July 2019
Volume 60, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2019
Elevated IOP increases Shear Stress in Schlemm’s Canal
Author Affiliations & Notes
  • Fiona McDonnell
    Duke University, Durham, North Carolina, United States
  • Kristin Marie Perkumas
    Duke University, Durham, North Carolina, United States
  • Nicole E. Ashpole
    Duke University, Durham, North Carolina, United States
  • Joan Kalnitsky
    Duke University, Durham, North Carolina, United States
  • Joseph M Sherwood
    Imperial College London, London, United Kingdom
  • Darryl R Overby
    Imperial College London, London, United Kingdom
  • W Daniel Stamer
    Duke University, Durham, North Carolina, United States
  • Footnotes
    Commercial Relationships   Fiona McDonnell, None; Kristin Perkumas, None; Nicole Ashpole, None; Joan Kalnitsky, None; Joseph Sherwood, iPerfusion (E); Darryl Overby, iPerfusion (E); W Daniel Stamer, None
  • Footnotes
    Support  NIH Grant EY022359, NIH Grant 5P30EY005722-32, 2018 Unrestricted Grant from Research to Prevent Blindness
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 3181. doi:
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    • Get Citation

      Fiona McDonnell, Kristin Marie Perkumas, Nicole E. Ashpole, Joan Kalnitsky, Joseph M Sherwood, Darryl R Overby, W Daniel Stamer; Elevated IOP increases Shear Stress in Schlemm’s Canal. Invest. Ophthalmol. Vis. Sci. 2019;60(9):3181.

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

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Abstract

Purpose : Elevations in intraocular pressure (IOP) narrow Schlemm’s canal (SC) lumen, increasing shear stress within the canal. SC endothelial cells in culture produce nitric oxide (NO) in a shear stress-dependent manner through activation of endothelial nitric oxide synthase (eNOS). Using two engineered adenovirus constructs that contain the eNOS promoter driving either Secreted Alkaline Phosphatase (eNOS-SEAP) or GFP (eNOS-GFP), we tested the effects of shear stress on reporter expression by endothelial cells in vitro and ex vivo.

Methods : Human umbilical vein endothelial cells (HUVECs) or SC cells were transduced with eNOS-SEAP or eNOS-GFP adenovirus (5.5x108ifu/ml) for 8 hrs. Cells were then exposed to shear stress of 0.1 or 10 dynes/cm2. In parallel, human anterior segments were perfused at 2.5 µl/min for 24-48 hrs then retroperfused with both the eNOS-SEAP and eNOS-GFP adenoviruses (2x106ifu/ml). Perfusion was restarted at 2.5 µl/min in one anterior segment and 5 µl/min in the contralateral segment; giving low and high shear stress in the SC lumen, respectively. SEAP secretion was measured using a SEAP reporter assay kit. GFP expression was measured using Western blotting, confocal microscopy and flow cytometry. Nitrite concentration was measured using a Griess Reagent assay.

Results : In response to 10 dynes/cm2 shear stress, HUVECs produced 43±12-fold more SEAP and 6.8±0.9-fold more GFP by Western blotting than cells exposed to 0.1 dynes/cm2. Similarly, SC cells exposed to 10 dynes/cm2 shear stress increased SEAP secretion by 1.6±0.1-fold, and increased GFP by 1.5±0.7-fold by Western blotting. Fluorescence intensity of GFP by flow cytometry increased 1.7±1.1-fold in SC cells when compared to controls. Human anterior segments perfused at a higher flow rate, and therefore at an elevated IOP showed a 5.6±1.7-fold increase in SEAP secretion in effluent and qualitatively higher levels of GFP expression in SC lumen and across collector channel ostia compared to those of lower flow rates. Elevated IOPs corresponded to increased nitrite concentrations (1.8±1.1-fold) in perfusion effluent.

Conclusions : The eNOS-SEAP and eNOS-GFP adenoviruses demonstrated the capacity to quantify and localize shear stress of endothelial cells in culture, and SC cells in situ in an organ culture model. This is the first demonstration of IOP-induced shear stress in Schlemm’s canal and across collector channel ostia.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

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