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Background Aims: Ex vivo production of red blood cells (RBCs) represents a promising alternative for transfusion medicine. Several strategies have been described to generate erythroid cell lines from different sources, including embryonic, induced pluripotent, and hematopoietic stem cells. All these approaches have in common that they require elaborate differentiation cultures whereas the yield of enucleated RBCs is inefficient., Methods: We generated a human immortalized adult erythroid progenitor cell line derived from bone marrow CD71-positive erythroid progenitor cells (immortalized bone marrow erythroid progenitor adult, or imBMEP-A) by an inducible expression system, to shorten differentiation culture necessary for terminal erythroid differentiation. It is the first erythroid cell line that is generated from direct reticulocyte progenitors and demonstrates robust hemoglobin production in the immortalized state., Results: Morphologic analysis of the immortalized cells showed that the preferred cell type of the imBMEP-A line corresponds to hemoglobin-producing basophilic erythroblasts. In addition, we were able to generate a stable cell line from a single cell clone with the triple knockout of RhAG, RhDCE and KELL. After removal of doxycycline, part of the cells differentiated into normoblasts and reticulocytes within 5-7 days., Conclusions: Our results demonstrate that the imBMEP-A cell line can serve as a stable and straightforward modifiable platform for RBC engineering in the future., Competing Interests: Declaration of Competing Interest The authors have no commercial, proprietary or financial interest in the products or companies described in this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Radiation-induced morphea (RIM) is a rare complication of radiotherapy presenting as inflammatory fibrosis, most commonly reported in breast cancer patients. As underlying disease mechanisms are not well understood, targeted therapies are lacking. Since fibroblasts are the key mediators of all fibroproliferative diseases, this study aimed to characterize patient-derived fibroblasts to identify therapeutic targets. We studied primary human control and RIM-fibroblasts on a functional and molecular basis, analyzed peripheral blood and tissue samples and conducted, based on our findings, a treatment attempt in one patient. In RIM, we identified a distinct myofibroblast phenotype reflected by increased alpha-smooth-muscle-actin (αSMA) expression, reduced proliferation and migration rates, and overexpression of osteopontin (OPN). Our RNA sequencing identified aberrant Myc activation as a potential disease driver in RIM fibroblasts, similar to previous findings in systemic sclerosis. Treatment with the anti-inflammatory drug mesalazine reversed the myofibroblast phenotype by targeting Myc. Based on these findings, a patient with RIM was successfully treated with mesalazine, resulting in reduced inflammation and pain and tissue softening, while serum OPN was halved. The present study provides a comprehensive characterization of RIM fibroblasts, suggests a disease-driving role for Myc, demonstrates promising antifibrotic effects of mesalazine and proposes OPN as a biomarker for RIM., (© 2024. The Author(s).)

Background: MicroRNAs (miRNAs) are small, endogenous non-coding RNA molecules that inhibit gene expression through either destabilization of the target mRNA or translational repression. MiRNAs recognize target sites, most commonly found in the 3'-untranslated regions of cognate mRNAs. This review aims to provide a state-of-the-art overview of the role of miRNAs in the regulation of major blood group antigens such as ABH as well as cancer-specific glycans., Summary: Besides their known roles in the control of developmental processes, proliferation, apoptosis, and carcinogenesis, miRNAs have recently been identified to play a regulatory role during erythropoiesis and blood group antigen expression. Since only little is known about the function of the red cell membrane proteins carrying blood group antigens, it is of great interest to shed light on the regulatory mechanisms of blood group gene expression. Some carrier proteins of blood group antigens are not restricted to red blood cells and are widely expressed in other bodily fluids and tissues and quite a few play a crucial role in tumor cells, as either tumor suppressors or promoters., Key Message: All available data point at a tremendous physiological as well as pathophysiological relevance of miRNAs in context of blood group regulation. Furthermore, miRNAs are involved in the regulation of pleiotropic genetic pathways such as hematopoiesis and tumorigenesis and thus have to be studied in future research on this subject., Competing Interests: The authors have no conflicts of interest to declare., (© 2024 The Author(s). Published by S. Karger AG, Basel.)

