Published online before print January 25, 2007, doi: 10.1161/01.ATV.0000258794.57872.3f
© 2007 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
IAP Survivin Regulates Atherosclerotic Macrophage Survival
From Centre de Recherche Cardiovasculaire Inserm-Lariboisère U689, (O.P.B.-B., Z.M., A.T.) Hôpital Lariboisière, Paris, France; INSERM U325 (E.T., B.S.), Institut Pasteur de Lille, Lille, France; Service de Chirurgie Vasculaire et Thoracique (Y.C., G.L.), Hôpital Bichat-Claude Bernard, Paris, France; Cardiovascular Research Institute Maastricht (CARIM) (A.-P.B., M.D.), University of Maastricht, Maastricht, The Netherlands.
Correspondence to Olivier Blanc-Brude, PhD, Centre de Recherche Cardiovasculaire Inserm-Lariboisère U689, Hôpital Lariboisière, 41 boulevard de la Chapelle, F-75475, Paris CEDEX 10, France. E-mail olivier.blanc-brude{at}larib.inserm.fr
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Abstract |
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Objectives— Inflammatory macrophage apoptosis is critical to atherosclerotic plaque formation, but its mechanisms remain enigmatic. We hypothesized that inhibitor of apoptosis protein (IAP) survivin regulates macrophage death in atherosclerosis.
Methods and Results— Western blot analysis revealed discrete survivin expression in human aorta lipid streaks but virtually none in advanced atherosclerotic plaques, despite increased XIAP and cIAP2 levels. Survivin was detected in CD68-positive macrophages infiltrating human lipid streaks by immunohistochemistry. In advanced atherosclerotic plaques, only rare macrophages outside the necrotic core or occasional fibrous cap smooth muscle cells expressed survivin. In vitro, macrophage colony-stimulating factor-stimulated mouse macrophage survivin expression, proliferation and resistance to apoptosis. Conversely, prolonged oxidized low-density lipoprotein treatment abolished macrophage survivin expression and triggered apoptosis after 12 hours, despite enhanced XIAP and cIAP2 expression. Adenoviral overexpression of survivin conferred macrophages with sustained resistance to apoptosis after oxidized low-density lipoprotein, tumor necrosis factor-
, or staurosporine challenge.
Conclusions— Survivin is a critical modulator of atherosclerotic macrophage apoptosis under dual control by growth factors and oxidized lipids accumulating in atheroma. In early lipid streaks, growth factor-stimulated survivin expression may contribute to macrophage accumulation and survival, but dysregulation of survivin expression caused by recurrent oxidized low-density lipoprotein exposure may favor apoptosis in advanced atherosclerotic plaques, despite upregulated cIAP2 and XIAP.
Macrophages express inhibitor of apoptosis (IAP) survivin in human aorta lipid streaks, or after growth factors stimulation, but not in advanced atherosclerotic plaques, or after loading with lipid degradation products. Adenoviral transfection showed that survivin is a predominant modulator of macrophage apoptosis in atherosclerotic conditions, regardless of other IAP proteins.
Key Words: apoptosis • atherosclerosis • inflammation • leukocytes
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Introduction |
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Atherosclerosis is an inflammatory disease of the vascular wall that leads to myocardial infarction, stroke, and sudden death. Critical to the disease is the infiltration and accumulation of low-density lipoproteins (LDL) into the subendothelial space where they oxidize, leading to the recruitment of circulating inflammatory cells to the vascular wall, including monocytes. Increasing evidence show that the atherosclerotic plaque hosts widespread apoptotic reactions targeting all cell types, including foam cells of macrophage origin, endothelial cells, smooth muscle cells (SMCs), and T-lymphocytes.1–4 Physiological apoptosis may serve to eliminate superfluous cells, but its deregulation may promote atherogenesis and life-threatening manifestations of the disease, such as necrotic lipid core formation,2,5,6 weakening of the fibrous cap,7,8 and sustained prothrombotic inflammatory reactions.9,10
Atheroma hosts apoptotic stimuli such as lipid degradation products and radical oxygen species.10–12 Atherosclerotic plaque apoptosis6,8 is associated with predominant caspase-3 activation in SMCs13 and macrophages.7,12,14,15 Atherosclerotic SMCs and macrophages express pro-apoptotic regulators of mitochondrial integrity Bax and Bad, but they lack cytoprotective Bcl-xL and Bcl-2,16,17 resulting in defective mitochondrial integrity and apoptosis. Moreover, the endogenous caspase inhibitor, cellular inhibitor of apoptosis (IAP) protein-2 (cIAP2) is thought to be expressed in atherosclerotic endothelial cells,18 but these cells are prone to apoptosis in atherosclerosis. Thus, the mechanisms activated in atherosclerotic cells and the role of IAP remain unclear.
