Effects Of Rosiglitazone On Proliferation And Apoptosis In Rat Thoracic Aorta Smooth Muscle Cells Induced By High Glucose

Objective: Vascular complications are the leading cause of death in diabetic patients, atherosclerosis is accelerated by the coexistence of diabetes mellitus. Poor control of diabetes is associated with the development of micro- and macroangiopathy, hyperglycemia in diabetic patients is believed to play a major role in the development of these vascular complications. Increased proliferation of vascular smooth muscle cells (VSMCs) is a key feature in the atherosclerotic lesion. It is well established that cell growth is a fundamental feature of intimal hyperplasia, and it is widely accepted that perturbations in the regulation of apoptosis are equally important. Excessive accumulation of VSMCs in atherosclerosis suggests reduced apoptosis and excessive cell proliferation in the lesions. Several findings support the concept that hyperglycemia accelerates the development of atherosclerosis. High concentration of glucose enhances growth rate in cultured VSMCs. Treatment with a high glucose not only significantly attenuate apoptosis in response to serum withdrawal in cultured rat VSMCs, but also markedly increased the expression of Bcl-2 and Bcl-xl. Another reports shows that there was a significant decrease in the DNA fragmentation ratio of human coronary artery smooth muscle cells cultured at high glucose concentration. Peroxisome proliferator-activated receptor (PPARγ) is a transcription factor belonging to the nuclear hormone receptor gene super-family. PPARγis expressed in VSMCs and prominently upregulated in response to mechanical injury of the arterial wall. Thiazolidinediones (TZDs) are synthetic ligands for PPARγand are commonly used as insulin-sensitizing agents in the treatment of type 2 diabetes. Data suggest that TZDs may also retard atherosclerotic disease progression. Treatment with rosiglitazone (RSG) decreased mean common carotid artery (CCA) intima-media thickness (IMT) progression, a surrogate index of atherosclerotic disease progression. Rosiglitazone attenuated total plaque area in diabetic mice and suppressd macrophage accumulation in diabetic aortic plaques. Although some reports indicated that RSG can inhibit cell proliferation and promote apoptosis in human VSMCs in vitro, little is known about the effect of rosiglitazone on proliferation and apoptosis and its related mechanisms of VSMCs treated with high glucose. To elucidate the precise mechanisms of RSG on AS, the effect of RSG on VSMCs proliferation, apoptosis and expression of Bcl-2, Bcl-xl in rat thoractic aorta smooth muscle cells were examined.Methods: 1 The primary and transfer culture were done by modified tissue-piece inoculation and trypsin digestion respectively. The cells were purified by natural passage transfer. The cultured cells were identified by morphological characteristics and immunocytochemistry with antibody toα-actin.2 The fifth to sixth passage purified cells were harvested for the following experiment. The effect of RSG on the cell proliferating viability in high concentration glucose was observed by MTT assay.3 The fifth to sixth passage purified cells were harvested for the following experiment. Flow cytometry was preformed to analyze the influences of RSG on cell cycle distribution, apoptosis rate and Bcl-2, Bcl-xl expression in high concentration glucose.4 The fifth to sixth passage purified cells were harvested for the following experiment. The effect of RSG on Bcl-2, Bcl-xl expression in VSMCs incubated in high concentration glucose was detected by Western blot.Results: 1 Approximately 80% inoculated tissue pieces survived in primary culture. The cultured cells possessed"peak and valley"characteristics for VSMCs Immunocytochemical staining with specific antibody against SM-α-actin demonstrated these cells were positive.2 MTT assays were used to characterize the viability of VSMCs. The proliferating viability of VSMCs induced by high concentration glucose was remarkably increased when compared with the normal glucose group (P<0.05) , and treatment of the cells with mannitol, used as an osmotic control, had no significant effect on apoptosis in rat VSMCs; RSG(30,100μmol/L)can inhibited the proliferation of VSMCs by high glucose treatment (P<0.05), and displayed in concention-dependent; the effects of RSG was attenuated partly after pretreated with GW9662 (10μmol/L).3 To detect the cell cycle progression of VSMCs, flow cytometric analysis was performed. High glucose can induce cell cycle progression from G0/G1 phase to S phase, and the cell cycle progression was not altered by mannitol treatment compared with the control; RSG (30～100μmol/L) pretreated for 48h can inhibit VSMCs cell cycle progression from G0/G1 phase to S phase, the cell number significantly decreased in G0/G1 phase and increased in S phase(P<0.05); GW9662 can suppress the function of RSG.4 We used flow cytometric analysis to determine the apoptosis rate and Bcl-2, Bcl-xl expression in VSMCs. Treatment with a high glucose significantly attenuated apoptosis rate and increased expression of Bcl-2 and Bcl-xl protein in cultured rat VSMCs when compared with the normal glucose group(P<0.05), but the mannitol have no effect on apoptosis rate and expression of Bcl-2 and Bcl-xl protein; Treatment with RSG(30,100μmol/L) can promoted apoptosis of VSMCs and reduced expression of Bcl-2 and Bcl-xl protein in high glucose group in a dose-dependent manner; the effect of proapoptosis can be suppressed partly by GW9662. 5 Western blot demonstrated that high glucose upregulated Bcl-xL protein and depended in glucose concentration, and expression of Bcl-xl protein had no change when treated with mannitol; the expression of Bcl-xl protein induced by high glucose was decreased by RSG (30,100μmol/L) and increased when pretreated with GW9662. However, Bcl-2 was not detected in VSMCs.Conclusions: 1 High glucose can induce the proliferation and inhibit apoptosis by increasing the expression of Bcl-xl and Bcl-2 protein in rat VSMCs; treatment with mannitol had no effect on VSMCs, indicating that high glucose condition may represent one of the mechanisms for atherosclerosis observed in T2DM.2 RSG (30～100μmol/L) can inhibit VSMCs cell cycle progression from G0/G1 phase to S phase, decrease expression of Bcl-2, Bcl-xl protein and promote VSMCs to apoptosis induced by high glucose in cultured VSMCs. These finding suggested that RSG may play a protective role against diabetic angiopathy through the intervention of proliferation and apoptosis in VSMCs.