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. Author manuscript; available in PMC: 2015 Sep 1.
Published in final edited form as: Arterioscler Thromb Vasc Biol. 2014 May 15;34(9):1888–1899. doi: 10.1161/ATVBAHA.114.303791

Myeloid Cell-Specific ABCA1 Deletion Has Minimal Impact on Atherogenesis in Atherogenic Diet-Fed LDL Receptor Knockout Mice

Xin Bi 1, Xuewei Zhu 1, Chuan Gao 1, Swapnil Shewale 1, Qiang Cao 1, Mingxia Liu 1, Elena Boudyguina 1, Abraham K Gebre 1, Martha D Wilson 1, Amanda L Brown 1, John S Parks 1,2
PMCID: PMC4140956  NIHMSID: NIHMS593146  PMID: 24833800

Abstract

Objective

Transplantation studies suggest that bone marrow (BM) cell ABCA1 protects against atherosclerosis development. However, the in vivo impact of macrophage ABCA1 expression on atherogenesis is not fully understood because BM contains other leukocytes and hematopoietic stem and progenitor cells. Myeloid-specific ABCA1 knockout (MSKO) mice in the LDL receptor knockout (LDLrKO) C57BL/6 background were developed to address this question.

Approach and Results

Chow-fed MSKO/LDLrKO (DKO) vs. LDLrKO (SKO) mice had similar plasma lipid concentrations, but atherogenic diet (AD)-fed DKO mice had reduced plasma VLDL/LDL concentrations resulting from decreased hepatic VLDL triglyceride secretion. Resident peritoneal macrophages from AD-fed DKO vs. SKO mice had significantly higher cholesterol content, but similar proinflammatory gene expression. Atherosclerosis extent was similar between genotypes after 10–16 wks of AD, but increased modestly in DKO mice by 24 wks of AD. Lesional macrophage content was similar, likely due to higher monocyte flux through aortic root lesions in DKO vs. SKO mice. After transplantation of DKO or SKO BM into SKO mice and 16 wk of AD feeding, atherosclerosis extent was similar and plasma apoB lipoproteins was reduced in mice receiving DKO BM. When differences in plasma VLDL/LDL concentrations were minimized by maintaining mice on chow for 24 wks, DKO mice had modest, but significantly more, atherosclerosis compared to SKO mice.

Conclusions

Myeloid cell ABCA1 increases hepatic VLDL triglyceride secretion and plasma VLDL/LDL concentrations in AD-fed LDLrKO mice, offsetting its atheroprotective role in decreasing macrophage cholesterol content, resulting in minimal increase in atherosclerosis.

Keywords: cardiovascular disease, atherosclerosis, lipids, lipoproteins, cholesterol

Introduction

ATP binding cassette transporter A1 (ABCA1) is a membrane transporter that facilitates the movement of cellular free cholesterol (FC) and phospholipid across the plasma membrane to combine with apolipoproteins, principally apoA-I, forming nascent HDL particles that are subsequently converted in plasma to mature HDL. Studies have documented an inverse association between plasma HDL cholesterol (HDL-C) concentrations and cardiovascular disease (CVD) risk 1. The apparent protective effect of elevated plasma HDL-C concentrations likely results from the role of HDL in facilitating macrophage reverse cholesterol transport (RCT). During RCT, excess macrophage cholesterol in atherosclerotic plaques is effluxed to HDL particles, which then transport cholesterol back to the liver for excretion2, 3. Recent studies suggest that plasma HDL’s capacity to efflux cholesterol better predicts prevalent CVD risk than static HDL-C concentrations4, 5.

ABCA1 is a key transporter in facilitating macrophage RCT6. Individuals with Tangier disease, a rare genetic disorder caused by functional mutations in ABCA1, have <5% of normal plasma HDL and massive storage of cholesteryl ester (CE) in macrophages throughout the body. Based on the Tangier disease plasma and macrophage lipid phenotype, one would predict increased CVD in these subjects. However, CVD is similar to controls in many Tangier subjects 7 and in animal models with global deficiency of ABCA1 8, 9. This paradoxical finding is likely due to the 50% decrease in plasma apoB lipoproteins (apoB Lp; i.e., VLDL and LDL) that accompanies whole-body deficiency of ABCA1. Tissue-specific roles of ABCA1 expression on atherogenesis and CVD are difficult to determine because ABCA1 is expressed to a variable extent in nearly all tissues10. In particular, the presumed atheroprotective role of macrophage ABCA1, based primarily on the well-accepted function of ABCA1 to stimulate macrophage FC efflux, lacks sufficient supporting evidence.

Attempts to identify and isolate the role of macrophage ABCA1 in atherosclerosis development have relied on bone marrow (BM) transplantation experiments to date. Several such studies have uniformly identified an atheroprotective role for hematopoietic ABCA1 8, 11–15. Nevertheless, BM contains multiple cell populations, many of which have been implicated in atherogenesis. For instance, hematopoietic stem and progenitor cells16, T and B lymphocytes 17, 18, natural killer cells19, and dendritic cells 20 all may affect atherosclerosis, making it difficult to distinguish the specific contribution of macrophage ABCA1 expression to atherogenesis.

