Localization of Human ATP-Binding Cassette Transporter 1 (ABC1) in Normal and Atherosclerotic Tissues
Abstract—The present study examines the expression of ATP-binding cassette transporter 1 (ABC1) mRNA in normal and atherosclerotic tissues by using in situ hybridization in an effort to better understand the function of this cholesterol transport protein. Samples of normal baboon tissues as well as human normal and atherosclerotic aortas were hybridized with 35S-labeled ABC1 sense and antisense riboprobes. Widespread expression of ABC1 was observed generally in tissues containing inflammatory cells and lymphocytes. Other noninflammatory cells that were also sites of ABC1 synthesis included the ductal cells of the kidney medulla, Leydig cells in the testis, and glial cells in the baboon cerebellum. Although normal veins and arteries did not express ABC1 mRNA, it was found to be upregulated in the setting of atherosclerosis, where widespread expression was found in macrophages within atherosclerotic lesions. These results are consistent with the proposed role of ABC1 in cholesterol transport in inflammatory cells. The specific upregulation of ABC1 mRNA in the setting of atherosclerosis probably reflects the response of leukocytes to cholesterol loading. However, the presence of ABC1 in ductal cells of the kidney medulla and in the small intestine suggest a more general role for this protein in cholesterol transport in other cell types.
- ATP-binding cassette transporter 1 (ABC1)
- ATB-binding cassette transporter A1 (ABCA1)
- Received October 5, 2000.
- Accepted December 5, 2000.
Low plasma HDL concentration is a major risk factor for cardiovascular disease, yet the mechanisms that control the flux of cholesterol through HDL transport in plasma remain poorly understood. The biochemical understanding of this pathway was enhanced by the identification of ATP-binding cassette transporter 1 (ABC1, also known as ABCA1), an ATP-dependent sterol transporter, as the genetic defect in Tangier disease, a monogenic disorder marked by near-zero levels of circulating HDL.1 2 3 4 ABC1 is a member of the family of ATP-binding cassette proteins that transport ligands across the plasma membrane. This family includes the cystic fibrosis transporter (CFTR), several multidrug resistance P glycoproteins, and the ocular protein ABC-R.5 Intracellular roles are also likely for some of the ABC proteins. Ever since its identification as the defective gene in Tangier disease, most investigations have focused on the role of ABC1 in apolipoprotein-mediated efflux of cholesterol and phospholipids from macrophages. To date, cell culture studies using inhibition and overexpression suggest that ABC1 may play a rate-limiting step in that process.4 6 Similarly, the intermediate levels of HDL in humans and knockout mice heterozygous for ABC1 gene deficiency suggest that ABC1 is a vital determinant of plasma HDL concentration.7 8 9 Because a hallmark of Tangier disease is massive cholesterol deposition in tissue macrophages, the initial focus on this cell type seems justified.10 However, ABC1 message has been detected in various tissues, including liver, lung, adrenals, small intestine, and brain.11
Because tissue macrophages may not represent the source of ABC1 in all organs, we used in situ hybridization to localize its expression in human and baboon tissues. Although its presence in macrophages in arterial lesions supports an expected role in the process of atherosclerosis, detection of ABC1 mRNA in liver hepatocytes, villi of the small intestine, tubule cells in the kidney, and cells of the central nervous system serves notice that ABC1 plays a number of roles in lipid transport and homeostasis in addition to the early lipidation of nascent HDL particles in the vascular wall.
Protocols for obtaining tissue samples were approved by the Emory University Hospital Human Investigation Committee and were in accordance with the principles of the Declaration of Helsinki. Human aorta samples representative of different stages of atherosclerosis were obtained from 9 transplant donors. Human internal mammary arteries (n=2) were used as nonatherosclerotic controls and were obtained from patients undergoing coronary artery bypass surgery. The stages of atherosclerotic development were based on the degree of intimal lesion formation, the presence of inflammatory cells, and regions of necrosis as previously described.12 Normal vessel segments (n=3) had almost no intimal development and no inflammatory cells, as defined by CD68 immunohistochemistry. Atherosclerotic aortas (n=6) had either minor intimal development with scattered macrophage staining just under the luminal surface or were advanced atherosclerotic lesions with a thickened intima, numerous CD-68–positive macrophages in the intima and media, and regions of necrosis and cholesterol deposits. The normal tissue distribution of ABC1 mRNA was determined by using tissues obtained from male baboons, which were collected in the course of other studies. All primate tissues were obtained under protocols approved by the Emory University Institutional Animal Care and Use Committee.
