Retina expresses microsomal triglyceride transfer protein: implications for age-related maculopathy
2005; Elsevier BV; Volume: 46; Issue: 4 Linguagem: Inglês
10.1194/jlr.m400428-jlr200
ISSN1539-7262
AutoresChuanming Li, Jennifer B. Presley, Xueming Zhang, Nassrin Dashti, Byong Hong Chung, Nancy E. Medeiros, Clyde Guidry, Christine A. Curcio,
Tópico(s)Retinal Imaging and Analysis
ResumoThe principal extracellular lesions of age-related maculopathy (ARM), the leading cause of vision loss in the elderly, involve Bruch's membrane (BrM), a thin vascular intima between the retinal pigment epithelium (RPE) and its blood supply. With age, 80–100 nm solid particles containing esterified cholesterol (EC) accumulate in normal BrM, and apolipoprotein B (apoB) immunoreactivity is detectable in BrM- and ARM-associated lesions. Yet little evidence indicates that increased plasma cholesterol is a risk factor for ARM. To determine if RPE is capable of assembling its own apoB-containing lipoprotein, we examined RPE for the expression of microsomal triglyceride transfer protein (MTP), which is required for this process. Consistent with previous evidence for apoB expression, MTP is expressed in RPE, the ARPE-19 cell line, and, unexpectedly, retinal ganglion cells, which are neurons of the central nervous system. De novo synthesis and secretion of neutral lipid by ARPE-19 was supported by high levels of radiolabeled EC and triglyceride in medium after supplementation with oleate. Lipoprotein assembly and secretion is implicated as a constitutive retinal function and a plausible candidate mechanism involved in forming extracellular cholesterol-containing lesions in ARM.The pigmentary retinopathy and neuropathy of abetalipoproteinemia (Mendelian Inheritance of Man 200100; Bassen-Kornzwieg disease), which is caused by mutations in the MTP gene, may involve loss of function at the retina. The principal extracellular lesions of age-related maculopathy (ARM), the leading cause of vision loss in the elderly, involve Bruch's membrane (BrM), a thin vascular intima between the retinal pigment epithelium (RPE) and its blood supply. With age, 80–100 nm solid particles containing esterified cholesterol (EC) accumulate in normal BrM, and apolipoprotein B (apoB) immunoreactivity is detectable in BrM- and ARM-associated lesions. Yet little evidence indicates that increased plasma cholesterol is a risk factor for ARM. To determine if RPE is capable of assembling its own apoB-containing lipoprotein, we examined RPE for the expression of microsomal triglyceride transfer protein (MTP), which is required for this process. Consistent with previous evidence for apoB expression, MTP is expressed in RPE, the ARPE-19 cell line, and, unexpectedly, retinal ganglion cells, which are neurons of the central nervous system. De novo synthesis and secretion of neutral lipid by ARPE-19 was supported by high levels of radiolabeled EC and triglyceride in medium after supplementation with oleate. Lipoprotein assembly and secretion is implicated as a constitutive retinal function and a plausible candidate mechanism involved in forming extracellular cholesterol-containing lesions in ARM. The pigmentary retinopathy and neuropathy of abetalipoproteinemia (Mendelian Inheritance of Man 200100; Bassen-Kornzwieg disease), which is caused by mutations in the MTP gene, may involve loss of function at the retina. Embryologically part of the central nervous system, the retina (Fig. 1A)converts light energy to electrochemical signals for transmission to the brain. The photoreceptors are supported by the retinal pigment epithelium (RPE), a monolayer with diverse functions including