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• W2023404968 abstract "Transcription factor Ets-1 has been reported to regulate angiogenesis in vascular endothelial cells. Here, we investigated a mechanism that may regulate the expression of Ets-1 in vascular endothelial growth factor (VEGF)- and hypoxia-induced retinal neovascularization and that may have potential to inhibit ocular neovascular diseases. VEGF and hypoxia increased Ets-1 expression in cultured bovine retinal endothelial cells. The VEGF-induced mRNA increase of Ets-1 was suppressed by a tyrosine kinase inhibitor (genistein), by inhibitors of MEK (mitogen-activated protein and extracellular signal-regulated kinase kinase) (PD98059 and UO126), and by inhibitors of protein kinase C (GF109203X, staurosporine, and Gö6976). Dominant-negative Ets-1 inhibited VEGF-induced cell proliferation, tube formation, and the expression of neuropilin-1 and angiopoietin-2. In a mouse model of proliferative retinopathy, Ets-1 mRNA was up-regulated. Intravitreal injection of dominant-negative Ets-1 suppressed retinal angiogenesis in a mouse model of proliferative retinopathy. In conclusion, VEGF induces Ets-1 expression in bovine retinal endothelial cells and its expression is protein kinase C/ERK pathway-dependent. Ets-1 up-regulation is involved in the development of retinal neovascularization, and inhibition of Ets-1 may be beneficial in the treatment of ischemic ocular diseases. Transcription factor Ets-1 has been reported to regulate angiogenesis in vascular endothelial cells. Here, we investigated a mechanism that may regulate the expression of Ets-1 in vascular endothelial growth factor (VEGF)- and hypoxia-induced retinal neovascularization and that may have potential to inhibit ocular neovascular diseases. VEGF and hypoxia increased Ets-1 expression in cultured bovine retinal endothelial cells. The VEGF-induced mRNA increase of Ets-1 was suppressed by a tyrosine kinase inhibitor (genistein), by inhibitors of MEK (mitogen-activated protein and extracellular signal-regulated kinase kinase) (PD98059 and UO126), and by inhibitors of protein kinase C (GF109203X, staurosporine, and Gö6976). Dominant-negative Ets-1 inhibited VEGF-induced cell proliferation, tube formation, and the expression of neuropilin-1 and angiopoietin-2. In a mouse model of proliferative retinopathy, Ets-1 mRNA was up-regulated. Intravitreal injection of dominant-negative Ets-1 suppressed retinal angiogenesis in a mouse model of proliferative retinopathy. In conclusion, VEGF induces Ets-1 expression in bovine retinal endothelial cells and its expression is protein kinase C/ERK pathway-dependent. Ets-1 up-regulation is involved in the development of retinal neovascularization, and inhibition of Ets-1 may be beneficial in the treatment of ischemic ocular diseases. Pathological growth of new blood vessels is the common final pathway in ocular neovascular diseases such as diabetic retinopathy, retinopathy of prematurity, and age-related macular degeneration, and often leads to catastrophic loss of vision. Vascular endothelial growth factor (VEGF) has been proven to be a predominant angiogenic factor that mediates such ocular neovascularization. VEGF is increased by hypoxia,1Shweiki D Itin A Soffer D Keshet E Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis.Nature. 