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Extracellular matrix (ECM) stiffening with downstream activation of mechanosensitive pathways is strongly implicated in fibrosis. We previously reported that altered collagen nanoarchitecture is a key determinant of pathogenetic ECM structure-function in human fibrosis (Jones et al., 2018). Here, through human tissue, bioinformatic and ex vivo studies we show that hypoxia-inducible factor (HIF) pathway activation is a critical pathway for this process regardless of oxygen status (pseudohypoxia). Whilst TGFβ increased rate of fibrillar collagen synthesis, HIF pathway activation was required to dysregulate post-translational modification of fibrillar collagen, promoting ‘bone-type’ cross-linking, altering collagen nanostructure, and increasing tissue stiffness. In vitro, knock down of Factor Inhibiting HIF (FIH) or oxidative stress caused pseudohypoxic HIF activation in normal fibroblasts. In contrast, endogenous FIH activity was reduced in fibroblasts from patients with lung fibrosis in association with significantly increased normoxic HIF pathway activation. In human lung fibrosis tissue, HIF mediated signalling was increased at sites of active fibrogenesis whilst subpopulations of IPF lung mesenchymal cells had increases in both HIF and oxidative stress scores. Our data demonstrate that oxidative stress can drive pseudohypoxic HIF pathway activation which is a critical regulator of pathogenetic collagen structure-function in fibrosis.

Idiopathic pulmonary fibrosis (IPF) is the prototypic progressive fibrotic lung disease with a median survival of 2 to 4 years. Injury to and/or dysfunction of the alveolar epithelium is strongly implicated in IPF disease initiation, but the factors that determine whether fibrosis progresses rather than normal tissue repair occurs remain poorly understood. We previously demonstrated that zinc finger E-box-binding homeobox 1-mediated epithelial-mesenchymal transition in human alveolar epithelial type II (ATII) cells augments transforming growth factor-β-induced profibrogenic responses in underlying lung fibroblasts via paracrine signaling. Here, we investigated bidirectional epithelial-mesenchymal crosstalk and its potential to drive fibrosis progression. RNA-Seq of lung fibroblasts exposed to conditioned media from ATII cells undergoing RAS-induced epithelial-mesenchymal transition identified many differentially expressed genes including those involved in cell migration and extracellular matrix regulation. We confirmed that paracrine signaling between RAS-activated ATII cells and fibroblasts augmented fibroblast recruitment and demonstrated that this involved a zinc finger E-box-binding homeobox 1-tissue plasminogen activator axis. In a reciprocal fashion, paracrine signaling from transforming growth factor-β-activated lung fibroblasts or IPF fibroblasts induced RAS activation in ATII cells, at least partially through the secreted protein acidic and rich in cysteine, which may signal via the epithelial growth factor receptor via epithelial growth factor-like repeats. Together, these data identify that aberrant bidirectional epithelial-mesenchymal crosstalk in IPF drives a chronic feedback loop that maintains a wound-healing phenotype and provides self-sustaining profibrotic signals.

