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• W2128668040 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 rapamycin. After 8 weeks of treatment, aortas were isolated for immunohistochemical analysis, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), and Oil Red O staining. All animal protocols were reviewed and approved by the University of Oklahoma Institutional Animal Care and Use Committee. HASMCs were maintained in Medium 231 with smooth muscle cell growth supplement. All culture media were supplemented with 100 units/mL penicillin and 100 μg/mL streptomycin. HASMCs at passages 3 to 8 and grown to 70% to 80% confluence were used for the experiments. All cells were incubated in a humidified atmosphere of 5% CO2 and 95% air at 37°C. Stock solutions of 7-KC (20 mmol/L) were freshly prepared by dissolving 7-KC in dimethyl sulfoxide. To exclude the toxic effect of dimethyl sulfoxide, control cells were treated with an equal amount of solvent. HASMCs were infected with adenovirus encoding catalase at a multiplicity of infection of 100 in medium with 5% fetal calf serum for 48 hours, and an adenovirus encoding green fluorescent protein (GFP) was used as a control. Under these conditions, infection efficiency was >80% as determined by GFP expression.14Xie Z. Dong Y. Zhang J. Scholz R. Neumann D. Zou M.H. Identification of the serine 307 of LKB1 as a novel phosphorylation site essential for its nucleocytoplasmic transport and endothelial cell angiogenesis.Mol Cell Biol. 2009; 29: 3582-3596Crossref PubMed Scopus (77) Google Scholar, 15Xie Z. Dong Y. Scholz R. Neumann D. Zou M.H. Phosphorylation of LKB1 at serine 428 by protein kinase C-zeta is required for metformin-enhanced activation of the AMP-activated protein kinase in endothelial cells.Circulation. 2008; 117: 952-962Crossref PubMed Scopus (226) Google Scholar Total mRNA was extracted from the cultured cells with Trizol reagent (Invitrogen, Grand Island, NY). For reverse transcription, 1 μg of the total mRNA was converted to first strand complementary DNA in a 20 μL reaction volume using a cDNA synthesis Kit (Promega, Madison, WI). Expression levels of human Nox1, Nox4, Nox5, and housekeeping β-actin mRNAs were determined by using the specific primer as follows: Nox1, forward 5′-GTACAAATTCCAGTGTGCAGACCAC-3′, reverse 5′-CAGACTGGAATATCGGTGACAGCA-3′; Nox4, forward 5′-CTCAGCGGAATCAATCAGCTGTG-3′, reverse 5′-AGAGGAACACGACAATCAGCCTTAG-3′; Nox5, forward 5′-ATCAAGCGGCCCCCTTTTTTTCAC3′, reverse 5′-CTCATTGTCACACTCCTCGACAGC-3′; and β-actin, forward 5′-GCAGCCCAGCCAGCACTGTCAGG-3′, reverse 5′-AGCCCAGAGCCATTGTCACACACCAA-3′. Quantitative real-time-PCR reactions were performed as described.16Tedesco L. Valerio A. Cervino C. Cardile A. Pagano C. Vettor R. Pasquali R. Carruba M.O. Marsicano G. Lutz B. Pagotto U. Nisoli E. Cannabinoid type 1 receptor blockade promotes mitochondrial biogenesis through endothelial nitric oxide synthase expression in white adipocytes.Diabetes. 2008; 57: 2028-2036Crossref PubMed Scopus (126) Google Scholar Quantifications were performed by a comparative method (2-ΔΔCt) using β-actin transcripts as an internal control.17Pfaffl M.W. A new mathematical model for relative quantification in real-time RT-PCR.Nucleic Acids Res. 2001; 29: e45Crossref PubMed Scopus (25810) Google Scholar HASMCs were plated at 5 × 104 cells per well and were allowed to adhere to the plate overnight. Cells were then transduced with LC3B-GFP using Premo autophagy sensors (LC3B-FP) from Invitrogen. After 24 hours of transfection, the cells were incubated with 5 μmol/L of chloroquine (CQ) for 16 hours in the presence or absence of 7-KC. Fluorescence images were obtained by using an inverted fluorescent microscope (Olympus America, Melville, NY). Autophagy was measured by quantifying the average number of autophagosomes per cell for each sample. A minimum of 100 cells per sample was counted.18He C. Zhu H. Li H. Zou M.H. Xie Z. Dissociation of Bcl-2-Beclin1 complex by activated AMPK enhances cardiac autophagy and protects against cardiomyocyte apoptosis in diabetes.Diabetes. 