title: Hydrogen peroxide induces overexpression of angiotensin-converting enzyme in human umbilical vein endothelial cells.
abstract: Oxidative stress has been linked to endothelial dysfunction in atherosclerosis and hypertension. The present study was designed to investigate the effect of hydrogen peroxide (H2O2) on angiotensin-converting enzyme (ACE), a key regulator of the renin-angiotensin system, and the mechanisms underlying ACE regulation in human umbilical vein endothelial cells (HUVECs). We used Tetrazolium bromide (MTT) assay for cell viability, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay for cell apoptosis, enzyme-linked immunosorbent assay (ELISA) for cAMP measurement, real-time PCR for mRNA detection, and Western blot for protein analysis in the study. Our results demonstrated that H2O2 (50-1000 μM) decreased HUVECs viability by inducing apoptosis. Notably, H2O2 upregulated ACE expression in a concentration-dependent manner. H2O2 100 μM significantly enhanced cyclic adenosine monophosphate (cAMP) expression by 1.48-fold (P<0.05). Additionally, forskolin 10 μM, a cAMP agonist, was also found to enhance ACE expression by 1.78-fold (P<0.05); in contrast, H-89 10 μM, a protein kinase A (PKA) inhibitor, abolished H2O2-induced ACE expression and prevented the enhancing effect of forskolin-induced ACE expression. Similar effects on ACE mRNA were also observed. cAMP-response element-specific decoy oligodeoxynucleotides (CRE-dODN) containing binding sites for cAMP-response element-binding protein (CREB) inhibited ACE expression at both the mRNA and protein levels. Negative control CRE-dODN had no effect on ACE expression. We conclude that H2O2 upregulates the expression of ACE through the activation of cAMP/PKA/CREB signal pathway in HUVECs, indicating a role of oxidative stress in the pathophysiology of hypertension.
authors.affiliation: Department of Pharmacology, Harbin Medical University, Harbin, PR China.
authors.forename: Xiaoqin
authors.initials: X
authors.lastname: Mu
authors.forename: Kaiwen
authors.initials: K
authors.lastname: He
authors.forename: Hui
authors.initials: H
authors.lastname: Sun
authors.forename: Xin
authors.initials: X
authors.lastname: Zhou
authors.forename: Lingling
authors.initials: L
authors.lastname: Chang
authors.forename: Xin
authors.initials: X
authors.lastname: Li
authors.forename: Wenfeng
authors.initials: W
authors.lastname: Chu
authors.forename: Guofen
authors.initials: G
authors.lastname: Qiao
authors.forename: Yanjie
authors.initials: Y
authors.lastname: Lu
mesh_terms: D017209:Apoptosis; D050882:CREB-Binding Protein / Q000378:metabolism; D002460:Cell Line; D002470:Cell Survival; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000037:antagonists & inhibitors / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D042783:Endothelial Cells / Q000378:metabolism; D061307:Human Umbilical Vein Endothelial Cells / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D006973:Hypertension; D020287:In Situ Nick-End Labeling; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D018384:Oxidative Stress; D007703:Peptidyl-Dipeptidase A / Q000096:biosynthesis / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D015533:Transcriptional Activation
chemical_list: D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D005576:Colforsin; D006861:Hydrogen Peroxide; D000242:Cyclic AMP; D050882:CREB-Binding Protein; D017868:Cyclic AMP-Dependent Protein Kinases; D007703:Peptidyl-Dipeptidase A