Effects of heparin on the production of homocysteine-induced extracellular matrix metalloproteinase-2 in cultured rat vascular smooth muscle cells

BASIC RESEARCH

Effects of heparin on the production of homocysteineinduced extracellular matrix metalloproteinase-2 in cultured rat vascular smooth muscle cells Hangyuan Guo MD PhD1,2, Jong-Dae Lee MD2, Hiroyasu Uzui MD2, Hong Yue MD2, Ping Wang MD2, Kiyohiro Toyoda MD2, Tooru Geshi MD2, Takanori Ueda MD2

H Guo, J-D Lee, H Uzui, et al. Effects of heparin on the production of homocysteine-induced extracellular matrix metalloproteinase-2 in cultured rat vascular smooth muscle cells. Can J Cardiol 2007;23(4):275-280. OBJECTIVE: To study the effects of heparin on the production of homocysteine-induced extracellular matrix metalloproteinase-2 (MMP-2) in cultured rat vascular smooth muscle cells. METHODS: The effects of different homocysteine levels (0 μmol/L to 1000 μmol/L) on MMP-2 production and the effects of different heparin concentrations (0 μg/mL to 100 μg/mL) on homocysteineinduced MMP-2 in cultured rat vascular smooth muscle cells were examined using gelatin zymography and Western blotting. The changes in MMP-2 were further compared with various treatments for 24 h, 48 h and 72 h. RESULTS: Homocysteine (50 μmol/L to 1000 μmol/L) increased the production of MMP-2 significantly in a dose-dependent manner. Increased production of MMP-2 induced by homocysteine was reduced by the extracellular addition of heparin in a dose-dependent manner. Production of MMP-2 with various treatment regimens for 72 h was greater than for 24 h and 48 h. CONCLUSIONS: Extracellular addition of heparin decreased homocysteine-induced MMP-2 secretion. Data suggest a mechanism by which hyperhomocysteinemia is involved in the pathogenesis of coronary artery disease and demonstrate a beneficial effect of heparin on these conditions.

Key Words: Atherosclerosis; Heparin; Homocysteine; Matrix metalloproteinase; Smooth muscle cell

igration of vascular smooth muscle cells (VSMCs) into the vascular intima and their subsequent proliferation, as well as extracellular matrix (ECM) remodelling, are important events in the pathogenesis of atherosclerosis. These events are known to be regulated by various cytokines and growth factors secreted by platelets and vascular cells (1). Zempo et al (2) reported that the processes of migration and proliferation of VSMCs that contribute to the morphogenesis of atherosclerotic plaque require ECM remodelling caused by matrix metalloproteinases (MMPs). Brown et al (3) identified MMPs in human coronary atherosclerotic lesions and suggested that MMPs are closely related to the progression of atherosclerosis.

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Les effets de l’héparine sur la production de métalloprotéinase-2 de la matrice extracellulaire induite par l’homocystéine dans les cellules des muscles lisses vasculaires de rats de culture OBJECTIF : Étudier les effets de l’héparine sur la production de métalloprotéinase-2 (MMP-2) de la matrice extracellulaire induite par l’homocystéine dans les cellules des muscles lisses de rats de culture. MÉTHODOLOGIE : On a examiné les effets de divers taux d’homocystéine (de 0 μmol/mL à 1 000 μmol/mL) sur la production de MMP-2 et les effets de diverses concentrations d’héparine (de 0 μg/mL à 100 μg/mL) sur la MMP-2 induite par l’homocystéine dans les cellules des muscles lisses vasculaires de rats de culture par zymographie à la gélatine et par transfert Western. On a ensuite comparé les modifications de la MMP-2 avec divers traitements pendant 24 heures, 48 heures et 72 heures. RÉSULTATS : L’homocystéine (de 50 μmol/mL à 1 000 μmol/mL) augmentait considérablement la production de MMP-2 selon la dose. Une hausse de la production de MPP-2 induite par l’homocystéine était réduite par l’ajout extracellulaire d’héparine selon la dose. La production de MMP 2 selon diverses posologies était plus élevée au bout de 72 heures qu’au bout de 24 heures et de 48 heures. CONCLUSIONS : L’ajout extracellulaire d’héparine réduit la sécrétion de MMP-2 induite par l’homocystéine. Selon les données, un mécanisme engagerait l’hyperhomocystéinémie dans la pathogenèse de la coronaropathie et démontrerait l’effet bénéfique de l’héparine sur ces maladies.

