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

Eduard N. Lavrentyev, Kafait Malik

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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 (∼4.1 and ∼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-βII dependent, and PKC-βII protein levels were reduced in the presence of HG (0.32 ± 0.03-fold); however, the PKC-βII 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-βII dependent. Nox1-derived superoxides reduce PKC-βII expression, which lowers ACE2 mRNA and protein levels and consequently decreases ANG(1-7) formation.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Heart and Circulatory Physiology
Volume296
Issue number1
DOIs
StatePublished - Jan 1 2009

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Vascular Smooth Muscle
Superoxides
Smooth Muscle Myocytes
Down-Regulation
Glucose
NADPH Oxidase
Small Interfering RNA
Radioimmunodetection
Messenger RNA
Proteins
Real-Time Polymerase Chain Reaction
Animal Models
Antioxidants
Western Blotting
Enzymes

All Science Journal Classification (ASJC) codes

  • Physiology
  • Cardiology and Cardiovascular Medicine
  • Physiology (medical)

Cite this

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title = "High glucose-induced Nox1-derived superoxides downregulate PKC-βII, 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 (∼4.1 and ∼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-βII dependent, and PKC-βII protein levels were reduced in the presence of HG (0.32 ± 0.03-fold); however, the PKC-βII 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-βII dependent. Nox1-derived superoxides reduce PKC-βII expression, which lowers ACE2 mRNA and protein levels and consequently decreases ANG(1-7) formation.",
author = "Lavrentyev, {Eduard N.} and Kafait Malik",
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T1 - High glucose-induced Nox1-derived superoxides downregulate PKC-βII, which subsequently decreases ACE2 expression and ANG(1-7) formation in rat VSMCs

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AU - Malik, Kafait

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N2 - 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 (∼4.1 and ∼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-βII dependent, and PKC-βII protein levels were reduced in the presence of HG (0.32 ± 0.03-fold); however, the PKC-βII 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-βII dependent. Nox1-derived superoxides reduce PKC-βII expression, which lowers ACE2 mRNA and protein levels and consequently decreases ANG(1-7) formation.

AB - 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 (∼4.1 and ∼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-βII dependent, and PKC-βII protein levels were reduced in the presence of HG (0.32 ± 0.03-fold); however, the PKC-βII 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-βII dependent. Nox1-derived superoxides reduce PKC-βII expression, which lowers ACE2 mRNA and protein levels and consequently decreases ANG(1-7) formation.

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