EphrinA1 inhibits vascular endothelial growth factor-induced intracellular signaling and suppresses retinal neovascularization and blood-retinal barrier breakdown

The Eph receptor/ephrin system is a recently discovered regulator of vascular development during embryogenesis. Activation of EphA2, one of the Eph receptors, reportedly suppresses cell proliferation and adhesion in a wide range of cell types, including vascular endothelial cells. Vascular endothelial growth factor (VEGF) plays a primary role in both pathological angiogenesis and abnormal vascular leakage in diabetic retinopathy. In the study described herein, we demonstrated that EphA2 stimulation by ephrinA1 in cultured bovine retinal endothelial cells inhibits VEGF-induced VEGFR2 receptor phosphorylation and its downstream signaling cascades, including PKC (protein kinase C)-ERK (extracellular signal-regulated kinase) 1/2 and Akt. This inhibition resulted in the reduction of VEGF-induced angiogenic cell activity, including migration, tube formation, and cellular proliferation. These inhibitory effects were further confirmed in animal models. Intraocular injection of ephrinA1 suppressed ischemic retinal neovascularization in a dose-dependent manner in a mouse model. At a dose of 125 ng/eye, the inhibition was 36.0 +/- 14.9% (P < 0.001). EphrinA1 also inhibited VEGF-induced retinal vascular permeability in a rat model by 46.0 +/- 10.0% (P < 0.05). These findings suggest a novel therapeutic potential for EphA2/ephrinA1 in the treatment of neovascularization and vasopermeability abnormalities in diabetic retinopathy.     

The inhibition of retinal neovascularization by gold nanoparticles via suppression of VEGFR-2 activation

The pathological angiogenesis in the retina is the major cause of vision loss at all ages. In particular, retinopathy of prematurity (ROP) is a leading cause of blindness in children. This study investigated whether gold nanoparticle (GNP) could inhibit retinal neovascularization in the animal model of ROP. Intravitreal injection of GNP significantly inhibited retinal neovascularization in the mouse model of ROP. In addition, GNP effectively suppressed VEGF-induced in vitro angiogenesis of retinal microvascular endothelial cells including proliferation, migration and capillary-like networks formation. GNP blocked VEGF-induced auto-phosphorylation of VEGFR-2 to inhibit consequently ERK 1/2 activation. GNP never affected on the cellular viability of retinal microvascular endothelial cells and induced no retinal toxicity. Our data suggest that GNP could be a potent inhibitor to retinal neovascularization without retinal toxicity. Furthermore, GNP could be extensively applied to variable vaso-proliferative retinopathies mediated by VEGF.     

Expression of thrombospondin-1 in ischemia-induced retinal neovascularization

Thrombospondin-1 is an extracellular matrix protein that inhibits endothelial cell proliferation, migration, and angiogenesis. This study was performed to investigate the role of thrombospondin-1 in ischemic retinal neovascularization. In a murine model of retinal neovascularization, thrombospondin-1 mRNA was increased from postnatal day 13 (P13), with a threefold peak response observed on P15, corresponding to the time of development of retinal neovascularization. Prominent expression of thrombospondin-1 was observed in neovascular cells, specifically, cells adjacent to the area of nonperfusion. It has been suggested that vascular endothelial growth factor (VEGF) plays a major role in ischemia-induced retinal neovascularization of this model, so we studied the effects of VEGF on thrombospondin-1 expression. In bovine retinal microcapillary endothelial cells, VEGF induced a biphasic response of thrombospondin-1 expression; VEGF decreased thrombospondin-1 mRNA 0.41-fold after 4 hours, whereas it increased, with a threefold peak response, after 24 hours. VEGF-induced endothelial cell proliferation was completely inhibited by exogenous thrombospondin-1 and increased by 37.5% with anti-thrombospondin-1 antibody. The present findings suggest that, in the ischemic retina, retinal neovascular cells increase thrombospondin-1 expression, and VEGF may stimulate endogenous thrombospondin-1 induction, which inhibits endothelial cell growth. VEGF-mediated thrombospondin-1 induction in ischemia-induced angiogenesis may be a negative feedback mechanism.     

