Phenotypic Characterization of Segmental Human Trabecular Meshwork Cells

Purpose: Aqueous humor outflow is segmental in the trabecular meshwork (TM) with regions of high-flow (HF) or low-flow (LF) that vary in molecular signature, biomechanics, and response to pressure in both normal and glaucoma eyes. We sought to investigate whether cells in these regions (1) deposit distinct extracellular matrices (ECM); (2a) exhibit phenotypes that vary as a function of substrate stiffness; and (2b) differ in their phagocytic abilities. Methods: (1) Primary human TM, HF and LF cells [n=3 non-glaucomatous donors] were treated with/without DEX for 30 days. Cultures were decellularized to obtain vehicle control ECM (VehM) and glucocorticoid-induced ECM (GIM) and elastic moduli were measured. Expression of ECM proteins: fibronectin (FN), collagen IV (COLIV) and laminin (LAM) was assessed. (2a) Relative expressions of COLIV, TIMP1, SPARC, CTGF, and MMP1 mRNA in segmental cells seeded on soft and stiff hydrogels, and on glass (control) were assessed; (2b) and phagocytic ability with/without DEX for 72 h, was quantified. Results: (1) LF VehM was 2-fold stiffer than HF VehM (p < 0.05). Moduli of HF and LF GIM were greater than corresponding VehM (p < 0.001). LF GIM was stiffer than HF GIM (1.75-fold; p < 0.001). FN, COLIV, and LAM expressions were upregulated in HF and LF GIMs in comparison to VehMs. (2a) CTGF mRNA were upregulated in HF compared to LF cells, and on stiffer substrates. MMP1 and TIMP1 were overexpressed in LF cells relative to HF cells and in both cells, the expression was higher on softer substrates. SPARC was upregulated in HF cells compared with LF cells on soft substrates (2b) HF and LF cells showed increased phagocytosis on softer substrates while DEX significantly impaired phagocytosis on all substrates except glass. LF cells showed ~25% reduction in phagocytic uptake when compared to HF cells. Conclusions: That substratum stiffness differentially mediated phagocytosis and expression of ECM genes in these cells highlights an intricate dynamic relationship between biophysical and biochemical cues in mechanobiology of segmental TM cells. Investigating this relationship further could inform new strategies to rejuvenate dysfunctional TM cells towards a healthier phenotype to lower ocular hypertension.