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Pulmonary hypertension (PH) is a general term used to describe high blood pressure in the lungs from any cause. Pulmonary arterial hypertension (PAH) is a progressive, and fatal disease that causes the walls of the pulmonary arteries to tighten and stiffen. One of the major characteristics of PAH is the hyperproliferation and resistance to apoptosis of vascular cells, which trigger excessive pulmonary vascular remodeling and vasoconstriction. The death-associated protein DAP-kinase (DAPK) is a tumor suppressor and Ser/Thr protein kinase, which was previously shown to regulate the hypoxia inducible factor (HIF)-1α. Against this background, we now show that DAPK1 regulates human pulmonary arterial smooth muscle cell (hPASMC) proliferation and energy metabolism in a HIF-dependent manner. DAPK1 expression is downregulated in pulmonary vessels and PASMCs of human and experimental PH lungs. Reduced expression of DAPK1 in hypoxia and non-hypoxia PAH-PASMCs correlates with increased expression of HIF-1/2α. RNA interference-mediated depletion of DAPK1 leads to fundamental metabolic changes, including a significantly decreased rate of oxidative phosphorylation associated with enhanced expression of both HIF-1α and HIF-2α and glycolytic enzymes, as hexokinase 2 (HK2), lactate dehydrogenase A (LDHA), and an integrator between the glycolysis and citric acid cycle, pyruvate dehydrogenase kinase 1 (PDK1). DAPK1 ablation in healthy donor hPASMCs leads to an increase in proliferation, while its overexpression provides the opposite effects. Together our data indicate that DAPK1 serves as a new inhibitor of the pro-proliferative and glycolytic phenotype of PH in PASMCs acting via HIF-signaling pathway., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Background: Fibrosis is often associated with aberrant repair mechanisms that ultimately lead to organ failure. In the lung, idiopathic pulmonary fibrosis (IPF) is a fatal form of interstitial lung disease (ILD) to which there is currently no curative therapy. From the cell biology point of view, the cell of origin and eventual fate of activated myofibroblasts (aMYFs) have taken center stage as these cells are believed to drive structural remodeling and lung function impairment. While aMYFs are now widely believed to originate from resident fibroblasts, the heterogeneity of aMYFs and ultimate fate during fibrosis resolution remain elusive. We have previously shown that aMYFs dedifferentiation and acquisition of a lipofibroblast (LIF)-like phenotype represent a route of fibrosis resolution., Methods: In this study, we combined genetic lineage tracing and single-cell transcriptomics in mice, and data mining of human IPF datasets to decipher the heterogeneity of aMYFs and investigate differentiation trajectories during fibrosis resolution. Furthermore, organoid cultures were utilized as a functional readout for the alveolar mesenchymal niche activity during various phases of injury and repair in mice., Results: Our data demonstrate that aMYFs consist of four subclusters displaying unique pro-alveologenic versus profibrotic profiles. Alveolar fibroblasts displaying a high LIF-like signature largely constitute both the origin and fate of aMYFs during fibrogenesis and resolution respectively. The heterogeneity of aMYFs is conserved in humans and a significant proportion of human aMYFs displays a high LIF signature., Conclusion: Our work identifies a subcluster of aMYFs that is potentially relevant for future management of IPF., (Copyright ©The authors 2024.)

Vasodilation in response to low oxygen (O 2 ) tension (hypoxic vasodilation) is an essential homeostatic response of systemic arteries that facilitates O 2 supply to tissues according to demand. However, how blood vessels react to O 2 deficiency is not well understood. A common belief is that arterial myocytes are O 2 -sensitive. Supporting this concept, it has been shown that the activity of myocyte L-type Ca 2+ channels, the main ion channels responsible for vascular contractility, is reversibly inhibited by hypoxia, although the underlying molecular mechanisms have remained elusive. Here, we show that genetic or pharmacological disruption of mitochondrial electron transport selectively abolishes O 2 modulation of Ca 2+ channels and hypoxic vasodilation. Mitochondria function as O 2 sensors and effectors that signal myocyte Ca 2+ channels due to constitutive Hif1α-mediated expression of specific electron transport subunit isoforms. These findings reveal the acute O 2 -sensing mechanisms of vascular cells and may guide new developments in vascular pharmacology., (© 2024. The Author(s).)

The molecular mechanisms of progressive right heart failure are incompletely understood. In this study, we systematically examined transcriptomic changes occurring over months in isolated cardiomyocytes or whole heart tissues from failing right and left ventricles in rat models of pulmonary artery banding (PAB) or aortic banding (AOB). Detailed bioinformatics analyses resulted in the identification of gene signature, protein and transcription factor networks specific to ventricles and compensated or decompensated disease states. Proteomic and RNA-FISH analyses confirmed PAB-mediated regulation of key genes and revealed spatially heterogeneous mRNA expression in the heart. Intersection of rat PAB-specific gene sets with transcriptome datasets from human patients with chronic thromboembolic pulmonary hypertension (CTEPH) led to the identification of more than 50 genes whose expression levels correlated with the severity of right heart disease, including multiple matrix-regulating and secreted factors. These data define a conserved, differentially regulated genetic network associated with right heart failure in rats and humans., (© 2024. The Author(s).)

Subtle changes in the membrane potential of pulmonary arterial smooth muscle cells (PASMCs) are pivotal for controlling pulmonary vascular tone, e.g., for initiating Hypoxic Pulmonary Vasoconstriction, a vital mechanism of the pulmonary circulation. In our study, we evaluated the ability of the fluorescence resonance energy transfer (FRET)-based voltage-sensor Mermaid to detect such subtle changes in membrane potential. Mouse PASMCs were isolated and transduced with Mermaid-encoding lentiviral vectors before the acceptor/donor emission ratio was assessed via live cell FRET-imaging. Mermaid's sensitivity was tested by applying specific potassium chloride (KCl) concentrations. These KCl concentrations were previously validated by patch clamp recordings to induce depolarization with predefined amplitudes that physiologically occur in PASMCs. Mermaid's emission ratio dose-dependently increased upon depolarization with KCl. However, Mermaid formed unspecific intracellular aggregates, which limited the usefulness of this voltage sensor. When analyzing the membrane rim only to circumvent these unspecific signals, Mermaid was not suitable to resolve subtle changes in the membrane potential of ≤10 mV. In summary, we found Mermaid to be a suitable alternative for reliably detecting qualitative membrane voltage changes of more than 10 mV in primary mouse PASMCs. However, one should be aware of the limitations associated with this voltage sensor.