daily phagocytosis of the distal tips of photoreceptor outer segments (OS), and the choroid, a vascular bed with the body's highest blood flow. The choriocapillaris is a dense capillary plexus in the innermost choroid, and Bruch's membrane (BrM) is a thin vascular intima between the RPE and the choriocapillaris (Fig. 1B, arrowheads). In human retina, the macula subserves high-acuity vision and is vulnerable to age-related maculopathy (ARM), the major cause of vision loss among the elderly of industrialized societies. The most prominent histopathologic and clinical signs of ARM are extracellular lesions [drusen (Fig. 1E, F) and basal linear deposits (not shown)] in the RPE/BrM complex that ultimately impact vision by the photoreceptors (1Sarks S.H. Ageing and degeneration in the macular region: a clinico-pathological study.Br. J. Ophthalmol. 1976; 60: 324-341Crossref PubMed Scopus (709) Google Scholar, 2Green W.R. Enger C. Age-related macular degeneration histopathologic studies: the 1992 Lorenz E. Zimmerman Lecture.Ophthalmology. 1993; 100: 1519-1535Abstract Full Text PDF PubMed Scopus (679) Google Scholar). Choroidal neovascularization, an invasion of choriocapillaries across BrM and lateral spread within the plane of drusen and basal linear deposit (see 3Curcio C.A. Millican C.L. Basal linear deposit and large drusen are specific for early age-related maculopathy.Arch. Ophthalmol. 1999; 117: 329-339Crossref PubMed Scopus (309) Google Scholar), is the principal sight-threatening complication of ARM's obscure underlying degeneration.Recent findings highlight a role for lipids and lipoproteins in this degeneration. These include a protective effect of the apolipoprotein E4 (apoE4) genotype in populations and the presence of apoB and apoE and histochemically identified lipids in aging- and ARM-associated drusen and deposits in human tissues (4Pauleikhoff D. Zuels S. Sheraidah G.S. Marshall J. Wessing A. Bird A.C.C. 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The lipid composition of drusen, Bruch's membrane, and sclera by hot stage polarizing microscopy.Invest. Ophthalmol. Vis. Sci. 2001; 42: 1592-1599PubMed Google Scholar, 10Pauleikhoff D. Harper C.A. Marshall J. Bird A.C. Aging changes in Bruch's membrane: a histochemical and morphological study.Ophthalmology. 1990; 97: 171-178Abstract Full Text PDF PubMed Scopus (344) Google Scholar, 11Holz F.G. Sheraidah G. Pauleikhoff D. Bird A.C. Analysis of lipid deposits extracted from human macular and peripheral Bruch's membrane.Arch. Ophthalmol. 1994; 112: 402-406Crossref PubMed Scopus (205) Google Scholar, 12Curcio C.A. Millican C.L. Bailey T. Kruth H.S. Accumulation of cholesterol with age in human Bruch's membrane.Invest. Ophthalmol. Vis. Sci. 2001; 42: 265-274PubMed Google Scholar). Furthermore, EC is associated with 80–100 nm diameter solid particles (Fig. 1C) that have surface and core substructure (12Curcio C.A. Millican C.L. Bailey T. Kruth H.S. Accumulation of cholesterol with age in human Bruch's membrane.Invest. Ophthalmol. Vis. Sci. 2001; 42: 265-274PubMed Google Scholar, 13Ruberti J.W. Curcio C.A. Millican C.L. Menco B.P.M. Huang J-D. Johnson M.M. Quick-freeze/deep-etch visualization of age-related lipid accumulation in Bruch's membrane.Invest. Ophthalmol. Vis. Sci. 2003; 44: 1753-1759Crossref PubMed Scopus (105) Google Scholar, 14Curcio C.A. Presley J.B. Millican C.L. Medeiros N.E. Basal deposits and drusen in eyes with age-related maculopathy: evidence for solid lipid particles.Exp. Eye Res. 2005; In pressGoogle Scholar). Understanding the pathway(s) that lead to neutral lipid deposition in normal human BrM is critical for understanding the pathway(s) that lead to drusen and basal linear deposits in ARM. Studies of BrM lipid composition (11Holz F.G. Sheraidah G. Pauleikhoff D. Bird A.C. Analysis of lipid deposits extracted from human macular and peripheral Bruch's membrane.Arch. Ophthalmol. 