7-Ketocholesterol Induces Autophagy in Vascular Smooth Muscle Cells through Nox4 and Atg4B

Abstract

Oxidized lipoproteins stimulate autophagy in advanced atherosclerotic plaques. However, the mechanisms underlying autophagy induction and the role of autophagy in atherogenesis remain to be determined. This study was designed to investigate the mechanisms by which 7-ketocholesterol (7-KC), a major component of oxidized lipoproteins, induces autophagy. This study was also designed to determine the effect of autophagy induction on apoptosis, a central event in the development of atherosclerosis. Exposure of human aortic smooth muscle cells to 7-KC increased autophagic flux. Autophagy induction was suppressed by treating the cells with either a reactive oxygen species scavenger or an antioxidant. Administration of 7-KC concomitantly up-regulated Nox4 expression, increased intracellular hydrogen peroxide levels, and inhibited autophagy-related gene 4B activity. Catalase overexpression to remove hydrogen peroxide or Nox4 knockdown with siRNA reduced intracellular hydrogen peroxide levels, restored autophagy-related gene 4B activity, and consequently attenuated 7-KC–induced autophagy. Moreover, inhibition of autophagy aggravated both endoplasmic reticulum (ER) stress and cell death in response to 7-KC. In contrast, up-regulation of autophagic activity by rapamycin had opposite effects. Finally, activation of autophagy by chronic rapamycin treatment attenuated ER stress, apoptosis, and atherosclerosis in apolipoprotein E knockout (ApoE−/−) mouse aortas. In conclusion, we demonstrate that up-regulation of autophagy is a cellular protective response that attenuates 7-KC–induced cell death in human aortic smooth muscle cells. Oxidized lipoproteins stimulate autophagy in advanced atherosclerotic plaques. However, the mechanisms underlying autophagy induction and the role of autophagy in atherogenesis remain to be determined. This study was designed to investigate the mechanisms by which 7-ketocholesterol (7-KC), a major component of oxidized lipoproteins, induces autophagy. This study was also designed to determine the effect of autophagy induction on apoptosis, a central event in the development of atherosclerosis. Exposure of human aortic smooth muscle cells to 7-KC increased autophagic flux. Autophagy induction was suppressed by treating the cells with either a reactive oxygen species scavenger or an antioxidant. Administration of 7-KC concomitantly up-regulated Nox4 expression, increased intracellular hydrogen peroxide levels, and inhibited autophagy-related gene 4B activity. Catalase overexpression to remove hydrogen peroxide or Nox4 knockdown with siRNA reduced intracellular hydrogen peroxide levels, restored autophagy-related gene 4B activity, and consequently attenuated 7-KC–induced autophagy. Moreover, inhibition of autophagy aggravated both endoplasmic reticulum (ER) stress and cell death in response to 7-KC. In contrast, up-regulation of autophagic activity by rapamycin had opposite effects. Finally, activation of autophagy by chronic rapamycin treatment attenuated ER stress, apoptosis, and atherosclerosis in apolipoprotein E knockout (ApoE−/−) mouse aortas. In conclusion, we demonstrate that up-regulation of autophagy is a cellular protective response that attenuates 7-KC–induced cell death in human aortic smooth muscle cells. Autophagy is a highly conserved cellular process for degradation of cytoplasmic components, such as long-lived proteins and damaged organelles in lysosomes.1Klionsky D.J. Autophagy: from phenomenology to molecular understanding in less than a decade.Nat Rev Mol Cell Biol. 2007; 8: 931-937Crossref PubMed Scopus (1577) Google Scholar The process is essential for the maintenance of cellular homeostasis and survival because the degradation of cytosolic components can provide amino acids and substrates for intermediary metabolism.2Yang Y. Fukui K. Koike T. Zheng X. Induction of autophagy in neurite degeneration of mouse superior cervical ganglion neurons.Eur J Neurosci. 2007; 26: 2979-2988Crossref PubMed Scopus (103) Google Scholar Although dysregulation of autophagy has been implicated in many human diseases, including neurodegeneration, cancer, and cardiomyopathy,3Xie Z. Lau K. Eby B. Lozano P. He C. Pennington B. Li H. Rathi S. Dong Y. Tian R. Kem D. Zou M.H. Improvement of cardiac functions by chronic metformin treatment is associated with enhanced cardiac autophagy in diabetic OVE26 mice.Diabetes. 