Nox4 Facilitates TGFβ1-Induced Fibrotic Response in Human Tenon's Fibroblasts and Promotes Wound Collagen Accumulation in Murine Model of Glaucoma Filtration Surgery.

Manisha H Shah,Nicole J Van Bergen,Jonathan G Crowston,Sze Ng,Elsa C Chan,Hitesh M Peshavariya,Surinder S Pandav

doi:10.3390/antiox9111126

Abstract

Collagen accumulation in sub-conjunctival tissue at the surgical wound is one of the major complications associated with glaucoma filtration surgery (GFS). This process often leads to unwanted fibrotic scar formation at the lesion site and dysfunction of tissues. Previously, we demonstrated that NADPH oxidase 4 (Nox4) is implicated in transforming growth factor-beta (TGFβ)-induced collagen production in ocular fibroblasts and scarring responses in a mouse model of corneal injury. Here, we propose that Nox4 is an important facilitator of TGFβ-induced responses. We tested this hypothesis in human Tenon’s fibroblasts (HTF) and also assessed a role of Nox4 in an experimental mouse model of GFS. TGFβ1 induced Nox4 mRNA expression but downregulated Nox5 in HTF. Targeting Nox4 gene expression with an adenovirus carrying a Nox4 small interfering RNA (siRNA) (Ad-Nox4i) or removal of hydrogen peroxide (H2O2) with EUK-134 (25 μM) in HTFs significantly reduced TGFβ1-induced Nox4 expression, H2O2 production, and collagen synthesis (p < 0.05, n = 3–6). SIS3 (5 μM) that prevents Smad3 phosphorylation is found to suppress TGFβ1-induced collagen production in HTFs. Furthermore, Ad-Nox4i and EUK-134 both abolished TGFβ1-stimulated proliferation of HTFs. We also compared collagen deposition at the wound arising from GFS between wildtype (WT) and Nox4 knockout (KO) mice. Both collagen deposition and fibrovascularization at the wound were significantly decreased in Nox4 KO mice at 14 days after GFS. Our results provide comprehensive evidence that Nox4 is an important mediator for TGFβ1-induced responses in HTFs and collagen deposition in surgical wound following GFS in mice. As such, pharmacological inhibition of Nox4 would be a viable therapeutic strategy for the control of scarring after glaucoma surgery.

Highlights

Glaucoma is the leading cause of vision impairment and blindness [1], and it results from degeneration of the optic nerve, often exacerbated by increasing pressure within the eye (intraocular pressure (IOP))
We assessed collagen production by evaluating the absorbance readings of the eluents from human Tenon’s fibroblasts (HTF) stained with picrosirius red
There was statistically significant accumulation of collagen in HTFs at both 6 and 24 h following TGFβ1 stimulation (Figure 1C, p < 0.05, n = 3–5)

Summary

Introduction

Glaucoma is the leading cause of vision impairment and blindness [1], and it results from degeneration of the optic nerve, often exacerbated by increasing pressure within the eye (intraocular pressure (IOP)). If IOP lowering drugs or laser treatment fails, glaucoma filtration surgery (GFS) is the gold standard operation widely used to treat glaucoma and manage IOP by creating an opening that bypasses the trabecular meshwork and allows aqueous to drain into the sub-conjunctival space [2]. Trabeculectomy procedure can sometimes stimulate excessive scarring and fibrosis in the sub-conjunctival tissue, which is the principal cause of GFS failure and inadequate control of IOP [3]. To reduce the risk of bleb scarring, anti-mitotic agents mitomycin C (MMC) and fluorouracil (5-FU) are often applied intra-operatively or post-operatively [4,5] to inhibit fibroblasts activation and improve surgical outcomes. The cytotoxic nature of these drugs poses limitation and risk on their applications as an adjunct to GFS. A safer and more effective means has always been explored to improve the long-term success of GFS

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