Macrophage-specific transgenic expression of cholesteryl ester hydrolase significantly reduces atherosclerosis and lesion necrosis in Ldlr mice

Abstract

Accumulation of cholesteryl esters (CEs) in macrophage foam cells, central to atherosclerotic plaque formation, occurs as a result of imbalance between the cholesterol influx and efflux pathways. While the uptake, or influx, of modified lipoproteins is largely unregulated, extracellular acceptor-mediated free cholesterol (FC) efflux is rate limited by the intracellular hydrolysis of CE. We previously identified and cloned a neutral CE hydrolase (CEH) from human macrophages and demonstrated its role in cellular CE mobilization. In the present study, we examined the hypothesis that macrophage-specific overexpression of CEH in atherosclerosis-susceptible Ldlr(-/-) mice will result in reduction of diet-induced atherosclerosis. Transgenic mice overexpressing this CEH specifically in the macrophages (driven by scavenger receptor promoter/enhancer) were developed and crossed into the Ldlr(-/-) background (Ldlr(-/-)CEHTg mice). Macrophage-specific overexpression of CEH led to a significant reduction in the lesion area and cholesterol content of high-fat, high-cholesterol diet-induced atherosclerotic lesions. The lesions from Ldlr(-/-)CEHTg mice did not have increased FC, were less necrotic, and contained significantly higher numbers of viable macrophage foam cells. Higher CEH-mediated FC efflux resulted in enhanced flux of FC from macrophages to gall bladder bile and feces in vivo. These studies demonstrate that by enhancing cholesterol efflux and reverse cholesterol transport, macrophage-specific overexpression of CEH is antiatherogenic.

Figure 1. Macrophage-specific expression of the human CEH transgene.

Total RNA was isolated from liver, lung, spleen, kidney, heart, and thioglycollate-elicited peritoneal macrophages. CEH mRNA expression was determined by real time PCR as described in Methods. Data (mean ± SD, n = 3) from founder line in C57BL/6 background are expressed as CEH copy no./ng total RNA. Similar results were obtained from the 2 founder lines in Balb-c/C57BL/6 hybrid background that were also tested.

Figure 2. Distribution of cholesterol among plasma lipoprotein fractions.

Plasma was obtained from non-fasted Ldlr^–/– and Ldlr^–/–CEHTg mice after 16 weeks of feeding. An aliquot of the plasma from each animal was applied to Superose 6 FPLC column (flow rate, 0.4 ml/min) and cholesterol content of individual lipoproteins determined by an online assay as described in Methods. A representative FPLC profile is presented showing a continuous online measurement of cholesterol (expressed as millivolts). n = 9 per genotype.

Figure 3. Ldlr^–/–CEHTg mice have reduced atherosclerosis compared with Ldlr^–/– controls.

Ldlr^–/– and Ldlr^–/–CEHTg mice were fed a Western diet for 16 weeks. Aorta (from aortic sinus to the iliac bifurcation) was isolated, opened and pinned to expose the lesions, and imaged on a black background. (A) Representative images of the aorta. The white horizontal line marks the place where the aortic arch was separated from the thoracic and abdominal aortae. (B) A magnified image of the aortic arch. (C) The quantification of the surface area occupied by the lesions in the aortic arch and the thoracic and abdominal aortae. Data (% area occupied by the lesions) are expressed as mean ± SEM for the indicated number of animals in each group. Original magnification, ×3. *P = 0.0008; **P = 0.004.

Figure 4. Aortic lesions in Ldlr^–/–CEHTg mice have significantly lower EC than lesions in Ldlr^–/– mice fed a Western diet for 16 weeks.

Aortae (from aortic sinus to the iliac bifurcation) were isolated, opened, and pinned to expose the lesions. After fixing in buffered formaldehyde for 24 hours, the aortic arch was cut along the line as shown in Figure 3 and lipids extracted from the resulting 2 pieces corresponding to the arch and the thoracic and abdominal aortae. FC and EC concentration (μg/mg protein) was determined as described in Methods. The concentration of total cholesterol is indicated at the top of the bars. Data are expressed as mean ± SEM (n = 6). *P < 0.004.

Figure 5. Lesions in Ldlr^–/–CEHTg mice are more cellular.

Hearts from Western diet–fed mice were fixed, paraffin embedded, and sectioned as described in Methods. (A) Representative aortic sinus lesion on 1 of the leaflets, stained with Masson’s Trichrome reagent. Original magnification, ×200. The lumen (L) of the aortic sinus is so marked for orientation. The red staining of the cytoplasm illustrates the highly cellular nature of the lesion in Ldlr^–/–CEHTg mice. Limited blue staining in the core of the plaque indicates reduced collagen levels in the lesions of Ldlr^–/– compared with Ldlr^–/–CEHTg mice. (B) Magnification of the boxed areas in A. Serial sections were stained with H&E. Arrowheads mark the nuclei, arrows point to the cholesterol clefts, and asterisks indicate foam cells. (C) A second serial section was immunostained with antibody to macrophage marker MOMA-2. Intact foam cells (asterisks) containing intracellular lipid droplets and stained brown with MOMA-2 antibody are visible throughout the lesion from Ldlr^–/–CEHTg but not in lesion from Ldlr^–/– mice. Original magnification, ×600 (B and C).

Figure 6. Decreased necrosis/apoptosis of macrophages in lesions from Ldlr^–/–CEHTg compared with Ldlr^–/– mice.

H&E-stained sections were used to determine the percentage of acellular area/necrotic area of the lesions (A), and the number of macrophages in the lesions (B) as well as the percentage of TUNEL-positive nuclei/total nuclei (C) were determined as described in Methods. Data are expressed as mean ± SEM for 5 mice per genotype, and 3 lesions were analyzed per animal. P = 0.00002 (A); P = 0.001 (B); P = 0.000008 (C).

Figure 7. Peritoneal macrophages from CEH-transgenic mice have lower EC content.

Thioglycollate-elicited peritoneal macrophages from chow-fed mice were isolated and loaded with AcLDL. After a 24-hour equilibration period, 1 set was incubated in serum-free medium (no extracellular acceptor) or in medium containing 10% FBS as the extracellular acceptor. Lipids were extracted after 24 hours and cellular FC and EC levels determined and normalized to cellular protein. Data are expressed as mean ± SD; n = 3. The percent decrease in FC and EC in the presence of extracellular acceptor is significantly higher in macrophages from Ldlr^–/–CEHTg mice (*P = 0.05 and **P = 0.048) compared with the corresponding decrease in macrophages from Ldlr^–/– mice.

Figure 8. Similar expression of genes involved in cholesterol efflux in peritoneal macrophages isolated from Ldlr^–/– and Ldlr^–/–CEHTg mice.

Thioglycollate-elicited peritoneal macrophages from chow-fed mice were isolated and plated in 60-mm culture plates. One set of cells was loaded with AcLDL for 48 hours. Total RNA was isolated from nonloaded as well AcLDL-loaded cells and expression of the indicated genes determined by real time RT-PCR. The level of expression in AcLDL-loaded cells was normalized to that in the corresponding nonloaded cells. Data are expressed as mean ± SD; n = 3. ND, not detectable.

Figure 9. Significantly higher movement of cholesterol from CEHTg macrophages to bile acids.

In vivo movement of [³H]-cholesterol from macrophages to gall bladder bile and feces was monitored as described in Methods. Total radioactivity (dpm) of [³H]-cholesterol was determined and data (mean ± SD; n = 3) are presented as percentage of the total dpm injected. *P = 0.0007; **P = 0.02.