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Pritesh P Jain,1 Regina Leber,1,2 Chandran Nagaraj,1 Gerd Leitinger,3 Bernhard Lehofer,4 Horst Olschewski,1,5 Andrea Olschewski,1,6 Ruth Prassl,1,4 Leigh M Marsh11Ludwig Boltzmann Institute for Lung Vascular Research, 2Biophysics Division, Institute of Molecular Biosciences, University of Graz, 3Research Unit Electron Microscopic Techniques, Institute of Cell Biology, Histology, and Embryology, 4Institute of Biophysics, 5Division of Pulmonology, Department of Internal Medicine, 6Department of Anesthesiology and Intensive Care Medicine, Medical University of Graz, Graz, AustriaAbstract: Prostacyclin analogues are standard therapeutic options for vasoconstrictive diseases, including pulmonary hypertension and Raynaud’s phenomenon. Although effective, these treatment strategies are expensive and have several side effects. To improve drug efficiency, we tested liposomal nanoparticles as carrier systems. In this study, we synthesized liposomal nanoparticles tailored for the prostacyclin analogue iloprost and evaluated their pharmacologic efficacy on mouse intrapulmonary arteries, using a wire myograph. The use of cationic lipids, stearylamine, or 1,2-di-(9Z-octadecenoyl)-3-trimethylammonium-propane (DOTAP) in liposomes promoted iloprost encapsulation to at least 50%. The addition of cholesterol modestly reduced iloprost encapsulation. The liposomal nanoparticle formulations were tested for toxicity and pharmacologic efficacy in vivo and ex vivo, respectively. The liposomes did not affect the viability of human pulmonary artery smooth muscle cells. Compared with an equivalent concentration of free iloprost, four out of the six polymer-coated liposomal formulations exhibited significantly enhanced vasodilation of mouse pulmonary arteries. Iloprost that was encapsulated in liposomes containing the polymer polyethylene glycol exhibited concentration-dependent relaxation of arteries. Strikingly, half the concentration of iloprost in liposomes elicited similar pharmacologic efficacy as nonencapsulated iloprost. Cationic liposomes can encapsulate iloprost with high efficacy and can serve as potential iloprost carriers to improve its therapeutic efficacy.Keywords: prostacyclin, cationic liposomes, pulmonary hypertension, wire myograph

RATIONALE: Idiopathic pulmonary arterial hypertension (IPAH) is characterized by medial hypertrophy due to pulmonary artery smooth muscle cell (paSMC) hyperplasia. Inflammation is proposed to play a role in vessel remodeling associated with IPAH. IL-13 is emerging as a regulator of tissue remodeling; however, the contribution of the IL-13 system to IPAH has not been assessed. OBJECTIVES: The objective of this study was to assess the possible contribution of the IL-13 system to IPAH. METHODS: Expression and localization of IL-13, and IL-13 receptors IL-4R, IL-13R[alpha]1, and IL-13R[alpha]2 were assessed by real-time reverse transcription-polymerase chain reaction, immunohistochemistry, and flow cytometry in lung tissue, paSMC, and microdissected vascular lesions from patients with IPAH, and in lung tissue from rodents with hypoxia- or monocrotaline-induced pulmonary hypertension. A whole-genome microarray analysis was used to study IL-13-regulated genes in paSMC. MEASUREMENTS AND MAIN RESULTS: Pulmonary expression of the IL-13 decoy receptor IL-13R[alpha]2 was up-regulated relative to that of the IL-13 signaling receptors IL-4R and IL-13R[alpha]1 in patients with IPAH and in two animal models of IPAH. IL-13, signaling via STAT3 and STAT6, suppressed proliferation of paSMC by promoting G(0)/G(1) arrest. Whole-genome microarrays revealed that IL-13 suppressed endothelin-1 production by paSMC, suggesting that IL-13 controlled paSMC growth by regulating endothelin production. Ectopic expression of the il13ra2 gene resulted in partial loss of paSMC growth control by IL-13 and blunted IL-13 suppression of endothelin-1 production by paSMC, whereas small-interfering RNA knockdown of il13ra2 gene expression had the opposite effects. CONCLUSIONS: The IL-13 system is a novel regulator of paSMC growth. Dysregulation of IL-13 receptor expression in IPAH may partially underlie smooth muscle hypertrophy associated with pathological vascular remodeling in IPAH. [ABSTRACT FROM AUTHOR]

Pulmonary fibrosis (PF) can be a fatal disease characterized by progressive lung scarring. It is still poorly understood how the pulmonary endothelium is involved in the disease pathogenesis. Differences of the pulmonary vasculature between patients and donors were analyzed using transmission electron microscopy, immunohistochemistry, and single-cell RNA sequencing. Vascular barrier resistance, endothelial-immune cell adhesion, and sensitivity to an inflammatory milieu were studied in vitro . Integrity and activation markers were measured by ELISA in human plasma. Transmission electron microscopy demonstrated abnormally swollen endothelial cells (ECs) in fibrotic lungs compared with donors. A more intense CD31 and von Willebrand Factor (vWF) and patchy vascular endothelial (VE)-Cadherin staining in fibrotic lungs supported the presence of a dysregulated endothelium. Integrity markers CD31, VE-Cadherin, Thrombomodulin, and VEGFR-2 (vascular endothelial growth factor receptor-2) and activation marker vWF gene expression was increased in different endothelial subpopulations (e.g., arterial, venous, general capillary, aerocytes) in PF. This was associated with a heightened sensitivity of fibrotic ECs to TNF-α or IFN-γ and elevated immune cell adhesion. The barrier strength was overall reduced in ECs from fibrotic lungs. vWF and IL-8 were increased in the plasma of patients, whereas VE-Cadherin, Thrombomodulin, and VEGFR-2 were decreased. VE-Cadherin staining was also patchy in biopsy tissue and was decreased in plasma samples of patients with PF 6 months after the initial diagnosis. Our data demonstrate highly abnormal ECs in PF. The vascular compartment is characterized by hyperactivation and increased immune cell adhesion, as well as dysfunctional endothelial barrier function. Reestablishing EC homeostasis and function might represent a new therapeutic option for fibrotic lung diseases.

Changes in the extracellular matrix of pulmonary arteries (PAs) are a key aspect of vascular remodeling in pulmonary hypertension (PH). Yet, our understanding of the alterations affecting the proteoglycan (PG) family remains limited. We sought to investigate the expression and spatial distribution of major vascular PGs in PAs from healthy individuals and various PH groups (chronic obstructive pulmonary disease: PH-COPD, pulmonary fibrosis: PH-PF, idiopathic: IPAH). PG regulation, deposition, and synthesis were notably heightened in IPAH, followed by PH-PF, with minor alterations in PH-COPD. Single-cell analysis unveiled cell-type and disease-specific PG regulation. Agrin expression, a basement membrane PG, was increased in IPAH, with PA endothelial cells (PAECs) identified as a major source. PA smooth muscle cells (PASMCs) mainly produced large-PGs, aggrecan and versican, and small-leucine-like proteoglycan (SLRP) biglycan, whereas the major PGs produced by adventitial fibroblasts were SLRP decorin and lumican. In IPAH and PF-PH, the neointima-forming PASMC population increased the expression of all investigated large-PGs and SLRPs, except fibroblast-predominant decorin (DCN). Expression of lumican, versican, and biglycan also positively correlated with collagen 1α1/1α2 expression in PASMCs in patients with IPAH and PH-PF. We demonstrated that transforming growth factor-beta (TGF-β) regulates versican and biglycan expression, indicating their contribution to vessel fibrosis in IPAH and PF-PH. We furthermore show that certain circulating PG levels display a disease-dependent pattern, with increased decorin and lumican across all patient groups, while versican was elevated in PH-COPD and IPAH and biglycan reduced in IPAH. These findings suggest unique compartment-specific PG regulation in different forms of PH, indicating distinct pathological processes. NEW & NOTEWORTHY Idiopathic pulmonary arterial hypertension (IPAH) pulmonary arteries (PAs) displayed the greatest proteoglycan (PG) changes, with PH associated with pulmonary fibrosis (PH-PF) and PH associated with chronic obstructive pulmonary disease (PH-COPD) following. Agrin, an endothelial cell-specific PG, was solely upregulated in IPAH. Among all cells, neo-intima-forming smooth muscle cells (SMCs) displayed the most significant PG increase. Increased levels of circulating decorin, lumican, and versican, mainly derived from SMCs, and adventitial fibroblasts, may serve as systemic indicators of pulmonary remodeling, reflecting perivascular fibrosis and neointima formation.

