Different effects of angiopoietin-2 in different vascular beds: new vessels are most sensitive.

In this study, we used double transgenic mice with inducible expression of angiopoietin-2 (Ang2) to investigate the role of Ang2 in the retinal and choroidal circulations and in three models of ocular neovascularization (NV). Mice with induced expression of Ang2 ubiquitously, or specifically in the retina, survived and appeared grossly normal. They also had normal-appearing retinal and choroidal circulations, demonstrating that high levels of Ang2 did not induce regression of mature retinal or choroidal vessels. When Ang2 expression was induced soon after birth, there was increased density of the deep capillary bed on postnatal day (P) 11 that returned to normal by P18, the time that retinal vascular development is usually completed. In mice with ischemic retinopathy, induction of Ang2 during the ischemic period resulted in a significant increase in retinal NV, but induction of Ang2 at a later time point when ischemia (and vascular endothelial growth factor [VEGF]) was less, hastened regression of NV. In triple transgenic mice that coexpressed VEGF and Ang2, the increased expression of Ang2 inhibited VEGF-induced NV in the retina. Increased expression of Ang2 also resulted in regression of choroidal neovascularization. These data suggest that ocular neovascularization, but not mature retinal or choroidal vessels, is sensitive to Ang2; a high Ang2/VEGF ratio promotes regression, while high Ang2 in the setting of hypoxia and/or concomitantly high Ang2 and VEGF stimulate neovascularization.

Serous macular detachment associated with retinal arterial macroaneurysm.

PURPOSE: To report optical coherence tomography (OCT) findings of macular edema secondary to retinal arterial macroaneurysms. METHODS: Six eyes with retinal arterial macroaneurysms were retrospectively examined by OCT. All eyes had macular edema without massive macular hemorrhage. Fluorescein angiography was performed in six eyes, and indocyanine green angiography was done in four eyes. Five eyes underwent direct laser photocoagulation to the retinal arterial macroaneurysms. The foveal thickness, height of the serous macular detachment, and visual acuity (VA) were evaluated from the initial examination until the macular edema resolved. RESULTS: In all six eyes, the macular edema secondary to retinal arterial macroaneurysms comprised a serous macular detachment with retinal swelling. No cystoid macular edema was observed in any eyes. Dye leakage occurred only from the macroaneurysms in all eyes. OCT showed complete resolution of the serous macular detachment and retinal swelling 1 to 4 months after the initial examination. Macular hard exudates developed during absorption of the serous macular detachment in all eyes. VA improved more than two lines in all eyes after the macular edema resolved. CONCLUSION: Retinal arterial macroaneurysms may leak extravasated fluid into the subretinal space, which may result in a serous macular detachment.

Intraocular expression of endostatin reduces VEGF-induced retinal vascular permeability, neovascularization, and retinal detachment.

Endostatin, a proteolytic fragment of collagen XVIII, is an endogenous inhibitor of tumor angiogenesis that also inhibits choroidal neovascularization. In this study, we assessed the effects of increased intraocular expression of endostatin on vascular endothelial growth factor (VEGF)-induced changes in the retina. After subretinal injection of a pair of gutless adenoviral vectors (AGV) designed to provide tamoxifen-inducible expression of endostatin, diffuse endostatin immunoreactivity was induced thoroughout the retina by administration of tamoxifen. Induction of endostatin in double transgenic mice with doxycycline-induced expression of VEGF in the retina resulted in significant suppression of leakage of intravascular [3H]mannitol into the retina. The ability of endostatin to reduce VEGF-induced retinal vascular permeability was confirmed by using [3H]mannitol leakage and two other parameters, fluorescein leakage and retinal thickness, after subretinal injection of a bovine immunodeficiency lentiviral vector coding for endostatin (BIV-vectored endostatin, or BIVendostatin). Subretinal injection of BIVendostatin resulted in more discrete, less intense staining for endostatin in the retina than that seen with the inducible AGV system, which suggested lower levels and allowed visualization of sites where endostatin was concentrated. Endostatin staining outlined retinal blood vessels, which suggested endostatin binding to a component of vessel walls. More prolonged or higher level expression of VEGF in the retina resulted in neovascularization and retinal detachment, both of which were also significantly reduced by BIVendostatin. These data suggest that endostatin may be an endogenous inhibitor of vasopermeability as well as neovascularization. In patients with diabetic retinopathy, endostatin gene transfer may provide a way to decrease the risk of three causes of visual loss: macular edema, neovascularization, and retinal detachment.

