Girdin and Its Phosphorylation Dynamically Regulate Neonatal Vascular Development and Pathological Neovascularization in the Retina
2012; Elsevier BV; Volume: 182; Issue: 2 Linguagem: Inglês
10.1016/j.ajpath.2012.10.012
ISSN1525-2191
AutoresTakanori Ito, Keiichi Komeima, Tetsuhiro Yasuma, Atsushi Enomoto, Naoya Asai, Masato Asai, Sayoko Iwase, Masahide Takahashi, Hiroko Terasaki,
Tópico(s)Retinal Development and Disorders
ResumoVascular endothelial growth factor (VEGF) is recognized as a principal mediator of vessel growth. VEGF regulates various endothelial cellular processes, including cell migration, proliferation, and survival, through the serine threonine protein kinase Akt. The Akt substrate girdin, an actin-binding protein, is known to regulate VEGF-mediated postnatal angiogenesis. However, the role of girdin and its phosphorylation in neonatal retinal vascular development and ocular pathological neovascularization in vivo has not been elucidated. In the present study, therefore, we investigated these processes using Girdin+/− mice lacking one copy of the girdin gene and girdin S1416A knockin (Girdin-KISA/SA) mice in which the phosphorylation site of girdin is completely disrupted. We used three mouse models of pathological ocular neovascularization: oxygen-induced retinopathy (a mouse model of ischemic retinopathies), laser-induced choroidal neovascularization, and a human VEGF transgenic mouse that overexpresses human VEGF specifically in photoreceptor cells and generates pathological neovascularization in the retina. Neonatal vascular development was delayed and pathological neovascularization was decreased in both Girdin+/− mice and Girdin-KISA/SA mice. These results demonstrate that girdin and its phosphorylation play an important role in neonatal vascular development and in pathological neovascularization in the retina. Vascular endothelial growth factor (VEGF) is recognized as a principal mediator of vessel growth. VEGF regulates various endothelial cellular processes, including cell migration, proliferation, and survival, through the serine threonine protein kinase Akt. The Akt substrate girdin, an actin-binding protein, is known to regulate VEGF-mediated postnatal angiogenesis. However, the role of girdin and its phosphorylation in neonatal retinal vascular development and ocular pathological neovascularization in vivo has not been elucidated. In the present study, therefore, we investigated these processes using Girdin+/− mice lacking one copy of the girdin gene and girdin S1416A knockin (Girdin-KISA/SA) mice in which the phosphorylation site of girdin is completely disrupted. We used three mouse models of pathological ocular neovascularization: oxygen-induced retinopathy (a mouse model of ischemic retinopathies), laser-induced choroidal neovascularization, and a human VEGF transgenic mouse that overexpresses human VEGF specifically in photoreceptor cells and generates pathological neovascularization in the retina. Neonatal vascular development was delayed and pathological neovascularization was decreased in both Girdin+/− mice and Girdin-KISA/SA mice. These results demonstrate that girdin and its phosphorylation play an important role in neonatal vascular development and in pathological neovascularization in the retina. CME Accreditation Statement: This activity (“ASIP 2013 AJP CME Program in Pathogenesis”) has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians.The ASCP designates this journal-based CME activity (“ASIP 2013 AJP CME Program in Pathogenesis”) for a maximum of 48 AMA PRA Category 1 Credit(s)™. Physicians should only claim credit commensurate with the extent of their participation in the activity.CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. CME Accreditation Statement: This activity (“ASIP 2013 AJP CME Program in Pathogenesis”) has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians. The ASCP designates this journal-based CME activity (“ASIP 2013 AJP CME Program in Pathogenesis”) for a maximum of 48 AMA PRA Category 1 Credit(s)™. Physicians should only claim credit commensurate with the extent of their participation in the activity. CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. Pathological ocular neovascularization is a leading causes of blindness in humans and is found in various eye diseases, including diabetic retinopathy, retinal vein occlusion, retinopathy of prematurity, and age-related macular degeneration (AMD).1Bradley J. Ju M. Robinson G.S. Combination therapy for the treatment of ocular neovascularization.Angiogenesis. 