Hypoxia Up-regulates CD36 Expression and Function via Hypoxia-inducible Factor-1- and Phosphatidylinositol 3-Kinase-dependent Mechanisms
2009; Elsevier BV; Volume: 284; Issue: 39 Linguagem: Inglês
10.1074/jbc.m109.033480
ISSN1083-351X
AutoresBupe R. Mwaikambo, Chun Yang, Sylvain Chemtob, Pierre Hardy,
Tópico(s)interferon and immune responses
ResumoNeovascular and degenerative diseases of the eye are leading causes of impaired vision and blindness in the world. Hypoxia or reduced oxygen tension is considered central to the pathogenesis of these disorders. Although the CD36 scavenger receptor features prominently in ocular homeostasis and pathology, little is known regarding its modulation by hypoxia. Herein we investigated the role and regulation of CD36 by hypoxia and by the major hypoxia effector, hypoxia-inducible factor (HIF)-1. In vivo, hypoxia markedly induced CD36 mRNA in corneal and retinal tissue. Subsequent experiments on human retinal pigment epithelial cells revealed that hypoxia time-dependently increased CD36 mRNA, protein, and surface expression; these responses were reliant upon reactive oxygen species production. As an important novel finding, we demonstrate that hypoxic stimulation of CD36 is mediated by HIF-1; HIF-1α down-regulation abolished CD36 induction by both hypoxia and cobalt chloride. Sequence analysis of the human CD36 promoter region revealed a functional HIF-1 binding site. A luciferase reporter construct containing this promoter fragment was activated by hypoxia, whereas mutation at the HIF-1 consensus site decreased promoter activation. Specific binding of HIF-1 to this putative site in hypoxic cells was detected by a chromatin immunoprecipitation assay. Interestingly, inhibition of the phosphatidylinositol 3-kinase pathway blocked the hypoxia-dependent induction of CD36 expression and promoter activity. Functional ramifications of CD36 hypoxic accumulation were evinced by CD36-dependent increases in scavenging and anti-angiogenic activities. Together, our findings indicate a novel mechanism by which hypoxia induces CD36 expression via activation of HIF-1 and the phosphatidylinositol 3-kinase pathway. Neovascular and degenerative diseases of the eye are leading causes of impaired vision and blindness in the world. Hypoxia or reduced oxygen tension is considered central to the pathogenesis of these disorders. Although the CD36 scavenger receptor features prominently in ocular homeostasis and pathology, little is known regarding its modulation by hypoxia. Herein we investigated the role and regulation of CD36 by hypoxia and by the major hypoxia effector, hypoxia-inducible factor (HIF)-1. In vivo, hypoxia markedly induced CD36 mRNA in corneal and retinal tissue. Subsequent experiments on human retinal pigment epithelial cells revealed that hypoxia time-dependently increased CD36 mRNA, protein, and surface expression; these responses were reliant upon reactive oxygen species production. As an important novel finding, we demonstrate that hypoxic stimulation of CD36 is mediated by HIF-1; HIF-1α down-regulation abolished CD36 induction by both hypoxia and cobalt chloride. Sequence analysis of the human CD36 promoter region revealed a functional HIF-1 binding site. A luciferase reporter construct containing this promoter fragment was activated by hypoxia, whereas mutation at the HIF-1 consensus site decreased promoter activation. Specific binding of HIF-1 to this putative site in hypoxic cells was detected by a chromatin immunoprecipitation assay. Interestingly, inhibition of the phosphatidylinositol 3-kinase pathway blocked the hypoxia-dependent induction of CD36 expression and promoter activity. Functional ramifications of CD36 hypoxic accumulation were evinced