7-Ketocholesterol Induces Protein Ubiquitination, Myelin Figure Formation, and Light Chain 3 Processing in Vascular Smooth Muscle Cells
http://www.100md.com
《动脉硬化血栓血管生物学》
From the Division of Pharmacology (W.M., M.D.B., D.M.S., G.R.Y.D., A.G.H., M.M.K.), University of Antwerp, Wilrijk, Belgium; and the Department of Pathology (M.M.K.), General Hospital Middelheim, Antwerp, Belgium.
ABSTRACT
Objective— Oxysterols such as 7-ketocholesterol (7-KC) are important mediators of cell death in atherosclerosis. Therefore, in vitro studies of human smooth muscle cell (SMC) death in response to 7-KC were undertaken to investigate the potential mechanisms.
Methods and Results— Human aortic SMCs treated with 7-KC showed enhanced immunoreactivity for the oxidative stress marker 4-hydroxy-2-nonenal and upregulated several stress genes (70-kDa heat shock protein 1, heme oxygenase 1, and growth arrest and DNA damage–inducible protein 153) at the mRNA but not at the protein level. 7-KC–treated SMCs rapidly underwent cell death as determined by neutral red, counting of adherent cells, and depolarization of the mitochondrial inner membrane. Cell death was associated with upregulation of ubiquitin mRNA and ubiquitination of cellular proteins. Inhibition of the proteasome by lactacystin potentiated considerably the toxicity of 7-KC. Transmission electron microscopy revealed formation of myelin figures, extensive vacuolization, and depletion of organelles. Formation of autophagosomes was suggested by labeling cells with LysoTracker and monitoring processing of microtubule-associated protein 1 light chain 3 (LC3). Analogous to our in vitro studies, human atherosclerotic plaques showed signs of ubiquitination in SMCs.
Conclusions— 7-KC activates the ubiquitin–proteasome system and induces a complex mode of cell death associated with myelin figure formation and processing of LC3 evocating autophagic processes.
7-Ketocholesterol induces a complex mode of cell death in human aortic smooth muscle cells associated with accumulation of ubiquitinated proteins in the cytoplasm, myelin figure formation, and LC3 processing, evocating autophagic processes.
Key Words: 7-ketocholesterol ? ubiquitination ? smooth muscle cells ? myelin figures ? LC3 ? autophagy ? atherosclerosis
Introduction
Cell death is a major event in the progression of atherosclerosis. Indeed, a large body of evidence suggests that apoptosis or type I programmed cell death1 frequently occurs in advanced human plaques (1% to 2% TUNEL-positive nuclei).2–5 However, results from electron microscopy studies showed that the majority of dying cells have an ultrastructure typical of cells undergoing "accidental" cell death or oncosis6 (type III programmed cell death).1 Furthermore, it is important to note that there are multiple pathways leading to cell death. Cells sometimes die with a morphology that is intermediate between apoptosis and oncosis (eg, aponecrosis and paraptosis), or they undergo cell death with a less clear morphology or mechanism such as lysosome-mediated cell death or autophagy (type II programmed cell death).1 It is presently unknown whether cells in human atherosclerotic plaques die by mechanisms distinct from apoptosis or oncosis, as shown recently for myocytes in failing human hearts.7,8
Oxidative processes, particularly oxidation of low-density lipoprotein (LDL), are thought to play a pivotal role in atherogenesis.9 Oxidized LDL (oxLDL) exerts its proatherogenic effects in several ways, including stimulation of inflammatory responses, foam cell formation, and induction of cell death.9 The mechanism of oxLDL-induced cell death is unclear, as oncosis and apoptosis have been reported,10 and largely depends on the oxidation degree, exposure time, and concentration of oxLDL.3 In macrophages, oxLDL induces mitochondrial dysfunction and lysosomal damage,11,12 indicating that oxLDL uptake is essential for its cytotoxic properties. In addition, it has been suggested that oxysterols such as 7?-hydroxycholesterol and 7-ketocholesterol (7-KC) are the primary cytotoxins of oxLDL. Although their role in oxLDL-mediated cytotoxicity remains unclear, oxysterol-induced cell death is associated with an enhanced production of reactive oxygen species and formation of myelin figures evocative of autophagic vacuoles.13,14
In the present study, we show that 7-KC induces a complex mode of cell death in human vascular smooth muscle cells (SMCs) associated with myelin figure formation and light chain 3 (LC3) processing, evocating autophagic processes. We also demonstrate that exposure of SMCs to 7-KC is associated with accumulation of ubiquitinated proteins in the cytoplasm. Furthermore, we provide evidence that ubiquitination also occurs in SMCs of advanced human atherosclerotic plaques.
Methods
The Methods sections is available online at http://atvb.ahajournals.org.