Survivin is a caspase inhibitor of the IAP family, also comprising XIAP, cIAP1, cIAP2, NAIP, livin, and apollon. However, survivin shows unique features.19,20 It is the only anti-apoptotic mediator transcribed specifically during mitosis and thought to be expressed during development and cancer, but mostly is absent from normal adult tissues.21–23 Survivin’s anti-apoptotic function is doubly controlled during G2/M via phosphorylation by the CDC2 kinase.24,25 Survivin plays a key role in a p53-dependent cell cycle checkpoint that rules over cell survival during mitosis.26,27 Survivin is expressed in proliferating endothelial cells and SMCs during angiogenesis28,29 and vascular wall remodeling.30 Survivin is thought to block effector caspase activation via apoptosome inhibition,25 whereas XIAP, cIAP1, and cIAP2 are thought to suppress effector caspase-3 and caspase-7 activity through direct interactions.31,32
Here, we studied survivin versus XIAP and cIAP2 expression in human atherosclerotic plaques and macrophages exposed to growth factors and oxidized low-density lipoprotein (oxLDL) in vitro.
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Methods |
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Histological Analysis
Human endarterectomy specimens of atherosclerotic carotids (n=8) and aorta wall samples containing lipid streaks (n=4) were obtained in full accordance with French law and the Inserm ethics guidelines. Segments of thoracic aorta near the origin of the Ductus Arteriosus (on the concavity of the posterior segment of the aortic arch, in the isthmus) were obtained from 4 male multi-organ donors aged between 25 and 45. Samples were immediately snap-frozen after dissection, and preparation of the heart and individual lungs for transplantation was performed. Eight-µm-thick serial cryosections of snap-frozen tissues were assayed for lipid deposition with Oil Red-O, or immunoreacted with rabbit polyclonal antibodies to survivin29,30 (Novus Biologicals), CD68 (Sigma Chemicals), monoclonal mouse antibody against human
-smooth muscle actin (clone 1A4; Sigma), or matched control antibodies (Sigma).
Culture and Adenoviral Transduction of Bone Marrow–Derived Macrophages
Primary macrophages were derived from mouse bone marrow as described.33 Tibias and femurs of C57/Bl6J male mice were dissected, their marrow flushed out. Cells were grown for 7 to 10 days at 37°C in RPMI 1640 medium, 20% neonatal calf serum (NCS), and 20% macrophage–colony-stimulating factor (M-CSF)-rich L929-conditioned medium. For survivin overexpression experiments, a replication-deficient adenovirus encoding wild-type survivin (pAd-Survivin) or control GFP (pAd-GFP) was generated using the pAd-Easy system, as described previously.29,30,34 Viruses were propagated in HEK293 cells followed by purification via cesium chloride banding. With this protocol, no replication-competent adenovirus particles were generated.34 For adenoviral transduction, subconfluent macrophage monolayers were incubated with pAd-GFP or pAd-Survivin at multiplicity of infection of 100 in RPMI-1640 medium plus 20% serum for 16 hours, washed with phosphate-buffered saline, pH 7.4, and placed in fresh medium plus 20% serum at 37°C for another 12 hours before NCS and growth factor deprivation. Survivin overexpression with this transduction protocol does not affect expression of other IAPs.29,30,34 For Western blot and apoptosis analysis, cultured macrophages were quiesced by NCS-deprivation for 24 hours, and treated for 36 hours with 0.1 to 10 ng/mL recombinant mouse granulocyte–macrophage colony-stimulating factor or M-CSF (Calbiochem, San Diego, Calif). Alternatively, M-CSF–stimulated or adenovirus-transduced macrophages were incubated for 48 hours with 1 to 100 µg/mL of human oxLDL prepared by complete CuSO4 oxidation, as described (215.2±32 nmol peroxides/mg protein, 46±4 nmol TBARS/mg protein).35
Western Blotting
Macrophages or biopsy samples were lysed in 0.5% Triton X100 buffer with protease inhibitors.29 Protein content-normalized lysates were separated by SDS gel electrophoresis and immunoblotted with antibodies to survivin (1:1000; Novus), XIAP (1:250; Santa Cruz Biotechnologies, Santa Cruz, Calif), cIAP-2 (1:250, Santa Cruz Biotechnologies), caspase-3 (1:5000; Transduction Laboratories), or ß-actin (1:10 000; Sigma), followed by chemiluminescence. Immunostaining was quantified by image analysis and normalized against ß-actin expression.
