Endothelial Scaffolding Protein ENH Promotes PHLPP2-mediated Dephosphorylation Of AKT1 And ENOS Promoting Vascular Remodeling

Rationale: Vascular remodeling is a pathological feature of multiple vascular diseases including atherosclerosis.A decrease in nitric oxide(NO),leading to the proliferation of vascular smooth muscle cells(VSMCs),is a common pathological feature of pathogenic vascular remodeling.The regulation of NO synthesis by endothelial nitric oxide synthase(eNOS)is precisely executed by phosphorylation and protein kinases including AKT1(protein kinase B).ENH(enigma homolog protein),containing PDZ and three LIM domains,is a scaffolding protein for multiple protein kinases.Whether ENH regulates eNOS activation and vascular remodeling remains unknown.The aim of this study was to study the role of endothelial ENH in vascular remodeling.Methods and Results: ENH was upregulated in both injured mouse arteries and human atherosclerotic plaques.Increased expression of ENH was associated with coronary artery disease.Neointima formation in carotid arteries induced by carotid artery ligation or wire injury was greatly decreased in endothelium-specific ENH knockout mice.Vascular ligation reduced the phosphorylation of AKT and eNOS and NO production in endothelium of control but not ENH knockout mice.ENH was found to interact with AKT1 and its phosphatases PHLPP2.AKT and eNOS activation were prolonged in VEGF-induced endothelial cells with either ENH or PHLPP2 deficiency.In addition,inhibitors of either AKT or eNOS effectively restored ligation-induced neointima formation in ENH deficient mice.Moreover,endothelium-specific PHLPP2-knockout mice displayed reduced ligation-induced neointima formation.Finally,immunofluorescence staining showed that PHLPP2 were increased in the endothelia of human atherosclerotic plaques.Publically available database(GEO)showed that PHLPP2 were increased in the human atherosclerotic vessels.Quantitative PCR showed that m RNA encoding PHLPP2 was increased in blood cells from patients with coronary artery disease.Conclusions: ENH forms a complex with AKT1 and its phosphatases PHLPP2 to negatively regulate AKT1 activation in the artery endothelium.AKT1 deactivation and the subsequent suppression of NO generation induced by vascular injury are mediated by ENH and PHLPP2.ENH and PHLPP2 are thus new pro-atherosclerotic factors that could be therapeutically targeted.