Results
Induction of Complex Mode of Cell Death in 7-KC–Treated SMCs
Treatment of human aortic SMCs with 100 μmol/L 7-KC disturbed the typical cell spreading of SMCs (acquisition of an elongated shape followed by rounding up of cells) and rapidly induced cell death as assessed by neutral red and counting of adherent cells (Figure 1A). The majority of cells did not contain cleaved caspase-3 (Figure 1B) and were negative for annexin V labeling and propidium iodide (PI) incorporation (Figure 1A), albeit a small but significant increase in annexin V and PI labeling was noticed after 12 hours of treatment (Figure 1A). Cells treated with 7-KC showed enhanced immunoreactivity for 4-hydroxy-2-nonenal (Figure I, available online at http://atvb.ahajournals.org), a major product of endogenous lipid peroxidation and an important marker of oxidative stress. We also observed a significant depolarization of the mitochondrial inner membrane potential as early as 2 hours after 7-KC administration (Figure 1A). However, internucleosomal DNA fragmentation typical of apoptosis could not be detected, and labeling of cells with Hoechst revealed neither a significant rise in chromatin condensation nor fragmentation of the nucleus (Figure II, available online at http://atvb.ahajournals.org). Transmission electron microscopic analysis of cells treated with 7-KC for 12 hours revealed formation of membranous whorls (also called myelin figures) in different stages of development, extensive vacuolization, and depletion of organelles (Figure 2 C through 2F). Several of these features, such as myelin figure formation and vacuolization, initiated within 2 hours after 7-KC administration (Figure III, available online at http://atvb.ahajournals.org). Of note, untreated controls also showed presence of small vesicles, especially in older cells (passage >5), suggesting that these vesicles are formed spontaneously to remove damaged intracellular components or protein aggregates (Figure 2A and 2B). To exclude the possibility that the inclusions in the vacuoles of dying cells represent heterophagocytized remnants of dead cells rather than autophagocytized intracellular components, SMCs were treated with 7-KC in the presence of fluorescent beads. After 12 hours, a small group of cells (2% to 3%) contained one single bead (Figure IV, available online at http://atvb.ahajournals.org). However, uptake of multiple beads, as shown in control J774 macrophages incubated with beads for 1 hour, could not be demonstrated (Figure IV). Accumulation of acidic organelles in 7-KC–treated cells was confirmed by labeling cells with LysoTracker (Figure 3 A and 3B). Moreover, initiation of 7-KC–induced cell death was associated with the conversion of the cleaved 18-kDa protein microtubule-associated protein 1 LC3 (LC3-I) into the 16-kDa protein LC3-II (Figure 3C), which is considered a reliable marker of autophagosome formation.15,16
Figure 1. Characterization of 7-KC–induced cell death in human aortic SMCs. Cells were treated with 100 μmol/L 7-KC for up to 24 hours. A, Cell death was examined by a neutral red viability assay, analysis of cell adherence, and disruption of the mitochondrial transmembrane potential (m), PI incorporation, and annexin V labeling. B, Western blot analysis of procaspase-3 (procasp-3) cleavage. Monocytic U937 cells treated with 50 μmol/L etoposide (+) for 5 hours served as a positive control. Results are representative of at least 3 independent experiments. **P<0.01; ***P<0.001 vs 0 hours (ANOVA followed by Dunnett test).
Figure 2. Ultrastructural features of SMCs treated with 100 μmol/L 7-KC for 12 hours. A and B show untreated controls with normal cell morphology. Note formation of small vesicles (arrowheads) that may arise under normal physiological conditions to remove abnormal proteins and other cytoplasmic macromolecules. However, treatment of SMCs with 7-KC causes severe cellular injury so that the cell is no longer able to process damaged cellular components. This results in cell death evocating autophagy, as shown in C through F, and is characterized by the accumulation of autophagic vesicles, extensive vacuolization (D and E), degradation of membranous cellular components that rearrange in membranous whorls called myelin figures (arrows), depletion of organelles, and finally complete disintegration of the cell (F). Data are representative of 2 separate experiments. Bar=1 μm. N indicates nucleus.
Figure 3. Formation of acidic vacuoles in 7-KC–treated SMCs. A and B, Labeling of untreated controls (A) or SMCs treated with 100 μmol/L 7-KC for 12 hours (B) with LysoTracker. Images represent adherent cells only. C, Western blot analysis of SMCs before and after treatment with 7-KC showing processing of the 18-kDa microtubule-associated LC3-I into the 16-kDa protein LC3-II, a marker of autophagosome formation. Data are representative of 3 separate experiments.
To further characterize 7-KC–induced cell death, transcript levels of 205 apoptosis-related and 234 stress-related genes were analyzed using cDNA expression arrays. Twelve hours after exposure to 7-KC, 4 stress-related genes with a differential steady-state expression level of >5-fold could be identified: 70-kDa heat shock protein 1 (HSP-70; 25.3-fold), heme oxygenase 1 (HO1; 7-fold), and growth arrest and DNA damage-inducible protein 153 (GADD153; 5.9-fold) were upregulated in 7-KC–treated SMCs versus untreated cells, whereas proliferating cyclic nuclear antigen (PCNA; 5.2-fold) was downregulated. Furthermore, it is noteworthy that the housekeeping gene ubiquitin was moderately upregulated (2.5-fold). Neither ubiquitin-like protein Nedd8 nor typical apoptosis-related genes such as Bcl-2 family members, caspases, or death receptors showed differential gene expression. Array results could be confirmed by real-time RT-PCR (12.3±1.3 , P<0.05; 20.6±3.0 , P<0.05; 28.3±2.8 , P<0.01; 6.6±3.3 , P=0.23;and 1.8±0.1 , P<0.01; n=3). However, differential expression of HSP-70 and PCNA at the protein level could not be observed at the different time points studied (0 to 24 hours; data not shown). Expression of HO1 protein did not increase but significantly decreased after 2 hours of 7-KC stimulation (data not shown), possibly as a result of protein degradation.
7-KC–Treated SMCs Accumulate Ubiquitinated Proteins
In addition to upregulation of ubiquitin mRNA, Western blots for ubiquitin demonstrated that SMCs accumulated various ubiquitinated proteins or protein aggregates of high molecular weight (>100 kDa) after 6 to 12 hours of exposure to 7-KC (Figure 4A). Protein ubiquitination was accompanied by a progressive increase in the expression of E2 ubiquitin–conjugating enzymes UbcH6, UbcH7, Ubc9, and the E3 ubiquitin ligase Itch (Figure 4A). Immunostaining of 7-KC–treated SMCs for ubiquitin showed an intense granular signal in the cytoplasm (Figure 4C and 4D), whereas in untreated SMCs, ubiquitin was difficult to detect (Figure 4B). Accumulation of ubiquitinated proteins could already be detected in cells treated with 6.25 μmol/L 7-KC for 12 hours (Figure 4E), but significant induction of cell death was only observed within 24 hours if cells were exposed to 7-KC concentrations 50 μmol/L. Besides 7-KC–treated SMCs, SMCs treated with the peroxynitrite-donor SIN1A (1 mmol/L) showed accumulation of ubiquitinated protein (Figure 4F), but this was not the case with SMCs undergoing amino acid deprivation (data not shown), suggesting that acute oxidative stress rather than autophagy is responsible for protein ubiquitination. Because oxidative damage induced by SIN1A occurs almost instantly, maximal levels of ubiquitinated protein were obtained after 1 to 2 hours of treatment (Figure 4F).
Figure 4. Accumulation of polyubiquitinated proteins in 7-KC–treated SMCs. A, Western blot analysis of ubiquitin, E2 ubiquitin–conjugating enzymes UbcH6, UbcH7, and Ubc9, and the E3 ubiquitin ligase Itch in SMCs before and after 7-KC treatment. ?-actin served as a loading control. B through D, Immunohistochemical detection of ubiquitin (brown) in untreated SMCs (B) and SMCs treated with 100 μmol/L 7-KC for 12 hours (C and D). E, Western blot analysis of ubiquitin in SMCs treated for 12 hours with different concentrations of 7-KC. F, Western blot analysis of ubiquitin in SMCs treated with 1 mmol/L SIN1A for up to 1 hour. Results are representative of 3 independent experiments. Bar=50 μm.
