Adrenomedullin-RAMP2 System Is Crucially Involved in Retinal Angiogenesis
2013; Elsevier BV; Volume: 182; Issue: 6 Linguagem: Inglês
10.1016/j.ajpath.2013.02.015
ISSN1525-2191
AutoresYasuhiro Iesato, Yuichi Toriyama, Takayuki Sakurai, Akiko Kamiyoshi, Yuka Ichikawa‐Shindo, Hisaka Kawate, Takahiro Yoshizawa, Teruhide Koyama, Ryuichi Uetake, Lei Yang, Akihiro Yamauchi, Megumu Tanaka, Kyoko Igarashi, Toshinori Murata, Takayuki Shindo,
Tópico(s)Chemokine receptors and signaling
ResumoAdrenomedullin (ADM) is an endogenous peptide first identified as a strong vasodilating molecule. We previously showed that in mice, homozygous knockout of ADM (ADM−/−) or its receptor regulating protein, RAMP2 (RAMP2−/−), is embryonically lethal due to abnormal vascular development, thereby demonstrating the importance of ADM and its receptor signaling to vascular development. ADM expression in the retina is strongly induced by ischemia; however, its role in retinal pathophysiology remains unknown. Here, we analyzed oxygen-induced retinopathy (OIR) using heterozygous ADM and RAMP2 knockout mice models (ADM+/− or RAMP2+/−, respectively). In addition, we analyzed the role of the ADM-RAMP2 system during earlier stages of retinal angiogenesis using an inducible endothelial cell-specific RAMP2 knockout mouse line (DI-E-RAMP2−/−). Finally, we assessed the ability of antibody-induced ADM blockade to control pathological retinal angiogenesis in OIR. In OIR, neovascular tufts, avascular zones, and hypoxic areas were all smaller in ADM+/− retinas compared with wild-type mice. ADM+/− retinas also exhibited reduced levels of VEGF and eNOS expression. DI-E-RAMP2−/− showed abnormal retinal vascular patterns in the early stages of development. However, ADM enhanced the proliferation and migration of retinal endothelial cells. Finally, we found intravitreal injection of anti-ADM antibody reduced pathological retinal angiogenesis. In conclusion, the ADM-RAMP2 system is crucially involved in retinal angiogenesis. ADM and its receptor system are potential therapeutic targets for controlling pathological retinal angiogenesis. Adrenomedullin (ADM) is an endogenous peptide first identified as a strong vasodilating molecule. We previously showed that in mice, homozygous knockout of ADM (ADM−/−) or its receptor regulating protein, RAMP2 (RAMP2−/−), is embryonically lethal due to abnormal vascular development, thereby demonstrating the importance of ADM and its receptor signaling to vascular development. ADM expression in the retina is strongly induced by ischemia; however, its role in retinal pathophysiology remains unknown. Here, we analyzed oxygen-induced retinopathy (OIR) using heterozygous ADM and RAMP2 knockout mice models (ADM+/− or RAMP2+/−, respectively). In addition, we analyzed the role of the ADM-RAMP2 system during earlier stages of retinal angiogenesis using an inducible endothelial cell-specific RAMP2 knockout mouse line (DI-E-RAMP2−/−). Finally, we assessed the ability of antibody-induced ADM blockade to control pathological retinal angiogenesis in OIR. In OIR, neovascular tufts, avascular zones, and hypoxic areas were all smaller in ADM+/− retinas compared with wild-type mice. ADM+/− retinas also exhibited reduced levels of VEGF and eNOS expression. DI-E-RAMP2−/− showed abnormal retinal vascular patterns in the early stages of development. However, ADM enhanced the proliferation and migration of retinal endothelial cells. Finally, we found intravitreal injection of anti-ADM antibody reduced pathological retinal angiogenesis. In conclusion, the ADM-RAMP2 system is crucially involved in retinal angiogenesis. ADM and its receptor system are potential therapeutic targets for controlling pathological retinal angiogenesis. Pathological retinal angiogenesis plays