Adrenomedullin–Receptor Activity-Modifying Protein 2 System Ameliorates Subretinal Fibrosis by Suppressing Epithelial-Mesenchymal Transition in Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is a leading cause of visual impairment. Anti–vascular endothelial growth factor drugs used to treat AMD carry the risk inducing subretinal fibrosis. We investigated use adrenomedullin (AM), vasoactive peptide, and its receptor activity-modifying protein 2, RAMP2, which regulate vascular homeostasis suppress The therapeutic potential AM-RAMP2 system was evaluated after laser-induced choroidal neovascularization (LI-CNV), mouse model AMD. Neovascular formation, fibrosis, macrophage invasion were all enhanced in both AM RAMP2 knockout mice compared with those wild-type mice. These pathologic changes suppressed by intravitreal injection AM. Comprehensive gene expression analysis choroid LI-CNV or without administration revealed that fibrosis-related molecules, including Tgfb, Cxcr4, Ccn2, Thbs1, down-regulated In retinal pigment epithelial cells, co-administration transforming factor-? tumor necrosis factor-? induced epithelial-mesenchymal transition, also prevented Finally, C-X-C chemokine type 4 (CXCR4) inhibitors eliminated difference fibrosis between findings suggest suppresses suppressing transition. blindness, global prevalence about 8.7%.1Wong W.L. Su X. Li Cheung C.M.G. Klein R. Cheng C.-Y. Wong T.Y. Global age-related disease burden projection for 2020 2040: systematic review meta-analysis.Lancet Glob Health. 2014; 2: e106-e116Abstract Full Text PDF PubMed Scopus (1990) Google Scholar It associated (CNV), develops from subretina, where it causes bleeding oozing, loss vision affected eye.2Fine S.L. Berger J.W. Maguire M.G. 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Jekyll Hyde blood cells during wound repair.J Invest Dermatol. 2007; 127: 1009-1017Abstract (182) CNV, hemorrhage lesions accompanied accumulation extracellular matrix components, such as collagen fibrinogen, infiltration differentiation (RPE) into fibroblast-like via transition (EMT), excessive poorly ordered displacement normal structures, intramacular and/or surrounding photoreceptor cells. This often formation fibrovascular tissue. Thus, suppression an important issue long-term prognosis patients. Adrenomedullin (AM) first identified vasodilating peptide isolated pheochromocytoma. However, subsequent studies exerts variety physiological effects has antioxidative, anti-inflammatory, antifibrotic, antiapoptotic properties.11Kato Tsuruda T. Kita Kitamura Eto Adrenomedullin.Arterioscler Thromb Vasc Biol. 2005; 25: 2480-2487Crossref (134) 12Shimosawa Ogihara Matsui Asano Ando Fujita Deficiency induces insulin resistance oxidative stress.Hypertension. 2003; 41: 1080-1085Crossref (87) 13Brain S.D. Grant A.D. Vascular actions calcitonin gene-related adrenomedullin.Physiol Rev. 2004; 84: 903-934Crossref (596) Homozygous (KO) embryonically lethal because systemic edema bleeding, caused mainly abnormal development.14Iimuro Shindo Moriyama Amaki Niu Takeda Iwata Zhang Ebihara A. Nagai Angiogenic ischemia growth.Circ Res. 95: 415-423Crossref (110) Scholar,15Shindo Kurihara Nishimatsu Kakoki Wang Imai Kuwaki Ju K.H. 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To overcome limitation, focusing AM's system. member superfamily acts G-protein–coupled seven-transmembrane domain receptor, receptor-like (CLR).24McLatchie L.M. Fraser N.J. Main M.J. Wise Thompson Solari Lee Foord S.M. RAMPs transport ligand specificity calcitonin-receptor-like receptor.Nature. 1998; 393: 333-339Crossref (1798) CLR ligands regulated three proteins, RAMP1, RAMP3. complex RAMP3 have high affinity AM.25Shindo Regulation development adrenomedullin-RAMP system.Peptides. 