This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. A. The proposed Specific Aims are: 1: To examine the role of VEGF signaling in cone photoreceptor survival. 2: To examine the role of VEGF signaling in rod photoreceptor survival. B. Studies and Results 1) In vitro analysis of VEGF signaling in a cone cell line. Since cones only consist of 3 percent of photoreceptors, it is not possible to investigate the biochemical events using whole retinal homogenates. Therefore, we have been using an existing cone cell line (661W). 661W cells expressed VEGF, VEGF receptor 1 (R1), and VEGF receptor 2 (R2). As significant ( 90%) retinal photoreceptor cells death was observed in experimental model of acute hypoxia, we used a hypoxia model and treated the cells with chemical inducer of hypoxia cobalt chloride (CoCl2) and receptor-specific inhibitors. While CoCl2 induces cell death, further VEGFR2 inhibition leads to an increased cell death. We also observed a slight but significant increase of VEGFR1 levels. Inhibition of VEGFR1 rescued hypoxia-induced cell death. Our results also indicated that VEGFR2 and AKT (and its down stream target) were activated under hypoxic conditions, suggesting that VEGFR2/AKT survival pathway is required for cone photoreceptor survival under hypoxia, which is negatively regulated by VEGFR1 activation. These results formed a working model for our mechanistic investigation for cone survival. 2) An acute model for studying diabetes-induced photoreceptor death. As diabetes-induced alternations in retinal cells are slow. We decided to use an acute model that produces various degree of retinal ischemia with cobalt chloride in mice. Various concentrations of cobalt chloride were used to identify suitable conditions to produce ischemia-induced photometer death in mice. The animals were subjected to functional analysis with electroretinography (ERG) and morphological analysis in retinal sections. We have identified conditions for various degrees of photoreceptor loss. This model mimics late stage of diabetes and can be regarded as """"""""acute diabetic animal model"""""""" which will allows us to address questions in vivo within 2 weeks. Therefore, these assays will be used in our in vivo analysis of our rod or cone photoreceptor-specific VEGFR2 knockout mice. 3) Conditional VEGFR2 knockout mice In order to perform Cre/lox-based conditional gene expression studies in cone and rod photoreceptors, I have generated cone- or rod-specific Cre mice. They have been successfully used in a number of studies. The floxed VEGF-R2 mice were mated with rod or cone photoreceptor-specific Cre mice to generate the conditional VEGF-R2 knockout mice. We are in the process of generating sufficient animals for phenotypic analysis. A few rod-specific VEGF-R2 knockout mice were subjected to ischemia. Our preliminary results indicated that loss of VEGF-R2 accelerated rod photoreceptor death under ischemic conditions. C. Significance Anti-VEGF treatments have become a major therapeutic strategy for vascular complication in diabetic retinopathy. However, VEGF may also be a survival factor for retinal neurons, including photoreceptors. Revealing the role of VEGF in retinal neuron survival is paramount to the safety of long-term anti-VEGF therapies. In addition, it is important to identify VEGF signaling intermediates and their isoforms that may play a role only in neuron survival but not in vascular leakage will be very useful for preserving vision in diabetes. These molecules will be potential agents for retinal neuron survival in diabetes. D. Future Plan In the next year, we will focus on identifying the role of other VEGF signaling components, such as VEGF-B and its downstream VEGF co-receptor, neuropilin 1 and 2 in the survival of photoreceptors. We will also perform functional analysis in rod or cone-photoreceptor-specific VEGFR2 knockout mice.
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