a key role in various retinal diseases, including diabetic retinopathy (DR), retinal vein occlusion, and retinopathy of prematurity. Retinal ischemia is thought to be a common precursor to pathological retinal angiogenesis, and ischemia-induced overexpression of vascular endothelial growth factor (VEGF) is one of the major causes of the resultant abnormal vessel growth. Intravitreal anti-VEGF antibody (bevacizumab or ranibizumab) administration targeting angiogenesis and increases in vessel permeability is widely used to treat exudative age-related macular degeneration,1Wong T.Y. Liew G. Mitchell P. Clinical update: new treatments for age-related macular degeneration.Lancet. 2007; 370: 204-206Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar DR,2Nicholson B.P. Schachat A.P. A review of clinical trials of anti-VEGF agents for diabetic retinopathy.Graefes Arch Clin Exp Ophthalmol. 2010; 248: 915-930Crossref PubMed Scopus (215) Google Scholar retinal vein occlusion,3Wong T.Y. Scott I.U. Clinical practice. Retinal-vein occlusion.N Engl J Med. 2010; 363: 2135-2144Crossref PubMed Scopus (179) Google Scholar and retinopathy of prematurity.4Mintz-Hittner H.A. Kennedy K.A. Chuang A.Z. Efficacy of intravitreal bevacizumab for stage 3+ retinopathy of prematurity.N Engl J Med. 2011; 364: 603-615Crossref PubMed Scopus (1023) Google Scholar However, this approach has several associated drawbacks. One is the recurrence of macular edema; another is tachyphylaxis, which can occur after long-term anti-VEGF administration.5Schaal S. Kaplan H.J. Tezel T.H. Is there tachyphylaxis to intravitreal anti-vascular endothelial growth factor pharmacotherapy in age-related macular degeneration?.Ophthalmology. 2008; 115: 2199-2205Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 6Keane P.A. Liakopoulos S. Ongchin S.C. Heussen F.M. Msutta S. Chang K.T. Walsh A.C. Sadda S.R. Quantitative subanalysis of optical coherence tomography after treatment with ranibizumab for neovascular age-related macular degeneration.Invest Ophthalmol Vis Sci. 2008; 49: 3115-3120Crossref PubMed Scopus (118) Google Scholar Therefore, other therapeutic targets to control pathological angiogenesis are greatly needed. Adrenomedullin (ADM) is a vasodilating polypeptide originally isolated from human pheochromocytoma,7Kitamura K. Kangawa K. Kawamoto M. Ichiki Y. Nakamura S. Matsuo H. Eto T. Adrenomedullin: a novel hypotensive peptide isolated from human pheochromocytoma.Biochem Biophys Res Commun. 1993; 192: 553-560Crossref PubMed Scopus (2074) Google Scholar though it is now known to be widely distributed in numerous tissues and organs and to exert a variety of physiological effects in addition to vasodilatation. Expression of ADM is up-regulated under ischemic conditions.8Fujita Y. Mimata H. Nasu N. Nomura T. Nomura Y. Nakagawa M. Involvement of adrenomedullin induced by hypoxia in angiogenesis in human renal cell carcinoma.Int J Urol. 2002; 9: 285-295Crossref PubMed Scopus (35) Google Scholar, 9Wang X. Yue T.L. Barone F.C. White R.F. Clark R.K. Willette R.N. Sulpizio A.C. Aiyar N.V. Ruffolo Jr., R.R. Feuerstein G.Z. Discovery of adrenomedullin in rat ischemic cortex and evidence for its role in exacerbating focal brain ischemic damage.Proc Natl Acad Sci U S A. 1995; 92: 11480-11484Crossref PubMed Scopus (179) Google Scholar, 10Nguyen S.V. Claycomb W.C. Hypoxia regulates the expression of the adrenomedullin and HIF-1 genes in cultured HL-1 cardiomyocytes.Biochem Biophys Res Commun. 