Apoptosis Measurements
Macrophages were incubated with tumor necrosis factor- (10 ng/mL) and cycloheximide (10 µmol/L) for 6 hours, or staurosporine (100 nM) for 12 hours, fixed in 70% ethanol and DNA was stained (50 µg/mL Propidium Iodide, 0.05% Triton X-100, 100 µg/mL RNAse A, 45 minutes), before fluorescence-assisted cell sorting (fluorescence-activated-cell sorter). Data were shown as apoptotic cell percentages (sub-G1 phase; n
3). Alternatively, macrophages were fixed with in 4% paraformaldehyde and DNA was stained with DAPI (Calbiochem). Nuclear morphology was examined by fluorescence microscopy. Apoptotic nuclei showing condensed chromatin or fragmented nuclear bodies (karyorrhexis, pyknosis) were counted in 5 random fields per treatment (n3). After adenoviral transduction, macrophages were individually examined for GFP fluorescence before morphology analysis of DAPI-stained nuclei. Alternatively, fixed cells were permeabilized with Triton X-100 (0.1%) for 20 minutes before degraded DNA was stained by TUNEL with the ApopDetek kit (AbCys) and amino ethyl carbazole as chromogen, following the manufacturer’s instructions, resulting in red staining of apoptotic cells by phase contrast microscopy. Results were expressed as cell percentages±SEM; statistics evaluated with unpaired Student t tests, and significance was achieved when P<0.05.
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Survivin Expression in Human Atherosclerotic Lesions
In human aorta lipid streaks identified by Oil Red-O staining, immunohistochemistry revealed macrophages co-expressing survivin and CD68 (Figure 1A), and infiltrating sites of lipid accumulation. There was no positive signal in underlying media, overlaying endothelium, or in adjacent aorta segments that were negative for Oil Red-O and CD68 staining (data not shown). Control antibodies produced no staining (Figure 1C). Most sections of advanced atherosclerotic plaques were devoid of survivin staining. However, survivin was occasionally observed in
-smooth muscle actin-positive SMCs of the fibrous cap (Figure 1B), but absent from the underlying smooth muscle. In relatively rare sections, sporadic survivin-expressing cells were localized to the periphery of the necrotic core (Figure 1C). In contrast with previous studies in mice and rabbits that reported undetectable survivin in vascular walls, our Western blots revealed discrete levels of survivin in human aorta lipid streaks (Figure 1D), but virtually none in advanced atherosclerotic plaques of human carotid arteries (80.6% reduction in plaques versus aorta; P<0.05; n=3). Nevertheless, atherosclerotic plaques showed increased levels of XIAP and cIAP2 (154.5% and 442.3% increase in plaques versus aorta, respectively; P<0.05; n=3; Figure 1E).
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M-CSF Upregulation of Survivin Expression
After NCS and growth factor deprivation, control mouse macrophages expressed negligible levels of survivin. Stimulation with recombinant mouse M-CSF for 36-hour induced strong survivin re-expression (166% increase over control; P<0.05; n=4), and stimulated moderate XIAP expression (51% increase over control; P<0.05; n=4). granulocyte–macrophage colony-stimulating factor, like IL-1ß, IL-6, IL-8, or tumor necrosis factor-, had no significant effects (Figure 2A, 2B; and data not shown). DNA content analysis by fluorescence-activated cell sorter (Figure 2C) showed that M-CSF stimulation reduced spontaneous apoptosis due to NCS deprivation (47% decrease; P<0.05; n=3) and stimulated proliferation (43% increase; P<0.05; n=3). Treatment with M-CSF (36 hours) restricted apoptosis in response to transient (8-hour) exposure to oxLDL (78% decrease; P<0.05; n=4), the combination of tumor necrosis factor- and cycloheximide (66% decrease; P<0.05; n=4), or staurosporine (67% decrease; P<0.05; n=4) compared with unstimulated control macrophages in DAPI experiments (Figure 2D, 2E). Transient cytoprotection by M-CSF against oxLDL was confirmed (80% decrease; P<0.05; n=4) by TUNEL staining (Figure 2F, 2G).
