The Eyes Absent Proteins in Developmental and Pathological Angiogenesis
2016; Elsevier BV; Volume: 186; Issue: 3 Linguagem: Inglês
10.1016/j.ajpath.2015.10.031
ISSN1525-2191
AutoresYuhua Wang, Emmanuel Tadjuidje, Ram Naresh Pandey, James A. Stefater, Lois E.H. Smith, Richard A. Lang, Rashmi S. Hegde,
Tópico(s)Ocular Oncology and Treatments
ResumoManagement of neoangiogenesis remains a high-value therapeutic goal. A recently uncovered association between the DNA damage repair pathway and pathological angiogenesis could open previously unexplored possibilities for intervention. An attractive and novel target is the Eyes absent (EYA) tyrosine phosphatase, which plays a critical role in the repair versus apoptosis decision after DNA damage. This study examines the role of EYA in the postnatal development of the retinal vasculature and under conditions of ischemia-reperfusion encountered in proliferative retinopathies. We find that the ability of the EYA proteins to promote endothelial cell (EC) migration contributes to a delay in postnatal development of the retinal vasculature when Eya3 is deleted specifically in ECs. By using genetic and chemical biology tools, we show that EYA contributes to pathological angiogenesis in a model of oxygen-induced retinopathy. Both in vivo and in vitro, loss of EYA tyrosine phosphatase activity leads to defective assembly of γ-H2AX foci and thus to DNA damage repair in ECs under oxidative stress. These data reveal the potential utility of EYA tyrosine phosphatase inhibitors as therapeutic agents in inhibiting pathological neovascularization with a range of clinical applications. Management of neoangiogenesis remains a high-value therapeutic goal. A recently uncovered association between the DNA damage repair pathway and pathological angiogenesis could open previously unexplored possibilities for intervention. An attractive and novel target is the Eyes absent (EYA) tyrosine phosphatase, which plays a critical role in the repair versus apoptosis decision after DNA damage. This study examines the role of EYA in the postnatal development of the retinal vasculature and under conditions of ischemia-reperfusion encountered in proliferative retinopathies. We find that the ability of the EYA proteins to promote endothelial cell (EC) migration contributes to a delay in postnatal development of the retinal vasculature when Eya3 is deleted specifically in ECs. By using genetic and chemical biology tools, we show that EYA contributes to pathological angiogenesis in a model of oxygen-induced retinopathy. Both in vivo and in vitro, loss of EYA tyrosine phosphatase activity leads to defective assembly of γ-H2AX foci and thus to DNA damage repair in ECs under oxidative stress. These data reveal the potential utility of EYA tyrosine phosphatase inhibitors as therapeutic agents in inhibiting pathological neovascularization with a range of clinical applications. Pathological neovascularization (NV) is associated with common human diseases such as proliferative retinopathies, wet age-related macular degeneration, cancer, atherosclerotic plaque rupture, and rheumatoid arthritis.1Chung A.S. Ferrara N. Developmental and pathological angiogenesis.Annu Rev Cell Dev Biol. 2011; 27: 563-584Crossref PubMed Scopus (549) Google Scholar Much evidence points to a role for vascular endothelial growth factor (VEGF) signaling in both pathological and developmental angiogenesis, and drugs targeting the VEGF pathway have been extensively tested as potential anti-angiogenic agents.2Campochiaro P.A. Ocular neovascularization.J Mol Med (Berl). 2013; 91: 311-321Crossref PubMed Scopus (287) Google Scholar, 3Welti J. Loges S. Dimmeler S. Carmeliet P. Recent molecular discoveries in angiogenesis and antiangiogenic therapies in cancer.J Clin Invest. 