2007; 10: 141-148Crossref PubMed Scopus (90) Google Scholar, 2Chen J. Smith L.E. Retinopathy of prematurity.Angiogenesis. 2007; 10: 133-140Crossref PubMed Scopus (480) Google Scholar, 3Friedlander M. Dorrell M.I. Ritter M.R. Marchetti V. Moreno S.K. El-Kalay M. Bird A.C. Banin E. Aguilar E. Progenitor cells and retinal angiogenesis.Angiogenesis. 2007; 10: 89-101Crossref PubMed Scopus (52) Google Scholar Over the past decade, our understanding of the molecular mechanisms of angiogenesis has increased at an explosive rate and has led to the approval of antiangiogenic drugs for ocular diseases.4Gragoudas E.S. Adamis A.P. Cunningham Jr., E.T. Feinsod M. Guyer D.R. VEGF Inhibition Study in Ocular Neovascularization Clinical Trial GroupPegaptanib for neovascular age-related macular degeneration.N Engl J Med. 2004; 351: 2805-2816Crossref PubMed Scopus (2103) Google Scholar, 5Ferrara N. Damico L. Shams N. Lowman H. Kim R. Development of ranibizumab, an anti-vascular endothelial growth factor antigen binding fragment, as therapy for neovascular age-related macular degeneration.Retina. 2006; 26: 859-870Crossref PubMed Scopus (691) Google Scholar Although many patients have benefited from blockers of the angiogenic protein vascular endothelial growth factor (VEGF), limited efficacy and disease recurrence remain a problem.6Carmeliet P. Jain R.K. Molecular mechanisms and clinical applications of angiogenesis.Nature. 2011; 473: 298-307Crossref PubMed Scopus (3721) Google Scholar Therefore, to improve therapeutic outcomes for ocular neovascular diseases, anti-VEGF agents should be combined with agents that target another molecule. VEGF signaling affects a number of vital processes during development in adult physiology and pathology, including cancer progression.7Olsson A.K. Dimberg A. Kreuger J. Claesson-Welsh L. VEGF receptor signalling–in control of vascular function.Nat Rev Mol Cell Biol. 2006; 7: 359-371Crossref PubMed Scopus (2440) Google Scholar, 8Bergers G. Hanahan D. Modes of resistance to anti-angiogenic therapy.Nat Rev Cancer. 2008; 8: 592-603Crossref PubMed Scopus (2340) Google Scholar VEGF denotes a family of five related mammalian growth factors. One family member, VEGF-A, is alternatively spliced to generate VEGF-A121, VEGF-A145, VEGF-A165, and VEGF-A189 in humans.9Koch S. Tugues S. Li X. Gualandi L. Claesson-Welsh L. Signal transduction by vascular endothelial growth factor receptors.Biochem J. 2011; 437: 169-183Crossref PubMed Scopus (631) Google Scholar VEGF-A is notably regulated by hypoxia-inducible factor (HIF), which leads to increased expression during tissue growth both in health (embryonic development, wound healing) and disease (cancer, ischemic retinopathy).10Fong G.H. Regulation of angiogenesis by oxygen sensing mechanisms.J Mol Med (Berl). 2009; 87: 549-560Crossref PubMed Scopus (104) Google Scholar In humans, three structurally related VEGF receptor tyrosine kinases (VEGFRs) have been identified: VEGFR-1, VEGFR-2, and VEGFR-3. VEGFR-2 is known to transduce the full range of VEGF responses in endothelial cells, mainly through the PI3K/Akt and ERK pathways.11Zachary I. VEGF signalling: integration and multi-tasking in endothelial cell biology.Biochem Soc Trans. 2003; 31: 1171-1177Crossref PubMed Google Scholar Akt, or protein kinase B, is a multifunctional serine-threonine protein kinase implicated in a diverse range of endothelial cellular functions, including differentiation, proliferation, migration, and vascular tube formation.12Ackah E. Yu J. Zoellner S. Iwakiri Y. Skurk C. Shibata R. Ouchi N. Easton R.M. Galasso G. Birnbaum M.J. Walsh K. Sessa W.C. Akt1/protein kinase Balpha is critical for ischemic and VEGF-mediated angiogenesis.J Clin Invest. 