Department of Physiology and Biophysics, University of Mississippi Medical Center, Jackson, Mississippi 39216
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ABSTRACT |
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Dynamic changes in the reduction-oxidation (redox) state of the tissue lead to the pathophysiological condition. Reduced homocysteine causes dysfunctions in endothelium. The proliferation of smooth muscle cells may lead to occlusive vascular disease, ischemia, and heart failure, but whether fibrosis and hypertension are a consequence of smooth muscle proliferation is unclear. Redox changes during hyperhomocyst(e)inemia may be one of the causes of premature atherosclerotic heart disease. To examine the effect of homocystine on human vascular smooth muscle cells (HVSMC), we isolated HVSMC from idiopathic dilated cardiomyopathic hearts. Coronaries in these hearts were apparently normal. HVSMC numbers in culture were measured by hemocytometer in the presence and absence of homocystine. Results show that homocystine induced cellular proliferation. This proliferation was reversed by the addition of the antioxidant N-acetylcysteine (NAC). Homocystine induces collagen expression in a dose- and time-dependent manner, as measured by Northern blot (mRNA) analysis. The 50% inhibitory concentration of 5 µM for collagen was estimated. The induction of collagen was reversed by the addition of NAC and reduced glutathione. To localize the receptor for homocystine on HVSMC, we synthesized fluorescamine-labeled homocystine conjugate. Incubation of labeled homocystine with HVSMC demonstrated membrane and cytosol localization of homocystine binding. The receptor-ligand binding was disrupted by NAC. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis fluorography, we observed a 40- to 25-kDa homocystine redox receptor in HVSMC. Our results suggested that the redox homocysteine induces HVSMC proliferation by binding to the redox receptor and may exacerbate atherosclerotic lesion formation by inducing collagen expression.
collagen; actin; vascular occlusive disease; gene expression; signal transduction; vascular remodeling
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INTRODUCTION |
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THE PHYSIOLOGICAL ROLE of reduction-oxidation (redox) in maintaining the metabolic hemostasis and cardiovascular function is a poorly understood phenomenon. Homocysteine is a sulfur-containing amino acid generated during the metabolism of methionine. Homocystine is two homocysteine residues linked by a disulfide bond. Extracellular homocysteine is readily oxidized to disulfide homocystine. Homocyst(e)ine is composed of a reduced form, oxidized form, and oxidative mixed disulfide form (mixture of reduced, oxidized, and thiolac-tone), both free and protein bound
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Homocyst(e)ine is one of the causes of coronary atherosclerosis and ischemic heart disease (31). Abnormalities in the homocystine levels have been found in 40% of patients presenting with premature peripheral vascular disease or stroke (7). Recent data suggest that increased plasma homocystine levels are an independent risk factor for the development of arterial disease (12, 22). This may be related to the lack of reducing power and/or conversion of homocystine to homocysteine. Homocysteine readily oxidizes to homocystine in human plasma. A high level of homocysteine is associated with endothelial cell damage and an increase in oxidative tension (12, 22), leading to smooth muscle cell (SMC) proliferation.
Previously, high levels of collagen and proteoglycans in the primary atherosclerotic and secondary restenotic lesions (28) have been shown. The level of elastin was reduced (28). This indicated differential regulation of extracellular matrix (ECM) components during the development of atherogenic lesions (27-29). The loss of elasticity, or "hardening," of arteries has long been thought to be an inevitable consequence of aging and an early sign of atherosclerosis. However, Demer et al. (5) reported that this is no more an inevitable degenerative change than is the development of other components of the inflammatory reaction (e.g., oxidative stress). Hypoxia, homocyst(e)inemia, acute coronary occlusion, and ischemic/reperfusion conditions increase oxidative homocystine and oxidized glutathione (GSSG) in the coronary vasculature (20), which leads to a change in the redox state of tissue surrounding the vascular SMC. Endothelium is the innermost layer of vessel wall between blood and the basement membrane that resides on the top of the inner elastic laminae and communicates signals generated from blood to the interstitium (10). Detachment of endothelium exposes vascular SMC to the oxidative conditions in blood. This may, in turn, lead to SMC proliferation and ECM induction. An oxidative environment induces normal fibroblast cell proliferation, and a reducing agent decreases normal fibroblast cell proliferation by inducing matrix proteinase and repressing tissue inhibitor gene transcription (25). It is possible that changes in the oxidative environment around human vascular smooth muscle cells (HVSMC) induce expression of ECM components.