To define the role of macrophage ABCA1 expression in atherosclerosis, we generated myeloid (i.e., macrophage and neutrophil) cell-specific ABCA1 knockout (MSKO) mice21. Macrophages from these mice have defective FC efflux and respond to Toll-like receptor agonists with an exaggerated production of inflammatory cytokines21. An initial study using these mice crossed into the LDLrKO background suggested a minimal role for myeloid cell-specific ABCA1 in atherogenesis22, in contrast to other reports8, 11–15. However, this study only measured effects at one time point of AD feeding (16 wks) and used with MSKO/LDLrKO mice in a mixed genetic background (90% C57BL/6, 10% 129 SVEV). Since 129 mice strains are resistant to atherosclerosis relative to C57BL/6 mice23, the 10% 129 SVEV background in MSKO/LDLrKO mice may have lessened atherogenesis. Thus, results of our previous study did not definitively elucidate the role of macrophage ABCA1 in atherogenesis.

To address this deficiency, we have backcrossed MSKO/LDLrKO mice to >99% in the C57BL/6 background and investigated early- to late-stage atherosclerosis progression. Myeloid cell-specific deletion in AD-fed LDLrKO mice did not result in the expected increase in atherosclerosis relative to LDLrKO mice, due to a considerable reduction in plasma apoB Lp concentrations in MSKO/LDLrKO mice, resulting from reduced hepatic VLDL triglyceride (TG) production. However, when differences in plasma apoB Lp concentrations between genotypes of mice were minimized by maintaining mice on a chow diet, expression of myeloid cell ABCA1 was atheroprotective.

Methods

Materials and Methods are available in the online-only Data Supplement.

Results

Myeloid-specific ABCA1 deletion reduces plasma lipids in AD-fed LDLrKO mice

To study atherosclerosis development in a stage-specific manner (i.e., early to advanced), MSKO/LDLrKO and LDLrKO mice were switched from chow at 7–9 wks of age to an AD for 10, 16, or 24 wks with periodic measurements of TPC, FC, and TG levels during disease progression. Chow-fed (i.e., baseline) MSKO/LDLrKO and LDLrKO mice displayed similar plasma lipid concentrations (Figure 1A–B; 0 wks), consistent with previous results from chow-fed MSKO and wild-type mice 21. Two weeks of AD consumption increased TPC and FC concentrations in both genotypes, but the increase was significantly attenuated in MSKO/LDLrKO mice throughout the 16 wk disease progression phase (area under curve; P< 0.05) (Figure 1A–B). Plasma TG concentrations varied little during AD feeding but were lower in MSKO/LDLrKO vs. LDLrKO mice (Figure 1A–B). FPLC fractionation of plasma showed that lipoprotein cholesterol distribution was similar for both groups of chow-fed mice (Figure 1C). However, after 16 wks of AD feeding, plasma VLDL-C and LDL-C concentrations were significantly lower for both genders of MSKO/LDLrKO mice relative to their LDLrKO counterparts (Figure 1D; Supplemental Table I). Collectively, these results suggest a novel role for myeloid cell ABCA1 in apoB Lp metabolism under atherogenic hyperlipidemia conditions and further support its minimal contribution to the plasma HDL pool21.

Figure 1. Plasma lipid and lipoprotein concentrations.

Figure 1

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Fasting (4h) plasma total cholesterol (TPC), free cholesterol (FC), and triglyceride (TG) concentrations in male (A) and female (B) mice during a 16 wk atherosclerosis progression phase were measured by enzymatic assays (n=5–11). Plasma cholesterol distribution among lipoproteins before (C; chow-fed at 7–9 wks of age) or after (D) 16 wks of AD feeding were determined after fractionation of plasma by FPLC (n=4–14). Data expressed as mean ± SEM. * P<0.05. MSKO, Myeloid-specific ABCA1 knockout.

VLDL TG secretion is decreased in AD-fed MSKO/LDLrKO mice

To determine the potential mechanism for decreased plasma apoB Lp concentrations in AD-fed MSKO/LDLrKO mice, we measured plasma TG accumulation in vivo using the Triton block procedure, which we use as a surrogate for VLDL TG secretion. VLDL TG mass accumulation was significantly reduced in MSKO/LDLrKO vs. LDLrKO mice (0.045 mmol/L vs. 0.035 mmol/L, P<0.05) (Figure 2A–B). VLDL particles isolated 3 h after triton injection from MSKO/LDLrKO mice had a significantly lower TC/TG ratio compared to their LDLrKO counterparts (Supplemental Figure I). Although hepatic expression of genes involved in de novo lipogenesis and fatty acid oxidation was similar between genotypes of mice (Supplemental Figure II), hepatic TG content was significantly lower in MSKO/LDLrKO vs. LDLrKO mice after 16 wks of AD feeding (Figure 2C), whereas TC, FC, and CE were not (Supplemental Figure III A–C). Gonadal fat pad mass was similar between genotypes of mice, but plasma non-esterified fatty acid (NEFA) levels were significantly lower in MSKO/LDLrKO mice (Supplemental Figure III D–F). Although the reason for decreased plasma NEFA is unknown, this would result in less NEFA substrate for hepatic TG synthesis, likely contributing to reduced hepatic VLDL TG production in MSKO/LDLrKO mice. Acute Kupffer cell ablation did not affect plasma TPC or TG concentrations (Supplemental Figure IV), suggesting local cytokine production from Kupffer cells was not reducing hepatic VLDL TG production in MSKO/LDLrKO mice. Food intake, fractional cholesterol absorption, and fecal neutral sterol were also similar between genotypes of mice (Supplemental Figure V). Overall, these data suggest a novel role for myeloid cell ABCA1 in inducing VLDL TG production during atherogenesis in LDLrKO mice.

