The Role And Mechanism Of Endothelial Peli1 Involved In Diabetes Induced Cardiac Microvascular Dysfunction

BackgroundIn diabetes,myocardial microvascular damage often occurs before large vessels and myocardial cells,which is the primary factor of myocardial structure and dysfunction.Meanwhile,it is also an important factor leading to diabetic cardiovascular complications and high mortality in diabetes.The endoplasmic reticulum stress induced by accumulation of metabolic products such as hyperglycemia and AGEs in diabetic plays an important role in the pathophysiological mechanism of myocardial microvascular damage.Previously study found phosphorylation of inositol requiring enzyme-1?(IRE1?)could down-regulate the expression of VE-cadherin and other adhesion factors to disrupt the microvascular permeability of the brain.In addition,the activation of IRE1?could induce the apoptosis of microvascular endothelial cells by down-regulating p38 and JNK signal pathway.Moreover,the reduction of microvascular damage and the number of micro vessels could lead to microcirculatory disturbances of myocardial tissue.However,the molecular mechanism regulating the activation of IRE1? and its downstream signaling in the damage of microvascular in diabetic myocardium has not been precisely understood.It has been reported that as toll like receptors endogenous ligand(AGEs?Hsp60?Hsp70)could induce the activation of TLR signal pathway,and then activated the endoplasmic reticulum receptor kinase IRE 1?.Furthermore,TLR-mediated activation of intracellular signal could up-regulate Peli1 and activate its E3 ubiquitin ligase activity,and Peli1 could regulate TLRs-mediated intracellular signal pathway.In particular,Peli1 could promote the ubiquitination of TRAF6,and it could directly inhibit the phosphorylation of IRE1?.The study suggested that Peli1 may regulate IRE la and its downstream signal activation in diabetes.However,whether Peli1 may play an important role in the development of diabetic micro vascular injury?Especially in the regulation of Pelil in endothelial cells?Is its mechanism related to regulating endoplasmic reticulum stress-sensing kinase IRE1? and its downstream signals?How Peli1 regulate IRE1? and its downstream pathways?Other issues have yet to be clarified.Therefore,this study was designed to explore the role of Peli1 in the regulation of myocardial microvascular damage in diabetic.Objective1.Clarify whether endothelial Peli1 participate in the process of diabetes-induced cardiac microvascular dysfunction.2.Clarify whether Peli1 regulate the cardiac microvascular dysfunction through IRE la and its downstream pathways.3.Clarify the underlying mechanism of Peli1 in AGEs-induced cardiac microvascular dysfunction.Methods1.Animal modelsWe simulated the process of diabetes-induced microvascular injury by intraperitoneal injection of streptozotocin to induce diabetes for 8weeks.We observed the protein level of Peli1 in the CMECs stimulated by STZ.To clarify the impact of endothelial Peli1,we generated endothelial conditional knockout Peli1 mice(Peli1 ?EC).We adopted 6 to 8 weeks Peli1F/F and Peli1 ?EC male mice which were divided into 4 groups at random,including PeliF/F,PeliF/F+STZ,Peli1 ?EC and Peli1 ?EC+STZ.We observed the improvement of diabetic microvascular injury at 8 weeks of diabetes by injecting STZ or the same volume of citrate buffer in PeliF/F and Peli1 ?EC mice and also observed the effects on ventricular remodeling and cardiac function at 16 weeks of diabetes.2.Cell modelsThe apotosis of CMECs and the reduction of intercellular connectivity proteins are both the mian reason for endothelial damage.On the condition of long-term diabetes,there is going to be a lot of irreversible accumulation of AGEs inducing the function change of CMECs.Therefore we stimulated CMECs with AGEs for 8h to imitate the endothelial damage in vitro.In addition,CMECs and CFs were cultured under this stimulation condition to observe the effects of paracrine of CFs after the