1999; 265: 382-386Crossref PubMed Scopus (101) Google Scholar Furthermore, we previously showed that homozygous ADM knockout (KO) mice (ADM−/−) die in utero due to lethally abnormal vascular development; that ADM also possesses angiogenic activity; and that ADM exerts its angiogenic effects, not only during development, but also in adulthood in regions experiencing ischemia.11Shindo T. Kurihara Y. Nishimatsu H. Moriyama N. Kakoki M. Wang Y. Imai Y. Ebihara A. Kuwaki T. Ju K.H. Minamino N. Kangawa K. Ishikawa T. Fukuda M. Akimoto Y. Kawakami H. Imai T. Morita H. Yazaki Y. Nagai R. Hirata Y. Kurihara H. Vascular abnormalities and elevated blood pressure in mice lacking adrenomedullin gene.Circulation. 2001; 104: 1964-1971Crossref PubMed Scopus (257) Google Scholar, 12Iimuro S. Shindo T. Moriyama N. Amaki T. Niu P. Takeda N. Iwata H. Zhang Y. Ebihara A. Nagai R. Angiogenic effects of adrenomedullin in ischemia and tumor growth.Circ Res. 2004; 95: 415-423Crossref PubMed Scopus (113) Google Scholar The main body of the ADM receptor is calcitonin receptor-like receptor (CLR), a seven-transmembrane domain G protein-coupled receptor (GPCR). CLR associates with one of the three subtypes of receptor activity-modifying protein (RAMP), which determines the affinity of CLR for its ligands.13McLatchie L.M. Fraser N.J. Main M.J. Wise A. Brown J. Thompson N. Solari R. Lee M.G. Foord S.M. RAMPs regulate the transport and ligand specificity of the calcitonin-receptor-like receptor.Nature. 1998; 393: 333-339Crossref PubMed Scopus (1853) Google Scholar, 14Parameswaran N. Spielman W.S. RAMPs: the past, present and future.Trends Biochem Sci. 2006; 31: 631-638Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar We also showed that homozygous RAMP2KO (RAMP2−/−) is lethal, with a phenotype similar to that of ADM−/−, suggesting the ADM-RAMP2 system is specifically involved in vascular development.15Ichikawa-Shindo Y. Sakurai T. Kamiyoshi A. Kawate H. Iinuma N. Yoshizawa T. Koyama T. Fukuchi J. Iimuro S. Moriyama N. Kawakami H. Murata T. Kangawa K. Nagai R. Shindo T. The GPCR modulator protein RAMP2 is essential for angiogenesis and vascular integrity.J Clin Invest. 2008; 118: 29-39Crossref PubMed Scopus (158) Google Scholar Expression of ADM has been detected in the eye. In earlier reports, ADM was shown to act as a vasodilator in the retinal arteries and to increase choroidal blood flow and ophthalmic arterial flow velocity.16Okamura T. Ayajiki K. Kangawa K. Toda N. Mechanism of adrenomedullin-induced relaxation in isolated canine retinal arteries.Invest Ophthalmol Vis Sci. 1997; 38: 56-61PubMed Google Scholar, 17Dorner G.T. Garhofer G. Huemer K.H. Golestani E. Zawinka C. Schmetterer L. Wolzt M. Effects of adrenomedullin on ocular hemodynamic parameters in the choroid and the ophthalmic artery.Invest Ophthalmol Vis Sci. 2003; 44: 3947-3951Crossref PubMed Scopus (21) Google Scholar In addition, peripheral administration of ADM causes dose-dependent conjunctival hyperemia in rabbits.18Clementi G. Floriddia M.L. Prato A. Marino A. Drago F. Adrenomedullin and ocular inflammation in the rabbit.Eur J Pharmacol. 2000; 400: 321-326Crossref PubMed Scopus (24) Google Scholar ADM also lowers intraocular pressure19Taniguchi T. Kawase K. Gu Z.B. Kimura M. Okano Y. Kawakami H. Tsuji A. Kitazawa Y. Ocular effects of adrenomedullin.Exp Eye Res. 1999; 69: 467-474Crossref PubMed Scopus (23) Google Scholar and relaxes iris sphincter smooth muscle via a cAMP-dependent pathway.20Yousufzai S.Y. Ali N. Abdel-Latif A.A. Effects of adrenomedullin on cyclic AMP formation and on relaxation in iris sphincter smooth muscle.Invest Ophthalmol Vis Sci. 1999; 40: 3245-3253PubMed Google Scholar Collectively, these data suggest that ADM could be involved in the pathophysiology of ocular diseases. Consistent with that idea, ADM levels are elevated in the vitreous fluid of patients with DR,21Er H. Doganay S. Ozerol E. Yurekli M. Adrenomedullin and leptin levels in diabetic retinopathy and retinal diseases.Ophthalmologica. 