Pulmonary abnormalities due to ABCA1 deficiency in mice

Sandra R. Bates,1,2 Jian-Qin Tao,1 Heidi L. Collins,3 Omar L. Francone,4 and George H. Rothblat3

1Institute for Environmental Medicine, 2Department of Physiology, University of Pennsylvania, 3Children's Hospital of Philadelphia, Philadelphia, Pennsylvania; and 4Pfizer, Groton, Connecticut

Submitted 31 May 2005 ; accepted in final form 17 July 2005


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Mice gene targeted for ATP-binding cassette transporter A1 (ABCA1; Abca1–/–) have been shown to have low-serum high-density lipoprotein and abnormal lung morphology. We examined alterations in the structure and function of lungs from –/– mice (DBA1/J). Electron microscopy of the diseased mouse lung revealed areas of focal disease confirming previous results (47). Lipid analysis of the lung tissue of –/– mice showed a 1.2- and 1.4-fold elevation in total phospholipid (PL) and saturated phosphatidylcholine, respectively, and a marked 50% enrichment in total cholesterol content predominately due to a 17.5-fold increase in cholesteryl ester compared with wild type (WT). Lung surfactant in the –/– mice was characterized by alveolar proteinosis (161%), a slight increase in total PL (124%), and a marked increase in free cholesterol (155%) compared with WT. Alveolar macrophages were enriched in cholesterol (4.8-fold) due to elevations in free cholesterol (2.4-fold) and in cholesteryl ester (14.8-fold) compared with WT macrophages. More PL mass was cleared from the alveolar space of –/– mice lungs, measured using intratracheal installation of 3H-PL liposomes. Compared with WT mice, the Abca1–/– mice demonstrated respiratory distress with rapid, shallow breathing. Thus the lungs of mice lacking ABCA1 protein demonstrated abnormal morphology and physiology, with alveolar proteinosis and cholesterol enrichment of tissue, surfactant, and macrophages. The results indicate that the activity of ABCA1 is important for the maintenance of normal lung lipid composition, structure, and function.

lung; surfactant; type II cells; alveolar macrophages; cholesterol


MAINTENANCE OF APPROPRIATE surface tension in the lung is the primary function of pulmonary surfactant, the lipid-protein mixture that lines the alveoli (for review, Ref. 33). Surfactant proteins (SP) are divided into two groups, the hydrophilic SP-A and SP-D and the hydrophobic SP-C and SP-B (for review, Ref. 64). The lipid moiety of surfactant is predominantly phospholipid with 10% cholesterol by weight (34); cholesterol represents 58% of the neutral lipid fraction (26). The relative distribution of cholesterol differs between the various subfractions of alveolar surfactant (24). In rat surfactant, cholesterol represents 18% of the total lipid in the large aggregate heavy subfraction that pellets after centrifugation at 60,000 g but is a major component (60% of total lipid) of the small aggregate light subfraction isolated at 100,000 g (24). The large aggregate is the most surface-active fraction of surfactant and contains most of the SPs (22). Although the lung is capable of de novo synthesis of cholesterol, the bulk of pulmonary cholesterol is provided by serum lipoproteins. Radiolabeled cholesterol from very low-density (VLDL), low-density (LDL), and high-density (HDL) lipoproteins is taken up promptly by the perfused rat lung and, subsequently, recovered from the surfactant fraction (26, 51, 56). Type II cells, known to be responsible for the synthesis and secretion of surfactant, bind and incorporate gold-conjugated VLDL, LDL, and HDL as shown by immunoelectron microscopy (23, 56).

The mechanism for the removal of cholesterol from the lung and transport to the liver for excretion from the body may occur through a process termed reverse cholesterol transport (RCT) (16), the same route used by other peripheral organs. The ATP-binding cassette transporter A1 (ABCA1), a member of the ABC superfamily of membrane-associated ATPase transporters, is thought to be of major importance in RCT. ABCA1 mediates the transport of cholesterol and phospholipid from tissue through interaction with lipid-poor apolipoproteins. Interaction of ABCA1 with apolipoprotein AI (apoAI), the principal protein component of HDL, results in the formation of nascent HDL discs (for review, Ref. 49). Patients lacking ABCA1 have Tangier disease (TD), which is characterized by excess tissue cholesterol, especially in macrophages; marked reduction in plasma HDL due to the catabolism of unlipidated nascent apoAI; peripheral neuropathy; lipid-soluble vitamin deficiency; and an increased risk of atherosclerosis (8, 11, 39, 53). Fibroblasts from TD patients are unable to release cholesterol and phospholipid to apoAI (19, 52). To understand the role of ABCA1 in cholesterol turnover, mice were generated in which the Abca1 gene was mutated (14, 47, 50). The phenotype observed in the ABCA1 null mice varied in severity, possibly due to genetic background and diet (2). However, all mice lacked HDL and demonstrated difficulty in breeding, the latter probably due to the lack of cholesterol necessary for steroidogenesis confounded by fat-soluble vitamin deficiency (14, 47, 50). The DBA1/J Abca1–/– mice used in the present study had low plasma cholesterol and phospholipids (reduced ~70%), markedly altered plasma phospholipid composition, virtually no HDL, and undetectable apoAI protein (47). Plasma triglycerides and VLDL levels were normal, but LDL cholesterol and apoB content were decreased. Analysis of the brain and central nervous system revealed defective apolipoprotein E metabolism (27, 61). However, the most predominant pathological change in these mice was focal lesions in the pulmonary parenchyma (47).

Abundant levels of ABCA1 have been found in the lung, in alveolar macrophages, and, as described by our recent report, in pneumocytes (9, 38, 65). We demonstrated that ABCA1 mRNA and protein were present and could be induced in isolated type II cells (9). Similar to other cell types, liver X receptor/retinoid X receptor agonists upregulated ABCA1 with a resultant stimulation in the release of phospholipid and cholesterol to apoAI from type II cells (9). Furthermore, the increase in ABCA1 protein was paralleled by a decrease in surfactant secretion from pneumocytes (9). On the basis of our findings and the previous reports of abnormal lung morphology in Abca1–/– gene-targeted mice, the aim of this study was to examine the role of ABCA1 in the lipid metabolism of lung surfactant. We compared Abca1+/+ and Abca1–/– mice with regard to morphology, lung and pneumocyte phospholipid and cholesterol content, rates of lipid clearance from the alveolar space, and respiratory parameters. We found significant alterations in lung lipid composition and function in the mice deficient in ABCA1 expression, indicating that ABCA1 plays an important role in the maintenance of a healthy pulmonary system.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Mice. The ABCA1 gene-targeted [Abca1 knockout (KO), –/–] mice (DBA1/J) were bred by McNeish et al. (47) and maintained at Pfizer Central Research (Groton, CT) according to protocols approved by the Pfizer Institutional Animal Care and Use Committee (47). The Abca1–/– and age-matched wild-type (WT) DBA1/J mice were sent to the University of Pennsylvania and used according to procedures approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania. All protocols adhered to the principles of the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Histology. The lungs were fixed in 10% neutral buffered formalin, cryopreserved with sucrose, and embedded in optimum cutting temperature compound. Frozen sections were cut and stained with Oil red O.

