Loss of SR-A and CD36 activity reduces atherosclerotic lesion complexity without abrogating foam cell formation in hyperlipidemic mice

Abstract

Objective: The scavenger receptors SR-A and CD36 have been implicated in macrophage foam cell formation during atherogenesis and in the regulation of inflammatory signaling pathways, including those leading to lesional macrophage apoptosis and plaque necrosis. To test the impact of deleting these receptors, we generated Apoe(-/-) mice lacking both SR-A and CD36 and fed them a Western diet for 12 weeks.

Methods and results: We analyzed atheroma in mice, assessing lesion size, foam cell formation, inflammatory gene expression, apoptosis, and necrotic core formation. Aortic root atherosclerosis in Apoe(-/-)Cd36(-/-)Msr1(-/-) mice, as assessed by morphometry, electron microscopy, and immunohistochemistry, showed no decrease in lesion area or in vivo foam cell formation when compared to Apoe(-/-) mice. However, Apoe(-/-)Cd36(-/-)Msr1(-/-) lesions showed reduced expression of inflammatory genes and morphological analysis revealed a approximately 30% decrease in macrophage apoptosis and a striking approximately 50% decrease in plaque necrosis in aortic root lesions of these mice.

Conclusions: Although targeted deletion of SR-A and CD36 does not abrogate macrophage foam cell formation or substantially reduce atherosclerotic lesion area in Apoe(-/-) mice, loss of these pathways does reduce progression to more advanced necrotic lesions. These data suggest that targeted inhibition of these pathways in vivo may reduce lesional inflammation and promote plaque stability.

Figure 1

Disruption of Cd36 and Msr1 does not alter the extent of aortic root atherosclerosis in hyperlipidemic Apoe^−/− mice. Aortic root atherosclerotic lesion area was quantified from 12 serial sections per mouse. Data are presented as (A) the mean lesion area of individual mice. N.S.; ANOVA. B, Representative photographs of oil red O–stained aortic root lesions. Magnification, × 100.

Figure 2

Immunohistochemical characterization of aortic root atherosclerosis. Aortic root lesions were stained for macrophage (MOMA-2) or smooth muscle cell content (α-smooth muscle actin) and quantified as a percentage of lesion area (±SEM) using 3 sections per mouse. N.S.; Student t test. Magnification, ×100 (upper panel, middle panel), × 250 (B, lower panel).

Figure 3

Morphometric analysis of lesion area measured in the aorta en face. A, Total aortic lesion area in male and female Apoe^−/− and Apoe^−/− Cd36^−/− Msr1^−/− mice and representative photographs of en face aortae. B, Regional analysis of lesion distribution in the aorta. Data are expressed as mean % lesion area ± SEM.

Figure 4

Analysis of lesional foam cell and cholesterol accumulation in the absence of CD36 and SR-A. A, Electron photomicrographs demonstrating intracellular lipid accumulation in the intima of aortic root lesions of Apoe^−/− and Apoe^−/− Cd36^−/− Msr1^−/− mice. Magnification, × 5000. B, Aortic free and esterified cholesterol content. **P<0.005; ANOVA.

Figure 5

Reduced apoptotic cell death and necrotic core formation in lesions of Apoe^−/− Cd36^−/− Msr1^−/− mice. A, Lesional apoptotic cells in aortic root lesions of female mice were detected by TUNEL staining. Representative fluorescent images (×100 magnification) and quantitative data are shown. Red, TUNEL positive nuclei; white, DAPI-stained nuclei. *P<0.05; Student t test. B, Quantitative analysis of necrotic core size relative to total lesion area; n=9 to 11 per group; **P<0.005, *P<0.05; Student t test.