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Flow cytometry and fluorescence-activated cell sorting are widely used to study endothelial cells, for which the generation of viable single-cell suspensions is an essential first step. Two enzymatic approaches, collagenase A and dispase, are widely employed for endothelial cell isolation. In this study, the utility of both enzymatic approaches, alone and in combination, for endothelial cell isolation from juvenile and adult mouse lungs was assessed, considering the number, viability, and subtype composition of recovered endothelial cell pools. Collagenase A yielded an 8-12-fold superior recovery of viable endothelial cells from lung tissue from developing mouse pups, compared to dispase, although dispase proved superior in efficiency for epithelial cell recovery. Single-cell RNA-Seq revealed that the collagenase A approach yielded a diverse endothelial cell subtype composition of recovered endothelial cell pools, with broad representation of arterial, capillary, venous, and lymphatic lung endothelial cells; while the dispase approach yielded a recovered endothelial cell pool highly enriched for one subset of general capillary endothelial cells, but poor representation of other endothelial cells subtypes. These data indicate that tissue dissociation markedly influences the recovery of endothelial cells, and the endothelial subtype composition of recovered endothelial cell pools, as assessed by single-cell RNA-Seq., (© 2024 The Authors. Cytometry Part A published by Wiley Periodicals LLC on behalf of International Society for Advancement of Cytometry.)

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

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

The morphometric analysis of lung structure using the principles of stereology has emerged as a powerful tool to describe the structural changes in lung architecture that accompany the development of lung disease that is experimentally modelled in adult mice. These stereological principles are now being applied to the study of the evolution of the lung architecture over the course of prenatal and postnatal lung development in mouse neonates and adolescents. The immature lung is structurally and functionally distinct from the adult lung, and has a smaller volume than does the adult lung. These differences have raised concerns about whether the inflation fixation of neonatal mouse lungs with the airway pressure (P aw ) used for the inflation fixation of adult mouse lungs may cause distortion of the neonatal mouse lung structure, leading to the generation of artefacts in subsequent analyses. The objective of this study was to examine the impact of a P aw of 10, 20 and 30 cmH 2 O on the estimation of lung volumes and stereologically assessed parameters that describe the lung structure in developing mouse lungs. The data presented demonstrate that low P aw (10 cmH 2 O) leads to heterogeneity in the unfolding of alveolar structures within the lungs, and that high P aw (30 cmH 2 O) leads to an overestimation of the lung volume, and thus, affects the estimation of volume-dependent parameters, such as total alveoli number and gas-exchange surface area. Thus, these data support the use of a P aw of 20 cmH 2 O for inflation fixation in morphometric studies on neonatal mouse lungs.

Along with nitric oxide (NO), the gasotransmitters carbon monoxide (CO) and hydrogen sulfide (H 2 S) are emerging as potentially important players in newborn physiology, as mediators of newborn disease, and as new therapeutic modalities. Several recent studies have addressed H 2 S in particular in animal models of bronchopulmonary dysplasia (BPD), a common complication of preterm birth where oxygen toxicity stunts lung development. In those studies, exogenous H 2 S attenuated the impact of oxygen toxicity on lung development, and two H 2 S-generating enzymes were documented to affect pulmonary vascular development. H 2 S is directly generated endogenously by three enzymes, one of which, 3-mercaptopyruvate sulfurtransferase (MPST), has not been studied in the lung. In a hyperoxia-based animal model of BPD, oxygen exposure deregulated MPST expression during post-natal lung development, where MPST was localized to the smooth muscle layer of the pulmonary vessels in developing lungs. siRNA-mediated abrogation of MPST expression in human pulmonary artery smooth muscle cells in vitro limited baseline cell migration and cell proliferation, without affecting apoptosis or cell viability. In vivo, MPST was dispensable for normal lung development in Mpst -/- mice, and MPST did not contribute to stunted lung development driven by hyperoxia exposure, assessed by design-based stereology. These data demonstrate novel roles for MPST in pulmonary vascular smooth muscle cell physiology. The potential caveats of using Mpst -/- mice to study normal and aberrant lung development are also discussed, highlighting the possible confounding, compensatory effects of other H 2 S-generating enzymes that are present alongside MPST in the smooth muscle compartment of developing pulmonary vessels., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Various forms of diffuse parenchymal lung disease have been proposed as potential consequences of severe COVID‑19. We describe the clinical, radiological and histological findings of patients with COVID‑19-associated acute respiratory distress syndrome who later developed severe organising pneumonia including longitudinal follow-up. Our findings may have important implications for the therapeutic modalities in the late-phase of severe COVID‑19 and might partially explain why a subgroup of COVID‑19 patients benefits from systemic corticosteroids., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. No commercial re-use. See rights and permissions. Published by BMJ.)

The lysyl hydroxylases (procollagen-lysine 5-dioxygenases) PLOD1, PLOD2, and PLOD3 have been proposed as pathogenic mediators of stunted lung development in bronchopulmonary dysplasia (BPD), a common complication of preterm birth. In affected infants, pulmonary oxygen toxicity stunts lung development. Mice lacking Plod1 exhibit 15% mortality, and mice lacking Plod2 or Plod3 exhibit embryonic lethality. Therefore, to address any pathogenic role of lysyl hydroxylases in stunted lung development associated with BPD, minoxidil was administered to newborn mice in an oxygen toxicity-based BPD animal model. Minoxidil, which has attracted much interest in the management of systemic hypertension and androgenetic alopecia, can also be used to reduce lysyl hydroxylase activity in cultured cells. An in vivo pilot dosing study established 50 mg⋅kg -1 ⋅day -1 as the maximum possible minoxidil dose for intraperitoneal administration in newborn mouse pups. When administered at 50 mg⋅kg -1 ⋅day -1 to newborn mouse pups, minoxidil was detected in the lungs but did not impact lysine hydroxylation, collagen crosslinking, or lysyl hydroxylase expression in the lungs. Consistent with no impact on mouse lung extracellular matrix structures, minoxidil administration did not alter the course of normal or stunted lung development in newborn mice. At doses of up to 50 mg⋅kg⋅day -1 , pharmacologically active concentrations of minoxidil were not achieved in neonatal mouse lung tissue; thus, minoxidil cannot be used to attenuate lysyl hydroxylase expression or activity during mouse lung development. These data also highlight the need for new and specific lysyl hydroxylase inhibitors. SIGNIFICANCE STATEMENT: Extracellular matrix crosslinking is mediated by lysyl hydroxylases, which generate hydroxylated lysyl residues in procollagen peptides. Deregulated collagen crosslinking is a pathogenic component of a spectrum of diseases, and thus, there is interest in validating lysyl hydroxylases as pathogenic mediators of disease and potential "druggable" targets. Minoxidil, administered at the maximum possible dose, did not inhibit lysyl hydroxylation in newborn mouse lungs, suggesting that minoxidil was unlikely to be of use in studies that pharmacologically target lysyl hydroxylation in vivo., (Copyright © 2020 by The American Society for Pharmacology and Experimental Therapeutics.)

