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Pharmacological induction of vascular extracellular superoxide dismutase expression in vivo.

Oppermann M, Balz V, Adams V, Dao VT, Bas M, Suvorava T, Kojda G - J. Cell. Mol. Med. (2008)

Bottom Line: These effects are associated with decreased vascular superoxide production, but the underlying molecular mechanisms remain unknown.A similar increase was found in aortic homogenates. eNOS(++) lung cytosols showed an increase of ecSOD protein level of 142 +/- 10.5% as compared with transgene-negative littermates (P < 0.05), which was abolished by N(omega)-nitro-L-arginine treatment.Up-regulation of vascular ecSOD may contribute to the reported antioxidative and anti-atherosclerotic effects of PETN.

View Article: PubMed Central - PubMed

Affiliation: Institute for Pharmacology and Clinical Pharmacology, Heinrich-Heine-University, Duesseldorf, Germany.

ABSTRACT
Pentaerythritol tetranitrate (PETN) treatment reduces progression of atherosclerosis and endothelial dysfunction and decreases oxidation of low-density lipoprotein (LDL) in rabbits. These effects are associated with decreased vascular superoxide production, but the underlying molecular mechanisms remain unknown. Previous studies demonstrated that endogenous nitric oxide could regulate the expression of extracellular superoxide dismutase (ecSOD) in conductance vessels in vivo. We investigated the effect of PETN and overexpression of endothelial nitric oxide synthase (eNOS(++)) on the expression and activity of ecSOD. C57BL/6 mice were randomized to receive placebo or increasing doses of PETN for 4 weeks and eNOS(++) mice with a several fold higher endothelial-specific eNOS expression were generated. The expression of ecSOD was determined in the lung and aortic tissue by real-time PCR and Western blot. The ecSOD activity was measured using inhibition of cytochrome C reduction. There was no effect of PETN treatment or eNOS overexpression on ecSOD mRNA in the lung tissue, whereas ecSOD protein expression increased from 2.5-fold to 3.6-fold (P < 0.05) by 6 mg PETN/kg body weight (BW)/day and 60 mg PETN/kg BW/day, respectively. A similar increase was found in aortic homogenates. eNOS(++) lung cytosols showed an increase of ecSOD protein level of 142 +/- 10.5% as compared with transgene-negative littermates (P < 0.05), which was abolished by N(omega)-nitro-L-arginine treatment. In each animal group, the increase of ecSOD expression was paralleled by an increase of ecSOD activity. Increased expression and activity of microvascular ecSOD are likely induced by increased bioavailability of vascular nitric oxide. Up-regulation of vascular ecSOD may contribute to the reported antioxidative and anti-atherosclerotic effects of PETN.

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ecSOD protein expression. (A) Increased ecSOD protein expression in lung cytosolic fractions of C57Bl/6 mice treated with 0, 6 or 60 mg/kg/day PETN (PETN-0, PETN-6 or PETN-60, respectively, *=P < 0.05 for PETN-60 versus PETN-0). (B) Increased ecSOD protein expression in aortic homogenates of PETN-treated mice (*=P < 0.01 versus PETN-0). (C) Increased ecSOD protein expression in lung cytosols of eNOS++versus eNOSn mice (P < 0.05). Treatment with L-NA significantly lowered ecSOD expression in both groups (eNOSn/L-NA and eNOS++/L-NA; each *=P < 0.05 versus eNOSn) and blunted the difference between both groups (N.S. =P > 0.05). (D) ecSOD protein in blood plasma of eNOS++ mice was significantly increased compared with transgene-negative littermates (eNOSn, *=P < 0.01).
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fig03: ecSOD protein expression. (A) Increased ecSOD protein expression in lung cytosolic fractions of C57Bl/6 mice treated with 0, 6 or 60 mg/kg/day PETN (PETN-0, PETN-6 or PETN-60, respectively, *=P < 0.05 for PETN-60 versus PETN-0). (B) Increased ecSOD protein expression in aortic homogenates of PETN-treated mice (*=P < 0.01 versus PETN-0). (C) Increased ecSOD protein expression in lung cytosols of eNOS++versus eNOSn mice (P < 0.05). Treatment with L-NA significantly lowered ecSOD expression in both groups (eNOSn/L-NA and eNOS++/L-NA; each *=P < 0.05 versus eNOSn) and blunted the difference between both groups (N.S. =P > 0.05). (D) ecSOD protein in blood plasma of eNOS++ mice was significantly increased compared with transgene-negative littermates (eNOSn, *=P < 0.01).

Mentions: PETN increased ecSOD protein expression in the lungs to 251 ± 91.0% (n= 5) in the PETN-6 group and to 362 ± 84.9% (n= 6) in the PETN-60 group (n= 6, P < 0.0479 versus PETN-0; Fig. 3A). A comparable result was obtained with a polyclonal ecSOD antibody (data not shown). A post-hoc analysis revealed statistical significance for PETN-60 versus PETN-0 but not for PETN-6 versus PETN-0 (Fig. 3A). Similar data were obtained in the aortas of the PETN-treated mice (Fig. 3B). The expression in PETN-6 mice was 152 ± 18.9% (n= 6) and that in PETN-60 was 207 ± 34.7% (n= 5) as compared with PETN-0 mice (P < 0.0116, for anova; *=P < 0.01 for PETN-0 versus PETN-60, post-hoc analysis).