Advanced glycation end-products increase monocyte adhesion to retinal endothelial cells through vascular endothelial growth factor-induced ICAM-1 expression: inhibitory effect of antioxidants

Accumulating evidence indicates a role for advanced glycation end-products (AGEs) in the development of diabetic retinopathy. In the present study, we examined the in vitro effect of AGEs on human monocyte adhesion to bovine retinal endothelial cells (BRECs) and the molecular mechanisms involved in this effect. Treatment of cultured BRECs with AGEs led to a significant increase in monocyte adhesion and intercellular cell adhesion molecule-1 (ICAM-1) expression. These effects were inhibited by antioxidants including gliclazide and vitamins C and E. On the basis of the stimulatory effect of AGEs on vascular endothelial growth factor (VEGF) secretion by retinal endothelial cells, the role of this growth factor as mediator of AGE-induced monocyte adhesion to BRECs was next investigated. Incubation of BRECs with VEGF increased monocyte adhesion to these cells and enhanced ICAM-1 expression. Treatment of BRECs with an anti-VEGF antibody abrogated AGE-induced monocyte adhesion and ICAM-1 expression. Finally, incubation of BRECs with protein kinase C (PKC) and nuclear factor (NF)-kappaB inhibitors suppressed monocyte adhesion and ICAM-1 expression elicited by AGEs and VEGF. Taken together, these data indicate that AGEs increase monocyte adhesion to BRECs and that this effect is mediated through VEGF-induced ICAM-1 expression. They also demonstrate that this effect is oxidative stress-sensitive and involves PKC and NF-kappaB-dependent signaling pathways.     

CAPE suppresses VEGFR-2 activation, and tumor neovascularization and growth

The growth and metastasis of human solid tumors and the development of conditions such as diabetic retinopathy, rheumatoid arthritis, inflammatory psoriasis, and others are regulated by the balance between angiogenic stimulators and inhibitors released in the angiogenic–pathological microenvironment. Vascular endothelial growth factor (VEGF), an angiogenic factor, is a potent endothelial-specific mitogen that activates endothelial cells in pathological angiogenesis. Recently, we demonstrated that caffeic acid phenethyl ester (CAPE) inhibits tumor growth, invasion, and metastasis. However, the precise molecular mechanism underlying the inhibitory effect of CAPE on VEGF-mediated angiogenesis remains unknown. Here, we show that CAPE suppressed VEGF-induced proliferation, tube formation, migration, the formation of actin stress fibers and loss of VE-cadherin at cell–cell contacts in endothelial cells, indicating the inhibition of VEGF-mediated VEGF receptor-2 (VEGFR-2) and its downstream signal activation in vitro. CAPE blocked VEGF-stimulated neovascularization in the Matrigel plugs assay, and reduced vascular permeability in mouse skin capillaries in vivo. CAPE inhibited the growth and neovascularization of primary tumor cells in C57BL/6 and BALB/c mice inoculated with Lewis lung carcinoma, colon carcinoma, and melanoma cells. These results suggest that CAPE negatively modulates VEGF-induced angiogenesis by suppressing VEGFR-2 activation, and might be a therapeutic avenue for anti-angiogenesis.     

Glial cell expression of hepatocyte growth factor in vitreoretinal proliferative disease

The hepatocyte growth factor (HGF) has been crucially implicated in the development of proliferative retinal diseases; however, it is unclear whether retinal glial cells express or respond to HGF. Therefore, we examined the expression of HGF and of the receptor for HGF, c-Met, by immunohistochemical costaining with glial fibrillary acidic protein (GFAP) in epiretinal membranes of patients with proliferative vitreoretinopathy (PVR) and proliferative diabetic retinopathy (PDR), respectively. Furthermore, it was determined whether cells of the human retinal glial cell line, MIO-M1, secrete HGF protein, and whether HGF stimulates proliferation and chemotaxis, and secretion of the vascular endothelial growth factor (VEGF). Neuroretinas of patients with PVR express elevated mRNA level for HGF in comparison to control retinas. In epiretinal membranes of patients with PVR or PDR, immunoreactivity for HGF and for c-Met, respectively, partially colocalized with immunoreactivity for GFAP. Fetal bovine serum and basic fibroblast growth factor, but not heparin-binding epidermal or platelet-derived growth factors, evoked HGF secretion by cultured retinal glial cells. HGF displayed only a marginal effect on cell proliferation while it stimulated chemotaxis. HGF promoted the secretion of VEGF, via activation of the phosphatidylinositol-3 kinase. It is concluded that glial cells in epiretinal membranes express both HGF protein and c-Met receptors. The results suggest an autocrine/paracrine role of HGF in glial cell responses during proliferative vitreoretinal disorders as well as in retinal neovascularization, by stimulating of VEGF release.     

