Smooth Muscle Cells Contribute the Majority of Foam Cells in ApoE (Apolipoprotein E)-Deficient Mouse Atherosclerosis

Abstract

Objective- Smooth muscle cells (SMCs) are the most abundant cells in human atherosclerotic lesions and are suggested to contribute at least 50% of atheroma foam cells. In mice, SMCs contribute fewer total lesional cells. The purpose of this study was to determine the contribution of SMCs to total foam cells in apolipoprotein E-deficient (ApoE^-/-) mice, and the utility of these mice to model human SMC foam cell biology and interventions. Approach and Results- Using flow cytometry, foam cells in the aortic arch of ApoE^-/- mice were characterized based on the expression of leukocyte-specific markers. Nonleukocyte foam cells increased from 37% of total foam cells in 27-week-old to 75% in 57-week-old male ApoE^-/- mice fed a chow diet and were ≈70% in male and female ApoE^-/- mice following 6 weeks of Western diet feeding. A similar contribution to total foam cells by SMCs was found using SMC-lineage tracing ApoE^-/- mice fed the Western diet for 6 or 12 weeks. Nonleukocyte foam cells contributed a similar percentage of total atheroma cholesterol and exhibited lower expression of the cholesterol exporter ABCA1 (ATP-binding cassette transporter A1) when compared with leukocyte-derived foam cells. Conclusions- Consistent with previous studies of human atheromas, we present evidence that SMCs contribute the majority of atheroma foam cells in ApoE^-/- mice fed a Western diet and a chow diet for longer periods. Reduced expression of ABCA1, also seen in human intimal SMCs, suggests a common mechanism for formation of SMC foam cells across species, and represents a novel target to enhance atherosclerosis regression.

Keywords: atherosclerosis; cholesterol; foam cells; macrophages; smooth muscle.

Figure 1.. Absence of DIT and challenges quantitating foam cells of specific lineage in the intima of ApoE^−/− mice by fluorescence microscopy.

(A) Movat’s pentachrome stain of a human coronary artery from a 47-year-old male showing typical diffuse intimal thickening (DIT, upper panel), and the aortic root of an 8-week-old ApoE^−/− mouse fed a WD for 6 weeks showing absence of DIT (lower panel). Scale bar, 50 μm. (B) Representative fluorescence microscopy of foam cells in the aortic root of a 36-week-old ApoE^−/− mouse fed a chow diet. Lipid fixation was performed followed by Oil Red O (red) and smooth muscle α-actin (SMA, green) staining. The internal elastic lamina is indicated as a yellow dashed line dividing the intima and media. Yellow arrows point to foam cells expressing SMA (green). Scale bar, 23 μm. (C) Cryostat section of the aortic root of an 8-week-old male ApoE^−/− mouse fed a WD for 6 weeks showing lipid droplets stained by BODIPY (red) and either CD45 (cyan, upper inset boxes) in the proximal intima or SMA (green, lower boxes) in the deep intima. Nuclei were stained by DAPI (blue). Scale bar, 16 μm.

Figure 2.. The majority of foam cells in ApoE^−/− mice fed a Western diet are non-leukocyte in origin.

(A) Representative immunostaining of lesion-free descending aorta (left panel) and lesion-bearing aortic arch (right panel) of 8-week-old ApoE^−/− mice after 6 weeks of WD feeding. Foam cells were labeled with BODIPY (red). The yellow dashed line indicates the internal elastic lamina separating the medial layer exhibiting absence of lipid staining and high levels of SMA (green), and intimal layer containing foam cells (red) and much lower levels of SMA. (B) Histogram identifying foam cells from the aortic arch. Nucleated single events from lesion-bearing aortas (b) have a cell population with higher BODIPY intensity comparing to lesion-free descending aortas (a). Cells from lesion-free aortas were gated as the Lipid^low population (c), and cells above this threshold in atherosclerotic aortic arch were gated as the Lipid^high population (d). Cholesterol content in cells in the Lipid^high population was much higher than cells in the Lipid^low population. N=4 using cells sorted from 4 different animals, *P < 0.05 using paired one-sided Student’s t test (e). (C) Lipid^high cells in the aortic arch of another mouse were identified by comparison to lipid content in lesion-free descending aorta of the same mouse (a), and further separated by their expression of CD45 and I-A/I-E (e). A small aliquot of the same cell suspension was stained in the absence of either CD45 (“fluorescence minus one”, FMO, b) or I-A/I-E (c) antibodies as negative controls to set the gates positive for CD45 (Q3 and Q4) and I-A/I-E (Q1 and Q4). Lipid^high foam cells were separated by I-A/I-E and CD45 expression at the same time based on the position of the antibody-negative control gates (d and e). (D) Foam cell numbers in the lesion-bearing aortas of individual male and female mice. N=9 mice/group. No statistical differences were found by Mann-Whitney U test between the sexes. (E) Percentage of non-leukocyte foam cells negative for both macrophage markers (Q2) and leukocyte-derived foam cells (Q3+Q4) out of total BODIPY+ events in male and female ApoE^−/− mice. N=9 mice/group, **P < 0.01 using 2-way ANOVA with Bonferroni post hoc comparisons.

