Abstract
Purpose :
The limitations of current retinopathy therapies reveal a shortcoming in our understanding of retinal angiogenesis. Previous studies have shown that certain caspases may be activated in the course of vascular development, but the specific roles of these proteases remains unclear. In this study, we investigate previously unexplored mechanisms and signaling pathways involved in the outgrowth and subsequent remodeling of the retinal vasculature. We hypothesize that caspases play a key non-apoptotic role in the signaling pathways involved in retinal angiogenesis.
Methods :
Retinas were isolated from C57BL/6 pups at intervals between P2-P21. Mice were perfused with saline and retinas were post-fixed overnight with 4% paraformaldehyde. Whole retinal flatmounts were prepared and stained for caspase-8, -9, -7, -3 and -1 and TUNEL assay was performed. Cdh5(PAC)-CreERT2 mice were crossed with caspase-8 floxed mice to produce inducible endothelial cell specific caspase-8 knock out mice (Casp8 KO). Recombination was induced by daily intraperitoneal injection with 2mg/ml tamoxifen from P1-P3. Retinas were isolated at P7 and whole flatmounts were prepared, staining for vasculature using Isolectin. Vascular outgrowth, vessel area and vessel density were measured and compared between Casp8 KO (N=17) and littermate controls (N=17). One-way ANOVA was used for statistical analysis.
Results :
Caspase-8, -9, -7 and -1 were expressed in endothelial cells and microglia during retinal vascular development, while caspase-3 expression/activation was localized to microglia. TUNEL assay revealed that caspase expression did not promote cell death. At P7, Casp8 KO resulted in a significant decease in vascular outgrowth (66.90±2.52% in Casp8 KO vs. 83.92±1.76% in littermate controls; p<0.0001), reduced vessel area (59.96± 2.75% in Casp8 KO vs. 78.45±2.09% in littermate controls; p<0.0001) and reduced vascular density (49.23±1.90% in Casp8 KO vs. 49.23±1.33% in littermate controls; p<0.0001).
Conclusions :
While many caspases were expressed in the course of retinal angiogenesis, this did not lead to cell death indicating a non-apoptotic role for caspase expression. Loss of endothelial caspase-8 resulted in delayed vascular outgrowth and premature vascular remodeling, suggesting a novel pathway in the regulation of retinal angiogenesis. Further investigation will be needed in order to understand this signaling pathway.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.