Brief Report: Increased Apoptosis in Advanced Atherosclerotic Lesions of Apoe−/− Mice Lacking Macrophage Bcl-2
Objective— Macrophage apoptosis plays important roles in atherosclerosis. Bcl-2 is a key cell survival molecule, but its role in macrophage apoptosis in atherosclerosis is not known. The goal herein was to determine the effect of macrophage-targeted deletion of Bcl-2 on macrophage apoptosis in atherosclerotic lesions of Apoe−/− mice.
Methods and Results— Bcl2flox-LysMCre mice were created as a model of macrophage Bcl-2 deficiency. Macrophages from these mice were more susceptible to apoptosis than those from control Bcl2WT-LysMCre mice. The mice were bred onto the Apoe−/− background and fed a Western-type diet for 4 or 10 weeks. Apoptotic cells were equally very rare in the lesions of both groups of the 4-week-diet mice, and there was no difference in lesion area. However, Bcl2flox-LysMCre;Apoe−/− plaques from the 10-week-diet protocol had a 40% to 45% increase in apoptotic cells and, in female mice, a ≈25% increase in plaque necrosis (P<0.05) compared with Bcl2WT-LysMCre lesions.
Conclusions— Macrophage Bcl-2 plays a protective role against macrophage apoptosis specifically in advanced atherosclerotic lesions of Apoe−/− mice.
Macrophage apoptosis can be a critical event in atherosclerosis and occurs at all stages of disease development.1 In early lesions, macrophage apoptosis is associated with a decrease in lesion cellularity and plaque progression,2,3 which could be attribtuable to rapid phagocytic clearance or egress of the apoptotic cells or decreased influx of macrophages. In advanced lesions, however, clearance of apoptotic cells is defective, which leads to secondary necrosis of apoptotic cells.4–6 Accordingly, we have proposed that macrophage apoptosis in advanced lesions leads to plaque necrosis, which is thought to promote plaque disruption and acute clinical events in humans.7 In support of this idea, vulnerable necrotic human plaques have increased macrophage apoptosis.8 Moreover, genetic or pharmacological manipulations that decrease macrophage apoptosis in advanced murine lesions decrease plaque necrosis, and vice versa.9
See accompanying article on page 153
Bcl-2 has been on the forefront of cell survival signaling since its discovery more than 20 years ago, yet there is an absence of in vivo causation studies assessing the role of Bcl-2 in atherosclerosis using genetically altered mouse models. This point is critical, because there are a number of other cell survival molecules in lesional cells, including Bcl-xL, apoptosis inhibitor expressed by macrophages (AIM; SPα), Mcl-1, and members of the inhibitor-of-apoptosis (IAP) family, and so redundancy might negate the effect of deletion of any one cell survival molecule. Because Bcl-2 is expressed in other lesional cell types and holo-Bcl2−/− mice have multiple abnormalities at birth,10 we created macrophage-targeted Bcl-2–deficient mice and studied the effect of this mutation on atherosclerosis in Western diet–fed Apoe−/− mice. Our studies indicate that macrophage Bcl-2 plays a protective role against macrophage apoptosis specifically in advanced atherosclerotic lesions. Moreover, in the more advanced lesions of female mice, macrophage Bcl-2 also has a modest protective effect against plaque necrosis.
Materials and Methods
Materials and methods related to cultured macrophages, genetically altered mice, plasma lipid analysis, laser capture microdissection, quantification of atherosclerotic lesions, and statistics appear in the supplemental materials (available online at http://atvb.ahajournals.org).
