ABCA1 and Scavenger Receptor Class B, Type I, Are Modulators of Reverse Sterol Transport at an in Vitro Blood-Brain Barrier Constituted of Porcine Brain Capillary Endothelial Cells
2002; Elsevier BV; Volume: 277; Issue: 45 Linguagem: Inglês
10.1074/jbc.m207601200
ISSN1083-351X
AutoresUte Panzenboeck, Zoltán Balázs, Andrea Sovic, Andelko Hrzenjak, Sanja Levak‐Frank, Andrea Wintersperger, Ernst Malle, Wolfgang Sattler,
Tópico(s)Peroxisome Proliferator-Activated Receptors
ResumoThe objective of the present study was to investigate the involvement of key players in reverse cholesterol/24(S)OH-cholesterol transport in primary porcine brain capillary endothelial cells (pBCEC) that constitute the BBB. We identified that, in addition to scavenger receptor class B, type I (SR-BI), pBCEC express ABCA1 and apolipoprotein A-I (apoA-I) mRNA and protein. Studies on the regulation of ABCA1 by the liver X receptor agonist 24(S)OH-cholesterol revealed increased ABCA1 expression and apoA-I-dependent [3H]cholesterol efflux from pBCEC. In unpolarized pBCEC, high density lipoprotein, subclass 3 (HDL3)-dependent [3H]cholesterol efflux, was unaffected by 24(S)OH-cholesterol treatment but was enhanced 5-fold in SR-BI overexpressing pBCEC. Efflux of cellular 24(S)-[3H]OH-cholesterol was highly efficient, independent of ABCA1, and correlated with SR-BI expression. Polarized pBCEC were cultured on porous membrane filters that allow separate access to the apical and the basolateral compartment. Addition of cholesterol acceptors to the apical compartment resulted in preferential [3H]cholesterol efflux to the basolateral compartment. HDL3 was a better promoter of basolateral [3H]cholesterol efflux than lipid-free apoA-I. Basolateral pretreatment with 24(S)OH-cholesterol enhanced apoA-I-dependent basolateral cholesterol efflux up to 2-fold along with the induction of ABCA1 at the basolateral membrane. Secretion of apoA-I also occurred preferentially to the basolateral compartment, where the majority of apoA-I was recovered in an HDL-like density range. In contrast, 24(S)-[3H]OH-cholesterol was mobilized efficiently to the apical compartment of the in vitro BBB by HDL3, low density lipoprotein, and serum. These results suggest the existence of an autoregulatory mechanism for removal of potentially neurotoxic 24(S)OH-cholesterol. In conclusion, the apoA-I/ABCA1- and HDL/SR-BI-dependent pathways modulate polarized sterol mobilization at the BBB. The objective of the present study was to investigate the involvement of key players in reverse cholesterol/24(S)OH-cholesterol transport in primary porcine brain capillary endothelial cells (pBCEC) that constitute the BBB. We identified that, in addition to scavenger receptor class B, type I (SR-BI), pBCEC express ABCA1 and apolipoprotein A-I (apoA-I) mRNA and protein. Studies on the regulation of ABCA1 by the liver X receptor agonist 24(S)OH-cholesterol revealed increased ABCA1 expression and apoA-I-dependent [3H]cholesterol efflux from pBCEC. In unpolarized pBCEC, high density lipoprotein, subclass 3 (HDL3)-dependent [3H]cholesterol efflux, was unaffected by 24(S)OH-cholesterol treatment but was enhanced 5-fold in SR-BI overexpressing pBCEC. Efflux of cellular 24(S)-[3H]OH-cholesterol was highly efficient, independent of ABCA1, and correlated with SR-BI expression. Polarized pBCEC were cultured on porous membrane filters that allow separate access to the apical and the basolateral compartment. Addition of cholesterol acceptors to the apical compartment resulted in preferential [3H]cholesterol efflux to the basolateral compartment. HDL3 was a better promoter of basolateral [3H]cholesterol efflux than lipid-free apoA-I. Basolateral pretreatment with 24(S)OH-cholesterol enhanced apoA-I-dependent basolateral cholesterol