Pulmonary hypertension (PH) is a progressive, severe and to date not curable disease of the pulmonary vasculature. Alterations of the insulin-like growth factor 1 (IGF-1) system are known to play a role in vascular pathologies and IGF-binding proteins (IGFBPs) are important regulators of the bioavailability and function of IGFs. In this study, we show that circulating plasma levels of IGFBP-1, IGFBP-2 and IGFBP-3 are increased in idiopathic pulmonary arterial hypertension (IPAH) patients compared to healthy individuals. These binding proteins inhibit the IGF-1 induced IGF-1 receptor (IGF1R) phosphorylation and exhibit diverging effects on the IGF-1 induced signaling pathways in human pulmonary arterial cells (i.e. healthy as well as IPAH-hPASMCs, and healthy hPAECs). Furthermore, IGFBPs are differentially expressed in an experimental mouse model of PH. In hypoxic mouse lungs, IGFBP-1 mRNA expression is decreased whereas the mRNA for IGFBP-2 is increased. In contrast to IGFBP-1, IGFBP-2 shows vaso-constrictive properties in the murine pulmonary vasculature. Our analyses show that IGFBP-1 and IGFBP-2 exhibit diverging effects on IGF-1 signaling and display a unique IGF1R-independent kinase activation pattern in human pulmonary arterial smooth muscle cells (hPASMCs), which represent a major contributor of PAH pathobiology. Furthermore, we could show that IGFBP-2, in contrast to IGFBP-1, induces epidermal growth factor receptor (EGFR) signaling, Stat-3 activation and expression of Stat-3 target genes. Based on our results, we conclude that the IGFBP family, especially IGFBP-1, IGFBP-2 and IGFBP-3, are deregulated in PAH, that they affect IGF signaling and thereby regulate the cellular phenotype in PH., Competing Interests: Declaration of competing interest The following authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Beate Christiane Schlueter, Karin Quanz, Julia Baldauf, Aleksandar Petrovic, Clemens Ruppert, Andreas Guenther, Henning Gall, Khodr Tello, Friedrich Grimminger, Hossein-Ardeschir Ghofrani, Norbert Weissmann, Ralph Theo Schermuly, Astrid Weiss. The authors declare the following financial interests/personal relationships which may be considered as potential competing interests. Werner Seeger received consulting fees from United Therapeutics, Tiakis Biotech AG, Liquidia, Pieris Pharmaceuticalsm Abivax, Pfitzer, Medspray BV., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Bleomycin (BLM)-induced lung injury in mice is a valuable model for investigating the molecular mechanisms that drive inflammation and fibrosis and for evaluating potential therapeutic approaches to treat the disease. Given high variability in the BLM model, it is critical to accurately phenotype the animals in the course of an experiment. In the present study, we aimed to demonstrate the utility of microscopic computed tomography (µCT) imaging combined with an artificial intelligence (AI)-convolutional neural network (CNN)-powered lung segmentation for rapid phenotyping of BLM mice. µCT was performed in freely breathing C57BL/6J mice under isoflurane anesthesia on days 7 and 21 after BLM administration. Terminal invasive lung function measurement and histological assessment of the left lung collagen content were conducted as well. µCT image analysis demonstrated gradual and time-dependent development of lung injury as evident by alterations in the lung density, air-to-tissue volume ratio, and lung aeration in mice treated with BLM. The right and left lung were unequally affected. µCT-derived parameters such as lung density, air-to-tissue volume ratio, and nonaerated lung volume correlated well with the invasive lung function measurement and left lung collagen content. Our study demonstrates the utility of AI-CNN-powered µCT image analysis for rapid and accurate phenotyping of BLM mice in the course of disease development and progression. NEW & NOTEWORTHY Microscopic computed tomography (µCT) imaging combined with an artificial intelligence (AI)-convolutional neural network (CNN)-powered lung segmentation is a rapid and powerful tool for noninvasive phenotyping of bleomycin mice over the course of the disease. This, in turn, allows earlier and more reliable identification of therapeutic effects of new drug candidates, ultimately leading to the reduction of unnecessary procedures in animals in pharmacological research.

Background: The precise origin of newly formed ACTA2+ (alpha smooth muscle actin-positive) cells appearing in nonmuscularized vessels in the context of pulmonary hypertension is still debatable although it is believed that they predominantly derive from preexisting vascular smooth muscle cells (VSMCs)., Methods: Gli1 Cre-ERT2 ; tdTomato flox mice were used to lineage trace GLI1+ (glioma-associated oncogene homolog 1-positive) cells in the context of pulmonary hypertension using 2 independent models of vascular remodeling and reverse remodeling: hypoxia and cigarette smoke exposure. Hemodynamic measurements, right ventricular hypertrophy assessment, flow cytometry, and histological analysis of thick lung sections followed by state-of-the-art 3-dimensional reconstruction and quantification using Imaris software were used to investigate the contribution of GLI1+ cells to neomuscularization of the pulmonary vasculature., Results: The data show that GLI1+ cells are abundant around distal, nonmuscularized vessels during steady state, and this lineage contributes to around 50% of newly formed ACTA2+ cells around these normally nonmuscularized vessels. During reverse remodeling, cells derived from the GLI1+ lineage are largely cleared in parallel to the reversal of muscularization. Partial ablation of GLI1+ cells greatly prevented vascular remodeling in response to hypoxia and attenuated the increase in right ventricular systolic pressure and right heart hypertrophy. Single-cell RNA sequencing on sorted lineage-labeled GLI1+ cells revealed an Acta2 high fraction of cells with pathways in cancer and MAPK (mitogen-activated protein kinase) signaling as potential players in reprogramming these cells during vascular remodeling. Analysis of human lung-derived material suggests that GLI1 signaling is overactivated in both group 1 and group 3 pulmonary hypertension and can promote proliferation and myogenic differentiation., Conclusions: Our data highlight GLI1+ cells as an alternative cellular source of VSMCs in pulmonary hypertension and suggest that these cells and the associated signaling pathways represent an important therapeutic target for further studies., Competing Interests: Disclosures None.

