Vascular Endothelial Cell Growth Factor-A: Not Just for Endothelial Cells Anymore

Abstract

When vascular endothelial cell growth factor-A (VEGF-A) was first identified as an endothelial cell mitogen and vascular permeability factor, it was thought to be a specific mitogen for endothelial cells, and its main signaling receptors, VEGFR2/Flk1/KDR and VEGFR1/Flt-1, were reported to be expressed solely by the endothelium. On what was this claim based? First, early studies with newly described growth factors are usually focused almost exclusively in the initial field of discovery (eg, VEGF-A and the vasculature). Second, in the early days of a growth factor's characterization, reagents are limiting—they may not be very accessible or of particularly good quality. Once a growth factor is better characterized and reagents improve in quality and sensitivity (eg, antisera for immunohistochemical localization of receptors and development of gene-reporter transgenic mice), more comprehensive and reliable analyses of expression and actions are possible. Finally, and perhaps more importantly, growth factors are often named for the activity on which they were first identified: for VEGF-A, its ability to induce vascular endothelial cell proliferation. The naming of a factor to reflect its identifying activity is an historical pattern in the designation of peptide growth factors and cytokines including, for example, fibroblast growth factor, epidermal growth factor, and hepatocyte growth factor. It seems then that the simple labeling of a factor limits, at least initially, consideration of activities outside of that implied by its name. For VEGF-A, the belief that its activities were specific to the vasculature were likely to have been further enforced by the observation of its critical and central role during vascular development: targeted disruption of even a single VEGF-A allele leads to the total disruption of vascular development and early embryologic lethality.1Carmeliet P Ferriera V Breier G Pollefeyt S Kieckens L Gertsenstein M Fahrig M Vandenhoeck A Harpal K Eberhardt C Declercq C Pawling J Moons L Collen D Risau W Nagy A Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele.Nature. 1996; 380: 435-439Crossref PubMed Scopus (3427) Google Scholar, 2Ferrara N Carver-Moore K Chen H Dowd M Lu L O'Shea K Powell-Braxton L Hillan KJ Moore MW Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene.Nature. 1996; 380: 439-442Crossref PubMed Scopus (3021) Google ScholarPerhaps, then, it was just a matter of time before the pluripotent nature of VEGF-A began to be revealed. The most significant findings outside of the vasculature relates to the role of VEGF-A in the neural system. VEGF-A has been previously demonstrated to be neuroprotective in models of ischemic/hypoxic injury in the central nervous system (see reviews3Zachary I Neuroprotective role of vascular endothelial growth factor: signalling mechanisms, biological function, and therapeutic potential.Neurosignals. 2005; 14: 207-221Crossref PubMed Scopus (239) Google Scholar, 4Jin KL Mao XO Greenberg DA Vascular endothelial growth factor: direct neuroprotective effect in in vitro ischemia.Proc Natl Acad Sci USA. 2000; 97: 10242-10247Crossref PubMed Scopus (631) Google Scholar) and now in the retina, as described in the article by Nishijima et al5Nishijima K Ng Y-S Zhong L Bradley J Shubert W Jo N Akita J Sameulsson SJ Robinson GS Adamis AP Shima DT Vascular endothelial growth factor-A is a survival factor for retinal neurons and a critical neuroprotectant during the adaptive response to ischemic injury.Am J Pathol. 2007; 171: 53-67Abstract Full Text Full Text PDF PubMed Scopus (573) Google Scholar in this issue of The American Journal of Pathology. A variety of activities have been suggested to account for the observed neuroprotection, including enhancement of axonal regeneration,6Sondell M Lundborg G Kanje M Vascular endothelial growth factor has neurotrophic activity and stimulates axonal outgrowth, enhancing cell survival and Schwann cell proliferation in the peripheral nervous system.J Neurosci. 