Retinal degenerative diseases are diseases that may severely affect vision of people at different ages. These include retinitis pigmentosa (RP) and age-related macular degeneration (AMD). The current treatments for these diseases are limited. Since dysfunction and atrophy of the RPE are the key factors in the development of retinal degenerative diseases, transplantation of healthy retinal pigment epithelial (RPE) cells might be a promising therapeutic strategy. However, homologous RPE cells may lead to host immune rejection and harvesting autologous RPE cells may cause severe complications. Autologous iris pigment epithelial (IPE) cells, which are relatively easy to obtain, possess the same embryonic origin and share similar characteristics as RPE cells. Therefore, they may be used as a substitute of RPE cells for transplantation. Increasing interests have been demonstrated with the use of substrate to support cell attachment, proliferation and differentiation, so that transplanted cells could maintain the differentiated phenotype and perform their normal functions. However, degradation of biodegradable substrates may cause the breakdown of functional cell monolayer and produce toxic byproducts. Therefore, the aim of current study is to investigate the in vitro characteristics of rat IPE cells cultured on surface modified non-degradable expanded-polytetrafluorethylene (ePTFE) substrates and host response to the substrates without cells. Primary pure IPE cells were successfully isolated from rat eyes, which provided abundant cells for subsequent experiments. IPE cells harvested from both Long Evans rats and Dark Agouti rats proliferated and reached confluence on fibronectin coated n-heptylamine modified (F-HA) ePTFE substrates. These cells exhibited cuboidal or polygonal morphology with heavy pigmentation. In addition to the typical epithelial cell morphology, rat IPE cells grown on F-HA ePTFE substrates were able to form a cell monolayer with functional formation of tight junctional complex between neighboring cells. The IPE cell monolayers also demonstrated increased phagocytosis of photoreceptor outer segments (POS) with time and expression of cellular retinylaldehyde-binding protein (CRALBP) that served an important role in the conversion of all-trans-retinal to 11-cis-retinal in visual cycle. In the in vivo study, F-HA ePTFE substrate was successfully transplanted into the subretinal space of Royal College of Surgeons (RCS) rat, which is a well-recognized animal model of retinal degeneration. The F-HA ePTFE substrate remained flat up to 4 weeks after transplantation and did not induce significant up-regulation of pro-inflammatory cytokines TNFα and IL1β as well as activation of Müller cells and astrocytes which occurred in response to retinal inflammation. In conclusion, rat IPE cells that were grown on F-HA ePTFE substrate were able to establish a monolayer with functional tight junctions and RPE-specific functions. The F-HA ePTFE substrate demonstrated good biocompatibility in the subretinal space of RCS rats. These findings provide a potential therapeutic strategy for retinal degeneration. / published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

Age-related macular degeneration (AMD) is the leading cause of blindness in the western world for people over 60 years of age. The most severe pathological event of AMD is choroidal neovascularization (CNV), the process of new vessel formation emerging from the choroid. The new vessels extend into the normally avascular photoreceptor cell layer, where they leak fluid and cause photoreceptor cell death. CNV is thought to be initiated by hypoxia and chronic inflammation, which occur due to abnormal, age-related changes within the retinal pigmented epithelium (RPE). These events cause increased expression of the angiogenic protein vascular endothelial growth factor (VEGF) via hypoxiainducible factor-1 (HIF-1), a transcription factor that is vital in regulation of cellular responses to hypoxic and inflammatory conditions. Increased VEGF signaling stimulates proliferation and migration of vascular endothelial cells and facilitates the neovascular process. To target the early pathological events that lead to CNV, we have engineered a novel gene therapy vector that uses HIF-1 regulation to stimulate production of an angiostatic protein, endostatin from the RPE. The purpose of this study was to characterize the activity of our hypoxiaregulated, RPE-specific promoter in vitro, and investigate the effects of regulated endostatin expression, driven by our regulated promoter, on CNV in a mousemodel. We found the regulated promoter construct has robust activity in vitro only in RPE cells, and is conditionally responsive in hypoxic conditions. / In the laserinduced CNV model, CNV area was 80% smaller (P<0.0001) in eyes treated with the hypoxia-regulated, RPE-specific endostatin vector than in untreated eyes. CNV area was equally reduced in eyes treated with an unregulated endostatin vector (CMV-endostatin). However, less endostatin protein was detected in eyes treated with the regulated vector. Since it is unknown whether broad and constitutive endostatin expression will have damaging effects within the retina, it may be safer to limit its expression to pathological conditions. We have demonstrated that local, hypoxia-regulated expression of endostatin can effectively inhibit CNV, and thus, offers the further possibility of a prophylactic treatment for neovascular AMD. / by George Wesley Tyler Smith. / Thesis (Ph.D.)--Florida Atlantic University, 2010. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2010. Mode of access: World Wide Web.

