An EZH2-dependent transcriptional complex promotes aberrant epithelial remodelling after injury.

Unveiling the molecular mechanisms of tissue remodelling following injury is imperative to elucidate its regenerative capacity and aberrant repair in disease. Using different omics approaches, we identified enhancer of zester homolog 2 (EZH2) as a key regulator of fibrosis in injured lung epithelium. Epithelial injury drives an enrichment of nuclear transforming growth factor-ß-activated kinase 1 (TAK1) that mediates EZH2 phosphorylation to facilitate its liberation from polycomb repressive complex 2 (PRC2). This process results in the establishment of a transcriptional complex of EZH2, RNA-polymerase II (POL2) and nuclear actin, which orchestrates aberrant epithelial repair programmes. The liberation of EZH2 from PRC2 is accompanied by an EZH2-EZH1 switch to preserve H3K27me3 deposition at non-target genes. Loss of epithelial TAK1, EZH2 or blocking nuclear actin influx attenuates the fibrotic cascade and restores respiratory homeostasis. Accordingly, EZH2 inhibition significantly improves outcomes in a pulmonary fibrosis mouse model. Our results reveal an important non-canonical function of EZH2, paving the way for new therapeutic interventions in fibrotic lung diseases.

Screening for Inhibitors of YAP Nuclear Localization Identifies Aurora Kinase A as a Modulator of Lung Fibrosis.

Idiopathic pulmonary fibrosis is a progressive lung disease with limited therapeutic options that is characterized by pathological fibroblast activation and aberrant lung remodeling with scar formation. YAP (Yes-associated protein) is a transcriptional coactivator that mediates mechanical and biochemical signals controlling fibroblast activation. We previously identified HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase inhibitors (statins) as YAP inhibitors based on a high-throughput small-molecule screen in primary human lung fibroblasts. Here we report that several Aurora kinase inhibitors were also identified from the top hits of this screen. MK-5108, a highly selective inhibitor for AURKA (Aurora kinase A), induced YAP phosphorylation and cytoplasmic retention and significantly reduced profibrotic gene expression in human lung fibroblasts. The inhibitory effect on YAP nuclear translocation and profibrotic gene expression is specific to inhibition of AURKA, but not Aurora kinase B or C, and is independent of the Hippo pathway kinases LATS1 and LATS2 (Large Tumor Suppressor 1 and 2). Further characterization of the effects of MK-5108 demonstrate that it inhibits YAP nuclear localization indirectly via effects on actin polymerization and TGFß (Transforming Growth Factor ß) signaling. In addition, MK-5108 treatment reduced lung collagen deposition in the bleomycin mouse model of pulmonary fibrosis. Our results reveal a novel role for AURKA in YAP-mediated profibrotic activity in fibroblasts and highlight the potential of small-molecule screens for YAP inhibitors for identification of novel agents with antifibrotic activity.

Fine-tuning of FOXO3A in cHL as a survival mechanism and a hallmark of abortive plasma cell differentiation.

We recently found that FOXO1 repression contributes to the oncogenic program of classical Hodgkin lymphoma (cHL). Interestingly, FOXO3A, another member of the FOXO family, was reported to be expressed in the malignant Hodgkin and Reed-Sternberg cells of cHL at higher levels than in non-Hodgkin lymphoma subtypes. We thus aimed to investigate mechanisms responsible for the maintenance of FOXO3A as well as the potential role of FOXO3A in cHL. Here, we show that high FOXO3A levels in cHL reflect a B-cell-differentiation-specific pattern. In B cells, FOXO3A expression increases during the process of centroblast to plasma cell (PC) differentiation. FOXO3A levels in cHL were found higher than in germinal center B cells, but lower than in terminally differentiated PCs. This intermediate FOXO3A expression in cHL might manifest the "abortive PC differentiation" phenotype. This assumption was further corroborated by the finding that overexpression of FOXO3A in cHL cell lines induced activation of the master PC transcription factor PRDM1α. As factors attenuating FOXO3A expression in cHL, we identified MIR155 and constitutive activation of extracellular signal-regulated kinase. Finally, we demonstrate the importance of FOXO3A expression in cHL using an RNA interference approach. We conclude that tightly regulated expression of FOXO3A contributes to the oncogenic program and to the specific phenotype of cHL.

Ureteroenteric strictures: a single center experience comparing Bricker versus Wallace ureteroileal anastomosis in patients after urinary diversion for bladder cancer.

