Di-Tyrosine Crosslinking and NOX4 Expression as Oxidative Pathological Markers in the Lungs of Patients with Idiopathic Pulmonary Fibrosis.

Sanja Blaskovic,Yves Donati,Isabelle Ruchonnet-Metrailler,Vincent Jaquet,Jean-Claude Pache,Tamara Seredenina,Karl-Heinz Krause,Constance Barazzone-Argiroffo,Dan Adler

doi:10.3390/antiox10111833

Sanja Blaskovic, Yves Donati + Show 7 more

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https://doi.org/10.3390/antiox10111833

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Abstract

Idiopathic pulmonary fibrosis (IPF) is a noninflammatory progressive lung disease. Oxidative damage is a hallmark of IPF, but the sources and consequences of oxidant generation in the lungs are unclear. In this study, we addressed the link between the H2O2-generating enzyme NADPH oxidase 4 (NOX4) and di-tyrosine (DT), an oxidative post-translational modification in IPF lungs. We performed immunohistochemical staining for DT and NOX4 in pulmonary tissue from patients with IPF and controls using validated antibodies. In the healthy lung, DT showed little or no staining and NOX4 was mostly present in normal vascular endothelium. On the other hand, both markers were detected in several cell types in the IPF patients, including vascular smooth muscle cells and epithelium (bronchial cells and epithelial cells type II). The link between NOX4 and DT was addressed in human fibroblasts deficient for NOX4 activity (mutation in the CYBA gene). Induction of NOX4 by Transforming growth factor beta 1 (TGFβ1) in fibroblasts led to moderate DT staining after the addition of a heme-containing peroxidase in control cells but not in the fibroblasts deficient for NOX4 activity. Our data indicate that DT is a histological marker of IPF and that NOX4 can generate a sufficient amount of H2O2 for DT formation in vitro.

Highlights

Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease leading to lung tissue stiffness and fibrosis, decreased lung function and eventually respiratory failure
The nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 (NOX4) antibody used in this study was a rabbit monoclonal antibody directed against the intracellular C terminus of human NOX4 produced in the laboratory of Professor
In order to address the specificity of the DT antibody and the biological significance of NOX4 in DT formation, we used an in vitro fibroblast experimental system inspired by the methodology described by Larios et al [25]

Summary

Introduction

Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease leading to lung tissue stiffness and fibrosis, decreased lung function and eventually respiratory failure. It is estimated that three million people are affected by IPF, worldwide. The median survival of IPF is estimated at 2–3 years after diagnosis [1,2]. IPF is characterized by the aberrant proliferation and activation of fibroblasts as well as the presence of myofibroblasts that secrete excessive collagen and extracellular matrix (ECM) leading to the scarring of lung tissue and destruction of the alveolar epithelium (reviewed in [3]). Lung remodeling is thought to occur following repetitive injuries leading to alveolar epithelial cells (AEC) apoptosis, proliferation of fibroblasts and Transforming growth factor beta 1 (TGFβ1)-induced myofibroblast differentiation as well as endothelial dysfunction. Knowledge of the underlying molecular mechanisms leading to IPF is scarce and the clinically approved

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