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Peroxynitrite mediates diabetes-induced endothelial dysfunction: possible role of Rho kinase activation.

El-Remessy AB, Tawfik HE, Matragoon S, Pillai B, Caldwell RB, Caldwell RW - Exp Diabetes Res (2010)

Bottom Line: Diabetic coronary arteries showed significant reduction in ACh-mediated maximal relaxation compared to controls.Diabetic vessels showed also significant increases in lipid-peroxides, nitrotyrosine, and active RhoA and 50% reduction in eNOS mRNA expression.Treatment of diabetic animals with FeTTPS blocked these effects.

View Article: PubMed Central - PubMed

Affiliation: College of Pharmacy, The University of Georgia, 1120 15th Street, HM-1200, Augusta, GA 30912, USA. aelremessy@mcg.edu

ABSTRACT
Endothelial dysfunction is characterized by reduced bioavailability of NO due to its inactivation to form peroxynitrite or reduced expression of eNOS. Here, we examine the causal role of peroxynitrite in mediating diabetes-induced endothelial dysfunction. Diabetes was induced by STZ-injection, and rats received the peroxynitrite decomposition catalyst (FeTTPs, 15 mg/Kg/day) for 4 weeks. Vasorelaxation to acetylcholine, oxidative-stress markers, RhoA activity, and eNOS expression were determined. Diabetic coronary arteries showed significant reduction in ACh-mediated maximal relaxation compared to controls. Diabetic vessels showed also significant increases in lipid-peroxides, nitrotyrosine, and active RhoA and 50% reduction in eNOS mRNA expression. Treatment of diabetic animals with FeTTPS blocked these effects. Studies in aortic endothelial cells show that high glucose or peroxynitrite increases the active RhoA kinase levels and decreases eNOS expression and NO levels, which were reversed with blocking peroxynitrite or Rho kinase. Together, peroxynitrite can suppress eNOS expression via activation of RhoA and hence cause vascular dysfunction.

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FeTPPs inhibited diabetes-induced RhoA activation in rat vessels and BAEC. (a) Pull-down assay showed significant increases in active RhoA in diabetic aortic homogenate compared to that of controls that were blocked by cotreatment with FeTPPs (n = 6). Values are expressed as means ± SEM, *P < .05 versus control. (b) Bovine aortic endothelial cells (BAECs) were incubated with normal (NG, 5 mM) and high glucose (HG, 25 mM) for 3 days or exogenous peroxynitrite (PN, 100 μM) for 18 hours (n = 4 in each group). High glucose significantly increased active RhoA, which was prevented by cotreatment with FeTPPs (Fe, 2.5 μM). Peroxynitrite exerted similar effects to high-glucose in increasing active RhoA (~2-fold) compared to decomposed peroxynitrite (DPN). Values are expressed as means ± SEM, *P < .05 versus control.
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fig4: FeTPPs inhibited diabetes-induced RhoA activation in rat vessels and BAEC. (a) Pull-down assay showed significant increases in active RhoA in diabetic aortic homogenate compared to that of controls that were blocked by cotreatment with FeTPPs (n = 6). Values are expressed as means ± SEM, *P < .05 versus control. (b) Bovine aortic endothelial cells (BAECs) were incubated with normal (NG, 5 mM) and high glucose (HG, 25 mM) for 3 days or exogenous peroxynitrite (PN, 100 μM) for 18 hours (n = 4 in each group). High glucose significantly increased active RhoA, which was prevented by cotreatment with FeTPPs (Fe, 2.5 μM). Peroxynitrite exerted similar effects to high-glucose in increasing active RhoA (~2-fold) compared to decomposed peroxynitrite (DPN). Values are expressed as means ± SEM, *P < .05 versus control.

Mentions: Activation of small GTPases such as RhoA has been shown to modulate eNOS expression at the mRNA level. Therefore, we measured the levels of active RhoA in aortic endothelial homogenate from various groups. The results showed that diabetes causes significant activation of RhoA (2-fold) compared to controls that blocked by treatment with FeTPPs (Figure 4(a)). The specific role of peroxynitrite in activating RhoA was further examined by comparing active RhoA levels in BAEC cultured in high glucose (25 mM) to cultures treated with exogenous peroxynitrite (100 μM). The results showed that both high glucose and exogenous peroxynitrite can directly activate RhoA in BAEC (Figure 4(b)). The effects of high glucose in inducing active RhoA were blunted by cotreatment of BAEC with FeTPPs (2.5 mM).