BACKGROUND: To evaluate the outcome and complication rate in a single institution experience using the two most commonly used techniques of ureteroenteric anastomosis, the Bricker and Wallace anastomosis. METHODS: A total of 137 patients underwent ileal conduit for bladder cancer. Ureters were anastomosed by two experienced surgeons, one performing a Bricker and the other, a Wallace anastomosis. Stricture was identified during clinical follow-up. RESULTS: Seventy-five patients underwent a Bricker anastomotic, and 65 received a Wallace anastomosis. The average age was 70 in both groups, males were predominant (66% Bricker, 70% Wallace). Follow up period was 36.5 months in Bricker group and 17 months in Wallace group. In both groups, the body mass index (BMI) was similar (26.1 kg/m2 Bricker and 26.4 kg/m2 Wallace). We observed that the stricture rate after performing the Bricker anastomosis technique was 25.3% (19/75) as compared to 7.7% (5/65) after Wallace anastomosis technique, which was statistically significant (p = 0.001). In the Bricker group, patients with strictures had higher BMI (28.3 vs. 25.7 kg/m2, p = 0.05). On average it took 8.5 months in the Bricker group and three months in the Wallace group (p = 0.6) to develop stricture. CONCLUSIONS: The stricture rate was significantly higher when Bricker technique was applied. Although the BMI was not different in both groups, patients with a higher BMI were more likely to develop stricture. We believe that the approach of the separate and refluxing technique of Bricker anastomosis especially in obese patients poses a higher risk for anastomotic stricture formation.

Tyrosine kinase inhibitor BIBF1000 does not hamper right ventricular pressure adaptation in rats.

BIBF1000 is a small molecule tyrosine kinase inhibitor targeting vascular endothelial growth factor receptor (VEGFR), fibroblast growth factor receptor (FGFR), and platelet-derived growth factor receptor (PDGFR) and is a powerful inhibitor of fibrogenesis. BIBF1000 is very similar to BIBF1120 (nintedanib), a drug recently approved for the treatment of idiopathic pulmonary fibrosis (IPF). A safety concern pertaining to VEGFR, FGFR, and PDGFR inhibition is the possible interference with right ventricular (RV) responses to an increased afterload, which could adversely affect clinical outcome in patients with IPF who developed pulmonary hypertension. We tested the effect of BIBF1000 on the adaptation of the RV in rats subjected to mechanical pressure overload. BIBF1000 was administered for 35 days in pulmonary artery-banded (PAB) rats. RV adaptation was assessed by echocardiography, pressure volume loop analysis, histology, and determination of atrial natriuretic peptide (ANP) expression. BIBF1000 treatment resulted in growth attenuation but had no effects on RV function after PAB, given absence of changes in cardiac index, end-systolic elastance, connective tissue disposition, and capillary density. We conclude that, in this experimental model of increased afterload, combined VEGFR, FGFR, and PDGFR inhibition does not hamper RV adaptation to pressure overload.

Direct nkx2-5 transcriptional repression of isl1 controls cardiomyocyte subtype identity.

During cardiogenesis, most myocytes arise from cardiac progenitors expressing the transcription factors Isl1 and Nkx2-5. Here, we show that a direct repression of Isl1 by Nkx2-5 is necessary for proper development of the ventricular myocardial lineage. Overexpression of Nkx2-5 in mouse embryonic stem cells (ESCs) delayed specification of cardiac progenitors and inhibited expression of Isl1 and its downstream targets in Isl1(+) precursors. Embryos deficient for Nkx2-5 in the Isl1(+) lineage failed to downregulate Isl1 protein in cardiomyocytes of the heart tube. We demonstrated that Nkx2-5 directly binds to an Isl1 enhancer and represses Isl1 transcriptional activity. Furthermore, we showed that overexpression of Isl1 does not prevent cardiac differentiation of ESCs and in Xenopus laevis embryos. Instead, it leads to enhanced specification of cardiac progenitors, earlier cardiac differentiation, and increased cardiomyocyte number. Functional and molecular characterization of Isl1-overexpressing cardiomyocytes revealed higher beating frequencies in both ESC-derived contracting areas and Xenopus Isl1-gain-of-function hearts, which associated with upregulation of nodal-specific genes and downregulation of transcripts of working myocardium. Immunocytochemistry of cardiomyocyte lineage-specific markers demonstrated a reduction of ventricular cells and an increase of cells expressing the pacemaker channel Hcn4. Finally, optical action potential imaging of single cardiomyocytes combined with pharmacological approaches proved that Isl1 overexpression in ESCs resulted in normally electrophysiologically functional cells, highly enriched in the nodal subtype at the expense of the ventricular lineage. Our findings provide an Isl1/Nkx2-5-mediated mechanism that coordinately regulates the specification of cardiac progenitors toward the different myocardial lineages and ensures proper acquisition of myocyte subtype identity.