The mitochondrial adaptor protein p66Shc has been suggested to control life span in mice via the release of hydrogen peroxide. However, the role of p66Shc in lung aging remains unsolved. Thus, we investigated the effects of p66Shc -/- on the aging of the lung and pulmonary circulation. In vivo lung and cardiac characteristics were investigated in p66Shc -/- and wild type (WT) mice at 3, 12, and 24 months of age by lung function measurements, micro-computed tomography (µCT), and echocardiography. Alveolar number and muscularization of small pulmonary arteries were measured by stereology and vascular morphometry, respectively. Protein and mRNA levels of senescent markers were measured by western blot and PCR, respectively. Lung function declined similarly in WT and p66Shc -/- mice during aging. However, µCT analyses and stereology showed slightly enhanced signs of aging-related parameters in p66Shc -/- mice, such as a decline of alveolar density. Accordingly, p66Shc -/- mice showed higher protein expression of the senescence marker p21 in lung homogenate compared to WT mice of the corresponding age. Pulmonary vascular remodeling was increased during aging, but aged p66Shc -/- mice showed similar muscularization of pulmonary vessels and hemodynamics like WT mice. In the heart, p66Shc -/- prevented the deterioration of right ventricular (RV) function but promoted the decline of left ventricular (LV) function during aging. p66Shc -/- affects the aging process of the lung and the heart differently. While p66Shc -/- slightly accelerates lung aging and deteriorates LV function in aged mice, it seems to exert protective effects on RV function during aging.

Background: COPD is an incurable disease and a leading cause of death worldwide. In mice, fibroblast growth factor (FGF)10 is essential for lung morphogenesis, and in humans, polymorphisms in the human FGF10 gene correlate with an increased susceptibility to develop COPD., Methods: We analysed FGF10 signalling in human lung sections and isolated cells from healthy donor, smoker and COPD lungs. The development of emphysema and PH was investigated in Fgf10 +/- and Fgfr2b +/- (FGF receptor 2b) mice upon chronic exposure to cigarette smoke. In addition, we overexpressed FGF10 in mice following elastase- or cigarette smoke-induced emphysema and pulmonary hypertension (PH)., Results: We found impaired FGF10 expression in human lung alveolar walls and in primary interstitial COPD lung fibroblasts. In contrast, FGF10 expression was increased in large pulmonary vessels in COPD lungs. Consequently, we identified impaired FGF10 signalling in alveolar walls as an integral part of the pathomechanism that leads to emphysema and PH development: mice with impaired FGF10 signalling ( Fgf10 +/- and Fgfr2b +/- ) spontaneously developed lung emphysema, PH and other typical pathomechanistic features that generally arise in response to cigarette smoke exposure., Conclusion: In a therapeutic approach, FGF10 overexpression successfully restored lung alveolar and vascular structure in mice with established cigarette smoke- and elastase-induced emphysema and PH. FGF10 treatment triggered an initial increase in the number of alveolar type 2 cells that gradually returned to the basal level when the FGF10-mediated repair process progressed. Therefore, the application of recombinant FGF10 or stimulation of the downstream signalling cascade might represent a novel therapeutic strategy in the future., Competing Interests: Conflict of interest: The authors have no potential conflicts of interest to disclose., (Copyright ©The authors 2023.)

The recently published new European guidelines for diagnosis and treatment of pulmonary hypertension now offer the so far most extensive description of genetic testing and counselling for pulmonary arterial hypertension patients. In addition, the importance of a clinical screening of healthy mutation carriers is highlighted as well as the genetic testing of patients with a suspicion of pulmonary veno-occlusive disease. We frame the respective parts of the guidelines on genetic testing and counselling in the context of recent data and provide comments. Finally, we give an outlook on novel molecular approaches starting from Sotatercept, addressing ion channels and novel therapeutic developments., Competing Interests: C.A.E. und K.H. sind Erfinderinnen des europäischen Patentens (EP3507380) “Gene panel specific for pulmonary hypertension and its uses”. CAE hat von MSD Vortragshonorare erhalten, unabhängig von dieser Arbeit.N.S. hat Berater- und Vortragstätigkeiten für MSD und Janssen.R.T.S. erhielt Forschungsförderung von Gossamer.S.S.P. erhielt Forschungsgelder und Honorare von Gossamer Bio, Inc.N.W. ist Besitzer des Patentes: L-NIL als Inhibitor zur Regeneration der Lunge von an COPD leidenden Patienten (EP2591777B1).L.H. erhielt Vortragshonorare von Janssen-Cilag und AOP Health sowie Forschungsmittel von MSD.C.T. erhielt finanzielle Förderung für Forschung von der Deutschen Gesellschaft für Pneumologie und Beatmungsmedizin e. V., Bayer HealthCare, Boehringer Ingelheim, und für Vorträge und Beratungstätigkeit von Actelion Pharmaceuticals, AstraZeneca, Berlin-Chemie, Boehringer Ingelheim, GlaxoSmithKline, und nicht-finanzielle Förderung von Actelion, ALK-Abelló, Bayer HealthCare, Boehringer Ingelheim und GlaxoSmithKline.M.W. erhielt Vortragshonorare von Janssen.V.F. erhielt Reisekosten und Vortragstätigkeiten von Boehringer Ingelheim, BMS, Chiesi, Janssen, MSD, alle nicht im Zusammenhang mit der aktuellen Übersichtsarbeit.A.O. ist Erfinderin des Patentes (WO2017153472A1 priority date 09.03.2016, erteilt in US, KR, JP, pending in CA, EP, AU) “Biomarker for the diagnosis of pulmonary hypertension (PH)”, ohne daraus persönlichen Gewinn zu erhalten. A.O. hat Honorare für Präsentationen und Reiseförderung von MSD erhalten, unabhängig von dieser Arbeit.O.B., W.M.K., G.K. haben keinen Interessenkonflikt., (Thieme. All rights reserved.)