efflux up to 2-fold along with the induction of ABCA1 at the basolateral membrane. Secretion of apoA-I also occurred preferentially to the basolateral compartment, where the majority of apoA-I was recovered in an HDL-like density range. In contrast, 24(S)-[3H]OH-cholesterol was mobilized efficiently to the apical compartment of the in vitro BBB by HDL3, low density lipoprotein, and serum. These results suggest the existence of an autoregulatory mechanism for removal of potentially neurotoxic 24(S)OH-cholesterol. In conclusion, the apoA-I/ABCA1- and HDL/SR-BI-dependent pathways modulate polarized sterol mobilization at the BBB. high density lipoproteins ATP-binding cassette transporter A1 apolipoprotein A-I blood-brain barrier diisothiocyanostilbene-2,2-disulfonic acid low density lipoproteins liver X receptor porcine brain capillary endothelial cells phosphate-buffered saline reverse cholesterol transport retinoid X receptor scavenger receptor class B, type I horseradish peroxidase human umbilical vein endothelial cells reverse transcriptase In the past few years substantial evidence has accumulated for some neurodegenerative disorders being tightly coupled to lipid and/or lipoprotein metabolism in the peripheral circulation. At the same time it has become generally accepted that high density lipoproteins (HDL)1 protect against atherosclerosis and possibly against neurodegenerative diseases by modulating sterol flux. For instance, a defect in intracellular cholesterol trafficking might be etiologically important in progressive neurodegeneration observed in Niemann-Pick type C disease (1Xie C. Turley S.D. Dietschy J.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11992-11997Crossref PubMed Scopus (83) Google Scholar). Studies performed in cell culture, animal models, and on human post-mortem material indicate that cholesterol is a major determinant affecting the severity of Alzheimer's disease and the deposition of intraneuronal amyloid β (reviewed in Ref. 2Sparks D.L. Martin T.A. Gross D.R. Hunsaker III, J.C. Microsc. Res. Tech. 2000; 50: 287-290Crossref PubMed Scopus (130) Google Scholar). In line with this, the outcome of retrospective studies demonstrated a strong decrease in the incidence of Alzheimer's disease and dementia for patients that were treated with statins, inhibitors of endogenous cholesterol biosynthesis (3Wolozin B. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5371-5373Crossref PubMed Scopus (177) Google Scholar,4Jick H. Zornberg G.L. Jick S.S. Seshadri S. Drachman D.A. Lancet. 2000; 356: 1627-1631Abstract Full Text Full Text PDF PubMed Scopus (1563) Google Scholar). Moreover, decreased serum HDL cholesterol and apolipoprotein A-I (apoA-I) concentrations correlate with the severity of Alzheimer's disease (5Merched A. Xia Y. Visvikis S. Serot J.M. Siest G. Neurobiol. Aging. 2000; 21: 27-30Crossref PubMed Scopus (206) Google Scholar). Several subtypes of neuropathies observed in patients suffering Tangier disease, a disorder characterized by severe deficiency or the absence of circulating HDL, further underline the importance of functional HDL metabolism for normal function of the central nervous system. Tangier disease is caused by mutations in the ATP-binding cassette transporter (ABC) A1 gene, and the absence of HDL is because of defective assembly of cholesterol and phospholipids with apoA-I (reviewed in Refs. 6Schmitz G. Langmann T. Curr. Opin. Lipidol. 2001; 12: 129-140Crossref PubMed Scopus (179) Google Scholar and 7Santamarina-Fojo S. Remaley A.T. Neufeld E.B. Brewer Jr., H.B. J. Lipid Res. 2001; 42: 1339-1345Abstract Full Text Full Text PDF PubMed Google Scholar). One of the most striking differences between cholesterol metabolism in the brain and the periphery is the slow turnover of cerebral cholesterol, accounting for 0.1–1% of the turnover observed in the periphery (8Dietschy J.M. Turley S.D. Curr. Opin. Lipidol. 