High density lipoprotein deficiency and foam cell accumulation in mice with targeted disruption of ATP-binding cassette transporter-1

Abstract

Recently, the human ATP-binding cassette transporter-1 (ABC1) gene has been demonstrated to be mutated in patients with Tangier disease. To investigate the role of the ABC1 protein in an experimental in vivo model, we used gene targeting in DBA-1J embryonic stem cells to produce an ABC1-deficient mouse. Expression of the murine Abc1 gene was ablated by using a nonisogenic targeting construct that deletes six exons coding for the first nucleotide-binding fold. Lipid profiles from Abc1 knockout (-/-) mice revealed an approximately 70% reduction in cholesterol, markedly reduced plasma phospholipids, and an almost complete lack of high density lipoproteins (HDL) when compared with wild-type littermates (+/+). Fractionation of lipoproteins by FPLC demonstrated dramatic alterations in HDL cholesterol (HDL-C), including the near absence of apolipoprotein AI. Low density lipoprotein (LDL) cholesterol (LDL-C) and apolipoprotein B were also significantly reduced in +/- and -/- compared with their littermate controls. The inactivation of the Abc1 gene led to an increase in the absorption of cholesterol in mice fed a chow or a high-fat and -cholesterol diet. Histopathologic examination of Abc1-/- mice at ages 7, 12, and 18 mo demonstrated a striking accumulation of lipid-laden macrophages and type II pneumocytes in the lungs. Taken together, these findings demonstrate that Abc1-/- mice display pathophysiologic hallmarks similar to human Tangier disease and highlight the capacity of ABC1 transporters to participate in the regulation of dietary cholesterol absorption.

Figure 1

Gene targeting of the murine Abc1 gene. (A) Strategy to disrupt Abc1 expression. The targeting vector construct was designed to replace exons 17–22 that encode the first nucleotide-binding fold. The resulting targeted allele has pMC-neo replacing the six exons deleting an endogenous BamHI (B) site resulting in a restriction fragment length polymorphism, from 6.8 kb to 4.4 kb, with a downstream BamHI (B′) site by using an external 5′ probe. (B) Southern analysis of offspring from Abc1+/− matings. All three genotypes are observed by using the genomic probe strategy described above. Lanes A, C, and E, samples from +/+ mice; lanes B and D, samples from +/− mice; lanes F and G, samples from −/− mice. (C) RT-PCR results from Abc1+/+ and −/− mice. Lanes B and C are DNA and no DNA controls, respectively. Lane D is RT-PCR of hepatic RNA for Abc1 from wild-type mouse. Lane D is RT-PCR from Abc1−/− mice. Lane A is a 100-bp molecular weight marker.

Figure 2

Lipid analysis of +/+, +/−, and −/− male (solid columns), and female (shadowed columns) mice fed a chow diet. Bar graph represents the means ± standard deviations from the number of animals in parentheses of each group. *, **, and ***: P < 0.05, P < 0.005, and P < 0.0001 compared with wild-type mice, respectively.

Figure 3

Plasma lipoprotein cholesterol, apoB and apoAI distribution in mice of different genotypes fed a chow diet, determined by FPLC. Two hundred microliters of pooled mouse plasma from the different genotypes was fractionated by FPLC as described under Methods. Cholesterol, apoAI and apoB content was determined and plotted as a function of FPLC fractions. The positions at which known lipoproteins eluted from the column are indicated.

Figure 4

Plasma lipoprotein cholesterol distribution in Abc1−/− and control mice fed a Western-type diet ad libitum for 4 wk. Two hundred microliters of pooled plasma from six mice was analyzed as described in Methods.

Figure 5

Pulmonary lesions in Abc1−/− mice. A contains a section of normal lung from a 12-mo-old +/+ mouse with a normal terminal bronchiole and alveoli lined predominantly with flattened type I pneumocytes (hematoxylin/eosin stain, ×400). B demonstrates an early lesion from a 12-mo-old −/−mouse including numerous foamy cells (arrows) and cholesterol clefts (arrowheads). Alveolar septae are focally expanded by mild type II pneumocyte hypertrophy, macrophages, and aggregates of lymphocytes and plasma cells. An Oil red O-stained section of lung from a 7-mo-old −/− mouse demonstrates lipid accumulation within alveolar cells (arrow, C). By electron microscopy, these cells are identified primarily as type II pneumocytes (D, arrows) and intraalveolar macrophages (arrowhead, ×3,500).