Pathophysiological Function of Endogenous Calcitonin Gene–Related Peptide in Ocular Vascular Diseases
2015; Elsevier BV; Volume: 185; Issue: 6 Linguagem: Inglês
10.1016/j.ajpath.2015.02.017
ISSN1525-2191
AutoresYuichi Toriyama, Yasuhiro Iesato, Akira Imai, Takayuki Sakurai, Akiko Kamiyoshi, Yuka Ichikawa‐Shindo, Hisaka Kawate, Akihiro Yamauchi, Kyoko Igarashi, Megumu Tanaka, Tian Liu, Xian Xian, Liuyu Zhai, Shinji Owa, Toshinori Murata, Takayuki Shindo,
Tópico(s)Chemokine receptors and signaling
ResumoCalcitonin gene–related peptide (CGRP; official name CALCA) has a variety of functions and exhibits both angiogenic and anti-inflammatory properties. We previously reported the angiogenic effects of the CGRP family peptide adrenomedullin in oxygen-induced retinopathy; however, the effects of CGRP on ocular angiogenesis remain unknown. Herein, we used CGRP knockout (CGRP−/−) mice to investigate the roles of CGRP in ocular vascular disease. Observation of pathological retinal angiogenesis in the oxygen-induced retinopathy model revealed no difference between CGRP−/− and wild-type mice. However, much higher levels of the CGRP receptor were present in the choroid than the retina. Laser-induced choroidal neovascularization (CNV), a model of exudative age-related macular degeneration, revealed more severe CNV lesions in CGRP−/− than wild-type mice, and fluorescein angiography showed greater leakage from CNV in CGRP−/−. In addition, macrophage infiltration and tumor necrosis factor (TNF)-α production were enhanced within the CNV lesions in CGRP−/− mice, and the TNF-α, in turn, suppressed the barrier formation of retinal pigment epithelial cells. In vivo, CGRP administration suppressed CNV formation, and CGRP also dose dependently suppressed TNF-α production by isolated macrophages. From these data, we conclude that CGRP suppresses the development of leaky CNV through negative regulation of inflammation. CGRP may thus be a promising therapeutic agent for the treatment of ocular vascular diseases associated with inflammation. Calcitonin gene–related peptide (CGRP; official name CALCA) has a variety of functions and exhibits both angiogenic and anti-inflammatory properties. We previously reported the angiogenic effects of the CGRP family peptide adrenomedullin in oxygen-induced retinopathy; however, the effects of CGRP on ocular angiogenesis remain unknown. Herein, we used CGRP knockout (CGRP−/−) mice to investigate the roles of CGRP in ocular vascular disease. Observation of pathological retinal angiogenesis in the oxygen-induced retinopathy model revealed no difference between CGRP−/− and wild-type mice. However, much higher levels of the CGRP receptor were present in the choroid than the retina. Laser-induced choroidal neovascularization (CNV), a model of exudative age-related macular degeneration, revealed more severe CNV lesions in CGRP−/− than wild-type mice, and fluorescein angiography showed greater leakage from CNV in CGRP−/−. In addition, macrophage infiltration and tumor necrosis factor (TNF)-α production were enhanced within the CNV lesions in CGRP−/− mice, and the TNF-α, in turn, suppressed the barrier formation of retinal pigment epithelial cells. In vivo, CGRP administration suppressed CNV formation, and CGRP also dose dependently suppressed TNF-α production by isolated macrophages. From these data, we conclude that CGRP suppresses the development of leaky CNV through negative regulation of inflammation. CGRP may thus be a promising therapeutic agent for the treatment of ocular vascular diseases associated with inflammation. In acquired blindness, retinal or choroidal neovascularization (CNV) is the main causative factor. Retinal neovascularization is induced by retinal hypoxia and is observed in diabetic retinopathy, retinal vein occlusion, and retinopathy of prematurity. CNV occurs as a result of abnormalities in Bruch membrane and the retinal pigment epithelium (RPE)1Lopez P.F. Grossniklaus H.E. Lambert H.M. Aaberg T.M. Capone Jr., A. Sternberg Jr., P. L'Hernault N. Pathologic features of surgically excised subretinal neovascular membranes in age-related macular degeneration.Am J Ophthalmol. 