Macrophage Apoptosis Exerts Divergent Effects on Atherogenesis as a Function of Lesion Stage
Background— Because apoptotic cell clearance appears to be defective in advanced compared with early atherosclerotic plaques, macrophage apoptosis may differentially affect plaque progression as a function of lesion stage.
Methods and Results— We first evaluated the impact of targeted protection of macrophages against apoptosis at both early and advanced stages of atherosclerosis. Increased resistance of macrophages to apoptosis in early atherosclerotic lesions was associated with increased plaque burden; in contrast, it afforded protection against progression to advanced lesions. Conversely, sustained induction of apoptosis in lesional macrophages of advanced lesions resulted in a significant increase in lesion size. Such enhanced lesion size occurred as a result not only of apoptotic cell accumulation but also of elevated chemokine expression and subsequent intimal recruitment of circulating monocytes.
Conclusions— Considered together, our data suggest that macrophage apoptosis is atheroprotective in fatty streak lesions, but in contrast, defective clearance of apoptotic debris in advanced lesions favors arterial wall inflammation and enhanced recruitment of monocytes, leading to enhanced atherogenesis.
Received July 11, 2008; accepted February 2, 2009.
Atherosclerosis is an inflammatory vascular disease characterized by the intimal accumulation of macrophage foam cells, cell death, and chronic arterial inflammation.1 Macrophage apoptosis has been identified as a prominent feature of atherosclerotic plaques because macrophage cell death is believed to support necrotic core growth. The apoptotic process is controlled by intracellular levels of proapoptotic and antiapoptotic proteins such as those of the Bcl-2 family. Indeed, the relative expression of proapoptotic (eg, Bax and Bak) and antiapoptotic proteins (eg, Bcl-2 and Bcl-xL) of the Bcl-2 family determines the overall sensitivity of the cell to apoptotic stimuli. In macrophages of atherosclerotic lesions, the proapoptotic Bax and Bak proteins predominate, whereas the antiapoptotic Bcl-2 and Bcl-xL are deficient,2,3 thereby arguing for their enhanced susceptibility to apoptosis. However, the impact of macrophage apoptosis on plaque progression remains to be specifically investigated.
Clinical Perspective p 1804
Recent studies have shed light on the potential impact of apoptosis on atherosclerotic lesion progression. Indeed, disruption of either the proapoptotic molecule Bax in bone marrow–derived cells4 or the antiapoptotic factor AIM5 has revealed that apoptosis attenuates early plaque formation. However, because apoptotic cells accumulate preferentially in advanced rather than in early lesions,6,7 macrophage apoptosis may differentially affect plaque progression as a function of lesion stage.8 In addition, apoptotic cell clearance appears to be defective in advanced lesions but efficient in early ones.9 Moreover, apoptotic cells may possess proinflammatory properties, in part as a result of the presence of oxidized phospholipids (oxPLs) at their surface,10,11 which are known triggers of inflammatory responses in arterial tissues.12 In this setting, studies of mice in which components of the apoptotic cell clearance machinery have been deleted13 or of lupus-prone mice characterized by ineffective apoptotic cell clearance14 have revealed that defective apoptotic cell clearance is associated with enhanced atherosclerotic plaque progression.
To understand the impact of macrophage apoptosis on atherosclerosis at both early and advanced stages of lesion progression, we first used a transgenic approach allowing specific protection of macrophages against apoptosis (CD68-hBcl-2 mice). With this approach, we demonstrated that macrophage apoptosis was antiatherogenic during the early stages of atherosclerosis, whereas macrophage cell death accelerated plaque progression in more advanced lesions. To mirror this effect, we applied a complementary approach based on sustained induction of lesional macrophage apoptosis in CD11c-DTR transgenic mice and demonstrated that apoptotic cell accumulation in advanced lesions enhances plaque progression. Finally, short-term induction of macrophage apoptosis provided evidence that apoptotic cell accumulation in advanced atherosclerotic lesions promotes inflammatory gene expression, circulating monocyte recruitment, and accumulation of newly recruited macrophages.
