Role And Mechanism Of KPC1 In Endothelial-Mesenchymal Transition Of Human Coronary Artery Endothelial Cells

Background: Endothelial-mesenchymal transition(EndMT)refers to the process in which the structure and function of endothelial cells are significantly changed under pathological or physiological conditions,and the characteristics of fibroblasts are demonstrated.EndMT is involved in the process of myocardial fibrosis(MF),which ultimately leads to decreased heart function,heart failure,arrhythmia,and even sudden death.Ring-finger proteins are widely expressed in eukaryotes and are involved in many physiological processes,such as apoptosis,cell cycle regulation and signaling.Kip1 ubiquitination-promoting complex 1(KPC1)is a class of ubiquitin-protein ligating enzymes containing a ring-finger domain.Precious researchs have confirmed that the expression of KPC1 was significantly decreased in the rat carotid artery balloon injury model,and KPC1 mRNA level was markedly increased in TGF-?1-induced endothelial cells.Multiple factors,therefore,that reduce the expression of KPC1 could correspondingly reduce myocardial fibrosis by inhibiting endothelial-mesenchymal transition.Objective: To investigate the expression characteristics and mechanisms of KPC1 during endothelial-mesenchymal transition of human coronary artery endothelial cells.Methods: Endothelial-mesenchymal transition model was constructed using HCAECs induced by transforming growth factor-?1(TGF-?1).The expression of KPC1 was detected by quantitative real-time PCR(qRT-PCR),Western Blot(WB)and immunofluorescence.PDTC,a nuclear factor kappaB(NF-?B)pathway inhibitor,was utilized to explore whether KPC1 regulated the NF-?B pathway to promote endothelial-mesenchymal transition.The effects of KPC1 on the proliferation and migration of HCAECs were evaluated by CCK8 cell proliferation assay and wound healing assay.Results: The expression of KPC1 was showed to increase in the endothelial-mesenchymal transition model compared to the control group usingquantitative real-time PCR,Western Blot(WB)and immunofluorescence.The expression of endothelial marker vascular endothelial cadherin(VE-cadherin)was decreased in the Ad-KPC1 group compared to the control group,and in contrast,the expression of mesenchymal cell marker ?-smooth muscle actin(?-SMA)was increased.However,the expression of VE-cadherin in Ad-KPC1+PDTC group was higher than that in Ad-KPC1 group,and the expression of?-SMA was lower than that in Ad-KPC1 group.In addition,the results of wound healing assay showed that the migration of HCAECs in Ad-KPC1 group was accelerated than that in the control group,Ad-Null group and Ad-shKPC1 group.Conversely,the results of CCK8 cell proliferation assay demonstrated that the proliferation of HCAECs in Ad-KPC1 group was not significantly different from that in the control group,Ad-Null group and Ad-shKPC1 group.Conclusion: HCAECs endothelial mesenchymal transition model was successfully established after 7-day induction of TGF-?1 with concentration of10 ng/mL.The expression of KPC1 was significantly increased in the TGF-?1-induced EndMT model.KPC1 promoted EndMT by regulating NF-?B pathway.KPC1 promoted the migration of HCAECs,but no significant effect was detected on the proliferation.