1991; 112: 647-656Abstract Full Text PDF PubMed Scopus (230) Google Scholar and is observed in exudative age-related macular degeneration (AMD), angioid streaks, and high myopia. Calcitonin gene–related peptide (CGRP; official symbol CALCA) is a 37–amino acid peptide produced by alternative splicing of the primary transcript of the calcitonin gene (CALCA). CGRP was originally identified as a sensory neurotransmitter,2Rosenfeld M.G. Mermod J.J. Amara S.G. Swanson L.W. Sawchenko P.E. Rivier J. Vale W.W. Evans R.M. Production of a novel neuropeptide encoded by the calcitonin gene via tissue-specific RNA processing.Nature. 1983; 304: 129-135Crossref PubMed Scopus (1955) Google Scholar and is now known to be structurally similar to the angiogenic factor adrenomedullin (AM).3Kitamura 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 (2073) Google Scholar Moreover, CGRP and AM share the same receptor, calcitonin receptor-like receptor (CLR), a seven transmembrane G protein–coupled receptor.4McLatchie 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 (1852) Google Scholar The affinity of CLR for CGRP or AM is determined by three accessory proteins called receptor activity–modifying proteins (RAMPs 1 to 3).4McLatchie 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 (1852) Google Scholar When associated with RAMP1, CLR has high affinity for CGRP; association with RAMP2 or RAMP3 gives CLR a high affinity for AM. Through analysis of knockout mice, we were able to demonstrate the novel angiogenic functions of AM and RAMP2.5Shindo 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, 6Iimuro 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, 7Ichikawa-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, 8Koyama T. Ochoa-Callejero L. Sakurai T. Kamiyoshi A. Ichikawa-Shindo Y. Iinuma N. Arai T. Yoshizawa T. Iesato Y. Lei Y. Uetake R. Okimura A. Yamauchi A. Tanaka M. Igarashi K. Toriyama Y. Kawate H. Adams R.H. Kawakami H. Mochizuki N. Martinez A. Shindo T. Vascular endothelial adrenomedullin-RAMP2 system is essential for vascular integrity and organ homeostasis.Circulation. 2013; 127: 842-853Crossref PubMed Scopus (71) Google Scholar Recently, CGRP was also reported to possess angiogenic functionality.9Mishima T. Ito Y. Hosono K. Tamura Y. Uchida Y. Hirata M. Suzsuki T. Amano H. Kato S. Kurihara Y. Kurihara H. Hayashi I. Watanabe M. Majima M. Calcitonin gene-related peptide facilitates revascularization during hindlimb ischemia in mice.Am J Physiol Heart Circ Physiol. 2011; 300: H431-H439Crossref PubMed Scopus (57) Google Scholar, 10Ohno T. Hattori Y. Komine R. Ae T. Mizuguchi S. Arai K. Saeki T. Suzuki T. Hosono K. Hayashi I. Oh-Hashi Y. Kurihara Y. Kurihara H. Amagase K. Okabe S. Saigenji K. Majima M. Roles of calcitonin gene-related peptide in maintenance of gastric mucosal integrity and in enhancement of ulcer healing and angiogenesis.Gastroenterology. 2008; 134: 215-225Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 11Toda M. Suzuki T. Hosono K. Hayashi I. Hashiba S. Onuma Y. Amano H. Kurihara Y. Kurihara H. Okamoto H. Hoka S. Majima M. Neuronal system-dependent facilitation of tumor angiogenesis and tumor growth by calcitonin gene-related peptide.Proc Natl Acad Sci U S A. 2008; 105: 13550-13555Crossref PubMed Scopus (83) Google Scholar, 12Toda M. Suzuki T. Hosono K. Kurihara Y. Kurihara H. Hayashi I. Kitasato H. Hoka S. Majima M. Roles of calcitonin gene-related peptide in facilitation of wound healing and angiogenesis.Biomed Pharmacother. 