Simvastatin inhibits TNFα-induced invasion of human cardiac myofibroblasts via both MMP-9-dependent and -independent mechanisms

Abstract 

Statins can reduce adverse myocardial remodeling independently of their cholesterol-lowering ability. We have previously reported that simvastatin inhibits tumor necrosis factor-α (TNFα)-induced cardiac myofibroblast invasion and MMP-9 secretion, key events in this remodeling process. The aim of the present study was to investigate the mechanisms underlying this effect. Selective MMP-9 gene silencing with siRNA oligonucleotides revealed that myofibroblast invasion through a Matrigel barrier (Boyden chamber assay) was MMP-9-dependent. In contrast, cell migration (in the absence of Matrigel) was MMP-9-independent. Simvastatin, a commonly prescribed statin, inhibited both invasion and migration of myofibroblasts and disrupted the actin cytoskeleton as determined by confocal microscopy of rhodamine–phalloidin staining. All these effects of simvastatin were mimicked by the Rho-kinase inhibitor Y27632. TNFα activated the ERK-1/2, p38 MAPK, PI-3-kinase and NF-κB pathways but not the JNK pathway, as determined by immunoblotting with phospho-specific antibodies. Quantitative RT-PCR revealed that TNFα-induced MMP-9 mRNA expression was substantially reduced by pharmacological inhibitors of the ERK-1/2, PI-3-kinase and NF-κB pathways. However, none of the signal transduction pathways studied was influenced by simvastatin treatment. Moreover, despite reducing MMP-9 secretion, simvastatin had no effect on MMP-9 promoter activity (luciferase reporter assay) and actually increased MMP-9 mRNA levels. In summary, simvastatin reduces TNFα-induced invasion of human cardiac myofibroblasts through two distinct mechanisms: (i) by attenuating cell migration via Rho-kinase inhibition and subsequent cytoskeletal disruption, and (ii) by decreasing MMP-9 secretion via a post-transcriptional mechanism.

Keywords: Statin, Tumor necrosis factor alpha, Cardiac fibroblast, Myofibroblast, MMP-9, Invasion, Migration, Rho kinase, Signal transduction pathways, siRNA