In Situ TUNEL Assays and Plaque Necrosis
Apoptotic cells in atherosclerotic lesions were detected by the TUNEL (TdT-mediated dUTP nick end labeling) technique using the TMR red in situ cell death detection kit (Roche). Nuclei were stained with DAPI. TUNEL-positive nuclei were counted under an Olympus IX-70 inverted fluorescent microscope. For assessment of macrophage colocalization, macrophages were detected using a rabbit antimacrophage antibody (AIA31240) from Accurate Chemical and Scientific Corporation, and nuclei were stained with Hoechst. Plaque necrosis was quantified by measuring the area of hematoxylin and eosin-negative acellular areas in the intima, as described previously.11
Intimal Cell Apoptosis Is Increased in Lesions of Bcl2flox-LysMCre;Apoe−/− Mice Fed A Western Diet for 10 Weeks But Not 4 Weeks
Mice with Bcl-2 deficiency in macrophages using the cre-lox strategy (Bcl2flox-LysMCre) were created as described in the supplemental materials (supplemental Results and Figure IA and IB). Peritoneal macrophages from these mice were more susceptible than control Bcl2flox-LysMCre mice to a variety of apoptosis inducers (supplemental Results and Figure IC and ID). The 2 groups of mice were bred onto the Apoe−/− background and fed a Western-type diet for 4 or 10 weeks, and the aortic root was examined. The 4-week lesions showed similar area between the 2 groups of mice, and only rare examples of lesional macrophage apoptosis (supplemental Results).
Background data for the 10-week-diet study are described in supplemental Results and supplemental Figure II. Figure 1A demonstrates and increase in TUNEL-positive nuclei, a measure of apoptosis, in Bcl2flox-LysMCre;Apoe−/− versus Bcl2WT-LysMCre;Apoe−/− lesions. As expected, the areas of TUNEL staining correlated with macrophage-rich areas in these lesions (Figure 1B). The quantified data for the full cohort of mice is shown in Figure 1C. Examples of hematoxylin and eosin-stained aortic root sections of from female Bcl2WT-LysMCre;Apoe−/− and Bcl2flox-LysMCre;Apoe−/− mice are shown in Figure 2A. Total lesion area per se was not affected by macrophage Bcl-2 depletion (Figure 2B). Also not affected by macrophage Bcl2 deficiency were plasma levels of tumor necrosis factor (TNF)α and interleukin (IL)-6 (data not shown).
Increases in macrophage apoptosis are translated into increases in plaque necrosis only after further lesion progression.12 In the current study, the female mice as a group had larger and more advanced lesions than male mice, and female Bcl2flox-LysMCre;Apoe−/− lesions appeared to have more acellular necrotic areas (black arrows in Figure 2A) as well as necrotic areas with cholesterol crystals (red arrows in Figure 2A). Quantification showed a ≈25% increase in necrotic area in Bcl2flox-LysMCre;Apoe−/− versus Bcl2WT-LysMCre;Apoe−/− female mice (P<0.05) (Figure 2C). Neither fibrous cap thickness nor lesional collagen content were affected by macrophage Bcl-2 deficiency (data not shown).
The difference in the affect of macrophage Bcl-2 deficiency on plaque necrosis could be attributable to a direct effect of sex differences, eg, sex steroids, or to the fact that female lesions were more advanced (see Figure 2B).12 In an attempt to sort out these possibilities, individual plaques from the female mice were divided into 2 subgroups based on plaque area: <200 000 and >200 000 μm2. We found a statistically significant difference in necrotic area between the genotypes only in the larger plaque subgroup (Table in Figure 2D). This finding is consistent with the conclusion that macrophage Bcl-2 deficiency has a selective effect on more advanced lesions. There were not enough larger plaques among the male lesions for analysis, and so pending future studies with more advanced male lesions, there is also the possibility of a direct sex effect as well. In summary, macrophage Bcl-2 deficiency is associated with increased advanced lesional macrophage apoptosis and, in large lesions in female mice, an increase in plaque necrosis.
See supplemental materials for a discussion of the findings in this report in relationship to relevant articles in the literature.
Sources of Funding
This work was supported by an American Heart Association Scientist Development Grant 0435364T (to Y.L.), an American Heart Association Postdoctoral Training Grant (to. E.T.), NIH grant HL084312 (to E.A.F.), and NIH grants HL54591 and HL75662 (to I.T.).
Received February 16, 2008; revision accepted October 21, 2008.
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