Pulmonary fibrosis is characterized by irreversible remodeling of the lung parenchyma, which leads to a progressive deterioration in lung function. Pro-fibrotic fibroblasts contribute to uncontrolled extracellular matrix deposition and progression of fibrosis. Emerging evidence implicates metabolic dysregulation - particularly enhanced glycolysis and impaired fatty acid metabolism - in the pathogenesis of pulmonary fibrosis. Here, we investigated whether modulating fatty acid metabolism could counteract fibroblast-driven fibrosis in experimental models. Treatment with pharmacological agents (metformin, rosiglitazone), or genetic ablation of the LPL inhibitory protein, angiopoietin like 4, markedly reduced lung fibrosis and ECM production in bleomycin-induced lung fibrosis mouse model. Similarly, in vitro, provision of albumin-bound fatty acids (FA) in the growth medium reduced TGFβ1-induced collagen production in lung fibroblasts. Mechanistically, excess fatty acid reduced glycolysis, while enhancing mitochondrial function and overall metabolic activity in TGFβ1-treated fibroblasts. This metabolic shift, but not the collagen reduction, was driven by increased fatty acid oxidation via CPT1, as confirmed by the use of the CPT1 inhibitor etomoxir. Importantly, in vivo metabolic interventions downregulated preferentially PDGFRα protein level, while in vitro provision of albumin-bound FA reduced both the amount of collagen produced by αSMA-positive cells and the amount of PDGFRα-positive cells, suggesting a dual role of FA on both pro-fibrotic fibroblasts. Collectively, our findings identify fatty acid oxidation as a potent metabolic checkpoint in fibrotic fibroblasts and support FA availability as a promising strategy to limit lung fibrosis progression.

"Reply to: Targeting Pulmonary Hypertension Caused by Pulmonary Fibrosis: A Promising NKT Cell-based Therapy." American Journal of Respiratory and Critical Care Medicine, 0(ja), pp. –

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.

Objective To analyze the quantity of cervical smears, also designated Papanicolaou tests, between 2006 and 2015 in all the Federal units of Brazil, as well as to verify the quantity of exams collected outside the recommended age range and the economic impact of such excess. Methods The data was collected from the Ministry of Health's database called Sistema de Informação do Câncer do Colo de Útero (SISCOLO), which contains all the test results collected nationwide by the Unified Health System (SUS, in the Portuguese acronym). From that, the number of exams and the age range of the women who underwent them were analyzed; besides, these numbers were stratified according to the state of where the exam was performed. The quantity of exams collected outside the recommended age range was verified, and, so, the economic impact generated was noted. Results Between 2006 and 2015, 87,425,549 Papanicolaou tests were collected in Brazil. Of these, 20,215,052 tests were collected outside the age range recommended by the Brazilian Ministry of Health; this number corresponded to 23.12% of all exams. From such data, considering that each Pap smear collected by SUS generates a cost of BRL 7.30 to the government, according to the information in the Tabela SUS dated September 2018, there was a total charge of BRL 147,569,880 for tests collected outside the protocol. Conclusion In Brazil, according to the Ministry of Health's protocol about the recommended practices on collecting Pap smears, whose newest edition dates of 2016, it is recommended that Pap smears are collected in women from a specific age range, in whom the potential diagnosing advantages overcome the onus of overdiagnosis or of a lesion with great regression potential. However, such protocols have not been correctly followed, promoting more than 20 million tests in excess, and an exorbitant cost for the Brazilian public health system. It is relevant to take measures to correctly use the official protocol, reducing the patients risks, as well as the economic impact for SUS.

RATIONALE Pulmonary hypertension (PH) is a common, yet severe comorbidity in interstitial lung diseases (ILD) such as pulmonary fibrosis (PF), with limited treatment options. Excessive vascular fibrosis and inflammation are often present in PH, but the underlying mechanisms are still not well understood. OBJECTIVE 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 immuno-histological analysis were complemented by pharmacological NKT-cell activation in-vivo, in-vitro and ex-vivo. MEASUREMENTS AND MAIN RESULTS In pulmonary vessels of PF-PH patients increased collagen deposition was linked to a local NKT cell deficiency and decreased interleukin-15 levels. In a mouse model of PH due to 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 (hPASMC) and in ex-vivo precision-cut lung slices of end-stage PF-PH patients. Co-culture with activated NKT cells induced STAT1 signaling in hPASMC. Secretome analysis of peripheral blood mononuclear cells (PBMCs) identified CXCL9 and CXCL10 as indicators of NKT cell activation. Pharmacologically, CXCL9, but not CXCL10, potently inhibited collagen deposition in hPASMC via the chemokine receptor, CXCR3. CONCLUSION 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 ILD.

Background and Aim: The basement membrane (BM), a specialized subset of extracellular matrix proteins underlying endothelial cells (ECs), is an emerging active player in the pathophysiology of the pulmonary hypertension (PH). However, function of BM matrikines in pulmonary vasculature is understudied. Here, we aimed to elucidate the role of type IV collagen α5 (Col4α5) matrikine, pentastatin, in PH by investigating its function on pulmonary arterial ECs (PAECs). Methods: Gene expression levels of type IV collagen isoforms were analysed from laser-microdissected pulmonary arteries (PAs) in different forms of PH (Group 1 and Group 3) patients. Localisation and quantity of Col4α5 were detected with immunostaining and immunoblotting. Functional impact of the matrikine, pentastatin, on PAECs was assessed in vitro and in vivo. Results: We observed a positive correlation between COL4A5 gene expression levels and mean pulmonary arterial pressure (mPAP) in patients with different forms of PH. Pronounced levels of Col4α5 fragments (matrikines) were found in PAs of PH patients. Treatment of PAECs with synthetic pentastatin, induced apoptosis, led to VE-cadherin disruption, stress fiber formation and barrier disruption in PAEC, while, inhibiting transendothelial migration of peripheral blood mononuclear cells. In the ex-vivo isolated perfused mouse lung (IPL) model, PS increased the mPAP and caused pulmonary edema formation. Conclusion: Our findings demonstrated that pentastatin might be involved in pulmonary hypertension by disturbing endothelial barrier function and increasing pressure. Thus, modulation of matrikines levels might serve as possible therapeutic option in these patients.

