Aling Dong

Oxidative damage is a potential cause of cone cell death in retinitis pigmentosa

Retinitis pigmentosa (RP) is a prevalent cause of blindness caused by a large number of different mutations in many different genes. The mutations result in rod photoreceptor cell death, but it is unknown why cones die. In this study, we tested the hypothesis that cones die from oxidative damage by performing immunohistochemical staining for biomarkers of oxidative damage in a transgenic pig model of RP. The presence of acrolein‐ and 4‐hydroxynonenal‐adducts on proteins is a specific indicator that lipid peroxidation has occurred, and there was strong immunofluorescent staining for both in cone inner segments (IS) of two 10‐month‐old transgenic pigs in which almost all rods had died, compared to faint staining in two 10‐month‐old control pig retinas. In 22‐ and 24‐month‐old transgenic pigs in which all rods and many cones had died, staining was strong in cone axons and some cell bodies as well as IS indicating progression in oxidative damage between 10 and 22 months. Biomarkers for oxidative damage to proteins and DNA also showed progressive oxidative damage to those macromolecules in cones during the course of RP. These data support the hypothesis that the death of rods results in decreased oxygen consumption and hyperoxia in the outer retina resulting in gradual cone cell death from oxidative damage. This hypothesis has important therapeutic implications and deserves rapid evaluation.

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.

Superoxide dismutase 1 protects retinal cells from oxidative damage

Bolstering the endogenous oxidative damage defense system is a good strategy for development of treatments to combat neurodegenerative diseases in which oxidative damage plays a role. A first step in such treatment development is to determine the role of various components of the defense system in cells that degenerate. In this study, we sought to determine the role of superoxide dismutase 1 (SOD1) in two models of oxidative damage‐induced retinal degeneration. In one model, paraquat is injected into the vitreous cavity and then enters retinal cells and generates reactive oxygen species (ROS) that cause progressive retinal damage. Assessment of retinal function with serial electroretinograms (ERGs) showed that sod1−/− mice were much more sensitive than sod1+/+ mice to the damaging effects of paraquat, while sod1+/− mice showed intermediate sensitivity. Compared to sod1+/+ mice, sod1−/− mice showed greater paraquat‐induced oxidative damage and apoptosis. In the second model, mice were exposed to hyperoxia for several weeks, and sod1−/− mice showed significantly greater reductions in ERG amplitudes than sod1+/+ mice. In both of these models, transgenic mice carrying a sod1 transgene driven by a β‐actin promoter showed less oxidative stress‐induced reduction in ERG amplitudes. These data demonstrate that SOD1 protects retinal cells against paraquat‐ and hyperoxia‐induced oxidative damage and suggest that overexpression of SOD1 should be considered as one component of ocular gene therapy to prevent oxidative damage‐induced retinal degeneration. J. Cell. Physiol. 208: 516–526, 2006.

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.

Blockade of Sphingosine-1-Phosphate Reduces Macrophage Influx and Retinal and Choroidal Neovascularization

Sphingosine‐1‐phosphate (S1P) is a bioactive lipid molecule that stimulates endothelial cell migration, proliferation, and survival in vitro, and tumor angiogenesis in vivo. In this study, we used a humanized monoclonal antibody (sonepcizumab) that selectively binds S1P to investigate its role in retinal and choroidal neovascularization (NV). Intraocular injection of sonepcizumab significantly reduced macrophage influx into ischemic retina and strongly suppressed retinal NV in mice with oxygen‐induced ischemic retinopathy. In mice with laser‐induced rupture sites in Bruchs membrane, intraocular injection of sonepcizumab significantly reduced the area of choroidal NV and concomitantly reduced fluorescein leakage from the remaining choroidal NV. Four weeks after intraocular injection of up to 1.8 mg of the sonepcizumab in non‐human primates, electroretinograms and fluorescein angiograms were normal, and light microscopy of ocular sections showed no evidence of structural damage. These data show for the first time that S1P stimulates both choroidal and retinal NV and suggest that sonepcizumab could be considered for evaluation in patients with choroidal or retinal NV. J. Cell. Physiol. 218: 192–198, 2009.

