The retina is one of the most metabolically active tissue in the body, rendering it sensitive to vascular dysfunction. Consequently, diseases that disrupt normal retinal blood supply, including retinal vein occlusions (RVO) and diabetic retinopathy, are the leading causes of blindness in working-age adults. Despite available therapies, an estimated 50% of patients do not respond to treatment. We employed a mouse model of retinal vein occlusion (RVO), achieved by tail-vein injection of rose bengal, followed by laser photocoagulation of retinal veins. In vivo analyses – optical coherence tomography (OCT), fluorescein angiography, and electroretinograms (ERGs) - were conducted with the Micron IV system (Phoenix Research Labs). RVO induces acute retinal edema, which peaks during the first 24 hours following injury. Over a 7 day time course the edema resolves, revealing a permanent retinal thinning due to death of retinal neurons. We identified caspase-9, a protease traditionally associated with apoptosis, as an essential mediator of edema. Increased levels of activated caspase-9 were detected in vascular endothelial cells 1 hour following RVO. We tested RVO in mice with inducible endothelial-cell-specific deletion of caspase-9 (iC9 ECKO). Compared to littermate controls, iC9 ECKO mice develop less edema, and sustain less retinal degeneration after RVO injury. ERG analysis showed preservation of retinal function in iC9 ECKO mice. To study whether inhibiting caspase-9 would provide protection against RVO we utilized a highly specific caspase-9 inhibitor, which we can deliver to the retina using simple eyedrops. Treatment of wildtype mice with the caspase-9 inhibitor immediately after induction of RVO provided morphologic, biochemical and functional retinal protection. Inhibition of caspase-9 reduces edema, protects retinal morphology, and helps prevent vision loss following RVO injury. Our studies indicate that endothelial caspase-9 plays an essential role in regulating edema pathogenesis. Moreover, our novel cell permeant caspase-9 inhibitor abrogates the edema and may be a potential therapy for individuals suffering from vascular eye disease.

The retina is one of the most metabolically active tissues in the body and the high energetic demand is met by a well-organized vascular network. Aberrant vasculature is a prominent feature of many vision-threatening diseases, and although angiogenic pathways have been extensively studied the limited efficacy of therapies currently available for the treatment of these diseases suggests that there is more to be elucidated. The caspase family of proteases is best known for their roles in programmed cell death and inflammation, however members of this family have been found to have essential functions independent of cell death. Caspase-8, in particular, has been previously shown to be essential for embryonic vascular development, however, a requirement for caspase-8 in postnatal vascular development has not been established and it is unclear how caspase-8 exerts its function. In this study, we investigate the cell specific roles of caspase-8 in the development of the retinal vasculature using the postnatal mouse retina as our model and identified endothelial caspase-8 as a mediator of canonical Wnt signaling. Inducible endothelial cell-specific caspase-8 knockout (Casp8 iECKO) resulted in a delay in early angiogenesis and barrier establishment, and an increase in inflammation and premature vascular remodeling compared to littermate controls. Assessment of Lef1, a downstream effector of the Wnt pathway, confirmed that this phenotype was a result of inhibited Wnt signaling. We additionally show that caspase-8 mediates this pathway through degradation of its substrate HDAC7. HDAC7 has been shown previously to bind to β-catenin blocking its nuclear translocation. Caspase-8 mediated HDAC7 degradation restores β-catenin translocation and downstream Wnt signaling. We also explore the function of caspase-8 in myeloid cells – microglia and macrophages – during angiogenesis. We used an inducible myeloid-specific caspase-8 knockout (Casp8 imGKO) mouse and found that loss of caspase-8 in these cells did not affect angiogenesis. However, Casp8 imGKO resulted in a reduction in microglia number and a change in their morphology specifying a role for caspase-8 in mediating cell survival and activation in microglia. Altogether we show that caspase-8 exerts cell specific functions during retinal angiogenesis that are independent of cell death. We elucidate a novel role of caspase-8 in mediating Wnt/β-catenin signaling, and implicate caspase-8 as a potential therapeutic target in pathological angiogenesis.