2001; 12: 105-112Crossref PubMed Scopus (705) Google Scholar). Because the blood-brain barrier (BBB) restricts exchange with plasma lipoproteins, the brain covers a major part of its own cholesterol demand by de novo synthesis (9Turley S.D. Burns D.K. Dietschy J.M. Am. J. Physiol. 1998; 274: E1099-E1105Crossref PubMed Google Scholar). In addition, the integrity of the BBB itself strongly depends on cellular cholesterol homeostasis. Another major difference is a unique strategy of the brain to secrete cholesterol. The removal of excess cholesterol from the brain is partly accounted for by the conversion to the more polar metabolite 24(S)OH-cholesterol by cytochrome P46 (cholesterol 24(S)-hydroxylase) (10Lund E.G. Guileyardo J.M. Russell D.W. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7238-7243Crossref PubMed Scopus (513) Google Scholar) and subsequent secretion across the BBB for elimination by the liver (11Björkhem I. Andersson U. Ellis E. Alvelius G. Ellegard L. Diczfalusy U. Sjovall J. Einarsson C. J. Biol. Chem. 2001; 276: 37004-37010Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Consistent with the conversion to 24(S)OH-cholesterol being the major pathway for the maintenance of brain cholesterol homeostasis, 24(S)OH-cholesterol levels are elevated in cerebrospinal fluid of Alzheimer's patients (12Papassotiropoulos A. Lutjohann D. Bagli M. Locatelli S. Jessen F. Buschfort R. Ptok U. Bjorkhem I. von Bergmann K. Heun R. J. Psychiatr. Res. 2002; 36: 27-32Crossref PubMed Scopus (193) Google Scholar) and in plasma correlate with the severity of dementia (13Lütjohann D. Papassotiropoulos A. Bjorkhem I. Locatelli S. Bagli M. Oehring R.D. Schlegel U. Jessen F. Rao M.L. von Bergmann K. Heun R. J. Lipid Res. 2000; 41: 195-198Abstract Full Text Full Text PDF PubMed Google Scholar), probably as a consequence of increased cholesterol turnover. Thus far, the underlying mechanisms that contribute to sterol transport and homeostasis in the brain and at the BBB are relatively obscure. In contrast, in peripheral tissues many steps of the protective pathway that prevent the excess accumulation of cholesterol, a process termed reverse cholesterol transport (RCT), have been elucidated. ABCA1 has been identified as the primary gatekeeper for eliminating tissue cholesterol, because it mediates the apolipoprotein-dependent transfer of intracellular cholesterol and phospholipid to lipid-free apoA-I (14Bodzioch M. Orso E. Klucken J. Langmann T. Bottcher A. Diederich W. Drobnik W. Barlage S. Buchler C. Porsch-Ozcurumez M. Kaminski W.E. Hahmann H.W. Oette K. Rothe G. Aslanidis C. Lackner K.J. Schmitz G. Nat. Genet. 1999; 22: 347-351Crossref PubMed Scopus (1328) Google Scholar, 15Lawn R.M. Wade D.P. Garvin M.R. Wang X. Schwartz K. Porter J.G. Seilhamer J.J. Vaughan A.M. Oram J.F. J. Clin. Invest. 1999; 104: R25-R31Crossref PubMed Scopus (648) Google Scholar, 16Brooks-Wilson A. Marcil M. Clee S.M. Zhang L.H. Roomp K. van Dam M., Yu, L. Brewer C. Collins J.A. Molhuizen H.O. Loubser O. Ouelette B.F. Fichter K. Ashbourne-Excoffon K.J. Sensen C.W. Scherer S. Mott S. Denis M. Martindale D. Frohlich J. Morgan K. Koop B. Pimstone S. Kastelein J.J. Hayden M.R. et al.Nat. Genet. 1999; 22: 336-345Crossref PubMed Scopus (1481) Google Scholar). Partially lipidated apoA-I matures into spherical HDL via esterification of cholesterol by plasma lecithin-cholesterol acyltransferase, and HDL particles are processed and remodeled by the combined actions of cholesteryl ester and phospholipid transfer proteins and of hepatic lipase (17Fielding C.J. Fielding P.E. J. Lipid Res. 1995; 36: 211-228Abstract Full Text PDF PubMed Google Scholar). Scavenger receptor class B, type I (SR-BI), highly expressed in liver parenchymal cells, takes up cholesteryl esters selectively, i.e. without concomitant HDL particle endocytosis (18Krieger M. Annu. Rev. Biochem. 