endothelial damage induced by AGEs.At the same time,we transfected CMECs with Adenovirus packaging Peli1 interfering plasmid(adv-shPeli1)to suppress the protein level of Peli1 in CMECs.Before stimulated by AGEs,we transfected adv-shPeli1 or adv-Scr for 48h.Groups of experiment were control,AGEs,adv-shPeli1+AGEs and adv-Scr+AGEs.This paper focuses on the following aspects.Firstly,the protective effects in endothelial damage after interfering the Peli1 expression in CMECs.Secondly,the influence of improvement in ER stress mediated by the phosphorylation of IRE1? after interfering the Peli1 expression in CMECs.Lastly,the specific molecular mechanisms of the phosphorylation of IRE1 a affected by Peli1.3.Observing targetsWe sorted endothelial cells using gentle MACS dissociator from mice under diabetic and non-diabetic condition and then measured the protein level of Peli1 in ECs of diabetic mice induced by STZ using Western blot.The integrity of microvessel was detected by scanning electron microscope and the permeability were tested by transmission electron microscope.The change of microvessel number were detected by immunofluorescence and meanwhile the size of cardiomyocytes were observed by WGA staining.HE staining was used to test the infiltration level of inflammatory cells in the mouse heart tissues.The deposition of perivascular and interstitial collagen was observed by Sirius red staining.Echocardiography was performed to evaluate the systolic and diastolic function of mouse heart.We evaluate the mRNA expression of connective molecules in ECs through RT-PCR.Tube capacity in CMECs through Tube formation assay.CMECs apoptosis levels were detected through flow cytometric analysis.To evaluate the degree of CMECs apoptosis,we used the caspase 12/3 enzymatic activity Detection Kit.Western Blot was used to calculate the expression of cleaved-caspase 12/3 and observe the protein level of p-IRE1?,p-perk and ATF6.Co-immunoprecipitation analysis was performed to detecte the combination of IRE1? and TRAF2.To observe the activity of downstream p-P38 and p-JNK induced by the phosphorylation of IRE1?,we used Western Blot analysis.To evaluate the level of p-IRE1? in the microvessels,we used immunofluorescent staining.We also used MACS to esitimate the phosphorylation of IREla and its downstream p38 in the ECs of PeliF/F,PeliF/F+STZ,Peli1?EC and Peli1 ?EC+STZ.Tunel staining was used to evaluate myocardial microvessel apoptosis.The mass spectrometry analysis was carried out to find the combined protein of Peli1.Protein dynamics simulated the possible binding sites of Hsp90 and Peli1 protein.Co-immunoprecipitation was performed to detecte the combination of endogenous and exogenous Hsp90 and Peli1.Western blot was used to detect whether the activation of Peli1-mediated IRE1 was dependent on Hsp90.Results1.Endothelial Peli1 prevents myocardial microvascular dysfunction in diabetes and reduces ventricular remodeling through regulating IRE1? and its downstream pathways.The expression of Peli1 protein dramatically increased in CMECs at 8weeks of diabetes induced by STZ.In the meantime,the microvessels' integrity was broken after diabetes.The permeability of lanthanum nitrate was detected by transmission electron microscope,which illustrated the permeability of microvessels changed.Immunofluorescence also showed a significant decrease in the number of microvessels,which indicated the injury of microvascular at 8 weeks of diabetes.However,the integrity and permeability and the number of microvessels were significantly reversed in endothelial conditional knockout Peli1 mice.It revealed that blocking Peli1 expression in ECs could prevent myocardial microvascular dysfunction in diabetes.Under diabetic conditions for 16 weeks,the cardiomyocyte size was markedly extensive larger in PeliF/F mice.HE staining showed that the recruited inflammatory cells were increased in the heart of PeliF/F mice.Myocardial fibrosis around perivascular and interstitial regions,viewed by Sirius Red