Retinol Binding Protein4Promotes Hyperinsulinism-induced Proliferation Of Vascular Smooth Muscle And Mechanism

Chapter1The plasma level of RBP4in coronary heart disease patients with hyperinsulinemiaObjective:Coronary heart disease is a group of clinical syndrome which induced by myocardial ischemia that because of coronary atherosclerotic plaque rupture associated with platelet aggregation, thrombosis, different degrees of stenosis or even complete occlusion of coronary artery. Insulin resistance is considered as an independent risk factor for atherosclerosis and cardiovascular and cerebrovascular diseases. Insulin levels are not only associated with the risk of coronary heart disease, but also the severity and prognosis of coronary heart disease. Retinol-binding protein4(RBP4) as new discoveried adipocytokines which is participated in insulin resistance, has been found a close association with obesity, insulin resistance, coronary heart disease and the metabolic syndrome. This chapter measured the change of plasma level of RBP4in patients with coronary heart disease and hyperinsulinemia plus, analyzed the correlation between RBP4and glucose, lipid metabolism, and insulin resistance, exlored the effect of hyperinsulinemia on it.Methods:This study was carried out at Xiangya Hospital of Central South University, China, from September2009to May2010. Thirty patients with coronary artery disease (the CAD group) were confirmed by coronary angiography,29patients with CAD plus hyperinsulinemia (the CAD+HIns group), and30healthy subjects were enrolled as controls (the control group). The peripheral blood sample from the anticubital vein was collected aseptically in all the subjects to measure the RBP4by enzyme linked immunosorbent-assay (ELISA). The height, weight, body mass index (BMI) the waist-to-hip ratio (WHR), the blood pressure, the fasting plasma glucose (FPG), the fasting insulin (Fins), the2-hour postprandial inslulin (2hPIns), and the homeostasis model assessment-insulin resistance index (HOMA-IR) was measured. The lipids, high sensitivity C reactive protein (hsCRP), uric acid(UA), free fatty acids (FFA) were all examined.Results:1. Comparison of clinical data among the groups:age, systolic blood pressure, diastolic blood pressure and waist/hip ratio was no significant difference among the three groups,(P>0.05). BMI of the CAD+HIns group is significantly higher than that in the CAD group and the control group (P<0.01).2. Comparison of biochemical criterion among the groups:FIns,2hPIns, HOMA-IR, UA in CAD+HIns group were significantly higher than those in other two groups (P<0.01)。TG, hs-CRP in CAD+HIns group and CAD group were significantly higher than that of the control group, HDL-C was significantly lower than the control group (P<0.01)。 The CAD group LDL-C was significantly higher than that in the control group (P<0.01). There is no statistically significant difference in the meas level of FPG, TC, FFA among the three groups.3、Comparison of RBP4among the three groups:The level of plasma RBP4in the CAD+HIns group was higher than that in the CAD group and the control group (both P<0.01), with no significant difference of plasma RBP4between the CAD group and the control group (P>0.05).4、Correlation analysis showed that the plasma RBP4level was significantly correlated with BMI, FPG, FIns,2hPIns, HOMA-IR, TG, HDL-C, UA, and hsCRP (r=0.259,0.331,0.582,0.452,0.600,0.236,-0.290,0.243,0.231, respectively, all P<0.05). Multiple regression analysis showed that BMI,2hPIns, and HOMA-IR were the independent factors related to RBP4(r2=0.181, P＜0.05; r2=0.273、r2=0.516, respectively, all P＜0.01)。Conclusion:The plasma level of RBP4is high in the CAD combined with hyperinsulinemia group. RBP4level is related to BMI, ipids, UA, and other cardiovascular risk factors. BMI,2hPIns, and HOMA-IR are the independent factors associated with RBP4. Chapter2Relationship between plasma RBP4and proliferation of vascular smooth muscle in Hyperinsulinemia ratsObjective:The first chapter has found RBP4level is related to the cardiovascular risk factors such as BMI, lipids and the inflammatory factor—hsCRP. Among the pathogenesis which insulin resistance induced atherosclerosis (AS) and coronary heart disease, the close relationship between RBP4and lipids and inflammatory cytokines may play a driving role in the occurrence and development of AS.Hyperinsulinemia can strengthen the effect that insulin stimulates proliferation of vascular smooth muscle cell, increases collagen synthesis, and promots the synthesis of growth factor synthesis, through the Shc/Raf/MAPK pathway. That manifested as effect which hyperinsulinemia induced atherosclerosis was strengthend. This chapter hopes to make sure. whether there is association between RBP4and vascular smooth muscle cells proliferation and the signal transduction pathway of it, through the hyperinsulinemia SD rat model. Then provide the new role of targets for the prevention and treatment of high insulin promote vascular smooth muscle cell proliferation.Methods:Ten-week-old male SD(Sprague Dawley) rats were selected for the experiment. They were randomly divided into two groups (n=7):the control group (Con) and Hyperinsulinemia group(HIns). The blood sample from the rats’tail vein was collected to measure the fasting plasma glucose using Roche glucometer. When the experiment was completed, the rats were anesthetized and blood was collected from the right ventricle for determination of relevant indicators. The total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), high sensitivity C reactive protein (hsCRP) were all examined. Fasting insulin (FIns) and RBP4was detected by enzyme linked immunosorbent-assay (ELISA). And the homeostasis model assessment-insulin resistance index (HOMA-IR) was measured. The thoracic aorta were quickly removed. Slices of