Paul Andrew Yates

EPHRIN-A5 (AL-1/RAGS) IS ESSENTIAL FOR PROPER RETINAL AXON GUIDANCE AND TOPOGRAPHIC MAPPING IN THE MAMMALIAN VISUAL SYSTEM

Ephrin-A5 (AL-1/RAGS), a ligand for Eph receptor tyrosine kinases, repels retinal axons in vitro and has a graded expression in the superior colliculus (SC), the major midbrain target of retinal ganglion cells. These properties implicate ephrin-A5 in the formation of topographic maps, a fundamental organizational feature of the nervous system. To test this hypothesis, we generated mice lacking ephrin-A5. The majority of ephrin-A5-/- mice develop to adulthood, are morphologically intact, and have normal anterior-posterior patterning of the midbrain. However, within the SC, retinal axons establish and maintain dense arborizations at topographically incorrect sites that correlate with locations of low expression of the related ligand ephrin-A2. In addition, retinal axons transiently overshoot the SC and extend aberrantly into the inferior colliculus (IC). This defect is consistent with the high level of ephrin-A5 expression in the IC and the finding that retinal axon growth on membranes from wild-type IC is inhibited relative to that on membranes from ephrin-A5-/- IC. These findings show that ephrin-A5 is required for the proper guidance and mapping of retinal axons in the mammalian midbrain.

Topographic Mapping from the Retina to the Midbrain Is Controlled by Relative but Not Absolute Levels of EphA Receptor Signaling

Topographic maps are a fundamental feature of sensory representations in nervous systems. The formation of one such map, defined by the connection of ganglion cells in the retina to their targets in the superior colliculus of the midbrain, is thought to depend upon an interaction between complementary gradients of retinal EphA receptors and collicular ephrin-A ligands. We have tested this hypothesis by using gene targeting to elevate EphA receptor expression in a subset of mouse ganglion cells, thereby producing two intermingled ganglion cell populations that express distinct EphA receptor gradients. We find that these two populations form separate maps in the colliculus, which can be predicted as a function of the net EphA receptor level that a given ganglion cell expresses relative to its neighbors.

Intravitreal bevacizumab (Avastin) as treatment for subfoveal choroidal neovascularisation secondary to pathological myopia

Objective: To evaluate the safety and efficacy of intravitreal bevacizumab (Avastin) as treatment for subfoveal choroidal neovascularisation (CNV) due to pathological myopia. Methods: Consecutive series of primary or recurrent subfoveal CNV secondary to myopia treated with intravitreal bevacizumab 1.25 mg between August 2005 and January 2006 at the New England Eye Center, Boston, Massachusetts, USA, were reviewed retrospectively. Data from clinical examination, fundus photography, fluorescein angiography, optical coherence tomography and visual acuity were collected. Results: There were 11 eyes of 9 patients. 5 of 11 eyes had been treated previously with photodynamic therapy. Pre-injection visual acuity measured 20/50 to 20/100 in 6 eyes and 20/200 or worse in 5 eyes. After a mean follow-up of 153 (range 35–224) days, post-injection visual acuity measured 20/20 to 20/40 in 7 eyes, 20/50 to 20/100 in 1 eye and 20/200 or worse in 3 eyes. Three eyes received two bevacizumab injections and eight eyes received one injection. Visual acuity improved by a mean of +3.5 (range −1 to +8 lines) lines, and 8 of 11 eyes achieved 20/50 or better at the last follow-up. Central foveal thickness improved from 340 (range 253–664) μm to 234 (range 142–308) μm, representing an average reduction of 103 (range +4 to −356) μm. No injection complications or drug-related side effects were observed. Conclusions: In this small series of eyes with limited follow-up, intravitreal bevacizumab seems to be safe and potentially efficacious in eyes with subfoveal CNV secondary to pathological myopia.

A POU domain transcription factor-dependent program regulates axon pathfinding in the vertebrate visual system.