2011; 60: 1770-1778Crossref PubMed Scopus (391) Google Scholar, 4Zou M.H. Xie Z. Regulation of interplay between autophagy and apoptosis in the diabetic heart: new role of AMPK.Autophagy. 2013; 9: 624-625Crossref PubMed Scopus (81) Google Scholar, 5Xie Z. He C. Zou M.H. AMP-activated protein kinase modulates cardiac autophagy in diabetic cardiomyopathy.Autophagy. 2011; 7: 1254-1255Crossref PubMed Scopus (111) Google Scholar little information exists about the role of autophagy in the development of atherosclerosis. Recently, oxidized lipoproteins have been demonstrated to stimulate autophagy in advanced atherosclerotic plaques,6Martinet W. De Meyer G.R. Autophagy in atherosclerosis: a cell survival and death phenomenon with therapeutic potential.Circ Res. 2009; 104: 304-317Crossref PubMed Scopus (317) Google Scholar leading us to hypothesize that oxidized lipoproteins may induce autophagy in vascular cells through increasing intracellular reactive oxygen species (ROS). In support of this model, a strong correlation between oxidative stress and the development of atherosclerosis has been established7Stocker R. Keaney Jr., J.F. Role of oxidative modifications in atherosclerosis.Physiol Rev. 2004; 84: 1381-1478Crossref PubMed Scopus (2122) Google Scholar and starvation-induced ROS have been demonstrated to trigger autophagy by oxidizing a critical cysteine residue in autophagy-related gene 4 (Atg4) protein.8Scherz-Shouval R. Shvets E. Fass E. Shorer H. Gil L. Elazar Z. Reactive oxygen species are essential for autophagy and specifically regulate the activity of Atg4.EMBO J. 2007; 26: 1749-1760Crossref PubMed Scopus (1669) Google Scholar Atherosclerosis is characterized by the accumulation of oxidized lipoproteins in large arteries. Oxidation of lipoproteins leads to the formation of dozens of new lipids, such as oxysterols, aldehydes, and oxidized fatty acids.7Stocker R. Keaney Jr., J.F. Role of oxidative modifications in atherosclerosis.Physiol Rev. 2004; 84: 1381-1478Crossref PubMed Scopus (2122) Google Scholar These oxidized lipoproteins could promote the progression of atherosclerosis by stimulating an inflammatory response, increasing foam cell formation, and inducing vascular cell apoptosis.9Chatterjee S. Role of oxidized human plasma low density lipoproteins in atherosclerosis: effects on smooth muscle cell proliferation.Mol Cell Biochem. 1992; 111: 143-147Crossref PubMed Scopus (110) Google Scholar Oxysterols, such as 7β-hydoxycholesterol and 7-ketocholesterol (7-KC), are major components of oxidized lipoproteins in human atherosclerotic plaques.10Brown A.J. Jessup W. Oxysterols and atherosclerosis.Atherosclerosis. 1999; 142: 1-28Abstract Full Text Full Text PDF PubMed Scopus (767) Google Scholar The oxysterol 7-KC has been shown to accelerate ROS production and induce complex modes of cell death,11Martinet W. De B.M. Schrijvers D.M. De Meyer G.R. Herman A.G. Kockx M.M. 7-ketocholesterol induces protein ubiquitination, myelin figure formation, and light chain 3 processing in vascular smooth muscle cells.Arterioscler Thromb Vasc Biol. 2004; 24: 2296-2301Crossref PubMed Scopus (111) Google Scholar including necrosis,12Ghelli A. Porcelli A.M. Zanna C. Rugolo M. 7-Ketocholesterol and staurosporine induce opposite changes in intracellular pH, associated with distinct types of cell death in ECV304 cells.Arch Biochem Biophys. 2002; 402: 208-217Crossref PubMed Scopus (25) Google Scholar apoptosis (type I cell death),13Miguet-Alfonsi C. Prunet C. Monier S. Bessede G. Lemaire-Ewing S. Berthier A. Menetrier F. Neel D. Gambert P. Lizard G. Analysis of oxidative processes and of myelin figures formation before and after the loss of mitochondrial transmembrane potential during 7beta-hydroxycholesterol and 7-ketocholesterol-induced apoptosis: comparison with various pro-apoptotic chemicals.Biochem Pharmacol. 