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

Open ArchivePublished:December 29, 2020DOI:https://doi.org/10.1016/j.ajpath.2020.12.012
      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 of inducing subretinal fibrosis. We investigated the use of adrenomedullin (AM), a vasoactive peptide, and its receptor activity-modifying protein 2, RAMP2, which regulate vascular homeostasis and suppress fibrosis. The therapeutic potential of the AM-RAMP2 system was evaluated after laser-induced choroidal neovascularization (LI-CNV), a mouse model of AMD. Neovascular formation, subretinal fibrosis, and macrophage invasion were all enhanced in both AM and RAMP2 knockout mice compared with those in wild-type mice. These pathologic changes were suppressed by intravitreal injection of AM. Comprehensive gene expression analysis of the choroid after LI-CNV with or without AM administration revealed that fibrosis-related molecules, including Tgfb, Cxcr4, Ccn2, and Thbs1, were all down-regulated by AM. In retinal pigment epithelial cells, co-administration of transforming growth factor-β and tumor necrosis factor-α induced epithelial-mesenchymal transition, which was also prevented by AM. Finally, transforming growth factor-β and C-X-C chemokine receptor type 4 (CXCR4) inhibitors eliminated the difference in subretinal fibrosis between RAMP2 knockout and wild-type mice. These findings suggest the AM-RAMP2 system suppresses subretinal fibrosis in LI-CNV by suppressing epithelial-mesenchymal transition.
      Age-related macular degeneration (AMD) is a leading cause of blindness, with a global prevalence of about 8.7%.
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      reported that in laser-induced choroidal neovascularization (LI-CNV), a model of AMD, CNV size was significantly greater in heterozygous AM KO than in wild-type (WT) mice.
      Given these observations, the clinical application of AM has been much anticipated
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      ; however, AM is a peptide with a short half-life in the bloodstream, which limits its usefulness for the treatment of chronic diseases. To overcome that limitation, our group has been focusing on AM's receptor system. AM is a member of the calcitonin superfamily and acts via a G-protein–coupled seven-transmembrane domain receptor, calcitonin receptor-like receptor (CLR).
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      This suggests that RAMP2 is the key determinant of the vascular function of AM, and that it may be possible to modulate the vascular function of AM by modulating RAMP2. In the present study, subretinal fibrosis was analyzed in LI-CNV model mice, and the possibility that the AM-RAMP2 system may serve as a therapeutic target for subretinal fibrosis associated with AMD was evaluated.

      Materials and Methods

      Animals

      AM, RAMP2, and RAMP3 KO mice were generated by our group previously.
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      • 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.
      ,
      • Ichikawa-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.
      ,
      • Yamauchi A.
      • Sakurai T.
      • Kamiyoshi A.
      • Ichikawa-Shindo Y.
      • Kawate H.
      • Igarashi K.
      • Toriyama Y.
      • Tanaka M.
      • Liu T.
      • Xian X.
      • Imai A.
      • Zhai L.
      • Owa S.
      • Arai T.
      • Shindo T.
      Functional differentiation of RAMP2 and RAMP3 in their regulation of the vascular system.
      In this study, because of the embryonic lethality of homozygous KO, heterozygous KO of AM and RAMP2 were used, which reduced expression of these genes to about half that in WT mice. Homozygous RAMP3 KO mice were used, as loss of RAMP3 is not lethal, and adult mice are available. Male mice, aged 9 to 12 weeks, were used. WT littermates from each KO line were used as control.
      Before the procedure, mice were anesthetized by an i.p. injection of a mixture of 0.3 mg/kg medetomidine (Nippon Zenyaku Kogyo Co Ltd, Koriyama, Japan), 4.0 mg/kg midazolam (Astellas Pharma Inc., Tokyo, Japan), and 5.0 mg/kg butorphanol (Meiji Seika Pharma Co Ltd, Tokyo, Japan).
      All animal handling complied with protocols approved by the Ethics Committee of Shinshu University School of Medicine. All experiments were performed according to the statements of the Society of Vision and Ophthalmology on the use of animals in ophthalmic and visual research and our institutional guidelines.

      Indocyanine Green Angiography

      Indocyanine green angiography was performed using a confocal scanning-laser ophthalmoscope, according to a previous study,
      • Kumar S.
      • Berriochoa Z.
      • Jones A.D.
      • Fu Y.
      Detecting abnormalities in choroidal vasculature in a mouse model of age-related macular degeneration by time-course indocyanine green angiography.
      to confirm no apparent choroidal and retinal vascular change in AM KO, RAMP2 KO, and RAMP3 KO mice under baseline conditions (Supplemental Figure S1). While keeping the cornea moist with saline, mice were manually held in front of a Heidelberg Retina Angiograph 2 confocal scanning-laser ophthalmoscope (Heidelberg Engineering GmbH, Heidelberg, Germany). Indocyanine green angiography was performed after tail vein injection of 2 mg/kg indocyanine green (Santen Pharmaceutical, Osaka, Japan). Images were taken at 10 minutes after injection.

      Continuous Administration of AM to Mice

      Male C57BL/6J mice, aged 9 to 12 weeks (Charles River Laboratories Japan, Kanagawa, Japan), were used. Human AM (Peptide Institute, Inc., Osaka, Japan) dissolved in phosphate-buffered saline (PBS) was infused subcutaneously using an osmotic pump (Alzet; DURECT Co, Cupertino, CA). The infusion rate was 29 μg/kg per day, and the infusion duration was 7 or 14 days. PBS was used as a control. The effectiveness of human AM in mice is well established,
      • Hobara N.
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      • Kawasaki H.
      Adrenomedullin facilitates reinnervation of phenol-injured perivascular nerves in the rat mesenteric resistance artery.
      ,
      • Uetake R.
      • Sakurai T.
      • Kamiyoshi A.
      • Ichikawa-Shindo Y.
      • Kawate H.
      • Iesato Y.
      • Yoshizawa T.
      • Koyama T.
      • Yang L.
      • Toriyama Y.
      • Yamauchi A.
      • Igarashi K.
      • Tanaka M.
      • Kuwabara T.
      • Mori K.
      • Yanagita M.
      • Mukoyama M.
      • Shindo T.
      Adrenomedullin-RAMP2 system suppresses ER stress-induced tubule cell death and is involved in kidney protection.
      and the dosage used was selected on the basis of prior studies.
      • Imai A.
      • Toriyama Y.
      • Iesato Y.
      • Hirabayashi K.
      • Sakurai T.
      • Kamiyoshi A.
      • Ichikawa-Shindo Y.
      • Kawate H.
      • Tanaka M.
      • Liu T.
      • Xian X.
      • Zhai L.
      • Dai K.
      • Tanimura K.
      • Liu T.
      • Cui N.
      • Yamauchi A.
      • Murata T.
      • Shindo T.
      Adrenomedullin suppresses vascular endothelial growth factor-induced vascular hyperpermeability and inflammation in retinopathy.
      ,
      • Hirabayashi K.
      • Tanaka M.
      • Imai A.
      • Toriyama Y.
      • Iesato Y.
      • Sakurai T.
      • Kamiyoshi A.
      • Ichikawa-Shindo Y.
      • Kawate H.
      • Tanaka M.
      • Dai K.
      • Cui N.
      • Wei Y.
      • Nakamura K.
      • Iida S.
      • Matsui S.
      • Yamauchi A.
      • Murata T.
      • Shindo T.
      Development of a novel model of central retinal vascular occlusion and the therapeutic potential of the adrenomedullin-receptor activity-modifying protein 2 system.

      Intravitreal Administration of AM to Mice

      Human AM (10−7 mol/L; 1.0 μL) in PBS was injected intravitreally into anesthetized mice under a surgical microscope at the corneal scleral junction using a Hamilton syringe equipped with a 32-gauge needle. PBS (1.0 μL) was used as a control. After operative procedures, mice were administered moxifloxacin hydrochloride (Vegamox Ophthalmic Solution; Alcon, Fudenberg, Switzerland).

      LI-CNV Model

      After anesthesia, both eyes were dilated with 0.5% tropicamide and 0.5% phenylephrine (Mydrine P; Santen, Osaka, Japan). Laser injury to the retina was performed using a green laser slit lamp (GYC-1000; NIDEK, Gamagori, Japan), and, a cover glass and a viscoelastic substance were used as contact lenses. A laser with wavelength 532 μm, power output 200 mW, lasing duration 0.05 seconds, spot size 50 μm was used, and laser injuries were generated in an area where there were no obvious retinal vessels around the optic nerve. Injury to the Bruch membrane was confirmed by the appearance of air bubbles. Three shots per eye were used to evaluate the sizes of the CNV and fibrotic area, and five shots per eye were used before extracting mRNA from the choroid. After treatment, 3.0 mg/kg of atipamezole (ZEOAQ, Fukushima, Japan) was intraperitoneally injected to reverse the anesthesia.
      In the LI-CNV experiment, the procedure for intravitreal injection, itself, affects the degree of CNV formation. Therefore, in the studies of intravitreal injection, the effects in each experiment were evaluated only in comparison with their control. As LI-CNV lesions appeared to be decreased toward 3 weeks, samples from 1 to 2 weeks were analyzed.

