title: NADPH oxidase 4 regulates vascular inflammation in aging and atherosclerosis.
abstract: 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 MTT and p<0.0001 vs. Lipofectamine in LDH assays) in VSMCs. TGFβ1 significantly upregulates Nox4 mRNA (p<0.01) and protein (p<0.01) expression in VSMCs. p5RHH-mediated Nox4 siRNA transfection greatly attenuated TGFβ1-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 Ccl2, Ccl5, Il6, and Vcam1 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 TGFβ1-treated mouse VSMCs, with the highest degree of inhibition in the expression of Il6. p5RHH peptide-mediated knockdown of TGFβ-activated kinase 1 (TAK1, also known as Map3k7), Jun, and Rela, but not Nfkb2, downregulated TGFβ1-induced Nox4 expression in VSMCs. Together, these data demonstrate that increased expression and activation of NOX4, which might result from increased TGFβ1 levels seen during aging, induces a proinflammatory phenotype in VSMCs, enhancing atherosclerosis.
authors.affiliation: Frankel Cardiovascular Center, Department of Internal Medicine, University of Michigan, Ann Arbor 48109, MI, USA.
authors.forename: Andrey
authors.initials: A
authors.lastname: Lozhkin
authors.affiliation: Frankel Cardiovascular Center, Department of Internal Medicine, University of Michigan, Ann Arbor 48109, MI, USA.
authors.forename: Aleksandr E
authors.initials: AE
authors.lastname: Vendrov
authors.affiliation: Division of Cardiology, Department of Medicine, Washington University School of Medicine, Saint Louis 63110, MO, USA.
authors.forename: Hua
authors.initials: H
authors.lastname: Pan
authors.affiliation: Division of Cardiology, Department of Medicine, Washington University School of Medicine, Saint Louis 63110, MO, USA.
authors.forename: Samuel A
authors.initials: SA
authors.lastname: Wickline
authors.affiliation: Frankel Cardiovascular Center, Department of Internal Medicine, University of Michigan, Ann Arbor 48109, MI, USA.
authors.forename: Nageswara R
authors.initials: NR
authors.lastname: Madamanchi
authors.affiliation: Frankel Cardiovascular Center, Department of Internal Medicine, University of Michigan, Ann Arbor 48109, MI, USA. Electronic address: mrunge@med.umich.edu.
authors.forename: Marschall S
authors.initials: MS
authors.lastname: Runge
mesh_terms: D000375:Aging / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D050197:Atherosclerosis / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D015415:Biomarkers; D002339:Carotid Arteries / Q000378:metabolism / Q000473:pathology; D002470:Cell Survival / Q000235:genetics; D016207:Cytokines / Q000235:genetics / Q000378:metabolism; D004195:Disease Models, Animal; D055785:Gene Knockdown Techniques; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D018836:Inflammation Mediators / Q000378:metabolism; D020930:MAP Kinase Kinase Kinases / Q000378:metabolism; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D008928:Mitochondria / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000378:metabolism; D032389:Myocytes, Smooth Muscle / Q000378:metabolism; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000235:genetics* / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D017382:Reactive Oxygen Species / Q000378:metabolism; D014657:Vasculitis / Q000209:etiology* / Q000378:metabolism / Q000473:pathology; D000099151:MAP Kinase Kinase Kinase 7
chemical_list: D015415:Biomarkers; D016207:Cytokines; D006861:Hydrogen Peroxide; D018836:Inflammation Mediators; D020930:MAP Kinase Kinase Kinases; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; D016328:NF-kappa B; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D000099151:MAP Kinase Kinase Kinase 7; C422943:Nox4 protein, mouse
keywords: Atherosclerosis; Cytokines; Inflammation; Nanoparticles; Reactive oxygen species