The Role of Matrix Metalloproteinases in Atherosclerosis

Autor: Tao-Cheng Wu, 吳道正
Rok vydání: 2008
Druh dokumentu: 學位論文 ; thesis
Popis: 96
英文摘要（Summary） Background Coronary artery disease (CAD) is a human atherosclerosis disease in which vascular remodelling may proceed with the deposition of lipids, macrophages, smooth muscle cells and extracellular matrix proteins in conduit coronary artery walls. Matrix metalloproteinases (MMPs) can degrade extracellular matrix and weaken the stability of plaques. Increased MMP expression has been identified in atherosclerotic lesions and has been shown to play an important role in extracellular remodelling, atheroma formation and especially plaque rupture. Thus, in clinical studies, we examined the expression and prognostic significance of different circulating MMPs in patients with or without angiographically proven CAD. In basic studies, we tested that pharmacological antioxidants such as carvedilol and probucol could inhibit vascular MMPs expression in experimental atherosclerosis, the study was conducted with an in vivo atherosclerosis model with balloon injury in hypercholesterolemic rabbits and with the in vitro culture system of human aortic smooth muscle cells (HASMCs) stimulated by tumor necrosis factor-�� (TNF-��), an inflammatory cytokine critical to atherogenesis. It was revealed that both carvedilol and probucol reduced oxidative damage and the expression and activity of MMP-2 and MMP-9 in vascular neointima. Materials and Methods Clinical studies: Between March 1999 and June 2003, The patients who were initially stable and who preferred a noninvasive strategy for their significant CAD (> 50% of luminal diameter stenosis in at least one major coronary artery) proven on coronary angiogram were included. All these patients were diviede into different treatment strategies included medical treatment or coronary revascularization, and were follow-up regularly at outpatient clinics by their own choice. In addition, the consecutive patients with exercise-induced ischaemia and normal coronary angiograms (cardiac syndrome X) and another normal subjects without clinical evidence of cardiovascular or other major systemic disease (normal control) were also studied for comparison. During follow-up period, the development of any major cardiovascular events including cardiac death, nonfatal MI, unscheduled coronary revascularization such as percutaneous coronary intervention (PCI) and coronary artery bypass graft surgery (CABG), and hospitalization as a result of unstable angina were recorded. Biochemical parameters including total cholesterol levels, triglyceride levels, and high-density lipoprotein cholesterol (HDL), plasma hsCRP level, MMP-2, MMP-3, MMP-9 were also analyzed. Basic Studies: In vivo study: The adult male New Zealand White rabbits were provided were then randomly divided into four groups included control, high-cholesterol (HC), HC +probucol and HC +carvedilol groups. At the end of the third week after studies, denudation procedure of abdominal artery was performed for these four groups. They were sacrified at 6th week. The abdominal arterial tissue was sent for morphometry and immunohistochemistry. The blood samples were collected before and 6 weeks after the high-cholesterol diet was fed. The plasma cholesterol and triglyceride were measured. Morphometric analyses and double-label immunostaining of MMP-2 andα-actin or MMP-9 andα-actin were performed. The double-label immunofluorescence microscopy of MMPs and 8-OhdG were also performed. Western blot analysis was conducted to determine the levels of MMPs in arterial segments and MMPs activities were determined by gelatin zymography. The levels of MMP-2 and MMP-9 mRNA expression were performed by Quantitative real time-PCR. In vitro study: HASMCs (Cascade Biologics, OR, USA) were grown and passaged as described previously. Cells were used at passages 3-8. Before treatment or stimulation with reagents, the cells were serum starved overnight. HASMCs were pretreated with probucol, carvedilol, propranolol or prazocin (an ��-blocker, Sigma, Saint Louis, Missouri, USA) for 18 h followed by TNF-�� (20 ng/ml, PeproTech, Inc, Rocky Hill, NJ, USA) stimulation for 24 h. The conditioned media was collected and western blot and gelatin zymography analysis were performed for MMP-2 and MMP-9. Results Clinical study: In stable CAD patients with non-invasive treatment, plasma MMP-2 levels were significantly higher in CAD patients than in cardiac syndrome X patients or normal subjects. Plasma MMP-3 and -9 levels were also significantly increased in CAD patients as compared with cardiac syndrome X patients or normal subjects. By univariate analysis, the predictors of major cardiovascular events included the number of diseased coronary arterial vessels, plasma hsCRP levels and plasma MMP-3 levels. By multivariate analysis, the adjusted risk of plasma hsCRP levels for future cardiovascular events was borderline significant (HR = 1.91, 95% CI 0.98–3.74, P= 0.059). Plasma MMP-3 levels≧103.7 ng mL was the only independent predictor of major cardiovascular events during the follow-up period. The patients with high MMP risk scores had increased incidence of adverse events during follow-up (P < 0.01). The CAD patients with revascularization therapy showed that baseline serum MMP-9 level was significantly associated with future cardiovascular events (P=0.026) whereas serum hsCRP level (P=0.065) and age (P=0.052) also had the trend. In multivariate Cox proportional hazards analysis, serum MMP-9 level >34.88 ng/ml was still the only independent predictor of future cardiovascular events after coronary revascularization (relative risk [RR], 3.178; 95% confidence interval [CI], 1.14-8.89; P=0.028). The Kaplan-Meier analysis was consistent with that by multivariate Cox proportional hazards analysis, patients with a baseline serum MMP-9 level ＞34.88 ng/ml had more future cardiovascular events than those with a MMP level ≦34.88 ng/ml (P=0.021 ) Basic study: In vivo study, Animals treated with carvedilol (HC+carvedilol) or probucol (HC+probucol) had significantly reduced neointima formation and the expressions of MMP-2 and MMP-9 in neointima. The expression of 8-OHdG was observed in the HC animals, but it was only limited to few smooth muscle cells in the probucol- (HC+carvedilol) or probucol- (HC+probucol) treated animals. Gelatin zymography showed that the activities of vascular pro-MMP-2, MMP2 and MMP-9 were significantly increased in the HC animals; pretreatment with carvedilol (HC+carvedilol) or probucol (HC+probucol) significantly decreased their activities Western blot analysis and RT-PCR showed that vascular expressions of MMP-2 and -9 in abdominal aortas were significantly increased in the HC animals as compared to control animals. Pretreatment with carvedilol and probucol significantly inhibited the protein and mRNA expressions of MMP-2 and -9. In the in vitro study, the expressions and activities of MMP-2 and -9 that were increased by TNF-�� stimulation and were significantly suppressed by pretreatment with carvedilol or probucol. Conclusions Plasma MMP-3 is a novel prognostic factor for future adverse cardiovascular events in stable CAD patients and MMP-9 level is an independent predictor for future cardiovascular events after PCI or CABG, suggesting its universal role in risk stratification before different revascularization strategies. Carvedilol as an antioxidant may inhibit oxidative DNA damage and the activities as well as expressions of MMP-2 and MMP-9, together with neointimal hyperplasia after balloon injury in hypercholesterolemic rabbits. At last, the MMPs play a important role in clinical and experimental atherosclerosis.
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