Background/Purpose: Excessive deposition of collagen and other connective tissue components in the skin and multiple internal organs is characteristic of Systemic Sclerosis (SSc). Besides the well characterized profibrotic effects of TGF-β, oxidative stress and other factors have been implicated in SSc pathogenesis. NADPH oxidase 4 (NOX4) is one of seven NADPH oxidases responsible for the production of reactive oxygen species (ROS) which are critical mediators of oxidative stress. TGF-β potently stimulates NOX4 expression, making NOX4 a critical downstream regulator of TGF-β profibrotic effects. Unlike other NOX proteins, NOX4 does not require cofactors to mediate its effects and therefore alterations in its effects are due to changes in its expression and production. The purpose of the studies described here was to evaluate the effect of the genetic deletion of Nox4 in a murine model of TGF-β-induced tissue fibrosis.

Methods: Male and female C57BL6/J control mice and Nox4 knockout mice in which Nox4 expression has been eliminated by replacement of exon 4 of the Nox4 gene with a neomycin cassette were implanted subcutaneously in the right intrascapular region at 4 weeks of age with osmotic pumps containing either saline or 2.5 µg TGF-β1. C57BL6/J mice with normal Nox4were similarly implanted with osmotic pumps. These pumps deliver a daily dose of 150 ng TGF-β/day for 28 days. Mice were sacrificed 28 days after the pumps had dispersed their contents. Skin adjacent to the pump implantation site and both lungs were isolated for analysis. A portion of each tissue was fixed in formalin and processed for histopathologic analysis (hematoxyllin/eosin and Masson’s trichrome stains). Another sample of each tissue was hydrolyzed for measurement of hydroxyproline content.

Results: Histopathology studies in samples from control C57BL6/J TGF-β-treated mice showed peribronchial fibrosis and diffuse interstitial lung fibrosis. In contrast, Nox4 knockout mice showed normal lung histology. Similarly, profound dermal fibrosis was evident in skin from C56BL6/J mice implanted with TGF-β pumps but not in skin isolated from Nox4 knockout mice. Hydroxyproline levels in C57BL6/ control mice skin demonstrated a 104% increase in female control mice and a 59% increase in male control mice in response to TGF-β however TGF-β failed to increase the hydroxyproline content of Nox4 knockout mice tissues of either sex. In the lung, TGF-β caused increased hydroxyproline levels in female control mice by 83% and by 116% in male control mice whereas no significant changes were observed in Nox4knockout mice of either sex.

Conclusion: Genetic deletion of Nox4 in mice abrogated the ability of TGF-β delivered by subcutaneously implanted osmotic pumps to induce skin or lung tissue fibrosis. This result confirms several recent in vitro studies demonstrating a role for Nox4 as an important mediator of the profibrotic effects of TGF-β-induced tissue fibrosis. Since Nox4 is constitutively active, increased levels of its expression induced by TGF-β are likely to contribute to the pathogenesis of fibrotic diseases. The results of this study highlight the potential of Nox4 inhibition as a potential therapeutic target in the treatment of SSc and other fibroproliferative disorders.

ACR Meeting Abstracts - https://acrabstracts.org/abstract/tgf-%ce%b2-induced-tissue-fibrosis-is-abrogated-in-mice-containing-a-constitutive-genetic-deletion-of-nox4-nox4-knockout/