In atrial fibrillation (AF), multifactorial pathologic atrial alterations are manifested by structural and electrophysiological changes known as atrial remodeling. AF frequently develops in the context of underlying cardiac abnormalities. A critical mechanistic role played by atrial stretch is played by abnormal substrates in a number of conditions that predispose to AF, including obesity, heart failure, hypertension, and sleep apnea. The significant role of overweight and obesity in the development of AF is known; however, the differential effect of overweight, obesity, cardiovascular comorbidities, lifestyle, and other modifiable risk factors on the occurrence and recurrence of AF remains to be determined. Reverse remodeling of the atrial substrate and subsequent reduction in the AF burden by conversion into a typical sinus rhythm has been associated with weight loss through lifestyle changes or surgery. This makes it an essential pillar in the management of AF in obese patients. According to recently published research, microRNAs (miRs) may function as post-transcriptional regulators of genes involved in atrial remodeling, potentially contributing to the pathophysiology of AF. The focus of this review is on their modulation by both weight loss and catheter ablation interventions to counteract atrial remodeling in AF. Our analysis outlines the experimental and clinical evidence supporting the synergistic effects of weight loss and catheter ablation (CA) in reversing atrial electrical and structural remodeling in AF onset and in recurrent post-ablation AF by attenuating pro-thrombotic, pro-inflammatory, pro-fibrotic, arrhythmogenic, and male-sex-associated hypertrophic remodeling pathways. Furthermore, we discuss the promising role of miRs with prognostic potential as predictive biomarkers in guiding approaches to AF recurrence prevention.

COVID-19 disease, caused by the severe acute respiratory syndrome virus 2 (SARS-CoV-2), induces a broad spectrum of clinical symptoms ranging from asymptomatic cases to fatal outcomes. About 10-35% of all COVID-19 patients, even those with mild COVID-19 symptoms, continue to show symptoms, i. e., fatigue, shortness of breath, cough, and cognitive dysfunction, after initial recovery. Previously, we and others identified red blood cell precursors as a direct target of SARS-CoV-2 and suggested that SARS-CoV-2 induces dysregulation in hemoglobin- and iron-metabolism contributing to the severe systemic course of COVID-19. Here, we put particular emphasis on differences in parameters of clinical blood gas analysis and hematological parameters of more than 20 healthy and Long-COVID patients, respectively. Long-COVID patients showed impaired oxygen binding to hemoglobin with concomitant increase in carbon monoxide binding. Hand in hand with decreased plasma iron concentration and transferrin saturation, mean corpuscular hemoglobin was elevated in Long-COVID patients compared to healthy donors suggesting a potential compensatory mechanism. Although blood pH was within the physiological range in both groups, base excess- and bicarbonate values were significantly lower in Long-COVID patients. Furthermore, Long-COVID patients displayed reduced lymphocyte levels. The clinical relevance of these findings, e. g., as a cause of chronic immunodeficiency, remains to be investigated in future studies. In conclusion, our data suggest impaired erythrocyte functionality in Long-COVID patients, leading to diminished oxygen supply. This in turn could be an explanation for the CFS, dyspnea and anemia. Further investigations are necessary to identify the underlying pathomechanisms., Competing Interests: The authors declare that they have no conflict of interest., (Thieme. All rights reserved.)

Background: Radiation-induced morphea is a fibro-inflammatory remodelling process of the subcutaneous connective tissue caused by ionising radiation, most commonly in the context of breast cancer treatment. The underlying pathomechanisms and putative risk factors are unknown. Therefore, misdiagnosis and inappropriate treatment pose a significant problem in the care of those patients., Objectives: The aim of the study was to provide an overview as well as guidance for the diagnosis and treatment of radiation-induced morphea based on current case reports and review articles., Results and Conclusions: Radiation-induced morphea is a rare condition that represents an interdisciplinary challenge for (gynaecological) oncology, radiotherapy and dermatology. Frequent misdiagnoses include infection (erysipelas), cancer recurrence or radiation dermatitis. Early histological diagnosis and the initiation of anti-inflammatory therapy using topical glucocorticoids or calcineurin inhibitors in combination with phototherapy and/or methotrexate are the most relevant success factors for an adequate clinical response., (© 2024. The Author(s), under exclusive licence to Springer Medizin Verlag GmbH, ein Teil von Springer Nature.)