2008; 62: 352-359Crossref PubMed Scopus (76) Google Scholar However, the precise roles of CGRP in ocular vascular development and disease remain totally unknown. To investigate pathological angiogenesis in the eye, two major animal models are frequently applied: the oxygen-induced retinopathy (OIR) model, which is designed to investigate retinal neovascularization under hypoxic exposure, and the laser-induced CNV model, which is recognized as a model of exudative AMD. Both models are characterized by uncontrolled production of fragile and leaky capillaries in the retina. In OIR, the transition from hyperoxia to normoxia causes relative ischemia within the retina, which may be the precursor of pathological angiogenesis. On the other hand, both angiogenic and inflammatory processes play important roles in the pathophysiology of CNV. Interestingly, evidence now suggests that CGRP may be involved in the regulation of inflammatory responses13Matsuda R. Kezuka T. Nishiyama C. Usui Y. Matsunaga Y. Okunuki Y. Yamakawa N. Ogawa H. Okumura K. Goto H. Suppression of murine experimental autoimmune optic neuritis by mature dendritic cells transfected with calcitonin gene-related Peptide gene.Invest Ophthalmol Vis Sci. 2012; 53: 5475-5485Crossref PubMed Scopus (19) Google Scholar, 14Kamiyoshi A. Sakurai T. Ichikawa-Shindo Y. Fukuchi J. Kawate H. Muto S. Tagawa Y. Shindo T. Endogenous alphaCGRP protects against concanavalin A-induced hepatitis in mice.Biochem Biophys Res Commun. 2006; 343: 152-158Crossref PubMed Scopus (20) Google Scholar and, thus, could be involved in the ocular neovascularization associated with inflammation. In the present study, therefore, we investigated the pathophysiological activities of endogenous CGRP in retinal and choroidal neovascularization. For this purpose, we induced OIR or CNV in CGRP knockout (CGRP−/−) mice. CGRP−/− mice were generated in our group using a targeting DNA construct that replaced exon 5 encoding a CGRP-specific region.15Oh-hashi Y. Shindo T. Kurihara Y. Imai T. Wang Y. Morita H. Imai Y. Kayaba Y. Nishimatsu H. Suematsu Y. Hirata Y. Yazaki Y. Nagai R. Kuwaki T. Kurihara H. Elevated sympathetic nervous activity in mice deficient in alphaCGRP.Circ Res. 2001; 89: 983-990Crossref PubMed Scopus (135) Google Scholar The CGRP−/− strain was on a pure C57BL/6J background and had undergone backcross breeding to C57BL/6J using the speed congenic method. All animal handling procedures were in accordance with a protocol approved by the ethics committee of Shinshu University School of Medicine (Nagano, Japan). 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. On postnatal day 7 (P7) and P10, mice were evaluated using flat-mounted specimens of retina stained with isolectin B4 (isolectin GS-IB4 from Griffonia simplicifolia, Alexa Fluor 594 conjugate I21413; 1:200 dilution; Invitrogen, Carlsbad, CA). Superficial vascular development in the retina was quantified on P7, and deep vascular development was evaluated on P10. Vascular progression was measured by defining a straight line from the angiogenic front to the center of the retina for each retinal quadrant under low magnification. The number of vessel branches and the vessel density in the area near the developing vascular front on P7 were quantified in 400 × 400-μm2 fields. Ischemic retinopathy and pathological neovascularization were induced using the OIR model established by Smith et al.16Smith L.E. Wesolowski E. McLellan A. Kostyk S.K. D'Amato R. Sullivan R. D'Amore P.A. Oxygen-induced retinopathy in the mouse.Invest Ophthalmol Vis Sci. 1994; 35: 101-111PubMed Google Scholar Beginning on P7, mice were exposed to 75% oxygen for 5 days (P12) and then allowed to recover in room air to induce retinal neovascularization. Retinal angiogenesis was quantified, as described previously, with slight modification.17Connor 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 Briefly, on P17, the eyes were fixed in 4% paraformaldehyde for 1 hour at 4°C and then washed with phosphate-buffered saline (PBS). Retinas were then isolated and stained overnight at 4°C with isolectin B4 in PBS with 0.3% Triton X-100. After washing three times in PBS, the retinas were whole mounted onto microscope slides with the photoreceptor side down and then embedded in fluorescent mounting medium (Dako, Glostrup, Denmark). Images of whole-mounted retinas were taken at ×40 magnification using a fluorescence