Transgenic Animals and Atherosclerosis Studies Design
Transgenic mice overexpressing the antiapoptotic protein hBcl-2 in macrophages (Mø-hBcl-2 mice) were generated using homologous recombination in embryonic stem cells to produce a single-copy transgene insertion at the hypoxanthine phosphoribosyl transferase (Hprt) locus located on the X chromosome. The transgene consists of the human hBcl-2 cDNA from pORF-hBcl-2 (InvivoGen, San Diego, Calif) cloned downstream of the mouse macrophage-specific promoter CD68 from pDRIVE-mCD68 (Invivogen). Mø-hBcl-2 and Apoe−/− mice were crossed to obtain Apoe−/− and Mø-hBcl-2 Apoe−/− mice, and littermates were used for all experiments. Because the Hprt locus is located on the X chromosome, all experiments were performed with male littermates to avoid the potential variability of transgene expression related to random inactivation of the X chromosome in females. At 6 weeks of age, mice were fed a Western diet (WD; 0.15% cholesterol and 20% saturated fat) for 5 or 15 weeks.
Apoe−/− and CD11c-DTR-GFP15 mice on the C57BL/6J background were obtained from Charles River Laboratories (Wilmington, Mass) and the European Mouse Mutant Archive (Centre National de la Recherche Scientifique, Centre de Distribution, de Typage et d’Archivage animal, Orléans, France), respectively, and crossed to obtain CD11c-DTRApoe−/− mice. To evaluate the impact of sustained induction of macrophage apoptosis in atherosclerotic plaques, Apoe−/− mice (8 weeks old) were lethally irradiated, transplanted with 3×106 bone marrow cells from CD11c-DTRApoe−/− mice, and fed a WD after a 4-week recovery period. After 5 weeks of WD feeding, mice were injected with either diphtheria toxin (DT; 4 ng/g; Sigma, St Louis, Mo) or PBS every 10 days for 50 days and killed 48 hours after the last injection. To study the impact of short-term induction of lesional macrophage apoptosis, 6-week-old Apoe−/− and CD11c-DTRApoe−/− mice were fed a WD for 8 weeks to develop atherosclerotic lesions, injected intravenously with DT (4 ng/g) or vehicle, and killed 48 hours later. All animal procedures were performed with accreditation from the French government and under strict compliance with animal welfare regulations.
Plasma Lipid Analyses, Quantification of Atherosclerotic Plaques, Immunohistochemistry, Terminal Deoxynucleotidyl Transferase-Mediated dUTP-Biotin Nick-End Label Staining, Analysis of Gene Expression by Quantitative Polymerase Chain Reaction, and Flow Cytometry
All these procedures were performed as previously described14,16 (see the Methods section in the online-only Data Supplement).
Quantification of Anti–Malondialdehyde-Modified Low-Density Lipoprotein, Anti–Oxidized Low-Density Lipoprotein, E06 Antibodies, and Serum-Oxidized Phospholipids
Antibody titers to oxidation-specific epitopes of malondialdehyde-modified (MDA) low-density lipoprotein (LDL) or copper-oxidized LDL (oxLDL) and T15 clonotypic (E06) natural antibodies were determined as described.17
Labeling of Blood Monocytes and Recruitment Assays
Monocyte labeling was performed as previously described.18 To assess the recruitment of blood monocytes, 6-week-old Apoe−/− and CD11c-DTRApoe−/− mice were fed a WD for 8 weeks, injected intravenously with 200 ng DT or vehicle and 250 μL of yellow green beads, and killed 48 hours later.
Statistical calculations were performed with GraphPad Prism, version 4.03. Results were analyzed by Student unpaired t tests with the Welch correction if variances were unequal or Mann–Whitney U test as indicated in the figure legends. The statistical significance of the differences between >2 groups was compared by ANOVA followed by the Newman-Keuls multiple-comparison test. Values of P<0.05 were considered significant.
The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.
Generation and Characterization of Mø-hBcl-2 Mice
To selectively overexpress hBcl-2 cDNA in macrophages, we constructed a vector in which the expression of the human form of the antiapoptotic Bcl-2 gene is driven by the macrophage-specific promoter CD68 (Figure 1A). The CD68-hBcl2 (Mø-hBcl-2) transgene transmitted with the expected frequency, and Mø-hBcl-2 or Mø-hBcl-2 Apoe−/− mice exhibited no obvious developmental or morphological abnormalities on the C57BL/6J genetic background (data not shown). To confirm expression of the transgene, hBcl-2 mRNA and protein were measured in thioglycollate-elicited peritoneal macrophages from Mø-hBcl-2 and control mice by reverse-transcription polymerase chain reaction (Figure 1B) and flow cytometry (Figure 1C). To evaluate the impact of hBcl-2 expression on apoptosis of macrophages, ox-LDL–induced caspase-3 activity was measured. Macrophages derived from Mø-hBcl-2 mice were markedly protected against oxLDL-induced apoptosis compared with nontransgenic controls (P<0.01; Figure 1D).