Organoids derived from mouse and human stem cells have recently emerged as a powerful tool to study organ development and disease. We here established a three-dimensional (3D) murine bronchioalveolar lung organoid (BALO) model that allows clonal expansion and self-organization of FACS-sorted bronchioalveolar stem cells (BASCs) upon co-culture with lung-resident mesenchymal cells. BALOs yield a highly branched 3D structure within 21 days of culture, mimicking the cellular composition of the bronchioalveolar compartment as defined by single-cell RNA sequencing and fluorescence as well as electron microscopic phenotyping. Additionally, BALOs support engraftment and maintenance of the cellular phenotype of injected tissue-resident macrophages. We also demonstrate that BALOs recapitulate lung developmental defects after knockdown of a critical regulatory gene, and permit modeling of viral infection. We conclude that the BALO model enables reconstruction of the epithelial-mesenchymal-myeloid unit of the distal lung, thereby opening numerous new avenues to study lung development, infection, and regenerative processes in vitro., (© 2020 The Authors. Published under the terms of the CC BY 4.0 license.)

Idiopathic pulmonary fibrosis (IPF) is a fatal disease in which the intricate alveolar network of the lung is progressively replaced by fibrotic scars. Myofibroblasts are the effector cells that excessively deposit extracellular matrix proteins thus compromising lung structure and function. Emerging literature suggests a correlation between fibrosis and metabolic alterations in IPF. In this study, we show that the first-line antidiabetic drug metformin exerts potent antifibrotic effects in the lung by modulating metabolic pathways, inhibiting TGFβ1 action, suppressing collagen formation, activating PPARγ signaling and inducing lipogenic differentiation in lung fibroblasts derived from IPF patients. Using genetic lineage tracing in a murine model of lung fibrosis, we show that metformin alters the fate of myofibroblasts and accelerates fibrosis resolution by inducing myofibroblast-to-lipofibroblast transdifferentiation. Detailed pathway analysis revealed a two-arm mechanism by which metformin accelerates fibrosis resolution. Our data report an antifibrotic role for metformin in the lung, thus warranting further therapeutic evaluation.

Background: The laboratory mouse is widely used in preclinical models of bronchopulmonary dysplasia, where lung alveolarization is stunted by exposure of pups to hyperoxia. Whether the diverse genetic backgrounds of different inbred mouse strains impacts lung development in newborn mice exposed to hyperoxia has not been systematically assessed., Methods: Hyperoxia (85% O 2 , 14 days)-induced perturbations to lung alveolarization were assessed by design-based stereology in C57BL/6J, BALB/cJ, FVB/NJ, C3H/HeJ, and DBA/2J inbred mouse strains. The expression of components of the lung antioxidant machinery was assessed by real-time reverse transcriptase polymerase chain reaction and immunoblot., Results: Hyperoxia-reduced lung alveolar density in all five mouse strains to different degrees (C57BL/6J, 64.8%; FVB/NJ, 47.4%; BALB/cJ, 46.4%; DBA/2J, 45.9%; and C3H/HeJ, 35.9%). Hyperoxia caused a 94.5% increase in mean linear intercept in the C57BL/6J strain, whilst the C3H/HeJ strain was the least affected (31.6% increase). In contrast, hyperoxia caused a 65.4% increase in septal thickness in the FVB/NJ strain, where the C57BL/6J strain was the least affected (30.3% increase). The expression of components of the lung antioxidant machinery in response to hyperoxia was strain dependent, with the C57BL/6J strain exhibiting the most dramatic engagement. Baseline expression levels of components of the lung antioxidant systems were different in the five mouse strains studied, under both normoxic and hyperoxic conditions., Conclusion: The genetic background of laboratory mouse strains dramatically influenced the response of the developing lung to hyperoxic insult. This might be explained, at least in part, by differences in how antioxidant systems are engaged by different mouse strains after hyperoxia exposure., (© 2019 Wiley Periodicals, Inc.)

