Functional loss of ABCA1 in mice causes severe placental malformation, aberrant lipid distribution, and kidney glomerulonephritis as well as high-density lipoprotein cholesterol deficiency

Abstract

Tangier disease (TD) and familial HDL deficiency (FHA) have recently been linked to mutations in the human ATP-binding cassette transporter 1 (hABCA1), a member of the ABC superfamily. Both diseases are characterized by the lowering or lack of high-density lipoprotein cholesterol (HDL-C) and low serum cholesterol. The murine ABCA1-/- phenotype corroborates the human TD linkage to ABCA1. Similar to TD in humans, HDL-C is virtually absent in ABCA1-/- mice accompanied by a reduction in serum cholesterol and lipid deposition in various tissues. In addition, the placenta of ABCA1-/- mice is malformed, resulting in severe embryo growth retardation, fetal loss, and neonatal death. The basis for these defects appears to be altered steroidogenesis, a direct result of the lack of HDL-C. By 6 months of age, ABCA1-/- animals develop membranoproliferative glomerulonephritis due to deposition of immunocomplexes followed by cardiomegaly with ventricular dilation and hypertrophy, ultimately succumbing to congestive heart failure. This murine model of TD will be very useful in the study of lipid metabolism, renal inflammation, and cardiovascular disease and may reveal previously unsuspected relationships between them.

Figure 1.

Disruption of the ABCA1 gene. A: Design of ABCA1 knockout construct. A mouse genomic DNA fragment containing exons 18–22 of the ABCA1 gene is shown. Exons 19 and 20 and part of exon 21 of the ABCA1 gene, which encode most of the first ATP binding cassette of the ABCA1 protein, was replaced with a neomycin resistance gene cassette containing stop codons (*) in all three frames at the 5′ end. Arrows denote PCR primers used to verify genotypes. The hybridization probe used in Southern blot analysis is also shown as a gray bar. E, EcoRI. B: Southern blot confirmation of the disrupted ABCA1 gene. DNA extracted from the ES cell lines 55.2 and 64.7 was digested with EcoRI and hybridized with the DNA probe shown in A. A 2.2-kb DNA band from the functional ABCA1 gene and a 2.6-kb DNA band from the disrupted ABCA1 gene hybridized to the probe. C: PCR genotyping of blood lysates from mice. The functional ABCA1 gene was amplified by oligonucleotides 3057 and 210R and resulted in a 1.3-kb PCR band. The disrupted ABCA1 gene was detected by oligonucleotides 1455 and 210R to yield a 1.0-kb PCR band. D: Northern Blot analysis of ABCA1 expression in ABCA1+/+ and ABCA1−/− mice. An ABCA1 cDNA probe (LM243) covering exons 26–31 of the ABCA1 gene, a region downstream of the disruption site, was used to detect the 7.8-kb ABCA1 mRNA in ABCA1+/+ and ABCA1−/− peritoneal macrophages. E: Detection of truncated ABCA1 mRNA in ABCA1−/− mice. ABCA1 expression was detected by reverse transcription-PCR amplification of RNA from ABCA1+/+ and ABCA1−/− placenta. A region covering exons 15–18, which is 5′ to the disrupted site, was detected as a 456-bp PCR band. A region covering exons 20–22, which is 3′ to the disrupted site, was detected by PCR as a 455-bp PCR band.

Figure 2.

Lipid deposition in ABCA1−/− thymic, hepatic, and testicular tissues. A: Oil red O staining of lipid in thymus derived from ABCA1+/+ (left) and ABCA1−/− mice (right). Original magnification, ×250. Arrows indicate lipid-stained macrophages in ABCA1+/+ thymus. C, cortex; M, medulla; n = 6 ABCA1+/+ and 9 ABCA1−/− mice. B: Oil red O staining of lipid in liver derived from ABCA1+/+ (left) and ABCA1−/− (right) mice. Original magnification, ×400. n = 4 ABCA1+/+ and 4 ABCA1−/− mice. C: Oil red O staining of lipid in testes derived from ABCA1+/+ (left) and ABCA1−/− mice (right). Original magnification, ×400. L, Leydig cells; S, Sertoli cells; n = 4 ABCA1+/+ and 3 ABCA1−/− mice. Sections were cut from frozen, unfixed tissue.

Figure 3.

Neonatal lungs of ABCA1−/− mice are severely congested. Lungs from 1-day-old neonates, wild type (left), and ABCA1−/− (right) mice were H&E stained. Original magnification, ×100.

Figure 4.

ABCA1 mRNA distribution in maternal deciduae and placenta. A: In situ hybridization showed that ABCA1 mRNA was expressed in the lining of maternal decidual blood vessel derived from a 64.7 ABCA1+/− female at day 14 gestation. Original magnification, ×250. B: Autoradiograph of ABCA1+/+ (top) and ABCA1−/− (bottom) placentas harvested from the same 64.7 ABCA1+/− female, hybridized to LM243 ABCA1 cDNA probe. L, labyrinth; D, deciduae.