Because polyubiquitination leads to protein degradation via the 26S proteasome, we investigated whether the proteasome inhibitor lactacystin could affect 7-KC–induced cell death. As shown in Figure V (available online at http://atvb.ahajournals.org), ubiquitinated proteins accumulated much faster in the cytoplasm (2 hours) compared with 7-KC–treated cells (Figure 4A). This resulted in a strong accumulation of the proapoptotic proteins p53, Bax, and Bid, as well as the nuclear factor B (NF-B) inhibitor IB (Figure V) and a more pronounced decrease in cell viability (15±2% cell viability versus 29±2% in 7-KC–treated cells 12 hours after treatment) without significant induction of apoptosis or necrosis. None of these proteins accumulated in 7-KC–treated SMCs (data not shown). Treatment of SMCs with lactacystin alone stimulated accumulation of ubiquitinated proteins (Figure V) but did not induce cell death.
Advanced Human Atherosclerotic Plaques Show Signs of Ubiquitination
To extend our in vitro observations described above to advanced human atherosclerotic plaques, we examined ubiquitin expression by immunohistochemistry in advanced plaques (thin fibrous cap atheromata) from carotid endarterectomy specimens. The majority of -SMC actin–positive cells in the plaque showed a strong nuclear staining for ubiquitin, whereas SMCs in adjacent margins of normal media were negative (Figure 5 A through 5C). CD68-positive macrophages showed an occasional nuclear staining (Figure 5D). Because cells in advanced stages of atherosclerosis often lose specific markers such as CD68 and -SMC actin, double immunostainings could not always be used to establish the identity of ubiquitin-immunoreactive cells. Therefore, we also combined immunodetection of ubiquitin with a periodic acid Schiff (PAS) stain. SMCs, particularly those present in the fibrous cap, but not macrophages were surrounded by a cage of PAS-positive basal lamina. By using this technique, we found macrophages (PAS-negative) around the necrotic core that stained negative for CD68 and positive for ubiquitin (only nuclear staining). Moreover, we could detect immunoreactivity for ubiquitin in the cytoplasm of -SMC actin–negative SMCs that were located in the fibrous cap and surrounded by a prominent cage of PAS-positive material (Figure 5E). Immunoreactivity for ubiquitin colocalized with Ubc9 expression (Figure 5F) but not with HSP-70 and HO1 expression (Figure VI, available online at http://atvb.ahajournals.org). HSP-70 and HO1 were detected predominantly in macrophages of the plaque showing weak nuclear staining for ubiquitin. HO1 expression was located specifically around microvessels in the plaque. Interestingly, 1% to 4% ubiquitin-immunoreactive SMCs located in the fibrous cap and enclosed by PAS-positive thickened basal laminae did not contain an intact nucleus and completely disintegrated into myriad vesicles. To distinguish apoptotic SMCs from SMCs undergoing nonapoptotic death, we combined ubiquitin immunostainings with TUNEL. Although 1% to 2% TUNEL-positive SMCs with numerous vesicles and surrounded by cages of basal laminae could be found, these cells were always ubiquitin negative (Figure 5G and 5H), indicating that ubiquitin-positive disintegrated SMCs may die an apoptosis-unrelated death. Importantly, this type of death coexists with apoptosis of SMCs in the same specimen.
Figure 5. Immunohistochemical detection of ubiquitin in atherosclerotic plaques of human carotid endarterectomy specimens. A, Low-power photomicrograph of endarterectomy specimen immunohistochemically stained for ubiquitin (brown) and -SMC actin (blue). SMCs in the plaque (PL) but not in the media (M) were positive for ubiquitin. B, High-power photomicrograph of the boxed area in A showing nuclear and cytoplasmic ubiquitin staining in SMCs of the plaque (arrows). C, High-power photomicrograph of medial SMCs as present in the boxed area of A. D, Double immunohistochemical staining for ubiquitin (brown) and CD68 (blue) in the plaque. E, Ubiquitin immunolabeling (brown) combined with PAS staining. Many SMCs that are surrounded by a prominent cage of PAS-positive basal laminae show cytoplasmic immunoreactivity for ubiquitin (white arrowheads). F, Double immunohistochemical staining for ubiquitin (blue) and Ubc9 (brown, arrowheads) in the plaque. G and H, Ubiquitin immunolabeling (blue) combined with TUNEL (brown) in the plaque. Ubiquitin-positive cells were TUNEL negative. Micrographs are representative of all carotid endarterectomy specimens studied (n=12). Bars=100 μm (A) and 10 μm (B through H).
