Role Of Epigenetic Modification On The Insulin Mediated Proliferation Of Vascular Smooth Muscle Cells From Spontaneously Hypertensive Rats

1. BackgroundPrevious studies show that the risk of cardiovascular complications in hypertensive patients with diabetes and hyperinsulinemia is much high. Animal experiments and basic researches have documented that 1) atherosclerosis could be induced by insulin in laboratory animals; 2) the proliferation and migration of vascular smooth muscle cells (VSMC) are increased by insulin at super-physiological concentrations; 3) long-term application of insulin leads to abnormal lipid metabolism and thickening of artery vessel wall; 4) infusion of insulin accelerates the progress of atherosclerosis in the infused artery. All the above-mentioned studies indicate that insulin resistance and compensatory hyperinsulinemia are the major risk factors for coronary heart disease. Moreover, prevention and treatment of coronary heart disease and vascular proliferative disease also show the importance of hyperinsulinemia in the process of vascular proliferative lesions. Improvement of the insulin sensitivity would be benefit to protect from coronary artery disease and other vascular diseases.2. ObjectivesThis study is to identify the effects of epigenetic modification on proliferation and phenotype conversion of vascular smooth muscle cells, to investigate the role of cell proliferation mediated by insulin-induced epigenetic modification, the internal relations and the mechanisms of different expressions between epigenetic modification and cell proliferation related genes, and to reveal the mechanisms of proliferation of vascular smooth muscle cells, to explore new theoretical and experimental basis for prevention and control of vascular proliferative diseases. 3. Study contents1) The role of MAPK in the proliferation of VSMCs induced by insulin.2) Effect of histone super-acetylation on proliferation of VSMCs after blockade of HDAC with sodium butyrate.3) Effect of insulin on histone acetylation and investigation of histone acetylation after blockade of MAPK with PD98059.4) Effect of insulin on HDAC1 with or without blockade of MAPK with PD98059.5) Effect of histone super-acetylation-induced by sodium butyrate on MAPK pathway.6) Effect of insulin on genetic transcription and protein expression of proliferation related gene PDGF before and after treatment of PD98059 and sodium butyrate.7) Effect of insulin on expressions ofα-SM actin and OPN before and after treatment of PD98059 and sodium butyrate.8) Effect of insulin, PD98059 and sodium butyrate on promoter methylation of PDGF andα-SM actin DNA.4. Methods and materials1) Primary culture of VSMCs from SHRs.2) Proliferation of VSMCs determined by H3-TdR, MTT, cytometry and flow cytometry.3) Establishment of concentration curve of insulin-mediated proliferation of VSMCs.4) To observe the effects of insulin and PD98059 on VSMCs proliferation and MAPK expression determined by immunoblotting and RT-PCR.5) Effect of sodium butyrate on proliferation of VSMCs induced by insulin.6) Effect of insulin, PD98059 and sodium butyrate on HDAC1 and histone acetylization of VSMCs determined by immunoblotting.7) Effect of insulin, PD98059 and sodium butyrate on MAPK protein and mRNA expressions determined by immunoblotting and RT-PCR.8) Effect of insulin, PD98059 and sodium butyrate on PDGF andα-SM actin protein and mRNA expressions determined by immunoblotting and RT-PCR.9) Effect of insulin, PD98059 and sodium butyrate on promoter methylation of PDGF andα-SM actin DNA determined by specific DNA methylation PCR. 5. Results1) Insulin increased MAPK expression and proliferation of VSMCs, which was blocked by PD98059.2) Sodium butyrate inhibited the insulin-mediated proliferation of VSMCs.3) Insulin increased, while sodium butyrate decreased HDAC1 expression, PD98059 had no effect on HDAC1 expression.4) Insulin increased, while PD98059 decreased the acetylation of histone H3 determined by immunoblotting.5) Insulin up-regulated the expression of PDGF, down-regulatedα-SM actin expression, which was blocked by PD98059 and sodium butyrate.6) Insulin increased the genetic transcription of MAPK and PDGF, decreased the gene transcription ofα-SM actin, which was blocked by PD98059 and sodium butyrate.7) PDGF DNA promoter was non-methylation in physical condition in VSMCs from SHRs; sodium butyrate, instead of PD98059 and insulin, increased methylation of PDGF DNA.8)α-SM actin DNA promoter was non-methylation status in physical condition in VSMCs from SHRs; sodium butyrate, PD98059 and insulin had no effect on this non-methoylation status.6. Conclusions1) The MAPK pathway is involved in the insulin-mediated proliferation of VSMCs from SHRs2) Histone acetylation is engaged into the insulin-mediated proliferation of VSMCs from SHRs, MAPK pathway is involved into this action.3) Proliferation of VSMCs from SHRs induced by insulin is not related toα-SM actin and PDGF DNA promoter methylation but effect of sodium butyrate on proliferation of VSMCs from SHRs might be related to PDGF DNA promoter methylation.