microscope (model BZ-9000; Keyence, Osaka, Japan). Avascular zones and neovascular tuft formation (regarded as pathological angiogenesis) were quantified using digital imaging/photoediting software (Adobe Photoshop CS5; Adobe Systems, San Jose, CA). Male mice between 9 and 12 weeks of age were used. After mice were anesthetized by i.p. injection of 2,2,2-tribromoethanol (240 mg/kg; Wako, Osaka, Japan), the pupil was dilated with 1 drop of 0.5% tropicamide and 0.5% phenylephrine (Mydrine P; Santen, Osaka, Japan). CNV was then induced, as described previously, with some modification.18Izumi-Nagai K. Nagai N. Ozawa Y. Mihara M. Ohsugi Y. Kurihara T. Koto T. Satofuka S. Inoue M. Tsubota K. Okano H. Oike Y. Ishida S. Interleukin-6 receptor-mediated activation of signal transducer and activator of transcription-3 (STAT3) promotes choroidal neovascularization.Am J Pathol. 2007; 170: 2149-2158Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar Laser injury was induced using a slit-lamp delivery system (model GYC-1000; NIDEK, Gamagori, Japan) with a coverslip serving as a contact lens. The wavelength was 532 nm, the power was 200 mW, the duration was 50 milliseconds, and the spot size was 50 μm. In each eye, four laser spots were placed around the optic disc. Only eyes that exhibited subretinal bubbles, which indicated rupture of the Bruch membrane, were used for studies. At 1, 3, or 7 days after laser injury, RPE-choroid-sclera complexes (choroidal complexes) were isolated for real-time RT-PCR. The sizes of the CNV lesions were measured in RPE-choroid-sclera flat mounts (choroidal flat mounts), as previously described.19Yu H.G. Liu X. Kiss S. Connolly E. Gragoudas E.S. Michaud N.A. Bulgakov O.V. Adamian M. DeAngelis M.M. Miller J.W. Li T. Kim I.K. Increased choroidal neovascularization following laser induction in mice lacking lysyl oxidase-like 1.Invest Ophthalmol Vis Sci. 2008; 49: 2599-2605Crossref PubMed Scopus (52) Google Scholar On day 14 after laser application, mice were anesthetized and perfused with 1 mL of PBS containing 50 mg/mL fluorescein-labeled dextran (molecular weight, 2 × 106) (fluorescein isothiocyanate–dextran; Sigma-Aldrich, St. Louis, MO) via the left ventricle. The eyes were then enucleated and fixed for 1 hour in 4% paraformaldehyde, after which the cornea and lens were removed, and the entire retina was carefully dissected from the eyecup. Four radial cuts were then made from the edge to the equator, and the eyecup with the choroidal complex was flat mounted with the sclera facing down and examined using a fluorescence microscope. The area of CNV in the choroidal flat mounts was then measured using an image analysis application (BZ-H2A; Keyence). CNV lesions were identified as fluorescent blood vessels at the choroidal/retinal interface circumscribed by regions lacking fluorescence, which were measured as the laser photocoagulation scar area. For immunohistochemistry of CLR, we used wild-type (WT) albino mice (BALB/c) to observe the staining of pigment epithelia. The eyes were enucleated and fixed in 4% paraformaldehyde for 1 hour at 4°C, washed with PBS, and then embedded in paraffin. Cross sections (10 μm thick) were deparaffinized and immunostained using polyclonal antibodies raised against CLR (dilution 1:200, rabbit; Bioss, Woburn, MA). After rinsing with PBS, the sections were incubated with Alexa Fluor 568–labeled secondary antibodies. For immunohistochemistry of CGRP, we used eyes from WT C57BL/6J mice soon (within 10 minutes) after the laser radiation. The sections were immunostained using polyclonal antibodies raised against CGRP (dilution 1:300, rabbit; Sigma-Aldrich). Rabbit IgG was used for the negative control. The sections were incubated with Alexa Fluor 488–labeled secondary antibodies. Images were obtained with a BZ-9000 microscope. In the laser-induced CNV model, fluorescein angiography (FA) was performed 14 days after the laser injury. To evaluate vascular leakage, we photographed the fluorescence at early (2 to 3 minutes) and late (5 to 6 minutes) times after i.p. injection of 0.1 mL of 2.5% fluorescein (Alcon, Tokyo, Japan), and compared the severity of the leakage between them. We graded the severity of the leakage, as described previously20Hoerster R. Muether P.S. Vierkotten S. Schroder S. Kirchhof B. Fauser S. In-vivo and ex-vivo characterization of laser-induced choroidal neovascularization variability in mice.Graefes Arch Clin Exp Ophthalmol. 