Inhibition Of Endothelial NADPH Oxidase 4 Exacerbates Atherosclerosis Through Induction Of Endoplasmic Reticulum Stress And Soluble Epoxide Hydrolase

Background:The morbidity and mortality rates of cardiovascular diseases are are higher than neoplasms and other diseases,and accounting for more than 40 percent of deaths from disease in the population.Atherosclerosis is an important pathological basis for cardiovascular disease,often leading to stroke and coronary heart disease.The onset of atherosclerosis is dysfunction of the endothelium located in the vascular lining,causing mononuclear cell infiltration.Monocyte-releasing cytokines stimulate the proliferation and crawling of smooth muscle cells located in the vascular mesentery,promoting the production of new endothelium.In addition,monocytes activate into macrophages along with smooth muscle cells in the neointima to ingest lipids that turn into foam cells and form atherosclerotic plaques.Nicotinamide adenine dinucleotide phosphate（NADPH）oxidase is a membrane protein widely distributed in tissues and organs in vivo,and NADPH oxidase 4（Nox4）is a major isoform of the Nox family of proteins in blood vessels and is abundantly expressed in both endothelial cells and smooth muscle cells.It has been previously reported by our group that atherosclerosis susceptibility conditions（high fat,diabetes,blood turbulence,etc.）down-regulate endothelial Nox4 expression and cause endothelial Nox4 dysfunction.Endothelial Nox4dysfunction exacerbates atherosclerosis by up-regulating soluble epoxide hydrolase 2（sEH,encoding the gene EPHX2）,a pro-inflammatory factor that is presumably inhibited by the in vivo administration of the sEH inhibitor TPPU,thereby ameliorating endothelial Nox4 dysfunction-induced atherosclerosis.Nox4 is a key protein that directly catalyzes the production of hydrogen peroxide（H₂O₂）,regulates the redox state of cells,and is involved in cellular physiological and pathological processes.Because ER requires an oxidized environment to maintain its normal function,it is speculated that endothelial Nox4 dysfunction may induce endoplasmic reticulum stress（ERS）,which in turn affects endothelial function.OBJECTIVE:This paper builds on previous research to further explore the role of upregulated sEH in the promotion of atherosclerosis by endothelial Nox4 dysfunction and whether endothelial Nox4 dysfunction exacerbates atherosclerosis by inducing ERS.To explore whether ERS mediates the regulation of sEH expression by endothelial Nox4 dysfunction and whether upregulation of sEH expression can also cause ERS.METHODS:Endothelial Nox4 Dominate Negative（EDN）mice were used to mimic downregulation of vascular endothelial Nox4 expression and dysfunction in atherosclerosis susceptible sites.1.High-fat-fed Apo E-/-mice and EDN/Apo E-/-mice were divided into control and sEH inhibitor TPPU-administered groups,respectively,to observe the effect of sEH inhibition on atherosclerotic lesions.2.In isolated Apo E-/-mice with EDN/Apo E-/-mice vascular endothelial cells,expression of ERS-associated proteins such as phosphorylated protein kinase R-like endoplasmic reticulum kinase（Phospho Protein Kinase R-like ER Kinase,p-PERK）,serine/threonine protein kinase/endonuclease inositol-requiring enzyme 1（Inositol-Requiring Enzyme 1α,IRE-1α）,glucose regulated protein 78（GRP78）also known as immunoglobulin heavy chain binding protein（BIP）,activating transcription factor 6（ATF6）,etc.was detected.3.In EDN/Apo E-/-endothelial cells,the ERS inhibitor sodium 4-phenylbutyrate（4-PBA）or a solvent control was given to detect the expression of sEH and Vascular Cell Adhesion Molecule 1（VCAM1）and the adhesion of macrophages to endothelial cells.4.EDN/Apo E-/-endothelial cell were given the sEH inhibitor TPPU or a solvent control to detect the expression levels of ERS-associated proteins.Endothelial cells of Apo E-/-mice,overexpressing wild-type sEH or null virus controls,were examined for expression levels of ERS-associated proteins.5.High-fat feeding of EDN/Apo E-/-mice to accelerate atherosclerosis formation while administering the ERS inhibitor 4-PBA or a solvent control to test whether inhibition of ERS improves atherosclerosis exacerbated by endothelial Nox4dysfunction.RESULTS:1)In EDN/Apo E-/-endothelial cells,the sEH inhibitor TPPU decreased the expression of inflammatory factors.2)In high-fat-fed EDN/Apo E-/-mice,TPPU significantly inhibited atherosclerotic plaque formation compared with solvent controls,but did not affect the proportion of necrotic nuclei in the plaque,suggesting that upregulation of sEH mediates the regulation of atherosclerosis by endothelial Nox4dysfunction.3)Nox4 dysfunction in endothelial cells induced ERS.4)ERS mediated the regulation of sEH and inflammatory responses by endothelial Nox4 dysfunction.5)sEH and ERS were positively regulated by each other and co-regulated inflammatory responses.6)ERS inhibitor 4-PBA significantly improved atherosclerosis exacerbated by endothelial Nox4 dysfunction.CONCLUSION:Endothelial Nox4 dysfunction induced by atherosclerosis susceptibility conditions promotes inflammatory responses by inducing ERS and upregulating sEH,thereby accelerating the atherosclerotic process.Endothelial sEH and ERS are positively regulated by each other.Inhibition of either sEH or ERS in vivo can improve atherosclerosis caused by endothelial Nox4 dysfunction.