High Glucose Induces Endothelial Progenitor Cells Apoptosis Via TNF Pathway Activated By Oxidative Stress

Objective:The exact mechanism contributing to diabetic trunk and capillaries complication is not determined. Increasing evidence suggests that the dysfounction of EPCs in both type 1 and type 2 diabetes which is likely to be involved in the pathogenesis of vascular complications. In the high sugar environment, the quantity and the function of endothelial progenitor cells (EPC) reduced which be regarded to maintaining the endothelial cell's integrity, promoting the neovasculorization. Meanwhile, high glucose (HG) determines the overproduction of reactive oxygen species (ROS) which can reduced the number of EPCs contribute to an impaired angiogenic by up-regulate the expression of apoptotic gene and actived death receptor. TNF-a is a cytokine produced mainly by activated macrophages, and is the major extrinsic mediator of binary hipaloptic apoptosis. It is able to induce apoptotic cell death and inflammation, and play an important role in signal transduction path of cell apoptosis. So we suppose that ROS can active TNFR1 to induced EPC apoptosis and reduce the function of EPC.further more, it also be responsible for the digression of angiogenesis. Therefore, the major aims of the present study were to evaluate the effect of hyperglycemia on the apoptosis of EPCs by observeting the number of EPCs and TNFR1 expression to determine whether HG induced EPC apoptotic through ROS activatly the TNF pathway. Methods:1.Isolation,cultivate and identification of the EPC in bone marrow, Observe the shape/growth/multiplication condition consequently by inverted phase contrast microscope. Separate the EPCs after 7 days, Identify EPC with CD34,CD133,VEGFR-2,ac-LDL and UEA-1 by immunohistochemistry and immunofluorescence.2. EPCs were treated with different concentrations (glucose concentration of medium terminal 15mmol/L,30mmol/L, 60mmol/L) of high glucose intervention, respectively At 24h,48h,96h for EPCs oxidative stress markers to assess oxidative stress level of EPCs in high glucose. MDA,GSH, antiO2- were observed by colorimetry method.3. EPCs were divided into 4 groups:(30 mmollL high glucose,30mmol/L mannitol,30mmol/L high glucose+4 mmol/L Tempol,5.5mmol/L glucose), At 24h,48h,96h,EPCs apoptosis detection by floweytomete.4. The mRNA expression of TNF-αby RT-PCR. Results:1. Cell morphology of rats EPCs,3 days colony formation, showing spindle cells and cell clusters, the line-like arrangement of spindle cells, and with incubation time increased, the cells become bigger, showing a typical cobblestone-like changes.Identify EPC with CD34,CD133,VEGFR-2,ac-LDL and UEA-1 by immunohistochemistry and immunofluorescence and shows positive staining.2. After high glucose intervention 24h, compared with the normal group, high glucose intervention group EPCs MDA,GSH concentrations had no significant difference, after high glucose intervention48h,96h, EPCs with high glucose intervention group, MDA GSHconcentration were significantly increased (P<0.05) compared with the normal group in concentration and time dependent manner. after high glucose intervention 48h,96h, EPCs with high glucose intervention group significantly reduced anti-O2--concentration (P<0.05) compared with the normal group in dose and time dependent manner.3. EPCs aoptosis detection by flow cytometer show that BM-derived EPCs significantly inereased apoptosis compared with control group and Tempol group after high glucose intervention 48h,96h (8.29±0.33)%, (13.08±0.68)% vs (2.54±0.47)%, (2.76±0.44)% vs (4.41±0.72)%, (5.12±0.53)%,P<0.05.4. The mRNA expression of TNFR-lsignifieantly inereased in high glucose intervention EPC (1.413±0.115,1.656±0.133 vs 0.982±0.099,0.975±0.113,P<0.05), antioxidant treatment can prevent this trend (1.072±0.126,1.035±0.114 vs 1.413±0.115,1.656±0.133 P<0.01).Conclusion:1. High glucose can induce oxidative stress increased and anti-oxidation decreased significantly in a concentration and time dependent manner in EPCs.2. EPCs significantly increase apoptosis after treated by high glucose, antioxidant treatment can prevent this trend.3. The mRNA expression of TNFR-1 significantly inereased after treated by high glucose in EPC, antioxidant treatment can prevent this trend.It may suggest that high glucose can modulate EPCs apoptosis via activating TNF pathway through oxidative stress.