2005; 219: 107-111Crossref PubMed Scopus (17) Google Scholar, 22Ito S. Fujisawa K. Sakamoto T. Ishibashi T. Elevated adrenomedullin in the vitreous of patients with diabetic retinopathy.Ophthalmologica. 2003; 217: 53-57Crossref PubMed Scopus (16) Google Scholar, 23Lu Y. Xu Y. Tang C. Changes in adrenomedullin in patients with proliferative diabetic retinopathy.Curr Eye Res. 2011; 36: 1047-1052Crossref PubMed Scopus (6) Google Scholar suggesting ADM may be associated with pathological retinal angiogenesis. Our initial aim in the present study was to clarify the pathophysiological roles of the ADM-RAMP2 system in the retina using an oxygen-induced retinopathy (OIR) model in heterozygous ADM and RAMP2 KO mice. We next generated an inducible endothelial cell-specific RAMP2 KO mouse to analyze the roles of the ADM-RAMP2 system during the early stages of retinal vascular development. Finally, we assessed the ability of antibody-induced ADM blockade to control pathological retinal angiogenesis. Wild-type (WT) C57BL/6J mice were purchased from Charles River Laboratories Japan (Yokohama, Japan). ADM and RAMP2 KO mice were originally generated in our group.11Shindo T. Kurihara Y. Nishimatsu H. Moriyama N. Kakoki M. Wang Y. Imai Y. Ebihara A. Kuwaki T. Ju K.H. Minamino N. Kangawa K. Ishikawa T. Fukuda M. Akimoto Y. Kawakami H. Imai T. Morita H. Yazaki Y. Nagai R. Hirata Y. Kurihara H. Vascular abnormalities and elevated blood pressure in mice lacking adrenomedullin gene.Circulation. 2001; 104: 1964-1971Crossref PubMed Scopus (257) Google Scholar, 15Ichikawa-Shindo Y. Sakurai T. Kamiyoshi A. Kawate H. Iinuma N. Yoshizawa T. Koyama T. Fukuchi J. Iimuro S. Moriyama N. Kawakami H. Murata T. Kangawa K. Nagai R. Shindo T. The GPCR modulator protein RAMP2 is essential for angiogenesis and vascular integrity.J Clin Invest. 2008; 118: 29-39Crossref PubMed Scopus (158) Google Scholar Because homozygous ADM and RAMP2 KO mice are embryonically lethal, we used heterozygous KO mice (ADM+/−, RAMP2+/−), in which ADM or RAMP2 expression is reduced to half that seen in WT mice. To generate inducible vascular endothelial cell-specific RAMP2 KO mice, we crossed a mouse line expressing tamoxifen-inducible Cre recombinase (Cre-ERT2) under the regulation of VE-Cadherin24Kogata N. Arai Y. Pearson J.T. Hashimoto K. Hidaka K. Koyama T. Somekawa S. Nakaoka Y. Ogawa M. Adams R.H. Okada M. Mochizuki N. Cardiac ischemia activates vascular endothelial cadherin promoter in both preexisting vascular cells and bone marrow cells involved in neovascularization.Circ Res. 2006; 98: 897-904Crossref PubMed Scopus (27) Google Scholar with floxed RAMP2 mice. Cre activation and gene deletion were induced by 1 mg/mL intragastric injections of tamoxifen (T5648; Sigma-Aldrich, St. Louis, MO). For analysis of P6 neonates, 50 μg of tamoxifen was injected on three consecutive days (postnatal day [P] 1 to P3); for analysis of P11 neonates, 100 μg of tamoxifen was injected on four consecutive days (P5 to P8).25Pitulescu M.E. Schmidt I. Benedito R. Adams R.H. Inducible gene targeting in the neonatal vasculature and analysis of retinal angiogenesis in mice.Nat Protoc. 2010; 5: 1518-1534Crossref PubMed Scopus (262) Google Scholar Control mice were injected with corn oil (Sigma-Aldrich) on P1 to P3 or P5 to P8. All animal handling procedures were in accordance with a protocol approved by the ethics committee of Shinshu University School of Medicine. All experiments were performed in accordance with the Association for Research in Vision and Ophthalmology’s Statement for the Use of Animals in Ophthalmic and Vision Research and our institutional guidelines. Mice were euthanized and retinas were removed on P17. Total RNA was extracted from each sample using Trizol Reagent (Invitrogen, Carlsbad, CA), after which the extracted RNA was treated