by CD36-dependent increases in scavenging and anti-angiogenic activities. Together, our findings indicate a novel mechanism by which hypoxia induces CD36 expression via activation of HIF-1 and the phosphatidylinositol 3-kinase pathway. Hypoxia, a reduction in cellular oxygen tension, is a key determinant of tissue pathology and survival during tumor development and ischemic diseases including retinopathies, myocardial infarction, and atherosclerosis. In response to hypoxia, mammalian cells express a variety of gene products important for erythropoiesis, angiogenesis, and glycolysis, thereby improving tissue oxygenation and facilitating metabolic demands (1Chandel N.S. Budinger G.R. Free Radic. Biol. Med. 2007; 42: 165-174Crossref PubMed Scopus (200) Google Scholar, 2Semenza G.L. Science. 2007; 318: 62-64Crossref PubMed Scopus (559) Google Scholar). These adaptive responses require the concerted activation of various transcription factors including hypoxia-inducible factor-1 (HIF-1), 4The abbreviations used are: HIF-1hypoxia-inducible factor-1HREhypoxia response elementROSreactive oxygen speciesPI3Kphosphatidylinositol 3-kinasemTORmammalian target of rapamycinPOSphotoreceptor outer segmentsActDactinomycin DFITCfluorescein isothiocyanateCHXcycloheximideDiI1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorateoxLDLoxidized lipoprotein lipaseqRTquantitative real timeARPEhuman retinal pigment epithelial cellssiRNAsilencing RNAmAbmonoclonal antibodyFACSfluorescence-activated cell sorting. 4The abbreviations used are: HIF-1hypoxia-inducible factor-1HREhypoxia response elementROSreactive oxygen speciesPI3Kphosphatidylinositol 3-kinasemTORmammalian target of rapamycinPOSphotoreceptor outer segmentsActDactinomycin DFITCfluorescein isothiocyanateCHXcycloheximideDiI1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorateoxLDLoxidized lipoprotein lipaseqRTquantitative real timeARPEhuman retinal pigment epithelial cellssiRNAsilencing RNAmAbmonoclonal antibodyFACSfluorescence-activated cell sorting. which is generally considered the master regulator of oxygen homeostasis (2Semenza G.L. 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It has become increasingly evident that reactive oxygen species (ROS) and the phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) pathway play a crucial role in regulating HIF-1 activity (10Lin X. David C.A. Donnelly J.B. Michaelides M. Chandel N.S. Huang X. Warrior U. Weinberg F. Tormos K.V. Fesik S.W. Shen Y. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 174-179Crossref PubMed Scopus (85) Google Scholar, 11Belaiba R.S. Bonello S. Zähringer C. Schmidt S. Hess J. Kietzmann T. Görlach A. Mol. Biol. Cell. 2007; 18: 4691-4697Crossref PubMed Scopus (329) Google Scholar, 12Sun H.L. Liu Y.N. Huang Y.T. Pan S.L. Huang D.Y. Guh J.H. Lee F.Y. Kuo S.C. Teng C.M. Oncogene. 2007; 26: 3941-3951Crossref PubMed Scopus (154) Google Scholar, 13Mottet D. Dumont V. Deccache Y. Demazy C. Ninane N. Raes M. Michiels C. J. Biol. Chem. 2003; 278: 31277-31285Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar, 14Chandel N.S. McClintock D.S. Feliciano C.E. Wood T.M. 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Sci. 2006; 47: 4356-4364Crossref PubMed Scopus (51) Google Scholar), whereas others have documented an up-regulation of CD36 during cerebral ischemia (29Cho S. Park E. Febbraio M. Anrather J. Park L. Racchumi G. Silverstein R. Iadecola C. J. Neuro. 2005; 25: 2504-2512Crossref Scopus (173) Google Scholar) and an attenuation of its expression and activity following antioxidant treatment (30Ricciarelli R. Zingg J. Azzi A. Circulation. 