1992; 359: 843-845Crossref PubMed Scopus (4186) Google Scholar which is one of the primary stimuli for ocular neovascularization. VEGF inhibition by soluble VEGF receptor 1 protein or adenovirus vector-encoding soluble VEGF receptor 1 have been reported to reduce retinal neovascularization effectively.2Aiello LP Pierce EA Foley ED Takagi H Chen H Riddle L Ferrara N King GL Smith LE Suppression of retinal neovascularization in vivo by inhibition of vascular endothelial growth factor (VEGF) using soluble VEGF-receptor chimeric proteins.Proc Natl Acad Sci USA. 1995; 92: 10457-10461Crossref PubMed Scopus (1175) Google Scholar, 3Bainbridge JW Mistry A De Alwis M Paleolog E Baker A Thrasher AJ Ali RR Inhibition of retinal neovascularization by gene transfer of soluble VEGF receptor sFlt-1.Gene Ther. 2002; 9: 320-326Crossref PubMed Scopus (157) Google Scholar The Ets gene family conserves an 85-amino acid DNA-binding ETS domain that binds the consensus sequence 5′-GGA(A/T)-3′ in the promoter region of the target genes,4Wasylyk B Hahn SL Giovane A The Ets family of transcription factors.Eur J Biochem. 1993; 211: 7-18Crossref PubMed Scopus (812) Google Scholar and have various biological functions, including cellular growth, differentiation, and organ development.5Sharrocks AD The ETS-domain transcription factor family.Nat Rev Mol Cell Biol. 2001; 2: 827-837Crossref PubMed Scopus (832) Google Scholar Ets-1, first identified among the Ets gene family, has been shown to also be associated with pathological angiogenesis. Increased Ets-1 expression is observed in cultured endothelial cells and in endothelial cells of new vessels during tumor angiogenesis in the adult.6Wernert N Raes MB Lassalle P Dehouck MP Gosselin B Vandenbunder B Stehelin D c-ets1 proto-oncogene is a transcription factor expressed in endothelial cells during tumor vascularization and other forms of angiogenesis in humans.Am J Pathol. 1992; 140: 119-127PubMed Google Scholar, 7Iwasaka C Tanaka K Abe M Sato Y Ets-1 regulates angiogenesis by inducing the expression of urokinase-type plasminogen activator and matrix metalloproteinase-1 and the migration of vascular endothelial cells.J Cell Physiol. 1996; 169: 522-531Crossref PubMed Scopus (274) Google Scholar A number of angiogenesis-related molecules, including matrix metalloproteinase (MMP)-1, MMP-3, MMP-9, urokinase-type plasminogen activator, integrin β3, vascular endothelial-cadherin (VE-cadherin), and neuropilin-1 (NRP1) are reported to be targets of Ets-1 in endothelial cells.7Iwasaka C Tanaka K Abe M Sato Y Ets-1 regulates angiogenesis by inducing the expression of urokinase-type plasminogen activator and matrix metalloproteinase-1 and the migration of vascular endothelial cells.J Cell Physiol. 1996; 169: 522-531Crossref PubMed Scopus (274) Google Scholar, 8Lelievre E Mattot V Huber P Vandenbunder B Soncin F ETS1 lowers capillary endothelial cell density at confluence and induces the expression of VE-cadherin.Oncogene. 2000; 19: 2438-2446Crossref PubMed Scopus (76) Google Scholar, 9Oda N Abe M Sato Y ETS-1 converts endothelial cells to the angiogenic phenotype by inducing the expression of matrix metalloproteinases and integrin beta3.J Cell Physiol. 1999; 178: 121-132Crossref PubMed Scopus (196) Google Scholar, 10Teruyama K Abe M Nakano T Takahashi S Yamada S Sato Y Neuropilin-1 is a downstream target of transcription factor Ets-1 in human umbilical vein endothelial cells.FEBS Lett. 2001; 504: 1-4Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar Receptor tyrosine kinases such as VEGF receptor 1, VEGF receptor 2, and TIE1/2 (tyrosine kinase that contains immunoglobulin-like loops and epidermal growth factor-similar domains), have been reported to have ETS-binding motif in their promoter regions.11Wakiya K Begue A Stehelin D Shibuya M A cAMP response element and an Ets motif are involved in the transcriptional regulation of flt-1 tyrosine kinase (vascular endothelial growth factor receptor 1) gene.J Biol Chem. 1996; 271: 30823-30828Crossref PubMed Scopus (135) Google Scholar, 12Kappel A Schlaeger TM Flamme I Orkin SH Risau W Breier G Role of SCL/Tal-1, GATA, and ets transcription factor binding sites for the regulation of flk-1 expression during murine vascular development.Blood. 