2005; 115: 2119-2127Crossref PubMed Scopus (328) Google Scholar However, the mechanism underlying how Akt regulates such cellular processes has long been a mystery. In 2005, Enomoto et al13Enomoto A. Murakami H. Asai N. Morone N. Watanabe T. Kawai K. Murakumo Y. Usukura J. Kaibuchi K. Takahashi M. Akt/PKB regulates actin organization and cell motility via Girdin/APE.Dev Cell. 2005; 9: 389-402Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar identified the Akt-binding protein girdin and demonstrated that girdin is essential for the integrity of the actin cytoskeleton and cell migration and directly links Akt to cell motility. Girdin is a large protein of 1871 amino acids, with a predicted relative molecular mass of 220 kDa, which binds directly to actin filaments through its carboxyl terminal domain. The Akt binding site maps to the carboxyl terminal domain of girdin, and Akt phosphorylates girdin at Ser1416 to regulate its subcellular localization and cell migration in fibroblasts. In 2008, Kitamura et al14Kitamura T. Asai N. Enomoto A. Maeda K. Kato T. Ishida M. Jiang P. Watanabe T. Usukura J. Kondo T. Costantini F. Murohara T. Takahashi M. Regulation of VEGF-mediated angiogenesis by the Akt/PKB substrate Girdin.Nat Cell Biol. 2008; 10: 329-337Crossref PubMed Scopus (191) Google Scholar revealed that the Akt/girdin signaling pathway is essential to VEGF-mediated neonatal angiogenesis. They found that exogenously delivered adenovirus harboring girdin small interfering RNA (siRNA) in Matrigel (BD Biosciences, San Jose, CA) embedded in mice markedly inhibited VEGF-mediated angiogenesis. They also showed that targeted disruption of the girdin gene (Ccdc88a) in mice impaired vessel remodeling in the neonatal mouse retina and angiogenesis from aortic rings, whereas girdin was dispensable for embryonic vasculogenesis. Girdin is also considered to be a key regulator for cancer progression and pathological neovascularization.15Weng L. Enomoto A. Ishida-Takagishi M. Asai N. Takahashi M. Girding for migratory cues: roles of the Akt substrate Girdin in cancer progression and angiogenesis.Cancer Sci. 2010; 101: 836-842Crossref PubMed Scopus (64) Google Scholar Moreover, girdin is also expressed in the central nervous system and is reported to be associated with postnatal neurogenesis in the dentate gyrus and neuroblast chain migration along the rostral migratory stream.16Enomoto A. Asai N. Namba T. Wang Y. Kato T. Tanaka M. Tatsumi H. Taya S. Tsuboi D. Kuroda K. Kaneko N. Sawamoto K. Miyamoto R. Jijiwa M. Murakumo Y. Sokabe M. Seki T. Kaibuchi K. Takahashi M. Roles of disrupted-in-schizophrenia 1-interacting protein girdin in postnatal development of the dentate gyrus.Neuron. 2009; 63: 774-787Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 17Wang Y. Kaneko N. Asai N. Enomoto A. Isotani-Sakakibara M. Kato T. Asai M. Murakumo Y. Ota H. Hikita T. Namba T. Kuroda K. Kaibuchi K. Ming G.L. Song H. Sawamoto K. Takahashi M. Girdin is an intrinsic regulator of neuroblast chain migration in the rostral migratory stream of the postnatal brain.J Neurosci. 2011; 31: 8109-8122Crossref PubMed Scopus (59) Google Scholar In the present study, we focused on investigating the contribution of the actin-binding protein girdin and its phosphorylation to neonatal vascular development and pathological neovascularization in AMD and in ischemic retinal diseases such as diabetic retinopathy, retinal vein occlusion, and retinopathy of prematurity. Human retinal endothelial cells (HRECs) from Cell Systems (Kirkland, WA) and human umbilical vein endothelial cells (HUVECs) from ATCC (Manassas, VA) were cultured with EGM-2 medium (Lonza, Tokyo, Japan) in an incubator with 5% CO2-enriched air. Studies were conducted with cells from passage 4 to 7. Quantitative real-time RT- PCR (RT-qPCR) was performed to measure levels of human girdin mRNA. RNA was isolated from retinas using TRIzol reagent (Invitrogen; Life Technologies, Carlsbad, CA). After quantification of RNA concentration, cDNA was synthesized with a SuperScript first-strand kit (Invitrogen; Life Technologies). RT-qPCR was performed and analyzed using a QuantiTect SYBR Green RT-PCR kit (Qiagen, Valencia, CA) and a LightCycler 2.0 PCR system (Roche Applied