VEGF regulates local inhibitory complement proteins in the eye and kidney

10.1172/JCI86418Journal of Clinical Investigation1271199-21

Selective vulnerability of the intermediate retinal capillary plexus precedes retinal ganglion cell loss in ocular hypertension

Introduction: Glaucoma, a disease of retinal ganglion cell (RGC) injury and potentially devastating vision loss, is associated with both ocular hypertension (OHT) and reduced ocular blood flow. However, the relationship between OHT and retinal capillary architecture is not well understood. In this project, we studied microvasculature damage in mice exposed to mild levels of induced OHT.Methods: Mild OHT was induced with the microbead model for 2 weeks. At this time point, some retinas were immunostained with CD31 (endothelium), Collagen IV (basement membrane), and RBPMS (RGCs) for z-stack confocal microscopy. We processed these confocal images to distinguish the three retinal capillary plexi (superficial, intermediate, and deep). We manually counted RGC density, analyzed vascular complexity, and identified topographical and spatial vascular features of the retinal capillaries using a combination of novel manual and automated workflows. Other retinas were dissociated and immunopanned to isolate RGCs and amacrine cells (ACs) for hypoxia gene array analysis.Results: RGC counts were normal but there was decreased overall retinal capillary complexity. This reduced complexity could be explained by abnormalities in the intermediate retinal capillary plexus (IRCP) that spared the other plexi. Capillary junction density, vessel length, and vascular area were all significantly reduced, and the number of acellular capillaries was dramatically increased. ACs, which share a neurovascular unit (NVU) with the IRCP, displayed a marked increase in the relative expression of many hypoxia-related genes compared to RGCs from the same preparations.Discussion: We have discovered a rapidly occurring, IRCP-specific, OHT-induced vascular phenotype that precedes RGC loss. AC/IRCP NVU dysfunction may be a mechanistic link for early vascular remodeling in glaucoma

PhD

dissertationThe RPE is a polarized pigmented epithelial monolayer that is critical for vision and development of the neural retina. Considering its diverse and required roles, it is surprising how little is known about how it develops. Two transcription factors Mitf and Otx2 have been identified. Both are required for RPE development by activating many RPE terminal differentiation genes. The loss of either factor is catastrophic and induces RPE cells to dedifferentiate and to instead express retina-specific genes in a process termed RPE-to-retina transdifferentiation. Therefore, it is imperative that Mitf and Otx2 expression levels be maintained during development. A few other intrinsic and extrinsic factors have been identified that regulate RPE fate, but only one gene family (Pax genes) has been demonstrated to directly regulate Mitf. In Chapter 2,1 present results demonstrating a previously unknown role for Wnt/B-catenin signaling in the murine RPE. The pathway is active during development, and is required for RPE differentiation by directly regulating both Mitf and Otx2. Conditional inactivation of B-catenin results in many defects including RPE-to-retina transdifferentiation, adhesion defects, and coloboma with optic nerve defects. In Chapter 3,1 show evidence (in collaboration with Shinichi Nagakawa), that the role of Wnt/B-catenin in mouse and chicken is conserved. I also show that Otx2 and ficatenin, when co-transfected into the chicken retina, ectopically induces Mitf expression. This finding is key, since neither factor is sufficient alone to do so. 1 also confirm findings and provide novel evidence that both Otx2 and Mitf-D are able to autoregulate their enhancers to further enhance their expression levels during development In the final chapter, I will emphasize the importance of maintaining RPE fate during development. Maintaining Mitf and Otx2 expression levels is imperative. Consequently, my findings demonstrating that Wnt/B-catenin signaling regulates both are significant. Mitf expression can also be enhanced through integrated Otx2 and Wnt/Bcatenin signaling to enhance Mitf expression even higher (and both can autoregulate). I also provide a model of RPE development incorporating some of the known factors, and describe future directions to further elucidate the role of Wnt/B-catenin signaling in RPE formation

Age-Related Macular Degeneration and Diabetic Retinopathy

More than 15 years ago, the results of the pivotal trials supporting the intravitreal use of ranibizumab were published [...

Ectopic Mitf in the Embryonic Chick Retina by Co-transfection of β-Catenin and Otx2

The present study shows, by electroporation of chick embryonic eyes, that co-transfection of activated β-catenin and Otx2 can convert retinal progenitor cells into presumptive RPE cells expressing Mitf

TGFβ1 Induces Senescence and Attenuated VEGF Production in Retinal Pericytes

Diabetic retinopathy (DR) is a microvascular disease of the retina and a serious complication of type I and type II diabetes mellitus. DR affects working-age populations and can cause permanent vision loss if left untreated. The standard of care for proliferative DR is inhibiting VEGF. However, the mechanisms that induce excessive VEGF production in the retina remain elusive, although some evidence links elevated VEGF in the diabetic retina with local and systemic TGFβ1 upexpression. Here, we present evidence from animal models of disease suggesting that excessive TGFβ1 production in the early DR is correlated with VEGF mRNA and protein production by senescent pericytes and other retinal cells. Collectively, these results confirm that TGFβ1 is strongly implicated in the vascular complications of DR