Effect Of Homocysteine On The Production Of Matrix Metalloproteinase-2 In Rats Cluture Vascular Smooth Muscle Cells

1, BackgroundHomocysteine (Hcy) is a non-protein-forming, sulfur-containing amino acid that stands at the crossroads of two metabolic pathways . Elevated plasma levels of Hcy have been associated with vascular disease since the initial descriptions of classical homocysteinuria in children . Indeed, the last decade has witnessed an exponential increase in studies defining plasma homocysteine as an independent risk factor, similar to smoking or hyperlipidemia, for atherosclerotic cardiovascular, cerebrovascular and peripheral vascular diseases . Comparatively little is known about Hcy-modulated smooth muscle cell function. Woo DK et al. suggested that D,L-Hcy stimulation of bovine aortic smooth muscle cells proliferation involves mitogen-activated protein (MAP) kinase activation . Desai A et al. suggested that hyperhomocysteinemia is associated with a significant decrease of the smooth muscle cell/extracellular matrix ratio of the media of muscular femoral arteries without significant changes in medial thickness . The extracellular matrix metalloproteinases (MMPs) are a family of distinct proteases with differing specificities of cleavaging toward various extracellular matrix components . Only MMP-2 and MMP-9 are expressed as latent proenzymes by aortic smooth muscle cells and both are involved inarterial diseases, such as atherosclerosis and abdominal aortic aneurysms. It has been reported that the processes of migration and proliferation of vascular smooth muscle cells (VSMCs) that contribute to the morphogenesis of atherosclerotic plaques require the extracellular matrix remodeling caused by MMPs . Zempo et al. reported that the processes of migration and proliferation of VSMCs that contribute to the morphogenesis of atherosclerotic plaque require the extracellular matrix remodeling caused by matrix metalloproteinases(MMPs) . Bescond A et al. suggested that Hcy was proved to exert a dual effect, activating proMMP-2 at low molar ratio (MR 10:1) and inhibiting active MMP-2 at high molar ratio (MR> 1000:1). All these studies suggested that MMP-2 plays an important role in the formation and progression of atherosclerotic lesion.2, ObjectiveTo study the effect of different concentration homocysteine(Hcy) on the production of matrix metalloproteinase-2(MMP-2) in cultured rats vascular smooth muscle cells(VSMCs) and explore its regulatory atherosclerosis mechanism.3, Methods3.1 MaterialsThe chemicals used in this study were obtained from the following sources: 6-week-old male rats were purchased from Medical College of Zhejiang University, D,L-Homocysteine and Rabbit anti-MMP-2 antibody were purchased from Sigma ,USA. All other chemicals were obtained from Sir Run Run Shaw Hospital Central Labratory.3.2 Preparation of smooth muscle cellsRat aortic smooth muscle cells (SMCs) were isilated by enzymatic digestion from the thoracic aortic of 6-week-old male Sprague-Dawleyrats . The cells were cultured in DMEM supplemented with 10% fetal calf serum at 37℃ in a humidified 5% CO2 to 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 . All SMCs cultures used in this study were between passages 4 and 7. At the subconfluent stage, the culture medium was replaced with serum-free medium and then cells were exposed to various treatments. The experiments were performed an 80% confluent state on five passages from primary culture.Cells were culture with DMEM without 10% fetal calf serum in order to cells in one-phase for 24h.These cells could be used with different treatments.3.3 Preparation of sample culture mediumThe aboved cells were cultured in six-well palate with 1 * 10~5 per well with DMEM including 10% fetal calf serum. Hcy was added into every well and the final Hcy concentrations were0,50, 100,500, 1000, 5000, 10000μmol/L. They were incubated for 24,48,72h.Different medium samples of 24h,48h,72h were harvested, centrifug...