Figure 2. VLDL secretion was determined after in vivo inhibition of TG lipolysis with Tyloxapol administration.

Figure 2

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Plasma TG levels were measured by enzymatic assay before (0 min) and after (30, 60, 120, 180 min) intravenous Tyloxapol injection. (A) Plasma TG concentration (mean ± SEM; n=6–7) was plotted for each genotype of mice and the line of best fit was determination by linear regression analysis. (B) TG secretion rate during the 3h experiment was calculated for each animal as the slope of the regression line. Results were then plotted for both groups of mice as mean ± SEM, n=6–7. (C) Hepatic TG content was measured by enzymatic assay and normalized to liver protein. Results are from two separate atherosclerosis studies (chow and 16-wk AD). Data are expressed as mean ± SEM (n=6–12). * P<0.05.

Macrophages ABCA1 deletion results in massive cellular cholesterol accumulation

Macrophage ABCA1 is critical in preventing excess cellular cholesterol accumulation 21, 24. To determine the degree of in vivo macrophage cholesterol accumulation during atherosclerosis progression, we measured cellular cholesterol content in resident peritoneal macrophages (PMs). Despite the significant reduction of plasma apoB Lp in MSKO/LDLrKO mice, PMs from these mice had dramatically higher TC, FC, and CE content after 16 wks of AD feeding compared to LDLrKO mice (732 vs. 9 µg CE/mg protein in females; P<0.01) (Figure 3A). A similar trend was observed for 16 wk AD-fed male mice (data not shown) and female mice fed the AD for 24 wks, with even greater differences between genotypes (1496 vs. 48 µg TC/mg protein in females, P<0.01) (Figure 3B). Resident PM ABCG1 gene expression was similar for both genotypes of mice (data not shown). These data suggest that PM ABCA1 deficiency results in massive CE accumulation that is progressive in the face of continued but stable hyperlipidemia (Figure 1) and that no other macrophage cholesterol efflux system can compensate for ABCA1 loss over an extended (10–24 wk) period of hyperlipidemia.

Figure 3. Resident peritoneal macrophage cholesterol content.

Figure 3

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Cholesterol content of resident PMs from female mice fed AD for 16wks (A) and 24 wks (B). Resident macrophages were isolated by peritoneal lavage from AD-fed female mice. After a 2h culture in RPMI medium containing 1% Nutridoma and removal of non-adherent cells, the adherent macrophages were lipid extracted and the cellular total cholesterol (TC) and free cholesterol (FC) content were quantified by gas-liquid chromatography. CE was calculated as (TC-FC) ×1.67. Data are normalized for cellular protein and expressed as mean ± SEM (n=5–11). * P<0.05.

ABCA1 deletion in macrophages results in comparable plasma cytokine levels

Macrophages lacking ABCA1 expression secreted more proinflammatory cytokines upon Toll-like receptor 4 stimulation with lipopolysaccharide compared with wild-type macrophages 21, 24. Dietary saturated fatty acids also activate Toll-like receptor 4 signaling in vitro and in vivo 25. To determine whether AD-fed MSKO/LDLrKO mice exhibit greater chronic low-grade inflammation in vivo than LDLrKO mice, we examined plasma cytokine protein expression semi-quantitatively using a cytokine array. Plasma proinflammatory cytokine and chemokine levels appeared similar between genotypes of mice fed chow or the AD for 16 or 24 wks (Supplemental Figure VI A), suggesting in vivo inflammatory response was not enhanced in MSKO/LDLrKO mice. In addition, proinflammatory gene expression in resident PMs was similar between MSKO/LDLrKO and LDLrKO mice fed the AD 10, 16, or 24 wks (Supplemental Figure VI B). These data suggest that the combination of AD and resulting hyperlipidemia did not result in an exacerbated inflammatory state in vivo in MSKO/LDLrKO vs. LDLrKO mice during atherosclerosis progression.

Myeloid-specific ABCA1 deficiency does not affect blood leukocyte distribution

Previous studies have suggested that deletion of BM cell ABC transporters (ABCA1 and ABCG1) results in blood monocytosis, neutrophilia, and increased numbers of Ly6-Chi monocytes, likely through increased expansion of hematopoietic stem and multipotential progenitor cells 16 Transplantation of BM from myeloid cell-specific ABCA1 and ABCG1 double KO donor mice into LDLrKO recipient mice resulted in monocytosis and neutrophilia when mice were fed a Western-type diet and neutrophilia when fed chow26. In our study, the distribution of blood leukocytes was similar for both genotypes of chow and AD-fed mice (Supplement Figure VII). The different outcome may be attributed to more restrictive deletion of ABC transporters (i.e., ABCA1 only). In addition, the anticipated increase in percentage of Ly6-Chi monocytes that accompanies diet-induced hyperlipidemia occurred and was similar for MSKO/LDLrKO and LDLrKO mice (Supplemental Figure VII E).