Massive macular hard exudates associated with branch retinal vein occlusion.

PURPOSE: To report four cases of branch retinal vein occlusion (BRVO) with the complication of serous retinal detachment (SRD). METHODS: We retrospectively studied four eyes of four patients with macular edema and macular hard exudates associated with midperipheral BRVO. Visual acuity, ophthalmoscopy, fluorescein angiography, and optical coherence tomography findings were evaluated. Three of the four eyes underwent laser photocoagulation in the BRVO area 1 month after the initial visit. RESULTS: Macular edema consisted of SRD without cystoid macular edema in all eyes. Late-phase fluorescein angiography showed extensive dye leakage in the BRVO area. When SRD was resolved 4 months after the initial examination, hard exudates had increased in the macular area. Although macular hard exudates decreased 1 year after the initial examination, visual acuity remained under 20/20 because of macular atrophy. CONCLUSIONS: SRD is one type of macular edema observed in BRVO. In macula-spared midperipheral BRVO, the SRD originates from a vascular leaking area, and there is a high risk that massive macular hard exudates will develop, which may affect visual recovery.

[Serous macular detachment combined with branch retinal vein occlusion].

PURPOSE: To report frequency, clinical characteristics, treatment, and the complications of branch retinal vein occlusion (BRVO) with serous macular detachment. PATIENTS AND METHODS: We retrospectively studied 22 eyes of 22 patients in 111 eyes with acute BRVO, whose eyes had serous macular detachment that was detected by optical coherence tomography (OCT). Fluorescein angiography was conducted in 19 of the 22 eyes. Fourteen of the 22 eyes underwent scatter laser photocoagulation of the BRVO area. We observed serial OCT findings before and after treatment. RESULTS: Approximately 20% of the BRVO eyes had serous macular detachment. The superotemporal vein was occluded in 15 eyes and the inferotemporal vein was involved in 7 eyes. Four eyes were ischemic and 15 eyes were not ischemic. Extensive dye leakage was observed in the BRVO area in all examined eyes (19 eyes). The occlusion area of perifoveal capillary network ranged from 5 to 60%, with an average of 40%. OCT demonstrated pure serous macular detachment in 13 eyes and the remaining 9 eyes had both serous macular detachment and cystoid macular edema(CME). The occlusion area of perifoveal capillaries in these 9 eyes was more than 20%. Serous macular detachment was resolved in 11 of 14 eyes (80%) 6 months after laser treatment. The average period for resolution of macular detachment was 3.4 months after treatment. Visual acuity was improved more than 2 lines in 8 of the treated 11 eyes (73%). Hard exudates appeared in the posterior fundus in 13 of 14 treated eyes (93%) and in 16 of the total of 22 eyes (73%) in the follow-up period. Massive macular hard exudates and ensuing macular atrophy resulted in poor visual outcome. CONCLUSIONS: Serous macular detachment is one of the patterns of macular edema in BRVO. OCT is an essential tool to detect it. Leakage from the entire BRVO area seems to travel via subretinal space by gravity or other factors and may form serous detachment in the macular area. Laser photocoagulation is indicated for early resolution of serous macular detachment. The major complication of serous detachment is the deposit of macular hard exudates, which may result in poor visual outcome.

Sustained transduction of ocular cells with a bovine immunodeficiency viral vector.

