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Maria Avrutsky, Ying Jean, Guy Salvesen, Carol M Troy; Caspase-9 inhibition by Pen1-XBir3 abrogates retinal edema. Invest. Ophthalmol. Vis. Sci. 2016;57(12):5351.
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© ARVO (1962-2015); The Authors (2016-present)
Macular edema results from fluid accumulation in the macula due to leaky blood vessels, and is a common complication of diabetes and retinal vein occlusion (RVO). Despite available therapies, an estimated 50% of patients do not respond to treatment. We identified caspase-9, a protease traditionally associated with apoptosis, as an essential component in edema formation. To test the therapeutic potential of modulating caspase-9 activity in edema, we developed a highly-specific caspase-9 inhibitor by utilizing the XBir3 domain of XIAP.
We induced retinal edema in 2-4 month-old C57Bl/6J mice, and endothelial cell specific caspase-9 knockout (caspase-9Flox/Tek-Cre) mice. We employed two models of retinal edema: intravitreal injection of hypertonic saline (5μl, 10X PBS), and RVO. RVO was achieved by tail-vein injection of rose bengal (150μl, 5mg/mL), followed by laser photocoagulation of retinal veins using 50mW output and 2.5 sec laser duration. In vivo analyses – optical coherence tomography (OCT) and fluorescein angiography - were conducted with the Micron IV system (Phoenix Research Labs). We generated Pen1-XBir3 by crosslinking XBir3 to a cell permeant peptide, Pen1 (1:2 molar ratio, incubated at 37°C for 2 hrs). Mice received eyedrops containing 10μg Pen1-XBir3 immediately following retinal injury.
Both injury models induce acute retinal edema as measured by OCT (injection model N=3, P<0.01; RVO model N=5, P<.001), fluorescein angiography, fluorescein-dextran leakage in retinal flatmounts (N=4, P<.05), and H&E staining of retinal cross-sections (injection model N=3, P<0.001). Concomitantly, caspase-9 and VEGF levels increase after injury, as measured by western blot and immunohistochemistry. Caspase-9 Flox/Tek-Cre mice develop substantially less edema compared to wildtype animals (injection model N=3, P<.05), confirming the importance of endothelial expression of caspase-9 in edema formation. Our findings reveal that Pen1-XBir3 inhibits caspase-9 activity, and can be delivered to the retina via eyedrop. Pen1-XBir3 reduces the severity of retinal edema (injection model N=3, P<0.01; RVO model N=10, P<.001) and prevents the induction of VEGF (N=4, P<0.05) in both models.
Our studies indicate that endothelial caspase-9 plays an essential role in regulating edema pathogenesis. Moreover, Pen1-XBir3 abrogates the edema and may be a potential novel therapy for individuals suffering from macular edema.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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