Abstract
Purpose :
Rare missense and nonsense variants in the Angiopoietin-like 7 (ANGPTL7) gene confer protection from primary open-angle glaucoma (POAG), though the functional mechanism remains uncharacterized. Here, we investigate the underlying mechanism using in silico and in vitro models.
Methods :
Protein stability of wild-type (WT) ANGPTL7 and five protective variants was estimated using FoldX software and correlated with variant effects on corneal-compensated intraocular pressure (IOPcc) in the UK Biobank. In parallel, primary cultures of human trabecular meshwork (TM) cells were transfected with an expression vector containing either WT ANGPTL7 or one of five protective variants (2 TM strains each tested in duplicate). Cells were processed for immunohistochemistry 24 hours post-transfection to assess Angptl7 localization. Cell lysate and conditioned media were collected 48 hours post-transfection to measure levels of Angptl7 and endoplasmic reticulum (ER) stress markers (Grp78 and Atf4). Human TM and Schlemm’s canal (SC) cells were subjected to 48 hours of cyclic mechanical stress (CMS) using the Flexcell strain unit (15% stretch at a frequency of 1Hz); control cells were plated on Flexcell plates but were not subjected to CMS. Real-time PCR was performed to measure changes in ANGPTL7 expression (3 TM and 3 SC strains each tested in triplicate).
Results :
More efficacious IOPcc lowering strongly correlates with in silico predictions of increased protein instability (r= -0.98, p<0.01). Missense and nonsense variants cause aggregation of mutant Angptl7 protein in the ER and decreased levels of secreted protein in TM cells. A lower secreted:intracellular protein ratio strongly correlates with variant effects on IOPcc in the UK Biobank (r=0.87, p=0.04), suggesting that protective variants lower Angptl7 protein levels. Interestingly, accumulation of mutant protein in the ER does not increase expression of ER stress markers (p>0.05 for all). CMS, a glaucoma-relevant physiologic stressor, also significantly lowers ANGPTL7 expression in SC cells (-2.4 fold-change, p=0.01).
Conclusions :
These data suggest that the protective effects of ANGPTL7 variants in POAG stem from lower levels of secreted protein, which may modulate responses to physiologic and pathologic ocular cell stressors. Downregulation of ANGPTL7 expression may therefore serve as a viable preventative and therapeutic strategy for this common blinding disease.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.