Diabetic retinopathy is an ischemic retinal neovascular disease causing vision loss among adults. The studies presented involve the design and testing of a gene therapy vector to inhibit retinal revascularization, similar to that found in diabetic retinopathy. Gene therapy has proven to be an effective method to introduce therapeutic proteins to treat retinal diseases. Targeting a specific cell type and expression of therapeutic proteins according to the tissue microenvironment should have an advantage over traditional gene therapy by avoiding unwanted transgene expression. Hypoxia plays a significant role in the pathophysiology of many retinal ischemic diseases. Retinal Mèuller cells provide structural and functional support to retinal neurons, as well as playing a significant role in retinal neovascularization. Targeting Mèuller cells may be an effective strategy to prevent retinal neovascularization under pathological conditions. ... The hypoxia regulated, glial specific vector successfully reduced the abnormal neovascularization in the periphery by 93% and reduced the central vasobliterated area by 90%. A substantial amount of exogenous endostatin was produced in the retinas of P17 OIR mice. A significant increase in human endostatin protein and reduced vascular endothelial growth factor (VEGF) were identified by Western blot and ELISA, respectively. These findings suggest hypoxia-regulated, glial cell-specific scAAV mediated gene expression may be useful to prevent blindness found in devastating retinal diseases involving neovascularization. / by Manas Ranjan Biswal. / Thesis (Ph.D.)--Florida Atlantic University, 2012. / Includes bibliography. / Mode of access: World Wide Web. / System requirements: Adobe Reader.

Indiana University-Purdue University Indianapolis (IUPUI) / Prevention of pathological angiogenesis is a key strategy for treatment of common blinding ocular diseases such as retinopathy of prematurity, proliferative diabetic retinopathy, and wet age-related macular degeneration. The current treatment strategies are associated with partial vision loss and are ineffective in a significant patient population. Hence novel drugs as well as new ways to target ocular angiogenesis are needed for treating these diseases. I pursued a natural antiangiogenic compound, cremastranone, to develop novel drug leads and to find new targets. The objective of my doctoral thesis project was to elucidate cremastranone’s molecular mechanism of action and optimize its structureactivity relationship (SAR). In order to achieve this goal, with the help of chemistry collaborators cremastranone was synthesized for the first time. I showed that cremastranone has 50-fold more potency against endothelial cells as compared to nonendothelial cells, and also tested a novel active isomer, SH-11052. By SAR studies I identified a potent molecule, SH-11037, that has 10-fold more selectivity against retinal endothelial cells as compared to macrovascular endothelial cells. I then elucidated cremastranone’s molecular mechanism using a chemical proteomic approach. I identified ferrochelatase (FECH) as a specific interacting protein partner of cremastranone using photoaffinity chromatography. Hence, I hypothesized that cremastranone exerts its antiangiogenic activities through modulation of the functions of FECH. Cremastranone inhibited the enzymatic activity FECH in endothelial cells. Therefore, I investigated the role of FECH in ocular angiogenesis. Partial loss of FECH, using a siRNA-based knock down approach, decreased retinal angiogenesis both in vitro and in vivo in mouse models. Knock down of FECH decreased the expression levels of key proangiogenic proteins HIF-1α, eNOS, and VEGFR2. This work suggests that ferrochelatase plays an important, previously undocumented role in angiogenesis and that targeting of this enzyme by cremastranone might be exploited to inhibit pathological angiogenesis in ocular diseases.