1999; 19: 5731-5740Crossref PubMed Google Scholar inhibition of caspase-3, and enhancement of neurogenesis in the subventricular and subgranular zones7Jin K Mao XO Batteur SP McEachron E Leahy A Greenberg DA Caspase-3 and the regulation of hypoxic neuronal death by vascular endothelial growth factor.Neuroscience. 2001; 108: 351-358Crossref PubMed Scopus (146) Google Scholar as well as activation of Akt.8Wick A Wick W Waltenberger J Weller M Dichgans J Schulz JB Neuroprotection by hypoxic preconditioning requires sequential activation of vascular endothelial growth factor receptor and Akt.J Neurosci. 2002; 22: 6401-6407Crossref PubMed Google Scholar In addition, decreased VEGF-A levels via deletion of the hypoxia-response element in the VEGF-A promoter led to progressive degeneration of motor neurons due, at least in part, to reduced action on VEGFR2-expressing motor neurons.9Oosthuyse B Moons L Storkebaum E Beck H Nuyens D Brusselmans K Van Dorpe J Hellings P Gorselink M Heymans S Theilmeier G Dewerchin M Laudenbach V Vermylen P Raat H Acker T Vleminckx V Van Den Bosch L Cashman N Fujisawa H Drost MR Sciot R Bruyninckx F Hicklin DJ Ince C Gressens P Lupu F Plate KH Robberecht W Herbert JM Collen D Carmeliet P Deletion of the hypoxia-response element in the vascular endothelial growth factor promoter causes motor neuron degeneration.Nat Genet. 2001; 28: 131-138Crossref PubMed Scopus (890) Google Scholar Despite this growing body of data demonstrating significant effects of VEGF-A on neural cells, very little is known about the role of VEGF-A in neural retina.Considering the widespread use of VEGF-A-neutralizing therapies in the treatment of macular edema and choroidal neovascularization associated with the wet form of age-related macular degeneration (AMD),10Rosenfeld PJ Brown DM Heier JS Boyer DS Kaiser PK Chung CY Kim RY Ranibizumab for neovascular age-related macular degeneration.N Engl J Med. 2006; 355: 1419-1431Crossref PubMed Scopus (4683) Google Scholar the lack of knowledge regarding the role of VEGF-A on neural retina represents a particularly serious information gap. VEGF-A clearly plays a role in the developmental vascularization of the retina, where it both directs angiogenic ingrowth11Stone 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 and acts as a survival factor.12Alon T Hemo I Itin A Pe'er J Stone J Keshet S 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 (1406) Google Scholar A few early reports also suggested a nonvascular role for VEGF-A in the retina. Retinal progenitors express VEGFR2,13Yang 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 and exogenously added VEGF-A induces the differentiation of photoreceptor cells in vitro14Yourey 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 as well as neurite outgrowth from postnatal retinal explants.15Böcker-Meffert S Rosenstiel P Rohl C Warneke N Held-Feindt J Sievers J Lucius R Erythropoietin and VEGF promote neural outgrowth from retinal explants in postnatal rats.Invest Ophthalmol Vis Sci. 2002; 43: 2021-2026PubMed Google Scholar In the adult retina, VEGF-A is produced by pericytes and astrocytes,16Darland DC Massingham LJ Smith SR Piek E Saint-Geniez M D'Amore PA Pericyte production of cell-associated VEGF is differentiation-dependent and is associated with endothelial survival.Dev Biol. 2003; 264: 275-288Crossref PubMed Scopus (314) Google Scholar Müller glial cells, amacrine cells, and ganglion cells17Famiglietti EV Stopa EG McGookin ED Song P LeBlanc V Streeten BW Immunocytochemical localization of vascular endothelial growth factor in neurons and glial cells of human retina.Brain Res. 2003; 969: 195-204Crossref PubMed Scopus (79) Google Scholar as well as by the retinal pigment epithelial cells.18Adamis AP Shima DT Yeo K-T Yeo T-K Brown LF Berse B D'Amore PA Folkman J Synthesis and secretion of vascular permeability factor/vascular endothelial growth factor by human retinal pigment epithelial cells.Biochem Biophys Res Commun. 1993; 193: 631-638Crossref PubMed Scopus (371) Google Scholar However, data regarding a function for VEGF-A in the adult—on either vascular or nonvascular cells—are lacking.In the article by Nishijima et al5Nishijima K Ng Y-S Zhong L Bradley J Shubert W Jo N Akita J Sameulsson SJ Robinson GS Adamis AP Shima DT Vascular endothelial growth factor-A is a survival factor for retinal neurons and a critical neuroprotectant during the adaptive response to ischemic injury.Am J Pathol. 