Effect And Mechanism Of Mammalian Target Of Rapamycin Complex 1(mTORC1) On Hindlimb Ischemia Injury In Diabetic Mice

Background: Peripheral arterial disease(PAD)still remains a major cause of disability worldwide.The presence of diabetes mellitus(DM)multiplied the possibility of having PAD or even critical limb ischemia(CLI).Endothelial cell dysfunction plays an important role in this pathological process.Autophagy acts as a pivotal character in maintaining intracellular homeostasis,whose dysregulation is involved in lots of diseases,particularly in DM.The mechanistic target of rapamycin complex 1(mTORC1)is a ubiquitous serine/threonine kinase known to control autophagy.This study was designed to explore the effects of endothelial-specific Raptor(mTORC1)knockout(KO)mice in diabetic hindlimb ischemia model and the underlying mechanisms involved in a focus on autophagy.Method: Eight week-old endothelial-specific Raptor KO(Tie2-mTORC1ko)mice and their wild-type littermates were subjected to intraperitoneal injection of streptozotocin(STZ)for inducing DM.The mice blood glucose levels above 16.7mmol/L were selected to administrate the following experiment.After 12 weeks ad libitum feeding,the PAD model was operated in control group(PAD group),DM group and Raptor-/-+DM group.Sham-operated mice received incision without artery ligation.Laser Doppler perfusion imaging(LDPI)was performed to serially monitor the blood perfusion of the ischemic hindlimb at postoperative day(POD)0、3、7 and 14.Left adductor muscle was harvested and made frozen sections to perform DHE and TUNEL staining for detecting reactive oxygen species(ROS)and apoptosis.Immunofluorescence staining was utilized to determinate the expression of CD31 and LC3 B for evaluating the relationship between angiogenesis and autophagy.HUVECs cultured in medium containing 33.3 mmol/L glucose for 48 hours were served as the high glucose group(HG group),while those cultured in 5.5 mmol/L glucose for equivalent period of time were conceived as control group.After transfected with or without small interfering RNA,HUVECs were then exposed to a 48-hour high glucose medium or control medium in the presence or absence of the autophagy inhibitor 3-methyladenine or the autophagy inducer rapamycin,followed with or without a 12 hours hypoxia and serum deprivation(H/SD)in the presence or absence of 3-MA or rapamycin.Apoptosis was evaluated by Annexin V-FITC/PI apoptosis detection kit.The expressions of LC-3A/B,STSQM1,Raptor,ULK1,P70S6 K,and mTOR were analyzed by Western blot.Mitochondrial membrane potential(MMP)was detected by JC-1.Intracellular ROS was detected by an oxidation-sensitive fluorescent probe DCFH-DA.Result: The index of perfusion ratio(the average LDPI of ischemic/nonischemic hindlimb PR)was quantified to evaluate the hindlimb blood perfusion recovery.There was rarely difference in PR among groups on POD 0(p> 0.05).PR ascended gradually in all groups over the next few days.Mice exhibited a higher perfusion ratio in ischemic hindlimb in Raptor-/-+DM+PAD groups compared with that of DM+PAD group at POD7 and POD14(p< 0.05).There was no obviously difference between Raptor-/-+DM+PAD group and PAD group at POD7 and POD14.Histological staining analysis indicated that PAD increased both CD31-positive tubular structures and α-SMA-positive capillary density as compared to sham group,the effect of which was attenuated by DM+PAD.Moreover,endothelial-specific raptor KO could rescue the impaired angiogenesis in DM+PAD as evidenced by increased α-SMA and CD31 expression in Raptor-/-+DM+PAD group(p < 0.05).In addition,PAD stimulated the ischemic gastrocnemius autophagy,yet,this beneficial effect was weaken under diabetic state.Moreover,endothelial-specific raptor KO could restore the autophagy as demonstrated by the expression of LC3 B in Raptor-/-+DM+PAD group(p< 0.05 vs.DM+PAD group).Interestingly,the expression of CD31 was accompanied by green LC3 B dots,merging as yellow puncta,which suggested that hindlimb angiogenesis could relate to autophagy.The result of DHE and TUNEL indicated that Raptor-/-+DM+PAD groups decreased the level of ROS and apoptosis compared with that of DM group(p< 0.05).In vitro study,flow cytometry analysis of Annexin V-FITC/PI staining indicated that HG+H/SD greatly increased the percentage of apoptotic endothelial cells,whereas raptor siRNA inhibited HG+H/SD-induced endothelial cells apoptosis(vs.the HG+H/SD group,P<0.05).In contrast,mTORC1 overexpression by TSC2 siRNA further exacerbated endothelial cells apoptosis in response to HG+H/SD(vs.the HG+H/SD group,P<0.05).Furthermore,autophagy inducer rapamycin and autophagy inhibitor 3-MA were shown to mimic the effects of raptor siRNA and TSC2 siRNA,respectively.Next,LC3-II protein levels and decreased SQSTM1(p62)protein levels were observed in H/SD group as compared to the control group,however,HG mitigated this increase and autophagy in HG+H/SD group was significantly lower than H/SD group.As expected,mTORC1 silencing rescued the impaired autophagy while mTORC1 overexpression aggravated the impaired autophagy in HG+H/SD treated HUVECs.And then,the protein levels of mTORC1 downstream molecules were also assessed by Western blot.The result showed that high glucose increased mTORC1 activity in both control and H/SD condition,as evidenced by elevated protein level of p-P70S6k(Thr 389),raptor and decreased protein level of p-ULK1(Ser555)in HG and H/SD+HG groups in comparison with control and H/SD group,respectively.While raptor siRNA reversed this trend triggered by high glucose,an opposite pattern was observed in TSC2 siRNA treated group,with reduced level of p-ULK1(Ser555)and enhanced level of p-P70S6k(Thr 389),raptor and p-mTOR(Ser 2448).The result of DCF fluorescence intensity showed that mTORC1 silencing with raptor siRNA decreased the level of intracellular ROS in HG+H/SD context.In contrast,an opposite trend was observed in TSC2 siRNA treated group,with significantly increased level of intracellular ROS.Furthermore,mTORC1 silencing restored the level of MMP in HG+H/SD as evidenced by increased the proportion of J-aggregate/J-monomer,yet the trend was reversed and even exacerbated by mTORC1 overexpression.Conclusion: Our current work demonstrated a beneficial role of endothelial-specific raptor knockout in promoting blood perfusion and functional recovery in diabetic PAD mice mainly through improving endothelial viability and function as well as promoting angiogenesis.Furthermore,in vitro studies in HUVECs unveiled that m TORC1 silencing protected endothelial cells against HG injury,possibly via induction of autophagy and resultant suppression of oxidative stress.Those findings may guide the prospective clinical trial to evaluate the latent therapeutic effect of mTORC1 inhibition for patients with diabetic vascular complications.