2013; 62: 1270-1281Crossref PubMed Scopus (282) Google Scholar Scrambled siRNA (5′-CUUACGCUGAGUACUUCGATT-3′), Beclin1 siRNA (5′-CTCAGGAGAGGAGCCATTT-3′), and Nox4 siRNA (5′-GTCAACATCCAGCTGTACC-3′) were obtained from Applied Biosystems (Foster City, CA). Transfection was performed according to the manufacturer’s instructions. The efficiency of siRNA-silenced genes was evaluated by Western blot analysis of the targeted proteins with specific antibodies. Western blot analysis was performed with specific antibodies. Optical density of bands was quantified by AlphaEase (α Innotech Corporation, San Leandro, CA) and expressed as arbitrary units as previously described.14Xie Z. Dong Y. Zhang J. Scholz R. Neumann D. Zou M.H. Identification of the serine 307 of LKB1 as a novel phosphorylation site essential for its nucleocytoplasmic transport and endothelial cell angiogenesis.Mol Cell Biol. 2009; 29: 3582-3596Crossref PubMed Scopus (77) Google Scholar, 19He C. Choi H.C. Xie Z. Enhanced tyrosine nitration of prostacyclin synthase is associated with increased inflammation in atherosclerotic carotid arteries from type 2 diabetic patients.Am J Pathol. 2010; 176: 2542-2549Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 20Li H. Xu M. Lee J. He C. Xie Z. Leucine supplementation increases SIRT1 expression and prevents mitochondrial dysfunction and metabolic disorders in high-fat diet-induced obese mice.Am J Physiol Endocrinol Metab. 2012; 303: E1234-E1244Crossref PubMed Scopus (101) Google Scholar Hydrogen peroxide was detected by using the hydrogen peroxide Cell-based Assay kit (Cayman Chemical Company, Ann Arbor, MI) in which hydrogen peroxide is detected with 10-acetyl-3,7-dihydrosyphsnoxazine, a highly sensitive and stable probe for hydrogen peroxide.21Zhou M. Diwu Z. Panchuk-Voloshina N. Haugland R.P. A stable nonfluorescent derivative of resorufin for the fluorometric determination of trace hydrogen peroxide: applications in detecting the activity of phagocyte NADPH oxidase and other oxidases.Anal Biochem. 1997; 253: 162-168Crossref PubMed Scopus (1125) Google Scholar Cells were grown in a 96-well plate at a density of 6 × 104 cells per well overnight. After the treatments, hydrogen peroxide was measured according to the manufacturer’s instructions. Cell viability assays were performed by using the Cell Counting kit-8 (Dojindo Molecular Technologies, Rockville, MD) according to the manufacturer’s protocol. Absorbance was measured with a Bio-Rad Benchmark microplate reader (Hercules, CA) at 450 nm. Apoptosis was analyzed by using a fluorescein isothiocyanate Annexin V Apoptosis Detection kit (BD Biosciences, San Jose, CA) according to the supplier’s instructions. Briefly, HASMCs were collected by using a brief trypsin treatment and were labeled for 15 minutes with Alexa 488-conjugated Annexin V and propidium iodide (PI). Labeled cells were analyzed with FACScan flow cytometry (Becton-Dickinson, Bedford, MA) and CellQuest software (version 5.1; Becton-Dickinson, Bedford, MA). His-tagged LC3B-PLA2 plasmid was kindly provided by Dr. John C. Reed (Sanford-Burnham Medical Research Institute, La Jolla, CA). The LC3B-PLA2 fusion protein was produced and purified as described.22Shu C.W. Drag M. Bekes M. Zhai D. Salvesen G.S. Reed J.C. Synthetic substrates for measuring activity of autophagy proteases: autophagins (Atg4).Autophagy. 