Background: In patients with end-stage chronic obstructive pulmonary disease (COPD), severe pulmonary hypertension (PH) is frequently associated with less severe airway obstruction as compared to mild or no PH. However, the histologic correlate of this finding is not clear. We aimed to quantify remodeling of pulmonary arteries, airways, and parenchyma in random samples of explanted end-stage COPD lungs., Methods: We quantified remodeling of small pulmonary arteries, small airways, and the degree of emphysema (mean interseptal distance [MID]) with dedicated software. As primary objective, we compared COPD patients with severe PH (SevPH-COPD) with age- and sex-matched MildPH-COPD. For comparison, we also investigated COPD lungs with no PH (NoPH-COPD), idiopathic PAH (IPAH), and healthy donors., Results: We included n = 17 SevPH-COPD (mPAP = 43 [39-45]mm Hg), n = 17 MildPH-COPD (mPAP = 28 [24-31]mm Hg), n = 5 NoPH-COPD (mPAP = 18 [16-19]mm Hg), n = 10 IPAH (mPAP = 72 [65-91]mm Hg), and n = 10 healthy donor lungs. SevPH-COPD versus MildPH-COPD was characterized by better preserved forced vital capacity (51% vs 40% predicted, p < 0.05), less emphysema (MID 169 µm vs 279 µm, p < 0.001), and less PAS-positive and CD45-positive mucosa cells (15% vs 22%, p = 0.063% and 5% vs 7%, p = 0.058) suggesting less airway inflammation. In COPD patients, intimal and medial thickening were strongly correlated with mPAP (r = 0.676, p < 0.001 and r = 0.595, p < 0.001). MID was negatively correlated with mPAP (r = -0.556, p < 0.001) and was highest in NoPH-COPD (mean 281 µm), suggesting that emphysema per se is not associated with PH., Conclusions: End-stage COPD with severe PH is characterized by pronounced pulmonary vascular remodeling, less inflammation of small airways, and less emphysema as compared to COPD with mild PH or no PH, suggesting that COPD with severe PH may represent a unique phenotype of COPD., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Deposition of basement membrane components, such as collagen IVα5, is associated with altered endothelial cell function in pulmonary hypertension. Collagen IVα5 harbors a functionally active fragment within its C-terminal noncollageneous (NC1) domain, called pentastatin, whose role in pulmonary endothelial cell behavior remains unknown. Here, we demonstrate that pentastatin serves as a mediator of pulmonary endothelial cell dysfunction, contributing to pulmonary hypertension. In vitro, treatment with pentastatin induced transcription of immediate early genes and proinflammatory cytokines and led to a functional loss of endothelial barrier integrity in pulmonary arterial endothelial cells. Mechanistically, pentastatin leads to β1-integrin subunit clustering and Rho/ROCK activation. Blockage of the β1-integrin subunit or the Rho/ROCK pathway partially attenuated the pentastatin-induced endothelial barrier disruption. Although pentastatin reduced the viability of endothelial cells, smooth muscle cell proliferation was induced. These effects on the pulmonary vascular cells were recapitulated ex vivo in the isolated-perfused lung model, where treatment with pentastatin-induced swelling of the endothelium accompanied by occasional endothelial cell apoptosis. This was reflected by increased vascular permeability and elevated pulmonary arterial pressure induced by pentastatin. This study identifies pentastatin as a mediator of endothelial cell dysfunction, which thus might contribute to the pathogenesis of pulmonary vascular disorders such as pulmonary hypertension. NEW & NOTEWORTHY This study is the first to show that pentastatin, the matrikine of the basement membrane (BM) collagen IVα5 polypeptide, triggers rapid pulmonary arterial endothelial cell barrier disruption, activation, and apoptosis in vitro and ex vivo. Mechanistically, pentastatin partially acts through binding to the β1-integrin subunit and the Rho/ROCK pathway. These findings are the first to link pentastatin to pulmonary endothelial dysfunction and, thus, suggest a major role for BM-matrikines in pulmonary vascular diseases such as pulmonary hypertension.

Rationale and Objective: Plasma extracellular vesicles (EVs) represent a vital source of molecular information about health and disease states. Due to their heterogenous cellular sources, EVs and their cargo may predict specific pathomechanisms behind disease phenotypes. Here we aimed to utilize EV microRNA (miRNA) signatures to gain new insights into underlying molecular mechanisms of obesity-associated low type-2 asthma., Methods: Obese low type-2 asthma (OA) and non-obese low type-2 asthma (NOA) patients were selected from an asthma cohort conjointly with healthy controls. Plasma EVs were isolated and characterised by nanoparticle tracking analysis. EV-associated small RNAs were extracted, sequenced and bioinformatically analysed., Results: Based on EV miRNA expression profiles, a clear distinction between the three study groups could be established using a principal component analysis. Integrative pathway analysis of potential target genes of the differentially expressed miRNAs revealed inflammatory cytokines (e.g., interleukin-6, transforming growth factor-beta, interferons) and metabolic factors (e.g., insulin, leptin) signalling pathways to be specifically associated with OA. The miR-17-92 and miR-106a-363 clusters were significantly enriched only in OA. These miRNA clusters exhibited discrete bivariate correlations with several key laboratory (e.g., C-reactive protein) and lung function parameters. Plasma EV miRNA signatures mirrored blood-derived CD4 + T-cell transcriptome data, but achieved an even higher sensitivity in identifying specifically affected biological pathways., Conclusion: The identified plasma EV miRNA signatures and particularly the miR-17-92 and -106a-363 clusters were capable to disentangle specific mechanisms of the obesity-associated low type-2 asthma phenotype, which may serve as basis for stratified treatment development., (© 2023 The Authors. Allergy published by European Academy of Allergy and Clinical Immunology and John Wiley & Sons Ltd.)

The pulmonary vasculature has been frequently overlooked in acute and chronic lung diseases, such as acute respiratory distress syndrome (ARDS), pulmonary fibrosis (PF), and chronic obstructive pulmonary disease (COPD). The primary emphasis in the management of these parenchymal disorders has largely revolved around the injury and aberrant repair of epithelial cells. However, there is increasing evidence that the vascular endothelium plays an active role in the development of acute and chronic lung diseases. The endothelial cell network in the capillary bed and the arterial and venous vessels provides a metabolically highly active barrier that controls the migration of immune cells, regulates vascular tone and permeability, and participates in the remodeling processes. Phenotypically and functionally altered endothelial cells, and remodeled vessels, can be found in acute and chronic lung diseases, although to different degrees, likely because of disease-specific mechanisms. Since vascular remodeling is associated with pulmonary hypertension, which worsens patient outcomes and survival, it is crucial to understand the underlying vascular alterations. In this Review, we describe the current knowledge regarding the role of the pulmonary vasculature in the development and progression of ARDS, PF, and COPD; we also outline future research directions with the hope of facilitating the development of mechanism-based therapies.

Pulmonary hypertension (PH) has a high mortality and few treatment options. Adaptive immune mediators of PH in mice challenged with antigen/particulate matter (antigen/PM) has been the focus of our prior work. We identified key roles of type-2- and type-17 responses in C57BL/6 mice. Here, we focused on type-2-response-related cytokines, specifically resistin-like molecule (RELM)α, a critical mediator of hypoxia-induced PH. Because of strain differences in the immune responses to type 2 stimuli, we compared C57BL/6J and BALB/c mice. A model of intraperitoneal antigen sensitization with subsequent, intranasal challenges with antigen/PM (ovalbumin and urban ambient PM 2.5 ) or saline was used in C57BL/6 and BALB/c wild-type or RELMα-/- mice. Vascular remodeling was assessed with histology; right ventricular (RV) pressure, RV weights and cytokines were quantified. Upon challenge with antigen/PM, both C57BL/6 and BALB/c mice developed pulmonary vascular remodeling; these changes were much more prominent in the C57BL/6 strain. Compared to wild-type mice, RELMα-/- had significantly reduced pulmonary vascular remodeling in BALB/c, but not in C57BL/6 mice. RV weights, RV IL-33 and RV IL-33 -receptor were significantly increased in BALB/c wild-type mice, but not in BALB/c-RELMα-/- or in C57BL/6-wild-type or C57BL/6-RELMα-/- mice in response to antigen/PM 2.5 . RV systolic pressures (RVSP) were higher in BALB/c compared to C57BL/6J mice, and RELMα-/- mice were not different from their respective wild-type controls. The RELMα-/- animals demonstrated significantly decreased expression of RELMβ and RELMγ, which makes these mice comparable to a situation where human RELMβ levels would be significantly modified, as only humans have this single RELM molecule. In BALB/c mice, RELMα was a key contributor to pulmonary vascular remodeling, increase in RV weight and RV cytokine responses induced by exposure to antigen/PM 2.5 , highlighting the significance of the genetic background for the biological role of RELMα.

Objective: The management of delayed cerebral ischemia after aneurysmal subarachnoid hemorrhage (aSAH) remains one of the most important targets for neurocritical care. Advances in monitoring technology have facilitated a more thorough understanding of the pathophysiology and therapeutic approaches, but interventions are generally limited to either systemic therapies or passive CSF drainage. The authors present a novel approach that combines a multimodal monitoring bolt-based system with an irrigating ventricular drain capable of delivering intrathecal medications and describe their early experience in patients with aSAH., Methods: The authors performed a retrospective review of cases treated with the combined Hummingbird multimodal bolt system and the IRRAflow irrigating ventriculostomy., Results: Nine patients were treated with the combined multimodal bolt system with irrigating ventriculostomy approach. The median number of days to clearance of the third and fourth ventricles was 3 days in patients with obstructive intraventricular hemorrhage. Two patients received intrathecal alteplase for intraventricular hemorrhage clearance, and 2 patients received intrathecal nicardipine as rescue therapy for severe symptomatic angiographic vasospasm., Conclusions: Combined CSF drainage, irrigation, multimodality monitoring, and automated local drug delivery are feasible using a single twist-drill hole device. Further investigation of irrigation settings and treatment approaches in high-risk cases is warranted.