Background: The antitumor activity of natural killer (NK) cells can be enhanced by specific targeting with therapeutic antibodies that trigger antibody-dependent cell-mediated cytotoxicity (ADCC) or by genetic engineering to express chimeric antigen receptors (CARs). Despite antibody or CAR targeting, some tumors remain resistant towards NK cell attack. While the importance of ICAM-1/LFA-1 interaction for natural cytotoxicity of NK cells is known, its impact on ADCC induced by the ErbB2 (HER2)-specific antibody trastuzumab and ErbB2-CAR-mediated NK cell cytotoxicity against breast cancer cells has not been investigated., Methods: Here we used NK-92 cells expressing high-affinity Fc receptor FcγRIIIa in combination with trastuzumab or ErbB2-CAR engineered NK-92 cells (NK-92/5.28.z) as well as primary human NK cells combined with trastuzumab or modified with the ErbB2-CAR and tested cytotoxicity against cancer cells varying in ICAM-1 expression or alternatively blocked LFA-1 on NK cells. Furthermore, we specifically stimulated Fc receptor, CAR and/or LFA-1 to study their crosstalk at the immunological synapse and their contribution to degranulation and intracellular signaling in antibody-targeted or CAR-targeted NK cells., Results: Blockade of LFA-1 or absence of ICAM-1 significantly reduced cell killing and cytokine release during trastuzumab-mediated ADCC against ErbB2-positive breast cancer cells, but not so in CAR-targeted NK cells. Pretreatment with 5-aza-2'-deoxycytidine induced ICAM-1 upregulation and reversed NK cell resistance in ADCC. Trastuzumab alone did not sufficiently activate NK cells and required additional LFA-1 co-stimulation, while activation of the ErbB2-CAR in CAR-NK cells induced efficient degranulation independent of LFA-1. Total internal reflection fluorescence single molecule imaging revealed that CAR-NK cells formed an irregular immunological synapse with tumor cells that excluded ICAM-1, while trastuzumab formed typical peripheral supramolecular activation cluster (pSMAC) structures. Mechanistically, the absence of ICAM-1 did not affect cell-cell adhesion during ADCC, but rather resulted in decreased signaling via Pyk2 and ERK1/2, which was intrinsically provided by CAR-mediated targeting. Furthermore, while stimulation of the inhibitory NK cell checkpoint molecule NKG2A markedly reduced FcγRIIIa/LFA-1-mediated degranulation, retargeting by CAR was only marginally affected., Conclusions: Downregulation of ICAM-1 on breast cancer cells is a critical escape mechanism from trastuzumab-triggered ADCC. In contrast, CAR-NK cells are able to overcome cancer cell resistance caused by ICAM-1 reduction, highlighting the potential of CAR-NK cells in cancer immunotherapy., Competing Interests: Competing interests: TT and WSW are named as inventors on patents in the field of cancer immunotherapy owned by their respective institutions. HK and LB are employed by ImmunityBio, California, USA. Other authors declare that they have no competing interests., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Natural killer (NK) cells are attractive effectors for adoptive immunotherapy of cancer. Results from first-in-human studies using chimeric antigen receptor (CAR)-engineered primary NK cells and NK-92 cells are encouraging in terms of efficacy and safety. In order to further improve treatment strategies and to test the efficacy of CAR-NK cells in a personalized manner, preclinical screening assays using patient-derived tumor samples are needed. Zebrafish ( Danio rerio ) embryos and larvae represent an attractive xenograft model to study growth and dissemination of patient-derived tumor cells because of their superb live cell imaging properties. Injection into the organism's circulation allows investigation of metastasis, cancer cell-to-immune cell-interactions and studies of the tumor cell response to anti-cancer drugs. Here, we established a zebrafish larval xenograft model to test the efficacy of CAR-NK cells against metastatic breast cancer in vivo by injecting metastatic breast cancer cells followed by CAR-NK cell injection into the Duct of Cuvier (DoC). We validated the functionality of the system with two different CAR-NK cell lines specific for PD-L1 and ErbB2 (PD-L1.CAR NK-92 and ErbB2.CAR NK-92 cells) against the PD-L1-expressing MDA-MB-231 and ErbB2-expressing MDA-MB-453 breast cancer cell lines. Injected cancer cells were viable and populated peripheral regions of the larvae, including the caudal hematopoietic tissue (CHT), simulating homing of cancer cells to blood forming sites. CAR-NK cells injected 2.5 hours later migrated to the CHT and rapidly eliminated individual cancer cells throughout the organism. Unmodified NK-92 also demonstrated minor in vivo cytotoxicity. Confocal live-cell imaging demonstrated intravascular migration and real-time interaction of CAR-NK cells with MDA-MB-231 cells, explaining the rapid and effective in vivo cytotoxicity. Thus, our data suggest that zebrafish larvae can be used for rapid and cost-effective in vivo assessment of CAR-NK cell potency and to predict patient response to therapy., Competing Interests: TT and WW are named as inventors on patents in the field of cancer immunotherapy owned by their respective institutions. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Murali Shankar, Ortiz-Montero, Kurzyukova, Rackwitz, Künzel, Wels, Tonn, Knopf and Eitler.)