Science, Indianapolis, IN). A total volume of 20 μL was loaded into LightCycler capillaries, which contained 2 μL of cDNA sample and 0.5 μmol/L of primers specific for human girdin (forward: 5′-TGGAGCAGTTCATGACCGC-3′; reverse: 5′-GAGCATGACCTGGTTCAAGAA-3′) or human VEGFR-2 (forward: 5′-ATCCCTGTGGATCTGAAACG3′; reverse: 5′-CCAAGAACTCCATGCCCTTA-3′). For normalization, human GAPDH was amplified (forward: 5′-GAAGGTGAAGGTCGGAGT-3′; reverse: 5′-GAAGATGGTGATGGGATTTC-3′). Titrations were performed to ensure that PCR reactions were performed in the linear range of amplification. The PCR reaction quality and specificity were verified by melting-curve dissociation analysis. For quantification, a standard curve was generated from a cDNA template for each gene. Relative transcript levels of each gene were calculated using the second-derivative maximum values from the linear regression of cycle number versus log concentration of the amplified gene. Retinas or cultured cells were lysed with radioimmunoprecipitation assay buffer (Pierce; Thermo Fisher Scientific, Rockford, IL). Western blots were performed with anti-girdin (R&D Systems, Minneapolis, MN) and anti–β-actin (Cell Signaling Technology, Danvers, MA) antibodies. HRECs were fixed in 3.7% formalin and stained with primary antibodies [anti-girdin, 1:100 (IBL Immuno-Biological Laboratories, Gunma, Japan) and anti–β-actin, 1:300 (Sigma-Aldrich, St. Louis, MO)], followed by secondary antibodies [Alexa Fluor 488 goat anti-rabbit IgG and Alexa Fluor 594 goat anti-mouse IgG (Invitrogen; Life Technologies)]. Fluorescence was examined under a C1si confocal laser-scanning microscope (Nikon, Tokyo, Japan). The eyes of mice were enucleated at postnatal day 10 (P10) and fixed in 4% paraformaldehyde overnight. Isolated retinas were cut into 50-μm sections on a microslicer (VT1200S; Leica Microsystems, Wetzlar, Germany) and immunostained with anti-girdin (1:100) (Abcam, Cambridge, UK) and anti-CD31 (1:100) (Dianova, Hamburg, Germany) antibodies. Fluorescence was examined under a fluorescence microscope (Observer Z1; Zeiss, Göttingen, Germany). The staining procedure was based on the protocol of the Nagy laboratory.18Lobe C.G. Koop K.E. Kreppner W. Lomeli H. Gertsenstein M. Nagy A. Z/AP, a double reporter for Cre-mediated recombination.Dev Biol. 1999; 208: 281-292Crossref PubMed Scopus (450) Google Scholar Briefly, mice were perfused with PBS containing 2 mmol/L MgCl2, 0.2% glutaraldehyde, and 30% sucrose in PBS, in series. Dissected tissues were soaked in 30% sucrose in PBS overnight at 4°C and in optimal cutting temperature compound (OCT; Sakura Finetek, Tokyo, Japan) for 1 hour at room temperature before freezing at −80°C. Frozen tissues were cryosectioned at 10 μm and thaw-mounted on Matsunami adhesive silane-coated glass slides (Matsunami Glass, Osaka, Japan). Sections on slides were fixed in PBS containing 2 mmol/L MgCl2 and 0.2% glutaraldehyde for 10 minutes, rinsed twice in PBS containing 2 mmol/L MgCl2, and soaked in LacZ wash buffer (PBS containing 2 mmol/L MgCl2, 0.01% sodium deoxycholate, and 0.02% Igepal CA-630 surfactant) twice. Slides were soaked in LacZ staining buffer (Wako Pure Chemical Industries, Osaka, Japan) for 48 hours at room temperature. Stained slides were rinsed in PBS, postfixed in 4% paraformaldehyde for 10 minutes at 4°C, counterstained with eosin, and dehydrated with increasing concentrations of ethanol and 100% xylene. Glass coverslips were mounted with xylene-based medium (Entellan Neu; Merck Millipore, Darmstadt, Germany). siRNA-mediated depletion (knockdown) of girdin was performed. The 21-nucleotide synthetic duplexes were prepared by Qiagen. Cells were transfected with girdin siRNA or a 21-nucleotide control RNA (Qiagen) using Lipofectamine 2000 reagent (Invitrogen; Life Technologies) according to the manufacturer’s protocol. For shRNA-mediated knockdown of girdin, a set of single-stranded oligonucleotides encoding the girdin target shRNA and its complement was synthesized: 5′-GAAGGAGAGGCAACTGGAT-3′ (nt 4166-4184). The oligonucleotide was directed against a sequence selected and provided by Dragon Genomics (Takara Bio, Otsu, Japan). The oligonucleotide pair was