2000; 96: 3078-3085Crossref PubMed Google Scholar, 13Dube A Akbarali Y Sato TN Libermann TA Oettgen P Role of the Ets transcription factors in the regulation of the vascular-specific Tie2 gene.Circ Res. 1999; 84: 1177-1185Crossref PubMed Scopus (93) Google Scholar Despite reports of the role of Ets-1 in angiogenesis of various tissues, its role in ocular angiogenesis has not been investigated. In this study, we investigated Ets-1 regulation and its function in VEGF- and ischemia-induced retinal neovascularization. Human recombinant VEGF was obtained from Genzyme (Cambridge, MA). Goat anti-human VEGF neutralizing antibody was purchased from R&D Systems (Minneapolis, MN). Rabbit polyclonal anti-Ets-1 antibody and rabbit polyclonal anti-extracellular signal-regulated kinase 1 (ERK1) antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal anti-phospho-p44/p42 antibody was purchased from New England Biolabs (Beverly, MA). PD98059, wortmannin, genistein, GF109203X, staurosporine, rottlerin, and Gö6976 were obtained from Calbiochem (La Jolla, CA). UO126 was obtained from Cell Signaling Technology (Beverly, MA). All other materials were obtained from Sigma (St. Louis, MO). Bovine retinal endothelial cells (BRECs) were grown under previously described condition.14Oh H Takagi H Suzuma K Otani A Matsumura M Honda Y Hypoxia and vascular endothelial growth factor selectively up-regulate angiopoietin-2 in bovine microvascular endothelial cells.J Biol Chem. 1999; 274: 15732-15739Crossref PubMed Scopus (427) Google Scholar BRECs were cultured in Dulbecco's modified Eagle's medium with 5.5 mmol/L glucose, 10% plasma-derived horse serum (Wheaton, Pipersville, PA), 50 mg/ml heparin, and 50 U/ml endothelial cell growth factor (Roche Diagnostics, Indianapolis, IN). Cells were characterized for their homogeneity by immunoreactivity with anti-factor VIII antibody, and remained morphologically unchanged under these conditions, as confirmed by light microscopy. BRECs were exposed to human recombinant VEGF or exposed to hypoxic conditions as described.14Oh H Takagi H Suzuma K Otani A Matsumura M Honda Y Hypoxia and vascular endothelial growth factor selectively up-regulate angiopoietin-2 in bovine microvascular endothelial cells.J Biol Chem. 1999; 274: 15732-15739Crossref PubMed Scopus (427) Google Scholar For hypoxic study, cells were exposed to 1 ± 0.5% oxygen using a water-jacketed mini-CO2/multigas incubator with reduced oxygen control (model BL-40M; Jujikagaku, Tokyo, Japan). All cells were maintained at 37°C in a constant carbon dioxide atmosphere with oxygen deficit induced by nitrogen replacement. To determine the signaling pathways involved in VEGF-induced Ets-1 mRNA expression, BRECs were treated for 4 hours with VEGF (25 ng/ml) after pretreatment for 30 minutes with genistein, a tyrosine kinase inhibitor (200 μmol/L); GF109203X, a general protein kinase C (PKC) inhibitor (5 μmol/L); staurosporine, a general PKC inhibitor (100 nmol/L); rottlerin, an inhibitor of PKCδ (5 μmol/L); Gö6976, an inhibitor of classical PKC (5 μmol/L); PD98059, an inhibitor of MEK (mitogen-activated protein and ERK kinase) (50 μmol/L); UO126, an inhibitor of MEK (10 μmol/L); or wortmannin, a phosphatidylinositol 3-kinase (PI3-kinase) inhibitor (100 nmol/L), respectively. To investigate the effects of VEGF on ERK phosphorylation, BRECs were treated for 10 minutes with VEGF (10 ng/ml) after pretreatment with GF109203X (5 μmol/L) or PD98059 (50 μmol/L) for 30 minutes. The sequence encoding the Ets domain and lacking the transactivation domain of murine Ets-1 was amplified from murine c-ets-1 cDNA by the polymerase chain reaction with oligonucleotides CCG CTC GAG CCA CCA TGG CTC CTG CTG CTG CCC T and GGC CTC GAG CTA AGC ATA ATC TGG AAC ATC ATA TGG ATA GTC AGC ATC CGG CT having XhoI restriction sites and HA epitope. The amplified product was digested with XhoI and subcloned into pGEM11zf(+), as described previously.15Nakano T Abe M Tanaka K Shineha R Satomi S Sato Y Angiogenesis inhibition by transdominant mutant Ets-1.J Cell Physiol. 