Electron microscopy. DBA1/J WT or Abca1–/– mice (4–6 mo old) were transcardially perfused with ice-cold 5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4, for 15 min. Next, the lungs were injected with the same fixative through the trachea, removed, and immersed in the same fixative at 4°C. After 30 min, the lungs were cut into 1 x 1-mm pieces and continually fixed for another 2.5 h. The tissue was further fixed in 2% OsO4 in 0.1 M sodium cacodylate buffer on ice for 1 h and then dehydrated using an acetone process. Tissue pieces were embedded in EM-bed 812 resin and polymerized at 60°C for 48 h. Ultrathin sections (70 nm) were prepared on a Leica Ultracut UCT (Vienna, Austria) with diamond knives, counterstained with uranyl acetate and lead citrate, and imaged with a JEOL electron microscope model 100 CX (Tokyo, Japan). The samples are typical for the three mice lungs/group examined.

Analysis of lung tissue and alveolar lavage. Mice were given a lethal dose of pentobarbital sodium and exsanguinated by distal aorta transaction. The trachea was cannulated with a 20-gauge blunt needle, and the lungs were lavaged with five 1-ml aliquots of 0.9% saline, as described (28). The bronchoalveolar lavage (BAL) fluid was centrifuged at 400 g for 10 min at 4°C to remove cells and debris, and the lungs were homogenized. Aliquots of the lung homogenate and BAL were analyzed for protein (10, 43), and lipids were extracted with chloroform:methanol using the Bligh and Dyer procedure (7). Lipid samples were analyzed for phospholipid (3) or cholesterol (35). The disaturated phospholipid fraction was separated from total phospholipid on a neutral alumina column after osmication of the lipids (46). When this method is used for lung tissue or lavage, 94% of the phosphorus eluted is disaturated phosphatidylcholine. Phospholipids were fractionated by thin-layer chromatography on silica gel plates using chloroform:methanol:ammonia:water (65:35:2.5:2.5, vol/vol) as the solvent system (17), and bands of interest were scraped and analyzed.

The cholesterol mass content of tissue and cells was quantitated before [free cholesterol (FC)] and after saponification (total cholesterol) (30) of the lipid extract using gas-liquid chromatography with cholesteryl methyl ether as an internal standard, as described previously (35). The esterified cholesterol (EC) was determined by subtraction of the FC from the total cholesterol and represents the cholesterol in the cholesteryl ester.

SP content of the large aggregate fraction. BAL fluid was collected from rodents and centrifuged at 1,000 g for 10 min to remove cells. Aliquots were analyzed for phospholipid (6) and protein (43) content, and the remainder was centrifuged at 48,000 g (4°C, 1 h, Sorvall) to pellet large surfactant aggregates. Aliquots were taken for phospholipid and protein content and subjected to Western blot analysis. Samples were run on 12% Novex Tris-glycine gels or 10–20% Tricine gels with Tricine SDS sample buffer (Invitrogen, Carlsbad, CA) under reducing conditions (25) for SP-A or SP-B, respectively, transferred to nitrocellulose (59), and probed with antibody to SP-A (5) or SP-B (25).

Cells. Type II cells were isolated from DBA1/J mouse lungs using a modification of the procedure described by Warshamana et al. (63) using dispase as previously described (9). Perfused lungs were instilled with dispase (100 units) via the trachea and incubated for 45 min at room temperature in a tube containing dispase (50 units). Digested lung tissue was treated with 0.01% DNase I for 10 min 37°C, filtered through nylon mesh (100-, 40-, and 25-µm size), centrifuged at 130 g for 10 min at 4°C, and then plated on mouse IgG (0.75 mg/ml)-coated petri dishes at 37°C for 1 h to remove macrophages. The nonadherent cells were centrifuged at 130 g for 10 min. The cell pellet was resuspended in MEM with 10% FBS and seeded on 100-mm cell culture dishes at 37°C for 1 h twice to remove fibroblasts. The final cell isolates were seeded on type I collagen-coated 35-mm dishes in Ham's F-12 culture medium supplemented with 15 mM HEPES, 0.8 mM CaCl2, 0.25% BSA, 5 µg/ml insulin, 5 µg/ml transferrin, 5 ng/ml sodium selinite, and 2% mouse serum. After 24 h, the isolated type II cells were 95% pure as judged by cytokeratin staining and the presence of Nile red-positive vacuoles.

Alveolar macrophages were isolated by BAL and were incubated on 35-mm plastic tissue culture dishes (Costar) for 1 h in MEM without serum. Before analysis, the cells were washed three times to remove nonadherent cells. The resultant preparation was >98% macrophages (4). The plated macrophages from three WT and three KO mice were photographed and counted (4–8 fields/mouse). The numbers of enlarged, foamy macrophages were expressed as a % of the total macrophage cell count. For BAL cell count, cells were isolated from the BAL after centrifugation for 10 min at 3,000 g. The resultant cell pellet was resuspended, and aliquots were taken for cell count and cytospun onto glass slides. The slides were stained with Diff-Quik, and the cell types were counted.

Preparation of liposomes. L-{alpha}-dipalmitoyl phosphatidylcholine (DPPC), egg phosphatidylcholine (PC), egg phosphatidylglycerol (PG), and cholesterol were mixed in a molar ratio of 10:5:2:3 with trace amounts of [choline-methyl-3H]DPPC ([3H]DPPC) and dried under nitrogen (17). The phospholipid mixture was resuspended in PBS (pH 7.4) and subjected to three cycles of freezing under liquid nitrogen and thawing at 50°C. The phospholipids were then extruded under pressure through a 100-µm pore-size filter. The liposomes were stored at 4°C and used within a week of preparation.

Clearance of DPPC liposomes from mice lungs. Liposomes (10 nmol of DPPC in 20 µl of saline) were instilled intratracheally using a Hamilton syringe into mice previously anesthetized by intraperitoneal injection of xylazine (5.2 mg/kg) plus ketamine (40 mg/kg). Mice were killed using pentobarbital sodium (50 mg/kg body wt) after 5 min or after recovery from anesthesia 4–42 h later. The lungs were removed and lavaged five times with 1-ml aliquots of saline, and lavage from each mouse was pooled. Lungs were then homogenized, and aliquots of lung homogenate and lavage were counted for radioactivity. The data are expressed as a % of the radioactivity of the DPPC originally instilled.

Ventilation parameters. Respiratory characteristics during air or CO2 breathing were measured using whole body plethysmography (Buxco Electronics, Sharon, CT) (31). Briefly, each mouse was placed in a plethysmography chamber, and the chamber pressure-time wave was measured continuously via a transducer connected to a computerized data acquisition system. After baseline measurements for 40 min to ensure a steady state, the frequency of breathing, tidal volume, and minute volume were recorded at 5-min intervals for 30 min during exposure either to air or 5% CO2 in air.

