Regulation of Pathologic Retinal Angiogenesis in Mice and Inhibition of VEGF-VEGFR2 Binding by Soluble Heparan Sulfate

Development of the retinal vascular network is strictly confined within the neuronal retina, allowing the intraocular media to be optically transparent. However, in retinal ischemia, pro-angiogenic factors (including vascular endothelial growth factor-A, VEGF-A) induce aberrant guidance of retinal vessels into the vitreous. Here, we show that the soluble heparan sulfate level in murine intraocular fluid is high particularly during ocular development. When the eyes of young mice with retinal ischemia were treated with heparan sulfate-degrading enzyme, the subsequent aberrant angiogenesis was greatly enhanced compared to PBS-injected contralateral eyes; however, increased angiogenesis was completely antagonized by simultaneous injection of heparin. Intraocular injection of heparan sulfate or heparin alone in these eyes resulted in reduced neovascularization. In cell cultures, the porcine ocular fluid suppressed the dose-dependent proliferation of human umbilical vein endothelial cells (HUVECs) mediated by VEGF-A. Ocular fluid and heparin also inhibited the migration and tube formation by these cells. The binding of VEGF-A and HUVECs was reduced under a high concentration of heparin or ocular fluid compared to lower concentrations of heparin. In vitro assays demonstrated that the ocular fluid or soluble heparan sulfate or heparin inhibited the binding of VEGF-A and immobilized heparin or VEGF receptor 2 but not VEGF receptor 1. The recognition that the high concentration of soluble heparan sulfate in the ocular fluid allows it to serve as an endogenous inhibitor of aberrant retinal vascular growth provides a platform for modulating heparan sulfate/heparin levels to regulate angiogenesis

Increased Expression of Catalase and Superoxide Dismutase 2 Reduces Cone Cell Death in Retinitis Pigmentosa

Oxidative and nitrosative damage are major contributors to cone cell death in retinitis pigmentosa (RP). In this study, we explored the effects of augmenting components of the endogenous antioxidant defense system in models of RP, rd1, and rd10 mice. Unexpectedly, overexpression of superoxide dismutase 1 (SOD1) in rd1 mice increased oxidative damage and accelerated cone cell death. With an elaborate mating scheme, genetically engineered rd10 mice with either inducible expression of SOD2, Catalase, or both in photoreceptor mitochondria were generated. Littermates with the same genetic background that did not have increased expression of SOD2 nor Catalase provided ideal controls. Coexpression of SOD2 and Catalase, but not either alone, significantly reduced oxidative damage in the retinas of postnatal day (P) 50 rd10 mice as measured by protein carbonyl content. Cone density was significantly greater in P50 rd10 mice with coexpression of SOD2 and Catalase together than rd10 mice that expressed SOD2 or Catalase alone, or expressed neither. Coexpression of SOD2 and Catalase in rd10 mice did not slow rod cell death. These data support the concept of bolstering the endogenous antioxidant defense system as a gene-based treatment strategy for RP, and also indicate that coexpression of multiple components may be needed

Increased Expression of Glutathione Peroxidase 4 Strongly Protects Retina from Oxidative Damage

Oxidative damage contributes to cone cell death in retinitis pigmentosa and death of rods, cones, and retinal pigmented epithelial (RPE) cells in age-related macular degeneration. In this study, we explored the strategy of overexpressing components of the endogenous antioxidant defense system to combat oxidative damage in RPE cells and retina. In transfected cultured RPE cells with increased expression of superoxide dismutase1 (SOD1) or SOD2, there was increased constitutive and stress-induced oxidative damage measured by the level of carbonyl adducts on proteins. In contrast, RPE cells with increased expression of glutathione peroxidase 1 (Gpx1) or Gpx4 did not show an increase in constitutive oxidative damage. An increase in Gpx4, and to a lesser extent Gpx1, reduced oxidative stress-induced RPE cell damage. Co-expression of Gpx4 with SOD1 or 2 partially reversed the deleterious effects of the SODs. Transgenic mice with inducible expression of Gpx4 in photoreceptors were generated, and in three models of oxidative damage-induced retinal degeneration, increased expression of Gpx4 provided strong protection of retinal structure and function. These data suggest that gene therapy approaches to augment the activity of Gpx4 in the retina and RPE should be considered in patients with retinitis pigmentosa or age-related macular degeneration. Antioxid. Redox Signal. 11, 715–724