2007; 171: 53-67Abstract Full Text Full Text PDF PubMed Scopus (573) Google Scholar in this issue of the AJP, this topic is addressed with the demonstration of a very compelling and timely role for VEGF-A in neural retinal survival. In a retinal model of ischemia-reperfusion injury, the authors demonstrate a dose-dependent ability of VEGF-A to prevent neural cell death. They further show that ischemic preconditioning leads to elevated VEGF-A, which in turn decreases neural retinal cell death, and that blocking VEGF-A in this model leads to increased neural cell death. Finally, the authors report that neutralization of VEGF-A by both systemic and local delivery of inhibitors for 8 weeks leads to retinal ganglion cell death. The report by Nishijima et al5Nishijima K Ng Y-S Zhong L Bradley J Shubert W Jo N Akita J Sameulsson SJ Robinson GS Adamis AP Shima DT Vascular endothelial growth factor-A is a survival factor for retinal neurons and a critical neuroprotectant during the adaptive response to ischemic injury.Am J Pathol. 2007; 171: 53-67Abstract Full Text Full Text PDF PubMed Scopus (573) Google Scholar highlights two evolving stories that relate to the biological activity of VEGF-A: first, that it acts on cells outside of the vasculature, and second, that it plays a significant role in the adult.Effect of VEGF-A on Nonvascular CellsA growing number of reports point to a major role for VEGF-A on nonvascular cells. VEGFR1 and/or VEGFR2 have been reported to be expressed by a variety of nonvascular cell types, including type 2 pneumocytes, monocytes, mast cells, megakaryocytes, dendritic cells, hematopoietic stem cells, lens epithelial cells, and a variety of neural cells. Despite these observations, data indicating a direct biological effect of VEGF-A on nonvascular cells have been relatively slow to accumulate. The exception is the nervous system, where there is now significant evidence indicating both neurotrophic and neuroprotective effects on neural cells both in vitro and in vivo (see review3Zachary I Neuroprotective role of vascular endothelial growth factor: signalling mechanisms, biological function, and therapeutic potential.Neurosignals. 2005; 14: 207-221Crossref PubMed Scopus (239) Google Scholar). VEGF-A has been shown to act on astrocytes and microglia, as well as a variety of neurons (sensory, cortical, hippocampal, etc), with neurogenic, neuroprotective, and/or neurotrophic effects. In addition, VEGF-A induces the proliferation of neural progenitors in parts of the brain capable of neurogenesis (hippocampus, olfactory, and subventricular zone). Despite quite a large body of literature describing the neuroprotective and neurotrophic effects in the brain, parallel studies regarding VEGF-A effects in the retina had not been reported. However, the fact that the retina is an extension of the central nervous system supports the idea that neural retina might also be a target for VEGF-A.Interpreting an effect of VEGF-A manipulation (either addition or subtraction) as being directly on the nonvascular cell is complicated by the role of VEGF-A in the vasculature of the tissue of interest. Thus, two specific criteria should be met before such a conclusion can be drawn. First, it is important to demonstrate the expression of VEGF-A receptors (VEGFR1 and/or VEGFR2) on the putative target cells. Although in vitro studies providing such evidence would be suggestive, it is also the case that establishment of cells in culture often leads to the expression of genes not observed in vivo.19Shima DT Saunders KB Gougos A D'Amore PA Alterations in gene expression associated with changes in the state of endothelial differentiation.Differentiation. 