      FITC Dextran Perfusion and Retinal Flat Mount

      Seven days after LI-CNV induction, mice were anesthetized, a thoracotomy was performed, and 1 mL of PBS containing 50 mg/mL fluorescein isothiocyanate (FITC)–labeled dextran (molecular weight, 2 × 106; Sigma-Aldrich, St. Louis, MO) was systemically administered from the left ventricle. Eyes were then enucleated and fixed with 4% paraformaldehyde for 1 hour, after which the cornea, lens, and retina were removed, and flat mounts of the scleral choroid complex were prepared. Eight pieces were cut radially from the rim toward the optic disc and mounted onto slide glass. FITC-positive areas represent CNV (patent vessels but not collapsed vessels). For immunostaining, after blocking with 1% bovine serum albumin, a rabbit anti-mouse α-smooth muscle actin (α-SMA) antibody (Abcam, Cambridge, UK), rat anti-mouse F4/80 antibody (Bio-Rad, Hercules, CA), and rabbit anti-mouse rho-associated, coiled-coil-containing protein kinase 1 (ROCK1) antibody (Cell Signaling Technology, Danvers, MA) were applied, followed by appropriate secondary antibodies. Flat mounts were then embedded in fluorescence mounting medium (Agilent Technologies, Santa Clara, CA) and observed using a fluorescence microscope (BZ-9000; Keyence, Osaka, Japan). The sizes of the CNV, α-SMA–positive, and ROCK1-positive areas were quantified using an analytic application, BZ analyzer (Keyence). Since only the areas and not the fluoresce intensity and thickness were evaluated, this may lead to underestimating the actual volume of the lesions. Numbers of F4/80-positive cells per CNV lesion were counted. A double-blinded method was used for quantification. In the flat mount analysis, n represents the number of CNV lesions.

      Pathologic Sections

      Seven days after laser irradiation, the eyes were enucleated, fixed in 4% paraformaldehyde overnight, and embedded in paraffin, after which sections (5 μm thick) were prepared for histologic analysis. Sections were stained with hematoxylin/eosin and Masson trichrome stain and immunostained using anti–α-SMA (Agilent Technologies, Santa Clara, CA), anti-AM (Thermo Fisher Scientific, Waltham, MA), and anti-CLR (Abcam) antibodies with diaminobenzidine and fluorescent anti-RhoA antibody (Abcam).

      Quantitative Real-Time RT-PCR

      Mice were sacrificed, and their eyes were enucleated. The cornea, iris, lens, vitreous, and surrounding soft tissues were removed. Finally, the retina was peeled off by pushing the RPE-choroid-sclera complex (choroidal complex) from behind. Total RNA was extracted from choroidal complexes or ARPE19 cells using TRIzol Reagent (Thermo Fisher Scientific). The extracted RNA was then treated with DNA-free (Thermo Fisher Scientific) to remove any contaminating DNA, and a 2-μg sample was reverse transcribed using a PrimeScript RT Reagent Kit (Takara Bio, Shiga, Japan) to produce cDNA. Quantitative real-time RT-PCR was performed using a StepOnePlus real-time PCR system (Thermo Fisher Scientific), SYBR Green (Toyobo, Osaka, Japan), and real-time PCR master mix (Toyobo). Glyceraldehyde-3-phosphate dehydrogenase (Thermo Fisher Scientific) expression was the endogenous control. Table 1 shows the primers used. In the gene expression analysis of the choroidal complexes, n represents the number of mice.
      Table 1Primers Used for Real-Time PCR
      GenePrimer
      Mouse Calcrl (CLR) forward5′-AGGCGTTTACCTGCACACACT-3′
      Mouse Calcrl (CLR) reverse5′-CAGGAAGCAGAGGAAACCCC-3′
      Mouse AM forward5′-GGACACTGCAGGGCCAGAT-3′
      Mouse AM reverse5′-GTAGTTCCCTCTTCCCACGACTTA-3′
      Mouse Ramp2 forward5′-ACTGAGGACAGCCTTGTGTCAAA-3′
      Mouse Ramp2 reverse5′-CCTTGACAGAGTCCATGCAACTC-3′
      Mouse Ramp3 forward5′-AAAGCCTTCGCTGACATGATG-3′
      Mouse Ramp3 reverse5′-ATCTCGGTGCAGTTAGTGAAGCT-3′
      Mouse Tgfb1 forward5′-CCCGAAGCGGACTACTATGC-3′
      Mouse Tgfb1 reverse5′-TAGATGGCGTTGTTGCGGT-3′
      Mouse Tgfb2 forward5′-TAAAATCGACATGCCGTCCC-3′
      Mouse Tgfb2 reverse5′-GAGACATCAAAGCGGACGAT-3′
      Mouse Tgfb3 forward5′-GATCACCACAACCCACACCT-3′
      Mouse Tgfb3 reverse5′-ATAAAGGGGGCGTACACAGC-3′
      Mouse Cxcr4 forward5′-TCAGTGGCTGACCTCCTCTT-3′
      Mouse Cxcr4 reverse5′-TTTCAGCCAGCAGTTTCCTT-3′
      Mouse Cxcl12 (SDF-1) forward5′-AGAGCCAACGTCAAGCATCT-3′
      Mouse Cxcl12 (SDF-1) reverse5′-TAATTTCGGGTCAATGCACA-3′
      Mouse Thbs1 forward5′-CGCCTTCCGCATTGAGAATG-3′
      Mouse Thbs1 reverse5′-CATCTGCCTCAAGGAAGCCA-3′
      Mouse Ccn2 (CTGF) forward5′-CAGAGGTGGTGGGGTAGAGA-3′
      Mouse Ccn2 (CTGF) reverse5′-CATTGCCACTCACAATGTCC-3′
      Mouse Tjp1 (ZO-1) forward5′-GCCACTACAGTATGACCATCC-3′
      Mouse Tjp1 (ZO-1) reverse5′-AATGAATAATATCAGCACCATGCC -3′
      Mouse Tagln (SM22α) forward5′-ACCAAAAACGATGGAAACTACCG-3′
      Mouse Tagln (SM22α) reverse5′-CATTTGAAGGCCAATGACGTG -3′
      Mouse Rhoa forward5′-GCTACCAGTATTTAGAAGCCAACCAC-3′
      Mouse Rhoa reverse5′-GCTGTTAGAGCAGTGTCAGAAGGAC-3′
      Mouse Rock1 forward5′-CAAAGCACGCCTAACTGACA -3′
      Mouse Rock1 reverse5′-TCTGCCTTCTCTCGAGCTTC-3′
      Mouse Icam1 forward5′-CCTAAAATGACCTGCAGACGG-3′
      Mouse Icma1 reverse5′-TTTGACAGACTTCACCACCCC-3′
      Mouse Ccl2 (MCP1) forward5′-GCAGTTAACGCCCCACTCA-3′
      Mouse Ccl2 (MCP1) reverse5′-CCTACTCATTGGGATCATCTTGCT-3′
      Mouse Tnfa forward5′-ACGGCATGGATCTCAAAGAC-3′
      Mouse Tnfa reverse5′-AGATAGCAAATCGGCTGACG-3′
      Mouse Il1b forward5′-CTACAGGCTCCGAGATGAACAAC-3′
      Mouse Il1b reverse5′-TCCATTGAGGTGGAGAGCTTTC-3′
      Mouse Smad2 forward5′-ATGTCGTCCATCTTGCCATTC-3′
      Mouse Smad2 reverse5′-AACCGTCCTGTTTTCTTTAGCTT-3′
      Mouse Smad3 forward5′-CATTACCATCCCCAGGTCAC-3′
      Mouse Smad3 reverse5′-CGTAATTCATGGTGGCTGTG-3′
      Human Tgfb1 forward5′-GTGGAAACCCACAACGAAAT-3′
      Human Tgfb1 reverse5′-CGGAGCTCTGATGTGTTGAA-3′
      Human Cxcr4 forward5′-CTCCAAGCTGTCACACTCCA-3′
      Human Cxcr4 reverse5′-TCGATGCTGATCCCAATGTA-3′
      Human Tjp1 (ZO-1) forward5′-TCACCTACCACCTCGTCGTCTG-3′
      Human Tjp1 (ZO-1) reverse5′-ATGAGCACTGCCCACCCATCT-3′
      Human Tagln (SM22α) forward5′-GATTTTGGACTGCACTTCGC-3′
      Human Tagln (SM22α) reverse5′-GTCCGAACCCAGACACAAGT-3′
      Human Rhoa forward5′-CTGGTGATTGTTGGTGATGG-3′
      Human Rhoa reverse5′-GCGATCATAATCTTCCTGCC-3′
      Human Rock1 forward5′-AACATGGCATCTTCGACACTC-3′
      Human Rock1 reverse5′-CAAAATCACAAAGGCCATGA-3′
      CLR, calcitonin receptor-like receptor; CTGF, connective tissue growth factor; MCP1, monocyte chemoattractant protein 1; SDF-1, stromal cell–derived factor-1; ZO-1, zonula occludens protein 1.