Objectives: Myocardial infarction (MI) initiates a complex reparative response during which damaged cardiac muscle is replaced by connective tissue. While the initial repair is essential for survival, excessive fibrosis post-MI is a primary contributor to progressive cardiac dysfunction, and ultimately heart failure. Currently, there are no approved drugs for the prevention or the reversal of cardiac fibrosis. Therefore, we tested the therapeutic potential of repurposed mesalazine as a post-MI therapy, as distinct antifibrotic effects have recently been demonstrated., Methods: At 8 weeks of age, MI was induced in male C57BL/6J mice by LAD ligation. Mesalazine was administered orally at a dose of 100 μg/g body weight in drinking water. Fluid intake, weight development, and cardiac function were monitored for 28 days post intervention. Fibrosis parameters were assessed histologically and via qPCR., Results: Compared to controls, mesalazine treatment offered no survival benefit. However, no adverse effects on heart and kidney function and weight development were observed, either. While total cardiac fibrosis remained largely unaffected by mesalazine treatment, we found a distinct reduction of perivascular fibrosis alongside reduced cardiac collagen expression., Conclusions: Our findings warrant further studies on mesalazine as a potential add-on therapy post-MI, as perivascular fibrosis development was successfully prevented., (© 2023 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

Background: Ventricular arrhythmia and sudden cardiac death are the most common lethal complications after myocardial infarction. Antiarrhythmic pharmacotherapy remains a clinical challenge and novel concepts are highly desired. Here, we focus on the cardioprotective CNP (C-type natriuretic peptide) as a novel antiarrhythmic principle. We hypothesize that antiarrhythmic effects of CNP are mediated by PDE2 (phosphodiesterase 2), which has the unique property to be stimulated by cGMP to primarily hydrolyze cAMP. Thus, CNP might promote beneficial effects of PDE2-mediated negative crosstalk between cAMP and cGMP signaling pathways., Methods: To determine antiarrhythmic effects of cGMP-mediated PDE2 stimulation by CNP, we analyzed arrhythmic events and intracellular trigger mechanisms in mice in vivo, at organ level and in isolated cardiomyocytes as well as in human-induced pluripotent stem cell-derived cardiomyocytes., Results: In ex vivo perfused mouse hearts, CNP abrogated arrhythmia after ischemia/reperfusion injury. Upon high-dose catecholamine injections in mice, PDE2 inhibition prevented the antiarrhythmic effect of CNP. In mouse ventricular cardiomyocytes, CNP blunted the catecholamine-mediated increase in arrhythmogenic events as well as in I CaL , I NaL , and Ca 2+ spark frequency. Mechanistically, this was driven by reduced cellular cAMP levels and decreased phosphorylation of Ca 2+ handling proteins. Key experiments were confirmed in human iPSC-derived cardiomyocytes. Accordingly, the protective CNP effects were reversed by either specific pharmacological PDE2 inhibition or cardiomyocyte-specific PDE2 deletion., Conclusions: CNP shows strong PDE2-dependent antiarrhythmic effects. Consequently, the CNP-PDE2 axis represents a novel and attractive target for future antiarrhythmic strategies.