with DNA-Free (Ambion, Austin, TX) to remove contaminating DNA, and 2-μg samples were subjected to reverse transcription using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Carlsbad, CA). Quantitative real-time RT-PCR was performed using an Applied Biosystems 7300 real-time PCR System (Applied Biosystems) with SYBR Green (Toyobo, Osaka, Japan) or Realtime PCR Master Mix (Toyobo) and TaqMan probe (MBL International, Woburn, MA). Values were normalized to mouse GAPDH (TaqMan Pre-Developed Assay Reagents; Applied Biosystems). The primers and probes used are listed in Table 1.Table 1Primers and Probes Used for Quantitative Real-Time RT-PCR AnalysisGeneSequenceADMForward5′-GGACACTGCAGGGCCAGAT-3′Reverse5′-GTAGTTCCCTCTTCCCACGACTTA-3′RAMP2Forward5′-GCAGCCCACCTTCTCTGATC-3′Reverse5′-AACGGGATGAGGCAGATGG-3′Probe5′-CCCAGAGGATGTGCTCCTGGCCAT-3′VEGF-AForward5′-CATCTTCAAGCCGTCCTGTGT-3′Reverse5′-CTCCAGGGCTTCATCGTTACA-3′VEGFR1Forward5′-CAAGGACGGCTTTGCAGATC-3′Reverse5′-GCTCATGAATTTGAAAGCGTTTAC-3′VEGFR2Forward5′-ACTGCAGTGATTGCCATGTTCT-3′Reverse5′-TCATTGGCCCGCTTAACG-3′eNOSForward5′-AGGCACTGCTGAGCCGAGT-3′Reverse5′-TTCTCCAGTTGTTCCACAGCC-3′EPOForward5′-CTACGTAGCCTCACTTCACTGCTT-3′Reverse5′-AGAGCTTGCAGAAAGTATCCACTGT-3′Tie-2Forward5′-GGAACCTGACCTCGGTGCTA-3′Reverse5′-CTGCGCCTTGGTGTTGACT-3′ Open table in a new tab Retinal tissue was lysed in an ice-cold RIPA Lysis Buffer System (Santa Cruz Biotechnology, Santa Cruz, CA) supplemented with PhosSTOP phosphatase inhibitor (Roche Applied Science, Indianapolis, IN) and then sonicated. Samples of the resultant lysate were subjected to SDS-PAGE, and the resolved proteins were transferred to cellulose nitrate membranes (GE Healthcare, Chalfont St Giles, UK). After blocking in 5% skim milk, the membranes were incubated with primary antibodies against β-tubulin (Santa Cruz Biotechnology), VEGF (Abcam, Cambridge, UK), endothelial nitric oxide synthase (eNOS) (Cell Signaling Technology, Danvers, MA), and phospho-eNOS (Ser1177; Cell Signaling Technology), followed by appropriate secondary antibodies (Santa Cruz Biotechnology). The bound antibodies were visualized using chemiluminescent horseradish peroxidase substrate (Millipore, Billerica, MA), and the chemiluminescence was analyzed using an ImageQuant LAS 4000 imager system (GE Healthcare). Levels of eNOS activation were determined based on the ratio of band intensities after blotting with antibodies specific for the phosphorylated and unphosphorylated proteins. For quantification, Western blot images were captured and analyzed using Scion Image software version 4.0.3 (Scion Corporation, Frederick, MD). Physiological and pathological retinal angiogenesis were quantified using previously reported protocols26Connor K.M. Krah N.M. Dennison R.J. Aderman C.M. Chen J. Guerin K.I. Sapieha P. Stahl A. Willett K.L. Smith L.E. Quantification of oxygen-induced retinopathy in the mouse: a model of vessel loss, vessel regrowth and pathological angiogenesis.Nat Protoc. 2009; 4: 1565-1573Crossref PubMed Scopus (478) Google Scholar with slight modification. Briefly, on P17, the eyes were fixed in 4% paraformaldehyde for 1 hour at 4°C and then washed with PBS. Retinas were then isolated and stained overnight at 4°C with Alexa Fluor 594–conjugated Griffonia simplicifolia (formerly Bandeiraea simplicifolia) isolectin B4 (isolectin GS-IB4 from G. simplicifolia, Alexa Fluor 594 conjugate I21413; 1:200 dilution; Invitrogen) in 0.3% PBS with Tween 20. After washing three times in PBS, the retinas were mounted whole onto microscope slides with the photoreceptor side down and embedded in fluorescent mounting medium (Dako, Glostrup, Denmark). Images of whole-mount retinas were taken at ×4 magnification using a fluorescence microscope (BZ-9000; Keyence, Osaka, Japan). Avascular zones