PDE4B inhibition by nerandomilast: Effects on lung fibrosis and transcriptome in fibrotic rats and on biomarkers in human lung epithelial cells.

BACKGROUND AND PURPOSE: The PDE4 family is considered a prime target for therapeutic intervention in several fibro-inflammatory diseases. We have investigated the molecular mechanisms of nerandomilast (BI 1015550), a preferential PDE4B inhibitor. EXPERIMENTAL APPROACH: In addition to clinically relevant parameters of idiopathic pulmonary fibrosis (IPF; lung function measurement/high-resolution computed tomography scan/AI-Ashcroft score), whole-lung homogenates from a therapeutic male Wistar rat model of pulmonary fibrosis were analysed by next-generation sequencing (NGS). Data were matched with public domain data derived from human IPF samples to investigate how well the rat model reflected human IPF. We scored the top counter-regulated genes following treatment with nerandomilast in human single cells and validated disease markers discovered in the rat model using a human disease-relevant in vitro assay of IPF. KEY RESULTS: Nerandomilast improved the decline of lung function parameters in bleomycin-treated animals. In the NGS study, most transcripts deregulated by bleomycin treatment were normalised by nerandomilast treatment. Most notably, a significant number of deregulated transcripts that were identified in human IPF disease were also found in the animal model and reversed by nerandomilast. Mapping to single-cell data revealed the strongest effects on mesenchymal, epithelial and endothelial cell populations. In a primary human epithelial cell culture system, several disease-related (bio)markers were inhibited by nerandomilast in a concentration-dependent manner. CONCLUSIONS AND IMPLICATIONS: This study further supports the available knowledge about the anti-inflammatory/antifibrotic mechanisms of nerandomilast and provides novel insights into the mode of action and signalling pathways influenced by nerandomilast treatment of lung fibrosis.

Corrigendum: BI 1015550 is a PDE4B inhibitor and a clinical drug candidate for the oral treatment of idiopathic pulmonary fibrosis.

[This corrects the article DOI: 10.3389/fphar.2022.838449.].

Tbx5 overexpression favors a first heart field lineage in murine embryonic stem cells and in Xenopus laevis embryos.

The T-box transcription factor Tbx5 is involved in several developmental processes including cardiogenesis. Early steps of cardiac development are characterised by the formation of two cardiogenic lineages, the first (FHF) and the second heart field (SHF) lineage, which arise from a common cardiac progenitor cell population. To further investigate the function of Tbx5 during cardiogenesis, we generated a murine embryonic stem cell line constitutively overexpressing Tbx5. Differentiation of these cells is characterised by an earlier and increased appearance of contracting cardiomyocytes that beat with a higher frequency than control cells. In semi-quantitative and quantitative RT-PCR analyses, we observed an up-regulation of cardiac marker genes such as Troponin T, endogenous Tbx5, and Nkx2.5 and a down-regulation of others like BMP4 and Hand2. Similar data were gained in Xenopus laevis arguing for a conserved function of Tbx5. Furthermore, markers of the conduction system and atrial cardiomyocytes were increased.

BI 1015550 is a PDE4B Inhibitor and a Clinical Drug Candidate for the Oral Treatment of Idiopathic Pulmonary Fibrosis.