1994; 112: 402-406Crossref PubMed Scopus (205) Google Scholar, 12Curcio C.A. Millican C.L. Bailey T. Kruth H.S. Accumulation of cholesterol with age in human Bruch's membrane.Invest. Ophthalmol. Vis. Sci. 2001; 42: 265-274PubMed Google Scholar, 15Sheraidah G. Steinmetz R. Maguire J. Pauleikhoff D. Marshall J. Bird A.C.C. Correlation between lipids extracted from Bruch's membrane and age.Ophthalmology. 1993; 100: 47-51Abstract Full Text PDF PubMed Scopus (107) Google Scholar) implicate extravasation of plasma lipoproteins from the choroid or deposition from intraocular cells as candidate mechanisms. The RPE processes abundant lipids from ingested OS membranes (16Nguyen-Legros J. Hicks D. Renewal of photoreceptor outer segments and their phagocytosis by the retinal pigment epithelium.Int. Rev. Cytol. 2000; 196: 245-313Crossref PubMed Google Scholar). It synthesizes and secretes BrM constituents (17Campochiaro P.A. Jerdan J.A. Glaser B.M. 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An integrated hypothesis that considers drusen as biomarkers of immune-mediated processes at the RPE-Bruch's membrane interface in aging and age-related macular degeneration.Prog. Retin. Eye Res. 2001; 20: 705-732Crossref PubMed Scopus (1066) Google Scholar), including apoE (6Anderson D.H. Ozaki S. Nealon M. Neitz J. Mullins R.F. Hageman G.S. Johnson L.V.V. Local cellular sources of apolipoprotein E in the human retina and retinal pigmented epithelium: implications for the process of drusen formation.Am. J. Ophthalmol. 2001; 131: 767-781Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 21Ishida B.Y. Bailey K.R. Duncan K.G. Chalkley R.J. Burlingame A.L. Kane J.P. Schwartz D.M.M. Regulated expression of apolipoprotein E by human retinal pigment epithelial cells.J. Lipid Res. 2004; 45: 263-271Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). However, a mechanism to account for the abundant EC and other neutral lipids in normal BrM is unknown.Atherosclerotic cardiovascular disease and ARM feature apoB- and cholesterol-containing extracellular lesions in a vascular intima and shared risk factors of smoking and hypertension (22Smith W. Assink J. Klein R. Mitchell P. Klaver C.C.W. Klein B.E.K. Hofman A. Jensen S. Wang J.J. de Jong P.T.V.M.M. Risk factors for age-related macular degeneration. Pooled findings from three continents.Ophthalmology. 2001; 108: 697-704Abstract Full Text Full Text PDF PubMed Scopus (810) Google Scholar, 23Age-Related Eye Disease Study GroupRisk factors associated with age-related macular degeneration. A case-control study in the Age-Related Eye Disease Study: Age-Related Eye Disease Study Report Number 3.Ophthalmology. 2000; 107: 2224-2232Abstract Full Text Full Text PDF PubMed Scopus (711) Google Scholar), among others. However, only 1 epidemiologic study of 23 since 1963 (24Eye Disease Case-Control Study GroupRisk factors for neovascular age-related macular degeneration.Arch. Ophthalmol. 1992; 110: 1701-1708Crossref PubMed Scopus (616) Google Scholar) links ARM with increased total plasma cholesterol (see supplementary table), a defining risk factor for atherosclerosis (25Verschuren W.M. Jacobs D.R. Bloemberg B.P. Kromhout D. Menotti A. Aravanis C. Blackburn H. Buzina R. Dontas A.S. Fidanza F.F. et al.Serum total cholesterol and long-term coronary heart disease mortality in different cultures. Twenty-five-year follow-up of the seven countries study.J. Am. Med. Assoc. 