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Regulation of Macrophage Apoptosis by Survivin and oxLDL
Western blot analysis showed that prolonged oxLDL treatment reduced macrophage survivin expression in a concentration-dependent manner, with complete inhibition at 10 µg/mL (Figure 3A). XIAP and cIAP2 were not modulated. Loss of survivin expression was inversely proportional to caspase activation, as monitored with active caspase-3 p17 fragment generation. Macrophage survivin expression was reduced after treatment with 100 ng/mL oxLDL for 24 hours (54% decrease versus control; P<0.05, n=4) (Figure 3C). XIAP and cIAP2 expression were not modulated significantly (Figure 3C). Survivin expression was reduced after oxLDL treatment of M-CSF–stimulated macrophages as early as 12 hours (data not shown) and totally blocked by 24 hours (Figure 3B). Loss of survivin expression thus coincided with gradual caspase activation, and preceded apoptosis implementation and DNA degradation by at least 12 to 24 hours, as measured by fluorescence-activated cell sorter (Figure 3D).
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To evaluate the anti-apoptotic role of survivin, NCS-deprived and growth factor-deprived macrophages were transduced with adenoviruses encoding either GFP alone under the SV40 promoter (pAd-GFP), or both GFP and survivin (pAd-Survivin). Analyzing GFP expression by fluorescence microscopy revealed comparable transduction rates (
25% to 30% cells) with either adenovirus, and allowed clear identification of successfully transduced cells (Figure 3E). Western blot analysis confirmed overexpression of survivin with pAd-survivin, and virtually no survivin in pAd-GFP–transduced or nontransduced macrophages (Figure 3F). Adenoviral transduction preserved viability, but somewhat sensitized macrophages to cell death. Macrophages challenged with oxLDL or staurosporine became strongly resistant to apoptosis after transduction with pAd-survivin compared with pAd-GFP (17.3±0.7% versus 36.0±5.0% after oxLDL, respectively; P<0.05; n=3), as shown by apoptotic nuclei quantification (Figure 3G).
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Discussion |
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Expression of Survivin in Human Atherosclerotic Lesions
It was previously suggested that survivin is not expressed in the normal adult vascular wall of mice and rabbits.21,30 Survivin was long considered specific to highly proliferating cancer cells,19,20,22 or to a few self-renewing adult tissues such as testis, bone marrow, or intestinal epithelium.21,23 Here, we showed detectable survivin expression, although low, in human aorta lipid streaks by Western blotting and immunohistochemistry. In lipid streaks, survivin expression seemed confined to CD68-positive monocyte/macrophage infiltrates. In contrast, we found that survivin expression was barely detectable in advanced atherosclerotic plaques, and most sections were entirely negative by immunohistochemistry. In some sections, survivin could however be detected in rare isolated macrophages, distal to the necrotic core, or in some fibrous cap SMCs. These occasional survivin-positive cells may account for the faint levels of survivin detected by Western blot. Western blot and immunochemical results converged for early and advanced lesions. Expression of survivin in lipid streak macrophages but not in advanced plaques suggested a modification in their apoptotic status during disease progression. Moreover, the pattern of survivin expression is reminiscent of the localization of proliferating cells in the outer regions of the atherosclerotic plaque.36–38 This would be consistent with survivin expression during mitosis in cancer cells,20,23 SMCs,30 and endothelial cells.29
In contrast, XIAP and particularly cIAP2 were both strongly upregulated in advanced plaques (5.5-fold increase), in agreement with the previous detection of cIAP2 in atherosclerosis,18 and XIAP in activated macrophages.39 Structural differences might potentially contribute to different IAP expression patterns in aorta and carotid cells. However, significant differences in the intracellular machinery of apoptosis in aorta and carotid cells have not yet been reported at this time, allowing the comparison of these vessels. Furthermore, the vascular pattern of survivin expression, limited to inflamed macrophage-rich areas, suggests that it results from atherogenesis. The survivin pathway may thus be activated in monocytes/macrophages early during atherogenic processes, but downregulated during plaque progression, contrary to other IAP.
