Retinal pigment epithelial cell expression of active Rap 1a by scAAV2 inhibits choroidal neovascularization.
To test the hypothesis that increased Rap1a activity specifically in retinal pigment epithelial cells resists choroidal neovascularization (CNV), self-complementary adeno-associated virus 2 (scAAV2) with RPE65-promoter-driven GFP vectors were generated and introduced subretinally into Rap1b-deficient mice. Six-week-old mice that received subretinal control (scAAV2-Con) or constitutively active Rap1a (scAAV2-CARap1a) showed strong GFP at the 5 × 10(8) viral particle/µl dose 5 weeks later without altering retinal morphology or function. Compared to scAAV2-Con- or phosphate-buffered saline (PBS)-injected, eyes injected with scAAV2-CARap1a had increased Rap1 in retinal pigment epithelial (RPE)/choroidal lysates and a significant reduction in CNV volume 7 days after laser, comparable to eyes that received intravitreal anti-VEGF versus IgG control. scAAV2-CARap1a-, but not anti-VEGF-, injected eyes had increased pan-cadherin in RPE/choroids. In cultured RPE cells, increased active Rap1a inhibited TNFα-induced disassociation of junctional pan-cadherin/β-catenin complexes, increased transepithelial electrical resistance through an interaction of β-catenin with phosphorylated scaffold protein, IQGAP1, and inhibited choroidal endothelial cell (CEC) transmigration of an RPE monolayer. This evidence shows that increased Rap1a activity specifically in RPE cells is sufficient to reduce CEC transmigration and CNV and involves IQGAP1-mediated protection of RPE junctional complexes.
The John Moran Eye Center, University of Utah , Salt Lake City, Utah, USA.
Haibo
H
Wang
The John Moran Eye Center, University of Utah, Salt Lake City, Utah, USA; Department of Ophthalmology, The Fourth Affiliated Hospital of China Medical University, Eye Hospital of China Medical University, Key Lens Research Laboratory of Liaoning Province, Shenyang, P.R. China.
Xiaokun
X
Han
The John Moran Eye Center, University of Utah , Salt Lake City, Utah, USA.
Colin A
CA
Bretz
The John Moran Eye Center, University of Utah , Salt Lake City, Utah, USA.
Silke
S
Becker
The John Moran Eye Center, University of Utah , Salt Lake City, Utah, USA.
Deeksha
D
Gambhir
The John Moran Eye Center, University of Utah , Salt Lake City, Utah, USA.
George W
GW
Smith
UNC Vector Core, University of North Carolina at Chapel Hill , Chapel Hill, North Carolina, USA.
R Jude
RJ
Samulski
Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill , Chapel Hill, North Carolina, USA.
Erika S
ES
Wittchen
Department of Biochemistry and Molecular Biology, Indiana University School of Medicine , Indianapolis, Indiana, USA.
Lawrence A
LA
Quilliam
Blood Research Institute, Blood Center of Wisconsin , Milwaukee, Wisconsin, USA.
Magdalena
M
Chrzanowska-Wodnicka
The John Moran Eye Center, University of Utah , Salt Lake City, Utah, USA.
M Elizabeth
ME
Hartnett