Reactive oxygen species (ROS) and lipid metabolism in idiopathic pulmonary fibrosis - role of peroxisomes in the pathogenesis of this devastating disease
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Idiopathic pulmonary fibrosis (IPF) is a chronic devastating disease, and its pathogenic mechanisms remain incompletely understood. In this disease, the lung undergoes dramatic pathological remodelling and myofibroblasts with high alpha-smooth muscle actin (alpha-SMA) content secrete huge amounts of extracellular matrix. However, altered peroxisome functions in IPF pathogenesis have never been investigated. Proinflammatory mediators and reactive oxygen species (ROS) accumulation were shown as pathogenetic mechanisms of this yet incurable disease. Since peroxisomes are involoved in both the degradation of proinflammatory lipid mediators (eicosanoids) as well as ROS metabolism, alterations in the protective capacity of this organelle might contribute to the pathogenesis of IPF. In addition, children with Zellweger Syndrome, the most severe peroxisomal biogenesis defect, develop chronic liver fibrosis and cirrhosis, suggesting that the peroxisomal metabolism is essential for the protection against fibrotic organ degeneration. In the experimental part of this thesis the hypothesis was tested, 1) whether the peroxisomal compartment and corresponding gene expression is altered in IPF, 2) whether the downregulation of peroxisomal biogenesis and metabolism in IPF promotes further excessive secretion of extracellular matrix proteins and proinflammatory mediators and 3) whether proinflammatory and profibrotic cytokines influence peroxisomal abundance and metabolism. Moreover, the molecular mechanisms leading to the alterations of the peroxisomal compartment were investigated. In addition, a bleomycin induced lung fibrosis mouse model was used to analyze peroxisomal biogenesis, antioxidative and lipid metabolic proteins at various time-points after bleomycin treatment. The molecular mechanisms and specific involvement of peroxisomal proteins in TGF-beta1 induced ECM production were investigated. Finally the effect of TGF-beta1 on the peroxisomal compartment in TGF-beta receptor II knockout (TbetaRII) and Smad3 knockout mice as well in transgenic constitutively active TbetaRICA overexpressing mice was studied. In this thesis, the peroxisomal compartment as well as peroxisomal metabolic proteins were analyzed in parallel to several cell type-specific markers in paraffin sections of different lung tissue samples of human controls in comparison to IPF patients. In addition, primary cultures of lung fibroblasts of the same individuals were used for morphological, biochemical and molecular biological analysis of peroxisomal protein alterations. Moreover, control and IPF fibroblast were challenged with TGF-beta1,TNF-alpha, IL-6 and PEX13 siRNA to analyze the impact of peroxisomes on the molecular pathogenesis of IPF. To confirm this in vitro findings the bleomycin induced lung fibrosis mouse model and TßRII mice were used to study peroxisome biogenesis and metabolism in situ in these animal models. By comparing peroxisome-related protein and gene expression in lung tissue and isolated lung fibroblasts between human control and IPF patients, we found that IPF lungs exhibited a significant down-regulation of peroxisomal biogenesis and metabolism (e.g. PEX13p, catalase, ABCD3 and acyl-CoA oxidase 1). Moreover, in vivo the bleomycin-induced downregulation of peroxisomes was abrogated in TßRII mice indicating a role for TGF-beta1signaling in the regulation of peroxisomes. Furthermore, in vitro treatment of IPF fibroblasts with the pro-fibrotic factors TGF-beta1 or TNF-alpha was found to downregulate peroxisomes via the AP-1 signaling pathway. Therefore, the molecular mechanisms by which reduced peroxisomal functions contribute to enhanced fibrosis were further studied. Direct down-regulation of PEX13 mRNA by RNAi 1) induced the activation of Smad-dependent TGF-beta signaling, accompanied by increased ROS production, 2) resulted in the release of cytokines (e.g. IL-6, TGF-beta) and excessive production of collagen I and III. In contrast, treatment of fibroblasts with ciprofibrate or WY14643, PPAR-alpha activators, induced peroxisome proliferation and reduced the TGF-beta-induced myofibroblast differentiation and collagen protein in IPF cells.Overall in this thesis it could be proven that the peroxisomal compartment is severely affected and compromised in IPF, mediated by TGF-beta1 signaling and the action of proinflammatory cytokines (TNF-alpha and IL-6) via AP-1 signal transduction. TGF-beta1 downregulates peroxisomes via the Smad-dependent pathway, which results in reduced ability of cells to scavenge ROS, degrade proinflammatory lipid mediators (eicosanoids) and subsequently inducing a vicious cycle, leading to the aggravation of IPF. Thus, TGF-beta1 and TNF-alpha may exacerbate the clinical conditions and intensify the fibrotic response in patients with IPF upon inflammation and lung injury by the downregulation of peroxisomal biogenesis and metabolism (e.g. PEX13, ACOX1).Verknüpfung zu Publikationen oder weiteren Datensätzen
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Giessen : VVB Laufersweiler Verlag