Cellular crosstalk regulates the aqueous humor outflow pathway and provides new targets for glaucoma therapies

Abstract

Primary congenital glaucoma (PCG) is a severe disease characterized by developmental defects in the trabecular meshwork (TM) and Schlemm’s canal (SC), comprising the conventional aqueous humor outflow pathway of the eye. Recently, heterozygous loss of function variants in TEK and ANGPT1 or compound variants in TEK/SVEP1 were identified in children with PCG. Moreover, common variants in ANGPT1and SVEP1 have been identified as risk alleles for primary open angle glaucoma (POAG) in GWAS studies. Here, we show tissue-specific deletion of Angpt1 or Svep1 from the TM causes PCG in mice with severe defects in the adjacent SC. Single-cell transcriptomic analysis of normal and glaucomatous Angpt1 deficient eyes allowed us to identify distinct TM and SC cell populations and discover additional TM-SC signaling pathways. Furthermore, confirming the importance of angiopoietin signaling in SC, delivery of a recombinant ANGPT1-mimetic promotes developmental SC expansion in healthy and Angpt1 deficient eyes, blunts intraocular pressure (IOP) elevation and RGC loss in a mouse model of PCG and lowers IOP in healthy adult mice. Our data highlight the central role of ANGPT1-TEK signaling and TM-SC crosstalk in IOP homeostasis and provide new candidates for SC-targeted glaucoma therapy.

Differential roles of neural crest- and endothelial-derived FOXC2 in trabecular meshwork and Schlemm’s Canal in glaucomatous pathology

“…Lyve1 and Pdpn (Figure 7C), which are absent in the SC, implying that this cluster predominately consists of SC Ecs with few limbal lymphatic Ecs that were not independently clustered during analysis. In support of this observation, the SC and lymphatic EC cluster exhibited high expression of Npnt, Nts, Pgf, and Postn and modest expression of Itga9 and Nts (Figure 7C and D) and other endothelial marker genes including Flt1, Kdr, Plvap, Ptprb, and Tek (Figure 7-figure supplement 1D), which were recently shown to be characteristic of the SC endothelial transcriptional profile by Thomson et al (40).…”

“…The BEC-1 cluster exhibited the strongest expression of these genes, potentially indicating that this cluster may be characteristic of collector channel vessels that exhibited a similarly high expression of these genes in a human dataset (38). The other BEC clusters showed stronger expression for markers recently reported to be associated with arterial limbal endothelium (40), with the exception of BEC-3, which exhibited higher expression of Ihh , a recently identified marker of choriocapillaris (40, 48) that may have been incorporated during dissection and tissue dissociation ( Figure 7-figure supplement 1B and C ). The SC and lymphatic EC cluster showed high expression of several markers identified in SC Ecs such as Ccl21a , Flt4 , and Prox1 .…”

“…As NC-derived cells do not contribute to the development of the SC vasculature (19), we focused our analyses on assessment of uvea meshwork and TM cell populations provided their anatomical proximity to SC and positive Foxc2 expression detected in these clusters. Previously reported single cell analyses of the outflow pathway have identified uniquely expressed markers within different cell populations comprising the TM, such as fibroblast-like trabecular “beam” cells comprising the inner part of the posterior filtering region of the TM, the juxtacanalicular tissue (JCT) comprising the outer part of the posterior filtering region located adjacent to the SC, and the possible presence of Cd34 -positive corneal stromal cells/fibroblasts clustering within TM cell populations as recently characterized by Thomson et al (37, 38, 40). Our initial analysis showed that known marker genes of TM, such as Myoc and Chil1, were predominately expressed in the Trabecular meshwork 3 (TM-3) cluster, with less prominent expression in the Trabecular meshwork 1 and 2 (TM-1 and TM-2) clusters ( Figure 4-figure supplement 1C, Figure 4-figure supplement 2A and D ).…”

“…Young mice after weaning were utilized to optimize the total cell number utilized for single-cell sequencing, and NC-Foxc2 -/mice with moderate phenotypes were selected so that the anterior eye segment could be definitively dissected from the posterior segment. Tdistributed stochastic neighbor embedding (t-SNE) visualization and clustering analysis of cells from both Foxc2 fl/fl Control and NC-Foxc2 -/mice identified 22 transcriptionally distinct cell populations including uveal meshwork, in which aqueous humor first traverses through the TM, 3 transcriptionally distinct trabecular meshwork populations, and endothelial cells among other cell populations (Figure 4, A -D, Figure 4-figure supplement 1) characterized from previously reported scRNA-seq analysis studies of the murine conventional outflow pathway and anterior eye segment (36)(37)(38)(39)(40) . Foxc2 expressing cells were detected in several cell clusters, but were primarily localized to trabecular meshwork, corneal stromal keratocyte, and pericyte populations (Figure 4E).…”

“…Moreover, sTIE2 is capable of binding angiopoietins to prevent them from activating TIE2 signaling (29). Provided the increase expression of MMPs in NC-Foxc2 -/mice and the crucial role of ANGPT/TIE2 signaling in SC development (25)(26)(27)40), we hypothesized that increased MMP activity in the TM may mediate cleavage of TIE2 in SC endothelium, resulting in higher production of sTIE2, impairment of ANGPT/TIE2 signaling, and impaired SC morphogenesis. To investigate this mechanism, we collected conditioned media from cultured human dermal LECs (HDLECs) treated with either DMSO vehicle or the MMP inhibitor GM-6001 at dosages shown to inhibit sTIE2 shedding in cultured HUVECs (29) or impair tubulogenesis in three-dimensional culture assays (47).…”