Human immunodeficiency viral (HIV) vectors mediate long-term transduction of many types of nondividing cells in vivo. Bovine immunodeficiency virus (BIV) is a lentivirus that shares many characteristics with HIV, but does not cause human disease. In this study, we investigated the potential of BIV vectors for ocular gene therapy. An enhanced green fluorescent protein (eGFP)-encoding reporter gene was packaged in recombinant BIV vector (BIV.eGFP). Adult C57BL/6 mice were given an intravitreous (5 x 10(4) or 5 x 10(5) transducing units [TU]) or subretinal (5 x 10(5) TU) injection of BIV.eGFP and then GFP expression was assessed at several time points. In vivo examinations of mice showed that subretinal injection of BIV.eGFP resulted in strong expression of GFP from the first examination at 1 week through the final examination at 20 weeks. Only a few mice that received intravitreous injection of BIV.eGFP showed GFP expression by ocular examinations until 11-12 weeks, when most showed small areas of expression. Postmortem examinations showed prominent GFP expression in retinal pigmented epithelial (RPE) cells throughout the region of subretinal injection of vector, although occasional negatively staining RPE cells were scattered among the much more numerous, brilliantly staining cells. Ciliary epithelial cells frequently expressed GFP, as did occasional Müller cells and rarely other retinal cells. The expression was stable from the first time point (2 weeks) to the last (20 weeks). Postmortem examination of eyes given an intravitreous injection of BIV.eGFP showed transduction of cells in the corneal endothelium and a few scattered retinal cells. There was no evidence of inflammation or toxicity in any eyes. These data show that BIV vectors mediate rapid and sustained transduction of RPE cells, suggesting that they may be useful for ocular gene therapy targeting RPE cells.

The kinase inhibitor PKC412 suppresses epiretinal membrane formation and retinal detachment in mice with proliferative retinopathies.

PURPOSE: Platelet-derived growth factor (PDGF) is an important stimulatory factor for proliferative retinopathies. Expression of PDGF-B in the retinas of transgenic mice (hemizygous rho/PDGF-B mice) results in rapid-onset retinal detachment caused by proliferation of glial cells, endothelial cells, and pericytes, whereas expression of PDGF-AA (homozygous rho/PDGF-A or PDGF-AA mice) causes slowly progressive retinal detachment from proliferation of glial cells. In this study, we investigated the effect in rho/PDGF-B and rho/PDGF-AA mice of several different receptor kinase inhibitors. METHODS: Hemizygous rhoPDGF-B or homozygous rho/PDGF-A mice were treated orally with PKC412 (an inhibitor of PDGF, VEGF, and c-kit receptor kinases and several isoforms of PKC), PTK787 (an inhibitor of PDGF, VEGF, and c-kit receptor kinases), SU1498 (an inhibitor of VEGF receptor kinases), imatinib mesylate (an inhibitor of PDGF, c-kit, and v-abl receptor kinases), or vehicle, and at appropriate time points epiretinal membrane (ERM) formation and retinal detachment were quantified. RESULTS: In either rho/PDGF-B or rho/PDGF-A mice, oral administration of PKC412 or PTK787, but not SU1498 or imatinib mesylate, significantly reduced ERM formation. PKC412 reduced the incidence of severe retinal detachments in both models and PTK787 did so in homozygous rho/PDGF-A mice. CONCLUSIONS: These data indicate that PKC412 (and possibly PTK787) has appropriate activity and sufficient intraocular bioavailability after oral administration to prevent retinal detachment in models of proliferative retinopathy. PKC412 should be considered for treatment of vascular and nonvascular proliferative retinopathies in humans.

Topical nepafenac inhibits ocular neovascularization.