55-61Crossref (11) Interestingly, among homozygous RAMP only die utero abnormalities, similar observed mice.26Ichikawa-Shindo Iinuma Yoshizawa Koyama Fukuchi Iimuro GPCR modulator essential integrity.J Clin Invest. 29-39Crossref (151) suggests determinant function AM, may be possible modulate modulating RAMP2. present analyzed possibility serve target evaluated. generated previously.15Shindo Scholar,26Ichikawa-Shindo Scholar,27Yamauchi Igarashi Owa Functional their regulation system.J Mol Cell Cardiol. 77: 73-85Abstract (24) this embryonic lethality KO, used, reduced genes half WT lethal, adult available. Male aged 9 12 weeks, used. littermates each line control. Before procedure, anesthetized i.p. mixture 0.3 mg/kg medetomidine (Nippon Zenyaku Kogyo Co Ltd, Koriyama, Japan), 4.0 midazolam (Astellas Pharma Inc., Tokyo, 5.0 butorphanol (Meiji Seika Japan). All animal handling complied protocols approved Ethics Committee Shinshu University School Medicine. experiments performed according statements Society Vision Ophthalmology animals ophthalmic research institutional guidelines. Indocyanine green angiography using confocal scanning-laser ophthalmoscope, previous study,28Kumar Berriochoa Z. Jones Fu Detecting vasculature time-course indocyanine angiography.J Vis Exp. e51061Google confirm no apparent change under baseline conditions (Supplemental Figure S1). While keeping cornea moist saline, manually held front Heidelberg Retina Angiograph ophthalmoscope (Heidelberg Engineering GmbH, Heidelberg, Germany). tail (Santen Pharmaceutical, Osaka, Images taken at 10 minutes injection. C57BL/6J weeks (Charles River Laboratories Japan, Kanagawa, Human (Peptide Institute, Japan) dissolved phosphate-buffered saline (PBS) infused subcutaneously osmotic pump (Alzet; DURECT Co, Cupertino, CA). infusion rate 29 ?g/kg per day, duration 7 14 days. PBS effectiveness human well established,29Hobara Goda Sendou Gomita Kawasaki facilitates reinnervation phenol-injured perivascular nerves mesenteric artery.Neuroscience. 144: 721-730Crossref (12) Scholar,30Uetake Yang Kuwabara Yanagita Mukoyama Adrenomedullin-RAMP2 ER stress-induced tubule cell death involved kidney protection.PLoS One. 9: e87667Crossref dosage selected basis prior studies.17Imai (10?7 mol/L; 1.0 ?L) injected intravitreally surgical microscope corneal scleral junction Hamilton syringe equipped 32-gauge needle. (1.0 After operative procedures, administered moxifloxacin hydrochloride (Vegamox Ophthalmic Solution; Alcon, Fudenberg, Switzerland). anesthesia, eyes dilated 0.5% tropicamide phenylephrine (Mydrine P; Santen, Laser injury retina laser slit lamp (GYC-1000; NIDEK, Gamagori, and, cover glass viscoelastic substance contact lenses. A wavelength 532 ?m, power output 200 mW, lasing 0.05 seconds, spot 50 ?m injuries area obvious vessels around optic nerve. Injury Bruch membrane confirmed appearance air bubbles. Three shots evaluate sizes fibrotic area, five before extracting mRNA choroid. treatment, 3.0 atipamezole (ZEOAQ, Fukushima, intraperitoneally reverse anesthesia. experiment, procedure injection, itself, affects degree Therefore, experiment As appeared decreased toward 3 samples 1 analyzed. Seven days induction, anesthetized, thoracotomy performed, mL containing mg/mL fluorescein isothiocyanate (FITC)–labeled dextran (molecular weight, × 106; Sigma-Aldrich, St. Louis, MO) systemically left ventricle. Eyes then enucleated fixed 4% paraformaldehyde hour, cornea, lens, removed, flat mounts prepared. Eight pieces cut radially rim disc mounted onto slide glass. FITC-positive areas represent (patent but collapsed vessels). For immunostaining, blocking 1% bovine serum albumin, rabbit anti-mouse ?-smooth muscle actin (?-SMA) antibody (Abcam, Cambridge, UK), F4/80 (Bio-Rad, Hercules, CA), rho-associated, coiled-coil-containing kinase (ROCK1) (Cell Signaling Technology, Danvers, MA) applied, followed appropriate secondary antibodies. Flat embedded fluorescence mounting medium (Agilent Technologies, Santa Clara, CA) (BZ-9000; Keyence, ?