Fetal liver cells or bone marrow from mice lacking ACAT1 (13
) were transplanted into LDLR–/–
), to generate mice with one of three ACAT1 genotypes: +/+, +/–, and –/–. Four weeks after transplantation, the mice were fed a western-type diet (containing 21% fat, 0.15% cholesterol, and no cholic acid) for 12 additional weeks. PCR analysis of bone marrow DNA extracted from recipient mice 8 weeks after transplantation showed a complete conversion of the ACAT genotype to the donor’s type, indicating that the marrow population had been reconstituted (Figure a). Immunohistochemical studies of the artery wall confirmed the absence of ACAT1 immunoreactivity in mice transplanted with knockout cells compared with recipients of wild-type cells (Figure , b and c). As shown in Table , no differences were observed in plasma cholesterol and triglyceride levels between study groups, although a trend toward lower cholesterol levels was detected among recipients of ACAT1–/–
= 0.073). Similarly, no differences were observed between groups in the distribution of cholesterol and triglycerides among the lipoproteins, including HDL (not shown).
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 (more ...)
Plasma lipid levels in transplanted mice
Unexpectedly, the atherosclerotic lesion area in cross sections of the aortic root (μm2/section/mouse ± SEM) was larger in recipients of ACAT1–/– marrow than in recipients of ACAT1+/+ marrow (2.3 × 105 ± 2.9 × 104 and 1.4 × 105 ± 1.4 × 104, respectively; P = 0.007). Lesion area in recipients of ACAT1+/– marrow was not different compared with recipients of control marrow, suggesting a lack of gene-dosage effect and no measurable consequences of a 50% reduction in cholesterol esterification in macrophages. Also, the area covered by plaque in pinned-out aortae (Figure a) was increased between two- and threefold in recipients of ACAT1–/– macrophages. Figure b shows a representative example of the increased aortic atherosclerosis in LDLR–/– mice that received ACAT1–/– marrow. The free cholesterol content in whole aortae was increased in ACAT1–/– marrow recipients (17.6 ± 1.6 vs. 11.5 ± 1.8 μg/mg protein in ACAT+/+ recipients, P = 0.013), consistent with the lack of macrophage ACAT activity resulting in the accumulation of unesterified cholesterol. Importantly neutral lipids were detectable in the sinus and aortic lesions of recipients of ACAT1-null marrow, indicating the presence of cholesterol esters in the plaque. As described below, this effect can be due to the migration of ACAT1+/+ smooth muscle cells from the media.
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 (more ...)
To confirm the atherosclerosis findings, a second experiment was performed in which male LDLR–/– mice were transplanted with bone marrow from LDLR–/– mice that were either wild-type or homozygous negative at the ACAT1 gene locus. Four weeks after transplantation, the mice were fed a butterfat diet (16% fat, 1.25% cholesterol, 0.5% cholic acid) for 10 weeks. As in the first experiment, there were no differences in plasma cholesterol levels among the study groups (Table ), and significantly larger lesions were found in recipients of ACAT1–/– marrow than in recipients of ACAT1+/+ marrow (3.5 × 105 ± 7.4 × 104 and 1.6 × 105 ± 2.7 × 104 P < 0.03; Figure c).
Immunohistochemical staining was performed on a subset of mice to analyze the effects of ACAT1 deficiency on lesion composition. Immunostaining for macrophages showed less staining in ACAT1–/– recipient mice (27.2% of the total lipid-stained area compared with 70.5% in recipients of ACAT1+/+ marrow, a 61% difference; P < 0.001; n = 10 for ACAT1+/+ recipients, and n = 9 for ACAT1–/– recipients). The sections shown in Figure , a and b, are not representative of the difference in lesion size between groups, but show the difference in macrophage staining distribution and the loss of macrophages in the deeper portions of the plaque in mice recipient of ACAT1–/– marrow (Figure a) compared with controls (Figure b).
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). (more ...)
Because in vitro studies have demonstrated that cholesterol toxicity associated with ACAT inhibition can cause cell death in macrophages (36
), we examined the extent of apoptosis and necrosis in arterial lesions by TUNEL staining of aortic cross sections from study animals. The proportion of TUNEL-stained cells was threefold higher in ACAT1–/–
recipient mice than in controls (18% vs. 52%, P
= 0.037), indicating that necrotic and apoptotic cell death contributed to the diminished numbers of macrophages in lesions (Figure , c and d). The TUNEL-stained cells did not stain with antisera that recognizes either macrophage or smooth muscle cell antigens (not shown). However, we presume that the TUNEL-stained cells were macrophages because these cells were markedly reduced in the ACAT1–/–
recipients and ACAT1 deficiency causes macrophage death in vitro (36
In recipients of ACAT1–/– marrow, the areas of intense staining were concentrated in the deeper portions of the plaque, whereas viable macrophages were observed closer to the endothelial surface. This pattern suggests that macrophages that have recently migrated from the lumen are healthy and remain active until, with deeper migration and increasing residence time in the lesion, the cholesterol overload induces severe accumulation of unesterified cholesterol and promotes cell death. To determine whether the macrophage-poor lesions in recipients of ACAT–/– marrow had other unique morphologic and compositional features, additional evaluations of the remaining sections were done using the Movat’s pentachrome stain and an anti-myosin antibody. Staining for smooth muscle cells was higher in recipients of ACAT1–/– marrow than in recipients of control marrow as absolute area (185,144 vs. 88,038 μm2, respectively; n = 5 per group; P = 0.05), but it was no different than in controls when expressed as percentage of total lesion area. However, the lesion area occupied by acellular material was 37.6% in the ACAT1–/– recipient mice (n = 6) and 1.8% in the ACAT1+/+ recipient controls (P < 0.001). Figure shows a representative set of Movat’s pentachrome stains, which demonstrate a large expansion of the core ground substance containing glycosaminoglycans and proteoglycans (blue stain) in ACAT1–/– recipients (Figure , right) relative to controls (Figure , left). Also, the picture shows the reduced cellularity of the lesion (nuclei are stained black), as well as the lack of elastic fibers (black) or collagen (yellow), and the relative increase in smooth muscle (red) in ACAT1–/– mice. Because proteoglycans are the secretory product of smooth muscle cells, the excess blue staining in aortic cross sections of ACAT–/– recipients is indicative of the preponderance of smooth muscle cells relative to macrophages in these lesions.
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 (more ...)