Pathophysiology and Mechanisms of Severe Retinopathy of Prematurity
2014; Elsevier BV; Volume: 122; Issue: 1 Linguagem: Inglês
10.1016/j.ophtha.2014.07.050
ISSN1549-4713
Autores Tópico(s)Retinal Diseases and Treatments
ResumoRetinopathy of prematurity (ROP) affects only premature infants, but as premature births increase in many areas of the world, ROP has become a leading cause of childhood blindness. Blindness can occur from aberrant developmental angiogenesis that leads to fibrovascular retinal detachment. To treat severe ROP, it is important to study normal developmental angiogenesis and the stresses that activate pathologic signaling events and aberrant angiogenesis in ROP. Vascular endothelial growth factor (VEGF) signaling is important in both physiologic and pathologic developmental angiogenesis. Based on studies in animal models of oxygen-induced retinopathy (OIR), exogenous factors such as oxygen levels, oxidative stress, inflammation, and nutritional capacity have been linked to severe ROP through dysregulated signaling pathways involving hypoxia-inducible factors and angiogenic factors like VEGF, oxidative species, and neuroprotective growth factors to cause phases of ROP. This translational science review focuses on studies performed in animal models of OIR representative of human ROP and highlights several areas: mechanisms for aberrant growth of blood vessels into the vitreous rather than into the retina through over-activation of VEGF receptor 2 signaling, the importance of targeting different cells in the retina to inhibit aberrant angiogenesis and promote physiologic retinal vascular development, toxicity from broad and targeted inhibition of VEGF bioactivity, and the role of VEGF in neuroprotection in retinal development. Several future translational treatments are discussed, including considerations for targeted inhibition of VEGF signaling instead of broad intravitreal anti-VEGF treatment. Retinopathy of prematurity (ROP) affects only premature infants, but as premature births increase in many areas of the world, ROP has become a leading cause of childhood blindness. Blindness can occur from aberrant developmental angiogenesis that leads to fibrovascular retinal detachment. To treat severe ROP, it is important to study normal developmental angiogenesis and the stresses that activate pathologic signaling events and aberrant angiogenesis in ROP. Vascular endothelial growth factor (VEGF) signaling is important in both physiologic and pathologic developmental angiogenesis. Based on studies in animal models of oxygen-induced retinopathy (OIR), exogenous factors such as oxygen levels, oxidative stress, inflammation, and nutritional capacity have been linked to severe ROP through dysregulated signaling pathways involving hypoxia-inducible factors and angiogenic factors like VEGF, oxidative species, and neuroprotective growth factors to cause phases of ROP. This translational science review focuses on studies performed in animal models of OIR representative of human ROP and highlights several areas: mechanisms for aberrant growth of blood vessels into the vitreous rather than into the retina through over-activation of VEGF receptor 2 signaling, the importance of targeting different cells in the retina to inhibit aberrant angiogenesis and promote physiologic retinal vascular development, toxicity from broad and targeted inhibition of VEGF bioactivity, and the role of VEGF in neuroprotection in retinal development. Several future translational treatments are discussed, including considerations for targeted inhibition of VEGF signaling instead of broad intravitreal anti-VEGF treatment. Retinopathy of prematurity (ROP) was described in 1942 by Terry1Terry T.L. Extreme prematurity and fibroblastic overgrowth of persistent vascular sheath behind each crystalline lens: (1) preliminary report.Am J Ophthalmol. 1942; 25: 203-204Abstract Full Text PDF Google Scholar as “retrolental fibroplasia,” which likely represents the most severe form of ROP, stage 5. Earlier stages of ROP were not well described because the Schepens/Pomerantzeff binocular indirect ophthalmoscope2Schepens C.L. A new ophthalmoscope demonstration.Trans Am Acad Ophthalmol Otolaryngol. 