Aberrant growth of blood vessels in the choroid layer of the eye, termed choroidal neovascularization (CNV), is the pathological hallmark of exudative age-related macular degeneration (AMD), causing irreversible blindness among the elderly. Co-localization of proangiogenic factors and hypoxia inducible factors (HIF) in neovascular membranes from AMD eyes suggests the role of hypoxia in pathogenesis of CNV. In order to utilize hypoxic conditions in RPE for therapeutic purposes, we developed an optimized hypoxia regulated, RPE cell-specific gene therapy to inhibit choroidal neovascularization. An adeno-associated virus (AAV2) vector comprising a RPE-specific promoter and HIF-1 response elements (HRE) was designed to regulate production of human endostatin (a powerful angiostatic protein) in RPE. The vector was tested in a mouse model of laser-induced CNV using subretinal delivery. Spectral domain optical coherence tomography (SD-OCT) images from live mice and confocal images from lectin stained RPE flat mount sections demonstrated reduction in CNV areas by 80% compared to untreated eyes. Quantitative real-time polymerase chain reaction (qPCR) confirmed exogenous endostatin mRNA expression from the regulated vector that was significantly elevated 3, 7, and 14 days following laser treatment, but its expression was completely shut off after 45 days. Thus, RPE-specific, hypoxia-regulated delivery of anti-angiogenic proteins could be a valuable therapeutic approach to treat neovascular AMD at the time and in the ocular space where it arises. Key points An optimized gene therapy vector targeting hypoxia and tissue-specific expression has been designed. The inhibitory role of gene therapy vector was tested in a mouse model of laser-induced CNV. An 80% reduction in choroidal neovascularization was achieved by the optimized vector. The expression of endostatin was limited to retinal pigment epithelium and regulated by hypoxia.

Significance Oxidative stress-induced damage to retinal pigmented epithelial (RPE) cells is implicated in the progression of age-related macular degeneration (AMD), which is one of the primary causes of vision loss in the elderly. The present studies show that sulindac, a known nonsteroidal antiinflammatory drug, can protect an established RPE cell line, low-passage human fetal RPE, and polarized primary human fetal RPE cells against oxidative damage. The results with the RPE cell line indicate that the protective response is similar to that seen with ischemic preconditioning. Our results suggest that preventing oxidative damage in RPE cells by this drug-induced protective mechanism could be an inexpensive and relatively nontoxic therapeutic approach for AMD treatment.

PURPOSE Müller cells, the major glial cell in the retina, play a significant role in retinal neovascularization in response to tissue hypoxia. We previously designed and tested a vector using a hypoxia-responsive domain and a glial fibrillary acidic protein (GFAP) promoter to drive green fluorescent protein (GFP) expression in Müller cells in the murine model of oxygen-induced retinopathy (OIR). This study compares the efficacy of regulated and unregulated Müller cell delivery of endostatin in preventing neovascularization in the OIR model. METHODS Endostatin cDNA was cloned into plasmids with hypoxia-regulated GFAP or unregulated GFAP promoters, and packaged into self-complementary adeno-associated virus serotype 2 vectors (scAAV2). Before placement in hyperoxia on postnatal day (P)7, mice were given intravitreal injections of regulated or unregulated scAAV2, capsid, or PBS. Five days after return to room air, on P17, neovascular and avascular areas, as well as expression of the transgene and vascular endothelial growth factor (VEGF), were compared in OIR animals treated with a vector, capsid, or PBS. RESULTS The hypoxia-regulated, glial-specific, vector-expressing endostatin reduced neovascularization by 93% and reduced the central vaso-obliteration area by 90%, matching the results with the unregulated GFAP-Endo vector. Retinas treated with the regulated endostatin vector expressed substantial amounts of endostatin protein, and significantly reduced VEGF protein. Endostatin production from the regulated vector was undetectable in retinas with undamaged vasculature. CONCLUSIONS These findings suggest that the hypoxia-regulated, glial cell-specific vector expressing endostatin may be useful for treatment of neovascularization in proliferative diabetic retinopathy.

This is a novel attempt to produce a rigorous mathematical model of a complex system. The complex system under study is the relationship between therapists and their clients.The success of psychotherapy depends on the nature of the relationship between a therapist and a client. We use dynamical systems theory to model the dynamics of the emotional interaction between a therapist and client. We determine how the therapeutic endpoint and the dynamics of getting there depend on the parameters of the model.Previously Gottman et al. [26] used a very similar approach (physical-sciences paradigm) for modeling and making predictions about husband-wife relationships. They modeled interactions using difference equations and then compared the behavior of those equations to the experimental affect data coded from video of married couples in a 15-minute discussion. The parameters they determined in this way had high predictive value of whether the marriages were stable and also gave new insights into the dynamics of how couples interact. Since that novel approach shed light on the dyadic interaction between couples we thought that it also had the possibility to give us new insights into the relationship between therapist and client.We describe the emotional state of both therapist and client with coupled, first order, nonlinear ordinary differential equations (ODE's). The rate of change of the emotional state of the therapist and client is proportional to their previous state, their uninfluenced state when alone, and an influence function which depends on the state of the other person. We formulated influence functions based on the research literature on psychotherapy and the therapeutic alliance. We then determined the critical points from the intersection of the nullclines and used a numerical ODE solver (Matlab ODE113) to compute the trajectories from different initial conditions.To empirically validate this approach, 73 unique therapy sessions were video-recorded. Four of these interactions (chosen by our psychotherapy expert) were selected to be modeled and were coded using Gottman's Specific Affect Coding System. The results validate this prototypical approach to psychotherapy; we have shown that human interaction (in the context of psychotherapy) can be quantified and modeled using differential equations.

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 hypoxia-inducible 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.

PURPOSE Retinal Müller cells span the retina and secrete several trophic factors and represent the functional link between blood vessels and neurons, making them attractive targets for gene therapy. Therefore, a hypoxia-regulated, retinal glial cell-specific vector was constructed and tested for its response to hypoxia. METHODS A hybrid promoter containing domains of human glial fibrillary acidic protein (GFAP) and several hypoxia-responsive and aerobically silenced elements (HRSE) was incorporated separately into plasmid vectors for generation of self-complementary adeno-associated virus. Müller cells trasfected with plasmids or virus were compared with other cell lines using standard METHODS The mouse model of oxygen-induced retinopathy (OIR) was used to analyze retinas from mice exposed to high oxygen or room air to evaluate the induction of the regulated promoter. RESULTS The regulated promoter was silenced under aerobic conditions in comparison with unregulated promoter in Müller cells. Hypoxia induced a 12-fold and 16-fold increase in promoter activity in primary Müller cells and human Müller cell lines, respectively. In the OIR model, intravitreal injection of the regulated promoter at postnatal day 7 (P7) resulted in high levels of green fluorescent protein expression only in retinal Müller cells at P17. GFP expression was absent in retinas of mice only exposed to room air. In vivo studies confirm normoxia silencing, hypoxic induction, and cell specificity of the regulated promoter in the mouse retina. CONCLUSIONS This hypoxia-regulated, retinal glial cell-specific AAV vector provides a platform for gene therapy within regions of retinal hypoxia which are found in diabetic retinopathy and age-related macular degeneration.