2010; 6: 936-947Crossref PubMed Scopus (49) Google Scholar After the treatment, cells were harvested and cell lysates were centrifuged at 16,000 × g for 15 minutes. The supernatant was used for the LC3B-PLA2 reporter assay to measure Atg4B activity. Briefly, lysates were mixed with 100 nmol/L LC3B-PLA2 fusion protein in 20 μL PLA2 reaction buffer containing 20 mmol/L Tris-HCl, pH 8.0, 2 mmol/L CaCl2, 1 mmol/L DTT, and 20 μmol/L 2-(6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino) hexanoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine (NBD-C6-HPC) (N-3786; Invitrogen). Fluorescence intensity was measured within 30 to 60 minutes by using a Bio-Tek Instrument (Winooski, VA) at room temperature with excitation and emission wavelength of 485 and 530, respectively. Quantitative data were presented as the means ± SEM. Statistical analyses were performed by using t-tests (2 groups) or one-way analysis of variance with the Bonferroni’s procedure for multiple comparison tests (≥3 groups). P < 0.05 was considered statistically significant. To establish whether 7-KC induces autophagy in VSMCs, HASMCs were exposed to 20 μmol/L of 7-KC for up to 24 hours, a concentration comparable to the mean serum level of 7-KC in normal cholesterolemic human subjects (16.9 μmol/L),23Schroepfer Jr., G.J. Oxysterols: modulators of cholesterol metabolism and other processes.Physiol Rev. 2000; 80: 361-554Crossref PubMed Scopus (828) Google Scholar and the conversion of LC3-I to LC3-II was then determined. LC3-II protein started to accumulate at 8 hours after treatment and reached a peak at 24 hours (Figure 1A). Next, autophagy induction was measured in HASMCs treated with different concentrations (0 to 40 μmol/L) of 7-KC. An increase in LC3-II levels was first observed at 10 μmol/L and was elevated to sixfold at 40 μmol/L (Figure 1B). To study the effect of 7-KC on autophagic flux, HASMCs were treated with 7-KC in the presence or absence of 5 μmol/L lysosomal inhibitor CQ. Administration of CQ stimulated LC3-II accumulation. Treatment of cells with both 7-KC and CQ induced a further increase in LC3-II protein levels (Figure 1C). Then we performed immunofluorescence analysis in HASMCs expressing GFP-LC3 to confirm the effect of 7-KC on autophagosome formation, because the number of punctate LC3 and GFP-LC3 structures per cell is usually an accurate measure of autophagosome.24Mizushima N. Yoshimori T. Levine B. Methods in mammalian autophagy research.Cell. 2010; 140: 313-326Abstract Full Text Full Text PDF PubMed Scopus (3584) Google Scholar In the absence of CQ, 7-KC treatment enhanced the number and distribution of GFP-LC3 punctate structures. Addition of CQ induced a further increase in autophagosome formation in the cells treated with 7-KC (Figure 1, D and E). These results indicate that 7-KC acts by inducing autophagosome formation rather than by disrupting its maturation into the autophagolysosome. To determine whether ROS mediates 7-KC–induced autophagy, we detected 7-KC–induced autophagy after treating the cells with an ROS scavenger and an antioxidant. Pretreatment of cells with N-acetyl cysteine, which can act both as a precursor for reduced glutathione and as a direct ROS scavenger,25de G.R. Tintu A. Stassen F. Kloppenburg G. Bruggeman C. Rouwet E. N-acetylcysteine prevents neointima formation in experimental venous bypass grafts.Br J Surg. 2009; 96: 941-950Crossref PubMed Scopus (10) Google Scholar attenuated 7-KC–enhanced LC3-II accumulation (Figure 1F). Similarly, administration of apocynin, an antioxidant in the vascular system,26Heumuller S. Wind S. Barbosa-Sicard E. Schmidt H.H. Busse R. Schroder K. Brandes R.P. Apocynin is not an inhibitor of vascular NADPH oxidases but an antioxidant.Hypertension. 