Major advances in pulmonary arterial hypertension, pulmonary hypertension (PH) associated with lung disease, and chronic thromboembolic PH cast new light on the pathogenetic mechanisms, epidemiology, diagnostic approach, and therapeutic armamentarium for pulmonary vascular disease. Here, we summarize key basic, translational, and clinical PH reports, emphasizing findings that build on current state-of-the-art research. This review includes cutting-edge progress in translational pulmonary vascular biology, with a guide to the diagnosis of patients in clinical practice, incorporating recent PH definition revisions that continue emphasis on early detection of disease. PH management is reviewed including an overview of the evolving considerations for the approach to treatment of PH in patients with cardiopulmonary comorbidities, as well as a discussion of the groundbreaking sotatercept data for the treatment of pulmonary arterial hypertension.

Background: Electronic cigarette (e-cigarette) vapour is gaining popularity as an alternative to tobacco smoking and can induce acute lung injury. However, the specific role of nicotine in e-cigarette vapour and its long-term effects on the airways, lung parenchyma and vasculature remain unclear., Results: In vitro exposure to nicotine-containing e-cigarette vapour extract (ECVE) or to nicotine-free e-cigarette vapour extract (NF ECVE) induced changes in gene expression of epithelial cells and pulmonary arterial smooth muscle cells (PASMCs), but ECVE in particular caused functional alterations ( e.g. a decrease in human and mouse PASMC proliferation by 29.3±5.3% and 44.3±8.4%, respectively). Additionally, acute inhalation of nicotine-containing e-cigarette vapour (ECV) but not nicotine-free e-cigarette vapour (NF ECV) increased pulmonary endothelial permeability in isolated lungs. Long-term in vivo exposure of mice to ECV for 8 months significantly increased the number of inflammatory cells, in particular lymphocytes, compared to control and NF ECV in the bronchoalveolar fluid (BALF) (ECV: 853.4±150.8 cells·mL -1 ; control: 37.0±21.1 cells·mL -1 ; NF ECV: 198.6±94.9 cells·mL -1 ) and in lung tissue (ECV: 25.7±3.3 cells·mm -3 ; control: 4.8±1.1 cells·mm -3 ; NF ECV: 14.1±2.2 cells·mm -3 ). BALF cytokines were predominantly increased by ECV. Moreover, ECV caused significant changes in lung structure and function ( e.g. increase in airspace by 17.5±1.4% compared to control), similar to mild tobacco smoke-induced alterations, which also could be detected in the NF ECV group, albeit to a lesser degree. In contrast, the pulmonary vasculature was not significantly affected by ECV or NF ECV., Conclusions: NF ECV components induce cell type-specific effects and mild pulmonary alterations, while inclusion of nicotine induces significant endothelial damage, inflammation and parenchymal alterations., Competing Interests: Conflict of interest: All authors have nothing to disclose., (Copyright ©The authors 2023.)

Rationale: Tobacco smoking and air pollution are primary causes of chronic obstructive pulmonary disease (COPD). However, only a minority of smokers develop COPD. The mechanisms underlying the defense against nitrosative/oxidative stress in nonsusceptible smokers to COPD remain largely unresolved. Objectives: To investigate the defense mechanisms against nitrosative/oxidative stress that possibly prevent COPD development or progression. Methods: Four cohorts were investigated: 1 ) sputum samples (healthy, n  = 4; COPD, n  = 37), 2 ) lung tissue samples (healthy, n  = 13; smokers without COPD, n  = 10; smoker+COPD, n  = 17), 3 ) pulmonary lobectomy tissue samples (no/mild emphysema, n  = 6), and 4 ) blood samples (healthy, n  = 6; COPD, n  = 18). We screened 3-nitrotyrosine (3-NT) levels, as indication of nitrosative/oxidative stress, in human samples. We established a novel in vitro model of a cigarette smoke extract (CSE)-resistant cell line and studied 3-NT formation, antioxidant capacity, and transcriptomic profiles. Results were validated in lung tissue, isolated primary cells, and an ex vivo model using adeno-associated virus-mediated gene transduction and human precision-cut lung slices. Measurements and Main Results: 3-NT levels correlate with COPD severity of patients. In CSE-resistant cells, nitrosative/oxidative stress upon CSE treatment was attenuated, paralleled by profound upregulation of heme oxygenase-1 (HO-1). We identified carcinoembryonic antigen cell adhesion molecule 6 (CEACAM6) as a negative regulator of HO-1-mediated nitrosative/oxidative stress defense in human alveolar type 2 epithelial cells (hAEC2s). Consistently, inhibition of HO-1 activity in hAEC2s increased the susceptibility toward CSE-induced damage. Epithelium-specific CEACAM6 overexpression increased nitrosative/oxidative stress and cell death in human precision-cut lung slices on CSE treatment. Conclusions: CEACAM6 expression determines the hAEC2 sensitivity to nitrosative/oxidative stress triggering emphysema development/progression in susceptible smokers.

Background: The brain-derived neurotrophic factor (BDNF) may promote development of pulmonary hypertension and right ventricular (RV) failure. However, BDNF plasma levels were decreased in patients with left ventricular (LV) failure. Therefore, we investigated BDNF plasma levels in pulmonary hypertension patients and the role of BDNF in mouse models of pulmonary hypertension and isolated RV failure., Methods: BDNF plasma levels were correlated to pulmonary hypertension in two patient cohorts, including either post- and pre-capillary pulmonary hypertension patients (first cohort) or only pre-capillary pulmonary hypertension patients (second cohort). In the second cohort, RV dimensions and load-independent function were determined by imaging and pressure-volume catheter measurements, respectively. For induction of isolated RV pressure overload, heterozygous Bdnf knockout ( Bdnf +/- ) mice were subjected to pulmonary arterial banding (PAB). For induction of pulmonary hypertension, mice with inducible knockout of BDNF in smooth muscle cells ( Bdnf / Smmhc knockout) were exposed to chronic hypoxia., Results: Plasma BDNF levels were decreased in patients with pulmonary hypertension. Following adjustment for covariables, BDNF levels negatively correlated in both cohorts with central venous pressure. In the second cohort, BDNF levels additionally negatively correlated with RV dilatation. In animal models, BDNF downregulation attenuated RV dilatation in Bdnf + / - mice after PAB or hypoxic Bdnf / Smmhc knockout mice, although they developed pulmonary hypertension to a similar extent., Conclusions: Similar to LV failure, circulating levels of BDNF were decreased in pulmonary hypertension patients, and low BDNF levels were associated with right heart congestion. Decreased BDNF levels did not worsen RV dilatation in animal models, and thus, may be the consequence, but not the cause of RV dilatation., Competing Interests: Conflict of interest: No disclosures., (Copyright ©The authors 2023.)

