| Abstract |
Atherosclerosis is not just the process of passive lipid accumulation in the intima of the arterial wall but an active inflammatory disease. Endothelial cells, monocytes/macrophages and smooth muscle cells react to hypoxic stress and various cytokines and growth factors such us transforming growth factor beta 1 (TGF-β1), angiotensin II, basic fibroblast growth factor (bFGF), interleukin-1, and produce vascular endothelial growth factor (VEGF), a powerful pro-inflammatory agiogenic and mitogenic factor. In previous studies this factor was found in high levels in the serum of patients with coronary artery disease and in atherosclerotic lesions of coronary and peripheral arteries. Purpose of the present study is to determine the role of agiogenic factors in the process of coronary artery disease and contribute to better understanding of the molecular mechanisms involved in establishment and progression of atherogenesis. For this purpose we tested expression of VEGF-A, TGF-1β and bFGF from monocytes/macrophages and lymphocytes of the peripheral blood from patients suffering from coronary artery disease. Also expression of these factors from macrophages/foam cells, vascular smooth muscle cells and endothelial cells of human atherosclerotic coronary arteries was studied. Furthermore we investigated the effect of fluvastatin and glucose on expression levels of those factors in peripheral monocytes of healthy donors. Finally expression of two newly discovered angiogenic growth factors, Heregulin (HRG1-β1), and CYR61 was studied in atherosclerotic lesions in order to identify their possible implication in coronary artery disease. The effect of HRG1-β1 in expression levels of CYR61 by endothelial cells was also tested. In further details monocytes and lymphocytes were isolated from peripheral blood of 53 donors, just before coronary angiography. Seventeen donors had normal coronary arteries, while the remaining 36 were proved to suffer from coronary artery disease of various severities. According to the clinical manifestation of the disease subjects were separated into three subgroups: a) normal donors b) patients with coronary artery disease and stable angina and c) patients with coronary artery disease and unstable angina. mRNAs were isolated from monocytes and lymphocytes of all the donors and analyzed by the RTPCR method for the semi-quantitive detection of VEGF-A, TGF-β1 and bFGF expression levels. Furthermore fifty ml of peripheral blood was obtained from two healthy donors (23 and 27 years of age respectively) and monocytes/macrophages were isolated and placed in plastic cell-culture containers. Then isolated cells were activated with glucose and fluvastatin and mRNA was obtained in several time periods (30 min, 1 h, 2 h, 4 h, 24 h και 48 h). The samples were analyzed by the RT-PCR method motioned before. During the second part of the study 26 necrotomic coronary artery samples from 8 different donors were obtained during autopsy in less than 8 hours since death occurred. All samples were separated into two. The first part was placed in 10% paraphormaldeide solution for further immunochemical study while the second part was frozen in -70 οC for RNA analysis by the RT-PCR method. After pathological inspection the samples were categorized into four groups a) normal coronary arteries b) Fatty Streak Lesions c) Fibrous plaques and d) complicated lesions. In order to perform immunochemical study of the angiogenic factors specific antibodies were used. Finally we examine the ability of HRG-β1 to induce CYR61 mRNA expression by using the EAhy926 cell line. Cells were activatedwith HGR1-β1 and mRNA was isolated in various time periods (6h, 12h, 24h and 48h). CYR61 mRNA expression was measured. Statistical analysis yielded a significant rise of VEGF-A and bFGF mRNA expression levels by monocytes/macrophages of patients suffering of coronary artery disease, diabetes mellitus and stable angina compared with mRNA expression levels of normal donors (p=0,041 and p=0,022 respectively) but also compared with patients with coronary artery disease and unstable angina (p=0,024 and p=0,005 respectively). A time depended reduction of mRNA levels of those factors was also detected in monocytes/macrophages activated with fluvastatin and glucose. Expression of TGF-β1 was not observed. A time-depended rise of VEGF-A, TGF-β1, HRG1-β1 and CYR61 mRNA expression levels was also observed in the intima of all the types of atherosclerotic lesions. Finally after activation of endothelial cells with HRG1-β1 a rise of CYR61 mRNA expression levels was noticed. Our findings suggest that there is a strong correlation between coronary artery disease and all the studied inflammatory factors. They seem to implicate in the actions of monocytes of the peripheral blood, monocytes/macrophages and smooth muscle cells of atherosclerotic lesions. Our study also showed that fluvastatin (a widely used statin) downregulates expression of VEGF-A and bFGF. In addition our in vitro experimental procedure showed that high levels of blood glucose -such as those found in patients suffering from diabetes mellitus- dont seem to up-regulate the expression levels of those factors by monocytes/macrophages. All the studied growth factors are implicated in the establishment and progression of coronary artery disease by activating endothelial cells and inducing the production of several other cytokines and angiogenic factors. They also contribute in the process of migration of monocytes of peripheral blood and smooth muscle cells of the media, into the intima. Finally those factors could be possible implicated in the expansion of the atherosclerotic lesion by inducing the production and accumulation of extracellular matrix. Those findings revealed new data concerning the role of monocytes/macrophages in coronary artery disease and the molecular and biochemical mechanisms implicated in the process of atherosclerosis.
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Σύγκριση επιπέδων έκφρασης των αυξητικών παραγόντων TGF-β, bFGF, EGF, IGF-I και VEGF, στο αίμα ασθενών με στεφανιαία νόσο και φυσιολογικών ατόμων