2008; 51: 211-217Crossref PubMed Scopus (625) Google Scholar depressed 7-KC–elevated LC3-II protein levels (Figure 1G). These data indicate that 7-KC–induced autophagy is ROS-dependent. NADPH oxidases are major sources of ROS under physiological and pathological conditions.27Bedard K. Krause K.H. The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology.Physiol Rev. 2007; 87: 245-313Crossref PubMed Scopus (5015) Google Scholar In the vascular system, VSMCs express mainly Nox1, Nox4, and Nox5.28Lassegue B. Sorescu D. Szocs K. Yin Q. Akers M. Zhang Y. Grant S.L. Lambeth J.D. Griendling K.K. Novel gp91(phox) homologues in vascular smooth muscle cells: nox1 mediates angiotensin II-induced superoxide formation and redox-sensitive signaling pathways.Circ Res. 2001; 88: 888-894Crossref PubMed Scopus (760) Google Scholar, 29Pedruzzi E. Guichard C. Ollivier V. Driss F. Fay M. Prunet C. Marie J.C. Pouzet C. Samadi M. Elbim C. O’dowd Y. Bens M. Vandewalle A. Gougerot-Pocidalo M.A. Lizard G. Ogier-Denis E. NAD(P)H oxidase Nox-4 mediates 7-ketocholesterol-induced endoplasmic reticulum stress and apoptosis in human aortic smooth muscle cells.Mol Cell Biol. 2004; 24: 10703-10717Crossref PubMed Scopus (365) Google Scholar To verify whether NADPH oxidases contribute to 7-KC–induced ROS production, we assessed the expression of NADPH oxidases in HASMCs exposed to 7-KC. As shown in Figure 2A–C, 7-KC treatment increased expression of Nox4 at mRNA and protein levels, but did not affect mRNA and protein expression of Nox1 and Nox5 (Figure 2A–C). Several recent studies demonstrated that Nox4 generates predominantly hydrogen peroxide rather than superoxide.30Dikalov S.I. Dikalova A.E. Bikineyeva A.T. Schmidt H.H. Harrison D.G. Griendling K.K. Distinct roles of Nox1 and Nox4 in basal and angiotensin II-stimulated superoxide and hydrogen peroxide production.Free Radic Biol Med. 2008; 45: 1340-1351Crossref PubMed Scopus (323) Google Scholar, 31Schroder K. Zhang M. Benkhoff S. Mieth A. Pliquett R. Kosowski J. Kruse C. Luedike P. Michaelis U.R. Weissmann N. Dimmeler S. Shah A.M. Brandes R.P. Nox4 is a protective reactive oxygen species generating vascular NADPH oxidase.Circ Res. 2012; 110: 1217-1225Crossref PubMed Scopus (479) Google Scholar Therefore, we determined whether induction of Nox4 by 7-KC enhanced hydrogen peroxide generation. As expected, the up-regulation of Nox4 protein levels (Figure 2D) was accompanied by higher levels of intracellular hydrogen peroxide (Figure 2E). The increases in Nox4 and hydrogen peroxide occurred before the induction of autophagy, suggesting that Nox4-derived hydrogen peroxide is involved in 7-KC–induced autophagy. Indeed, removal of hydrogen peroxide by overexpression of adenovirus encoding catalase attenuated 7-KC–induced autophagy, whereas transfection of GFP adenovirus failed to diminish autophagy induced by 7-KC (Figure 2F). Thus, Nox4-derived hydrogen peroxide was required for 7-KC–induced autophagy. Currently, more than 30 autophagic-related genes have been identified.32Klionsky D.J. Cregg J.M. Dunn Jr., W.A. Emr S.D. Sakai Y. Sandoval I.V. Sibirny A. Subramani S. Thumm M. Veenhuis M. Ohsumi Y. A unified nomenclature for yeast autophagy-related genes.Dev Cell. 2003; 5: 539-545Abstract Full Text Full Text PDF PubMed Scopus (1022) Google Scholar Among them Beclin1, the mammalian ortholog of yeast Atg6, is a critical factor required for the initiation of autophagy.33Liang X.H. Kleeman L.K. Jiang H.H. Gordon G. Goldman J.E. Berry G. Herman B. Levine B. Protection against fatal Sindbis virus encephalitis by beclin, a novel Bcl-2-interacting protein.J Virol. 1998; 72: 8586-8596Crossref PubMed Google Scholar First we determined whether Beclin1 participates in the regulation of 7-KC–induced autophagy by transfecting HASMCs with control or Beclin1-specific siRNA. Beclin1 siRNA, but not control siRNA, significantly reduced Beclin1 protein levels. The reduction of Beclin1 expression did not affect 7-KC–induced LC3-II accumulation (Figure 3A), suggesting that Beclin1 may not be involved in 7-KC–induced autophagy. Next we investigated whether Atg4 is involved in 7-KC–induced autophagy because a recent report showed that starvation-induced ROS, specifically hydrogen peroxide, acted as signaling molecules in autophagy through the inhibition of Atg4 activity and consequently reduced LC3-II delipidation.