1947; 51: 298-301PubMed Google Scholar had not been adopted universally to examine the peripheral retina. To understand potential causes of ROP, investigators exposed newborn animals, which vascularize their retinas postnatally, to conditions similar to what human premature infants then experienced. From early studies in animals and later a clinical trial in human infants by Arnall Patz3Hartnett M.E. Penn J.S. Mechanisms and management of retinopathy of prematurity.N Engl J Med. 2012; 367: 2515-2526Crossref PubMed Scopus (327) Google Scholar, it became recognized that high oxygen at birth damaged fragile, newly formed retinal capillaries, causing “vaso-obliteration.” After animals were removed from supplemental oxygen to ambient air, “vasoproliferation” occurred at junctions of vascular and avascular retina. Thus, the 2-phase hypothesis was developed, almost 30 years before the classification of human ROP into zones and stages. With advances in neonatal care, including the ability to monitor and regulate oxygen, and in funduscopic imaging of the peripheral retina in preterm infants before the development of stage 5 ROP, several changes in our understanding of ROP occurred. First, the hypothesis of ROP has been revised in that there is a delay in physiologic retinal vascular development and some hyperoxia-induced, vasoattenuation in phase 1, followed by vasoproliferation into the vitreous as intravitreal neovascularization (IVNV) in phase 2 (Fig 1).3Hartnett M.E. Penn J.S. Mechanisms and management of retinopathy of prematurity.N Engl J Med. 2012; 367: 2515-2526Crossref PubMed Scopus (327) Google Scholar Second, it is recognized that the phenotype of ROP differs throughout the world in association with resources for prenatal care and oxygen regulation. Preterm infants of older gestational ages and larger birth weights than those screened in the United States now are demonstrating severe ROP in some regions with insufficient nutrition and neonatal or prenatal resources and care, and where high, unregulated oxygen is used.4Gilbert C. Retinopathy of prematurity: a global perspective of the epidemics, population of babies at risk and implications for control.Early Hum Dev. 2008; 84: 77-82Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar, 5Shah P.K. Narendran V. Kalpana N. Aggressive posterior retinopathy of prematurity in large preterm babies in South India.Arch Dis Child Fetal Neonatal Ed. 2012; 97: F371-F375Crossref PubMed Scopus (70) Google Scholar Finally, heritable causes are recognized as important,6Bizzarro M.J. Hussain N. Jonsson B. et al.Genetic susceptibility to retinopathy of prematurity.Pediatrics. 2006; 118: 1858-1863Crossref PubMed Scopus (107) Google Scholar but candidate gene studies often have been small and have not replicated findings potentially because of phenotypic variability. The International Classification of ROP describes stages and zones of ROP severity.7International Committee for the Classification of Retinopathy of PrematurityThe International Classification of Retinopathy of Prematurity revisited.Arch Ophthalmol. 2005; 123: 991-999Crossref PubMed Scopus (2130) Google Scholar Because human retinal vasculature is not complete until term birth, an infant born prematurely initially has incomplete vascular coverage of the retina. The zones of ROP define the area of retina covered by physiologic retinal vascularization. The stages often progress sequentially and describe the severity of disease. Stages 1 and 2 represent early ROP, and stage 3 represents the vascular phase in which IVNV occurs (Fig 1). Stages 4 and 5 ROP represent the fibrovascular phase with retinal detachment.8Hartnett M.E. Studies on the pathogenesis of avascular retina and neovascularization into the vitreous in peripheral severe retinopathy of prematurity (an American Ophthalmological Society thesis).Trans Am Ophthalmol Soc. 2010; 108: 96-119PubMed Google Scholar Laser treatment for severe ROP, now described as type 1 ROP in the Early Treatment for Retinopathy of Prematurity Study,9Early Treatment for Retinopathy of Prematurity Cooperative GroupRevised indications for the treatment of retinopathy of prematurity: results of the Early Treatment for Retinopathy of Prematurity randomized trial.Arch Ophthalmol. 