Abstract
Purpose :
The profibrotic cytokine, transforming growth factor beta (TGF-β), is found in increased concentrations in the aqueous of glaucomatous eyes. We therefore aimed to see what effect TGF-β has on normal trabecular meshwork (TM) cells with regards to markers of myofibroblast activation and ECM production which are features of fibrosis. NADPH oxidases (NOXs) have been implicated in ECM synthesis and myofibroblast differentiation in the fibrosis of various organ systems. We therefore also aimed to demonstrate the role that NOX4 has in TGF-β induced fibrosis.
Methods :
Primary and transformed normal human TM cells were cultured and treated with recombinant TGF-β1. Expression of α-SMA, Collagen 1α1 and NOX4 was measured using real time quantitative PCR with and without treatment with the NOX1/4 inhibitor, GKT137831.
Results :
Normal primary TM cells treated with TGF-β1 showed a 14-fold increase in the expression of Collagen 1α1 [p<0.01], a 2.8-fold increase in the expression of α-SMA [p<0.05] and a 31-fold increase in the expression of NOX4 [p<0.01] (n=3) after treatment. Transformed normal TM cells (NTM5) treated with TGF-β1 also showed an increase in Collagen 1α1 which peaked with 10ng/ml. α-SMA expression was also increased and 10ng/ml of TGF-β1 produced the largest increase in NOX4 expression in these cells [p<0.01] (n=3). NTM5 cells treated with TGF-β1 and the NOX4/1 inhibitor, GKT137831 showed a significantly reduced the expression of Collagen 1α1, α-SMA and NOX4 [p<0.01] (n=3). Our results show that TGF-β increases markers of myofibroblasts and ECM production in normal TM cells. We demonstrated that when NOX4 (which is increased in normal TM cells treated with TGF-β) is inhibited with GKT137831 these markers of fibrosis are decreased.
Conclusions :
The data shows that NOX4 inhibition with GKT137831 blocks TGF-β induced fibrotic gene expression in human TM cells, which suggests a potential role for NOX4 inhibition therapy in glaucoma.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.