Figure 7.

Kidney glomerulonephritis and immunoglobulin deposition in ABCA1−/− mice. A: H&E-stained kidney sections from healthy ABCA1+/+ (left), inflammatory cell-infiltrated ABCA1−/− (middle), and diseased 55.2 ABCA1−/− (right) mice. Original magnification, ×250. Scarring of glomeruli was visible when sections were stained with trichrome (data not shown). G, glomerulus. B: Heavy deposition of immunoglobulin was found in glomeruli of 64.7 ABCA1−/− animal (right) but was not present in ABCA1+/+ glomeruli (left). Original magnification, ×100.

Figure 5.

Placenta malformation and fetal distress in ABCA1−/− mice. A: Placental labyrinth from day 14 of gestation was immunostained with anti-CD71 (specific for trophoblasts). Placentas were ABCA1+/+ (left) and 64.7 ABCA1−/− (right). Original magnification, ×100. B: Placental labyrinth from day 14 of gestation was H&E stained. Placentas were ABCA1+/+ (left), ABCA1+/− (middle), and ABCA1−/− (right). Original magnification, ×63. L, labyrinth; S, spongiotrophoblast; D, deciduae; n = 20 ABCA1−/−, 13 ABCA1+/−, and 22 ABCA1+/+ placentas assayed. C: Intrauterine growth retardation of 64.7 ABCA1−/− embryos. Embryos at gestation day 14 were collected from an ABCA1−/− cross and are arranged by size. Sizes of the ABCA1−/− embryos vary from normal (top left) to resorption (bottom). D: Brown-stained amniotic sac observed in ABCA1−/− (right) but not ABCA1+/+ (left) embryos at day 19 gestation. E: Placenta labyrinth from ABCA1+/+ (left), ABCA1+/− (middle), and ABCA1−/− (right) day 19 gestation embryos was H&E stained. Original magnification, ×63. L, labyrinth; S, spongiotrophoblast; D, deciduae; n = 4 ABCA1−/−, 5 ABCA1+/−, and 7 ABCA1+/+ placentas. F: Resorbing embryos were collected from ABCA1+/+ (left) or ABCA1−/− (right) cross at day 19 of gestation. G: Resorbing placenta from ABCA1+/+ (left) or ABCA1−/− (right) embryos at day 19 of gestation were H&E stained. Original magnification, ×100.

Figure 5.

Placenta malformation and fetal distress in ABCA1−/− mice. A: Placental labyrinth from day 14 of gestation was immunostained with anti-CD71 (specific for trophoblasts). Placentas were ABCA1+/+ (left) and 64.7 ABCA1−/− (right). Original magnification, ×100. B: Placental labyrinth from day 14 of gestation was H&E stained. Placentas were ABCA1+/+ (left), ABCA1+/− (middle), and ABCA1−/− (right). Original magnification, ×63. L, labyrinth; S, spongiotrophoblast; D, deciduae; n = 20 ABCA1−/−, 13 ABCA1+/−, and 22 ABCA1+/+ placentas assayed. C: Intrauterine growth retardation of 64.7 ABCA1−/− embryos. Embryos at gestation day 14 were collected from an ABCA1−/− cross and are arranged by size. Sizes of the ABCA1−/− embryos vary from normal (top left) to resorption (bottom). D: Brown-stained amniotic sac observed in ABCA1−/− (right) but not ABCA1+/+ (left) embryos at day 19 gestation. E: Placenta labyrinth from ABCA1+/+ (left), ABCA1+/− (middle), and ABCA1−/− (right) day 19 gestation embryos was H&E stained. Original magnification, ×63. L, labyrinth; S, spongiotrophoblast; D, deciduae; n = 4 ABCA1−/−, 5 ABCA1+/−, and 7 ABCA1+/+ placentas. F: Resorbing embryos were collected from ABCA1+/+ (left) or ABCA1−/− (right) cross at day 19 of gestation. G: Resorbing placenta from ABCA1+/+ (left) or ABCA1−/− (right) embryos at day 19 of gestation were H&E stained. Original magnification, ×100.

Figure 6.

Dramatic decrease of lipid storage in ABCA1−/− adrenals and ovaries. Sections of adrenal glands and ovaries from ABCA1 +/+ (left) and ABCA1−/− (right) are shown. A: Oil red O staining of lipid in adrenal gland. Original magnification, ×100. C, cortex; M, medulla. B: Oil red O staining of lipid in ovary. Original magnification, ×63. F, follicle; CL, corpus luteum. C: PAN staining of cholesteryl esters in adrenal gland. Original magnification, ×63. D: PAN staining of cholesteryl esters in ovary. Original magnification, ×100; n = 4 ABCA1+/+ and 5 ABCA1−/− females. E: Birefringence of free cholesterol crystals in adrenal gland. Original magnification, ×100. The minor amount of birefringence that is visible at the edge of the ABCA1−/− adrenal gland is from attached fat. Sections were cut from frozen, unfixed tissue.