The Secondary Effect Of Endothelial Cell Injury Induced By High Glucose On Mesenchymal Stem Cell Proliferation And Apoptosis

Background and purposeDiabetes is a metabolic disorder characterized by hyperglycemia.The persistent high glucose in these patients would lead to a series of macrovascular and microvascular diseases,such as diabetic cardiomyopathy,diabetic retinopathy,diabetic peripheral neuropathy,diabetic foot,etc.,which severely affect the quality of life of these patients,and contribute to the high morbidity and mortality related to diabetes.The main goal of current treatment for diabetes is to control blood sugar and improve insulin deficiency and insulin resistance,which,however,couldn’t effectively prevent occurrence of the complications of diabetes.Mesenchymal stem cell(MSC)transplantation is a recently developed treatment for diabetic complications.MSCs are pluripotent stem cells that exists in almost all body tissues and organs,which exhibit great potential to proliferate and differentiate into mesodermal cells.It has been proved by numerous studies that MSCs play an important role in immune response and tissue repair,and show satisfactory efficacy for a variety of diabetic complications in some of the patients,but not the others.Studies have revealed that,for some unknown reason,MSCs are very fragile when transplanted into the diabetes patients.In addition,compared to healthy people of the same age,number of endogenous MSCs in patients with advanced diabetes also significantly decreased.It has been established that high glucose induces oxidative stress and calcium overload in vascular endothelial cells,which promote apoptosis,inhibit proliferation and migration,and leads to endothelial dysfunction of these cells.And our previous studies have demonstrated that high glucose promoted,instead of inhibited MSC proliferation.So,it was suspected that high glucose in diabetes patients doesn’t directly damage endogenous or exogenous MSCs,instead,it damages endothelial cells first,which then release certain cytokines that damage MSCs.This study intends to investigate the indirect impact of high glucose on proliferation and apoptosis of MSCs via damage to endothelial cells,and the underlying molecular mechanism,to provide theoretical basis to guide efforts to improve efficacy of MSC transplantation for diabetes and diabetic complications,and to protect endogenous MSCs in diabetes patients.Methods: human umbilical vein endothelial cells were isolated by enzyme digestion or tissue block culture,whose function and surface marker were verified.A model of high glucose-induced endothelial damage was established,to evaluate the effect of high glucose on endothelial cell function,and the conditioned medium of the endothelial cells cultured in high-glucose environment were then collected to stimulated MSCs,followed by proliferation and apoptosis analyses of the MSCs,including expression of relevant proteins by Western blot and assessment of calcium concentration by calcium staining.Results:1.Human umbilical cord endothelial cells and mesenchymal stem cells were successfully isolated and cultured,and their surface markers and functional characteristics were in line with relevant standards.2.A cell model of high glucose-induced endothelial cell injury was successfully established,and it was observed that high glucose induced apoptosis of the endothelial cells and impaired their vascular formation capacity.Conditioned medium of the endothelial cells under different culture conditions were collected.3.Real-time cell analysis and flow cytometry results indicated that the conditioned medium of endothelial cells under high-glucose condition inhibited proliferation and induced apoptosis of the MSCs;Western blot results revealed changes in expression of apoptosis-related proteins(Bax,Bcl-2,Cl-Caspase-3);Calcium staining indicated calcium influx and overload that contributed to apoptosis of the MSCs.Conclusion:High glucose promotes apoptosis of endothelial cells and impair their vascular formation capacity.And the conditioned medium of endothelial cells under high glucose increased calcium influx,upregulated Bax and Cl-caspase-3 levels and downregulated Bcl-2 expression of the MSCs,leading to decreased proliferation and apoptosis.These results suggest that after high glucose-induced injury,endothelial cells may release certain factors that cause damage to endogenous or transplanted endothelial cells in diabetes patients.