THE EFFECT OF CONSTITUTIVELY ACTIVE RAP1A IN CHOROIDAL NEOVASCULARIZATION
Brooke Romine1, M.E. Hartnett2, Deeksha Gambhir2, Lori Fotheringham2, Sadiki Deane2.
1Oklahoma State University, Stillwater, OK, 2Moran Eye Center, University of Utah, Salt Lake City, UT.
Rap1a enhances barrier integrity of the retinal pigment epithelium (RPE), which makes up the outer blood-retinal barrier that is important to visual acuity. In neovascular age-related macular degeneration (AMD), choroidal endothelial cells invade the retina after migrating through the RPE to become vision-threatening choroidal neovascularization (CNV). Broadly activating Rap1 in the retina reduced CNV in a murine laser model. We formulated the hypothesis that activating Rap1a in RPE would be sufficient to reduce CNV. To introduce active Rap1a into RPE in vivo, self-complementary adeno-associated viral vectors with green-fluorescent-protein (GFP) and constitutively active Rap1a driven by an RPE65 promoter (CARap1a-GFP-RPE65-scAAV2) were created. Controls were GFP-RPE65-scAAV2 and phosphate-buffered saline (PBS). Four-week old C57Bl/6 mice received 1-microliter subretinal injections of CARap1a-GFP-RPE65-scAAV2, GFP-RPE65-scAAV2 or PBS. Viral infection and toxicity were assessed using in vivo imaging for GFP and retinal structure (Micron IV, Phoenix-Research-Labs). Laser was delivered at 6 weeks and eyes were processed as choroidal flat-mounts 1 week later and labeled with anti-GFP and lectin. Images through CNV were captured as 3-micron optical sections within Z-stacks on a confocal microscope (Olympus), summed, and analyzed by ANOVA. GFP increased in both scAAV2 groups, but not in PBS. No viral toxicity was noted on GFP-labeled RPE mosaics in scAAV2-treated groups and or by assessing retinal structure in all groups. A pattern of reduced CNV volume occurred in scAAV2-treated groups, but no significant difference was found compared to PBS. CARap1a-GFP-RPE65-scAAV2 and GFP-RPE65-scAAV2 successfully transduced RPE following subretinal injections. We did not find that CARap1a-GFP-RPE65-scAAV2 reduced CNV compared to GFP-RPE65-scAAV2.