Statistical analysis. Results are reported as means ± SE unless stated otherwise. Statistical analysis was made using unpaired or paired Student's t-test using SigmaStat for Windows (Jandel, San Rafael, CA). Multiple group comparisons were done using one-way ANOVA. Results were statistically significant at P < 0.05.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Morphology. The reduction in plasma cholesterol and HDL and the morphological changes in the lungs of ABCA1–/– mice at 12 mo of age have been reported previously (47). Evaluation of the lungs of ABCA1–/– DBA1/J mice (~4 mo old) confirmed previous observations of focal areas of abnormal morphology seen in 12-mo-old mice (47), although the alterations were not as severe, likely due to the younger age of our animals (Figs. 13). Light microscopy of Oil red O-stained lungs, shown in Fig. 1, revealed a focal lesion of lipid-filled cells in the ABCA1–/– mouse (Fig. 1, C and D). To characterize the effects of ABCA1 ablation on lung pneumocytes, we performed a detailed morphological examination of the lungs of diseased animals at the electron microscopic level (Figs. 2 and 3). Figure 2A demonstrates the normal pulmonary morphology of a WT mouse. Lung pathology in the severely affected sections of the 4-mo-old Abca1–/– mice, shown in Fig. 2, B, D, and E, was characterized by thickened interalveolar septa and by the presence of large, lipid-filled macrophages in the alveolar space (Fig. 2, B and D). Type II cell hyperplasia (Fig. 2C) was evident throughout the lung with many cells containing large vacuoles (Fig. 3, B and D). Occasional type II cells had greatly enlarged lamellar bodies (Fig. 2E), as shown previously (47). Most of the lamellar bodies and microvilli appeared normal. Occasional type I cells had abnormal cytoplasmic extensions, but, on average, type I cells and endothelial cells did not demonstrate significant ultrastructural abnormalities. The areas of milder lung disease (Fig. 2C) exhibited vacuoles within type II cells as well as in the interstitium. Fewer alveolar macrophages were found, and the macrophages appeared normal. Evidence of typical surfactant structures resembling newly secreted lamellar bodies and tubular myelin was found in both WT (Fig. 3A) and ABCA1–/– mice (Fig. 3C). Thus the absence of ABCA1 produced a gradient of pathology affecting most of the predominant cell types of the alveoli.



View larger version (153K):
[in this window]
[in a new window]
 
Fig. 1. Light microscopy of ABCA1–/– mouse lung stained with Oil red O. Lungs from DBA1/J wild-type (WT) (A and B) and ABCA1–/– (C and D) 2-mo-old mice. A and C: x10 magnification. Arrows indicate areas in x10 field that is enlarged for the x20 view. B and D: x20 magnification. A focal lesion of lipid-filled cells is visible in the ABCA1–/– lung.

 


View larger version (121K):
[in this window]
[in a new window]
 
Fig. 3. Abnormal type II morphology in cells from ABCA1–/– mouse. A: type II cell from normal mouse lung with a lamellar body (LB) in the alveolar space. B–E: type II cells from ABCA1–/– mouse lung. B: 2 juxtaposed type II cells with vacuoles (V) and LB. C: high power view of type II cell with normal mitochondria (Mit), LB, tubular myelin (TM), and a secreted LB in the alveolar space. D: type II cells with many vacuoles. E: hypertrophy of a type II cell.

 


View larger version (146K):
[in this window]
[in a new window]
 
Fig. 2. Morphological abnormalities in lungs of ABCA1–/– mice at the electron micrographic level. Lungs from DBA1/J WT (A) and ABCA1–/– mice (B–E) are shown. Overview of WT (A) and severely affected foci of ABACA1–/– mouse lung (B) showing enlarged septa, foamy macrophages (d, enlarged view in D), and lipid-filled, abnormal type II cells (e, enlarged view in E). C: ABCA1–/– mouse, area with moderate changes demonstrating type II cell hyperplasia. D: enlargement of alveolar macrophages shown in B. E: enlargement of type II cell shown in B.

 
Lipid content of lung. Lungs were isolated from the mice and lavaged to remove surfactant, and the lipid content of the lung and surfactant was analyzed. The weights of the animals did not differ significantly (WT = 26.4 ± 0.8 g vs. –/– 28.5 ± 0.9 g, P = 0.123, n = 6 or 5, respectively), and the amount of protein in the lung tissue/g mouse did not differ among groups (Table 1). Thus the data are expressed as micrograms of lipid or protein per gram mouse or micromoles of saturated PC per kilogram of mouse. The phospholipid content of the lung tissue of the ABCA1–/– mice was slightly (1.2-fold), but significantly (P = 0.03), enriched in total phospholipid and saturated phospholipid (1.2-fold) vs. WT mice (Table 1). The relative composition of the various phospholipid species in the WT DBA1/J mice lungs after lavage expressed as a % of the total phospholipids was 46, 22, 13, 12, 4, and 3% for PC, phosphatidylethanolamine, phosphatidylserine-phosphatidylinositol, sphingomyelin, lyso-PC, and PG, respectively, and did not differ among mice groups (mean of duplicate determinations from a pool of 3 mice lungs/group). Analysis of the surfactant lavaged from the lungs demonstrated the presence of mild alveolar proteinosis in the ABCA1–/– mouse, as manifested by a 75% increase in the protein content (Table 1, lavage). In addition, the total phospholipid content of the BAL fluid was slightly increased (20%) over that of WT mice, whereas there was no difference in saturated PC levels (Table 1). As demonstrated by the data in Fig. 4, the cholesterol content of the ABCA1–/– mice lungs was enriched. The lung tissue of the ABCA1–/– mouse showed a 1.6-fold increase in the total cholesterol content compared with WT, predominantly due to a marked increase in EC. In the lavage of the diseased animals, there was a 43% enrichment in FC levels compared with WT, with no EC in either animal group.


View this table:
[in this window]
[in a new window]
 
Table 1. Protein and phospholipid content of the wild-type and ABCA1–/– mouse lung

 


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 4. Cholesterol concentration in the ABCA1–/– mouse lung is enriched. Lung tissue (A) and alveolar lavage (B) were extracted, and the cholesterol content was analyzed by gas-liquid chromatography. Total and free cholesterol were measured, and cholesteryl ester (esterified) was determined by subtraction. No cholesteryl ester was found in the alveolar lavage from either group of mice. *Significantly different from WT, P < 0.05, n = 5–6. KO, knockout.

 
To further characterize the surfactant, we isolated and analyzed the composition of the large aggregate fraction. The amount of total phospholipid recovered in the large aggregate fraction as a percentage of the total surfactant phospholipid did not differ between WT (67.3 ± 13.8%, n = 4) and ABCA1–/– (58.2 ± 13.3%, n = 4) mice. In addition, the protein to phospholipid ratios were identical (WT, 0.22 ± 0.01 vs. ABCA1–/–, 0.22 ± 0.06 µg protein/µg phospholipid, n = 4 per group). The relative abundance of SP-A and SP-B in the large aggregate, in relation both to protein or to phospholipid mass, tended to be lower in the ABCA1–/– mice, but the differences did not reach statistical significance. Quantitation of the amount of SP in arbitrary densitometry units normalized to total protein loaded and expressed relative to WT SP run on the same gel showed: for SP-A, WT = 100.0 ± 9.0% (n = 8) vs. ABCA1–/– = 84.1 ± 4.5% (n = 4), P = 0.26, not significant; for SP-B, WT = 100.0 ± 5.2% (n = 4) vs. ABCA1–/– = 84.5 ± 6.7% (n = 4), P = 0.12, not significant.

