Increased atherosclerosis in LDL receptor-null mice lacking ACAT1 in macrophages

Abstract

During atherogenesis, circulating macrophages migrate into the subendothelial space, internalize cholesterol-rich lipoproteins, and become foam cells by progressively accumulating cholesterol esters. The inhibition of macrophage acyl coenzyme A:cholesterol acyltransferase (ACAT), which catalyzes the formation of cholesterol esters, has been proposed as a strategy to reduce foam cell formation and to treat atherosclerosis. We show here, however, that hypercholesterolemic LDL receptor-deficient (LDLR(-/-)) mice reconstituted with ACAT1-deficient macrophages unexpectedly develop larger atherosclerotic lesions than control LDLR(-/-) mice. The ACAT1-deficient lesions have reduced macrophage immunostaining and more free cholesterol than control lesions. Our findings suggest that selective inhibition of ACAT1 in lesion macrophages in the setting of hyperlipidemia can lead to the accumulation of free cholesterol in the artery wall, and that this promotes, rather than inhibits, lesion development.

Figure 1

Reconstitution of recipient mice with donor marrow. (a) ACAT1 genotyping in recipients of fetal liver cell transplants. DNA was prepared from the bone marrow of LDLR^–/– recipient mice at least 4 weeks after transplantation. The genomic DNA was amplified in a PCR reaction. The absence of the wild-type band in recipients of ACAT1^–/– cells indicates complete repopulation of the marrow with cells of donor origin. (b and c) Immunocytochemical analyses of the proximal aorta. Cryosections (5 μm) were treated according to our previously published method (46) and stained for ACAT1 using a rabbit antibody to recombinant human ACAT1 (31).

Figure 2

Development of atherosclerosis in transplanted mice. (a) Aortic area covered by plaque, as percent of total. Aortae were dissected from the aortic valve to the iliac bifurcation, fixed in 4% paraformaldehyde, opened longitudinally, and pinned flat on a black surface. The aortae were stained with Sudan IV for computerized assessment of the extent of atherosclerosis. (b) A representative sample of the difference in atherosclerosis between groups. The analysis was done on seven consecutive pairs by two blinded operators, and no overlap was found in the distribution of the data from the two study groups. (c) Quantitation of cross sections of the proximal aorta in recipients of bone marrow. The heart and aorta were embedded in OCT and snap-frozen in liquid nitrogen. Cryosections (10 μm) were taken from the region of the proximal aorta starting from the end of the aortic sinus and for 300 μm distally, as described (46).

Figure 3

Immunocytochemical analyses of the proximal aorta in LDLR^–/– mice after transplantation with ACAT1^+/+ (a and c) or ACAT1^–/– cells (b and d). a and b show staining with MOMA 2 (a macrophage-specific marker). c and d show TUNEL staining. Sections were permeabilized in 0.1% Triton X-100, 0.1% Na citrate. After incubation with deoxynucleotidyl transferase and fluorescein-labeled nucleotide mixture, sections were treated with alkaline phosphatase–conjugated anti-fluorescein antibody. Fast Red TR/naphthol was used for color development. Controls for each experiment included serial sections treated with DNase (positive controls) or incubated with the labeling solution without terminal transferase (negative controls).

Figure 4

Movat’s pentachrome staining of aortic cross sections from representative LDLR^–/– mice after western-type diet. The left panel shows a representative section from a recipient of ACAT1^+/+ wild-type marrow, whereas the right panel shows a recipient of ACAT1^–/– marrow. The lesion area in the control animal is highly cellular, and amorphous material is detected in the deeper portion of the plaque. This phenomenon is exaggerated in the ACAT1^–/– recipient mouse, where lesions are thicker, less cellular, and more enriched in extracellular material than in controls. Nuclei are stained black, and smooth muscle cells are stained red. The blue stain indicates ground substance and mucins. The lack of yellow stain indicates the absence of significant collagen deposition. In each panel, the section is oriented with the lumen on top and the media at the bottom.