Autophagy plays a protective role in free cholesterol overload-induced death of smooth muscle cells

Smooth muscle cells (SMC) make up most of the vascular system. In advanced atherosclerotic plaques, dying SMCs undergo a complex death mode. In the present study, we examined the activation of autophagy in SMCs overloaded with excess free cholesterol (FC) and investigated the possible role which autophagy plays during the FC-induced cell death. After incubation with excess FC, a robust expression of autophagic vacuoles (AV) was detected using both fluorescence microscopy and transmission electron microscopy (TEM). The results revealed that FC induced a time-dependent upregulation of microtubule-associated protein-1 light chain 3-II (LC3-II). Inhibition of autophagy by 3-methyladenine (3-MA) enhanced both cell apoptosis and necrosis, while on the contrary, rapamycin inhibited cell death following cholesterol application. Furthermore, the impact of the colocalization of fragmented mitochondria with AVs was observed after cholesterol treatment. Our results also revealed that the modulation of autophagy directly influenced the cellular organellar stress. In conclusion, our findings demonstrated that excess FC induced the activation of autophagy in SMCs as a cellular defense mechanism, possibly through the degradation of dysfunctional organelles such as mitochondria and endoplasmic reticulum. Smooth muscle cells (SMC) make up most of the vascular system. In advanced atherosclerotic plaques, dying SMCs undergo a complex death mode. In the present study, we examined the activation of autophagy in SMCs overloaded with excess free cholesterol (FC) and investigated the possible role which autophagy plays during the FC-induced cell death. After incubation with excess FC, a robust expression of autophagic vacuoles (AV) was detected using both fluorescence microscopy and transmission electron microscopy (TEM). The results revealed that FC induced a time-dependent upregulation of microtubule-associated protein-1 light chain 3-II (LC3-II). Inhibition of autophagy by 3-methyladenine (3-MA) enhanced both cell apoptosis and necrosis, while on the contrary, rapamycin inhibited cell death following cholesterol application. Furthermore, the impact of the colocalization of fragmented mitochondria with AVs was observed after cholesterol treatment. Our results also revealed that the modulation of autophagy directly influenced the cellular organellar stress. In conclusion, our findings demonstrated that excess FC induced the activation of autophagy in SMCs as a cellular defense mechanism, possibly through the degradation of dysfunctional organelles such as mitochondria and endoplasmic reticulum. Smooth muscle cells (SMCs) are the major components of the vascular system. The loss of SMCs in advanced atherosclerotic lesions promotes lesional necrosis, compromises plaque stability, and ultimately triggers thrombotic vascular occlusion (1Kockx M.M. Apoptosis in the atherosclerotic plaque: quantitative and qualitative aspects.Arterioscler. Thromb. Vasc. Biol. 1998; 18: 1519-1522Crossref PubMed Scopus (211) Google Scholar). In the late stage of atherosclerosis, a great number of SMCs and macrophages that phagocytose lipoproteins accumulate in the intimal layer of arterial wall and develop into “foam cells.” It is proposed that typical apoptosis of macrophages' original foam cell is caused by the intracellular accumulation of unesterified cholesterol (2Feng B. Yao P.M. Li Y. Devlin C.M. Zhang D. Harding H.P. Sweeney M. Rong J.X. Kuriakose G. Fisher E.A. et al.The endoplasmic reticulum is the site of cholesterol-induced cytotoxicity in macrophages.Nat. Cell Biol. 2003; 5: 781-792Crossref PubMed Scopus (718) Google Scholar). However, SMCs are more resistant to the accumulation of free cholesterol (FC) than macrophages (3Rong J.X. Kusunoki J. Oelkers P. Sturley S.L. Fisher E.A. Acyl-coenzymeA (CoA):cholesterol acyltransferase inhibition in rat and human aortic smooth muscle cells is nontoxic and retards foam cell formation.Arterioscler. Thromb. Vasc. Biol. 2005; 25: 122-127Crossref PubMed Scopus (29) Google Scholar). Significant increase of apoptotic cell death is not observed until SMCs are incubated with a higher concentration of cholesterol (4Kedi X. Ming Y. Yongping W. Yi Y. Xiaoxiang Z. Free cholesterol overloading induced smooth muscle cells death and activated both ER- and mitochondrial-dependent death pathway.Atherosclerosis. 