Homocysteine (Hcy) is an intermediate sulphur-containing amino acid formed during intracellular metabolism of methionine. Circulating Hcy levels may be increased by a genetic deficiency of enzymatic pathways involved in its catabolism, as well as by environmental factors, including nutritional deficiencies, lifestyle, physiological conditions, drugs and some diseases, which mainly induce a deficiency of folic acid, and vitamins B12 and B6. Therefore, plasma Hcy levels may be reduced by interventional therapy with folic acid and B vitamins (4). Hyperhomocysteinemia is an independent risk factor for coronary artery disease (5). Heparin, an anticoagulant, has been shown to reduce neointimal proliferation and restenosis after vascular injury in

of Cardiology, Shaoxing People’s Hospital, The First Affiliated Hospital of Shaoxing University, Shaoxing, China; 2First Department of Internal Medicine, Fukui Medical University, Fukui, Japan Correspondence and reprints: Dr Hangyuan Guo, Department of Cardiology, Shaoxing People’s Hospital, The First Affiliated Hospital of Shaoxing University, Shaoxing, Zhejiang 312000, China. Telephone 86-571-869-97949, fax 86-575-522-8899, e-mail [email protected] Received for publication September 6, 2005. Accepted May 25, 2006

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experimental studies. A number of studies have shown that heparin inhibits VSMC proliferation and migration in vivo and in culture (6-8). Tyagi et al (6) demonstrated the role of heparin in ECM remodelling and concluded that it has implications for angiogenesis and atherosclerosis. Caenazzo et al (7) suggested that heparin may have the potential to correct this high MMP abnormality. Kazi et al (8) showed that heparins with varying anticoagulant activities and molecular weights, but similar sulphate contents, can retain antiproliferative properties, while the effect on some other biological processes, such as cell adhesion, is lost. Such chemical alterations may yield useful substances for the prevention of smooth muscle cell (SMC) proliferation after arterial injury. However, clinical trials that have used heparin in coronary balloon angioplasty have been negative. Doucet et al (9) showed that intravenous nitroglycerin is highly effective in preventing adverse ischemic events (recurrent or refractory angina) in patients with unstable angina secondary to restenosis, whereas heparin has no effect. Karsch et al (10) showed that low-molecular-weight heparin (reviparin) use during and after coronary angioplasty does not reduce the occurrence of major clinical events or the incidence of angiographic restenosis over 30 weeks. Thus, clinical trials are negative despite positive basic research; perhaps this is due to different methods, doses of heparin, duration of therapy, etc. We therefore sought to determine effects of heparin on the production of Hcy-induced ECM MMP-2 in the cultured rat VSMC. Most authors think that heparin inhibits VSMC proliferation, but the mechanisms remain elusive. The effects of heparin on Hcy-induced MMP-2 in cultured VSMCs have not previously been studied. The aim of the present study was to test whether extracellular addition of heparin alters MMP-2 secretion. We investigated the effects of heparin on production of MMP-2 in VSMCs at basal and in Hcy-stimulated conditions.

MATERIALS AND METHODS Materials The chemicals used in the present study were obtained from the following sources: Dulbecco’s modified Eagle’s medium (DMEM) without magnesium was obtained from GIBCO (USA); fetal calf serum (FCS) was purchased from Filtron Pty Ltd (Japan); D,L-Hcy was purchased from Nacalai Tesque Inc (Japan); heparin (sodium salt, 185.5 USP units/mg) was supplied by Sigma-Aldrich Co (USA); and rabbit anti-MMP-2 antibody (NeoMarkers’ antibody line, Ab-7, RB-1537-PO) was obtained from Lab Vision Corp (USA). All other chemicals were of reagent grade or the of highest grade commercially available.

with a serum-free medium, and cells were then exposed to various treatments.