annealed and inserted into pcPURU6β (Dragon Genomics) or the pNAMA retroviral shRNA expression vector. To produce retroviral supernatants, GP-293 packaging cells were transfected with 24 μg of control or girdin shRNA-containing pNAMA vectors, 2 μg of pVSVG, and 60 μL of Lipofectamine 2000 reagent (Invitrogen; Life Technologies) in 100-mm cell culture dishes containing Opti-MEM medium (Invitrogen; Life Technologies) without fetal bovine serum or antibiotics. The medium was replaced 24 hours later, and virus-containing supernatants were harvested 48 hours after transfection. To infect cells with retrovirus plasmids, 1 × 105 cells were mixed with 3 mL of virus-containing supernatant and were seeded in 100-mm cell culture dishes. The supernatant was replaced after 24 hours of incubation. The cell lysates were analyzed by Western blotting using anti-girdin and anti–β-actin antibodies. Clones in which the expression of girdin was effectively suppressed were selected and used for further study. HRECs were starved in serum-free medium, and then 1 × 104 cells were seeded with or without 1 μg/mL VEGF, 50 μmol/L LY294002 (PI3K inhibitor; Merck Millipore), or 1.0 mmol/L U0126 (ERK inhibitor; Merck Millipore). In the girdin knockdown study, HRECs were cotransfected with either control or girdin siRNA. Transfected cells were starved in serum-free medium, and then 1.25 × 104 cells were seeded with 100 ng/mL VEGF. After 24 hours of incubation, 3-(4,5-dimethylthiazol-2-yl)-2,5′-diphenyltetrazolium bromide (MTT) labeling solution (Cell Proliferation Kit I; Roche Diagnostics, Mannheim, Germany) was added and incubated for 4 hours. Then 10% SDS solution in 0.1 mol/L HCl was added, and the absorbance was measured at 570 nm. Cell migration assays were performed using the Cultrex 96-well cell migration assay kit (Trevigen, Gaithersburg, MD) according to the manufacturer’s instructions. In brief, 5 × 104 HRECs were seeded onto polyethylene terephthalate membranes with 8-μm pores in the top chambers. In the top and bottom chamber, LY294002 (50 or 200 μmol/L) or U0126 (1.0 or 4.0 mmol/L) were added, and in the bottom chamber, 5 ng/mL VEGF was added as a chemoattractant. In the girdin knockdown study, HRECs were cotransfected with either control or girdin siRNA, and 1.25 × 104 transfected HRECs were seeded onto the top chamber. In the bottom chamber, 5 ng/mL VEGF was added as a chemoattractant. After 4 hours of incubation, migrated cells were labeled with Calcein AM (Wako Pure Chemical Industries) solution, and plates were read at 485 nm (excitation) and 520 nm (emission). Extracellular matrix gels were prepared with a Chemicon in vitro angiogenesis assay kit (EMD Millipore, Billerica, MA). Gels were solidified over a 96-well microplate, and 30 minutes later, 1 × 104 HRECs were added with 1 ng/mL VEGF and 50 μmol/L LY294002 or 1.0 mmol/L U0126. In the girdin knockdown study, HRECs were cotransfected with either control or girdin siRNA. Using this kit, 1.25 × 104 HRECs were added onto the surface of gels. After 4 hours of incubation, tubes were labeled by Calcein-AM solution and photographed. Girdin-deficient Girdin+/− and Girdin−/− mice were generated as described previously.14Kitamura T. Asai N. Enomoto A. Maeda K. Kato T. Ishida M. Jiang P. Watanabe T. Usukura J. Kondo T. Costantini F. Murohara T. Takahashi M. Regulation of VEGF-mediated angiogenesis by the Akt/PKB substrate Girdin.Nat Cell Biol. 2008; 10: 329-337Crossref PubMed Scopus (191) Google Scholar Generation of Girdin-KISA/SA mice has also been described previously in detail.19Miyake H. Maeda K. Asai N. Shibata R. Ichimiya H. Isotani-Sakakibara M. Yamamura Y. Kato K. Enomoto A. Takahashi M. Murohara T. The actin-binding protein Girdin and its Akt-mediated phosphorylation regulate neointima formation after vascular injury.Circ Res. 2011; 108: 1170-1179Crossref PubMed Scopus (53) Google Scholar The Akt phosphorylation site of girdin (Ser1416) was completely disrupted in Girdin-KISA/SA mice. These mice had normal weight and expressed the same level of girdin protein as wild-type (WT) mice. WT C57BL/6 mice were purchased from the Jackson Laboratory (Bar Harbor, ME). V-6+ transgenic mice