      Real-Time RT-PCR Array Assay Analysis

      Genes in the mouse choroid were comprehensively evaluated using a PCR array assay (RT2 Profiler PCR Array; Qiagen, Hilden, Germany). After converting 1 μg of total choroidal complex RNA into cDNA using a RT2 First Strand Kit (Qiagen), a PCR array of fibrosis-related factors was performed according to the manufacturer's protocol. All PCRs were performed using the StepOnePlus real-time PCR system. RT2 profiler PCR array data were analyzed using RT2 profiler array data analysis software version 3.5 (Thermo Fisher Scientific).

      Western Blot

      Western blot was performed using protein extract from choroidal complex. The lysates were subjected to electrophoresis in TGX gel (Bio-Rad Laboratories), transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories), and probed using anti-Smad2, anti-Smad3, anti–phosphorylated Smad2, anti–phosphorylated Smad3 (Cell Signaling Technology), and anti-AM (Thermo Fisher Scientific) antibodies. Anti–β-actin antibody (Abcam) served as a loading control. The blots were developed using an ImageQuant LAS 4000 (GE Healthcare, Chicago, IL). In the Western blot analysis of the choroidal complexes, n represents the number of mice.

      Inhibitor Administration

      After laser irradiation, SB431542 (Cayman Chemical, Ann Arbor, MI), a transforming growth factor-β (TGF-β) inhibitor, was intraperitoneally injected daily at 10 mg/kg in 5% dimethyl sulfoxide, as described previously.
      • Davies M.R.
      • Liu X.
      • Lee L.
      • Laron D.
      • Ning A.Y.
      • Kim H.T.
      • Feeley B.T.
      TGF-beta small molecule inhibitor SB431542 reduces rotator cuff muscle fibrosis and fatty infiltration by promoting fibro/adipogenic progenitor apoptosis.
      Dimethyl sulfoxide at 5% served as the control. Plerixafor (Sigma-Aldrich), a C-X-C chemokine receptor type 4 (CXCR4) inhibitor, was injected once into the posterior vitreous cavity (200 mmol/L; 1 μL), as described previously.
      • Lyu Y.
      • Xu W.Q.
      • Sun L.J.
      • Pan X.Y.
      • Zhang J.
      • Wang Y.S.
      Effect of integrin alpha5beta1 inhibition on SDF-l/CXCR4-mediated choroidal neovascularization.
      PBS served as the control. Y27632 (Enzo Life Science, Inc., Farmingdale, NY), a ROCK inhibitor, was injected into the vitreous body (30 μmol/L; 1 μL) every 3 days, as described previously.
      • Zhang J.
      • Liu W.
      • Zhang X.
      • Lin S.
      • Yan J.
      • Ye J.
      Sema3A inhibits axonal regeneration of retinal ganglion cells via ROCK2.
      PBS served as the control.

      Human RPE Cells

      Immortalized human RPE cells, ARPE19, were purchased from ATCC (Manassas, VA). The cells were cultured in medium supplemented with 10% fetal bovine serum and penicillin/streptomycin at 37°C and under 5% CO2.
      TGF-β2 (5 ng/mL) and tumor necrosis factor-α (TNF-α; 10 ng/mL) were added before the cells became confluent. AM (10−7 or 10−9 mol/L) was added 24 hours before the addition of TGF-β2 plus TNF-α. The dosage and treatment period were chosen on the basis of prior studies.
      • Shimekake Y.
      • Nagata K.
      • Ohta S.
      • Kambayashi Y.
      • Teraoka H.
      • Kitamura K.
      • Eto T.
      • Kangawa K.
      • Matsuo H.
      Adrenomedullin stimulates two signal transduction pathways, cAMP accumulation and Ca2+ mobilization, in bovine aortic endothelial cells.
      • Horio T.
      • Kohno M.
      • Kano H.
      • Ikeda M.
      • Yasunari K.
      • Yokokawa K.
      • Minami M.
      • Takeda T.
      Adrenomedullin as a novel antimigration factor of vascular smooth muscle cells.
      • Tanaka M.
      • Koyama T.
      • Sakurai T.
      • Kamiyoshi A.
      • Ichikawa-Shindo Y.
      • Kawate H.
      • Liu T.
      • Xian X.
      • Imai A.
      • Zhai L.
      • Hirabayashi K.
      • Owa S.
      • Yamauchi A.
      • Igarashi K.
      • Taniguchi S.
      • Shindo T.
      The endothelial adrenomedullin-RAMP2 system regulates vascular integrity and suppresses tumour metastasis.
      The plate was dipped in 4% paraformaldehyde for 10 minutes, blocked, and immunostained with anti–zonula occludens protein 1 (ZO-1; 1:200 dilution; BD Biosciences, Franklin Lakes, NJ), anti-SM22α (1:200 dilution; Abcam), and anti–collagen α1 (1:200 dilution; Novus Biologicals, Littleton, CO) antibodies. Cells were also stained with phalloidin (Thermo Fisher Scientific) to visualize actin fibers. Cells were then examined under a microscope (BZ-9000). Positive areas were determined using Hybrid Cell Count (BZ analyzer) under the same conditions. Phalloidin-positive intracellular fiber concentrations were determined, as described previously.
      • Peacock J.G.
      • Miller A.L.
      • Bradley W.D.
      • Rodriguez O.C.
      • Webb D.J.
      • Koleske A.J.
      The Abl-related gene tyrosine kinase acts through p190RhoGAP to inhibit actomyosin contractility and regulate focal adhesion dynamics upon adhesion to fibronectin.
      Phalloidin-stained cells were randomly selected, and their images were enlarged for binarization using ImageJ software version 1.53f (NIH, Bethesda, MD; http://imagej.nih.gov/ij). Fluorescence density was measured along a line perpendicular to the stress fibers at the longest intracellular distance. The obtained average values served as fiber density.

      Statistical Analysis

      Statistical analysis was performed using GraphPad Prism 7.00 (GraphPad Software Inc., La Jolla, CA). Differences were assessed using t-test, one-way analysis of variance with the Tukey test, or two-way analysis of variance with the Tukey test. P < 0.05 was considered significant. Values are represented as means ± SEM.

      Results

      Pathology of LI-CNV and Expression of AM and Its Related Genes

      First, pathologic changes were analyzed in the retina after laser injury in C57BL/6J WT mice. LI-CNV was confirmed under the retina (Figure 1A) in the sections prepared 7 days after laser irradiation. Consistent with LI-CNV formation, immunostaining for α-SMA, a marker of activated myofibroblasts, Masson trichrome staining were positive. Although Masson trichrome also stained extracellular matrix in retinal areas outside the LI-CNV lesion, the immunostaining for α-SMA was largely limited to the lesion and is thought to more selectively reflect the progression of fibrosis. Thereafter, subretinal fibrosis was evaluated on the basis of α-SMA immunostaining.
      Figure thumbnail gr1
      Figure 1Pathology of the laser-induced choroidal neovascularization (LI-CNV) lesion and expression of adrenomedullin (AM) and its related genes. A: Hematoxylin-eosin staining, Masson trichrome staining, and immunostaining for α-smooth muscle actin (α-SMA; diaminobenzidine) in sections of retina from C57BL/6J wild-type (WT) mice 7 days after laser irradiation. Dotted circles show the LI-CNV. Masson trichrome staining and α-SMA immunostaining show the subretinal fibrosis in LI-CNV. B: Quantitative real-time PCR analysis of the expression of AM and its related genes in the choroid of untreated C57BL/6J WT mice on days 1, 3, and 7 after the laser irradiation. The mean of the control group without laser irradiation (Ctrl) was assigned a value of 1. Data are expressed as means ± SEM (B). n = 5 to 6 in each group (B). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P<0.0001 (one-way analysis of variance with the Tukey test). Scale bars = 100 μm (A). CLR, calcitonin receptor-like receptor.
      To clarify the involvement of the AM-RAMP2 system, we analyzed the expression of AM and its receptor components (Figure 1B). Following laser irradiation, expression levels of AM, RAMP2, and RAMP3 reached their peaks on day 1, and then gradually returned to basal levels by day 7. On the other hand, CLR expression gradually increased from day 1 to day 7. The significant up-regulation of AM and its receptor components strongly suggests involvement of AM in the pathogenesis of LI-CNV. AM and CLR protein expression within LI-CNV lesions was also analyzed by immunohistostaining and Western blot (Supplemental Figure S2).