2003; 121: 1684-1694Crossref PubMed Scopus (1518) Google Scholar can reduce the risk of a poor outcome in approximately 90% of eyes. In some infants, aggressive posterior ROP occurs, in which stage 3 and severe plus disease develops—without prior stages 1 or 2—in zone 1 or posterior zone 2. It is important to consider human retinal vascular development when studying what goes awry in ROP. Because of the difficulty in obtaining intact human preterm infant eyes, studies on human retinal vascular development have been limited, but reports indicate that the initial retinal vasculature develops through vasculogenesis in the posterior pole from precursor cells that migrate out of the deep retina and into inner layers.10Chan-Ling T. McLeod D.S. Hughes S. et al.Astrocyte-endothelial cell relationships during human retinal vascular development.Invest Ophthalmol Vis Sci. 2004; 45: 2020-2032Crossref PubMed Scopus (105) Google Scholar, 11McLeod D.S. Hasegawa T. Prow T. et al.The initial fetal human retinal vasculature develops by vasculogenesis.Dev Dyn. 2006; 235: 3336-3347Crossref PubMed Scopus (70) Google Scholar At approximately 15 weeks of gestation11McLeod D.S. Hasegawa T. Prow T. et al.The initial fetal human retinal vasculature develops by vasculogenesis.Dev Dyn. 2006; 235: 3336-3347Crossref PubMed Scopus (70) Google Scholar until at least 22 weeks of gestation, these precursors become angioblasts and form the inner vascular plexus that extends to approximately zone 1. After 22 weeks of gestation, when it is difficult to obtain fetal human tissue, the ensuing development of the vascular plexi is based on studies carried out in other species and believed to occur through budding angiogenesis, that is, the proliferation and growth of blood vessels from existing blood vessels. In several species, astrocytes sense a physiologic hypoxia12Chan-Ling T. Gock B. Stone J. The effect of oxygen on vasoformative cell division: evidence that “physiological hypoxia” is the stimulus for normal retinal vasculogenesis.Invest Ophthalmol Vis Sci. 1995; 36: 1201-1214PubMed Google Scholar and upregulate vascular endothelial growth factor (VEGF). Ensuing endothelial cells proliferate and migrate toward the gradient of VEGF and thereby extend the inner vascular plexus toward the ora serrata. Angiogenesis also is important in the development of the deep retinal plexi. Besides astrocytes, glia, like Müller cells, and neurons, such as ganglion cells, are also important.13Bai Y. Ma J.X. Guo J. et al.Müller cell-derived VEGF is a significant contributor to retinal neovascularization.J Pathol. 2009; 219: 446-454Crossref PubMed Scopus (175) Google Scholar, 14Jiang Y. Wang H. Culp D. et al.Targeting Muller cell–derived VEGF164 to reduce intravitreal neovascularization in the rat model of retinopathy of prematurity.Invest Ophthalmol Vis Sci. 2014; 55: 824-831Crossref PubMed Scopus (35) Google Scholar, 15Rivera J.C. Sapieha P. Joyal J.S. et al.Understanding retinopathy of prematurity: update on pathogenesis.Neonatology. 2011; 100: 343-353Crossref PubMed Scopus (88) Google Scholar The process is complex and requires interactions between different cell types and regulation of signaling pathways through several family members of VEGF and other pathways, including delta-like 4/notch and robo/slit, as examples, which regulate interactions between the sensing, endothelial tip cells and the proliferating stalk cells.16Gerhardt H. Golding M. Fruttiger M. et al.VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia.J Cell Biol. 2003; 161: 1163-1177Crossref PubMed Scopus (2075) Google Scholar Of all the factors involved in physiologic retinal vascular development, it is clear that VEGF is essential. It is not safe to experiment on human preterm infant eyes because of risks of bleeding and retinal detachment. Therefore, models of oxygen-induced retinopathy (OIR) are performed in animals that vascularize their retinas postnatally to study disease mechanisms. Most OIR models recreate only some aspects of human ROP. All models have limitations because they use newborn, instead of premature, animals. Newborn animals are healthy and do not have the comorbidities of human preterm infants, such as necrotizing enterocolitis, sepsis, bronchopulmonary dysplasia, shunting of oxygenated and deoxygenated blood, and