2009; 207: 123-130Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Moreover, the studies using TUNEL technique reported that lipid-laden SMCs rarely undergo apoptosis in early atherosclerotic plaques (5Hegyi L. Skepper J.N. Cary N.R. Mitchinson M.J. Foam cell apoptosis and the development of the lipid core of human atherosclerosis.J. Pathol. 1996; 180: 423-429Crossref PubMed Scopus (139) Google Scholar). The investigation of human carotid plaques and plaques from cholesterol-fed rabbits also showed that dying SMCs exhibit typical ultrastructures of autophagic and necrotic cell death (6Kockx M.M. De Meyer G.R. Buyssens N. Knaapen M.W. Bult H. Herman A.G. Cell composition, replication, and apoptosis in atherosclerotic plaques after 6 months of cholesterol withdrawal.Circ. Res. 1998; 83: 378-387Crossref PubMed Scopus (150) Google Scholar). These results indicate that SMCs may undergo a complex cell death mode after FC stimulation and that autophagy may provide an alternative mechanism of cell death in lipid-laden SMCs. Autophagy (specifically macroautophagy) is an evolutionarily conserved catabolic process for subcellular degradation of proteins, organelles, and other cytoplasmic components (7Baehrecke E.H. Autophagy: dual roles in life and death?.Nat. Rev. Mol. Cell Biol. 2005; 6: 505-510Crossref PubMed Scopus (843) Google Scholar). As the degradation of cellular organelles generates free fatty acids, amino acids, and nucleotides that can be reused to fuel mitochondrial ATP production and maintain protein synthesis, autophagy is considered primarily a cell survival mechanism, especially during nutrient deprivation (8Yang 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 (102) Google Scholar). However, it has also been argued that autophagy may act as an initiator of cell death (9Levine B. Yuan J. Autophagy in cell death: an innocent convict?.J. Clin. Invest. 2005; 115: 2679-2688Crossref PubMed Scopus (1476) Google Scholar). Excessive autophagic activity may directly destroy a major proportion of the cytosol and organelles, or alternatively, it may trigger apoptosis rather than play a direct role in the death process. For instance, stress in certain organelles, such as mitochondria and endoplasmic reticulum (ER), were indicated as stimulators of autophagic response because autophagy could mediate the specific removal of dysfunctional or damaged cytoplasmic organelles (10Yorimitsu T. Nair U. Yang Z. Klionsky D.J. Endoplasmic reticulum stress triggers autophagy.J. Biol. Chem. 2006; 281: 30299-30304Abstract Full Text Full Text PDF PubMed Scopus (808) Google Scholar, 11Kiffin R. Bandyopadhyay U. Cuervo A.M. Oxidative stress and autophagy.Antioxid. Redox Signal. 2006; 8: 152-162Crossref PubMed Scopus (422) Google Scholar). Several pathways that link the apoptotic and autophagic mechanisms also have been described at the molecular level (12Maiuri M.C. Zalckvar E. Kimchi A. Kroemer G. Self-eating and self-killing: crosstalk between autophagy and apoptosis.Nat. Rev. Mol. Cell Biol. 2007; 8: 741-752Crossref PubMed Scopus (2844) Google Scholar). Thus, the result of a common stimulation may be decided by the crosstalk between autophagy and apoptosis or as a result of a cellular “decision” between these two responses. In our present study, we found that overloading FC triggered autophagy in SMCs. This was confirmed by the conversion of microtubule-associated protein-1 light chain 3-I (LC3-I) to a phosphatidylethanolamine-conjugated form (LC3-II) and the accumulation of autophagic vacuoles (AV) under excess FC conditions. We further discovered that 3-methyladenine (3-MA) suppressed autophagy induced by FC overloading and led to large-scale cell death, whereas preincubation of rapamycin, an autophagy stimulator that inactivates mammalian target of rapamycin (mTOR), protected SMCs from death induced by excess FC. Moreover, the modulation of autophagy influenced the cellular organellar function, suggesting that autophagy played an important role in cell survival after FC overloading. Unless otherwise noted, all reagents and chemicals were purchased from Sigma. Annexin V/propidium iodide (PI), H2DCFDA, and JC-1 were from Invitrogen (CA). Antibody against KDEL (anti-GRP78/GRP94 SPA-827) was purchased from Stressgen (Victoria, Canada). The other antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Primary SMCs were obtained from the aortic media of a male Sprague-Dawley rat with the explants