Background: Smooth muscle cell (SMC) expansion is one key morphological hallmark of pathologically altered vasculature and a characteristic feature of pulmonary vascular remodeling in pulmonary hypertension. Normal embryonal vessel maturation requires successful coverage of endothelial tubes with SMC, which is dependent on ephrin-B2 and EphB4 ligand-receptor guidance system. In this study, we investigated the potential role of ephrin-B2 and EphB4 on neomuscularization in adult pulmonary vascular disease., Methods and Results: Ephrin-B2 and EphB4 expression is preserved in smooth muscle and endothelial cells of remodeled pulmonary arteries. Chronic hypoxia-induced pulmonary hypertension was not ameliorated in mice with SMC-specific conditional ephrin-B2 knockout. In mice with global inducible ephrin-B2 knockout, pulmonary vascular remodeling and right ventricular hypertrophy upon chronic hypoxia exposure were significantly diminished compared to hypoxic controls, while right ventricular systolic pressure was unaffected. In contrast, EphB4 receptor kinase activity inhibition reduced right ventricular systolic pressure in hypoxia-induced pulmonary hypertension without affecting pulmonary vascular remodeling. Genetic deletion of ephrin-B2 in murine pulmonary artery SMC, and pharmacological inhibition of EphB4 in human pulmonary artery smooth muscle cells, blunted mitogen-induced cell proliferation. Loss of EphB4 signaling additionally reduced RhoA expression and weakened the interaction between human pulmonary artery smooth muscle cells and endothelial cells in a three-dimensional coculture model., Conclusions: In sum, pulmonary vascular remodeling was dependent on ephrin-B2-induced Eph receptor (erythropoietin-producing hepatocellular carcinoma receptor) forward signaling in SMC, while EphB4 receptor activity was necessary for RhoA expression in SMC, interaction with endothelial cells and vasoconstrictive components of pulmonary hypertension.

Hemodynamic factors have long been associated with clinical outcomes in the treatment of cerebral aneurysms. Computational studies of cerebral aneurysm hemodynamics have provided valuable estimates of the mechanical environment experienced by the endothelium in both the parent vessel and aneurysmal dome walls and have correlated them with disease state. These computational-clinical studies have recently been correlated with the response of endothelial cells (EC) using either idealized or patient-specific models. Here, we present a robust workflow for generating anatomic-scale aneurysm models, establishing luminal cultures of ECs at physiological relevant flow profiles, and comparing EC responses to curvature mediated flow. We show that flow patterns induced by parent vessel curvature produce changes in wall shear stress (WSS) and wall shear stress gradients (WSSG) that are correlated with differences in cell morphology and cellular protein localization. Cells in higher WSS regions align better with the flow and display strong Notch1-extracellular domain (ECD) polarization, while, under low WSS, differences in WSSG due to curvature change were associated with less alignment and attenuation of Notch1-ECD polarization in ECs of the corresponding regions. These proof-of-concept results highlight the use of engineered cellularized aneurysm models for connecting computational fluid dynamics to the underlying endothelial biology that mediates disease., (Copyright © 2023 by ASME.)

A central feature of progressive vascular remodeling is altered smooth muscle cell (SMC) homeostasis; however, the understanding of how different cell populations contribute to this process is limited. Here, we utilized single-cell RNA sequencing to provide insight into cellular composition changes within isolated pulmonary arteries (PAs) from pulmonary arterial hypertension and donor lungs. Our results revealed that remodeling skewed the balanced communication network between immune and structural cells, in particular SMCs. Comparative analysis with murine PAs showed that human PAs harbored heterogeneous SMC populations with an abundant intermediary cluster displaying a gradient transition between SMCs and adventitial fibroblasts. Transcriptionally distinct SMC populations were enriched in specific biological processes and could be differentiated into 4 major clusters: oxygen sensing (enriched in pericytes), contractile, synthetic, and fibroblast-like. End-stage remodeling was associated with phenotypic shift of preexisting SMC populations and accumulation of synthetic SMCs in neointima. Distinctly regulated genes in clusters built nonredundant regulatory hubs encompassing stress response and differentiation regulators. The current study provides a blueprint of cellular and molecular changes on a single-cell level that are defining the pathological vascular remodeling process.

Background: Systemic sclerosis (SSc) is an autoimmune disease characterised by severe vasculopathy and fibrosis of various organs including the lung. Targeted treatment options for SSc-associated interstitial lung disease (SSc-ILD) are scarce. We assessed the effects of pirfenidone in a mouse model of SSc-ILD., Methods: Pulmonary function, inflammation and collagen deposition in response to pirfenidone were assessed in Fra-2-overexpressing transgenic (Fra-2 TG) and bleomycin-treated mice. In Fra-2 TG mice, lung transcriptome was analysed after pirfenidone treatment. In vitro , pirfenidone effects on human eosinophil and endothelial cell function were analysed using flow cytometry-based assays and electric cell-substrate impedance measurements, respectively., Results: Pirfenidone treatment attenuated pulmonary remodelling in the bleomycin model, but aggravated pulmonary inflammation, fibrosis and vascular remodelling in Fra-2 TG mice. Pirfenidone increased interleukin (IL)-4 levels and eosinophil numbers in lung tissue of Fra-2 TG mice without directly affecting eosinophil activation and migration in vitro . A pronounced immune response with high levels of cytokines/chemokines and disturbed endothelial integrity with low vascular endothelial (VE)-cadherin levels was observed in pirfenidone-treated Fra-2 TG mice. In contrast, eosinophil and VE-cadherin levels were unchanged in bleomycin-treated mice and not influenced by pirfenidone. In vitro , pirfenidone exacerbated the IL-4 induced reduction of endothelial barrier resistance, leading to higher leukocyte transmigration., Conclusion: This study shows that antifibrotic properties of pirfenidone may be overruled by unwanted interactions with pre-injured endothelium in a setting of high T-helper type 2 inflammation in a model of SSc-ILD. Careful ILD patient phenotyping may be required to exploit benefits of pirfenidone while avoiding therapy failure and additional lung damage in some patients., Competing Interests: Conflict of interest: A. Heinemann has received remunerations from AstraZeneca for lecture activities; grants from Austrian Science Funds, FWF. H. Olschewski received consulting fees from Actelion, Bayer AG, Böhringer Ingelheim, Inventiva, Janssen Pharmaceutica, MSD (Merck Sharp & Dohme), payment for educational events from Böhringer Ingelheim and remunerations for participation on advisory boards from Bayer AG, Pfizer, MSD (Merck Sharp & Dohme) and IQVIA Biotech LLC, and is Deputy Director of the Ludwig Boltzmann Institute for Lung Vascular Research. G. Kwapiszewska is Director of LBI-LVR. All other authors have nothing to disclose., (Copyright ©The authors 2022.)

Rationale: Pulmonary hypertension (PH) is a common, severe comorbidity in interstitial lung diseases such as pulmonary fibrosis (PF), and it has limited treatment options. Excessive vascular fibrosis and inflammation are often present in PH, but the underlying mechanisms are still not well understood. Objectives: To identify a novel functional link between natural killer T (NKT) cell activation and vascular fibrosis in PF-PH. Methods: Multicolor flow cytometry, secretome, and immunohistological analyses were complemented by pharmacological NKT cell activation in vivo , in vitro , and ex vivo . Measurements and Main Results: In pulmonary vessels of patients with PF-PH, increased collagen deposition was linked to a local NKT cell deficiency and decreased IL-15 concentrations. In a mouse model of PH caused by lung fibrosis, pharmacological NKT cell activation using a synthetic α-galactosylceramide analog (KRN7000) restored local NKT cell numbers and ameliorated vascular remodeling and right ventricular systolic pressure. Supplementation with activated NKT cells reduced collagen deposition in isolated human pulmonary arterial smooth muscle cells (hPASMCs) and in ex vivo precision-cut lung slices of patients with end-stage PF-PH. Coculture with activated NKT cells induced STAT1 signaling in hPASMCs. Secretome analysis of peripheral blood mononuclear cells identified CXCL9 and CXCL10 as indicators of NKT cell activation. Pharmacologically, CXCL9, but not CXCL10, potently inhibited collagen deposition in hPASMCs via the chemokine receptor CXCR3. Conclusions: Our results indicate that the absence of NKT cells impairs the STAT1-CXCL9-CXCR3 axis in PF-PH and that restoration of this axis by NKT cell activation may unravel a novel therapeutic strategy to target vascular fibrosis in interstitial lung disease.