Tissue samples were fixed overnight in 4% (wt/vol) paraformaldehyde in 0.1 mol/L sodium phosphate (pH 7.4) at 4°C and then processed by using standard paraffin techniques. Other tissue samples were collected, immediately immersed in 4% (wt/vol) paraformaldehyde in 0.1 mol/L sodium phosphate (pH 7.4) at 4°C for 3 to 4 hours, cryoprotected in 15% (wt/vol) sucrose/isotonic PBS overnight at 4°C, and processed for frozen sections as previously described. Equivalent in situ hybridization results were obtained with both methods.
In Situ Hybridization
In situ hybridization was performed on paraffin sections with the use of human-specific 35S-labeled riboprobes as previously described.13 In situ hybridization results were photographed by polarized light epiluminescence microscopy (Leitz) so that the silver grains appeared white. The results were evaluated by 2 individuals and graded (−, +, ++, and +++) on the basis of the number of cells expressing ABC1 mRNA in each tissue type.
A plasmid template for the synthesis of sense and antisense riboprobes to ABC1 mRNA was constructed by the ligation of sequences from base pairs 350 to 1805 of the ABC1 cDNA (GenBank AF285167) into vector pGEM 3Zf- (Promega). RNA probes constituting either strand of this sequence were synthesized by in vitro transcription from the T7 or SP6 RNA polymerase promoters flanking the insert after linearization of the plasmid by restriction digestion. Probe specificity was confirmed by hybridization of a 32P-labeled probe containing this insert sequence to Southern blots of human genomic DNA that had been digested with KpnI, BamHI, or EcoRI. Only the bands expected from restriction enzyme fragment prediction based on the human ABC1 gene sequence (GenBank accession numbers AF287262 and AF287263) were observed.
Immunohistochemistry was used on serial sections to identify cells containing ABC1 mRNA. The following antibodies were used: CD68 for macrophages (Dako, 1:50 dilution), CD20 for B cells (Pharmagen, 1:125 dilution), CD3 for T cells (Dako, 1:25 dilution), and SM1 for smooth muscle cell α-actin (Sigma Chemical Co, 1:800 dilution). Sections were predigested with proteinase K (1 mg/mL, Sigma) or pronase E (1 mg/mL, Sigma), the primary antibodies were applied at the indicated dilutions, and the slides were stained by using ABC-AP (Vector Labs) as described.14 Serial sections treated with secondary antibodies only or with nonimmune IgG did not show any staining.
The cell types expressing ABC1 mRNA in a series of tissues taken from adult male baboons were determined by in situ hybridization and are summarized in Table 1⇓. Generally, expression of ABC1 was found in tissues involved in cholesterol transport and utilization and in inflammatory cells, including macrophages, T cells, and B cells.
