Increased ABCA1 activity protects against atherosclerosis

Abstract

The ABC transporter ABCA1 plays a key role in the first steps of the reverse cholesterol transport pathway by mediating lipid efflux from macrophages. Previously, it was demonstrated that human ABCA1 overexpression in vivo in transgenic mice results in a mild elevation of plasma HDL levels and increased efflux of cholesterol from macrophages. In this study, we determined the effect of overexpression of ABCA1 on atherosclerosis development. Human ABCA1 transgenic mice (BAC(+)) were crossed with ApoE(-/-) mice, a strain that spontaneously develop atherosclerotic lesions. BAC(+)ApoE(-/-) mice developed dramatically smaller, less-complex lesions as compared with their ApoE(-/-) counterparts. In addition, there was increased efflux of cholesterol from macrophages isolated from the BAC(+)ApoE(-/-) mice. Although the increase in plasma HDL cholesterol levels was small, HDL particles from BAC(+)ApoE(-/-) mice were significantly better acceptors of cholesterol. Lipid analysis of HDL particles from BAC(+)ApoE(-/-) mice revealed an increase in phospholipid levels, which was correlated significantly with their ability to enhance cholesterol efflux.

Figure 1

Expression of ABCA1 in BAC⁺ApoE^–/– mice. Increased ABCA1 protein was detected in the liver, kidney, brain, aorta, gonad, large intestine (lg. int.), and in peritoneal macrophage cells isolated from the BAC⁺ApoE^–/– mice (a). An anti-ABCA1 mAb raised against the second nucleotide-binding domain was used (top panel) to detect ABCA1. To ensure equal protein loading, the Western blots were probed with anti-GAPDH Ab (lower panel). (b) Human-specific ABCA1 transcript was detected in all tissues tested using RT-PCR. For each tissue, lane 1 is human specific, lane 2 is mouse specific, and lane 3 is an 18S control. The last panel is from a control mouse and contains no human transcript.

Figure 2

Analysis of efflux levels in primary macrophages from BAC⁺ApoE^–/– mice. Primary peritoneal macrophage cultures were established from the BAC⁺ApoE^–/– and control mice as described (n = 4) (17). A 38.4% increase (P < 0.001) in efflux in the BAC⁺ApoE^–/– macrophages is evident when compared with the ApoE^–/– littermates. Upon stimulation of the cultures with 9(cis)-retinoic acid and 22(R)-hydroxy cholesterol, there was a 23.7% increase (P < 0.001) in cholesterol efflux between BAC⁺ApoE^–/– versus control littermates. There was a further 46.8% increase (P < 0.001) in efflux in the BAC⁺ApoE^–/– macrophages after stimulation with 9(cis)-retinoic acid and 22(R)-hydroxy cholesterol when compared with unstimulated cells.

Figure 3

Assessment of lesions in BAC⁺ApoE^–/– mice. (a) Lesions in aortic roots of BAC⁺ApoE^–/– mice (left) and ApoE^–/– (right) mice (×5) were stained with ORO to detect the accumulation of lipids. BAC⁺ApoE^–/– mice showed 2.7 times decrease in lesion size compared with the ApoE^–/– mice (b). ORO-stained sections were used for the assessment of lesions (c and d) (×24; inset ×4, transversely sectioned aortic root). Lesions were markedly smaller and less well-developed in BAC⁺ApoE^–/– mice as compared with ApoE^–/– mice. Markedly less ORO positivity was seen in foam cells from BAC⁺ApoE^–/– mice as compared with ApoE^–/– mice. A color overlay based on hue, saturation, and intensity further shows a dramatic decrease in ORO positivity (green color) in lesions from BAC⁺ApoE^–/– mice (e), compared with ApoE^–/– mice (f) (×24). For the assessment of lesion complexity in BAC⁺ApoE^–/– mice, lesions in aortic roots of BAC⁺ApoE^–/– mice (g) and ApoE^–/– mice (h) were stained with Movat’s pentachrome to visualize extracellular matrix deposition within the lesions (×24; inset ×4, transversely sectioned aortic root). Small amounts of matrix rich in proteoglycans, especially versican (reflected here as sea-green color corresponding to glycosaminoglycan content), is observed in lesions from BAC⁺ApoE^–/– mice, particularly near the noduli Arantii. Furthermore, these early lesions have only a very faint intimal positivity. However, in lesions of the ApoE^–/– mice, glycosaminoglycan is observed in deep intimal areas of the lesions underlying the foam cell–rich regions. Also, extracellular matrix deposition is observed interwoven with extracellular cholesterol clefts and near the noduli Arantii. A color overlay reflecting extracellular matrix based on hue, saturation, and intensity shows differential localization of these components (green color) in BAC⁺ApoE^–/– (i) and ApoE^–/– mice (j) (×24). An aortic valve cusp is denoted in g (arrow).

Figure 4

Correlation between the phospholipid content of HDL and its ability to elicit efflux. HDL (density between 1.063 and 1.21 g/ml) isolated from BAC⁺ApoE^–/– and control ApoE^–/– mice was used for the quantification of phospholipid content and its ability to elicit cholesterol efflux. Phospholipid was quantified using a previously characterized method (21). Efflux was measured using Fu5AH cells, using protocols previously described (25). Linear regression analysis was performed on the percentage of phospholipid content of the HDL and the percentage of efflux elicited. The phospholipid content of HDL accounts for 68% of its ability to induce cholesterol efflux. PL, phospholipid.