Whether the effects of oxidative disulfide homocystine are due to redox reactions involving direct modification by an oxygen radical or to intracellular glutathione-dependent oxidation-reduction reactions is not yet known. The determination of cellular permeability of antioxidants and their half-lives under tissue culture conditions and determination of redox-receptor are the critical parameters to be determined to develop a relationship between redox-sensitive ECM remodeling in vivo versus in vitro. The binding of antithrombin III to endothelial cell membrane was decreased after preincubation with homocysteine, cysteine, or 2-mercaptoethanol (19). This suggested the possibility that reducing agents decrease the disulfide exchange reaction between membrane receptor and antithrombin. However, this raises the possibility that oxidative disulfide homocystine may bind to receptor directly by a disulfide exchange reaction. To this end, an identification of a redox receptor may be an ideal development. Nothing is known about the redox receptor(s) in vivo or in vitro. Here we report that the HVSMC are sensitive to redox state and there is a redox-sensitive receptor on HVSMC.
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MATERIALS AND METHODS |
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Materials. Reduced glutathione (GSH), GSSG, tris(hydroxymethyl)aminomethane (Tris) base, N-acetylcysteine (NAC), homocystine, homocysteine, cysteine, cystine, and trypsin were obtained from Sigma (St. Louis, MO). Fetal calf serum (FCS), normal rabbit serum, minimum essential medium (MEM) with Earle's salts, collagen- and laminin-coated culture plates, and Hanks' balanced salt solution were all obtained from Collaborative Research (Bedford, MA). Elastase was obtained from Elastin Products (St. Louis, MO). Bacterial collagenase was obtained from Worthington (Freehold, NJ). N-terminal propeptide (TMRKPRCGNPDVAN) of matrix metalloproteinases (32) was synthesized at the Protein Core Facility at the University of Missouri-Columbia and was kept under vacuum conditions before use.
Human arterial tissue. Normal arteries were isolated from idiopathic dilated cardiomyopathic explanted heart (24). The reasons for heart failure in idiopathic patients were other than coronary. The hearts with idiopathic dilated cardiomyopathy have apparently normal coronary arteries. A waiver for using explanted human tissue was obtained from the Institutional Review Board before collecting the tissue. The coronaries were stripped of adventitial and external tissue before isolating the cells.
Isolation of HVSMC. HVSMC were isolated by a modification of the combined collagenase and elastase digestion. The endothelium was removed by gently scraping the luminal side of the vessel with a cotton swab and washing the swab in MEM. Medial SMC were isolated by mincing the rest of the vascular tissue in the presence of collagenase and elastase (10 µM) and were grown in 10% FCS (26). Isolated cells were free of endothelial cell contamination, as determined by positive staining with human anti-smooth muscle actin-related antigen and negative staining for von Willebrand factor (26). The cells were characteristically "hill and valley" in morphology. Contamination by nonspecific esterase-positive cells or cells with morphological feature and growth characteristics of monocytes, macrophages, or endothelial cells was not observed in our HVSMC culture preparation. The early passage cells were used for cellular characterization and receptor labeling. Cultures were routinely checked for the presence of mycoplasma.
HVSMC cells were cultured on laminin-coated (BioCoat) plates in medium that was supplemented with 10% FCS, 0.1% collagen suspension (Vitrogen 100; Celtrix, Santa Clara, CA), 2% normal rabbit serum, 4.5 mg/ml glucose, gentamycin, and fungizone (10 µg/ml), and 2 mM glutamine. For most experiments, cells were washed two times with serum-free Dulbecco's modified Eagle's medium (DMEM) and deprived of serum for 24 h to arrest the cell growth before the experimental treatment. Passage 9 cells were used for Northern blot (mRNA) experiments.
Biological assays. HVSMC cell-conditioned media was prepared as follows. Duplicate sets of confluent HVSMC monolayers in 35-mm petri dishes were briefly rinsed in 37°C serum-free medium followed by incubation in 1 ml test medium containing 2% serum plus homocystine or homocystine plus NAC. The test medium was replaced each day with fresh conditioned medium. The cells were counted every other day by hemocytometer. Total genomic DNA was isolated by lysing the cells directly on the plate after removing the culture medium and by adding the lysis solution, followed by incubation with ribonuclease A and protein precipitatation solution (DNAzol; Molecular Research Center). The samples were centrifuged at 2,000 g for 10 min, and the supernatant was transferred to new tubes. Precipitated DNA was resuspended in Tris-borate-EDTA buffer and quantitated by an ultraviolet spectrophotometer using an optical density of 1 at 260 nm for 50 µg/ml double-stranded DNA.