2012; 250: 1579-1586Crossref PubMed Scopus (36) Google Scholar: grade 0, no fluorescence; 1, no increase of fluorescent area or intensity between the two times; 2A, increase in fluorescent intensity between the two times; and 2B, increase of both fluorescent area and intensity between the two times. To investigate the effect of CGRP deficiency on macrophage infiltration, macrophages infiltrating into CNV lesions were counted. Seven days after the laser photocoagulation, mice were sacrificed and choroidal flat mounts were made as described above. After blocking with 1% bovine serum albumin, the flat mounts were stained with rat anti-mouse F4/80 antibody (Bio-Rad, Hercules, CA) and Alexa 568–conjugated secondary antibody. The immunostained preparations were then examined using a fluorescence microscope, and F4/80-positive cells around CNV were counted. Human αCGRP (Peptide Institute, Inc., Osaka, Japan) and its antagonist, αCGRP 8-37 (Peptide Institute, Inc.), dissolved in PBS were infused into the s.c. tissues of WT mice at a rate of 50 nmol per day using osmotic pumps (Alzet; DURECT Co, Cupertino, CA). The delivery rate was 0.5 μL/hour, and the mice received αCGRP or αCGRP 8-37 for 7 to 14 days. One day after implantation of the pumps, mice were subjected to laser injury. Mice treated with PBS served as controls. CNV area was evaluated 14 days after, and macrophage infiltration was evaluated 7 days after, the laser injury. CGRP−/− and WT mice were i.p. injected with lenalidomide (Chemscene Chemicals, Monmouth Junction, NJ) at a dose of 10 mg/kg per day from 1 day before to 3 days after the laser photocoagulation. The dose and administration method were determined according to a previous study.21Rozewski D.M. Herman S.E. Towns 2nd, W.H. Mahoney E. Stefanovski M.R. Shin J.D. Yang X. Gao Y. Li X. Jarjoura D. Byrd J.C. Johnson A.J. Phelps M.A. Pharmacokinetics and tissue disposition of lenalidomide in mice.AAPS J. 2012; 14: 872-882Crossref PubMed Scopus (22) Google Scholar Mice treated with PBS served as controls. Seven days after the laser injury, CNV area and macrophage infiltration were evaluated. ARPE-19 human RPE cells were purchased from the ATCC (Manassas, VA). For preparation of isolated macrophages, mice were i.p. injected with 2 mL of 3% thioglycolate medium (DIFCO; Biobrás, Montes Claros, Brazil). Three days later, macrophages were harvested by peritoneal lavage using cold PBS and placed in culture. In some cases, lipopolysaccharide (Sigma-Aldrich) and/or human αCGRP (Peptide Institute, Inc.) were used to stimulate and/or suppress cytokine production by the cultured macrophages. Total RNA was extracted 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 reverse transcribed 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, Nagoya, Japan). Values were normalized to mouse glyceraldehyde-3-phosphate dehydrogenase (Pre-Developed TaqMan 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 and RT-PCRQuantitative real-time RT-PCR mCGRP forward5′-GGAGCAGGAGGAAGAGCAG-3′ mCGRP reverse5′-TGCCAGCCGATGGGTCACA-3′ mCLR forward5′-AGGCGTTTACCTGCACACACT-3′ mCLR reverse5′-CAGGAAGCAGAGGAAACCCC-3′ mCLR probe5′-ATCGTGGTGGCTGTGTTTGCGGAG-3′ mRAMP1 forward5′-GCACTGGTGGTCTGGAGGA-3′ mRAMP1 reverse5′-CCCTCATCACCTGGGATACCT-3′ mRAMP1 probe5′-CAAGCGCACAGAGGGCATCGTG-3′ mTNF-α forward5′-ACGGCATGGATCTCAAAGAC-3′ mTNF-α reverse5′-AGATAGCAAATCGGCTGACG-3′RT-PCR hTNFR1 forward5′-GGTGCTAACCCCTCGATGTA-3′ hTNFR1 reverse5′-GCTTGCTATGTGCTTGTCCA-3′hTNFR, human tumor necrosis factor receptor; mCGRP, mouse calcitonin gene–related peptide; mCLR, mouse calcitonin receptor-like