Secondary infections contribute significantly to covid-19 mortality but driving factors remain poorly understood. Autopsies of 20 covid-19 cases and 14 controls from the first pandemic wave complemented with microbial cultivation and RNA-seq from lung tissues enabled description of major organ pathologies and specification of secondary infections. Lethal covid-19 segregated into two main death causes with either dominant diffuse alveolar damage (DAD) or secondary pneumonias. The lung microbiome in covid-19 showed a reduced biodiversity and increased prototypical bacterial and fungal pathogens in cases of secondary pneumonias. RNA-seq distinctly mirrored death causes and stratified DAD cases into subgroups with differing cellular compositions identifying myeloid cells, macrophages and complement C1q as strong separating factors suggesting a pathophysiological link. Together with a prominent induction of inhibitory immune-checkpoints our study highlights profound alterations of the lung immunity in covid-19 wherein a reduced antimicrobial defense likely drives development of secondary infections on top of SARS-CoV-2 infection., Competing Interests: H.L. is founder of Alacris Theranostics GmbH and M.S. and L.O. are employees of Alacris Theranostics GmbH. K. Z. is CEO and founder of Zatloukal Innovations GmbH. All other authors declare no conflicts of interest., (© 2022 The Authors.)

Natural killer (NK) cells play an important role in recognizing and killing pathogen-infected or malignant cells. Changes in their numbers or activation can contribute to several diseases and pathologies including systemic sclerosis (SSc), an autoimmune disease characterized by inflammation and tissue remodeling. In these patients, increased expression of the AP-1 transcription factor, Fra-2 was reported. In mice ectopic overexpression of Fra-2 (TG) leads to SSc with strong pulmonary fibrosis, pulmonary hypertension, and inflammation. Analysis of the underlying immune cell profile in the lungs of young TG mice, which do not yet show any signs of lung disease, revealed increased numbers of eosinophils and T cells but strongly reduced NK numbers. Therefore, we aimed to identify the cause of the absence of NK cells in the lungs of these mice and to determine the potential role of Fra-2 in NK development. Examination of inflammatory cell distribution in TG mice revealed similar NK deficiencies in the spleen, blood, and bone marrow. Deeper analysis of the WT and TG bone marrow revealed a potential NK cell developmental defect beginning at the preNKP stage. To determine whether this defect was cell-intrinsic or extrinsic, mixed bone marrow chimera and in vitro differentiation experiments were performed. Both experiments showed that the defect caused by Fra-2 was primarily cell-intrinsic and minimally dependent on the environment. Closer examination of surface markers and transcription factors required for NK development, revealed the expected receptor distribution but changes in transcription factor expression. We found a significant reduction in Nfil3, which is essential for the transition of common lymphoid cells to NK committed precursor cells and an AP-1 binding site in the promotor of this gene. In Summary, our data demonstrates that regulation of Fra-2 is essential for NK development and maturation, and suggests that the early NK dysfunction plays an important role in the pathogenesis of systemic sclerosis., 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 Schnoegl, Hochgerner, Gotthardt and Marsh.)

The lung airways are constantly exposed to inhaled toxic substances, resulting in cellular damage that is repaired by local expansion of resident bronchiolar epithelial club cells. Disturbed bronchiolar epithelial damage repair lies at the core of many prevalent lung diseases, including chronic obstructive pulmonary disease, asthma, pulmonary fibrosis, and lung cancer. However, it is still not known how bronchiolar club cell energy metabolism contributes to this process. Here, we show that adipose triglyceride lipase (ATGL), the rate-limiting enzyme for intracellular lipolysis, is critical for normal club cell function in mice. Deletion of the gene encoding ATGL, Pnpla2 (also known as Atgl), induced substantial triglyceride accumulation, decreased mitochondrial numbers, and decreased mitochondrial respiration in club cells. This defect manifested as bronchiolar epithelial thickening and increased airway resistance under baseline conditions. After naphthalene‑induced epithelial denudation, a regenerative defect was apparent. Mechanistically, dysfunctional PPARα lipid-signaling underlies this phenotype because (a) ATGL was needed for PPARα lipid-signaling in regenerating bronchioles and (b) administration of the specific PPARα agonist WY14643 restored normal bronchiolar club cell ultrastructure and regenerative potential. Our data emphasize the importance of the cellular energy metabolism for lung epithelial regeneration and highlight the significance of ATGL-mediated lipid catabolism for lung health.

Background: High-altitude therapy has been used as add-on treatment for allergic asthma with considerable success. However, the underlying mechanisms remain unclear. In order to investigate the possible therapeutic effects of high-altitude therapy on allergic asthma, we utilized a new in vivo mouse model., Methods: Mice were treated with house dust mite (HDM) extract over 4 weeks and co-exposed to 10% oxygen (Hyp) or room air for the final 2 weeks. Experimental asthma was assessed by airway hyper-responsiveness, mucus hypersecretion and inflammatory cell recruitment. Isolated immune cells from mouse and allergic patients were stimulated in vitro with HDM under Hyp and normoxia in different co-culture systems to analyse the adaptive immune response., Results: Compared to HDM-treated mice in room air, HDM-treated Hyp-mice displayed ameliorated mucosal hypersecretion and airway hyper-responsiveness. The attenuated asthma phenotype was associated with strongly reduced activation of antigen-presenting cells (APCs), effector cell infiltration and cytokine secretion. In vitro, hypoxia almost completely suppressed the HDM-induced adaptive immune response in both mouse and human immune cells. While hypoxia did not affect effector T-cell responses per-se, it interfered with antigen-presenting cell (APC) differentiation and APC/effector cell crosstalk., Conclusions: Hypoxia-induced reduction in the Th2-response to HDM ameliorates allergic asthma in vivo. Hypoxia interferes with APC/T-cell crosstalk and confers an unresponsive phenotype to APCs., (© 2021 The Authors. Allergy published by European Academy of Allergy and Clinical Immunology and John Wiley & Sons Ltd.)

Numerous studies assess intracranial aneurysm rupture risk based on morphological and hemodynamic parameter analysis in addition to clinical information such as aneurysm localization, age, and sex. However, intracranial aneurysms mostly occur with a saccular shape located either lateral to the parent artery or at a bifurcation. In contrast, fusiform intracranial aneurysms (FIAs), i.e., aneurysms with a non-saccular, dilated form, occur in approximately 3-13% of all cases and therefore have not yet been as thoroughly studied. To improve the understanding of FIA hemodynamics, this pilot study contains morphological analyses and image-based blood flow simulations in three patient-specific cases. For a precise and realistic comparison to the pre-pathological state, each dilation was manually removed and the time-dependent blood flow simulations were repeated. Additionally, a validated fast virtual stenting approach was applied to evaluate the effect of virtual endovascular flow-diverter deployment focusing on relevant hemodynamic quantities. For two of the three patients, post-interventional information was available and included in the analysis. The results of this numerical pilot study indicate that complex flow structures, i.e., helical flow phenomena and the presence of high oscillating flow features, predominantly occur in FIAs with morphologically differing appearances. Due to the investigation of the individual healthy states, the original flow environment could be restored which serves as a reference for the virtual treatment target. It was shown that the realistic deployment led to a considerable stabilization of the individual hemodynamics in all cases. Furthermore, a quantification of the stent-induced therapy effect became feasible for the treating physician. The results of the morphological and hemodynamic analyses in this pilot study show that virtual stenting can be used in FIAs to quantify the effect of the planned endovascular treatment., Competing Interests: DB is a Proctor/Advisor: Phenox, Balt, Acandis, Thromb X, Perflow, Kaneka, Penumbra, outside of the submitted work. 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 © 2022 Saalfeld, Stahl, Korte, Miller Marsh, Preim, Beuing, Cherednychenko, Behme and Berg.)

