Purpose: The multifunctional profibrotic cytokine TGF-β2 is implicated in the pathophysiology of primary open angle glaucoma (POAG). While the underlying cause of POAG remains unclear, TGF-β2 dependent remodeling of the extracellular matrix (ECM) within the trabecular meshwork (TM) microenvironment is considered an early pathologic consequence associated with impaired aqueous humor (AH) outflow and elevated IOP. Mitochondrial-targeted antioxidants have been recently shown by our group to markedly attenuate TGF-β2 profibrotic responses, strongly implicating oxidative stress as a key facilitator of TGF-β2 signaling in human TM cells. In this study, we determined the mechanism by which oxidative stress facilitates TGF-β2 profibrotic responses in cultured primary human TM cells.
Methods: Semiconfluent cultures of primary or transformed human TM cells were conditioned overnight in serum-free media and subsequently challenged without or with TGF-β2 (5 ng/mL). Relative changes in the mRNA content of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) isoforms, connective tissue growth factor (CTGF), collagen 1α1 and 4α1 isoforms or relative changes in the protein content of Nox4, phospho- and total-Smad2 and -Smad3, collagens I and IV were determined in the absence or presence of GKT137831, a Nox1-Nox4 dual enzyme inhibitor, and quantified by real-time qPCR or by immunoblot, respectively. Relative in situ changes in collagens I and IV and in alpha smooth muscle actin (αSMA) were semiquantified by immunocytochemistry, whereas relative changes in filamentous actin stress fiber formation was semiquantified by phalloidin staining.
Results: Quiescent primary human TM cells cultured in the presence of TGF-β2 exhibited a marked selective increase in endogenous Nox4 mRNA and Nox4 protein expression. Actinomycin D prevented TGF-β2 mediated increases in Nox4 mRNA expression. TM cells reverse transfected with siRNA against Smad3 prevented TGF-β2 mediated increases in Nox4 mRNA expression. Pre-incubating TM cells with GKT137831 attenuated TGF-β2 mediated increases in intracellular reactive oxygen species (ROS), in COL1A1, COL4A1, and CTGF mRNA expression, in Smad3 protein phosphorylation, in collagens I, collagens IV, and αSMA protein expression, and in filamentous actin stress fiber formation.
Conclusions: TGF-β2 promotes oxidative stress in primary human TM cells by selectively increasing expression of NADPH oxidase 4. Dysregulation of redox equilibrium by induction of NADPH oxidase 4 expression appears to be a key early event involved in the pathologic profibrotic responses elicited by TGF-β2 canonical signaling, including ECM remodeling, filamentous actin stress fiber formation, and αSMA expression. Selective inhibition of Nox4 expression/activation, in combination with mitochondrial-targeted antioxidants, represents a novel strategy by which to slow the progression of TGF-β2 elicited profibrotic responses within the TM.