Blood outer retina barrier regulation

Abstract As with other neurons, photoreceptor metabolism is significantly focused on glucose, which is transported to the outer retina from the choroid circulation via the RPE that serves as the blood-outer retinal barrier. The RPE also transports other key metabolites back-and-forth between the outer retina and the choroid circulation, and it metabolizes circulating compounds into important metabolites that are passed on to photoreceptors, providing evidence that the RPE and choroid vasculature act as a metabolic unit. Mounting evidence suggests that retinal diseases, including retinitis pigmentosa (RP) and AMD, are linked to metabolic changes. Taking advantage of perfusion differences in retinal compartments during the previous grant period, we have developed a protocol to selectively deliver labeled metabolites in vivo to the pig RPE and in turn outer retina, and to quantify uptake and secretion of metabolites from the RPE and choroid circulation by comparing levels in entering arteries to draining veins using mass spectroscopy. These findings were coupled with single cell RNA sequencing analysis to generate metabolomes of pig and human retina and retina-related cells. We utilized these metabolomes to follow metabolism in the WT retina and RPE in vivo, and to examine how metabolic changes contribute to pathology in progression of RP, while addressing each of the Aims in the original grant. We have uncovered new pathways and unanticipated roles for established pathways in vivo that highlight bidirectional interplay between the RPE and outer retinal photoreceptors and emphasize the key role of the choroid vasculature and RPE as a metabolic unit, and we propose to investigate these pathways in this renewal. We concluded from these studies that the complex interplay between retinal and extraretinal cell types and the notion of a choroid circulation-RPE metabolic unit emphasize the importance of vivo studies where cell and vascular architecture is intact. Because of the key role of the choroid vasculature in RPE and outer retinal metabolism, we expanded our studies during the previous grant period to include new vascular sprouting in response to disease and damage, examining endothelial tip cells that lead sprout migration and macrophages that are crucial for new vessel formation. At the molecular level, we show formation and migration of new vascular sprouts in multiple models of neovascularization are linked to mesenchymal transition using lineage tracing and depend upon induction of the mesenchymal-driving transcription factor ZEB1 using cell-specific gene knockouts. In the outer retina such sprouts from the choroid vasculature bypass the RPE to invade the subretinal space and eventually enter the retina, and these vessels also show altered metabolite permeability. This lack of an RPE barrier and permeability differences suggest altered delivery of metabolites from these new sprouts affect retinal and RPE metabolic pathways, which we propose to investigate.