Katsutoshi Yokoi

The SDF-1/CXCR4 ligand/receptor pair is an important contributor to several types of ocular neovascularization

Hypoxia causes increased expression of several proteins that have the potential to promote neovascularization. Vascular endothelial growth factor (VEGF) is up‐regulated by hypoxia in the retina and plays a central role in the development of several types of ocular neovascularization, but the effects of other hypoxia‐regulated proteins are less clear. Stromal‐de‐rived factor‐1 (SDF‐1) and its receptor, CXCR4, have hypoxia response elements in the promoter regions of their genes and are increased in hypoxic liver and heart. In this study, we found that SDF‐1 and CXCR4 are increased in hypoxic retina, with SDF‐1 localized in glial cells primarily near the surface of the retina and CXCR4 localized in bone marrow‐derived cells. Glial cells also expressed CXCR4, which suggested the possibility of autocrine stimulation, but influx of bone marrow‐derived cells is the major source of increased levels of CXCR4. High levels of VEGF in the retina in the absence of hypoxia also increased levels of Cxcr4 and Sdf1 mRNA. CXCR4 antagonists reduced influx of bone marrow‐derived cells into ischemic retina and strongly suppressed retinal neovascularization, VEGF‐induced subretinal neovascularization, and choroidal neovascularization. These data suggest that SDF‐1 and CXCR4 contribute to the involvement of bone marrow‐derived cells and collaborate with VEGF in the development of several types of ocular neovascularization. They provide new targets for therapeutic intervention that may help to bolster and supplement effects obtained with VEGF antagonists.—Lima e Silva, R., Shen, J., Hackett, S. F., Kachi, S., Akiyama, H., Kiuchi, K., Yokoi, K., Hatara, M. C., Lauer, T., Aslam, S., Gong, Y. Y., Xiao, W‐H., Khu, N. H., Thut, C., Campochiaro, P. A. The SDF‐1/CXCR4 ligand/receptor pair is an important contributor to several types of ocular neo‐vascularization. FASEB J. 21, 3219–3230 (2007)

Oxidative Stress Promotes Ocular Neovascularization

Mice deficient in superoxide dismutase 1 (Sod1−/− mice) develop many features seen in patients with age‐related macular degeneration (AMD) including choroidal neovascularization (NV). We sought to determine if the absence of SOD1 contributes to the pro‐angiogenic environment in the subretinal space or whether it is completely secondary to other changes in Bruchs membrane and the retinal pigmented epithelium (RPE) that precede the development of choroidal NV. In an ischemic retinopathy model or a transgenic model in which the rhodopsin promoter drives expression of vascular endothelial growth factor (VEGF) in photoreceptor there was significantly more NV in Sod1−/− compared to Sod1+/+ mice. The compromised antioxidant defense system in Sod1−/− mice contributes to the pro‐angiogenic environment, because treatment of Sod1−/− mice with a mixture of antioxidants caused a significant reduction in ischemia‐induced retinal NV. Wild‐type mice treated with the same antioxidants also showed reduced ischemia‐induced retinal NV, reduced VEGF‐induced subretinal NV, and reduced choroidal NV at Bruchs membrane rupture sites. These data suggest that reactive oxygen species contribute to several types of ocular NV. This could explain why in the Age‐Related Eye Disease Trial, antioxidant treatment reduced conversion from non‐neovascular to neovascular AMD and severe vision loss, and suggest that potent antioxidants should be considered for other diseases complicated by ocular NV. J. Cell. Physiol. 219: 544–552, 2009.

Topical Administration of a Multi-Targeted Kinase Inhibitor Suppresses Choroidal Neovascularization and Retinal Edema

Age‐related macular degeneration, diabetic retinopathy, and retinal vein occlusions are complicated by neovascularization and macular edema. Multi‐targeted kinase inhibitors that inhibit select growth factor receptor tyrosine kinases and/or components of their down‐stream signaling cascades (such as Src kinases) are rationale treatment strategies for these disease processes. We describe the discovery and characterization of two such agents. TG100572, which inhibits Src kinases and selected receptor tyrosine kinases, induced apoptosis of proliferating endothelial cells in vitro. Systemic delivery of TG100572 in a murine model of laser‐induced choroidal neovascularization (CNV) caused significant suppression of CNV, but with an associated weight loss suggestive of systemic toxicity. To minimize systemic exposure, topical delivery of TG100572 to the cornea was explored, and while substantial levels of TG100572 were achieved in the retina and choroid, superior exposure levels were achieved using TG100801, an inactive prodrug that generates TG100572 by de‐esterification. Neither TG100801 nor TG100572 were detectable in plasma following topical delivery of TG100801, and adverse safety signals (such as weight loss) were not observed even with prolonged dosing schedules. Topical TG100801 significantly suppressed laser‐induced CNV in mice, and reduced fluorescein leakage from the vasculature and retinal thickening measured by optical coherence tomography in a rat model of retinal vein occlusion. These data suggest that TG100801 may provide a new topically applied treatment approach for ocular neovascularization and retinal edema. J. Cell. Physiol. 216: 29–37, 2008.