Targeting VE-PTP activates TIE2 and stabilizes the ocular vasculature

Retinal and choroidal neovascularization (NV) and vascular leakage contribute to visual impairment in several common ocular diseases. The angiopoietin/TIE2 (ANG/TIE2) pathway maintains vascular integrity, and negative regulators of this pathway are potential therapeutic targets for these diseases. Here, we demonstrated that vascular endothelial-protein tyrosine phosphatase (VE-PTP), which negatively regulates TIE2 activation, is upregulated in hypoxic vascular endothelial cells, particularly in retinal NV. Intraocular injection of an anti-VE-PTP antibody previously shown to activate TIE2 suppressed ocular NV. Furthermore, a small-molecule inhibitor of VE-PTP catalytic activity (AKB-9778) activated TIE2, enhanced ANG1-induced TIE2 activation, and stimulated phosphorylation of signaling molecules in the TIE2 pathway, including AKT, eNOS, and ERK. In mouse models of neovascular age-related macular degeneration, AKB-9778 induced phosphorylation of TIE2 and strongly suppressed NV. Ischemia-induced retinal NV, which is relevant to diabetic retinopathy, was accentuated by the induction of ANG2 but inhibited by AKB-9778, even in the presence of high levels of ANG2. AKB-9778 also blocked VEGF-induced leakage from dermal and retinal vessels and prevented exudative retinal detachments in double-transgenic mice with high expression of VEGF in photoreceptors. These data support targeting VE-PTP to stabilize retinal and choroidal blood vessels and suggest that this strategy has potential for patients with a wide variety of retinal and choroidal vascular diseases.

Lysosomal-mediated waste clearance in retinal pigment epithelial cells is regulated by CRYBA1/βA3/A1-crystallin via V-ATPase-MTORC1 signaling

In phagocytic cells, including the retinal pigment epithelium (RPE), acidic compartments of the endolysosomal system are regulators of both phagocytosis and autophagy, thereby helping to maintain cellular homeostasis. The acidification of the endolysosomal system is modulated by a proton pump, the V-ATPase, but the mechanisms that direct the activity of the V-ATPase remain elusive. We found that in RPE cells, CRYBA1/βA3/A1-crystallin, a lens protein also expressed in RPE, is localized to lysosomes, where it regulates endolysosomal acidification by modulating the V-ATPase, thereby controlling both phagocytosis and autophagy. We demonstrated that CRYBA1 coimmunoprecipitates with the ATP6V0A1/V0-ATPase a1 subunit. Interestingly, in mice when Cryba1 (the gene encoding both the βA3- and βA1-crystallin forms) is knocked out specifically in RPE, V-ATPase activity is decreased and lysosomal pH is elevated, while cathepsin D (CTSD) activity is decreased. Fundus photographs of these Cryba1 conditional knockout (cKO) mice showed scattered lesions by 4 months of age that increased in older mice, with accumulation of lipid-droplets as determined by immunohistochemistry. Transmission electron microscopy (TEM) of cryba1 cKO mice revealed vacuole-like structures with partially degraded cellular organelles, undigested photoreceptor outer segments and accumulation of autophagosomes. Further, following autophagy induction both in vivo and in vitro, phospho-AKT and phospho-RPTOR/Raptor decrease, while pMTOR increases in RPE cells, inhibiting autophagy and AKT-MTORC1 signaling. Impaired lysosomal clearance in the RPE of the cryba1 cKO mice also resulted in abnormalities in retinal function that increased with age, as demonstrated by electroretinography. Our findings suggest that loss of CRYBA1 causes lysosomal dysregulation leading to the impairment of both autophagy and phagocytosis.