-SMA–positive, ROCK1-positive quantified analytic application, BZ analyzer (Keyence). Since fluoresce intensity thickness evaluated, lead underestimating actual volume lesions. Numbers F4/80-positive lesion counted. double-blinded method quantification. mount analysis, n represents number irradiation, enucleated, overnight, paraffin, sections (5 thick) prepared histologic analysis. Sections stained hematoxylin/eosin Masson trichrome stain immunostained anti–?-SMA anti-AM (Thermo Fisher Scientific, Waltham, MA), anti-CLR (Abcam) antibodies diaminobenzidine fluorescent anti-RhoA (Abcam). Mice sacrificed, enucleated. iris, vitreous, soft removed. peeled off pushing RPE-choroid-sclera (choroidal complex) behind. Total RNA extracted complexes ARPE19 TRIzol Reagent Scientific). treated DNA-free Scientific) remove any contaminating DNA, 2-?g sample transcribed PrimeScript RT Kit (Takara Bio, Shiga, produce cDNA. Quantitative real-time RT-PCR StepOnePlus PCR Scientific), SYBR Green (Toyobo, master mix (Toyobo). Glyceraldehyde-3-phosphate dehydrogenase endogenous Table shows primers complexes, mice.Table 1Primers Used Real-Time PCRGenePrimerMouse Calcrl (CLR) forward5?-AGGCGTTTACCTGCACACACT-3?Mouse reverse5?-CAGGAAGCAGAGGAAACCCC-3?Mouse forward5?-GGACACTGCAGGGCCAGAT-3?Mouse reverse5?-GTAGTTCCCTCTTCCCACGACTTA-3?Mouse Ramp2 forward5?-ACTGAGGACAGCCTTGTGTCAAA-3?Mouse reverse5?-CCTTGACAGAGTCCATGCAACTC-3?Mouse Ramp3 forward5?-AAAGCCTTCGCTGACATGATG-3?Mouse reverse5?-ATCTCGGTGCAGTTAGTGAAGCT-3?Mouse Tgfb1 forward5?-CCCGAAGCGGACTACTATGC-3?Mouse reverse5?-TAGATGGCGTTGTTGCGGT-3?Mouse Tgfb2 forward5?-TAAAATCGACATGCCGTCCC-3?Mouse reverse5?-GAGACATCAAAGCGGACGAT-3?Mouse Tgfb3 forward5?-GATCACCACAACCCACACCT-3?Mouse reverse5?-ATAAAGGGGGCGTACACAGC-3?Mouse Cxcr4 forward5?-TCAGTGGCTGACCTCCTCTT-3?Mouse reverse5?-TTTCAGCCAGCAGTTTCCTT-3?Mouse Cxcl12 (SDF-1) forward5?-AGAGCCAACGTCAAGCATCT-3?Mouse reverse5?-TAATTTCGGGTCAATGCACA-3?Mouse Thbs1 forward5?-CGCCTTCCGCATTGAGAATG-3?Mouse reverse5?-CATCTGCCTCAAGGAAGCCA-3?Mouse Ccn2 (CTGF) forward5?-CAGAGGTGGTGGGGTAGAGA-3?Mouse reverse5?-CATTGCCACTCACAATGTCC-3?Mouse Tjp1 (ZO-1) forward5?-GCCACTACAGTATGACCATCC-3?Mouse reverse5?-AATGAATAATATCAGCACCATGCC -3?Mouse Tagln (SM22?) forward5?-ACCAAAAACGATGGAAACTACCG-3?Mouse reverse5?-CATTTGAAGGCCAATGACGTG Rhoa forward5?-GCTACCAGTATTTAGAAGCCAACCAC-3?Mouse reverse5?-GCTGTTAGAGCAGTGTCAGAAGGAC-3?Mouse Rock1 forward5?-CAAAGCACGCCTAACTGACA reverse5?-TCTGCCTTCTCTCGAGCTTC-3?Mouse Icam1 forward5?-CCTAAAATGACCTGCAGACGG-3?Mouse Icma1 reverse5?-TTTGACAGACTTCACCACCCC-3?Mouse Ccl2 (MCP1) forward5?-GCAGTTAACGCCCCACTCA-3?Mouse reverse5?-CCTACTCATTGGGATCATCTTGCT-3?Mouse Tnfa forward5?-ACGGCATGGATCTCAAAGAC-3?Mouse reverse5?-AGATAGCAAATCGGCTGACG-3?Mouse Il1b forward5?-CTACAGGCTCCGAGATGAACAAC-3?Mouse reverse5?-TCCATTGAGGTGGAGAGCTTTC-3?Mouse Smad2 forward5?-ATGTCGTCCATCTTGCCATTC-3?Mouse reverse5?-AACCGTCCTGTTTTCTTTAGCTT-3?Mouse Smad3 forward5?-CATTACCATCCCCAGGTCAC-3?Mouse reverse5?-CGTAATTCATGGTGGCTGTG-3?Human forward5?-GTGGAAACCCACAACGAAAT-3?Human reverse5?-CGGAGCTCTGATGTGTTGAA-3?Human forward5?-CTCCAAGCTGTCACACTCCA-3?Human reverse5?-TCGATGCTGATCCCAATGTA-3?Human forward5?-TCACCTACCACCTCGTCGTCTG-3?Human reverse5?-ATGAGCACTGCCCACCCATCT-3?Human forward5?-GATTTTGGACTGCACTTCGC-3?Human reverse5?-GTCCGAACCCAGACACAAGT-3?Human forward5?-CTGGTGATTGTTGGTGATGG-3?Human reverse5?-GCGATCATAATCTTCCTGCC-3?Human forward5?-AACATGGCATCTTCGACACTC-3?Human reverse5?-CAAAATCACAAAGGCCATGA-3?CLR, receptor; CTGF, connective tissue factor; MCP1, monocyte chemoattractant 1; SDF-1, stromal cell–derived factor-1; ZO-1, zonula occludens 1. Open table new tab CLR, Genes comprehensively array assay (RT2 Profiler Array; Qiagen, Hilden, converting ?g total c