immature lung development. Animals experience much higher arterial oxygen levels when given similar inspired oxygen levels as premature infants with these comorbidities. Neonatologists strive to avoid high oxygen in the perinatal period, but most animal models use high oxygen, making them less representative of human ROP. These are important considerations when choosing an OIR model to study a scientific question. The 2 most commonly used OIR models are in the mouse and rat. Also important is the beagle OIR model. None of these species is premature; rather, they complete retinal vascular development after term birth. The use of transgenic mice makes the mouse OIR model most helpful to study molecular mechanisms of high oxygen-induced vascular loss followed by regrowth of vessels either into the retina or into the vitreous during relative hypoxia.17Smith L.E. Wesolowski E. McLellan A. et al.Oxygen-induced retinopathy in the mouse.Invest Ophthalmol Vis Sci. 1994; 35: 101-111PubMed Google Scholar However, there are a few ways in which the model does not represent human ROP. First, oxygen levels used do not recreate what human preterm infants experience. The arterial oxygen (PaO2) in healthy newborn mice can approach very high levels (500 mmHg) with 75% inspired oxygen, oxygen levels that are avoided in preterm infants. Day 7 pups placed into 75% constant inspired oxygen experience vaso-obliteration of newly formed capillaries in the central retina and then are placed into room air and form intravitreal vascular buds at the junctions of vascular and avascular retina (Fig 2). Thus, the model is not similar to the phases of human ROP in that complete inner plexus vascularization has occurred already when the pups are placed into high oxygen, unlike the preterm infant whose retina is incompletely vascularized. Nonetheless, several signaling pathways important in human ROP have been identified using the mouse model. The model also may reflect aspects of ROP in the United States and the United Kingdom in the 1940s or in places currently that lack resources to regulate oxygen and provide prenatal and perinatal care.5Shah P.K. Narendran V. Kalpana N. Aggressive posterior retinopathy of prematurity in large preterm babies in South India.Arch Dis Child Fetal Neonatal Ed. 2012; 97: F371-F375Crossref PubMed Scopus (70) Google Scholar The most representative model of human ROP in the era of oxygen regulation is the rat OIR model, which has aspects of both vasoattenuation centrally and delayed physiologic retinal vascularization peripherally18Penn J.S. Henry M.M. Tolman B.L. Exposure to alternating hypoxia and hyperoxia causes severe proliferative retinopathy in the newborn rat.Pediatr Res. 1994; 36: 724-731Crossref PubMed Scopus (222) Google Scholar (Fig 2). Shortly after birth, pups and dams are placed into a controlled oxygen environment that changes inspired oxygen levels from 50% to 10% every 24 hours for 14 days. This oxygen profile recreates transcutaneous arterial oxygen extremes similar to those in a human preterm infant with severe ROP.19Cunningham S, Fleck BW, Elton RA, Mclntosh N. Transcutaneous oxygen levels in retinopathy of prematurity. Lancet 1995;346:1464-5.Google Scholar The notion of oxygen fluctuations, including intermittent episodes of hypoxia, has been associated with ROP.20Di Fiore J.M. Kaffashi F. Loparo K. et al.The relationship between patterns of intermittent hypoxia and retinopathy of prematurity in preterm infants.Pediatr Res. 2012; 72: 606-612Crossref PubMed Scopus (75) Google Scholar However, the duration of the fluctuations in oxygenation in the rat model is 24 hours, whereas in the human preterm infant, minute-to-minute fluctuations occur. The rat pups experience extrauterine growth restriction, a factor associated with severe ROP. The appearance of first delayed physiologic retinal vascular development followed by IVNV at the junction of vascular and avascular retina at day 18 is similar in appearance to type 1 severe ROP.9Early Treatment for Retinopathy of Prematurity Cooperative GroupRevised indications for the treatment of retinopathy of prematurity: results of the Early Treatment for Retinopathy of Prematurity randomized trial.Arch Ophthalmol. 