Discussion
An increasing body of evidence suggests that SMC death and the progressive thinning of the fibrous cap are major events in the progression of atherosclerosis.2–5 Infiltration of inflammatory cells and intracellular accumulation of oxidized lipids, particularly oxysterols, are considered the main triggers responsible for initiation of cell death.3 Recently, Zahm et al17 reported that 7-KC is a potent inducer of SMC death characterized by loss of cell adhesion, modification of actin organization, decrease in mitochondrial transmembrane potential, and condensation or fragmentation of the nucleus. According to previous reports from several groups, 7-KC cytotoxicity in SMCs is mediated by apoptosis.10,18,19 In this study, we present evidence that human SMCs treated with 7-KC undergo a complex mode of cell death with some characteristics of autophagy (type II cell death). However, we do not rule out the possibility that apoptosis and necrosis occurred in a small subgroup of treated cells because a small increase in annexin V labeling and PI incorporation was noticed. Therefore, it is reasonable to assume that 7-KC may induce simultaneously various types of cell death but at different rates. Although this hypothesis may seem contradictory to published literature, it should be noted that apoptosis can be easily detected with various techniques, whereas at present, autophagy requires electron microscopy for unambiguous detection. On the other hand, it is of note that the 7-KC concentration used in different studies varies from 12.5 to 200 μmol/L. This might have an important impact on the type of death and the incubation period before cell death becomes imminent. We used a concentration of 100 μmol/L 7-KC, which is 1000-fold higher than the 7-KC levels in human plasma20 and 25-fold higher than the plasma 7-KC levels after a fat-rich meal.21
Autophagy is a general manifestation of injury that involves the sequestration of intracellular components and their subsequent degradation in secondary lysosomes.16,22 This process occurs in at least 2 steps: first, organelles or portions of cytosol are sequestrated by an enveloping membrane, resulting in the formation of an autophagosome. The latter subsequently fuses with a primary lysosome to form an autolysosome where the cell content is digested by lysosomal enzymes. We could demonstrate that 7-KC–treated SMCs contain myelin figures evocative of autophagic vacuoles, as shown previously for 7-KC–treated monocytes and endothelial cells.13,14 Moreover, other markers of autophagy such as extensive vacuolization and depletion of organelles were present. Formation of acidic vacuoles was confirmed by labeling cells with LysoTracker and by demonstrating LC3 processing. These features were never observed in apoptosis or oncosis but are considered characteristic of autophagy.16,22 In addition, 7-KC–induced cell death was characterized by a rapid depolarization of the mitochondrial inner membrane potential, which is a common mechanism in apoptotic, oncotic, and autophagic cell death.23 If cellular injury is severe, a major proportion of the cytosol and organelles (most noticeably the mitochondria and endoplasmic reticulum) will be destroyed, and this eventually leads to autophagic cell death. Importantly, autophagy does not directly destroy the plasma membrane (typical of necrosis) or the nucleus (absence of nuclear or internucleosomal DNA fragmentation), as shown in 7-KC–treated SMCs, perhaps because the nucleus is too large to be engulfed.
7-KC–induced SMC death was associated with protein ubiquitination, as described previously for cells treated with aggregated LDL24 or oxLDL.25,26 Ubiquitin targets proteins for degradation by a multisubunit, ATP-dependent protease termed the proteasome, and thus fulfills an important function in elimination of damaged or unneeded proteins.27 Indeed, SMCs treated with the peroxynitrite donor SIN1A showed increased ubiquitination, most likely to remove oxidatively modified proteins, but this was not the case with autophagic SMCs undergoing starvation. Because 7-KC–treated cells showed enhanced immunoreactivity for the lipid peroxidation marker 4-hydroxy-2-nonenal, it is tempting to speculate that oxidative stress is the major cause of protein ubiquitination during 7-KC stimulation. Furthermore, ubiquitination is one of the major mechanisms that regulate apoptotic cell death.28 Inhibition of the proteasome function by lactacystin as shown in this study leads to accumulation of several proapoptotic proteins including p53, Bax, and Bid, as well as the NF-B inhibitor IB. Accumulation of ubiquitin/protein conjugates potentiated considerably the toxicity of 7-KC, which confirms previous studies with oxLDL.25 However, accumulation of ubiquitin-conjugated proapoptotic proteins did not give rise to a higher incidence of apoptotic cell death. There is a possibility that certain proapoptotic proteins have a function in apoptosis and autophagic cell death, as proposed recently for Bax by Camougrand et al.29 Interestingly, the core protein machinery necessary to drive formation of autophagosomes includes a ubiquitin-like protein conjugation system.16,22 Moreover, Kostin et al8 reported that autophagic vacuoles are positive for ubiquitin immunolabeling. Therefore, it is reasonable to assume that ubiquitination of proteins and autophagic cell death are closely interacting and self-supporting phenomena during 7-KC–mediated cell death.
In analogy to our in vitro studies, SMCs in advanced human atherosclerotic plaques showed signs of ubiquitination, apoptosis, and nonapoptotic cell death. Similar findings were reported for the cellular degeneration of myocytes in failing human hearts.8 Moreover, ubiquitin immunoreactivity of SMCs is enhanced in unstable, infarct-related coronary plaques, predominantly in plaque regions of tissue degeneration,30 indicating that the ubiquitin–proteasome system might play an essential role in the destabilization and rupture of atherosclerotic plaques. In contrast to medial SMCs, most SMCs in the plaque showed nuclear staining for ubiquitin, which could indicate changes in nuclear functioning such as regulation of transcription, DNA repair, and replication.31 However, a significant number of SMCs in the fibrous cap showed cytoplasmic immunoreactivity for ubiquitin. These SMCs are surrounded by a cage of thickened basal laminae and often disintegrate into numerous cytoplasmic fragments. Previous work in our group revealed that this form of cell death has several characteristics of apoptotic cell death (DNA fragmentation and upregulation of Bax).2 However, it is important to note that we could detect disintegrated SMCs enclosed by thickened basal laminae, which were immunoreactive for ubiquitin in their cytoplasm and TUNEL negative, suggesting that they died a nonapoptotic death. Interestingly, lipid-laden SMCs in human plaques but not macrophages upregulate death-associated protein (DAP) kinase,32 a positive mediator of apoptotic cell death that also mediates membrane blebbing and the formation of autophagic vesicles.33 Because autophagy and proteasome-mediated degradation are the major cellular pathways for turnover of damaged protein and organelles,16,22 there is a possibility that ubiquitination of cytoplasmic proteins in SMCs of atherosclerotic plaques together with enhanced levels of DAP kinase and cellular disintegration into TUNEL-negative remnants point to autophagic cell death. If autophagy would occur in SMCs of advanced human plaques, its significance remains to be elucidated. Indeed, several lines of evidence support the hypothesis that autophagy is not a death pathway but a survival mechanism activated during cellular distress.34
In summary, treatment of SMCs with 7-KC may induce oxidative damage, activation of the ubiquitin–proteasome system, and induction of cell death. Although we do not exclude apoptosis or oncosis, our data indicate that the majority of 7-KC–treated SMCs preferentially undergo a complex mode of cell death associated with the formation of myelin figure and LC3 processing, evocating autophagy. Because the ubiquitin–proteasome system could be a unique target for therapeutic intervention,35,36 additional work may reveal the precise role of ubiquitin in plaque destabilization.
Acknowledgments
Research was supported by the Fund for Scientific Research-Flanders (projects G.0080.98, G.0180.01, and G.0427.02). W.M. is a postdoctoral fellow of the Fund for Scientific Research-Flanders. M.M.K. held a fund for fundamental clinical research of the Fund for Scientific Research-Flanders. The authors are indebted to Jeff Thielemans, Hermine Fret, and André Van Daele for excellent technical assistance, Dr L. Andries (HistoGeneX, Belgium) for fluorescence microscopy, and Dr T. Yoshimori (National Institute of Genetics, Mishima, Japan) for providing anti-LC3 antibody.
