Abstract
Purpose :
Rare coding variants in the Angiopoietin-like 7 (ANGPTL7) gene are associated with lower intraocular pressure (IOP) and decreased odds of developing primary open-angle glaucoma (POAG), likely through an impact on conventional aqueous outflow. To further assess the protective mechanism in vivo, we characterized glaucoma-relevant phenotypes in an ANGPTL7 zebrafish knockout line.
Methods :
Commercially available ANGPTL7 knockout fish (ZFIN ZDB-ALT-131217-15866) were outcrossed to AB genetic background fish to generate wild-type (WT), heterozygous (HET), and homozygous knockout (KO) lines. Survival at 5 days post-fertilization (dpf), eye area and visual motor response using the ZebraBox system were compared between WT, HET and KO lines using two-way ANOVA (n=96 for each). WT, HET, and KO fish were also euthanized at 5, 10, and 20 dpf, preserved in Davidson’s fixative and submitted for histology using toluidine blue (n=15 for each). Additionally, immunohistochemistry was performed to evaluate Angptl7 localization in cryopreserved WT and KO fish at 5, 10 and 20 dpf (n=3 for each). IOP was measured using the Tonolab device (iCare USA) in adult (9 months old) WT (n=13) and KO (n=8) fish following induction of anesthesia with MS-222 and compared using a two-tailed t-test.
Results :
Compared to WT fish at 5 dpf, HET fish and KO fish had 48% and 96% lower ANGPTL7 expression, respectively (p<0.001). There were no significant differences in survival, eye area or visual motor response among the three lines at 5dpf (p>0.05 for all). Immunohistochemistry revealed specific localization of ANGPTL7 to the developing zebrafish outflow tract. Compared to WT fish, HET and KO fish demonstrated reduced accumulation of fluid in the developing outflow tract on histology, suggesting that reduced ANGPTL7 expression either increases aqueous outflow or reduces production. There was a trend towards reduced IOP in the KO line compared to WT (10.5 vs 12.5 mm Hg), though this did not reach statistical significance (p=0.12).
Conclusions :
Knockout of ANGPTL7 leads to histological changes in the developing outflow pathway and reduced IOP in vivo, further suggesting that reduced ANGPTL7 expression may serve as a viable therapeutic strategy for ocular hypertension and glaucoma. This study further highlights the utility of zebrafish as a high throughput model system for functional characterization of glaucoma-associated genes.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.