Preparation of the culture medium Hcy was added to DMEM without magnesium to final Hcy concentrations of 0 μmol/L to 1000 μmol/L. The final concentrations of heparin were 0 μg/mL to 100 μg/mL; 6.25 μmol/L to 12.5 μmol/L Hcy is a normal range, 12.5 μmol/L to 25 μmol/L indicates a subpathological state and more than 25 μmol/L indicates a pathological state. The present study aimed to assess the effects of heparin on the production of hyperhomocysteinemia (homocysteine concentration of 50 μmol/L to 1000 μmol/L)-induced ECM MMP-2 in the cultured rat VSMC. All treatments of heparin were sustained in the 500 μM Hcy-treated media for 72 h.

Analysis of gelatinase production After treatments for 24 h, 48 h and 72 h, medium samples were harvested, centrifuged at 2000 g for 10 min and normalized for cell protein content using the Bio-Rad assay (Bio-Rad Laboratories, USA) (11). The samples were applied without reduction to a 7.5% polyacrylamide slab gel impregnated with 1 mg/mL gelatin (15). After electrophoresis, the gel was washed at room temperature for 30 min in a washing buffer (50 mmol/L Tris chloride, pH 7.5, 15 mmol/L calcium chloride, 1 mol/L zinc chloride, and 2.5% Triton X-100 [Nichitionogene Tenside, Marke von Union Carbide, USA]); it was then incubated overnight at 37°C and shaken in the same buffer, but containing 1% rather than 2.5% Triton X-100. The gel was stained with a solution of 0.1% Coomassie Brilliant Blue R-250. Clear zones against the blue background indicated the presence of gelatinase. To quantify the amount of gelatinase expression, the stained zymograms were scanned on a densitograph (ATTO, Japan).

Western blot analysis After 24 h, 48 h and 72 h, medium samples were harvested with the protease inhibitors phenylmethanesulfonyl fluoride (0.1 mmol/L) and leupeptin (10 μg/mL) from cells, centrifuged at 2000 g for 10 min and separated by electrophoresis on 7.5% sodium dodecyl sulphate polyacrylamide gels, followed by transfer to polyvinylidene difluoride membranes (Immobilon-P, Millipore, USA; 0.22 μm pore size). The membranes were blocked in 5% skim milk in phosphatebuffered saline solution containing 0.1% Tween 20 at room temperature for 1 h and were probed with anti-MMP-2 monoclonal antibodies overnight. After washing three times with the phosphate-buffered saline containing 0.1% Tween 20, the membranes were incubated with a secondary antibody conjugated with horseradish peroxidase for 1 h, as described previously (16). Finally, the blots were washed and scanned on a densitograph.

Preparation of SMCs Rat aortic SMCs were isolated by enzymatic digestion from the thoracic aorta of six-week-old male Sprague-Dawley rats (Charles River, Japan) as described previously (11). All surgical interventions and anesthesia were conducted in conformity with institutional guidelines and in compliance with international laws and policies (12,13). The cells were cultured in DMEM supplemented with 10% FCS at 37°C in a humidified 5% CO2 and 95% air atmosphere. At confluence, cells displayed a ‘hill and valley’ growth pattern and abundant myofilaments in their cytoplasm. They were identified as VSMCs by immunocytochemistry using HHF35, a monoclonal antibody that recognizes muscle-specific actin (14). All VSMC cultures used in the present study were performed between passages 4 and 7. At the subconfluent stage, the culture medium was replaced 276

Statistical analysis Results are presented as percentages of the control and represent the mean ± SEM for five separate experiments performed in duplicate. Differences among all data were analyzed for statistical significance by ANOVA followed by unpaired Student’s t test. Differences of P<0.05 were considered statistically significant.

