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 ( 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. 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 - 0.1mM) 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. 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 O-2(-) production, probably via a direct interaction with the NAD( P) H oxidase pathway.