Cells isolated from ABCA1–/– mice are enriched in cholesterol. Alveolar macrophages (by lavage) and type II cells (by enzymatic digestion, see MATERIALS AND METHODS) were isolated from the WT and ABCA1–/– mice and placed in culture, and the cellular cholesterol content was analyzed. Before culture, the total number of cells in the lung lavage fluid from the WT or ABCA1–/– mice was the same (WT, 1.7 x 105 cells/lung vs. ABCA1–/–, 2.2 x 105 cells/lung, n = 3, P = 0.43, not significant). More than 90% of the cells were macrophages with no difference between WT and ABCA1–/– mice. After 1 h in culture, the macrophages were photographed and counted. Of alveolar macrophages, 8.9 ± 0.9% (n = 3) were derived from ABCA1–/– mice were greatly enlarged and foamy in appearance. Examples of the macrophages are shown in Fig. 5A. The total cholesterol content was 4.8-fold higher in macrophages from ABCA1–/– vs. the WT mice due to a 2.4-fold increase in FC and a 15-fold increase in EC (Fig. 5B).



View larger version (52K):
[in this window]
[in a new window]
 
Fig. 5. Alveolar macrophages from ABCA1–/– mice have an elevated cholesterol content. Alveolar macrophages were isolated from the lavage and placed in culture (1 h). A: phase photographs of macrophages from WT and ABCA1–/– mouse showing large, foamy macrophages in the latter. B: cholesterol content of the macrophages. Macrophages from the plates shown in A were lipid extracted. Total and free cholesterol were analyzed by gas-liquid chromatography, and the cholesteryl ester (esterified) content was determined by subtraction. *Significantly different from WT, n = 3.

 
The type II cells isolated from the lungs of WT or ABCA1–/– mice were placed in culture with 10% serum for 24 h before analysis. Culture may have affected the cholesterol content of the cells but served to remove macrophages. Phase-contrast microscopy did not reveal substantial differences in morphology between the type II cells from the two groups of mice (not shown), a possible indication that many of the abnormal cells did not survive the isolation procedure. Although the FC content did not differ (WT 8.2 ± 0.4 vs. ABCA1–/– 7.8 ± 0.5 µg FC/mg cell protein, not significantly different), the type II cells from ABCA1–/– mice demonstrated an elevation in total cholesterol content (WT 8.2 ± 0.2 vs. ABCA1–/– 10.5 ± 0.2 µg total cholesterol/mg cell protein, P < 0.01) due to the presence of EC (WT 0.0 ± 0.3 vs. ABCA1–/– 2.7 ± 0.6 µg EC/mg cell protein, P < 0.02, n = 3). EC was not detected in the pneumocytes from WT mice.

Lipid clearance. Because of the morphological changes observed in the type II cells and macrophages in the ABCA1–/– mouse and the enrichment in phospholipid content of the lungs, it was possible that clearance of surfactant from mice lungs had been affected. Figure 6, A and B, shows the time course of the clearance of labeled [3H]DPPC liposomes from the lungs of WT mice after intratracheal instillation. The lipid composition of the liposomes was made to reflect that of surfactant (see MATERIALS AND METHODS). [3H]DPPC was cleared exponentially from the alveolar lavage (the surfactant pool, Fig. 6A) and the lungs (Fig. 6B) over the 40-h period. Analysis of the recovery of DPPC dpm after each sequential lavage, shown in Fig. 6A, inset, indicated that five (1 ml) lavages were sufficient to remove the instilled DPPC remaining in the alveolar space. With the use of this same protocol, clearance of [3H]DPPC liposomes from WT and ABCA1–/– mice was compared. Because of the limited numbers of mice available, comparisons were made after 0.5, 16, or 42 h. After 42 h, >90% of the instilled liposomes had been cleared from the alveolar space, and >75% had been removed from the total lung (Fig. 7, A and B). Although clearance of liposomes tended to be more rapid in the ABCA1–/– mice, differences in the clearance of DPPC between WT and ABCA1–/– mice as a % of instilled liposomes in the lavage or total lung did not reach statistical significance at any time point. However, since there was more phospholipid in the lavage of ABCA1–/– mice, the net clearance of phospholipid from the lavage of the gene-targeted mice lungs was significantly greater than the WT mice (Fig. 7C).



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 6. Time course of clearance of [choline-methyl-3H]DPPC ([3H]DPPC) liposomes by WT mice. [3H]DPPC liposomes (10 nmol) were instilled intratracheally into anesthetized mice. At the indicated time, the lungs were removed, lavaged, and homogenized (31). Aliquots of lung were lavaged and analyzed for radioactivity. A: bronchoalveolar lavage. Inset: recovery of DPPC dpm after each sequential lavage as a % of total lavage dpm. B: total lung = lung homogenate + lavage. Data are means ± range, n = 2 mice per data point and are % of instilled dpm remaining in the lung.

 


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 7. Lipid clearance of [3H]DPPC liposomes by WT and ABCA1–/– mice. The experimental protocol was identical to Fig. 6, A and B. Data are dpm of [3H]DPPC as % of instilled and are means ± SE, n = 3–4 mice per data point except ABCA1–/– at 16 h where n = 2. There were no significant differences between WT and ABCA1–/– mice. C: data are micrograms of phospholipid (PL)/lung and are means ± SE for the same animals in A and B. The mass uptake of PL from the lung can be calculated using the dpm from the liposomes and the microgram of PL in the lavage and assuming an even distribution of liposomes with the surfactant pool of the lung. Equal distribution of liposomes throughout the lung using our procedure has been demonstrated (18). *Significantly different from WT.

 
Respiratory characteristics. To determine whether the absence of ABCA1 affected respiratory parameters during normal breathing or with hyperventilation, whole body plethysmography (Buxco Electronics, Ref. 31) was performed. Frequency of breathing, tidal volume (TV), and minute ventilation were recorded at 5-min intervals for 30 min during exposure either to air or 5% CO2. In air, the ABCA1–/– mice demonstrated respiratory distress due to lower TV (Fig. 8A) and higher respiratory frequency (Fig. 8B) compared with WT, as exemplified by the increase in the rapid shallow breathing index (frequency/TV; Fig. 8D) in the ABCA1–/– mice. The end result was similar minute ventilation between the WT and ABCA1–/– mice (Fig. 8C). Challenge with 5% CO2 resulted in hyperventilation in both types of mice characterized by a greater TV and an elevation in minute ventilation, although the change in TV in WT mice was greater than in ABCA1–/– mice (4.4 ± 0.3 WT vs. 2.7 ± 0.2 ABCA1–/–, µl/g mouse, P < 0.05, n = 4).



