Rosuvastatin upregulates the antioxidant defense protein heme oxygenase-1ā˜†

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Abstract

Cholesterol-independent, pleiotropic actions of HMG-CoA reductase inhibitors (statins) lead to anti-inflammatory and antioxidant actions by as yet unidentified mechanisms. This study explores the role of heme oxygenase-1 (HO-1) as target and potential mediator of rosuvastatin. In cultured human endothelial cells (ECV 304), rosuvastatin increased HO-1 mRNA and protein levels in a concentration-dependent fashion. HO-1 induction by rosuvastatin remained unaffected by mevalonate and N-nitro-l-arginine-methylester, showing that isoprenoid- and NO-dependent pathways were not involved. Pretreatment of endothelial cells with rosuvastatin reduced NADPH-dependent production of oxygen radicals. The HO-1 metabolite bilirubin, when added exogenously to the cells, virtually abolished NADPH-dependent oxidative stress. Rosuvastatin-induced inhibition of free radical formation was rescued in the presence of the HO inhibitor, tin protoporphyrin-IX. Our results demonstrate that HO-1 is a target site and antioxidant mediator of rosuvastatin in endothelial cells. This novel pathway may contribute to and partially explain the pleiotropic antiatherogenic actions of rosuvastatin.

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Materials and methods

Materials. Fetal bovine serum, cell culture media, and penicillinā€“streptomycin were obtained from Gibco (Eggenstein, FRG). The Chemiluminescence Western Blotting Kit was from Amersham (Freiburg, FRG) and the Random Primed DNA Labeling Kit was from Roche (Penzberg, FRG). HO-1 primary antibody and anti-rabbit peroxidase-conjugated secondary antibody were obtained from Alexis (GrĆ¼nberg, FRG). Rosuvastatin was provided by AstraZeneca UK. For HO-1 probes, the template was an EcoRI restriction

HO-1 mRNA and protein analysis

An 8-h incubation with rosuvastatin led to a concentration-dependent increase of HO-1 mRNA expression by up to twofold over basal values (Fig. 1). Rosuvastatin-dependent HO-1 induction was also observed at the protein level (Fig. 2). Concentration-dependent increases in HO-1 protein occurred between 3 and 300Ā Ī¼M rosuvastatin (Fig. 2). Induction of HO-1 protein was detectable after 4Ā h of exposure and reached a maximum after 12Ā h (not shown). The HMG-CoA product mevalonate was used to investigate

Discussion

It is generally accepted that mechanisms beyond reduction of plasma cholesterol contribute significantly to the antiatherogenic and tissue protective properties of statins. Pleiotropic, cholesterol-independent effects of statins previously reported include elevations in eNOS and tissue-type plasminogen activator expression as well as reduction of pro-inflammatory signaling such as cytokine and oxygen radical formation [7], [8].

This study demonstrates that the antioxidant defense protein heme

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    ā˜†

    Abbreviations: HO-1, heme oxygenase-1; HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; NO, nitric oxide; NOS, NO synthase; l-NAME, N-nitro-l-arginine-methylester; ROS, reactive oxygen species; SnPP, tin protoporphyrin-IX.

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