In the era of personalized medicine, insights into the molecular mechanisms that differentially contribute to disease phenotypes, such as asthma phenotypes including obesity-associated asthma, are urgently needed. Peripheral blood was drawn from 10 obese, non-atopic asthmatic adults with a high body mass index (BMI; 36.67 ± 6.90); 10 non-obese, non-atopic asthmatic adults with normal BMI (23.88 ± 2.73); and 10 healthy controls with normal BMI (23.62 ± 3.74). All asthmatic patients were considered to represent a low type-2 asthma phenotype according to selective clinical parameters. RNA sequencing (RNA-Seq) was conducted on peripheral blood CD4 + T cells. Thousands of differentially expressed genes were identified in both asthma groups compared with heathy controls. The expression of interferon (IFN)-stimulated genes associated with IFN-related signaling pathways was specifically affected in obese asthmatics, while the gap junction and G protein-coupled receptor (GPCR) ligand binding pathways were enriched in both asthma groups. Furthermore, obesity gene markers were also upregulated in CD4 + T cells from obese asthmatics compared with the two other groups. Additionally, the enriched genes of the three abovementioned pathways showed a unique correlation pattern with various laboratory and clinical parameters. The specific activation of IFN-related signaling and viral infection pathways might provide a novel view of the molecular mechanisms associated with the development of the low type-2 obesity-associated asthma phenotype, which is a step ahead in the development of new stratified therapeutic approaches.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a serious threat to healthcare systems worldwide. Binding of the virus to angiotensin-converting enzyme 2 (ACE2) is an important step in the infection mechanism. However, it is unknown if ACE2 expression in patients with chronic lung diseases (CLDs), such as chronic obstructive pulmonary disease (COPD), idiopathic pulmonary arterial hypertension (IPAH), or pulmonary fibrosis (PF), is changed as compared to controls. We used lung samples from patients with COPD (n = 28), IPAH (n = 10), and PF (n = 10) as well as healthy control donor (n = 10) tissue samples to investigate the expression of ACE2 and related cofactors that might influence the course of SARS-CoV-2 infection. Expression levels of the ACE2 receptor, the putative receptor CD147/BSG, and the viral entry cofactors TMPRSS2 (transmembrane serine protease 2), EZR, and FURIN were determined by quantitative PCR and in open-access RNA sequencing datasets. Immunohistochemical and single-cell RNA sequencing (scRNAseq) analyses were used for localization and coexpression, respectively. Soluble ACE2 (sACE2) plasma levels were analyzed by enzyme-linked immunosorbent assay. In COPD as compared to donor, IPAH, and PF lung tissue, gene expression of ACE2, TMPRSS2, and EZR was significantly elevated, but circulating sACE2 levels were significantly reduced in COPD and PF plasma compared to healthy control and IPAH plasma samples. Lung tissue expressions of FURIN and CD147/BSG were downregulated in COPD. None of these changes were associated with changes in pulmonary hemodynamics. Histological analysis revealed coexpression of ACE2, TMPRSS2, and Ezrin in bronchial regions and epithelial cells. This was confirmed by scRNAseq analysis. There were no significant expression changes of the analyzed molecules in the lung tissue of IPAH and idiopathic PF as compared to control. In conclusion, we reveal increased ACE2 and TMPRSS2 expression in lung tissue with a concomitant decrease of protective sACE2 in COPD patients. These changes represent the possible risk factors for an increased susceptibility of COPD patients to SARS-CoV-2 infection., (© 2021 The Authors. The Journal of Pathology: Clinical Research published by The Pathological Society of Great Britain and Ireland & John Wiley & Sons, Ltd.)

The regulator of G protein signaling (RGS) represents a widespread system of controllers of cellular responses. The activities of the R4 subfamily of RGSs have been elucidated in allergic pulmonary diseases. However, the R4 signaling in other inflammatory lung diseases, with a strong cellular immune response, remained unexplored. Thus, our study aimed to discern the functional relevance of the R4 family member, RGS5, as a potential modulating element in this context. Gene profiling of the R4 subfamily showed increased RGS5 expression in human fibrosing lung disease samples. In line with this, RGS5 was markedly increased in murine lungs following bleomycin injury. RGS knock-out mice (RGS-/-) had preserved lung function while control mice showed significant combined ventilatory disorders three days after bleomycin application as compared to untreated control mice. Loss of RGS5 was associated with a significantly reduced neutrophil influx and tissue myeloperoxidase expression. In the LPS lung injury model, RGS5-/- mice also failed to recruit neutrophils into the lung, which was accompanied by reduced tissue myeloperoxidase levels after 24 h. Our in-vitro assays showed impaired migration of RGS5-/- neutrophils towards chemokines despite preserved Ca 2+ signaling. ERK dephosphorylation might play a role in reduced neutrophil migration in our model. As a conclusion, loss of RGS5 preserves lung function and attenuates hyperinflammation in the acute phase of bleomycin-induced pulmonary fibrosis and LPS-induced lung injury. Targeting RGS5 might alleviate the severity of exacerbations in interstitial lung diseases.

The coronavirus disease-2019 (COVID-19) pandemic has caused an enormous loss of lives. Various clinical trials of vaccines and drugs are being conducted worldwide; nevertheless, as of today, no effective drug exists for COVID-19. The identification of key genes and pathways in this disease may lead to finding potential drug targets and biomarkers. Here, we applied weighted gene co-expression network analysis and LIME as an explainable artificial intelligence algorithm to comprehensively characterize transcriptional changes in bronchial epithelium cells (primary human lung epithelium (NHBE) and transformed lung alveolar (A549) cells) during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Our study detected a network that significantly correlated to the pathogenicity of COVID-19 infection based on identified hub genes in each cell line separately. The novel hub gene signature that was detected in our study, including PGLYRP4 and HEPHL1 , may shed light on the pathogenesis of COVID-19, holding promise for future prognostic and therapeutic approaches. The enrichment analysis of hub genes showed that the most relevant biological process and KEGG pathways were the type I interferon signaling pathway, IL-17 signaling pathway, cytokine-mediated signaling pathway, and defense response to virus categories, all of which play significant roles in restricting viral infection. Moreover, according to the drug-target network, we identified 17 novel FDA-approved candidate drugs, which could potentially be used to treat COVID-19 patients through the regulation of four hub genes of the co-expression network. In conclusion, the aforementioned hub genes might play potential roles in translational medicine and might become promising therapeutic targets. Further in vitro and in vivo experimental studies are needed to evaluate the role of these hub genes in COVID-19.

Idiopathic pulmonary fibrosis (IPF) is a deadly condition characterized by progressive respiratory dysfunction. Exacerbations due to airway infections are believed to promote disease progression, and presence of Streptococcus in the lung microbiome has been associated with the progression of IPF and mortality. The aim of this study was to analyze the effect of lung fibrosis on susceptibility to pneumococcal pneumonia and bacteremia. The effects of subclinical (low dose) infection with Streptococcus pneumoniae were studied in a well characterized fos-related antigen-2 (Fra-2) transgenic (TG) mouse model of spontaneous, progressive pulmonary fibrosis. Forty-eight hours after transnasal infection with S. pneumoniae , bacterial load was assessed in lung tissue, bronchoalveolar lavage (BAL), blood, and spleen. Leukocyte subsets and cytokine levels were analyzed in BAL and blood. Lung compliance and arterial blood gases were assessed. In contrast to wildtype mice, low dose lung infection with S. pneumoniae in Fra-2 TG mice resulted in substantial pneumonia including weight loss, increased lung bacterial load, and bacteremia. BAL alveolar macrophages were reduced in Fra-2 TG mice compared to the corresponding WT mice. Proinflammatory cytokines and chemokines (IL-1β, IL-6, TNF-α, and CXCL1) were elevated upon infection in BAL supernatant and plasma of Fra-2 TG mice. Lung compliance was decreased in Fra-2 TG mice following low dose infection with S. pneumoniae . Pulmonary fibrosis increases susceptibility to pneumococcal pneumonia and bacteremia possibly via impaired alveolar bacterial clearance.

Life-threatening inflammatory conditions such as acute respiratory distress syndrome or sepsis often go hand in hand with severe vascular leakage. During inflammation, endothelial cell integrity and intact barrier function are crucial to limit leukocyte and plasma extravasation. Prostaglandin D 2 (PGD 2 ) is a potent inflammatory lipid mediator with vasoactive properties. Previous studies suggest that PGD 2 is involved in the regulation of endothelial barrier function; however, it is unclear whether this is also true for primary human pulmonary microvascular endothelial cells. Furthermore, as PGD 2 is a highly promiscuous ligand, we set out to determine which receptors are important in human pulmonary endothelial cells. In the current study, we found that PGD 2 and the DP1 agonist BW245c potently strengthened pulmonary and dermal microvascular endothelial cell barrier function and protected against thrombin-induced barrier disruption. Yet surprisingly, these effects were mediated only to a negligible extent via DP1 receptor activation. In contrast, we observed that the EP4 receptor was most important and mediated the barrier enhancement by PGD 2 and BW245c. Stimulation with PGE 2 or PGD 2 reduced AKT phosphorylation which could be reversed by prior blockade of EP4 receptors. These data demonstrate a novel mechanism by which PGD 2 may modulate inflammation and emphasizes the role of EP4 receptors in human endothelial cell function., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

The bleomycin mouse model is the extensively used model to study pulmonary fibrosis; however, the inflammatory cell kinetics and their compartmentalization is still incompletely understood. Here we assembled historical flow cytometry data, totaling 303 samples and 16 inflammatory-cell populations, and applied advanced data modeling and machine learning methods to conclusively detail these kinetics. Three days post-bleomycin, the inflammatory profile was typified by acute innate inflammation, pronounced neutrophilia, especially of SiglecF + neutrophils, and alveolar macrophage loss. Between 14 and 21 days, rapid responders were increasingly replaced by T and B cells and monocyte-derived alveolar macrophages. Multicolour imaging revealed the spatial-temporal cell distribution and the close association of T cells with deposited collagen. Unbiased immunophenotyping and data modeling exposed the dynamic shifts in immune-cell composition over the course of bleomycin-triggered lung injury. These results and workflow provide a reference point for future investigations and can easily be applied in the analysis of other datasets., Competing Interests: The authors declare that they have no competing interests., (© 2020 The Author(s).)

