The Role And Mechanism Of Which Kallikerin-8 Promotes Endothelial-Mesenchymal Transition In Myocardial And Renal Interstitial Fibrosis Of Diabetes Mellitus

Tissue kallikrein-related peptidases(KLKs)are a family of serine proteases with trypsin and chymotrypsin-like activities.KLKs could specifically cleaved low-molecular weight kininogen to form kinins,which could selectively stimulate bradykinin B1 and B2 receptors and thus exert extensive biological role.Our previous research indicated that upregulation of KLK8 could induce myocardial hypertrophy and progressive cardiac dysfunction,which may be not dependent on a kinin receptor pathway but the EGF and PAR1/2 signaling pathway.At the same time,we also found that KLK8 overexpression could induce myocardial fibrosis,and endothelial dysfunction triggered endothelial-mesenchymal transition(EndMT)participated in KLK8 induced myocardial fibrosis.Diabetes mellitus is a metabolic disorder syndrome characterized by long-term hyperglycemia and systemic lesions of multiple organs.According to statistics,the number of global diabetes patients in 2017 was as high as 435 million,and it is estimated that the number will be more than 600 million to 2045.Now,diabetes mellitus is becoming a global disease and it could trigger a variety of complications,including diabetic cardiomyopathy and diabetic nephropathy without timely diagnosis and treatment.Diabetic cardiomyopathy and nephropathy were characterized by organ dysfunction and tissue fibrosis.Researches demonstrated that kallikrein-kinin system(KKS)was involved in the pathogenesis and development of diabetes mellitus and its complications,but the mechanism has not been fully elucided.So,that whether KLK8 is involved in the occurrence of organ dysfunction and tissue fibrosis in diabetic cardiomyopathy and nephropathy deserves further research.Therefore,this study was aimed to use type 1 diabetes mice model in vivo and coronary artery and glomerular endothelial cells treated with high glucose in vitro to measure the expression of KLK8 firstly and then employ the two kinds of animal models KLK8 transgenic and gene knockout to confirm the critical role of KLK8 in the pathological development of EndMT and fibrosis in diabetic myocardium and kidney in vivo and vitro and clarify the molecular mechanism.Main results:1.The role and mechanism of KLK8 promoted EndMT in myocardialinterstitial fibrosis in diabetic cardiomyopathy1.1.High glucose significantly increased KLK8 expression in diabetic heart and coronary endothelial cells1.1.1.Mice were intraperitoneally injected with streptozocin(STZ)to construct type 1 diabetic mice,and KLK8 mRNA level was detected at 3 and 6 months after the model was successfully constructed.Compared with the controlc group,KLK8 mRNA level in diabetic heart was significantly increased and exhibited obvious time-dependent.1.1.2.HCAECs were treated with 5.5mM,15 mM and 25 mM glucose for 120 h,while the control group was treated with mannitol.Compared with the control group,high glucose could significantly increase KLK8 expression in mRNA levels,showing a significant concentration dependence.1.2.The role and mechanism of KLK8 promoted EndMT in myocardial interstitial fibrosis in diabetic cardiomyopathy1.2.1.KLK8 overexpression induces EndMT in human coronary endothelial cells1.2.1.1.Human coronary artery endothelial cells(HCAECs)were transfected with KLK8 adenovirus for 72 h to increase the KLK8 expression.Compared with the adenovirus vector group,KLK8 overexpression significantly increased the expression of interstitial cell markers ?-SMA and vimentin,and decreased the expression of endothelial cell markers CD31 and VE-cadherin.1.2.1.2.TGF-?1 neutralizing antibody partially blocked KLK8-induced EndMT,namely suppressing KLK8-induced up-regulation of ?-SMA and vimentin,and down-regulation of CD31 and VE-cadherin.1.2.2.Up-regulation of KLK8 was involved in hyperglycemic-induced endothelial dysfunction,EndMT and cardiac fibrosis1.2.2.1.Up-regulation KLK8 contributed to high glucose induced endothelial dysfunction and EndMTKLK8 siRNA to used to knock down KLK8 expression in endothelial cells and the control group was given equivalent control siRNA.Compared with the control group,KLK8 siRNA could significantly reverse endothelial dysfunction induced by high glucose,namely decreased the endothelial cell damage markers vWF,sTM and Eselectin level,and a significant increase in endothelial cell vitality.And separate transfection KLK8 siRNA had no effect on endothelial cell function.KLK8 siRNA could significantly reverse the down-regulation of VE-cadherin and CD31,and the up-regulation of ?