and neovascular tuft formation (represented as pathological angiogenesis) were quantified using digital imaging/photo editing software (Adobe Photoshop CS5 version 12.0.3; Adobe Systems, San Jose, CA). Pathological angiogenesis was analyzed by counting the nuclei extending beyond the internal limiting membrane. The eyes were enucleated on P17 and fixed in 4% paraformaldehyde for 24 hours, and then embedded in paraffin. Six sections were selected within 300 μm of the optic nerve in serial 5-μm-thick sagittal sections of whole eyes and stained with H&E, after which photomicrographs were taken (BZ-9000 microscope). All evaluations of retinal angiogenesis were done using double-blind methods. Retinal ischemia was assessed using pimonidazole hydrochloride (Hypoxyprobe-1 [H-1]; Chemicon, Temecula, CA) according to the manufacturer’s protocol. Briefly, mice were intraperitoneally injected with 50 mg/kg H-1 90 minutes before sacrifice. Detection of H-1–positive (hypoxic) areas was achieved through immunohistochemical analysis of retinal flat mounts after fixation in 4% paraformaldehyde using a primary antibody against the H-1 probe (provided in the kit) and fluorescein isothiocyanate–conjugated secondary antibodies. Images were captured using a fluorescence microscope (BZ-9000) and subjected to morphometric analysis using Adobe Photoshop. The RF/6A135 chorioretinal endothelial cell (CREC) line was obtained from the Riken Cell Bank, Tsukuba, Japan, and cultured in RPMI 1640 medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS). The RAMP2-overexpressing (RAMP2O/E) endothelial cell line was created using EAhy926 endothelial cells, which is an immortal, clonally pure, human endothelial cell line (kindly provided by C.J. Edgell, University of North Carolina). Human RAMP2 cDNA was inserted into the cloning site of pcDNA3.1+ (Invitrogen), which was then transfected into EAhy926 cells to create the RAMP2 O/E cell line.15Ichikawa-Shindo Y. Sakurai T. Kamiyoshi A. Kawate H. Iinuma N. Yoshizawa T. Koyama T. Fukuchi J. Iimuro S. Moriyama N. Kawakami H. Murata T. Kangawa K. Nagai R. Shindo T. The GPCR modulator protein RAMP2 is essential for angiogenesis and vascular integrity.J Clin Invest. 2008; 118: 29-39Crossref PubMed Scopus (158) Google Scholar EAhy926 cells transfected with empty pcDNA3.1+ vector served as control. EAhy926 endothelial cells were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 10% FBS. Human retinal endothelial cells (Cell Systems, Kirkland, WA) were cultured according to the manufacturer’s protocol. To determine whether ADM promotes CREC proliferation and migration, we performed scratch-wound assays as described by Liang et al,27Liang C.C. Park A.Y. Guan J.L. In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro.Nat Protoc. 2007; 2: 329-333Crossref PubMed Scopus (3102) Google Scholar with some modifications. CRECs were grown in 60-mm dishes with medium containing 10% FBS until 90% confluent, after which they were starved for 24 hours in 1% FBS + medium. The center of the cultured dish was then scratched using a P200 pipet tip, washed 2 times with serum-free medium, and the culture was continued for 16 hours in 10% FBS + medium, with or without ADM or VEGF (Invitrogen). Images of the scratched areas at 0 hours and after 16 hours were captured using a light microscope (Olympus, Tokyo, Japan). The cell-covered area at 16 hours was determined as a percentage of the cell-free area at 0 hours measured in three fields per well using ImageJ software version 1.46e (NIH, Bethesda, MD; http://rsb.info.nih.gov/ij/index.html). The experiment was repeated three times on separate occasions. For the study of EAhy926 cells and human retinal endothelial cells, they were grown in 35-mm dishes, and the coverage of the scratch