RESULTS Effects of Hcy on MMP-2 in VSMCs Gelatin zymograms of VSMC-conditioned media showed that the major MMP expressed under these conditions was MMP-2. Hcy (50 μmol/L to 1000 μmol/L) increased the production of MMP-2 significantly in a dose-dependent manner (Figure 1A). Can J Cardiol Vol 23 No 4 March 15, 2007

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Figure 1) Effects of homocysteine (Hcy) on production of matrix metalloproteinase-2 (MMP-2) in cultured vascular smooth muscle cells. Hcy (50 μmol/L to 1000 μmol/L) significantly increased the production of MMP-2 as determined by zymography (A) and Western blot analysis (B) in a dose-dependent manner. Clear zones against the blue background indicated the presence of MMP-2. Columns indicate data of gelatinolytic activity as percentages of the amount of control (100 μmol/L Hcy) and represent the mean ± SEM for five separate experiments performed in duplicate. *P<0.01 versus Hcy 0 μmol/L; †P<0.05 versus Hcy 50 μmol/L; ‡P<0.01 versus Hcy 50 μmol/L; §P<0.05 versus Hcy 100 μmol/L; ¶P<0.01 versus Hcy 100 μmol/L

Figure 2) Effects of heparin on the production of matrix metalloproteinase-2 (MMP-2) stimulated by homocysteine (Hcy) (500 μmol/L, indicated by ‘+’) in cultured vascular smooth muscle cells. Heparin significantly and dose-dependently decreased the production of MMP-2 as determined by zymography (A) and Western blot analysis (B) under conditions of stimulation by Hcy. Clear zones against the blue background indicate the presence of MMP-2. Columns indicate data of gelatinolytic activity as percentages of the amount of control (100 μmol/L Hcy) and represent the mean ± SEM for five separate experiments performed in duplicate. Treatment with 10 μg/mL heparin, together with Hcy, was regarded as the control. *P<0.01 versus heparin 0 μg/mL; †P<0.05 versus heparin 1.0 μg/mL; ‡P<0.01 versus heparin 1.0 μg/mL; §P<0.01 versus heparin 10 μg/mL

MMP-2 protein was shown to be expressed by Western blots of culture medium probed using an anti-MMP-2 antibody. Hcy induced a significant and dose-dependent upgrade in MMP-2 production (Figure 1B).

manner. Heparin significantly and dose-dependently decreased the production of Hcy-induced MMP-2.

Effects of heparin on the production of Hcy-induced MMP-2 in VSMCs Next, the effects of heparin on cells stimulated with Hcy (500 μmol/L) were examined. Heparin decreased MMP-2 production significantly under these conditions in a dosedependent manner (Figure 2A). The MMP-2 protein was shown to be expressed by Western blots of culture medium probed using anti-MMP-2 antibody. Heparin induced a significant and dose-dependent reduction in MMP-2 production by cells stimulated with Hcy (Figure 2B). These effects were not toxic, as determined by Trypan Blue exclusion. Changes in MMP-2 production after VSMCs were cultured for 24 h, 48 h and 72 h Production of MMP-2 with treatment for three days increased to levels greater than at one and two days (Figure 3). Hcy significantly increased the production of MMP-2 in a time-dependent Can J Cardiol Vol 23 No 4 March 15, 2007

Effects of heparin on the zymography system To detect the effects of heparin on the zymography system, subconfluent VSMCs were treated with 0 mmol/L of heparin for 24 h, and this culture medium was then adjusted to different heparin concentrations (0 μg/mL to 100 μg/mL) before zymography. Heparin did not influence MMP detection under these conditions. The lack of an inhibitory effect of heparin on Hcyinduced MMP expression after removal of cells demonstrated the feasibility of studying the effects of heparin.