After myocardial infarction the innate immune response is pivotal in clearing of tissue debris as well as scar formation, but exaggerated cytokine and chemokine secretion with subsequent leukocyte infiltration also leads to further tissue damage. Here, we address the value of targeting a previously unknown a disintegrin and metalloprotease 10 (ADAM10)/CX3CL1 axis in the regulation of neutrophil recruitment early after MI. We show that myocardial ADAM10 is distinctly upregulated in myocardial biopsies from patients with ischemia-driven cardiomyopathy. Intriguingly, upon MI in mice, pharmacological ADAM10 inhibition as well as genetic cardiomycyte-specific ADAM10 deletion improves survival with markedly enhanced heart function and reduced scar size. Mechanistically, abolished ADAM10-mediated CX3CL1 ectodomain shedding leads to diminished IL-1β-dependent inflammation, reduced neutrophil bone marrow egress as well as myocardial tissue infiltration. Thus, our data shows a conceptual insight into how acute MI induces chemotactic signaling via ectodomain shedding in cardiomyocytes., (© 2022. The Author(s).)

The combination of the human induced pluripotent stem cell (hiPSC) and organoid technology enables the generation of human 3D culture systems, providing the opportunity to model human tissue-like structures in vitro . This protocol offers the details to generate and characterize self-assembling 3D cardiac organoids in a controlled and efficient manner based on hiPSC-derived cardiomyocytes. Cardiac organoids can be used as 3D-based assay systems and offer a wide range of applications in pharmacological and toxicological research as well as an alternative to animal experiments., Competing Interests: The authors declare no conflict of interest., (© 2022 The Author(s).)

Skin fibrosis is a complex biological remodeling process occurring in disease like systemic sclerosis, morphea, or eosinophilic fasciitis. Since the knowledge about the underlying pathomechanisms is still incomplete, there is currently no therapy, which prevents or reverses skin fibrosis sufficiently. The present study investigates the role of polo-like kinase 2 (PLK2) and the pro-fibrotic cytokine osteopontin (OPN) in the pathogenesis of cutaneous fibrosis and demonstrates the antifibrotic effects of systemic mesalazine treatment in vivo. Isolated primary dermal fibroblasts of PLK2 wild-type (WT) and knockout (KO) mice were characterized in vitro. Skin thickness and histoarchitecture were studied in paraffin-embedded skin sections. The effects of mesalazine treatment were examined in isolated fibroblasts and PLK2 KO mice, which were fed 100 µg/g mesalazine for 6 months via the drinking water. Compared to WT, PLK2 KO fibroblasts displayed higher spontaneous myofibroblast differentiation, reduced proliferation rates, and overexpression of the fibrotic cytokine OPN. In vitro, 72 h of treatment with 10 mmol/L mesalazine induced phenotype conversion in PLK2 KO fibroblasts and attenuated OPN expression by inhibiting ERK1/2. In vivo, dermal myofibroblast differentiation, collagen accumulation, and skin thickening were prevented by mesalazine in PLK2 KO. Plasma creatinine levels indicated good tolerability of systemic long-term mesalazine treatment. The current study reveals a spontaneous fibrotic skin phenotype and ERK1/2-dependent OPN overexpression in PLK2 KO mice. We provide experimental evidence for the antifibrotic effectiveness of systemic mesalazine treatment to prevent fibrosis of the skin, suggesting further investigation in experimental and clinical settings., (© 2021. The Author(s).)

[Figure: see text].