Growth Factor Regulation of Survivin Expression in Atherosclerosis
Because atherosclerotic plaque formation involves the chronic recruitment of circulating inflammatory cells and macrophages in response to lipid accumulation and vascular inflammation,40 we hypothesized that macrophage survivin expression may be stimulated during this process. M-CSF is an inflammatory growth factor released by the atherosclerotic vessel wall, known to stimulate monocyte/macrophage proliferation41,42 and macrophage differentiation.41 We showed that M-CSF stimulated survivin expression and reduced macrophage apoptosis in vitro, whereas other nonmitogenic inflammatory cytokines had no effect. Moreover, it is known that disrupting M-CSF function in mouse models of atherosclerosis (ApoE-deficient or LDL receptor-deficient mice) reduces plaque size, implying that M-CSF is a critical contributor to atherogenesis.43–45 M-CSF may thus contribute to atherogenesis via the activation the cytoprotective survivin pathway in macrophages.
Inhibition of the Survivin Pathway by Oxidized Lipoproteins
The rapid loss of survivin expression in proliferating macrophages after oxLDL treatment not only coincided with increasing caspase-3 activation but also preceded apoptosis implementation by >12 hours. Near-complete absence of survivin-immunoreactive cells in the core of human plaques rich in degraded lipoproteins may reflect the downregulation of survivin by oxLDL. In vitro, macrophage apoptosis inversely correlated with survivin expression, and occurred despite increasing or sustained levels of XIAP and cIAP2. This positioned oxLDL-induced survivin downregulation upstream of macrophage death.
A previous study reported that oxLDL repressed cIAP1 expression in cultured endothelial cells after 12 to 24 hours, with maximal downregulation between 40 and 80 µg/mL oxLDL.46 This paralleled the inhibition of survivin in our macrophages in terms of kinetics and dose-response. Dysregulation of IAP may thus occur in several cell types during vascular inflammation and atherosclerosis. The lectin-like endothelial oxLDL receptor-1 was essential in endothelial cIAP1 downregulation by oxLDL, and similar scavenger receptors47 may potentially mediate survivin downregulation in macrophages and foam cell apoptosis.
M-CSF stimulation of survivin afforded macrophages with resistance to apoptosis triggered by transient exposure to oxLDL (8 hours), whereas prolonged oxLDL exposure resulted in survivin downregulation and cell collapse. However, enforced expression of survivin by adenovirus afforded macrophages with enhanced and sustained resistance to apoptosis triggered by oxLDL and other challenges in vitro. In contrast, raising XIAP and cIAP2 expression was not sufficient to block oxLDL-induced caspase activation or apoptosis, although XIAP is essential to macrophage survival.39
Taken together, our data suggest that survivin expression functions as an anti-apoptotic switch essential to macrophage survival after exposure to oxLDL. Supporting evidence was recently provided by observations in cancer cells that survivin can directly interact with XIAP, inhibit its ubiquitination and degradation, and favor the caspase-inhibitory function of this and other IAPs.48
Moreover, experimental disruption of the survivin pathway with dominant-negative survivin mutants in proliferating cancer cells,49,50 SMCs,30 or endothelial cells,29,49 or via siRNA targeting in proliferating SMCs,51 resulted in apoptosis sensitization via caspase-9 and caspase-3 activation.25,52,53 It is thus tempting to speculate that macrophages targeted by mitogenic signals while survivin expression is blocked by oxLDL, will face p53-dependent and cdc2-dependent checkpoints unfavorably at the G2/M transition.25–27 In the sustained absence of survivin, proliferating macrophages unable to oppose caspase activation will fail to traverse these checkpoints and succumb to apoptosis.50,52,53
In summary, we have shown that IAP survivin is expressed in macrophages infiltrating human lipid streaks, but not in advanced atherosclerotic lesions. Cultured macrophages re-expressed survivin on growth factor stimulation, but oxLDL blocked this effect. Survivin may thus have a biphasic role in atherosclerotic disease. It may promote macrophage accumulation in the vascular wall and plaque progression, but loss of survivin expression under recurrent exposure to oxidized lipid degradation products may contribute to apoptosis and atherosclerotic plaque vulnerability.
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Acknowledgments |
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Sources of Funding
This work was supported by funding from the CNRS, the INSERM, and the European Vascular Genomics Network, a network of excellence granted European Commission through the Sixth Framework Programme initiative (Contract LSHM-CT-2003-503254).
Disclosures
None.
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Footnotes |
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Original received August 24, 2006; final version accepted December 21, 2006.
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