2013; 123: 3190-3200Crossref PubMed Scopus (472) Google Scholar Antibodies to VEGF have achieved clinical success in the treatment of wet age-related macular degeneration, clinically significant macular edema, and retinopathy of prematurity, as well as in improving the odds of progression-free survival in certain cancers. However, intrinsic and induced resistance to anti-VEGF treatment poses limitations to their use in cancer treatment.3Welti J. Loges S. Dimmeler S. Carmeliet P. Recent molecular discoveries in angiogenesis and antiangiogenic therapies in cancer.J Clin Invest. 2013; 123: 3190-3200Crossref PubMed Scopus (472) Google Scholar, 4Bergers G. Hanahan D. Modes of resistance to anti-angiogenic therapy.Nat Rev Cancer. 2008; 8: 592-603Crossref PubMed Scopus (2340) Google Scholar Furthermore, side effects associated with normal angiogenic processes such as wound healing are a concern. It is in these clinical contexts that novel anti-angiogenic agents capable of discriminating between developmental and pathological NV and complementing anti-VEGF treatment would be valuable. Several recent studies have begun to delineate mechanisms that differentiate between developmental and pathological angiogenesis.5Okuno Y. Nakamura-Ishizu A. Otsu K. Suda T. Kubota Y. Pathological neoangiogenesis depends on oxidative stress regulation by ATM.Nat Med. 2012; 18: 1208-1216Crossref PubMed Scopus (116) Google Scholar, 6Economopoulou M. Langer H.F. Celeste A. Orlova V.V. Choi E.Y. Ma M. Vassilopoulos A. Callen E. Deng C. Bassing C.H. Boehm M. Nussenzweig A. Chavakis T. Histone H2AX is integral to hypoxia-driven neovascularization.Nat Med. 2009; 15: 553-558Crossref PubMed Scopus (109) Google Scholar, 7Alitalo K. A radical view of pathological vasculature.Cell Metab. 2012; 16: 287-288Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar, 8Rankin E.B. Giaccia A.J. Hammond E.M. Bringing H2AX into the angiogenesis family.Cancer Cell. 2009; 15: 459-461Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, 9Shen J. Frye M. Lee B.L. Reinardy J.L. McClung J.M. Ding K. Kojima M. Xia H. Seidel C. Lima e Silva R. Dong A. Hackett S.F. Wang J. Howard B.W. Vestweber D. Kontos C.D. Peters K.G. Campochiaro P.A. Targeting VE-PTP activates TIE2 and stabilizes the ocular vasculature.J Clin Invest. 2014; 124: 4564-4576Crossref PubMed Scopus (146) Google Scholar Notably, Okuno et al5Okuno Y. Nakamura-Ishizu A. Otsu K. Suda T. Kubota Y. Pathological neoangiogenesis depends on oxidative stress regulation by ATM.Nat Med. 2012; 18: 1208-1216Crossref PubMed Scopus (116) Google Scholar showed that the ataxia telangiectasia mutated (ATM) pathway is activated by tissue hypoxia but is not active during normal angiogenesis, and Economopoulou et al6Economopoulou M. Langer H.F. Celeste A. Orlova V.V. Choi E.Y. Ma M. Vassilopoulos A. Callen E. Deng C. Bassing C.H. Boehm M. Nussenzweig A. Chavakis T. Histone H2AX is integral to hypoxia-driven neovascularization.Nat Med. 2009; 15: 553-558Crossref PubMed Scopus (109) Google Scholar report that the H2AX-dependent DNA damage response (DDR) is activated and promotes NV specifically under hypoxic conditions. The Eyes absent (EYA) proteins are protein tyrosine phosphatases (PTPs) that hydrolyze the terminal phosphotyrosine of H2AX, permitting the assembly of the DNA damage response machinery and preventing apoptosis.10Cook P.J. Ju B.G. Telese F. Wang X. Glass C.K. Rosenfeld M.G. Tyrosine dephosphorylation of H2AX modulates apoptosis and survival decisions.Nature. 