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 Although 7-KC did not affect the expression of Atg4A and Atg4B (Figure 3B), it significantly inhibited Atg4B activity (Figure 3B). To determine whether Nox4-mediated hydrogen peroxide formation contributes to 7-KC–induced autophagy through inhibition of Atg4B activity, we inhibited Nox4 expression by using a gene silencing technique. Compared with control siRNA, Nox4-specific siRNA significantly reduced Nox4 protein levels without affecting the expression of the other related proteins, Nox1 and Nox5 (Figure 3, C and D). Knockdown of Nox4 diminished hydrogen peroxide levels under basal conditions and attenuated 7-KC–enhanced hydrogen peroxide formation (Figure 3E). The reduction of Nox4 concomitantly increased Atg4B activity and prevented 7-KC–inhibited Atg4B activity (Figure 3F). Accordingly, enhanced accumulation of LC3-II protein by 7-KC treatment was suppressed by silencing of the Nox4 gene (Figure 3G). These findings are consistent with the observation that hydrogen peroxide reduces Atg4 activity, which promotes lipidation of Atg8 (LC-3) and increases autophagic capacity.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 To determine the physiological role of 7-KC–induced autophagy, we evaluated the effect of autophagy on cell death in HASMCs. As assessed by inverted phase microscopy, 7-KC treatment was associated with cell rounding and detachment from the plate substrate (Figure 4A). The cell viability was significantly lower in HASMCs exposed to 7-KC than in the control cells (Figure 4B). Western blot analysis showed that administration of 7-KC significantly enhanced the apoptosis markers, such as cleavages of caspase-3 and poly (ADP-ribose) polymerase (Figure 4C), suggesting that 7-KC induces apoptosis. Next, we analyzed the effect of 7-KC on cell death by using annexin V-fluorescein isothiocyanate and PI double labeling and flow cytometry. Early apoptosis with intact membrane (annexin V+/PI−), end-stage apoptosis and dead cells (annexin V+/PI+), and damaged cells (annexin V−/PI+) were distinguished on the basis of double labeling for annexin V-fluorescein isothiocyanate and PI. 7-KC treatment led to dramatic increases in the percentages of apoptotic cells (annexin V+/PI−) and end-stage apoptosis (annexin V+/PI+), and to a lesser extent, in the percentage of damaged cells (annexin V−/PI+) (Figure 4, D and E). We further analyzed the effect of 7-KC on cell death after treating the cells with either caspase-3 inhibitor, Ac-DEVD-CMK, or the receptor-interacting protein kinase inhibitor, necrostatin. Inhibition of caspase-3 reduced 7-KC–enhanced annexin V+/PI− cells (Figure 4F), whereas inhibition of receptor interacting protein kinase reduced 7-KC–enhanced annexin V+/PI+ cells (Figure 4G), suggesting that 7-KC induces cell death through enhancing early apoptosis (annexin V+/PI−), as well as end-stage apoptosis and dead cells (annexin V+/PI+). To test the role of Nox4 and hydrogen peroxide in 7-KC–induced cell death, HASMCs were transfected with control or Nox4 siRNA and treated with 7-KC. After the treatment, cell death was analyzed by using flow cytometry. As shown in Figure 4H, Nox4 siRNA, which prevented 7-KC–enhanced intracellular hydrogen peroxide levels, reduced apoptotic cells (ann" @default.
• W2128668040 created "2016-06-24" @default.
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• W2128668040 date "2013-08-01" @default.
• W2128668040 modified "2023-10-01" @default.
• W2128668040 title "7-Ketocholesterol Induces Autophagy in Vascular Smooth Muscle Cells through Nox4 and Atg4B" @default.
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