FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2102329287> ?p ?o ?g. }

• W2102329287 endingPage "1149" @default.
• W2102329287 startingPage "1140" @default.
• W2102329287 abstract "Mice deficient in scavenger receptor class B type I (SR-BI) and apolipoprotein E (apoE) [double knockout (DKO) mice] develop dyslipidemia, accelerated atherosclerosis, and myocardial infarction, and die prematurely. We examined effects of apoE and SR-BI deficiency on macrophage cholesterol homeostasis. DKO macrophages had increased total cholesterol (TC) stores (220–380 μg/mg protein) compared with apoE−/− cells (40 μg/mg), showed significant lysosomal lipid engorgement, and increased their TC by 34% after exposure to HDL. DKO macrophages from apoE−/− mice reconstituted with DKO bone marrow showed less cholesterol accumulation (89 μg/mg), suggesting that the dyslipidemia of DKO mice explains part of the cellular cholesterol defect. However, analyses of DKO and apoE−/− macrophages from transplanted apoE−/− mice revealed a role for macrophage SR-BI, inasmuch as the TC in DKO macrophages increased by 10% in the presence of HDL, whereas apoE−/− macrophage TC decreased by 33%. After incubation with HDL, the free cholesterol (FC) increased by 29% in DKO macrophages, and decreased by 8% in apoE−/− cells, and only DKO cells had FC in large peri-nuclear pools. Similar trends were observed with apoA-I as an acceptor. Thus, the abnormal cholesterol homeostasis of DKO macrophages is due to the plasma lipid environment of DKO mice and to altered trafficking of macrophage cholesterol. Both factors are likely to contribute to the accelerated atherosclerosis in DKO mice. Mice deficient in scavenger receptor class B type I (SR-BI) and apolipoprotein E (apoE) [double knockout (DKO) mice] develop dyslipidemia, accelerated atherosclerosis, and myocardial infarction, and die prematurely. We examined effects of apoE and SR-BI deficiency on macrophage cholesterol homeostasis. DKO macrophages had increased total cholesterol (TC) stores (220–380 μg/mg protein) compared with apoE−/− cells (40 μg/mg), showed significant lysosomal lipid engorgement, and increased their TC by 34% after exposure to HDL. DKO macrophages from apoE−/− mice reconstituted with DKO bone marrow showed less cholesterol accumulation (89 μg/mg), suggesting that the dyslipidemia of DKO mice explains part of the cellular cholesterol defect. However, analyses of DKO and apoE−/− macrophages from transplanted apoE−/− mice revealed a role for macrophage SR-BI, inasmuch as the TC in DKO macrophages increased by 10% in the presence of HDL, whereas apoE−/− macrophage TC decreased by 33%. After incubation with HDL, the free cholesterol (FC) increased by 29% in DKO macrophages, and decreased by 8% in apoE−/− cells, and only DKO cells had FC in large peri-nuclear pools. Similar trends were observed with apoA-I as an acceptor. Thus, the abnormal cholesterol homeostasis of DKO macrophages is due to the plasma lipid environment of DKO mice and to altered trafficking of macrophage cholesterol. Both factors are likely to contribute to the accelerated atherosclerosis in DKO mice. Apolipoprotein E (apoE) and scavenger receptor class B type I (SR-BI) are essential for maintenance of normal cholesterol homeostasis. ApoE regulates plasma cholesterol levels by facilitating the uptake of remnant lipoproteins (1Mahley R.W. Huang Y. Apolipoprotein E: from atherosclerosis to Alzheimer's disease and beyond..Curr. Opin. Lipidol. 1999; 10: 207-217Crossref PubMed Scopus (324) Google Scholar), and hepatic SR-BI functions in reverse cholesterol transport by mediating the uptake of HDL cholesterol (2Arai T. Wang N. Bezouevski M. Welch C. Tall A.R. Decreased atherosclerosis in heterozygous low density lipoprotein receptor-deficient mice expressing the scavenger receptor BI transgene..J. Biol. Chem. 1999; 274: 2366-2371Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar, 3Ueda Y. Gong E. Royer L. Cooper P.N. Francone O.L. Rubin E.M. Relationship between expression levels and atherogenesis in scavenger receptor class B, type I transgenics..J. Biol. Chem. 2000; 275: 20368-20373Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). In addition to these important functions, apoE and SR-BI may be relevant in peripheral cell cholesterol homeostasis as