In the liver, ABC1 was expressed in some, but not all, hepatocytes (Figure 1⇓). Strong consistent hybridization was seen in scattered CD68-positive macrophages and Kupffer cells. At least some of the hybridizing cells in the liver were likely to be B cells, as determined by serial section immunohistochemistry with the CD20 antibody (Figure 1⇓, available online at http://atvb.ahajournals.org). Strong hybridization was found in the region surrounding the portal vein and appeared to consist of connective tissue cells and CD68- and CD20-positive lymphocytes found in this region. The small intestine was found to have numerous ABC1-positive macrophages in the lamina propria of the intestinal villi. ABC1 mRNA was not detected in the epithelial cells lining the small intestine. In the spleen and lymph nodes, hybridization was found in regions consistent with the localization of macrophages and B cells. Some hybridization to T cells was likely in the marginal zone but could not be confirmed. In the testis, ABC1-positive cells were found in the Leydig cells surrounding the seminiferous tubules. In the kidney, strong hybridization that was localized to scattered tissue macrophages as well as cells lining the proximal and distal tubules in the cortex and medulla (Figure 2⇓, available online at http://atvb.ahajournals.org) was found. In a section of the baboon cerebellum, ABC1 mRNA was detected in glial cells in the white matter as well as in cells within the granular layer. Purkinje cells did not express ABC1 mRNA (Figure 3⇓, available online at http://atvb.ahajournals.org). In the lung, ABC1 was expressed by pulmonary microphages (Figure 4, available online at http: //atvb.ahajournals.org).
Normal baboon aorta and carotid artery did not express ABC1 mRNA. Consistent with this result, no ABC1-expressing cells were found in samples of human internal mammary artery or normal nonatherosclerotic aortas (Table 2⇓). Atherosclerotic vessels demonstrated expression of ABC1 mRNA in scattered lipid-filled macrophages in early fatty streaks (Figure 2⇑) as well as in the shoulder region of advanced lesions (see http://atvb.ahajournals.org). Nonatherosclerotic regions of these same vessel cross sections contained no ABC1-hybridizing cells. In at least 1 vessel, strong hybridization was found in an inflammatory zone filled with scattered macrophages without lipids (Figure 3⇑). In the same tissue section, a portion of the vessel had a diffuse intimal thickening without inflammatory cells, which showed no ABC1 hybridization. In all cases, serial section staining of these vessels with CD68 suggested that ABC1 hybridization was to macrophages in the atherosclerotic vessels. No CD20-positive cells were found in the vascular tissues, so it is unlikely that B cells are the source of ABC1 mRNA; scattered CD3-positive T cells were present but differed morphologically from the ABC1-positive cells (Figures 2⇑ and 3⇑). However, it should be emphasized that not all CD68-positive macrophages hybridized with the ABC1 riboprobe. ABC1 mRNA was not detected in a number of non–lipid-associated CD68-positive cells found scattered in other parts of the vessel, especially in the adventitia.
The genetic basis for understanding the function of ABC1 was its establishment as the defect in the monogenic disorder Tangier disease. These patients have near-zero levels of circulating HDL. Sequence analysis and metabolic and cell culture studies in the last decade have established that apolipoprotein components of HDL are normal in sequence and synthesis in these patients but that HDL is catabolized an increased rate.15 16 Francis et al17 have shown that apolipoprotein-mediated cholesterol efflux is deficient in cells derived from patients with Tangier disease. Hence, the defect in Tangier disease represents an initial step in reverse cholesterol transport. The current understanding is that apolipoproteins or nascent lipid-poor HDL particles fail to acquire a “mature” lipid content and are relatively quickly cleared from the circulation. ABC1 is likely to function as a cell membrane transporter that facilitates the transfer of cholesterol and phospholipids to poorly lipidated apolipoproteins at an exofacial pore, which is ringed by 12 predicted trans-membrane domains.18 Although this may describe the role of ABC1 in resident macrophages coping with excessive cholesterol import, the potential functions of ABC1 in other settings remain unclear. Involvement in activities as diverse as apoptotic cell engulfment and secretion of interleukin-1β has been proposed,19 20 with the former linked to the calcium-induced exposure of phosphatidylserine at the outer surface of the plasma membrane by a hypothesized “flippase” activity of ABC1.21
Patients with Tangier disease experience cholesterol deposition in many tissues, not only in the enlarged orange tonsils, noted as the hallmark of this disease, but in liver, spleen, intestinal mucosa, lymph nodes, cornea, and Schwann cells.10 22 Major clinical symptoms include not only HDL deficiency and increased incidence of cardiovascular disease but splenomegaly and neuropathy as well. The results in the present study support the role of ABC1 in the development of arterial lesions as well as a broader function in lipid distribution. Previous studies detected the presence of ABC1 message by blot hybridization to whole-organ RNA samples from heart, liver, spleen, kidney, lung, small intestine, and brain.11 23 In situ hybridization data have now confirmed and extended these observations to the level of individual cells within these tissues.
