Jikui Shen

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.

MicroRNAs Regulate Ocular Neovascularization

In this study, we used ischemia-induced retinal neovascularization (NV) as a model to investigate the possible role of microRNAs in a clinically important disease process. Microarray analysis demonstrated seven microRNAs (miR-106a, -146, -181, -199a, -214, -424, and -451) that were substantially increased and three microRNAs (miR-31, -150, and -184) that were substantially decreased in ischemic retina. Potential targets for the upregulated microRNAs were not identified, but bioinformatic analysis suggested target genes for the downregulated microRNAs, and these were confirmed using a luciferase reporter assay. Real-time reverse transcriptase PCR confirmed that the substantial levels of miR-31, -150, and -184 present in normal retina were significantly reduced in ischemic retina. Interestingly, constitutive levels of miR-31 and -184 are high in the cornea and lens, two avascular tissues. Intraocular injection of pre-miR-31, -150, or -184 significantly reduced ischemia-induced retinal NV, and injection of pre-miR-31 or -150 also significantly reduced choroidal NV. These data suggest that alteration of microRNA levels contributes to two types of ocular NV, and that injection or enhanced expression of microRNAs is a potential therapeutic strategy.

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)

Vasohibin is up-regulated by VEGF in the retina and suppresses VEGF receptor 2 and retinal neovascularization

Vasohibin is a recently identified protein that is up‐regulated in cultured vascular endothelial cells by vascular endothelial growth factor and fibroblast growth factor 2. It inhibits endothelial cell migration, proliferation, and tube formation, and suppresses angiogenesis in chick chorioallantoic membrane, after subcutaneous implantation of matrigel, and in a tumor xenograft model. This has led to the hypothesis that vasohibin functions as a negative feedback inhibitor of angiogenesis. In this study, we tested that hypothesis in a well‐characterized model of retinal neovascularization. In ischemic retina, increased expression of VEGF was accompanied by elevation of vasohibin mRNA and blocking of the increase in vegf mRNA with vegf siRNA significantly attenuated the rise in vasohibin mRNA. In transgenic mice in which the rhodopsin promoter drives expression of VEGF in the retina, there was also a significant increase in vasohibin mRNA. In mice with ischemic retinopathy, there was increased expression of vasohibin in vascular endothelial cells, and vasohibin knockdown caused an increase in neovascularization. Conversely, intraocular injection of recombinant vasohibin or an adenoviral vector containing a vasohibin expression cassette strongly suppressed retinal neovascularization in mice with ischemic retinopathy. Knockdown of vasohibin mRNA in ischemic retina had no significant effect on vegf or vegf receptor 1 mRNA levels but caused a significant elevation in the level of vegf receptor 2 mRNA. These data support the hypothesis that vasohibin acts as a negative feedback regulator of neovascularization in the retina and suggest that suppression of VEGF receptor 2 may play some role in mediating its activity.

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.

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.

Implication of the hypoxia response element of the vegf promoter in mouse models of retinal and choroidal neovascularization, but not retinal vascular development

Retinal neovascularization (NV) and macular edema, resulting from blood‐retinal barrier (BRB) breakdown, are major causes of visual loss in ischemic retinopathies. Choroidal NV (CNV) occurs in diseases of the retinal pigmented epithelium/Bruchs membrane complex and is another extremely prevalent cause of visual loss. We used mice in which the hypoxia response element (HRE) is deleted from the vascular endothelial growth factor (vegf) promoter (Vegfδ/δ mice) to explore the role of induction of VEGF through the HRE in these disease processes. Compared to wild type (Vegf+/+) mice with oxygen‐induced ischemic retinopathy (OIR) in which vegf mRNA levels were increased and prominent retinal NV and BRB breakdown occurred, Vegfδ/δ littermates with OIR failed to increase vegf mRNA levels in the retina and had significantly less retinal NV and BRB breakdown, but showed prominent dilation of some superficial retinal vessels. Vegf+/δ littermates with ischemic retinopathy developed comparable retinal NV to Vegf+/+ mice, exhibited intermediate levels of BRB breakdown, and did not show vasodilation. In a mouse model of CNV, due to laser‐induced rupture of Bruchs membrane, the area of CNV at Bruchs membrane rupture sites was more than tenfold greater in Vegf+/+ mice than in Vegfδ/δ littermates. In contrast to these dramatic differences in pathologic ocular NV, Vegfδ/δ mice showed subtle differences in retinal vascular development compared to Vegf+/+ mice; it was slightly delayed, but otherwise normal. These data suggest that induction of VEGF through the HRE in its promoter is critical for retinal and CNV, but not for retinal vascular development. J.Cell.Physiol.

Digoxin inhibits retinal ischemia-induced HIF-1α expression and ocular neovascularization

Digoxin and other cardiac glycosides in‐hibit hypoxia‐inducible factor‐1 (HIF‐1) transcriptional activity in cultured cells and suppress tumor xenograft growth. We tested the hypothesis that digoxin reduces HIF‐1 levels in ischemic tissue in vivo and suppresses neovascularization. Well‐established murine models of ocular neovascularization were used to test our hypothesis. In mice with ischemic retinopathy, intraocular or intraperitoneal injection of digoxin markedly reduced retinal levels of HIF‐1α protein and mRNAs encoding multiple hypoxia‐regulated proangiogenic proteins and their receptors. Daily intraperitoneal injection of 2 mg/kg starting at postnatal day (P) 12 or a single intravitreous injection of 100 ng of digoxin at P12 reduced retinal neovascularization by >70% at P17. Digoxin also reduced the number of CXCR4+ cells and F4/80+ macrophages in ischemic retina and significantly reduced choroidal neovascularization at Bruchs membrane rupture sites. Digoxin suppresses retinal and choroidal neovascularization by reducing HIF‐1α levels, which blocks several proangiogenic pathways. Since digoxin suppresses multiple pathways in addition to VEGF signaling, it may provide advantages over specific VEGF antagonists for treatment of patients with retinal and choroidal diseases complicated by neovascularization and/or excessive vascular permeability. It may also be useful for treatment of neovascular diseases in other tissues.—Yoshida, T., Zhang, H., Iwase, T., Shen, J., Semenza, G. L., Campochiaro, P. A. Digoxin inhibits retinal ischemia‐induced HIF‐1α expression and ocular neovascularization. FASEB J. 24, 1759–1767 (2010). www.fasebj.org

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.