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ABSTRACT
Objective— Oxysterols such as 7-ketocholesterol (7-KC) are important mediators of cell death in atherosclerosis. Therefore, in vitro studies of human smooth muscle cell (SMC) death in response to 7-KC were undertaken to investigate the potential mechanisms.
Methods and Results— Human aortic SMCs treated with 7-KC showed enhanced immunoreactivity for the oxidative stress marker 4-hydroxy-2-nonenal and upregulated several stress genes (70-kDa heat shock protein 1, heme oxygenase 1, and growth arrest and DNA damage–inducible protein 153) at the mRNA but not at the protein level. 7-KC–treated SMCs rapidly underwent cell death as determined by neutral red, counting of adherent cells, and depolarization of the mitochondrial inner membrane. Cell death was associated with upregulation of ubiquitin mRNA and ubiquitination of cellular proteins. Inhibition of the proteasome by lactacystin potentiated considerably the toxicity of 7-KC. Transmission electron microscopy revealed formation of myelin figures, extensive vacuolization, and depletion of organelles. Formation of autophagosomes was suggested by labeling cells with LysoTracker and monitoring processing of microtubule-associated protein 1 light chain 3 (LC3). Analogous to our in vitro studies, human atherosclerotic plaques showed signs of ubiquitination in SMCs.
Conclusions— 7-KC activates the ubiquitin–proteasome system and induces a complex mode of cell death associated with myelin figure formation and processing of LC3 evocating autophagic processes.
7-Ketocholesterol induces a complex mode of cell death in human aortic smooth muscle cells associated with accumulation of ubiquitinated proteins in the cytoplasm, myelin figure formation, and LC3 processing, evocating autophagic processes.
Key Words: 7-ketocholesterol ? ubiquitination ? smooth muscle cells ? myelin figures ? LC3 ? autophagy ? atherosclerosis
Introduction
Cell death is a major event in the progression of atherosclerosis. Indeed, a large body of evidence suggests that apoptosis or type I programmed cell death1 frequently occurs in advanced human plaques (1% to 2% TUNEL-positive nuclei).2–5 However, results from electron microscopy studies showed that the majority of dying cells have an ultrastructure typical of cells undergoing "accidental" cell death or oncosis6 (type III programmed cell death).1 Furthermore, it is important to note that there are multiple pathways leading to cell death. Cells sometimes die with a morphology that is intermediate between apoptosis and oncosis (eg, aponecrosis and paraptosis), or they undergo cell death with a less clear morphology or mechanism such as lysosome-mediated cell death or autophagy (type II programmed cell death).1 It is presently unknown whether cells in human atherosclerotic plaques die by mechanisms distinct from apoptosis or oncosis, as shown recently for myocytes in failing human hearts.7,8
Oxidative processes, particularly oxidation of low-density lipoprotein (LDL), are thought to play a pivotal role in atherogenesis.9 Oxidized LDL (oxLDL) exerts its proatherogenic effects in several ways, including stimulation of inflammatory responses, foam cell formation, and induction of cell death.9 The mechanism of oxLDL-induced cell death is unclear, as oncosis and apoptosis have been reported,10 and largely depends on the oxidation degree, exposure time, and concentration of oxLDL.3 In macrophages, oxLDL induces mitochondrial dysfunction and lysosomal damage,11,12 indicating that oxLDL uptake is essential for its cytotoxic properties. In addition, it has been suggested that oxysterols such as 7?-hydroxycholesterol and 7-ketocholesterol (7-KC) are the primary cytotoxins of oxLDL. Although their role in oxLDL-mediated cytotoxicity remains unclear, oxysterol-induced cell death is associated with an enhanced production of reactive oxygen species and formation of myelin figures evocative of autophagic vacuoles.13,14
In the present study, we show that 7-KC induces a complex mode of cell death in human vascular smooth muscle cells (SMCs) associated with myelin figure formation and light chain 3 (LC3) processing, evocating autophagic processes. We also demonstrate that exposure of SMCs to 7-KC is associated with accumulation of ubiquitinated proteins in the cytoplasm. Furthermore, we provide evidence that ubiquitination also occurs in SMCs of advanced human atherosclerotic plaques.
Methods
The Methods sections is available online at http://atvb.ahajournals.org.
Results
Induction of Complex Mode of Cell Death in 7-KC–Treated SMCs
Treatment of human aortic SMCs with 100 μmol/L 7-KC disturbed the typical cell spreading of SMCs (acquisition of an elongated shape followed by rounding up of cells) and rapidly induced cell death as assessed by neutral red and counting of adherent cells (Figure 1A). The majority of cells did not contain cleaved caspase-3 (Figure 1B) and were negative for annexin V labeling and propidium iodide (PI) incorporation (Figure 1A), albeit a small but significant increase in annexin V and PI labeling was noticed after 12 hours of treatment (Figure 1A). Cells treated with 7-KC showed enhanced immunoreactivity for 4-hydroxy-2-nonenal (Figure I, available online at http://atvb.ahajournals.org), a major product of endogenous lipid peroxidation and an important marker of oxidative stress. We also observed a significant depolarization of the mitochondrial inner membrane potential as early as 2 hours after 7-KC administration (Figure 1A). However, internucleosomal DNA fragmentation typical of apoptosis could not be detected, and labeling of cells with Hoechst revealed neither a significant rise in chromatin condensation nor fragmentation of the nucleus (Figure II, available online at http://atvb.ahajournals.org). Transmission electron microscopic analysis of cells treated with 7-KC for 12 hours revealed formation of membranous whorls (also called myelin figures) in different stages of development, extensive vacuolization, and depletion of organelles (Figure 2 C through 2F). Several of these features, such as myelin figure formation and vacuolization, initiated within 2 hours after 7-KC administration (Figure III, available online at http://atvb.ahajournals.org). Of note, untreated controls also showed presence of small vesicles, especially in older cells (passage >5), suggesting that these vesicles are formed spontaneously to remove damaged intracellular components or protein aggregates (Figure 2A and 2B). To exclude the possibility that the inclusions in the vacuoles of dying cells represent heterophagocytized remnants of dead cells rather than autophagocytized intracellular components, SMCs were treated with 7-KC in the presence of fluorescent beads. After 12 hours, a small group of cells (2% to 3%) contained one single bead (Figure IV, available online at http://atvb.ahajournals.org). However, uptake of multiple beads, as shown in control J774 macrophages incubated with beads for 1 hour, could not be demonstrated (Figure IV). Accumulation of acidic organelles in 7-KC–treated cells was confirmed by labeling cells with LysoTracker (Figure 3 A and 3B). Moreover, initiation of 7-KC–induced cell death was associated with the conversion of the cleaved 18-kDa protein microtubule-associated protein 1 LC3 (LC3-I) into the 16-kDa protein LC3-II (Figure 3C), which is considered a reliable marker of autophagosome formation.15,16
Figure 1. Characterization of 7-KC–induced cell death in human aortic SMCs. Cells were treated with 100 μmol/L 7-KC for up to 24 hours. A, Cell death was examined by a neutral red viability assay, analysis of cell adherence, and disruption of the mitochondrial transmembrane potential (m), PI incorporation, and annexin V labeling. B, Western blot analysis of procaspase-3 (procasp-3) cleavage. Monocytic U937 cells treated with 50 μmol/L etoposide (+) for 5 hours served as a positive control. Results are representative of at least 3 independent experiments. **P<0.01; ***P<0.001 vs 0 hours (ANOVA followed by Dunnett test).