Mecamylamine suppresses basal and nicotine- stimulated choroidal neovascularization

PURPOSE Nicotinic acetylcholine receptors (nAChR) are best known for their role in neurotransmission, but they have recently been demonstrated on vascular endothelial cells. Acetylcholine is their endogenous ligand, but they are also stimulated by nicotine. By stimulating nAChR, nicotine promotes tumor angiogenesis as well as atherosclerotic plaque neovascularization. In this study, the authors investigated the role of nAChR in the pathogenesis of choroidal neovascularization (CNV). METHODS The effect of the nonselective nAChR antagonist mecamylamine was tested on human retinal and choroidal endothelial cells in vitro and in a murine model of CNV. RESULTS Several nAChR isoforms were identified in retinal and choroidal microvascular endothelial cells, and the ability of these cells to form tubules when grown in growth factor-reduced basement membrane matrix and supplemented with VEGF was suppressed by the nAChR antagonist mecamylamine. Supplementation of the drinking water of mice with nicotine increased the size of CNV lesions at Bruch membrane rupture sites, an effect that was blocked by subcutaneous administration of mecamylamine (50 mg/kg/d) by an osmotic pump. In the absence of nicotine, CNV formation was suppressed by the infusion of 50 mg/kg/d mecamylamine or by topical application 0.1 or 1% mecamylamine to the cornea. CONCLUSIONS These data suggest that endogenous activation of nAChR promotes CNV and that activation of nAChR by nicotine may contribute to the increased incidence of CNV seen in smokers with age-related macular degeneration (AMD). Topically administered mecamylamine could provide an appealing new treatment approach for CNV.

Equine Infectious Anemia Viral Vector-Mediated Codelivery of Endostatin and Angiostatin Driven by Retinal Pigmented Epithelium-Specific VMD2 Promoter Inhibits Choroidal Neovascularization

Equine infectious anemia virus (EIAV) is a nonprimate lentivirus that does not cause human disease. Subretinal injection into mice of a recombinant EIAV lentiviral vector in which lacZ is driven by a CMV promoter (EIAV CMV LacZ) resulted in rapid and strong expression of LacZ in retinal pigmented epithelial (RPE) cells and some other cells including ganglion cells, resulting in the presence of 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside within the optic nerve. Substitution of the RPE-specific promoter from the vitelliform macular dystrophy (VMD2) gene for the CMV promoter resulted in prolonged (at least 1 year) expression of LacZ that was restricted to RPE cells, albeit reduced 6- to 10-fold compared with the CMV promoter. Similarly, the amount of FLAG-tagged endostatin detected in eyes injected with the EIAV VMD2 Endo(FLAG) vector was similar to that seen in eyes injected with a vector that expressed both endostatin and angiostatin [EIAV VMD2 Endo(FLAG)/Angio]; expression was approximately 6-fold lower than with identical vectors in which the CMV promoter drove expression. Compared with murine eyes treated with a control EIAV vector, subretinal injection of EIAV vectors expressing murine endostatin alone or in combination with angiostatin driven by either the CMV or VMD2 promoter caused significant suppression of choroidal neovascularization (NV) at laser-induced rupture sites in Bruchs membrane. These data support proceeding toward clinical studies with EIAV-based gene therapy for choroidal NV, using the VMD2 promoter to selectively drive expression of a combination of endostatin and angiostatin in RPE cells.

Development of prodrug 4-chloro-3-(5-methyl-3-{[4-(2-pyrrolidin-1-ylethoxy) phenyl]amino}-1,2,4-benzotriazin-7-yl)phenyl benzoate (TG100801): A topically administered therapeutic candidate in clinical trials for the treatment of age-related macular degeneration

Age-related macular degeneration (AMD) is one of the leading causes of loss of vision in the industrialized world. Attenuating the VEGF signal in the eye to treat AMD has been validated clinically. A large body of evidence suggests that inhibitors targeting the VEGFr pathway may be effective for the treatment of AMD. Recent studies using Src/YES knockout mice suggest that along with VEGF, Src and YES play a crucial role in vascular leak and might be useful in treating edema associated with AMD. Therefore, we have developed several potent benzotriazine inhibitors designed to target VEGFr2, Src, and YES. One of the most potent compounds is 4-chloro-3-{5-methyl-3-[4-(2-pyrrolidin-1-yl-ethoxy)phenylamino]benzo[1,2,4]triazin-7-yl}phenol ( 5), a dual inhibitor of both VEGFr2 and the Src family (Src and YES) kinases. Several ester analogues of 5 were prepared as prodrugs to improve the concentration of 5 at the back of the eye after topical administration. The thermal stability of these esters was studied, and it was found that benzoyl and substituted benzoyl esters of 5 showed good thermal stability. The hydrolysis rates of these prodrugs were studied to analyze their ability to undergo conversion to 5 in vivo so that appropriate concentrations of 5 are available in the back-of-the-eye tissues. From these studies, we identified 4-chloro-3-(5-methyl-3-{[4-(2-pyrrolidin-1-ylethoxy)phenyl]amino}-1,2,4-benzotriazin-7-yl)phenyl benzoate ( 12), a topically administered prodrug delivered as an eye drop that is readily converted to the active compound 5 in the eye. This topically delivered compound exhibited excellent ocular pharmacokinetics and poor systemic circulation and showed good efficacy in the laser induced choroidal neovascularization model. On the basis of its superior profile, compound 12 was advanced. It is currently in a clinical trial as a first in class, VEGFr2 targeting, topically applied compound for the treatment of AMD.