Generation of functional transcripts requires transcriptional initiation at regular start sites, avoiding production of aberrant and potentially hazardous aberrant RNAs. The mechanisms maintaining transcriptional fidelity and the impact of spurious transcripts on cellular physiology and organ function have not been fully elucidated. Here we show that TET3, which successively oxidizes 5-methylcytosine to 5-hydroxymethylcytosine (5hmC) and other derivatives, prevents aberrant intragenic entry of RNA polymerase II pSer5 into highly expressed genes of airway smooth muscle cells, assuring faithful transcriptional initiation at canonical start sites. Loss of TET3-dependent 5hmC production in SMCs results in accumulation of spurious transcripts, which stimulate the endosomal nucleic-acid-sensing TLR7/8 signaling pathway, thereby provoking massive inflammation and airway remodeling resembling human bronchial asthma. Furthermore, we found that 5hmC levels are substantially lower in human asthma airways compared with control samples. Suppression of spurious transcription might be important to prevent chronic inflammation in asthma., (© 2022. The Author(s).)

Background: Chronic obstructive pulmonary disease (COPD) is the third leading cause of death worldwide. In addition to chronic bronchitis and emphysema, patients often develop at least mild pulmonary hypertension (PH). We previously demonstrated that inhibition of inducible nitric oxide synthase (iNOS) prevents and reverses emphysema and PH in mice. Interestingly, strong iNOS upregulation was found in alveolar epithelial type II cells (AECII) in emphysematous murine lungs, and peroxynitrite, which can be formed from iNOS-derived NO, was shown to induce AECII apoptosis in vitro. However, the specific cell type(s) that drive(s) iNOS-dependent lung regeneration in emphysema/PH has (have) not been identified yet., Aim: we tested whether iNOS knockout in AECII affects established elastase-induced emphysema in mice., Methods: four weeks after a single intratracheal instillation of porcine pancreatic elastase for the induction of emphysema and PH, we induced iNOS knockout in AECII in mice, and gave an additional twelve weeks for the potential recovery., Results: iNOS knockout in AECII did not reduce elastase-induced functional and structural lung changes such as increased lung compliance, decreased mean linear intercept and increased airspace, decreased right ventricular function, increased right ventricular systolic pressure and increased pulmonary vascular muscularization. In vitro, iNOS inhibition did not reduce apoptosis of AECII following exposure to a noxious stimulus., Conclusion: taken together, our data demonstrate that iNOS deletion in AECII is not sufficient for the regeneration of emphysematous murine lungs, and suggest that iNOS expression in pulmonary vascular or stromal cells might be critically important in this regard.

Chronic hypoxia-induced pulmonary hypertension (CHPH) is a severe disease that is characterized by increased proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs) leading to pulmonary vascular remodeling. The resulting increase in pulmonary vascular resistance (PVR) causes right ventricular hypertrophy and ultimately right heart failure. In addition, increased PVR can also be a consequence of hypoxic pulmonary vasoconstriction (HPV) under generalized hypoxia. Increased proliferation and migration of PASMCs are often associated with high intracellular Ca 2+ concentration. Recent publications suggest that Ca 2+ -permeable nonselective classical transient receptor potential (TRPC) proteins-especially TRPC1 and 6-are crucially involved in acute and sustained hypoxic responses and the pathogenesis of CHPH. The aim of our study was to investigate whether the simultaneous deletion of TRPC proteins 1, 3 and 6 protects against CHPH-development and affects HPV in mice. We used a mouse model of chronic hypoxia as well as isolated, ventilated and perfused mouse lungs and PASMC cell cultures. Although right ventricular systolic pressure as well as echocardiographically assessed PVR and right ventricular wall thickness (RVWT) were lower in TRPC1, 3, 6-deficient mice, these changes were not related to a decreased degree of pulmonary vascular muscularization and a reduced proliferation of PASMCs. However, both acute and sustained HPV were almost absent in the TRPC1, 3, 6-deficient mice and their vasoconstrictor response upon KCl application was reduced. This was further validated by myographical experiments. Our data revealed that 1) TRPC1, 3, 6-deficient mice are partially protected against development of CHPH, 2) these changes may be caused by diminished HPV and not an altered pulmonary vascular remodeling., Competing Interests: The 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 © 2022 Malkmus, Brosien, Knoepp, Schaffelhofer, Grimminger, Rummel, Gudermann, Dietrich, Birnbaumer, Weissmann and Kraut.)

Plants and other organisms, but not insects or vertebrates, express the auxiliary respiratory enzyme alternative oxidase (AOX) that bypasses mitochondrial respiratory complexes III and/or IV when impaired. Persistent expression of AOX from Ciona intestinalis in mammalian models has previously been shown to be effective in alleviating some metabolic stresses produced by respiratory chain inhibition while exacerbating others. This implies that chronic AOX expression may modify or disrupt metabolic signaling processes necessary to orchestrate adaptive remodeling, suggesting that its potential therapeutic use may be confined to acute pathologies, where a single course of treatment would suffice. One possible route for administering AOX transiently is AOX-encoding nucleic acid constructs. Here we demonstrate that AOX-encoding chemically-modified RNA (cmRNA), sequence-optimized for expression in mammalian cells, was able to support AOX expression in immortalized mouse embryonic fibroblasts (iMEFs), human lung carcinoma cells (A549) and primary mouse pulmonary arterial smooth muscle cells (PASMCs). AOX protein was detectable as early as 3 h after transfection, had a half-life of ~4 days and was catalytically active, thus supporting respiration and protecting against respiratory inhibition. Our data demonstrate that AOX-encoding cmRNA optimized for use in mammalian cells represents a viable route to investigate and possibly treat mitochondrial respiratory disorders., (© 2021. The Author(s).)

