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Enhua H. Zhou, Ramaswamy Krishnan, W Daniel Stamer, Kristin M. Perkumas, Kavitha Rajendran, Jeffrey J. Fredberg, Mark Johnson; Mechanical Responsiveness Of The Human Schlemm's Canal Endothelial Cell In Vitro. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4812.
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© ARVO (1962-2015); The Authors (2016-present)
In primary open angle glaucoma, elevated intraocular pressure (IOP) arises from increased resistance to aqueous humor outflow. Before exiting the eye, aqueous humor passes across the continuous barrier formed by the Schlemm’s canal (SC) endothelium in the basal to apical direction. We hypothesize that mechanical characteristics of this endothelium regulates outflow resistance.
We isolated human SC endothelial cells from 8 human donors and used them for in vitro testing at passages 2 and 3. We quantified cell stiffness using optical magnetic twisting cytometry (OMTC) and cell contractility using traction force microscopy (TFM). We further assessed mechanical responsiveness of these cells to seven drugs: lysophosphatidic acid (LPA, 0.1 microM), sphingosine-1-phosphate (S1P, 0.1 microM), thrombin (0.1 micro-NIH unit/ml), latrunculin A (0.1 microM), Y-27632 (4 doses from 10 to 200 microM),dibutyryl cyclic-AMP (DBcAMP, 4 doses from 1 to 10 mM), and isoproterenol (4 doses from 0.01 to 10 microM). These drugs were chosen because their effects on outflow resistance had been previously established: the first 3 drugs increased and the latter 4 decreased outflow resistance in perfused eyes.
5 min exposure to LPA, thrombin, or S1P caused a stiffness increase of 20-100%. By contrast, 5 min latrunculin A treatment caused a stiffness decrease of 40-60%. On average, DBcAMP, Y-27632, or isoproterenol caused a dose-dependent softening; however, cells from 3 donors did not soften in response to isoproterenol. Across donors, those cells that exhibited larger responses to Y-27632 also exhibited larger responses to latrunculin A, and vice versa. Similarly, responses to DBcAMP were positively correlated with responses to isoproterenol. Those donor lines (3 out of 8) that were not responsive to isoproterenol also lacked expression of beta2 adrenergic receptor as determined by Western blotting.
Overall, our data suggest that those drugs that increase outflow resistance also increase SC cell stiffness, and, conversely, that those drugs that decrease outflow resistance also decrease SC cell stiffness. Although the mechanism remains unclear, this finding supports the hypothesis that aqueous humor outflow resistance is controlled by mechanical properties of the SC endothelium.
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