2000; 102: 82-87Crossref PubMed Scopus (259) Google Scholar, 31Cho S. Szeto H.H. Kim E. Kim H. Tolhurst A.T. Pinto J.T. J. Biol. Chem. 2007; 282: 4634-4642Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Collectively, these studies led us to hypothesize that hypoxia modulates CD36 expression and function, with the objective of characterizing the mechanisms behind its regulation. Indeed our findings describe a novel mechanism in which hypoxia induces CD36 expression and promoter activity by stimulating ROS production in a HIF-1- and PI3K-dependent manner. Actinomycin D (ActD), allopurinol, angiotensin II, cobalt chloride (CoCl2), fluorescein isothiocyanate (FITC), myxothiazol, rotenone, isotype control antibodies anti-human IgA, anti-mouse IgG1, and anti-mouse IgM (Sigma); cycloheximide (CHX) and rapamycin (Calbiochem); monoclonal β-actin, CD36 monoclonal antibody (mAb) clone SMO, and anti-HIF-1α antibody (Abcam); CD36 rabbit polyclonal antibody, PI 3-kinase p85α mouse monoclonal antibody, and horseradish peroxidase-linked IgG (Santa Cruz Biotechnology); monoclonal CD36-FITC (Serotec, Oxford, UK); CD36 mAb clone JC63.1 (aCD36 (oxLDL)) and YC-1 (Cayman Chemical, Ann Arbor, MI); U74389G (Biomol); Tempol (Fluka Biochemica); 1, 1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI)-oxidized LDL (DiI-oxLDL; Intracel, Frederick, MD); wortmannin and LY294002 (Alomone Labs Ltd.); CD36 mAb clone FA6-152 (aCD36 (TSP-1) (Beckman Coulter, Fullerton, CA)); 4′,6-diamidino-2-phenylindole, dichlorofluorescein diacetate, annexin V-FITC/PI staining (Molecular Probes, Eugene, OR); CellTracker Green 5-fluoromethylfluorescein diacetate (Cambrex Corp.); and Lipofectamine 2000 (Invitrogen Corp.) were used. Six-week-old male C57BL/6 mice purchased from Charles River (St. Constant, Quebec, Canada) were used according to a protocol approved by the Research Center of CHU Sainte-Justine Animal Care Committee. Mice were exposed to ambient room air or placed in an OxyCycler (BioSpherix, Ltd.) and exposed to hypoxia (8% O2) for 6 h. At the end of the experiments, animals were immediately sacrificed and tissues were processed for quantitative real time-PCR (qRT-PCR). Human dermal microvascular endothelial cells, human pulmonary artery smooth muscle cells (Cambrex, Walkersville, MD), human retinal pigment epithelial (ARPE-19), and human promyelocytic leukemia (HL60) cells (ATCC, Manassas, VA) were maintained according to standard procedures. For the majority of experiments, ARPEs were used because of their relevance to our ocular studies. For hypoxia exposure, culture dishes were placed in a hypoxia chamber (Billups Rothenburg Inc.) allowing the establishment of a hypoxic environment of 2% O2, 5% CO2, and 93% N2; unless otherwise stated, this hypoxic level (24 h) was used in all experiments. The airtight incubator was kept at 37 °C for preset time periods, whereas normoxic cells were placed at 37 °C in a 21% O2, 5% CO2, and 74% N2 humidified incubator. Total RNA was extracted using the standard TRIzol RNA isolation protocol (Invitrogen) and treated with DNase I (Qiagen, Hilden, Germany). cDNA was synthesized from 1 μg of RNA with Moloney murine leukemia virus reverse transcriptase (Promega Corp.) according to the manufacturer's instructions and amplified using SYBR Green I (Stratagene) in a sequence detection system (MxPro 3000 QPCR systems, Stratagene). Primers for CD36, HIF-1α, and β-Actin were synthesized by Invitrogen as follows: human CD36, forward 5′-TCTTTCCTGCAGCCCAATG-3′, reverse 5′-AGCCTCTGTTCCAACTGATAGTGA-3′; human HIF-1α, forward 5′-TGCTTGGTGCTGATTTGTGA-3′, reverse 5′-GGTCAGATGATCAGAGTCCA-3′; human β-actin, forward 5′-GGGRCAGAAGGATTCCTATG-3′, reverse 5′-GGTCTCAAACATGATCTGGG-3′; mouse CD36, forward 5′-TCCTCTGACATTTGCAGGTCTATC-3′, reverse 5′-AAAGGCATTGGCTGGAAGAA-3′; and mouse β-actin, forward 5′-ACTATTGGCAACGACCGGTTT-3′, reverse, 