Peroxynitrite mediates diabetes-induced endothelial dysfunction: possible role of Rho kinase activation.

El-Remessy AB, Tawfik HE, Matragoon S, Pillai B, Caldwell RB, Caldwell RW - Exp Diabetes Res (2010)

FeTPPs inhibited diabetes-induced RhoA activation in rat vessels and BAEC. (a) Pull-down assay showed significant increases in active RhoA in diabetic aortic homogenate compared to that of controls that were blocked by cotreatment with FeTPPs (n = 6). Values are expressed as means ± SEM, *P < .05 versus control. (b) Bovine aortic endothelial cells (BAECs) were incubated with normal (NG, 5 mM) and high glucose (HG, 25 mM) for 3 days or exogenous peroxynitrite (PN, 100 μM) for 18 hours (n = 4 in each group). High glucose significantly increased active RhoA, which was prevented by cotreatment with FeTPPs (Fe, 2.5 μM). Peroxynitrite exerted similar effects to high-glucose in increasing active RhoA (~2-fold) compared to decomposed peroxynitrite (DPN). Values are expressed as means ± SEM, *P < .05 versus control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig4: FeTPPs inhibited diabetes-induced RhoA activation in rat vessels and BAEC. (a) Pull-down assay showed significant increases in active RhoA in diabetic aortic homogenate compared to that of controls that were blocked by cotreatment with FeTPPs (n = 6). Values are expressed as means ± SEM, *P < .05 versus control. (b) Bovine aortic endothelial cells (BAECs) were incubated with normal (NG, 5 mM) and high glucose (HG, 25 mM) for 3 days or exogenous peroxynitrite (PN, 100 μM) for 18 hours (n = 4 in each group). High glucose significantly increased active RhoA, which was prevented by cotreatment with FeTPPs (Fe, 2.5 μM). Peroxynitrite exerted similar effects to high-glucose in increasing active RhoA (~2-fold) compared to decomposed peroxynitrite (DPN). Values are expressed as means ± SEM, *P < .05 versus control.
Mentions: Activation of small GTPases such as RhoA has been shown to modulate eNOS expression at the mRNA level. Therefore, we measured the levels of active RhoA in aortic endothelial homogenate from various groups. The results showed that diabetes causes significant activation of RhoA (2-fold) compared to controls that blocked by treatment with FeTPPs (Figure 4(a)). The specific role of peroxynitrite in activating RhoA was further examined by comparing active RhoA levels in BAEC cultured in high glucose (25 mM) to cultures treated with exogenous peroxynitrite (100 μM). The results showed that both high glucose and exogenous peroxynitrite can directly activate RhoA in BAEC (Figure 4(b)). The effects of high glucose in inducing active RhoA were blunted by cotreatment of BAEC with FeTPPs (2.5 mM).

Bottom Line: Diabetic coronary arteries showed significant reduction in ACh-mediated maximal relaxation compared to controls.Diabetic vessels showed also significant increases in lipid-peroxides, nitrotyrosine, and active RhoA and 50% reduction in eNOS mRNA expression.Treatment of diabetic animals with FeTTPS blocked these effects.

View Article: PubMed Central - PubMed

Affiliation: College of Pharmacy, The University of Georgia, 1120 15th Street, HM-1200, Augusta, GA 30912, USA. aelremessy@mcg.edu

ABSTRACT
Endothelial dysfunction is characterized by reduced bioavailability of NO due to its inactivation to form peroxynitrite or reduced expression of eNOS. Here, we examine the causal role of peroxynitrite in mediating diabetes-induced endothelial dysfunction. Diabetes was induced by STZ-injection, and rats received the peroxynitrite decomposition catalyst (FeTTPs, 15 mg/Kg/day) for 4 weeks. Vasorelaxation to acetylcholine, oxidative-stress markers, RhoA activity, and eNOS expression were determined. Diabetic coronary arteries showed significant reduction in ACh-mediated maximal relaxation compared to controls. Diabetic vessels showed also significant increases in lipid-peroxides, nitrotyrosine, and active RhoA and 50% reduction in eNOS mRNA expression. Treatment of diabetic animals with FeTTPS blocked these effects. Studies in aortic endothelial cells show that high glucose or peroxynitrite increases the active RhoA kinase levels and decreases eNOS expression and NO levels, which were reversed with blocking peroxynitrite or Rho kinase. Together, peroxynitrite can suppress eNOS expression via activation of RhoA and hence cause vascular dysfunction.

Show MeSH
Related in: MedlinePlus