expressing human VEGF through rhodopsin promoter in photoreceptors (hVEGF Tg mice) were a generous gift from Peter A. Campochiaro (Johns Hopkins University). These mice demonstrate onset of hVEGF expression at P7 and, starting at P10, develop sprouts of neovascularization from the deep capillary bed of the retina that grow through the photoreceptor layer and form an extensive network of new vessels in the subretinal space.20Okamoto N. Tobe T. Hackett S.F. Ozaki H. Vinores M.A. LaRochelle W. Zack D.J. Campochiaro P.A. Transgenic mice with increased expression of vascular endothelial growth factor in the retina: a new model of intraretinal and subretinal neovascularization.Am J Pathol. 1997; 151: 281-291PubMed Google Scholar All experiments were performed in compliance with Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Visual Research and the Nagoya University School of Medicine guidelines for the use of animals. The eyes of mice were enucleated at P5, P7, or P10. The enucleated eyes were fixed in 4% paraformaldehyde for 2 hours. For flat-mount analyses, four radial incisions were made to relax the eyecups, and the vitreous was carefully removed. The samples were permeabilized for 30 minutes, blocked with 10% normal goat serum in PBS at room temperature, and incubated with fluorescein isothiocyanate-conjugated lectin from Bandeiraea simplicifolia (Sigma-Aldrich) for 12 hours. Retinal flat mounts were examined under a C1si scanning laser confocal microscope (Nikon), and vascular area was measured. Oxygen-induced retinopathy (OIR) was induced by exposing mice to 80% oxygen using an oxygen box with a ProOx 110 controller (BioSpherix, Lacona, NY) from P7 to P12, which resulted in extensive obstruction of the retinal vessels. Returning the mice to room air at P12 triggered outgrowth of pathological retinal neovascularization. To visualize retinal vascular regression, the eyes of mice were enucleated at P12 in the OIR model. Retinal pathological neovascularization and revascularization after hypoxic injury was compared at P17. Enucleated eyes were fixed and treated as described above. Retinal flat mounts were examined under a C1si scanning laser confocal microscope (Nikon), and neovascular area or avascular area was measured as described previously.21Guaiquil V. Swendeman S. Yoshida T. Chavala S. Campochiaro P.A. Blobel C.P. ADAM9 is involved in pathological retinal neovascularization.Mol Cell Biol. 2009; 29: 2694-2703Crossref PubMed Scopus (77) Google Scholar To induce choroidal neovascularization (CNV), 5-week-old female mice were anesthetized with 50 mg/kg Avertin formulation (2,2,2-tribromoethanol-amylene hydrate), and the pupils were dilated with eye drops containing 0.5% tropicamide and 0.5% phenylephrine hydrochloride (Mydrin-P; Santen Pharmaceuticals, Osaka, Japan). Three burns of 532-nm diode laser photocoagulation (75 μm spot size, 150 mW intensity, and 100 ms duration) were delivered to the retina of each eye using a photocoagulator slit-lamp delivery system and a handheld coverslip as a contact lens. Burns were performed in the three directions of the posterior pole of the retina. Production of a bubble at the time of laser indicates rupture of Bruch’s membrane, which is an important factor in obtaining CNV, and so only burns in which a bubble was produced were included in the present study. For visualization of laser-induced CNV, mice were sacrificed after 1 week, and the complex of vitreous and retina was carefully peeled off the remaining ocular structures. The eyecups were stained with lectin as described above. Retinal and choroidal flat mounts were examined under a C1si scanning laser confocal microscope (Nikon), and CNV area was measured as described previously.21Guaiquil V. Swendeman S. Yoshida T. Chavala S. Campochiaro P.A. Blobel C.P. ADAM9 is involved in pathological retinal neovascularization.Mol Cell Biol. 2009; 29: 2694-2703Crossref PubMed Scopus (77) Google Scholar hVEGF Tg mice were crossed with Girdin-KISA/SA mice to obtain hVEGF+/−Girdin-KISA/SA mice. The hVEGF+/−Girdin-KISA/SA mice were compared with hVEGF+/− mice as a control. These mice were sacrificed at P21, to compare intraretinal