Myeloid-specific ABCA1 deficiency enhances advanced, but not early or intermediate, atherogenesis

We evaluated the effect of myeloid ABCA1 expression on atherosclerosis development using several measurements. MSKO/LDLrKO and LDLrKO mice fed the AD for 10 wks or 16 wks had similar levels of aortic TC, FC, and CE content, although FC concentrations were slightly, but significantly, lower for 10 wk diet-fed MSKO/LDLrKO mice (Figure 4A–B). After 24 wks of AD, aortic TC and FC levels were increased in MSKO/LDLrKO mice relative to LDLrKO mice and CE was higher on average (Figure 4C). Aortic root lesions stained with Oil red O were comparable in size for both genotypes of mice fed the AD for 16 wks (Figure 4D–E), supporting the aortic cholesterol measurements. In addition, no significant differences were observed in lesional macrophages (Figure 4F–G), T cells (Supplemental Figure VIII A–B), collagen content (Supplemental Figure VIII C–D), or lesion classification (data not shown), suggesting similar aortic root lesion composition. Thus, myeloid cell ABCA1 expression had minimal impact on early/intermediate atherogenesis, but mildly exacerbated late-stage atherosclerosis in AD-fed LDLrKO mice.

Figure 4. Atherosclerosis in AD-fed mice.

Figure 4

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Aortas were lipid extracted for quantification of total cholesterol (TC) and free cholesterol (FC) content by gas-liquid chromatography. Cholesterol ester (CE) content was calculated as (TC-FC) ×1.67. Aortic cholesterol of mice fed AD for 10 wks (A), 16 wks (B) and 24 wks (C). (D) Representative aortic root sections stained with Oil Red O from LDLrKO (left) and MSKO/LDLrKO (right) mouse. (E). Aortic root lesion area for mice. Each point represents the average lesion area of 3 sections per mouse. Horizontal lines denote mean ± SEM for each genotype of mice. (F) Representative LDLrKO (left) and MSKO/LDLrKO (right) mouse aortic root sections immunostained for CD68. (G) Percentage aortic root lesional area occupied by CD68+ cells. Each point represents the average lesion area of 4 sections per mouse. Horizontal lines denote mean ± SEM for each genotype of mice. All data in panels D-G are from female mice fed the AD for 16 wks. * p<0.05. Statistical analyses of data in panels A-C were performed using Student’s t-test (MSKO/LDLrKO vs. LDLrKO at each time point) and two-way ANOVA (genotype vs. time on diet) with the same outcome.

Myeloid-specific ABCA1 deficiency significantly increases monocyte/macrophage flux through atherosclerotic lesions

Macrophage ABCA1 has been implicated previously in chemotaxis 27, 28. The massive cholesterol accumulation in MSKO/LDLrKO resident PMs and similar extent of aortic cholesterol accumulation between genotypes of mice led us to speculate that fewer macrophages accumulate in MSKO/LDLrKO mouse atherosclerotic lesions, assuming PMs are phenotypically similar to aortic lesional macrophages. However, aortic root lesional macrophage content, measured by CD68 staining, was similar between genotypes (Figure 4F–G). In vivo recruitment of fluorescent bead-labeled monocytes to aortic root lesions was measured over a 5 day period after 16 wks of diet-induced atherogenesis and surprisingly, we found a four-fold increase in number of monocytes recruited into MSKO/LDLrKO vs. LDLrKO mouse lesions (Figure 5A), compatible with previous studies showing increased in vitro and in vivo chemotaxis of ABCA1-deficient macrophages 27, 29. The extent of apoptosis in atherosclerotic lesions was similar for both genotypes of mice (Figure 5B). We then repeated the monocyte recruitment study and examined aortic root lesional bead content 5 and 28 days after fluorescent bead labeling. Blood monocyte bead labeling efficiency 24 h after bead injection was similar for both genotypes of mice (Supplemental Figure IX A). Consistent with the first study (Figure 5A), significantly more beads were observed in lesions of MSKO/LDLrKO vs. LDLrKO mice five days (baseline) after labeling (Figure 5C). However, sustained lesion progression from 5 to 28 days after bead injection resulted in increased bead accumulation in LDLrKO, but not MSKO/LDLrKO mice (Figure 5C). The increase in bead labeling between 5 and 28 days in LDLrKO mice likely resulted from low but measurable bead labeled monocytes remaining in blood after 5 days 30. Similar trends were observed using total number of beads per section, without normalizing to blood monocyte bead labeling efficiency (Supplemental Figure IX B–C). The lower bead content of aortic lesions from MSKO/LDLrKO mice at 28 days may have occurred through egress of bead-labeled cells, counterbalancing the greater influx, resulting in similar steady-state aortic root macrophage content in MSKO/LDLrKO and LDLrKO mice.

Figure 5. Monocyte recruitment and lesional monocyte/macrophage flux.

Figure 5

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Fluorescent beads were intravenously injected into 15 wk AD-fed female mice to label monocytes. One day later, a blood sample was taken to quantify blood monocyte labeling efficiency and specificity by flow cytometry. (A) Five days after bead injection, mice were sacrificed, hearts were harvested and frozen in OCT, frozen sections were cut (8 µm), and the number of fluorescent beads in aortic root intima was quantified manually and normalized to blood monocyte labeling efficiency. (B) Aortic root sections from 16 wk AD-fed female mice were immunostained with cleaved caspase 3 for apoptotic cell quantification. (C) Lesional monocyte/macrophage flux was evaluated by comparing bead-labeled monocyte accumulation 5 and 28 days after intravenous fluorescent bead injection. (D) Gene expression in whole aortas of mice from monocyte/macrophage flux study (i.e., panel C). (E) Flow cytometry analysis of aortas from male mice fed the AD for 34 wks (n=4–6). (F–G) Flow cytometry analysis of peripheral lymph node cells from monocyte flux study mice (n= 3–4 for 5 day data, panel F; n=4–6 for 28 day data, panel G). Data expressed as mean ± SEM. * P<0.05.