We next evaluated whether hBcl2 overexpression in macrophages exerted an impact on foam cell formation. Macrophages from Mø-hBcl-2 or control mice displayed similar cholesterol-loading capacity on incubation with acetylated LDL (Figure 1E). In addition, because CD68 also is expressed in circulating monocytes, we assessed the expression of the transgene in this cell type. Cytometric analysis revealed that splenic CD19+ B cells, CD4+ and CD8+ T cells, and CD11chigh dendritic cells from Mø-hBcl-2 mice did not express hBcl2, whereas blood CD115+ monocytes did at a low level (Figure I of the online-only Data Supplement). Consistent with this finding, we found that monocyte count was 40% higher in the Mø-hBcl-2 Apoe−/− mice than in wild-type Apoe−/− mice on both chow and WD (Figure 1F).
Impact of Macrophage Resistance to Apoptosis on Atherogenesis
To assess the impact of macrophage resistance to apoptosis on the progression of early versus advanced atherosclerotic lesions, Mø-hBcl-2 Apoe−/− and Apoe−/− littermates were fed a WD for 5 or 15 weeks. No significant differences in plasma lipid levels or body weight were found between Mø-hBcl-2 Apoe−/− and Apoe−/− mice at either time point (Table 1). After 5 weeks of diet, aortic root lesion area was 50% larger in Mø-hBcl-2 Apoe−/− mice than in Apoe−/− mice (P<0.03; Figure 2A). As expected, 10 additional weeks on diet shifted the early cellular lesions to larger (>10-fold) and more complicated lesions with signs of necrotic core formation. In contrast to the 5-week time point, the mean area of the aortic root lesion in Mø-hBcl-2 Apoe−/− mice was 25% smaller than that in controls after 15 weeks of diet (P<0.04; Figure 2B). At this time point, en face analysis of the descending aorta confirmed this finding; the lesion area was 50% smaller in Mø-hBcl-2 Apoe−/− mice compared with controls (P<0.04; Figure 2C).
To determine whether lesional macrophage number was equally influenced in Mø-hBcl-2 Apoe−/− mice, CD68-positive area was quantified by immunohistochemistry on aortic sinus sections. After 5 weeks of diet, macrophage area was 50% greater in Mø-hBcl-2 Apoe−/− mice than in littermate controls (P<0.01; Figure 3A), whereas after 15 weeks of this diet, the macrophage area was 35% lower in Mø-hBcl-2 Apoe−/− mice (P<0.01; Figure 3B). We next quantified apoptotic cell accumulation in aortic sinus lesions. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end label (TUNEL)–positive cells were detectable only in advanced lesions (15 weeks) but not in early ones (5 weeks), consistent with earlier reports.6,7 At 15 weeks of diet, apoptotic cells in plaques from Mø-hBcl-2 Apoe−/− mice were 3-fold less abundant than in controls (P<0.003; Figure 3C), corresponding to a 3-fold reduction in apoptotic cell content per 1 mm2 lesion (P<0.003; Figure 3D). Because macrophages undergoing apoptosis in advanced plaque express tissue factor,19 we investigated its pattern of expression in early and advanced lesions of Mø-hBcl-2 Apoe−/− mice and controls. Tissue factor staining localized at the site of lipid accumulation mainly in acellular areas of advanced lesions (online-only Data Supplement Figures II and III). However, no difference in staining was revealed between Mø-hBcl-2 Apoe−/− mice lesions and controls at this advanced stage of plaque growth.