Pharmacological induction of vascular extracellular superoxide dismutase expression in vivo.

Oppermann M, Balz V, Adams V, Dao VT, Bas M, Suvorava T, Kojda G - J. Cell. Mol. Med. (2008)

ecSOD protein expression. (A) Increased ecSOD protein expression in lung cytosolic fractions of C57Bl/6 mice treated with 0, 6 or 60 mg/kg/day PETN (PETN-0, PETN-6 or PETN-60, respectively, *=P < 0.05 for PETN-60 versus PETN-0). (B) Increased ecSOD protein expression in aortic homogenates of PETN-treated mice (*=P < 0.01 versus PETN-0). (C) Increased ecSOD protein expression in lung cytosols of eNOS++versus eNOSn mice (P < 0.05). Treatment with L-NA significantly lowered ecSOD expression in both groups (eNOSn/L-NA and eNOS++/L-NA; each *=P < 0.05 versus eNOSn) and blunted the difference between both groups (N.S. =P > 0.05). (D) ecSOD protein in blood plasma of eNOS++ mice was significantly increased compared with transgene-negative littermates (eNOSn, *=P < 0.01).
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fig03: ecSOD protein expression. (A) Increased ecSOD protein expression in lung cytosolic fractions of C57Bl/6 mice treated with 0, 6 or 60 mg/kg/day PETN (PETN-0, PETN-6 or PETN-60, respectively, *=P < 0.05 for PETN-60 versus PETN-0). (B) Increased ecSOD protein expression in aortic homogenates of PETN-treated mice (*=P < 0.01 versus PETN-0). (C) Increased ecSOD protein expression in lung cytosols of eNOS++versus eNOSn mice (P < 0.05). Treatment with L-NA significantly lowered ecSOD expression in both groups (eNOSn/L-NA and eNOS++/L-NA; each *=P < 0.05 versus eNOSn) and blunted the difference between both groups (N.S. =P > 0.05). (D) ecSOD protein in blood plasma of eNOS++ mice was significantly increased compared with transgene-negative littermates (eNOSn, *=P < 0.01).
Mentions: PETN increased ecSOD protein expression in the lungs to 251 ± 91.0% (n= 5) in the PETN-6 group and to 362 ± 84.9% (n= 6) in the PETN-60 group (n= 6, P < 0.0479 versus PETN-0; Fig. 3A). A comparable result was obtained with a polyclonal ecSOD antibody (data not shown). A post-hoc analysis revealed statistical significance for PETN-60 versus PETN-0 but not for PETN-6 versus PETN-0 (Fig. 3A). Similar data were obtained in the aortas of the PETN-treated mice (Fig. 3B). The expression in PETN-6 mice was 152 ± 18.9% (n= 6) and that in PETN-60 was 207 ± 34.7% (n= 5) as compared with PETN-0 mice (P < 0.0116, for anova; *=P < 0.01 for PETN-0 versus PETN-60, post-hoc analysis).

Bottom Line: These effects are associated with decreased vascular superoxide production, but the underlying molecular mechanisms remain unknown.A similar increase was found in aortic homogenates. eNOS(++) lung cytosols showed an increase of ecSOD protein level of 142 +/- 10.5% as compared with transgene-negative littermates (P < 0.05), which was abolished by N(omega)-nitro-L-arginine treatment.Up-regulation of vascular ecSOD may contribute to the reported antioxidative and anti-atherosclerotic effects of PETN.

View Article: PubMed Central - PubMed

Affiliation: Institute for Pharmacology and Clinical Pharmacology, Heinrich-Heine-University, Duesseldorf, Germany.

ABSTRACT
Pentaerythritol tetranitrate (PETN) treatment reduces progression of atherosclerosis and endothelial dysfunction and decreases oxidation of low-density lipoprotein (LDL) in rabbits. These effects are associated with decreased vascular superoxide production, but the underlying molecular mechanisms remain unknown. Previous studies demonstrated that endogenous nitric oxide could regulate the expression of extracellular superoxide dismutase (ecSOD) in conductance vessels in vivo. We investigated the effect of PETN and overexpression of endothelial nitric oxide synthase (eNOS(++)) on the expression and activity of ecSOD. C57BL/6 mice were randomized to receive placebo or increasing doses of PETN for 4 weeks and eNOS(++) mice with a several fold higher endothelial-specific eNOS expression were generated. The expression of ecSOD was determined in the lung and aortic tissue by real-time PCR and Western blot. The ecSOD activity was measured using inhibition of cytochrome C reduction. There was no effect of PETN treatment or eNOS overexpression on ecSOD mRNA in the lung tissue, whereas ecSOD protein expression increased from 2.5-fold to 3.6-fold (P < 0.05) by 6 mg PETN/kg body weight (BW)/day and 60 mg PETN/kg BW/day, respectively. A similar increase was found in aortic homogenates. eNOS(++) lung cytosols showed an increase of ecSOD protein level of 142 +/- 10.5% as compared with transgene-negative littermates (P < 0.05), which was abolished by N(omega)-nitro-L-arginine treatment. In each animal group, the increase of ecSOD expression was paralleled by an increase of ecSOD activity. Increased expression and activity of microvascular ecSOD are likely induced by increased bioavailability of vascular nitric oxide. Up-regulation of vascular ecSOD may contribute to the reported antioxidative and anti-atherosclerotic effects of PETN.

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Related in: MedlinePlus