Macrophage Mal1 deficiency suppresses atherosclerosis in low-density lipoprotein receptor-null mice by activating peroxisome proliferator-activated receptor-γ-regulated genes

Abstract

Objective: The adipocyte/macrophage fatty acid-binding proteins aP2 (FABP4) and Mal1 (FABP5) are intracellular lipid chaperones that modulate systemic glucose metabolism, insulin sensitivity, and atherosclerosis. Combined deficiency of aP2 and Mal1 has been shown to reduce the development of atherosclerosis, but the independent role of macrophage Mal1 expression in atherogenesis remains unclear.

Methods and results: We transplanted wild-type (WT), Mal1(-/-), or aP2(-/-) bone marrow into low-density lipoprotein receptor-null (LDLR(-/-)) mice and fed them a Western diet for 8 weeks. Mal1(-/-)→LDLR(-/-) mice had significantly reduced (36%) atherosclerosis in the proximal aorta compared with control WT→LDLR(-/-) mice. Interestingly, peritoneal macrophages isolated from Mal1-deficient mice displayed increased peroxisome proliferator-activated receptor-γ (PPARγ) activity and upregulation of a PPARγ-related cholesterol trafficking gene, CD36. Mal1(-/-) macrophages showed suppression of inflammatory genes, such as COX2 and interleukin 6. Mal1(-/-)→LDLR(-/-) mice had significantly decreased macrophage numbers in the aortic atherosclerotic lesions compared with WT→LDLR(-/-) mice, suggesting that monocyte recruitment may be impaired. Indeed, blood monocytes isolated from Mal1(-/-)→LDLR(-/-) mice on a high-fat diet had decreased CC chemokine receptor 2 gene and protein expression levels compared with WT monocytes.

Conclusion: Taken together, our results demonstrate that Mal1 plays a proatherogenic role by suppressing PPARγ activity, which increases expression of CC chemokine receptor 2 by monocytes, promoting their recruitment to atherosclerotic lesions.

Figure 1

Changes in body weight (A), serum lipoprotein profiles (B) and atherosclerotic lesion area in the proximal aorta (C) of LDLR^-/- mice reconstituted with wild type (●), Mal1^-/- (○), or aP2^-/- ( ) bone marrow cells. Data from FPLC analysis (B) are represented as the average (n=3 per group) of the percent of total cholesterol for each fraction. Fractions 14-17 contain VLDL; fractions 18-24 contain IDL/LDL; and fractions 25-30 contain HDL. Note the differences (* p<0.05) between mice reconstituted with WT marrow vs. mice transplanted with Mal1^-/- or aP2^-/- marrow determined by One Way ANOVA.

Figure 2

Expression of PPARγ (A) and CD36 (B) genes, PPARγ (C,D and CD36 (E,F) protein expression levels in WT and Mal1^-/- macrophages treated with the PPARγ agonist, ciglitazone (A-E) alone or together with the specific PPARγ antagonist, GW9662 (A). A, B. Thioglycollate-elicited WT (■) and Mal1^-/- (□) macrophages were treated with lipid-free DMEM media containing the PPARγ agonist, ciglitazone (Cigl., 15μM), alone or together with the specific PPARγ antagonist, GW9662 (GW, 10μM) at 37°C for 24 hours. Then RNA was extracted from the macrophages and analyzed by real-time PCR. Graphs represent data (Mean ± SEM) of analysis the same number (n=3) of mice per group (*p<0.05 between untreated and treated with the ciglitazone macrophages of the same group) C-F. WT and Mal1^-/- macrophages were treated with ciglitazone (15μM), alone or together with GW9662 (30μM) for 24 hours. Extracted proteins (50μg/well) were resolved and analyzed by Western Blot. The data of PPARγ/β-actin and CD36/β-actin ratios are presented as average (Mean+SEM) assay of three separate experiments. *Differences between the groups with the same dose (D; p<0.05) or between control cells and treated with ciiglitasone macrophages (.F; p<0.001).

Figure 3

Visualization (A) and quantified uptake of human DiI-OxLDL (B) or DiI-AcLDL (C) by macrophages from WT (■) and Mal1^-/- (□) mice. A. Peritoneal macrophages were incubated with human DiI-oxLDL (10μg/mL) or DiI-acLDL (10μg/mL) for 2 hours and examined under the microscope (Olympus BX-40) (Magnification x20; insert x60). B, C. Peritoneal macrophages were incubated with indicated doses of human DiI-OxLDL or DiI-AcLDL for 1 hour and analyzed by FACS.

Figure 4

Treatment with PA-BSA (A,B) or LPS (C-F) significantly increases activity of Akt and slightly suppresses NF-κB-related protein and genes in Mal1 deficient macrophages. A, B. Macrophages were incubated with media alone or with palmitic acid complexed with PBS (PA-BSA, 0.5mM) for 3 and 6 hours. Extracted proteins were resolved (100μg/well) and analyzed by Western Blot using antibodies to Akt, p-Akt or β-actin (A); or extracted proteins (20μg/well) were analyzed by Western Blot using antibodies to p-65, c-Rel, IκBα or β-actin (B). C-F. Peritoneal macrophages from WT (●) and Mal1^-/-(○) mice were treated with LPS (50ng/ml) for the indicated time periods and the expression of Akt-1 (C), c-Jun(D), COX-2 (E) or IL6 (F) genes were analyzed by real-time PCR

Figure 5

Mal 1 deficiency decreases the number of nuclei in MOMA-2+ area of atherosclerotic lesions (A-G), and suppresses CCR2 gene (H) and protein (I,K) expression levels in blood monocytes. A-F. Serial sections from the proximal aorta of LDLR^-/- mice reconstituted with WT (A-C; ■) and Mal1^-/-(D-F; □) marrow and fed the Western diet for 12 weeks. Sections were stained with antibodies to mouse macrophage, MOMA-2 (A,D) and nuclear stain, DAPI (B,E). After merging of the images, the number of nuclei was analyzed in MOMA-2-positive area. Note, number of nuclei per standard lesion area (G; Mean ± SEM; *p<0.05 between mice with WT and Mal1^-/- marrow). A-C. Blood monocytes were isolated from WT→LDLR^-/-(■) and Mal1^-/-→LDLR^-/-(□) mice on the Western diet. CCR2 gene (H) and protein (I) expression levels were analyzed by real-time PCR and FACS. Graphs represent data (Mean ± SEM) analysis of the same number (n=3) mice per group (*p<0.05 between WT and Mal1^-/- cells).