View larger version (28K):
[in this window]
[in a new window]
 
Fig. 8. Respiratory parameters of WT or ABCA1–/– mice. Parameters were measured during a 30-min exposure to air or 5% CO2 in air. A: tidal volume. B: frequency of breaths. C: minute ventilation = tidal volume x frequency. D: rapid shallow breathing index = frequency/tidal volume. Where appropriate, data are expressed per killigram of mouse and are shown as medians (lines) and 25th-75th percentile (boxes). The ranges (whiskers) are within the boxes. *Significantly different from WT. #Significantly different from air. N = 4 mice/group.

 

    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The important contribution of cholesterol to the physiological properties of lung surfactant in the reduction in surface tension is well known (37, 57). A major role for ABCA1 in the cholesterol metabolism of the lung has been suggested by the substantial levels of ABCA1 expression in pneumocytes (9, 38, 65), the role of ABCA1 in cholesterol efflux (49), and the observed abnormalities in the lungs of ABCA1–/– mice (47). The aim of this study was to perform a detailed evaluation of the morphology and surfactant metabolism of ABCA1-deficient mice to identify the importance of reverse cholesterol transport to lung function. We found that the lack of ABCA1 resulted in focal pathology of the lung, abnormalities in the lung and surfactant lipid content, accumulation of cholesterol in the tissue, surfactant, and isolated cells, greater lipid clearance from the alveoli, and abnormal respiratory characteristics. Our findings support an important role for ABCA1 in lung lipid homeostasis.

Changes in the phospholipid content of tissue and surfactant in 4-mo-old ABCA1–/– mice examined in this report, although significant, were modest. Pathological changes were only focal, although alveolar proteinosis, indicative of loss of alveolar capillary membrane integrity, was observed. However, minor differences over time can result in more severe disease as demonstrated by the more significant pathology seen in the 12-mo-old ABCA1–/– animals (47) compared with our younger mice. Age-dependent changes in severity of pathology also have been demonstrated in ABCA1 and apoE double KO mice as well as SP-D-deficient mice (1, 29).

In the ABCA1-deficient mice, plasma lipids and lipoproteins are substantially reduced due to the severe depression of HDL (47). Serum apolipoprotein B and LDL cholesterol are also low, whereas VLDL concentrations remain unchanged. The lack of normal cholesterol and phospholipid removal from the lung due to the absence of ABCA1 provides a further insult. The remarkable ability of the lung to maintain fairly normal cholesterol and phospholipid levels in the face of these conditions is impressive. It has been shown that depressing the hepatic secretion of all serum lipoproteins for 3 days with 4-aminopyrazolo(3,4d)pyrimidine results in a parallel reduction of phospholipids in both the surfactant and lamellar body fractions, whereas cholesterol content remains unaltered (15, 58). However, despite the profound reduction in plasma phospholipid and cholesterol over the lifetime of the ABCA1–/– mouse, there was a moderate enrichment in phospholipid levels in lung tissue and a significant cholesterol enrichment accompanied by pathological alterations. Thus, while lowering all serum lipoprotein lipids for a brief period produced a reduction in lung phospholipid in other studies, the retention of VLDL and the lack of the biological functioning of ABCA1 allowed for maintenance of fairly normal lung phospholipids in the ABCA1 gene-targeted mice.

It also was surprising that the lung tissue phospholipid species did not differ between the groups given the notable differences in the plasma phospholipid levels and composition in the ABCA1–/– mice (21). However, the ability of the lung to maintain proper pulmonary phospholipid composition under stress, such as food deprivation, has been observed previously (12). Thus it seems that alternate pathways are compensating to control lung lipid homeostasis and may explain the lack of reports of respiratory abnormalities in TD patients. The type II cell hyperplasia observed may function to provide additional cells for the production of surfactant lipids. Besides lipid synthesis, the lung may also be upregulating other lipoprotein receptors to maximize the use of the low levels of circulating lipoproteins remaining. The lung does contain substantial levels of other members of the ABC superfamily, ABCG1 (32) and ABCA7 (62), as well as scavenger receptor class B type I (36) and the LDL receptor (60), all known to play a role in lipid turnover.

Although the pathways responsible for the enrichment in tissue phospholipid levels seen in the present study are unclear, candidates include increased phospholipid uptake, decreased phospholipid secretion, blocked degradation, or increased phospholipid synthesis. Our data on the clearance of instilled phospholipid liposomes from the alveoli indicate that the ABCA1–/– lung not only maintains similar rates of surfactant phospholipid clearance on a % basis as WT but actually removes more phospholipids/lung than seen with WT mice lungs. The normal SP-A and SP-B and phospholipid levels in the alveolar space would argue that SP production and surfactant secretion have not been compromised. As for the synthetic pathway, it has been shown that lipoprotein deprivation of type II epithelial-like cells in culture or removal of lipid from the diet of mice results in an increase in surfactant phosphatidylcholine production through stimulation of CTP:phosphocholine cytidylyltransferase (CCT), the rate limiting enzyme in phospholipid synthesis (45, 54). In fact, overexpression of CCT in mice resulted in an increase in tissue and total disaturated phospholipid content (41) similar to that seen in the ABCA1–/– mice (25%), supporting the hypothesis that enhancement of lipid biosynthesis likely contributes to the observed elevated lung phospholipid content.

In the present report, ABCA1–/– mice, with only modest changes in lung lipids, demonstrated rapid shallow breathing without challenge. Upon exposure to 5% CO2, the mice were unable to increase lung TV to the same extent as WT. The rapid shallow breathing is compatible with an infiltrative or obliterative lung disease, but not diagnostic, as there are many other causes. Whether respiratory distress was due to altered lung lipids or to other factors or organ systems that were affected by the lack of ABCA1 remains to be determined.

One of the most dramatic affects of the absence of ABCA1 in mice or in TD patients is the development of cholesterol accumulation in tissue and peritoneal macrophages resulting in "foamy" macrophages (14, 20, 47). Although tissue macrophages, which reside in lung parenchyma, and alveolar macrophages, isolated from the alveolar space, share many functional properties, they exhibit some quantitative differences in cytokine and reactive oxygen species production in response to stimuli and in metabolism of SP-A after priming (40, 42). As demonstrated in this report, alveolar macrophages isolated by BAL from the ABCA1 gene-targeted mice had a similar response as other types of macrophages, an enlargement in size, and an enrichment in intracellular cholesterol with lipid droplets. Cholesterol loading may affect the function of the alveolar macrophages in view of the recent report that the cholesteryl ester-enriched peritoneal macrophages isolated from ABCA1-deficient mice demonstrated elevated accumulation of cholesterol from altered LDL, probably due to a high expression of several scavenger receptors (20). In addition, the ABCA1-deficient peritoneal macrophages showed enhanced responsiveness to chemotactic factors compared with WT macrophages (20).