2002; 64: 527-541Crossref PubMed Scopus (106) Google Scholar and autophagic type II cell death. Martinet et al11Martinet W. De B.M. Schrijvers D.M. De Meyer G.R. Herman A.G. Kockx M.M. 7-ketocholesterol induces protein ubiquitination, myelin figure formation, and light chain 3 processing in vascular smooth muscle cells.Arterioscler Thromb Vasc Biol. 2004; 24: 2296-2301Crossref PubMed Scopus (111) Google Scholar demonstrated that 7-KC activates the ubiquitin proteasome system and induces the formation of a myelin figure and the processing of microtubule-associated protein light chain 3 (LC3). However, the molecular mechanisms by which 7-KC induces autophagy and the role of autophagy induction in the development of atherosclerosis remain undefined. Therefore, we investigated the mechanism by which 7-KC induces autophagy and the effect of autophagy on apoptosis, an important process in the development of atherosclerosis. We found that 7-KC increased Nox4-mediated hydrogen peroxide formation, which triggered autophagy through the inhibition of Atg4B activity. The induction of autophagy mitigated vascular smooth muscle cell (VSMC) death by suppressing the endoplasmic reticulum (ER) stress–apoptosis pathway. We used the following antibodies: antibodies against Beclin1, LC3B, Atg4B, and phosphorylated eukaryotic initiation factor-2 α (P-eIF2α) (Cell Signaling Inc., Beverly, MA), antibodies against Atg4A and 78 kDa glucose-regulated protein (GRP78) (Abcam, Cambridge, MA), antibodies against catalase and reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase (Nox) subunits (Nox4, catalog number sc-55142; Nox5, catalog number sc-67006) (Santa Cruz Biotechnology, Santa Cruz, CA), antibody against Nox1 (catalog number NBP1-69573; Novus Biologicals, Littleton, CO), and the antibody against activating transcription factor 6 (Imgenex, San Diego, CA). Human aortic smooth muscle cells (HASMCs) and cell culture media were purchased from Cascade Biologics (Portland, OR). The hydrogen peroxide cell-based assay kit was obtained from Cayman Chemical Company (Ann Arbor, MI). Chemicals, along with 7-KC, were obtained from Sigma-Aldrich (St. Louis, MO). Male apolipoprotein E knockout (ApoE−/−) mice used for these experiments were obtained from Jackson Laboratories (Bar Harbor, ME). Six-week-old mice that were maintained on a high fat diet (1.3% cholesterol, 0.5% cholic acid, TD 02028; Harlan Teklad, Madison, WI) were treated with or without 8 mg/kg/day, subcutaneous injection of 8 of were for and were and by the of and were maintained in with smooth muscle cell culture media were with and 3 to 8 and to to were used for the cells were in a of and of 7-KC were by 7-KC in the effect of cells were treated with an of were with catalase a of of in with for and an protein was used as a these was as by Z. Dong Y. J. R. D. Zou M.H. of the of as a essential for and cell Cell Biol. 2009; PubMed Scopus Google Scholar, Z. Dong Y. R. D. Zou M.H. of by protein kinase is for activation of the AMP-activated protein kinase in PubMed Scopus Google Scholar was from the cells with of the was to in a a Madison, of human Nox5, and were by the as Nox5, and were as L. A. C. A. C. R. R. G. B. E. type mitochondrial through in PubMed Scopus Google Scholar were by a as an new for in Res. PubMed Scopus Google Scholar were cells and were to to the were with autophagy from of the cells were with of for in the or of 7-KC. were obtained by an Autophagy was by the number of cell for of cells was C. H. Li H. Zou M.H. Xie Z. of complex by cardiac autophagy and against apoptosis in 2013; PubMed Scopus Google Scholar siRNA siRNA and Nox4 siRNA were obtained from CA). was to the The of was by of the proteins with was with density of was by San and as as Z. Dong Y. J. R. D. Zou M.H. of the of as a essential for and cell Cell Biol. 2009; PubMed Scopus Google Scholar, C. Xie Z. of is associated with increased in atherosclerotic from type diabetic J Full Text Full Text PDF PubMed Scopus Google Scholar, H. M. J. He C. Xie Z. and mitochondrial and in J PubMed Scopus Google Scholar peroxide was by the hydrogen peroxide kit Chemical Arbor, in which hydrogen peroxide is with a highly and for hydrogen M. Z. of for the of hydrogen in the activity of oxidase and Biochem. PubMed Scopus Google Scholar were in a a density of 6 cells the hydrogen peroxide was to the Cell were by the Cell to the was with a was by a kit San to the were by a treatment and were for with and cells were with and was by C. CA). The protein was and as M. M. 