Endogenous expression of Notch pathway molecules in human trabecular meshwork cells. (March 2022)

• Record Type: Journal Article
• Authors: Dhamodaran, Kamesh; Baidouri, Hasna; Nartey, Andrews; Staverosky, Julia; Keller, Kate; Acott, Ted; Vranka, Janice A; Raghunathan, Vijay Krishna
• Is Part Of: Experimental eye research. Volume 216(2022)
• Publication Date: 2022-03
• Subjects: HF High Flow -- LF Low Flow -- IF Intermediate Flow -- TM Trabecular Meshwork -- ECM Extracellular Matrix -- qRT-PCR Quantitative Real Time Polymerase Chain Reaction -- IOP Intra Ocular Pressure -- DEX Dexamethasone -- GR Glucocorticoid Receptor -- kPa Kilo Pascal -- PBS Phosphate Buffered Saline -- ANOVA Analysis of Variance -- NTM Non-Glaucomatous Trabecular Meshwork -- GTM Glaucomatous Trabecular Meshwork; Cyclical strain -- Trabecular meshwork -- Notch -- Mechanotransduction -- Cell-cell communication; Ophthalmology -- Periodicals; Eye -- Periodicals; Œil -- Périodiques; Ophthalmology; Periodicals; Electronic journals; 612.8405
• DOI: 10.1016/j.exer.2022.108935
• Languages: English
• ISSNs: 0014-4835

Abstract

Abstract: Purpose: Cells in the trabecular meshwork sense and respond to a myriad of physical forces through a process known as mechanotransduction. Whilst the effect of substratum stiffness or stretch on TM cells have been investigated in the context of transforming growth factor (TGF-β), Wnt and YAP/TAZ pathways, the role of Notch signaling, an evolutionarily conserved pathway, recently implicated in mechanotransduction, has not been investigated in trabecular meshwork (TM) cells. Here, we compare the endogenous expression of Notch pathway molecules in TM cells from glaucomatous and non-glaucomatous donors, segmental flow regions, and when subjected to cyclical strain, or grown on hydrogels of varying rigidity. Methods: Primary TM from glaucomatous (GTM), non-glaucomatous (NTM) donors, and from segmental flow regions [high flow (HF), low flow (LF)], were utilized between passages 2–6. Cells were (i) plated on tissue culture plastic, (ii) subjected to cyclical strain (6 h and 24 h), or (iii) cultured on 3 kPa and 80 kPa hydrogels. mRNA levels of Notch receptors/ligands/effectors in the TM cells was determined by qRT-PCR. Phagocytosis was determined as a function of substratum stiffness in NTM-HF/LF cells in the presence or absence of 100 nM Dexamethasone treatment. Results: Innate expression of Notch pathway genes were significantly overexpressed in GTM cells with no discernible differences observed between HF/LF cells in either NTM or GTM cells cultured on plastic substrates. With 6 h of cyclical strain, a subset of Notch pathway genes presented with altered expression. Expression of Notch receptors/ligands/receptors/inhibitors progressively declined with increasing stiffness and this correlated with phagocytic ability of NTM cells. Dexamethasone treatment decreased phagocytosis regardless of stiffness or cells isolated from segmental outflow regions. Conclusions: We demonstrate here that the Notch expression in cultured TM cells differ intrinsically between GTM vs NTM, and by substratum cues (cyclical strain and stiffness). Of import, the most apparent differences in gene expression were observed as a function of substratum stiffness which closely followed phagocytic ability of cells. Interestingly, on soft substrates (mimicking normal TM stiffness) Notch expression and phagocytosis was highest, while both expression and phagocytosis was significantly lower on stiffer substrates (mimicking glaucomatous stiffness) regardless of DEX treatment. Such context dependent changes suggest Notch pathway may play differing roles in disease vs homeostasis. Studies focused on understanding the mechanistic role of Notch (if any) in outflow homeostasis are thus warranted. Highlights: Endogenous gene expression of Notch receptors, ligands, targets, & inhibitors differ between normal & glaucomatous trabecular meshwork cells. Gene expression of Notch receptors, ligands, targets, & inhibitors are elevated in normal trabecular meshwork cells subjected to cyclical strain. Notch receptors, ligands, targets, & inhibitors mRNA exression in normal trabecular meshwork cells are mediated by substratum stiffness.