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Kamesh Dhamodaran, Janice A Vranka, Vijaykrishna Raghunathan; Expression of Notch signal in primary human trabecular meshwork segmental flow cells. Invest. Ophthalmol. Vis. Sci. 2020;61(7):3443.
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Primary open-angle glaucoma (POAG) is associated with elevations in intraocular pressure (IOP) and increased resistance to aqueous humor (AH) outflow at the trabecular meshwork (TM). AH outflow is segmental and the causative differences between high or low outflow regions of the TM are not identified. Notch signaling is an evolutionarily conserved pathway that dictates cell fate and differentiation. Here we investigate if Notch expression differs between segmental regions of the TM.
Human eye whole globes (n=6 donors) were perfused with CellMask™ Orange to label the segmental regions. Primary human TM (hTM) cells from high (HF) and low (LF) flow regions (n=3) were isolated, cultured, and treated with/without 100nM dexamethasone (dex) for 72hrs to determine Myocilin expression. Presence of Notch proteins in the hTM tissue (n=3) were determined by immunofluorescence. Receptors, ligands, and downstream targets of the Notch pathway were evaluated by RT-qPCR, and Notch activity was determined using western blot for cleaved Notch 1(Val1744) and downstream target Hes1 in hTM HF/LF cells in vitro.
Notch receptors/ligands and downstream target expression was prevalent in hTM tissues ex vivo. In vitro, Notch receptors: Notch1 (~1.4, p=0.299), Notch3 (~1.4 fold) and Notch ligands: Jagged1 (~2.8 fold, p=0.0002) and DLL4 (~3.4 fold, p=0.0003) were overexpressed in LF cells, while the expression of Notch2 (~0.8 fold), Jagged2 (~0.7 fold, p=0.0327), DLL1 (~0.4 fold, p=0.0092), and DLL3 (~0.7 fold) were downregulated in LF cells. Also, expression of Notch downstream targets, Hey1 (~2.5 fold, p=0.0012), Hes1 (~5.6 fold, p=0.0001), Hes3 (~3.8 fold, p=0.0001) and Hes5 (~1.2 fold) were significantly greater in LF cells.
For the first time, our data demonstrates that proteins involved in Notch signaling are (i) expressed in the TM, and (ii) that they are differentially expressed in cells from segmental flow regions. Interestingly, downstream targets of Notch pathway were overexpressed in LF cells. The implications of such differential expression in ECM remodeling and how it contributes to outflow resistance warrants further studies.
This is a 2020 ARVO Annual Meeting abstract.
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