Accurate estimation of the absolute number of a particular cell-type in whole organs is increasingly important in studies on organogenesis, and the remodelling and repair of diseased tissues. The unbiased estimation of the absolute number of cells in an organ is complicated, and design-based stereology remains the method of choice. This has led investigators to explore alternative approaches - such as flow cytometry - as a faster and less labour-intensive replacement for stereology. To address whether flow cytometry might substitute stereology, design-based stereology was compared with microfluorosphere-controlled flow cytometry, for estimation of the absolute number of alveolar epithelial type 2 cells (AEC2) in the lungs of two mouse strains: wild-type C57BL/6J mice and Sftpc-YFP mice. Using design-based stereology, ≈10.7 million and ≈9.0 million AEC2 were estimated in the lungs of wild-type C57BL/6J mice and Sftpc-YFP mice, respectively. Substantially fewer AEC2 were estimated using flow cytometry. In wild-type C57/BL6J mouse lungs, 59% of the AEC2 estimated by design-based stereology were estimated by flow cytometry (≈6.3 million), using intracellular staining for pro-surfactant protein C. Similarly, in Sftpc-YFP mouse lungs, 23% of the AEC2 estimated by design-based stereology were estimated by flow cytometry (≈2.1 million), using yellow fluorescent protein fluorescence. Our data suggest that flow cytometry underestimates AEC2 number, possibly due to impaired recoverability of AEC2 from dissociated lung tissue. These data suggest design-based stereology as the method of choice for the unbiased estimation of the absolute number of cells in an organ. LAY DESCRIPTION: There is much interest in studies on the pathological changes that accompany disease, to be able to count or estimate the number of a particular cell-type in solid tissue, such as an organ. The easiest way to do this is to make liquid suspensions of single cells from solid tissue, and then to count the number of cells of interest, using either a microscope, or automated cell counting (for example, a flow cytometer). Alternatively, solid tissue may be examined microscopically, where the cell-type of interest might also be counted 'by eye' or in an automated manner using software (called planimetry). All of these approaches to counting cells in solid organs come with serious drawbacks, and estimation of the cell number may thus be inaccurate. To overcome this, we have employed a combination of mathematical tools and statistical principles together with microscopy (called 'design-based stereology') that permits the unbiased counting of cells in microscopic fields, which can then be extrapolated to the entire solid tissue volume, to accurately estimate the number of a cell-type of interest in the solid tissue. We have compared this method with the estimation of cell number using a flow cytometer. Our data reveal that flow cytometry appreciably underestimates the total number of cells in solid tissue, where we used the lung as an example of solid tissue, and estimated the number of a unique cell-type in the lung: the alveolar epithelial type 2 cell, to compare stereology with flow cytometry. We believe that flow cytometry underestimates the cell number due to the difficulty of breaking up solid tissue into single cells, and being able to recover all of those single cells for analysis. Our data supports the recommendation to use stereology, not flow cytometry, to accurately estimate the number of a particular cell-type in solid tissue. Accurate estimation of the absolute number of a particular cell-type in whole organs is increasingly important in studies on organogenesis, and the remodelling and repair of diseased tissues. Although estimation of the relative number of cells might be straightforward, unbiased estimation of the absolute number of cells in an organ is complicated, and design-based stereology remains the method of choice. This has led investigators to explore alternative approaches - such as flow cytometry - as a faster and less labour-intensive replacement for stereology. To address whether flow cytometry might substitute stereology, design-based stereology was compared with microfluorosphere-controlled flow cytometry, for estimation of the absolute number of alveolar epithelial type 2 cells (AEC2) in the lungs of two mouse strains: wild-type C57BL/6J mice and Sftpc-YFP mice. Using design-based stereology, ≈10.7 million and ≈9.0 million AEC2 were estimated in the lungs of wild-type C57BL/6J mice and Sftpc-YFP mice, respectively. Substantially fewer AEC2 were estimated using flow cytometry. In wild-type C57/BL6J mouse lungs, 59% of the AEC2 estimated by design-based stereology were estimated by flow cytometry (≈6.3 million), using intracellular staining for pro-surfactant protein C. Similarly, in Sftpc-YFP mouse lungs, 23% of the AEC2 estimated by design-based stereology were estimated by flow cytometry (≈2.1 million), using yellow fluorescent protein fluorescence. Our data suggest that flow cytometry underestimates AEC2 number, possibly due to impaired recoverability of AEC2 from dissociated lung tissue. These data suggest design-based stereology as the method of choice for the unbiased estimation of the absolute number of cells in an organ., (© 2019 The Authors Journal of Microscopy © 2019 Royal Microscopical Society.)

There is currently much interest in understanding the mechanisms of normal and aberrant lung alveolarization, particularly in the context of bronchopulmonary dysplasia, a common complication of preterm birth where alveolarization is impeded. To this end, the parenteral administration of pharmacological agents that modulate biochemical pathways, or facilitate modulation of gene expression in transgenic animals, has facilitated the discovery and validation of mechanisms that direct lung development. Such studies include control interventions, where the solvent vehicle, perhaps containing an inactive form of the agent applied, is administered; thereby providing a well-controlled point of reference for the analysis of the partner experiment. In the present study, the impact of several widely used control interventions in developing C57Bl/6J mouse pups was examined for effects on lung structure and the lung transcriptome. Parenteral administration of scrambled microRNA inhibitors (called antagomiRs) that are used to control in vivo microRNA neutralization studies, impacted lung volume, septal thickness, and the transcriptome of developing mouse lungs; with some effects dependent upon nucleotide sequence. Repeated intraperitoneal isotonic saline injections altered lung volume, with limited impact on the transcriptome. Parenteral administration of the tamoxifen solvent Miglyol accelerated mouse pup growth, and changed the abundance of 73 mRNA transcripts in the lung. Tamoxifen applied in Miglyol-in the absence of Cre recombinase-decreased pup growth, lung volume, and lung alveolarization and changed the abundance of 298 mRNA transcripts in the lung. These data demonstrate that widely used control interventions can directly impact lung alveolarization and the lung transcriptome in studies on lung development. Anat Rec, 302:346-363, 2019. © 2018 Wiley Periodicals, Inc., (© 2018 Wiley Periodicals, Inc.)

Background: Gas exchange represents the key physiological function of the lung, and is dependent upon proper formation of the delicate alveolar structure. Malformation or destruction of the alveolar gas-exchange regions are key histopathological hallmarks of diseases such as bronchopulmonary dysplasia (BPD), chronic obstructive pulmonary disease (COPD), and pulmonary fibrosis; all of which are characterized by perturbations to the alveolo-capillary barrier structure. Impaired gas-exchange is the primary initial consequence of these perturbations, resulting in severe clinical symptoms, reduced quality of life, and death. The pronounced morbidity and mortality associated with malformation or destruction of alveoli underscores a pressing need for new therapeutic concepts. The re-induction of alveolarization in diseased lungs is a new and exciting concept in a regenerative medicine approach to manage pulmonary diseases that are characterized by an absence of alveoli., Main Text: Mechanisms of alveolarization first need to be understood, to identify pathways and mediators that may be exploited to drive the induction of alveolarization in the diseased lung. With this in mind, a variety of candidate cell-types, pathways, and molecular mediators have recently been identified. Using lineage tracing approaches and lung injury models, new progenitor cells for epithelial and mesenchymal cell types - as well as cell lineages which are able to acquire stem cell properties - have been discovered. However, the underlying mechanisms that orchestrate the complex process of lung alveolar septation remain largely unknown., Conclusion: While important progress has been made, further characterization of the contributing cell-types, the cell type-specific molecular signatures, and the time-dependent chemical and mechanical processes in the developing, adult and diseased lung is needed in order to implement a regenerative therapeutic approach for pulmonary diseases.