DISCUSSION The ECM MMPs are a family of distinct proteases with differing specificities for cleaving various ECM components (17). The following four MMPs are known as elastolytic proteinases: gelatinase A (MMP-2), gelatinase B, metalloelastase and matrilysin. Only MMP-2 and gelatinase B are expressed as latent proenzymes by aortic SMCs, and both are involved in arterial diseases, such as atherosclerosis and abdominal aortic aneurysms. It has been reported that the migration and 277

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Figure 3) Changes in metalloproteinase-2 (MMP-2) levels after vascular smooth muscle cells cultured under various treatments for 24 h, 48 h and 72 h. Homocysteine (Hcy) (indicated by ‘+’) significantly increased the production of MMP-2 in a time-dependent manner. Heparin significantly and dose-dependently decreased the production of Hcy-induced MMP-2 as determined by zymography (A) and Western blot analysis (B). Clear zones against the blue background indicate the presence of MMP-2. Treatment with 500 μmol/L Hcy (second day) was regarded as control. Columns indicate data of gelatinolytic activity as percentages of the amount of control (100 μmol/L Hcy) and represent the mean ± SEM for five separate experiments performed in duplicate. *P<0.05 versus 24 h; †P<0.01 versus 24 h; ‡P<0.05 versus 48 h; §P<0.01 versus 48 h; compared with Hcy: ¶P<0.01 versus 24 h; **P<0.01 versus 48 h; ††P<0.01 versus 72 h

proliferation of VSMCs, which contribute to the morphogenesis of atherosclerotic plaques, require the ECM remodelling caused by MMPs (2). The production of MMPs in VSMCs is known to be regulated by a number of cytokines and growth factors, such as platelet-derived growth factor secreted by platelets and vascular cells (1,11,18). Among the MMPs, MMP-2 has the widest distribution and plays an important role in the turnover of basement membrane type IV collagen and in controlling cell proliferation (19). The proliferation and migration of VSMCs were demonstrated to be closely related to the stimulation of MMP-2 production (11). Increased expression of MMP-2 has been shown to be present in atherosclerotic plaques (20). It has also been shown that the binding of MMP-2 to insoluble elastin induces fast autoactivation of the proenzyme, suggesting that this mechanism could be relevant to the focal elastolysis in the arterial wall during arteriosclerosis. All these studies suggest that MMP-2 plays an important role in the formation and progression of atherosclerotic lesions. Hcy is a nonprotein-forming, sulphur-containing amino acid that stands at the crossroads of two metabolic pathways (21). Hcy, which is not a dietary constituent, is formed 278

exclusively on demethylation of methionine and is eliminated through one of two vitamin-dependent pathways. Elevated plasma levels of Hcy have been associated with vascular disease since the initial descriptions of classical homocysteinuria in children (22). Indeed, the past decade has witnessed an exponential increase in studies defining plasma Hcy as an independent risk factor, similar to smoking or hyperlipidemia, for atherosclerotic cardiovascular, cerebrovascular and peripheral vascular diseases (5,23). However, little is known about the pathogenic mechanisms underlying the actions of Hcy. The mechanisms by which hyperhomocysteinemia causes coronary artery disease may include injury to the endodermis of the vessels, activation of platelets, improvement in the congregation of platelets, enhancement in the production of fibrinogen, and the promotion of migration and proliferation of SMCs. Hcy can also activate protein kinase C and promote the expression of c-fos and c-myb genes in VSMCs (5,24-26). Hcy is known to increase reactive oxygen species and can cause an increase in nitrotyrosine concentration. This tyrosine concentration, in turn, is resolved by the activation of MMP-2. The exact mechanism of cause and effect is not known. Although in vitro studies have revealed a number of Hcymediated alterations in the thromboregulatory properties of endothelial cells, comparatively little is known about Hcymodulated SMC function. Woo et al (27) suggested that D,LHcy stimulation of bovine aortic SMC proliferation involves mitogen-activated protein kinase activation. An initial effect of Hcy is to induce release of intracellular calcium in VSMCs, which may induce vascular reactivity. The transient in calcium correlates with the effect on ECM associated with Hcy (28). Desai et al (29) suggested that Hcy may increase monocyte recruitment into developing atherosclerotic lesions by upregulating human monocyte chemoattractant protein-1 and interleukin-8 expression in VSMCs. Hyperhomocysteinemia is associated with a significant decrease in the SMC/ECM ratio of the media of muscular femoral arteries without significant changes in medial thickness (30). Taha et al (31) suggested that Hcy induces SMC growth by a hydrogen peroxideindependent pathway, and that the effects of Hcy may combine with the known initiating events produced by oxidative stress to accelerate the progression of atherosclerosis. Tsai et al (32) showed that Hcy and serum increased DNA synthesis synergistically in both human and rat aortic SMCs. In the present study, we found that Hcy (from a concentration of 50 μmol/L to 1000 μmol/L) activated the production of MMP-2. However, the major finding of the study was that extracellular heparin supplementation reduced the production of Hcy-induced MMP-2 in rat VSMCs. To our knowledge, this is the first report describing the effects of heparin on the production of MMP-2 in cultured rat VSMCs. Heparin has been shown to reduce intimal thickening after arterial wall injury by inhibiting VSMC proliferation and migration, but its mechanisms of action remain unclear. Although heparin has been well recognized as the representative molecule suppressing VSMCs growth in vitro, attempts to use heparin as a therapeutic antirestenosis drug have not favourably influenced the angiographic or clinical outcome after angioplasty in clinical trials. Zhao et al (33) concluded that phosphatase activation is not a direct mechanism of suppression of multiple kinase cascades by heparin. Mori et al (34) suggested that histidine-rich glycoprotein, in combination Can J Cardiol Vol 23 No 4 March 15, 2007