2000; 184: 255-262Crossref PubMed Scopus (60) Google Scholar Adenovirus vector encoding dominant-negative Ets-1 was constructed by homologous recombination in 293 cells between the transfer cassette bearing the expression unit of dominant-negative Ets-1 and almost the entire adenovirus genome and restriction enzyme-digested adenovirus genome tagged with terminal protein.15Nakano T Abe M Tanaka K Shineha R Satomi S Sato Y Angiogenesis inhibition by transdominant mutant Ets-1.J Cell Physiol. 2000; 184: 255-262Crossref PubMed Scopus (60) Google Scholar The adenovirus was applied at a concentration of 1 × 108 plaque-forming units/ml, and adenovirus with the genome carrying an enhanced green fluorescent protein gene (GFP) (Clontech, Palo Alto, CA) or lacZ were used as controls as described.16Suzuma K Takahara N Suzuma I Ishiki K Ueki K Leitegs M Aiello LP King GL Characterization of protein kinase c β isoform's action on retinoblastoma protein phosphorylation, vascular endothelial growth factor-induced endothelial cell proliferation, and retinal neovascularization.Proc Natl Acad Sci USA. 2002; 22: 721-726Crossref Scopus (164) Google Scholar Infection efficiency was monitored by fluorescence, which showed expression in >80% of cells. Expression of recombinant protein was confirmed by Western blot analysis. The study adhered to the Association for Research in Vision and Ophthalmology (ARVO) Standards for the Use of Animals in Ophthalmic and Vision Research. The well-established mouse model of proliferative retinopathy was created as described.17Smith LE Wesolowski E McLellan A Kostyk SK D'Amato R Sullivan R D'Amore PA Oxygen-induced retinopathy in the mouse.Invest Ophthalmol Vis Sci. 1994; 35: 101-111PubMed Google Scholar Briefly, litters of 7-day-old (postnatal day 7, P7) C57BL/6J mice were exposed to 75% oxygen for 5 days and then were returned to room air at P12 to produce retinal neovascularization. Mice of the same age, maintained in room air, served as controls. Total RNA was isolated from cells and retinas of mice using guanidinium thiocyanate, and Northern blot analysis was performed as described.18Otani A Takagi H Suzuma K Honda Y Angiotensin II potentiates vascular endothelial growth factor-induced angiogenic activity in retinal microcapillary endothelial cells.Circ Res. 1998; 82: 619-628Crossref PubMed Scopus (261) Google Scholar Total RNA (20 μg) was electrophoresed through 1% formaldehyde-agarose gels and then transferred to a nylon membrane (Pall BioSupport, East Hills, NY). After ultraviolet cross-linking, blots were prehybridized, and hybridized with 32P-labeled cDNA. All signals were analyzed using a densitometer (BAS-2000 II; Fuji Photo Film, Tokyo, Japan). The signal for each sample was normalized by reprobing the same blot using 36B4 cDNA control probe. cDNA template of murine Ets-1, human NRP1, and human angiopoietin-2 (Ang-2) were prepared by reverse transcriptase-polymerase chain reaction using the following primer pairs: 5′-CCC TGG GTA AAG AAT GCT TTC TCG-3′ (Ets-1 sense), 5′-GGA CTG ACA AGA CTT ATC AGT GAG-3′ (Ets-1 anti-sense), 5′-CAC ATT GGG CGT TAC TGT GGA C-3′ (NRP1 sense), 5′-CCT TTG TGG TTG GGG TGT CTA C-3′ (NRP1 anti-sense), 5′-AGC TGT GAT CTT GTC TTG GC-3′ (Ang-2 sense), and 5′-GTT CAA GTC TCG TGG TCT GA-3′ (Ang-2 anti-sense). For the animal studies, total RNA was isolated from retinas of mice at different time points (10 retinas from five mice at each time point: P12 immediately after return to room air, P13, P15, P17, P19, P21, P23, and P26). Total protein from cells was assessed by Western blot analysis as described.19Suzuma I Suzuma K Ueki K Hata Y Feener EP King GL Aiello LP Stretch-induced retinal vascular endothelial growth factor expression is mediated by phosphatidylinositol 3-kinase and protein kinase C (PKC)-ζ but not by stretch-induced ERK1/2, Akt, Ras, or classical/novel PKC pathways.J Biol Chem. 2002; 277: 1047-1057Crossref PubMed Scopus (69) Google Scholar BRECs were washed with ice-cold phosphate-buffered saline (PBS) and lysed in 1× Laemmli buffer (50 mmol/L Tris, pH 6.8, 2% sodium dodecyl sulfate, 10% glycerol) containing protease inhibitors (10 mmol/L sodium pyrophosphate, 100 mmol/L NaF, 1 mmol/L Na3VO4, 1 μg/ml aprotinin, 1 μg/ml leupeptin, and 2mmol/L phenylmethyl sulfonyl fluoride). Total protein (30 μg) was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and was transferred to nitrocellulose membrane (Bio-Rad Laboratories, Richmond, CA). The blots were incubated overnight at 4°C with primary antibodies followed by incubation for 2 hours with horseradish peroxidase-conjugated secondary antibody (1:2000 dilution) (Amersham International, Buckinghamshire, UK). Primary antibodies specific for Ets-1, phospho-p44/p42, and ERK1 were used at 1:500, 1:2000, and 1:2000 dilutions, respectively. Visualization was performed by enhanced chemiluminescence detection system (Amersham International). Nuclear cell extracts were prepared by the freezing-thawing method as described.20Schreiber E Matthias P Muller MM Schaffner W Rapid detection of octamer binding proteins with ‘mini-extracts,’ prepared from a small number of cells.Nucleic Acids Res. 1989; 17: 6419Crossref PubMed Scopus (3921) Google Scholar Synthetic oligonucleotide was as follows: Ets-1 consensus, 5′-GTC AGT TAA GCA GGA AGT GAC TAA C-3′.21Hultgardh-Nilsson A Cercek B Wang JW Naito S Lovdahl C Sharifi B Forrester JS Fagin JA Regulated expression of the ets-1 transcription factor in vascular smooth muscle cells in vivo and in vitro.Circ Res. 1996; 78: 589-595Crossref PubMed Scopus (85) Google Scholar The sequences of the double-strand oligonucleotide probes labeled with T4 kinase and γ-32P ATP were generated by use of labeling kits (Promega, Madison, WI). Reactions of nuclear cell extract and 32P-labeled consensus oligonucleotides were electrophoresed through a 4% nondenaturing polyacrylamide gel, and the signal was analyzed using a densitometer (BAS-2000 II; Fuji Photo Film). Cell growth assay was studied by DNA concentration measured by a fluorometer as described.16Suzuma K Takahara N Suzuma I Ishiki K Ueki K Leitegs M Aiello LP King GL Characterization of protein kinase c β isoform's action on retinoblastoma protein phosphorylation, vascular endothelial growth factor-induced endothelial cell proliferation, and retinal neovascularization.Proc Natl Acad Sci USA. 2002; 22: 721-726Crossref Scopus (164) Google Scholar BRECs were plated in 12-well plates at a density of 1 × 104 cells/well in Dulbecco's modified Eagle's medium containing 5% calf serum. After 24 hours of incubation, cells were infected with adenovirus, and the medium was replaced with Dulbecco's modified Eagle's medium containing 5% calf serum with or without VEGF (10 ng/ml). Four days later, the cells were lysed in 0.1% sodium dodecyl sulfate, and DNA concentrations in each well were measured using Hoechst 33258 dye (Calbiochem) and a fluorometer (model DyNA Quant 200; Hoefer, San Francisco, CA). In vitro tube formation assays were performed as described.18Otani A Takagi H Suzuma K Honda Y Angiotensin II potentiates vascular endothelial growth factor-induced angiogenic activity in retinal microcapillary endothelial cells.Circ Res. 1998; 82: 