2009; 458: 591-596Crossref PubMed Scopus (406) Google Scholar The tyrosine phosphatase activity of EYA is also necessary for endothelial cell (EC) migration and tube formation.11Pandey R.N. Wang T.S. Tadjuidje E. McDonald M.G. Rettie A.E. Hegde R.S. Structure-activity relationships of benzbromarone metabolites and derivatives as EYA inhibitory anti-angiogenic agents.PLoS One. 2013; 8: e84582Crossref PubMed Scopus (18) Google Scholar, 12Tadjuidje E. Wang T.S. Pandey R.N. Sumanas S. Lang R.A. Hegde R.S. The EYA tyrosine phosphatase activity is pro-angiogenic and is inhibited by benzbromarone.PLoS One. 2012; 7: e34806Crossref PubMed Scopus (34) Google Scholar Taken together, these observations led us to postulate that the EYA proteins could contribute to angiogenesis by multiple mechanisms. They have a specific role in DDR-dependent pathological NV following ischemia-associated DNA damage and an EC migration–associated role in early development of the retinal vasculature. Hence, inhibition of the EYA tyrosine phosphatase activity could be a novel target for development of anti-angiogenic drugs. Here we report on the viability of this new therapeutic mechanism by using both genetic and chemical biology tools. These studies open the door for the development of EYA inhibitors with potential uses in the treatment of a host of vascularization-associated disorders, including cancer and proliferative retinopathies. The following antibodies were used in these studies: anti-cleaved caspase-3 (9664S; Cell Signaling Technology, Danvers, MA), anti-CD31 (550274, BD Biosciences, San Jose, CA), isolectin 488 (121411; Life Technologies, Carlsbad CA), bromodeoxyuridine (BrdU) mouse monoclonal Alexa 647 (B35133, Invitrogen, Carlsbad, CA), donkey anti-rabbit Alexa Fluo-594 and goat anti-mouse Alexa Fluo-488 (A21207 and A-11001, Life Technologies, Carlsbad, CA), anti-MDC1 (AB1169; Abcam, Cambridge, MA), anti-γH2AX (MABE205; Millipore, Billerica, MA), VEGF antibody (VG1, Thermo Fisher, Rockford, IL). Human retinal microvascular ECs (HRMECs) were obtained from Cell Systems (Kirkland, WA), and mouse retinal microvascular ECs (MRMECs) were obtained from Cell Biologics (Chicago, IL). This study adhered to the Association for Research in Vision and Ophthalmology statement for the use of animals in ophthalmic and vision research. C57Bl/6 mice obtained from Jackson Laboratories (Bar Harbor, ME) were bred in the Cincinnati Children's Hospital Medical Center facility (animal protocol 3D09062). We generated a conditional loss-of-function allele, Eya3flox, by conventional gene targeting. We targeted exon 6 for conditional deletion. This design left open the possibility that exons 1 to 5 might produce a functional truncated protein. Incorporation of sequences from the C-terminus of ornithine decarboxylase as a signal for rapid degradation13Matsuzawa S. Cuddy M. Fukushima T. Reed J.C. Method for targeting protein destruction by using a ubiquitin-independent, proteasome-mediated degradation pathway.Proc Natl Acad Sci U S A. 2005; 102: 14982-14987Crossref PubMed Scopus (43) Google Scholar was designed to eliminate any expressed Eya3 N-terminal sequence. The allele design incorporated loxP sites into an artificial exon that is initially in reverse orientation. Through two steps of recombination mediated by a pair of wild-type and a pair of variant loxP sequences that are incompatible, the C-terminus of ornithine decarboxylase degradation signal was spliced into the mRNA, simultaneously deleting exon 6. This allele design strategy has been used previously.14Schnutgen F. Doerflinger N. Calleja C. Wendling O. Chambon P. Ghyselinck N.B. A directional strategy for monitoring Cre-mediated recombination at the cellular level in the mouse.Nat Biotechnol. 