[3H]thymidine incorporation. To substantiate the effect of homocysteine on proliferation, 104 HVSMC were grown to subconfluence by incubation in DMEM with 10% FCS. The medium was changed to serum-free medium for 24 h, followed by incubation in 0.4% serum in the presence or absence of DL-homocysteine (10 µM). The HVSMC were labeled with [methyl-3H]thymidine (NEN) at 1 µCi/ml (1 µCi = 37 kBq) during the last 3 h of homocysteine treatment. After labeling, the cells were washed with phosphate-buffered saline, fixed in cold 10% trichloroacetic acid, and then washed with 95% ethanol. Incorporated [3H]thymidine was extracted in 0.2 M NaOH and measured in a liquid scintillation counter. The values were expressed as means ± SD from 10 wells and 3 separate experiments.
Enzyme-linked immunosorbent assay. HVSMC were homogenized, and protein fractions in homogenates were analyzed for collagen type I using enzyme-linked immunosorbent assay as described previously (33). A similar protocol was employed for ![]() |
RESULTS |
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Proliferation of HVSMC by homocystine and inhibition by NAC. HVSMC were isolated from normal vessels. The spindle phenotype characteristic of SMC and growth in close contact to each other is shown in Fig. 1A. To examine whether oxidative mixed-disulfide homocystine induces cellular proliferation, HVSMC were seeded at day 0 with 2.5 × 104 cells in 2% serum. At day 1, 2% serum, serum plus homocystine, or serum plus homocystine plus NAC was added and changed every day (Fig. 1B). The number of cells in 2% serum increased 140% to 6 × 104 in 7 days. However, in the presence of serum plus 10 µM homocystine, the number of cells increased 680% to 19.5 × 104 over the 7-day period. The addition of 50 µM NAC inhibited 62% of the homocystine-induced growth of HVSMC. To determine whether the growth induced by homocystine (10 µM) is mediated by disulfide or thiol, we cultured HVSMC in the presence of homocystine plus homocysteine (50 µM). We observed that homocysteine has some inhibitory effect on homocystine-induced proliferation (Fig. 1, B and E; P < 0.005). The dose dependence of homocystine treatment on VSMC growth and DNA synthesis suggested a maximal effect at 10-12 µM (Fig. 1C). The treatment of cystine, cysteine, and NAC demonstrated no significant proliferation on HVSMC (Fig. 1D; P > 0.01).
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To determine the actual mitogenic effect of homocysteine in HVSMC, incorporation into the HVSMC in the presence and absence of homocysteine of radiolabeled precursor of DNA, [3H]thymidine, was carried out. The radioactivity incorporated into the cells was 210 ± 30 and 380 ± 35 disintegrations/min in the absence and presence of homocysteine, respectively. The results suggested that homocysteine stimulated the de novo DNA synthesis.
Collagen and -actin induction in HVSMC by
homocystine. ECM collagen is the primary component of
atherosclerotic plaque structure. To determine whether homocystine
induces both collagen and contractile actin expression simultaneously,
confluent HVSMC were cultured in the presence and absence of
homocystine at various time intervals (Fig.
2). Incubation of HVSMC with 10 µM
homocystine induces time-dependent expression of collagen and
-actin. Treatment with cysteine (Fig. 2, lane
5), cystine, and homocysteine has no effect on HVSMC
collagen and actin expression. The scanned intensity data for collagen and actin mRNA were normalized with the 18SR gene. From the plots of
normalized data and time, the half-time
(t1/2) for the
homocystine effect on HVSMC was estimated to be 1 and 2.5 h for actin
and collagen expressions, respectively. Based on the concentration of
homocystine (10 µM) and
t1/2, a
second-order rate constant for homocystine action was calculated. The
results suggested induction of actin with a rate of ~1 × 104
min
1 · M
1.
The rate of collagen expression was ~6 × 102
min
1 · M
1
(Fig. 2B). This suggested that the
rate of collagen synthesis is slower than the synthesis of actin. To
further evaluate that homocysteine induces a contractile phenotype
before its expression of ECM components, we assessed protein levels of
collagen and actin after homocystine treatment (Fig.