To explore this idea further, we examined whole aorta gene expression. As anticipated, ABCA1 expression was significantly lower in MSKO/LDLrKO aortas, suggesting macrophage gene expression likely dominated overall aortic gene expression. We also observed significantly decreased CD11c expression in MSKO/LDLrKO vs. LDLrKO, but similar F4/80 expression (Figure 5D), suggesting that dendritic cell content may be reduced in MSKO/LDLrKO aortas. In a separate experiment, we analyze cells isolated from aortic digests of mice fed the AD for 34 weeks and observed a trend towards deceased CD11C+ cells in MKSO/LDLrKO vs. LDLrKO mice (Figure 5E). Since dendritic cells migrate to peripheral lymph nodes for antigen presentation, we measured peripheral lymph node CD11c content in mice used for the second bead labeling experiment and observed significantly increased percentage of CD11c+ and Ly6Chi-CD11C+ cells at 5 (Figure 5F) and 28 (Figure 5G) days after bead injection in MSKO/LDLrKO mice. The combined results are compatible with the interpretation that MSKL/LDLrKO monocytes flux through atherosclerotic lesions at a greater rate than LDLrKO monocytes.

Similar extent of atherosclerosis in LDLrKO mice transplanted with MSKO/LDLrKO or LDLrKO bone marrow

Previous BM transplantation studies uniformly found a significant atheroprotective role of hematopoietic ABCA1, and macrophage ABCA1 expression was assumed to mediate this effect. However, AD-fed MSKO/LDLrKO mice did not display accelerated atherosclerosis under similar conditions (i.e., AD for 10 or 16 wks). To determine whether the different outcomes were due to different experimental strategies or to a more specific deletion of ABCA1, we transplanted male MSKO/LDLrKO (DKO) or LDLrKO (SKO) BM into lethally-irradiated female LDLrKO recipient mice and measured atherosclerosis extent after 16 wks of AD feeding. Successful replacement of hematopoietic cells was confirmed 4 wks after transplantation (Supplemental Figure X). The plasma phenotype of BM transplant mice resembled that of the non-transplanted counterparts in the 16 wk atherosclerosis experiment (i.e., Figure 1). TPC and plasma TG levels in DKO BM recipients were ~40–50% lower than those of SKO BM recipients as soon as 2 wks after initiation of the AD, and remained lower throughout the 16 wk progression phase (Figure 6A). There was only a 5% difference in basal TPC (i.e. chow diet) with indistinguishable cholesterol distribution among lipoproteins (Figure 6B, time 0), whereas VLDL-C and LDL-C contributed to the decreased TPC in AD-fed DKO BM recipient mice (Figure 6B, 2–16 wks). In agreement with the non-transplant 16-week study, cholesterol content of resident PMs was greatly increased (Figure 6C), but whole aorta cholesterol content was similar between DKO vs. SKO BM recipient mice (Figure 6D). These data suggest that the difference in outcome between our study and previous BM transplantation studies is due to the more restrictive myeloid cell ABCA1deletion in our study.

Figure 6. Bone marrow transplantation.

Figure 6

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Seven weeks after transplantation of LDLrKO (SKO) or MSKO/LDLrKO (DKO) bone marrow, LDLrKO recipient mice were switched from chow to an AD for 16 wks. (A) Periodic TPC, plasma FC, and TG measurements were made during the atherosclerosis progression phase using enzymatic assays. (B) Pooled plasma from subgroups of mice (n=3 plasma pools from 5 mice) was fractionated by FPLC to measure lipoprotein cholesterol distribution. (C) Cholesterol content (TC, total cholesterol; FC, free cholesterol, CE, cholesteryl ester) of resident PMs from recipient mice fed AD for 16 wks was determined by gas-liquid chromatography. (D) Aortic cholesterol content of mice fed AD for 16 wks. Data expressed as mean ± SEM (n=15). *p<0.05