      Evaluation of Flat Mount LI-CNV Specimens from AM, RAMP2, or RAMP3 KO

      Next, the pathophysiological functions of the endogenous AM-RAMP2 system were evaluated using AM KO and RAMP2 KO mice and their WT littermates. Seven days after laser irradiation, FITC-dextran was systemically administered via the left ventricle. Thereafter, eyes were collected, and choroidal flat mounts prepared. FITC-positive vascular area was evaluated as the size of the CNV. α-SMA immunostaining was used to evaluate the degree of subretinal fibrosis, and for F4/80, a macrophage marker, to evaluate the degree of inflammatory cell infiltration. FITC imaging revealed the average size of the CNV to be significantly larger in AM KO than WT mice (15,508 μm2 versus 8309 μm2) (Figure 2, A and B). The α-SMA–positive fibrotic area was also significantly larger in AM KO mice (23,023 μm2 versus 10,638 μm2). Moreover, the difference after subtraction of the CNV area from the α-SMA–positive area, which reveals the fibrosis spreading beyond the CNV, was significantly larger in AM KO than WT mice (7515 μm2 versus 2328 μm2).
      Figure thumbnail gr2
      Figure 2Choroidal flat mount analysis of laser-induced choroidal neovascularization (LI-CNV), fibrosis, and macrophage invasion in adrenomedullin (AM) knockout (KO) and RAMP2 KO mice. A: Comparison of LI-CNV between AM KO and wild-type (WT) mice. On day 7 after laser-induced injury to the Bruch membrane, choroidal flat mounts were prepared, the areas of the fluorescein isothiocyanate (FITC)–positive CNV and α-smooth muscle actin (α-SMA)–immunopositive fibrotic areas were measured, and the number of F4/80-positive macrophages were counted. B: Bar graphs comparing CNV and fibrotic areas and macrophage invasion between AM KO and WT mice. C: Comparison of LI-CNV between RAMP2 KO and WT mice. D: Bar graphs comparing the CNV and fibrotic areas and macrophage invasion between RAMP2 KO and WT mice. Data are given as means ± SEM (B and D). n = 10 in WT mice (B and D); n = 11 in AM KO mice (B); n = 14 in RAMP2 KO mice (D). ∗P < 0.05 (t-test). Scale bars = 100 μm (A and C).
      The number of infiltrating F4/80-positive macrophages was also significantly higher in AM KO mice (52 versus 29). Similarly, comparison of WT and RAMP2 KO mice showed that the average CNV was significantly larger in RAMP2 KO than in the WT mice (18,070 μm2 versus 6655 μm2), as was fibrotic area (26,462 μm2 versus 9678 μm2) and the number of infiltrating F4/80-positive macrophages (55 versus 34) (Figure 2, C and D). These observations indicate that the endogenous AM-RAMP2 system works to suppress CNV formation, fibrosis, and inflammation after laser injury. On the other hand, ratio of α-SMA–positive area/CNV area was not different in either AM KO or RAMP2 KO compared with WT mice, which may indicate that larger CNV accompanies lager fibrosis (Supplemental Figure S3).
      The involvement of RAMP3 after laser injury was also evaluated. Unlike AM and RAMP2 KO, RAMP3 KO did not significantly affect CNV size, degree of fibrosis, or inflammation (Supplemental Figure S4). Thus, the AM-RAMP2 system, but not the AM-RAMP3 system, works to suppress the pathologic changes associated with laser injury.

      Effect of the Intravitreal Injection of AM in Flat Mount Specimens of LI-CNV

      Given that the endogenous AM-RAMP2 system appears to act to suppress the pathologic changes associated with LI-CNV, we next assessed the effect of exogenous administration of AM (Figure 3). After laser irradiation of C57BL/6J WT mice, control PBS or AM was administered intraocularly. Because intraocular injection itself causes damage and results in the enlargement of the LI-CNV in mice,
      • Yuda K.
      • Takahashi H.
      • Inoue T.
      • Ueta T.
      • Iriyama A.
      • Kadonosono K.
      • Tamaki Y.
      • Aburatani H.
      • Nagai R.
      • Yanagi Y.
      Adrenomedullin inhibits choroidal neovascularization via CCL2 in the retinal pigment epithelium.
      the effects of AM injection were compared with control PBS injection. The size of the CNV in mice administered PBS was significantly larger than in those administered AM (18,779 μm2 versus 9770 μm2). Likewise, the size of the α-SMA–positive fibrotic area was significantly larger in the control than in the AM group (27,265 μm2 versus 22,185 μm2). Although there were an average of 65 F4/80-positive macrophages inside or around each laser photocoagulation site in the control group, they were significantly reduced to 44 in the AM group.
      Figure thumbnail gr3
      Figure 3Choroidal flat mount analysis of laser-induced choroidal neovascularization (LI-CNV), fibrosis, and macrophage invasion in adrenomedullin (AM)–administered mice. A: Comparison of LI-CNV in the laser injury model between AM-administered and control mice. After the laser irradiation, mice were injected once into the posterior vitreous with phosphate-buffered saline (PBS; 1 μmol/L; 1 μL; control) or AM (10−7 mol/L; 1 μL). On day 7 after the laser-induced injury, choroidal flat mounts were prepared, the fluorescein isothiocyanate (FITC)–positive CNV and α-smooth muscle actin (α-SMA)–immunopositive fibrotic areas were measured, and the number of F4/80-immunopositive macrophages were counted. B: Bar graphs comparing the CNV and fibrotic areas and macrophage invasion between control and AM-administered mice. Data are given as means ± SEM (B). n = 10 in each group (B). ∗∗P < 0.01, ∗∗∗∗P < 0.0001 (t-test). Scale bars = 100 μm (A).
      These results indicate that, like the endogenous AM-RAMP2 system, exogenous AM administration effectively suppresses the pathologic changes associated with LI-CNV.

      Comprehensive Analysis of the Changes in Gene Expression in LI-CNV Induced by AM

      To understand the mechanism underlying the beneficial effect of AM, a comprehensive gene expression analysis was performed using an RT2 Profiler PCR Array of mouse fibrosis-related factors. To evaluate the changes of fibrosis-related genes, PBS or AM was continuously administered to C57BL/6J WT mice using subcutaneously implanted osmotic pumps. Samples were then collected from the choroid 14 days after laser irradiation, and the gene expression profiles were analyzed. The expression of fibrosis-related genes (Tgif1, Ccn2, Cxcr4, Mmp3, and Timp1) was down-regulated by AM administration (Figure 4A). Focusing on the fibrosis-related molecules, quantitative real-time PCR analysis confirmed that expression levels of genes encoding the TGF-β family and its receptors, connective tissue growth factor, CXCR4, tissue inhibitor of metalloproteinase 1 (TIMP1), and thrombospondin 1 (Thbs1), were all significantly down-regulated by AM administration (Figure 4B). Although Smads have been identified as the canonical downstream targets of TGF-β, no significant effect of AM administration on the expression of Smads was detected (Supplemental Figure S5).
      Figure thumbnail gr4
      Figure 4Comprehensive gene expression analysis in the choroids after laser irradiation with or without adrenomedullin (AM) administration. A: Following laser irradiation, AM or phosphate-buffered saline (PBS) was administered using subcutaneously implanted osmotic pumps. On day 14, choroids were collected for comprehensive gene expression analysis of mouse fibrosis-related factors. The dot plot shows the results of the real-time PCR array analysis. The horizontal axis shows the fold change [log2 (fold change)], and the vertical axis shows the P value [–log10 (P value)]. Dashed lines indicate the distribution of unchanged genes. B: Quantitative real-time PCR analysis of the expression of fibrosis-related genes in the choroids after 14 days of laser irradiation with administration of PBS or AM. The mean of the untreated group (without laser irradiation) was assigned a value of 1. Data are expressed as means ± SEM (B). n = 4 in each group (B). ∗P < 0.05, ∗∗P < 0.01 (one-way analysis of variance with the Tukey test). CNV, choroidal neovascularization.

      Evaluation of the Gene Expression Changes in LI-CNV from AM KO or RAMP2 KO

      Because suppression of fibrosis-related factors may explain the beneficial effects of AM in LI-CNV, the gene expression of fibrosis-related factors in AM KO and RAMP2 KO mice on days 7 and 14 after the laser irradiation was evaluated. Some of the examined molecules showed significant up-regulation in AM KO and RAMP2 KO mice compared with WT mice (Figure 5). In particular, expression of Tgfb3 (TGF-β3), Ccn2 (connective tissue growth factor), Thbs1 (Thbs1), Cxcr4 (CXCR4), and Tagln (SM22α) was significantly up-regulated in RAMP2 KO compared with WT mice, whereas expression of Tgfb1 (TGF-β1), Cxcr4 (CXCR4), and Tagln (SM22α) was significantly up-regulated in AM KO compared with WT mice.
      Figure thumbnail gr5
      Figure 5Up-regulation of fibrosis-related genes in the choroids of adrenomedullin (AM) knockout (KO) and RAMP2 KO mice after laser irradiation. A: Quantitative real-time PCR analysis of the expression of fibrosis-related genes. Gene expression was compared between choroids from wild-type (WT) and AM KO mice untreated (Ctrl) and on days 7 and 14 after laser irradiation. The mean of the untreated WT mice was assigned a value of 1. B: Quantitative real-time PCR analysis of the expression of fibrosis-related genes. Gene expression was compared between choroids from WT and RAMP2 KO untreated (Ctrl) and on days 7 and 14 after laser irradiation. Data are expressed as means ± SEM (A and B). n = 4 in each group (A and B). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.01, and ∗∗∗∗P < 0.001 versus AM WT (two-way analysis of variance with the Tukey test); P < 0.05, P < 0.01, ††P < 0.01, and ††P < 0.001 versus RAMP2 WT (two-way analysis of variance with the Tukey test). CTGF, connective tissue growth factor; SDF-1, stromal cell–derived factor-1; ZO-1, zonula occludens protein 1.
      As CXCR4 is known to be the receptor for stromal cell–derived factor-1, the expression of stromal cell–derived factor-1 was analyzed and it was found to be unchanged in AM KO or RAMP2 KO mice. In RAMP2 KO mice, expression of Tgfb3 (TGF-β3), Ccn2 (connective tissue growth factor), and Thbs1 (Thbs1) continued to be elevated on day 14, whereas the up-regulation of Cxcr4 (CXCR4) was transient, reaching a peak at 7 days and returning to basal levels by day 14.
      Because the AM-RAMP2 system has been shown to suppress inflammation,
      • Tanaka M.
      • Koyama T.
      • Sakurai T.
      • Kamiyoshi A.
      • Ichikawa-Shindo Y.
      • Kawate H.
      • Liu T.
      • Xian X.
      • Imai A.
      • Zhai L.
      • Hirabayashi K.
      • Owa S.
      • Yamauchi A.
      • Igarashi K.
      • Taniguchi S.
      • Shindo T.
      The endothelial adrenomedullin-RAMP2 system regulates vascular integrity and suppresses tumour metastasis.
      gene expression of various inflammation-related molecules in LI-CNV samples was also analyzed. Expression of some of the genes, especially the gene encoding IL-1β, was actually up-regulated in AM KO and RAMP2 KO mice, but was suppressed by AM administration (Supplemental Figure S6). This suggests that inflammation is also involved in the antifibrotic function of the AM-RAMP2 system. In addition, there was no significant difference between the activation levels of Smad2 and Smad3, two receptor-activated Smads, in WT and RAMP2 KO mice (Supplemental Figure S7).