-SMA and vimentin in protein level induced by high glucose.1.3.KLK8 deficiency significantly attenuates EndMT and tissue fibrosis in diabetic hearts1.3.1.KLK8 deficiency significantly attenuates diabetic cardiac endothelial dysfunction and cardiac dysfunction1.3.1.1.Mice were intraperitoneally injected with streptozocin(STZ)to construct type 1 diabetic mice,and the blood glucose level was detected 6 months after the model was successfully constructed.Compared with the wild-type(WT)diabetic group,the blood glucose level in the diabetic group was significantly increased,while that in the KLK8 deficiency diabetic group was not significantly decreased.3.1.2.Compared with the WT control group,the serum level of vWF,sTM and E-selectin in diabetic mice were significantly increased,while they were significantly decreased in KLK8 deficiency group.1.3.2.KLK8 deficiency significantly attenuates EndMT in diabetic heartCompared with WT group,VE-cadherin and CD31 expression were significantly reduced and a significant rise in ?-SMA and vimentin expression in diabetic mice heart.And KLK8 deficiency significantly reverse the increased endothelial cell markers and decreased interstitial cell markers induced by high glucose.The separate KLK8 deficiency group had no effect on the expression of endothelial and interstitial cell markers in myocardium.The immunofluorescence double dye also showed that compared with control group,CD31 expression in myocardium of WT mice was significantly reduced and ?-SMA,vimentin and fsp1 expression were significantly increased.Moreover,a large number of CD31 were co-stained with ?-SMA,vimentin and fsp1,while KLK8 deficiency significantly increased the expression of CD31 and decreased the expression of ?-SMA,vimentin and fsp1.1.3.3.KLK8 deficiency significantly attenuated cardiac fibrosis and dysfunction in diabetic heart tissue1.3.3.1.Compared with the WT control group,the blood pressure increased and heart rate decreased in the 6-month diabetic mice.However,KLK8 deficiency could significantly reverse the increased blood pressure and decreased heart rate and cardiac systolic dysfunction induced by hyperglycemia,while the blood pressure,heart rate and cardiac function were not significantly abnormal in KLK8 deficient mice.3.3.2.Masson staining showed that KLK8 deficiency could also significantly reduce the fibrosis in diabetic heart tissue.1.4.KLK8 degrades VE-cadherin,thus promoting plakoglobin nuclear translocation1.4.1.KLK8 overexpression induced endothelial cell injury and EndMT is not through the kinin B1 or B2 receptors,but its enzyme activity1.4.1.1.Kinin B1 and B2 receptor siRNA were used to down-regulate the expression of kinin B1 and B2 receptors.MTT and westernblot results demonstrated that B1 R and B2 R siRNA could not block KLK8 overexpression induced endothelial cell injury and EndMT respectively.1.4.1.2.Two protease inhibitors,antipain and zinc sulfate,were used to block KLK8 proteolysis.In comparison with the control group,antipain and zinc sulfate partially blocked KLK8-induced endothelial injury and EndMT,which increased endothelial cell viability and the expression of VE-cadherin and CD31 as well as decreased the expression of ?-SMA and vimentin.1.4.2.KLK8 degraded VE-cadherin,thus promoting ?-catenin nuclear translocation1.4.2.1.Western blot combined with N-terminal sequencing confirmed that KLK8 could shear VE-cadherin to form an extracellular fragment of about 30kDa;1.4.2.2.?-catenin of endothelial cells was located in cell membrane and part co-located with VE-cadherin in adenovirus vector treated group.However,?-catenin and VE-cadherin in KLK8 adenovirus treated endothelial cell were significantly reduced,and plenty of ?-catenin was expressed in nuclear.Moreover,compared with adenovirus vector group,KLK8 overexpression resulted in decreased expression of ?-catenin in the cell membrane and cytoplasm as well as increased expression in the nucleus.However,transfection of VE-cadherin lentivirus reversed KLK8-induced ?-catenin nuclear translocation and the expression in the cell membrane and cytoplasm.1.5.?