Abstract
Purpose :
Schlemm's canal (SC) plays a crucial role in the outflow of aqueous humor, and a hypo-morphic SC can lead to elevated intraocular pressure and the development of glaucoma. We have previously reported the significance of trabecular meshwork (TM)-SC crosstalk in ANGPT/TIE/TEK vascular signaling for SC formation. However, the roles of their binding partner SVEP1 and its receptor ITGA9 remain unclear. Previously, we demonstrated that specific deletion of SVEP1 in the TM hinders SC formation (Thomson BR, et al., Nat Comm, 2021). Accordingly, this study investigates the expression of ITGA9 in the angle (TM and SC) and examines the consequences of its deletion on SC formation in mice.
Methods :
Following our established protocol (Thomson BR and Quaggin SE, Bio Protoc, 2022), we collected angle tissues from 6-week-old WT mice (n = 6 angle tissues per sample) for single-cell RNA sequence analysis. We analyzed two samples using the Seurat package in R software, annotating TM and SC clusters using canonical markers such as Myoc, Chil1, Cdh5, Prox1, and Lyve1. Itga9 fl/fl mice were crossed with Rosa26-rtTA and TetO-Cre mouse lines to create a doxycycline-inducible ITGA9 knockout model. Pups were generated, and doxycycline water was administered from E 15.5 for two weeks to induce ITGA9 deletion. We harvested angle tissues from 4-week-old mice, staining SC with anti-PECAM1 antibody. We then analyzed the effect of ITGA9 deletion on SC morphology using Nikon A1C confocal microscopy (control: n = 7 and mutant: n = 4). We calculated the SC area as a percentage of the 20x image and made comparisons.
Results :
Cell clusters positive for Myoc or Chil1 were identified as TM, and those positive for Prox1 and Cdh5 but negative for Lyve1 as SC endothelial cells. Svep1 was expressed in TM but not in SC. Itga9 expression was not expressed in TM but was expressed in SC. Deletion of ITGA9 resulted in approximately a 40-50% reduction in SC area; WT: 3.1 ± 0.2 vs. Itga9 mutant: 2.0 ± 0.3 104 μm2 per 20x field (P = 0.020, Student's t-test).
Conclusions :
These findings and our previous reports support a model of molecular crosstalk between the TM and SC during SC development, mediated by SVEP1 from TM and ITGA9 from SC. These data provide new insights in understanding the molecular interactions involved in SC formation and function.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.