2C). Results suggested significant
(P < 0.001) induction of actin at 6 h compared with collagen expression at 6 h, suggesting that homocystine
induces a contractile phenotype before its expression of ECM
components.
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To determine the effective dose and affinity of homocystine to HVSMC and its induction of collagen in HVSMC, we incubated cells with various concentrations of homocystine. The mRNA for collagen was normalized with the 18SR gene (Fig. 3A). The plot between homocystine concentration and mRNA level of collagen demonstrated a 50% effective concentration of ~5 µM for homocystine on HVSMC (Fig. 3B). Disulfide homocystine interacts with thiol to produce disulfide exchange. This may reduce the homocystine concentration. Therefore, thiol-containing reagents may reduce homocystine binding to HVSMC. To determine whether reducing agent, glutathione, GSH, inhibits the homocystine effect on HVSMC, various concentrations of GSH were added to homocystine-activated HVSMC. The collagen mRNA normalized with the 18SR gene was measured (Fig. 3B). Results suggested dose-dependent inhibition of homocystine-induced collagen expression. At a concentration of 40 µM, GSH inhibited collagen expression by 85%. From the plot between the decrease in collagen mRNA levels and the concentration of GSH (Fig. 3B), a 50% inhibition constant of ~15 µM was estimated (Fig. 3B).
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To determine whether other reducing agents inhibit homocystine-induced collagen expression, we cultured HVSMC in the presence of cysteine, NAC, and the thiol-containing propeptide (prop) of matrix metalloproteinase. The propeptide keeps matrix metalloproteinase in its inactive/latent form. The results suggested that cysteine, NAC, and propeptide at a concentration of 50 µM completely inhibited the homocystine-induced collagen expression in HVSMC. On the other hand, thiol-containing homocysteine at a concentration of ~100 µM partially inhibits the collagen expression (Fig. 4). The GSH, NAC, and propeptide alone do not induce collagen expression (data not shown). This suggested a differential effect of oxidative thiols on the homocystine-induced collagen expression in HVSMC. Inhibition of collagen expression by propeptide may also suggest the following dual effects of activation of matrix metalloproteinases: 1) active metalloproteinase will degrade collagen, and 2) liberated propeptide will inhibit collagen synthesis. These events will increase ECM turnover during remodeling. However, the reverse of these events will lead to ECM accumulation and atherosclerotic stage. The results (Fig. 4) further suggest that reducing agent can reverse the effect of homocystine on HVSMC as well as collagen expression. The binding of homocystine was displaced by GSH and NAC. This raised the possibility that HVSMC contains the receptor for homocystine.
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Receptor of homocystine on HVSMC. To localize the receptor for homocystine, I labeled homocystine with fluorescamine. Disulfide homocystine contains no free thiol. It is therefore not possible to label at thiol. However, homocystine contains two primary amine, NH2, groups. Fluorescamine conjugates with primary amines to produce an amide linkage and fluorescent product (Fig. 5). Free fluorescamine has no intrinsic fluorescence. We synthesized homocystine-fluorescamine conjugate by a 1:4 stoichiometry reaction between homocystine and fluorescamine. The reaction product gives fluorescence at 480 nm when excited at 380 nm. Therefore, we used fluorescamine to label homocystine and used fluorescence-labeled homocystine as the probe to measure receptor labeling on the HVSMC. Also, to determine the specificity of binding to HVSMC, we synthesized another fluorescamine analog, the fluorescamine-cystine complex.
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In Fig. 6, binding of labeled homocystine to HVSMC is demonstrated. The fluorescence labeling of homocystine in HVSMC was localized at the membrane. Incubation of homocystine with fluorescamine-homocystine produced bright crispy fluorescent granules in and around the cell membrane (Fig. 6A). Some of the homocystine appears to have entered into the cytoplasm as well. Whether this diffusion or transport of homocystine into the cell is mediated through receptor internalization or redox channel mechanisms remains to be elucidated. On the other hand, fluorescamine-cystine has minimal binding under these conditions (Fig. 6B). Pretreatment of homocysteine has no effect on receptor labeling. However, in the presence of NAC, the homocystine ligand binding was abolished (Fig. 6C). These results suggested that NAC was able to prevent the homocystine binding to HVSMC.