Figure 2. Ultrastructural features of SMCs treated with 100 μmol/L 7-KC for 12 hours. A and B show untreated controls with normal cell morphology. Note formation of small vesicles (arrowheads) that may arise under normal physiological conditions to remove abnormal proteins and other cytoplasmic macromolecules. However, treatment of SMCs with 7-KC causes severe cellular injury so that the cell is no longer able to process damaged cellular components. This results in cell death evocating autophagy, as shown in C through F, and is characterized by the accumulation of autophagic vesicles, extensive vacuolization (D and E), degradation of membranous cellular components that rearrange in membranous whorls called myelin figures (arrows), depletion of organelles, and finally complete disintegration of the cell (F). Data are representative of 2 separate experiments. Bar=1 μm. N indicates nucleus.
Figure 3. Formation of acidic vacuoles in 7-KC–treated SMCs. A and B, Labeling of untreated controls (A) or SMCs treated with 100 μmol/L 7-KC for 12 hours (B) with LysoTracker. Images represent adherent cells only. C, Western blot analysis of SMCs before and after treatment with 7-KC showing processing of the 18-kDa microtubule-associated LC3-I into the 16-kDa protein LC3-II, a marker of autophagosome formation. Data are representative of 3 separate experiments.
To further characterize 7-KC–induced cell death, transcript levels of 205 apoptosis-related and 234 stress-related genes were analyzed using cDNA expression arrays. Twelve hours after exposure to 7-KC, 4 stress-related genes with a differential steady-state expression level of >5-fold could be identified: 70-kDa heat shock protein 1 (HSP-70; 25.3-fold), heme oxygenase 1 (HO1; 7-fold), and growth arrest and DNA damage-inducible protein 153 (GADD153; 5.9-fold) were upregulated in 7-KC–treated SMCs versus untreated cells, whereas proliferating cyclic nuclear antigen (PCNA; 5.2-fold) was downregulated. Furthermore, it is noteworthy that the housekeeping gene ubiquitin was moderately upregulated (2.5-fold). Neither ubiquitin-like protein Nedd8 nor typical apoptosis-related genes such as Bcl-2 family members, caspases, or death receptors showed differential gene expression. Array results could be confirmed by real-time RT-PCR (12.3±1.3 , P<0.05; 20.6±3.0 , P<0.05; 28.3±2.8 , P<0.01; 6.6±3.3 , P=0.23;and 1.8±0.1 , P<0.01; n=3). However, differential expression of HSP-70 and PCNA at the protein level could not be observed at the different time points studied (0 to 24 hours; data not shown). Expression of HO1 protein did not increase but significantly decreased after 2 hours of 7-KC stimulation (data not shown), possibly as a result of protein degradation.
7-KC–Treated SMCs Accumulate Ubiquitinated Proteins
In addition to upregulation of ubiquitin mRNA, Western blots for ubiquitin demonstrated that SMCs accumulated various ubiquitinated proteins or protein aggregates of high molecular weight (>100 kDa) after 6 to 12 hours of exposure to 7-KC (Figure 4A). Protein ubiquitination was accompanied by a progressive increase in the expression of E2 ubiquitin–conjugating enzymes UbcH6, UbcH7, Ubc9, and the E3 ubiquitin ligase Itch (Figure 4A). Immunostaining of 7-KC–treated SMCs for ubiquitin showed an intense granular signal in the cytoplasm (Figure 4C and 4D), whereas in untreated SMCs, ubiquitin was difficult to detect (Figure 4B). Accumulation of ubiquitinated proteins could already be detected in cells treated with 6.25 μmol/L 7-KC for 12 hours (Figure 4E), but significant induction of cell death was only observed within 24 hours if cells were exposed to 7-KC concentrations 50 μmol/L. Besides 7-KC–treated SMCs, SMCs treated with the peroxynitrite-donor SIN1A (1 mmol/L) showed accumulation of ubiquitinated protein (Figure 4F), but this was not the case with SMCs undergoing amino acid deprivation (data not shown), suggesting that acute oxidative stress rather than autophagy is responsible for protein ubiquitination. Because oxidative damage induced by SIN1A occurs almost instantly, maximal levels of ubiquitinated protein were obtained after 1 to 2 hours of treatment (Figure 4F).
Figure 4. Accumulation of polyubiquitinated proteins in 7-KC–treated SMCs. A, Western blot analysis of ubiquitin, E2 ubiquitin–conjugating enzymes UbcH6, UbcH7, and Ubc9, and the E3 ubiquitin ligase Itch in SMCs before and after 7-KC treatment. ?-actin served as a loading control. B through D, Immunohistochemical detection of ubiquitin (brown) in untreated SMCs (B) and SMCs treated with 100 μmol/L 7-KC for 12 hours (C and D). E, Western blot analysis of ubiquitin in SMCs treated for 12 hours with different concentrations of 7-KC. F, Western blot analysis of ubiquitin in SMCs treated with 1 mmol/L SIN1A for up to 1 hour. Results are representative of 3 independent experiments. Bar=50 μm.
