Abstract
Purpose :
Treatment with corticosteroid hormone Dexamethasone (DEX) results in increased fibrogenic stimulus, extracellular matrix (ECM) accumulation in the trabecular meshwork (TM) and elevated intraocular pressure (IOP) leading to steroid induced glaucoma (SIG). Notch signaling pathway is an evolutionarily conserved signaling mechanism contributing to the pathophysiological processes like tissue fibrosis. Here, we investigated the effects of DEX on Notch signaling and ECM remodeling in the TM.
Methods :
Primary Human TM (HTM) cells were treated with 100nM DEX in serum free media alone or in combination with- a) N-(N-[3, 5-difluorophenacetyl])-l- alanyl)-S-phenylglycine t-butyl ester - DAPT (Notch signaling inhibitor), b) Phenethyl isothiocyanate - PEITC (Notch signaling activator), and c) RU486 (Glucocorticoid receptor inhibitor) - and investigated for changes in protein expression by immunoblotting for- canonical notch signaling including Notch receptor - Notch1, Notch ligand - Jagged-1, Notch effector - Hes1, ECM proteins - Fibronectin (FN) and Collagen1A (COL1A), pro-fibrogenic marker - α-Smooth Muscle Actin (α-SMA), and pro-fibrotic growth factor - Transforming growth factor β2 (TGFβ2). Student’s t-test and/or one-way ANOVA was used for statistical analysis and results were significant if p≤0.05 with a sample size of n≥3 in each experiment.
Results :
DEX treatment significantly - downregulated - Notch1 (p=0.008), Jagged1 (p=0.050), Hes1 (p=0.001) and upregulated - a) α-SMA (p=0.001), b) TGFβ2 (p=0.048), and c) ECM- COL1A (p=0.018) and FN (p=0.024) proteins. Inhibition of Notch signaling using DAPT mimicked the effect of DEX in upregulating COL1A and FN. Conversely, activation of Notch signaling using PEITC downregulated ECM proteins. Finally, inhibition of DEX signaling by RU486 restored the Notch1 (p=0.016) and Hes1 (p=0.001) and decreased FN (p=0.024) levels similar to PEITC treatment in presence of DEX.
Conclusions :
Our preliminary data in TM demonstrates that - 1) canonical Notch signaling pathway is downstream of DEX mediated events, 2) DEX induces ECM production by downregulating the Notch pathway, and 3) activation of Notch signaling mimics inhibition of DEX receptor-mediated signaling. From this study, we speculate that notch signaling plays an important role in ECM remodeling in TM outflow pathway and help maintain the IOP homeostasis. The activation of notch signaling pathway can be a potential therapeutic target for SIG.
This is a 2021 ARVO Annual Meeting abstract.