1995; 58: 217-226Crossref PubMed Scopus (20) Google Scholar That said, direct demonstration of VEGF-A receptor expression by immunohistochemistry in the cells of interest in vivo would be critical to conclude convincingly that VEGF-A could act directly on the nonvascular cells. Nishijima and coworkers5Nishijima K Ng Y-S Zhong L Bradley J Shubert W Jo N Akita J Sameulsson SJ Robinson GS Adamis AP Shima DT Vascular endothelial growth factor-A is a survival factor for retinal neurons and a critical neuroprotectant during the adaptive response to ischemic injury.Am J Pathol. 2007; 171: 53-67Abstract Full Text Full Text PDF PubMed Scopus (573) Google Scholar demonstrate the presence of VEGFR2 by both indirect and direct methods. Administration of VEGF-E, a ligand for VEGFR2, reduced cell death in a model of ischemia-reperfusion, whereas placental-derived growth factor, which does not bind VEGFR2, did not prevent cell death, providing evidence of a neuroprotective role for VEGFR2 signaling. In a more direct demonstration, they used immunohistochemistry to demonstrate the presence of VEGFR2, not only on endothelial cells but also on a population of nonendothelial cells. Although they did not investigate the identity of these cells further by immunohistochemistry, some of them are probably neuronal cells of the retina.A second requirement is the demonstration, in the absence of a vascular supply, of a relevant biological effect on the target nonvascular cells. Nishijima and his coworkers5Nishijima K Ng Y-S Zhong L Bradley J Shubert W Jo N Akita J Sameulsson SJ Robinson GS Adamis AP Shima DT Vascular endothelial growth factor-A is a survival factor for retinal neurons and a critical neuroprotectant during the adaptive response to ischemic injury.Am J Pathol. 2007; 171: 53-67Abstract Full Text Full Text PDF PubMed Scopus (573) Google Scholar addressed the second of these criteria by culturing a fragment of the neonatal (and as yet unvascularized) rat retina in culture. In the absence of added factors, there was significant retinal ganglion cell death, which was prevented by the addition of exogenous VEGF-A. This definitive effect, in the absence of a vascular supply, provides strong evidence for a direct role of VEGF-A on neural retinal cells.Role of VEGF-A in the AdultEarly reports suggested that the actions of VEGF-A were limited to developmental and postnatal periods. One report used a mouse model of inducible Cre to systemically delete VEGF-A as well as systemic VEGF-A neutralization and concluded that “the dependence on VEGF-A was lost sometime after the 4th postnatal week.”20Gerber H-P Hillan KJ Ryan AM Kowalski J Keller GA Rangell L Wright BD Radtke F Aguet M Ferrara N VEGF is required for growth and survival in neonatal mice.Development. 1999; 126: 1149-1159Crossref PubMed Google Scholar However, the continued expression of VEGF-A in virtually all adult tissues, in the absence of active angiogenesis, suggests otherwise.21Ng Y-S Rohan R Sunday M deMello DE D'Amore PA Differential expression of VEGF isoforms in mouse during development and in the adult.Dev Dyn. 2001; 220: 112-121Crossref PubMed Scopus (319) Google Scholar Indeed, a series of studies using a variety of approaches to block the action of VEGF-A in adult mice has revealed a dramatic dependence of the microvasculature on local exogenous VEGF-A. The neutralization of VEGF-A led to the disappearance of fenestrations, specializations that characterize the microvasculature of secretory and filtering organs, as well as to regression of capillaries.22Kamba T Tam BY Hashizume H Haskell A Sennino B Mancuso MR Norberg SM O'Brien SM Davis RB Gowen LC Anderson KD Thurston G Joho S Springer ML Kuo CJ McDonald DM VEGF-dependent plasticity of fenestrated capillaries in the normal adult microvasculature.Am J Physiol. 2006; 290: H560-H576Crossref PubMed Scopus (646) Google Scholar, 23Baffert F Le T Sennino B Thurston G Kuo CJ Hu-Lowe D McDonald DM Cellular changes in normal blood capillaries undergoing regression after inhibition of VEGF signaling.Am J Physiol. 2006; 290: H547-H559Google ScholarThe consequences of systemic VEGF-A neutralization in the adult human can be seen in preeclampsia, a pathology of pregnancy that is characterized by elevated levels of soluble VEGFR1 (sFlt-1) (see review24Lam C Lim K-H Karumanchi SA Circulating angiogenic factors in the pathogenesis and prediction of preeclampsia.Hypertension. 