5′-AAGGAAGGCTGGAAAAGAGGG-3′. Primers for PI3K p85α were supplied by Santa Cruz Biotechnology. PCR amplification protocol involved 40 cycles of denaturation at 95 °C for 30 s, primer annealing at 55 °C, and primer extension at 72 °C for 60 s. Each sample was analyzed in triplicate along with RT and no template controls. ARPEs were grown to 40% confluence and transfected using Lipofectamine 2000 with scrambled Silencer Negative Control 1 silencing RNA (siRNA; Ambion) or sequence-specific siRNA targeting PI3K p85α (Santa Cruz Biotechnology) or HIF-1α (Ambion; pre-designed siRNA ID 106498 and 106500 both showed similar knock-down effectiveness and the former was used in all experiments). Cells were incubated with HIF-1α or PI3K p85α siRNA for 24 h after which they were subjected to hypoxia followed by analysis by qRT-PCR, Western blot, or luciferase assay. Protein extraction from cells and Western blots were performed as previously described (19Sennlaub F. Valamanesh F. Vazquez-Tello A. El-Asrar A.M. Checchin D. Brault S. Gobeil F. Beauchamp M.H. Mwaikambo B. Courtois Y. Geboes K. Varma D.R. Lachapelle P. Ong H. Behar-Cohen F. Chemtob S. Circulation. 2003; 108: 198-204Crossref PubMed Scopus (145) Google Scholar) using 40 μg of total protein and anti-CD36 (1:400), anti-p85α (1:300), anti-HIF-1α (1:250), or anti-β-Actin (1:10000) antibodies. Protein expression was quantified via densitometry (Image-Pro Plus software, version 4.1; Media Cybernetics, Silver Spring, MD). ARPEs at 70% confluence were exposed to normoxia or hypoxia followed by analysis of CD36 expression via fluorescence-activated cell sorting (FACS) (FACScan; BD Biosciences) using an anti-CD36-FITC antibody. A minimum of 10,000 cells/sample were assessed. Data were acquired and analyzed using CellQuest software. ARPEs were pre-exposed to 21 or 2% O2 followed by addition of ActD (4.5 μg/ml) to block transcription and subsequent return to normoxia or hypoxia. Cells were harvested at various time points after ActD treatment and processed for qRT-PCR. CD36 mRNA stability was determined as the percentage of initial mRNA remaining after ActD exposure. To elucidate the contribution of ongoing protein synthesis and protein stability, CHX was added to block translation before (25 μm) or after (100 μm) hypoxia. CD36 protein stability was assessed as the proportion of the initial protein remaining after CHX exposure. Intracellular ROS generation was measured using the fluorescent probe dichlorofluorescein diacetate. ARPEs were cultured in 24-well plates and exposed to hypoxia at the indicated time points or angiotensin II (100 nm) for 45 min. Cells were subsequently incubated with dichlorofluorescein diacetate (10 μm) for 30 min at 37 °C followed by measurements using a multiwell fluorescent plate reader (Wallac 1420 VICTOR Multilabel Counter) set at 485 nm excitation and 535 nm emission wavelengths. 8-Isoprostanes (8-Iso-prostaglandin F2α) were measured in normoxia and hypoxia were exposed ARPEs by enzyme immunoassay (Cayman Chemical, Ann Arbor, MI) as previously described (18Mwaikambo B.R. Sennlaub F. Ong H. Chemtob S. Hardy P. Invest. Ophthalmol. Vis. Sci. 2006; 47: 4356-4364Crossref PubMed Scopus (51) Google Scholar). A human bacterial artificial chromosome (clone RP11-542H16 from chromosome 7) carrying the human gene for the CD36 promoter (NCBI accession number DD115563) was obtained from the BACPAC Resources Center (Children's Hospital Oakland Research Institute, Oakland, CA). The identity of the DNA clone was verified by restriction digest. A fragment containing the 5′-untranslated region (∼3.5 kbp) of the putative CD36 promoter was generated from the bacterial artificial chromosome by PCR using the following primers: forward, 