technique. Briefly, thoracic aorta was isolated and cleaned of adventitia and endothelium. The aorta was then cut into 1 mm sections, transferred to a culture dish, and maintained in growth medium of DMEM supplemented with 10% FBS, 2 mM L-glutamine, 4 mM sodium pyruvate, 100 U/ml streptomycin, and 100 U/ml penicillin. The initial migration of SMCs was observed within the first 6 days. After 9 days of migration, these cells were transferred to a new culture dish. All experiments were conducted using SMCs between passage 2 and 5. SMCs were identified by immunocytochemical analysis with anti-α-actin antibody. The purity of the cultures was over 90%. Cholesterol was delivered to SMCs by a cholesterol-cyclodextrin complex (Chol:MβCD) that contained approximately 50 mg of cholesterol/g solid (C4951, Sigma). All treatment concentrations were calculated based on cholesterol weight. SMCs were plated on the culture dish until the cells reached 90% confluence. To load excess FC, the monolayer was then incubated with 30 µg/ml Chol:MβCD and the acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor sandoz58035 (58035) (10 µg/ml) in the culture medium for 24 h (4Kedi X. Ming Y. Yongping W. Yi Y. Xiaoxiang Z. Free cholesterol overloading induced smooth muscle cells death and activated both ER- and mitochondrial-dependent death pathway.Atherosclerosis. 2009; 207: 123-130Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 13Rong J.X. Shapiro M. Trogan E. Fisher E.A. Transdifferentiation of mouse aortic smooth muscle cells to a macrophage-like state after cholesterol loading.Proc. Natl. Acad. Sci. USA. 2003; 100: 13531-13536Crossref PubMed Scopus (351) Google Scholar). In some experiments, SMCs were preincubated with 10 mM 3-MA or 1 µM rapamycin for 4 h, followed by the incubation with FC in the presence of 3-MA or rapamycin to modulate the autophagy activity. SMCs were collected using a brief trypsin treatment and labeled for 15 min with 5 µg/ml Alexa 488-conjugated Annexin V and 10 µg/ml PI according to the manufacture's protocol. Labeled cells were analyzed with FACScan flow cytometry (Becton-Dickinson, Bedford, MA) using CellQuest software. The percentages of Annexin V- and PI-positive cells were calculated and presented as apoptosis and death percentage, respectively. To evaluate the concentration of cellular ROS, SMCs were collected and stained with 5 µM H2DCFDA at 37°C for 20 min according to the manufacture's protocol. The mean fluorescence (FL1 channel, excitation 488 nm, emission 530 nm) of labeled cells was determined by flow cytometry. The pEGFP-LC3 expression vector was a gift from Dr. Yoshimori. Mitochondria were visualized by expression of pDsRed2-mito (Clontech) in SMCs. Transfection was performed one day after subculture with Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. Fluorescence images were obtained using an inverted fluorescent microscope (Olympus IX81; 60×; NA 1.45). SMCs were lysed in an appropriate volume of ice-cold radioimmune precipitation assay buffer containing 1% Triton X-100, 50 mM Tris/HCl, pH 7.4, 300 mM NaCl, 1:100 protease inhibitor cocktail, and 1:100 phosphatase inhibitor cocktail. Equal amounts of protein were loaded to 10% to 15% SDS-polyacrylamide gel. After electrophoresis, proteins were electrotransferred onto polyvinylidene difluoride membrane (Millipore). Membranes were blocked in 5% (w/v) nonfat dry milk for 1 h and probed overnight at 4°C with each primary antibody, followed by incubation with the secondary antibody for 2 h at room temperature. Antibody detection was developed using 3,3,5,5-Tetramethylbenzidine Liquid Substrate kit (Amresco). Samples were fixed in 0.1 M sodium cacodylatebuffered (pH 7.4) for 2 h and postfixed in 0.1 M sodium cacodylatebuffered (pH 7.4) 1% OsO4 solution for 1 h. After dehydration in an ethanol gradient [70% (v/v) ethanol (15 min), 80% (v/v) ethanol (15 min), 90% (v/v) ethanol (15 min), 100% ethanol (2 × 20 min)], samples were incubated with propylenoxid (2 × 10 min), impregnated with a mixture of propylenoid/LX-112 (Ladd Research Industries, 1:1), and embedded in LX-112. Ultrathin sections were stained with uranyl acetate and were examined in a To evaluate the of SMCs were collected and stained with 5 µM JC-1 at 37°C for 20 min according to the manufacture's protocol. JC-1 within the mitochondrial a JC-1 as fluorescence of flow excitation 488 nm, emission whereas the of JC-1 as fluorescence (FL1 the concentration of JC-1 and increase the fluorescence M.W. membrane and and Neurosci. Full Text Full Text PDF PubMed Scopus Google Scholar). Thus, mean and fluorescence were determined by flow cytometry for of mean which was as a of The were as mean from were by followed by was considered In our treatment of SMCs with excess FC induced cell death the mitochondrial and death with cellular (4Kedi X. Ming Y. Yongping W. Yi Y. Xiaoxiang Z. Free cholesterol overloading induced smooth muscle cells death and activated both ER- and mitochondrial-dependent death pathway.Atherosclerosis. 