1999; 68: 523-558Crossref PubMed Scopus (457) Google Scholar), and cholesterol and its catabolites are finally excreted into bile. Depending on the concentration gradient of cholesterol, SR-BI also promotes cholesterol efflux from peripheral cells to HDL but not to lipid-free apoA-I (19Ji Y. Jian B. Wang N. Sun Y. Moya M.L. Phillips M.C. Rothblat G.H. Swaney J.B. Tall A.R. J. Biol. Chem. 1997; 272: 20982-20985Abstract Full Text Full Text PDF PubMed Scopus (629) Google Scholar, 20Kellner-Weibel G. de La Llera-Moya M. Connelly M.A. Stoudt G. Christian A.E. Haynes M.P. Williams D.L. Rothblat G.H. Biochemistry. 2000; 39: 221-229Crossref PubMed Scopus (131) Google Scholar). The endothelial cell lineage of the BBB is able to synthesize a number of proteins exhibiting key functions during RCT. As recently reported by our group (21Goti D. Hammer A. Galla H.J. Malle E. Sattler W. J. Neurochem. 2000; 74: 1374-1383Crossref PubMed Scopus (53) Google Scholar, 22Goti D. Hrzenjak A. Levak-Frank S. Frank S. van Der Westhuyzen D.R. Malle E. Sattler W. J. Neurochem. 2001; 76: 498-508Crossref PubMed Scopus (131) Google Scholar), primary porcine brain capillary endothelial cells (pBCEC) express SR-BI, and SR-BI contributes to selective uptake of HDL-associated lipids by these cells. In addition, porcine brain endothelial cells have been reported to synthesize apoA-I (23Möckel B. Zinke H. Flach R. Weiss B. Weiler-Guttler H. Gassen H.G. J. Neurochem. 1994; 62: 788-798Crossref PubMed Scopus (69) Google Scholar), and apoA-I abundantly present in the central nervous system is obviously transported across the BBB (8Dietschy J.M. Turley S.D. Curr. Opin. Lipidol. 2001; 12: 105-112Crossref PubMed Scopus (705) Google Scholar). With the present study we aimed to elucidate the mechanisms underlying cholesterol and 24(S)OH-cholesterol transport at the BBB by studying the functions and regulation of key players in RCT that are expressed by pBCEC, i.e. SR-BI, apoA-I, and ABCA1 (Refs. 21Goti D. Hammer A. Galla H.J. Malle E. Sattler W. J. Neurochem. 2000; 74: 1374-1383Crossref PubMed Scopus (53) Google Scholarand 23Möckel B. Zinke H. Flach R. Weiss B. Weiler-Guttler H. Gassen H.G. J. Neurochem. 1994; 62: 788-798Crossref PubMed Scopus (69) Google Scholar and this study). We investigated the regulation of ABCA1 and apoA-I-dependent cholesterol efflux by 24(S)OH-cholesterol, which, like other oxysterols, represents a specific ligand for the nuclear receptor that regulates ABCA1 expression, liver X receptor (LXR; Ref. 24Lu T.T. Repa J.J. Mangelsdorf D.J. J. Biol. Chem. 2001; 276: 37735-37738Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar). We studied the impact of SR-BI expression levels on HDL-dependent efflux of cholesterol and of 24(S)OH-cholesterol. To verify results obtained with pBCEC monolayers, we investigated polarized sterol flux in the presence of apically added acceptors and the polarized regulation of ABCA1 expression by 24(S)OH-cholesterol, using an in vitro model of the BBB (25Goti D. Balazs Z. Panzenboeck U. Hrzenjak A. Reicher H. Wagner E. Zechner R. Malle E. Sattler W. J. Biol. Chem. 2002; 277: 28537-28544Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Earle's medium M199, Dulbecco's modified Eagle's/Ham's F-12 (1:1, v/v) medium, penicillin/streptomycin, gentamycin, l-glutamine, and trypsin were obtained from Biochrom (Berlin, Germany). Pronase and dispase were purchased from Sigma. Ox serum was from PAA Laboratories (Linz, Austria). Plasticware for cell culture and Transwell® inserts (polycarbonate membrane, 0.4-μm pore size) were