ABC1 RNA is readily detected in fatty macrophages in early atherosclerotic lesions (Figure 2⇑). However, not all macrophages in the plaque specimens are positive. This raises the intriguing issue of the signals and responses that control its synthesis. Cell culture studies have demonstrated sterol-induced ABC1 transcription in parallel with enhancement of cholesterol efflux from such cells.24 This may be an adaptive response to maintain cholesterol homeostasis in cells containing scavenger receptors that allow unregulated uptake of oxidized or otherwise modified LDL. Cultured THP-1 macrophages show a steady increase of ABC1 RNA during 4 days of exposure to oxidized LDL.25 It remains to be determined whether some plaque macrophages fail to express ABC1 as a result of an insufficient signaling input or a lack of response due to signal overload or because they represent a unique subclass of macrophages. At this point, such questions remain unanswered, yet it is suggestive that we have detected less ABC1 RNA in macrophage-rich regions of plaques considered to be of an advanced nature. We speculate that these macrophages may represent foam cells incapable of ABC1-mediated cholesterol efflux.
A substantial portion of the ABC1 RNA detected in spleen, lung, liver, and small intestine can be attributed to resident macrophages. Splenomegaly with lipid-filled macrophages occurs in some patients with Tangier disease, supporting a role of ABC1 in sterol efflux from macrophages that must cope with a large flux of cholesterol derived from the uptake by the spleen of phagocytic cells and senescent cells.10 Expression of ABC1 in alveolar macrophages in the lung is consistent with a role in lipid homeostasis in these cells that are involved in the uptake and clearance of surfactant phospholipids and cholesterol, as well as diseased and damaged cells.
Much of the expression of ABC1 in liver resides in macrophages and Kupffer cells. However, cells lining the portal vein, B cells, and some hepatocytes are also positive. It can be suggested that in hepatocytes the primary function of ABC1 is to mediate the formation of nascent HDL through the provision of cholesterol and phospholipid to lipid-free apolipoproteins rather than to promote the efflux of excess cholesterol as in macrophages. Such divergent functions may require ABC1 gene regulation to differ in these tissue types. Indeed, Costet et al26 have recently provided evidence that Hep G2 cells produce a variant of ABC1 mRNA that uses a distinct 5′ exon, containing a different transcription and translation start site than that expressed in macrophages and cultured fibroblasts.24 26 27 , Because only the initial 22 amino acids of the protein are predicted to differ in the liver form of ABC1,26 potential differences in transcriptional response of the gene may be a more important consequence of the alternatively spliced variants.
ABC1 expression in Leydig cells of the testes implies a role in cholesterol homeostasis in tissues that synthesize steroid hormones, as also noted by the detection of ABC1 message by blot hybridization to adrenal RNA.11 Synthesis of ABC1 in the kidney suggests a role in sterol transport during filtration and reabsorption. Because apoA-I is known to be filtered in the kidney, renal clearance of poorly lipidated apoA-I–containing particles may play a key role in their scarcity in the plasma of patients with Tangier disease and potentially other individuals with low levels of HDL.28 29 The HDL-binding proteins megalin and cubilin are expressed in the kidney and have been proposed to mediate the uptake of apoA-I dissociated from the lipoprotein particle.30 Our detection of ABC1 gene expression in tubules and collecting ducts in the kidney medulla suggest a renal function for ABC1 as well as these other proteins in secreting or recapturing cholesterol and not merely the dissociated protein components of HDL.