Increased expression of glial cell line-derived neurotrophic factor protects against oxidative damage-induced retinal degeneration

Oxidative damage contributes to retinal cell death in patients with age‐related macular degeneration or retinitis pigmentosa. One approach to treatment is to identify and eliminate the sources of oxidative damage. Another approach is to identify treatments that protect cells from multiple sources of oxidative damage. In this study, we investigated the effect of increased expression of glial cell line‐derived neurotrophic factor (GDNF) in three models of oxidative damage‐induced retinal degeneration. Double transgenic mice with doxycycline‐inducible expression of GDNF in the retina were exposed to paraquat, FeSO4, or hyperoxia, all sources of oxidative damage and retinal cell death. Compared to controls, mice with increased expression of GDNF in the retina showed significant preservation of retinal function measured by electroretinograms, reduced thinning of retinal cell layers, and fewer TUNEL‐positive cells indicating less retinal cell death. Mice over‐expressing GDNF also showed less staining for acrolein, nitrotyrosine, and 8‐hydroxydeoxyguanosine, indicating less oxidative damage to lipids, proteins, and DNA. This suggests that GDNF did not act solely to allow cells to tolerate higher levels of oxidative damage before initiation of apoptosis, but also reduced damage from oxidative stress to critical macromolecules. These data suggest that gene transfer of Gdnf should be considered as a component of therapy for retinal degenerations in which oxidative damage plays a role.

Vascular cell-adhesion molecule-1 plays a central role in the proangiogenic effects of oxidative stress

Oxidative stress exacerbates neovascularization (NV) in many disease processes. In this study we investigated the mechanism of that effect. Mice deficient in superoxide dismutase 1 (Sod1−/− mice) have increased oxidative stress and show severe ocular NV that is reduced to baseline by antioxidants. Compared with wild-type mice with ischemic retinopathy, Sod1−/− mice with ischemic retinopathy had increased expression of several NF-κB–responsive genes, but expression of vascular cell-adhesion molecule-1 (Vcam1) was particularly high. Intraocular injection of anti–VCAM-1 antibody eliminated the excessive ischemia-induced retinal NV. Elements that contributed to oxidative stress-induced worsening of retinal NV that were abrogated by blockade of VCAM-1 included increases in leukostasis, influx of bone marrow-derived cells, and capillary closure. Compared with ischemia alone, ischemia plus oxidative stress resulted in increased expression of several HIF-1–responsive genes caused in part by VCAM-1–induced worsening of nonperfusion and, hence, ischemia, because anti–VCAM-1 significantly reduced the increased expression of all but one of the genes. These data explain why oxidative stress worsens ischemia-induced retinal NV and may be relevant to other neovascular diseases in which oxidative stress has been implicated. The data also suggest that antagonism of VCAM-1 provides a potential therapy to combat worsening of neovascular diseases by oxidative stress.

An Adam15 amplification loop promotes vascular endothelial growth factor-induced ocular neovascularization

Proteins with a disintegrin and a metalloproteinase domain (ADAMs) are a family of membrane‐bound proteinases that bind integrins through their disintegrin domain. In this study, we have found modest expression of ADAM15 in pericytes in normal retina and strong up‐regulation of ADAM15 in retinal vascular endothelial cells in ischemic retina. Increased expression of vascular endothelial growth factor (VEGF) in the retina in the absence of ischemia also increased ADAM15 levels, and knockdown of Vegf mRNA in ischemic retina reduced Adam15 mRNA. Mice deficient in ADAM15 showed a significant reduction in ischemia‐induced retinal neovascularization, choroidal neovascularization at rupture sites in Bruchs membrane, and VEGF‐induced subretinal neovascularization. ADAM15‐deficient mice also showed reduced levels of VEGF164, VEGF receptor 1, and VEGF receptor 2 in ischemic retina. These data suggest that ADAM15 and VEGF participate in an amplification loop; VEGF increases expression of ADAM15, which in turn increases expression of VEGF and its receptors. Perturbation of the loop by elimination of ADAM15 suppresses ocular neovascularization in 3 different model systems, and thus ADAM15 provides a new therapeutic target for diseases complicated by neovascularization.—Xie, B., Shen, J., Dong, A., Swaim, M., Hackett, S. F., Wyder, L., Worpenberg, S., Barbieri, S., Campochiaro, P. A. An Adam15 amplification loop promotes VEGF‐induced ocular neovascularization. FASEB J. 22, 2775–2783 (2008)