2003; 21: 562-565Crossref PubMed Scopus (262) Google Scholar Successful Flp recombination was confirmed by PCR analysis with Eya3fl forward, 5′-CCACTTGGAGTAAGCATCCAGTC-3′ and reverse, 5′-AAGCAAAACGTCCTAGGTGCTC-3′. The protocol for oxygen-induced retinopathy was one previously described in studies associating DNA damage repair pathway with NV.5Okuno Y. Nakamura-Ishizu A. Otsu K. Suda T. Kubota Y. Pathological neoangiogenesis depends on oxidative stress regulation by ATM.Nat Med. 2012; 18: 1208-1216Crossref PubMed Scopus (116) Google Scholar, 6Economopoulou M. Langer H.F. Celeste A. Orlova V.V. Choi E.Y. Ma M. Vassilopoulos A. Callen E. Deng C. Bassing C.H. Boehm M. Nussenzweig A. Chavakis T. Histone H2AX is integral to hypoxia-driven neovascularization.Nat Med. 2009; 15: 553-558Crossref PubMed Scopus (109) Google Scholar Briefly, neonatal mice and their nursing mothers were kept in room air from postnatal day 0 (P0) to P8. At P8 each pup and its nursing mother were moved to a chamber maintained at 85% oxygen. At P11 the pups and their nursing mothers were returned to room air (surrogate mothers were used if necessary). When appropriate, intravitreal injection of EYA inhibitors [1 μL of either 100 μmol/L stock solution of benzarone (BZ) or 250 μmol/L stock solution of benzbromarone (BBR)] was performed at P12. To examine the effect of genetic deletion of Eya3 in the oxygen-induced retinopathy (OIR) protocol, tamoxifen was administered i.p. at 300 μg per pup daily between P11 and P13 to both Eya3flox/flox, pdgf-iCre, and Eya3flox/flox mice. At various time-points the eyes were enucleated while the pups were maintained under anesthesia. Immunofluorescence imaging and Western blot analysis of ECs demonstrated the effectiveness of this protocol in lowering EYA3 levels in ECs. There was no compensatory up-regulation of other Eyas (Supplemental Figure S1). Enucleated eyes were fixed for 1 hour in 4% paraformaldehyde/phosphate-buffered saline at room temperature and dissected. Retinas were permeabilized at room temperature for 30 minutes and then incubated with specific antibodies (γ-H2AX, EYA3, or cleaved caspase-3) overnight at 4°C. Antigen was detected with secondary antibodies conjugated to Alexa Fluor 594 or Alexa Fluor 488. To visualize vasculature, retinas were stained with fluoresceinated Griffonia (Bandeiraea) simplicifolia (isolectin B4–Alexa Fluor 594 conjugate, 1:500 dilution; Invitrogen) in phosphate-buffered saline with Tween (PBST). For the BrdU incorporation studies, 10 mg/kg BrdU was administered i.p. at P13. Isolated retinas were treated with 2N HCl for 30 minutes and then extensively washed with PBS, blocked with 10% fetal bovine serum, and incubated with BrdU Alexa Fluor 647 for 1 hour at room temperature. Images were taken at ×400 magnification on a Zeiss microscope (Zeiss, Jena, Germany). Standard published protocols were used to quantitate NV and VO. The number of pixels in the pathological tufts was quantified and compared with the number of pixels in the entire retinal area by a computer-aided method (SWIFT-NV15Stahl A. Connor K.M. Sapieha P. Willett K.L. Krah N.M. Dennison R.J. Chen J. Guerin K.I. Smith L.E. Computer-aided quantification of retinal neovascularization.Angiogenesis. 2009; 12: 297-301Crossref PubMed Scopus (116) Google Scholar) that utilizes a series of macros in ImageJ version 1.48 (NIH, Bethesda, MD; http://imagej.nih.gov/ij). Percentage of NV and VO in total retinas was compared between inhibitor- and vehicle-treated eyes, or Eya3VEC-KO and Eya3fl/fl mice. In each case, n was the number of eyes quantified. Each experiment included three independent litters. To determine expression of Eya transcripts in mouse retinal microvascular ECs or retinal ECs from genetically engineered mice, total RNA was extracted from 1 million ECs, and cDNA was synthesized with the Primescript RT reagent kit (Takara Bio, Shiga, Japan). PCR product was analyzed on a 1.5% agarose gel to confirm that amplified products were of