      AM Suppresses EMT in RPE Cells

      The in vivo study clearly showed a protective effect of the AM-RAMP2 system against subretinal fibrosis. Recently, there has been growing interest in EMT as the mechanism involved in various fibrosis-related diseases, including subretinal fibrosis.
      • Nieto M.A.
      • Huang R.Y.
      • Jackson R.A.
      • Thiery J.P.
      EMT: 2016.
      ,
      • Kimura K.
      • Orita T.
      • Liu Y.
      • Yang Y.
      • Tokuda K.
      • Kurakazu T.
      • Noda T.
      • Yanai R.
      • Morishige N.
      • Takeda A.
      • Ishibashi T.
      • Sonoda K.H.
      Attenuation of EMT in RPE cells and subretinal fibrosis by an RAR-γ agonist.
      Among the retinal cell components, RPE cells are thought to be the most susceptible to EMT. For that reason, EMT was evaluated in ARPE19, an immortalized human RPE cell line. To induce EMT, ARPE19 cells were stimulated for 48 hours with TGF-β (5 ng/mL) plus TNF-α (10 ng/mL). Immunostaining was performed to assess expression of ZO-1 (an epithelial cell marker detected in the cell membrane) and SM22α (a cytosolic marker of mesenchymal cells) (Figure 6A). Stimulation with TGF-β plus TNF-α resulted in down-regulation of ZO-1 and up-regulation of SM22α, which suggests induction of EMT (Figure 6B). Using this protocol, the cells were pretreated for 24 hours with or without AM (10−9 or 10−7 mol/L) before stimulation. AM (10−7 mol/L) significantly up-regulated ZO-1 and down-regulated SM22α, apparently preventing the effect of TGF-β plus TNF-α. In addition, phalloidin-positive actin filament formation (indicating myofibroblast-like changes in ARPE19 cells) and collagen-positive area (indicating accumulation of extracellular matrix) were both enhanced by TGF-β plus TNF-α stimulation, which suggests induction of EMT. Those changes were prevented by AM (10−7 mol/L) (Figure 7). This suppression of EMT may explain at least some of the protective effects of AM against subretinal fibrosis.
      Figure thumbnail gr6
      Figure 6Suppression of epithelial-mesenchymal transition (EMT) in cultured retinal pigment epithelial cells by adrenomedullin (AM). A: Immunostaining for zonula occludens protein 1 (ZO-1; top row) and SM22α (bottom row) in ARPE19 human retinal pigment epithelial cells. Cells were stimulated with transforming growth factor-β (TGF-β; 5 ng/mL) plus tumor necrosis factor-α (TNF-α; 10 ng/mL) for 48 hours to induce EMT. AM (10−9 or 10−7 mol/L) was added 24 hours before EMT induction. ZO-1 and SM22α were used as epithelial and mesenchymal markers, respectively. B: Bar graphs showing the ZO-1– and SM22α-positive areas/field. For ZO-1, only the cell boundary region was measured. Data are expressed as means ± SEM (B). n = 6 in the AM group (B); n = 10 in the other groups (B). ∗∗P < 0.01, ∗∗∗∗P < 0.0001 (one-way analysis of variance with the Tukey test). Scale bars = 100 μm (A). Original magnification, ×200 (A, top row); ×100 (A, bottom row). Ctrl, control.
      Figure thumbnail gr7
      Figure 7Changes in actin filaments and collagen in cultured retinal pigment epithelial cells after epithelial-mesenchymal transition (EMT) induction. A: ARPE19 cells were stained with phalloidin (top row) or immunostained for collagen-1 (bottom row) to evaluate the changes in the actin cytoskeleton and extracellular matrix induced by EMT, which was elicited by incubation with transforming growth factor-β (TGF-β; 5 ng/mL) plus tumor necrosis factor-α (TNF-α; 10 ng/mL) for 48 hours. Adrenomedullin (AM; 10−9 or 10−7 mol/L) or phosphate-buffered saline was added 24 hours before EMT induction. B: Bar graphs showing the percentage of phalloidin-positive actin filament area/cell and collagen-1 immunostaining-positive area/field. Data are expressed as means ± SEM (B). n = 10 in each group (B). ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001 (one-way analysis of variance with the Tukey test). Scale bars = 100 μm (A). Original magnification, ×200 (A). Ctrl, control.
      Gene expression analysis in the EMT model in ARPE19 cells also showed that stimulation with TGF-β plus TNF-α resulted in down-regulation of genes encoding ZO-1 and up-regulation of SM22α, effects prevented by AM (Figure 8). The EMT stimulation also up-regulated expression of genes encoding TGF-β, CXCR4, RhoA, and ROCK, also effects prevented by AM.
      Figure thumbnail gr8
      Figure 8Changes in gene expression in cultured retinal pigment epithelial cells after induction of epithelial-mesenchymal transition (EMT). Quantitative real-time PCR analysis of gene expression in ARPE19 cells. Cells were stimulated with transforming growth factor-β (TGF-β; 5 ng/mL) plus tumor necrosis factor-α (TNF-α; 10 ng/mL) for 48 hours to induce EMT. Adrenomedullin (AM; 10−7 mol/L) or phosphate-buffered saline was added 24 hours before EMT induction. The mean of the control (Ctrl) group was assigned a value of 1. Data are expressed as means ± SEM. n = 12 in Cxcr4 and Tjp1; n = 8 in others. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗∗P < 0.0001 (one-way analysis of variance with the Tukey test). ZO-1, zonula occludens protein 1.

