Atorvastatin inhibition of cytokine-inducible nitric oxide synthase expression in native endothelial cells in situ

Abstract

1. Animal experimental studies have demonstrated that inducible nitric oxide synthase (iNOS) expression correlates with neointima formation and is prevented by HMG-CoA reductase inhibitors (statins). In the present study we have investigated the underlying mechanism of action of these drugs in isolated segments of the rat aorta. 2. Western blot analysis and immunohistochemistry revealed that tumour necrosis factor α (TNFα) plus interferon-γ (IFNγ) synergistically induce iNOS gene expression in the endothelium but not in the smooth muscle of these segments while constitutive endothelial NO synthase (eNOS) abundance was markedly reduced. 3. Pre-treatment with 1 - 10 μM atorvastatin, cerivastatin or pravastatin decreased TNFα plus IFNγ stimulated iNOS expression in the endothelium irrespective of the presence of the HMG-CoA reductase product mevalonate (400 μM). 4. Electrophoretic mobility shift assay experiments confirmed that the combination of TNFα plus IFNγ causes activation of the transcription factors STAT-1 and NF-κB in native endothelial cells. Neutralization of these transcription factors by employing the corresponding decoy oligonucleotides confirmed their involvement in TNFα plus IFNγ stimulated iNOS expression. Translocation of both transcription factors was attenuated by atorvastatin, and this effect was insensitive to exogenous mevalonate. 5. The present findings thus demonstrate a specific HMG-CoA reductase-independent inhibitory effect of statins, namely atorvastatin, on cytokine-stimulated transcription factor activation in native endothelial cells in situ and the subsequent expression of a gene product implicated in vascular inflammation. This effect may be therapeutically relevant and in addition provide an explanation for the reported rapid onset of action of these drugs in humans.