Targeting of NADPH oxidase in vitro and in vivo suppresses fibroblast activation and experimental skin fibrosis

Abstract

Although there is increasing evidence that oxidative stress is involved in collagen synthesis and myofibroblast activation, the NADPH oxidase (Nox) system is incompletely investigated in the context of human dermal fibroblasts (HDFs) and skin fibrosis. Using the pan-Nox inhibitor diphenyleneiodonium (DPI) as an initial tool, we show that gene expression of collagen type I, α-smooth muscle actin (α-SMA) and fibronectin 1 is suppressed in HDFs. Detailed expression analysis of all Nox isoforms and adaptors revealed expression of RNA and protein expression of Nox4, p22 and Poldip2 but neither Nox1 nor Nox2. Nox4 could be immunolocalized to the endoplasmic reticulum. Importantly, TGF-β had a dose- and time-dependent upregulating effect on NADH activity and Nox4 gene expression in HDFs. Genetic silencing of Nox4 as demonstrated by siRNA in HDFs as well as in murine fibroblasts established from Nox4 knockout mice confirmed that TGF-β -mediated collagen type I gene, α-SMA and fibronectin 1 gene expressions were Nox4-dependent. This TGF-β effect was mediated by Smad3 as shown by in silico promoter analysis, pharmacological inhibition and gene silencing of Smad3. The relevance of these findings is highlighted in the bleomycin-induced scleroderma mouse model. DPI treatment attenuated skin fibrosis and myofibroblast activation. Moreover, Nox4 knockdown by siRNA reduced skin collagen synthesis, α-SMA and fibronectin 1 expression in vivo. Finally, analyses of HDFs from patients with systemic sclerosis confirmed the expression of Nox4 and its adaptors, whereas Nox1 and Nox2 were not detectable. Our findings indicate that Nox4 targeting is a promising future treatment for fibrotic skin diseases.

Oxidative stress in cancer and fibrosis: Opportunity for therapeutic intervention with antioxidant compounds, enzymes, and nanoparticles

“…Currently, a few nonspecific NOX inhibitors have been identified (which target more than one NOX isoforms) [88], [142], [143], [144], [145], [146], [147]. Some issues with specificity, potency, and toxicity of these inhibitors have limited the use of these compounds in clinical studies [148].…”

“…It inhibits a number of flavoproteins including eNOS, xanthine oxidase, and cholinesterases and the internal calcium pump [150]. Although at least two pre-clinical studies using DPI have shown positive results on lung cancer inhibition [142] as well as skin fibrosis reduction [143], the off-target effects of DPI against other flavoproteins will prevent its translation into clinical use.…”

Polymerase δ-interacting Protein 2: A Multifunctional Protein

“…Poldip2 is fairly ubiquitously expressed, having been reported in aorta, 3, 4 lung, 3 kidney, 3, 5, 6 heart 7 and thyroid, 8 as well as many cell types including mouse aortic smooth muscle cells, 9 astrocytes, 10 vascular smooth muscle cells (VSMC), 3, 11 brain endothelial cells, 12 epithelial cells, 12 HeLa, 1, 12-15 fibroblasts, 2, 13, 16, 17 kidney fibroblasts, 18 myoblasts, 19 human umbilical vein endothelial cells, 20 human embryonic kidney cells 293 (HEK 293), 13 MRC5 cells, 21, 22 and cortical neurons. 23 There is also evidence of Poldip2 expression in human breast cancer, 24 human breast adenocarcinoma cells 13 and human lung adenocarcinoma cells.…”

Oxidative stress in the pathogenesis of systemic scleroderma: An overview

“…[10][11][12] In addition, targeting of ROS-generating NADPH oxidase in vitro and in vivo suppresses fibroblast activation and experimental skin fibrosis. 13 15,16 monocytes, 17 T lymphocytes 18 and erythrocytes. 19 Likewise, the total oxidant status of serum from SSc patients has been found to be higher with respect to control samples.…”