Mechanism And Role Of Nrf2 In Regulation Of Endothelial Progenitor Cell Function In Vascular Aging

Background: Aging and diabetic mellitus are associated with an increased risk for cardiovascular disease.Decreased endothelial progenitor cells(EPCs)number and function play a pivotal role in reduced endothelial repair and development of cardiovascular disease.The purpose of the present study was to explore the protective effect of the nuclear factor(erythroid-derived 2)-like 2(Nrf2)on EPCs dysfunction during aging and in diabetic mellitus and mechanism underlying.Methods:(1)Biological functions of EPCs from young(3~4 month),middle-aged(12~13 month)and aged mice(22~24 month)were evaluated,including migration,proliferation and secretion of VEGF,stromal-derived growth factor(SDF)and NO.Laser Doppler perfusion imaging was conducted to demonstrate the blood flow reperfusion.Capillary density was measured in the ischemic gastrocnemius muscle.Oxidative stress levels in forementioned EPCs were also detected with intracellular reactive oxygen species(ROS),superoxide dismutase(SOD)and malondialdehyde(MDA).Furthermore,the functions of EPCs and the levels of oxidative stress were assessed after the Nrf2 silence by si RNA or activation by tert-butylhydroquinone.Western blot was used to determine the protein expression of Nrf2,keap1,NLRP3,TRX1,TXNIP,p65.RT-PCR was used to determine the m RNA expression of Nrf2,HO-1,NOQ-1,TRX-1,keap1,NLRP3,TXNIP,p65.Immunofluorescence is also used to detect Nrf2 expressions.(2)The biological functions of EPCs in streptozotocin-induced diabetic mice were evaluated,including migration,proliferation,angiogenesis and the secretion of vascular endothelial growth factor(VEGF),stromal-derived growth factor(SDF)and nitric oxide(NO).Oxidative stress levels in diabetic EPCs were also assessed by detecting intracellular reactive oxygen species(ROS),superoxide dismutase(SOD)and malondialdehyde(MDA).EPCs senescence was evaluated by measuring p16 and β-gal expression and observing the senescence-associated secretory phenotype.In addition,the function of EPcs and level of oxidative stress were assessed following Nrf2 silencing or activation.Results:(1)Biological functions of EPCs both in vitro and in vivo decreased during aging,accompanied with declined Nrf2 expression and its target gene(HO-1,NQO-1,TRX-1)and increased levels of NLRP3 expression and oxidative stress by increasing ROS and MDA content and decreasing SOD activity.Moreover,Nrf2 silence obviously damaged the biological functions of EPCs and enhanced the oxidative stress in EPCs from young mice.While Nrf2 activation protected the impaired EPCs from aged mice against oxidative stress and ameliorated the biological dysfunction of EPCs derived from aged mice by downregulating NLRP3 inflammasome.(2)DM reduced the expression of Nrf2,which was accompanied by enhanced oxidative stress,senescence and dysfunction in EPCs.Nrf2 silencing resulted in a decrease of EPCs biological functions,accelerated cell senescence and increased oxidative stress,as indicated by ROS and MDA upregulation accompanied with decreased SOD activity.Furthermore,Nrf2 silencing inhibited migration,proliferation and secretion in EPCs,while it increased oxidative stress and cell senescence.Nrf2 activation protected diabetic EPcs against the effects of oxidative stress and cell senescence,ameliorating the biological dysfunction of EPcs derived from mice with diabetes.Conclusion: The biological function of EPCs decreased and level of oxidative stress increased during aging and in diabetic mice.Nrf2 protects the biological functions of EPCs during aging and in diabetic mice from oxidative stress.Nrf2 could protect the functional damage of EPCs by downregulating NLRP3 inflammasome though NF-κB signal during aging.Nrf2 overexpression protected against oxidative stress-induced functional damage in EPCs derived from diabetic mice by regulating cell senescence.