Impact of Macrophage Resistance to Apoptosis on Circulating Levels of Autoantibodies Against Oxidation-Specific Epitopes and Levels of Oxidized Phospholipids
Apoptotic cells contain bioactive oxPL and other oxidized lipids on their cell surface and are highly immunogenic, leading primarily to an enhanced IgM response to oxidation-specific epitopes in immunized mice.11 In this context, we evaluated serum levels of autoantibodies directed against oxidation-specific epitopes and the quantity of circulating oxPL on apoB-100 lipoproteins. After 5 weeks of WD, levels of IgG1, IgG2c, IgG3, and IgM anti–MDA-LDL and anti-oxLDL antibodies were comparable in Apoe−/− and Mø-hBcl-2 Apoe−/− mice (Figure 4A and 4B). Plasma levels of these IgG subspecies were not different between the 2 groups at 15 weeks of diet (Figure 4C and 4D); however, a significant increase in the titer of IgM anti–MDA-LDL antibodies (P<0.05; Figure 4C) and a trend toward higher IgM anti-oxLDL titers (Figure 4D) were observed in Mø-hBcl-2 Apoe−/− mice compared with Apoe−/− mice. Moreover, titers of the T15/E06 natural antibody, which binds to oxPL, were more elevated in Mø-hBcl-2 Apoe−/− mice after 15 weeks (Figure 4E). Without reaching statistical significance, a similar trend could be observed at the 5-week time point (Figure 4E). Next, because atherosclerotic lesions, including the apoptotic cells within them, are a likely source of oxPLs with proinflammatory and immunogenic properties, we measured circulating levels of oxPLs bound to apoB-100 lipoproteins in both groups of mice. After 5 and 15 weeks of the diet, the relative amounts of oxPL/apoB-100 were significantly lower in Mø-hBcl-2 Apoe−/− mice compared with controls at each time point (Figure 4F).
Effect of Sustained Induction of Apoptosis of Lesional Macrophages on Atherosclerotic Plaque Progression
Because increased resistance to apoptosis of macrophages was associated with a reduced progression of advanced plaques, we designed an experimental strategy to evaluate whether sustained induction of apoptosis in established lesions would mirror this effect. To address this point, we used CD11c-DTR-GFP transgenic mice that were developed to allow conditional depletion of CD11c-positive cells in vivo through administration of DT.15 The expression of the DT receptor (DTR)–green fluorescent protein (GFP) fusion protein driven by the CD11c promoter was evaluated on aortic root lesions of CD11c-DTRApoe−/− mice by immunostaining and revealed that GFP-expressing cells represent a subpopulation of CD68+ lesional cells (Figure 5A). In this context, administration of DT should lead to the induction of apoptosis in a subpopulation of lesional phagocytes, including macrophages in CD11c-DTRApoe−/− mice lesions. Indeed, a marked accumulation of TUNEL+ cells was observed in lesions of CD11c-DTRApoe−/− mice compared with controls 48 hours after DT injection (Figure 5B).
Because ongoing apoptosis of macrophages is thought to occur continuously during atherogenesis, we evaluated the impact of sustained induction of apoptosis of lesional cells on plaque progression. However, long-term repeated injection of DT in CD11c-DTRApoe−/− mice was previously shown to be lethal.15 To circumvent this problem, the hematopoietic system of mice was selectively reconstituted with CD11c-DTR cells by lethally irradiating Apoe−/− mice, followed by reconstitution with CD11c-DTRApoe−/− bone marrow cells. Plasma total cholesterol and triglyceride levels were similar in DT- and PBS-treated animals fed a WD for 12 weeks. However, we observed a 25% increase in plasma free cholesterol concentration 48 hours after the final DT injection (Table 2). We further examined this phenomenon in another set of animals and found that this effect was transient, lasting 48 to 72 hours, and correlated with DT-induced depletion of CD11c-positive cells (data not shown). Importantly, although the proportion of splenic dendritic cells was markedly decreased in Apoe−/− mice reconstituted with bone marrow cells from CD11c-DTRApoe−/− mice 48 hours after DT injection, the percentage of blood monocytes remained unchanged (online-only Data Supplement Figure IV). Apoptotic cell accumulation was markedly increased in lesions of chimeric mice treated with DT compared with PBS-injected controls (P<0.0001; Figure 5C). Moreover, aortic root lesion area was 20% larger in transplanted mice treated with DT compared with animals treated with PBS (P=0.01, Figure 5D), whereas a larger difference was observed in the descending thoracic and abdominal aorta (5-fold) in the DT group compared with controls (P<0.0001; Figure 5E). Surprisingly, the degree of CD68+ macrophage immunoreactivity was similar in both groups (data not shown) 48 hours after the final DT injection and may reflect an increased recruitment of circulating monocytes (see below), an effect that could mask the loss of lesional macrophages in the DT-treated group.