      Effects of TGF-β, CXCR4, and ROCK Inhibitors on Subretinal Fibrosis

      The protective effects of the AM-RAMP2 system against subretinal fibrosis in LI-CMV may be explained by suppression of fibrosis-related factors such as TGF-β and CXCR4, which is reportedly a downstream target of TGF-β.
      • Feng Y.F.
      • Yuan F.
      • Guo H.
      • Wu W.Z.
      TGF-beta1 enhances SDF-1-induced migration and tube formation of choroid-retinal endothelial cells by up-regulating CXCR4 and CXCR7 expression.
      ,
      • Chen S.
      • Tuttle D.L.
      • Oshier J.T.
      • Knot H.J.
      • Streit W.J.
      • Goodenow M.M.
      • Harrison J.K.
      Transforming growth factor-beta1 increases CXCR4 expression, stromal-derived factor-1alpha-stimulated signalling and human immunodeficiency virus-1 entry in human monocyte-derived macrophages.
      To confirm the involvement of the TGF-β–CXCR4 pathway in subretinal fibrosis, the effects of TGF-β and CXCR4 inhibitors on LI-CNV in RAMP2 KO and WT mice were analyzed.
      The effect of i.p. injection of the TGF-β inhibitor, SB431542 was first studied. In the control 5% dimethyl sulfoxide groups, CNV size was significantly larger in RAMP2 KO than in WT mice (23,797 μm2 versus 14,389 μm2) (Figure 9, A and B ). By contrast, SB431542 administration did not result in a difference in CNV size between RAMP2 KO and WT mice (8461 μm2 versus 8475 μm2) (Figure 9, A and B). In the control groups, α-SMA–positive fibrotic area was also significantly larger in RAMP2 KO than in WT mice (26,711 μm2 versus 13,262 μm2) (Figure 9, C and D). On the other hand, the fibrotic area was no larger in RAMP2 KO than in WT mice (7750 μm2 versus 8225 μm2) after SB431542 administration. Similar results were obtained with intravitreal injection of the CXCR4 inhibitor, plerixafor (Figure 10).
      Figure thumbnail gr9
      Figure 9Transforming growth factor-β (TGF-β) inhibition eliminates the enhanced subretinal fibrosis in RAMP2 knockout (KO) mice. Experiments with SB431542 (TGF-β inhibitor). Mice were peritoneally injected SB431542 or 5% dimethyl sulfoxide (DMSO; control) every day after the laser irradiation. Flat mounts were prepared 7 days after laser injury. A: Fluorescein isothiocyanate–positive choroidal neovascularization (CNV). B: Comparison of CNV area between RAMP2 KO and wild type (WT) with or without the inhibitor. C: α-Smooth muscle actin (α-SMA)–immunopositive fibrotic area. D: Comparison of α-SMA–positive fibrotic area between RAMP2 KO and WT with or without the inhibitor. Data are expressed as means ± SEM (B and D). n = 7 to 9 in each group (B and D). ∗∗P < 0.01, ∗∗∗P < 0.001 versus RAMP2 WT (two-way analysis of variance with the Tukey test). Scale bars = 100 μm (A and C).
      Figure thumbnail gr10
      Figure 10CXCR4 inhibition eliminates the enhanced subretinal fibrosis in RAMP2 knockout (KO) mice. Experiments with plerixafor (CXCR4 inhibitor). Mice were injected with plerixafor or phosphate-buffered saline (PBS; control) into the posterior vitreous immediately after laser irradiation. Flat mounts were prepared 7 days after laser injury. A: Fluorescein isothiocyanate–positive choroidal neovascularization (CNV). B: Comparison of CNV area between RAMP2 KO and wild type (WT) with or without the inhibitor. C: α-Smooth muscle actin (α-SMA)–immunopositive fibrotic area. D: Comparison of α-SMA–positive fibrotic area between RAMP2 KO and WT with or without the inhibitor. Data are expressed as means ± SEM (B and D). n = 12 in WT with PBS (B and D); n = 7 in RAMP2 KO with PBS and in RAMP2 KO with inhibitor (B and D); n = 6 in WT with inhibitor (B and D). ∗P < 0.05, ∗∗P < 0.01 versus RAMP2 WT (two-way analysis of variance with the Tukey test). Scale bars = 100 μm (A and C).
      TGF-β and CXCR4 may interact with the RhoA-ROCK1 pathway, which is reportedly a downstream target of TGF-β
      • Fleming Y.M.
      • Ferguson G.J.
      • Spender L.C.
      • Larsson J.
      • Karlsson S.
      • Ozanne B.W.
      • Grosse R.
      • Inman G.J.
      TGF-beta-mediated activation of RhoA signalling is required for efficient (V12)HaRas and (V600E)BRAF transformation.
      and involved in subretinal fibrosis.
      • Hollanders K.
      • Van Bergen T.
      • Kindt N.
      • Castermans K.
      • Leysen D.
      • Vandewalle E.
      • Moons L.
      • Stalmans I.
      The effect of AMA0428, a novel and potent ROCK inhibitor, in a model of neovascular age-related macular degeneration.
      As described above, immunostaining retinal sections from WT and RAMP2 KO mice for α-SMA after LI-CNV revealed subretinal fibrosis to be present in both groups, although larger in RAMP2 KO mice (Figure 11A). Correspondingly, intense immunostaining for RhoA was detected in RAMP2 KO mice (Figure 11B). In flat mounts, ROCK1-positive areas were significantly larger in RAMP2 KO than in WT mice (Figure 11, C and D). Gene expression analysis showed significant up-regulation of RhoA and ROCK1 in RAMP2 KO compared with WT mice (Figure 11E).
      Figure thumbnail gr11
      Figure 11Immunostaining and gene expression of RhoA and rho-associated, coiled-coil-containing protein kinase 1 (ROCK1) within laser-induced choroidal neovascularization (LI-CNV) lesions. A and B: Immunostaining of α-smooth muscle actin (α-SMA; A) and RhoA (B) in pathologic sections of retina in wild-type (WT) and RAMP2 knockout (KO) mice 7 days after laser irradiation. Dotted circles show the LI- CNV. C: Immunostaining of ROCK1 in flat mounts from WT and RAMP2 KO mice 7 days after laser irradiation. D: Comparison of the ROCK1-positive area between RAMP2 KO and WT. E: Comparison of RhoA and Rock1 gene expression between RAMP2 KO and WT mice. Data are expressed as means ± SEM (D and E). n = 8 in WT (D); n = 6 in RAMP2 KO (D); n = 4 in each mouse category (E). ∗∗P < 0.01, ∗∗∗P < 0.001 versus RAMP2 WT (t-test). Scale bars = 100 μm (AC).
      To further confirm the relationship between TGF-β, ROCK1, and CXCR4, the effect of the TGF-β inhibitor, SB431542, and ROCK inhibitor, Y27632, on gene expression in LI-CNV samples was analyzed. TGF-β inhibition suppressed expression of the genes encoding ROCK1 and CXCR4, suggesting TGF-β is upstream of both ROCK1 and CXCR4 (Figure 12A). Similarly, ROCK inhibition suppressed expression of the gene encoding CXCR4, suggesting ROCK1 is upstream of CXCR4 (Figure 12B).
      Figure thumbnail gr12
      Figure 12Down-regulation of Cxcr4 and Rock1 expression within laser-induced choroidal neovascularization (CNV) lesions by transforming growth factor-β (TGF-β) or ROCK inhibitor. Following laser irradiation, wild-type (WT) mice were peritoneally injected with SB431542 (TGF-β inhibitor) or 5% dimethyl sulfoxide (DMSO; control) daily (A), or intravitreally injected with Y27632 (ROCK inhibitor) or phosphate-buffered saline (PBS; control) every 3 days (B). Choroids were prepared 7 days after laser irradiation, and quantitative real-time PCR analysis was performed. The mean of the untreated WT mice (no CNV) was assigned a value of 1. Data are expressed as means ± SEM (A and B). n = 4 in each group (A and B). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001 (one-way analysis of variance with the Tukey test).
      These data suggest that enhanced subretinal fibrosis in RAMP2 KO mice is associated with up-regulation of activity in the TGF-β–ROCK1–CXCR4 pathway, and that inhibition of this pathway eliminates the difference in fibrosis between RAMP2 KO and WT.

