September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Bidirectional interactions between trabecular meshwork cells and their extracellular matrix in the presence of dexamethasone
Author Affiliations & Notes
  • Vijaykrishna Raghunathan
    Surgical & Radiological Sciences, University of California Davis, Davis, California, United States
  • Christopher J Murphy
    Ophthalmology & Vision Science, University of California Davis, Davis, California, United States
    Surgical & Radiological Sciences, University of California Davis, Davis, California, United States
  • Paul Russell
    Surgical & Radiological Sciences, University of California Davis, Davis, California, United States
  • Footnotes
    Commercial Relationships   Vijaykrishna Raghunathan, None; Christopher Murphy, None; Paul Russell, None
  • Footnotes
    Support  Funded by Bright Focus Foundation grant (G2015078), NIH Grant P30EY12576 and an unrestricted grant from Research to Prevent Blindness.
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 3544. doi:
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      Vijaykrishna Raghunathan, Christopher J Murphy, Paul Russell; Bidirectional interactions between trabecular meshwork cells and their extracellular matrix in the presence of dexamethasone. Invest. Ophthalmol. Vis. Sci. 2016;57(12):3544.

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

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Abstract

Purpose : Treatment with corticosteroids can result in ocular hypertension, and steroid induced glaucoma. The extent to which biomechanical changes in trabecular meshwork (TM) cells and extracellular matrix (ECM) contribute towards this dysfunction is poorly understood. Further, the impact that dysregulated ECM has on cellular function in exacerbating disease is unknown. We wanted to determine how (i) dexamethasone (dex) modulated cell & ECM biomechanics, and ECM composition & morphology, and (ii) ECM derived from cells treated with dex might further influence cell biomechanics and cytoskeletal dynamics.

Methods : Primary and hTERT immortalized human trabecular meshwork (HTM & htHTM respectively) cells (25000/cm2) were each plated on aminated glass and were cultured in 10-7M dex or vehicle control (ethanol) for 4 weeks. htHTM cells were then dissociated and the mechanics of the ECM that was deposited by the treated or the control cells was measured by AFM. Decellularization was verified by immunostaining. ECM derived from cells was solubilized for further analyses. To determine the impact that these ECM had on cellular behavior, htHTM cells were plated on matrices derived, after chronic control or dex treatment, for 3days. Cell mechanics, immunocytochemistry, qPCR and Western blotting were performed to ascertain cytoskeletal dynamics.

Results : Dex treatment resulted in 2 fold increases in HTM cell stiffness. The matrix deposited after chronic dex treatment was 4 fold stiffer than that deposited by vehicle control cells. Changes in ECM mechanics were associated with significant differences in fibronectin morphology with no changes in their expression level. htHTM cells cultured on ECM derived after dex treatment were ~2 fold stiffer, than those cultured on ECM derived from cells without steroid treatment, accompanied by elevated mRNA expression of myocilin.

Conclusions : We have demonstrated in vitro ECM derived from dex treated cells are stiffer than ECM deposited by control HTM cells associated with compositional changes and fibronectin morphology the most significantly altered. Further, ECM derived from cells with steroid treatment significantly altered cytoskeletal dynamics and gene expression. These results demonstrate glucocorticoid treatment can initiate substantial changes in the biophysical and biochemical environment of cells which can further alter cellular phenotype.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

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