Abstract
Purpose :
Elevated intraocular pressure (IOP) is the only treatable risk factor for primary open-angle glaucoma (POAG). Our previous pathway analysis of IOP-associated loci identified 2 networks focused on an unspecified estrogen receptor and estrogen receptor 1 (ESR1), respectively (Liu 2020). Here we seek to confirm the role of ESR1 signaling in the aqueous humor (AH) outflow pathway.
Methods :
We examined which 17 IOP-associated genes in the estrogen receptor networks were specifically expressed in a single-cell RNA sequencing (scRNA-Seq) dataset of human outflow tissue (van Zyl 2020) as well as an RNA-Seq dataset of nonglaucomatous human TM (n=4) and Schlemms Canal (SC) (n=2) cells. To confirm ESR1 activity in the outflow pathway, we examined its protein expression in nonglaucomatous human (n=4) and mouse outflow tissues (n=2) by antibody-based immunofluorescence, using tissue from an Esr1-/- mouse as a negative control (n=1). We also measured 17β-estradiol (E2) concentration in glaucomatous (n=4) and nonglaucomatous (n=4) human AH using an ELISA kit (Enzo Life Sciences).
Results :
Seven IOP genes from the estrogen receptor networks (i.e., CAV2, FBXO32, SIX3, FOXC1, SPTBN1, TCF7L2, and TNXB) were expressed in ≥1 cell clusters defined by scRNA-Seq, including cell clusters localized to the TM, SC, collector channels, ciliary muscle, Schwalbe’s line, vascular endothelium, etc. As confirmation, in a separate RNA-Seq study of human TM and SC cells, 8 genes of interest (i.e., CAV2, ETS1, FBXO32, VEGFC, FOXC1, SPTBN1, TCF7L2, and TMEM119) were highly expressed (i.e., FPKM ≥ 10) in either TM or SC cells, with SPTBN1 having the highest expression (i.e., 85.85 FPKM and 85.83 FPKM). Esr1 protein was expressed in the iridocorneal angle of female B6J wild-type, but not Esr1-/- mice. Similarly, ESR1 protein was expressed in human TM/SC. E2 was present in both glaucomatous (34.46 ± 7.08 pg/mL) and nonglaucomatous human (37.90 ± 14.80 pg/mL) AH (p>0.05).
Conclusions :
Many IOP-associated genes within the estrogen receptor networks are highly expressed in the outflow pathway including in the TM, SC, ciliary muscle, and vascular endothelium. The confirmation of ESR1 protein expression in human and mouse outflow tissues and the presence of E2 in AH further supports a possible function of ESR1 signaling in IOP regulation through modulation of AH outflow.
This is a 2021 ARVO Annual Meeting abstract.