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. 2013 May 24;112(11):1456-65.
doi: 10.1161/CIRCRESAHA.113.301086. Epub 2013 Apr 9.

Deficiency of ATP-binding cassette transporters A1 and G1 in macrophages increases inflammation and accelerates atherosclerosis in mice

Marit Westerterp  1 , Andrew J Murphy, Mi Wang, Tamara A Pagler, Yuliya Vengrenyuk, Mojdeh S Kappus, Darren J Gorman, Prabhakara R Nagareddy, Xuewei Zhu, Sandra Abramowicz, John S Parks, Carrie Welch, Edward A Fisher, Nan Wang, Laurent Yvan-Charvet, Alan R Tall
Affiliations

Deficiency of ATP-binding cassette transporters A1 and G1 in macrophages increases inflammation and accelerates atherosclerosis in mice

Marit Westerterp et al. Circ Res. .

Abstract

Rationale: Plasma high-density lipoprotein levels are inversely correlated with atherosclerosis. Although it is widely assumed that this is attributable to the ability of high-density lipoprotein to promote cholesterol efflux from macrophage foam cells, direct experimental support for this hypothesis is lacking.

Objective: To assess the role of macrophage cholesterol efflux pathways in atherogenesis.

Methods and results: We developed mice with efficient deletion of the ATP-binding cassette transporters A1 and G1 (ABCA1 and ABCG1) in macrophages (MAC-ABC(DKO) mice) but not in hematopoietic stem or progenitor populations. MAC-ABC(DKO) bone marrow (BM) was transplanted into Ldlr(-/-) recipients. On the chow diet, these mice had similar plasma cholesterol and blood monocyte levels but increased atherosclerosis compared with controls. On the Western-type diet, MAC-ABC(DKO) BM-transplanted Ldlr(-/-) mice had disproportionate atherosclerosis, considering they also had lower very low-density lipoprotein/low-density lipoprotein cholesterol levels than controls. ABCA1/G1-deficient macrophages in lesions showed increased inflammatory gene expression. Unexpectedly, Western-type diet-fed MAC-ABC(DKO) BM-transplanted Ldlr(-/-) mice displayed monocytosis and neutrophilia in the absence of hematopoietic stem and multipotential progenitor cells proliferation. Mechanistic studies revealed increased expressions of machrophage colony stimulating factor and granulocyte colony stimulating factor in splenic macrophage foam cells, driving BM monocyte and neutrophil production.

Conclusions: These studies show that macrophage deficiency of ABCA1/G1 is proatherogenic likely by promoting plaque inflammation and uncover a novel positive feedback loop in which cholesterol-laden splenic macrophages signal BM progenitors to produce monocytes, with suppression by macrophage cholesterol efflux pathways.

Keywords: atherosclerosis; high-density lipoprotein; inflammation; macrophages; monocytes.