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Affinity cross-linking. The study was conducted to determine the homocystine distribution, protein/peptide subunits, and their apparent molecular weights in SMC. Fluorography comparing SDS-PAGE patterns obtained after affinity labeling homocystine receptors, with fluorescamine-labeled homocystine as reporter, with and without unlabeled fluorescamine is shown in Fig. 7. A major fluorogenic 40-kDa band and a less fluorogenic band at 25 kDa were observed, suggesting cross-reactivity of homocystine with SMC membrane proteins. This labeling was displaced by NAC, suggesting a disulfide nature of the interactions between the receptor and homocystine.
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DISCUSSION |
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The mechanisms by which homocystine induces coronary atherosclerotic and fibrotic lesions are largely unknown. ECM collagen is the primary component of atherosclerotic lesions (31). Here, we demonstrated that oxidative disulfide homocystine induces collagen expression in HVSMC. This induction of collagen was blocked by GSH and NAC. Homocystine interacted with HVSMC via a membrane receptor. The binding of homocystine to receptor was reverse by reduced NAC.
Oxidative mixed disulfide and GSSG induce a proliferative stage in the vessel wall (16). Hypertension, but not total serum lipoproteins, cholesterol, and fibrinogen, was significantly associated with atherosclerosis in homocyst(e)inemic subjects (2). This may also suggest a role of intimal SMC proliferation after homocyst(e)inemia. Homocyst(e)ine has been shown to promote SMC growth and cause dysfunctions in endothelium (22). The SMC growth was linked to cyclin expression and kinase activity (23), suggesting a role of homocysteine in signal transduction. Previously, we demonstrated that oxidative serum conditions induce cellular proliferation and that a reducing condition inhibits cell proliferation (25). Furthermore, we demonstrated that the redox state around the cell modulates ECM expression by regulating its turnover (25). In this study, we determined that oxidative disulfide homocystine induces HVSMC proliferation and ECM expression (Figs. 1 and 2). These data suggested that oxidative homocystine induces the redox state around the vascular SMC, which in turn induces cellular proliferation and ECM expression.
Thiol autooxidation of reduced homocysteine forms stable homocystine, and it had a direct cytotoxic effect on the cells (14). Cell-detachment studies indicated that sulfydryl-containing amino acids may have marginal relevance to endothelial cell detachment and mechanisms of atherosclerosis in homocystinuria (6). However, oxidative mixed disulfide may induce cell detachment by inducing expression of ECM components. In fact, a role of ECM components in endothelial cell detachment has been suggested (8). In vivo atherogenic lesions contain high levels of collagen (31). It was demonstrated that homocystine induces collagen expression in HVSMC in a dose-dependent and time-dependent manner (Figs. 2 and 3). This is the first study that shows a role of homocystine in ECM collagen expression and suggests a link to the development of atherosclerotic-fibrotic lesion. Our data further suggest that the effect of homocystine can be reversed by the excess amount of GSH or cysteine (NAC; Fig. 4). To our surprise, we did not observe the reverse effect of reduced homocysteine over homocystine. This may in part be due to the fact that, at ambient conditions, most of the free thiols are autooxidized and form stable disulfides and, therefore, decrease the potency of free thiols. In fact, in plasma, most of the homocystine is in the form of oxidative mixed disulfide and is protein bound, and only a very small fraction is in the reduced form.
Homocystine detaches endothelial cells by its cytotoxic effect and
therefore gets access to the SMC (6, 8, 14). Thereafter, homocystine
induces proliferation in HVSMC. We have shown induction at the
transcription level and the role of signal transduction (i.e., actin
expression) in matrix metalloproteinase and their inhibitor expression
in cardiac fibroblast cells by serum (26). It is not clear whether
homocystine induces cellular migration/contraction before synthesizing
ECM components. We show that contractile actin expression is induced
before the expression of ECM collagen in HVSMC (Fig. 2). These data
suggested that homocystine may also induce migration in HVSMC by
inducing -actin. This induction may be associated with the
transformation of SMC to myofibroblast-like cells. In fact, in the
atherosclerotic lesion, SMC are transformed and differentiated to
myofibroblast-like cells (21), and myofibroblasts synthesize collagen
(1).