neovascularization. The eyes were enucleated and fixed, and the vitreous was removed. The eyecups were stained with lectin as described above. The complex of sclera, choroid, and retina was mounted on glass slides and was pressed gently, to visualize intraretinal neovascularization clearly. Intraretinal neovascularization was examined under a fluorescence microscope (BIOREVO BZ-9000; Keyence, Osaka, Japan), and the number of neovascular bulges in each retinal flat mount was counted. We first examined girdin mRNA and protein expression in HRECs. We performed RT-qPCR and Western blot analysis of both HRECs and HUVECs. The relative expression level of girdin mRNA in HRECs was approximately 73% of that in HUVECs, whereas VEGFR-2 mRNA was expressed almost equally in HRECs and HUVECs (Figure 1, A and B). Western blot analysis revealed that girdin protein expression in HRECs was also approximately 75% of that in HUVECs (Figure 1, C and D). In HUVECs, girdin has been reported to localize in actin stress fibers and, to a lesser extent, in peripheral cortical actin filaments.14Kitamura T. Asai N. Enomoto A. Maeda K. Kato T. Ishida M. Jiang P. Watanabe T. Usukura J. Kondo T. Costantini F. Murohara T. Takahashi M. Regulation of VEGF-mediated angiogenesis by the Akt/PKB substrate Girdin.Nat Cell Biol. 2008; 10: 329-337Crossref PubMed Scopus (191) Google Scholar We performed immunostaining for HRECs using anti-girdin antibody and found that girdin was partially localized to peripheral cortical actin filaments in the same manner as HUVECs (Figure 2A).Figure 2Localization of girdin in HRECs and mouse retinas. A: HRECs were stained with anti–β-actin monoclonal and anti-girdin polyclonal antibodies. Girdin was expressed in peripheral cortical actin filaments (arrows). B: Retinal sections from a WT and a Girdin−/− P12 mouse were stained for LacZ. Sections were counterstained with eosin (red). Blue-stained nuclei indicate expression of girdin-LacZ fusion protein containing nuclear localization signal. In Girdin−/− retina, girdin-LacZ-positive cells were present in the ganglion cell layer (GCL), the inner nuclear layer (INL), and a retinal blood vessel (arrow) in the inner plexiform layer (IPL). In WT retina, however, no blue-stained cells were visible in WT retina, including a retinal blood vessel (arrow). C: Retinal sections from a WT P10 mouse were double-stained with anti-girdin and anti-CD31 antibodies. Girdin expression was observed in retinal vessel plexuses and in sprouting tip cells (arrowheads). OPL, outer plexiform layer; ONL, outer nuclear layer. Original magnification, ×40 (A); ×10 (B and C).View Large Image Figure ViewerDownload Hi-res image Download (PPT) We also investigated localization of girdin in the mouse retina using girdin-deficient (Girdin−/−) mice for LacZ staining and WT mice for immunohistochemistry with anti-girdin antibody. As reported previously, in generating girdin-deficient mice, a knockin strain of mice expressing LacZ in place of girdin was established,14Kitamura T. Asai N. Enomoto A. Maeda K. Kato T. Ishida M. Jiang P. Watanabe T. Usukura J. Kondo T. Costantini F. Murohara T. Takahashi M. Regulation of VEGF-mediated angiogenesis by the Akt/PKB substrate Girdin.Nat Cell Biol. 2008; 10: 329-337Crossref PubMed Scopus (191) Google Scholar and LacZ staining was performed in retinal sections of Girdin−/− mice. LacZ immunostaining suggested that girdin was expressed in endothelial cells, ganglion cells, and some neuronal cells in the inner nuclear layer of the mouse retina (Figure 2B). We also performed immunostaining of WT mouse retina using anti-girdin polyclonal antibody and confirmed that girdin was expressed in retinal vessel plexuses and sprouting tip cells (Figure 2C). We next investigated the effect of PI3K inhibitor LY294002 or ERK inhibitor U0126 on the proliferation, migration, and tube formation of HRECs. MTT assay was performed to quantify HREC proliferation.22Schomber T. Kopfstein L. Djonov V. Albrecht I. Baeriswyl V. Strittmatter K. Christofori G. Placental growth factor-1 attenuates vascular endothelial growth factor-A-dependent tumor an
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