The anti-inflammatory and immunomodulatory abilities of oral selective phosphodiesterase 4 (PDE4) inhibitors enabled the approval of roflumilast and apremilast for use in chronic obstructive pulmonary disease and psoriasis/psoriatic arthritis, respectively. However, the antifibrotic potential of PDE4 inhibitors has not yet been explored clinically. BI 1015550 is a novel PDE4 inhibitor showing a preferential enzymatic inhibition of PDE4B. In vitro, BI 1015550 inhibits lipopolysaccharide (LPS)-induced tumor necrosis factor-α (TNF-α) and phytohemagglutinin-induced interleukin-2 synthesis in human peripheral blood mononuclear cells, as well as LPS-induced TNF-α synthesis in human and rat whole blood. In vivo, oral BI 1015550 shows potent anti-inflammatory activity in mice by inhibiting LPS-induced TNF-α synthesis ex vivo and in Suncus murinus by inhibiting neutrophil influx into bronchoalveolar lavage fluid stimulated by nebulized LPS. In Suncus murinus, PDE4 inhibitors induce emesis, a well-known gastrointestinal side effect limiting the use of PDE4 inhibitors in humans, and the therapeutic ratio of BI 1015550 appeared to be substantially improved compared with roflumilast. Oral BI 1015550 was also tested in two well-known mouse models of lung fibrosis (induced by either bleomycin or silica) under therapeutic conditions, and appeared to be effective by modulating various model-specific parameters. To better understand the antifibrotic potential of BI 1015550 in vivo, its direct effect on human fibroblasts from patients with idiopathic pulmonary fibrosis (IPF) was investigated in vitro. BI 1015550 inhibited transforming growth factor-ß-stimulated myofibroblast transformation and the mRNA expression of various extracellular matrix proteins, as well as basic fibroblast growth factor plus interleukin-1ß-induced cell proliferation. Nintedanib overall was unremarkable in these assays, but interestingly, the inhibition of proliferation was synergistic when it was combined with BI 1015550, leading to a roughly 10-fold shift of the concentration-response curve to the left. In summary, the unique preferential inhibition of PDE4B by BI 1015550 and its anticipated improved tolerability in humans, plus its anti-inflammatory and antifibrotic potential, suggest BI 1015550 to be a promising oral clinical candidate for the treatment of IPF and other fibro-proliferative diseases.

Nintedanib improves cardiac fibrosis but leaves pulmonary vascular remodelling unaltered in experimental pulmonary hypertension.

Aims: Pulmonary arterial hypertension (PAH) is associated with increased levels of circulating growth factors and corresponding receptors such as platelet derived growth factor, fibroblast growth factor and vascular endothelial growth factor. Nintedanib, a tyrosine kinase inhibitor targeting primarily these receptors, is approved for the treatment of patients with idiopathic pulmonary fibrosis. Our objective was to examine the effect of nintedanib on proliferation of human pulmonary microvascular endothelial cells (MVEC) and assess its effects in rats with advanced experimental pulmonary hypertension (PH). Methods and results: Proliferation was assessed in control and PAH MVEC exposed to nintedanib. PH was induced in rats by subcutaneous injection of Sugen (SU5416) and subsequent exposure to 10% hypoxia for 4 weeks (SuHx model). Four weeks after re-exposure to normoxia, nintedanib was administered once daily for 3 weeks. Effects of the treatment were assessed with echocardiography, right heart catheterization, and histological analysis of the heart and lungs. Changes in extracellular matrix production was assessed in human cardiac fibroblasts stimulated with nintedanib. Decreased proliferation with nintedanib was observed in control MVEC, but not in PAH patient derived MVEC. Nintedanib treatment did not affect right ventricular (RV) systolic pressure or total pulmonary resistance index in SuHx rats and had no effects on pulmonary vascular remodelling. However, despite unaltered pressure overload, the right ventricle showed less dilatation and decreased fibrosis, hypertrophy, and collagen type III with nintedanib treatment. This could be explained by less fibronectin production by cardiac fibroblasts exposed to nintedanib. Conclusion: Nintedanib inhibits proliferation of pulmonary MVECs from controls, but not from PAH patients. While in rats with experimental PH nintedanib has no effects on the pulmonary vascular pathology, it has favourable effects on RV remodelling.

Model-based identification of TNFα-induced IKKß-mediated and IκBα-mediated regulation of NFκB signal transduction as a tool to quantify the impact of drug-induced liver injury compounds.

Drug-induced liver injury (DILI) has become a major problem for patients and for clinicians, academics and the pharmaceutical industry. To date, existing hepatotoxicity test systems are only poorly predictive and the underlying mechanisms are still unclear. One of the factors known to amplify hepatotoxicity is the tumor necrosis factor alpha (TNFα), especially due to its synergy with commonly used drugs such as diclofenac. However, the exact mechanism of how diclofenac in combination with TNFα induces liver injury remains elusive. Here, we combined time-resolved immunoblotting and live-cell imaging data of HepG2 cells and primary human hepatocytes (PHH) with dynamic pathway modeling using ordinary differential equations (ODEs) to describe the complex structure of TNFα-induced NFκB signal transduction and integrated the perturbations of the pathway caused by diclofenac. The resulting mathematical model was used to systematically identify parameters affected by diclofenac. These analyses showed that more than one regulatory module of TNFα-induced NFκB signal transduction is affected by diclofenac, suggesting that hepatotoxicity is the integrated consequence of multiple changes in hepatocytes and that multiple factors define toxicity thresholds. Applying our mathematical modeling approach to other DILI-causing compounds representing different putative DILI mechanism classes enabled us to quantify their impact on pathway activation, highlighting the potential of the dynamic pathway model as a quantitative tool for the analysis of DILI compounds.