      Discussion

      Currently, AMD patients are treated with photodynamic therapy, retinal photocoagulation, or anti-VEGF therapy.
      • Miller J.W.
      Treatment of age-related macular degeneration: beyond VEGF.
      Among those, anti-VEGF therapy has good short-term results and is becoming a standard treatment for AMD.
      • Rosenfeld P.J.
      • Shapiro H.
      • Tuomi L.
      • Webster M.
      • Elledge J.
      • Blodi B.
      MARINA and ANCHOR Study Groups
      Characteristics of patients losing vision after 2 years of monthly dosing in the phase III ranibizumab clinical trials.
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      • Korobelnik J.F.
      • Brown D.M.
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      • Sandbrink R.
      • Heier J.S.
      Intravitreal aflibercept injection for neovascular age-related macular degeneration: ninety-six-week results of the VIEW studies.
      • Group C.R.
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      • Maguire M.G.
      • Ying G.S.
      • Grunwald J.E.
      • Fine S.L.
      • Jaffe G.J.
      Ranibizumab and bevacizumab for neovascular age-related macular degeneration.
      However, the beneficial effects of anti-VEGF drugs on the eye are time limited and require regular intravitreal injections.
      • Sawada O.
      • Ohji M.
      Retinal vein occlusion.
      Furthermore, chronic usage of anti-VEGF therapy is becoming a subject of concern, as it may promote subretinal fibrosis.
      • Cohen S.Y.
      • Oubraham H.
      • Uzzan J.
      • Dubois L.
      • Tadayoni R.
      Causes of unsuccessful ranibizumab treatment in exudative age-related macular degeneration in clinical settings.
      • Hwang J.C.
      • Del Priore L.V.
      • Freund K.B.
      • Chang S.
      • Iranmanesh R.
      Development of subretinal fibrosis after anti-VEGF treatment in neovascular age-related macular degeneration.
      • Daniel E.
      • Toth C.A.
      • Grunwald J.E.
      • Jaffe G.J.
      • Martin D.F.
      • Fine S.L.
      • Huang J.
      • Ying G.S.
      • Hagstrom S.A.
      • Winter K.
      • Maguire M.G.
      Comparison of Age-related Macular Degeneration Treatments Trials Research Group: Risk of scar in the comparison of age-related macular degeneration treatments trials.
      Clarifying more precisely the mechanism and molecular participants in subretinal fibrosis may reveal novel candidates that may serve as the basis for new therapeutic approaches.
      The bioactive peptide, AM, which was originally identified as a vasodilating and antihypertensive mediator that contributed to circulatory homeostasis, was studied. However, subsequent studies revealed that AM also possesses anti-oxidative, anti-inflammatory, anti-apoptotic, and antifibrotic properties, and that it is present in the eyes.
      • Udono-Fujimori R.
      • Udono T.
      • Totsune K.
      • Tamai M.
      • Shibahara S.
      • Takahashi K.
      Adrenomedullin in the eye.
      It has been speculated that AM may serve as a possible therapeutic agent for treatment of AMD. However, an important limitation of AM is its short half-life in the bloodstream, which limits its usefulness for treatment of chronic diseases. As an alternative, we propose that the AM receptor system may be a more useful target for the treatment of retinal vascular diseases. The complex of AM's receptor, CLR, with RAMP2 or RAMP3 has high affinity for AM.
      • Shindo T.
      • Tanaka M.
      • Kamiyoshi A.
      • Ichikawa-Shindo Y.
      • Kawate H.
      • Yamauchi A.
      • Sakurai T.
      Regulation of cardiovascular development and homeostasis by the adrenomedullin-RAMP system.
      Moreover, the earlier finding that only homozygous RAMP2 KO die in utero of vascular abnormalities, similar to those observed in homozygous AM KO
      • Ichikawa-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.
      mice, suggests RAMP2 is the key determinant of the vascular function of AM. Here, we focused on the AM-RAMP2 system and its actions in subretinal fibrosis associated with AMD.
      In this study, LI-CNV size, fibrosis, and inflammation were exacerbated in AM KO and RAMP2 KO compared with their WT littermates. By contrast, no difference was found between RAMP3 KO and WT mice. This suggests that the beneficial effects of AM in LI-CNV were mediated via RAMP2 rather than RAMP3. In contrast to the findings with AM and RAMP2 KO, exogenous AM administration suppressed LI-CNV size, fibrosis, and inflammation. In the gene expression analysis, fibrosis-related molecules were up-regulated in AM KO and RAMP2 KO mice and down-regulated in AM-administered mice. In fact, AM suppressed expression of some fibrosis-related molecules to levels below their baseline levels, which is consistent with the strong antifibrotic properties of AM. Ratio of α-SMA–positive area/CNV area was not different in either AM KO or RAMP2 KO compared with WT mice, which may indicate that larger CNV accompanies lager fibrosis, and suppression of CNV formation may reduce the apparent levels of fibrosis.
      Sakimoto et al
      • Sakimoto S.
      • Kidoya H.
      • Kamei M.
      • Naito H.
      • Yamakawa D.
      • Sakaguchi H.
      • Wakabayashi T.
      • Nishida K.
      • Takakura N.
      An angiogenic role for adrenomedullin in choroidal neovascularization.
      used AM22-52 (a partial AM peptide that acts as a competitive antagonist of AM) and AM antibody in a similar LI-CNV model. They showed that intravitreal injection of AM22-52 or AM antibody reduced the size of the CNV. Their results appear to be opposite the present result obtained with AM KO mice, and we cannot be certain what accounts for the difference. However, one possibility is the difference in the methods used to suppress AM. Whereas Sakimoto et al
      • Sakimoto S.
      • Kidoya H.
      • Kamei M.
      • Naito H.
      • Yamakawa D.
      • Sakaguchi H.
      • Wakabayashi T.
      • Nishida K.
      • Takakura N.
      An angiogenic role for adrenomedullin in choroidal neovascularization.
      used AM inhibitors, we used AM KO and RAMP2 KO mice. Moreover, Sakimoto et al
      • Sakimoto S.
      • Kidoya H.
      • Kamei M.
      • Naito H.
      • Yamakawa D.
      • Sakaguchi H.
      • Wakabayashi T.
      • Nishida K.
      • Takakura N.
      An angiogenic role for adrenomedullin in choroidal neovascularization.
      intravitreally injected the inhibitors. In our experience, intravitreal injection, itself, affects the degree of CNV formation in mice. We therefore suggest the results of the present study more accurately reflect the role of endogenous AM. Indeed, using a similar LI-CNV model, Yuda et al
      • Yuda K.
      • Takahashi H.
      • Inoue T.
      • Ueta T.
      • Iriyama A.
      • Kadonosono K.
      • Tamaki Y.
      • Aburatani H.
      • Nagai R.
      • Yanagi Y.
      Adrenomedullin inhibits choroidal neovascularization via CCL2 in the retinal pigment epithelium.
      showed that CNV is larger in AM KO than WT mice, which is consistent with the present findings. The limitation of using the LI-CNV model for studying retinal fibrosis is that it was evaluated up to 2 weeks, although pathology of fibrosis is in fact chronic in nature.
      EMT is the process by which epithelial cells lose their adhesive function and transform into mesenchymal-like cells. It is known to be important in embryonic development, organogenesis, wound healing, and cancer invasion and metastasis. In addition, EMT has also been implicated in fibrosis,
      • Thiery J.P.
      • Sleeman J.P.
      Complex networks orchestrate epithelial-mesenchymal transitions.
      and is therefore a potential therapeutic target in fibrosis-related diseases. It was recently suggested that RPE cells are mainly affected by EMT in subretinal fibrosis associated with AMD.
      • Ishikawa K.
      • Kannan R.
      • Hinton D.R.
      Molecular mechanisms of subretinal fibrosis in age-related macular degeneration.
      ,
      • Hirasawa M.
      • Noda K.
      • Noda S.
      • Suzuki M.
      • Ozawa Y.
      • Shinoda K.
      • Inoue M.
      • Ogawa Y.
      • Tsubota K.
      • Ishida S.
      Transcriptional factors associated with epithelial-mesenchymal transition in choroidal neovascularization.
      In vitro, cellular stimulation with TGF-β plus TNF-α has been used to induce EMT.
      • Yoshimatsu Y.
      • Watabe T.
      Roles of TGF-beta signals in endothelial-mesenchymal transition during cardiac fibrosis.
      ,
      • Matoba R.
      • Morizane Y.
      • Shiode Y.
      • Hirano M.
      • Doi S.
      • Toshima S.
      • Araki R.
      • Hosogi M.
      • Yonezawa T.
      • Shiraga F.
      Suppressive effect of AMP-activated protein kinase on the epithelial-mesenchymal transition in retinal pigment epithelial cells.
      As a result of EMT, the epithelial cell markers E-cadherin, claudin-1, ZO-1, and cytokeratin-18 are all down-regulated, whereas the mesenchymal markers α-SMA, fibronectin, fibroblast-specific protein-1, vimentin, collagen 1, and SM22α are all up-regulated.
      • Yoshimatsu Y.
      • Watabe T.
      Roles of TGF-beta signals in endothelial-mesenchymal transition during cardiac fibrosis.
      In the present study, EMT was induced in ARPE19 cells, an RPE cell line, by exposing the cells to TGF-β plus TNF-α. Subsequent evaluation of ZO-1 and SM22α levels confirmed that AM suppresses EMT in ARPE19 cells.
      Using real-time PCR analysis, expression level of genes encoding TGF-β and CXCR4 were found to be elevated in AM KO and RAMP2 KO mice and decreased by AM administration. TGF-β has been identified as the main factor driving the inflammation and fibrosis associated with AMD in humans.
      • Tosi G.M.
      • Orlandini M.
      • Galvagni F.
      The controversial role of TGF-beta in neovascular age-related macular degeneration pathogenesis.
      On the other hand, AM has been shown to suppress expression of inflammatory cytokines, including TGF-β, by inhibiting phosphorylation of c-Jun N-terminal kinase (JNK), extracellular signal–regulated kinase, and p38 mitogen-activated protein kinase.
      • Mandal J.
      • Roth M.
      • Papakonstantinou E.
      • Fang L.
      • Savic S.
      • Tamm M.
      • Stolz D.
      Adrenomedullin mediates pro-angiogenic and pro-inflammatory cytokines in asthma and COPD.
      • Hu W.
      • Shi L.
      • Li M.Y.
      • Zhou P.H.
      • Qiu B.
      • Yin K.
      • Zhang H.H.
      • Gao Y.
      • Kang R.
      • Qin S.L.
      • Ning J.Z.
      • Wang W.
      • Zhang L.J.
      Adrenomedullin protects Leydig cells against lipopolysaccharide-induced oxidative stress and inflammatory reaction via MAPK/NF-kappaB signalling pathways.
      • Wang Y.
      • Zhang J.S.
      • Qian J.
      • Huang G.C.
      • Chen Q.
      Adrenomedullin regulates expressions of transforming growth factor-beta1 and beta1-induced matrix metalloproteinase-2 in hepatic stellate cells.
      CXCR4 is thought to be a downstream target of TGF-β.
      • Feng Y.F.
      • Yuan F.
      • Guo H.
      • Wu W.Z.
      TGF-beta1 enhances SDF-1-induced migration and tube formation of choroid-retinal endothelial cells by up-regulating CXCR4 and CXCR7 expression.
      ,
      • Chen S.
      • Tuttle D.L.
      • Oshier J.T.
      • Knot H.J.
      • Streit W.J.
      • Goodenow M.M.
      • Harrison J.K.
      Transforming growth factor-beta1 increases CXCR4 expression, stromal-derived factor-1alpha-stimulated signalling and human immunodeficiency virus-1 entry in human monocyte-derived macrophages.
      CXCR4 is well known as the receptor required for HIV to enter cells,
      • Feng Y.
      • Broder C.C.
      • Kennedy P.E.
      • Berger E.A.
      HIV-1 entry cofactor: functional cDNA cloning of a seven-transmembrane, G protein-coupled receptor.
      and it is also reportedly involved in the formation of LI-CNV.
      • Lyu Y.
      • Xu W.Q.
      • Sun L.J.
      • Pan X.Y.
      • Zhang J.
      • Wang Y.S.
      Effect of integrin alpha5beta1 inhibition on SDF-l/CXCR4-mediated choroidal neovascularization.
      ,
      • Lee E.
      • Rewolinski D.
      Evaluation of CXCR4 inhibition in the prevention and intervention model of laser-induced choroidal neovascularization.
      In recent years, CXCR4 has become an attractive target for the treatment of fibrosis in many organs, including the lung,
      • Chen Y.
      • Yu X.
      • He Y.
      • Zhang L.
      • Huang X.
      • Xu X.
      • Chen M.
      • Chen X.
      • Wang L.
      Activation of A(2a)R attenuates bleomycin-induced pulmonary fibrosis via the SDF-1/CXCR4 axis-related pathway.
      heart,
      • Chu P.Y.
      • Walder K.
      • Horlock D.
      • Williams D.
      • Nelson E.
      • Byrne M.
      • Jandeleit-Dahm K.
      • Zimmet P.
      • Kaye D.M.
      CXCR4 antagonism attenuates the development of diabetic cardiac fibrosis.
      liver,
      • Chow L.N.
      • Schreiner P.
      • Ng B.Y.
      • Lo B.
      • Hughes M.R.
      • Scott R.W.
      • Gusti V.
      • Lecour S.
      • Simonson E.
      • Manisali I.
      • Barta I.
      • McNagny K.M.
      • Crawford J.
      • Webb M.
      • Underhill T.M.
      Impact of a CXCL12/CXCR4 antagonist in bleomycin (BLM) induced pulmonary fibrosis and carbon tetrachloride (CCl4) induced hepatic fibrosis in mice.
      and kidney,
      • Yuan A.
      • Lee Y.
      • Choi U.
      • Moeckel G.
      • Karihaloo A.
      Chemokine receptor Cxcr4 contributes to kidney fibrosis via multiple effectors.
      and CXCR4 inhibition using plerixafor suppresses fibrosis. Thus, the beneficial effects of the AM-RAMP2 system may be explained by suppression of the TGF-β–CXCR4 pathway, although it is also possible that plerixafor can directly suppress CNV and fibrosis independent of AM-RAMP2 system. Although Smads have been identified as the canonical downstream targets of TGF-β, no significant effect of AM on Smads was observed.
      To confirm that suppression of the TGF-β–CXCR4 pathway explains the beneficial effects of the AM-RAMP2 system against subretinal fibrosis, the effects of anti–TGF-β or anti-CXCR4 agents in LI-CNV between RAMP2 KO and WT mice were compared. Although both CNV and subretinal fibrosis were greater in RAMP2 KO than WT mice, inhibition of TGF-β using SB431542 or inhibition of CXCR4 using plerixafor eliminated the difference between the responses in the two groups. This suggests up-regulation of activity in the TGF-β–CXCR4 pathway is the main cause of the enhanced pathologic features seen in RAMP2 KO mice.
      The levels of RhoA and ROCK1 expression were up-regulated in RAMP2 KO mice after induction of LI-CMV. The RhoA-ROCK1 pathway is known to be a downstream target of TGF-β
      • Fleming Y.M.
      • Ferguson G.J.
      • Spender L.C.
      • Larsson J.
      • Karlsson S.
      • Ozanne B.W.
      • Grosse R.
      • Inman G.J.
      TGF-beta-mediated activation of RhoA signalling is required for efficient (V12)HaRas and (V600E)BRAF transformation.
      and is also reported to enhance EMT.
      • Hollanders K.
      • Van Bergen T.
      • Kindt N.
      • Castermans K.
      • Leysen D.
      • Vandewalle E.
      • Moons L.
      • Stalmans I.
      The effect of AMA0428, a novel and potent ROCK inhibitor, in a model of neovascular age-related macular degeneration.
      ROCK inhibition using Y27632 blocked enlargement of the subretinal fibrotic area in RAMP2 KO mice, whereas gene expression analysis of LI-CNV samples suggested TGF-β is upstream of both ROCK1 and CXCR4, and ROCK1 is upstream of CXCR4.
      Figure 13 summarizes the actions of the AM-RAMP2 system in subretinal fibrosis. EMT of RPE cells plays a crucial role in the progression of subretinal fibrosis in AMD. Downstream TGF-β signaling molecules, including RhoA-ROCK and CXCR4, promote EMT. In addition to its suppression of CNV formation, the AM-RAMP2 system also suppresses EMT and subretinal fibrosis through inhibition of TGF-β, RhoA-ROCK, and CXCR4. We therefore propose that the AM-RAMP2 system has the potential to serve as a novel therapeutic target for suppression of subretinal fibrosis in the treatment of AMD.
      Figure thumbnail gr13
      Figure 13Proposed mechanism by which the adrenomedullin (AM)–RAMP2 system suppresses subretinal fibrosis within laser-induced choroidal neovascularization (LI-CNV) lesions. Epithelial-mesenchymal transition (EMT) of retinal pigmental epithelial cells plays a crucial role in the progression of subretinal fibrosis within LI-CNV lesions. Downstream transforming growth factor-β (TGF-β) signaling molecules, including RhoA-ROCK and CXCR4, promote EMT. The AM-RAMP2 system suppresses subretinal fibrosis by inhibiting EMT as well as CNV formation.