2005; 46: 1077-1085Crossref PubMed Scopus (306) Google Scholar). The hallmarks of preeclampsia, hypertension and proteinuria, are at least partially the result of the blockade of VEGF-A on vascular endothelial cells, the hypertension resulting from diminished nitric oxide, and the proteinuria from the disruption of the paracrine interactions between podocyte-derived VEGF-A and its target glomerular endothelium. Although it is tempting to extrapolate these findings to what might be observed in therapeutic VEGF-A neutralization, it is important to keep in mind that preeclampsia is distinct in several ways, including the complex physiological effects of pregnancy, the fact that sFlt1 is not specific for VEGF-A (sFlt1 also binds placental-derived growth factor), and the recent finding that preeclampsia is also associated with elevated soluble endoglin.25Venkatesha S Toporsian M Lam C Hanai J Mammoto T Kim YM Bdolah Y Lim KH Yuan HT Libermann TA Stillman IE Roberts D D'Amore PA Epstein FH Sellke FW Romero R Sukhatme VP Letarte M Karumanchi SA Soluble endoglin contributes to the pathogenesis of preeclampsia.Nat Med. 2006; 12: 642-649Crossref PubMed Scopus (1438) Google Scholar In contrast, the therapeutic inhibition of VEGF-A for antitumor angiogenesis is achieved with systemic infusion of bevacizumab (Avastin; Genentech, Inc., South San Francisco, CA), a humanized recombinant VEGF-A-neutralizing monoclonal antibody, not complicated by these caveats. Interestingly, however, the two most common side effects reported with bevacizumab administration are hypertension and proteinuria, the hallmarks of preeclampsia.Clinical SignificanceThe observations of Nishijima and coworkers5Nishijima K Ng Y-S Zhong L Bradley J Shubert W Jo N Akita J Sameulsson SJ Robinson GS Adamis AP Shima DT Vascular endothelial growth factor-A is a survival factor for retinal neurons and a critical neuroprotectant during the adaptive response to ischemic injury.Am J Pathol. 2007; 171: 53-67Abstract Full Text Full Text PDF PubMed Scopus (573) Google Scholar combine these two new evolving fields of VEGF-A actions—that of its effects outside of the vasculature and of its effects in the adult. So what do the observations of Nishijima et al5Nishijima K Ng Y-S Zhong L Bradley J Shubert W Jo N Akita J Sameulsson SJ Robinson GS Adamis AP Shima DT Vascular endothelial growth factor-A is a survival factor for retinal neurons and a critical neuroprotectant during the adaptive response to ischemic injury.Am J Pathol. 2007; 171: 53-67Abstract Full Text Full Text PDF PubMed Scopus (573) Google Scholar imply for the therapeutic manipulation of VEGF-A? The studies of the effect of VEGF-A neutralization on neural retinal response to ischemic injury and survival are clearly quite relevant to the current widespread use of anti-VEGF-A therapy for the treatment of the wet form of AMD. AMD, a pathology of the retina that affects central vision, is the leading cause of vision loss in Americans older than 55. Current estimates indicate that there are close to 2 million US residents with significant symptoms related to AMD. The disease is diagnosed either as dry (or non-neovascular) or wet macular degeneration. The dry form accounts for a majority (90%) of the diagnoses and is associated with deposits of complex insoluble materials beneath the retinal pigment epithelium (RPE) or within Bruch's membrane, the elastic lamina that separates the RPE from the underlying choroidal circulation. The wet (or exudative) form accounts for only 10% of the cases but nearly 90% of the vision loss and is characterized by the growth of new blood vessels beneath the retina, particularly in the macula, the site of central vision. As is the case for virtually all nascent vessels associated with pathologies, these vessels are highly permeable, and the fluid that accumulates under the retina can cause retinal detachments, photoreceptor atrophy, and vision loss.Prior to the introduction of ocular anti-VEGF-A therapies such as pegaptanib sodium (Macugen; OSI/Eyetech, Melville, NY), an aptamer reportedly specific for VEGF-A164, and ranibizumab (Lucentis; Genentech, Inc.), a Fab