Receptor(s) of homocystine on HVSMC have not been demonstrated previously. However, one study has suggested that a membrane-associated assembly of plasminogen and tissue plasminogen activator (tPA) involved a membrane protein of 40 kDa in ternary complex (11). The addition of homocystine to ternary complex and endothelial cells dissociated 40-kDa protein, and tPA and has no effect on tPA plasminogen interaction (11), suggesting that 40-kDa membrane protein may be involved in homocystine binding. However, this study does not reveal directly the receptor of homocystine on SMC. We have synthesized and purified fluorescent homocystine as a probe for binding to the HVSMC membrane (Fig. 5). Incubation of labeled homocystine with HVSMC localized homocystine receptors on HVSMC (Figs. 6 and 7). Based on photo cross-linking experiments, we observed the molecular weight of homocystine receptor on vascular SMC of 40-25 kDa (Fig. 7). This receptor binding was sensitive to the reducing agent (NAC), suggesting reversibility of this binding. The redox cycle plays an important role as an endogenous antioxidant defense mechanism in cultured endothelial cells (13). To our knowledge, this is the first report on the receptor of homocystine. Furthermore, it is the first receptor that is sensitive to the redox state on the cell membrane (i.e., redox receptor). It is of great interest to identify and to characterize this receptor.
In early atherogenic lesions, oxidative stress is manifested by an
elevated production of reactive oxygen species by neutrophils, macrophages, and endothelial cells that results in the oxidative modification of low-density lipoprotein. Oxidized low-density lipoprotein is a potent stimulator of inflammation. In this oxidative environment, vascular cell adhesion molecular expression and monocyte accumulation have been observed in the early atherogenic process (18).
We demonstrated that oxidative homocystine induces the metabolic
changes in HVSMC. In normal HVSMC, homocystine induces the expression
of collagen. After the treatment with homocystine, the -actin level
was induced at the gene transcription level. The mechanism for collagen
induction by homocystine in HVSMC is mediated through the
redox-sensitive cell membrane receptor. The effect of homocystine was
reversed by the treatment of cells with reducing agent. These results
suggested that an antioxidant therapy may benefit against
atherogenesis.
Perspective. In normal HVSMC, collagen
was induced by redox homocystine. Although an effect on a redox
mechanism is inferred, this study does not identify the specific redox
biochemistry mediating the antioxidant regulatory effect. However, our
studies indirectly suggest that thiol-antioxidants function through the
mechanism related to its thiol group properties. It will be of great
interest to elaborate on other ECM components and the signal
transduction mechanisms involved in response to homocystine-induced
collagen and other ECM components. For example, transforming growth
factor-1 is a strong stimulator of collagen (4), and decorin, a
physiological transforming growth factor-
1 antagonist, neutralizes
its effect (15). It is not known whether homocystine induces
transforming growth factor-
1 and represses decorin expression in the
cardiovascular system. These studies are in progress.
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ACKNOWLEDGEMENTS |
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The author acknowledges the expert technical assistance of Susan Borders and Suresh Kumar in this work. A part of this study was carried out at the University of Missouri, Columbia, MO.
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FOOTNOTES |
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This work was supported in part by National Institutes of Health Grants GM-46366 and HL-51971 and by a Grant-In-Aid from the American Heart Association.
A preliminary account of this study was presented at the 69th conference of the American Heart Association on November 9-13, 1996, New Oreleans, LA, and at Experimental Biology '97 on April 5-9, 1997, New Orleans, LA.
Address for reprint requests: S. C. Tyagi, Univ. of Mississippi Medical Center, Dept. of Physiology and Biophysics, Jackson, MS 39216-4505.
Received 24 April 1997; accepted in final form 23 October 1997.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Arora, P. D.,
and
C. A. McCulloch.
Dependence of collagen remodeling on alpha-smooth muscle actin expression by fibroblasts.
J. Cell. Physiol.
159:
161-175,
1994[Medline].
2.
Bachmann, J.,
M. Tepel,
H. Raidt,
R. Riezler,
U. Graefe,
K. Langer,
and
W. Zidek.
Hyperhomocysteinemia, and the risk for vascular disease in hemodialysis.
J. Am. Soc. Nephrol.
6:
121-125,
1995[Abstract].
3.
Chen, R. F.,
P. D. Smith,
and
M. Maly.
The fluorescence of fluorescamine-amino acids.
Arch. Biochem. Biophys.
189:
241-250,
1978[Medline].
4.
Chua, C. C.,
B. H. L. Chua,
Z. Y. Zhao,
C. Krebs,
C. Diglio,
and
E. Perrin.
Effect of growth factor on collagen metabolism in cultured human heart fibroblasts.