facilitators of cholesterol efflux to a number of acceptors, including HDL (4Mazzone T. Reardon C. Expression of heterologous human apolipoprotein E by J774 macrophages enhances cholesterol efflux to HDL3..J. Lipid Res. 1994; 35: 1345-1353Abstract Full Text PDF PubMed Google Scholar, 5Ji Y. Jian B. Wang N. Sun Y. Moya M.L. Phillips M.C. Rothblat G.H. Swaney J.B. Tall A.R. Scavenger receptor BI promotes high density lipoprotein-mediated cellular cholesterol efflux..J. Biol. Chem. 1997; 272: 20982-20985Abstract Full Text Full Text PDF PubMed Scopus (635) Google Scholar) and apoA-I (6Huang H. Gu D. Lange Y. Mazzone T. Expression of scavenger receptor BI facilitates sterol movement between the plasma membrane and the endoplasmic reticulum in macrophages..Biochemistry. 2003; 42: 3949-3955Crossref PubMed Scopus (32) Google Scholar). Numerous studies have demonstrated that both SR-BI and apoE are important in minimizing atherosclerotic lesion development. Single-gene deletion of either apoE (7Nakashima Y. Plump A.S. Raines E.W. Breslow J.L. Ross R. ApoE-deficient mice develop lesions of all phases of atherosclerosis throughout the arterial tree..Arterioscler. Thromb. 1994; 14: 133-140Crossref PubMed Google Scholar, 8Reddick R.L. Zhang S.H. Maeda N. Atherosclerosis in mice lacking apoE. Evaluation of lesional development and progression..Arterioscler. Thromb. 1994; 14: 141-147Crossref PubMed Scopus (550) Google Scholar) or SR-BI (9Huszar D. Varban M.L. Rinninger F. Feeley R. Arai T. Fairchild-Huntress V. Donovan M.J. Tall A.R. Increased LDL cholesterol and atherosclerosis in LDL receptor-deficient mice with attenuated expression of scavenger receptor B1..Arter. Thromb. Vasc. Biol. 2000; 20: 1068-1073Crossref PubMed Scopus (150) Google Scholar) in mice accelerates atherosclerotic lesion development, whereas low-level transgenic expression of either SR-BI (3Ueda Y. Gong E. Royer L. Cooper P.N. Francone O.L. Rubin E.M. Relationship between expression levels and atherogenesis in scavenger receptor class B, type I transgenics..J. Biol. Chem. 2000; 275: 20368-20373Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar) or apoE (10Thorngate F. Rudel L. Walzem R. Williams D. Low levels of extrahepatic nonmacrophage apoE inhibit atherosclerosis without correcting hypercholesterolemia in apoE-deficient mice..Arterioscl Thromb Vasc Biol. 2000; 8: 1852-1853Google Scholar, 11Zhang S. Picard M.H. Vasile E. Zhu Y. Raffai R.L. Weisgraber K.H. Krieger M. Diet-induced occlusive coronary atherosclerosis, myocardial infarction, cardiac dysfunction, and premature death in scavenger receptor class B type I-deficient, hypomorphic apolipoprotein ER61 mice..Circulation. 2005; 111: 3457-3464Crossref PubMed Scopus (109) Google Scholar) reduces the extent of atherosclerosis. Furthermore, the combined deficiency of SR-BI and apoE produces the only mouse model of accelerated and fatal atherosclerosis. SR-BI and apoE double knockout (DKO) mice display a toxic dyslipidemia, develop early occlusive atherosclerotic coronary artery disease, and die prematurely at 6–8 weeks of age with evidence of myocardial infarction, mimicking features of human atherosclerosis (12Braun A. Trigatti B.L. Post M.J. Sato K. Simons M. Edelberg J.M. Rosenberg R.D. Schrenzel M. Krieger M. Loss of SR-BI expression leads to the early onset of occlusive atherosclerotic coronary artery disease, spontaneous myocardial infarctions, severe cardiac dysfunction, and premature death in apolipoprotein E-deficient mice. [comment].Circ. Res. 2002; 90: 270-276Crossref PubMed Scopus (0) Google Scholar). SR-BI (13Chinetti G. Gbaguidi F.G. Griglio S. Mallat Z. Antonucci M. Poulain P. Chapman J. Fruchart J.C. Tedgui A. Najib-Fruchart J. et al.CLA-1/SR-BI is expressed in atherosclerotic lesion macrophages and regulated by activators of peroxisome proliferator-activated receptors..Circulation. 