Hematopoietic Cell-Specific SLC37A2 Deficiency Accelerates Atherosclerosis in LDL Receptor-Deficient Mice

Figures

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  Figure 1. Hematopoietic cell-specific SLC37A2 deletion increases plasma lipids in WD-fed Ldlr−/− mice. Hematopoietic SLC37A2 knockout (SLC37A2Δhema) and control (WTΔhema) Ldlr−/− mice were switched from a standard laboratory diet to high-fat WD for 16 wks to promote the development of advanced atherosclerosis. (A–D) Fasting (4 h) plasma total cholesterol (TC), free cholesterol (FC), cholesterol ester (CE), and triglyceride (TG) concentrations were measured by enzymatic assays over time (0-16 wks) (n = 12-16). (E) Plasma cholesterol distribution among lipoproteins after 16 wks of WD feeding was determined after fractionation of plasma by fast protein liquid chromatography (n = 6-8). Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. The mixed-effects models were used to compare lipids variables between genotype groups at each time point. HDL indicates high-density lipoprotein; LDL, low-density lipoprotein; and VLDL, very-low-density lipoprotein.

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  Figure 2. Hematopoietic cell-specific SLC37A2 deletion increases liver cholesterol ester and reduces hepatic macrophage activation in WD-fed LDLrKO mice. Irradiated Ldlr−/− mice transplanted with WT or SLC37A2KO bone marrow were fed a high-fat western diet for 16 wks. (A) Fasting (4 h) total cholesterol (TC), free cholesterol (FC), cholesterol ester (CE), and triglyceride (TG) concentrations in the liver were measured by enzymatic assays (n = 12 per genotype). (B) Slc37a2 transcript expression in liver (n = 12 per genotype). (C) Relative transcript levels for cholesterol efflux, cholesterol uptake, cholesterol synthesis, fatty acid synthesis, and transcriptional regulators controlling these pathways in the liver (n = 12 per genotype). (D) Relative transcript levels for genes encoding key enzymes in fatty acid uptake, β-oxidation, and transcriptional regulators controlling these pathways in the liver (n = 12 per genotype). (E) Relative transcript levels for genes encoding macrophage markers in the liver (n = 12 per genotype). (F,G) Liver cytokine and chemokine protein concentrations (n = 12 per genotype). (H) Relative transcript levels for genes encoding macrophage anti-inflammatory markers…

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  Figure 3. Hematopoietic cell-specific SLC37A2 deletion primarily impairs anti-inflammatory responses in WD-fed Ldlr−/− mice. Hematopoietic SLC37A2 knockout (SLC37A2Δhema) and control (WTΔhema) Ldlr−/− mice were fed a high-fat western diet for 16 wks. (A–C) Plasma pro-inflammatory cytokine (A), chemokine (B), and anti-inflammatory cytokine IL-10 (C) concentrations were quantified by multiplex assay. (D–F) Relative transcript levels of Slc37a2, pro- and anti-inflammatory markers in resident peritoneal macrophages. Each symbol represents an individual mouse. Data are expressed as mean ± SEM. *p < 0.05; ***p < 0.001, unpaired, two-tailed Student's t-test.

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  Figure 4. Hematopoietic cell-specific SLC37A2 deficiency promotes atherosclerosis in WD-fed Ldlr−/− mice. Hematopoietic SLC37A2 knockout (SLC37A2Δhema) and control (WTΔhema) Ldlr−/− mice were fed a high-fat western diet for 16 wks (n = 12-16 mice per genotype). (A,B) Quantification of Oil Red O positive intimal area in the aortic root. (C,D) Quantification of CD68+ cells (macrophages) in the aortic root intimal area. (E) Linear regression analysis of aortic root intimal area vs. plasma total cholesterol of 16-wk diet-fed mice. Each point represents an individual animal of the denoted diet group and the correlation coefficient is shown for the entire data set. (F) Linear regression analysis of aortic root intimal area vs. plasma IL-10 of 16-wk diet-fed mice. Each point represents an individual animal of the denoted diet group and the correlation coefficient is shown for the entire data set. (G,H) Quantification of the aortic necrotic intimal area (total necrotic area) in H&E stained aortic root sections. White dash line circled lesion areas. White stars indicate the necrotic area in the plaques. (I,J) Quantification of apoptosis (TUNEL-positive cells) and efferocytosis (the ratio of…

