Loss of receptor-mediated lipid uptake via scavenger receptor A or CD36 pathways does not ameliorate atherosclerosis in hyperlipidemic mice

Abstract

Macrophage internalization of modified lipoproteins is thought to play a critical role in the initiation of atherogenesis. Two scavenger receptors, scavenger receptor A (SR-A) and CD36, have been centrally implicated in this lipid uptake process. Previous studies showed that these receptors mediated the majority of cholesterol ester accumulation in macrophages exposed to oxidized LDL and that mice with deletions of either receptor exhibited marked reductions in atherosclerosis. This work has contributed to an atherosclerosis paradigm: scavenger receptor-mediated oxidized lipoprotein uptake is required for foam cell formation and atherogenesis. In this study, Apoe-/- mice lacking SR-A or CD36, backcrossed into the C57BL/6 strain for 7 generations, were fed an atherogenic diet for 8 weeks. Hyperlipidemic Cd36-/-Apoe-/- and Msr1-/-Apoe-/- mice showed significant reductions in peritoneal macrophage lipid accumulation in vivo; however, in contrast with previous reports, this was associated with increased aortic sinus lesion areas. Characterization of aortic sinus lesions by electron microscopy and immunohistochemistry showed abundant macrophage foam cells, indicating that lipid uptake by intimal macrophages occurs in the absence of CD36 or SR-A. These data show that alternative lipid uptake mechanisms may contribute to macrophage cholesterol ester accumulation in vivo and suggest that the roles of SR-A and CD36 as proatherosclerotic mediators of modified LDL uptake in vivo need to be reassessed.

Figure 1

Distribution of cholesterol in plasma lipoproteins from Apoe^–/–, Cd36^–/–Apoe^–/–, and Msr1^–/–Apoe^–/– mice. Pooled plasma samples (n = 3 mice) from male Apoe^–/–, Cd36^–/–Apoe^–/–, and Msr1^–/–Apoe^–/– mice fed a Western diet for 8 weeks were separated by FPLC, and fractions were assayed for cholesterol. (A) FPLC profile demonstrating the concentration of cholesterol in each fraction. (B) Normalized FPLC profile demonstrating a similar distribution of cholesterol to lipoproteins in all 3 genotypes when the cholesterol in each fraction is expressed as a percentage of the total cholesterol recovered from the column. Closed circles, Apoe^–/– mice; open squares, Cd36^–/–Apoe^–/– mice; open triangles, Msr1^–/–Apoe^–/– mice.

Figure 2

Disruption of Cd36 or Msr1 increases aortic sinus atherosclerosis in Apoe^–/– mice. Aortic sinus atherosclerotic lesion area was quantified from serial sections (12 sections per mouse) cut through the aorta at the origins of the aortic valve leaflets that were stained with oil red O and hematoxylin. (A) Dot plot showing aortic sinus lesion area of individual mice, with mean lesion area shown in (B). *P ≤ 0.05; significantly different from Apoe^–/– control mice. (C) Representative photographs of the aortic sinus from male Apoe^–/–, Cd36^–/–Apoe^–/–, and Msr1^–/–Apoe^–/– mice stained with H&E. Magnification, ×60 (top row); ×200 (bottom row).

Figure 3

Morphometric analysis of lesion area in the aortic tree. Aortae were stained en face with Sudan IV, and lesion area was measured as a percentage of total aortic area. (A) Lesion area in male and female mice. *P ≤ 0.05; significantly different from Apoe^–/– control mice. (B) Representative photographs of the en face aortae from female Apoe^–/–, Cd36^–/–Apoe^–/–, and Msr1^–/–Apoe^–/– mice. The boxed region indicates the area of reduced lesions observed in the Cd36^–/–Apoe^–/– female mice.

Figure 4

Immunohistochemical characterization of aortic sinus atherosclerosis. Aortic sinus lesions from Apoe^–/–, Cd36^–/–Apoe^–/–, and Msr1^–/–Apoe^–/– mice were stained for neutral lipid (oil red O), SMC (α-actin) and macrophage content (MOMA-2). Staining was quantified using IP Lab Spectrum image analysis software and is presented graphically. (A) Representative photographs of aortic sinus lesions from male Apoe^–/–, Cd36^–/–Apoe^–/–, and Msr1^–/–Apoe^–/– mice stained with oil red O and anti-smooth muscle actin (α-SMA). Magnification, ×400. (B) Representative photographs of aortic sinus lesions from male and female Apoe^–/–, Cd36^–/–Apoe^–/–, and Msr1^–/–Apoe^–/– mice stained with anti–MOMA-2 antibody to detect macrophages. Fewer macrophages were detected in female Cd36^–/–Apoe^–/– mouse lesions than in Apoe^–/– mouse lesions. *P ≤ 0.05; ANOVA. Magnification, ×100.

Figure 5

Histological staining of aortic sinus lesions from female Apoe^–/–, Cd36^–/–Apoe^–/–, and Msr1^–/–Apoe^–/– mice with (A) Movat’s pentachrome stain (top panel) to identify cells (red), collagen (yellow), and glycosaminoglycans (blue), and picrosirius red stain (bottom panel) for type 3 collagen. Magnification, ×200 (top panel); ×100 (bottom panel). (B) Classification of aortic sinus lesions according to severity: (a) early lesions contained only foam cells; (b) moderate lesions contained additional smooth muscle cells in the intima and cap; and (c) advanced lesions contained large extracellular cholesterol clefts or lipid cores and exhibited disruption of the coherence of the media and thick layers of fibrous connective tissue.

Figure 6

Abundant foam cell formation occurs in the absence of SR-A and CD36. (A) Electron photomicrographs demonstrating cellular lipid accumulation in the intima of aortic sinus lesions from Apoe^–/–, Cd36^–/–Apoe^–/–, and Msr1^–/–Apoe^–/– mice. Magnification, ×3900. (B) Aggregated, but not native, LDL (AgLDL) induces abundant cholesterol ester accumulation in Apoe^–/– macrophages that is independent of Cd36 or Msr1. Cellular cholesterol ester was measured by GC-MS in triplicate samples and is expressed as the mean ± SD.