2000; 101: 2411-2417Crossref PubMed Scopus (390) Google Scholar) and apoE (14Basu S.K. Brown M.S. Ho Y.K. Havel R.J. Goldstein J.L. Mouse macrophages synthesize and secrete a protein resembling apolipoprotein E..Proc. Natl. Acad. Sci. USA. 1981; 78: 7545-7549Crossref PubMed Scopus (201) Google Scholar) are expressed in macrophages. Macrophage apoE has an antiatherogenic effect independent of its effect on plasma lipids (15Fazio S. Babaev V. Murray A. Hasty A. Carter K. Gleaves L. Atkinson J. Linton M. Increased atherosclerosis in C57BL/6 mice transplanted with apo E null bone marrow..Proc. Natl. Acad. Sci. USA. 1997; 94: 4647-4652Crossref PubMed Scopus (249) Google Scholar, 16Bellosta S. Mahley R.W. Sanan D.A. Murata J. Newland D.L. Taylor J.M. Pitas R.E. Macrophage-specific expression of human apolipoprotein E reduces atherosclerosis in hypercholesterolemic apolipoprotein E-null mice..J. Clin. Invest. 1995; 96: 2170-2179Crossref PubMed Scopus (251) Google Scholar). Studies utilizing bone marrow (BM) transplants have substantiated the protective roles of macrophage SR-BI and apoE in atherosclerotic lesion development. Introduction of BM with low expression levels of apoE reduces atherosclerosis development in apoE-deficient mice (17Hasty A.H. Linton M.F. Brandt S.J. Babaev V.R. Gleaves L.A. Fazio S. Retroviral gene therapy in apoE-deficient mice: apoE expression in the artery wall reduces early foam cell lesion formation..Circulation. 1999; 99: 2571-2576Crossref PubMed Scopus (105) Google Scholar). A study by Covey and colleagues (18Covey S. Krieger M. Wang W. Penman M. Trigatti B. Scavenger receptor class B type-I-mediated protection against atherosclerosis in LDL receptor-negative mice involves its expression in bone marrow-derived cells..Arterioscl Thromb Vasc Biol. 2003; 23: 1589-1594Crossref PubMed Scopus (195) Google Scholar) demonstrated that transplantation of SR-BI-deficient BM into LDL receptor-deficient mice accelerates atherosclerosis development. Similarly, we have recently shown that transplantation of DKO marrow into apoE-null mice enhances atherosclerotic lesion formation, indicating that macrophage SR-BI expression is antiatherogenic (19Zhang W. Yancey P.G. Su Y. Babaev V. Zhang Y. Fazio S. Linton M. Inactivation of macrophage scavenger receptor class B type I promotes atherosclerotic lesion development in apolipoprotein E-deficient mice..Circulation. 2003; 108: 2258-2263Crossref PubMed Scopus (173) Google Scholar). Macrophages are critical determinants of atherosclerotic lesion progression through internalization of intimal lipoproteins and transformation into foam cells. The degree of cholesterol accumulation in the macrophage, obviously a function of lipoprotein entry, is finely regulated by genes coordinating cholesterol efflux. In recent years, many efflux mechanisms have been identified that may operate to minimize macrophage foam cell formation. These include ABCA1 (20Lawn R. Wade D. Garvin M. Wang X. Schwartz D. Porter J. Seillhamer J. Vaughn A. Oram J. The Tangier disease gene product ABC1 controls the cellular apolipoprotein-mediated lipid removal pathway..J. Clin. Invest. 1999; 104: R25-R31Crossref PubMed Scopus (653) Google Scholar, 21Oram J. Lawn R. Garvin M. Wade D. ABCA1 is the cAMP-inducible apolipoprotein receptor that mediates cholesterol secretion from macrophages..J. Biol. Chem. 2000; 275: 34508-34511Abstract Full Text Full Text PDF PubMed Scopus (474) Google Scholar), ABCG1 (22Kennedy M. Barrera G. Nakamura K. Baldan A. Tarr P. Fishbein M. Frank J. Francone O. Edwards P. ABCG1 has a critical role in mediating cholesterol efflux to HDL and preventing cellular lipid accumulation..Cell Metab. 2005; 1: 121-131Abstract Full Text Full Text PDF PubMed Scopus (684) Google Scholar, 23Wang N. Lan D. Chen W. Matsuura F. Tall A. ATP-binding cassette transporters G1 and G4 mediate cellular cholesterol efflux to high-density lipoproteins..Proc. Natl. Acad. Sci. USA. 2004; 101: 9774-9779Crossref PubMed Scopus (886) Google Scholar), SR-BI (24Jian B. de la Llera-Moya M. Ji Y. Wang N. Phillips M.C. Swaney J. Tall A. Rothblat G.H. Scavenger receptor class B type I as a mediator of cellular cholesterol efflux to lipoproteins and phospholipid acceptors..J. Biol. Chem. 1998; 273: 5599-5606Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar, 25Yancey P.G. de la Llera-Moya M. Swarnakar S. Monza P. Klein S. Connelly M. Johnson W. Williams D. Rothblat G.H. High density lipoprotein phospholipid composition is a major determinant of the bi-directional flux and net movement of cellular free cholesterol mediated by scavenger receptor BI..J. Biol. Chem. 2000; 275: 36596-36604Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar), and apoE (26Mazzone T. Reardon