2009; 207: 123-130Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). To further the mechanism of FC cell death, SMCs were with a inhibitor that cell and incubated with FC in the presence of for 24 h. As in and the inhibitor on the of SMCs under the lipid-laden as it led to a in the of apoptotic V cells increase in the necrotic Furthermore, FC death was with an upregulation of cellular which we to be a major of cell death. and were to the production of cellular could SMCs from FC-induced cell death, both the cellular level These results that apoptosis not be the in FC death and that other such as autophagy and necrosis, to complex mode of cell death. the of AVs is by the most important of of these by electron microscopy is considered the for we examined the in SMCs cholesterol treatment. AVs were rarely detected in SMCs or SMCs incubated with Chol:MβCD After overloading SMCs with FC for 24 h, a number of AVs and were the and of the AVs contained the of cellular organelles in to autophagic were also observed in SMCs and that in the was presented in The in cells was determined by fluorescence As in in most protein was the a of SMCs contained fluorescence in SMCs after After treatment with excess FC, cytoplasmic were to of suggesting the activation of The of was in SMCs with the specific autophagy inhibitor Autophagy was further confirmed by analysis with antibody. SMCs with FC caused a and time-dependent increase in the expression of the The of was enhanced after a h and a robust production of was observed after a 24 h treatment These results further the that autophagy was activated in SMCs. To further the of autophagy as a or in both autophagy inhibitor and were 3-MA was reported to autophagic activity by the of AVs M. Li T. of autophagy and cell survival in the of 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). In our experiments, the expression of was in SMCs with 10 mM 3-MA for 4 h followed by 24 h incubation with FC in the presence of 3-MA 3-MA triggered cell apoptosis and death in SMCs for and for both with the we investigated of autophagy with rapamycin could have a from FC cell SMCs with rapamycin a to the excess FC as demonstrated by a V- and PI-positive cell However, was not and it was the cells were with 3-MA rapamycin, suggesting that the of rapamycin was blocked autophagy was inhibited possible mechanism for the of autophagy was that autophagy the of cellular To SMCs were with and As in in most protein was the cell colocalization of labeled mitochondria and The FC induced the of AVs and the of mitochondria The colocalization of AVs and mitochondria were more detected after 24 h treatment with FC in the We then the to the mitochondrial The inhibition of autophagy by 3-MA in an in the of to a large-scale mitochondrial SMCs incubated with FC rapamycin maintained a than overloaded with FC Furthermore, both and protein a of protein response activation and after rapamycin treatment and with 3-MA treatment These results that autophagy as a cellular defense mechanism and cell survival in possibly by the of cellular FC vascular cell death is considered an important in of reported that excess intracellular FC triggered death with the of apoptosis (2Feng B. Yao P.M. Li Y. Devlin C.M. Zhang D. Harding H.P. Sweeney M. Rong J.X. Kuriakose G. Fisher E.A. et al.The endoplasmic reticulum is the site of cholesterol-induced cytotoxicity in macrophages.Nat. Cell Biol. 2003; 5: 781-792Crossref PubMed Scopus (718) Google Scholar). In our study, FC was also found to be a of rat aortic both and apoptotic pathways were in FC-induced death (4Kedi X. Ming Y. Yongping W. Yi Y. Xiaoxiang Z. Free cholesterol overloading induced smooth muscle cells death and activated both ER- and mitochondrial-dependent death pathway.Atherosclerosis. 