The Mechanism Of Oxidized Low Density Lipoprotein And Insulin-like Growth Factor-1 On Rabbit Vascular Smooth Muscle Cell Biological Function And The Intervention Effect Of Atorvastatin And Rapamycin

Objective: To investigate the cellular signal transudation pathway of vascular smooth muscle cell (VSMC) proliferation and cytokins secretion stimulated by oxidized low density lipoprotein(oxLDL) and insulin-like growth factor-1(IGF-1) and the intervention effect of HMG-coA reductase inhibitor, atovastatin and immune supppressor .rapamycin.Methods: Rabbit aortic VSMC was cultured in 8 groups:control; native low density lipoprotein (LDL); mitogensCoxLDL or IGF-1); drugs(atorvastatin or rapamycin); PI3K(phosphatidylinositol 3-kinase) specific inhibitor , wortmannin(WT); drugs plus mitogens; WT plus mitogens; atorvastatin plus mevalonate(MVA). Cell proliferating ability was determined by measuring cell number and mitochondrial dehydrogenase (MD)activity(MTT assay).The level of tumor necrosis factore alpha(TNF a ), interleukin-6(IL-6) and interleukin~8(IL-8) were evaluated using ELISA method. Western blotting was used to detect the protein expression of protein kinase B(PKB) and lipid phosphatase PTEN . Immunoprecipitations and radioactivity of y 32P incorporated into its specific substrate histon HzB was utilized to determine the PKB activation. Phosphate concentration released from PTEN-specific substrat diC16PIP3 was assessed using green reagent method.Results: oxLDL(50 u g/ml)and IGF-1(lOOng/ml) increased cell number and MD activity to 1. 78~3. 8 fold, but the same concentration of LDL has no such mitogenic effect. WT itself decreased the above parameters to69%, 61.25%, 71.4% and 68.7% of control group (p<0.001) and completely reversed the proliferation-promoting effect of both oxLDL and IGF-1. oxLDL elevated PKB activity in a concentration-(5~50 P g/ml) and time-(3min. ~ 24h)dependent manner. 5 P g/ml of oxLDL caused a 1.95-fold increase in PKB activity,whereas the maximum dose of oxLDL(50 n g/ml) induced a 15.37-fold increase within 10 minutes. The time course was characterized by two peaks, the first appeared within 10 minutes, the second at 8 hours with a 10.1-fold increase and still as high as 6.92-fold in 24 hours. The concentration effect of IGF-1 on PKB activity emerged at 1.0 ng/ml and reached to the maximum of 57.25-fold at 100 ng/ml in 10 minutes, followed by continual decrease to 5.47-fold in 24 hours (p<0.001). The effect of WT on PKB activity was also time dependent, reaching to 76% after 20 minutes preincubation and keeping decreasing to 38% in 24 hours. Phosphatase activity detection show that the dephosphate action of PTEN was inhibited by oxLDL(20~50 u g/ml) and IGF-1(10 ~ lOOng/ml) in a concentration dependent manner, and this inhibiting effect was at its maximum within 10 minutes, lasting at least for 24 hours(p<0. 001). WT abolished the mitogenic effects of oxLDL and IGF~1 on PKB activity. Cytokins evaluation demenstrated that the promoting effect of oxLDL and IGF-1 on the secretion of TNF a ,IL-6 and IL-8 was rapid,causing a 1. 2~1.4-fold increase within Ih , reached to peak of 6. 4~ 8. 5-fold and sustained as high as 1.26~5.05-fold in 24 hours. WT decreased these cytokin's level to that of control group and abolish the above secretion promoting effects of the two mitogens. Drug intervention experiment show that both atorvastatin and rapamycin can markedly inhibit VSMC proliferation and completely abolish the mitogenic effect of oxLDL and IGF-1. In a concentration range of 0. 05~luM and 10~ lOOng/ml separetly, atorvastatin and rapamycin can decrease the PKB activity in a concentration dependent manner, with a more than 70% decrease at the highest concentration. Coincubation with cholesterol precurssor MVA completely reversed all the above effects of atorvastatin. Conclusions: PI3K/PTEN/PKB signal transduction system is one of the crucial signal pathway involved in rabbit VSMC proliferation andsecretion of TNF a , IL~6 and IL-8 stimulated with or without oxLDL and IGF-l.oxLDL and IGF-1 can also inhibit the phosphatase activity of PTEN which dephosphate PIPS and thus regulate the PKB activity adversely. Both atorvastatin and rapamycin can inhibit cell proliferation, cytok...