C. Expression of heterologous human apolipoprotein E by J774 macrophages enhances cholesterol effux to HDL3..J. Lipid Res. 1994; 35: 1345-1353Abstract Full Text PDF PubMed Google Scholar, 27Langer C. Yadong H. Cullen P. Wiesenhutter B. Mahley R.W. Assmann G. Eckardstein A. von Endogenous apolipoprotein E modulates cholesterol efflux and cholesteryl ester hydrolysis mediated by high-density lipoprotein-3 and lipid-free apoproteins in mouse peritoneal macrophages..J. Mol. Med. 2000; 78: 217-222Crossref PubMed Scopus (61) Google Scholar, 28Huang Z. Mazzone T. ApoE-dependent sterol efflux from macrophages is modulated by scavenger receptor class B type I expression..J. Lipid Res. 2002; 43: 375-382Abstract Full Text Full Text PDF PubMed Google Scholar). Given the vast redundancy of the efflux system and the minimal effects of macrophage SR-BI expression levels on cholesterol efflux (18Covey S. Krieger M. Wang W. Penman M. Trigatti B. Scavenger receptor class B type-I-mediated protection against atherosclerosis in LDL receptor-negative mice involves its expression in bone marrow-derived cells..Arterioscl Thromb Vasc Biol. 2003; 23: 1589-1594Crossref PubMed Scopus (195) Google Scholar, 19Zhang W. Yancey P.G. Su Y. Babaev V. Zhang Y. Fazio S. Linton M. Inactivation of macrophage scavenger receptor class B type I promotes atherosclerotic lesion development in apolipoprotein E-deficient mice..Circulation. 2003; 108: 2258-2263Crossref PubMed Scopus (173) Google Scholar, 28Huang Z. Mazzone T. ApoE-dependent sterol efflux from macrophages is modulated by scavenger receptor class B type I expression..J. Lipid Res. 2002; 43: 375-382Abstract Full Text Full Text PDF PubMed Google Scholar, 29Van Eck M. Bos I. Hildebrand R.B. Van Rij B.T. Van Berkel T.J. Dual role for scavenger receptor class B, type I on bone marrow-derived cells in atherosclerotic lesion development..Am. J. Pathol. 2004; 165: 785-794Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar), it is not clear what contribution macrophage SR-BI makes in preventing foam cell formation, particularly in relation to apoE. The goal of the present study is to examine the effect of combined deletion of SR-BI and apoE on cholesterol homeostasis in macrophages. We demonstrate that macrophages deficient in SR-BI and apoE accumulate substantial cholesterol stores in vivo. This cholesterol accumulation is due to a combination of the DKO mouse plasma lipid environment and defective intracellular trafficking of cholesterol in macrophages resulting from the absence of SR-BI. Thus, both toxic dyslipidemia and defective macrophage cholesterol mobilization are likely to contribute to the accelerated atherosclerotic lesion development in mice with combined deficiency of SR-BI and apoE. FBS, BSA, Dulbecco's phosphate-buffered saline (DPBS), penicillin, and streptomycin were purchased from Sigma. Tissue culture plasticware was obtained through Falcon (Lincoln, NJ). Lipid-free human apoA-I was obtained from Calbiochem. Human HDL was isolated by sequential ultracentrifugation as previously described. All other reagents and organic solvents were purchased from Fisher. SR-BI+/− (1:1 mixed C57BL/6×S129 genetic background) and apo E−/− mice on a C57BL/6 background were obtained from The Jackson Laboratory. SR-BI+/−apoE−/− mice on a C57BL/6 background were then generated by mating the SR-BI+/− mice with apoE−/− mice and backcrossing the resulting doubly heterozygous offspring with apoE−/− mice for nine generations. SR-BI+/−apoE−/− mice were then mated to generate SR-BI−/−apoE−/− mice on the C57BL/6 background (19Zhang W. Yancey P.G. Su Y. Babaev V. Zhang Y. Fazio S. Linton M. Inactivation of macrophage scavenger receptor class B type I promotes atherosclerotic lesion development in apolipoprotein E-deficient mice..Circulation. 