area was evaluated at 6 hours with or without ADM. All of the images shown were captured using a fluorescence microscope. The isolectin-B4–stained endothelial network was analyzed on flat-mount specimens. In P6, the numbers of filopodial extensions at the retinal angiogenic front were quantified in 16 fields (200 μm × 200 μm in each field) from four retinas per group. The total number of filopodia was normalized to a standard endothelial vessel length of 1000 μm, which was measured and defined according to previously reported protocols.25Pitulescu M.E. Schmidt I. Benedito R. Adams R.H. Inducible gene targeting in the neonatal vasculature and analysis of retinal angiogenesis in mice.Nat Protoc. 2010; 5: 1518-1534Crossref PubMed Scopus (262) Google Scholar, 28Benedito R. Rocha S.F. Woeste M. Zamykal M. Radtke F. Casanovas O. Duarte A. Pytowski B. Adams R.H. Notch-dependent VEGFR3 upregulation allows angiogenesis without VEGF-VEGFR2 signalling.Nature. 2012; 484: 110-114Crossref PubMed Scopus (271) Google Scholar The density of front vessel branches was calculated in 16 fields (200 μm × 400 μm in each field) from four retinas per group. Vascular progression was measured by defining a straight line from the angiogenic front to the center of the retina for each retinal quadrant in low magnification, and 24 quadrants from six retinas per group were used for quantification. In P11, the vascular branch point was counted in 16 fields (200 μm × 400 μm in each field) from four retinas per group. For the analysis of endothelial proliferation, Ki-67 (Abcam) immunostaining was performed in P10, and the numbers of Ki-67 and isolectin-B4 double-positive cells were counted in 12 cross sections from three retinas per group. Anti-ADM antibody kindly provided by Dr. Tanaka was used for intravitreal administration. In the OIR model, mice underwent intravitreal injection on P13; 1.5 μL of anti-ADM antibody (ADMab) was intravitreally injected at the temporal cornea–scleral junction using a Hamilton syringe equipped with a 32-G needle under a stereoscopic surgical microscope. As control, the eye received an intravitreal injection of 1.5 μL of mouse IgG (Abcam) at the same position. After the procedure, the mice were administered moxifloxacin hydrochloride antibiotic drops (Vegamox Ophthalmic Solution; Alcon, Fort Worth, TX). Avascular zone and neovascular tufts were analyzed on P17. Values are expressed as means ± SEM. Student’s t-test and one-way analysis of variance, followed by the Tukey or Dunnett test were used to determine the significance of differences. Values of P < 0.05 or P < 0.01 were considered significant. In the OIR model, mice were exposed to 75% oxygen for 5 days, from P7 to P12, the period (hyperoxic phase) during which oxygen-induced loss of retinal vessels (vaso-obliteration) occurs. After the mice were returned to normoxic conditions (room air) for 5 days, from P12 to P17 (hypoxic phase), the hyperoxia-induced vessel loss led to pathological angiogenesis. We initially compared the retinal angiogenesis at P12 among WT, ADM+/−, and RAMP2+/− mice. In retinal flat-mount specimens, we did not detect avascular zones in WT mice kept under normoxia. Mice kept under hyperoxia showed the existence of avascular zones in the central region of the retina; however, we detected no significant differences in the size of the avascular zones among the three mouse types (WT, 34.1 ± 1.3%; ADM+/−, 33.3 ± 1.3%; RAMP2+/−, 36.4 ± 1.8%), indicating no genotype-dependent differences in the oxygen-induced loss of retinal vessels (Figure 1, A and B). We also found no differences in the hypoxic area at this stage (Supplemental Figure S1). These data indicate that there was no difference in the vascular development and ischemic level among these mice at the