Transforming growth factor β1-induced NADPH Oxidase-4 expression and fibrotic response in conjunctival fibroblasts

Purpose: Fibrotic scarring after ocular surgeries and chemical burn injuries can impede clarity of the cornea and cause vision impairment. Transforming growth factor β (TGFβ) signaling pathway is known to mediate fibrotic scarring, and NADPH oxidase-derived reactive oxygen species has been shown to be an effector molecule that facilitates TGFβ1-mediated responses. The present study explores the expression profile and functional importance of NADPH oxidase (Nox) in conjunctival fibroblasts. In addition, the effect of curcumin on the TGFβ1-induced NADPH oxidase expression and collagen synthesis was also investigated.

Methods: The mRNA expression of Nox isoforms in rabbit conjunctival fibroblasts was measured by real-time PCR. The production of hydrogen peroxide (H₂O₂) and total collagen by these cells was measured by Amplex Red assay and Picro-Sirius red assay, respectively. Nox4 was knocked down by adenovirus-mediated siRNA targeting Nox4 (Adv-Nox4i).

Results: We describe for the first time that conjunctival fibroblasts express mRNA encoding for Nox2, Nox3, Nox4, and Nox5. TGFβ1 was found to induce Nox4 mRNA expression and total collagen release by these cells (P < 0.05; n = 4), and both responses are blocked by Smad3 inhibitor SIS3. Suppressing Nox4 gene transcription with Adv-Nox4i completely attenuated TGFβ1-stimulated H₂O₂ release and collagen production by conjunctival fibroblasts (P < 0.05; n = 4-6). Similarly, curcumin also inhibited TGFβ1-induced Smad3 phosphorylation, Nox4-derived H2O2 production, and total collagen synthesis by conjunctival fibroblasts (P < 0.05; n = 4-6).

Conclusions: The present study suggests that TGFβ1-mediated production of collagen by conjunctival fibroblasts involves Nox4-derived H₂O₂ pathway and this effect of Nox4 is abrogated by curcumin. This mechanism might be exploited to prevent fibrotic scarring after surgeries and chemical burn injuries in the eye.