor in platelet aggregation; therefore, increased concentrations may enhance thrombosis. We also found no effect of folic acid plus vitamin B-12 supplementation on fibrinogen. A randomized controlled trial of B-vitamin supplementation also found no significant effects of tHcy-lowering treatment on markers of clotting activation, although there was a trend for fibrin D-dimer to be reduced (24). Therefore, there is insufficient evidence to prove the hypothesis that an elevated tHcy concentration damages the endothelium and leads to a prothrombotic state. We did see a statistically significant reduction in fibrinogen in the group who received riboflavin alone, compared with placebo. However this was likely a chance finding. The marginal analysis, in which all patients who received riboflavin were compared with all who did not receive riboflavin, did not confirm any effect on fibrinogen.

Associations between tHcy and dementia (2, 25, 26)or cognitive impairment (27) have been reported. However, the relation of tHcy with cognitive decline is less certain (28). We found no significant effects on changes in cognition from tHcy lowering with folic acid plus vitamin B-12. Indeed, there was a trend for those receiving folic acid plus vitamin B-12 to do slightly worse than those not receiving this treatment, and the CIs for the effect were such that we are reasonably confident that these vitamins do not significantly improve cognition or protection against cognitive decline, at least in the short to medium term. Patients with dementia were excluded from our study; however, subjects with a mild degree of cognitive impairment (as indicated by the mean baseline MMSE score for the cohort) were included.

Several other randomized, double-blind, placebo-controlled trials have reported on the cognitive effects of folic acid with or without vitamin B-12 (29) and vitamin B-6 (30). Short-term (5 wk) treatment with folic acid, vitamin B-6, or vitamin B-12 showed no significant beneficial effects on cognition in 211 healthy women (31); this study included young and middle-aged subjects as well as 75 women aged >65 y. Similarly, no effects on cognition were seen in a 3-mo trial of vitamin B-6 supplementation in 76 healthy elderly men (30, 31). The VITAL (vitamins and acetyl-salicylic acid) trial examined the effects of 3 mo of treatment with folic acid plus vitamin B-12 in a factorial design (also including aspirin and antioxidant vitamins) in 149 subjects with dementia or mild cognitive impairment; no significant effects on global measures of cognition were seen (2). Two other studies of folic acid supplementation reported no beneficial effects on cognition in 11 (32) and 30 cognitively impaired patients (33); however, these studies were very underpowered because of the small sample sizes. Therefore, our study has extended knowledge on this issue by being the largest randomized controlled trial in older people to have examined the effects of folic acid and various B-vitamin supplements on cognition over the longest period of time.

No significant effect was seen on incident vascular events from tHcy lowering; however, this study was underpowered to detect any such effect. Two other randomized, double-blind, placebo-controlled trials have reported no significant effect of tHcy-lowering on the incidence of vascular events: the Vitamin Intervention for Stroke Prevention trial (34) and a study conducted in Cambridge in patients with ischemic heart disease (19). An open label study of folic acid in patients with coronary artery disease also found no effect (35). Other large randomized controlled trials (36) are due to report their findings soon, which should provide sufficient evidence to determine whether tHcy lowering prevents ischemic vascular disease.

Our study had several limitations. In particular, the duration was relatively short; therefore, longer-term cognitive benefits from tHcy lowering in older people cannot be excluded. Although our study, to date, is the largest randomized controlled trial to have assessed effects of these vitamins on cognition, it was not large enough to have the statistical power to exclude possible modest benefits. The need to co-prescribe folic acid with vitamin B-12 means that we cannot determine the relative effects of these vitamins on any of the outcomes; however, it was thought not to be ethical to give these vitamins separately because of the theoretical risk of harming those with covert vitamin B-12 deficiency by giving folic acid without vitamin B-12.

In conclusion we found that oral folic acid plus vitamin B-12 resulted in large reductions in plasma homocysteine in elderly patients with vascular disease. Neither riboflavin nor vitamin B-6 had any significant effects. The large reductions in tHcy achieved with folic acid plus vitamin B-12 were not accompanied by any demonstrable effects on endothelial or hemostatic function on the basis of circulating concentrations of fibrinogen and the von Willebrand factor. The lowering of tHcy concentrations with folic acid plus vitamin B-12 had no statistically significant effects on cognitive function over a 1-y period. Therefore, we found no evidence to support clinically important beneficial effects on cognitive function in older people with vascular disease from the short- to medium-term administration of these vitamins.

We thank Melanie Shields and Jackie Scott (research nurses) for their hard work in patient recruitment and assessment and Mark Barber, Tricia Moylan, and Martin Whitehead for assistance with patient recruitment.

