Abstract
Purpose: :
Rap1, a member of the Ras family of small GTPases, is important in cell-cell junctions and angiogenesis. Active Rap1 increases RPE barrier properties and when inactivated permits greater migration of choroidal endothelial cells across the RPE in a coculture model. We tested the hypothesis that a Rap1 activator, 8-CPT-2-O-Me-cAMP (8CPT), would reduce choroidal neovascularization (CNV) volume in wild type mice.
Methods: :
We used a well-accepted laser-induced model for CNV. A 532nm OcuLight GL laser (0.1sec, 100 µm, 150mW) (Iridex, CA) was used to cause laser burns in adult wild type mice (2 to 4 months old). We delivered 4 to 6 laser spots to each eye. Immediately following treatment, eyes were injected with 1µL of 8CPT to the vitreous. Based on several doses between (0.205, 2.05, 20.5µM), we chose 2.05µM for studies. Seven days after laser injury, retinal images were taken using a spectral domain optical coherence tomography unit (sd-OCT; Bioptogen, NC). Following this, animals were euthanized, and choroidal flat mounts were dissected and stained using isolectin B4 (GS-1B4, Alexa Flour 568, Invitrogen, CA). Each lesion was imaged using confocal microscopy (Olympus, Japan), and a z-series was collected. CNV volumes of each lesion were measured with the image-analysis software, Volocity (PerkinElmer, MA). Lesions with obvious hemorrhage or bridging of CNV were excluded from analysis. CNV volumes and the widths of lesions from each eye were averaged and analyzed using t-test or Mann-Whitney test. In some eyes, RPE-choroids were removed from eyes and measured for active Rap1 (Neweast Biosciences, PA) using western blot.
Results: :
8CPT increased active Rap1 in RPE-choroids from mice treated with laser compared to those injected with PBS. The average width of lesions measured on sd-OCTs from PBS-injected mice was significantly wider than comparable measurements from 8CPT injected mice (0.33±0.02 mm [PBS] vs. 0.25±0.01mm [8CPT]; P<0.01, t-test). 8CPT reduced CNV volume and was significant (0.29±0.04 x105µm^3 [8CPT] vs. 0.86±0.15 x105µm^3 [PBS], P<0.01, Mann-Whitney test).
Conclusions: :
Following laser injury in mice, intravitreal 8CPT caused an increase in active Rap1 in RPE-choroids and reduced CNV volume compared to PBS. Elevated active Rap1 may be important in reducing laser-induced CNV.
Keywords: choroid: neovascularization • imaging/image analysis: non-clinical • laser