these arteries were stained with Hematoxylineosin staining(HE). Media thickness(MT), lumen diameter(LD) were assessed with the compter-assisted image analysis system. To determine the expression of ERK1/2and Phosphorylation of ERK1/2(P-ERK1/2)in the thoracic aorta wall, the thoracic aorta were divided into two parts:one part was homogenized by Trizol reagent for RT-PCR analysis; The other part was homogenized in RIPA lysis buffer for immunoblotting analysis.Results1. The plasma glucose, insulin, HOMA-IR, and TG in HIns group were significantly higher than those in Control group(P<0.05). Model of hyperinsulinemia SD rats was successful.2. Triglyceride, hs-CRP in HIns rats were significantly higher than those in Con group, while high-density lipoprotein cholesterol (HDL-C) is lower,(P<0.05). There is no statistical difference of Cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) between the two groups.3. The level of plasma RBP4in the HIns group was higher than that in the control group (P<0.05).4. Proliferation of aortic smooth muscle occurd obviously in HIns group, media thickness (MT) and lumen diameter (LD) of thoracic aorta in HIns group were higher than control group, but without statistical significance (P>0.05)5. Both P-ERK1/2mRNA and protein expression in thoracic aorta of HIns group were much higher than Con group (P<0.05)6. Plasma RBP4had a significant positive correlation with MT and both P-ERK1/2mRNA and protein expression. After the correction of FIns value, plasma RBP4and P-ERK1/2protein expression continued to show a significant positive correlation.Conclusion:Plasma RBP4levels of Hyperinsulinemia SD rat were significantly increased, and circulating RBP4levels is positively correlated with proliferation of rat thoracic aorta smooth muscle, and also with phosphorylation level of EPK1/2which stands for smooth muscle proliferation signal transduction pathway. Chapter3RBP4promotes insulin-induced proliferation of vascular smooth muscle cells and its mechanismObjective:Hyperinsulinemia is an important risk factor for cardiovascular events.The first chapter has found that circulating RBP4levels in coronary heart disease combined with hyperinsulinism patients are significantly increased, and the RBP4level is related to the cardiovascular risk factors such as BMI, lipids and the inflammatory factor-hsCRP. BMI,2hPIns, HOMA-IR were independently associated with RBP4, which suggested that RBP4level may play a driving role in the occurrence and development of AS.The second chapter in the study of hyperinsulinemia model rats on blood specimens has once again proven that plasma RBP4was significantly positively related to HOMA-IR and inflammation-related factors hsCRP. Then have further study on rat aortic smooth muscle, for the first time found that the circulating RBP4level was positively correlated with the wall thickness of vascular smooth muscle as well as the phosphorylation of ERK1/2, which prompted that the RBP4is closely related to proliferation of vascular smooth muscle cell.Proliferation of vascular smooth muscle cells (VSMC) play a important role in coronary atherosclerosis and coronary heart disease. Proliferation and migration of VSMC included a variety of signal transduction pathways. This chapter makes high insulin-stimulated rat vascular smooth muscle cell (RVSMC) to observe proliferation of RVSMC, and for the first time uses RBP4as an intervention factors in the smooth muscle cells to clear the causal relationship between RBP4levels and the proliferation of VSMC, and to observe the influence on proliferation and proliferative signal transduction pathways of RVSMC, then to further clarify the influence and mechanism that RBP4on vascular smooth muscle cell proliferation.Methods:Cultured rat aorta vascular smooth muscle cells(RASMCs) were treated with insulin by different concentrations (0,10-9M,10-8M,10-7M,10-6M,10-5M,10-4M) for different time (3h,6h,12h,24h,48h). Cell proliferation was analyzed by using the MTT and cell cycle assays. The expression of ERK1/2, P-ERK1/2, JAK2, P-JAK2, STAT3, P-STAT3was determined by Werstern-Blot. To observe the effect of RBP4on cell proliferation and ERK1/2, P-ERK1/2, JAK2, P-JAK2, STAT3, P-STAT3expression in RASMCs,1μg/ml and4μg/ml RBP4was added to RASMCs1h before the treatment of insulin (10-5M) for24h, respectively. Cell proliferation was analyzed by using the MTT and cell cycle assays. The expression of ERK1/2, P-ERK1/2, JAK2, P-JAK2, STAT3, P-STAT3was determined by Werstern-Blot. The ERK1/2specific inhibitor PD980592and JAK-specific inhibitor AG490were added to RASMCs10min before the treatment of insulin (10-5M) plus RBP4(4μg/ml) for24h, respectively. Cell proliferation was analyzed by using the MTT and cell cycle assays.Results1. Insulin up-regulated proliferation of of RASCMs in a concentration-and time-dependent manner, an effect which was most significant after24h treatment with a concentration of10-5M.2. Insulin-induced proliferation of RASCMs could be increased by the pretreatment of RBP4for1h. 3. Insulin up-regulated ERK1/2, P-ERK1/2, JAK2, P-JAK2, STAT3, P-STAT3expression in a concentration-and time-dependent manner in RASCMs, an effect which was most significant after24h treatment with a concentration of10-5M.4. Insulin-induced P-ERK、P-JAK2expression of RASCMs could be increased by the pretreatment of RBP4for1h, while P-Stat3expression did not have obvious change.5. The above effects of RBP4could be inhibited by pretreament with ERK1/2specific inhibitor PD980592but not with JAK2specific inhibitor AG490.Conclusion:Insulin induced proliferation of rat vascular smooth muscle cells in a concentration-and time-dependent manner through the MAPK pathway and JAK2/STAT3pathway, RBP4can significantly enhanced hyperinsulinism-induced vascular smooth muscle cell proliferation through the promotion of the MAPK pathway.