VEGF-Mediated STAT3 Activation Inhibits Retinal Vascularization by Down-Regulating Local Erythropoietin Expression

Avascular, hypoxic retina has been postulated to be a source of angiogenic factors that cause aberrant angiogenesis and intravitreal neovascularization (IVNV) in retinopathy of prematurity. Vascular endothelial growth factor (VEGF) is an important factor involved. However, VEGF is also required for normal retinal vascular development, which raises concerns about inhibiting its activity to treat IVNV in retinopathy of prematurity. Therefore, understanding the effects that VEGF has on other factors in the development of avascular retina is important to prevent aberrant angiogenesis and IVNV. Here, we show that STAT3 was activated by increased retinal VEGF in the rat 50/10 oxygen-induced retinopathy model. Phospho-STAT3 colocalized with glutamine synthetase-labeled Müller cells. Inhibition of STAT3 reduced avascular retina and increased retinal erythropoietin (Epo) expression. Epo administered exogenously also reduced avascular retina in the model. In an in vitro study, hypoxia-induced VEGF inhibited Epo gene expression by STAT3 activation in rat Müller cells. The mechanism by which activated STAT3 regulated Epo was by inhibition of Epo promoter activity. Together, these findings show that increased retinal VEGF contributes to avascular retina by regulating retinal Epo expression through Janus kinase/STAT signaling. Our results suggest that rescuing Epo expression in the retina before the development of IVNV may promote normal developmental angiogenesis and, therefore, reduce the stimulus for later pathologic IVNV. Avascular, hypoxic retina has been postulated to be a source of angiogenic factors that cause aberrant angiogenesis and intravitreal neovascularization (IVNV) in retinopathy of prematurity. Vascular endothelial growth factor (VEGF) is an important factor involved. However, VEGF is also required for normal retinal vascular development, which raises concerns about inhibiting its activity to treat IVNV in retinopathy of prematurity. Therefore, understanding the effects that VEGF has on other factors in the development of avascular retina is important to prevent aberrant angiogenesis and IVNV. Here, we show that STAT3 was activated by increased retinal VEGF in the rat 50/10 oxygen-induced retinopathy model. Phospho-STAT3 colocalized with glutamine synthetase-labeled Müller cells. Inhibition of STAT3 reduced avascular retina and increased retinal erythropoietin (Epo) expression. Epo administered exogenously also reduced avascular retina in the model. In an in vitro study, hypoxia-induced VEGF inhibited Epo gene expression by STAT3 activation in rat Müller cells. The mechanism by which activated STAT3 regulated Epo was by inhibition of Epo promoter activity. Together, these findings show that increased retinal VEGF contributes to avascular retina by regulating retinal Epo expression through Janus kinase/STAT signaling. Our results suggest that rescuing Epo expression in the retina before the development of IVNV may promote normal developmental angiogenesis and, therefore, reduce the stimulus for later pathologic IVNV. Retinopathy of prematurity (ROP) is a leading cause of childhood blindness.1Chen J. Smith L.E. Retinopathy of prematurity.Angiogenesis. 2007; 10: 133-140Crossref PubMed Scopus (491) Google Scholar It is characterized first by a delay in developmental retinal angiogenesis and potentially some capillary constriction, resulting in the clinical appearance of avascular retina and then later by dilated and tortuous retinal vessels and intravitreous neovascularization (IVNV), that is, blood vessels that proliferate into the vitreous. Several clinical studies have found an association between large areas of peripheral avascular retina and worse outcomes in ROP.2The Early Treatment for Retinopathy of Prematurity Cooperative GroupThe Early Treatment for Retinopathy Of Prematurity Study: structural findings at age 2 years.Br J Ophthalmol. 2006; 90: 1378-1382Crossref PubMed Scopus (73) Google Scholar, 3Schaffer D.B. Palmer E.A. Plotsky D.F. Metz H.S. Flynn J.T. Tung B. Hardy R.J. Prognostic factors in the natural course of retinopathy of prematurity.Ophthalmology. 1993; 100: 230-237Abstract Full Text PDF PubMed Scopus (289) Google Scholar Besides ROP, proliferative, uncontrolled angiogenesis occurs as a result of avascular retina in other conditions, including proliferative diabetic retinopathy and retinal vein occlusions.4Aiello L.P. Clinical implications of vascular growth factors in proliferative retinopathies.Curr Opin Ophthalmol. 1997; 8: 19-31Crossref PubMed Scopus (64) Google Scholar Currently, efforts have been focused on inhibiting aberrant angiogenesis in these conditions5Nicholson B. Schachat A. A review of clinical trials of anti-VEGF agents for diabetic retinopathy.Graefes Arch Clin Exp Ophthalmol. 