Axon pathfinding relies on the ability of the growth cone to detect and interpret guidance cues and to modulate cytoskeletal changes in response to these signals. We report that the murine POU domain transcription factor Brn-3.2 regulates pathfinding in retinal ganglion cell (RGC) axons at multiple points along their pathways and the establishment of topographic order in the superior colliculus. Using representational difference analysis, we identified Brn-3.2 gene targets likely to act on axon guidance at the levels of transcription, cell-cell interaction, and signal transduction, including the actin-binding LIM domain protein abLIM. We present evidence that abLIM plays a crucial role in RGC axon pathfinding, sharing functional similarity with its C. elegans homolog, UNC-115. Our findings provide insights into a Brn-3.2-directed hierarchical program linking signaling events to cytoskeletal changes required for axon pathfinding.

Molecular Development of Sensory Maps: Representing Sights and Smells in the Brain

retinotectal and olfactory systems has clear distinctions, Introduction which relate in part to differences in their functional Defining the molecules and mechanisms that control requirements. In the retinotectal system, the main objecthe establishment of an orderly representation of the tive is to represent the visual world in the brain, that is, peripheral sense organs within the brain has long been to reconstruct a topographic representation of the world of interest to systems and developmental neurobiolothat projects onto the retina and is remapped in the gists. Classically, the projection from the retina to the tectum. To carry out this function requires the maintebrain has served as the model system for understanding nance of a precise spatial ordering of axonal connechow precise neural connections are formed. More retions within the tectum that reflects their origins in the cently, the molecular cloning of olfactory receptors retina. In contrast, in the olfactory system, since odors (ORs) has provided valuable insights into the functional have no relevant spatial component, there is no overridand anatomical organization of the olfactory system, ing need to maintain spatial continuity, either between including the projection of olfactory neurons (ONs) from cells expressing a given OR, and presumably respondthe olfactory epithelium to the olfactory bulb (OB). The ing to the same odors, or between glomeruli in the OB. mechanisms involved in establishing this projection, as This functional difference relates to differences in the well as its organization, are atypical and make for revealmapping strategies employed in the two systems. ing comparisons when juxtaposed to the development This article will review current knowledge of the mechof order in the visual system. anisms and molecules proposed to control mapping in Both the visual and olfactory systems represent senthe visual and olfactory systems and attempt a synthesis sory information within the brain through the use of to highlight differences and similarities in their organizasensory maps. The projection of sensory axons to the tions and the molecular mechanisms that may control brain forms these maps through the spatial segregation their development. We have focused almost exclusively and orderly termination of their axonal connections in on the projection of RGCs to the chick optic tectum, or specific target tissues. However, the visual map is funits equivalent in rodents, the superior colliculus (SC), as damentally different from the olfactory map in that it is well as the main olfactory system of mice. We have not strictly topographic: a two-dimensional sheet of retinal discussed the large bodies of excellent work on the ganglion cells (RGCs) in the retina is rerepresented in retinotectal system of fish and amphibians, the main the brain as more or less the same two-dimensional olfactory system of lower vertebrates or C. elegans, nor sheet through the orderly terminations of RGC axons. do we consider the projection from the vomeronasal In contrast, the olfactory map is formed by the converorgan to the accessory olfactory bulb (for reviews of gence of the axonal projections of a specific set of functhese topics see Roskies et al., 1995; Bargmann, 1997; tionally similar ONs that are randomly distributed in the Karlstrom et al., 1997; Ebrahimi and Chess, 1998). olfactory epithelium onto specific glomeruli, and in doing

Pericytes Derived from Adipose-Derived Stem Cells Protect against Retinal Vasculopathy