from Costar (Vienna, Austria). [3H]Cholesterol (1.48–2.22 TBq/mmol) was from PerkinElmer Life Sciences; 24(S)-[3H]OH-cholesterol (2.07 GBq/mmol) was from Biotrend (Köln, Germany), and 24(S)OH-cholesterol was from Steraloids (Newport, CT). Opti-Fluor® scintillation mixture was from Packard Canberra (Vienna, Austria). PD10 size-exclusion columns, dNTPs, RNAguard, and random hexamer primers were obtained fromAmersham Biosciences. RNeasy kit was from Qiagen (Vienna, Austria); PCR primers were from MWG Biotech (Ebersberg, Germany). Antibodies were from Santa Cruz Biotechnology (LXRα; Santa Cruz, CA), Abcam (SR-BI; Cambridge, UK), Genosphere Biotechnologies (ABCA1; Paris, France), and Behring Diagnostics, Inc. (apoA-I; Marburg, Germany). All solvents were purchased from Sigma in the highest quality available, and all other chemicals were from Roche Molecular Biochemicals. pBCEC were isolated by sequential enzymatic digestion and centrifugation steps according to Ref. 26Tewes B. Franke H. Hellwig S. Hoheisel D. Decker S. Griesche D. Tilling T. Wegener J. Galla H.-J. De Boer A.G. Sutanto W. Transport across the Blood-Brain Barrier: In Vitro and in Vivo Techniques. Harwood Academic Publishers, Amsterdam1997: 91-97Google Scholar and characterized as described (21Goti D. Hammer A. Galla H.J. Malle E. Sattler W. J. Neurochem. 2000; 74: 1374-1383Crossref PubMed Scopus (53) Google Scholar). pBCEC (from one brain) were cultured in 6 × 75-cm2 collagen-coated culture flasks with M199 containing 10% ox serum, 1% gentamycin, 1% penicillin/streptomycin, and 0.35% glutamine (v/v). After 3 days the cells were plated onto collagen-coated multiwell or Transwell® (6- or 12-well) cell culture dishes at a density of 40,000 or 80,000 cells/cm2, respectively. Transwell® cultures were grown for 2 or 3 days, depending on the trans-endothelial electrical resistance (≥70 ohms/cm2) prior to induction of tight junction formation in Dulbecco's modified Eagle's/Ham's F-12 medium, containing 150 nm hydrocortisone, 1% penicillin/streptomycin, and 0.35% glutamine (v/v). After 1–2 days of induction, dishes with 300–1000 ohms/cm2 were used for experiments. Human apoE-free HDL3 and low density lipoproteins (LDL) were prepared by density gradient ultracentrifugation of plasma obtained from normolipidemic human volunteers in a TL120 tabletop ultracentrifuge (350,000 × g; Beckman Instruments, Vienna, Austria) (27Sattler W. Mohr D. Stocker R. Methods Enzymol. 1994; 233: 469-489Crossref PubMed Scopus (283) Google Scholar). Lipoproteins recovered by direct aspiration were desalted by size-exclusion chromatography on PD-10 columns, and purity was confirmed via SDS-PAGE identification of associated proteins. ApoA-I was isolated as described (28Bergt C. Oettl K. Keller W. Andreae F. Leis H.J. Malle E. Sattler W. Biochem. J. 2000; 346: 345-354Crossref PubMed Scopus (47) Google Scholar). SDS-PAGE was performed on detergent-extracted (1% Triton X-100, 20 mm Tris, pH 7.5, 100 mm NaCl, 1 mmNa3VO4, 40 mm NaF, 5 mmEGTA, 0.2% SDS, 0.5% deoxycholate, and 0.2 mmphenylmethylsulfonyl fluoride) pBCEC proteins that were separated by 6% (ABCA1), 8% (SR-BI), 10% (LXRα), or 12% (apoA-I) SDS-PAGE under reducing conditions (150 V, 90 min). To analyze apoA-I secretion by pBCEC, cellular supernatants were centrifuged to remove cell debris. Proteins were precipitated with 3 m trichloroacetic acid (0.1 ml/ml supernatant, 30 min, 4 °C) and pelleted by centrifugation. Pellets were washed with 0.5 ml of acetone and resuspended in 60 μl of sample buffer (0.1 m Tris/HCl, pH 6.8, 4% SDS, 15% glycerol, and 1% mercaptoethanol) and incubated at 95 °C for 5 min before application to gels. For Western blotting, proteins were electrophoretically transferred to nitrocellulose membranes at 150 mA, 4 °C, for 120 (ABCA1) or 