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Figures

Figure 1
Figure 1
Characterization of MAC-ABCDKO mice. A and B. Protein levels of ABCA1 (A) and ABCG1 (B) in wild-type, control, and MAC-ABCDKO macrophages. Thioglycollate-elicited peritoneal macrophages were isolated, incubated with the LXR activator TO901317 (3 µM, 24 h), and ABCA1 and ABCG1 expression were assessed by Western blot. Bands were quantified using Image J and corrected for β-actin expression (bar graphs). C and D. Bone marrow (BM) cells were sorted into RLT-buffer by FACS sorting and the mRNA expression of ABCA1 (C) and ABCG1 (D) were assessed and corrected for the housekeeping gene m36B4. *P<0.05, **P<0.01, ***P<0.001.
Figure 1
Figure 1
Characterization of MAC-ABCDKO mice. A and B. Protein levels of ABCA1 (A) and ABCG1 (B) in wild-type, control, and MAC-ABCDKO macrophages. Thioglycollate-elicited peritoneal macrophages were isolated, incubated with the LXR activator TO901317 (3 µM, 24 h), and ABCA1 and ABCG1 expression were assessed by Western blot. Bands were quantified using Image J and corrected for β-actin expression (bar graphs). C and D. Bone marrow (BM) cells were sorted into RLT-buffer by FACS sorting and the mRNA expression of ABCA1 (C) and ABCG1 (D) were assessed and corrected for the housekeeping gene m36B4. *P<0.05, **P<0.01, ***P<0.001.
Figure 2
Figure 2
Leukocyte subsets and atherosclerosis in chow-fed Ldlr-/- mice transplanted with (A and C-E) MAC-ABCDKO (n=10) or control (n=12) BM or (B and C-E) Abca1-/- Abcg1-/- (n=9) or wild-type (n=7) BM. A and B. Blood leukocyte subsets as assessed by flow cytometry. Mono denotes monocytes and neutro denotes neutrophils. Levels of Ly6-Chi and Ly6-Clo monocyte subsets are shown. C. Atherosclerotic lesion area. Hematoxylin-eosin staining was performed on paraffin sections of the aortic root and atherosclerotic lesion area was assessed. In C, each data point represents an individual mouse. D. Atherosclerotic lesion type as percentage of total number of atherosclerotic lesions. Only segments containing atherosclerotic lesions were included in the analysis. *P<0.05, **P<0.01, ***P<0.001.
Figure 2
Figure 2
Leukocyte subsets and atherosclerosis in chow-fed Ldlr-/- mice transplanted with (A and C-E) MAC-ABCDKO (n=10) or control (n=12) BM or (B and C-E) Abca1-/- Abcg1-/- (n=9) or wild-type (n=7) BM. A and B. Blood leukocyte subsets as assessed by flow cytometry. Mono denotes monocytes and neutro denotes neutrophils. Levels of Ly6-Chi and Ly6-Clo monocyte subsets are shown. C. Atherosclerotic lesion area. Hematoxylin-eosin staining was performed on paraffin sections of the aortic root and atherosclerotic lesion area was assessed. In C, each data point represents an individual mouse. D. Atherosclerotic lesion type as percentage of total number of atherosclerotic lesions. Only segments containing atherosclerotic lesions were included in the analysis. *P<0.05, **P<0.01, ***P<0.001.
Figure 3
Figure 3
Leukocyte subsets in Ldlr-/- or Ldlr-/- APOA1TG mice transplanted with MAC-ABCDKO or control BM (n=13-17 per group) on a Western-type diet (6-8 weeks). Blood monocytes, Ly6-Chi and Ly6-Clo monocyte subsets, and neutrophils were measured by flow cytometry.*P<0.05, **P<0.01.
Figure 4
Figure 4
Monocytosis and neutrophilia in MAC-ABCDKO BM transplanted Ldlr-/- mice on the WTD are cell-extrinsic. Ldlr-/- mice were transplanted with a 1:1 mix of CD45.1 wild type + CD45.2 MAC-ABCDKO BM or 1:1 mix of CD45.1 wild-type + CD45.2 control BM. All parameters were assessed by flow cytometry. A. Total blood monocyte levels. B. CD45.2:CD45.1 monocyte ratio. C. Total blood neutrophil levels. D. CD45.2:CD45.1 neutrophil ratio. *P<0.05, **P<0.01, ***P<0.001.
Figure 4
Figure 4
Monocytosis and neutrophilia in MAC-ABCDKO BM transplanted Ldlr-/- mice on the WTD are cell-extrinsic. Ldlr-/- mice were transplanted with a 1:1 mix of CD45.1 wild type + CD45.2 MAC-ABCDKO BM or 1:1 mix of CD45.1 wild-type + CD45.2 control BM. All parameters were assessed by flow cytometry. A. Total blood monocyte levels. B. CD45.2:CD45.1 monocyte ratio. C. Total blood neutrophil levels. D. CD45.2:CD45.1 neutrophil ratio. *P<0.05, **P<0.01, ***P<0.001.
Figure 5
Figure 5