Effect of Short-Term Induction of Lesional Macrophage Apoptosis in Established Atherosclerotic Lesions
To further examine the molecular and cellular mechanisms that may underlie the enhanced plaque progression observed on sustained induction of lesional macrophage apoptosis, we evaluated the impact of short-term DT treatment on vascular inflammation in established lesions. For this purpose, Apoe−/− and CD11c-DTRApoe−/− mice with established atherosclerotic lesions were injected with DT and either PBS or DT, respectively, and the cellular response to lesional induction of apoptosis was evaluated 48 hours after injection. We first determined the content of apoptotic cells in the aortic sinus of the 3 groups of mice 2 days after injection. TUNEL staining revealed marked accumulation of apoptotic cells in CD11c-DTRApoe−/− mice treated with DT compared with the control groups, thereby confirming that DT injection had induced apoptosis in lesions (P<0.05; Figure 6A and 6B). The persistence of apoptotic cells 2 days after DT injection in CD11c-DTRApoe−/− mice is consistent with defective clearance of dead cells in advanced plaques as previously suggested20 (P<0.05; Figure 6A and 6B). By comparison, TUNEL staining was not observed in the spleens of CD11c-DTRApoe−/− mice 48 hours after DT treatment, suggesting efficient removal in this tissue (data not shown).
The accumulation of apoptotic cells in the lesions of the CD11c-DTRApoe−/− mice was associated with dense DAPI staining indicative of accumulation of cells in TUNEL+ areas (Figure 6A). These cells stained positively for the newly recruited macrophage marker CD11b16 (Figure 6A), and quantitative analysis revealed that these CD11b+ macrophages accumulated in the lesions of CD11c-DTRApoe−/− mice treated with DT to a greater degree compared with controls (P<0.05; Figure 6C). Interestingly, we noted that rare lesional TUNEL+ areas were equally associated with the presence of adjacent CD11b+ macrophages in Apoe−/− mice, thereby suggesting that apoptotic cell death, which occurred independently of DT-triggered apoptosis, equally induced newly recruited macrophage accumulation (data not shown).
To investigate the potential molecular mechanisms underlying the recruitment of new macrophages within lesions, we next evaluated the expression of monocyte markers and chemokines in the aorta. Levels of mRNA encoding the monocyte markers CD11b, CCR2, and CX3CR1 were significantly elevated (P<0.05 each) in the aortas of CD11c-DTRApoe−/− mice treated with DT compared with controls (Figure 7A through 7C, respectively). The expression of the activation marker VCAM-1 also was significantly elevated in DT-treated CD11c-DTRApoe−/− mice compared with controls (P<0.05; Figure 7D). Moreover, expression levels of the chemokines monocyte chemoattractant protein-1 (MCP-1) (Figure 7E), macrophage inflammatory protein (MIP)-1α (Figure 7F), MIP-1β (Figure 7G), and MIP-2 (Figure 7H) were significantly increased (P<0.05 each) in CD11c-DTRApoe−/− mice treated with DT compared with control groups.
Effect of Short-Term Induction of Lesional Macrophage Apoptosis in Established Atherosclerotic Lesions on Recruitment of Circulating Monocytes
The observation that induction of apoptosis within the lesion was associated with higher levels of chemokines and the presence of CD11b+ cells strongly suggests that apoptotic cell deposition triggered the recruitment of monocytes. Thus, we designed an experiment to dynamically measure the level of monocyte infiltration in response to accumulation of apoptotic cells in the atherosclerotic vascular bed. Systemic injection of fluorescein-labeled microspheres (FITC-M) was used to label monocytes in the circulation. Within 18 hours after intravenous injection of microspheres, ≈1% of leukocytes were FITC-M+, of which two thirds were monocytes (data not shown). Overall, ≈10% of blood monocytes were phagocytically labeled with this approach (Figure 8A). Only a few beads were detected in atherosclerotic lesions of Apoe−/− mice 48 hours after injection of FITC-M, reflecting the “basal” recruitment of monocytes into the plaque (Figure 8B). The majority of the beads that had not been phagocytosed by peripheral blood leukocytes were found in the spleen (data not shown). In CD11c-DTRApoe−/− mice, monocytes were labeled in vivo with FITC-M, whereas apoptosis was simultaneously induced in the lesions by DT injection. Forty-eight hours after injection, aortic root sections were prepared and stained both for TUNEL to detect apoptotic cells and with DAPI to visualize nuclei, whereas recruited monocytes were discriminated with green bead fluorescence. As shown in Figure 8C, DT injection induced apoptotic cell accumulation in lesions of CD11c-DTRApoe−/− mice, which in turn promoted recruitment of circulating monocytes as evidenced by detection of FITC-M+ cells in the lesions of these mice; in contrast, rare beads were detected in control groups.