60 min (SR-BI, LXRα, and apoA-I). Immunochemical detection of the respective proteins was performed using polyclonal rabbit anti-human/mouse ABCA1 antiserum raised against the C-terminal peptide 2177–2199 (1:500), polyclonal rabbit anti-human SR-BI (1:1500), anti-human apoA-I (1:2000), and goat anti-human LXRα (1:500) IgG as primary antibodies. Immunoreactive bands were visualized using peroxidase (HRP)-conjugated goat anti-rabbit (1:4000) or donkey anti-goat IgG (1:1000) and subsequent ECL development. Bands were quantified densitometrically using camera, scanner, and software from Herolab (Heidelberg, Germany). The relative percentage of plasma membrane ABCA1 was analyzed as described (29Marmorstein A. Zurzolo C. le Bivic A. Rodriguez-Boulan E. Celis J.E. Cell Biology: a Laboratory Handbook. 4. Academic Press, San Diego, CA1998: 341-350Google Scholar). In brief, ice-cold sulfo-NHS-biotin (0.5 mg/ml in phosphate-buffered saline (PBS) containing 1 mm MgCl2 and 1.3 mmCaCl2) was added twice either to the apical or basolateral chamber of 6-well Transwell® pBCEC cultures and incubated for 20 min (4 °C). The reaction was quenched by replacing the solution with 50 mm NH4Cl for 10 min; filters were then washed and excised, and cellular proteins were extracted in Tris-buffered saline containing 1% Triton X-100, 0.2% bovine serum albumin, and protease inhibitors. After a sham precipitating with preimmune serum, ABCA1 was immunoprecipitated with rabbit anti-human ABCA1 antiserum overnight (4 °C). Biotinylated ABCA1 was immunodetected using streptavidin-HRP and subsequent ECL development (29Marmorstein A. Zurzolo C. le Bivic A. Rodriguez-Boulan E. Celis J.E. Cell Biology: a Laboratory Handbook. 4. Academic Press, San Diego, CA1998: 341-350Google Scholar). Total polyadenylated RNA from pBCEC, RBE4 (immortalized rat brain endothelial cells, kindly provided by Neurotech SA, Evry, France), human umbilical vein endothelial cells (HUVEC, kindly provided by Dr. R. Heller, Erfurt, Germany), and lung carcinoma epithelial-HUVEC hybridoma cells (EAHY, kindly provided by Dr. W. F. Graier, Graz, Austria) was isolated according to the RNeasy protocol (Qiagen). Three μg of total RNA was treated with RQ1 RNase-free DNase I for 15 min at 37 °C and used as a template for first strand cDNA synthesis (the reaction mix contained 0.5 mm dNTPs, 15 units of RNAguard, 3.3 μm random hexamer primers, 10 mmdithiothreitol, 1× First Strand Buffer, and 200 units of Moloney murine leukemia virus-reverse transcriptase). Reverse transcription was performed for 1 h at 37 °C and stopped at 75 °C for 10 min. Fifty-μl PCRs contained 0.2 mm dNTPs, appropriate oligonucleotide primers at 10 μm, 1× PCR buffer, and 1 unit of Finnzyme DyNAzyme II DNA polymerase. The reaction mix was heated at 94 °C for 4 min, and amplification was carried out for 35 cycles (denaturation, 30 s at 94 °C; annealing, 30 s at 57 °C; extension 1 min at 72 °C). Oligonucleotide primers used for amplification of ABCA1 are as follows: forward primer 5′-GTTCTCAGATGCTCGGAGGCTTCTT and reverse primer 5′-GACAATACGAGACACAGCCTGGTAG (MWG Biotech; Ebersberg, Germany). A 609-bp fragment was obtained. cDNA for ABCA1 was amplified using human ABCA1-specific primers. Oligonucleotide primers used for amplification of apoA-I are as follows: forward primer 5′-CTGACCTTGGCTGTGCTCTT and reverse primer 5′-atccttctggcggtacgtctc (MWG Biotech; Ebersberg, Germany). A 410-bp cDNA fragment was obtained. RT-PCR products were separated on 1% agarose gels. Efflux of cellular sterols was analyzed as described previously (30Panzenböck U. Kritharides L. Raftery M. Rye K.A. Stocker R. J. Biol. Chem. 2000; 275: 19536-19544Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). In brief, sterol pools of pBCEC monolayers were metabolically labeled