2003; 108: 2258-2263Crossref PubMed Scopus (173) Google Scholar). ApoE genotypes were determined by using a PCR protocol from The Jackson Laboratory. SR-BI genotypes were determined by PCR analysis of DNA extracted from ear punches (30Rigotti A. Trigatti B. Penman M. Rayburn H. Herz J. Krieger M. A targeted mutation in murine gene encoding the high density lipoprotein (HDL) receptor scavenger receptor class B type I reveals its key role in HDL metabolism.Proc. Natl. Acad. Sci. USA. 1997; 94: 12610-12615Crossref PubMed Scopus (756) Google Scholar). Mouse peritoneal macrophages were harvested by peritoneal lavage 3 to 4 days after intraperitoneal injection of 3% thioglycollate. To enable comparisons of apoE−/− and SR-BI−/−apoE−/− macrophages that were exposed to the same lipid environment in vivo, cells were harvested from apo E−/− mice reconstituted with either apoE−/− or SR-BI−/−apoE−/− BM. Eight-week- old female apo E−/− mice were lethally irradiated with a single dose of 9 Gray using a cobalt-60 γ source. The same day, the recipient mice were injected with 5 × 106 BM cells via the retro-orbital venous complex. After transplantation, the apo E−/−-recipient mice were maintained on a chow diet for 4 weeks, and to obtain apo E−/− and SR-BI−/−apoE−/− macrophages loaded with cholesterol in vivo, recipient mice were then maintained for 8 weeks on a Western-type diet prior to elicitation of cells with thioglycollate. ApoE−/− or DKO macrophages were suspended in 5% FBS/DMEM and plated onto 12-well plates at a density of 1 × 106 cells/well. After 4 h, nonadherent cells were removed by washing two times with 1 ml of DMEM containing HEPES (25 mM). For cholesterol mobilization, the cells were incubated for 24 h or 48 h in DMEM alone or containing either lipid-free apoA-I (20 μg/ml) or human HDL (150 μg protein/ml). As a control for testing acceptor efflux capacity in experiments involving mobilization with macrophages isolated from DKO mice, apoE−/− macrophages were cholesterol enriched by incubation for 3 days with apoE−/− remnant lipoproteins (d = 1.006 to 1.019 g/ml, 20 μg protein/ml), and cholesterol mobilization was examined. The cells were then washed two times with DPBS, and the cell monolayer was air dried. Cell lipids were extracted by overnight incubation at room temperature in isopropanol containing cholesteryl methyl ether (2.5 μg/well) as an internal standard. The lipid extract free cholesterol (FC) and total cholesterol (TC) contents were then measured by gas-liquid chromatography following the procedure of Ishikawa et al. (31Ishikawa T.T. MacGee J. Morrison J.A. Glueck C.J. Quantitative analysis of cholesterol in 5 to 20 μl of plasma..J. Lipid Res. 1974; 15: 286-291Abstract Full Text PDF PubMed Google Scholar) as modified by Klansek and colleagues (32Klansek J.J. Yancey P.G. St. Clair R.W. Fischer R.T. Johnson W.J. Glick J.M. Cholesterol quantitation by GLC: artifactual formation of short-chain steryl esters..J. Lipid Res. 1995; 36: 2261-2266Abstract Full Text PDF PubMed Google Scholar). Cell proteins were solubilized by addition of 1 N NaOH to the wells, and the protein content measured using the method of Lowry et al. (33Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. Protein measurement with the Folin phenol reagent..J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) as modified by Markwell et al. (34Markwell M.A.K. Haas S.M. Bieber L.L. Tolbert N.E. A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples..Anal. Biochem. 1978; 87: 206-210Crossref PubMed Scopus (5332) Google Scholar). Microscopy was used to visualize macrophage lipids before and after efflux. Fluorescence microscopy was used to visualize cellular FC by filipin staining. Transmission electron microscopy (EM), in combination with acid phosphatase staining, was used to detect lipid-engorged lysosomes. For filipin staining of cell FC, macrophages were plated onto sterile glass coverslips placed in the bottom of 35 mm tissue culture wells. After incubations, the cells were washed, fixed for 15 min (2.5% paraformaldehyde), and stained at room temperature with filipin stain solution (1.25 mg of filipin dissolved in dimethyl sulfoxide and diluted in 25 ml of PBS). Filipin-stained FC was detected with ultraviolet filter excitation and viewed through a 510 nm barrier filter using a Zeiss Axioplan motorized fluorescent microscope (Oberkochen, Germany) equipped with a Photometrics Coolsnap charge-coupled device camera (Nikon Instruments; Melville, NY). Acid phosphatase staining of lysosomes and related organelles was done essentially as described previously (35Yancey P.G. Jerome W.G. Lysosomal sequestration of free and esterified cholesterol from oxidized low density lipoprotein in macrophages of different species..J. Lipid Res. 1998; 39: 1349-1361Abstract Full Text Full Text PDF PubMed Google Scholar). Briefly, the cells or arteries were first washed four times in 0.1 M cacodylate buffer (pH 7.4) containing 0.1 M sucrose at 4°C, and then washed two times in the same buffer at room temperature. The cells were then fixed for 2 min at 22°C and 8 min at 4°C in 4% glutaraldehyde in 0.1 M cacodylate/sucrose buffer, washed, and stained to show the presence of acid phosphatase using our standard modification of the Gomori (36Gomori G. An improved histochemical technique for acid phosphatase..Stain Technol. 