June 2021
Volume 62, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2021
Substrate stiffness modulates gene expression and phagocytosis in segmental human trabecular meshwork cells
Author Affiliations & Notes
  • Andrews Nartey
    College of Optometry, University of Houston, Houston, Texas, United States
  • Kamesh Dhamodaran
    College of Optometry, University of Houston, Houston, Texas, United States
  • Julia Staverosky
    Casey Eye Institute, Oregon Health & Science University, Portland, Oregon, United States
  • Janice A Vranka
    Casey Eye Institute, Oregon Health & Science University, Portland, Oregon, United States
  • VijayKrishna Raghunathan
    College of Optometry, University of Houston, Houston, Texas, United States
  • Footnotes
    Commercial Relationships   Andrews Nartey, None; Kamesh Dhamodaran, None; Julia Staverosky, None; Janice Vranka, None; VijayKrishna Raghunathan, None
  • Footnotes
    Support  NIH/NEI grants EY026048-01A1 (JAV, VKR), 5P30 EY007551-30 (NEI Core grant to UHCO), P30 EY010572 (NEI Core grant to OHSU), and by an unrestricted grant to the Casey Eye Institute from Research to Prevent Blindness, New York, NY.
Investigative Ophthalmology & Visual Science June 2021, Vol.62, 1632. doi:
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      Andrews Nartey, Kamesh Dhamodaran, Julia Staverosky, Janice A Vranka, VijayKrishna Raghunathan; Substrate stiffness modulates gene expression and phagocytosis in segmental human trabecular meshwork cells. Invest. Ophthalmol. Vis. Sci. 2021;62(8):1632.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Aqueous humor outflow in the trabecular meshwork (TM) is segmental with regions of high- (HF) or low-flow (LF) that vary in their molecular signature, biomechanics, and response to pressure. Whether these changes are driven by cells or their microenvironment is unclear. Recently, we isolated HF and LF TM cells and found differences in gene expression and extracellular matrix (ECM) stiffness. We hypothesize that cells from these regions are intrinsically different and exhibit phenotypes that vary as a function of substrate stiffness and differ in phagocytic function.

Methods : Using whole human globe perfusion, HF and LF TM were delineated using cell mask orange, a plasma membrane stain. Primary human TM (hTM) cells [n=3 non-glaucomatous donors] were isolated and primary cultures established. Cells were seeded on substrates of relevant stiffness: soft hydrogel (3kPa to mimic normal HF), stiff hydrogel (80kPa to mimic glaucomatous LF) or glass (>1GPa). RT-qPCR was used to determine relative expression levels of COLIV, TIMP1, SPARC, CTGF, and MMP1 genes. HF and LF hTM cells were cultured on these substrates with or without dexamethasone (DEX) for 72 h. Phagocytic ability was measured using quantitative phagocytosis assay.

Results : CTGF mRNA levels were upregulated in HF compared to LF cells (3-fold change) seeded on stiffer substrates. MMP1 and TIMP1 were overexpressed in LF relative to HF cells (1.5-fold change) and expression levels of both were higher on soft substrates. SPARC was upregulated in HF compared to LF cells on soft substrates (2.3-fold change). Phagocytic ability of HF and LF hTM cells was higher on soft substrates and attenuated on stiffer substrates (1.5-2.5-fold change). DEX impaired phagocytosis in both HF and LF cells on soft and stiff substrates (2-fold mean decrease), but not on glass. Following DEX treatment, LF cells seeded on soft and stiff substrates showed a decline in phagocytic uptake by 18-40% compared to their HF counterparts.

Conclusions : Our data demonstrate that HF and LF hTM cells have intrinsic phagocytic differences, a critical property in facilitating aqueous humor outflow and maintaining intraocular pressure homeostasis in TM tissues. That substratum stiffness can differentially mediate phagocytosis and expression of ECM genes in these cells highlights an intricate dynamic relationship between biophysical and biochemical cues in segmental TM cells.

This is a 2021 ARVO Annual Meeting abstract.

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