Figure 3.. Non-leukocyte foam cells are highly enriched with a SMC-specific epigenetic mark.

MicroChIP analysis of enrichment in the SMC-specific lineage mark in CD45⁻ and CD45⁺ foam cells from ApoE^−/− mice fed a WD for 6 weeks (isolated in Figure 2C panel e). Cultured MASMCs were measured as positive control. Cultured RAW 264.7 macrophages and sorted CD45⁻ foam cells immunoprecipitated with IgG alone are shown as negative controls. N=3/group, *P < 0.05 using Friedman test with Dunn’s multiple comparisons versus MASMCs.

Figure 4.. Percentage of non-leukocyte- and leukocyte-derived foam cells in spontaneous atherosclerosis of ApoE^−/− mice and their contribution to cholesterol accumulation in arteries.

(A) CD45⁻ and CD45⁺ foam cells in the aortic arch were quantified in male 27- (left panel, n=8) and 57-week-old (right panel, n=4) ApoE^−/− mice fed a chow diet. *P < 0.05 using a Student’s t test (left panel) or Mann–Whitney U test (right panel). (B) CD45⁺ and CD45⁻ foam cells were separated by flow cytometry from 27-week-old mice. Sorted foam cells from 1-3 animals were pooled to obtain sufficient cells for mass determination of cholesterol content (n=3 data sets from a total of 8 animals). There was no significant differences in the contribution of CD45⁺ and CD45⁻ foam cells to total cholesterol content in isolated foam cells by Wilcoxon matched pairs sign rank test.

Figure 5.. Reduced ABCA1 expression in non-leukocyte foam cells and cultured MASMCs.

(A) ABCA1 expression of CD45⁺ and CD45⁻ foam cells from ApoE^−/− mice fed a WD for 6 weeks (left panel) or 27-week-old male ApoE^−/− mice fed a chow diet (right panel) determined as the median fluorescence of ABCA1 intensity by flow cytometry. For WD-fed mice, **P < 0.01 using two-way ANOVA, n=9 mice/group; there was no statistical difference between the two sexes. For chow-fed mice, **P < 0.01 using Mann–Whitney U test, n=5 mice/group. (B) Fold change of ABCA1 mRNA level in RAW 264.7 macrophages and MASMCs treated with 100 g/ml agLDL as compared to fatty acid-free albumin alone for 24 h. *P < 0.05, using Welch’s t test. (C) ABCA1 protein levels and cholesterol efflux from RAW 264.7 macrophages and MASMCs treated with 100 μg/mL agLDL for 48 hrs followed by incubation with 0.3 mM 8Br-cAMP ± 10 μg/mL (for efflux) for 24h. Cholesterol efflux was determined by LC-MS and normalized by total cell protein levels *P < 0.05, **P < 0.01 using two-tailed Mann–Whitney U test, N=5.

Figure 6.. A model comparing early atherogenesis and foam cell formation in humans and ApoE^−/− mice.

In humans, a thick diffuse intimal thickening (DIT) layer of SMCs is present in atherosclerosis-prone arteries beginning from birth. At the onset of atherosclerosis lipoproteins that diffuse into the artery wall are trapped through a charge-charge interaction with SMC-secreted proteoglycans, primarily in the deep intima. These trapped lipoproteins are modified and aggregated, becoming a substrate for uptake by the surrounding SMCs. At this stage monocyte/macrophages are located primarily in the subendothelial region away from the deposited lipids. Over time both SMCs and macrophages take up modified lipoproteins to become foam cells independently and/or interactively. ABCA1 (red dots), the rate-limiting cholesterol efflux promoter, is robustly upregulated in macrophage foam cells, but less so in SMC foam cells. Increased ABCA1 expression facilitates cholesterol removal out of macrophage-derived foam cells, while impaired ABCA1 expression reduces cholesterol efflux from SMC foam cells, resulting in SMCs being the predominant lineage among total foam cells. In ApoE^−/− mice, no DIT SMC layer is present. Atherogenic lipoproteins induce endothelial injury and monocyte binding and infiltration into the intima, where they mature into macrophages that take up the modified intimal lipids. Cytokines released by injured endothelial cells and monocytes/macrophages, including PDGF, TNF-α, and IL-1β induce SMC migration from the media into the intima. SMCs proliferate in the intima and take up modified lipoproteins to become foam cells. Similar to human lesions, the SMC foam cells exhibit reduced ABCA1 expression compared to macrophages. Over time the total contribution to foam cells shifts from macrophages to SMCs, due at least in part to impaired ABCA1 expression by the SMC foam cells.