Background: Since 2009, IPF patients across Europe are recruited into the eurIPFreg, providing epidemiological data and biomaterials for translational research., Methods: The registry data are based on patient and physician baseline and follow-up questionnaires, comprising 1700 parameters. The mid- to long-term objectives of the registry are to provide clues for a better understanding of IPF phenotype sub-clusters, triggering factors and aggravating conditions, regional and environmental characteristics, and of disease behavior and management., Results: This paper describes baseline data of 525 IPF subjects recruited from 11/2009 until 10/2016. IPF patients had a mean age of 68.1 years, and seeked medical advice due to insidious dyspnea (90.1%), fatigue (69.2%), and dry coughing (53.2%). A surgical lung biopsy was performed in 32% in 2009, but in only 8% of the cases in 2016, possibly due to increased numbers of cryobiopsy. At the time of inclusion in the eurIPFreg, FVC was 68.4% ± 22.6% of predicted value, DLco ranged at 42.1% ± 17.8% of predicted value (mean value ± SD). Signs of pulmonary hypertension were found in 16.8%. Steroids, immunosuppressants and N-Acetylcysteine declined since 2009, and were replaced by antifibrotics, under which patients showed improved survival (p = 0.001)., Conclusions: Our data provide important insights into baseline characteristics, diagnostic and management changes as well as outcome data in European IPF patients over time., Trial Registration: The eurIPFreg and eurIPFbank are listed in ClinicalTrials.gov( NCT02951416 ).

The quantitative assessment of the lung architecture forms the foundation of many studies on lung development and lung diseases, where parameters such as alveoli number, alveolar size, and septal thickness are quantitatively influenced by developmental or pathological processes. Given the pressing need for robust data that describe the lung structure, there is currently much enthusiasm for the development and refinement of methodological approaches for the unbiased assessment of lung structure with improved precision. The advent of stereological methods highlights one such approach. However, design-based stereology is both expensive and time-demanding. The objective of this study was to examine whether 'limited' stereological analysis, such as the stereological analysis of a single mouse lung lobe, may serve as a surrogate for studies on whole, intact mouse lungs; both in healthy lungs and in diseased lungs, using an experimental animal model of bronchopulmonary dysplasia (BPD). This served the dual-function of exploring BPD pathobiology, asking whether there are regional (lobar) differences in the responses of developing mouse lungs to oxygen injury, by examining each mouse lung lobe separately in the BPD model. Hyperoxia exposure resulted in decreased alveolar density, alveoli number, and gas-exchange surface area in all five mouse lung lobes, and increased the arithmetic mean septal thickness in all mouse lung lobes except the lobus cardialis. The data presented here suggest that - in healthy developing mice - a single mouse lung lobe might serve as a surrogate for studies on whole, intact mouse lungs. This is not the case for oxygen-injured developing mouse lungs, where a single lobe would not be suitable as a surrogate for the whole, intact lung. Furthermore, as the total number of alveoli can only be determined by an analysis of the entire lung, and given regional differences in lung structure, particularly under pathological conditions, the stereological assessment of the whole, intact lung remains desirable., (© 2018 Anatomical Society.)

Rationale: Acute pulmonary oxygen sensing is essential to avoid life-threatening hypoxemia via hypoxic pulmonary vasoconstriction (HPV) which matches perfusion to ventilation. Hypoxia-induced mitochondrial superoxide release has been suggested as a critical step in the signaling pathway underlying HPV. However, the identity of the primary oxygen sensor and the mechanism of superoxide release in acute hypoxia, as well as its relevance for chronic pulmonary oxygen sensing, remain unresolved., Objectives: To investigate the role of the pulmonary-specific isoform 2 of subunit 4 of the mitochondrial complex IV (Cox4i2) and the subsequent mediators superoxide and hydrogen peroxide for pulmonary oxygen sensing and signaling., Methods and Results: Isolated ventilated and perfused lungs from Cox4i2 -/- mice lacked acute HPV. In parallel, pulmonary arterial smooth muscle cells (PASMCs) from Cox4i2 -/- mice showed no hypoxia-induced increase of intracellular calcium. Hypoxia-induced superoxide release which was detected by electron spin resonance spectroscopy in wild-type PASMCs was absent in Cox4i2 -/- PASMCs and was dependent on cysteine residues of Cox4i2. HPV could be inhibited by mitochondrial superoxide inhibitors proving the functional relevance of superoxide release for HPV. Mitochondrial hyperpolarization, which can promote mitochondrial superoxide release, was detected during acute hypoxia in wild-type but not Cox4i2 -/- PASMCs. Downstream signaling determined by patch-clamp measurements showed decreased hypoxia-induced cellular membrane depolarization in Cox4i2 -/- PASMCs compared with wild-type PASMCs, which could be normalized by the application of hydrogen peroxide. In contrast, chronic hypoxia-induced pulmonary hypertension and pulmonary vascular remodeling were not or only slightly affected by Cox4i2 deficiency, respectively., Conclusions: Cox4i2 is essential for acute but not chronic pulmonary oxygen sensing by triggering mitochondrial hyperpolarization and release of mitochondrial superoxide which, after conversion to hydrogen peroxide, contributes to cellular membrane depolarization and HPV. These findings provide a new model for oxygen-sensing processes in the lung and possibly also in other organs., (© 2017 American Heart Association, Inc.)

