Mechanosensitive channel inhibition attenuates TGFβ2-induced actin cytoskeletal remodeling in optic nerve head astrocytes.

Abstract

Purpose : Astrocytes within the optic nerve head undergo actin cytoskeletal rearrangement early in glaucoma, prior to the development of tissue stiffening. Elevated transforming growth factor (TGF)β2 levels within astrocytes have been described in glaucoma, and TGFβ signaling induces actin cytoskeletal remodeling and extracellular matrix (ECM) stiffening in many tissues. A key mechanism by which astrocytes sense and respond to external stimuli is via mechanosensitive ion channels. Here, we tested the hypothesis that inhibition of mechanosensitive channels will attenuate TGFβ2-mediated optic nerve head astrocyte actin cytoskeletal remodeling and ECM stiffening.

Methods : Primary optic nerve head astrocytes were isolated from C57BL/6J mice and cell purity was confirmed by immunostaining. Astrocytes were treated with control medium, 5 ng/ml TGFβ2, 500 nM GsMTx4 (a mechanosensitive channel inhibitor), or 5 ng/ml TGFβ2 + 500 nM GsMTx4 for 48 h. FITC-phalloidin staining was used to assess the formation of f-actin stress fibers and to quantify the presence of crosslinked actin networks (CLANs). Cell reactivity was determined by immunostaining for GFAP. 3D hydrogels were made by mixing primary astrocytes with functionalized ECM components, UV-crosslinked using a photoinitiator, and subjected to the above treatments for 7 d. Live/dead staining was used to confirm astrocyte viability and hydrogel stiffness was measured using rheometry.

Results : Primary optic nerve head astrocytes were positive for the astrocyte marker GFAP and negative for markers for microglia (Iba1) and oligodendrocytes (OSP1). Increased %CLAN-positive cells were observed after 48-h treatment with TGFβ2 vs. control (23.8% vs 5.3%, p<0.0001). Co-treatment with GsMTx4 decreased %CLAN-positive cells vs. TGFβ2 treatment (12.4% vs 23.8%, p<0.0001) and decreased the presence of f-actin stress fibers. TGFβ2 treatment increased GFAP fluorescence intensity, which was decreased when co-treated with GsMTx4 (p<0.05). Cells within hydrogels maintained >90% viability after 7 d. Hydrogels treated with GsMTx4 were less stiff when compared to control and TGFβ2-treated hydrogels (p<0.01).

Conclusions : Our data suggest inhibition of mechanosensitive channel activity as a potential therapeutic target to prevent actin cytoskeletal remodeling and tissue stiffening within the optic nerve head in glaucoma.

This is a 2021 ARVO Annual Meeting abstract.