Effects And Mechanisms Of Gliclazide On Endothelial Progenitor Cells Damage Induced By Hyperglycemia

Objective Endothelial dysfunction is thought to be a critical event in the pathogenesis of vasculopathy in type 2 diabetes and oxidant stress is a major etiological factor. Gliclazide, a second generation sulfonylurea, contains an azabicyclo-octyl ring, which has been described to have antioxidant properties. However, the effect of gliclazide on endothelial function is unknown. Therefore, in this study, we examined the effect of gliclazide on endothelial function in patients with newly diagnosed type 2 diabetes.Methods Between August 2007 and December 2008,33 Chinese patients with newly diagnosed type 2 diabetes who were outpatients of the Endocrinology Department at Wuhan Union Hospital, participated in this study. A further 25 control subjects (control group) who took part in health examination at the hospital were randomly selected. Diabetic patients (diabetic group) were treated with gliclazide,30-90 mg/day, for 12 weeks. At the first and the last visit, endothelial function, circulating endothelial progenitor cells (EPC) count, laboratory parameters and oxidant stress markers were determined in all participants. Assessments of flow-mediated vasodilation (FMD) and nitroglyceride-mediated vasodilation (NMD) were obtained using a high-resolution ultrasound machine. Serum malondialdehyde (MDA) level was measured by thiobarbituric acid adduct. The total superoxide dismutase (SOD) activity was assayed by xanthine oxidase. Serum nitric oxide (NO) concentration was measured using nitratase method.Results Gliclazide treatment could effectively reduce the blood glucose level and improve insulin resistance (P<0.05). FMD, circulating EPC count and superoxide dismutase activity were significantly lower in the diabetic group than in the control group at baseline (P<0.05), and improved significantly following gliclazide treatment (P<0.05).Furthermore, the percentage increase in FMD and FMD/NMD was positively correlated with the percentage increase of EPC count in the diabetic group (0.496,0.477,P<0.05). Malondialdehyde and nitric oxide levels were higher in the diabetic group than in the control group at baseline (P<0.05), and decreased following gliclazide treatment.Conclusion Compared with controls, FMD and circulating EPC count in diabetic patients were significantly lower, and the oxidative stress status in diabetic patients was severer. But treatment with gliclazide for 12 weeks significantly reversed endothelial dysfunction and improvements in oxidative stress status. These results suggest that gliclazide could improve endothelial function in diabetes, which may be related to its antioxidant properties. Objective Bone marrow, as the main pool for endothelial progenitor cells(EPC), the quantity of EPC in bone marrow indicates the ability to reserve the EPC. The effect of gliclazide on circulating EPC in diabetic patients has been studied, but its effect on EPC in bone marrow has been unknown. Furthermore, not only the quantity but the quality of EPC (the function of EPC) is crucial for re-endothelialization and neoangiogenesis. One of the vital reasons of EPC dysfunction is oxidative stress, and also the antioxidation of gliclazide in endothelial cells has been confirmed. Thus, our study mainly focused on the effect of gliclazide on the number and function of bone marrow-derived EPC in diabetic rats.Methods Forty Wistar rats were randomly equally divided into two groups:control group (CT group) and diabetes mellitus group (DM group). Diabetic rats were induced by intraperitoneal injection of streptozocin (STZ,60mg/kg) and confirmed by a blood glucose >300 mg/dl. Two weeks after STZ injection, all rats received vehicle or gliclazide (20mg/kg/day) by oral gavage until 6 weeks later. Thus rats were ultimately divided into four groups:control (CT), control treated with gliclazide (CT+Gliclazide), diabetes (DM) and diabetes treated with gliclazide (DM+Gliclazide). Bone marrow mononuclear cells were isolated by density gradient centrifugation and cultured in plates. After 7 days in culture, EPC were indentified and counted as double positive cells for Dil-ac-LDL and FITC-UEA-1. The proliferation and migration function were evaluated by MTT assay and transwell migration assay. Colony forming units were counted in day 11. Levels of serum malondialdehyde (MDA) in all rats were detected by thiobarbituric acid adduct.Results Gliclazide had no effect on blood glucose in diabetic rats or controls. Double positive cells for ac-LDL and UEA-1 and colony forming units were decreased in DM group (P<0.01), and gliclazide treatment could significant increase their quantities although they were stills lower than controls (P<0.05). The similar results could be found in EPC function tests. Compared to controls, proliferation and migration functions of bone marrow-derived EPC in diabetic group were impaired (P<0.05), and gliclazide treatment restored partly their impaired functions. But no such effect could be found by gliclazide in controls. As for levels of serum MDA, it is much higher in diabetic group than in controls (P<0.05), but it declined markedly after gliclazide treatment. Further study implied that MDA level was negatively correlated with the quantity and functions of bone marrow-derived EPC.Conclusion Gliclazide could augment the quantity and improve the impaired functions of bone marrow-derived EPC in diabetic rats, and this is not related to its hpyerglycemic effect and probably related to its antioxidation. This provides another way for gliclazide to protect vascular. Objective Endothelial progenitor cells (EPC) have been consider to involving in healing of endothelial damage and neovasculogenesis. The ablility of healing of damaged endothelium is impaired in diabetes, and diabetes is associated with reduction of EPC. But the mechanisms have been unknown, increased apoptosis and decreased survival of EPC may account for EPC reduction. Gliclazide has been shown to protect apoptosis in endothelial cells and islet beta cells, thus, we investigated the effect of gliclazide on high glucose-induced apoptosis of EPC.Methods Bone marrow-derived EPC were cultured in different conditions, including 5.5 Mm D-glucose (NG),25 mM D-glucose (HG), mannitol control (MAN) and 25 mM D-glucose with 10μmol/1 gliclazide (HG+Gli). The percentage of apoptotic cells was quantified using the Annexin-V/PI kit. Intracellular ROS generation was evaluated using DCFH-DA and analyzed by flow cytometry. The activities of MnSOD, catalase and NADPH oxidase were assessed using corresponding kits. Protein levels of t-Akt, p-Akt, t-FOXO3a and p-FOXO3a were measured by Western blot analysis. Real-time PCR was performed to quantify the mRNA expressions of MnSOD, catalase, Bim and FasL.Results The percentage of apoptotic EPC and ROS generation were significantly increased in HG group than in controls (P<0.05), and gliclazide reduced the apoptosis and ROS generation of EPC induced by high glucose. The activities and mRNA expressions of MnSOD and catalase were also restored by gliclazide. In addition, gliclazide reduced the activation of NADPH oxidase induced by high glucose. The ratios of p-Akt/t-Akt and p-FOXO3a/t-FOXO3a were decreased in HG group, gliclazide augment the ratios in HG group by way of a PI3K-dependent mechanism. Furthermore, gliclazide inhibited mRNA expressions of proapoptotic gene Bim and FasL.Conclusion Gliclazide improved oxidative stress induced by high glucose through increment in ROS clearance and reduction of ROS generation. It also inhibited the apoptosis of EPC by the PI3K/Akt pathway. FOXO3a may involve in the antiapoptotic effect of gliclazide.