The extracellular matrix (ECM) increasingly emerges as an active driver in several diseases, including idiopathic pulmonary arterial hypertension (IPAH). The basement membrane (BM) is a specialized class of ECM proteins. In pulmonary arteries, the BM is in close contact and direct proximity to vascular cells, including endothelial cells. So far, the role of the BM has remained underinvestigated in IPAH. Here, we aimed to shed light on the involvement of the BM in IPAH, by addressing its structure, composition, and function. On an ultrastructural level, we observed a marked increase in BM thickness in IPAH pulmonary vessels. BM composition was distinct in small and large vessels and altered in IPAH. Proteoglycans were mostly responsible for distinction between smaller and larger vessels, whereas BM collagens and laminins were more abundantly expressed in IPAH. Type IV collagen and laminin both strengthened endothelial barrier integrity. However, only type IV collagen concentration dependently increased cell adhesion of both donor and IPAH-derived pulmonary arterial endothelial cells (PAECs) and induced nuclear translocation of mechanosensitive transcriptional coactivator of the hippo pathway YAP (Yes-activated protein). On the other hand, laminin caused cytoplasmic retention of YAP in IPAH PAECs. Accordingly, silencing of COL4A5 and LAMC1, respectively, differentially affected tight junction formation and barrier integrity in both donor and IPAH PAECs. Collectively, our results highlight the importance of a well-maintained BM homeostasis. By linking changes in BM structure and composition to altered endothelial cell function, we here suggest an active involvement of the BM in IPAH pathogenesis.

Pulmonary fibrosis is characterized by pronounced collagen deposition and myofibroblast expansion, whose origin and plasticity remain elusive. We utilized a fate-mapping approach to investigate α-smooth muscle actin (αSMA)+ and platelet-derived growth factor receptor α (PDGFRα)+ cells in two lung fibrosis models, complemented by cell type-specific next-generation sequencing and investigations on human lungs. Our data revealed that αSMA+ and PDGFRα+ cells mark two distinct mesenchymal lineages with minimal transdifferentiation potential during lung fibrotic remodeling. Parenchymal and perivascular fibrotic regions were populated predominantly with PDGFRα+ cells expressing collagen, while αSMA+ cells in the parenchyma and vessel wall showed variable expression of collagen and the contractile protein desmin. The distinct gene expression profile found in normal conditions was retained during pathologic remodeling. Cumulatively, our findings identify αSMA+ and PDGFRα+ cells as two separate lineages with distinct gene expression profiles in adult lungs. This cellular heterogeneity suggests that anti-fibrotic therapy should target diverse cell populations.

Fos-related antigen-2 (Fra-2) belongs to the activator protein 1 (AP-1) family of transcription factors and is involved in a broad variety of cellular processes, such as proliferation or differentiation. Aberrant expression of Fra-2 or regulation can lead to severe growth defects or diverse pathologies. Elevated Fra-2 expression has been described in several chronic lung diseases, such as pulmonary fibrosis, chronic obstructive pulmonary disease and asthma. However, the pathomechanisms behind the Fra-2-induced pulmonary remodelling are still not fully elucidated. Fra-2 overexpressing mice were initially described as a model of systemic sclerosis associated organ fibrosis, with predominant alterations in the lung. High levels of Fra-2 expression give rise to profound inflammation with severe remodelling of the parenchyma and the vasculature, resulting in fibrosis and pulmonary hypertension, respectively, but also alters bronchial function. In this review we discuss the central role of Fra-2 connecting inflammation, cellular proliferation and extracellular matrix deposition underlying chronic lung diseases and what we can learn for future therapeutic options., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

The interleukin (IL)-1 family of cytokines is strongly associated with systemic sclerosis (SSc) and pulmonary involvement, but the molecular mechanisms are poorly understood. The aim of this study was to assess the role of IL-1α and IL-1β in pulmonary vascular and interstitial remodelling in a mouse model of SSc.IL-1α and IL-1β were localised in lungs of SSc patients and in the fos-related antigen-2 (Fra-2) transgenic (TG) mouse model of SSc. Lung function, haemodynamic parameters and pulmonary inflammation were measured in Fra-2 TG mice with or without 8 weeks of treatment with the IL-1 receptor antagonist anakinra (25 mg·kg -1 ·day -1 ). Direct effects of IL-1 on pulmonary arterial smooth muscle cells (PASMCs) and parenchymal fibroblasts were investigated in vitro Fra-2 TG mice exhibited increased collagen deposition in the lung, restrictive lung function and enhanced muscularisation of the vasculature with concomitant pulmonary hypertension reminiscent of the changes in SSc patients. Immunoreactivity of IL-1α and IL-1β was increased in Fra-2 TG mice and in patients with SSc. IL-1 stimulation reduced collagen expression in PASMCs and parenchymal fibroblasts via distinct signalling pathways. Blocking IL-1 signalling in Fra-2 TG worsened pulmonary fibrosis and restriction, enhanced T-helper cell type 2 (Th2) inflammation, and increased the number of pro-fibrotic, alternatively activated macrophages.Our data suggest that blocking IL-1 signalling as currently investigated in several clinical studies might aggravate pulmonary fibrosis in specific patient subsets due to Th2 skewing of immune responses and formation of alternatively activated pro-fibrogenic macrophages., Competing Interests: Conflict of interest: A. Birnhuber reports grants from Jubilee Foundation of the Austrian National Bank, during the conduct of the study. Conflict of interest: S. Crnkovic has nothing to disclose. Conflict of interest: V. Biasin has nothing to disclose. Conflict of interest: L.M. Marsh has nothing to disclose. Conflict of interest: B. Odler has nothing to disclose. Conflict of interest: A. Sahu-Osen has nothing to disclose. Conflict of interest: E. Stacher-Priehse has nothing to disclose. Conflict of interest: L. Brcic reports grants and personal fees from AstraZeneca, personal fees from Roche Austria, nonfinancial support from MSD Austria and Pfizer Austria, outside the submitted work. Conflict of interest: F. Schneider has nothing to disclose. Conflict of interest: N. Cikes has nothing to disclose. Conflict of interest: B. Ghanim has nothing to disclose. Conflict of interest: W. Klepetko has nothing to disclose. Conflict of interest: W. Graninger has nothing to disclose. Conflict of interest: Y. Allanore has nothing to disclose. Conflict of interest: R. Eferl has nothing to disclose. Conflict of interest: A. Olschewski has nothing to disclose. Conflict of interest: H. Olschewski reports personal fees and nonfinancial support from Bayer, MSD, Pfizer and Novartis, grants, personal fees and nonfinancial support from Actelion, grants from Inventiva, personal fees from Bellerophon, outside the submitted work; and is a part-time employee of the Ludwig Boltzmann Institute for Lung Vascular Research, Graz, Austria. Conflict of interest: G. Kwapiszewska reports grants from Jubilee Foundation of the Austrian National Bank (grant 16187), Austrian Science Fund (FWF) (P27848-B28), and Austrian Agency for International Cooperation in Education and Research (WTZ HR14/2012), during the conduct of the study., (Copyright ©ERS 2019.)

Background: Lung eosinophilia is a hallmark of asthma, and eosinophils are believed to play a crucial role in the pathogenesis of allergic inflammatory diseases. Short-chain fatty acids (SCFAs), such as acetate, propionate, and butyrate, are produced in high amounts in the gastrointestinal tract by commensal bacteria and can be absorbed into the bloodstream. Although there is recent evidence that SCFAs are beneficial in allergic asthma models, the effect on eosinophils has remained elusive., Objective: The role of SCFAs was investigated in human eosinophil function and a mouse model of allergic asthma., Methods: Eosinophils were purified from self-reported allergic or healthy donors. Migration, adhesion to the endothelium, and eosinophil survival were studied in vitro. Ca 2+ flux, apoptosis, mitochondrial membrane potential, and expression of surface markers were determined by using flow cytometry and in part by using real-time PCR. Allergic airway inflammation was assessed in vivo in an ovalbumin-induced asthma model by using invasive spirometry., Results: For the first time, we observed that SCFAs were able to attenuate human eosinophils at several functional levels, including (1) adhesion to the endothelium, (2) migration, and (3) survival. These effects were independent from GPR41 and GPR43 but were accompanied by histone acetylation and mimicked by trichostatin A, a pan-histone deacetylase inhibitor. In vivo butyrate ameliorated allergen-induced airway and lung eosinophilia, reduced type 2 cytokine levels in bronchial fluid, and improved airway hyperresponsiveness in mice., Conclusion: These in vitro and in vivo findings highlight the importance of SCFAs, especially butyrate as a promising therapeutic agent in allergic inflammatory diseases., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Rationale: Remodeling and fibrosis of the right ventricle (RV) may cause RV dysfunction and poor survival in patients with pulmonary hypertension. Objectives: To investigate the consequences of RV fibrosis modulation and the accompanying cellular changes on RV function. Methods: Expression of fibrotic markers was assessed in the RV of patients with pulmonary hypertension, the murine pulmonary artery banding, and rat monocrotaline and Sugen5416/hypoxia models. Invasive hemodynamic and echocardiographic assessment was performed on galectin-3 knockout or inhibitor-treated mice. Measurements and Main Results: Established fibrosis was characterized by marked expression of galectin-3 and an enhanced number of proliferating RV fibroblasts. Galectin-3 genetic and pharmacologic inhibition or antifibrotic treatment with pirfenidone significantly diminished RV fibrosis progression in the pulmonary artery banding model, without improving RV functional parameters. RV fibrotic regions were populated with mesenchymal cells coexpressing vimentin and PDGFRα (platelet-derived growth factor receptor-α), but generally lacked αSMA (α-smooth muscle actin) positivity. Serum levels of galectin-3 were increased in patients with idiopathic pulmonary arterial hypertension but did not correlate with cardiac function. No changes of galectin-3 expression were observed in the lungs. Conclusions: We identified extrapulmonary galectin-3 as an important mediator that drives RV fibrosis in pulmonary hypertension through the expansion of PDGFRα/vimentin-expressing cardiac fibroblasts. However, interventions effectively targeting fibrosis lack significant beneficial effects on RV function.

