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Karen Yud Torrejon, Ellen L Papke, Andrea Unser, Matthew Kerr, W Daniel Stamer, Feryan Ahmed; Nitric Oxide Induces Changes in the Cytoskeleton and ECM of TGFβ2-treated Trabecular Meshwork and Schlemm’s canal cells. Invest. Ophthalmol. Vis. Sci. 2018;59(9):4708.
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Nitric oxide synthase (NOS) and thereby the production of nitric oxide (NO) has been shown to increase aqueous humor outflow facility and subsequently lower intraocular pressure (IOP). The binding of NO to soluble guanylate cyclase (sGC) initiates a cascade of events such as the promotion of vasodilation, which could therefore increase outflow within the conventional outflow pathway involving both the trabecular meshwork and Schlemm’s canal. Using a 3D bioengineered construct of the human conventional outflow tract, we studied the effect of NO donating compounds on actin cytoskeleton and proteins that have been linked to glaucomatous conventional outflow tract.
3D conventional outflow tract constructs, comprised of HTM, HSC cells and their associated ECM, were treated with TGFb-2 (2.5 ng/mL) for at 9 days to induce a glaucomatous state. These constructs were then exposed to two different NO-donating compounds, SNP (100µM) or DETA-NO (100µM) for 48 hrs. Cells were stained with phalloidin-488 and the changes in actin cytoskeleton were analyzed by confocal microscopy. The protein and gene expression of a-smooth muscle actin, fibronectin, and collagen type IV (col-IV) were analyzed by western blot and qPCR. Statistical significance was determined using one-way ANOVA followed by Bonferroni post-tests.
Immunocytochemical analysis showed pronounced changes in the actin cytoskeleton of the cells after SNP and DETA-NO treatments. Both NO-donating agents relaxed the cells and induced actin depolymerization demonstrated by less pronounced and shorter F-actin fibers. Western blot analysis and immunocytochemistry demonstrated a significant decrease in expression of a- smooth muscle actin in both, SNP and DETA-NO treated 3D constructs (p<0.01 and p<0.05, respectively). Furthermore, fibronectin and col-IV protein and gene expression was significantly lowered (p<0.01 and 0.05, respectively). Confocal imaging further confirmed the decreased expression of fibronectin and col-IV in the NO-treated samples.
We report that NO induce changes in the actin cytoskeleton and ECM by decreasing F-actin, a- smooth muscle actin and ECM proteins, fibronectin and col-IV, expression. Decrease in expression of cytoskeletal and ECM proteins suggests a potential mechanism by which NO-donating agents sustain IOP-lowering effect through the conventional outflow pathway.
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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