619-628Crossref PubMed Scopus (261) Google Scholar Vitrogen 50 (Cohesion, Palo Alto, CA), 0.2 N NaOH, and 200 mmol/L HEPES (8:1:1, v/v/v) and 10× RPMI medium (Gibco BRL-Invitrogen, Carlsbad, CA) were made to 400 μl and added to 24-well plates. After polymerization of the gels, 1 × 105 BRECs were seeded and incubated with Dulbecco's modified Eagle's medium containing 5% plasma-derived horse serum for 24 hours. After infection with adenovirus, additional collagen gel was added, and then BRECs were incubated with medium containing 3% plasma-derived horse serum with or without VEGF (25 ng/ml). Five days later, five different fields (×4 objective) were chosen, and total tube-like structures were measured using public domain NIH image software (downloaded from http://rsb.info.nih.gov/nih-image/Default.html). A solution (1 μl) containing adenovirus vector encoding dominant-negative Ets-1 (1 × 1010 plaque-forming units/ml) was injected into the vitreous of one eye of anesthetized C57BL/6J mice with a 33-gauge needle (Ito, Hamamatsu, Japan) on P12, as previously described.2Aiello LP Pierce EA Foley ED Takagi H Chen H Riddle L Ferrara N King GL Smith LE Suppression of retinal neovascularization in vivo by inhibition of vascular endothelial growth factor (VEGF) using soluble VEGF-receptor chimeric proteins.Proc Natl Acad Sci USA. 1995; 92: 10457-10461Crossref PubMed Scopus (1175) Google Scholar, 3Bainbridge JW Mistry A De Alwis M Paleolog E Baker A Thrasher AJ Ali RR Inhibition of retinal neovascularization by gene transfer of soluble VEGF receptor sFlt-1.Gene Ther. 2002; 9: 320-326Crossref PubMed Scopus (157) Google Scholar, 22Mori K Gehlbach P Ando A Wahlin K Gunther V McVey D Wei L Campochiaro PA Intraocular adenoviral vector-mediated gene transfer in proliferative retinopathies.Invest Ophthalmol Vis Sci. 2002; 43: 1610-1615PubMed Google Scholar As a control, an equivalent amount of control adenovirus was injected into the contralateral eye. At P19, the mice were killed by cardiac perfusion of 1 ml 4% paraformaldehyde in PBS, and the eyes were enucleated and fixed in 4% paraformaldehyde overnight at 4°C before paraffin embedding. Serial 6-μm paraffin-embedded axial sections were obtained from the optic nerve and stained with hematoxylin and periodic acid-Schiff, according to a standard protocol. All retinal vascular nuclei anterior to the internal limiting membrane were counted in each section by a fully masked protocol. For each eye, 10 intact sections of equal length, each 30 μm apart, were evaluated. The mean number of neovascular nuclei per section per eye was then determined. Determinations were performed in triplicate, and experiments were repeated at least three times. Results are expressed as the mean ± SEM. One-way analysis of variance followed by the Fisher t-test was used to evaluate significant differences, and P < 0.05 was selected as the statistically significant value. For evaluation of in vivo retinal angiogenesis, the chi-square test for categorical data and the paired Student's t-test or the Mann-Whitney rank sum test for quantitative data with unequal variance are used. The effects of VEGF on the expression of Ets-1 mRNA were studied by Northern blot analysis in BRECs. VEGF (25 ng/ml) increased Ets-1 mRNA levels in a time-dependent manner, reaching a maximum after 4 hours (2.5 ± 0.2-fold, P < 0.01) (Figure 1A). The dose response to VEGF-induced Ets-1 mRNA expression was studied after 4 hours of VEGF stimulation. The expression of Ets-1 mRNA was up-regulated in a dose-dependent manner, with an EC50 of ∼0.25 ng/ml; maximal increase was observed at a VEGF concentration of 25 ng/ml (2.5 ± 0.1-fold, P < 0.001) (Figure 1B). Ets-1 protein expression was studied by Western blot analysis in BRECs using anti-Ets-1 