PURPOSE: Topical nepafenac readily penetrates the cornea and is metabolized to amfenac, a potent cyclooxygenase (COX)-1 and COX-2 inhibitor. In this study, we tested the effect of topical nepafenac in three murine models of ocular neovascularization (NV). METHODS: A masked trial was performed to compare the topical effects of vehicle with one of several concentrations of nepafenac (0.01%, 0.03%, 0.1%, or 0.5%), 0.1% diclofenac, or 0.5% ketorolac tromethamine in mice with oxygen-induced ischemic retinopathy, mice with choroidal NV (CNV) due to laser-induced rupture of Bruch's membrane, or transgenic mice with increased expression of vascular endothelial growth factor (VEGF) in photoreceptors (rho/VEGF transgenic mice). RESULTS: Mice treated with 0.1% or 0.5% nepafenac had significantly less CNV and significant less ischemia-induced retinal NV than did vehicle-treated mice. Nepafenac also blunted the increase in VEGF mRNA in the retina induced by ischemia. In rho/VEGF transgenic mice, nepafenac failed to inhibit neovascularization. In additional studies, compared with vehicle-treated mice, mice treated with 0.1% or 0.03% nepafenac had significantly less CNV, whereas eyes treated with 0.1% diclofenac showed no significant difference. Mice treated with 0.5% ketorolac tromethamine for 14 days had high mortality, but when evaluated after 7 days of treatment showed no difference from mice treated with vehicle for 7 days. CONCLUSIONS: Topical nepafenac inhibits CNV and ischemia-induced retinal neovascularization by decreasing production of VEGF. The absence of effect in rho/VEGF transgenic mice is consistent with this mechanism. Topical nepafenac may provide an effective new treatment for ocular neovascularization. The excellent corneal penetration of nepafenac certainly plays an important role in this effect. It is possible that other antiangiogenic agents are also amenable to topical application after formulations are identified that maximize their corneal penetration. Because of the many advantages of the topical route of delivery, this is a possible topic for exploration.

Inhibition of ocular angiogenesis by an adenovirus carrying the human von Hippel-Lindau tumor-suppressor gene in vivo.

PURPOSE: The purpose of this study was to investigate the effect of the von Hippel-Lindau (VHL) protein on VEGF gene expression in vitro and to determine whether adenovirus-mediated VHL intraocular gene transfer inhibits the development of angiogenesis in a monkey model of multiple branch retinal vein occlusion (BRVO). METHODS: A recombinant adenovirus vector adVHL was constructed to deliver the human VHL gene. Total RNA prepared from various kinds of cells transduced with adLacZ (control) or adVHL under normoxic or hypoxic conditions was subjected to Northern blot analyses. Either adLacZ or adVHL was delivered by preretinal injection in monkeys. The effects of adLacZ or adVHL on ocular neovascularization in laser-induced multiple BRVO was evaluated in color photographs and with fluorescein angiography (FA). RESULTS: VHL expression in adVHL-transduced cells was confirmed at the transcript and protein levels. VHL overexpression significantly decreased the levels of VEGF transcripts in human aortic endothelial cells (HAECs); retinal pigment epithelium (RPE) cells; and RCC 786-O cells, renal carcinoma cells lacking VHL expression under normoxia. In contrast, VHL had no effect on the hypoxia-mediated increase in VEGF expression in these cells, although basal levels of VEGF expression were substantially reduced. Color photographs and FA revealed that retinal neovascularization and iris rubeosis accompanied by multiple BRVO in a monkey model were obviously suppressed by VHL overexpression. Northern blot analysis and immunostaining for VHL and VEGF indicated that VHL transfer obviously suppressed VEGF gene expression in VHL-transduced tissues such as retina or RPE. CONCLUSIONS: The results showed that adenovirus expressing VHL led to a significant reduction in VEGF expression in vitro under normoxic or hypoxic conditions. adVHL effectively inhibited angiogenesis in retina and iris in laser-induced multiple BRVO in monkey eyes. These data suggest that gene therapy based on VHL gene delivery has potential in the treatment of human ocular neovascularization.

Optic nerve involvement in neuro-Behcet's disease.

BACKGROUND: To report the ocular manifestations of neuro-Behcet's disease. CASES: A 34-year-old woman had recurrent meningitis. She developed diplopia, headache, and ataxia, and was diagnosed as having neuro-Behcet's disease. OBSERVATIONS: Imaging revealed two infarct foci in the transitional midbrain and pons. After treatment with prednisolone and colchicine, the diplopia resolved. Two years later, a scotoma developed in the right eye, in which the best-corrected visual acuity was 4/200. Papillitis and a prepapillary vitreous opacity were seen in the right fundus. These findings disappeared 11 days after subconjunctival steroid injections and increased colchicine. Her vision gradually improved to 20/20 two months later. CONCLUSION: Neuro-Behcet's disease may manifest with transient optic neuritis and prepapillary vitreous opacity.