receptor; mRAMP, mouse receptor activity–modifying protein; mTNF, mouse tumor necrosis factor. Open table in a new tab hTNFR, human tumor necrosis factor receptor; mCGRP, mouse calcitonin gene–related peptide; mCLR, mouse calcitonin receptor-like receptor; mRAMP, mouse receptor activity–modifying protein; mTNF, mouse tumor necrosis factor. Tumor necrosis factor (TNF)-α production by macrophages isolated from mice was quantified using specific enzyme-linked immunosorbent assay kits (R&D Systems, Minneapolis, MN), according to the manufacturer's instructions. To evaluate the suppressive effect of CGRP on cytokine production by macrophages stimulated for 4 hours with 100 ng/mL lipopolysaccharide, 10−7 to 10−10 mol/L CGRP was added to the cells 1 hour before the assay. ARPE-90 human RPE cells were grown until confluent and further cultured for 24 hours with or without TNF-α (0.2 to 20 ng/mL). To visualize the tight junctions between RPE cells, we immunostained the cells using anti–zonula occludens protein 1 antibody (BD Biosciences–Pharmingen, Franklin Lakes, NJ). Values are expressed as means ± SEM. Student's t-test and one-way analysis of variance, followed by Fisher's partial least squares difference, were used to evaluate the significance of differences. P < 0.05 was considered significant. We initially compared the developing retinal vessels on P7 and P10 between WT and CGRP−/− neonates. On P7, the retinal vessels were elongating from the center of the retina toward the periphery in the superficial layer. Examination of lectin-stained whole retinas (Figure 1A) revealed that vascular progression did not differ between CGRP−/− and WT mice (Figure 1B). On P10, the vascular progression had reached the periphery of the retina; at that point, the vessels elongated into deeper layers. We detected no difference in the deep vessel progression between CGRP−/− and WT mice (Figure 1, C and D). We also quantified vessel density (Figure 1, E and F) and vessel branching on P7 (Figure 1, E and G) and found no significant differences between the two genotypes. These results indicate that retinal vessel development under physiological conditions is unaffected by CGRP deficiency. We next evaluated whether ischemia within the eyes evokes changes in retinal angiogenesis in CGRP−/− mice, because CGRP deficiency reportedly retarded angiogenesis in an ischemia model.9Mishima T. Ito Y. Hosono K. Tamura Y. Uchida Y. Hirata M. Suzsuki T. Amano H. Kato S. Kurihara Y. Kurihara H. Hayashi I. Watanabe M. Majima M. Calcitonin gene-related peptide facilitates revascularization during hindlimb ischemia in mice.Am J Physiol Heart Circ Physiol. 2011; 300: H431-H439Crossref PubMed Scopus (57) Google Scholar In the OIR model, mice were exposed to 75% oxygen for 5 days, from P7 to P12. During this period (hyperoxic phase), oxygen-induced loss of retinal vessels (vaso-obliteration) occurs. Then, after the mice are returned to normoxic conditions (room air) for 5 days, from P12 to P17 (hypoxic phase), the hyperoxia-induced vessel loss leads to pathological angiogenesis. Both WT and CGRP−/− mice exposed to hyperoxia exhibited avascular zones in the central region of the retina on P12 (Figure 2A), and we detected no significant differences in the size of the avascular zones between the two groups (Figure 2B). When we analyzed the retinas at the end of the hypoxic phase (P17), the avascular zones were similarly diminished because of progressive neovascularization in both groups (Figure 2, C and D). On P17, we detected neovascular tuft formation. This aneurysmal formation of retinal arteries is typical of pathological angiogenesis and did not differ between WT and CGRP−/− mice (Figure 2, E and F). These results are strikingly different from those obtained with heterozygous AM knockout (AM+/−; official gene name Adm) mice, which showed reduction of the avascular zone and reduced pathological angiogenesis.22Iesato Y. Toriyama Y. Sakurai T. Kamiyoshi A. Ichikawa-Shindo Y. Kawate H. Yoshizawa T. Koyama T. Uetake R. Yang L. Yamauchi A. Tanaka M. Igarashi K. Murata T. Shindo T. Adrenomedullin-RAMP2 system is crucially involved in retinal angiogenesis.Am J Pathol. 