Our data suggest that macrophage survival exerts proatherogenic effects during the early stages of atherosclerotic plaque progression, whereas it reduced plaque burden when lesions were at a more advanced stage. Second, we report that accumulation of apoptotic macrophages in established lesions has a major incidence on the vascular inflammatory response by promoting inflammatory gene expression, circulating monocyte recruitment, subsequent accumulation of newly recruited macrophages, and ultimately plaque progression.
The present experiments demonstrated that protection of macrophage cell death in Mø-hBcl-2 Apoe−/− mice enhanced lesion size compared with Apoe−/− littermate controls at early stages of plaque development (5 weeks of WD). At this early stage, lesions in both Apoe−/− and Mø-hBcl-2 Apoe−/− mice did not contain TUNEL-positive cells, consistent with previous studies that revealed that apoptotic cells are detectable only in advanced lesions.6,7 The absence of apoptotic cells in early lesions might reflect an efficient clearance mechanism at this stage rather than the absence of apoptosis8,9 because notably the free cholesterol–induced apoptosis pathway has been shown to be operative (unfolding protein response activation) in the early phases of plaque development.7,21 Furthermore, if lesions are larger in Mø-hBcl-2 Apoe−/− mice compared with Apoe−/− mice, that might suggest that hBcl-2 overexpression could protect the lesion from an apoptotic activity masked by an efficient clearance machinery, thereby increasing the overall cellularity of the plaque and its subsequent growth.
In contrast, at a more advanced stage of atherosclerosis (15 weeks on WD), we observed smaller lesion size in Mø-hBcl-2 Apoe−/− mice compared with Apoe−/− controls. This finding was associated with a lower abundance of apoptotic cells present in the lesions of Mø-hBcl-2 Apoe−/− mice. Because the opposite situation was noted after 5 weeks on the WD, our data indicate that a marked shift occurred within the following 10-week period. Therefore, we speculate that this period of plaque growth was associated with a growing impairment of apoptotic cell clearance mechanisms. Thus, in this context, enhanced macrophage resistance to apoptosis would confer protection against plaque progression. This hypothesis is consistent with studies in mice in which components of the apoptotic cell clearance machinery have been deleted13 as well as with other models of ineffective apoptotic cell clearance (lupus-prone mice)14 that suggested that lesional apoptotic cell accumulation from early lesion stages was associated with increased atherosclerotic plaque progression. Another potential explanation for such a dual effect was that overexpression of hBcl-2 preferentially protected macrophage foam cells from potential inducers of apoptosis present in advanced versus early lesions (such as high levels of oxLDL), thereby reflecting the various potential pathways that can trigger cell death in plaques.8,22 Alternatively, the presence of macrophages protected from apoptosis in advanced lesions of Mø-hBcl-2 Apoe−/− mice might favor the phagocytosis of plaque debris and might therefore delay or limit the adverse impact of apoptotic cell accumulation, thereby contributing to the limitation of plaque growth. It is noteworthy that monocytes were constitutively increased in Mø-hBcl-2 Apoe−/− mice, an effect most likely due to leakage of the CD68-hBcl2 transgene in this cell type. As a result, increased resistance to apoptosis of macrophages but equally, to a certain degree, monocytes may have contributed to plaque development in Mø-hBcl-2 Apoe−/− mice at an early stage. Alternatively, the increased monocyte count could also have participated in the observed early plaque progression in those mice. Nevertheless, such a scenario would not hold true in advanced lesions and/or would be markedly counterbalanced by the protected effect afforded by increased resistance of macrophage to apoptosis.