-catenin is required for KLK8-induced EndMT by cooperating with p531.5.1.?-catenin contributes to KLK8 induced EndMT?-catenin siRNA was used to down-regulate ?-catenin expression in endothelial cells.?-catenin siRNA could significantly reverse the reduced expression of VE-cadherin and CD31 and the increased expression of ?-SMA and vimentin in KLK8-induced endothelial cells.1.5.2.The role of ?-catenin in KLK8 induced EndMT is completed through the interaction with p531.5.2.1.CO-IP result showed that ?-catenin was correlated with p53 and TCF4 in endothelial cells,and KLK8 overexpression further enhanced the binding effect between?-catenin and p53 and TCF4.1.5.2.2.p53 inhibitor pifithrin-? reversed KLK8-inducedEndMT whereas TCF/?-catenin pathway inhibitor ICG-001 could not reverse KLK8-mediated EndMT;1.5.3.TGF-?1 was involved in KLK8-induced EndMT and cardiac fibrosis1.5.3.1.KLK8 significantly increased the mRNA expression of TGF-?1 in endothelial cells,which was partially reversed by ?-catenin siRNA and pifithrin-?.1.5.3.2.ChIP analysis indicated the binding of HIF-1? to the hypoxia response element of TGF-?1 promoter,which was significantly enhanced by KLK8 overexpression.HIF-1? inhibitor echinomycin not only inhibited basal TGF-?1 expression,but also blocked KLK8-induced TGF-?1 mRNA expression and release in endothelial cells.?-catenin siRNA and pifithrin-? could significantly reverse KLK8-induced HIF-1?binding to TGF-?1 promoter.1.5.4.The p53-Smad3 pathway is involved in mediating KLK8-induced EndMT and cardiac fibrosis1.5.4.1.KLK8 overexpression could significantly increase the binding of p53 and Smad3 in endothelial cells,which could be partially blocked by ?-catenin.1.5.4.2.KLK8 overexpression could also significantly increase the mRNA expression of TGF-?1-targeted pro-fibrosis transcription factors in endothelial cells,including Snail,Slug,Twist,Zeb1,Zeb2,which could be reversed by r-catenin siRNA and pifithrin-?.1.6.High glucose promotes ?-catenin-dependent cooperation of p53 with HIF1? and Smad,subsequently increasing the expression of TGF-?1 and its pro-fibrotic target genes in a KLK8-dependent manner1.6.1.KLK8 was involved in high glucose induced ?-catenin nuclear translocation and TGF-?1-dependent activation of pro-fibrotic signaling pathway1.6.1.1.High glucose treatment resulted in significant loss of membrane and cytosol?-catenin,whereas caused profound nuclear translocation of ?-catenin in endothelial cells.KLK8 siRNA reduced nuclear whereas increased membrane and cytosol ?-catenin levels in high glucose-treated endothelial cells.1.6.1.2.High glucose treatment resulted in significant increases in TGF-?1 mRNA expression and release as well as mRNA levels of TGF-?1 pro-fibrotic transcriptional targets in endothelial cells,which were largely reduced by siRNA targeting KLK8 and?-catenin.1.6.2 KLK8 was involved in mediating high glucose induced ?-catenin dependent association of p53 with Smad3 and HIF-1?1.6.2.1.High glucose led to a significant increase in the association of ?-catenin with p53 in endothelial cells,which was blocked by KLK8 siRNA.In addition,high glucose induced p53 binding to both HIF-1? and Smad? in endothelial cells,which was blocked by siRNA targeting KLK8 and ?-catenin.Using ChIP analysis,we found that the binding of HIF-1? to the hypoxia response element of TGF-?1 promoter was significantly enhanced by high glucose treatment.In addition,high glucose-induced HIF-1? binding to TGF-?1 promoter was largely blocked in the presence of KLK8 siRNA,?-catenin siRNA or p53 inhibitor pifithrin-?.1.6.2.2.Using STZ-induced type I diabetes model,we observed significant increases in TGF-?1 mRNA and protein levels as well as mRNA levels of TGF-?1pro-fibrotic transcriptional targets in heart tissues after 24 weeks of diabetes mellitus,which were significantly attenuated in KLK8 deficient mice.The association of ?