Background: Altered collagen architecture with increased “bone-type” pyridinoline collagen cross-linking, rather than collagen quantity, is a key determinant of abnormal tissue structure-function in Idiopathic Pulmonary Fibrosis (IPF). We recently identified that hypoxia inducible factor (HIF) pathway activation promotes induction of the bone-type collagen cross-linking enzymes lysyl hydroxylase 2 and lysyl oxidase-like 2, yet the consequences of this on collagen structure-function in lung fibrosis remain unknown. Methods: Using a long term 3D in vitro model of the fibroblastic focus we cultured primary lung fibroblasts from IPF donors without or with the HIF-stabilising compound IOX2. Hydroxyproline and mature pyridinium cross-links were measured by colorimetric assays, and collagen ultrastructure assessed by electron microscopy (EM). The biomechanical effects of HIF stabilisation were investigated by parallel plate compression testing. Results: IOX2 stabilised HIF within the 3D fibroblastic focus model, promoting HIF pathway activation to disproportionately induce collagen-modifying enzymes relative to collagen fibril synthesis. After 6 weeks of culture, mature pyridinium cross-links were significantly increased by IOX2 to levels we have previously observed in IPF tissue. Ultrastructural analysis of the collagen fibrils with EM identified that IOX2 significantly reduced fibril diameter to sizes comparable with collagen fibrils enzymatically extracted from IPF tissue. IOX2 induced a greater than 3-fold increase in tissue stiffness. Conclusions: HIF pathway activation is a core regulator of bone-type collagen fibrillogenesis and altered structure-function in lung fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic scarring disease in which aging, environmental exposure(s) and genetic susceptibility have been implicated in disease pathogenesis, however, the causes and mechanisms of the progressive fibrotic cascade are still poorly understood. As epithelial–mesenchymal interactions are essential for normal wound healing, through human 2D and 3D in vitro studies, we tested the hypothesis that IPF fibroblasts (IPFFs) dysregulate alveolar epithelial homeostasis. Conditioned media from IPFFs exaggerated the wound-healing response of primary human Type II alveolar epithelial cells (AECs). Furthermore, AECs co-cultured with IPFFs exhibited irregular epithelialization compared with those co-cultured with control fibroblasts (NHLFs) or AECs alone, suggesting that epithelial homeostasis is dysregulated in IPF as a consequence of the abnormal secretory phenotype of IPFFs. Secretome analysis of IPFF conditioned media and functional studies identified the matricellular protein, SPARC, as a key mediator in the epithelial–mesenchymal paracrine signaling, with increased secretion of SPARC by IPFFs promoting persistent activation of alveolar epithelium via an integrin/focal adhesion/cellular-junction axis resulting in disruption of epithelial barrier integrity and increased macromolecular permeability. These findings suggest that in IPF fibroblast paracrine signaling promotes persistent alveolar epithelial activation, so preventing normal epithelial repair responses and restoration of tissue homeostasis. Furthermore, they identify SPARC-mediated paracrine signaling as a potential therapeutic target to promote the restoration of lung epithelial homoestasis in IPF patients.

Background: Altered collagen architecture, rather than collagen quantity, is a key determinant of abnormal tissue structure-function in idiopathic pulmonary fibrosis (IPF), with the collagen cross-linking enzymes lysyl hydroxylase 2 (LH2) and lysyl oxidase-like 2 (LOXL2) promoting pathological bone-type collagen crosslinking and increased tissue stiffness (Jones et al eLife. 2018;7:e36354). However, the upstream mechanisms regulating this process remain unknown. Objective: To determine the cellular provenance of LOXL2 and LH2 in IPF tissue and identify the mechanisms promoting their dysregulation. Methods: Cellular provenance of LH2 and LOXL2 was analysed using mRNA in-situ hybridisation. IPF lung fibroblasts were cultured under a range of conditions that have been associated with IPF pathogenesis and their effects on LH2 and LOXL2 induction assessed by RTqPCR and Western Blotting. Small-interfering RNAs (siRNA) were transfected into IPF fibroblasts to investigate signalling pathways of interest and their role in regulating LH2 and LOXL2 expression. Results: LH2 and LOXL2 were co-expressed within fibroblastic foci in IPF tissue. Hypoxia-inducible factor (HIF) pathways were the strongest inducers of both LOXL2 and LH2 in IPF fibroblasts. siRNA mediated knockdown of HIF-1α but not HIF-2α prevented LH2 induction, whilst knockdown of both HIF-1α and HIF-2α was required to prevent LOXL2 induction. Conclusions: Our results suggest that HIF pathway activation via LH2/LOXL2 may be a core regulator of bone-type collagen fibrillogenesis in IPF.

