Leukocyte ABCA1 controls susceptibility to atherosclerosis and macrophage recruitment into tissues

Abstract

The ATP-binding cassette transporter 1 (ABCA1) has recently been identified as a key regulator of high-density lipoprotein (HDL) metabolism, which is defective in familial HDL-deficiency syndromes such as Tangier disease. ABCA1 functions as a facilitator of cellular cholesterol and phospholipid efflux, and its expression is induced during cholesterol uptake in macrophages. To assess the role of macrophage ABCA1 in atherosclerosis, we generated low-density lipoprotein (LDL) receptor knockout (LDLr(-/-)) mice that are selectively deficient in leukocyte ABCA1 (ABCA1(-/-)) by using bone marrow transfer (ABCA1(-/-) --> LDLr(-/-)). Here we demonstrate that ABCA1(-/-) --> LDLr(-/-) chimeras develop significantly larger and more advanced atherosclerotic lesions compared with chimeric LDLr(-/-) mice with functional ABCA1 in hematopoietic cells. Targeted disruption of leukocyte ABCA1 function did not affect plasma HDL cholesterol levels. The amount of macrophages in liver and spleen and peripheral blood leukocyte counts is increased in the ABCA1(-/-) --> LDLr(-/-) chimeras. Our results provide evidence that leukocyte ABCA1 plays a critical role in the protection against atherosclerosis, and we identify ABCA1 as a leukocyte factor that controls the recruitment of inflammatory cells.

Figure 1

Design of bone marrow transplantation experiment and verification of success of bone marrow transplantation. (a) One day before transplantation, mice were exposed to total body irradiation and subsequently engrafted with 0.5 × 10⁷ bone marrow cells from ABCA1-deficient mice or wild-type littermates. During the first 8 weeks, mice were maintained on a chow diet containing 5.7% fat and no cholesterol. At 8 weeks after bone marrow transplantation, the diet was switched to a high-cholesterol Western-type diet (WTD) containing 15% fat and 0.25% cholesterol. Serum lipid levels were carefully monitored throughout the experiment. After 12 weeks feeding WTD, the chimeras were killed for lesion assessment in the aortic root. (b) Verification of successful reconstitution with donor hematopoietic cells by PCR at 20 weeks posttransplant using genomic DNA isolated from bone marrow. Amplification of the wild-type ABCA1 gene resulted in a 1.3-kb PCR band, whereas the ABCA1 null mutant gene yielded a 1.0-kb band. (c) HDL mediated cellular cholesterol efflux using ³H-cholesterol-labeled peritoneal macrophages isolated from mice transplanted with either ABCA1^+/+ (○, n = 3) or ABCA1^−/− (●, n = 3) bone marrow at 20 weeks posttransplant.

Figure 2

Effect of leukocyte ABCA1 deficiency on the plasma cholesterol and triglycerides distribution. Blood samples were drawn after an overnight fast at 8 weeks posttransplant, while feeding regular chow diet (a and b) and at 16 weeks after bone marrow transplantation after 8 weeks of feeding a high-cholesterol Western-type diet (c and d). Sera from individual mice were loaded onto a Superose 6 column, and fractions were collected. Fractions 3–7 represent VLDL; fractions 8–14, LDL; and fractions 15–19, HDL, respectively. The distribution of cholesterol (a and c) and triglycerides (b and d) over the different lipoproteins in ABCA1^+/+ → LDLr^−/− (○) and ABCA1^+/+ → LDLr^−/− (●) chimeras is shown. Values represent the mean of 10–11 mice. SEM are shown only for fractions containing the top of the VLDL, LDL, and HDL peaks.

Figure 3

Leukocyte ABCA1 controls susceptibility to atherosclerosis. Formation of atherosclerotic lesions was determined at 20 weeks posttransplant at the aortic root of ABCA1^+/+ → LDLr^−/− and ABCA1^−/− → LDLr^−/− chimeras that were fed a high-cholesterol Western-type diet for 12 weeks. (a and b) The mean lesion area was calculated from oil red O-stained cross sections of the aortic root at the level of the tricuspid valves. Values represent the mean of 10–11 mice. Original magnification, ×50. (c and d) Immunohistochemical quantification of lesion macrophages by using the MOMA-2 antibody that identifies macrophages as well as freshly infiltrated monocytes. The macrophage lesion area is indicated as % of the total lesion area. Original magnification, ×200. Statistically significant difference of ***, P < 0.0001, and **, P < 0.005 compared with ABCA1^+/+ → LDLr^−/− mice.

Figure 4

Increased macrophage content in spleen and liver of mice lacking functional leukocyte ABCA1. At 20 weeks after bone marrow transplantation, the macrophage contents of spleen and liver were determined by immunohistochemistry by using F4/80, a specific marker for mature tissue macrophages. Values represent the mean of 10–11 mice. Original magnification, ×50. Statistically significant difference of *, P < 0.05, compared with ABCA1^+/+ → LDLr^−/− mice.

Figure 5

Effect of leukocyte ABCA1 deficiency on peripheral leukocyte and bone marrow progenitor counts. (a) Peritoneal leukocyte numbers of ABCA1^+/+ → LDLr^−/− mice (hatched bars, n = 8) and ABCA1^−/− → LDLr^−/− mice (closed bars, n = 7) bone marrow were determined by flushing the peritoneal cavity with sterilized PBS and subsequent cell counting. (b and c) Counts of peripheral leukocytes and bone marrow progenitors were assessed on May–Grünwald–Giemsa-stained smears and by flow cytometry of blood and bone marrow from ABCA1^+/+ → LDLr^−/− (hatched bars, n = 16) and ABCA1^−/− → LDLr^−/− (closed bars, n = 14) mice. Statistically significant difference of **, P < 0.01, ***, P < 0.001 compared with ABCA1^+/+ → LDLr^−/− mice.