Suppression and regression of choroidal neovascularization by the multitargeted kinase inhibitor pazopanib.

OBJECTIVE: To investigate pazopanib hydrochloride, a multitargeted kinase inhibitor, for treatment of choroidal neovascularization (CNV). METHODS: Choroidal neovascularization was induced in mice by rupture of Bruch membrane with laser photocoagulation. Mice were treated with pazopanib by gavage or periocular injection, and the area of CNV was measured. RESULTS: Twice-daily gavage of pazopanib, 100 mg/kg, suppressed the development of CNV by 93%. Treatment of established CNV between days 7 and 14 with 8, 40, or 200 mg/kg per day reduced CNV by 0%, 58%, and 71%, respectively. Substantial regression (40%) of CNV was also achieved after periocular injection of pazopanib. A single oral dose of 4 or 100 mg/kg resulted in an area under the curve from time 0 to the last quantifiable concentration of 129.6 and 752.0 microg x h/mL, respectively. After 7 days of 4, 20, or 100 mg/kg twice a day by gavage, plasma levels were 1300, 4900, and 5800 ng/mL and levels in the retina/choroid were 4800, 28 800, and 38 000 ng/g of tissue. CONCLUSIONS: Orally administered pazopanib has good bioavailability to the retina/choroid and strongly suppresses CNV in mice. Treatment with pazopanib after CNV is established causes dose-dependent regression of CNV. CLINICAL RELEVANCE: Pazopanib may be useful for treatment of CNV in humans.

Choroidal vascular occlusion in internal carotid artery obstruction.

PURPOSE: To determine choroidal vascular abnormalities in eyes with internal carotid artery obstruction (ICO). METHODS: We examined eight eyes with ICO with wide-angle indocyanine green (ICG) video-angiography beginning in the posterior fundus. Another angiography was performed in three eyes to observe the arterial phase of the temporal peripheral fundus. ICG angiography was done until the late choroidal venous phase or for more than 10 minutes. RESULTS: Slow dye filling in the choroidal arteries and delayed filling of the posterior watershed zone occurred in all eyes. Delayed perfusion or multiple patchy occlusions of choriocapillaris was seen in all eyes. There were delayed perfusion and/or occlusion in the peripheral watershed zone in the examined three eyes. The super-late-phase angiograms showed hyperfluorescent lines corresponding to choroidal veins. CONCLUSION: Elongated arm-to-choroid circulation and delayed intrachoroidal circulation times suggest severe choroidal hypoperfusion. Choroidal hypoperfusion resulted in multiple occlusions of the choriocapillaris and attenuated choroidal vessels. Slow filling or nonperfusion of the peripheral watershed zone was another characteristic finding. Linear hyperfluorescence may reflect severely damaged choroidal venous walls. These findings are useful to understand choroidal angiopathy in ICO or high-grade stenosis, and to diagnose ocular ischemic syndrome.

Inhibition of protein kinase C decreases prostaglandin-induced breakdown of the blood-retinal barrier.

Breakdown of the blood-retinal barrier (BRB) occurs in several retinal diseases and is a major cause of visual loss. Vascular endothelial growth factor (VEGF) has been implicated as a cause of BRB breakdown in diabetic retinopathy and other ischemic retinopathies, and there is evidence to suggest that other vasopermeability factors may act indirectly through VEGF. In this study, we investigated the effect of several receptor kinase inhibitors on BRB breakdown resulting from VEGF, tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), insulin-like growth factor-1 (IGF-1), prostaglandin E1 (PGE(1)), or PGE(2). Inhibitors of VEGF receptor kinase, including PKC412, PTK787, and SU1498, decreased VEGF-induced breakdown of the BRB. None of the inhibitors blocked leakage caused by TNF-alpha, IL-1beta, or IGF-1 and only PKC412, an inhibitor of protein kinase C (PKC) as well as VEGF and platelet-derived growth factor (PDGF) receptor kinases, decreased leakage caused by prostaglandins. Since the other inhibitors of VEGF and/or PDGF receptor kinases that do not also inhibit PKC had no effect on prostaglandin-induced breakdown of the BRB, these data implicate PKC in retinal vascular leakage caused by prostaglandins. PKC412 may be useful for treatment of post-operative and inflammatory macular edema, in which prostaglandins play a role, as well as macular edema associated with ischemic retinopathies.

