Anne Otto

Rosuvastatin treatment protects against nitrate-induced oxidative stress.

Nitrate tolerance is associated with an enhanced superoxide anion production and can be attenuated by statins, which interact with the 2 main [eNOS and NAD(P)H oxidase] pathways involved in producing this oxidative stress. Three groups of normocholesterolemic rats were treated: group 1 received rosuvastatin (10 mg/kg/d PO) for 5 weeks and in the last 3 days cotreatment with nitroglycerin (NTG 50 mg/kg/d, subcutaneous injections BID); group 2 received only NTG (50 mg/kg/d BID for the last 3 days); and group 3 served as control. Rings of thoracic aortas from these groups were studied in organ baths. Relaxations to NTG (0.1 nM to 0.1 mM) were determined on phenylephrine-preconstricted rings and O2− production (RLU/10 s/mg dry weight) was assessed by lucigenin and the luminol analogue (L-012) chemiluminescence technique. In group 2 (NTG), the concentration-response curves to NTG were significantly shifted to the right: the pD2 (−log NTG concentration evoking a half-maximal relaxation) was 6.75 ± 0.06 (n = 7) versus 7.75 ± 0.07 (n = 7) in group 3 (not exposed to NTG, P < 0.05); O2− production was enhanced (10,060 ± 1,205, n = 7 versus 5,235 ± 1,052, n = 7; P < 0.05). In contrast, in group 1, the rightward shift was attenuated: pD2 value was 7.20 ± 0.10 (n = 8), P < 0.05 versus group 2; O2− production was decreased (5911 ± 663; n = 9, P < 0.05 versus group 2). In addition, before NTG exposure, rosuvastatin treatment decreased p22phox [the essential NAD(P)H oxidase subunit] abundance in the aortic wall and decreased NAD(P)H oxidase activity. In contrast, this treatment did not alter either eNOS abundance or the basal release of endothelium-derived NO. Interestingly, in vivo treatment with apocynin, an NAD(P)H oxidase inhibitor, produced a protection similar to that with rosuvastatin. Long-term rosuvastatin treatment protects against nitrate tolerance in the rat aorta by counteracting NTG-induced increase in O2− production. This protection seems to involve a direct interaction with the NAD(P)H oxidase pathway rather than an up-regulation of the eNOS pathway.

Prevention of Nitrate Tolerance by Long-Term Treatment with Statins

AbstractRecent studies have shown that statins seem to upregulate the endothelial NO synthase pathway (eNOS) and may, therefore, enhance NO availability, a direct scavenger of O2− and an inhibitor of oxidative enzymes. Methods. To assess whether the oxidative stress produced by an in vivo exposure to nitroglycerin (NTG) is attenuated by statins, 4 groups of normocholesterolemic rats were treated; group 1 received pravastatin (20 mg/kg/d p.o) and group 2 atorvastatin (10 mg/kg/d) both for 5 weeks and the last 3 days, a cotreatment with the statin plus NTG (50 mg/kg/d, s.c. injections b.i.d.); group 3 (NTG) received only NTG (50 mg/kg/d, b.i.d. for 3 days) and group 4 served as control. Rings of thoracic aortas from these groups were studied in organ baths. Relaxations to NTG (0.1 nM to 0.1 mM) were determined on phenylephrine-preconstricted rings and O2− production (counts/10 s/mg) was assessed by lucigenin chemiluminescence technique. Results. In vivo NTG exposure induced a rightward shift of the concentration-response curves to NTG: the pD2 (−log NTG concentration evoking a half maximal relaxation) was 5.8 ± 0.3 (n = 7) vs. 7.2 ± 0.2 in the control group (not exposed to NTG, n = 7) and O2− production was enhanced (1259 ± 71 vs. 787 ± 76, (n = 5) P < .05). In contrast, groups 1 (n = 7) and 2 (n = 7) behaved as the control group (pD2 values were 7.4 ± 0.1 (n = 7) and 6.9 ± 0.1 (n = 7); O2− production was 721 ± 109 and 647 ± 121). The protective effect on nitrate tolerance disappeared when L-NAME (an eNOS inhibitor, 100 mg/kg/d) was co-administered with NTG in groups 1 and 2. Incubation of aortic rings with NAD(P)H (100 μM) also impaired the protective effect of both statins. Moreover, before NTG exposure, aortic cGMP content, reflecting EDNO availability, was significantly enhanced in group 1 (P < .05 vs. control). Conclusion. Long-term statin treatment protects against nitrate tolerance by counteracting NTG-induced increase in O2− production. Both eNOS pathway and NAD(P)H oxidases seem to be involved in this protective mechanism.