Introduction: The lung epithelium is a functional interface between our body and the outside environment and it plays a key role in the respiratory physiology and pathology. Recurring alveolar epithelial injury is a hallmark of idiopathic pulmonary fibrosis (IPF) that lead to aberrant fibroblast activation resulting in the progressive destruction of the lung parenchymal architecture and impairment of gas exchange. The complex interactions between the persistent injured epithelium and the abnormal activated fibroblasts seems to be the main factors determining initiation and progression of the disease in association with aging, environmental exposure and genetic susceptibility. A better understanding of the epithelial-mesenchymal signaling in IPF is needed for the development of more efficient therapeutic strategies that promote restoration of normal epithelium integrity and limit the progression of fibrosis. Aim: Characterize the paracrine signaling between alveolar cells (AECs) and lung fibroblasts (FFs) and investigate its role in injury/repair of the epithelium. Methods: we established and characterize an ex-vivo co-culture models of AECs and FFs to mimic the epithelial-mesenchymal paracrine interaction in injured alveoli. Results and Conclusions: we detected abnormal epithelialization in co-culture of AECs and IPF FFs compared to co-culture of AECs and healthy FFs. Mass spectrometry analysis of fibroblasts secretome identified different extracellular matrix (ECM) proteins that are involved in tissue repair and fibrosis. Gene silencing of the ECM protein osteonectin in IPF FFs restored the alveolar epithelium homeostasis. These results suggest that IPF FFs secrete factors that alter the normal epithelial repair responses preventing the restoration of tissue homeostasis.

Introduction: Idiopathic pulmonary fibrosis (IPF) is a progressive disease with a poor prognosis and limited therapeutic options. Increasing evidence suggests that alveolar epithelial damage and resulting abnormal epithelial-mesenchymal crosstalk may contribute to the aberrant wound healing response observed in the lungs of IPF patients. The epithelial-mesenchymal trophic unit (EMTU) describes the functional interactions between epithelial cells, mesenchymal cells and the ECM which are necessary to regulate lung development, repair of damaged tissue and inflammatory responses. Thus characterization of EMTU in IPF will help to understand the pathofisiology of the disease. Aim: Characterize the paracrine crosstalk between alveolar cells (AECs) and lung fibroblasts (FFs) and investigate its role in injury and repair of the epithelium. Methods: we enstablished an ex-vivo co-culture models of AECs and FFs to mimic the EMTU of the respiratory epithelium. Epithelial cell layer intergrity was assed by immunofluorescence. Oxidative stress and injury/repair response were analysed using qPCR, western blot and time-laspe microscopy. Results and Conclusions: we detected abnormal epithelialization in co-culture of AECs and IPF FFs compared to co-culture of AECs and healthy FFs while western blot and qPCR data show increase in markers of cell motility. These results suggest that IPF FFs alter the normal epithelial repair responses preventing the restoration of lung tissue homeostasis. Therefore characterization of the EMTU in IPF will help the development of more efficient therapeutic strategies to limit the progression of fibrosis and promote restoration of the normal lung epithelium in IPF patients.

Endometrial cancer is one of the most common gynaecological cancers in developed countries. Its incidence has increased 20% over the last decade and the death rate has increased >100% over the past two decades. Current models for prediction of prognosis and treatment response are suboptimal, and as such biomarkers to support clinical decision-making and contribute to individualised treatment are needed. In this study, we show that the E3-ubiquitin ligase PIR2/RNF144B is a potential targetable biomarker in endometrial cancer. At transcript level, it is expressed both in normal endometrium and tumour samples, but at protein level, it is expressed in tumours only. By using endometrial cancer cell lines, we demonstrated that PIR2/RNF144B is stabilised via phosphorylation downstream of GSK3β and this is necessary for the proliferation of endometrial cancer cells, in the absence of oestrogenic growth stimuli. Here, inactivation of GSK3β activity is associated with loss of PIR2/RNF144B protein and consequent inhibition of cell proliferation. Our results, therefore, substantiate PIR2/RNF144B as a novel candidate for targeted therapy in endometrial cancer.

The contribution of epithelial-mesenchymal transition (EMT) to human lung fibrogenesis is controversial. Here we provide evidence that ZEB1-mediated EMT in human alveolar epithelial type II (ATII) cells contributes to the development of lung fibrosis by paracrine signalling to underlying fibroblasts. Activation of EGFR-RAS-ERK signalling in ATII cells induced EMT via ZEB1. ATII cells had extremely low extracellular matrix gene expression even after induction of EMT, however conditioned media from ATII cells undergoing RAS-induced EMT augmented TGFβ-induced profibrogenic responses in lung fibroblasts. This epithelial-mesenchymal crosstalk was controlled by ZEB1 via the expression of tissue plasminogen activator (tPA). In human fibrotic lung tissue, nuclear ZEB1 expression was detected in alveolar epithelium adjacent to sites of extracellular matrix (ECM) deposition, suggesting that ZEB1-mediated paracrine signalling has the potential to contribute to early fibrotic changes in the lung interstitium. Targeting this novel ZEB1 regulatory axis may be a viable strategy for the treatment of pulmonary fibrosis.