The cholesterol content of type II pneumocytes isolated from the lungs of the ABCA1–/– mice and placed in culture for 24 h in fetal calf serum was composed of 25% EC relative to the total cholesterol content. Although it is possible that the minor macrophage contamination of the primary type II cell cultures (<5%) contributed to the cholesteryl ester content of the type II cells, the lack of change in type II cell FC content in the face of a doubling of macrophage FC content makes a significant contribution of macrophages to the data unlikely. To our knowledge, this is the first report of the cholesterol content of a cell type freshly isolated from the ABCA1–/– mouse, other than macrophages. One of the striking features of type II cells in intact lungs of ABCA1–/– mice was the presence of spherical, nonmembrane-bound cytoplasmic organelles containing amorphous material, typical in appearance to lipid bodies (48). In other cell types, lipid bodies represent a site for cholesteryl ester or lipid storage as is the case for smooth muscle cells (66) and macrophages (13) and may be the case for type II cells. Few are present in pneumocytes under normal conditions but are induced in response to stress such as ischemia and are felt to be the site of eicosanoid mediator synthesis (48). The role of lipid bodies in the type II cells of the lungs from ABCA1–/– mice is unknown.

Since the first report on ABCA1-deficient DBA1/J mouse, recent surveys of other organs have identified further abnormalities. Examination of the testis revealed accumulation of lipid droplets in Sertoli cells, resulting in reduced sperm count and testosterone levels, and was associated with a decrease in male fertility (55). The central nervous system of ABCA1–/– mice was dramatically affected by reduced apolipoprotein E levels in the brain and cerebrospinal fluid. Isolated astrocytes accumulated lipid and secreted abnormal nascent lipoproteins (27, 61). Our current findings of abnormal lung lipid content in the ABCA1–/– mice lungs support previous reports of lung damage (47) and add to the growing list of organ systems that rely on the activity of ABCA1 to maintain normal lipid turnover and organ function.

In summary, we demonstrate ABCA1 plays a role in lipid homeostasis of the lung. The pulmonary system of mice lacking ABCA1 shows abnormal morphology and physiology, with respiratory distress, alveolar proteinosis, and cholesterol enrichment of tissue, surfactant, and macrophages. Although the observed pathology may not be due solely to alterations in surfactant metabolism, results suggest that the ABCA1-dependent processes involved in reverse cholesterol transport are important for the maintenance of normal lung lipid composition, structure, and function.


    GRANTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
These studies were supported by National Heart, Lung, and Blood Institute Grants HL-22633 and HL-63768 and American Heart Association Grant 0355695U (S. R. Bates).


    ACKNOWLEDGMENTS
 
The authors thank Drs. Elena Atochina and Dr. Michael Beers for assistance with the lung plethysmography and SP analysis, Drs. Beers and Dr. Susan Guttentag for the supply of anti-SP-B antibodies, Dr. Dominique Brees and Germaine Boucher for the light microscopy, Kevin Yu for the electron microscopy, and Dr. Aron B. Fisher for helpful discussions.