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  Figure 5. SLC37A2 deficiency promotes oxidized LDL (OxLDL)-induced macrophage inflammation in vitro. (A) SLC37A2 protein expression analyzed by Western blotting in WT and Slc37a2−/− bone marrow-derived macrophages (BMDMs) treated with 50 μg/ml oxLDL for 0-24 h. (B) Quantification of cellar total cholesterol (TC), free cholesterol (FC), and cholesterol ester (CE) in WT and Slc37a2−/− BMDMs after treated with or without 25 or 50 μg/ml oxLDL for 24 h. (C) Relative transcript level of cytokines in WT and Slc37a2−/− BMDMs stimulated with 50 μg/ml oxLDL for 0-24 h, measured by qPCR. Data are representative of two independent experiments with three samples per group (mean ± SEM). *p < 0.05; unpaired, two-tailed Student's t-test.

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  Figure 6. SLC37A2 deficiency impairs IL-4-induced macrophage activation in vitro. (A,B) SLC37A2 expression in WT and Slc37a2−/− bone marrow-derived macrophages (BMDMs) stimulated with 20 ng/ml IL-4 for 0-24 h. (A) relative transcript expression and (B) protein expression of SLC37A2 measured by qPCR and western blotting, respectively. (C) Relative transcript level of macrophage alternative activation markers, including arginase−1 (Arg), mannose receptor, type I (Mrc1), Ym1, IL-10, and chemokine MCP-1, in WT and Slc37a2−/− BMDMs stimulated with 20 ng/ml IL-4 for 0-24 h. (D,E) The relative transcript level of PPARs and genes encoding transporters or enzymes involved in fatty acid uptake or β-oxidation in WT and Slc37a2−/− BMDMs stimulated with 20 ng/ml IL-4 for 0-24 h. (F,G) Seahorse analysis of oxygen consumption rate (OCR) in WT and Slc37a2−/− BMDMs treated with or without 20 ng/ml IL-4 for 24 h. (H,I) Seahorse analysis of extracellular acidification rates (ECAR) in WT and Slc37a2−/− BMDMs treated with or without 20 ng/ml IL-4 for 24 h. Data are representative of two independent experiments with three samples per group (mean ± SEM). *p < 0.05; **p < 0.01; unpaired, two-tailed Student's…

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  Figure 7. SLC37A2 deficiency impairs apoptotic cell-induced macrophage activation in vitro. (A) IL-10 transcript expression in WT and Slc37a2−/− BMDMs stimulated with apoptotic Jurkat T cells (ACs) (macrophages: ACs = 1:5) for 0-6 h. Jurkat T cells were incubated with 1 μM staurosporine for 4 h to induce apoptosis. (B) IL-10 protein secretion from WT and Slc37a2−/− BMDMs stimulated with ACs (macrophages: ACs = 1:5) for 4 h. (C) Efferocytosis index was measured by incubating WT and Slc37a2−/− BMDMs with cell tracker green CMFDA-labeled ACs (macrophages: ACs = 1:5) for 0-120 min. After the indicated time, macrophages were washed with cold PBS 3 times and cell dissociation buffer once before being analyzed by flow cytometry. Mean fluorescence intensity (MFI) of macrophages engulfing ACs was quantified. (D–F) IL-10 protein secretion from WT and Slc37a2−/− BMDMs pretreated with phagocytosis inhibitor cytochalasin D (Cyto D, 10 μM), fatty acid oxidation inhibitor etomoxir (100 μM), and hexokinase inhibitor 2-deoxy-D-glucose (2-DG; 10 mM) for 30 min, followed by co-culture with ACs (macrophages: ACs = 1:5) for 4 h. (G,H) Seahorse analysis of oxygen consumption rate (OCR) in WT and Slc37a2−/−…