It had been suggested that the neuropathology associated with Tangier disease could be linked to the low concentrations of apoA-I and HDL, which could impair the unloading of cholesterol from macrophages and Schwann cells during myelination.10 The detection of ABC1 synthesis in neuronal tissue suggests that this protein plays a direct role in the redistribution of cholesterol and phospholipid in the nervous system, as do other components of lipid transport pathways, such as apoE and members of the LDL receptor family.31
The role of ABC1 in the intestine was not appreciated before the discovery that mice with a targeted disruption of this gene showed an increased absorption of cholesterol from the gut.8 A more recent report of drug-induced increase in ABC1expression and reduction in cholesterol uptake in the mouse intestine supports this activity.32 This suggests that ABC1 acts as a unidirectional transporter to efflux some percentage of absorbed cholesterol back to the intestinal lumen. The cell type and the presumed acceptor apolipoprotein in this process remain to be determined. Although we detect a clear ABC1 RNA signal in intestinal villi, it appears to be uniformly distributed throughout the lamina propria, where it colocalizes with macrophages, not with epithelial cells. Thus, our data may not support a proposed role of ABC1 in cholesterol absorption by the intestine. Alternatively, they may suggest that the “barrier” to absorption may be occurring not in the epithelial layer but in macrophages within the lamina propria. This might be consistent with the report that cultured intestinal epithelioid CaCo2 cells did not show apoA-I–stimulated cholesterol efflux.33 However, until the full nature of the induction of this pathway in the intestine is elucidated, we cannot exclude the possibility that intestinal epithelial cells may not express ABC1 under certain conditions.
The discovery that ABC1 represents the defective gene in Tangier disease and plays a key role in the efflux of cholesterol and phospholipids from macrophages to nascent HDL particles was a key step in clarifying the initiating events in reverse cholesterol transport. Conditions that are known to increase cholesterol efflux from macrophages, including cholesterol loading, cAMP signaling, and treatment with ligands of nuclear hormone receptors, are now known to affect ABC1 transcription in parallel.26 24 33 34 Most cells regulate intracellular cholesterol by controlling its synthesis or its uptake by LDL receptor–mediated endocytosis. Macrophages that ingest cell debris and modified lipoproteins by scavenger receptors that are not downregulated are more dependent on the efflux pathway in which ABC1 may be a rate-limiting component.35 The studies of Chimini and colleagues19 21 add to this the involvement of ABC1 in membrane phospholipid distribution in the process of phagocytosis. However, the distribution of ABC1 gene expression suggests a function beyond that appropriate for phagocytic cells. It can be proposed that ABC1 serves not only to aid in the net efflux of lipids from nonpolarized cells, such as macrophages. It can be hypothesized that in polarized cells lining blood and lymphatic vessels or kidney tubules, ABC1 is involved in the physiological redistribution of lipids via transcytosis, delivering its cargo out of a luminal or basolateral surface. Further definition of all the functions of ABC1 will be gained by the development and use of additional tools, which include genetically manipulated mice, inhibiting antibodies, and selective drugs that alter the quantity or activity of this protein.
This work was support by CV Therapeutics and National Institutes of Health grant HL-58000 (J.N.W.).
- ↵Lawn RM, Wade DP, Garvin MR, Wang X, Schwartz K, Porter JG, Seilhamer JJ, Vaughan AM, Oram JF. The Tangier disease gene product ABC1 controls the cellular apolipoprotein-mediated lipid removal pathway. J Clin Invest. 1999;104:R25–R31.
- ↵Wang N, Silver DL, Costet P, Tall AR. Specific binding of ApoA-I, enhanced cholesterol efflux and altered plasma membrane morphology in cells expressing ABC1. J Biol Chem. 2000;275:33053–33058.
- ↵McNeish J, Aiello RJ, Guyot D, Turi T, Gabel C, Aldinger C, Hoppe KL, Roach ML, Royer LJ, de Wet J, et al. High density lipoprotein deficiency and foam cell accumulation in mice with targeted disruption of ATP-binding cassette transporter-1. Proc Natl Acad Sci U S A. 2000;97:4245–4250.
- ↵Assmann G, Schmitz G Jr. BHB: familial HDL deficiency: Tangier disease. In: Scriver CR, Beaudet AL, Sly WS, Valle D, eds. The Metabolic Basis of Inherited Disease. 6th ed. New York, NY: McGraw-Hill; 1989:1267–1282.
