Abstract 19752: Nox4 Expression Regulates Inflammation in Tgfβ-treated Vascular Smooth Muscle Cells

Abstract

NADPH oxidases (NOX) are a major source of reactive oxygen species in the cardiovascular system, contributing to the pathogenesis of disease. We recently reported that increased NOX4 expression in aging results in enhanced cellular and mitochondrial oxidative stress, vascular inflammation, dysfunction, and atherosclerosis. However, the role of NOX4 in the activation of inflammatory molecular pathways in vascular smooth muscle cells (VSMC) under pathophysiological conditions has not been elucidated. In this study, we investigated whether inhibition of NOX4 expression modulates proinflammatory gene expression in mouse VSMC by suppressing its expression with a novel peptide-mediated siRNA transfection approach. Using melittin derived peptide p5RHH, we achieved significantly higher transfection efficiency (92% vs 85% with Lipofectamine) and decreased toxicity (p<0.01 vs Lipofectamine in MTT assay). TGFβ, a proinflammatory cytokine, known to induce NOX4 expression significantly upregulated NOX4 mRNA (p<0.01) and protein expression (p<0.01) in VSMC. p5RHH mediated NOX4 siRNA transfection attenuated TGFβ induced upregulation of NOX4 mRNA (p<0.01) and protein (p<0.01) levels. Analysis of inflammatory marker genes, CCL2, CCL5, IL6, and VCAM-1, showed that NOX4 knockdown significantly attenuated the expression of these genes in TGFβ-treated VSMC, with the highest inhibition seen in the expression of IL6. Congruent with this, inflammatory marker gene expression, which increases with aging, was significantly lower in VSMC isolated from aged (16 months old) NOX4 knockout mice compared with the aged wild-type. TGFβ-activated kinase 1 (TAK1, also known as MAP3K7) is an upstream regulator of inflammatory signaling in cells treated with cytokines. p5RHH mediated TAK1 siRNA transfection downregulated AP1 and NFκB activation and inhibited NOX4 expression in wild-type VSMC, indicating that TAK1 regulates NOX4 expression via regulation of AP1 and NFκB. Because plasma TGFβ levels increase with aging, it may be of one of the factors contributing to increased NOX4 expression with aging. Our data indicate that NOX4 is a potential therapeutic target for modulation of inflammation in aging and aging-associated cardiovascular disease.