the expected sizes. Primers used included EYA1 forward 5′-CATAGCCGACTGAGTGGTAGT-3′ and reverse 5′-GCTCTGTTTTAACTTCGGTGCC-3′; EYA2 forward 5′-CACCGCTGGGCTCTATCAAG-3′ and reverse 5′-GGGGTAGGACGGATAATCCTG-3′; EYA3 forward 5′-CTCAAACCAGGATTATCCCACC-3′ and reverse 5′-CAGCATCACTGTTAGTCTGACC-3′; EYA4 forward 5′-TCCTTGGCCCTGCTAAGAG-3′ and reverse 5′-TGCCTATTTTTGTTGCGCTGT-3′; GAPDH forward 5′-AAGGCCGGGGCCCACTTGAA-3′ and reverse: 5′-CGGCCATCACGCCACAGCTT-3′. HRMECs were cultured in Complete Medium (Cell Systems) and used in the first nine passages. ECs from Eya3VEC-KO and control mice were isolated by using magnetic Dynabeads (Life Technologies) coated with anti–PECAM-1 antibody as previously described.16Su X. Sorenson C.M. Sheibani N. Isolation and characterization of murine retinal endothelial cells.Mol Vis. 2003; 9: 171-178PubMed Google Scholar For analysis of the formation of DNA repair complexes, cells were fixed with 4% paraformaldehyde at room temperature for 15 minutes, and then the coverslips were immunostained for DDR proteins with anti-γH2AX and anti-MDC1 antibodies. Cell proliferation was measured at 72 hours with the WST-8 assay (CCK-8 Kit, Dojindo Molecular Technologies, Rockville, MD) as previously described.12Tadjuidje E. Wang T.S. Pandey R.N. Sumanas S. Lang R.A. Hegde R.S. The EYA tyrosine phosphatase activity is pro-angiogenic and is inhibited by benzbromarone.PLoS One. 2012; 7: e34806Crossref PubMed Scopus (34) Google Scholar Proliferation studies were performed in Epithelial Cell Growth Medium 2 (Lonza, Allendale, NJ). Vehicle control contained 0.1% dimethyl sulfoxide, and both EYA inhibitors were dissolved in 0.1% dimethyl sulfoxide. For hypoxia experiments, the cells were maintained at 1% O2 with BioSpherix ProCO2 Model P120 and ProOx Model 110 controllers for C chambers (Biospherix, Parish, NY). Transwell migration experiments were performed as previously described.12Tadjuidje E. Wang T.S. Pandey R.N. Sumanas S. Lang R.A. Hegde R.S. The EYA tyrosine phosphatase activity is pro-angiogenic and is inhibited by benzbromarone.PLoS One. 2012; 7: e34806Crossref PubMed Scopus (34) Google Scholar Results are presented as the means ± SEM for the in vivo experiments and as means ± SD for the in vitro studies. Statistical analyses were performed with Graphpad PRISM version 5.0 for Mac OSX, (GraphPad Software, La Jolla, CA). A t-test was used when two samples and conditions were compared, and analysis of variance was used for more than two groups. Significance represents P < 0.05. The mouse retina is avascular at birth, with a monolayer of vessels extending out from the center to the periphery between birth and P7, providing an accessible and well-characterized system17Stahl A. Connor K.M. Sapieha P. Chen J. Dennison R.J. Krah N.M. Seaward M.R. Willett K.L. Aderman C.M. Guerin K.I. Hua J. Lofqvist C. Hellstrom A. Smith L.E. The mouse retina as an angiogenesis model.Invest Ophthalmol Vis Sci. 2010; 51: 2813-2826Crossref PubMed Scopus (439) Google Scholar to examine the role of EYA in developmental angiogenesis. RT-PCR analysis for Eya1, Eya2, Eya3, and Eya4 in mouse retinal ECs showed a transcript for only Eya3 (Supplemental Figure S1). To investigate the cell-autonomous function of EYA3 in NV, we deleted Eya3 from ECs by using the loxP recombination strategy and the EC-specific tamoxifen-inducible Cre-recombinase Pdgfb-iCreER18Claxton S. Kostourou V. Jadeja S. Chambon P. Hodivala-Dilke K. Fruttiger M. Efficient, inducible Cre-recombinase activation in vascular endothelium.Genesis. 