title: High glucose-induced Nox1-derived superoxides downregulate PKC-betaII, which subsequently decreases ACE2 expression and ANG(1-7) formation in rat VSMCs.
abstract: 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.
authors.affiliation: Dept. of Pharmacology, Univ. of Tennessee Health Science Center, 874 Union Ave., Crowe Bldg. 217, Memphis, TN 38163, USA.
authors.forename: Eduard N
authors.initials: EN
authors.lastname: Lavrentyev
authors.forename: Kafait U
authors.initials: KU
authors.lastname: Malik
mesh_terms: D000803:Angiotensin I / Q000096:biosynthesis*; D000085962:Angiotensin-Converting Enzyme 2; D000818:Animals; D015153:Blotting, Western; D015536:Down-Regulation / Q000187:drug effects; D005947:Glucose / Q000494:pharmacology*; D051272:Glucose Transporter Type 1 / Q000096:biosynthesis / Q000235:genetics; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D000074624:NADPH Oxidase 1; D019255:NADPH Oxidases / Q000378:metabolism; D016877:Oxidants / Q000378:metabolism*; D010446:Peptide Fragments / Q000096:biosynthesis*; D007703:Peptidyl-Dipeptidase A / Q000096:biosynthesis* / Q000235:genetics; D010797:Phthalimides / Q000494:pharmacology; D011493:Protein Kinase C / Q000096:biosynthesis* / Q000235:genetics; D064546:Protein Kinase C beta; D034741:RNA, Small Interfering / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013481:Superoxides / Q000378:metabolism*; D014162:Transfection
chemical_list: D051272:Glucose Transporter Type 1; D016877:Oxidants; D010446:Peptide Fragments; D010797:Phthalimides; D034741:RNA, Small Interfering; C495679:Slc2a1 protein, rat; D013481:Superoxides; D000803:Angiotensin I; D009247:NADH, NADPH Oxidoreductases; D000074624:NADPH Oxidase 1; D019255:NADPH Oxidases; C000614712:NOX1 protein, rat; D011493:Protein Kinase C; D064546:Protein Kinase C beta; D007703:Peptidyl-Dipeptidase A; C000705329:Ace2 protein, rat; D000085962:Angiotensin-Converting Enzyme 2; C118790:angiotensin I (1-7); D005947:Glucose; C098874:4,5-dianilinophthalimide