By using the CD11c-DTR system, we provide equal evidence that lesional accumulation of apoptotic cells could induce inflammatory signals and favor monocyte recruitment, thereby further demonstrating that advanced lesions progress when apoptotic cells accumulate. Indeed, we demonstrate that the accumulation of dead cells in plaques induces a proinflammatory milieu as assessed by elevated expression of small inducible chemokines (MCP-1, MIP-1α, MIP-1β, and MIP-2) and monocyte markers. Our results are consistent with recent studies showing that membranes of apoptotic cells contain significant amounts of active oxPLs,11 which are known inducers of inflammation in murine arteries.12 It is interesting to note that circulating oxPL levels were decreased in Mø-hBcl-2 Apoe−/− mice compared with controls after both 5 and 15 weeks on WD, potentially as a result of both decreased macrophage apoptosis and higher titers of IgM antibodies (particularly oxPL-specific E06 titers), which might facilitate oxPL clearance.
We provide dynamic evidence that the accumulation of CD11b+ newly recruited macrophages in areas of apoptotic cell deposition most probably arose from recruitment of circulating monocytes. Our data are consistent with in vitro experiments that revealed that apoptotic cells, through their oxPLs, can induce endothelial cell activation and subsequent monocyte adhesion10,11 and provide the first evidence in vivo that apoptotic cells can stimulate the migration of phagocytes, as previously demonstrated in vitro.11,23 Some discrepancies may exist between our results and those of Stoneman and colleagues,24 who reported that short-term DT treatment of CD11b-DTR-GFP mice triggered macrophage apoptosis in lesions without inducing plaque inflammation, whereas long-term DT treatment of atherosclerotic CD11b-DTR-GFP mice had no impact on plaque progression. The fact that monocytes are CD11bhigh cells that were massively depleted in DT-treated CD11b-DTR-GFP mice, thus significantly impairing recruitment to the lesion and consequently reducing the potential impact on vascular inflammation and plaque development, may partly explain the differences with our data. One limitation for the use of the CD11c-DTR model in the present study, however, was the elevation in plasma cholesterol observed after long-term DT treatment of these mice. We cannot exclude that such an effect may have accelerated plaque progression in DT-treated CD11c-DTRApoe−/− mice. Nevertheless, it is doubtful that a periodic, temporary, and moderate elevation in plasma cholesterol levels might explain the 4-fold increase in lesion size we observed in the aortas of these mice.
The present study provides strong support for a divergent impact of apoptosis on plaque progression as a function of the stage of lesion development, thereby providing a rationale for the association between apoptotic cell accumulation and plaque progression. Considered together, our data suggest that reducing apoptotic cell accumulation in advanced atherosclerotic plaques may be beneficial in attenuating either recruitment of monocytes, the resulting local inflammatory response, or both.
Sources of Funding
This work was funded by INSERM, Fondation de France, Leducq Foundation, PPG grant HL088093, and NIH HL086559 (Dr Witztum). Dr Gautier was supported by a fellowship from the Fondation pour la Recherche Médicale. Drs Lesnik, Huby, and Chapman are recipients of Contrat d’Interface from Assistance Publique-Hopitaux de Paris/INSERM.
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Acute ischemic syndromes are related primarily to rupture of unstable plaques, leading to thrombus formation and occlusive complications. Unstable plaques are typically constituted of the most prominent cell types in atherosclerosis: macrophages and macrophage-derived foam cells. Most of these macrophages are postapoptotic and form a graveyard of dead collapsed cells that might contribute to constitution of the necrotic core. Genetic manipulations of apoptotic genes have been shown to differentially alter atherosclerotic lesion size in murine models of atherosclerosis; however, the potential beneficial or detrimental role of apoptotic macrophage death in plaque development remains controversial. To address this question, we created transgenic mice in which both the lifespan of macrophages was increased in response to elevated resistance to apoptosis (CD68-hBcl2) and targeted induction of lesional macrophage apoptosis (CD11c-DTR) could be achieved. These data provide the first in vivo evidence that macrophage apoptosis is atheroprotective in fatty streak lesions; in contrast, however, defective clearance of apoptotic debris in advanced lesions favors arterial wall inflammation and enhanced recruitment of monocytes, thereby leading to enhanced atherogenesis. Considered together, these findings suggest that attenuating macrophage apoptosis in advanced plaques represents a promising therapeutic strategy.
The online-only Data Supplement is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.108.806158/DC1.