Lozhkin Andrey, Vendrov Aleksandr E, Pan Hua, Wickline Samuel A, Madamanchi Nageswara R, Runge Marschall S
Frankel Cardiovascular Center, Department of Internal Medicine, University of Michigan, Ann Arbor 48109, MI, USA.
Division of Cardiology, Department of Medicine, Washington University School of Medicine, Saint Louis 63110, MO, USA.
J Mol Cell Cardiol. 2017 Jan;102:10-21. doi: 10.1016/j.yjmcc.2016.12.004. Epub 2016 Dec 14.
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.
我们最近报道，衰老过程中NADPH氧化酶4（NOX4）表达和活性增加会导致细胞和线粒体氧化应激增强、血管炎症、功能障碍及动脉粥样硬化。本研究的目的是阐明这些效应的分子机制，并确定NOX4调节小鼠血管平滑肌细胞（VSMC）中促炎基因表达的重要性。采用一种新型肽介导的siRNA转染方法，以最小的细胞毒性抑制Nox4表达。使用蜂毒肽衍生肽p5RHH，我们在VSMC中实现了显著更高的转染效率（92%，而Lipofectamine为85%），并降低了毒性（MTT法中与Lipofectamine相比p<0.001，LDH测定中与Lipofectamine相比p<0.0001）。转化生长因子β1（TGFβ1）显著上调VSMC中Nox4 mRNA（p<0.01）和蛋白（p<0.01）表达。p5RHH介导的Nox4 siRNA转染极大地减弱了TGFβ1诱导的Nox4 mRNA（p<0.01）和蛋白（p<0.0001）水平上调，并降低了过氧化氢生成（p<0.0001）。在几种情况下，促炎基因Ccl2、Ccl5、Il6和Vcam1的表达在从老年与年轻野生型小鼠分离的VSMC、Apoe小鼠的动脉粥样硬化动脉以及动脉粥样硬化人类颈动脉中显著上调，且与NOX4表达相关。p5RHH介导的Nox4 siRNA转染显著减弱了TGFβ1处理的小鼠VSMC中这些促炎基因的表达，其中对Il6表达的抑制程度最高。p5RHH肽介导的转化生长因子β激活激酶-1（TAK1，也称为Map3k7）、Jun和Rela（而非Nfkb2）的敲低下调了TGFβ1诱导的VSMC中Nox4表达。总之，这些数据表明，衰老过程中TGFβ1水平升高可能导致的NOX4表达和激活增加，会在VSMC中诱导促炎表型，加重动脉粥样硬化。