DJS contributed to the study design, supervision of clinical data collection, analysis of data, and writing of the manuscript. GM contributed to the patient recruitment and clinical data collection. GDOL, DSJO, and RCT contributed to the study design, laboratory analysis, and writing of the manuscript. AR contributed to the laboratory analysis and the writing of the manuscript. ADM contributed to the study design, randomization schedule, analysis of the data, and writing of the manuscript. PL contributed to the study design and writing of the manuscript. EGS and JBM helped supervise the clinical data collection and contributed to the writing of the manuscript. PWM provided consultation and contributed to the writing of the manuscript. RGJW provided consultation and contributed to the study design and writing of the manuscript. None of the authors had any conflicting or competing interests, and the funder of the study had no role in data collection, analysis, or interpretation of the data or in the writing of the report.


Received for publication January 18, 2005. Accepted for publication July 11, 2005.

日期:2008年12月28日 - 来自[2005年82卷第6期]栏目

Links between food and vascular disease

John C Rutledge

1 From the University of California, Davis, Medical Center, the Division of Endocrinology, Clinical Nutrition and Vascular Medicine, Sacramento.

See corresponding article on page 119.

2 Reprints not available. Address correspondence to JC Rutledge, University of California, Davis, Medical Center, Division of Endocrinology, Clinical Nutrition and Vascular Medicine, 4150 V Street, PSSB, G400, Sacramento, CA 95817. E-mail: jcrutledge{at}ucdavis.edu.

The article by Toborek et al (1) in this issue of the Journal is this group's next logical step in a theme targeting the actions of ingested lipids on the vascular system (2,3). Over the past decade, these investigators have contributed a substantial body of literature investigating the dietary links to vascular physiology and pathophysiology. The current article reports that specific unsaturated dietary fatty acids, particularly linoleic acid, can stimulate a proinflammatory environment within the vascular endothelium. For many years this concept has remained elusive in the context of how a diet-vascular interaction could occur in this complex system. The authors have pursued this concept in a systematic and logically progressive fashion. Their work and this article specifically show that specific lipids can, without any modification, perturb vascular endothelial cells and promote a proinflammatory environment (4,5). This finding separates this study from much previous work indicating that lipids must be modified to stimulate and activate endothelial cells.

These studies provide part of the foundation for future studies of diet-vascular interactions in more complex organ systems and whole organisms, including humans. These future studies are essential for determining the pathophysiologic relevance of the present experiments performed in cell culture. Additionally, basic mechanistic studies are needed to determine the exact sequence in which certain ingested lipids activate endothelial cells (6) and the modalities and therapies by which the process of endothelial cell activation can be prevented or attenuated (7). Additional interesting experiments may determine the mechanisms by which endothelial cell activation is amplified by specific lipids, such as linoleic acid.

It is clear that multiple mechanisms exist by which the vascular endothelium can be activated and the vascular wall injured. The studies by Hennig et al (2) show that specific lipids, ie, fatty acids, can injure vascular endothelium without modification. Thus, it is clear that our reliance on measuring classic blood lipid indexes, such as total cholesterol, triacylglycerol, HDL cholesterol, and LDL cholesterol, remains rudimentary. Future analysis may include more comprehensive dietary phenotyping and plasma lipid composition, including specific phospholipids and fatty acids. Although the authors have identified a specific culprit in the activation of the vascular endothelium, many more of these "bad actors" may exist in the diet. In the coming years, our major challenge will be to identify additional pathogenic lipids and other blood components that activate endothelial cells, investigate their mechanisms, and develop treatment regimens to prevent the development of vascular disease, the most costly disease in our country in terms of both lives lost and health care dollars.


  1. Toborek M, Lee YW, Garrido R, Kaiser S, Hennig B. Unsaturated fatty acids selectively induce an inflammatory environment in human endothelial cells. Am J Clin Nutr 2002;75:119–25.
  2. Hennig B, Chung BH, Watkins BA, Alvarado A. Disruption of endothelial barrier function by lipolytic remnants of triglyceride-rich lipoproteins. Atherosclerosis 1992;95:235–47.
  3. Toborek M, Blanc EM, Kaiser S, Mattson MP, Hennig B. Linoleic acid potentiates TNF-mediated oxidative stress, disruption of calcium homeostasis, and apoptosis of cultured vascular endothelial cells. J Lipid Res 1997;38:2155–67.
  4. Gimbrone MA Jr, Topper JN, Nagel T, Anderson KR, Garcia-Cardena G. Endothelial dysfunction, hemodynamic forces, and atherogenesis. Ann N Y Acad Sci 2000;902:230–9.
  5. Ross R. Atherosclerosis is an inflammatory disease. Am Heart J 1999;138:S419–20.
  6. Zilversmit DB. Atherogenesis: a postprandial phenomenon. Circulation 1979;60:473–85.
  7. Rutledge JC, Mullick AE, Gardner G, Goldberg IJ. Direct visualization of lipid deposition and reverse lipid transport in a perfused artery: roles of VLDL and HDL. Circ Res 2000;86:768–73.

日期:2008年12月28日 - 来自[2002年75卷第1期]栏目
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