2013; 182: 2380-2390Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar Collectively, the similarity between the CGRP−/− and WT (CGRP+/+) phenotypes and the difference between the CGRP−/− and AM+/− phenotypes demonstrate that CGRP has little, if any, involvement in hypoxia-induced retinal neovascularization. We next analyzed the distribution of CGRP and its receptor system within the eyes. Real-time RT-PCR analysis of the retina and choroid from WT mice (Figure 3) revealed levels of CGRP expression to be equivalent in the two layers. On the other hand, the expression levels of CLR and RAMP1 (the CGRP receptor system) were, respectively, approximately 19- and 8-fold higher in the choroid than the retina. This prompted us to speculate that CGRP is much more involved in the physiology of the choroid than the retina. Because it is known to be a sensory neurotransmitter, we hypothesized that CGRP would be most strongly involved in angiogenesis within regions of the eye where sensory nerve terminals are present, such as the choroid. To test that idea, we applied a laser-induced CNV model of exudative AMD to WT and CGRP−/− mice. Examination of choroidal flat mounts revealed that on day 14 after the laser injury, CGRP−/− mice showed significantly larger areas of CNV (23,458 ± 1679 μm2) than WT mice (17,232 ± 1122 μm2) (Figure 4, A and B). Although the laser photocoagulation scar area did not differ between the two genotypes (Figure 4C), the CNV area/laser scar area ratio was significantly higher in CGRP−/− than WT mice (Figure 4D). This suggests that, with the same degree of laser injury, there was greater neovascularization in CGRP−/− than WT mice. To confirm the involvement of CGRP in laser-induced CNV, we analyzed the localization of CGRP and its receptor in sections of retina. Immunostaining was able to detect expression of the CGRP receptor CLR at the pigment epithelium (Figure 5A). The expression of CGRP was not clearly observed in the control mice; however, soon (within 10 minutes) after the laser radiation, we could detect a high level of immunostaining of CGRP at the laser-irradiated area (Figure 5B). We think that tissue injury after the laser radiation may enhance the secretion of CGRP from nerve endings at the choroid. To determine the severity of the CNV lesions, we analyzed the leakage from the CNV by performing FA on day 14 after the laser injury. Figure 6 shows typical fluorescence images obtained at earlier (2 to 3 minutes) (Figure 6A) and later (5 to 6 minutes) (Figure 6B) times after fluorescein injection. By using FA, we detected angiographic leakage from CNV lesions in both genotypes, but CGRP−/− mice developed larger and leakier CNV lesions than WT mice (Figure 6C). In particular, the numbers of grade 2B lesions were approximately twofold higher in CGRP−/− than WT mice (Figure 6D). These results demonstrate that, in CGRP−/− mice, not only is laser-induced CNV formation enhanced, the lesions are much leakier and fragile than in WT mice. Both angiogenesis and inflammation contribute to the pathophysiology of CNV. In earlier studies, we and others14Kamiyoshi A. Sakurai T. Ichikawa-Shindo Y. Fukuchi J. Kawate H. Muto S. Tagawa Y. Shindo T. Endogenous alphaCGRP protects against concanavalin A-induced hepatitis in mice.Biochem Biophys Res Commun. 2006; 343: 152-158Crossref PubMed Scopus (20) Google Scholar, 23Kamiyoshi A. Sakurai T. Ichikawa-Shindo Y. Iinuma N. Kawate H. Yoshizawa T. Koyama T. Muto S. Shindo T. Endogenous alpha-calcitonin gene-related peptide mitigates liver fibrosis in chronic hepatitis induced by repeated administration of concanavalin A.Liver Int. 2009; 29: 642-649Crossref PubMed Scopus (17) Google Scholar, 24Holzmann B. Antiinflammatory activities of CGRP modulating innate immune responses in health and disease.Curr Protein Pept Sci. 2013;
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