Macrophage-to-endothelial vitronectin/integrin avβ3 signaling promotes Schlemm’s canal formation and aqueous humor homeostasis

Abstract

Purpose : Schlemm's canal (SC), as the last governor of the aqueous humor outflow passageway, is one of the key players in controlling aqueous humor homeostasis. However, the exact mechanisms underlying the structural formation and maintenance of SC remain unclear. Here, we studied the role of integrin αvβ3 in SC development and intraocular pressure (IOP) homeostasis.

Methods : To obtain endothelial cell (EC), lymphatic endothelial cell or whole-body deletions of the indicated genes, floxed mice including integrin β3 (Itgb3) ^f/f, integrin aV (Itgav) ^f/f and vitronectin (Vtn) ^f/f were respectively intercrossed with Cdh5CreER^T2mice, Prox1CreER^T2mice or ROSA26CreER^T2mice. To deplete macrophage/microglia in neonatal mice,the pregnant females were fed on a special diet supplemented with PLX5622 from E17.5 until analysis. Whole-mount immunostaining was performed to examine SC formation in mice from P3 to P21. Q-PCR and Western blot analysis were performed to study the molecular mechanisms of SC transdifferentiation.

Results : EC-specific Itgb3 knockout (Itgb3iECko) mice displayed significantly impaired SC development, as indicated by reduced SC cross-sectional areas along with a reduced number of Erg⁺ in the SC on P3, P5, P9, P14, and P21 compared with control mice. In addition, Itgb3iECko mice showed moderately higher IOP at the fourth week, which was associated with decreased Tuj1-positive retinal nerve fiber density. Our immunostaining results revealed that VTN protein was highly expressed by the Iba1⁺ macrophages located near the developing SC between P1 and P21. Importantly, depletion of VTN or macrophages in mice resulted in retarded SC development similar to the Itgb3iECko mice.

Conclusions : Taken together, our results demonstrated that the integrin αvβ3 takes part in the regulation of SC development and IOP homeostasis, which may be mediated via VTN produced by the peri-SC macrophages. Our study presents the first evidence for the involvement of integrin αvβ3 in SC development and may provide insights for finding new targets for the treatment of open-angle glaucoma.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.