2003; 121: 1684-1694Crossref PubMed Scopus (1518) Google Scholar Thus, the rat OIR model closely represents human preterm infants with severe ROP. The study of molecular mechanisms or potential treatments had been limited to pharmacologic manipulations in the rat, because the availability of transgenic rats is limited. Now, other techniques have been developed to permit study of molecular mechanisms in the rat. One example is the use of gene therapy to introduce short-hairpin RNAs (shRNAs) or genetic mutations to silence or knock out certain genes. Different viruses or viral vectors are used in gene therapy and include adeno-associated virus, adenovirus, or lentivirus, as examples. Several valuable aspects of a lentiviral vector are that it is not infectious (is self-inactivating) and has a large cargo-carrying capacity. The lentiviral vector cassette contains the only genetic cargo delivered into the genome to allow stable transgene expression. Using lentivirus, cell-specific promoters have been linked with shRNAs to target certain cell types in the retina and to knock-down specific gene products in those cells only. This has been a novel and useful technique to determine the effects of angiogenic signaling in pathologic and physiologic retinal angiogenesis from knockdown of genes in specific retinal cells and to assess safety on transduced and other cells within the retina.14Jiang Y. Wang H. Culp D. et al.Targeting Muller cell–derived VEGF164 to reduce intravitreal neovascularization in the rat model of retinopathy of prematurity.Invest Ophthalmol Vis Sci. 2014; 55: 824-831Crossref PubMed Scopus (35) Google Scholar, 21Wang H. Smith G.W. Yang Z. et al.Short hairpin RNA-mediated knockdown of VEGFA in Muller cells reduces intravitreal neovascularization in a rat model of retinopathy of prematurity.Am J Pathol. 2013; 183: 964-974Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 22Wang H. Yang Z. Jiang Y. et al.Quantitative analyses of retinal vascular area and density after different methods to reduce VEGF in a rat model of retinopathy of prematurity.Invest Ophthalmol Vis Sci. 2014; 55: 737-744Crossref PubMed Scopus (28) Google Scholar In addition, techniques to delete genes have been developed that will permit gene knockout in many species besides mice to study molecular events in various models. The beagle OIR model23Lutty G.A. McLeod D.S. Bhutto I. Wiegand S.J. Effect of VEGF Trap on normal retinal vascular development and oxygen-induced retinopathy in the dog.Invest Ophthalmol Vis Sci. 2011; 52: 4039-4047Crossref PubMed Scopus (67) Google Scholar is especially useful to translate drug doses from the puppy eye to the human preterm infant eye because of greater similarity in size between eyes of the puppy and preterm infant than between those of the preterm infant and newborn rodent. The newborn beagle retina initially vascularizes through a process of vasculogenesis that is followed by angiogenesis similar to what occurs in premature human infant retinas. However, the model uses high oxygen to cause OIR, which differs from the pathogenesis of ROP in most premature infants. In the beagle model, newborn postnatal day 1 pups are placed into 100% oxygen for 4 days and then into ambient air to recreate the phases of OIR.23Lutty G.A. McLeod D.S. Bhutto I. Wiegand S.J. Effect of VEGF Trap on normal retinal vascular development and oxygen-induced retinopathy in the dog.Invest Ophthalmol Vis Sci. 2011; 52: 4039-4047Crossref PubMed Scopus (67) Google Scholar When comparing the phases of OIR (Fig 2) with what occurs in human ROP (Fig 1), it is helpful to clarify definitions.3Hartnett M.E. Penn J.S. Mechanisms and management of retinopathy of prematurity.N Engl J Med. 2012; 367: 2515-2526Crossref PubMed Scopus (327) Google Scholar, 8Hartnett M.E. Studies on the pathogenesis of avascular retina and neovascularization into the vitreous in peripheral severe retinopathy of prematurity (an American Ophthalmological Society thesis).Trans Am Ophthalmol Soc. 2010; 108: 96-119PubMed Google Scholar Phase 1 in the rat OIR reflects the Early Phase of human ROP, that is, delayed physiologic retinal vascular development. Phase 2 in rat and mouse models of OIR reflect vasoproliferative IVNV, similar to the Vascular Phase of human stage 3 ROP with plus disease. However, human