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

Abstract

Nitrate tolerance is associated with an enhanced superoxide anion (O(2)(-)) 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. 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. 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 O(2)(-) 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. 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. 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. Superoxide anion formation was increased after NTG treatment in both mice strains and the rosuvastatin + NTG cotreatment was able to reduce that production. 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. These findings suggest that long-term rosuvastatin treatment protects against nitrate tolerance by counteracting NTG-induced increase in O(2)(-) production, probably via a direct interaction with the NAD(P)H oxidase pathway.

Figure 1

Concentration–response curves to NTG in wt mice aortas. Preparations were precontracted with U-46619 (10⁻⁷ M) and NTG was given cumulatively. ^*P<0.05 versus control wt and ^†P<0.05 versus control eNOS^−/−. Results are expressed as mean±s.e.m.

Figure 2

(a) Concentration–response curves to NTG in wt mice aortas. Preparations were precontracted with U-46619 (10⁻⁷ M) and NTG was given cumulatively. ^*P<0.05 versus control and ^†P<0.05 versus rosuvastatin–NTG. Results are expressed as mean±s.e.m. (b) Concentration–effect curve to NTG in eNOS^−/− mice aortas. Preparations were precontracted with U-46619 (10⁻⁷ M) and NTG was given cumulatively. ^*P<0.05 versus control and ^†P<0.05 versus rosuvastatin–NTG. Results are expressed as mean±s.e.m.

Figure 3

(a) Concentration–effect curve to NTG in wt mice aortas. Preparations were precontracted with U-46619 (10⁻⁷ M) and NTG was given cumulatively. ^*P<0.05 versus control. Results are expressed as mean±s.e.m. (b) Concentration–effect curve to NTG in eNOS^−/− mice aortas. Preparations were precontracted with U-46619 (10⁻⁷ M) and NTG was given cumulatively. ^*P<0.05 versus control. Results are expressed as mean±s.e.m.

Figure 4

(a) NAD(P)H oxidase activity assay in aortas from wt mice. Data are expressed as RLU and presented as mean±s.e.m. NTG treatment for 4 days significantly increased the NAD(P)H oxidase activity, which was abolished by rosuvastatin treatment (rosuvastatin–NTG group). ^*P<0.05 versus all other groups. (b) NAD(P)H oxidase activity in aortas from eNOS^−/− mice. Data are expressed as RLU and presented as mean±s.e.m. NTG treatment for 4 days significantly increased the NAD(P)H oxidase activity, which was abolished by rosuvastatin treatment (rosuvastatin–NTG group). ^*P<0.05 versus all other groups.

Figure 5

RT–PCR results in aortas from wild-type (wt) mice. (a) The bar graph corresponds to the densitometric analysis and (b) the stained agarose gel contains the RT–PCR products. The intensity of expression of the three genes of interest (p22phox, gp91phox and eNOS) normalized to the intensity of expression of the internal standard gene GAPDH from the same aorta sample. The normalized mRNA of the three genes of interest from the control mice was set as 100%. ^*P<0.05 versus control wt.

Figure 6

RT–PCR analysis in aortas from eNOS^−/− mice. The intensity of expression of the two genes of interest (p22phox, gp91phox) was normalized to the intensity of expression of the internal standard gene GAPDH from the same aorta sample. The normalized mRNA expression of the two genes of interest from the control mice was set as 100%. ^*P<0.05 versus control eNOS^−/−.

Figure 7

RT–PCR analysis in aortas from wt mice. (a) The bar graph corresponds to the densitometric analysis and (b) the stained agarose gel contains the RT–PCR products. The intensity of expression of the three genes of interest (p22phox, gp91phox and eNOS) was normalized to the intensity of expression of the internal standard gene GAPDH from the same aorta sample. The normalized mRNA expressions of the three genes of interest from the control mice were set as 100%. ^*P<0.05 versus control wt.

Figure 8

RT–PCR analysis in aortas from eNOS^−/− mice. The intensity of expression of the two genes of interest (p22phox, gp91phox) was normalized to the intensity of expression of the internal standard gene GAPDH from the same aorta sample. The normalized mRNA expressions of the two genes of interest from the control mice were set as 100%. ^*P<0.05 versus control eNOS^−/−.

Figure 9

RT–PCR analysis in aortas from eNOS^−/− and wt mice. The intensity of expression of p22phox, gp91phox and eNOS mRNA expression was normalized to the intensity of expression of the internal standard gene GAPDH from the same aorta sample. The normalized mRNA expressions of p22phox, gp91phox and eNOS from the control mice were set as 100%. ^*P<0.05 versus the respective control.

Figure 10

(a) RT–PCR analysis in wt mice. The intensity of expression of rac-1 mRNA expression was normalized to the intensity of expression of the internal standard gene GAPDH from the same aorta sample. The normalized mRNA expression of rac-1 from the control mice was set as 100%. ^*P<0.05 versus control wt. (b) RT–PCR analysis in eNOS^−/− mice. The intensity of expression of rac-1 mRNA expression was normalized to the intensity of expression of the internal standard gene GAPDH from the same aorta sample. The normalized mRNA expression of rac-1 from the control mice was set as 100%. ^*P<0.05 versus control eNOS^−/−.