Connect. Tissue Res.
26:
271-281,
1991[Medline].
5.
Demer, L. L.,
K. E. Watson,
and
K. Bostrom.
Mechanism of calcification in atherosclerosis.
Trends Cardiovasc. Med.
4:
45-49,
1994.
6.
Dudman, N. P.,
C. Kicks,
J. Wang,
and
D. E. Wilcken.
Human arterial endothelial cell detachment in vitro: its promotion by homocysteine, and cysteine.
Atherosclerosis
91:
77-83,
1991[Medline].
7.
Genest, J. J.,
J. R. McNamara,
B. Upson,
D. N. Salem,
J. M. Ordovas,
E. J. Schaefer,
and
M.R. Malinow.
Prevalence of familial hyperhomocyst(e)inemia in men with premature coronary artery disease.
Arterioscler. Thromb.
11:
1129-1136,
1991[Abstract].
8.
Gordon, P. B.,
M. A. Levitt,
C. S. Jenkins,
and
V. B. Hatcher.
The effect of the extracellular matrix on the detachment of human endothelial cells.
J. Cell. Physiol.
121:
467-475,
1984[Medline].
9.
Guarda, E.,
L. C. Katwa,
S. C. Tyagi,
and
K. T. Weber.
Effect of endothelin-1, and -3 on rat cardiac fibroblast collagen turnover and DNA synthesis (Abstract).
Am. J. Hypertens.
6:
15A,
1993.
10.
Guarda, E.,
P. R. Myers,
C. G. Brilla,
S. C. Tyagi,
and
K. T. Weber.
Endothelial cell-induced modulation of cardiac fibroblast collagen metabolism.
Cardiovasc. Res.
27:
1004-1008,
1993[Medline].
11.
Hajjar, K. A.
Homocysteine-induced modulation of tissue plasminogen activator binding to its endothelial cell membrane receptor.
J. Clin. Invest.
91:
2873-2879,
1993[Medline].
12.
Harker, L. A.,
R. Ross,
S. J. Slichter,
and
C. R. Scott.
Homocystine-induced artherosclerosis. The role of endothelial cell injury, and platelet response in its genesis.
J. Clin. Invest.
58:
731-741,
1976[Medline].
13.
Harlan, J. M.,
J. D. Levine,
K. S. Callahan,
B. R. Schwartz,
and
L. A. Harker.
Glutathione redox-cycle protects cultured endothelial cells against lysis by extracellularly generated hydrogen peroxide.
J. Clin. Invest.
73:
706-713,
1984[Medline].
14.
Hultberg, B.,
A. Andersson,
and
A. Isaksson.
Metabolism of homocysteine, its relation to the other cellular thiols, and its mechanism of cell damage in a cell culture line (human histiocytic cell line U-937).
Biochim. Biophys. Acta
1269:
6-12,
1995[Medline].
15.
Isaka, Y.,
D. Brees,
K. Ikegaya,
Y. Kaneda,
E. Imai,
N. A. Noble,
and
W. A. Border.
Gene therapy by skeletal muscle expression of decorin prevents fibrotic disease in rat kidney.
Nat. Med.
2:
418-423,
1996[Medline].
16.
Janiszewski, M.,
C. A. Pasqualucci,
P. L. Daluz,
and
F. R. M. Launndo.
Oxidized glutathione markedly enhances cell proliferation after vascular injury in inact rabbit: possible role of metalloproteinase activation (Abstract).
Circulation
90 (4):
I-139,
1994.
17.
Katwa, L. C.,
E. Guarda,
and
K. T. Weber.
Endothelin receptors in cultured rat cardiac fibroblasts.
Cardiovasc. Res.
27:
2125-2129,
1993[Medline].
18.
Marui, N,
M. K. Offermann,
R. Swerlick,
C. Kunsch,
C. A. Rosen,
M. Ahmad,
R. W. Alexander,
and
R. M. Medford.
Vascular cell adhesion molecule-1 (VCAM-1) gene transcription, and expression are regulated through an antioxidant-sensitive mechanism in human vascular endothelial cells.
J. Clin. Invest.
92:
1866-1874,
1993[Medline].
19.
Nishinaga, M.,
T. Ozawa,
and
K. Shimada.
Homocysteine, a thrombogenic agent, suppresses anticoaguant heparan sulfate expression in cultured porcine aortic endothelial cells.