Background Retinal vasculopathies, including diabetic retinopathy (DR), threaten the vision of over 100 million people. Retinal pericytes are critical for microvascular control, supporting retinal endothelial cells via direct contact and paracrine mechanisms. With pericyte death or loss, endothelial dysfunction ensues, resulting in hypoxic insult, pathologic angiogenesis, and ultimately blindness. Adipose-derived stem cells (ASCs) differentiate into pericytes, suggesting they may be useful as a protective and regenerative cellular therapy for retinal vascular disease. In this study, we examine the ability of ASCs to differentiate into pericytes that can stabilize retinal vessels in multiple pre-clinical models of retinal vasculopathy. Methodology/Principal Findings We found that ASCs express pericyte-specific markers in vitro. When injected intravitreally into the murine eye subjected to oxygen-induced retinopathy (OIR), ASCs were capable of migrating to and integrating with the retinal vasculature. Integrated ASCs maintained marker expression and pericyte-like morphology in vivo for at least 2 months. ASCs injected after OIR vessel destabilization and ablation enhanced vessel regrowth (16% reduction in avascular area). ASCs injected intravitreally before OIR vessel destabilization prevented retinal capillary dropout (53% reduction). Treatment of ASCs with transforming growth factor beta (TGF-β1) enhanced hASC pericyte function, in a manner similar to native retinal pericytes, with increased marker expression of smooth muscle actin, cellular contractility, endothelial stabilization, and microvascular protection in OIR. Finally, injected ASCs prevented capillary loss in the diabetic retinopathic Akimba mouse (79% reduction 2 months after injection). Conclusions/Significance ASC-derived pericytes can integrate with retinal vasculature, adopting both pericyte morphology and marker expression, and provide functional vascular protection in multiple murine models of retinal vasculopathy. The pericyte phenotype demonstrated by ASCs is enhanced with TGF-β1 treatment, as seen with native retinal pericytes. ASCs may represent an innovative cellular therapy for protection against and repair of DR and other retinal vascular diseases.

Chronic Whole-body Hypoxia Induces Intussusceptive Angiogenesis and Microvascular Remodeling in the Mouse Retina

Currently, little is known about the response of the adult retinal microvasculature to hypoxia. To test the hypothesis that chronic systemic hypoxia induces angiogenesis and microvascular remodeling in the adult mouse retina, adult 10-week old female C57Bl/6 mice were exposed to 10% O(2) for 2 or 3 weeks. After hypoxia exposure, retinas were harvested, whole-mounted, and processed for immunohistochemistry. Retinas were stained with lectin, anti-smooth muscle alpha-actin antibody, and anti-NG2 antibody to visualize microvascular networks and their cellular components. Confocal microscopy was used to obtain images of superficial retinal networks. Images were analyzed to assess vessel diameter, vascular length density, branch point density, and the presence of vascular loops, a hallmark of intussusceptive angiogenesis. Both 2 and 3 weeks of hypoxia exposure resulted in a significant increase in the diameters of arterioles and post-arteriole capillaries (p<0.003). After 3 weeks of hypoxia, vascular length density and branch point density were significantly increased in retinas exposed to hypoxia as compared to normoxic controls (p<0.001). The number of vascular loops in the superficial retinal networks was significantly greater in hypoxia-exposed retinas (p < or = 0.001). Our results demonstrate, for the first time, intussusceptive angiogenesis as a tissue-level mechanism of vascular adaptation to chronic systemic hypoxia in the adult mouse retina and contribute to our understanding of hypoxia-induced angiogenesis and microvascular remodeling in the adult animal.

Adipose-Derived Stem Cells From Diabetic Mice Show Impaired Vascular Stabilization in a Murine Model of Diabetic Retinopathy

Diabetic retinopathy is characterized by progressive vascular dropout with subsequent vision loss. We have recently shown that an intravitreal injection of adipose‐derived stem cells (ASCs) can stabilize the retinal microvasculature, enabling repair and regeneration of damaged capillary beds in vivo. Because an understanding of ASC status from healthy versus diseased donors will be important as autologous cellular therapies are developed for unmet clinical needs, we took advantage of the hyperglycemic Akimba mouse as a preclinical in vivo model of diabetic retinopathy in an effort aimed at evaluating therapeutic efficacy of adipose‐derived stem cells (mASCs) derived either from healthy, nondiabetic or from diabetic mice. To these ends, Akimba mice received intravitreal injections of media conditioned by mASCs or mASCs themselves, subsequent to development of substantial retinal capillary dropout. mASCs from healthy mice were more effective than diabetic mASCs in protecting the diabetic retina from further vascular dropout. Engrafted ASCs were found to preferentially associate with the retinal vasculature. Conditioned medium was unable to recapitulate the vasoprotection seen with injected ASCs. In vitro diabetic ASCs showed decreased proliferation and increased apoptosis compared with healthy mASCs. Diabetic ASCs also secreted less vasoprotective factors than healthy mASCs, as determined by high‐throughput enzyme‐linked immunosorbent assay. Our findings suggest that diabetic ASCs are functionally impaired compared with healthy ASCs and support the utility of an allogeneic injection of ASCs versus autologous or conditioned media approaches in the treatment of diabetic retinopathy.