Thrombospondin-1-deficient mice exhibit increased vascular density during retinal vascular development and are less sensitive to hyperoxia-mediated vessel obliteration.

Thrombospondin-1 (TSP1) is a natural inhibitor of angiogenesis. Its expression is most prominent during the late stages of vascular development and in the adult vasculature. Our previous studies have shown that TSP1 expression promotes a quiescent, differentiated phenotype of vascular endothelial cells. However, the physiological role TSP1 plays during vascular development and neovascularization requires further delineation. Here, we investigated the role of TSP1 during development of retinal vasculature and retinal neovascularization during oxygen-induced ischemic retinopathy. The retinal vascular density was increased in TSP1-deficient (TSP1-/-) mice compared with wild-type mice. This finding was mainly attributed to increased number of retinal endothelial cells in TSP1-/- mice. During oxygen-induced ischemic retinopathy, the developing retinal vasculature of TSP1-/- mice was less sensitive to vessel obliteration induced by hyperoxia but exhibited a similar level of neovascularization induced by normoxia compared with wild-type mice. This finding is consistent with the similar pattern of VEGF expression detected in wild-type and TSP1-/- mice. Furthermore, the increased expression of TSP1 during development of retinal vasculature was not affected by oxygen-induced ischemic retinopathy. In addition, the regression of ocular embryonic (hyaloid) vessels, as well as the newly formed retinal vessels during oxygen-induced ischemic retinopathy, was delayed in TSP1-/- mice. Therefore, TSP1 is a modulator of vascular homeostasis and its expression is essential for appropriate remodeling and maturation of retinal vasculature.     

VEGF-binding aptides and the inhibition of choroidal and retinal neovascularization

Age-related macular degeneration and diabetic retinopathy are leading causes of blindness. Vascular endothelial growth factor (VEGF) is known to be the main factor that induces pathological angiogenesis in these diseases. In this study, we investigate the therapeutic potential and safety profiles of high-affinity peptides targeting VEGF which are identified using an ‘aptide’ technology. We show that two VEGF-binding aptides, APT_VEGF1 and APT_VEGF2, demonstrate high binding affinity and specificity to VEGF. Furthermore, they suppress VEGF-induced activation of VEGF receptor-2, in vitro angiogenesis, and in vivo pathological choroidal and retinal neovascularization. Despite potent anti-angiogenic effects, both VEGF-binding aptides do not induce any definite toxicity at the level of cellular viability, histological integrity, and gene expression. Our data show the therapeutic potential of VEGF-binding peptides for the treatment of choroidal and retinal neovascularization.     

Inhibition of NAD(P)H oxidase activity blocks vascular endothelial growth factor overexpression and neovascularization during ischemic retinopathy

Because oxidative stress has been strongly implicated in up-regulation of vascular endothelial growth factor (VEGF) expression in ischemic retinopathy, we evaluated the role of NAD(P)H oxidase in causing VEGF overexpression and retinal neovascularization. Dihydroethidium imaging analyses showed increased superoxide formation in areas of retinal neovascularization associated with relative retinal hypoxia in a mouse model for oxygen-induced retinopathy. The effect of hypoxia in stimulating superoxide formation in retinal vascular endothelial cells was confirmed by in vitro chemiluminescence assays. The superoxide formation was blocked by specific inhibitors of NAD(P)H oxidase activity (apocynin, gp91ds-tat) indicating that NAD(P)H oxidase is a major source of superoxide formation. Western blot and immunolocalization analyses showed that retinal ischemia increased expression of the NAD(P)H oxidase catalytic subunit gp91phox, which localized primarily within vascular endothelial cells. Treatment of mice with apocynin blocked ischemia-induced increases in oxidative stress, normalized VEGF expression, and prevented retinal neovascularization. Apocynin and gp91ds-tat also blocked the action of hypoxia in causing increased VEGF expression in vitro, confirming the specific role of NAD(P)H oxidase in hypoxia-induced increases in VEGF expression. In conclusion, NAD(P)H oxidase activity is required for hypoxia-stimulated increases in VEGF expression and retinal neovascularization. Inhibition of NAD(P)H oxidase offers a new therapeutic target for the treatment of retinopathy.     

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.     