antibody. VEGF increased significantly Ets-1 protein expression after 6 hours (1.7 ± 0.3-fold, P < 0.05), 12 hours (1.8 ± 0.3-fold, P < 0.05), and 24 hours (2.0 ± 0.4-fold, P < 0.05) (Figure 1C). The DNA-binding activity of nuclear proteins was studied by EMSA. After 12 hours and 24 hours of stimulation of BRECs with VEGF, significant increases of DNA-Ets-1 complex were observed. The DNA-Ets-1 complex disappeared by adding unlabeled competitor (Figure 1D). The effects of hypoxia on the expression of Ets-1 mRNA were studied by Northern blot analysis in BRECs. Ets-1 mRNA levels were increased significantly after 8 hours (2.0 ± 0.1-fold, P < 0.01) and 24 hours of hypoxia stimulation (2.6 ± 0.2-fold, P < 0.001) (Figure 2A). Because VEGF expression is up-regulated by hypoxia, we studied whether or not VEGF mediation is involved in the observed hypoxia regulation of Ets-1 by using anti-VEGF neutralizing antibody. The increased Ets-1 mRNA levels induced by hypoxia for 24 hours were almost completely reversed by anti-VEGF neutralizing antibody (Figure 2B). Using Northern blot analysis, we further studied regulation of the Ets-1 gene in an in vivo model of proliferative retinopathy. Total RNA was isolated from retinas of mice at different time points (10 retinas from five mice at each time point: P12 immediately after return to room air, P13, P15, P17, P19, P21, P23, and P26). In the animals in which retinal ischemia was induced, Ets-1 mRNA levels were lower compared with those in the control animals at P12, but a remarkable increase of the mRNA level was observed from P15 to P23. In contrast, in age-matched control animals, Ets-1 mRNA levels decreased gradually from P12 to P26 (Figure 3). A maximal 4.0-fold increase was detected at P19 compared with that in the normal age-matched controls. The effects of tyrosine kinase, PKC, MEK, and PI3-kinase inhibitors were determined by Northern blot analysis. Wortmannin, a PI3-kinase inhibitor caused no significant change in the effect of VEGF on Ets-1 mRNA expression in BRECs, but genistein, a tyrosine kinase inhibitor did inhibit the effects of VEGF by 71 ± 4.1% (P < 0.001). MEK inhibitors, PD98059 and UO126 inhibited the effects of VEGF by 65 ± 2.0% (P < 0.001) and 65 ± 2.8% (P < 0.001), respectively. General PKC inhibitors, GF109203X and staurosporine inhibited the effects of VEGF by 58 ± 4.5% (P < 0.001) and 61 ± 6.7% (P < 0.001), respectively. PKCδ inhibitor, rottlerin, and classical PKC inhibitor, Gö6976 inhibited the effects of VEGF by 35 ± 7.7% (P < 0.05) and 53 ± 3.9% (P < 0.01), respectively (Figure 4A). We investigated the involvement of PKC in ERK pathway by Western blot analysis. We investigated the effects of a PKC inhibitor, GF109203X, or a MEK inhibitor, PD98059, on ERK1/2 phosphorylation in BRECs. Both GF109203X and PD98059 decreased ERK1/2 phosphorylation by 40 ± 6.7% (P < 0.01), and 36 ± 6.8% (P < 0.01), respectively (Figure 4B). To further investigate the role of Ets-1 in retinal neovascularization, we determined effects of Ets-1 dominant-negative on expression of NRP1 and Ang-2 in BRECs stimulated by VEGF. Both NRP1 and Ang-2 mRNA expression were decreased by dominant-negative Ets-1 (Figure 5A). To investigate the role of Ets-1 in VEGF-dependent angiogenesis in vitro, we examined whether or not dominant-negative Ets-1 would affect cell proliferation and tube formation of BRECs. VEGF (10 ng/ml) increased DNA synthesis (2.0 ± 0.1-fold, P < 0.01). DNA synthesis of adenovirus encoding dominant-negative Ets-1 transfectants was significantly reduced in comparison with that of VEGF stimulation alone (42 ± 11%, P < 0.001) or GFP transfectants (Figure 5B). VEGF induced tube formation at 14.3 ± 0.3 mm/field, whereas treatment with dominant-negative Ets-1 transfectants inhibited