Abstract
Purpose :
The profibrotic cytokine, TGF-β2, is strongly implicated in the pathophysiology of primary open angle glaucoma (POAG). Recently published studies from our lab have demonstrated that TGF-β2 elicits marked increases in reactive oxygen species (ROS) in cultured human primary trabecular meshwork (TM) cells. In this study, we sought to determine the intracellular origin and mechanism by which TGF-β2 induces oxidative stress in TM cells.
Methods :
Human primary TM cells were conditioned in serum-free media overnight and subsequently incubated in the absence or presence of active TGF-β2 (5 ng/ml) and relative changes in ROS production (fluorogenic dyes), TGF-β2 dependent canonical signaling (immunoblot), NADPH oxidase (types 1-5) and collagen isoform (Col1A1, Col4A1) mRNA (qPCR) and protein expression (immunoblot) were quantified. In some experiments, serum starved cells were pretreated (2h) with the ALK5 inhibitor SB-431542 (10 µM) or with the selective Nox1/4 dual inhibitor GKT137381 (20 µM).
Results :
As we have previously reported, TGF-β2 (5 ng/ml) elicited a marked 2-fold increase in intracellular ROS production in serum-starved human primary TM cells. Coincident with ROS production was a marked time-dependent increase in NOX4, but not Nox 1-3 or Nox 5, mRNA (200-fold, p<0.05) and protein (1.5-fold) expression. TGF-β2 similarly elicited marked increases in phospho Smad2/3 content along with increases in COL1A1 and COL4A1 mRNA and collagens type 1 and type IV protein expression. Pretreating cells with either SB-431542 or GKT137831 significantly attenuated TGF-β2 mediated intracellular ROS generation, TGF-β2 induced Smad signaling, and TGF-β2 induced collagens mRNA and protein expression.
Conclusions :
TGF-β2 elicits oxidative stress in human primary TM cells, in part, through a NOX4-dependent mechanism. Subsequently generated ROS may potentiate, in a feed-forward manner, TGF-β2 signaling in TM cells thereby promoting and/or exacerbating induced ECM remodeling. Pharmacologic inhibition of Nox4 represents a novel therapeutic strategy by which to limit TGF-β2 induced oxidative injury to the TM.
This is a 2020 ARVO Annual Meeting abstract.