Acclimation to acute hypoxia through cardiorespiratory responses is mediated by specialized cells in the carotid body and pulmonary vasculature to optimize systemic arterial oxygenation and thus oxygen supply to the tissues. Acute oxygen sensing by these cells triggers hyperventilation and hypoxic pulmonary vasoconstriction which limits pulmonary blood flow through areas of low alveolar oxygen content. Oxygen sensing of acute hypoxia by specialized cells thus is a fundamental pre-requisite for aerobic life and maintains systemic oxygen supply. However, the primary oxygen sensing mechanism and the question of a common mechanism in different specialized oxygen sensing cells remains unresolved. Recent studies unraveled basic oxygen sensing mechanisms involving the mitochondrial cytochrome c oxidase subunit 4 isoform 2 that is essential for the hypoxia-induced release of mitochondrial reactive oxygen species and subsequent acute hypoxic responses in both, the carotid body and pulmonary vasculature. This review compares basic mitochondrial oxygen sensing mechanisms in the pulmonary vasculature and the carotid body., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The role of microparticles (MPs) and cold in high altitude pulmonary hypertension (HAPH) remains unexplored. We investigated the impact of long-term cold exposure on the pulmonary circulation in lowlanders and high-altitude natives and the role of MPs. Pulmonary hemodynamics were evaluated using Doppler echocardiography at the end of the colder and warmer seasons. We further examined the miRNA content of MPs isolated from the study participants and studied their effects on human pulmonary artery smooth muscle (hPASMCs) and endothelial cells (hPAECs). Long-term exposure to cold environment was associated with an enhanced pulmonary artery pressure in highlanders. Plasma levels of CD62E-positive and CD68-positive MPs increased in response to cold in lowlanders and HAPH highlanders. The miRNA-210 expression contained in MPs differentially changed in response to cold in lowlanders and highlanders. MPs isolated from lowlanders and highlanders increased proliferation and reduced apoptosis of hPASMCs. Further, MPs isolated from warm-exposed HAPH highlanders and cold-exposed highlanders exerted the most pronounced effects on VEGF expression in hPAECs. We demonstrated that prolonged exposure to cold is associated with elevated pulmonary artery pressures, which are most pronounced in high-altitude residents. Further, the numbers of circulating MPs are differentially increased in lowlanders and HAPH highlanders during the colder season.

Background: Pulmonary arterial hypertension (PAH) is a progressive disease characterised by pro-proliferative and anti-apoptotic phenotype in vascular cells, leading to pulmonary vascular remodelling and right heart failure. Peptidyl-prolyl cis / trans isomerase, NIMA interacting 1 (Pin1), a highly conserved enzyme, which binds to and catalyses the isomerisation of specific phosphorylated Ser/Thr-Pro motifs, acts as a molecular switch in multiple coordinated cellular processes. We hypothesised that Pin1 plays a substantial role in PAH, and its inhibition with a natural organic compound, Juglone, would reverse experimental pulmonary hypertension., Results: We demonstrated that the expression of Pin1 was markedly elevated in experimental pulmonary hypertension ( i.e. hypoxia-induced mouse and Sugen/hypoxia-induced rat models) and pulmonary arterial smooth muscle cells of patients with clinical PAH. In vitro Pin1 inhibition by either Juglone treatment or short interfering RNA knockdown resulted in an induction of apoptosis and decrease in proliferation of human pulmonary vascular cells. Stimulation with growth factors induced Pin1 expression, while its inhibition reduced the activity of numerous PAH-related transcription factors, such as hypoxia-inducible factor (HIF)-α and signal transducer and activator of transcription (STAT). Juglone administration lowered pulmonary vascular resistance, enhanced right ventribular function, improved pulmonary vascular and cardiac remodelling in the Sugen/hypoxia rat model of PAH and the chronic hypoxia-induced pulmonary hypertension model in mice., Conclusion: Our study demonstrates that targeting of Pin1 with small molecule inhibitor, Juglone, might be an attractive future therapeutic strategy for PAH and right heart disease secondary to PAH., Competing Interests: Conflict of interest: W. Seeger reports personal fees from Actelion, Bayer AG, Novartis, Vectura, Medspray and United Therapeutics, outside the submitted work. All other authors disclose no potential conflicts of interest., (Copyright ©The authors 2022.)

Mitochondrial and immune cell dysfunction contributes to the development of pulmonary arterial hypertension (PAH). We thus aimed to investigate mitochondrial respiration and mitochondrial gene expression patterns in the peripheral blood mononuclear cells (PBMC) of patients with idiopathic and hereditary PAH and their correlation to disease parameters. Mitochondrial respiration determined using high-resolution respirometry was not significantly different in PBMC when comparing an outpatient cohort of PAH patients with healthy controls. However, when directly comparing mitochondrial respiration to the hemodynamic parameters of an inpatient PAH cohort, mitochondrial respiration negatively correlated with pulmonary vascular resistance (PVR) and positively correlated with the cardiac index (CI). Furthermore, microarray analysis shows upregulation of mitochondrial erythroid-specific 5-aminolevulinate synthase 2 (ALAS2), as well as the regulation of genes involved in iron and heme metabolism, in the PBMC of patients with PAH, with ALAS2 upregulation in PAH patients being confirmed on the protein level. Multiple regression analysis with age and gender as confounders showed that both PVR and hemoglobin content negatively correlated with maximal respiration. Therefore, we conclude that mitochondrial function in the PBMC of PAH patients is affected by disease severity. However, further studies to investigate cell-type-specific alterations and functional consequences are necessary.

Chronic obstructive pulmonary disease (COPD) is a disease with an inflammatory phenotype with increasing prevalence in the elderly. Expanded population of mutant blood cells carrying somatic mutations is termed clonal hematopoiesis of indeterminate potential (CHIP). The association between CHIP and COPD and its relevant effects on DNA methylation in aging are mainly unknown. Analyzing the deep-targeted amplicon sequencing from 125 COPD patients, we found enhanced incidence of CHIP mutations (~20%) with a predominance of DNMT3A CHIP-mediated hypomethylation of Phospholipase D Family Member 5 ( PLD5 ), which in turn is positively correlated with increased levels of glycerol phosphocholine, pro-inflammatory cytokines, and deteriorating lung function.

