July 2018
Volume 59, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2018
Mechanical stretch increases connexin43 expression and cell death in human trabecular meshwork cells
Author Affiliations & Notes
  • Mary Mengrui Feng
    Ophthalmology, Western University, London, Ontario, Canada
  • Hong Liu
    Ophthalmology, Western University, London, Ontario, Canada
  • Cindy M L Hutnik
    Ophthalmology, Western University, London, Ontario, Canada
  • Footnotes
    Commercial Relationships   Mary Feng, None; Hong Liu, None; Cindy Hutnik, None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 4718. doi:https://doi.org/
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      Mary Mengrui Feng, Hong Liu, Cindy M L Hutnik; Mechanical stretch increases connexin43 expression and cell death in human trabecular meshwork cells. Invest. Ophthalmol. Vis. Sci. 2018;59(9):4718. doi: https://doi.org/.

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

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Abstract

Purpose : Primary open angle glaucoma is the most prevalent form of glaucoma and is characterized by an increase in intraocular pressure (IOP) due to buildup of aqueous humor. The trabecular meshwork is an important part of the aqueous humor outflow pathway. Increased IOP causes distention and stretching of trabecular meshwork cells, and influences cellular homeostatic mechanisms. Gap junctions are a group of proteins critical in maintaining cellular homeostasis, and are made of connexin units. Connexin 43 (Cx43) is most widely expressed in the body, and is present throughout the trabecular meshwork. The purpose of this study was to determine the effect of mechanical stress on human trabecular meshwork cells (HTMC) and to study the role of Cx43 in HTMC exposed to mechanical strain.

Methods : Primary HTMC were cultured on collagen IV coated Flexcell plates for 5 days in 10% FBS until 90% confluent, and switched to serum free medium immediately prior to mechanical stress. In addition, a group of HTMC were treated with Carbenoloxone at the time of mechanical stress. HTMC were exposed to 15% stretch for 48 hours at 1 Hz using the FX-5000 Tension system. Cell morphology and Cx43 protein expression after stretch were analyzed with fluorescent microscopy. LDH release was measured using the Cytotoxicity Detection Kit and levels of cytosolic DNA fragments were determined to assess apoptosis. Cx43 mRNA and protein levels were measured using real-time PCR and Western Blot respectively. Gap junction function was measured using fluorescent microscopy to assess the transit of Luciferase Yellow dye through cells.

Results : Mechanical stretch increased LDH release from HTMC (p<0.05). In addition, stretching increased HTMC apoptosis (p<0.05). Cx43 mRNA (p<0.01) and protein expression (p<0.01) increased significantly after mechanical strain, as did Luciferase Yellow dye transfer through stretched cells. Cx43 inhibition with Carbenoloxone mitigates the increase in LDH after stretch.

Conclusions : Mechanical stretch induces cell injury and apoptosis, and increases Cx43 expression in HTMC. When Cx43 function is chemically inhibited, there is reduced cellular injury. Further investigations will be required to determine whether Cx43 inhibition can be used to prevent trabecular meshwork cell death in an animal model of glaucoma.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

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