Lysyl oxidases are credited with pathogenic roles in lung diseases, including cancer, fibrosis, pulmonary hypertension, congenital diaphragmatic hernia, and bronchopulmonary dysplasia (BPD). Lysyl oxidases facilitate the covalent intra- and intermolecular cross-linking of collagen and elastin fibers, thereby imparting tensile strength to the extracellular matrix (ECM). Alternative ECM-independent roles have recently been proposed for lysyl oxidases, including regulation of growth factor signaling, chromatin remodeling, and transcriptional regulation, all of which impact cell phenotype. We demonstrate here that three of the five lysyl oxidase family members, Lox , Loxl1 , and Loxl2 , are highly expressed in primary mouse lung fibroblasts compared with other constituent cell types of the lung. Microarray analyses revealed that small interfering RNA knockdown of Lox , Loxl1 , and Loxl2 was associated with apparent changes in the expression of 134, 3,761, and 3,554 genes, respectively, in primary mouse lung fibroblasts. The impact of lysyl oxidase expression on steady-state Mmp3 , Mmp9 , Eln , Rarres1 , Gdf10 , Ifnb1 , Csf2 , and Cxcl9 mRNA levels was validated, which is interesting, since the corresponding gene products are relevant to lung development and BPD, where lysyl oxidases play a functional role. In vivo, the expression of these genes broadly correlated with Lox , Loxl1 , and Loxl2 expression in a mouse model of BPD. Furthermore, β-aminopropionitrile (BAPN), a selective lysyl oxidase inhibitor, did not affect the steady-state mRNA levels of lysyl oxidase target genes, in vitro in lung fibroblasts or in vivo in BAPN-treated mice. This study is the first to report that lysyl oxidases broadly influence the cell transcriptome., (Copyright © 2017 the American Physiological Society.)

Background: Airway smooth muscle cells (ASMCs) have been widely studied during embryonic lung development. These cells have been shown to control epithelial bifurcation during branching morphogenesis. Fibroblast growth factor 10-positive (FGF10 + ) cells, originally residing in the submesothelial mesenchyme, contribute to ASMC formation in the distal lung. The reported work aims at monitoring the response of FGF10 + progenitors and differentiated ASMCs to growth factor treatment in real time using lineage tracing in the background of an air-liquid interface (ALI) culture system., Results: FGF ligands impose divergent effects on iterative lung branching in vitro. Moreover, time-lapse imaging and endpoint analysis show that FGF9 treatment leads to amplification of the FGF10 + lineage and represses its differentiation to ASMCs. Sonic hedgehog (SHH) treatment reduces the amplification of this lineage and leads to decreased lung branching. Finally, differentiated ASMCs in proximal regions fail to expand upon FGF9 treatment., Conclusions: Our data demonstrate, in real time, that FGF9 is an important regulator of amplification, migration, and subsequent differentiation of ASMC progenitors during early lung development. The attained results agree with previous findings regarding ASMC formation and highlight the complexity of growth factor signaling networks in controlling mesenchymal cell-fate decisions in the developing mouse lung. Developmental Dynamics 246:531-538, 2017. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

Aims: Pulmonary hypertension is a progressive disease with poor prognosis, characterized by pathological inward remodelling and loss of patency of the lung vasculature. The right ventricle is co-affected by pulmonary hypertension, which triggers events such as hypoxia and/or increased mechanical load. Initially the right ventricle responds with 'adaptive' hypertrophy, which is often rapidly followed by 'maladaptive' changes leading to right heart decompensation and failure, which is the ultimate cause of death., Methods and Results: We report here that miR-223 is expressed in the murine lung and right ventricle at higher levels than in the left ventricle. Moreover, lung and right-ventricular miR-223 levels were markedly down-regulated by hypoxia. Correspondingly, increasing right-ventricular load by pulmonary artery banding, induced right-ventricular ischaemia, and the down-regulation of miR-223. Lung and right ventricle miR-223 down-regulation were linked with increased expression of the miR-223 target; insulin-like growth factor-I receptor (IGF-IR) and IGF-I downstream signalling. Similarly, miR-223 was decreased and IGF-IR increased in human pulmonary hypertension. Notably in young mice, miR-223 overexpression, the genetic inactivation or pharmacological inhibition of IGF-IR, all attenuated right-ventricular hypertrophy and improved right heart function under conditions of hypoxia or increased afterload., Conclusion: These findings highlight the early role of pulmonary and right-ventricular miR-223 and the IGF-IR in the right heart failure programme initiated by pulmonary hypoxia and increased mechanical load and may lead to the development of novel therapeutic strategies that target the development of PH and right heart failure., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2016. For permissions please email: journals.permissions@oup.com.)

Herein, we challenged the isolated lung (IL) technique to discriminate the performance of lung-delivered polymeric microparticles (MPs) having distinct drug release rates. For this purpose, sildenafil-loaded poly(lactide-co-glycolide) MPs were administered to the airspace of an IL model and the drug absorption profile was monitored. MPs (particle size of ~5μm) composed of PLGA of lower molecular weight (and glass transition temperature) manifested in the most rapid in vitro drug release (half-times ranging from <15 to ~200min). Moreover, microencapsulation resulted in a delayed sildenafil transfer over the air/perfusate barrier (half-times ranging from <5 to ~230min), where the actual ex vivo absorption profile depended on the release behavior of the utilized formulation. Finally, the obtained in vitro and ex vivo results were tested for level C, B and A correlations. The plotted data showed good agreement (R(2)>0.96) and the slopes of the resulting lines of regression (i.e., 0.80-0.85) indicated a slightly elevated in vitro drug release behavior. Overall, the IL model was able to differentiate between distinct microparticulate formulations and is, therefore, a valuable technique for early testing of potential inhalable controlled release medications., (Copyright © 2016 Elsevier B.V. All rights reserved.)

