Effect Of Fibrinogen On The Proliferation And Migration Of Human Vascular Smooth Muscle Cells And Intervention Effect Of Statins

Background and Objective A major step in the pathogenesis of atherosclerosis is the vectorial migration of vascular smooth muscle cells (VSMC) from the arterial media through the internal elastic lamina into the intima and their subsequent proliferation in the intima. The coagulation system, including fibrinogen and fibrin may be involved in the initiation and progression of atherosclerosis. The factors that stimulate restenosis following percutaneous coronary intervention are not clearly understood, and so far the only intervention regimes that have proved sufficiently successful in patients (and increasingly adopted) are drugs and antiplatelet antibodies that inhibit thrombus formation. Several epidemiological studies have produced longitudinal data identifying fibrinogen as a major cardiovascular risk factor. Fibrinogen strongly affects blood coagulation, blood theology and platelet aggregation; in addition fibrinogen and its metabolites have direct effects on the vascular wall. The discovery of 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor or statins has led to significant improvements in the management of hypercholesteremia. However recently a significant number of investigations have given evidence that beyond their lipid lowering capacitystatins exert various 'vasoprotective' effects. PAI-1 is the major physiological inhibitor of tissue type plasminogen activator (t-PA) and urokinase type plasminogen activator (u-PA) and therefore plays a key role in the regulation of fibrinolysis. High PAI-1 plasma levels and decreased levels of t-PA activity have been shown to be associated with coronary heart disease and PAI-1 mRNA has been found in human atherosclerotic lesions underlining its role in the development of these disorders. Several in vivo studies have also investigated the effect of statin therapy on this key regulator of the fibrinolytic system. The results, however, are inconclusive, hi this study we have investigated the effect of fibrinogen (Fg), fibrin (Fb) and fibrin degradation products (FDPs) on the proliferation and migration of human VSMC, meanwhile, compared the intervention effect of three different statins on the human VSMC and the fibrinolytic system of them.Methods (1) HITB5 VSMC is a human clone derived from adult internal thoracic artery and can stably passage and maintain its physiological characters.VSMC identity was confirmed morphologically and by positive immunostaining for SM a-actin, myosin and calponin. Cells for the present study were used with 9-16 passages. (2) The preparation of Fb monomer, crosslinked Fb and FDPs was performed using thrombin, factor XIII and plasmin refering Naito's reports.(3) The effects of Fg, Fb, FDPs and statins on the proliferation of VSMC were observed by means of cell counting and MTT test. (4) The migration assays were performed using the Sarkar's technique and the Transwell cell culture appatatus. (5) Total cellular RNA was prepared by the Trizol extraction from confluent cells. The t-PA, u-PA and PAI-1 mRNA levels expressed by VSMC were determined by RT-PCR method.Results (1) Fg itself did not stimulate the proliferation of VSMC, but stimulated VSMC migration in a dose-dependent manner. (2) Fibrin monomer can stimulate the proliferation of VSMC; both crosslinked Fb and noncrosslinked Fb simulated the migration of VSMC, however, at near all the concentrations approximately twice as many cells migrated into crosslinked fibrin. (3) FDPs stimulated both the proliferation and migration of VSMC , but the effect were diphasic characters, i.e. the effect was significant on specific concentrations. (4) Migration of VSMC to Fb monomer was partially inhibited by GRGDSP, but not by GRGESP. (5) Simvastatin and fluvastatin except pravastatin significantly inhibited the proliferation and migration of VSMC induced by 10% FBS. The inhibitory effects of two statins were dose-dependently. (6) The inhibitory effects of simvastatin and fluvastatin on migration of VSMC were reversed nearly completely by addition of mevalonate. Control culture...