start of hypoxia-induced neovascularization. We next analyzed the time course of ADM expression during retinal development in WT neonates, with and without OIR (Figure 1C). Under normoxic conditions, ADM expression was unchanged or declined slightly from P12 to P17 (0.94 ± 0.17-fold at P13 to 0.33 ± 0.03-fold at P17). By contrast, neonates subjected to OIR showed prominent up-regulation of ADM from P13 (5.45 ± 0.83-fold). This suggests ADM expression is induced by hypoxia in OIR. The up-regulated ADM expression persisted until P17 (3.17 ± 0.25-fold), though it declined somewhat over that period. However, RAMP2 expression was down-regulated once at P13, and its expression returned from P15 to P17 (Figure 1D). We then analyzed retinas at the end of the hypoxic phase (P17) and found that neovascular tuft formation, which is the aneurysmal formation of retinal arteries representing pathological angiogenesis, was significantly reduced in ADM+/− mice (7.0 ± 0.4%), as compared to WT mice (11.0 ± 1.3%) (Figure 2, A and B). In addition, in HE-stained cross sections prepared on P17, retinas from OIR model mice exhibited vascular nuclei extending into the vitreous from the retinal surface (Figure 2C). The numbers of cell nuclei on the vitreal side of the internal limiting membrane were significantly lower in ADM+/− mice (20.0 ± 3.3) than WT mice (36.8 ± 4.4) (Figure 2D), which is also indicative of a smaller degree of pathological angiogenesis in ADM+/− mice. We detected no significant difference in RAMP2+/− mice (neovascular tufts, 13.1 ± 1.4%; numbers of nuclei above internal limiting membrane, 33.1 ± 2.0). Using both lectin and H-1 staining, we evaluated the retinal vessels and hypoxic areas in P17 retinas. In ADM+/− and RAMP2+/− retinas, the hypoxic area was limited to the central region of the retina, with little or no hypoxia detected in the periphery (Figure 3A). We found that both the avascular zone and hypoxic area were significantly smaller in ADM+/− (14.2 ± 2.3% and 3.1 ± 0.6%, respectively) than WT mice (25.6 ± 3.2% and 9.9 ± 1.9%, respectively) (Figure 3, B and C). Similarly, in RAMP2+/− mice, the avascular zone tended to be smaller (19.8 ± 1.3%) (Figure 3B) and the hypoxic area was significantly smaller (5.2 ± 0.9%) than in WT mice (Figure 3C). The reductions in the avascular zone and hypoxic area suggest physiological angiogenesis is enhanced in both KO strains. In the WT retina, H-1–positive (hypoxic) areas were detected even within lectin-positive areas in the retinal periphery. However, as stated above, in ADM+/− and RAMP2+/− retinas, the hypoxic area was limited to the central region of the retina, with little or no hypoxia detected in the periphery (Figure 3A). This may indicate that in the WT retina, vascular formation in peripheral regions could not fully compensate for the ischemia caused by OIR, whereas it was almost entirely compensated for in ADM+/− and RAMP2+/− retinas. We also investigated retinal gene expression in the OIR model (Figure 4A). In WT mice, ADM and RAMP2 were up-regulated after OIR (P17). The expressions of VEGF, vascular endothelial growth factor receptor 1 (VEGFR1), and eNOS were all significantly up-regulated in WT OIR model; however, the up-regulation was suppressed in ADM+/− mice. In Western blot analysis (Figure 4, B and C), we also confirmed the reduction of VEGF protein level in ADM+/−. Activation of eNOS (represented by the p-eNOS/eNOS ratio) was also suppressed in ADM+/−. Apparently, the lower ADM in ADM+/− affected several angiogenic pathways under ischemic conditions. Compared with ADM+/−, changes in VEGF and eNOS expression were smaller in RAMP2+/− (Supplemental Figure S2). We directly assessed the effects of ADM on endothelia
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