2008; 46: 74-80Crossref PubMed Scopus (218) Google Scholar (Eya3VEC-KO). The targeting strategy and the resulting reduction in EYA3 protein levels in endothelial cells is shown in Figure 1. The developing retinal vasculature was examined at P5 and P8 by using flat mounts and staining with isolectin B4. Endothelial deletion of Eya3 caused a significant reduction in the extension of the superficial vascular plexus at P5, as measured by the ratio of the vascular plexus radius to the radius of the total retina (Figure 2, A and B). Branching density was quantitated by counting the number of branch points per unit area of vasculature at the angiogenic front. On average, 24% fewer branch points were observed in the Eya3VEC-KO retinas relative to control littermates (Figure 2, C and D). Both EC proliferation and migration could contribute to the vascular defects observed here. Each of these was examined in retinas of Eya3VEC-KO and control cre-negative littermates at P5. The proliferation of stalk cells plays a key role in the angiogenic process at the vascular front.19Gerhardt H. Golding M. Fruttiger M. Ruhrberg C. Lundkvist A. Abramsson A. Jeltsch M. Mitchell C. Alitalo K. Shima D. Betsholtz C. VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia.J Cell Biol. 2003; 161: 1163-1177Crossref PubMed Scopus (2100) Google Scholar When using bromo-deoxyuridine (BrdU) incorporation by isolectin B4–positive cells at P5 as a measure of EC proliferation, we did not observe a significant difference between retinas from Eya3VEC-KO and their cre-negative littermates (Figure 2E). In retinas from Eya3VEC-KO pups, however, we observed a 27% reduction in the number of filopodia per millimeter of vessel length at the vascular front, suggesting a likely defect in EC migration (Figure 2F). Despite the early phenotype observed at P5, analysis of the superficial retinal vascular plexus at P8 shows recovery of both extension and branching defects in Eya3VEC-KO animals (Figure 3A), suggesting that lack of EYA3 only delays development of the superficial vascular layer. To elucidate the role of EYA in pathological angiogenesis we used a modification of the well-established OIR model20Smith 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 in which pups are subjected to hyperoxia (75% O2 between P7 and P12) and then returned to room air for 4 to 5 days before analysis. This modified protocol (85% O2 chamber between P8 and P11) was previously described in the study showing that NV requires the DDR-associated minor histone protein H2AX.6Economopoulou M. Langer H.F. Celeste A. Orlova V.V. Choi E.Y. Ma M. Vassilopoulos A. Callen E. Deng C. Bassing C.H. Boehm M. Nussenzweig A. Chavakis T. Histone H2AX is integral to hypoxia-driven neovascularization.Nat Med. 2009; 15: 553-558Crossref PubMed Scopus (109) Google Scholar This protocol was validated by the observation of characteristic regression of immature capillaries in the center of the retina leading to a central area of VO upon return to room air (P11-OIR) (Figure 3B). The resulting effective hypoxia leads to EC proliferation and the formation of neovascular tufts (P17-OIR) (Figure 3B), similar to that seen with the 5-day hyperoxia protocol. Maximum severity of NV, the proliferative phase, typically occurs 5 days after return to room air (P16 in this protocol and P17 in the 5-day protocol). Clinically, NV is considered pathological, whereas revascularization is desirable. Eya3fl/fl (control) and Eya3VEC-KO pups were subjected to hyperoxia from P8 to P11 and injected with tamoxifen from P11 to P13 to induce Eya3 deletion. NV at P17 was quantitated by using an automated protocol.15Stahl A. Connor K.M. Sapieha P. Willett K.L. Krah N.M. Dennison R.J. Chen J. Guerin K.I. Smith L.E. Computer-aided quantification of retinal neovascularization.Angiogenesis. 