Olodaterol shows anti-fibrotic efficacy in in vitro and in vivo models of pulmonary fibrosis.

BACKGROUND AND PURPOSE: Idiopathic pulmonary fibrosis (IPF) is a fatal respiratory disease characterized by excessive fibroblast activation ultimately leading to scarring of the lungs. Although, the activation of ß2 -adrenoceptors (ß2 -AR) has been shown to inhibit pro-fibrotic events primarily in cell lines, the role of ß2 -adrenoceptor agonists has not yet been fully characterized. The aim of our study was to explore the anti-fibrotic activity of the long-acting ß2 -adrenoceptor agonist olodaterol in primary human lung fibroblasts (HLF) and in murine models of pulmonary fibrosis. EXPERIMENTAL APPROACH: We assessed the activity of olodaterol to inhibit various pro-fibrotic mechanisms, induced by different pro-fibrotic mediators, in primary HLF from control donors and patients with IPF (IPF-LF). The in vivo anti-fibrotic activity of olodaterol, given once daily by inhalation in either a preventive or therapeutic treatment regimen, was explored in murine models of lung fibrosis induced by either bleomycin or the overexpression of TGF-ß1. KEY RESULTS: In both HLF and IPF-LF, olodaterol attenuated TGF-ß-induced expression of α-smooth muscle actin, fibronectin and endothelin-1 (ET-1), FGF- and PDGF-induced motility and proliferation and TGF-ß/ET-1-induced contraction. In vivo olodaterol significantly attenuated the bleomycin-induced increase in lung weight, reduced bronchoalveolar lavage cell counts and inhibited release of pro-fibrotic mediators (TGF-ß, MMP-9 and tissue inhibitor of metalloproteinase-1). Forced vital capacity was increased only with the preventive treatment regimen. In the TGF-ß-overexpressing model, olodaterol additionally reduced the Col3A1 mRNA expression. CONCLUSION AND IMPLICATIONS: Olodaterol showed anti-fibrotic properties in primary HLF from control and IPF patients and in murine models of lung fibrosis.

A prospective study on radiation-induced changes in hearing function.

PURPOSE: To quantitate changes in hearing function after radiotherapy for head-and-neck tumors. METHODS AND MATERIALS: At the Department of Radiotherapy and Radiation Oncology, 32 patients were irradiated for head-and-neck tumors. Three-dimensional treatment planning was applied. Total tumor doses were 30.0-77.6 Gy, local doses to the inner ear (n = 64) ranged from 1.7 to 64.3 Gy. Audiometry was performed before the onset of radiotherapy (RT), at a tumor dose of 40 Gy or at the end of palliative treatment, at the end of curative RT, and 2-6 months post-RT. Assays applied were frequency-specific threshold measurements for air and bone conduction, measurements according to Weber and Rinne, tympanometry and assessment of the stapedius reflex. RESULTS: Age and prior disease significantly decreased, whereas previous or concurrent alcohol consumption significantly increased hearing ability. A significant reduction in hearing ability during RT was found for high frequencies (at 40 Gy) and low frequencies (at end of RT), which persisted after RT. No differences were observed for air or bone conduction. None of the other assays displayed time- or dose-dependent changes. Dose-effect analyses revealed an ED50 (dose at which a 50% incidence is expected) for significant changes in hearing thresholds (15 dB) in the range of 20-25 Gy, with large confidence limits. CONCLUSIONS: Radiation effects on hearing ability were confined to threshold audiogram values, which started during the treatment without reversibility during 6 months postradiotherapy.

A boolean model of the cardiac gene regulatory network determining first and second heart field identity.

Two types of distinct cardiac progenitor cell populations can be identified during early heart development: the first heart field (FHF) and second heart field (SHF) lineage that later form the mature heart. They can be characterized by differential expression of transcription and signaling factors. These regulatory factors influence each other forming a gene regulatory network. Here, we present a core gene regulatory network for early cardiac development based on published temporal and spatial expression data of genes and their interactions. This gene regulatory network was implemented in a Boolean computational model. Simulations reveal stable states within the network model, which correspond to the regulatory states of the FHF and the SHF lineages. Furthermore, we are able to reproduce the expected temporal expression patterns of early cardiac factors mimicking developmental progression. Additionally, simulations of knock-down experiments within our model resemble published phenotypes of mutant mice. Consequently, this gene regulatory network retraces the early steps and requirements of cardiogenic mesoderm determination in a way appropriate to enhance the understanding of heart development.