-catenin with p53 in diabetic heart tissues was significantly decrease in KLK8 deficient mice.Diabetes mellitus increased p53 binding to both HIF-1? and Smad3 in heart tissues,which was attenuated in KLK8 deficient mice.Using ChIP analysis,we found that KLK8 deficient mice exhibited significant less binding of HIF-1? to the hypoxia response element of TGF-?1 promoter in diabetic heart tissues compared with wild-type mice.Conclusion: High glucose promotes the expression of KLK8 in myocardial tissue,and then induces cardiac dysfunction and interstitial fibrosis through endothelial dysfunction triggered EndMT,which may be dependent on the role of KLK8 on VE-cadherin-r-catenin complex,and leads to VE-cadherin degradation and r-catenin nuclear translocation,and activating TGF-? signal pathway at last.2.The role and mechanism of KLK8 promoted End MT in renal fibrosis in diabetic nephropathy2.1.Transgenic KLK8 rats demonstrated EndMT and renal fibrosis2.1.1.KLK8 overexpression leads to renal fibrosis and renal injuryMasson staining showed that transgenic KLK8 rats demonstrated significant collagen deposition in the kidney compared with the WT rats of the same age,and showed an obvious time-dependence.In other words,the collagen accumulation of the kidney tissue of transgenic KLK8 rats at the age of 12 weeks was significantly higher than that at the age of 6 weeks.In addition,the levels of hydroxyproline,collagen and TGF-?1 in kidney were significantly higher than those in the control group.The urine protein test results also showed that the urine protein content of the transgenic KLK8 rats was significantly higher than that of the WT rats of the same age,and all of them showed significant time-dependent.2.1.2.KLK8 overexpression leads to End MT in kidneyCompared with WT rats,the expression of ?-SMA and vimentin in kidney of transgenic KLK8 rats were significantly increased,while the expression of VE-cadherin and CD31 was significantly decreased.Further IF result indicated that endothelial cell marker CD31 expression was decreased significantly,?-SMA and fsp1 were significantly increased in the kidney of 12 weeks transgenic KLK8 rat,and some of which were dyeing with CD31 positive cells.Confocal results also confirmed the co-location of CD31 and ?-SMA and fsp1.2.1.3.KLK8 overexpression leads to human glomerular endothelial cells dysfunctionHRGECs(Human glomerular endothelial cells)were transfected with KLK8 adenovirus to increase the KLK8 expression.KLK8 overexpression could result in significant decrease in cell viability and the result presented time and concentration dependence.Moreover,the release quantity of the biomarkers of endothelial injury E-selectin,s TM,v WF were robust increase,and endothelial cell permeability also increased significantly.2.1.4.KLK8 overexpression induces End MT in HRGECsKLK8 dose-dependently increased the expression of ?-SMA and vimentin,and decreased the expression of CD31 and VE-cadherin.2.2.Up-regulation KLK8 contributed to high glucose induced endothelial dysfunction,End MT and renal fibrosis2.2.1.Up-regulation of KLK8 contributed to high glucose induced endothelial dysfunctionKLK8 si RNA was used to knock down the expression of KLK8 in endothelial cells.MTT results showed that KLK8 si RNA could significantly reverse the dysfunction of endothelial cells induced by high glucose,while KLK8 si RNA transfection alone had no effect on the function of endothelial cells.2.2.2.Up-regulation of KLK8 contributed to high glucose induced EndMTCompared with the control group,high glucose significantly up-regulated the expression of ?-SMA and vimentin,and decreased the expression of VE-cadherin and CD31,while KLK8 si RNA reversed high glucose induced the up-regulation of ?-SMA and vimentin and down-regulation of VE-cadherin and CD31.2.3.?-catenin is required for KLK8-induced End MT2.3.1.?-catenin si RNA could significantly reverse the changed level of VE-cadherin and CD31 as well as ?-SMA and vimentin in KLK8-induced endothelial cells.In addition,?-catenin si RNA also reversed the KLK8-induced decrease in endothelial cell activity.2.3.2.KLK8 could significantly increase the expression of TGF-?1 in m RNA level,which can be partially reversed by ?-catenin si RNA.2.3.3.CHIP assay was used to confirm the binding of HIF-1? to the hypoxia response element of TGF-?1 promoter,which was significantly enhanced by KLK8 overexpression.HIF-1? inhibitor echinomycin not only inhibited basal TGF-?1 expression,but also blocked KLK8-induced TGF-?1 m RNA expression and release in endothelial cells.Conclusion: High glucose promotes the expression of KLK8 in renal tissue,and then induces renal dysfunction and fibrosis through endothelial dysfunction triggered End MT,which may be dependent on KLK8 mediated activation of the TGF signaling pathway.