Vascular Endothelial Growth Factor/Vascular Permeability Factor Is Temporally and Spatially Correlated with Ocular Angiogenesis in a Primate Model

Ischemia often precedes neovascularization. Inocular neovascularization, such as occurs in diabetic retinopathy, a diffusible angiogenic factor has been postulated to be produced by ischemicretina and to lead to neovascularization of theretina, optic nerve, or iris. However, no angiogenic factor has been conclusively identified that satisfies this hypothesis. Vascular endothelial growth factor/vascular permeability factor, hereafter referred to as VEGF, is a likely candidate for an ocular angiogenic factor because it is a secreted mitogen, specific for endothelial cells, and is upregulated by hypoxia. We investigated the association of VEGF with the development of experimental iris neovascularization in the cynomolgus monkey. Following theproduction of retinal ischemia by laser occlusion of all branch retinal veins, VEGF was increased in the aqueous fluid, and the aqueous VEGF levels changed synchronously and proportionally with the severity of iris neovascularization. Northern analysis and in situ hybridization revealed that VEGF messenger RNA is upregulated in the ischemic retina. These observations support the hypothesis that ocular neovascularization is regulated by a diffusible factor and identify VEGF as a likely candidate for a retina-derived vascular permeability and angiogenesis factor in vivo.     

Blockade of vascular endothelial cell growth factor receptor signaling is sufficient to completely prevent retinal neovascularization

Retinal vasculogenesis and ischemic retinopathies provide good model systems for study of vascular development and neovascularization (NV), respectively. Vascular endothelial cell growth factor (VEGF) has been implicated in the pathogenesis of retinal vasculogenesis and in the development of retinal NV in ischemic retinopathies. However, insulin-like growth factor-I and possibly other growth factors also participate in the development of retinal NV and intraocular injections of VEGF antagonists only partially inhibit retinal NV. One possible conclusion from these studies is that it is necessary to block other growth factors in addition to VEGF to achieve complete inhibition of retinal NV. We recently demonstrated that a partially selective kinase inhibitor, PKC412, that blocks phosphorylation by VEGF and platelet-derived growth factor (PDGF) receptors and several isoforms of protein kinase C (PKC), completely inhibits retinal NV. In this study, we have used three additional selective kinase inhibitors with different selectivity profiles to explore the signaling pathways involved in retinal NV. PTK787, a drug that blocks phosphorylation by VEGF and PDGF receptors, but not PKC, completely inhibited retinal NV in murine oxygen-induced ischemic retinopathy and partially inhibited retinal vascularization during development. CGP 57148 and CGP 53716, two drugs that block phosphorylation by PDGF receptors, but not VEGF receptors, had no significant effect on retinal NV. These data and our previously published study suggest that regardless of contributions by other growth factors, VEGF signaling plays a critical role in the pathogenesis of retinal NV. Inhibition of VEGF receptor kinase activity completely blocks retinal NV and is an excellent target for treatment of proliferative diabetic retinopathy and other ischemic retinopathies.     

Attenuation of retinal vascular development and neovascularization in transgenic mice over-expressing thrombospondin-1 in the lens.

Thrombospondin-1 (TSP1) is an endogenous inhibitor of angiogenesis and induces endothelial cell (EC) apoptosis. To study the role TSP1 plays during vascular development and neovascularization, we assessed the effects of ectopic TSP1 expression in the lens on retinal vascularization in transgenic mice. The TSP1 over-expressing mice showed abnormalities in the development of retinal vasculature. There was a dramatic decrease in the density of superficial and deep vascular plexuses of the retina in transgenic mice. The retinal vessels in TSP1 transgenic mice also appeared nonuniform and abnormal in maturation. We detected an increase in the number of EC undergoing apoptosis, which was compensated, in part, by an increase in cell proliferation in retinal vasculature of TSP1 transgenic mice. The TSP1 transgenic mice also exhibited increased levels of vessel obliteration and a limited preretinal neovascularization during oxygen-induced ischemic retinopathy (OIR). Our results indicate increased expression of TSP1 attenuates normal retinal vascularization and preretinal neovascularization during OIR. Therefore, modulation of TSP1 expression may provide an effective mechanism for regulation of ocular angiogenesis.     