1950; 25: 81-85Crossref Scopus (287) Google Scholar) lead precipitation method with β-glycerol phosphate as the substrate. After incubation with the substrate, the cells were postfixed in 1% osmium tetroxide, dehydrated, and embedded in epoxy resin. The sections were viewed without additional staining to verify the enzymatic reaction and were then stained with uranyl acetate. Transmission EM was performed using a Phillips CM-12 operated at 80 kiloelectron volts to ultrastructurally analyze cell lysosomal lipid volume. Images were processed with Metamorph (Universal Imaging Co.) and Photoshop (Adobe) software. The volume of lipid in foam cells within atherosclerotic lesions was estimated from EM sections of lesions as described previously (37Jerome W.G. Lewis J.C. Early atherogenesis in White Carneau pigeons. II. Ultrastructural and cytochemical observations..Am. J. Pathol. 1985; 119: 210-222PubMed Google Scholar). Briefly, lesions were identified and that area of the artery removed, fixed, and processed for acid phosphatase cytochemistry. Starting at an arbitrary point, the lesion was dissected by cross-sectioning into five equal parts. Five sections were taken from each part, and 20 random fields (50 μm × 50 μm) containing lesions were analyzed from each section. Volume of lysosomal lipids and inclusion of lipid per volume of cell were estimated using standard point count stereology (38Weibel E.R. Measuring through the microscope: development and evolution of stereological methods..J. Microsc. 1989; 155: 393-403Crossref PubMed Scopus (103) Google Scholar). Stereologic volume density determination in isolated macrophages was also done by stereologic point counting. Isolated macrophages were fixed and stained to demonstrate acid phosphatase as described above. Cross sections through 30 individual cells were visualized by EM, and the volume of lipid in lysosomes (acid phosphatase-positive) and cytoplasmic inclusions was estimated using point count stereology as described previously for acid phosphatase-stained cells (35Yancey P.G. Jerome W.G. Lysosomal sequestration of free and esterified cholesterol from oxidized low density lipoprotein in macrophages of different species..J. Lipid Res. 1998; 39: 1349-1361Abstract Full Text Full Text PDF PubMed Google Scholar). Protein extracts from macrophages were separated using 3% to 8% Tris-acetate gels (NuPAGE) and transferred to nitrocellulose membranes. Murine ABCA1 was detected with a primary antibody (Novus Biological) and visualized by chemiluminescence. Previous studies by Krieger and colleagues (39Braun A. Trigatti B. Post M. Sato K. Simons K. Edelberg J. Rosenburg R. Schrenzel M. Krieger M. Loss of SR-BI expression leads to the early onset of occlusive atherosclerotic coronary artery disease, spontaneous myocardial infarctions, severe cardiac dysfunction, and premature death in apolipoprotein E-deficient mice..Circ. Res. 2002; 90: 270-276Crossref PubMed Scopus (427) Google Scholar, 40Trigatti B. Rayburn H. Vinals M. Braun A. Miettinen H. Penman M. Hertz M. Schrenzel M. Amigo L. Rigotti A. et al.Influence of the high density lipoprotein receptor SR-BI on reproductive and cardiovascular pathophysiology..Proc. Natl. Acad. Sci. USA. 1999; 96: 9322-9327Crossref PubMed Scopus (441) Google Scholar, 41Yu H. Zhang W. Yancey P.G. Koury M. Zang Y. Fazio S. Linton M.F. Macrophage apolipoprotein E reduces atherosclerosis and prevents premature death in apolipoprotein E and scavenger receptor-class BI double-knockout mice..Arterioscl. Thromb. Vasc. Biol. 2006; 26: 150-156Crossref PubMed Scopus (35) Google Scholar) have shown that mice deficient in both SR-BI and apoE develop extreme dyslipidemia (worse than that presented by apoE−/− mice), even when maintained on a chow diet. DKO mice