2010; 248: 915-930Crossref PubMed Scopus (218) Google Scholar, 6Wolf-Schnurrbusch U.E. Ghanem R. Rothenbuehler S.P. Enzmann V. Framme C. Wolf S. Predictors of short-term visual outcome after anti-VEGF therapy of macular edema due to central retinal vein occlusion.Invest Ophthalmol Vis Sci. 2011; 52: 3334-3337Crossref PubMed Scopus (49) Google Scholar and less on reducing avascular retina,7Chen J. Connor K.M. Aderman C.M. Smith L.E. Erythropoietin deficiency decreases vascular stability in mice.J Clin Invest. 2008; 118: 526-533PubMed Google Scholar the causes of which are incompletely understood.8Lofqvist C. 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Google Scholar the of this we found that vascular endothelial growth factor of the angiogenic factors that causes aberrant retinal L.P. factors in diabetic mechanisms of for Ophthalmol PubMed Scopus Google Scholar, J. vascular endothelial growth factor in the of with proliferative diabetic J Ophthalmol. 118: Full Text PDF PubMed Scopus Google Scholar was increased in with avascular with in that vascularization of the retinal H. Hartnett M.E. of retinal vascular endothelial growth factor with avascular retina in a rat model of retinopathy of Ophthalmol. 2010; PubMed Scopus Google Scholar Therefore, a was the in retinal VEGF a in the of avascular Janus is a mechanism for and growth factor of binding and of cell which result in the activation of and of at on these by 2 then are on and then into the to gene and the in the 2010; PubMed Scopus Google Scholar resulting in cell and The mechanism of on binding and The activity of is by of a which with Therefore, to increased reduced of a Besides activated by a be by and effects of angiogenic as VEGF and J. S. S. T. J. H. R. H. and VEGF expression by growth PubMed Scopus Google Scholar that activation of and STAT3 the of retinopathy in a model of oxygen-induced retinopathy in S. Hartnett M.E. cause intravitreous neovascularization through in a model of retinopathy of Ophthalmol Vis Sci. PubMed Scopus Google Scholar In this study, we that activation of to the of avascular retina after by apoptosis inhibiting angiogenic factor expression. found that the angiogenic erythropoietin was by STAT3 activation and was in the of avascular with in vitro cell and the 50/10 model. animal in with the of and of for the and of and the for in and for the of in and A that regulates the an by was to in as J.S. to and causes proliferative retinopathy in the Res. PubMed Scopus Google Scholar of and into the which between and for in was and the by and between and for to in and weight of was found to be of other at the of is a that N. T. H. J.S. A. A. A. C.M. Inhibition of by a PubMed Scopus Google Scholar and was to the in Shaw S. H. M.B. activation of and in diabetic rat J PubMed Scopus Google Scholar inhibition angiogenesis and to avascular retina in the 50/10 model with in in by was on studies that an effect on IVNV in the 50/10 model in S. Hartnett M.E. cause intravitreous neovascularization through in a model of retinopathy of Ophthalmol Vis Sci. 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Inhibition of NAD(P)H oxidase reduces apoptosis and avascular retina in an animal model of retinopathy of prematurity.Mol Vis. 2007; 13: 840-853PubMed Google Scholar we found that inhibition of oxidase activation by reduced avascular retina and retinal a of the and of In with and Müller in the postulated that loss of these interfere with normal retinal vascular development and avascular retina by reducing endothelial cell growth factor expression. also found for endothelial cell apoptosis with of and therefore, to avascular retina in the 50/10 model by and the in the 2010; PubMed Scopus Google Scholar retinal and found between and rat The in 2 not apoptosis VEGF expression as of activated avascular therefore, other Epo has been to be important in retinal J. Connor K.M. Aderman C.M. Smith L.E. Erythropoietin deficiency decreases vascular stability in mice.J Clin Invest. 2008; 118: 526-533PubMed Google Scholar and has been with function later in V. Moseley and and function in Full Text Full Text PDF PubMed Scopus Google Scholar However, Epo has also been to cause pathologic IVNV in T. S. N. S. S. T. Y. is a factor in retinopathy of prematurity in association with J. 22: PubMed Scopus Google Scholar and, to treat of prematurity in Epo has been with Liu A. P. S. erythropoietin and the of retinopathy of a 2008; Full Text Full Text PDF PubMed Scopus Google Scholar STAT3 activation the expression of Epo in the we retinal Epo in in the 50/10 model that been administered found increased retinal Epo with Epo has been as in the and in other of J.E. 