Statins are lipid-lowering drugs that lower serum cholesterol levels by inhibiting the conversion of HMG-CoA to mevalonic acid, thereby attenuating the biosynthesis of cholesterol (Andrews et al. 2001). However, statins also possess pleiotropic effects unrelated to lipid lowering, which may influence atherosclerotic lesion development. One example of this is inhibition of leukocyte recruitment (Weitz-Schmidt et al. 2001). Nitric oxide may also influence leukocyte adhesion through alterations in expression of endothelial cell adhesion molecules and production of inflammatory cytokines (Lin et al. 2001; Burke-Gaffney et al. 1997). We hypothesised, therefore, that nitrated statins may have an enhanced ability to modulate leukocyte function compared to native statins. The aim was to assess the effect of the NicOx compound NO-pravastatin (NCX6550) in comparison with native pravastatin on leukocyte adhesion and ROS production ex vivo, using normal (C57Bl6) and atherosclerotic mice (ApoE knockout; Charles River Laboratories, France; bred in house).

C57Bl6 and ApoE K/O mice (n = 8-12) were dosed orally with a 200µL volume of pravastatin
(P; 40mgkg^-1), NO-pravastatin (N; equimolar dose) or vehicle (V; 10:7:2:1 H₂O: PEG-400: castor oil: DMSO) for 5 days. Mice were killed by CO₂ asphyxiation and the aorta removed and cut into 3 segments (aortic arch, thoracic and abdominal), and each segment was pinned out luminol side up. The homologous spleen was isolated and disrupted over a 200µm mesh; the resulting cell suspension was centrifuged, and erythrocytes lysed with H₂O. Leukocytes were resuspended at 1x10⁶ cells per ml and labelled for 1h at 37°C with 185kBq of ⁵¹Cr. The artery segments were stimulated with 10Uml^-1 thrombin for 10min and incubated with a 5µL aliquot of the labelled leukocytes for 30min at 37°C, washed, and assayed for 51Cr on a gamma counter. ROS production by PMA (phorbol myristate acetate)-stimulated leukocytes was measured by luminol-enhanced chemiluminescence.

NO-pravastatin significantly reduced leukocyte adhesion to the aortic arch of C57Bl6 (V 24±4%; P 24±5%; N 10±2%, p<0.05 vs. V & P, n = 8) and to the thoracic segment of ApoE K/O mice (V 23±5%; P 23±6%; N 9±3%, p<0.05 vs. V & P, n = 10). NO-pravastatin also significantly reduced ROS production by ApoE K/O leukocytes stimulated with 50µgml^-1 (V 3630±180; P 2070±1170; N 1610±523 arb. units, p<0.05 vs. V, n = 4) and 100µgml^-1 (V 485±345; P 2410±1140; N 2320±523 arb. units, p<0.01 vs. V, n = 4) of PMA.

This study has demonstrated that NO-pravastatin, in comparison to normal pravastatin, reduces leukocyte adhesion in both control and ApoE knockout mice and ROS production from ApoE-derived leukocytes. These results may have implications for the treatment and prevention of atherosclerosis. This study was supported by NicOx (Italy).

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Weitz-Schmidt et al. (2001). Nat. Med. 7: 687 - 692.