2009; 12: 297-301Crossref PubMed Scopus (116) Google Scholar Eya3VEC-KO retinas had nearly 40% less NV than did control retinas (Figure 3C). Interestingly, residual VO in the central avascular area was comparable in the control and Eya3VEC-KO retinas (Figure 3C). The OIR protocol is a VEGF-dependent model in which VEGF levels are suppressed during hyperoxia and elevated upon return to room air. Maximal VEGF levels are typically observed within 3 days after return to room air (P14 in the protocol used here) and remain high until P17. Interestingly, VEGF protein levels were comparable in both the control and Eya3VEC-KO retinas (Figure 3D), although reduced cell proliferation, as measured by BrdU incorporation, was seen in the Eya3VEC-KO retinas at P14 relative to the controls (Figure 3E). EYA proteins have multiple and separable biochemical activities (tyrosine phosphatase, threonine phosphatase, and transactivation).21Tadjuidje E. Hegde R.S. The eyes absent proteins in development and disease.Cell Mol Life Sci. 2013; 70: 1897-1913Crossref PubMed Scopus (100) Google Scholar We hypothesized that only the tyrosine phosphatase activity contributes to pathological angiogenesis through its role in DNA damage repair. To specifically target this enzymatic activity of the EYA proteins, we used a chemical biology approach. We have previously identified and validated EYA tyrosine phosphatase inhibitors that showed in vitro anti-angiogenic activity.11Pandey R.N. Wang T.S. Tadjuidje E. McDonald M.G. Rettie A.E. Hegde R.S. Structure-activity relationships of benzbromarone metabolites and derivatives as EYA inhibitory anti-angiogenic agents.PLoS One. 2013; 8: e84582Crossref PubMed Scopus (18) Google Scholar, 12Tadjuidje E. Wang T.S. Pandey R.N. Sumanas S. Lang R.A. Hegde R.S. The EYA tyrosine phosphatase activity is pro-angiogenic and is inhibited by benzbromarone.PLoS One. 2012; 7: e34806Crossref PubMed Scopus (34) Google Scholar Two of the best-characterized members of this inhibitor series, BBR and BZ, were used in these studies. These compounds show selectivity toward the EYA family of PTPs, and previous cell-based studies support their ability to target EYA3 in cells.12Tadjuidje E. Wang T.S. Pandey R.N. Sumanas S. Lang R.A. Hegde R.S. The EYA tyrosine phosphatase activity is pro-angiogenic and is inhibited by benzbromarone.PLoS One. 2012; 7: e34806Crossref PubMed Scopus (34) Google Scholar At P12 (24 hours after return to room air), each pup subjected to the OIR protocol was injected in one eye with an EYA inhibitor and in the other with a vehicle control. Retinas were dissected and flat-mounted at P17 and stained with isolectin B4 to visualize the vasculature (Figure 4A). There was significant normalization of the vasculature in the eyes treated with either BZ or BBR relative to the vehicle controls (54% and 49% reduction in NV, respectively) (Figure 4, A and B); however, VO was comparable to that in the vehicle-treated eye, suggesting a preferential effect on pathological NV (Figure 4C). Increased EC proliferation is known to contribute to NV as well as to vessel tortuosity in the OIR protocol.22Guaiquil V.H. Hewing N.J. Chiang M.F. Rosenblatt M.I. Chan R.V. Blobel C.P. A murine model for retinopathy of prematurity identifies endothelial cell proliferation as a potential mechanism for plus disease.Invest Ophthalmol Vis Sci. 2013; 54: 5294-5302Crossref PubMed Scopus (18) Google Scholar To determine whether EYA inhibitors had an effect on proliferation, we measured incorporation of the nucleotide analog BrdU. Low levels of BrdU incorporation were detected in control vehicle-treated OIR retinas in the proliferative phase (measured here at P14), with a 60% decrease in either the BZ- or BBR-treated retinas (Figure 4D). To establish the kinetics of DNA-damage repair subsequent to ischemic insult induced by the OIR protocol, we monitored γ-H2AX foci in the retina of mice at 48, 72, 96, 