1995; 274: 131-136Crossref PubMed Google Scholar). Although not the strongest indicator of cardiovascular risk, total plasma cholesterol was the one measure common to all of these investigations. Many studies were small and used different ARM end points. However, if plasma lipoproteins were the major source of cholesterol in BrM and sub-RPE deposits, then a more consistent association should have been detected despite these limitations. This paradox could be reconciled if an apoB-containing lipoprotein of intraocular origin were the major source of early-arriving EC in BrM- and ARM-associated lesions, in contrast to apoB-containing lipoprotein from plasma that arrives early in incipient atherosclerotic plaques (26Williams K.J. Tabas I. The response-to-retention hypothesis of early atherogenesis.Arterioscler. Thromb. Vasc. Biol. 1995; 15: 551-561Crossref PubMed Google Scholar, 27Kruth H.S. Cholesterol deposition in atherosclerotic lesions.Subcell. Biochem. 1997; 28: 319-362Crossref PubMed Scopus (30) Google Scholar). This idea is credible, because native human RPE contains apoB mRNA and protein (8Malek G. Li C-M. Guidry C. Medeiros N.E. Curcio C.A. Apolipoprotein B in cholesterol-containing drusen and basal deposits in eyes with age-related maculopathy.Am. J. Pathol. 2003; 162: 413-425Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar, 19Mullins R.F. Russell S.R. Anderson D.H. Hageman G.S. Drusen associated with aging and age-related macular degeneration contain proteins common to extracellular deposits associated with atherosclerosis, elastosis, amyloidosis, and dense deposit disease.FASEB J. 2000; 14: 835-846Crossref PubMed Scopus (739) Google Scholar), and the thermal behavior of birefringent EC crystals in BrM differs from that in sclera, which accumulates EC from plasma apoB-containing lipoprotein (28Smith E. Relationship between plasma lipids and arterial tissue lipids.Nutr. Metab. 1973; 15: 17-26Crossref PubMed Scopus (23) Google Scholar).If the RPE synthesizes, assembles, and secretes an apoB-containing lipoprotein with a neutral lipid core, it should also express microsomal triglyceride transfer protein (MTP). This soluble heterodimer in the endoplasmic reticulum lumen is required for apoB-containing lipoprotein assembly (29Wetterau J.R. Lin M.C. Jamil H. Microsomal triglyceride transfer protein.Biochim. Biophys. Acta. 1997; 1345: 136-150Crossref PubMed Scopus (283) Google Scholar, 30Gordon D.A. Jamil H. Progress towards understanding the role of microsomal triglyceride transfer protein in apolipoprotein-B lipoprotein assembly.Biochim. Biophys. Acta. 2000; 1486: 72-83Crossref PubMed Scopus (191) Google Scholar). Cells expressing apoB without MTP cannot secrete lipoproteins, and all cells expressing MTP also secrete apoB-containing lipoprotein (31Wetterau J.R. Aggerbeck L.P. Bouma M.E. Eisenberg C. Munck A. Hermier M. Schmitz J. Gay G. Rader D.J. 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Lipoprotein assembly capacity of the mammary tumor-derived cell line C127 is due to the expression of functional microsomal triglyceride transfer protein.J. Lipid Res. 2001; 42: 1897-1904Abstract Full Text Full Text PDF PubMed Google Scholar). MTP mutations resulting in a lack of functional protein cause abetalipoproteinemia (ABL; MIM 200100, Bassen-Kornzwieg disease), a rare autosomal recessive disorder. ABL features the absence of plasma apoB-containing lipoprotein, fat malabsorption, steatorrhea, acanthocytosis, ataxic neuropathy, and, importantly, a pigmentary retinopathy (31Wetterau J.R. Aggerbeck L.P. Bouma M.E. Eisenberg C. Munck A. Hermier M. Schmitz J. Gay G. Rader D.J. Gregg R.E.E. Absence of microsomal triglyceride transfer protein in individuals with abetalipoproteinemia.Science. 