      Supplemental Data

      • Supplemental Figure S1

        No apparent choroidal and retinal vascular abnormalities are found in any mouse without laser irradiation. Representative indocyanine green angiography images of knockout (KO) mice with their control mice are shown. Scale bars = 200 μm. AM, adrenomedullin; WT, wild type.

      • Supplemental Figure S2

        AdrenomedullinAM) and calcitonin receptor-like receptor (CLR) protein expression within laser-induced choroidal neovascularization (LI-CNV) lesions. A: Immunostaining for AM or CLR (diaminobenzidine) and negative control in sections of retina from C57BL/6J wild-type mice 7 days after laser irradiation. AM and CLR expression is detected within the LI-CNV lesion. B: Western blot analysis of AM in control (Ctrl) and laser-treated (day 1) choroidal complex. β-Actin was used as a loading control. Ratio of quantified OD is displayed. Data are given as means ± SEM (B). n = 6 each (B). ∗P < 0.05 versus control (t-test). Scale bars = 100 μm (A).

      • Supplemental Figure S3

        Ratio of α-smooth muscle actin (α-SMA)–positive area/choroidal neovascularization (CNV) area in adrenomedullin (AM) knockout (KO) and RAMP2 KO. Data are given as means ± SEM. n = 10 in AM wild-type (WT) and RAMP2 WT mice; n = 11 in AM KO mice; n = 14 in RAMP2 KO mice.

      • Supplemental Figure S4

        Choroidal neovascularization (CNV) size or the degree of fibrosis or inflammation remains unchanged in RAMP3 knockout (KO) mice. A: Comparison of CNV areas in the laser injury model between RAMP3 KO and wild-type (WT) mice in choroidal flat mounts. On day 7 after laser injury, choroidal flat mounts were prepared, fluorescein isothiocyanate (FITC)–positive CNV-immunopositive and α-smooth muscle actin (α-SMA)–immunopositive areas were measured, and numbers of F4/80-immunopositive macrophages were counted. B: Bar graphs comparing CNV and fibrotic areas and macrophage infiltration between RAMP3 KO and WT mice. P = 0.15 in CNV area, P = 0.34 in α-SMA–positive area, and P = 0.11 in F4/80-positive cells (t-test). Data are given as means ± SEM (B). n = 13 in WT mice (B); n = 17 in RAMP3 KO mice (B). Scale bars = 100 μm (A).

      • Supplemental Figure S5

        Smad2 and Smad3 gene expression remains unchanged by adrenomedullin (AM) administration. Quantitative real-time PCR analysis of Smad2 and Smad3 gene expression. Expression was compared between choroids from a phosphate-buffered saline (PBS) pump group and an AM pump group without laser irradiation (Ctrl), or on days 7 and 14 after laser irradiation. The mean of the untreated PBS pump group was assigned a value of 1. Data are expressed as means ± SEM. n = 4 in each group.

      • Supplemental Figure S6

        Gene expression analysis of inflammation-related molecules. Quantitative real-time PCR analysis of the expression of inflammation-related molecules in adrenomedullin (AM) knockout (KO; A), RAMP2 KO (B), and AM-treated (C) mice. Gene expression was compared in choroids from each mouse on day 7 after laser irradiation. The mean of untreated wild-type (WT) mice [no choroidal neovascularization (CNV)] was assigned a value of 1. Data are expressed as means ± SEM (AC). n = 4 in each group (AC). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001 (one-way analysis of variance with the Tukey test). MCP1, monocyte chemoattractant protein 1; PBS, phosphate-buffered saline.

      • Supplemental Figure S7

        Levels of Smad2 and Smad3 activation remains unchanged in RAMP2 knockout (KO) mice. Western blot analysis of Smads and phosphorylated Smads (p-Smads) in choroidal complex on day 7 after laser irradiation. β-Actin was used as a loading control. Ratios of quantified ODs of p-Smad/Smad (representing activation level of Smads) are displayed. The mean of the untreated wild-type (WT) mice [no choroidal neovascularization (CNV)] was assigned a value of 1. p-Smad2/Smad2: P = 0.165 (no CNV versus RAMP2 WT), P = 0.006 (no CNV versus RAMP2 KO), P = 0.06 (RAMP2 WT versus RAMP2 KO). p-Smad3/Smad3: P = 0.998 (no CNV versus RAMP2 WT), P = 0.211 (no CNV versus RAMP2 KO), P = 0.228 (RAMP2 WT versus RAMP2 KO) (one-way analysis of variance with the Tukey test). Data are given as means ± SEM. n = 3 in each.

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