Effects Of Multiple Factors On Expression Of Osteopontin And Matrix Metalloproteinase-2 Of Rat Vascular Smooth Muscle Cells

Background: In view of the pathological foundation of atherosclerosis and restenosis(RS) after percutaneous coronary intervention (PCI) is characterized by vascular smooth muscle cell (VSMC) adhesion, proliferation,migration to endometrium and secrete extracellular matrix(ECM) which caused by endothelium injuryed.The mechanism of it is complicated which numerous growth factors and cytokines involved. Up to now, very little is known. VSMC phenotype dictates the growth response and proliferation capability of it at the quiescent and proliferating state.During atherogenesis VSMCs are converted from a contractile into a synthetic phenotype characterized by enhanced matrix production. Dedifferentiation to a synthetic/proliferative phenotype has been hypothesized as a critical step in atherogenesis. Osteopontin (OPN), an arginine-glycine-aspartate (R.GD) containing extracellular matrix protein and a mark gene which make VSMC convert from a contractile into a synthetic phenotype, is associated with VSMC activation in vitro and in vivo and associated with neointima formation ,vascular calcification which plays a important role in the development of atherosclerosis, vascular remodeling, VSMC adhesion, spreading, invasion and RS after PCI in vivo. Matrix metalloproteinases (MMP), especially gelatinases (MMP-2) regulate the composition and stability of the ECM, which affects VSMC proliferation, migration, anddifferentiation. It also mediates neovascularization, inhibits apoptosis, and plays important roles in extracellular matrix remodeling and angiogenesis. Many cytokines, hormones ,extracellular nucleotides and growth factors involved in atherosclerosis and RS after PCI induce OPN and MMP-2 overexpression.Objective: To explore the pathogenesis of atherosclerosis and RS after PCI further, We are interested in understanding mechanisms regulating the OPN and MMP-2 steady state level in VSMC.This study the effect of multiple factors including endothelin-1 (ET-1), S-nitroso-N-acetyl-d,l-penicillamine (SNAP), hydrogen peroxide (H₂O₂). low frequency electromagnetic field (LFEMF)on OPN mRNA expression,protein synthesis and activity of MMP-2 were investigated in cultured rat VSMC in vitro which are not reported. Methods:(1) Rat VSMC were cultured in vitro with DMEM and 10 percent calf plasma. The VSMC were incubated with different factors respectively.(2) The numbers of VSMC proliferation was monitored by 3H-TdR incorporation and the changes in cell cycle progression of synchronized VSMC were examined by flow cytometric(FCM) analysis. (3) The expression of OPN mRNA were assessed by reverse transcription-polymerase chain reaction (RT-PCR). (4) The expression of OPN protein were assessed by Western blot analysis. (5) The expression of MMP-2 was monitored by enzyme image analysis way , SDS-PAGE contained gelatin was used to observe activity of MMP-2 .Results: (1) The VSMC proliferation is significantly promoted by ET-1 groups with different concentration and different time(P<0.05), H₂O₂ groups with different concentration in a dose-and time-dependent manner and is significantly inhibited by LFEMF with the different intensity exhibiting dose-dependent effect.Cpm numbers of VSMC proliferation of all factor groups are significantly different with respective controls (P<0.05). The cell cycle tested through FCM method show the same results.(2) RT-PCR and Western blot analysis demonstrated that different concentration and different...