1992; 258: 999-1001Crossref PubMed Scopus (629) Google Scholar, 35Berriot-Varoqueaux N. Aggerbeck L.P. Samson-Bouma M. Wetterau J.R. The role of the microsomal triglyceride transfer protein in abetalipoproteinemia.Annu. Rev. Nutr. 2000; 20: 663-697Crossref PubMed Scopus (261) Google Scholar). To begin evaluating our hypothesis, we determined whether MTP was present in human neurosensory retina, native RPE, and ARPE-19, a spontaneously arising transformed cell line with differentiated properties (36Dunn K.C. Aotaki-Keen A.E. Putkey F.R. Hjelmeland L.M. ARPE-19, a human retinal pigment epithelial cell line with differentiated properties.Exp. Eye Res. 1996; 62: 155-162Crossref PubMed Scopus (1041) Google Scholar, 37Philp N.J. Wang D. Yoon H. Hjelmeland L.M. Polarized expression of monocarboxylate transporters in human retinal pigment epithelium and ARPE-19 cells.Invest. Ophthalmol. Vis. Sci. 2003; 44: 1716-1721Crossref PubMed Scopus (133) Google Scholar, 38Blackburn J. Tarttelin E.E. Gregory-Evans C.Y. Moosajee M. Gregory-Evans K.K. Transcriptional regulation and expression of the dominant drusen gene FBLN3 (EFEMP1) in mammalian retina.Invest. Ophthalmol. Vis. Sci. 2003; 44: 4613-4621Crossref PubMed Scopus (31) Google Scholar). We then determined that ARPE-19 could secrete EC-containing particles, confirming these results in primary porcine RPE. Of a lipoprotein's surface and core components, we focused on EC, because it is localized exclusively within BrM (Fig. 1D). Our results have implications for ARM pathobiology, retinal cell biology, and ABL.MATERIALS AND METHODSHuman tissueHuman donor eyes with grossly normal maculas were obtained from the Alabama Eye Bank within 6 h of death (n = 8, 65–85 years for RT-PCR; n = 9, 30–92 years for Western blot; n = 9, 38–89 years for immunohistochemistry). For immunohistochemistry, anterior segments of donor globes were removed by encircling cuts at the corneoscleral limbus. Posterior segments were immersed in 4% paraformaldehyde in 0.1 M phosphate buffer for 6–16 h and stored in 1% paraformaldehyde at 4°C until used. To isolate retinal mRNA and protein, globes were incised at the equator, and anterior segments were removed with vitreous body attached. Small forceps were used to peel the retina from the RPE/choroid while the scleral eyecup was stabilized with another forceps.Cell cultureARPE-19 and HepG2 cell lines were obtained from the American Type Culture Collection at passage 22 and subjected to RT-PCR, Western blot, and secretion experiments after 2 passages. ARPE-19 cells were also provided by Dr. N. J. Philp (Thomas Jefferson University) (37Philp N.J. Wang D. Yoon H. Hjelmeland L.M. Polarized expression of monocarboxylate transporters in human retinal pigment epithelium and ARPE-19 cells.Invest. Ophthalmol. Vis. Sci. 2003; 44: 1716-1721Crossref PubMed Scopus (133) Google Scholar). For RT-PCR and Western blot, cells were plated in T-75 flasks or on six-well plates and grown for 4 weeks in DMEM/F12 (1:1) supplemented with 10% fetal calf serum as described (36Dunn K.C. Aotaki-Keen A.E. Putkey F.R. Hjelmeland L.M. ARPE-19, a human retinal pigment epithelial cell line with differentiated properties.Exp. Eye Res. 1996; 62: 155-162Crossref PubMed Scopus (1041) Google Scholar). Medium was changed twice weekly. HepG2 cells were grown on six-well plates in MEM containing 10% fetal calf serum for 5 days with a medium change every other day. Primary cultures of porcine RPE were prepared as described (39Mamballikalathil I. Mann C. Guidry C. Tractional force generation by porcine Müller cells: stimulation by retinal pigment epithelial cell-secreted growth factor.Invest. Ophthalmol. Vis. Sci. 2000; 41: 529-536PubMed Google Scholar) and used 24 h after isolation.RT-PCR and sequencingTotal RNA was isolated from human retina, RPE, ARPE-19 cells, and HepG2 cells as described (8Malek G. Li C-M. Guidry C. Medeiros N.E. Curcio C.A. Apolipoprotein B in cholesterol-containing drusen and basal deposits in eyes with age-related maculopathy.Am. J. Pathol. 