Aims: Atrial Fibrillation (AF) is an arrhythmia of increasing prevalence in the aging populations of developed countries. One of the important indicators of AF is sustained atrial dilatation, highlighting the importance of mechanical overload in the pathophysiology of AF. The mechanisms by which atrial cells, including fibroblasts, sense and react to changing mechanical forces, are not fully elucidated. Here, we characterise stretch-activated ion channels (SAC) in human atrial fibroblasts and changes in SAC- presence and activity associated with AF., Methods and Results: Using primary cultures of human atrial fibroblasts, isolated from patients in sinus rhythm or sustained AF, we combine electrophysiological, molecular and pharmacological tools to identify SAC. Two electrophysiological SAC- signatures were detected, indicative of cation-nonselective and potassium-selective channels. Using siRNA-mediated knockdown, we identified the cation-nonselective SAC as Piezo1. Biophysical properties of the potassium-selective channel, its sensitivity to calcium, paxilline or iberiotoxin (blockers), and NS11021 (activator), indicated presence of calcium-dependent 'big potassium channels' (BK Ca ). In cells from AF patients, Piezo1 activity and mRNA expression levels were higher than in cells from sinus rhythm patients, while BK Ca activity (but not expression) was downregulated. Both Piezo1-knockdown and removal of extracellular calcium from the patch pipette resulted in a significant reduction of BK Ca current during stretch. No co-immunoprecipitation of Piezo1 and BK Ca was detected., Conclusions: Human atrial fibroblasts contain at least two types of ion channels that are activated during stretch: Piezo1 and BK Ca . While Piezo1 is directly stretch-activated, the increase in BK Ca activity during mechanical stimulation appears to be mainly secondary to calcium influx via SAC such as Piezo1. During sustained AF, Piezo1 is increased, while BK Ca activity is reduced, highlighting differential regulation of both channels. Our data support the presence and interplay of Piezo1 and BK Ca in human atrial fibroblasts in the absence of physical links between the two channel proteins., (Copyright © 2021. Published by Elsevier Ltd.)

ATP-Citrate-Lyase is a key enzyme of cholesterol biosynthesis. Its liver-specific inhibition by the bempedoic acid opens new possibilities to effectively escalate a cholesterol-lowering therapy while avoiding muscle-related side effects. Herein, we present the properties of this new first-in-class pharmaceutical agent and discuss potential consequences for pharmacotherapy., Competing Interests: A. El-Armouche weist auf Forschungs- und/oder Vortragstätigkeiten hin für Boehringer Ingelheim, Novartis, Bayer, Novo Nordisk, Sanofi Aventis, Daiichi Sankyo und Berlin Chemie. Die anderen Autorinnen/Autoren geben keinen Interessenkonflikt an., (Thieme. All rights reserved.)

Pulmonary fibrosis is the chronic-progressive replacement of healthy lung tissue by extracellular matrix, leading to the destruction of the alveolar architecture and ultimately death. Due to limited pathophysiological knowledge, causal therapies are still missing and consequently the prognosis is poor. Thus, there is an urgent clinical need for models to derive effective therapies. Polo-like kinase 2 (PLK2) is an emerging regulator of fibroblast function and fibrosis. We found a significant downregulation of PLK2 in four different entities of human pulmonary fibrosis. Therefore, we characterized the pulmonary phenotype of PLK2 knockout (KO) mice. Isolated pulmonary PLK2 KO fibroblasts displayed a pronounced myofibroblast phenotype reflected by increased expression of αSMA, reduced proliferation rates and enhanced ERK1/2 and SMAD2/3 phosphorylation. In PLK2 KO, the expression of the fibrotic cytokines osteopontin and IL18 was elevated compared to controls. Histological analysis of PLK2 KO lungs revealed early stage remodeling in terms of alveolar wall thickening, increased alveolar collagen deposition and myofibroblast foci. Our results prompt further investigation of PLK2 function in pulmonary fibrosis and suggest that the PLK2 KO model displays a genetic predisposition towards pulmonary fibrosis, which could be leveraged in future research on this topic.