Chow diet-fed MSKO/LDLrKO mice are more susceptible to early-stage atherogenesis

The finding that AD-fed MSKO/LDLrKO mice had similar early and intermediate atherosclerosis compared with LDLrKO mice, but lower plasma VLDL and LDL concentrations and higher macrophage cholesterol content, led us to hypothesize that the deleterious effects of ABCA1 deletion in macrophages were offset by the substantial reduction of plasma atherogenic apoB Lp. To test this possibility, we fed female mice chow until they were 24 wks old to minimize the differences in plasma lipids and lipoproteins observed with AD feeding. This time course was similar to the previously described 16 wk AD study (16 wks of AD feeding starting at 8 wks of age) using mice with and without BM transplantation. As anticipated, chow-fed MSKO/LDLrKO vs. LDLrKO mice had similar TPC and plasma TG concentrations over time (Figure 7A–B). Although the chow-fed MSKO/LDLrKO mice had significantly lower TPC concentrations compared with chow-fed LDLrKO mice, the difference was minimal (~6–17%) throughout the study and likely achieved statistical significance due to the very low variability in measurements. Plasma lipoprotein cholesterol distribution of 12-wk-old mice revealed very minor differences between genotypes, with LDL-C and HDL-C being slightly decreased in MSKO/LDLrKO mice (2.93 mmol/L vs. 2.61 mmol/L LDL-C; 1.11 mmol/L vs. 0.94 mmol/L HDL-C) (Figure 7C). Resident PMs without ABCA1 expression exhibited 1.5-fold and 3-fold increases in cellular FC and TC content, respectively, and a striking 70 fold induction in CE accumulation (~1 vs. 71 µg CE/mg protein in females, P<0.05;) under mild hyperlipidemic conditions (Figure 7D). A similar trend was present in male mice (data not shown). Whole aorta chemical analysis by gas-liquid chromatography showed low, but detectable, amounts of cholesterol compared to AD-fed mice. A significant ~70% higher aortic CE content (0.77 vs. 1.31 µg CE/ mg protein, p<0.05) in MSKO/LDLrKO mice suggests increased very early-stage atherosclerosis compared with LDLrKO mice (Figure 7E). Aortic root Oil red O-stained sections from representative animals of each group showed ~74% larger lesions for MSKO/LDLrKO vs. LDLrKO mice (0.0299 mm2 vs. 0.0519 mm2, p=0.0227) (Figure 7F). Collectively, these results suggest that in the absence of major differences in plasma lipid concentrations, deletion of myeloid cell ABCA1 enhanced early atherosclerotic lesion development. These results support the conclusion that less atherogenic lipid profiles may counterbalance the proatherogenic effects present in MSKO/LDLrKO mice, preventing the expected increase in early/intermediate stage atherogenesis.

Figure 7. Atherosclerosis in chow-fed mice.

Figure 7

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(A) TPC and (B) plasma TG were measured by enzymatic assays in chow-fed female mice between 12–24 weeks of age. (C) Plasma lipoprotein cholesterol distribution was determined in chow-fed female mice at 12 wks of age. (D) Resident PM TC, FC and CE content were assayed by gas-liquid chromatography. Atherosclerosis was evaluated by two measurements: (E) aortic cholesterol content of female chow-fed mice (n=15–16) and (F) aortic root lesion area of representative animals from each genotype (n=13–16). Each point represents the average lesion area per mouse. Data expressed as mean ± SEM. * P<0.05.

Discussion

Macrophages are a major cellular constituent of atherosclerotic lesions and are involved in the uptake and breakdown of modified lipoproteins 31. This process can result in the formation of CE-enriched macrophages or foam cells. ABCA1 and other cholesterol export proteins (e.g., ABCG1 and SR-BI) counterbalance the unregulated accumulation of macrophage CE by effluxing excess cellular cholesterol 32. Studies using whole body ABCA1 knockout mouse BM suggest that genetic deletion of hematopoietic ABCA1 uniformly increases atherosclerosis8, 11–13. However, the role of macrophage ABCA1 in atherosclerosis development has never been formally tested, as BM used for transplantation studies contains other cell populations that are likely important in atherosclerosis progression 33. We addressed this question by performing atherosclerosis studies using mice with genetic deletion of ABCA1 in leukocytes of myeloid lineage crossed into the LDLrKO C57BL/6 background (i.e., MSKO/LDLrKO).

Contrary to expectation, AD-fed MSKO/LDLrKO mice did not have accelerated atherosclerosis relative to LDLrKO mice at early and intermediate time points, due to an unanticipated decrease in plasma VLDL and LDL concentrations. Only advanced atherosclerosis (24 wks of AD feeding) was modestly, but significantly, increased in MSKO/LDLrKO mice. Similar atherosclerosis (i.e., SKO and DKO not different) and plasma lipoprotein (i.e., lower VLDL/LDL for DKO mice) results were obtained for intermediate-stage atherosclerosis (i.e., 16 wks) using transplantation of SKO and DKO BM into LDLrKO recipient mice, suggesting that our unique finding was not due to a different study design compared with previous BM transplantation studies.

We hypothesize that the negative impact of myeloid cell ABCA1 deletion in vivo was offset by an atheroprotective reduction in plasma VLDL and LDL, significantly delaying (16 wks to 24 wks) and minimizing the anticipated increase in atherosclerosis in MSKO/LDLrKO vs. LDLrKO mice. In support of this hypothesis, minimizing the difference in plasma VLDL and LDL by maintaining mice on a chow diet resulted in a modest, but significant, increase in atherosclerosis in MSKO/LDLrKO mice. Overall, our study results suggest that myeloid ABCA1 expression is atheroprotective in similar states of hyperlipidemia and has a novel function in controlling plasma VLDL and LDL concentrations and in vivo monocyte trafficking during atherogenesis (see below).

