Lack of macrophage fatty-acid-binding protein aP2 protects mice deficient in apolipoprotein E against atherosclerosis

Abstract

The adipocyte fatty-acid-binding protein, aP2, has an important role in regulating systemic insulin resistance and lipid metabolism. Here we demonstrate that aP2 is also expressed in macrophages, has a significant role in their biological responses and contributes to the development of atherosclerosis. Apolipoprotein E (ApoE)-deficient mice also deficient for aP2 showed protection from atherosclerosis in the absence of significant differences in serum lipids or insulin sensitivity. aP2-deficient macrophages showed alterations in inflammatory cytokine production and a reduced ability to accumulate cholesterol esters when exposed to modified lipoproteins. Apoe-/- mice with Ap2+/+ adipocytes and Ap2-/- macrophages generated by bone-marrow transplantation showed a comparable reduction in atherosclerotic lesions to those with total aP2 deficiency, indicating an independent role for macrophage aP2 in atherogenesis. Through its distinct actions in adipocytes and macrophages, aP2 provides a link between features of the metabolic syndrome and could be a new therapeutic target for the prevention of atherosclerosis.

Fig. 1

Insulin sensitivity and atherosclerosis in Ap2^+/+Apoe^–/– and Ap2^–/–Apoe^–/– mice on chow diet. a and b, Insulin tolerance tests were performed in 15 male (a) and 7 female (b) 14-week-old Apoe^–/– (Δ) and Ap2^–/–Apoe^–/– (■), mice. Data are mean ± s.e.m. *, P < 0.05. c, Immunocytochemical detection of macrophages and aP2 expression in the proximal aorta of Ap2^+/+Apoe^–/– (left) and Ap2^–/–Apoe^–/– (right) mice. Macrophages are stained with MOMA-2 (upper), and aP2 is detected with polyclonal rabbit antiserum against mouse aP2 (lower). d and e, Quantification of atherosclerotic lesion area in the proximal (d) and en face aorta (e) in Ap2^+/+Apoe^–/– and Ap2^–/–Apoe^–/– mice on chow diet. Data are represented as the average mean lesion area for each group.

Fig. 2

Expression of fatty-acid binding proteins in macrophages. a and b, Human monocyte/macrophage cell lines THP-1 (a) and U-937 (b) were stimulated with PMA, and aP2 and mal1 expression was determined at the indicated times by northern-blot analysis. c, Primary mouse peritoneal macrophages were elicited with thioglycollate and cultured in the presence or absence of PMA or LPS. Positive controls are adipose tissue (A) and tongue (T). d, Human monocytes were isolated and differentiated into macrophages by PMA treatment and aP2 and mal1 protein determined by immunoblot analysis. Lanes: 1, positive control; 50 ng recombinant human aP2 (top) or mal1 (bottom); 2, human monocytes; 3, human macrophages; 4, THP-1 macrophages; 5, 50 ng mouse recombinant aP2 (top) or mal1 (bottom); 6 and 7, negative controls, human and mouse mal1 (top) and human and mouse aP2 (bottom), respectively. e, Transgene expression in the macrophages driven by the 5.4-kb aP2 promoter/enhancer. Peritoneal macrophages were obtained from 3 independent lines of transgenic mice expressing UCP1, agouti or TNF-α under the control of aP2 promoter/enhancer. Expression of the transgenes and control CD36 was determined by northern-blot analysis. RNA from mouse white adipose tissue (WAT) and brown adipose tissue (BAT) was used as controls. C, control non-transgenic; Tg, transgenic mice.

Fig. 3

Expression of inflammatory cytokines and cholesterol ester levels in Ap2^–/– macrophages. a, TNF-α, IL-1β and IL-6 mRNA levels were determined following PMA treatment. The bottom two blots show mal1 and aP2 mRNA expression. b and c, Accumulation of cholesterol esters (b) and cytokine secretion (c) in control (□) and Ap2^–/– (■) macrophages. Cholesterol ester levels were determined before and after treatment with Ac-LDL. Cytokine levels were determined by ELISA in the conditioned medium following treatment with Ac-LDL. The graph shows mean ± s.e. from 10 independent experiments. Immunoblot analysis of aP2 (L) and mal1 (R) protein expression is shown in b, lower. Lanes: 1, Ap2^+/+ without Ac-LDL; 2, Ap2^+/+ with Ac-LDL; 3, Ap2^–/– without Ac-LDL; 4, Ap2^–/– with Ac-LDL; 5&6, 20 and 40 ng of recombinant murine aP2 (left) or Mal1 (right) standards.

Fig. 4

Lipoprotein distribution, immunochemistry and atherosclerosis in Ap2^–/– BMT mice. a, Lipoprotein distribution in Apoe^–/– mice with transplanted marrow after 13 wk on standard chow diet. , female Ap2^–/–Apoe^–/–; , female Ap2^+/+Apoe^–/–; , male Ap2^–/–Apoe^–/–; , male Ap2^+/+Apoe^–/–. Data are represented as an average (n = 3) percent distribution of total cholesterol for each group. Fractions 14–17 contain VLDL; fractions 18–24 are IDL/LDL; and fractions 25–29 contain HDL. Fractions 30–40 are the non–lipoprotein-associated proteins. b, Immunocytochemical detection of macrophages and aP2 expression in the proximal aorta of Apoe^–/– mice transplanted with Ap2^+/+Apoe^–/– (left) or Ap2^–/–Apoe^–/– (right) marrow. Macrophages are stained as in Fig. 1c. c–f, Quantification of atherosclerotic lesion area in the proximal and the en face aorta, respectively for male (c and d) and female (e and f) Apoe^–/– mice 13 weeks after receiving Ap2^+/+Apoe^–/– or Ap2^–/–Apoe^–/– marrow. The atherosclerotic lesions were stained and quantified as noted in Fig. 1. Data are represented as the average mean lesion area for each group.