ROP also has a third Fibrovascular Phase, in which retinal detachment occurs in stages 4 and 5 ROP, and few animal models demonstrate this form of human ROP. However, the beagle OIR model shares some features seen in stage 4 ROP, such as retinal folding and dragging of vessels.23Lutty G.A. McLeod D.S. Bhutto I. Wiegand S.J. Effect of VEGF Trap on normal retinal vascular development and oxygen-induced retinopathy in the dog.Invest Ophthalmol Vis Sci. 2011; 52: 4039-4047Crossref PubMed Scopus (67) Google Scholar For clarity, the phases of OIR are described by the animal and phase. Phase 1 in the mouse is vaso-obliteration and in the rat is delayed physiologic retinal vascular development, and phase 2 is IVNV in both models. The 3 phases of human ROP are described as Early (delayed physiologic retinal vascular development and some vasoattenuation),3Hartnett M.E. Penn J.S. Mechanisms and management of retinopathy of prematurity.N Engl J Med. 2012; 367: 2515-2526Crossref PubMed Scopus (327) Google Scholar Vascular (IVNV) and Fibrovascular (retinal detachment; Fig 1). Most early investigations sought to understand causes of the vascular phase of human ROP by studying phase 2 OIR with IVNV, but several investigators24Saito 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 [serial online]. 2007; 13 (Available at:) (Accessed August 2, 2014): 840-853http://www.molvis.org/molvis/v13/a92/Google Scholar, 25Niesman M.R. Johnson K.A. Penn J.S. Therapeutic effect of liposomal superoxide dismutase in an animal model of retinopathy of prematurity.Neurochem Res. 1997; 22: 597-605Crossref PubMed Scopus (84) Google Scholar strove to understand the early phase of human ROP by studying phase 1 OIR. The thinking was that in facilitating vascularization of avascular retina, there would be less hypoxia-induced IVNV, and this line of thought aligned with clinical observations that infants with zone 1 ROP, compared with zone 2 ROP, were at greater risk of severe ROP developing and having poor outcomes.26Schaffer D.B. Palmer E.A. Plotsky D.F. et al.Prognostic factors in the natural course of retinopathy of prematurity.Ophthalmology. 1993; 100: 230-237Abstract Full Text PDF PubMed Scopus (281) Google Scholar Several exogenous stresses implicated in ROP—such as fluctuations in oxygenation, oxidative stress, nutritional factors, and poor infant growth—activate inflammatory, oxidative, and hypoxic signaling pathways.3Hartnett M.E. Penn J.S. Mechanisms and management of retinopathy of prematurity.N Engl J Med. 2012; 367: 2515-2526Crossref PubMed Scopus (327) Google Scholar Studies of phase 2 OIR focused on induced angiogenic factors from these activated signaling pathways. As with most biologic processes, it has become recognized that interactions and crosstalk exist within different signaling pathways. Hypoxia-inducible factors (HIFs) are transcription factors that bind DNA at the hypoxia-responsive element and enable transcription of a number of downstream genes that are angiogenic, including VEGF, angiopoietins, and erythropoietin, as examples. The classic mechanism involves hypoxia, which occurs in avascular retina as soon as a newborn pup is removed from supplemental oxygen and placed in ambient air. Hypoxia prevents HIFs from degradation by prolyl hydroxylases and thus allows them to translocate to the nucleus to cause angiogenic gene transcription.3Hartnett M.E. Penn J.S. Mechanisms and management of retinopathy of prematurity.N Engl J Med. 2012; 367: 2515-2526Crossref PubMed Scopus (327) Google Scholar Hypoxia-inducible factors also can be stabilized through oxidative compounds or inflammatory cytokines, mediated through NFkB, which can lead to downstream angiogenic effector compounds, including succinate or RTP801. Using mouse and rat OIR models, investigators studied prolyl hydroxylase inhibitors to stabilize HIF and promote physiologic retinal vascular development in phase 1 OIR models.27Sears J.E. Hoppe G. Ebrahem Q. Anand-Apte B. Prolyl hydroxylase inhibition during hyperoxia prevents oxygen-induced retinopathy.Proc Natl Acad Sci U S A. 2008; 105: 19898-19903Crossref PubMed Scopus (85) Google Scholar Others found that administration of HIF-induced growth factors, including erythropoietin or VEGF, reduced avascular