2003; 162: 413-425Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). Total RNA from porcine retina, RPE, and liver was isolated with Trizol Reagent (Invitrogen, Carlsbad, CA). Human ileum mRNA was purchased from Stratagene. Primers used for RT-PCR are listed in Table 1. To distinguish between amplified mRNA and genomic DNA, primers were designed to span intron boundaries except where noted. One-step RT-PCR was used for human apoB and porcine apoB and MTP, as described (8Malek G. Li C-M. Guidry C. Medeiros N.E. Curcio C.A. Apolipoprotein B in cholesterol-containing drusen and basal deposits in eyes with age-related maculopathy.Am. J. Pathol. 2003; 162: 413-425Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). Two-step RT-PCR was used for others. The first-strand cDNA was synthesized with Omniscript™ Reverse Transcriptase (Qiagen, Valencia, CA). The PCR Core System (Promega, Madison, WI) was used for PCR. The cDNA was denatured for 4 min at 94°C before cycling. The reaction was amplified through 35 cycles of 45 s at 94°C (denaturing), 45 s at 55–66°C (annealing), and 1 min at 72°C (extension), then incubated for 10 min at 72°C. Cross-contamination of retinal and RPE mRNA was assessed by checking the expression of the retina-specific gene RHO in RPE mRNA and the RPE-specific gene VMD2 in neurosensory retina mRNA. All results for neurosensory retina and RPE were obtained in at least one sample without contamination. The primers 5′-GAG AAA CTG ACT GCT CTC AC-3′ (sense) and 5′-ATG ATA GTG CTC ATC AAG ACT T-3′ (antisense) were used to amplify a 234 bp human apoB cDNA fragment containing the editing site of apoB-48. The cDNA sequence was analyzed as described (8Malek G. Li C-M. Guidry C. Medeiros N.E. Curcio C.A. Apolipoprotein B in cholesterol-containing drusen and basal deposits in eyes with age-related maculopathy.Am. J. Pathol. 2003; 162: 413-425Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar).TABLE 1The sequences of PCR primers and expected fragment sizesSpeciesGeneProteinPrimersExpected SizebpHumanAPOBApolipoprotein BF: 5′-GAG GTC ATC AGG AAG GGC TCA AAG-3′419R: 5′- GGG ATC ACC TCC GTT TTG GTG GTA-3′HumanAPOBApolipoprotein BF: 5′-GAG AAA CTG ACT GCT CTC AC-3′234R: 5′-ATG ATA GTG CTC ATC AAG ACT T-3′HumanMTPMicrosomal triglyceride transfer protein (large polypeptide, 88 kDa)F: 5′-GGA CTT TTT GGA TTT CAA AAG TGA C-3′ R: 5′-GGA GAA ACG GTC ATA ATT GTG-3′698HumanPDIProtein disulfide isomeraseF: 5′-TGA CCT TTG GCC TCA CAG ACC-3′715R: 5′-TAC TTC TCA GCC AAT GCC TCC-3′HumanAPOBEC1Apolipoprotein B mRNA-editing enzyme, catalytic polypeptide 1F: 5′-ACA CCA CCA ATC ACG TGG AAG-3′ R: 5′-TCA TCT CCA AGC CAC AGA AGG-3′542HumanSOAT1Sterol O-acyltransferase (acyl-CoA:cholesterol acyltransferase) 1F: 5′-AAG TTG ACA GCA GAG GCA GA-3′ R: 5′-ATC CAC CAG GTC CAA ACA AC-3′413HumanSOAT2Sterol O-acyltransferase (acyl-CoA:cholesterol acyltransferase) 2F: 5′-GCT GCT GCT GGA GTT TGA CC-3′ R: 5′-AGC AGG CAT AGA GCA CAC ATC-3′494PorcineAPOBApolipoprotein BF: 5′-TGG GAA CGA AGA TCA CAC CTA C-3′351R: 5′-GGG AAG CCA CAA AGT TCT TCA C-3′PorcineMTPMicrosomal triglyceride transfer proteinF: 5′-TGA CCT ACC AGG CTC ATC AA-3′336R: 5′-GGA TGG