Characterization of spleen and plasma of control and MAC-ABCDKO BM transplanted Ldlr-/- mice on the WTD (8 weeks). A. Mcp-1, M-csf, and G-csf mRNA expression in the spleen. B. Mcp-1 and M-csf mRNA expression in FACS-sorted splenic monocytes and macrophages. Mono and macro denote monocytes and macrophages, respectively. C. Monocytes and macrophages as % of total spleen cells assessed by flow cytometry. D-F. Plasma levels of MCP-1 (D), M-CSF (E), and G-CSF (F) assessed by ELISA in Ldlr-/- and Ldlr-/- APOA1TG mice transplanted with control or MAC-ABCDKO BM. n=6-12 mice per group. For A and B, RNA was extracted, and expression of Mcp-1, M-csf, and G-csf was assessed and corrected for the housekeeping gene m36B4. *P<0.05, **P<0.01, ***P<0.001.
Figure 5
Figure 5
Characterization of spleen and plasma of control and MAC-ABCDKO BM transplanted Ldlr-/- mice on the WTD (8 weeks). A. Mcp-1, M-csf, and G-csf mRNA expression in the spleen. B. Mcp-1 and M-csf mRNA expression in FACS-sorted splenic monocytes and macrophages. Mono and macro denote monocytes and macrophages, respectively. C. Monocytes and macrophages as % of total spleen cells assessed by flow cytometry. D-F. Plasma levels of MCP-1 (D), M-CSF (E), and G-CSF (F) assessed by ELISA in Ldlr-/- and Ldlr-/- APOA1TG mice transplanted with control or MAC-ABCDKO BM. n=6-12 mice per group. For A and B, RNA was extracted, and expression of Mcp-1, M-csf, and G-csf was assessed and corrected for the housekeeping gene m36B4. *P<0.05, **P<0.01, ***P<0.001.
Figure 6
Figure 6
Atherosclerosis in control and MAC-ABCDKO BM transplanted Ldlr-/- mice after 7.5 weeks of WTD (n=13-17 mice per group). A. Atherosclerotic lesion area. Haematoxylin-eosin staining was performed on paraffin sections of the aortic root and atherosclerotic lesion area was assessed. B. Relation between atherosclerotic lesion area and cholesterol levels. r denotes the correlation coefficient. In A and B, each data point represents an individual mouse. *P<0.05. C. Inflammatory gene expression in macrophages in atherosclerotic lesions. D. Inflammatory gene expression in macrophages in atherosclerotic lesions of Abca1-/- Abcg1-/- BM transplanted Ldlr+/- mice after 10-11 weeks of Paigen diet. For C and D, LCM was performed to collect macrophages, RNA was isolated and amplified and mRNA expression was assessed and corrected for the housekeeping gene m36B4. *P<0.05, ***P<0.01.
Figure 6
Figure 6
Atherosclerosis in control and MAC-ABCDKO BM transplanted Ldlr-/- mice after 7.5 weeks of WTD (n=13-17 mice per group). A. Atherosclerotic lesion area. Haematoxylin-eosin staining was performed on paraffin sections of the aortic root and atherosclerotic lesion area was assessed. B. Relation between atherosclerotic lesion area and cholesterol levels. r denotes the correlation coefficient. In A and B, each data point represents an individual mouse. *P<0.05. C. Inflammatory gene expression in macrophages in atherosclerotic lesions. D. Inflammatory gene expression in macrophages in atherosclerotic lesions of Abca1-/- Abcg1-/- BM transplanted Ldlr+/- mice after 10-11 weeks of Paigen diet. For C and D, LCM was performed to collect macrophages, RNA was isolated and amplified and mRNA expression was assessed and corrected for the housekeeping gene m36B4. *P<0.05, ***P<0.01.
Figure 6
Figure 6
Atherosclerosis in control and MAC-ABCDKO BM transplanted Ldlr-/- mice after 7.5 weeks of WTD (n=13-17 mice per group). A. Atherosclerotic lesion area. Haematoxylin-eosin staining was performed on paraffin sections of the aortic root and atherosclerotic lesion area was assessed. B. Relation between atherosclerotic lesion area and cholesterol levels. r denotes the correlation coefficient. In A and B, each data point represents an individual mouse. *P<0.05. C. Inflammatory gene expression in macrophages in atherosclerotic lesions. D. Inflammatory gene expression in macrophages in atherosclerotic lesions of Abca1-/- Abcg1-/- BM transplanted Ldlr+/- mice after 10-11 weeks of Paigen diet. For C and D, LCM was performed to collect macrophages, RNA was isolated and amplified and mRNA expression was assessed and corrected for the housekeeping gene m36B4. *P<0.05, ***P<0.01.
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