This is the first atherosclerosis study using MSKO/LDLrKO mice backcrossed into the C57BL/6 background. Brunham et al 22 previously reported that atherosclerosis development was similar for MSKO/LDLrKO and LDLrKO mice fed an AD for 16 wks, in agreement with our results (Figure 4). However, mice used in that study were in a mixed genetic background (~90 C57BL/6, ~10% 129 SVEV) and since the 129 background is atherosclerosis-resistant 23, these results may have been due to background effects. In addition, previous BM transplantation studies using different study designs (i.e., diets: chow, Western, Paigen; genetic backgrounds: apoE, LDLrKO) uniformly observed a 1.4–1.8-fold increase in early- to intermediate-stage (10–14 wks diet feeding) atherosclerosis development 8, 11–14. In two studies, atherosclerosis increased 40–60% despite significantly reduced plasma cholesterol concentrations 11, 13. The atheroprotective effect of ABCA1 observed was mainly attributed to macrophage ABCA1. However, our results, using a more restrictive deletion of ABCA1 in BM cells of myeloid origin, showed no difference in atherosclerosis at early (10 wks) and intermediate (16 wks) stages of progression. The different outcomes may have arisen from distinct experimental strategies (i.e. BM transplantation vs. myeloid cell-specific gene targeting) or the relative contribution of non-myeloid BM vs. myeloid cell ABCA1 expression in atherogenesis. The first possibility was eliminated when we found similar atherosclerosis and plasma lipoprotein outcomes between our non-transplant study and our BM transplantation study using AD-fed LDLrKO recipient mice transplanted with MSKO/LDLrKO or LDLrKO BM. The significantly accelerated atherosclerosis development in earlier studies is likely due to the impact of ABCA1 expression on non-myeloid BM cells, since more specific deletion of ABCA1 in only myeloid cells under similar experimental conditions failed to exacerbate atherogenesis during the same time frame.

Macrophages (PM and bone marrow derived macrophages) from chow-fed MSKO mice have defective FC and PL efflux 21. Although CE accumulation is minimal in chow-fed MSKO mouse macrophages, FC is significantly elevated (~10%), leading to increased membrane lipid raft content and hyperresponsiveness to proinflammatory agents like lipopolysaccharide. Previous studies showed that macrophage FC efflux is facilitated by ABCA1, ABCG1, SR-BI, and aqueous diffusion 6. In the hyperlipidemic background of LDLr deficiency, resident MSKO PMs had massive CE accumulation that increased in severity from early to late stages of atherogenesis. Even chow-fed MSKO/LDLrKO mice had elevated resident PM CE content. However, despite the massive cholesterol loading in resident MSKO/LDLrKO PMs, inflammatory gene expression was similar to that of LDLrKO macrophages (Supplemental Figure VI B). This outcome likely results from LXR-mediated downregulation of inflammatory gene expression by desmosterol as described by Glass and colleagues 34. Our data document that loss of myeloid cell ABCA1 expression in AD-fed LDLrKO mice results in progressive accumulation of PM CE, although plasma lipoprotein concentrations were relatively stable after 2 wks of AD feeding (Figure 1), suggesting that no other efflux mechanism can compensate for loss of PM ABCA1 in vivo to prevent massive macrophage CE accumulation.

Another novel finding in the present study was that plasma VLDL and LDL concentrations were ~50% lower in AD-fed MSKO/LDLrKO vs. LDLrKO mice, suggesting an in vivo role for macrophage ABCA1 expression in regulating plasma apoB Lp metabolism during atherogenesis. Similar observations were made in two BM transplantation studies after global ABCA1 knockout vs. wild-type BM was transplanted into LDLrKO mice 11, 13. Although the two BM transplantation studies did not address this finding with additional experiments, herein we show that reduced hepatic TG content, reduced hepatic VLDL TG secretion, and lower plasma NEFA concentrations may contribute to lower plasma VLDL in AD-fed MSKO/LDLrKO mice. Since cytokines can modulate lipid metabolism 35 and macrophages are an important source of cytokines, decreased hepatic VLDL TG secretion in AD-fed MSKO/LDLrKO mice may result from macrophage cytokine production, either locally (i.e., Kupffer cells) or systemically. Acute ablation of Kupffer cells did not normalize the differences in TPC and plasma TG between genotypes of mice (Supplemental Figure IV), suggesting that resident hepatic macrophages were not affecting hepatic lipogenesis and TG secretion. The fact that the plasma lipid phenotype in our BMT study recapitulated the 16 wk AD study also suggests that circulating leukocytes and hematopoietic stem and progenitor cells may affect hepatic lipid metabolism, since Kupffer cells are not efficiently deleted from liver over the time course of the BMT study 36, 37. The underlying mediators and mechanisms responsible for decreased hepatic TG production in AD-fed MSKO/LDLrKO mice are unknown and will require additional studies to identify.

Hyperlipidemic mouse models (i.e., LDLrKO and apoE KO) exhibit monocytosis and increased trafficking of monocytes into atherosclerotic lesions when fed cholesterol-containing ADs 38, 39. We also observed similar AD-induced blood Ly6Chi monocytosis for both genotypes of mice (Supplemental Figure VII E). Moreover, monocyte flux through atherosclerotic lesions was significantly increased in MSKO/LDLrKO mice (Figure 5). Subsequent analysis of aortic gene expression and lymph node cells suggested that monocytes from MSKO/LDLrKO mice enter lesions more rapidly, convert to dendritic-like cells (i.e., CD11C+), and exit lesions more rapidly than their LDLrKO counterparts. This concept is supported by decreased aortic CD11c expression and increased lymph node CD11C+, Ly6Chi cells. Macrophage ABCA1 deficiency enhances chemotaxis in vitro and in vivo in chow-fed mice 29 and our results in AD-fed MSKO/LDLrKO mice provide additional in vivo evidence of this effect. However, because aortic bead content progressed between 5 and 28 days in LDLrKO mice, but not MSKO/LDLrKO mice (Figure 5C), there may be alternative explanations that require cautious interpretation of our monocyte flux data. For instance, we observed similar blood monocyte bead labeling for both genotypes of mice one day after intravenous bead injection, but residual monocyte labeling may differ between genotypes between 5 and 28 days. In addition, MSKO/LDLrKO monocyte influx into lesions may decrease between 5–28 days after bead injection resulting in decreased influx and not necessarily increase egress from lesions. However, these limitations notwithstanding, we believe the sum total of evidence suggests strikingly different in vivo trafficking of monocytes into and out of atherosclerotic lesions in MSKO/LDLrKO vs. LDLrKO mice.

