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.

Keywords::

• oxidative stress
• ACE
• cAMP
• hypertension
• HUVECs