Harmine Alleviates Atherosclerosis Via Targeting Endothelial PTPN14/YAP Pathway

Atherosclerosis is a common cardiovascular disease and the main cause of myocardial infarction and stroke.An important pathological feature of atherosclerosis is the focal distribution of plaque.Although all arteries are in the same blood composition,the lesions occur in the bifurcations and bends of arterial blood vessels.The blood flow shear force in these parts is mainly turbulence without a defined direction.It plays an important role in the pathological progress of atherosclerosis.Endothelial dysfunction is the first step in atherosclerosis.When endothelial cells are exposed to different blood flow shear forces,they changes in endothelial function.OSS induces activation of YAP which triggers expression of pro-inflammatory factors and then promotes atherosclerosis.Therefore,finding available drugs for YAP can provide a research basis for clinical treatment of atherosclerosis.Harmine is a natural small-molecule alkaloid,and its effect on atherosclerosis has not yet been elucidated.Therefore,the purpose of this study was to clarify the role of Harmine in atherosclerosis mediated by OSS-induced YAP activation.As a natural?-tricyclic compound,Harmine has been reported exerting a variety of pharmacological properties.Harmine promotes pancreatic?-cell proliferation by inhibiting the non-receptor tyrosine kinase DYRK1A,which plays a role in improving type 2 diabetes.It suggested that Harmine has potential clinical significance for treatment of diseases related to metabolic syndrome.In order to explore the role of Harmine in atherosclerosis,we used LDLR^-/-and Apo E^-/-mice to induce atherosclerosis by feeding the Western diet.The staining results showed that Harmine inhibited the formation of plaques in the aorta and outflow tract of mice.Oil red staining of the aortic bundle showed that Harmine could only inhibit plaque formation in the arch of blood vessels.Also,Harmine reduced VCAM-1 expression in the intimal lining of the outflow tract and endothelial cells in vitro.Hippo signaling pathway can regulate the size of organs.YAP is an important downstream effector molecule.It was reported that turbulence promotes the phosphorylation of tyrosine 357(Y357)at YAP through c-Abl,leading to YAP nuclear translocation and activation of endothelial cells.Thus,we further investigated whether Harmine inhibits OSS induced endothelial activation through YAP^Y357.In vitro experiments found that Harmine inhibits OSS induced endothelial cell YAP^Y357phosphorylation,thus retaining YAP in the cytoplasm,but does not affect the expression of pc-Abl^Y412;The overexpressing YAP in Endothelial cells can reversedownregulation of VCAM-1 expression induced by Harmine.YAP overexpression in ECs blunted the anti-atherosclerosis effect of Harmine in Apo E^-/-micePTPN14 is a non-receptor tyrosine phosphatase that is known to play an important role in tumorigenesis,but its role in blood flow shear-mediated endothelial activation and atherosclerosis is unknown.Proteomics analysis found that PTPN14interacted with YAP;endogenous and exogenous co-immunoprecipitation experiments also confirmed that.In addition,Harmine can promote the protein expression of PTPN14 in endothelial cells while reduced the degradation rate of PTPN14.Treatment with proteasome inhibitor MG132 blocked PTPN14 protein level regulated by Harmine,suggesting that Harmine can stabilize PTPN14 protein and inhibit PTPN14 proteasome pathway degradation.Therefore we guess that PTPN14 is the middle link between Harmine and YAP^Y357.Knockdown of PTPN14 inhibited Harmine's regulation of turbulence-induced YAP^Y357 phosphorylation and VCAM-1expression;conversely,overexpression of PTPN14 mimicked Harmine's regulatory effects.In vivo experiments,it was found that over-expression of PTPN14 can mimic the effect of Harmine,inhibiting the progress of atherosclerosis in Apo E^-/-mice induced by partial ligation,and decrease the YAP^Y357 and VCAM-1 of the left common carotid artery intima.expression.To sum up,we found that when endothelial cells are stimulated by turbulence,Harmine promotes the dephosphorylation of YAP^Y357 by inhibiting the degradation of PTPN14,making it unable to aggregate to the nucleus and then activating,thereby improving endothelial dysfunction.This study clarified that Harmine inhibits the progress of atherosclerosis,and provides a research basis and new ideas for clinical prevention and treatment of atherosclerosis.