Rosuvastatin treatment protects against nitrate‐induced oxidative stress in eNOS knockout mice: implication of the NAD(P)H oxidase pathway

1 Nitrate tolerance is associated with an enhanced superoxide anion (O2−) production and may be attenuated by statins as they interact with the two main endothelial NO synthase (eNOS) and NAD(P)H oxidase pathways involved in this oxidative stress. 2 Groups of wild‐type (wt, C57Bl/6J) and eNOS knock‐out mice (eNOS−/−) received rosuvastatin (20 mg kg−1 day−1 p.o.) for 5 weeks and a cotreatment with the statin plus nitroglycerin (NTG; 30 mg kg−1 day−1, subcutaneous injections b.i.d.) for the last 4 days. Another group received only NTG (30 mg kg−1 d−1, b.i.d. for 4 days) and finally control mice from both strains received no treatment. 3 Rings of thoracic aortas from these groups were studied in organ baths. Relaxations to NTG (0.1 nM–0.1 mM) were determined on thromboxane analogue (U44619)‐precontracted rings and O2− production (RLU 5 s−1 mg−1 of total protein content) was assessed in aorta homogenates with the lucigenin‐enhanced chemiluminescence technique. Reverse transcriptase–polymerase chain reaction analysis was performed on aortas from both mice strains. 4 In vivo NTG treatment induced a significant rightward shift of the concentration–effect curve to NTG compared to control group. There was, however, no cross‐tolerance with non‐nitrate sources of NO (unaltered response to acetylcholine in wt group). The rosuvastatin+NTG cotreatment was able to protect against the development of nitrate tolerance in both mice strains and L‐mevalonate abolished this protective effect of rosuvastatin. 5 In vivo treatment with apocynin, a purported NAD(P)H oxidase inhibitor, also produced a similar protection to that observed with rosuvastatin in both strains. 6 Superoxide anion formation was increased after NTG treatment in both mice strains and the rosuvastatin+NTG cotreatment was able to reduce that production. 7 Moreover, rosuvastatin treatment abolished the increase in gp91phox mRNA (an endothelial membrane NAD(P)H oxidase subunit) expression induced by in vivo exposure to NTG. 8 These findings suggest that long‐term rosuvastatin treatment protects against nitrate tolerance by counteracting NTG‐induced increase in O2− production, probably via a direct interaction with the NAD(P)H oxidase pathway.

Ramipril treatment protects against nitrate-induced oxidative stress in eNOS-/- mice: An implication of the NADPH oxidase pathway

The development of nitrate tolerance has been found to be associated with vascular production of superoxide anion (O2−·), generated mainly by the eNOS and NADPH oxidase pathways. The aim of our study was to investigate whether long-term angiotensin-converting enzyme inhibition by ramipril is able to protect against nitrate tolerance in the aortas of eNOS-deficient (eNOS−/−) mice and to assess the implication of the NADPH oxidase pathway. Therefore, 3 types of treatment were given to wild-type (WT) and eNOS−/− mice: group 1 received ramipril for 5 weeks and a co-treatment with ramirpil plus nitroglycerine (NTG) during the last 4 days, group 2 received only NTG, and group 3 served as control. Relaxations to NTG (0.1 nmol/L to 0.1 mmol/L) were determined on U44619, a thromboxane analogue, precontracted rings, and O2−· production were assessed on aorta homogenates with the lucigenin-enhanced chemiluminescence technique. Cyclic guanosine monophosphate and reverse-transcriptase-polymerase chain reaction analyses were performed on whole mouse aortas. In WT group 2, the concentration-effect curves to NTG were significantly shifted to the right: the pD2 was 6.16 ± 0.17 (n = 6) vs 6.81 ± 0.10 (n = 6) in WT group 3 (not exposed to NTG; P < 0.05) and O2−· production was enhanced from 100% ± 11% (n = 9) to 191% ± 21% (n = 6; P < 0.01). In contrast, in WT group 1, the rightward shift was abolished: the pD2 value was 6.73 ± 0.13 (n = 6; NS vs group 3 WT) and O2−· production was 117% ± 6% (n = 7; NS vs group 3 WT). In eNOS−/− groups 1 and 3, similar data were observed: the pD2 values were 7.58 ± 0.08 and 7.38 ± 0.11 (NS) vs 6.89 ± 0.20 in eNOS−/− group 2 (n = 6; P < 0.01). In the WT mice aortas, ramipril treatment significantly increased the cyclic guanosine monophosphate levels (reflecting nitric oxide availability), which returned to control values after in vivo co-treatment with a bradykinin BK2 antagonist (Icatibant). In both strains, candesartan, an AT1 blocker, was also able to protect against the development of nitrate tolerance. Moreover, before NTG exposure, ramipril treatment decreased p22phox and gp91phox (essential NADPH oxidase subunits) mRNA expression in aortas from both mice strains. In conclusion, long-term ramipril treatment in mice protects against the development of nitrate tolerance by counteracting NTG-induced increase in O2−· production, which involves a direct interaction with the NADPH oxidase pathway and seems to be completely independent of the eNOS pathway.