Diazoxide Improves Impaired Endothelial Progenitor Cells Function In Streptozotocin-induced Diabetic Mice

Background and purpose:Diabetes mellitus (DM), or simply diabetes, is a group of metabolic diseases in whicha person has high blood sugar, either because the pancreas does not produce enoughinsulin, or because cells do not respond to the insulin that is produced. Vascular disease isone of the principal complications and the main cause of death of diabetes. Endothelialdysfunction is reported to initiate vascular disease, and is the inner cause of impairedwould healing ability and impaired artery relaxation. The activity of endothelial cells wasthe base of endothelial function, and abnormal apoptosis of endothelial cells is mortal forvessels. Endothelial progenitor cells (EPCs) are a population of rare cells that circulate inthe blood with the ability to differentiate into endothelial cells, the cells that make up thelining of blood vessels. The process by which blood vessels are born de novo fromendothelial progenitor cells is known as vasculogenesis. Thrombospondin-1is a subunitof a disulfide-linked homotrimeric protein. This protein is an adhesive glycoprotein thatmediates cell-to-cell and cell-to-matrix interactions. The thrombospondin-1protein is amember of the thrombospondin family, and encoded by THBS1gene. It is amulti-domain matrix glycoprotein that has been shown to be a natural inhibitor ofneovascularization and tumorigenesis in healthy tissue. Both positive and negativemodulation of endothelial cell adhesion, motility, and growth has been attributed toTSP-1. In diabetes mellitus and other cardiovascular diseases, transcription of TSP-1mRNA was augmented in EPCs. And TSP-1could be activated by the tumour suppressorgene p53. It is reported that p53inhibits vasculogenesis of fibroblast via activating TSP-1.Diazoxide is a potassium channel activator, which causes local relaxation in smoothmuscle by increasing membrane permeability to potassium ions. This switches offvoltage-gated calcium ion channels which inhibits the generation of an action potential.Diazoxide also inhibits the secretion of insulin from the pancreas through its action onpotassium channels. The efflux of potassium, as an effect of diazoxide, will lead tohyperpolarization of cell membrane that leads to decrease calcium influx. Therefore, theaim of this study is to investigate whether diazoxide could ameliorate diabetes inducedendothelial dysfunction and the involved mechanism in a EPCs-dependent manner. Methods and results:Streptozotocin-induced diabetic C57/BL6mouse model was adopted to investigate theeffect of diazoxide in the endothelial function specializing in EPCs function. For the cellmodel, high glucose-damaged HUVECs and BM-EPCs were used to investigate theeffect of diazoxide on the endothelial function. TSP-1and p53expression was observedfor the molecular target in the underlying mechanism. EPCs function was evaluated bythe adhesion, migration and tube formation function of the cells.1. Diazoxide significantly accelerated wound healing and improved in-vitroendothelial-dependent artery relaxation in STZ-induced diabetic miceMale C57/BL6mice were separated into three random groups: Control, STZ(streptozotocin)-induced diabetic mouse group and diazoxide-pretreated diabeticmousegroup. Dosage of diaoxide was determined according to clinical use and references,which was30mg/kg/d in gavage for4weeks. After anesthetized, a6-mm circular skinwas cut from the back of mice for the establishment of wound-healing mouse model.Wound closure was observed and taken photos for12consecutive days, andquantification of wound closure was conducted as previously described. Angiogenesiswas evaluated by immunohistochemical staining of CD31around the wounded skin area.It was shown that diazoxide pretreatment could accerlerate In STZ-induced diabetic miceimpaired wound healing and angiogenesis around the wounded area.After the adjacent fat and connective tissue were removed, the aorta was cut into ringsof2-3mm length. The rings were suspended in10mL organ bath chamber containingKrebs solution to determine the artery relaxation of each group. It was shown thatdiazoxide improved artery relaxation impaired by diabetes in mice.2. Diazoxide significantly improved BM-EPCs function in diabetes miceTo identify BM-EPCs, Dil-acLDL and FITC-UEA-1were used to specifically bind tosurface antigens of the cells. In addition, Hoechst33258was used to stain the nuclei.Flow cytometry was adopted to count peripheral EPCs that were identified as CD34,Flk-1double positive cells. Modified Boyden chamber method was adopted to evaluatethe migration ablility of BM-EPCs. Matrigel-based tube formation assay was adopted toevaluate angiogenesis of BM-EPCs in vitro. Statistics has shown that diazoxidesignificantly restored the function of BM-EPCs. 3. Diazoxide significantly inhibited STZ-induced diabetic mice p53and TSP-1expression in BM-EPCsExpression of p53and TSP-1in BM-EPCs isolated from mice was determined usingimmunobloting. It was shown that p53was activated in the diabetic mice, which wasreversed by diazoxide; TSP-1expression was augmented in the diabetic mice, which wasalso reduced by diazoxide.4. Diazoxide significantly improved BM-EPCs function and inhibited the expression ofp53and TSP-1by high glucoseAfter7days’ culture, isolated BM-EPCs from normal C57/BL6mice were separatedinto3random groups: Control, high glucose (33mM) and high glucose+diaoxide(30μM). Diaoxide was given for24hours. Statistics has shown that high glucoseimpaired adhesion, migration and tube formation function of BM-EPCs, and inducedhigh level of TSP-1and p53in the culture medium of BM-EPCs. Diazoxide treatmentreversed these effects, suggesting that diazoxide could significantly ameliorate impairedBM-EPCs function and was possibly related to the signaling pathway of p53and TSP-1.5. Diazoxide significantly improved impaired HUVECs function by high glucoseCultured HUVECs were separated into3random groups: Control, high glucose (33mM) and high glucose+diaoxide (30μM). Diaoxide was given for24hours. Statisticshas shown that high glucose impaired adhesion, migration and tube formation function ofHUVECs, which was restored by diazoxide.Conclusion:Diazoxide was able to improve wound healing and stimulate angiogensis inSTZ-induced diabetic mice, which was related to improve impaired BM-EPCs function.The underlying mechanism was possibly related to p53/TSP-1pathway.