Nitric oxide is proangiogenic in the retina and choroid.

Nitric oxide (NO) has been shown to have proangiogenic or antiangiogenic effects depending upon the setting. In this study, we used mice with targeted deletion of one of the three isoforms of nitric oxide synthase (NOS) to investigate the effects of NO in ocular neovascularization. In transgenic mice with increased expression of vascular endothelial growth factor (VEGF) in photoreceptors, deficiency of any of the three isoforms caused a significant decrease in subretinal neovascularization, but no alteration of VEGF expression. In mice with laser-induced rupture of Bruch's membrane, deficiency of inducible NOS (iNOS) or neuronal NOS (nNOS), but not endothelial NOS (eNOS), caused a significant decrease in choroidal neovascularization. In mice with oxygen-induced ischemic retinopathy, deficiency of eNOS, but not iNOS or nNOS caused a significant decrease in retinal neovascularization and decreased expression of VEGF. These data suggest that NO contributes to both retinal and choroidal neovascularization and that different isoforms of NOS are involved in different settings and different disease processes. A broad spectrum NOS inhibitor may have therapeutic potential for treatment of both retinal and choroidal neovascularization.

VEGF-TRAP(R1R2) suppresses choroidal neovascularization and VEGF-induced breakdown of the blood-retinal barrier.

Vascular endothelial growth factor (VEGF) plays a central role in the development of retinal neovascularization and diabetic macular edema. There is also evidence suggesting that VEGF is an important stimulator for choroidal neovascularization. In this study, we investigated the effect of a specific inhibitor of VEGF, VEGF-TRAP(R1R2), in models for these disease processes. VEGF-TRAP(R1R2) is a fusion protein, which combines ligand binding elements taken from the extracellular domains of VEGF receptors 1 and 2 fused to the Fc portion of IgG1. Subcutaneous injections or a single intravitreous injection of VEGF-TRAP(R1R2) strongly suppressed choroidal neovascularization in mice with laser-induced rupture of Bruch's membrane. Subcutaneous injection of VEGF-TRAP(R1R2) also significantly inhibited subretinal neovascularization in transgenic mice that express VEGF in photoreceptors. In two models of VEGF-induced breakdown of the blood-retinal barrier (BRB), one in which recombinant VEGF is injected into the vitreous cavity and one in which VEGF expression is induced in the retina in transgenic mice, VEGF-TRAP(R1R2) significantly reduced breakdown of the BRB. These data confirm that VEGF is a critical stimulus for the development of choroidal neovascularization and indicate that VEGF-TRAP(R1R2) may provide a new agent for consideration for treatment of patients with choroidal neovascularization and diabetic macular edema.

Intraocular gutless adenoviral-vectored VEGF stimulates anterior segment but not retinal neovascularization.

Vascular endothelial growth factor (VEGF) and insulin-like growth factor-1 (IGF-1) have been implicated as important stimulatory factors for retinal neovascularization. In this study, we used intraocular gene transfer with gutless adenoviral (AGV) vectors to determine the effect of increased intraocular expression of VEGF, IGF-1, or sphingosine kinase (SPK), which produces sphingosine-1-phosphate, another angiogenic factor. Retinal neovascularization did not occur from intravitreous AGV-vectored VEGF, IGF-1, SPK, or combined VEGF and IGF-1, except occasionally adjacent to the retinal penetration site from the injection. However, corneal and iris neovascularization occurred after 2 weeks in all eyes injected with AGV.VEGF, but not those injected with only AGV.IGF-1 or AGV.SPK. These data suggest that the superficial capillary bed of the retina is relatively insensitive to VEGF, IGF-1, or SPK in adult mice, except when combined with retinal trauma. However, AGV-vectored VEGF is sufficient to consistently cause severe corneal and iris neovascularization. This provides a model for anterior segment neovascularization, which unlike previous models is relatively inexpensive and is not plagued by spontaneous regression, and therefore, may be useful for identification of new treatments.