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

Lavrentyev EN, Malik KU. High glucose-induced Nox1-derived superoxides downregulate PKC-beta II, which subsequently decreases ACE2 expression and ANG(1-7) formation in rat VSMCs. Am J Physiol Heart Circ Physiol 296: H106-H118, 2009. First published October 31, 2008; doi: 10.1152/ajpheart.00239.2008. - 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 (similar to 4.1 and similar to 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-beta II dependent, and PKC-beta II protein levels were reduced in the presence of HG (0.32 +/- 0.03-fold); however, the PKC-beta II 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-beta II dependent. Nox1-derived superoxides reduce PKC-beta II expression, which lowers ACE2 mRNA and protein levels and consequently decreases ANG(1-7) formation.