retina in phase 1 OIR.3Hartnett M.E. Penn J.S. Mechanisms and management of retinopathy of prematurity.N Engl J Med. 2012; 367: 2515-2526Crossref PubMed Scopus (327) Google Scholar However, a potential concern with these strategies is that early and vascular phases of human ROP may not be sufficiently distinct in the individual preterm infant to determine a safe window of time to administer an angiogenic agonist to treat early ROP without causing vascular ROP. Oxidative stress has been proposed in ROP because of the susceptibility of the phospholipid-rich retina to reactive oxygen species that can be generated in high or low oxygen. Repeated oxygen fluctuations in the rat OIR model also lead to the generation of oxidative compounds. Although use of antioxidants, such as superoxide dismutase in liposomes25Niesman M.R. Johnson K.A. Penn J.S. Therapeutic effect of liposomal superoxide dismutase in an animal model of retinopathy of prematurity.Neurochem Res. 1997; 22: 597-605Crossref PubMed Scopus (84) Google Scholar or apocynin,24Saito 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 [serial online]. 2007; 13 (Available at:) (Accessed August 2, 2014): 840-853http://www.molvis.org/molvis/v13/a92/Google Scholar reduced avascular retina in phase 1 of the rat OIR model, these substances did not reduce IVNV in phase 2 in the rat OIR model. In addition, human clinical trials that tested n-acetyl cysteine, vitamin E, or lutein have not inhibited severe ROP successfully or safely to date.15Rivera J.C. Sapieha P. Joyal J.S. et al.Understanding retinopathy of prematurity: update on pathogenesis.Neonatology. 2011; 100: 343-353Crossref PubMed Scopus (88) Google Scholar, 28Wang H. Zhang S.X. Hartnett M.E. Signaling pathways triggered by oxidative stress that mediate features of severe retinopathy of prematurity.JAMA Ophthalmol. 2013; 131: 80-85Crossref PubMed Scopus (40) Google Scholar These findings may reflect the complexities in oxidative signaling and that reactive oxygen species can be damaging or beneficial to the retina. Besides direct interaction with the phospholipids in retina, some species act as signaling effectors that promote physiologic or pathologic events. Nitric oxide can be activated by nitric oxide synthetases, including endothelial nitric oxide synthetase, and can act as an endothelial relaxing agent in blood vessels, but in high oxygen, nitric oxide can form nitro-oxidative forms like peroxynitrite that lead to microvascular degeneration in phase 1 OIR. Oxidative stress can activate VEGF receptor 2 (VEGFR2) signaling that is needed in physiologic angiogenesis or overactivate VEGFR2 signaling in phase 2 OIR. In the immunocompromised preterm infant, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase can generate reactive oxygen species that defend against invading micro-organisms. However, NADPH oxidase–generated reactive oxygen species also can cause endothelial cell injury and avascular retina in phase 1 OIR through activation of isoforms NOX1 or NOX2 or can increase vasoproliferation in phase 2 OIR through activation of isoforms NOX1 or NOX2 or through NOX4-induced activation of the transcription factor, signal transducer and activator of transcription 3 (STAT3), in endothelial cells.28Wang H. Zhang S.X. Hartnett M.E. Signaling pathways triggered by oxidative stress that mediate features of severe retinopathy of prematurity.JAMA Ophthalmol. 2013; 131: 80-85Crossref PubMed Scopus (40) Google Scholar, 29Wang H. Yang Z. Jiang Y. Hartnett M.E. Endothelial NADPH oxidase 4 mediates vascular endothelial growth factor receptor 2-induced intravitreal neovascularization in a rat model of retinopathy of prematurity.Mol Vis [serial online]. 2014; 20 (Available at:) (Accessed August 2, 2014): 231-241http://www.molvis.org/molvis/v20/231/Google Scholar In contrast, activation of STAT3 in Müller cells inhibits the expression of erythropoietin and thus reduces angiogenesis in phase 1 OIR.30Wang H. Byfield G. Jiang Y. et al.VEGF-mediated STAT3 activation inhibits retinal vascularization by down-regulating local erythropoietin expression.Am J Pathol. 2012; 180: 1243-1253Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar Although exogenous erythropoieti
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