Our systematic analysis of anion channels and transporters in idiopathic pulmonary arterial hypertension (IPAH) showed marked upregulation of the Cl - channel TMEM16A gene. We hypothesised that TMEM16A overexpression might represent a novel vicious circle in the molecular pathways causing pulmonary arterial hypertension (PAH).We investigated healthy donor lungs (n=40) and recipient lungs with IPAH (n=38) for the expression of anion channel and transporter genes in small pulmonary arteries and pulmonary artery smooth muscle cells (PASMCs).In IPAH, TMEM16A was strongly upregulated and patch-clamp recordings confirmed an increased Cl - current in PASMCs (n=9-10). These cells were depolarised and could be repolarised by TMEM16A inhibitors or knock-down experiments (n=6-10). Inhibition/knock-down of TMEM16A reduced the proliferation of IPAH-PASMCs (n=6). Conversely, overexpression of TMEM16A in healthy donor PASMCs produced an IPAH-like phenotype. Chronic application of benzbromarone in two independent animal models significantly decreased right ventricular pressure and reversed remodelling of established pulmonary hypertension.Our findings suggest that increased TMEM16A expression and activity comprise an important pathologic mechanism underlying the vasoconstriction and remodelling of pulmonary arteries in PAH. Inhibition of TMEM16A represents a novel therapeutic approach to reverse remodelling in PAH., Competing Interests: Conflict of interest: C. Nagaraj has a patent (file number EP17169063.9): modulation of the calcium-activated chloride channel including TMEM16A represent a novel therapy for pulmonary hypertension, pending. Conflict of interest: D. Zabini has nothing to disclose. Conflict of interest: B.M. Nagy has a patent (file number EP17169063.9): modulation of the calcium-activated chloride channel including TMEM16A represent a novel therapy for pulmonary hypertension, pending. Conflict of interest: M. Lengyel has nothing to disclose. Conflict of interest: D. Skofic Maurer has nothing to disclose. Conflict of interest: N. Sharma has nothing to disclose. Conflict of interest: B. Egemnazarov has nothing to disclose. Conflict of interest: G. Kovacs reports personal fees and non-financial support from Actelion, Bayer, GSK, MSD, Boehringer Ingelheim, Novartis and Chiesi, and non-financial support from VitalAire, outside the submitted work. Conflict of interest: G. Kwapiszewska has nothing to disclose. Conflict of interest: L.M. Marsh has nothing to disclose. Conflict of interest: A. Hrzenjak has nothing to disclose. Conflict of interest: G. Höfler has nothing to disclose. Conflict of interest: M. Didiasova has nothing to disclose. Conflict of interest: M. Wygrecka has nothing to disclose. Conflict of interest: L.K. Sievers has nothing to disclose. Conflict of interest: P. Szucs has nothing to disclose. Conflict of interest: P. Enyedi has nothing to disclose. Conflict of interest: B. Ghanim has nothing to disclose. Conflict of interest: W. Klepetko has nothing to disclose. Conflict of interest: H. Olschewski reports grants, personal fees and non-financial support from Actelion, Bayer and Boehringer, personal fees and non-financial support from GSK, Chiesi, Menarini, TEVA, MSD and Ludwig Boltzmann Institute for Lung Vascular Research, personal fees from Novartis, AstraZeneca and Bellerophon, and grants and personal fees from Roche, outside the submitted work. Conflict of interest: A. Olshewski has a patent (file number EP17169063.9): modulation of the calcium-activated chloride channel including TMEM16A represent a novel therapy for pulmonary hypertension, pending. Conflict of interest: R. Papp has a patent (file number EP17169063.9): modulation of the calcium-activated chloride channel including TMEM16A represent a novel therapy for pulmonary hypertension, pending., (Copyright ©ERS 2019.)

Diastolic dysfunction is a prominent feature of right ventricular (RV) remodeling associated with conditions of pressure overload. However, the RV diastolic function is rarely quantified in experimental studies. This might be due to technical difficulties in the visualization of the RV in the apical four-chamber view in rodents. Here we describe two positions facilitating the visualization of the apical four-chamber view in mice to assess the RV diastolic function. The apical four-chamber view is enabled by tilting the mouse fixation platform to the left and caudally (LeCa) or to the right and cranially (RiCr). Both positions provide images of comparable quality. The results of the RV diastolic function obtained from two positions are not significantly different. Both positions are comparably easy to perform. This protocol can be incorporated into published protocols and enables detailed investigations of the RV function.

In idiopathic pulmonary arterial hypertension (IPAH), global transcriptional changes induce a smooth muscle cell phenotype characterised by excessive proliferation, migration, and apoptosis resistance. Long non-coding RNAs (lncRNAs) are key regulators of cellular function. Using a compartment-specific transcriptional profiling approach, we sought to investigate the link between transcriptional reprogramming by lncRNAs and the maladaptive smooth muscle cell phenotype in IPAH. Transcriptional profiling of small remodelled arteries from 18 IPAH patients and 17 controls revealed global perturbations in metabolic, neuronal, proliferative, and immunological processes. We demonstrated an IPAH-specific lncRNA expression profile and identified the lncRNA PAXIP1-AS1 as highly abundant. Comparative transcriptomic analysis and functional assays revealed an intrinsic role for PAXIP1-AS1 in orchestrating the hyperproliferative and migratory actions of IPAH smooth muscle cells. Further, we showed that PAXIP1-AS1 mechanistically interferes with the focal adhesion axis via regulation of expression and phosphorylation of its downstream target paxillin. Overall, we show that changes in the lncRNA transcriptome contribute to the disease-specific transcriptional landscape in IPAH. Our results suggest that lncRNAs, such as PAXIP1-AS1, can modulate smooth muscle cell function by affecting multiple IPAH-specific transcriptional programmes. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd., (Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Pulmonary siRNA delivery has attracted strong interest and has been reported to successfully mediate target gene knockdown in a number of disease models. However, the nature of the epithelial cells that eventually take up siRNA and the question if other lung cell types may also be transfected has so far been neglected. Therefore, we describe here a flow cytometry-based method using transgenic enhanced green fluorescence protein (EGFP) expressing mice for the differentiation of transfected lung cell populations based on their antigen expression.

Despite the beneficial effects of pirfenidone in treating idiopathic pulmonary fibrosis (IPF), it remains unclear if lung fibroblasts (FB) are the main therapeutic target.To resolve this question, we employed a comparative transcriptomic approach and analysed lung homogenates (LH) and FB derived from IPF patients treated with or without pirfenidone.In FB, pirfenidone therapy predominantly affected growth and cell division pathways, indicating a major cellular metabolic shift. In LH samples, pirfenidone treatment was mostly associated with inflammation-related processes. In FB and LH, regulated genes were over-represented in the Gene Ontology node "extracellular matrix". We identified lower expression of cell migration-inducing and hyaluronan-binding protein (CEMIP) in both LH and FB from pirfenidone-treated IPF patients. Plasma levels of CEMIP were elevated in IPF patients compared to healthy controls and decreased after 7 months of pirfenidone treatment. CEMIP expression in FB was downregulated in a glioma-associated oncogene homologue-dependent manner and CEMIP silencing in IPF FB reduced collagen production and attenuated cell proliferation and migration.Cumulatively, our approach indicates that pirfenidone exerts beneficial effects via its action on multiple pathways in both FB and other pulmonary cells, through its ability to control extracellular matrix architecture and inflammatory reactions., Competing Interests: Conflict of interest: A. Olschewski reports grants and honoraria for speaking from Pfizer, outside the submitted work. Conflict of interest: W. Seeger reports personal fees from Pfizer, Novartis, United Therapeutics, Actelion, Vectura, Savara, Medspray and Bayer AG, outside the submitted work. Conflict of interest: H. Olschewski reports grants from Intermune/Roche, and grants and personal fees from Boehringer, outside the submitted work. All other authors have nothing to disclose., (Copyright ©ERS 2018.)