Protein kinase B (akt) promotes pathological angiogenesis in murine model of oxygen-induced retinopathy

Akt, or protein kinase B, is an important signaling molecule that modulates many cellular processes such as cell growth, survival, and metabolism. However, the vivo roles and effectors of Akt in retinal angiogenesis are not explicitly clear. We therefore detected the expression of Akt using Western blotting or RT-PCR technologies in an animal model of oxygen-induced retinopathy, and investigated the effects of recombinant Akt on inhibiting vessels loss and Akt inhibitor on suppressing experimental retinal neovascularization in this model. We showed that in the hyperoxic phase of oxygen-induced retinopathy, the expression of Akt was greatly suppressed. In the hypoxic phase, the expression of Akt was increased dramatically. No significant differences were found in normoxic groups. Compared with control groups, administration of the recombinant Akt in the first phase of retinopathy markedly reduced capillary-free areas, while the administration of the Akt inhibitor in the second phase of retinopathy significantly decreased retinal neovascularization but capillary-free areas. These results indicate that Akt play a critical role in the pathological process (vessels loss and neovascularization) of mouse model of oxygen-induced retinopathy, which may provide a valubale therapeutic tool for ischemic-induced retinal diseases.     

Insulin-like growth factor-I plays a pathogenetic role in diabetic retinopathy

Diabetic retinopathy is a leading cause of blindness in the Western world. Aberrant intercellular adhesion molecule-1 expression and leukocyte adhesion have been implicated in its pathogenesis, raising the possibility of an underlying chronic inflammatory mechanism. In the current study, the role of insulin-like growth factor (IGF)-I in these processes was investigated. We found that systemic inhibition of IGF-I signaling with a receptor-neutralizing antibody, or with inhibitors of PI-3 kinase (PI-3K), c-Jun kinase (JNK), or Akt, suppressed retinal Akt, JNK, HIF-1alpha, nuclear factor (NF)-kappaB, and AP-1 activity, vascular endothelial growth factor (VEGF) expression, as well as intercellular adhesion molecule-1 levels, leukostasis, and blood-retinal barrier breakdown, in a relevant animal model. Intravitreous administration of IGF-I increased retinal Akt, JNK, HIF-1alpha, NF-kappaB, and AP-1 activity, and VEGF levels. IGF-I stimulated VEGF promoter activity in vitro, mainly via HIF-1alpha, and secondarily via NF-kappaB and AP-1. In conclusion, IGF-I participates in the pathophysiology of diabetic retinopathy by inducing retinal VEGF expression via PI-3K/Akt, HIF-1alpha, NF-kappaB, and secondarily, JNK/AP-1 activation. Taken together, these in vitro and in vivo signaling studies thus identify potential targets for pharmacological intervention to preserve vision in patients with diabetes.     

Inducible expression of vascular endothelial growth factor in adult mice causes severe proliferative retinopathy and retinal detachment

Transgenic mice with vascular endothelial growth factor (VEGF) driven by the rhodopsin promoter (rho/VEGF mice) develop neovascularization that originates from the deep capillary bed of the retina and grows into the subretinal space. In rho/VEGF mice, VEGF expression in photoreceptors begins between postnatal days 5 and 7, the period when the deep capillary bed is developing. An important question is whether or not the developmental stage of the deep capillary bed is critical for occurrence of neovascularization. Also, although rho/VEGF mice are extremely useful for the study of ocular neovascularization, there are some applications for which the early onset of VEGF expression is a disadvantage. In this study, we used the reverse tetracycline transactivator (rtTA) inducible promoter system coupled to either the rhodopsin or interphotoreceptor retinoid-binding protein (IRBP) promoter to control the time of onset of VEGF transgene expression in photoreceptors. In the absence of doxycycline, adult double-transgenic rho/rtTA-TRE/VEGF or IRBP/rtTA-TRE/VEGF mice showed little VEGF transgene expression and no phenotype. The addition of doxycycline to the drinking water resulted in prominent transgene expression and evidence of neovascularization within 3 to 4 days. Like rho/VEGF mice, the neovascularization originated from the deep capillary bed of the retina, but it was more extensive and caused outer retinal folds followed by total retinal detachment. Real-time polymerase chain reaction and enzyme-linked immunosorbent assay demonstrated that the mice with inducible expression of VEGF that developed retinal detachment had much higher ocular levels of VEGF mRNA and protein compared to rho/VEGF mice that manifest a much milder phenotype. These data demonstrate that regardless of developmental stage of the vascular bed, increased expression of VEGF in the retina is sufficient to cause neovascularization, and high levels of expression cause severe neovascularization and traction retinal detachment. Mice with inducible expression of VEGF in the retina provide a valuable new model of ocular neovascularization.