High glucose-induced Nox1-derived superoxides downregulate PKC-betaII, which subsequently decreases ACE2 expression and ANG(1-7) formation in rat VSMCs.

In rat diabetic animal models, ANG(1-7) treatment prevents the development of cardiovascular complications. Angiotensin-converting enzyme (ACE)2 is a major ANG(1-7)-generating enzyme in vascular smooth muscle cells (VSMCs), and its expression is decreased by a prolonged exposure to high glucose (HG), which is reflected by lower ANG(1-7) levels. However, the underlying mechanism of its downregulation is unknown and was the subject of this study. Rat aortic VSMCs were maintained in normal glucose (NG) or HG ( approximately 4.1 and approximately 23.1 mmol/l, respectively) for up to 72 h. Several PKC and NADPH oxidase inhibitors and short interfering (si)RNAs were used to determine the mechanism of HG-induced ACE2 downregulation. Cell lysates were subjected to Western blot analysis, real-time quantitative PCR, and ANG(1-7) radioimmunodetection. At 72 h of HG exposure, ACE2 mRNA, protein, and ANG(1-7) levels were decreased (0.17 +/- 0.01-, 0.47 +/- 0.03-, and 0.16 +/- 0.01-fold, respectively), and the expression of NADPH oxidase subunit Nox1 was increased (1.70 +/- 0.2-fold). The HG-induced ACE2 decrease was reversed by antioxidants and Nox1 siRNA as well as by inhibitors of glycotoxin formation. ACE2 expression was PKC-betaII dependent, and PKC-betaII protein levels were reduced in the presence of HG (0.32 +/- 0.03-fold); however, the PKC-betaII inhibitor CG-53353 prevented the HG-induced ACE2 loss and Nox1 induction, suggesting a nonspecific effect of the inhibitor. Our data suggest that glycotoxin-induced Nox1 expression is regulated by conventional PKCs. ACE2 expression is PKC-betaII dependent. Nox1-derived superoxides reduce PKC-betaII expression, which lowers ACE2 mRNA and protein levels and consequently decreases ANG(1-7) formation.