    FOOTNOTES
 

Address for reprint requests and other correspondence: S. R. Bates, 1 John Morgan Bldg., Institute for Environmental Medicine, 3620 Hamilton Walk, Univ. of Pennsylvania, Philadelphia, PA 19104 (e-mail: batekenn{at}mail.med.upenn.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Aiello RJ, Brees D, Bourassa PA, Royer L, Lindsey S, Coskran T, Haghpassand M, and Francone OL. Increased atherosclerosis in hyperlipidemic mice with inactivation of ABCA1 in macrophages. Arterioscler Thromb Vasc Biol 22: 630–637, 2002.[Abstract/Free Full Text]
  2. Aiello RJ, Brees D, and Francone OL. ABCA1-deficient mice: insights into the role of monocyte lipid efflux in HDL formation and inflammation. Arterioscler Thromb Vasc Biol 23: 972–980, 2003.[Abstract/Free Full Text]
  3. Bartlett G. Phosphorus assay in column chromatography. J Biol Chem 234: 466–468, 1959.[Free Full Text]
  4. Bates SR and Fisher AB. Surfactant protein A is degraded by alveolar macrophages. Am J Physiol Lung Cell Mol Physiol 271: L258–L266, 1996.[Abstract/Free Full Text]
  5. Bates SR, Gonzales LW, Tao JQ, Rueckert P, Ballard PL, and Fisher AB. Recovery of rat type II cell surfactant components during primary cell culture. Am J Physiol Lung Cell Mol Physiol 282: L267–L276, 2002.[Abstract/Free Full Text]
  6. Bates SR, Ibach PB, and Fisher AB. Phospholipids co-isolated with rat surfactant protein C account for the apparent protein-enhanced uptake of liposomes into lung granular pneumocytes. Exp Lung Res 15: 695–708, 1989.[ISI][Medline]
  7. Bligh EG and Dyer WJ. A rapid method of total lipid extraction and purification. Can J Med Sci 37: 911–917, 1959.[Medline]
  8. Bodzioch M, Orso E, Klucken J, Langmann T, Bottcher A, Diederich W, Drobnik W, Barlage S, Buchler C, Porsch-Ozcurumez M, Kaminski WE, Hahmann HW, Oette K, Rothe G, Aslanidis C, Lackner KJ, and Schmitz G. The gene encoding ATP-binding cassette transporter 1 is mutated in Tangier disease. Nat Genet 22: 347–351, 1999.[CrossRef][ISI][Medline]
  9. Bortnick AE, Favari E, Tao JQ, Francone OL, Reilly M, Zhang Y, Rothblat GH, and Bates SR. Identification and characterization of rodent ABCA1 in isolated type II pneumocytes. Am J Physiol Lung Cell Mol Physiol 285: L869–L878, 2003.[Abstract/Free Full Text]
  10. Bradford M. A rapid and sensitive method for quantitation of microgram quantities of proteins utilizing the principle of protein dye binding. Anal Biochem 72: 248–254, 1976.[CrossRef][ISI][Medline]
  11. Brooks-Wilson A, Marcil M, Clee SM, Zhang LH, Roomp K, van Dam M, Yu L, Brewer C, Collins JA, Molhuizen HO, Loubser O, Ouelette BF, Fichter K, Ashbourne-Excoffon KJ, Sensen CW, Scherer S, Mott S, Denis M, Martindale D, Frohlich J, Morgan K, Koop B, Pimstone SN, Kastelein JJ, Genest J, and Hayden MR. Mutations in ABC1 in Tangier disease and familial high-density lipoprotein deficiency. Nat Genet 22: 336–345, 1999.[CrossRef][ISI][Medline]
  12. Brown LA, Bliss AS, and Longmore WJ. Effect of nutritional status on the lung surfactant system: food deprivation and caloric restriction. Exp Lung Res 6: 133–147, 1984.[ISI][Medline]
  13. Brown MS, Goldstein JL, Krieger M, Ho YK, and Anderson RG. Reversible accumulation of cholesteryl esters in macrophages incubated with acetylated lipoproteins. J Cell Biol 82: 597–613, 1979.[Abstract]
  14. Christiansen-Weber TA, Voland JR, Wu Y, Ngo K, Roland BL, Nguyen S, Peterson PA, and Fung-Leung WP. Functional loss of ABCA1 in mice causes severe placental malformation, aberrant lipid distribution, and kidney glomerulonephritis as well as high-density lipoprotein cholesterol deficiency. Am J Pathol 157: 1017–1029, 2000.[Abstract/Free Full Text]
  15. Davidson KG, Acton SM, Barr HA, and Nicholas TE. Effect of lowering serum cholesterol on the composition of surfactant in adult rat lung. Am J Physiol Lung Cell Mol Physiol 272: L106–L114, 1997.[Abstract/Free Full Text]
  16. Fielding CJ and Fielding PE. Molecular physiology of reverse cholesterol transport. J Lipid Res 36: 211–228, 1995.[Abstract]
  17. Fisher AB and Dodia C. Lysosomal-type PLA2 and turnover of alveolar DPPC. Am J Physiol Lung Cell Mol Physiol 280: L748–L754, 2001.[Abstract/Free Full Text]
  18. Fisher AB, Dodia C, and Chander A. {beta}-Adrenergic mediators increase pulmonary retention of instilled phospholipids. J Appl Physiol 59: 743–748, 1985.[Abstract/Free Full Text]
  19. Francis GA, Knopp RH, and Oram JF. Defective removal of cellular cholesterol and phospholipids by apolipoprotein A-I in Tangier Disease. J Clin Invest 96: 78–87, 1995.[ISI][Medline]
  20. Francone OL, Royer L, Boucher G, Haghpassand M, Freeman A, Brees D, and Aiello RJ. Increased cholesterol deposition, expression of scavenger receptors, and response to chemotactic factors in Abca1-deficient macrophages. Arterioscler Thromb Vasc Biol 25: 1198–1205, 2005.[Abstract/Free Full Text]
  21. Francone OL, Subbaiah PV, van Tol A, Royer L, and Haghpassand M. Abnormal phospholipid composition impairs HDL biogenesis and maturation in mice lacking Abca1. Biochemistry 42: 8569–8578, 2003.[CrossRef][ISI][Medline]
  22. Gross NJ and Narine KR. Surfactant subtypes in mice: characterization and quantitation. J Appl Physiol 66: 342–349, 1989.[Abstract/Free Full Text]
  23. Guthmann F, Harrach-Ruprecht B, Looman AC, Stevens PA, Robenek H, and Rustow B. Interaction of lipoproteins with type II pneumocytes in vitro: morphological studies, uptake kinetics and secretion rate of cholesterol. Eur J Cell Biol 74: 197–207, 1997.[ISI][Medline]
  24. Guthmann F, Haupt R, Schlame M, Stevens PA, and Rustow B. Alveolar surfactant subfractions differ in their lipid composition. Int J Biochem Cell Biol 27: 1021–1026, 1995.[CrossRef][ISI][Medline]
  25. Guttentag SH, Beers MF, Bieler BM, and Ballard PL. Surfactant protein B processing in human fetal lung. Am J Physiol Lung Cell Mol Physiol 275: L559–L566, 1998.[Abstract/Free Full Text]
  26. Hass MA and Longmore WJ. Regulation of lung surfactant cholesterol metabolism by serum lipoproteins. Lipids 15: 401–406, 1980.[ISI][Medline]
  27. Hirsch-Reinshagen V, Zhou S, Burgess BL, Bernier L, McIsaac SA, Chan JY, Tansley GH, Cohn JS, Hayden MR, and Wellington CL. Deficiency of ABCA1 impairs apolipoprotein E metabolism in brain. J Biol Chem 279: 41197–41207, 2004.[Abstract/Free Full Text]
  28. Ikegami M, Ueda T, Hull W, Whitsett JA, Mulligan RC, Dranoff G, and Jobe AH. Surfactant metabolism in transgenic mice after granulocyte macrophage-colony stimulating factor ablation. Am J Physiol Lung Cell Mol Physiol 270: L650–L658, 1996.[Abstract/Free Full Text]
  29. Ikegami M, Whitsett JA, Jobe A, Ross G, Fisher J, and Korfhagen T. Surfactant metabolism in SP-D gene-targeted mice. Am J Physiol Lung Cell Mol Physiol 279: L468–L476, 2000.[Abstract/Free Full Text]
  30. Ishikawa TT, MacGee J, Morrison JA, and Glueck CJ. Quantitative analysis of cholesterol in 5 to 20 microliter of plasma. J Lipid Res 15: 286–291, 1974.[Abstract/Free Full Text]
  31. Jain D, Dodia C, Bates SR, Hawgood S, Poulain FR, and Fisher AB. SP-A is necessary for increased clearance of alveolar DPPC with hyperventilation or secretagogues. Am J Physiol Lung Cell Mol Physiol 284: L759–L765, 2003.[Abstract/Free Full Text]
  32. Kaplan R, Gan X, Menke JG, Wright SD, and Cai TQ. Bacterial lipopolysaccharide induces expression of ABCA1 but not ABCG1 via an LXR-independent pathway. J Lipid Res 43: 952–959, 2002.[Abstract/Free Full Text]
  33. King RJ. Pulmonary surfactant. J Appl Physiol 53: 1–8, 1982.[Abstract/Free Full Text]
