The Protective Role of the Sigma-1 Receptor in Trabecular Meshwork Cells in vitro

Abstract

Purpose : Trabecular meshwork (TM) is the main pathway for aqueous humor drainage from the eye. The fibrotic-like remodeling of the actin cytoskeleton in TM cells results in altered stiffness and impaired outflow which are the primary cause of increased intraocular pressure (IOP) leading to glaucoma. Thus, cytoskeletal-disrupting drugs could be a novel therapeutic approach to lower IOP. Recently, we proved that fluvoxamine (FLU), a specific Sigma-1 receptor agonist is antifibrotic in the kidney. Therefore, here we investigated whether FLU is effective in the prevention of cytoskeletal rearrangement and in vitro TM fibrosis in primary mouse- (MsTM) and non-glaucomatous human TM cells (HTM5).

Methods : Immunocytochemistry and Western blot were used to detect S1R on HTM5 and on MsTM cells. Primary MsTM cells from wild type (WT) and S1R knock-out (KO) mice and HTM5 cells were treated for 24h with 20 ng/mL platelet-derived growth factor (PDGF) combined with 10 µM of FLU. Cell proliferation was determined by thiazolyl blue tetrazolium bromide assay, cell toxicity by LDH assay, cell migration was measured by scratch assay, and F-actin was visualized by phalloidin staining and detected with fluorescent microscope. The fibrotic protein levels (fibronectin and αSMA) were measured by Western blot.

Results : S1R is present both in primary MsTM and HTM5 cells and is localized in the endoplasmic reticulum. Cell proliferation, migration, levels of fibrotic proteins (fibronectin, αSMA) and cytoskeletal rearrangement were induced by PDGF. FLU treatment was not toxic to the cells and ameliorated or even prevented the PDGF induced changes in all assays. Furthermore,upon PDGF treatment the integrated density of phalloidin staining increased with 19.73% in primary KO MsTM cells compared to WT and the formation of F-actin bundles and actin clumps was more pronounced in the absence of S1R suggesting its protective role in TM fibrosis.

Conclusions : FLU is non-toxic and effectively reduces profibrotic factor-induced cytoskeletal rearrangement and cell proliferation of TM cells. Therefore one can speculate that it might lead to lower outflow resistance also in animals and it could be an IOP-lowering drug candidate for glaucoma.
Grants: OTKA- K135398, LP2021-3/2021, 2020-4.1.1.-TKP2020-6183069269 (FIKP), 2020-4.1.1.-TKP2020-6183169273.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.