Background: Asthma is a complex chronic inflammatory disease characterised by airway inflammation, remodelling and hyperresponsiveness (AHR). Members of the AP-1 transcription factor family play important roles in the activation of the immune system and the control of cellular responses; however, their role in the development of asthma has not been well studied. We aimed to investigate the role of the lesser known AP-1 family member, Fra2 in experimental asthma. Methods: Phenotypic characterisation and gene expression profiling was performed on Fra2 (TG) overexpressing and wild-type mice. The efficacy of therapeutic interventions in regulating the Fra2 phenotype was determined. Results: Transcriptional profiling of TG mice revealed a high abundance of regulated genes associated with airway remodelling, inflammation and mucus production. A concomitant increase in peribronchial collagen deposition, smooth muscle thickening and mucus production was observed. TG mice possessed increased inflammatory infiltration in the lung, predominantly consisting of eosinophils and T-cells and elevated expression of Th2 cytokines and eotaxin. Furthermore, TG mice possessed severe AHR in response to increasing doses of methacholine. Glucocorticoid treatment led to a partial improvement of the asthma phenotype, whereas blockade of IL-13 via neutralising antibodies ameliorated AHR and mucus production, but had no effect on collagen deposition. Conclusion: We here describe a novel model for non-allergic asthma that does not require the application of exogenous allergens, which mimics several key features of the disease, such as airway inflammation, remodelling and hyperresponsiveness. Fra2 may represent a key molecule coordinating multiple aspects of asthma pathogenesis.

The multi-kinase inhibitor dasatinib is used for treatment of imatinib-resistant chronic myeloid leukemia, but is prone to induce microvascular dysfunction. In lung this can manifest as capillary leakage with pleural effusion, pulmonary edema or even pulmonary arterial hypertension. To understand how dasatinib causes endothelial dysfunction we examined the effects of clinically relevant concentrations of dasatinib on both human pulmonary arterial macro- and microvascular endothelial cells (ECs). The effects of dasatinib was compared to imatinib and nilotinib, two other clinically used BCR/Abl kinase inhibitors that do not inhibit Src. Real three-dimensional morphology and high resolution stiffness mapping revealed softening of both macro- and microvascular ECs upon dasatinib treatment, which was not observed in response to imatinib. In a dose-dependent manner, dasatinib decreased transendothelial electrical resistance/impedance and caused a permeability increase as well as disruption of tight adherens junctions in both cell types. In isolated perfused and ventilated rat lungs, dasatinib increased mean pulmonary arterial pressure, which was accompanied by a gain in lung weight. The Rho-kinase inhibitor Y27632 partly reversed the dasatinib-induced changes in vitro and ex vivo , presumably by acting downstream of Src. Co-administration of the Rho-kinase inhibitor Y27632 completely blunted the increased pulmonary pressure in response to dasatinib. In conclusion, a dasatinib-induced permeability increase in human pulmonary arterial macro- and microvascular ECs might explain many of the adverse effects of dasatinib in patients. Rho-kinase inhibition might be suitable to ameliorate these effects.

Pulmonary vascular remodeling is the main pathological hallmark of pulmonary hypertension disease. We undertook a comprehensive and multilevel approach to investigate the origin of smooth muscle actin-expressing cells in remodeled vessels. Transgenic mice that allow for specific, inducible, and permanent labeling of endothelial (Cdh5-tdTomato), smooth muscle (Acta2-, Myh11-tdTomato), pericyte (Cspg4-tdTomato), and fibroblast (Pdgfra-tdTomato) lineages were used to delineate the cellular origins of pulmonary vascular remodeling. Mapping the fate of major lung resident cell types revealed smooth muscle cells (SMCs) as the predominant source of cells that populate remodeled pulmonary vessels in chronic hypoxia and allergen-induced murine models. Combining in vivo cell type-specific, time-controlled labeling of proliferating cells with a pulmonary artery phenotypic explant assay, we identified proliferation of SMCs as an underlying remodeling pathomechanism. Multicolor immunofluorescence analysis showed a preserved pattern of cell type marker localization in murine and human pulmonary arteries, in both donors and idiopathic pulmonary arterial hypertension (IPAH) patients. Whilst neural glial antigen 2 (chondroitin sulfate proteoglycan 4) labeled mostly vascular supportive cells with partial overlap with SMC markers, PDGFRα-expressing cells were observed in the perivascular compartment. The luminal vessel side was lined by a single cell layer expressing endothelial markers followed by an adjacent and distinct layer defined by SMC marker expression and pronounced thickening in remodeled vessels. Quantitative flow cytometric analysis of single cell digests of diverse pulmonary artery layers showed the preserved separation into two discrete cell populations expressing either endothelial cell (EC) or SMC markers in human remodeled vessels. Additionally, we found no evidence of overlap between EC and SMC ultrastructural characteristics using electron microscopy in either donor or IPAH arteries. Lineage-specific marker expression profiles are retained during pulmonary vascular remodeling without any indication of cell type conversion. The expansion of resident SMCs is the major underlying and evolutionarily conserved paradigm of pulmonary vascular disease pathogenesis. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland., (© 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.)

Increasing evidence points towards an inflammatory component underlying pulmonary hypertension. However, the conclusive characterisation of multiple inflammatory cell populations in the lung is challenging due to the complexity of marker specificity and tissue inaccessibility. We used an unbiased computational flow cytometry approach to delineate the inflammatory landscape of idiopathic pulmonary arterial hypertension (IPAH) and healthy donor lungs.Donor and IPAH samples were discriminated clearly using principal component analysis to reduce the multidimensional data obtained from single-cell flow cytometry analysis. In IPAH lungs, the predominant CD45 + cell type switched from neutrophils to CD3 + T-cells, with increases in CD4 + , CD8 + and γδT-cell subsets. Additionally, diversely activated classical myeloid-derived dendritic cells (CD14 - HLA-DR + CD11c + CD1a +/- ) and nonclassical plasmacytoid dendritic cells (pDCs; CD14 - CD11c - CD123 + HLA-DR + ), together with mast cells and basophils, were more abundant in IPAH samples. We describe, for the first time, the presence and regulation of two cell types in IPAH, γδT-cells and pDCs, which link innate and adaptive immunity.With our high-throughput flow cytometry with multidimensional dataset analysis, we have revealed the interactive interplay between multiple inflammatory cells is a crucial part of their integrative network. The identification of γδT-cells and pDCs in this disease potentially provides a missing link between IPAH, autoimmunity and inflammation., Competing Interests: Conflict of interest: Disclosures can be found alongside this article at erj.ersjournals.com, (Copyright ©ERS 2018.)

Aim: Chronic airway diseases such as asthma are associated with increased production of reactive oxygen species (ROS) and oxidative stress. Endogenous NADPH oxidases are a major source of superoxide in lung, but their underlying role in asthma pathology is poorly understood. We sought to characterize the involvement of NADPH oxidase in allergic asthma by studying the role of CYBA (p22phox) in human asthma and murine house dust mite (HDM)-induced allergic airway inflammation., Results: Increased expression and localization of p22-PHOX were observed in biopsies of asthmatic patients. HDM-treated wild-type mice possessed elevated p22phox expression, corresponding with elevated superoxide production. p22phox knockout (KO) mice did not induce superoxide and were protected against HDM-induced goblet cell hyperplasia and mucus production and HDM-induced airway hyperresponsiveness (AHR). IL-13-induced tracheal hyperreactivity and signal transducer and activator of transcription (STAT)6 phosphorylation were attenuated in the absence of p22phox or catalase pretreatment., Innovation: Our study identifies increased expression of p22phox in lungs of asthmatic patients and in experimental model. The induced AHR and mucus hypersecretion are a result of increased ROS from the p22phox-dependent NADPH oxidase, which in turn activates STAT6 for the pathological feature of asthma., Conclusions: Together with the increased p22phox expression in lungs of asthmatic patients, these findings demonstrate a crucial role of p22phox-dependent NADPH oxidase for the development of mucus hypersecretion and AHR in HDM-induced model of asthma. This suggests that inhibition of functional NADPH oxidase by selective interference of p22phox might hold a promising therapeutic strategy for the management of asthma. Antioxid. Redox Signal. 27, 1460-1472.

Chronic obstructive pulmonary disease (COPD) is a heterogeneous disease in which the amount of emphysema and airway disease may be very different between individuals, even in end-stage disease. Emphysema formation may be linked to the involvement of the small pulmonary vessels. The NAPDH oxidase (Nox) family is emerging as a key disease-related factor in vascular diseases, but currently its role in hypoxia-induced pulmonary remodelling in COPD remains unclear.Here we investigate the role of p22phox, a regulatory subunit of Nox, in COPD lungs, hypoxic pulmonary vasoconstriction (HPV), hypoxia-induced pulmonary vascular remodelling and pulmonary hypertension.In COPD, compared to control lungs, p22phox expression was significantly reduced. The expression was correlated positively with mean pulmonary arterial pressure and oxygenation index and negatively with the diffusing capacity of the lung for carbon monoxide (p<0.02). This suggests a role of p22phox in ventilation/perfusion ratio matching, vascular remodelling and loss of perfused lung area. In p22phox -/- mice, HPV was significantly impaired. In the chronic hypoxic setting, lack of p22phox was associated with improved right ventricular function and decreased pulmonary vascular remodelling.p22phox-dependent Nox plays an important role in the COPD phenotype, by its action on phase II HPV and chronic vascular remodelling., Competing Interests: Conflict of interest: Disclosures can be found alongside this article at erj.ersjournals.com, (Copyright ©ERS 2017.)