120, and 144 hours after return to room air. Widespread γ-H2AX foci containing nuclei were observed within 48 hours and peaked between 120 and 144 hours (P16 to P17); further analyses were conducted at P17 to coincide with evaluation of NV. Although DNA foci were generally more frequent in non-ECs, foci were also observed in ECs at sites of NV (Figure 5A), which is consistent with previous reports that DNA damage repair is coincident with proliferating ECs.6Economopoulou M. Langer H.F. Celeste A. Orlova V.V. Choi E.Y. Ma M. Vassilopoulos A. Callen E. Deng C. Bassing C.H. Boehm M. Nussenzweig A. Chavakis T. Histone H2AX is integral to hypoxia-driven neovascularization.Nat Med. 2009; 15: 553-558Crossref PubMed Scopus (109) Google Scholar Genetic and chemical biology tools were used to examine the effect of either Eya3 deletion or EYA-PTP inhibition on the presence of γ-H2AX foci at P17 as a marker of DNA damage repair. Eya3VEC-KO and control mice were subjected to the P8 to P11 OIR protocol, and tamoxifen was administered after return to room air. There was more than a 50% reduction in the number of nuclei with γ-H2AX foci in the retinas of the Eya3VEC-KO mice relative to in the retinas of their cre-negative littermates (Figure 5C). Similarly, retinas treated with the EYA inhibitors BZ or BBR had significantly fewer nuclei with γ-H2AX foci relative to the vehicle-treated controls, which is suggestive of an impaired DNA damage response (Figure 5, A and B). To better define the molecular mechanism(s) by which the EYAs might play a role in retinal angiogenesis, we used primary HRMECs. We have previously reported that the EYA-PTP inhibitors BZ and BBR inhibit human umbilical vein EC migration in vitro.12Tadjuidje E. Wang T.S. Pandey R.N. Sumanas S. Lang R.A. Hegde R.S. The EYA tyrosine phosphatase activity is pro-angiogenic and is inhibited by benzbromarone.PLoS One. 2012; 7: e34806Crossref PubMed Scopus (34) Google Scholar Similarly, BZ and BBR inhibited Transwell migration of HRMECs in a dose-dependent manner (Figure 6A), confirming a role for the EYA-PTP in the motility of retinal ECs as seen in the P5 retina. We next examined the effect of EYA-PTP inhibition on HRMEC proliferation under hypoxic conditions. As expected, HRMECs were more proliferative under hypoxic conditions (1% O2) than in 21% O2. Increased proliferation under hypoxic stress distinguishes ECs from most other cell types that undergo cell cycle arrest and apoptosis in hypoxic conditions because of stalled replication forks. Both BZ and BBR attenuated proliferation in a dose-dependent fashion (Figure 6B). Analogous results were previously reported for human umbilical vein endothelial cells transfected with siRNA targeting H2AX: H2AX was required to maintain proliferation only under hypoxic conditions.6Economopoulou M. Langer H.F. Celeste A. Orlova V.V. Choi E.Y. Ma M. Vassilopoulos A. Callen E. Deng C. Bassing C.H. Boehm M. Nussenzweig A. Chavakis T. Histone H2AX is integral to hypoxia-driven neovascularization.Nat Med. 2009; 15: 553-558Crossref PubMed Scopus (109) Google Scholar Because the EYA tyrosine phosphatase targets the C-terminal tyrosine 142 of H2AX, these observations were consistent with a model in which the tyrosine 142–dephosphorylated form of H2AX supports EC proliferation in hypoxia. ATM/ATR-dependent stabilization of stalled replication forks is generally initiated by formation of γ-H2AX foci.23Bencokova Z. Kaufmann M.R. Pires I.M. Lecane P.S. Giaccia A.J. Hammond E.M. ATM activation and signaling under hypoxic conditions.Mol Cell Biol. 2009; 29: 526-537Crossref PubMed Scopus (168) Google Scholar In ECs this process is ATR and H2AX dependent, permits survival and proliferation und
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