| Part oneSimvastatin Inhibit Proliferation in Rat Vascular Smooth Muscle Cells in Association with Induction of PTEN and p27Objective Excessive VSMCs growth play a pivotal role in the pathophysiology of atherosclerosis and restenosis following percutaneus translumianl coronary angioplasty (PTCA). Proliferation of VSMCs is regulated by a complex interplay between cell surface signals and the expression products of intracellular genes such as the pro-oncogenes and tumor suppressor genes. Recently a new tumor suppressor gene PTEN has been found and is supposed to play an important role in the signal transduction of proliferation It has been demonstrated that PTEN inhibit cell proliferation associated with accumulation of cells in GO/GJ phase and increase in the protein level of the cyclin-CDK inhibitor p27. P27 played an important role in the regulation of VSMCs proliferation, but to the best of our knowledge no attentions have been paid to the role of PTEN in VSMCs.3-Hydroxy-3-methylglutaryl coenzyme A (HMG-COA) reductase inhibitors have been found to attenuate restenosis by mechanisms unrelated to lipid reduction. Recently some reports showed that HMG-COA reductase inhibitors could inhibit proliferation via cell cycle arrest in Go/Gi phase in a variety of cell types including VSMCs. The purpose of this study is to investigate whether Gj/S transfer-related genes such as p21, p27, c-myc and PTEN participate in simvastatin-induced growth arrest of VSMCs.MethodsDrugs and reagentsAnti-p27, p21 and c-myc monoclonal antibody were purchased from Santa Cruz, anti-PTEN rabbit polyclonal antibody was purchased from Neomarkers. Simvastatin (Merck Sharp&Dohme Research Laboratories.USA) was prepared as described by Llorente , mevalonate was from sigma, lipofectin regent DOSPER was from Roche company. Cell cultureVSMCs were routinely isolated from thoracic aorta of 200-250g Sprague-Dawley rats and cultured in Dulbecco's modified Eagle's medium( DMEM)(Gibco) supplemented with 10 % fetal bovine serum (FBS) , lOOU/ml penicillin and 100 u g/ml streptomycin .Cells were characterized as smooth muscle cells by their typical "hill-and-valley" morphology and by positive immunostaining for a -smooth muscle actin. Cells between passages 4 to 6 were used for all experiments. Measurement of DNA synthesisCell proliferation in terms of DNA synthesis was determined by measuring [ 3H ] thymidine incorporation. VSMCs were plated in 24-well plates at 1*10s cells per well and grown to 80 % confluence. After 24 hours of serum free, cells were cultured for 16 h with different concentrations of simvastatin (0 u mol/L 1 u mol/L, 5 u mol/L and 10 u mol/L) in 10 % FBS medium, followed by labeling with 1 u Ci/ml [3H] thymidine for additional 8 h. Cell preparation was performed by standard methods. The radioactivity of incorporated [ 3H ] thymidine was measured in a liquid scintillation counter. Experiments were performed 3 times in triplicate. Cell Cycle analysisCells were stimulated with different concentrations of simvastatin (Omol/L, Imol/L, Smol/L and lOmol/L) in 10 % FBS medium for 24 h, then trypsinized and harvested by centrifugation, washed with PBS and fixed in 70% ice-cold ethanol. 105cells were stained with propidium iodide (0.5 ml/L in PBS, containing with 100 V g/ml RNase A ) and subjected to flow cytometric analysis of DNA content using Coulter Berkman cytometer Immunoblot AnalysisAfter being stimulated for the indicated times, cell lysates were prepared using lysis buffer. Protein concentration was estimated by Lowry method. SDS sample buffer [Tris-HCl 0.33mol/L, SDS 10 % (w/v), glycerol 40 % (v/v), and dithiothreitol 20 % (v/v) containing bromophenol blue 0.4 % (w/v)] of 1/4 volume were added to cell lysates. After boiling for 3 mins, 30 U g of the sample was subjected to SDS-PAGE in a 10% SDS gel and transferred to PVDF membrane, which was followed by blocking for 1 h with 5 % non-fat milk in TEST. The blots were incubated for 3 h at 25 "C with the primary antibodies (at a 1 : 400 dilut...
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