Because polyubiquitination leads to protein degradation via the 26S proteasome, we investigated whether the proteasome inhibitor lactacystin could affect 7-KC–induced cell death. As shown in Figure V (available online at http://atvb.ahajournals.org), ubiquitinated proteins accumulated much faster in the cytoplasm (2 hours) compared with 7-KC–treated cells (Figure 4A). This resulted in a strong accumulation of the proapoptotic proteins p53, Bax, and Bid, as well as the nuclear factor B (NF-B) inhibitor IB (Figure V) and a more pronounced decrease in cell viability (15±2% cell viability versus 29±2% in 7-KC–treated cells 12 hours after treatment) without significant induction of apoptosis or necrosis. None of these proteins accumulated in 7-KC–treated SMCs (data not shown). Treatment of SMCs with lactacystin alone stimulated accumulation of ubiquitinated proteins (Figure V) but did not induce cell death.
Advanced Human Atherosclerotic Plaques Show Signs of Ubiquitination
To extend our in vitro observations described above to advanced human atherosclerotic plaques, we examined ubiquitin expression by immunohistochemistry in advanced plaques (thin fibrous cap atheromata) from carotid endarterectomy specimens. The majority of -SMC actin–positive cells in the plaque showed a strong nuclear staining for ubiquitin, whereas SMCs in adjacent margins of normal media were negative (Figure 5 A through 5C). CD68-positive macrophages showed an occasional nuclear staining (Figure 5D). Because cells in advanced stages of atherosclerosis often lose specific markers such as CD68 and -SMC actin, double immunostainings could not always be used to establish the identity of ubiquitin-immunoreactive cells. Therefore, we also combined immunodetection of ubiquitin with a periodic acid Schiff (PAS) stain. SMCs, particularly those present in the fibrous cap, but not macrophages were surrounded by a cage of PAS-positive basal lamina. By using this technique, we found macrophages (PAS-negative) around the necrotic core that stained negative for CD68 and positive for ubiquitin (only nuclear staining). Moreover, we could detect immunoreactivity for ubiquitin in the cytoplasm of -SMC actin–negative SMCs that were located in the fibrous cap and surrounded by a prominent cage of PAS-positive material (Figure 5E). Immunoreactivity for ubiquitin colocalized with Ubc9 expression (Figure 5F) but not with HSP-70 and HO1 expression (Figure VI, available online at http://atvb.ahajournals.org). HSP-70 and HO1 were detected predominantly in macrophages of the plaque showing weak nuclear staining for ubiquitin. HO1 expression was located specifically around microvessels in the plaque. Interestingly, 1% to 4% ubiquitin-immunoreactive SMCs located in the fibrous cap and enclosed by PAS-positive thickened basal laminae did not contain an intact nucleus and completely disintegrated into myriad vesicles. To distinguish apoptotic SMCs from SMCs undergoing nonapoptotic death, we combined ubiquitin immunostainings with TUNEL. Although 1% to 2% TUNEL-positive SMCs with numerous vesicles and surrounded by cages of basal laminae could be found, these cells were always ubiquitin negative (Figure 5G and 5H), indicating that ubiquitin-positive disintegrated SMCs may die an apoptosis-unrelated death. Importantly, this type of death coexists with apoptosis of SMCs in the same specimen.
Figure 5. Immunohistochemical detection of ubiquitin in atherosclerotic plaques of human carotid endarterectomy specimens. A, Low-power photomicrograph of endarterectomy specimen immunohistochemically stained for ubiquitin (brown) and -SMC actin (blue). SMCs in the plaque (PL) but not in the media (M) were positive for ubiquitin. B, High-power photomicrograph of the boxed area in A showing nuclear and cytoplasmic ubiquitin staining in SMCs of the plaque (arrows). C, High-power photomicrograph of medial SMCs as present in the boxed area of A. D, Double immunohistochemical staining for ubiquitin (brown) and CD68 (blue) in the plaque. E, Ubiquitin immunolabeling (brown) combined with PAS staining. Many SMCs that are surrounded by a prominent cage of PAS-positive basal laminae show cytoplasmic immunoreactivity for ubiquitin (white arrowheads). F, Double immunohistochemical staining for ubiquitin (blue) and Ubc9 (brown, arrowheads) in the plaque. G and H, Ubiquitin immunolabeling (blue) combined with TUNEL (brown) in the plaque. Ubiquitin-positive cells were TUNEL negative. Micrographs are representative of all carotid endarterectomy specimens studied (n=12). Bars=100 μm (A) and 10 μm (B through H).
Discussion
An increasing body of evidence suggests that SMC death and the progressive thinning of the fibrous cap are major events in the progression of atherosclerosis.2–5 Infiltration of inflammatory cells and intracellular accumulation of oxidized lipids, particularly oxysterols, are considered the main triggers responsible for initiation of cell death.3 Recently, Zahm et al17 reported that 7-KC is a potent inducer of SMC death characterized by loss of cell adhesion, modification of actin organization, decrease in mitochondrial transmembrane potential, and condensation or fragmentation of the nucleus. According to previous reports from several groups, 7-KC cytotoxicity in SMCs is mediated by apoptosis.10,18,19 In this study, we present evidence that human SMCs treated with 7-KC undergo a complex mode of cell death with some characteristics of autophagy (type II cell death). However, we do not rule out the possibility that apoptosis and necrosis occurred in a small subgroup of treated cells because a small increase in annexin V labeling and PI incorporation was noticed. Therefore, it is reasonable to assume that 7-KC may induce simultaneously various types of cell death but at different rates. Although this hypothesis may seem contradictory to published literature, it should be noted that apoptosis can be easily detected with various techniques, whereas at present, autophagy requires electron microscopy for unambiguous detection. On the other hand, it is of note that the 7-KC concentration used in different studies varies from 12.5 to 200 μmol/L. This might have an important impact on the type of death and the incubation period before cell death becomes imminent. We used a concentration of 100 μmol/L 7-KC, which is 1000-fold higher than the 7-KC levels in human plasma20 and 25-fold higher than the plasma 7-KC levels after a fat-rich meal.21
Autophagy is a general manifestation of injury that involves the sequestration of intracellular components and their subsequent degradation in secondary lysosomes.16,22 This process occurs in at least 2 steps: first, organelles or portions of cytosol are sequestrated by an enveloping membrane, resulting in the formation of an autophagosome. The latter subsequently fuses with a primary lysosome to form an autolysosome where the cell content is digested by lysosomal enzymes. We could demonstrate that 7-KC–treated SMCs contain myelin figures evocative of autophagic vacuoles, as shown previously for 7-KC–treated monocytes and endothelial cells.13,14 Moreover, other markers of autophagy such as extensive vacuolization and depletion of organelles were present. Formation of acidic vacuoles was confirmed by labeling cells with LysoTracker and by demonstrating LC3 processing. These features were never observed in apoptosis or oncosis but are considered characteristic of autophagy.16,22 In addition, 7-KC–induced cell death was characterized by a rapid depolarization of the mitochondrial inner membrane potential, which is a common mechanism in apoptotic, oncotic, and autophagic cell death.23 If cellular injury is severe, a major proportion of the cytosol and organelles (most noticeably the mitochondria and endoplasmic reticulum) will be destroyed, and this eventually leads to autophagic cell death. Importantly, autophagy does not directly destroy the plasma membrane (typical of necrosis) or the nucleus (absence of nuclear or internucleosomal DNA fragmentation), as shown in 7-KC–treated SMCs, perhaps because the nucleus is too large to be engulfed.