In addition to its renowned poisonous effects, carbon monoxide (CO) is being recognized for its beneficial actions on inflammatory and vasoregulatory pathways, particularly when applied at low concentrations via CO-releasing molecules (CO-RMs). In the lung, CO gas and CO-RMs are suggested to decrease pulmonary vascular tone and hypoxic pulmonary vasoconstriction (HPV). However, the direct effect of CO-RMs on the pulmonary vasoreactivity in isolated lungs has not yet been investigated. We assessed the effect of CORM-2 and CORM-3 on the pulmonary vasculature during normoxia and acute hypoxia (1% oxygen for 10 min) in isolated ventilated and perfused mouse lungs. The effects were compared with those of inhaled CO gas (10%). The interaction of CORM-2 or CO with cytochrome P-450 (CYP) was measured simultaneously by tissue spectrophotometry. Inhaled CO decreased HPV and vasoconstriction induced by the thromboxane mimetic U-46619 but did not alter KCl-induced vasoconstriction. In contrast, concentrations of CORM-2 and CORM-3 used to elicit beneficial effects on the systemic circulation did not affect pulmonary vascular tone. High concentration of CO-RMs or long-term application induced a continuous increase in normoxic pressure. Inhaled CO showed spectral alterations correlating with the inhibition of CYP. In contrast, during application of CORM-2 spectrophotometric signs of interaction with CYP could not be detected. Application of CO-RMs in therapeutic doses in isolated lungs neither decreases pulmonary vascular tone and HPV nor does it induce spectral alterations that are characteristic of CO-inhibited CYP. High doses, however, may cause pulmonary vasoconstriction., (Copyright © 2016 the American Physiological Society.)

Acute respiratory distress syndrome (ARDS) is clinical syndrome characterized by decreased lung fluid reabsorption, causing alveolar edema. Defective alveolar ion transport undertaken in part by the Na(+)/K(+)-ATPase underlies this compromised fluid balance, although the molecular mechanisms at play are not understood. We describe here increased expression of FXYD1, FXYD3 and FXYD5, three regulatory subunits of the Na(+)/K(+)-ATPase, in the lungs of ARDS patients. Transforming growth factor (TGF)-β, a pathogenic mediator of ARDS, drove increased FXYD1 expression in A549 human lung alveolar epithelial cells, suggesting that pathogenic TGF-β signaling altered Na(+)/K(+)-ATPase activity in affected lungs. Lentivirus-mediated delivery of FXYD1 and FXYD3 allowed for overexpression of both regulatory subunits in polarized H441 cell monolayers on an air/liquid interface. FXYD1 but not FXYD3 overexpression inhibited amphotericin B-sensitive equivalent short-circuit current in Ussing chamber studies. Thus, we speculate that FXYD1 overexpression in ARDS patient lungs may limit Na(+)/K(+)-ATPase activity, and contribute to edema persistence., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Objectives: This study investigated the controlled drug release potential of formulations revealing temperature-induced sol-gel transition following administration to the respiratory tract., Methods: Diverse sildenafil-containing aqueous poloxamer 407 preparations were evaluated for critical gelation temperature and rheological properties. The in-vitro drug release profiles of the in-situ forming formulations were studied in a Franz type cell, while the drug absorption characteristics were determined in an isolated lung model. Furthermore, the weight gain of isolated lungs was monitored and the bronchoalveolar lavage fluid was analysed for the total protein content., Key Findings: Poloxamer 407 solutions with concentrations of >12 wt.% revealed gelation upon temperature increase (>20°C). Compared with free sildenafil solution, sildenafil-containing polymer formulations showed a prolonged in-vitro drug release profile. Likewise, 17 and 21 wt.% of poloxamer 407 were characterized by a sustained sildenafil transfer from the lung into the perfusate. However, a 10 wt.% polymer solution displayed an immediate sildenafil absorption. Interestingly, increasing the poloxamer 407 concentration (21 and 17 vs. 10 wt.%) led to decreased organ weight gain kinetics and a lower total protein content found in the bronchoalveolar lavage fluid., Conclusions: In-situ forming controlled release hydrogels represent a viable approach for inhalative therapy., (© 2015 Royal Pharmaceutical Society.)

Limited literature sources implicate mast-cell mediator chymase in the pathologies of pulmonary hypertension and pulmonary fibrosis. However, there is no evidence on the contribution of chymase to the development of pulmonary hypertension associated with lung fibrosis, which is an important medical condition linked with increased mortality of patients who already suffer from a life-threatening interstitial lung disease.The aim of this study was to investigate the role of chymase in this particular pulmonary hypertension form, by using a bleomycin-induced pulmonary hypertension model.Chymase inhibition resulted in attenuation of pulmonary hypertension and pulmonary fibrosis, as evident from improved haemodynamics, decreased right ventricular remodelling/hypertrophy, pulmonary vascular remodelling and lung fibrosis. These beneficial effects were associated with a strong tendency of reduction in mast cell number and activity, and significantly diminished chymase expression levels. Mechanistically, chymase inhibition led to attenuation of transforming growth factor β1 and matrix-metalloproteinase-2 contents in the lungs. Furthermore, chymase inhibition prevented big endothelin-1-induced vasoconstriction of the pulmonary arteries.Therefore, chymase plays a role in the pathogenesis of pulmonary hypertension associated with pulmonary fibrosis and may represent a promising therapeutic target. In addition, this study may provide valuable insights on the contribution of chymase in the pulmonary hypertension context, in general, regardless of the pulmonary hypertension form., (Copyright ©ERS 2015.)