J. Clin. Invest.
92:
1381-1386,
1993[Medline].
20.
Park, Y.,
S. Kanekal,
and
J. P. Kehrer.
Oxidative changes in hypoxic rat heart tissue.
Am. J. Physiol.
260 (Heart Circ. Physiol. 29):
H1395-H1405,
1991
21.
Shi, Y.,
M. Pieniek,
A. Fard,
J. O'Brien,
J. D. Mannion,
and
A. Zalewski.
Adventitial remodeling after coronary arterial injury.
Circulation
93:
340-148,
1996
22.
Tsai, J.-C.,
M. A. Perrella,
M. Yoshizumi,
C.-M. Hsieh,
E. Harber,
R. Schlegel,
and
M.-E. Lee.
Promotion of vascular smooth muscle cell growth by homocysteine: a link to atherosclerosis.
Proc. Natl. Acad. Sci. USA
91:
6369-6373,
1994[Abstract].
23.
Tsai, J.-C.,
H. Wang,
M. A. Perrella,
M. Yoshizumi,
N. E. Sibinga,
L. C. Tan,
E. Haber,
T. H. Chang,
R. Schlegel,
and
M. E. Lee.
Induction of cyclin A gene expression by homocysteine in vascular smooth muscle cells.
J. Clin. Invest.
97:
146-153,
1996
24.
Tyagi, S. C.,
S. E. Campbell,
H. K. Reddy,
E. Tjahja,
and
D. J. Voelker.
Matrix metalloproteinase activity expression in infarcted, noninfarcted, and dilated cardiomyopathic human hearts.
Mol. Cell. Biochem.
155:
13-21,
1996[Medline].
25.
Tyagi, S. C.,
S. G. Kumar,
and
S. Borders.
Reduction-oxidation (redox) state regulation of extracellular matrix metalloproteinases, and tissue inhibitors in cardiac normal and transformed fibroblast cells.
J. Cell. Biochem.
61:
139-151,
1996[Medline].
26.
Tyagi, S. C.,
S. G. Kumar,
and
G. Glover.
Induction of tissue inhibitor, and matrix metalloproteinase by serum in human heart-derived fibroblast and endomyocardial endothelial cells.
J. Cell. Biochem.
58:
360-371,
1995[Medline].
27.
Tyagi, S. C.,
L. Meyer,
S. G. Kumar,
R. A. Schmaltz,
H. K. Reddy,
and
D. J. Voelker.
Induction of tissue inhibitor of metalloproteinase, and its mitogenic response to endothelial cells in human atherosclerotic and restenotic lesions.
Can. J. Cardiol.
12:
353-362,
1996[Medline].
28.
Tyagi, S. C.,
L. Meyer,
R. A. Schmaltz,
H. K. Reddy,
and
D. J. Voelker.
Proteinases, and restenosis in human coronary artery: extracellular matrix production exceeds the expression of proteolytic activity.
Atherosclerosis
116:
43-57,
1995[Medline].
29.
Tyagi, S. C.,
L. Meyer,
R. A. Schmaltz,
H. K. Reddy,
and
D. J. Voelker.
Proteinases, and restenosis: matrix metalloproteinase and their inhibitor and activator.
In: Cardiovascular Disease II: Cellular and Molecular Mechanisms, Prevention, Treatment, edited by L. L. Gallo. New York: Plenum, 1995, p. 19-31.
30.
Tyagi, S. C.,
and
S. R. Simon.
Hydrophobic binding sites of elastin-derived peptide on neutrophil elastase.
Biochem. Cell Biol.
72:
419-427,
1994[Medline].
31.
Wilcken, D. E.,
S. G. Reddy,
and
V. J. Gupta.
Homocysteinemia, ischemic heart disease, and the carrier state for homocystinuria.
Metabolism
32:
363-370,
1983[Medline].
32.
Woessner, J. F., Jr.
Matrix metalloproteinaes, and their inhibitors in connective tissue remodeling.
FASEB J.
5:
2145-2154,
1991
33.
Zhou, G,
J. C. Kandala,
S. C. Tyagi,
L. C. Katwa,
and
K. T. Weber.
Effects of Ang II, and aldosterone on collagen gene expression and protein turnover in cardiac fibroblasts.
Mol. Cell. Biochem.
154:
171-178,
1996[Medline].