2009; 207: 123-130Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). However, the present demonstrated that apoptosis may not be the of FC death. In to the FC death, a increase of V was after excess FC and the inhibitor could not SMCs from cell death. the SMCs presented a level of cellular by the loss of such as and to cell death. the other autophagy was also in FC cell death. The activation of autophagy was observed from the of AVs and conversion and was further confirmed by of cellular organelles, mitochondria and was observed after SMCs were incubated with excess FC. of the AVs in the SMCs contained the of these cellular organelles, which indicated that excess FC induced cellular and that these cellular organelles be through Thus, the present after overloaded with FC, SMCs a complex mode of cell death that and to some of have revealed the link between autophagy and apoptosis (12Maiuri M.C. Zalckvar E. Kimchi A. Kroemer G. Self-eating and self-killing: crosstalk between autophagy and apoptosis.Nat. Rev. Mol. Cell Biol. 2007; 8: 741-752Crossref PubMed Scopus (2844) Google Scholar). Autophagy and apoptosis can be by common such as cellular The of as an initial in cell is also identified as a trigger of Mitochondria can undergo the in which a increase in the of the membrane the loss of and may trigger the of mitochondria by autophagy to mitochondrial autophagy in rat 2006; PubMed Scopus Google Scholar). Several studies have that autophagy is activated for cell survival after stress and the of the (10Yorimitsu T. Nair U. Yang Z. Klionsky D.J. Endoplasmic reticulum stress triggers autophagy.J. Biol. Chem. 2006; 281: 30299-30304Abstract Full Text Full Text PDF PubMed Scopus (808) Google Scholar, J. M. B. E. The protein response is for endoplasmic reticulum and autophagy in mammalian PubMed Scopus Google Scholar). with these we found that the upregulation of and activation of in FC cell death. Several pathways may also be in the crosstalk between apoptosis and For the autophagy also as was identified as a The activity of could be inhibited by an between autophagy and apoptosis M.C. G. A. P. E. G. K. N. et and between and a in J. 2007; 26: PubMed Scopus Google Scholar). protein and which membrane during can also the autophagic B. G. Kimchi A. and mediate membrane and the of autophagic during cell Cell Biol. PubMed Scopus Google Scholar). Thus, in some autophagy and apoptosis could be induced by the could and and could be to complex crosstalk The role of autophagy in FC SMCs death is Autophagy is as an evolutionarily conserved mechanism for the degradation of cellular components that as a survival mechanism in cells N. Autophagy plays a role during necrotic cell PubMed Scopus (150) Google Scholar). 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However, a certain it could also cellular the of cellular In our study, large-scale of AVs was observed by in some most of the intracellular volume was by autophagic activity is of major of cellular organelles and may to cellular of that SMCs in advanced human atherosclerotic plaques of apoptotic and cell death. of FC can be observed in macrophages or SMCs in advanced plaques, these cells not undergo apoptotic cell death. to our activation of autophagy in SMCs was also reported in other in experiments W. De Meyer G.R. Autophagy in PubMed Scopus Google Scholar). to the of lipid such as activated autophagy in rat aortic and inhibition of autophagy by 3-MA caused cell death P. Y. A. lipid autophagy in vascular J. PubMed Scopus Google Scholar). one of the major present in triggered not also of cellular organelles, and conversion in SMCs W. De M. De Meyer G.R. Herman A.G. M.M. protein and light chain in vascular smooth muscle Thromb. Vasc. Biol. PubMed Scopus Google Scholar). 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De Meyer G.R. of macrophages in atherosclerotic plaques by autophagy.J. 2007; PubMed Scopus Google Scholar). All these results the that autophagy ultimately to process in the treatment for N. B. Cuervo A.M. Klionsky D.J. Autophagy through cellular PubMed Scopus Google Scholar, Klionsky D.J. of Rev. 2007; 6: PubMed Scopus Google Scholar). Our findings demonstrated that SMCs a complex mode of cell death. The excess intracellular FC led to large-scale cellular organelles which further activated the of AVs and enhanced autophagy in a mechanism that cell death in SMCs. possible for is that the of autophagy in the of cellular organelles and organellar stress that protected SMCs from the death

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