the VEGF-induced tube formation by 8.3 ± 0.4 mm/field compared to VEGF stimulation alone (P < 0.001) (Figure 5, C and D). To further determine the role of Ets-1 in pathological angiogenesis in vivo, we examined the effect of Ets-1 inhibition in the mouse model of proliferative retinopathy. The mice exposed to 75% oxygen from P7 to P12 exhibited extensive retinal capillary obliteration. When the mice were returned to room air, the inner retina became hypoxic, expression of VEGF was up-regulated, and retinal neovascularization occurred above the internal limiting membrane into the vitreous. These neovascular tufts were most prominent at P17 to P19. Adenovirus vector encoding dominant-negative Ets-1 treatment resulted in a significant reduction in retinal neovascularization compared with injections of control null adenovirus vector (45% ± 6%, P < 0.01) (Figure 6, A and B). Suppression of the neovascular response was evident in histological examination of paraffin-embedded ocular cross sections (Figure 6C, arrows). Expression of lacZ was prominent on the retinal surface and in the neovascular tufts (data not shown) as described previously.22Mori K Gehlbach P Ando A Wahlin K Gunther V McVey D Wei L Campochiaro PA Intraocular adenoviral vector-mediated gene transfer in proliferative retinopathies.Invest Ophthalmol Vis Sci. 2002; 43: 1610-1615PubMed Google Scholar No retinal detachment or other damage related to the needle puncture was observed. We also found no retinal toxicity by microscopy morphologically. VEGF inhibition effectively suppresses ischemia-induced retinal neovascularization.2Aiello LP Pierce EA Foley ED Takagi H Chen H Riddle L Ferrara N King GL Smith LE Suppression of retinal neovascularization in vivo by inhibition of vascular endothelial growth factor (VEGF) using soluble VEGF-receptor chimeric proteins.Proc Natl Acad Sci USA. 1995; 92: 10457-10461Crossref PubMed Scopus (1175) Google Scholar, 3Bainbridge JW Mistry A De Alwis M Paleolog E Baker A Thrasher AJ Ali RR Inhibition of retinal neovascularization by gene transfer of soluble VEGF receptor sFlt-1.Gene Ther. 2002; 9: 320-326Crossref PubMed Scopus (157) Google Scholar However, because VEGF is crucial for the physiological functions of the retina such as normal development of the vasculature through its effects on survival of vascular endothelial cells and retinal neurons,23Alon T Hemo I Itin A Pe'er J Stone J Keshet E Vascular endothelial growth factor acts as a survival factor for newly formed retinal vessels and has implications for retinopathy of prematurity.Nat Med. 1995; 1: 1024-1028Crossref PubMed Scopus (1427) Google Scholar, 24Stone J Itin A Alon T Pe'er J Gnessin H Chan-Ling T Keshet E Development of retinal vasculature is mediated by hypoxia-induced vascular endothelial growth factor (VEGF) expression by neuroglia.J Neurosci. 1995; 15: 4738-4747Crossref PubMed Google Scholar, 25Yang K Cepko CL Flk-1, a receptor for vascular endothelial growth factor (VEGF), is expressed by retinal progenitor cells.J Neurosci. 1996; 16: 6089-6099Crossref PubMed Google Scholar, 26Yourey PA Gohari S Su JL Alderson RF Vascular endothelial cell growth factors promote the in vitro development of rat photoreceptor cells.J Neurosci. 2000; 20: 6781-6788Crossref PubMed Google Scholar complete inhibition of VEGF itself could reduce basal level and cause undesirable effects on homeostasis of endothelial cell or neuron. 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• W2023404968 date "2004-05-01" @default.
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• W2023404968 title "Transcription Factor Ets-1 Mediates Ischemia- and Vascular Endothelial Growth Factor-Dependent Retinal Neovascularization" @default.
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