- ↵Wilcox JN, Subramanian RR, Sundell CL, Tracey WR, Pollock JS, Harrison DG, Marsden PA. Expression of multiple isoforms of nitric oxide synthase in normal and atherosclerotic vessels. Arterioscler Thromb Vasc Biol. 1997;17:2479–2488.
- ↵Wilcox JN. Fundamental principles of in situ hybridization. J Histochem Cytochem. 1993;41:1725–1733.
- ↵Scott NA, Ross CE, Dunn B, Martin FH, Simonet L, Wilcox JN. Identification of a potential role for the adventitia in vascular lesion formation after balloon overstretch injury of porcine coronary arteries. Circulation. 1996;93:2178–2187.
- ↵Law SW, Brewer HB Jr. Tangier disease: the complete mRNA sequence encoding for preproapo-A-I. J Biol Chem. 1985;260:12810–12814.
- ↵Francis GA, Knopp RH, Oram JF. Defective removal of cellular cholesterol and phospholipids by apolipoprotein A-I in Tangier disease. J Clin Invest. 1995;96:78–87.
- ↵Rosenberg MF, Callaghan R, Ford RC, Higgins CF. Structure of the multidrug resistance P-glycoprotein to 2.5 nm resolution determined by electron microscopy and image analysis. J Biol Chem. 1997;272:10685–10694.
- ↵Hamon Y, Luciani MF, Becq F, Verrier B, Rubartelli A, Chimini G. Interleukin-1beta secretion is impaired by inhibitors of the ATP binding cassette transporter, ABC1. Blood. 1997;90:2911–2915.
- ↵Schiffman D, Mikita T, Tai JTN, Wade DP, Porter JG, Seilhammer JJ, Somogyi R, Liang S, Lawn RM. Large scale gene expression analysis of cholesterol-loaded macrophages. J Biol Chem. 2000;275:37324–37332.
- ↵Costet P, Luo Y, Wang N, Tall AR. Sterol-dependent transactivation of the ABC1 promoter by the liver X receptor/retinoid X receptor. J Biol Chem. 2000;275:28240–28245.
- ↵Glass CK, Pittman RC, Keller GA, Steinberg D. Tissue sites of degradation of apoprotein A-I in the rat. J Biol Chem. 1983;258:7161–7167.
- ↵Horowitz BS, Goldberg IJ, Merab J, Vanni TM, Ramakrishnan R, Ginsberg HN. Increased plasma and renal clearance of an exchangeable pool of apolipoprotein A-I in subjects with low levels of high density lipoprotein cholesterol. J Clin Invest. 1993;91:1743–1752.
- ↵Hammad SM, Barth JL, Knaak C, Argraves WS. Megalin acts in concert with cubilin to mediate endocytosis of high density lipoproteins. J Biol Chem. 2000;275:12003–12008.
- ↵Ignatius MJ, Gebicke-Harter PJ, Skene JH, Schilling JW, Weisgraber KH, Mahley RW, Shooter EM. Expression of apolipoprotein E during nerve degeneration and regeneration. Proc Natl Acad Sci U S A. 1986;83:1125–1129.
- ↵Repa JJ, Turley SD, Lobaccaro JM, Medina J, Li L, Lustig K, Shan B, Heyman RA, Dietschy JM, Mangelsdorf DJ. Regulation of absorption and ABC1-mediated efflux of cholesterol by RXR heterodimers. Science. 2000;289:1524–1529.
- ↵Bornick AE, Rothblat GH, Stoudt G, Hoppe KL, Royer LJ, McNeish J, Francone OL. The correlation of ATP-binding cassette 1 mRNA levels with cholesterol efflux from various cell lines. J Biol Chem. 2000;275:28634–28640.
- ↵Oram JF, Lawn R, M., Garvin MR, Wade DP. ABCA1 is the cAMP-inducible apolipoprotein receptor that mediates cholesterol secretion from macrophages. J Biol Chem. 2000;275:34508–34511.