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.

The extracellular matrix (ECM) increasingly emerges as an active driver in several pulmonary diseases, including idiopathic pulmonary arterial hypertension (IPAH). The basement membrane (BM) is a specialized class of ECM proteins, which underlies all endothelial cells. Despite this close contact, the role of the BM in IPAH, and its impact on endothelial function, has remained under-investigated. In this study, we aimed to shed light on the involvement of the BM in PAH, 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, while BM collagens and laminins were more abundantly expressed in IPAH. Seeding donor or IPAH derived pulmonary arterial endothelial cells (PAEC) on collagen IV- or laminin coated surfaces strengthened endothelial barrier integrity as quantified by electrical impedance measurement, however only type IV collagen increased cell adhesion and induced nuclear translocation of mechanosensitive transcriptional co-activator of the hippo pathway Yes-activated protein (YAP). Opposingly, laminin induced cytoplasmic retention of YAP in IPAH PAEC, which, inherently, presented with increased YAP activation levels. Here we show that changes in BM structure and composition have a profound bearing on endothelial cell function, and suggest that changes of the BM may play a role in the pathogenesis of IPAH.

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.

The heterogeneous nature of most human organs and tissues represents a common challenge when analyzing specific structures or cells. Laser capture microdissection (LCM) enables isolation of pure cells from a mixed population of cells or tissue samples via usage of laser energy. Combined with high-throughput gene or protein techniques, compartment specific analysis elucidating the role of specialized cell types in physiological or pathophysiological activity can be performed. This chapter describes the crucial steps that have to be taken into consideration when designing and conducting a LCM project. Detailed protocols describing the workflow from project planning to high-throughput analysis of LCM material used in our laboratory are provided. Routinely occurring challenges and appropriate solutions, e.g., when working with fibrotic tissue are described.

Over past years, a critical role for the immune system and, in particular, for mast cells in the pathogenesis of pulmonary hypertension (PH) has emerged. However, the way in which mast cells promote PH is still poorly understood. Here, we investigated the mechanisms by which mast cells may contribute to PH, specifically focusing on the interaction between the innate and adaptive immune response and the role of B cells and autoimmunity. Experiments were performed in Sprague-Dawley rats and B cell-deficient JH-KO rats in the monocrotaline, Sugen/hypoxia, and the aortic banding model of PH. Hemodynamics, cell infiltration, IL-6 expression, and vascular remodeling were analyzed. Gene array analyses revealed constituents of immunoglobulins as most prominently regulated mast cell-dependent genes in the lung in experimental PH. IL-6 was shown to link mast cells to B cells, as 1) IL-6 was upregulated and colocalized with mast cells and was reduced by mast-cell stabilizers and 2) IL-6 or mast cell blockade reduced B cells in lungs of monocrotaline-treated rats. A functional role for B cells in PH was demonstrated in that either blocking B cells by an anti-CD20 antibody or B-cell deficiency in JH-KO rats attenuated right ventricular systolic pressure and vascular remodeling in experimental PH. We here identify a mast cell-B cell axis driven by IL-6 as a critical immune pathway in the pathophysiology of PH. Our results provide novel insights into the role of the immune system in PH, which may be therapeutically exploited by targeted immunotherapy.

Microarrays are a powerful and effective tool that allows the detection of genome-wide gene expression differences between controls and disease conditions. They have been broadly applied to investigate the pathobiology of diverse forms of pulmonary hypertension, namely group 1, including patients with idiopathic pulmonary arterial hypertension, and group 3, including pulmonary hypertension associated with chronic lung diseases such as chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis. To date, numerous human microarray studies have been conducted to analyse global (lung homogenate samples), compartment-specific (laser capture microdissection), cell type-specific (isolated primary cells) and circulating cell (peripheral blood) expression profiles. Combined, they provide important information on development, progression and the end-stage disease. In the future, system biology approaches, expression of noncoding RNAs that regulate coding RNAs, and direct comparison between animal models and human disease might be of importance.

Julia Hoffmann, Leigh M. Marsh, Mario Pieper, Elvira Stacher, Bahil Ghanim, Gabor Kovacs, Peter König, Heinrike Wilkens, Hans Michael Haitchi, Gerald Hoefler, Walter Klepetko, Horst Olschewski, Andrea Olschewski, and Grazyna Kwapiszewska Ludwig Boltzmann Institute for Lung Vascular Research, Graz, Austria; Institute of Anatomy, University Lübeck, Lübeck, Germany and Airway Research Center North, Member of the German Center for Lung Research (DZL), Grosshansdorf, Germany; Institute of Pathology, Medical University of Graz, Graz, Austria; Division of Thoracic Surgery, Department of Surgery, Medical University of Vienna, Vienna, Austria; Department of Pulmonology, Medical University of Graz, Graz, Austria; Department of Pulmonology, Faculty of Medicine, Saarland University, Homburg/Saar, Germany; Clinical and Experimental Sciences, Faculty of Medicine, University Southampton, UK; NIHR Southampton Respiratory BioMedical Research Unit at University Hospital Southampton, NHS Foundation Trust, UK; and Department of Experimental Anesthesiology, Medical University of Graz, Graz, Austria