Bronchopulmonary dysplasia (BPD) is a neonatal lung disease developing in premature babies characterized by arrested alveologenesis and associated with decreased Fibroblast growth factor 10 (FGF10) expression. One-week hyperoxia (HYX) exposure of newborn mice leads to a permanent arrest in alveologenesis. To test the role of Fgf10 signaling to promote de novo alveologenesis following hyperoxia, we used transgenic mice allowing inducible expression of Fgf10 and recombinant FGF10 (rFGF10) protein delivered intraperitoneally. We carried out morphometry analysis, and IF on day 45. Alveolospheres assays were performed co-culturing AT2s from normoxia (NOX) with FACS-isolated Sca1Pos resident mesenchymal cells (rMC) from animals exposed to NOX, HYX-PBS, or HYX-FGF10. scRNAseq between rMC-Sca1Pos isolated from NOX and HYX-PBS was also carried out. Transgenic overexpression of Fgf10 and rFGF10 administration rescued the alveologenesis defects following HYX. Alveolosphere assays indicate that the activity of rMC-Sca1Pos is negatively impacted by HYX and partially rescued by rFGF10 treatment. Analysis by IF demonstrates a significant impact of rFGF10 on the activity of resident mesenchymal cells. scRNAseq results identified clusters expressing Fgf10, Fgf7, Pdgfra, and Axin2, which could represent the rMC niche cells for the AT2 stem cells. In conclusion, we demonstrate that rFGF10 administration is able to induce de novo alveologenesis in a BPD mouse model and identified subpopulations of rMC-Sca1Pos niche cells potentially representing its cellular target., (© The Author(s) 2022. Published by Oxford University Press.)

Background: Pulmonary hypertension (PH) is a common complication of COPD, associated with increased mortality and morbidity. Intriguingly, pulmonary vascular alterations have been suggested to drive emphysema development. Previously, we identified inducible nitric oxide synthase (iNOS) as an essential enzyme for development and reversal of smoke-induced PH and emphysema, and showed that iNOS expression in bone-marrow-derived cells drives pulmonary vascular remodelling, but not parenchymal destruction. In this study, we aimed to identify the iNOS-expressing cell type driving smoke-induced PH and to decipher pro-proliferative pathways involved., Methods: To address this question we used 1) myeloid-cell-specific iNOS knockout mice in chronic smoke exposure and 2) co-cultures of macrophages and pulmonary artery smooth muscle cells (PASMCs) to decipher underlying signalling pathways., Results: Myeloid-cell-specific iNOS knockout prevented smoke-induced PH but not emphysema in mice. Moreover, iNOS deletion in myeloid cells ameliorated the increase in expression of CD206, a marker of M2 polarisation, on interstitial macrophages. Importantly, the observed effects on lung macrophages were hypoxia-independent, as these mice developed hypoxia-induced PH. In vitro , smoke-induced PASMC proliferation in co-cultures with M2-polarised macrophages could be abolished by iNOS deletion in phagocytic cells, as well as by extracellular signal-regulated kinase inhibition in PASMCs. Crucially, CD206-positive and iNOS-positive macrophages accumulated in proximity of remodelled vessels in the lungs of COPD patients, as shown by immunohistochemistry., Conclusion: In summary, our results demonstrate that iNOS deletion in myeloid cells confers protection against PH in smoke-exposed mice and provide evidence for an iNOS-dependent communication between M2-like macrophages and PASMCs in underlying pulmonary vascular remodelling., Competing Interests: Conflict of interest: M. Gredic has nothing to disclose. Conflict of interest: C-Y. Wu has nothing to disclose. Conflict of interest: S. Hadzic has nothing to disclose. Conflict of interest: O. Pak has nothing to disclose. Conflict of interest: R. Savai has nothing to disclose. Conflict of interest: B. Kojonazarov has nothing to disclose. Conflict of interest: S. Doswada has nothing to disclose. Conflict of interest: A. Weiss has nothing to disclose. Conflict of interest: A. Weigert has nothing to disclose. Conflict of interest: A. Guenther has nothing to disclose. Conflict of interest: R.P. Brandes has nothing to disclose. Conflict of interest: R.T. Schermuly has nothing to disclose. Conflict of interest: F. Grimminger has nothing to disclose. Conflict of interest: W. Seeger reports personal fees from Actelion, Bayer AG, Novartis, Vectura, Medspray and United Therapeutics, outside the submitted work. Conflict of interest: N. Sommer reports personal fees from Actelion, outside the submitted work. Conflict of interest: S. Kraut has nothing to disclose. Conflict of interest: N. Weissmann reports grants from the German Research Foundation, during the conduct of the study; and has a patent L-NIL as inhibitor for regenerating the lung of a patient suffering from COPD (EP2591777A2) issued., (Copyright ©The authors 2022.)

Background: Pulmonary hypertension (PH) is a life-threatening disease, characterized by excessive pulmonary vascular remodeling, leading to elevated pulmonary arterial pressure and right heart hypertrophy. PH can be caused by chronic hypoxia, leading to hyper-proliferation of pulmonary arterial smooth muscle cells (PASMCs) and apoptosis-resistant pulmonary microvascular endothelial cells (PMVECs). On reexposure to normoxia, chronic hypoxia-induced PH in mice is reversible. In this study, the authors aim to identify novel candidate genes involved in pulmonary vascular remodeling specifically in the pulmonary vasculature., Methods: After microarray analysis, the authors assessed the role of SPARC (secreted protein acidic and rich in cysteine) in PH using lung tissue from idiopathic pulmonary arterial hypertension (IPAH) patients, as well as from chronically hypoxic mice. In vitro studies were conducted in primary human PASMCs and PMVECs. In vivo function of SPARC was proven in chronic hypoxia-induced PH in mice by using an adeno-associated virus-mediated Sparc knockdown approach., Results: C57BL/6J mice were exposed to normoxia, chronic hypoxia, or chronic hypoxia with subsequent reexposure to normoxia for different time points. Microarray analysis of the pulmonary vascular compartment after laser microdissection identified Sparc as one of the genes downregulated at all reoxygenation time points investigated. Intriguingly, SPARC was vice versa upregulated in lungs during development of hypoxia-induced PH in mice as well as in IPAH, although SPARC plasma levels were not elevated in PH. TGF-β1 (transforming growth factor β1) or HIF2A (hypoxia-inducible factor 2A) signaling pathways induced SPARC expression in human PASMCs. In loss of function studies, SPARC silencing enhanced apoptosis and reduced proliferation. In gain of function studies, elevated SPARC levels induced PASMCs, but not PMVECs, proliferation. Coculture and conditioned medium experiments revealed that PMVECs-secreted SPARC acts as a paracrine factor triggering PASMCs proliferation. Contrary to the authors' expectations, in vivo congenital Sparc knockout mice were not protected from hypoxia-induced PH, most probably because of counter-regulatory proproliferative signaling. However, adeno-associated virus-mediated Sparc knockdown in adult mice significantly improved hemodynamic and cardiac function in PH mice., Conclusions: This study provides evidence for the involvement of SPARC in the pathogenesis of human PH and chronic hypoxia-induced PH in mice, most likely by affecting vascular cell function.