in medium containing 10% ox serum and 0.5 μCi/ml [3H]cholesterol or 0.1 μCi/ml 24(S)-[3H]OH-cholesterol for 24–48 h. The "labeling" medium was then replaced with medium containing 0.1% bovine serum albumin (w/v) for 16 h to equilibrate labeled cholesterol or for 1 h to equilibrate labeled 24(S)OH-cholesterol among cellular pools. Where indicated, 24(S)OH-cholesterol (10 μm) or 9-cis-retinoic acid (10 μm) was added during equilibration for 16 h. Subsequently, cells were washed twice with PBS before starting efflux incubations. Sterol acceptors (i.e. apoA-I, HDL3, LDL, serum, or albumin) were added in serum-free medium at the indicated concentrations. At the indicated times, aliquots of efflux media were collected and centrifuged to remove cell debris, and the radioactivity of the respective tracers was determined by liquid scintillation counting. The remaining intact monolayers were washed twice with ice-cold PBS and lysed in 0.3 m NaOH (2 h, 4 °C), and aliquots of the lysates were then used to count radioactivity and to determine the cellular protein content using the method of Lowryet al. (31Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). In order to metabolically label the sterol pools of polarized pBCEC grown on Transwell® filters, the tracer was added to the basolateral compartment (i.e. lower chamber, representing the "brain parenchymal side") for the duration of the inducing period or as otherwise indicated. Sterol acceptors were added to the apical compartment (i.e. upper chamber, representing the "microvessel lumenal side") and incubated at 37 °C for the indicated periods. Efflux media were collected from both chambers and treated as above; cells on filter inserts were lysed in 0.3m NaOH at 4 °C overnight prior to determining cell-associated radioactivity and cell protein. Sterol efflux was calculated as the percentage of the sum of the total medium and cellular counts/min. ABCA1-dependent cholesterol efflux was inhibited with DIDS, and P-glycoprotein function was inhibited with PSC833. Adenoviral plasmid shuttle vector (pAvCvSv) and pJM17 vectors were kindly supplied by L. Chan (Baylor College of Medicine, Houston, TX) and human SR-BI cDNA by H. Hauser (ETH, Zürich, Switzerland). Human SR-BI adenovirus and control β-galactosidase virus were amplified and purified exactly as described previously (22Goti D. Hrzenjak A. Levak-Frank S. Frank S. van Der Westhuyzen D.R. Malle E. Sattler W. J. Neurochem. 2001; 76: 498-508Crossref PubMed Scopus (131) Google Scholar). pBCEC cultivated in 12-well culture dishes at a density of 4 × 104 cells/cm2 were transfected with recombinant adenoviruses (multiplicity of infection = 1000 plaque-forming units/ml; 16 h) as described (22Goti D. Hrzenjak A. Levak-Frank S. Frank S. van Der Westhuyzen D.R. Malle E. Sattler W. J. Neurochem. 2001; 76: 498-508Crossref PubMed Scopus (131) Google Scholar). Expression levels of SR-BI were analyzed by densitometric evaluation of Western blots. Unless otherwise indicated in the individual figure legends, all data shown represent means ± S.D. of triplicate determinations. Two-tailed Student's t tests and two-way analysis of variance were performed using Prism software (Graphpad). Because ABCA1 and apoA-I represent key players in RCT, we initially analyzed their expression by pBCEC. Among the various endothelial cell types investigated by RT-PCR, pBCEC and HUVEC exhibited a prominent signal for ABCA1 mRNA, followed by EAHY hybridoma cells, whereas no mRNA was detected in RBE4 cells (Fig.1 A). The expression of apoA-I by microcapillary endothelial cells has been reported earlier (23Möckel B. Zinke H. Flach R. Weiss B. Weiler-Guttler H. Gassen H.G. J. Neurochem. 