Maturation of the lung extracellular matrix (ECM) plays an important role in the formation of alveolar gas exchange units. A key step in ECM maturation is cross-linking of collagen and elastin, which imparts stability and functionality to the ECM. During aberrant late lung development in bronchopulmonary dysplasia (BPD) patients and animal models of BPD, alveolarization is blocked, and the function of ECM cross-linking enzymes is deregulated, suggesting that perturbed ECM cross-linking may impact alveolarization. In a hyperoxia (85% O2)-based mouse model of BPD, blunted alveolarization was accompanied by alterations to lung collagen and elastin levels and cross-linking. Total collagen levels were increased (by 63%). The abundance of dihydroxylysinonorleucine collagen cross-links and the dihydroxylysinonorleucine-to-hydroxylysinonorleucine ratio were increased by 11 and 18%, respectively, suggestive of a profibrotic state. In contrast, insoluble elastin levels and the abundance of the elastin cross-links desmosine and isodesmosine in insoluble elastin were decreased by 35, 30, and 21%, respectively. The lung collagen-to-elastin ratio was threefold increased. Treatment of hyperoxia-exposed newborn mice with the lysyl oxidase inhibitor β-aminopropionitrile partially restored normal collagen levels, normalized the dihydroxylysinonorleucine-to-hydroxylysinonorleucine ratio, partially normalized desmosine and isodesmosine cross-links in insoluble elastin, and partially restored elastin foci structure in the developing septa. However, β-aminopropionitrile administration concomitant with hyperoxia exposure did not improve alveolarization, evident from unchanged alveolar surface area and alveoli number, and worsened septal thickening (increased by 12%). These data demonstrate that collagen and elastin cross-linking are perturbed during the arrested alveolarization of developing mouse lungs exposed to hyperoxia., (Copyright © 2015 the American Physiological Society.)

Chronic obstructive pulmonary disease (COPD) is a major disease of the lungs. It primarily occurs after a prolonged period of cigarette smoking. Chronic inflammation of airways and the alveolar space as well as lung tissue destruction are the hallmarks of COPD. Recently it has been shown that cellular senescence might play a role in the pathogenesis of COPD. Cellular senescence comprises signal transduction program, leading to irreversible cell cycle arrest. The growth arrest in senescence can be triggered by many different mechanisms, including DNA damage and its recognition by cellular sensors, leading to the activation of cell cycle checkpoint responses and activation of DNA repair machinery. Senescence can be induced by several genotoxic factors apart from telomere attrition. When senescence induction is based on DNA damage, senescent cells display a unique phenotype, which has been termed "senescence-associated secretory phenotype" (SASP). SASP may be an important driver of chronic inflammation and therefore may be part of a vicious cycle of inflammation, DNA damage, and senescence. This research perspective aims to showcase cellular senescence with relevance to COPD and the striking similarities between the mediators and secretory phenotype in COPD and SASP.

Octamer binding trascription factor 4 (Oct4) is a transcription factor of POU family specifically expressed in embryonic stem cells (ESCs). A role for maintaining pluripotency and self-renewal of ESCs is assigned to Oct4 as a pluripotency marker. Oct4 can also be detected in adult stem cells such as bone marrow-derived mesenchymal stem cells. Several studies suggest a role for Oct4 in sustaining self-renewal capacity of adult stem cells. However, Oct4 gene ablation in adult stem cells revealed no abnormalities in tissue turnover or regenerative capacity. In the present study we have conspicuously found pulmonary Oct4-positive cells closely resembling the morphology of telocytes (TCs). These cells were found in the perivascular and peribronchial areas and their presence and location were confirmed by electron microscopy. Moreover, we have used Oct4-GFP transgenic mice which revealed a similar localization of the Oct4-GFP signal. We also found that Oct4 co-localized with several described TC markers such as vimentin, Sca-1, platelet-derived growth factor receptor-beta C-kit and VEGF. By flow cytometry analyses carried out with Oct4-GFP reporter mice, we described a population of EpCAM(neg) /CD45(neg) /Oct4-GFP(pos) that in culture displayed TC features. These results were supported by qRT-PCR with mRNA isolated from lungs by using laser capture microdissection. In addition, Oct4-positive cells were found to express Nanog and Klf4 mRNA. It is concluded for the first time that TCs in adult lung mouse tissue comprise Oct4-positive cells, which express pluripotency-related genes and represent therefore a population of adult stem cells which might contribute to lung regeneration., (© 2014 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Background: Pulmonary hypertension (PH) is a life-threatening disease characterized by vascular remodeling and increased pulmonary vascular resistance. Chronic alveolar hypoxia in animals is often used to decipher pathways being regulated in PH. Here, we aimed to investigate whether chronic hypoxia-induced PH in mice can be reversed by reoxygenation and whether possible regression can be used to identify pathways activated during the reversal and development of PH by genome-wide screening., Methods and Results: Mice exposed to chronic hypoxia (21 days, 10% O2) were reoxygenated for up to 42 days. Full reversal of PH during reoxygenation was evident by normalized right ventricular pressure, right heart hypertrophy, and muscularization of small pulmonary vessels. Microarray analysis from these mice revealed s-adenosylmethionine decarboxylase 1 (AMD-1) as one of the most downregulated genes. In situ hybridization localized AMD-1 in pulmonary vessels. AMD-1 silencing decreased the proliferation of pulmonary arterial smooth muscle cells and diminished phospholipase Cγ1 phosphorylation. Compared with the respective controls, AMD-1 depletion by heterozygous in vivo knockout or pharmacological inhibition attenuated PH during chronic hypoxia. A detailed molecular approach including promoter analysis showed that AMD-1 could be regulated by early growth response 1, transcription factor, as a consequence of epidermal growth factor stimulation. Key findings from the animal model were confirmed in human idiopathic pulmonary arterial hypertension., Conclusions: Our study indicates that genome-wide screening in mice from a PH model in which full reversal of PH occurs can be useful to identify potential key candidates for the reversal and development of PH. Targeting AMD-1 may represent a promising strategy for PH therapy.