Reactive oxygen species (ROS) are important mediators of both physiological and pathophysiological signal transduction in the cardiovascular system. The effects of ROS on cellular processes depend on the concentration, localization, and duration of exposure. Cellular stress response mechanisms have evolved to mitigate the negative effects of acute oxidative stress. In this study, we investigate the short-term and long-term metabolic and transcriptomic response of human umbilical vein endothelial cells (HUVEC) to different types and concentrations of ROS. To generate intracellular H 2 O 2 , we utilized a lentiviral chemogenetic approach for overexpression of human D-amino acid oxidase (DAO). DAO converts D-amino acids into their corresponding imino acids and H 2 O 2 . HUVEC stably overexpressing DAO (DAO-HUVEC) were exposed to D-alanine (3 mM), exogenous H 2 O 2 (10 µM or 300 µM), or menadione (5 µM) for various timepoints and subjected to global untargeted metabolomics (LC-MS/MS) and RNAseq by MACE (Massive analysis of cDNA ends). A total of 300 µM H 2 O 2 led to pronounced changes on both the metabolic and transcriptomic level. In particular, metabolites linked to redox homeostasis, energy-generating pathways, and nucleotide metabolism were significantly altered. Furthermore, 300 µM H 2 O 2 affected genes related to the p53 pathway and cell cycle. In comparison, the effects of menadione and DAO-derived H 2 O 2 mainly occurred at gene expression level. Collectively, all types of ROS led to subtle changes in the expression of ribosomal genes. Our results show that different types and concentration of ROS lead to a different metabolic and transcriptomic response in endothelial cells.

Background: Obstructive sleep apnea (OSA) imposes vascular and metabolic risks through chronic intermittent hypoxia (CIH) and impairs skeletal muscle performance. As studies addressing limb muscles are rare, the reasons for the lower exercise capacity are unknown. We hypothesize that CIH-related morphological alterations in neuromuscular junctions (NMJ) and mitochondrial integrity might be the cause of functional disorders in skeletal muscles., Methods: Mice were kept under 6 weeks of CIH (alternating 7% and 21% O 2 fractions every 30 s, 8 h/day, 5 days/week) compared to normoxia (NOX). Analyses included neuromuscular junctions (NMJ) postsynaptic morphology and integrity, fiber cross-sectional area (CSA) and composition (ATPase), mitochondrial ultrastructure (transmission-electron-microscopy), and relevant transcripts (RT-qPCR). Besides wildtype (WT), we included inducible nitric oxide synthase knockout mice (iNOS -/- ) to evaluate whether iNOS is protective or risk-mediating., Results: In WT soleus muscle, CIH vs. NOX reduced NMJ size (- 37.0%, p < 0.001) and length (- 25.0%, p < 0.05) together with fiber CSA of type IIa fibers (- 14%, p < 0.05) and increased centronucleated fiber fraction (p < 0.001). Moreover, CIH vs. NOX increased the fraction of damaged mitochondria (1.8-fold, p < 0.001). Compared to WT, iNOS -/- similarly decreased NMJ area and length with NOX (- 55%, p < 0.001 and - 33%, p < 0.05, respectively) or with CIH (- 37%, p < 0.05 and - 29%, p < 0.05), however, prompted no fiber atrophy. Moreover, increased fractions of damaged (2.1-fold, p < 0.001) or swollen (> 6-fold, p < 0.001) mitochondria were observed with iNOS -/- vs. WT under NOX and similarly under CIH. Both, CIH- and iNOS -/- massively upregulated suppressor-of-cytokine-signaling-3 (SOCS3) > 10-fold without changes in IL6 mRNA expression. Furthermore, inflammatory markers like CD68 (macrophages) and IL1β were significantly lower in CIH vs. NOX. None of these morphological alterations with CIH- or iNOS -/- were detected in the gastrocnemius muscle. Notably, iNOS expression was undetectable in WT muscle, unlike the liver, where it was massively decreased with CIH., Conclusion: CIH leads to NMJ and mitochondrial damage associated with fiber atrophy/centronucleation selectively in slow-twitch muscle of WT. This effect is largely mimicked by iNOS -/- at NOX (except for atrophy). Both conditions involve massive SOCS3 upregulation likely through denervation without Il6 upregulation but accompanied by a decrease of macrophage density especially next to denervated endplates. In the absence of muscular iNOS expression in WT, this damage may arise from extramuscular, e.g., motoneuronal iNOS deficiency (through CIH or knockout) awaiting functional evaluation., (© 2022. The Author(s).)

The high incidence of thrombotic events suggests a possible role of the contact system pathway in COVID-19 pathology. In this study, we determined the altered levels of factor XII (FXII) and its activation products in critically ill patients with COVID-19 in comparison with patients with severe acute respiratory distress syndrome related to the influenza virus (acute respiratory distress syndrome [ARDS]-influenza). Compatible with those data, we found rapid consumption of FXII in COVID-19 but not in ARDS-influenza plasma. Interestingly, the lag phase in fibrin formation, triggered by the FXII activator kaolin, was not prolonged in COVID-19, as opposed to that in ARDS-influenza. Confocal and electron microscopy showed that increased FXII activation rate, in conjunction with elevated fibrinogen levels, triggered formation of fibrinolysis-resistant, compact clots with thin fibers and small pores in COVID-19. Accordingly, clot lysis was markedly impaired in COVID-19 as opposed to that in ARDS-influenza. Dysregulated fibrinolytic system, as evidenced by elevated levels of thrombin-activatable fibrinolysis inhibitor, tissue-plasminogen activator, and plasminogen activator inhibitor-1 in COVID-19 potentiated this effect. Analysis of lung tissue sections revealed widespread extra- and intravascular compact fibrin deposits in patients with COVID-19. A compact fibrin network structure and dysregulated fibrinolysis may collectively contribute to a high incidence of thrombotic events in COVID-19., (© 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)