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with the zinc ion, plays a role in modulating the SMC growth response in pathophysiological states and explains the lack of success of heparin as a therapeutic antirestenosis drug in clinical trials. Mishra-Gorur et al (35) showed that alterations in calcium-mediated mitogenic signalling pathways may be involved in the antiproliferative mechanism of action of heparin. He et al (36) showed that a heparinoid at 1.6 mg/mL to 0.05 mg/mL significantly inhibits VSMC proliferation induced by FCS (10%), basic fibroblast growth factor or interleukin-1. Kalmes et al (37) showed that the inhibitory effect of heparin on SMC migration induced by thrombin relies, at least in part, on a blockade of heparin-binding epidermal growth factor-like growth factor-mediated epidermal growth factor receptor transactivation. The identification of two heparinregulated tyrosine phosphoproteins (42 kDa and 200 kDa) suggested that they may be key mediators of the antiproliferative effect of heparin (38). Heparin inhibits growth of baboon SMCs by preventing prolonged mitogen-activated protein kinase activation elicited by ligands of seven transmembrane domain receptors and heterotrimeric G-proteins (39). Bono et al (40) showed that heparin inhibits the binding of basic fibroblast growth factor to cultured human aortic SMCs. Lake and Castellot (41) showed that Wnt-induced secreted protein-2 modulates the antiproliferative effect of heparin and regulates cell motility in VSMCs. Until now, there have been no controlled data on the effects of Hcy-lowering treatment on vascular function or clinical end points. The precise mechanisms by which Hcy mediates its adverse vascular effects are unknown, but they may relate to impaired SMC function.

CONCLUSIONS In cultured rat VSMCs, heparin significantly reduced the production of Hcy-induced MMP-2 in a dose-dependent manner. Our data suggest that the beneficial effect of heparin supplementation on vascular disease processes may be due, at least in part, to the inhibitory effect of heparin on the production of Hcy-induced MMP-2 in VSMCs. ACKNOWLEDGEMENTS: This study was supported in part by a Grant-in-Aid for General Scientific Research (No 08770492, No 12770339) from the Ministry of Education, Science and Culture of Japan. The authors thank Dr Hiromasa Shimizu, Hiroshi Tsutani, Yasuhiko Mitsuke, Hiromichi Iwasaki and Yangbo Xing for their data collection, assistance with statistical analysis and helpful discussions. The authors are also grateful to Hiromi Nishimura and Makoto Matsumoto for their technical assistance.

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Can J Cardiol Vol 23 No 4 March 15, 2007