fragment of a humanized monoclonal pan-VEGF-A antibody, the most common form of treatment for wet AMD involved photodynamic therapy in which a photosensitizer (verteporphin, Visudyne; Novartis, Basel, Switzerland) is injected and then activated with a laser. Although some response was observed with this treatment, the effects were short-term and there was no significant effect on disease progression. The Food and Drug Administration approval in December 2004 of pegaptanib sodium marked the first therapeutic for AMD with a “rational” target. Patient response to pegaptanib sodium was mixed, and the treatment was shown to slow the progression of vision loss from wet AMD. The introduction of pegaptanib sodium was followed about one and a half years later with the approval of ranibizumab. Intravitreal administration of ranibizumab has been shown to reduce disease progression in 95% of patients (versus 62% of sham-injected controls) and to lead to increased visual acuity in more than one-third of patients.10Rosenfeld PJ Brown DM Heier JS Boyer DS Kaiser PK Chung CY Kim RY Ranibizumab for neovascular age-related macular degeneration.N Engl J Med. 2006; 355: 1419-1431Crossref PubMed Scopus (4683) Google Scholar The treatment has been appropriately hailed as “miraculous.”26Stone EM A very effective treatment for neovascular macular degeneration.N Engl J Med. 2006; 355: 1493-1495Crossref PubMed Scopus (57) Google ScholarAre there reasons to be wary of the potential long-term effects of intraocular VEGF-A neutralization? The observations of Nishijima et al5Nishijima K Ng Y-S Zhong L Bradley J Shubert W Jo N Akita J Sameulsson SJ Robinson GS Adamis AP Shima DT Vascular endothelial growth factor-A is a survival factor for retinal neurons and a critical neuroprotectant during the adaptive response to ischemic injury.Am J Pathol. 2007; 171: 53-67Abstract Full Text Full Text PDF PubMed Scopus (573) Google Scholar as well as those emerging from other groups suggest that there may be. Evidence pointing to a role for VEGF-A as an endogenous neuroprotectant indicate that continued neutralization of retinal VEGF-A may have unintended consequences, including loss27Ishida S Usui T Yamashiro K Kaji Y Amano S Ogura Y Hida T Oguchi Y Ambati J Miller JW Gragoudas ES Ng YS D'Amore PA Shima DT Adamis AP VEGF164-mediated inflammation is required for pathological, but not physiological, ischemia-induced retinal neovascularization.J Exp Med. 2003; 198: 483-489Crossref PubMed Scopus (399) Google Scholar of neural retina cells. Furthermore, demonstration that the benefits of preconditioning are due, at least in part, to the up-regulation of VEGF-A indicate that VEGF-A blockage may interfere with physiological mechanisms that are intended to attenuate ischemic injury. Although the pathogenesis of AMD is not known, ischemia is often postulated as a component and the results of Nishijima et al5Nishijima K Ng Y-S Zhong L Bradley J Shubert W Jo N Akita J Sameulsson SJ Robinson GS Adamis AP Shima DT Vascular endothelial growth factor-A is a survival factor for retinal neurons and a critical neuroprotectant during the adaptive response to ischemic injury.Am J Pathol. 2007; 171: 53-67Abstract Full Text Full Text PDF PubMed Scopus (573) Google Scholar suggest that VEGF-A inhibition may exacerbate ischemia-induced neural damage.The existence of splice variants of VEGF (three in mice and at least five in humans) raises the possibility that the isoforms might serve distinct functions. In fact, an observation of mice that were engineered to express single isoforms supports the conclusion that the isoforms are not functionally equivalent; for instance, mice expressing only VEGF120 develop to term (although not in the predicted ratios) and die perinatally due to pulmonary and cardiac insufficiency.28Carmeliet P Ng YS Nuyens D Theilmeier G Brusselmans K Cornelissen I Ehler E Kakkar VV Stalmans I Mattot V Perriard JC Dewerchin M Flameng W Nagy A Lupu F Moons L Collen D D'Amore PA Shima DT Impaired myocardial angiogenesis and ischemic cardiomyopathy in mice lacking the vascular endothelial growth factor isoforms VEGF164 and VEGF188.Nat Med. 1999; 5: 