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.

Fig. 1.

Nox1, Nox4, PKC-βII, and glucose transporter 1 (GLUT1) short interfering (si)RNA efficiency. Subconfluent rat aortic vascular smooth muscle cells (VSMCs) were transfected in 6-well plates with siRNA for Nox1, Nox4, PKC-βII, GLUT1, or cyclophilin B (control siRNA) for 96 h. A: Nox1 expression; B: PKC-βII expression; C: Nox4 expression; D: GLUT1 expression. Data are shown as mRNA abundance and protein levels for each target gene (means ± SE; n = 4). *P < 0.05 vs. the corresponding value with control siRNA.

Fig. 2.

Effect of high glucose (HG) on angiotensin-converting enzyme (ACE)2 expression and ANG(1-7) formation. A: ACE2 mRNA expression (means ± SE; n = 9). B: ACE2 protein levels in cell lysates (means ± SE; n = 11). C: ACE2 protein levels in cultured media (means ± SE; n = 4). D: ACE2 protein (media/cell) ratio (means ± SE; n = 4). E: ANG(1-7) levels in cell lysates (means ± SE; n = 3). *P < 0.05 vs. the corresponding value in the presence of normal glucose (NG). F: ACE2 protein levels in cell lysates. Mannitol, l-glucose, and 2-deoxyglucose (2-DO-glucose) were used as an osmotic controls (means ± SE; n = 3). *P < 0.05 vs. vehicle.

Fig. 3.

Cytochalasin B, alrestatin, and aminoguanidine diminish the effect of HG on ACE2 expression and ANG(1-7) levels. A: effect of 1 μmol/l cytochalasin B on ACE2 expression in the presence of HG. B: effect of 10 μmol/l aminoguanidine on ACE2 expression in the presence of HG. C: effect of 10 μmol/l alrestatin on ACE2 expression in the presence of HG (means ± SE; n = 3). †P < 0.05 vs. 0 h; *P < 0.05 vs. HG alone at the corresponding time points. D: effect of inhibitors on endogenous ANG(1-7) formation (means ± SE; n = 3). †P < 0.05 vs. vehicle in the presence of NG; *P < 0.05 vs. vehicle in the presence of HG; #P < 0.05 vs. the corresponding treatment in the presence of NG.

Fig. 4.

GLUT1 siRNA diminishes the effect of HG on ACE2 mRNA expression and ACE2 protein levels. Shown are the effects of GLUT1 silencing on ACE2 expression in the presence of HG (means ± SE; n = 3). †P < 0.05 vs. 0 h; *P < 0.05 vs. the value at corresponding time points where cells were treated with HG alone.

Fig. 5.

NADPH oxidase inhibitors/general antioxidants and catalase diminished the effect of HG on ACE2 protein levels. A: ACE2 protein levels in VSMCs treated with 1 μmol/l diphenyleneiodonium (DPI) in the presence of HG (means ± SE; n = 5). B: ACE2 protein levels in VSMCs treated with 10 μmol/l apocynin in the presence of HG (means ± SE; n = 3). C: treatment with 150–200 U/ml catalase diminished the effect of HG on ACE2 protein levels (means ± SE; n = 7). *P < 0.05 vs. the corresponding value in the presence of HG alone; †P < 0.05 vs. the 0-h value.

Fig. 6.

Effect of NADPH oxidase inhibitors, Nox1 and Nox4 siRNA, or catalase on ACE2 expression and ANG(1-7) levels. A: ACE2 mRNA expression and protein levels (means ± SE; n = 3). B: ANG(1-7) levels (means ± SE; n = 3). *P < 0.05 vs. vehicle in the presence of HG; †P < 0.05 vs. the corresponding treatment in the presence of NG; #P < 0.05 vs. vehicle in the presence of NG.

Fig. 7.

Specificity of Nox1 and Nox4 siRNA. A: effect of Nox4 siRNA on Nox1 mRNA expression. B: effect of Nox1 siRNA on Nox4 mRNA expression. Data are means ± SE; n = 3.

Fig. 8.

Effect of PKC inhibitors and PKC-βII siRNA on ACE2 expression and ANG(1-7) levels in the presence of NG or HG. A: ACE2 mRNA expression (means ± SE; n = 3) and protein levels (means ± SE; n = 4). *P < 0.05 vs. vehicle in the presence of HG; †P < 0.05 vs. the corresponding treatment in the presence of NG; #P < 0.05 vs. vehicle in the presence of NG. B: ACE2 mRNA expression (means ± SE; n = 3) and protein levels (means ± SE; n = 5). *P < 0.05 vs. the corresponding time point in the presence of NG; †P < 0.05 vs. vehicle in the presence of NG. C: ANG(1-7) levels in cell lysates (means ± SE; n = 3). *P < 0.05 vs. vehicle in the presence of HG; †P < 0.05 vs. vehicle in the presence of NG; #P < 0.05 vs. vehicle in the presence of NG. D: effect of PKC-βII silencing on ACE2 mRNA expression and protein levels in the presence of NG or HG (means ± SE; n = 3). #P < 0.05 vs. vehicle in the presence of NG.

Fig. 9.

Effect of HG on Nox1 and Nox4 expression. A: Nox1 mRNA abundance and protein levels (means ± SE; n = 4). B: Nox4 mRNA abundance and protein levels (means ± SE; n = 3). CT, threshold cycle. *P < 0.05 vs. the corresponding value in the presence of NG.

Fig. 10.

Effect of cytochalasin B, alrestatin, aminoguanidine, PKC inhibitors, and PKC-β siRNA on Nox1 expression. A: Nox1 mRNA abundance and protein levels (means ± SE; n = 5). B: Nox1 mRNA and protein levels (means ± SE; n = 4). *P < 0.05 vs. vehicle in the presence of HG; †P < 0.05 vs. the corresponding value in the presence of NG.

Fig. 11.

CG-53353 diminished the effect of HG on PKC-β mRNA expression and PKC-β isoform splicing. A: PKC-β mRNA expression (means ± SE; n = 4). B: PKC-βI protein levels (means ± SE; n = 4). C: phospho- and total PKC-βII protein levels (means ± SE; n = 4). D: effect of CG-53353 on PKC-β mRNA expression in the presence of HG (means ± SE; n = 7). E and F: effect of CG-53353 on PKC-βI and PKC-βII protein levels in VSMCs in the presence of HG (means ± SE; n = 4). *P < 0.05 vs. the corresponding value in the presence of NG; #P < 0.05 vs. the 0-h value.

Fig. 12.

Effect of cytochalasin B, alrestatin, aminoguanidine, and NADPH oxidase inhibition on PKC-βII expression. A: PKC-β mRNA expression and PKC-βII protein levels (means ± SE; n = 5). B: PKC-β mRNA and PKC-βII protein levels (means ± SE; n = 3). *P < 0.05 vs. vehicle in the presence of HG; †P < 0.05 vs. the corresponding treatment in the presence of NG.

Fig. 13.

Proposed mechanism of HG-induced ACE2 downregulation and ANG(1-7) formation. AR, aldose reductase; AGEs, advanced glycation end-products.