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VEGF and expression after a of between and to clinical Vis. 10: Google Scholar therefore, postulated that which was in the 50/10 to Epo expression. we administered a antibody to as an intravitreal at to of in the 50/10 model. The and of the found to reduce VEGF and P. Peterson L.J. Martiniuk D. Uppal A. Saito Y. Hartnett M.E. 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NAD(P)H oxidase neovascularization of VEGF in retinopathy of prematurity Ophthalmol Vis Sci. 2008; PubMed Scopus (73) Google Scholar and Müller cell Therefore, we postulated that Epo gene expression was regulated by hypoxia-induced STAT3 activation in Müller cells. this to for in with was increased in in and a in Epo gene expression was also The of Epo in was by by with a antibody to However, in the of and VEGF Epo was in and was increased with and VEGF antibody the that Epo gene expression was by STAT3 activation in and also that be in the of Epo gene expression in STAT3 activation was STAT3 is a STAT3 the and gene by binding to of A the promoter to of the rat Epo gene to the STAT3 binding the that this STAT3 binding was in the of the Epo we first STAT3 was of binding to this the promoter of the Epo gene with the of a of the that in STAT3 to the promoter of the Epo gene However, in the of VEGF hypoxia-induced to the Epo promoter was reduced with However, some to the Epo potentially STAT3 activation was not inhibited by and the VEGF suggest that the binding of to the Epo promoter is by STAT3 the STAT3 binding was in the inhibition of Epo gene A gene the STAT3 binding the promoter of the rat Epo gene to was with a rat into for at In the of VEGF was to the and with a with VEGF inhibited Epo promoter activity. Treatment with VEGF and the activity of the Epo effect on Epo gene these results are with findings in we that after causes an in Müller cell VEGF expression that activation of and activated STAT3 Epo gene expression by binding to and inhibiting the activity of the Epo ROP is a that M.J. N. B. R. H. V. to retinopathy of prematurity.Pediatrics. 2006; 118: PubMed Scopus Google Scholar and including in and and R. P. J. A. Retinopathy of prematurity and 2006; PubMed Scopus Google Scholar, S. Inhibition of NAD(P)H oxidase activity vascular endothelial growth factor and neovascularization during J Full Text Full Text PDF PubMed Scopus Google Scholar avascular retina and IVNV are in It has been that retinal avascular retina by a delay in retinal vascular development and of retinal to pathologic angiogenesis in A. A. T. studies in J Ophthalmol. Full Text PDF PubMed Scopus Google Scholar, The of development of the vascular of the retina some on its for retinal Scholar, N. B. of on retinal vessels with to the of J Ophthalmol. PubMed Scopus Google Scholar Our has been in the causes of avascular have focused studies on effects of and a J.S. retinopathy in the of retinal to Ophthalmol Vis Sci. Google Scholar that to to with Our studies and other have that VEGF L.P. E.A. H. Chen H. N. Smith L.E. of retinal neovascularization in by inhibition of vascular endothelial growth factor (VEGF) Natl Acad Sci U S A. PubMed Scopus Google Scholar, M.E. Martiniuk Geisen P. Neutralizing VEGF decreases and endothelial cell in and in rat model of to Ophthalmol Vis Sci. 2008; PubMed Scopus (85) Google Scholar oxidase P. Peterson L.J. Martiniuk D. Uppal A. Saito Y. Hartnett M.E. Neutralizing antibody to VEGF reduces intravitreous neovascularization and does not interfere with vascularization of avascular retina in an ROP model.Mol Vis. 2008; 14: 345-357PubMed Google Scholar, Y. Uppal A. S. Hartnett M.E. NAD(P)H oxidase neovascularization of VEGF in retinopathy of prematurity Ophthalmol Vis Sci. 2008; PubMed Scopus (73) Google Scholar, S. Inhibition of NAD(P)H oxidase activity vascular endothelial growth factor and neovascularization during J Full Text Full Text PDF PubMed Scopus Google Scholar and S. Hartnett M.E. cause intravitreous neovascularization through in a model of retinopathy of Ophthalmol Vis Sci. PubMed Scopus Google Scholar, D. T. M.B. VEGF STAT3 in endothelial J. PubMed Scopus Google Scholar are important in the pathologic angiogenesis in the of studies that the 50/10 we found that in increased retinal VEGF in association with peripheral avascular retina before the development of H. Hartnett M.E. of retinal vascular endothelial growth factor with avascular retina in a rat model of retinopathy of Ophthalmol. 