staining,were aggravated in PeliF/F mice.Consistently,systolic and diastolic function,detected by echocardiography were significantly impaired in PeliF/F mice after diabetes.But pellinol depeletion in the endothelium prevented ventricular dysfunction,suggesting that abrogating Peli1 expression in ECs inhibits cardiac remodeling induced by diabetes and restore cardiac function.2.Peli1 regulates IREla and its downstream pathways-induced cardiac microvascular dysfunction through Hsp90.The protein of Peli1 was highly expressed in CMECs stimulated 8h with AGEs.At the same time,the expression of VE-Cadherin and PECAM-1,which were the connective protein of CMECs,were significantly reduced.Tube formation assay showed that the tube capacity declined sharply.Together,these results portend that AGEs could induce functional damages of CMECs,but the induction was dramatically inhibited when Peli1 was knocked down in endothelial cells.Flow cytometry assay indicated AGEs promoted apoptosis in CMECs for stimulating 48h.AGEs increased the activity of cleaved caspase 12 and cleaved 3 in endothelial cells.The caspase12/3 enzymatic activity Detection Kit showed that caspase12/3 enzymatic activity were markedly enhanced in CMECs.Western Blot were found the expression of cleaved-caspase 12/3 were elevate significantly,indicating that AGEs promoted apoptosis in CMECs.While this alternation was reversed by inhibited Peli1 expression in CMECs.Blocking Peli1 expression reduced the apotosis of CMECs.Western blot was carried out to detecte the phosphorylation of IRE1?,suggesting that AGEs enhanced IRE1? phosphorylation.This leads to the recruitment of TRAF2 in the cytoplasm region of IRE1?,concretely embodied in the increase of combination of IRE1 and TRAF2.And further induce the activation of the downstream MAPK signaling pathway.It indicated that the phosphorylation of p38 and JNK were highly enhanced,showing AGEs induced endoplasmic reticulum(ER)stress through IRE1? signaling branch in CMECs.But blocking Peli1 expression diminished AGEs-induced IRE1? phosphorylation thus inhibited ER stress.Through LC-Mas,we detected the protein binding to Peli1,namely Hsp90.The result of ZDOCK suggested that the Ring domain of Peli1 could interact with Hsp90.Analysis of molecular dynamics revealed that there are multiple binding sites to Hsp90 in the Ring domain of Peli1.The first five key amino acids binding to Hsp90 were ARG-279,ARG-222,ARG-326,LYS-229,LYS-374.Above results identify that it is Ring domain of Peli1 bind to Hsp90.The combination of two proteins was further verified by co-immunoprecipitation assay,indicating that Peli1 combined to Hsp90 in CMECs.We constructed adenovirus-mediated delivery of Flag-tagged Full length Peli1(Flag-Peli1),Peli1 with deletion at FHA domain(Flag-Peli1 ?FHA)and Peli1 with deletion at Ring domain(Flag-Peli1 ? Ring).Co-immunoprecipitation analysis revealed that Flag-Peli1 ?Ring abolished the interaction of Peli1 with Hsp90,indicated that Ring domain of Peli1 was a vital domain to bind with Hsp90.In order to clarify the effect on the phosphorylation of IRE1?,we transfected alone or co-transfected Flag-Peli1 and HA-Hsp90.As is shown in Western blot,the level of IRE1?phosphorylation was significantly improved when transfected Flag-Peli1 alone,whereas the phosphorylation of IRE 1a was inhibited after co-transfecting Flag-Peli1 and HA-Hsp90.It is suggest that IRE1a phosphorylation induced by Peli1 is independent of Hsp90.ConclusionIntegrated with the above experiments,we found that Peli1 is a main regulator protein during the development of diabetes-induced cardiac microvascular injury.Endothelial conditional knockout Peli1 could ameliorate cardiac microvascular dysfunction and ventricular remodeling,as well as restoring heart function.Moreover,blocking Peli1 expression in CMECs also reduced AGEs-induced cell damage.The mechanism may be related to the interaction between Peli1 and Hsp90,which influences the phosphorylation of IRE1? and its downstream signaling.