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Reactive oxygen species are essential for autophagy and specifically regulate the activity of Atg4.EMBO J. 2007; 26: 1749-1760Crossref PubMed Scopus (1669) Google Scholar have found that ROS are in starvation-induced autophagy as and have both cysteine residue in Atg4A and cysteine residue 78 in Atg4B are in this that the activity of is the of this R. Shvets E. Fass E. Shorer H. Gil L. Elazar Z. Reactive oxygen species are essential for autophagy and specifically regulate the activity of Atg4.EMBO J. 2007; 26: 1749-1760Crossref PubMed Scopus (1669) Google Scholar In 7-KC enhanced hydrogen reduced Atg4B activity, and increased autophagic of Nox4 attenuated intracellular hydrogen peroxide and restored Atg4B activity, which mitigated 7-KC–induced autophagy. These that 7-KC may intracellular hydrogen peroxide through up-regulation of This leads to the and inhibition of Atg4B, of and autophagy. The molecular mechanisms underlying these processes Oxidized have been to stimulate autophagy in advanced human atherosclerotic and in W. Kockx M.M. De Meyer G.R. Autophagy in Mol 2007; Full Text Full Text PDF PubMed Scopus Google Scholar in and human the of such as the formation of myelin figure and W. De B.M. Schrijvers D.M. De Meyer G.R. Herman A.G. Kockx M.M. 7-ketocholesterol induces protein ubiquitination, myelin figure formation, and light chain 3 processing in vascular smooth muscle cells.Arterioscler Thromb Vasc Biol. 2004; 24: 2296-2301Crossref PubMed Scopus (111) Google Scholar protein a of autophagic formation, is up-regulated in smooth muscle cells in human B. S. G. A. kinase and and the formation of autophagic during cell Cell Biol. 2002; PubMed Scopus Google Scholar Moreover, a activates autophagy in aortic smooth muscle P. Y. S. A. autophagy in vascular J. PubMed Scopus Google Scholar In the we autophagy and apoptosis in treated that between these important cellular processes in the development of atherosclerosis. These are with that 7-KC oxidative stress, autophagic G. G. Autophagy of vascular smooth muscle cells in atherosclerotic 2007; PubMed Scopus Google Scholar and of to in smooth muscle A. Porcelli A.M. Zanna C. Rugolo M. 7-Ketocholesterol and staurosporine induce opposite changes in intracellular pH, associated with distinct types of cell death in ECV304 cells.Arch Biochem Biophys. 2002; 402: 208-217Crossref PubMed Scopus (25) Google Scholar, W. Schrijvers D.M. H. between cell death by and 7-ketocholesterol in smooth muscle J Pharmacol. PubMed Scopus Google Scholar in to inducing However, induction of autophagy in to 7-KC is the of death or is an to support survival in response to cellular stress to be determined. Martinet et W. Schrijvers D.M. H. between cell death by and 7-ketocholesterol in smooth muscle J Pharmacol. PubMed Scopus Google Scholar that 7-KC–induced autophagy attenuated apoptosis through with study that autophagy a protective role in advanced X. Y. M. K. J. J. autophagy a protective role in advanced Full Text Full Text PDF PubMed Scopus Google Scholar In inhibition of autophagy by the of in and of or in mouse enhanced ER stress and cell death in the cells treated with 7-KC. up-regulation of autophagic activity by rapamycin inhibited 7-KC–induced ER stress and cell death. These that increased autophagy in response to 7-KC may be an survival that cellular components for intermediary and damaged such as J. G. autophagy to cell PubMed Scopus Google Scholar against ER stress and cell death. This is by in that the up-regulation of autophagy by chronic of rapamycin was associated with the of ER stress, apoptosis, and atherosclerosis in mouse aortas. have been used for the production of by In to human atherosclerotic the are of foam cells and have and such as and H. S. E. R. Atherosclerosis in and of smooth muscle cells of in response to Mol PubMed Scopus Google Scholar stress is by 7-KC, which leads to autophagic cell death in smooth muscle W. Schrijvers D.M. H. between cell death by and 7-ketocholesterol in smooth muscle J Pharmacol. PubMed Scopus Google Scholar However, 7-KC–induced autophagy in to be This type of that the of to in a than that of human H. S. E. R. Atherosclerosis in and of smooth muscle cells of in response to Mol PubMed Scopus Google Scholar In demonstrate that 7-KC enhanced Nox4-mediated hydrogen peroxide formation, which autophagy through inhibition of Atg4B activity. In of autophagy 7-KC–induced ER stress and cell death. up-regulation of autophagy mitigated ER stress and cell death by 7-KC. chronic rapamycin treatment attenuated ER stress, apoptosis, and atherosclerosis in mouse aortas. These that activation of autophagy is a response that ER stress and attenuates 7-KC–induced cell death. We Zou for