NADPH oxidase 4 regulates vascular inflammation in aging and atherosclerosis

We recently reported that increased NADPH oxidase 4 (NOX4) expression and activity during aging results in enhanced cellular and mitochondrial oxidative stress, vascular inflammation, dysfunction, and atherosclerosis. The goal of the present study was to elucidate the molecular mechanism(s) for these effects and determine the importance of NOX4 modulation of proinflammatory gene expression in mouse vascular smooth muscle cells (VSMCs). A novel peptide-mediated siRNA transfection approach was used to inhibit Nox4 expression with minimal cellular toxicity. Using melittin-derived peptide p5RHH, we achieved significantly higher transfection efficiency (92% vs. 85% with Lipofectamine) and decreased toxicity (p < 0.001 vs. Lipofectamine in MIT and p < 0.0001 vs. Lipofectamine in LDH assays) in VSMCs. TGF01 significantly upregulates Nox4 mRNA (p < 0.01) and protein (p < 0.01) expression in VSMCs. p5RHH-mediated Nox4 siRNA transfection greatly attenuated TGF beta 1-induced upregulation of Nox4 mRNA (p < 0.01) and protein (p < 0.0001) levels and decreased hydrogen peroxide production (p < 0.0001). Expression of pro-inflammatory genes Cc12, Cc15, 116, and Vcaml was significantly upregulated in VSMCs in several settings cells isolated from aged vs. young wild-type mice, in atherosclerotic arteries of Apoe(-/-) mice, and atherosclerotic human carotid arteries and correlated with NOX4 expression. p5RHH-mediated Nox4 siRNA transfection significantly attenuated the expression of these pro-inflammatory genes in TGF beta 1-treated mouse VSMCs, with the highest degree of inhibition in the expression of 116. p5RHH peptide-mediated knockdown of TGFO-activated kinase 1 (TAK1, also known as Map3k7),Jun, and Rela, but not Nfkb2, downregulated TGF beta 1-induced Nox4 expression in VSMCs. Together, these data demonstrate that increased expression and activation of NOX4, which might result from increased TGF beta 1 levels seen during aging, induces a proinflammatory phenotype in VSMCs, enhancing atherosclerosis. (C) 2016 Published by Elsevier Ltd.