In our study, resident PMs from MSKO/LDLrKO mice were massively loaded with cholesterol compared with LDLrKO mice, yet aortic lesions were similar in cholesterol content, Oil red O staining, and macrophage number, suggesting that resident PMs and macrophages in atherosclerotic lesions are phenotypically different. It is not likely that aortic lesional macrophages had reduced cholesterol content due to upregulation of alternative efflux pathways since aortic ABCG1 and SR-BI mRNA expression was similar for both genotypes of mice (Figure 5D). Recent studies suggest that monocyte recruitment to atherosclerotic lesions primarily contributes to macrophage accumulation in early lesion development, but local proliferation of lesional macrophages predominates in advanced atherosclerotic lesions 40, 41,42. Thus, it may not be surprising that resident PMs do not phenocopy aortic macrophages in intermediate to advanced stages of atherosclerosis and are not good surrogates for aortic lesional macrophages. Attempts to isolate F4/80+cells from aortas of mice fed the AD for 20 wks using fluorescence-activated cell sorting failed to yield sufficient cells for mass spectrometric quantification of FC and CE. Considering the likely distinct differences in local environment where macrophages reside (i.e. peritoneal cavity vs. atherosclerotic plaque), characterization of lesional macrophages, though difficult to approach, and the underlying mechanisms of altered monocyte/macrophage trafficking merit further investigation.

In conclusion, we investigated the effects of myeloid cell-specific ABCA1 deletion on early to advanced stage atherosclerosis development. Myeloid cell ABCA1 expression appeared to be modestly atheroprotective in chow-fed mice, in which plasma lipid changes are minimal, but its atheroprotective effect was eliminated or significantly delayed in AD-fed LDLrKO mice due to a paradoxical decrease in plasma VLDL and LDL concentrations, resulting from decreased hepatic production of VLDL TG. Our findings also indirectly support an atheroprotective role for ABCA1 expression in non-myeloid cells (i.e., BM HSPC, T cells) in atherogenesis given the difference in atherosclerosis outcome in BM transplantation studies using MSKO/LDLrKO vs. global ABCA1 knockout BM. Finally, this study highlights the multifaceted impact of myeloid ABCA1 expression on atherogenesis and lipoprotein metabolism, and the complex patterns of atherosclerosis progression.

Supplementary Material

Methods and materials
Supplemental material

Significance.

ATP binding cassette transporter A1 (ABCA1) functions in cellular cholesterol export and nascent HDL formation. Previous transplantation studies suggest that bone marrow (BM) cell ABCA1 protects against atherosclerosis development. However, the in vivo impact of macrophage ABCA1 expression on atherogenesis is not fully understood because BM contains other cells that are important in atherogenesis. We examined atherosclerosis using myeloid-specific ABCA1 knockout (MSKO) mice in the LDL receptor knockout (LDLrKO) background and did not find the anticipated increase in atherosclerosis at early (10 wks) and intermediate (16 wks) time points, presumably due to an unexpected decrease in plasma VLDL and LDL levels. When differences in plasma VLDL/LDL concentrations were minimized by maintaining mice on chow, MSKO/LDLrKO mice had a modest, but significant, increase in atherosclerosis compared to LDLrKO mice. Our results show that myeloid ABCA1 expression has novel and multifaceted effects on atherogenesis and lipoprotein metabolism.

Acknowledgments

The authors gratefully acknowledge Karen Klein (Translational Science Institute, Wake Forest School of Medicine) for editing the manuscript, Dr. J. Mark Cline (Professor of Pathology, Wake Forest School of Medicine) for advice on analysis of aortic root lesions, Kristi Brzoza-Lewis, Manal Zabalawi and Hermina Borgerink (Wake Forest School of Medicine) for advice on flow cytometry data analysis, assistance in lymph node isolation, and aortic root sectioning, respectively. We also gratefully acknowledge Drs. Alan Tall (Columbia University) and Gwendalyn Randolph (Washington University) for helpful discussions regarding our data. We acknowledge the Comprehensive Cancer Center of Wake Forest University Flow Cytometry shared resource supported by NCI CCSG P30CA012197 and the helpful advice from Dr. Martha Alexander-Miller, the Core lab director.

Sources of funding

This study was supported by National Institutes of Health Grants HL49373, HL94525 and HL119962.

Non-standard Abbreviations

ABC

ATP-binding cassette transporter

AD

Atherogenic diet

ApoB Lp

ApoB containing lipoproteins

BM

Bone marrow

CE

Cholesteryl ester

DKO

Double knockout (MSKO/LDLrKO)

FC

Free cholesterol

HSPC

Hematopoietic stem and progenitor cell

MSKO

Myeloid-specific ABCA1 knockout

PM

Peritoneal macrophages

SKO

Single knockout (LDLrKO)

TG

Triglyceride

TPC

Total plasma cholesterol

Footnotes

Disclosures- None

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Supplementary Materials

Methods and materials
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Supplemental material
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