1994; 62: 788-798Crossref PubMed Scopus (69) Google Scholar), and our data confirmed that pBCEC secrete substantial amounts of apoA-I into the culture medium, whereas only traces were detectable in cell lysates (Fig. 1 B). The identification of apoA-I mRNA in these cells further suggested that at least part of the secreted apoA-I is derived from endogenous biosynthesis (Fig. 1 C). ABCA1 mediates the transfer of cellular cholesterol to lipid-free apoA-I. Oxysterols, including 24(S)OH-cholesterol, are high affinity endogenous ligands for LXR, the nuclear receptor that upon dimerization with retinoid X receptor (RXR) induces ABCA1 gene transcription in macrophages and other cells (reviewed in Refs. 6Schmitz G. Langmann T. Curr. Opin. Lipidol. 2001; 12: 129-140Crossref PubMed Scopus (179) Google Scholar and 7Santamarina-Fojo S. Remaley A.T. Neufeld E.B. Brewer Jr., H.B. J. Lipid Res. 2001; 42: 1339-1345Abstract Full Text Full Text PDF PubMed Google Scholar). We therefore investigated apoA-I-mediated cholesterol efflux from pBCEC monolayers and the effect of 24(S)OH-cholesterol on cholesterol efflux and on the regulation of ABCA1 protein expression (Fig.2). As shown in Fig. 2 A, apoA-I dose-dependently mobilized cellular [3H]cholesterol from pBCEC. Cholesterol efflux was enhanced up to 1.6-fold (at concentrations between 5 and 50 μg apoA-I/ml culture medium) in response to physiological concentrations of 24(S)OH-cholesterol. The RXR ligand 9-cis-retinoic acid exhibited only a minor effect on cholesterol release, and the addition of both ligands exhibited an effect equivalent to 24(S)OH-cholesterol alone. It may be important to note that basal cholesterol efflux (i.e. in the absence of exogenous apoA-I) from pBCEC was relatively high as compared with RBE4 cells (2.9 ± 0.6% versus 0.9 ± 0.12% for pBCEC and RBE4, respectively; data for RBE4 not shown). Since, in contrast to pBCEC, RBE4 did not secrete apoA-I (as determined by immunoblotting, data not shown), it is reasonable to assume that endogenous secretion of apoA-I contributes to cholesterol removal from pBCEC under basal conditions. In line with increased cholesterol efflux to lipid-free apoA-I, pretreatment of pBCEC with 24(S)OH-cholesterol induced the expression of ABCA1 protein (4- and 6-fold, at 2.5 and 10 μm 24(S)OH-cholesterol, respectively) and of LXRα (3- and 4-fold, at 2.5 and 10 μm24(S)OH-cholesterol), as determined by densitometric evaluation of immunoblots (Fig. 2 B). To support the possibility that ABCA1 is responsible for apoA-I-dependent cholesterol removal, we tested the effect of the ABC transporter inhibitor DIDS that dose-dependently inhibited both basal (27.5 ± 12.9% inhibition) and 24(S)OH-cholesterol-induced (51.3 ± 6.7% inhibition) [3H]cholesterol efflux (Fig. 2 C). It is important to note that DIDS inhibits cholesterol efflux in the absence of exogenous apoA-I, indicating that the ABCA1 pathway in pBCEC is constitutively active. By contrast, the specific P-glycoprotein inhibitor PSC833 (0.1–10 μm) failed to inhibit [3H]cholesterol efflux (data not shown), confirming that P-glycoproteins are not involved. These results together are consistent with a major role for ABCA1 in apoA-I-mediated cholesterol efflux from pBCEC, a process that is regulated by 24(S)OH-cholesterol via an LXR-dependent pathway. In addition to ABCA1 and apoA-I, SR-BI plays a major role in RCT. SR-BI mediates selective uptake of HDL-associated lipids, but it may also mediate HDL-dependent net cholesterol efflux from peripheral tissues, presumably depending on the gradient of the chemical potential of the lipid between cell surface and acceptor particle (19Ji Y. Jian B. Wang N. Sun Y. Moya M.L. Phillips M.C. Rothblat G.H. Swaney J.B. Tall A.R. J. Biol. Chem. 1997; 272: 20982-20985Abstract Full Text Full Text PDF PubMed Scopus (6
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