7-KC–induced SMC death was associated with protein ubiquitination, as described previously for cells treated with aggregated LDL24 or oxLDL.25,26 Ubiquitin targets proteins for degradation by a multisubunit, ATP-dependent protease termed the proteasome, and thus fulfills an important function in elimination of damaged or unneeded proteins.27 Indeed, SMCs treated with the peroxynitrite donor SIN1A showed increased ubiquitination, most likely to remove oxidatively modified proteins, but this was not the case with autophagic SMCs undergoing starvation. Because 7-KC–treated cells showed enhanced immunoreactivity for the lipid peroxidation marker 4-hydroxy-2-nonenal, it is tempting to speculate that oxidative stress is the major cause of protein ubiquitination during 7-KC stimulation. Furthermore, ubiquitination is one of the major mechanisms that regulate apoptotic cell death.28 Inhibition of the proteasome function by lactacystin as shown in this study leads to accumulation of several proapoptotic proteins including p53, Bax, and Bid, as well as the NF-B inhibitor IB. Accumulation of ubiquitin/protein conjugates potentiated considerably the toxicity of 7-KC, which confirms previous studies with oxLDL.25 However, accumulation of ubiquitin-conjugated proapoptotic proteins did not give rise to a higher incidence of apoptotic cell death. There is a possibility that certain proapoptotic proteins have a function in apoptosis and autophagic cell death, as proposed recently for Bax by Camougrand et al.29 Interestingly, the core protein machinery necessary to drive formation of autophagosomes includes a ubiquitin-like protein conjugation system.16,22 Moreover, Kostin et al8 reported that autophagic vacuoles are positive for ubiquitin immunolabeling. Therefore, it is reasonable to assume that ubiquitination of proteins and autophagic cell death are closely interacting and self-supporting phenomena during 7-KC–mediated cell death.
In analogy to our in vitro studies, SMCs in advanced human atherosclerotic plaques showed signs of ubiquitination, apoptosis, and nonapoptotic cell death. Similar findings were reported for the cellular degeneration of myocytes in failing human hearts.8 Moreover, ubiquitin immunoreactivity of SMCs is enhanced in unstable, infarct-related coronary plaques, predominantly in plaque regions of tissue degeneration,30 indicating that the ubiquitin–proteasome system might play an essential role in the destabilization and rupture of atherosclerotic plaques. In contrast to medial SMCs, most SMCs in the plaque showed nuclear staining for ubiquitin, which could indicate changes in nuclear functioning such as regulation of transcription, DNA repair, and replication.31 However, a significant number of SMCs in the fibrous cap showed cytoplasmic immunoreactivity for ubiquitin. These SMCs are surrounded by a cage of thickened basal laminae and often disintegrate into numerous cytoplasmic fragments. Previous work in our group revealed that this form of cell death has several characteristics of apoptotic cell death (DNA fragmentation and upregulation of Bax).2 However, it is important to note that we could detect disintegrated SMCs enclosed by thickened basal laminae, which were immunoreactive for ubiquitin in their cytoplasm and TUNEL negative, suggesting that they died a nonapoptotic death. Interestingly, lipid-laden SMCs in human plaques but not macrophages upregulate death-associated protein (DAP) kinase,32 a positive mediator of apoptotic cell death that also mediates membrane blebbing and the formation of autophagic vesicles.33 Because autophagy and proteasome-mediated degradation are the major cellular pathways for turnover of damaged protein and organelles,16,22 there is a possibility that ubiquitination of cytoplasmic proteins in SMCs of atherosclerotic plaques together with enhanced levels of DAP kinase and cellular disintegration into TUNEL-negative remnants point to autophagic cell death. If autophagy would occur in SMCs of advanced human plaques, its significance remains to be elucidated. Indeed, several lines of evidence support the hypothesis that autophagy is not a death pathway but a survival mechanism activated during cellular distress.34
In summary, treatment of SMCs with 7-KC may induce oxidative damage, activation of the ubiquitin–proteasome system, and induction of cell death. Although we do not exclude apoptosis or oncosis, our data indicate that the majority of 7-KC–treated SMCs preferentially undergo a complex mode of cell death associated with the formation of myelin figure and LC3 processing, evocating autophagy. Because the ubiquitin–proteasome system could be a unique target for therapeutic intervention,35,36 additional work may reveal the precise role of ubiquitin in plaque destabilization.
Acknowledgments
Research was supported by the Fund for Scientific Research-Flanders (projects G.0080.98, G.0180.01, and G.0427.02). W.M. is a postdoctoral fellow of the Fund for Scientific Research-Flanders. M.M.K. held a fund for fundamental clinical research of the Fund for Scientific Research-Flanders. The authors are indebted to Jeff Thielemans, Hermine Fret, and André Van Daele for excellent technical assistance, Dr L. Andries (HistoGeneX, Belgium) for fluorescence microscopy, and Dr T. Yoshimori (National Institute of Genetics, Mishima, Japan) for providing anti-LC3 antibody.
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