Abstract
Purpose :
A number of studies have shown that aqueous humor outflow resistance and thereby intraocular pressure (IOP) are regulated by nitric oxide synthase (NOS). Recent studies by our group found that altered biomechanical behavior of cells in the glaucomatous conventional outflow pathway interferes with their proper functioning. Surprisingly, no previous study has examined the mechanical response of endothelial cells to NO. In the present study, we analyzed the effect of NO on the actin cytoskeleton and the biomechanical behavior of endothelial cells.
Methods :
Schlemm canal (SC) cell strains were isolated from 3 human donors and HUVECs were purchase from Cell Applications. HUVECs and SC cells were treated with 50µM and 100µM DETA-NO for time periods of 3 to 5 hours. HUVEC and SC cells were labeled with phalloidin-TRITC and changes of the actin cytoskeleton were analyzed by immunocytochemistry. Stiffnesses of vehicle and DETA-NO treated (50 µM) HUVECs and SC cells were measured by atomic force microscopy using spherical (10 µm) and sharp tips, and statistical significance was determined using a Student’s t-test.
Results :
The immunocytochemical analysis of HUVEC and SC cells showed marked changes in the actin cytoskeleton of both cell types after DETA-NO treatment. DETA-NO treatment induced a pronounced actin depolymerization in both cell types (figure below shows typical results for SC cells). Use of a rounded AFM tip, which probes subcortical stiffness, showed that DETA-NO decreased SC cell stiffness from 1.4±0.3 kPa (mean±SE) to 0.8±0.1 kPa (p<0.02) and decreased HUVEC stiffness from 1.3±0.2 kPa (mean±SE) to 0.8±0.2 kPa (p<0.05). Use of a sharp AFM tip, that probes cortical stiffness, showed that DETA-NO decreased stiffness from 6.2±1.1 kPa (mean±SE) to 4.0±0.6 kPa (p<0.005) in SC cells while studies on HUVECs are in progress.
Conclusions :
We report strong preliminary evidence that NO leads to marked changes in the biomechanical behavior of SC cells. We suggest that previous observed reduction of the conventional outflow resistance after NO treatment is in part linked to the observed alteration of the cellular stiffness in SC cells.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.