Anti-atherogenic effect of statins: role of nitric oxide, peroxynitrite and haem oxygenase-1

Abstract

Background and purpose: The pleiotropic effects of HMG-CoA inhibitors (statins), which include anti-inflammation, antioxidation and immunomodulation, are not yet fully understood. The present study was designed to elucidate the role of nitric oxide (NO), peroxynitrite (ONOO(-)) and haem oxygenase-1 (HO-1) in the anti-atherogenic effect of statins.

Experimental approach: Normal and atherosclerotic New Zealand rabbits were treated with atorvastatin or simvastatin in the presence or absence of inhibitors and promoters of endothelial nitric oxide synthase (eNOS) and HO-1. NO and ONOO(-) released from isolated aortae by calcium ionophore were measured with nanosensors placed 6 +/- 2 nm from aortic endothelium. Expression of eNOS and HO-1 protein, HO activity, plasma malondialdehyde (MDA) and vessel wall thickness were also measured.

Key results: Hypercholesterolaemia decreased eNOS expression by 31 +/- 3%, decreased NO (230 +/- 16 vs. 433 +/- 17 nmol x L(-1) control) and increased cytotoxic ONOO(-) (299 +/- 15 vs. 187 +/- 11 nmol x L(-1) control). The concentration ratio of [NO]/[ONOO(-)] decreased from 2.3 +/- 0.1 (normal) to 0.7 +/- 0.1 indicating an increase of nitroxidative stress in atherosclerotic endothelium. Expression of HO-1 protein increased by 20 +/- 8% in atherosclerosis and further increased (about 30%) after treatment with statins. Statins partially restored the [NO]/[ONOO(-)] balance (1.5 +/- 0.1 for atorvastatin and 1.4 +/- 0.1 simvastatin), decreased MDA and wall thickening. Promoters of eNOS and HO-1 (L-arginine and haemin) ameliorated the [NO]/[ONOO(-)] ratio while their inhibitors (L-NAME or tin-protoporphyrin) showed no improvement in these ratio.

Conclusions and implications: Atherosclerosis induced an endothelial [NO]/[ONOO(-)] balance indicative of endothelial dysfunction. Statins showed anti-atherosclerotic effects mediated by HO-1/eNOS, restoring the [NO]/[ONOO(-)] imbalance and reducing lipid peroxidation.

Figure 2

Total activity of HO, measured in the aortas of the different animal groups: (A) normal animals (control) and atherosclerotic animals treated with atorvastin or simvastatin; (B) atherosclerotic groups either treated with statins (atorvastatin or simvastatin) and L-NAME or SnPP or treated with L-arginine or haemin. n = 6 for each bar; *P < 0.05 versus control group and **P < 0.05 versus atherosclerotic group.

Figure 1

Immunoblots showing the expression of HO-1 protein from isolated aortas of: (A) normal and atherosclerotic rabbits treated with atorvastatin or simvastatin for 1 month; (B) atherosclerotic groups either co-treated with L-NAME and atorvastatin/simvastatin or treated with L-arginine; (C) atherosclerotic group either co-treated with SnPP and atorvastatin/simvastatin or treated with haemin. Representative Western blots and the bar graph show analysis of four separate experiments; *P < 0.05 versus control and **P < 0.05 versus atherosclerotic group.

Figure 4

Calcium ionophore-stimulated maximal NO and ONOO⁻ release from isolated aortas of atherosclerotic groups either co-treated with L-NAME and atorvastatin/simvastatin or treated with L-arginine (A), and from isolated aortas of atherosclerotic group either co-treated with SnPP and atorvastatin/simvastatin or treated with haemin (B). In C & D are shown the ratios of NO concentration to ONOO⁻ concentration, from the treatment groups shown in A and B. n = 6 for each bar; *P < 0.05 versus atherosclerotic group.

Figure 3

Typical amperograms of the changes in NO (A) and ONOO⁻ (B) concentrations, recorded by nanosensors from isolated aortas of normal and atherosclerotic rabbits treated with atorvastatin or simvastatin for 1 month. The release of NO and ONOO⁻ were stimulated by 1 µmol·L⁻¹ calcium ionophore A23187 (Cal at arrow) in control group and atherosclerotic groups, non-treated or treated with atorvastatin or simvastatin. (C) Calcium ionophore-stimulated maximal NO and ONOO⁻ release, and (D) ratio of NO concentration to ONOO⁻ concentration of the above corresponding groups. n = 6 for each bar; *P < 0.05 versus control group and **P < 0.05 versus atherosclerotic group.

Figure 5

Immunoblots showing the expression of eNOS protein from isolated aortas of: (A) normal and atherosclerotic rabbits treated with atorvastatin or simvastatin for 1 month; (B) atherosclerotic groups either co-treated with L-NAME and atorvastatin/simvastatin or treated with L-arginine; (C) atherosclerotic group either co-treated with SnPP and atorvastatin/simvastatin or treated with haemin. Representative Western blot and bar graph show analysis of four separate experiments; *P < 0.05 versus control group and **P < 0.05 versus atherosclerotic group.

Figure 6

Plasma malondialdehyde (MDA) levels on the different treatment groups: (A) control group and atherosclerotic animals treated with atorvastin or simvastatin; (B) MDA levels plotted against % changes (vs. control) in ONOO⁻ production in control, non-treated atherosclerotic group and atherosclerotic group treated with atorvastatin and simvastatin; (C) Atherosclerotic groups either co-treated with statins (atorvastatin or simvastatin) and L-NAME or SnPP or treated with L-arginine or haemin. n = 6 for each bar; *P < 0.05 versus control group and **P < 0.05 versus atherosclerotic group.

Figure 8

Representative microscopic analysis of lesion area of the aortic arch sections from atherosclerotic rabbits co-treated with atorvastatin and L-NAME or SnPP and treated with either L-arginine or haemin for one month; stained with haematoxylin and eosin (×100); L, lumen; FS, fatty streak; VW, vessel wall; F, adventitial fatty tissue. (B) % change of the vessel wall thickness of the aortic arch from normal control or atherosclerotic control was quantitated using an image analysis system. n = 6 for each bar; *P < 0.05 versus control group and **P < 0.05 versus atherosclerotic group.

Figure 7

Representative microscopic analysis of lesion area of the aortic arch sections from control and atherosclerotic rabbits treated with atorvastatin for 1 month and stained with haematoxylin and eosin (×100); L, lumen; FS, fatty streak; VW, vessel wall; F, adventitial fatty tissue. (B) % change of the vessel wall thickness of the aortic arch from normal control or from atherosclerotic control was quantitated using an image analysis system. n = 6 for each bar; *P < 0.05 versus control group and **P < 0.05 versus atherosclerotic group.