have total plasma cholesterol levels of 800–1,000 mg/dl (40Trigatti B. Rayburn H. Vinals M. Braun A. Miettinen H. Penman M. Hertz M. Schrenzel M. Amigo L. Rigotti A. et al.Influence of the high density lipoprotein receptor SR-BI on reproductive and cardiovascular pathophysiology..Proc. Natl. Acad. Sci. USA. 1999; 96: 9322-9327Crossref PubMed Scopus (441) Google Scholar, 41Yu H. Zhang W. Yancey P.G. Koury M. Zang Y. Fazio S. Linton M.F. Macrophage apolipoprotein E reduces atherosclerosis and prevents premature death in apolipoprotein E and scavenger receptor-class BI double-knockout mice..Arterioscl. Thromb. Vasc. Biol. 2006; 26: 150-156Crossref PubMed Scopus (35) Google Scholar) and show abnormally enlarged, FC-enriched HDL particles. We initially examined the effects of this abnormal lipid environment on cholesterol homeostasis in DKO macrophages. Measurement of the FC and cholesteryl ester (CE) content of peritoneal macrophages isolated from either apoE−/− or DKO mice (on chow diet) showed that DKO macrophages become extremely enriched in cholesterol in vivo (Fig. 1 ). DKO cells contained between 220 μg and 380 μg of cholesterol/mg cell protein, with CE representing 67% of the total. In contrast, macrophages from apoE−/− mice did not show any cholesterol enrichment, compared with controls, despite the hypercholesterolemic environment (TC = 475 ± 46 mg/dl). Moreover, the apoE−/− macrophages contained primarily FC, and only 7% of TC was CE. We used filipin staining to identify the location of the massive FC accumulation seen in DKO macrophages. FC in macrophages isolated from DKO mice occurred in large perinuclear pools (Fig. 2A ) typical of FC-engorged lysosomes (42Yancey P.G. Jerome W.G. Lysosomal cholesterol derived from mildly oxidized low density lipoprotein is resistant to efflux..J. Lipid Res. 2001; 42: 317-327Abstract Full Text Full Text PDF PubMed Google Scholar). The lysosomal location was confirmed by acid phosphatase staining (Fig. 2B). In contrast, FC was not detectable in intracellular vesicles in apoE−/− macrophages exposed to the plasma lipid environment of apoE−/− mice maintained on a chow diet (data not shown).Fig. 2.Fluorescence photomicrographs of filipin-stained cholesterol and electron micrographs of acid phosphatase-stained lysosomes in DKO macrophages. Peritoneal macrophages were isolated from DKO mice consuming a chow diet. After an overnight incubation in 5% FBS/DMEM, the cells were fixed and stained with filipin (A) or acid phosphatase (B) as described in Experimental Procedures. A: In addition to plasma membrane staining, bright fluorescence is seen in large perinuclear pools indicative of FC in lysosomes. Magnification, ×1,500; bar = 3 μm. B: DKO macrophages contain significant lipid within cytoplasmic droplets, but there is also major engorgement of lysosomes with lipid as evidenced by the acid phosphatase activity associated with lipid (arrow). Magnification, ×7,000; bar = 1 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We next examined whether the massive cholesterol stores of DKO macrophages could be mobilized to cholesterol acceptors (Fig. 3 ). Interestingly, incubation with human HDL did not induce a decrease in the cholesterol content of DKO macrophages (Fig. 3A). Indeed, by the end of 48 h, the FC and TC content of the cells increased by 82 ± 5% and 34 ± 2%, respectively. As a control, apoE−/− macrophages were cholesterol enriched by incubation with remnant lipoproteins and subsequently incubated with the same preparation of HDL. When incubated with apoE−/− mouse remnant lipoproteins in vitro, the cholesterol content of control apoE−/− macrophages reached 233 μg TC/mg cell protein (22 ± 1% of TC is FC) prior to initiation of efflux. In contrast to the DKO macrophages, the same preparation of HDL reduced the cholesterol content of control apoE−/− macrophages by 42 ± 1%, demonstrating the capacity of t" @default.
• W2102329287 created "2016-06-24" @default.
• W2102329287 creator A5014813522 @default.
• W2102329287 creator A5049038844 @default.
• W2102329287 creator A5056739581 @default.
• W2102329287 creator A5057828795 @default.
• W2102329287 creator A5060059885 @default.
• W2102329287 creator A5067797353 @default.
• W2102329287 creator A5071380920 @default.
• W2102329287 creator A5089000198 @default.