Aims: To (i) investigate the effects of Romidepsin in vitro using fibroblasts and alveolar cells (ATII); (ii) in vivo in Bleomycin treated mice; and (iii) to identify biomarkers in order to monitor response to therapy in a future clinical trial. Background: Idiopathic pulmonary fibrosis (IPF) is a complex chronic fibroproliferative disease of unknown aetiology and with limited therapeutic options.Numerous 9single pathway9 agents have failed to show efficacy in IPF clinical trial. By targeting multiple genes and pathways, HDAC inhibitors offer novel therapeutic strategies that could be used alone or in combination with existing agents (Pirfenidone and Nintedanib). Methods: Fibroblast and ATII cell proliferation were determined by cell counting and MTS assay. Myofibroblast differentiation was analysed by western blotting (WB) for α -SMA and Lysyl Oxidase (LOX). The antifibrotic effects of Romidepsin on bleomycin treated mice were assessed by RTqPCR, histology and WB. Broncho-alveolar-lavage (BAL) fluid from IPF patients was analysed by WB. Results: Romidepsin caused a strong inhibition of IPF fibroblast proliferation, myofibroblast differentiation and secretion of LOX. Comparison of the effect of Romidepsin on ATII cells and IPF fibroblasts showed that the ATII cells were significantly less sensitive. Romidepsin reduced bleomycin-induced lung fibrosis in mice and suppressed the expression of LOX. We detected elevated levels of LOX in the BALF of IPF patients. Conclusions: Romidepsin shows strong anti-fibrotic effects without harmful consequences on ATII cells. It is therefore a potential novel anti-fibrotic therapy and LOX may be a potential biomarker of response.

Aims: Investigate the effect of the HADCI, FK228, on IPF fibroblasts differentiation and migration. Background: IPF is the most severe form of interstitial pneumonias with an unknown aetiology and without a successful therapy. Recent studies have demonstrated that HDAC inhibitors are good candidate for the treatment of fibrosis. These enzymes are specifically druggable, providing important therapeutic targets for IPF. Methods: The expression of FAK in IPF fibroblasts during FK228 treatment was assessed by western blot and real time PCR. The migration ability of IPF fibroblasts was assessed by scratch wound assay and transwell migration assay. Results: During FK228 treatment, FAK protein levels are down regulated in IPF fibroblasts resulting in the reduction of cell motility. Conclusions: FK228 is a good candidate for the treatment of IPF exhibiting a potent inhibition of fibroblast migration. By target FAK expression, FK228 could reduce fibroblasts dissemination resulting in a slower progression of lung fibrosis.

p73 is a tumor suppressor belonging to the p53 family of transcription factors. Distinct isoforms are transcribed from the p73 locus. The use of 2 promoters at the N-terminus allows the expression of an isoform containing (TAp73) or not containing (ΔNp73) a complete N-terminal transactivation domain, with the latter isoform capable of a dominant negative effect over the former. In addition, both N-terminal variants are alternatively spliced at the C-terminus. TAp73 is a bona fide tumor suppressor, being able to induce cell death and cell cycle arrest; conversely, ΔNp73 shows oncogenic properties, inhibiting TAp73 and p53 functions. Here, we discuss the latest findings linking p73 to cancer. The generation of isoform specific null mice has helped in dissecting the contribution of TA versus ΔNp73 isoforms to tumorigenesis. The activity of both isoforms is regulated transcriptionally and by posttranslational modification. p73 dysfunction, particularly of TAp73, has been associated with mitotic abnormalities, which may lead to polyploidy and aneuploidy and thus contribute to tumorigenesis. Although p73 is only rarely mutated in cancer, the tumor suppressor actions of TAp73 are inhibited by mutant p53, a finding that has important implications for cancer therapy. Finally, we discuss the expression and role of p73 isoforms in human cancer, with a particular emphasis on the neuroblastoma cancer model. Broadly, the data support the hypothesis that the ratio between TAp73 and ΔNp73 is crucial for tumor progression and therapeutic response.