Cardiovascular diseases are exacerbated and driven by cardiac fibrosis. TGFβ induces fibroblast activation and differentiation into myofibroblasts that secrete excessive extracellular matrix proteins leading to stiffening of the heart, concomitant cardiac dysfunction, and arrhythmias. However, effective pharmacotherapy for preventing or reversing cardiac fibrosis is presently unavailable. Therefore, drug repurposing could be a cost- and time-saving approach to discover antifibrotic interventions. The aim of this study was to investigate the antifibrotic potential of mesalazine in a cardiac fibroblast stress model. TGFβ was used to induce a profibrotic phenotype in a human cardiac fibroblast cell line. After induction, cells were treated with mesalazine or solvent control. Fibroblast proliferation, key fibrosis protein expression, extracellular collagen deposition, and mechanical properties were subsequently determined. In response to TGFβ treatment, fibroblasts underwent a profound phenoconversion towards myofibroblasts, determined by the expression of fibrillary αSMA. Mesalazine reduced differentiation nearly by half and diminished fibroblast proliferation by a third. Additionally, TGFβ led to increased cell stiffness and adhesion, which were reversed by mesalazine treatment. Collagen 1 expression and deposition-key drivers of fibrosis-were significantly increased upon TGFβ stimulation and reduced to control levels by mesalazine. SMAD2/3 and ERK1/2 phosphorylation, along with reduced nuclear NFκB translocation, were identified as potential modes of action. The current study provides experimental pre-clinical evidence for antifibrotic effects of mesalazine in an in vitro model of cardiac fibrosis. Furthermore, it sheds light on possible mechanisms of action and suggests further investigation in experimental and clinical settings.

Atrial fibrillation (AF) is regularly accompanied by cardiac fibrosis and concomitant heart failure. Due to the heterogeneous nature and complexity of fibrosis, the knowledge about the underlying mechanisms is limited, which prevents effective pharmacotherapy. A deeper understanding of cardiac fibroblasts is essential to meet this need. We previously described phenotypic and functional differences between atrial fibroblasts from patients in sinus rhythm and with AF. Herein, we established and characterized a novel human atrial fibroblast line, which displays typical fibroblast morphology and function comparable to primary cells but with improved proliferation capacity and low spontaneous myofibroblast differentiation. These traits make our model suitable for the study of fibrosis mechanisms and for drug screening aimed at developing effective antifibrotic pharmacotherapy., (© 2020 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

Primary adult fibroblasts have become an important tool to study fibrosis, fibroblast interactions and inflammation in all body tissues. Since primary fibroblasts cannot divide indefinitely due to myofibroblast differentiation or senescence induction, new cultures must be established regularly. However, there are several obstacles to overcome during the processes of developing a reliable isolation protocol and primary fibroblast isolation itself: the method's degree of difficulty (especially for beginners), the risk of bacterial contamination, the required time until primary fibroblasts can be used for experiments, and subsequent cell quality and viability. In this study, a fast, reliable and easy-to-learn protocol to isolate and culture primary adult fibroblasts from mouse heart, lung, liver and kidney combining enzymatic digestion and ultrasonic agitation is provided.

There is growing interest in estimating and analyzing heterogeneous treatment effects in experimental and observational studies. We describe a number of metaalgorithms that can take advantage of any supervised learning or regression method in machine learning and statistics to estimate the conditional average treatment effect (CATE) function. Metaalgorithms build on base algorithms-such as random forests (RFs), Bayesian additive regression trees (BARTs), or neural networks-to estimate the CATE, a function that the base algorithms are not designed to estimate directly. We introduce a metaalgorithm, the X-learner, that is provably efficient when the number of units in one treatment group is much larger than in the other and can exploit structural properties of the CATE function. For example, if the CATE function is linear and the response functions in treatment and control are Lipschitz-continuous, the X-learner can still achieve the parametric rate under regularity conditions. We then introduce versions of the X-learner that use RF and BART as base learners. In extensive simulation studies, the X-learner performs favorably, although none of the metalearners is uniformly the best. In two persuasion field experiments from political science, we demonstrate how our X-learner can be used to target treatment regimes and to shed light on underlying mechanisms. A software package is provided that implements our methods., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)