• W2102329287 date "2007-05-01" @default.
• W2102329287 modified "2023-09-26" @default.
• W2102329287 title "Severely altered cholesterol homeostasis in macrophages lacking apoE and SR-BI" @default.
• W2102329287 cites W1040626293 @default.
• W2102329287 cites W1522830306 @default.
• W2102329287 cites W1578152528 @default.
• W2102329287 cites W1775749144 @default.
• W2102329287 cites W1799444595 @default.
• W2102329287 cites W1859624620 @default.
• W2102329287 cites W1920139491 @default.
• W2102329287 cites W1931983289 @default.
• W2102329287 cites W1964814612 @default.
• W2102329287 cites W1972960247 @default.
• W2102329287 cites W1976538330 @default.
• W2102329287 cites W1977504620 @default.
• W2102329287 cites W1981003642 @default.
• W2102329287 cites W1981886716 @default.
• W2102329287 cites W1986755882 @default.
• W2102329287 cites W1987525850 @default.
• W2102329287 cites W1991272474 @default.
• W2102329287 cites W1992980650 @default.
• W2102329287 cites W1993447679 @default.
• W2102329287 cites W2003300810 @default.
• W2102329287 cites W2013282518 @default.
• W2102329287 cites W2020892886 @default.
• W2102329287 cites W2022690810 @default.
• W2102329287 cites W2024863960 @default.
• W2102329287 cites W2031423487 @default.
• W2102329287 cites W2032513433 @default.
• W2102329287 cites W2035780860 @default.
• W2102329287 cites W2037105577 @default.
• W2102329287 cites W2040915824 @default.
• W2102329287 cites W2041298355 @default.
• W2102329287 cites W2044279966 @default.
• W2102329287 cites W2046921036 @default.
• W2102329287 cites W2053365325 @default.
• W2102329287 cites W2055382682 @default.
• W2102329287 cites W2058555221 @default.
• W2102329287 cites W2059059166 @default.
• W2102329287 cites W2062030534 @default.
• W2102329287 cites W2062535842 @default.
• W2102329287 cites W2069421633 @default.
• W2102329287 cites W2070732640 @default.
• W2102329287 cites W2075597895 @default.
• W2102329287 cites W2087250632 @default.
• W2102329287 cites W2093099491 @default.
• W2102329287 cites W2095153977 @default.
• W2102329287 cites W2111482014 @default.
• W2102329287 cites W2114199907 @default.
• W2102329287 cites W2120083869 @default.
• W2102329287 cites W2130086266 @default.
• W2102329287 cites W2134010411 @default.
• W2102329287 cites W2142957125 @default.
• W2102329287 cites W2144214566 @default.
• W2102329287 cites W2145532583 @default.
• W2102329287 cites W2153170868 @default.
• W2102329287 cites W2156886662 @default.
• W2102329287 cites W2161419248 @default.
• W2102329287 cites W2170417134 @default.
• W2102329287 cites W2184705469 @default.
• W2102329287 cites W2187715873 @default.
• W2102329287 cites W2188585907 @default.
• W2102329287 cites W2276906655 @default.
• W2102329287 cites W2315335674 @default.
• W2102329287 cites W2343798913 @default.
• W2102329287 doi "https://doi.org/10.1194/jlr.m600539-jlr200" @default.
• W2102329287 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/17299204" @default.
• W2102329287 hasPublicationYear "2007" @default.
• W2102329287 type Work @default.
• W2102329287 sameAs 2102329287 @default.
• W2102329287 citedByCount "35" @default.
• W2102329287 countsByYear W21023292872012 @default.
• W2102329287 countsByYear W21023292872013 @default.
• W2102329287 countsByYear W21023292872014 @default.
• W2102329287 countsByYear W21023292872015 @default.
• W2102329287 countsByYear W21023292872017 @default.
• W2102329287 countsByYear W21023292872018 @default.
• W2102329287 countsByYear W21023292872019 @default.
• W2102329287 countsByYear W21023292872020 @default.
• W2102329287 countsByYear W21023292872021 @default.
• W2102329287 countsByYear W21023292872022 @default.
• W2102329287 countsByYear W21023292872023 @default.
• W2102329287 crossrefType "journal-article" @default.
• W2102329287 hasAuthorship W2102329287A5014813522 @default.
• W2102329287 hasAuthorship W2102329287A5049038844 @default.
• W2102329287 hasAuthorship W2102329287A5056739581 @default.
• W2102329287 hasAuthorship W2102329287A5057828795 @default.
• W2102329287 hasAuthorship W2102329287A5060059885 @default.

• next