5′-GCGAGCTCCTGAGGAATACAATTGGGGATTGAG-3′ and reverse, 5′-GCGCTCGAGGCAGTCCTCAGTACATAAATGCGT-3′. This product was cloned into the SacI and XhoI sites of the pGL3-basic and pGL3-promoter vectors (Promega Corp.), and the generated plasmids were designated pCD36-luc1 and pCD36- luc2, respectively. In the pCD36-mutHRE construct, the putative HRE of pCD36-luc1 was replaced from 5′-CGTG-3′ to 5′-AAAG-3′ using the QuikChange site-directed mutagenesis kit (Stratagene). All constructs were verified by DNA sequencing. ARPEs at about 70% confluence in 12-well plates were transiently transfected with reporter plasmid (1 μg) using Lipofectamine 2000 according to the manufacturer's directions. To correct for variable transfection efficiency, cells were co-transfected with pcDNA3.1 LacZ (0.5 μg), which encodes for the β-galactosidase gene. Transfected cells were allowed to recover for 24 h in fresh medium, and then treated with CoCl2 (100 μm) or subjected to normoxia or hypoxia. Cells were lysed and luciferase activity determined using the Luciferase Reporter Assay system (Promega) via a multiwell luminescence reader (Wallac 1420 VICTOR Multilabel Counter). Chromatin immunoprecipitation was performed according to the chromatin immunoprecipitation assay kit protocol (Upstate Biotechnology, Lake Placid, NY). Briefly confluent cells were grown on 15-cm dishes and exposed to CoCl2, normoxia, or hypoxia (4% O2, 1 h). Cells were immediately fixed with 1% formaldehyde/phosphate-buffered saline, lysed, and sonicated to obtain 500 to 1000-base pair DNA fragments. Chromatin was immunoprecipitated using 5 μg of anti-HIF-1α (overnight, 4 °C) or rabbit IgG as a negative control. Immunoprecipitated DNA complex was amplified by PCR with primers for the CD36 promoter (forward, 5-TGAGTGTGTGAGAATTAAGGTTGA-3; and reverse, 5-GAAATGACCATGTGCAATCTCT-3) flanking the HIF-1 binding site. PCR products (∼239 bp) were separated by electrophoresis through 1.8% agarose gels and visualized by ethidium bromide staining. Analysis of DiI-oxLDL uptake was performed according to the manufacturer's protocol with minor modifications. Briefly, cells were pre-treated for 2 h with IgA or aCD36 (oxLDL) (clone JC63.1, 10 μg/ml) prior to normoxia or hypoxia exposure. Next, cells were incubated for 6 h with DiI-oxLDL (20 μg/ml) in serum-free medium at 37 °C, washed extensively with phosphate-buffered saline, 0.1% bovine serum albumin, pelleted, and resuspended in 400 μl of FACS flow for immediate FACS analysis. HL60 cells were pre-labeled with 5 μm CellTracker Green for 45 min at 37 °C according to the manufacturer's instructions. Cells were subsequently rendered apoptotic after treatment with 5 μm camptothecin (4 h), washed with media, and cultured for an additional 6 h before use. Apoptosis was confirmed by DNA laddering and annexin V-FITC/PI staining (>50% annexin V positive). POS were isolated on 25–60% sucrose gradients from porcine eyes obtained fresh from the slaughterhouse according to established protocols (24Finnemann S.C. Silverstein R.L. J. Exp. Med. 2001; 194: 1289-1298Crossref PubMed Scopus (111) Google Scholar, 32Sun M. Finnemann S.C. Febbraio M. Shan L. Annangudi S.P. Podrez E.A. Hoppe G. Darrow R. Organisciak D.T. Salomon R.G. Silverstein R.L. Hazen S.L. J. Biol. Chem. 2006; 281: 4222-4230Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 33Molday R.S. Hicks D. Molday L. Invest. Ophthalmol. Vis. Sci. 1987; 28: 50-61PubMed Google Scholar, 34Sun K. Cai H. Tezel T.H. Paik D. Gaillard E.R. Del Priore L.V. Mol. Vis. 2007; 13: 2310-2319PubMed Google Scholar). Before use, POS were labeled with 10 μg/ml FITC for 1 h at room temperature in the dark, washed, and resuspended in Dulbecco's modified Eagle's medium as previously described (24Finnemann S.C. Silverstein R.L. J. Exp. Med. 2001; 194: 1289-1298Crossref PubMed Scopus (111) Google Scholar, 34Sun K. Cai H. Tezel T.H. Paik D. Gaillard E.R. Del Priore L.V. Mol. Vis. 2007; 13: 2310-2319PubMed Google Scholar). We used modified versions of established phagocytosis assay protocols (24Finnemann S.C. Silverstein R.L. J. Exp. Med. 2001; 194: 1289-1298Crossref PubMed Scopus (111) Google Scholar, 32Sun M. Finnemann S.C. Febbraio M. Shan L. Annangudi S.P. Podrez E.A. Hoppe G. Darrow R. Organisciak D.T. Salomon R.G. Silverstein R.L. Hazen S.L. J. Biol. Chem. 2006; 281: 4222-4230Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 35Greenberg M.E. Sun M. Zhang R. Febbraio M. Silverstein R. Hazen S.L. J. Exp. Med. 2006; 203: 2613-2625Crossref PubMed Scopus (318) Google Scholar). ARPEs were seeded onto 15-mm glass coverslips and pre-treated for 2 h with IgA or aCD36 (oxLDL) (10 μg/ml) followed by hypoxic exposure. 5 × 105 apoptotic cells or 106 POS were allowed to incubate for 3 h on ARPE monolayers in serum-free medium. After incubation, unbound cells were removed by extensive washing with medium. Cells were fixed in ice-cold methanol (15 min at room temperature), washed with phosphate-buffered saline, and nuclei were counterstained with 4′,6-diamidino-2-phenylindole (1:3000, 5 min). Bound or ingested cells were detected by their green fluorescence and observed using a Nikon eclipse E800 epifluorescent microscope with a Nikon DXM 1200 digital camera. This assay was performed as described previously (18Mwaikambo B.R. Sennlaub F. Ong H. Chemtob S. Hardy P. Invest. Ophthalmol. Vis. Sci. 2006; 47: 4356-4364Crossref PubMed Scopus (51) Google Scholar). The aortic ring culture media was changed on day 4 with addition of the following test compounds: aCD36 (TSP-1) (clone FA6-152, 10 μg/ml), a CD36 agonist (clone SMO, 5 μg/ml), and their respective isotype controls IgG1 and IgM. Photographs were taken before (day 4) and after treatment (day 5) using an inverted microscope (Eclipse TE300; Nikon). The area of neovessel formation was determined using Image Pro Plus software. All experiments were repeated at least three times and values are presented as mean ± S.E. Data were analyzed by Student's t test, one-way or two-way analysis of variance followed by post-hoc Bonferroni tests for comparison among means. Statistical significance was set at p < 0.05. Based on previous reports eluding to the hypoxic regulation of CD36 (28Kwapiszewska G. Wilhelm J. Wolff S. Laumanns I. Koenig I.R. Ziegler A. Seeger W. Bohle R.M. Weissmann N. Fink L. Respir. Res. 2005; 6: 109Crossref PubMed Scopus (88) Google Scholar, 36Chabowski A. Górski J. Calles-Escandon J. Tandon N.N. Bonen A. FEBS Lett. 2006; 580: 3617-3623Crossref PubMed Scopus (55) Google Scholar), we first investigated the effects of hypoxia on CD36 expression in vivo. CD36 mRNA levels were determined in corneal and retinal tissue following mouse subjection to ambient room air or whole body hypoxia (8% O2, 92% N2 for 6 h). We focused our attention on the cornea and retina due to their abundant CD36 expression (18Mwaikambo B.R. Sennlaub F. Ong H. Chemtob S. Hardy P. Invest. Ophthalmol. Vis. Sci. 2006; 47: 4356-4364Crossref PubMed Scopus (51) Google Scholar, 19Sennlaub F. Valamanesh F. Vazquez-Tello A. El-Asrar A.M. Checchin D. Brault S. Gobeil F. Beauchamp M.H. Mwaikambo B. Courtois Y. Geboes K. Varma D.R. Lachapelle P. Ong H. Behar-Cohen F. Chemtob S. Circulation. 2003; 108: 198-204Crossref PubMed Scopus (145) Google Scholar, 23Ryeom S. Sparrow J. Silverstein R. J. Cell Sci. 1996; 108: 387-395Google Scholar) and relevance to neovascular eye diseases. As shown in Fig. 1, CD36 expression was ma
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