Attenuation of EphrinB2 Reverse Signaling Decreases Vascularized Area and Preretinal Vascular Tuft Formation in the Murine Model of Oxygen-Induced Retinopathy

PURPOSE EphB4 and ephrinB2 are known key regulators of retinal vascular development, but due to their capacity for bidirectional signaling, delineation of their individual roles in this process remains unclear. To better dissect out individual contributions, a model of proliferative retinopathy in mice with attenuated ephrinB2 reverse signaling was studied. It was hypothesized that endothelial ephrinB2 reverse signaling regulates hypoxia-induced capillary sprouting, as well as the pathologic formation of neovascular tufts in postnatal retinal microvascular networks. METHODS Genetically manipulated mice with attenuated ephrinB2 reverse signaling (ephrinB2(lacZ/+)), along with wild-type (WT) controls, were exposed to oxygen-induced retinopathy (OIR), a postnatal model of proliferative retinopathy. At peak disease (postnatal day 18), microvascular networks were analyzed to examine intraretinal revascularization, capillary sprouting, and pathologic neovascularization responses. EphB4 and phosphorylated ephrinB protein expression patterns along retinal microvessels were also assessed. RESULTS EphrinB2(lacZ/+) mice exhibited reduced hypoxia-induced revascularization (P ≤ 0.04) and reduced formation of neovascular tufts (P < 0.001), as compared with WT controls. Corresponding to the observed inhibition of retinal angiogenesis, ephrinB2(lacZ/+) retinas displayed an increased number of blind-ended capillary sprout tips (P < 0.02) and endothelial filopodial processes (P = 0.001). In WT and ephrinB2(lacZ/+) OIR-exposed retinas, ephrinB was confined to endothelial cells, with expression detected along angiogenic vascular processes including neovascular tufts and blind-ended capillary sprouts. CONCLUSIONS EphrinB2 reverse signaling is a regulator of key processes during retinal vascularization and controls pathologic retinal angiogenesis through direct effects on capillary sprouting and endothelial filopodia formation.

Disparities in recommended preventive care usage among persons living with diabetes in the Appalachian region

Objective To examine disparities in the receipt of preventive care recommended by the American Diabetes Association (ADA) between Appalachian and non-Appalachian counties and within Appalachian counties. Research design and methods Behavioral Risk Factor Surveillance System (BRFSS) data for 2008–2010 were used to identify individuals with diabetes and their preventive care usage. Each Appalachian respondent county of residence was categorised into one of the five economic levels: distressed, at-risk, transitional, competitive and attainment counties. Competitive and attainment counties were combined and designated as competitive counties. We used logistic regressions to compare receipt of ADA preventive care recommendations by county economic level, adjusting for respondent demographic, socioeconomic, health and access-to-care factors. Results Compared to the most affluent (competitive) counties, less affluent (distressed and at-risk) counties demonstrated equivalent or higher rates of self-care practices such as daily blood glucose monitoring and daily foot checks. But they showed 40–50% lower uptake of annual foot and eye examinations and 30% lower uptake of diabetes education and pneumococcal vaccinations compared to competitive counties. After adjusting for demographic factors, significant disparities still existed in the uptake of annual foot examinations, annual eye examinations, 2 or more A1c tests per year and pneumococcal vaccinations in distressed and at-risk counties compared to competitive counties. Appalachian counties as a whole were similar to non-Appalachian counties in the uptake of all recommendations with the absolute differences of ≤3%. Conclusions Our results show that there are significant disparities in the uptake of many recommended preventive services between less and more affluent counties in the Appalachian region.