CCG TGT ACT TAG AA-3′All primers for human genes were designed based on GenBank gene sequences. Primers for porcine MTP were from Lu et al. (79Lu S. Huffman M. Yao Y. Mansbach 2nd, C.M. Cheng X. Meng S. Black D.D.D. Regulation of MTP expression in developing swine.J. Lipid Res. 2002; 43: 1303-1311Abstract Full Text Full Text PDF PubMed Google Scholar). ACAT1 and ACAT2 are gene aliases for SOAT1 and SOAT2. F, forward primer; R, reverse primer. Open table in a new tab Western blotProtein extracts of human retina, RPE, ARPE-19 cells, and HepG2 cells were prepared and Western blot analysis was performed as described (8Malek G. Li C-M. Guidry C. Medeiros N.E. Curcio C.A. Apolipoprotein B in cholesterol-containing drusen and basal deposits in eyes with age-related maculopathy.Am. J. Pathol. 2003; 162: 413-425Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). Polyclonal rabbit anti-bovine MTP 97 kDa large subunit (40Levy E. Stan S. Delvin E. Menard D. Shoulders C. Garofalo C. Slight I. Seidman E. Mayer G. Bendayan M.M. Localization of microsomal triglyceride transfer protein in the Golgi. Possible role in the assembly of chylomicrons.J. Biol. Chem. 2002; 277: 16470-16477Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar) was a gift from Dr. J. R. Wetterau at Bristol-Meyers-Squibb (used at 1:500). Monoclonal anti-human apoB [1D1, recognizing an N-terminal epitope between amino acids 474 and 539 (41Wang X. Bucala R. Milne R. Epitopes close to the apolipoprotein B low density lipoprotein receptor-binding site are modified by advanced glycation end products.Proc. Natl. Acad. Sci. USA. 1998; 95: 7643-7647Crossref PubMed Scopus (34) Google Scholar)] was a gift from Dr. R. Milne at the University of Ottawa (used at 1:1,000). Secondary antibodies, horseradish peroxidase-conjugated donkey anti-rabbit IgG, and donkey anti-mouse IgG were purchased from Jackson Immunoresearch (West Grove, PA; used at 1:2,000). Immunoblotted proteins were detected by enhanced chemiluminescence (Amersham Biosciences, Piscataway, NJ).ImmunohistochemistryCryosections of human retina and choroid were prepared as described (8Malek G. Li C-M. Guidry C. Medeiros N.E. Curcio C.A. Apolipoprotein B in cholesterol-containing drusen and basal deposits in eyes with age-related maculopathy.Am. J. Pathol. 2003; 162: 413-425Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). Except where noted, reagents were purchased from Vector Laboratories (Burlingame, CA). For localizing apoB, MTP, and monocarboxylate transporter 3 (MCT3) in human RPE, sections were bleached before staining as described (42Bhutto I.A. Kim S.Y. McLeod D.S. Merges C. Fukai N. Olsen B.R. Lutty G.A.A. Localization of collagen XVIII and the endostatin portion of collagen XVIII in aged human control eyes and eyes with age-related macular degeneration.Invest. Ophthalmol. Vis. Sci. 2004; 45: 1544-1552Crossref PubMed Scopus (98) Google Scholar). Briefly, cryosections were removed from −20°C, heated for 30 min at 50–55°C, and rinsed with PBS. Slides were incubated in 0.05% potassium permanganate for 25 min, rinsed for 5 min in distilled water, flooded with 35% peracetic acid in a humidified container for 15 min, and washed in distilled water for 10 min, all at room temperature. Next, sections were heated at 50–55°C for 30 min and incubated with the primary antibodies anti-apoB (Biodesign, Saco, ME; 1:500), anti-MTP (1:100), and anti-MCT3 (1:100; a gift from Dr. N. J. Philp) followed by biotinylated secondary antibodies, peroxidase-conjugated streptavidin, and the VIP Peroxidase Substrate Kit. Appropriate controls were processed w
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