Pharmacological induction of vascular extracellular superoxide dismutase expression in vivo

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.

Figure 1

eNOS and blood pressure in eNOS-overexpressing mice. (A) Blood pressure in C57Bl/6 mice before (initial) and after treatment with PETN (not significant). (B) Untreated eNOS⁺⁺ mice have a significant lower blood pressure than eNOSⁿ (*=P < 0.05). After 25 days of treatment with the eNOS inhibitor L-NA, this difference disappeared (P > 0.05). (C) Upper panel: Western blot for eNOS in aortic homogenates of eNOS⁺⁺; lower panel (n= 4): a mean of 2.8 ± 0.5-fold greater eNOS expression than in eNOSⁿ (n= 4, *=P < 0.05).

Figure 2

ecSOD mRNA levels. (A) Treatment of C57Bl/6 mice with 6 mg/kg/day (PETN-6) or 60 mg/kg/day (PETN-60) PETN induced no changes in lung ecSOD mRNA expression relative to HPRT (P > 0.05 versus PETN-0). (B) eNOS⁺⁺ mice induced no changes in lung ecSOD mRNA expression relative to HPRT (P > 0.05 versus eNOSⁿ). (C) eNOS⁺⁺ mice induced no changes in aortic ecSOD mRNA expression relative to SMA (P > 0.05 versus eNOSⁿ), irrespective of oral treatment with L-NA.

Figure 3

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).

Figure 4

ecSOD activity. (A) The activity of extracellular superoxide dismu-tase (ecSOD) in lung tissue of eNOS⁺⁺ mice was significantly increased compared with eNOSⁿ mice (*=P < 0.05). (B) Treatment of C57Bl/6 mice with 6 or 60 mg/kg/day pentaerythritol tetranitrate (PETN-6 or PETN-60, respectively) similarly resulted in increased activities compared with control mice (PETN-0). *=P < 0.05 versus PETN-0.