495-502Crossref PubMed Scopus (557) Google Scholar More recently, based on results using a mouse model of ocular angiogenesis, it was suggested that VEGF164 mediates pathological but not physiological neovascularization.27Ishida S Usui T Yamashiro K Kaji Y Amano S Ogura Y Hida T Oguchi Y Ambati J Miller JW Gragoudas ES Ng YS D'Amore PA Shima DT Adamis AP VEGF164-mediated inflammation is required for pathological, but not physiological, ischemia-induced retinal neovascularization.J Exp Med. 2003; 198: 483-489Crossref PubMed Scopus (399) Google Scholar In light of these observations, one very promising observation from the work of Nishijima and colleagues5Nishijima K Ng Y-S Zhong L Bradley J Shubert W Jo N Akita J Sameulsson SJ Robinson GS Adamis AP Shima DT Vascular endothelial growth factor-A is a survival factor for retinal neurons and a critical neuroprotectant during the adaptive response to ischemic injury.Am J Pathol. 2007; 171: 53-67Abstract Full Text Full Text PDF PubMed Scopus (573) Google Scholar is that VEGF120 was as effective as VEGF164 in protecting the retina against hypoxic injury. This finding suggests the possibility that targeting specific VEGF isoforms block vessel growth while sparing the neuroprotective effects of VEGF. Because so little is known about the relative actions of the various VEGF isoforms in vitro or in vivo, the cellular, biochemical, and molecular basis of these observations a not clear. Clearly, additional work is necessary to determine whether this suggestion will be sustained in clinical settings.Furthermore, although Nishijima and colleagues5Nishijima K Ng Y-S Zhong L Bradley J Shubert W Jo N Akita J Sameulsson SJ Robinson GS Adamis AP Shima DT Vascular endothelial growth factor-A is a survival factor for retinal neurons and a critical neuroprotectant during the adaptive response to ischemic injury.Am J Pathol. 2007; 171: 53-67Abstract Full Text Full Text PDF PubMed Scopus (573) Google Scholar did not detect an effect of VEGF-A neutralization on the retinal vessels within the 8-week time course of their study, it is possible that extended inhibition may have revealed an effect. This possibility is supported by the observation that VEGF-A acts as a survival factor on developing retinal vessels12Alon T Hemo I Itin A Pe'er J Stone J Keshet S 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 (1406) Google Scholar and that VEGF-A continues to be produced by retinal pericytes and astrocytes in the vicinity of the inner retinal vessels in the adult retina.16Darland DC Massingham LJ Smith SR Piek E Saint-Geniez M D'Amore PA Pericyte production of cell-associated VEGF is differentiation-dependent and is associated with endothelial survival.Dev Biol. 2003; 264: 275-288Crossref PubMed Scopus (314) Google Scholar Finally, a fact that was not discussed by Nishijima et al5Nishijima K Ng Y-S Zhong L Bradley J Shubert W Jo N Akita J Sameulsson SJ Robinson GS Adamis AP Shima DT Vascular endothelial growth factor-A is a survival factor for retinal neurons and a critical neuroprotectant during the adaptive response to ischemic injury.Am J Pathol. 2007; 171: 53-67Abstract Full Text Full Text PDF PubMed Scopus (573) Google Scholar but which must be given serious consideration is the potential effect of VEGF-A neutralization on the choroidal circulation. The choroidal vasculature underlies the RPE and nourishes the outer retina, including the photoreceptor cells. Development of the choroidal circulation is dependent on RPE-produced VEGF-A.29Marneros AG Fan J Yokoyama Y Gerber HP Ferrara N Crouch RK Olsen BR Vascular endothelial growth factor expression in the retinal pigment epithelium is essential for choriocapillaris development and visual function.Am J Pathol. 2005; 167: 1451-1459Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar More importantly, the fact that RPE continues to express VEGF-A in the adult and that the endothelial cells of the choriocapillaris (the capillary plexus of the choroids) express VEGFR230Saint-Geniez M Maldonado AE D'Amore PA VEGF expression and receptor activation in the choroid during development and in the adult.Invest Ophthalmol Vis Sci. 2006; 47: 3135-3