Ejemplo de aplicaci´ on: sistema mec´ anico con fricci´ on de

The activation of embryological pathways are essential for the tissue repair process. There is increasing evidence that persistent irritation to the lung leads to dysregulation of some

embryological pathways, including Wnt/Wingless¹³⁷, phosphatase and tensin homologue (PTEN)¹³⁸, Sonic hedgehog (Shh)¹³⁹ and bone morphogenetic proteins (BMPs)¹⁴⁰, which may play a role in the abnormal behaviour of AECs and perhaps fibroblasts in IPF¹⁴¹. The dysregulation of the NOX4 redox signalling pathway has also been reported to participate in the pathogenesis of IPF¹⁴².

1.1.4.4.1 Wnt/Wingless pathway

The Wnt ligands comprise a large family of proteins that are essential to the morphogenetic processes. Results from several studies have indicated an overexpression of members of the Wnt/wingless pathway in the lungs of IPF patients103,143–145. In addition, there is an accumulation of Figure 1.4 Aberrantly activated AEC2s may facilitate fibroblast recruitment and activation in IPF.AEC2s may recruit and activate local fibroblasts to generate α-SMA+ myofibroblasts by secretion of PDGF. AEC2s may also recruit fibrocytes via CXCR4 from the bloodstream by their secretion of CXCL12. Fibrocytes express both hematopoietic (CD45, CD34) and mesenchymal (collagen 1, fibronectin) markers. Furthermore, AECs can undergo epithelial to mesenchymal transition (EMT) to give rise to fibroblasts. Fibroblasts arising from EMT express both epithelial (pro-surfactants) and also mesenchymal (α-SMA, N-cadherin) proteins.

β-catenin, a signal transducer of the Wnt signalling pathway, in AECs and fibroblasts, suggesting that the Wnt–β-catenin pathway is switched on in both cell types¹⁴⁶.

1

1.1.4.4.2 PTEN

PTEN is crucial for development. In adults, PTEN participates in the regulation of physiological processes such as cell polarity, proliferation and apoptosis¹⁴⁷. In IPF patients, myofibroblasts within the fibrotic foci downregulate PTEN expression, which may protect them from apoptosis¹⁴⁸. The interaction between β1-intergrin, a transmembrane receptor required for cell-cell or cell-ECM interactions, and collagen in normal fibroblasts activates PTEN, which is also a negative growth regulator; this negative feedback is however defective in fibroblasts in IPF patients¹⁴⁹.

1.1.4.4.3 Shh

Shh is an essential morphogen for patterning during embryogenesis. This developmental ligand allows the evasion of apoptosis and cell cycle arrest and promotes proliferation in cells. In the lungs of IPF patients an elevated level of Shh expression was observed, particularly in epithelial cells lining the honeycomb cysts^150,151. Shh overexpression may contribute to AEC2 hyperplasia observed in the lungs of IPF patients.

1.1.4.4.4 BMPs

BMPs belong to the TGF-β superfamily and have an essential role in embryonic and postnatal development¹⁵². In adults, reactivating the expression of BMP antagonists can contribute to the progression of some chronic degenerative diseases, such as IPF¹⁵³, liver¹⁵⁴ and renal¹⁵⁵ fibrosis and cervical cancer^156,157. BMP-4 is one of the key morphogens during embryonic lung development¹⁵⁸. BMP-4 inhibits the proliferation of human pulmonary fibroblasts and during embryogenesis induces the proliferation and differentiation of pulmonary epithelial cells¹⁵⁹. Gremlin is a strong BMP antagonist that can associate with BMP-2, -4 and -7 and inhibit their binding to cell surface

receptors¹⁶⁰. Increased expression of gremlin-1 has been reported in fibroblasts in lungs affected by IPF⁴². Increased concentrations of gremlin-1 could attenuate the mothers against decapentaplegic homolog (Smad)1/5/8 phosphorylation mediated by BMP-4 signalling in the lungs, leading to increased TGF-β1 induced EMT and reduced myofibroblast apoptosis^161–163.

In conclusion, some of embryological pathways are activated during normal tissue repair, but this process must be tissue specific and temporarily modulated. On the other hand, sustained activation of these programmes as a result of dysregulated wound healing may contribute to the pathology of IPF.

Chapter 1 | General Introduction

44 1

1.1.4.4.5 NOX4

NOX4 is a member of the NADPH oxidases, a group of transmembrane multiprotein complexes found in the plasma membrane and phagosomes, which generates superoxide anions by transferring electrons from NADPH across the membrane to molecular oxygen¹⁶⁴. Accumulating evidence highlights the involvement of NOX-dependent redox signalling, in particular NOX4^165–171, in the profibrotic responses mediated by TGF-β signalling¹⁶⁵. TGF-β1 induced the increased gene expression of NOX4, but not other NOX family members, in human fetal lung mesenchymal cells (hFLMCs).

Hydrogen peroxide (H2O2) generated by NOX4 induction is found to promote TGF-β1 induced differentiation of hFLMC into α-SMA+ myofibroblasts. Knockdown of NOX4 expression by small interfering RNA (siRNA) inhibited TGF-β1-induced H2O2 production and hFLMC differentiation into myofibroblasts. Removal of H2O2 by exogenous catalase activity also mimicked such TGF-β1 inhibitory activity¹⁶⁸. H2O2 may promote the conversion of latent TGF-β into its active form,

enhancing the phosphorylation and activation of the TGF-β receptor (TGF-βR) TGF-βR1/ ALK5 and/or the downstream signalling molecule Smad2/3, and increasing the activation of the transcription factors c-Jun N-terminal kinase (Jnk) and p38¹⁷².

NOX4 has been shown to play a role in pulmonary fibrosis. There is evidence showing increased NOX4 mRNA and protein levels in pulmonary fibroblasts isolated from IPF patients, and the NOX4 expression level correlates with expressions of procollagen I and α-SMA¹⁴². NOX4 was observed in immunohistochemistry staining to be highly expressed in the fibrotic foci of IPF patients¹⁶⁸. NOX4 was also found to mediate TGF-β1 signalling in human mesenchymal cells isolated from IPF patients that lead to fibronectin and α-SMA gene and protein expression, collagen secretion, and

proliferation. These processes were inhibited by NOX4 siRNA¹⁶⁸. In BLM-induced pulmonary fibrosis in mice, NOX4 was induced between days 7 to 21 post-challenge, and was found to accumulate around remodelled alveolar structure at day 14¹⁶⁸. Knockdown of NOX4 by siRNA at day 0 post BLM challenge reduced fibrosis that is observed by reduced staining for collagen, α-SMA and decreased acid soluble collagen in whole lung homogenate at days 14 and 21¹⁶⁸. Therapeutic treatment of BLM challenged mice at day 8 with diphenyleneiodonium (DPI), a NOX/flavoenzyme inhibitor, protected mice from pulmonary fibrosis at day 21¹⁶⁸. NOX4 knockout mice were also protected from BLM-induced pulmonary fibrosis, which was attributable to reduced TGF-β BLM-induced epithelial cell apoptosis¹⁷³. Oral treatment with a NOX4 inhibitor attenuated BLM-induced pulmonary fibrosis in rats and TGF-β-induced induction of procollagen and α-SMA expression in human pulmonary fibroblasts¹⁷⁴. Knockdown of NOX4 by siRNA similarly attenuated fluorescein isothiocyanate (FITC)-induced pulmonary fibrosis in mice¹⁶⁸.