  34. King RJ and Clements JA. Surface active materials from dog lung. II. Composition and physiological correlations. Am J Physiol 223: 715–726, 1972.[Free Full Text]
  35. Klansek JJ, Yancey P, St. Clair RW, Fischer RT, Johnson WJ, and Glick JM. Cholesterol quantitation by GLC: artifactual formation of short-chain steryl esters. J Lipid Res 36: 2261–2266, 1995.[Abstract]
  36. Kolleck I, Schlame M, Fechner H, Looman AC, Wissel H, and Rustow B. HDL is the major source of vitamin E for type II pneumocytes. Free Radic Biol Med 27: 882–890, 1999.[CrossRef][ISI][Medline]
  37. Ladbrooke BD, Williams RM, and Chapman D. Studies on lecithin-cholesterol-water interactions by differential scanning calorimetry and X-ray diffraction. Biochim Biophys Acta 150: 333–340, 1968.[ISI][Medline]
  38. Lawn RM, Wade DP, Couse TL, and Wilcox JN. Localization of human ATP-binding cassette transporter 1 (ABC1) in normal and atherosclerotic tissues. Arterioscler Thromb Vasc Biol 21: 378–385, 2001.[Abstract/Free Full Text]
  39. Lawn RM, Wade DP, Garvin MR, Wang X, Schwartz K, Porter JG, Seilhamer JJ, Vaughan AM, and Oram JF. The Tangier disease gene product ABC1 controls the cellular apolipoprotein-mediated lipid removal pathway. J Clin Invest 104: R25–R31, 1999.[ISI][Medline]
  40. Lehnert BE, Valdez YE, and Holland LM. Pulmonary macrophages: alveolar and interstitial populations. Exp Lung Res 9: 177–190, 1985.[ISI][Medline]
  41. Li J, Marsh JJ, and Spragg RG. Effect of CTP:phosphocholine cytidylyltransferase overexpression on the mouse lung surfactant system. Am J Respir Cell Mol Biol 26: 709–715, 2002.[Abstract/Free Full Text]
  42. Lohmann-Matthes M, Steinmuller C, and Franke-Ullmann G. Pulmonary macrophages. Eur Respir J 7: 1678–1689, 1994.[Abstract/Free Full Text]
  43. Lowry OH, Rosebrough NJ, Farr LA, and Randall RJ. Protein measurement with Folin phenol reagent. J Biol Chem 193: 265–275, 1951.[Free Full Text]
  44. Mallampalli RK, Ryan AJ, Carroll JL, Osborne TF, and Thomas CP. Lipid deprivation increases surfactant phosphatidylcholine synthesis via a sterol-sensitive regulatory element within the CTP:phosphocholine cytidylyltransferase promoter. Biochem J 362: 81–88, 2002.[CrossRef][ISI][Medline]
  45. Mason RJ, Nellenbogen J, and Clements JA. Isolation of disaturated phosphatidylcholine with osmium tetroxide. J Lipid Res 17: 281–284, 1976.[Abstract]
  46. McNeish J, Aiello RJ, Guyot D, Turi T, Gabel C, Aldinger C, Hoppe KL, Roach ML, Royer LJ, de Wet J, Broccardo C, Chimini G, and Francone OL. High density lipoprotein deficiency and foam cell accumulation in mice with targeted disruption of ATP-binding cassette transporter-1. Proc Natl Acad Sci USA 97: 4245–4250, 2000.[Abstract/Free Full Text]
  47. Ochs M, Fehrenbach H, and Richter J. Occurrence of lipid bodies in canine type II pneumocytes during hypothermic lung ischemia. Anat Rec 277A: 287–297, 2004.[CrossRef]
  48. Oram JF. ATP-binding cassette transporter A1 and cholesterol trafficking. Curr Opin Lipidol 13: 373–381, 2002.[CrossRef][ISI][Medline]
  49. Orso E, Broccardo C, Kaminski WE, Bottcher A, Liebisch G, Drobnik W, Gotz A, Chambenoit O, Diederich W, Langmann T, Spruss T, Luciani MF, Rothe G, Lackner KJ, Chimini G, and Schmitz G. Transport of lipids from golgi to plasma membrane is defective in tangier disease patients and Abc1-deficient mice. Nat Genet 24: 192–196, 2000.[CrossRef][ISI][Medline]
  50. Pietra GG, Spagnoli LG, Capuzzi DM, Sparks CE, Fishman AP, and Marsh JB. Metabolism of 125I-labeled lipoproteins by the isolated rat lung. J Cell Biol 70: 33–46, 1976.[Abstract]
  51. Remaley AT, Schumacher UK, Stonik JA, Farsi BD, Nazih H, and Brewer HB Jr. Decreased reverse cholesterol transport from Tangier disease fibroblasts. Acceptor specificity and effect of brefeldin on lipid efflux. Arterioscler Thromb Vasc Biol 17: 1813–1821, 1997.[Abstract/Free Full Text]
  52. Rust S, Rosier M, Funke H, Real J, Amoura Z, Piette JC, Deleuze JF, Brewer HB, Duverger N, Denefle P, and Assmann G. Tangier disease is caused by mutations in the gene encoding ATP-binding cassette transporter 1. Nat Genet 22: 352–355, 1999.[CrossRef][ISI][Medline]
  53. Ryan AJ, McCoy DM, Mathur SN, Field FJ, and Mallampalli RK. Lipoprotein deprivation stimulates transcription of the CTP:phosphocholine cytidylyltransferase gene. J Lipid Res 41: 1268–1277, 2000.[Abstract/Free Full Text]
  54. Selva DM, Hirsch-Reinshagen V, Burgess B, Zhou S, Chan J, McIsaac S, Hayden MR, Hammond GL, Vogl AW, and Wellington CL. The ATP-binding cassette transporter 1 mediates lipid efflux from Sertoli cells and influences male fertility. J Lipid Res 45: 1040–1050, 2004.[Abstract/Free Full Text]
  55. Sparks CE, Dehoff JL, Capuzzi DM, Pietra G, and Marsh JB. Proteolysis of very low density lipoprotein in perfused lung. Biochim Biophys Acta 529: 123–130, 1978.[ISI][Medline]
  56. Suzuki Y. Effect of protein, cholesterol, and phosphatidylglycerol on the surface activity of the lipid-protein complex reconstituted from pig pulmonary surfactant. J Lipid Res 23: 62–69, 1982.[Abstract]
  57. Suzuki Y and Tabata R. Selective reduction of phosphatidylglycerol and phosphatidylcholine in pulmonary surfactant by 4-aminopyrazolo(3,4d)pyrimidine in the rat. J Lipid Res 21: 1090–1096, 1980.[Abstract]
  58. Towbin H, Staehelin T, and Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76: 4350–4354, 1979.[Abstract/Free Full Text]
  59. Voyno-Yasenetskaya TA, Dobbs LG, Erickson SK, and Hamilton RL. Low density lipoprotein- and high density lipoprotein-mediated signal transduction and exocytosis in alveolar type II cells. Proc Natl Acad Sci USA 90: 4256–4260, 1993.[Abstract/Free Full Text]
  60. Wahrle SE, Jiang H, Parsadanian M, Legleiter J, Han X, Fryer JD, Kowalewski T, and Holtzman DM. ABCA1 is required for normal central nervous system ApoE levels and for lipidation of astrocyte-secreted apoE. J Biol Chem 279: 40987–40993, 2004.[Abstract/Free Full Text]
  61. Wang N, Lan D, Gerbod-Giannone M, Linsel-Nitschke P, Jehle AW, Chen W, Martinez LO, and Tall AR. ATP-binding cassette transporter A7 (ABCA7) binds apolipoprotein A-I and mediates cellular phospholipid but not cholesterol efflux. J Biol Chem 278: 42906–42912, 2003.[Abstract/Free Full Text]
  62. Warshamana GS, Corti M, and Brody AR. TNF-{alpha}, PDGF, and TGF-{beta}1 expression by primary mouse bronchiolar-alveolar epithelial and mesenchymal cells: TNF-{alpha} induces TGF-{beta}1. Exp Mol Pathol 71: 13–33, 2001.[CrossRef][ISI][Medline]
  63. Weaver TE and Whitsett JA. Function and regulation of expression of pulmonary surfactant-associated proteins. Biochem J 273: 249–264, 1991.[ISI][Medline]
  64. Wellington CL, Walker EK, Suarez A, Kwok A, Bissada N, Singaraja R, Yang YZ, Zhang LH, James E, Wilson JE, Francone O, McManus BM, and Hayden MR. ABCA1 mRNA and protein distribution patterns predict multiple different roles and levels of regulation. Lab Invest 82: 273–283, 2002.[CrossRef][ISI][Medline]
  65. Wolfbauer G, Glick JM, Minor LK, and Rothblat GH. Development of the smooth muscle foam cell: uptake of macrophage lipid inclusions. Proc Natl Acad Sci USA 83: 7760–7764, 1986.[Abstract/Free Full Text]




This Article
Abstract
Full Text (PDF)
All Versions of this Article:
289/6/L980    most recent
00234.2005v1
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Google Scholar
Articles by Bates, S. R.
Articles by Rothblat, G. H.
PubMed
PubMed Citation
Articles by Bates, S. R.
Articles by Rothblat, G. H.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2005 by the American Physiological Society.