2010; PubMed Scopus Google Scholar the increased VEGF was to retinal vascularization IVNV. In this study, a of that the 50/10 model was to increased VEGF by retinal vascularization and the mechanisms of in an delay in retinal an increased of avascular retina with and a activation in retinal STAT3 not other as and found that mechanism for increased avascular retinal in the 50/10 model was by Y. Geisen P. Uppal A. Hartnett M.E. Inhibition of NAD(P)H oxidase reduces apoptosis and avascular retina in an animal model of retinopathy of prematurity.Mol Vis. 2007; 13: 840-853PubMed Google Scholar Therefore, we postulated that apoptosis of vascular be in the of avascular also that VEGF be is an angiogenic growth factor important in retinal vascular T. B. J. The effect of on cell that is the stimulus for normal retinal Ophthalmol Vis Sci. Google Scholar, J. A. T. J. H. Chan-Ling T. of retinal is by hypoxia-induced vascular endothelial growth factor (VEGF) expression by PubMed Google Scholar inhibiting reduce However, we found the avascular retina of apoptosis VEGF expression. However, an important factor in retinal capillary J. Connor K.M. Aderman C.M. Smith L.E. Erythropoietin deficiency decreases vascular stability in mice.J Clin Invest. 2008; 118: 526-533PubMed Google Scholar was reduced by STAT3 It also that the inhibition of Epo was to the retina and not of reduced in by inhibition of signaling. The of Epo in the has been with of between of erythropoietin and for retinopathy of 2006; 10: Full Text Full Text PDF PubMed Scopus Google Scholar However, also has effects as a in that children Epo as found to on D. B. R. of erythropoietin and developmental outcomes in PubMed Scopus Google Scholar Epo was to retinal endothelial in a model of J.E. B. inhibition during oxygen-induced Natl Acad Sci U S A. 2008; PubMed Scopus Google Scholar in the to Epo at a reduced the of later J. Connor K.M. Aderman C.M. Smith L.E. of retinal neovascularization by erythropoietin in a model of proliferative Ophthalmol Vis Sci. PubMed Scopus Google Scholar Therefore, as to the of Epo in prematurity and its effects in increased as an angiogenic retinal Epo was to in the 50/10 model at and by Our results show that with increased Epo retinal vascularization and reduced avascular retinal the that inhibition of retinal Epo by in the 50/10 model be a cause of retinal vascularization and, therefore, increased avascular is a for a of growth factors and The that VEGF was increased in the 50/10 model to STAT3 activation reduce Epo expression. found to this after a antibody into the of in the 50/10 model. colocalized with Müller in of In we STAT3 at the and which are for STAT3 and Y. Y. S. Y. of STAT3 for vascular endothelial growth endothelial cell and PubMed Scopus Google Scholar, A. S. C. C. of in retinal J. 2006; PubMed Scopus Google Scholar, J.S. K.M. The Sci. PubMed Scopus Google Scholar Epo gene expression by in which hypoxia-induced Epo T. S. N. S. S. T. Y. is a factor in retinopathy of prematurity in association with J. 22: PubMed Scopus Google Scholar and may that Epo gene expression to cell and the activation of signaling. a STAT3 regulates gene expression by the promoter of In this the effect of activated STAT3 by VEGF on Epo gene expression may the of STAT3 to the Epo a STAT3 binding at a in the promoter of the Epo gene to show by in to STAT3 on the promoter of the Epo gene the STAT3 binding of STAT3 to the Epo promoter was reduced to that of in the of VEGF that the binding of STAT3 to the Epo promoter is by on and that activated is of binding to the promoter of the Epo that the Epo promoter activity was by and on the activation of The results that the effect of on Epo gene the Epo A may also be to the promoter of the Epo In we of STAT3 regulates its binding to the Epo promoter and the effects on of In we that an in retinal to to pathologic avascular retina and IVNV in the 50/10 STAT3 activation and inhibited retinal Epo expression. Epo at in the before the development of to reduced avascular However, the for Epo as not to the of pathologic as studies that the A. S. C. C. of in retinal J. 2006; PubMed Scopus Google Clinical studies have a in ROP after inhibition of VEGF with a of for of retinopathy of prematurity in 2008; PubMed Scopus Google Scholar However, the is in the course of ROP, and avascular retina a In the 50/10 and antibody retinal IVNV effect on avascular P. Peterson L.J. Martiniuk D. Uppal A. Saito Y. Hartnett M.E. Neutralizing antibody to VEGF reduces intravitreous neovascularization and does not interfere with vascularization of avascular retina in an ROP model.Mol Vis. 2008; 14: 345-357PubMed Google Scholar, S. Martiniuk D. Geisen P. Hartnett M.E. in endothelial cell to reduced intravitreous not vascularization in a model of Res. PubMed Scopus Google Scholar, H. Y. Y. Y. S. T. H. N. of of antibody vascular endothelial growth factor on the model of Sci. PubMed Scopus Google Scholar Our findings suggest that inhibiting VEGF may not be to promote retinal vascularization and that other angiogenic as may be However, and is to Epo expression during to later of IVNV.