Background: The high-mobility-group (HMG) proteins are the most abundant non-histone chromatin-associated proteins. HMG proteins are present at high levels in various undifferentiated tissues during embryonic development and their levels are strongly reduced in the corresponding adult tissues, where they have been implicated in maintaining and activating stem/progenitor cells. Here we deciphered the role of the high-mobility-group AT-hook protein 2 (HMGA2) during lung development by analyzing the lung of Hmga2-deficient mice (Hmga2(-/-))., Results: We found that Hmga2 is expressed in the mouse embryonic lung at the distal airways. Analysis of Hmga2(-/-) mice showed that Hmga2 is required for proper cell proliferation and distal epithelium differentiation during embryonic lung development. Hmga2 knockout led to enhanced canonical WNT signaling due to an increased expression of secreted WNT glycoproteins Wnt2b, Wnt7b and Wnt11 as well as a reduction of the WNT signaling antagonizing proteins GATA-binding protein 6 and frizzled homolog 2. Analysis of siRNA-mediated loss-of-function experiments in embryonic lung explant culture confirmed the role of Hmga2 as a key regulator of distal lung epithelium differentiation and supported the causal involvement of enhanced canonical WNT signaling in mediating the effect of Hmga2-loss-of-fuction. Finally, we found that HMGA2 directly regulates Gata6 and thereby modulates Fzd2 expression., Conclusions: Our results support that Hmga2 regulates canonical WNT signaling at different points of the pathway. Increased expression of the secreted WNT glycoproteins might explain a paracrine effect by which Hmga2-knockout enhanced cell proliferation in the mesenchyme of the developing lung. In addition, HMGA2-mediated direct regulation of Gata6 is crucial for fine-tuning the activity of WNT signaling in the airway epithelium. Our results are the starting point for future studies investigating the relevance of Hmga2-mediated regulation of WNT signaling in the adult lung within the context of proper balance between differentiation and self-renewal of lung stem/progenitor cells during lung regeneration in both homeostatic turnover and repair after injury.

The regulation of the balance between proliferation and differentiation in the mesenchymal compartment of the lung is largely uncharacterized, unlike its epithelial counterpart. In this study, we determined that miR-142-3p contributes to the proper proliferation of mesenchymal progenitors by controlling the level of WNT signaling. miR-142-3p can physically bind to adenomatous polyposis coli mRNA, functioning to regulate its expression level. In miR-142-3p loss-of-function experiments, proliferation of parabronchial smooth muscle cell progenitors is significantly impaired, leading to premature differentiation. Activation of WNT signaling in the mesenchyme, or Apc loss of function, can both rescue miR-142-3p knockdown. These findings show that in the embryonic lung mesenchyme, the microRNA machinery modulates the level of WNT signaling, adding an extra layer of control in the feedback loop between FGFR2C and β-catenin-mediated WNT signaling.

Glucocorticoids represent the mainstay therapy for many lung diseases, providing outstanding management of asthma but performing surprisingly poorly in patients with acute respiratory distress syndrome, chronic obstructive pulmonary disease, lung fibrosis, and blunted lung development associated with bronchopulmonary dysplasia in preterm infants. TGF-β is a pathogenic mediator of all four of these diseases, prompting us to explore glucocorticoid/TGF-β signaling cross-talk. Glucocorticoids, including dexamethasone, methylprednisolone, budesonide, and fluticasone, potentiated TGF-β signaling by the Acvrl1/Smad1/5/8 signaling axis and blunted signaling by the Tgfbr1/Smad2/3 axis in NIH/3T3 cells, as well as primary lung fibroblasts, smooth muscle cells, and endothelial cells. Dexamethasone drove expression of the accessory type III TGF-β receptor Tgfbr3, also called betaglycan. Tgfbr3 was demonstrated to be a "switch" that blunted Tgfbr1/Smad2/3 and potentiated Acvrl1/Smad1 signaling in lung fibroblasts. The Acvrl1/Smad1 axis, which was stimulated by dexamethasone, was active in lung fibroblasts and antagonized Tgfbr1/Smad2/3 signaling. Dexamethasone acted synergistically with TGF-β to drive differentiation of primary lung fibroblasts to myofibroblasts, revealed by acquisition of smooth muscle actin and smooth muscle myosin, which are exclusively Smad1-dependent processes in fibroblasts. Administration of dexamethasone to live mice recapitulated these observations and revealed a lung-specific impact of dexamethasone on lung Tgfbr3 expression and phospho-Smad1 levels in vivo. These data point to an interesting and hitherto unknown impact of glucocorticoids on TGF-β signaling in lung fibroblasts and other constituent cell types of the lung that may be relevant to lung physiology, as well as lung pathophysiology, in terms of drug/disease interactions.

Bronchopulmonary dysplasia (BPD) is a common and serious complication of premature birth, characterized by a pronounced arrest of alveolar development. The underlying pathophysiological mechanisms are poorly understood although perturbations to the maturation and remodeling of the extracellular matrix (ECM) are emerging as candidate disease pathomechanisms. In this study, the expression and regulation of three members of the lysyl hydroxylase family of ECM remodeling enzymes (Plod1, Plod2, and Plod3) in clinical BPD, as well as in an experimental animal model of BPD, were addressed. All three enzymes were localized to the septal walls in developing mouse lungs, with Plod1 also expressed in the vessel walls of the developing lung and Plod3 expressed uniquely at the base of developing septa. The expression of plod1, plod2, and plod3 was upregulated in the lungs of mouse pups exposed to 85% O2, an experimental animal model of BPD. Transforming growth factor (TGF)-β increased plod2 mRNA levels and activated the plod2 promoter in vitro in lung epithelial cells and in lung fibroblasts. Using in vivo neutralization of TGF-β signaling in the experimental animal model of BPD, TGF-β was identified as the regulator of aberrant plod2 expression. PLOD2 mRNA expression was also elevated in human neonates who died with BPD or at risk for BPD, compared with neonates matched for gestational age at birth or chronological age at death. These data point to potential roles for lysyl hydroxylases in normal lung development, as well as in perturbed late lung development associated with BPD.

The lung mesenchyme consists of a widely heterogeneous population of cells that play crucial roles during development and homeostasis after birth. These cells belong to myogenic, adipogenic, chondrogenic, neuronal and other lineages. Yet, no clear hierarchy for these lineages has been established. We have previously generated a novel Fgf10(iCre) knock-in mouse line that allows lineage tracing of Fgf10-positive cells during development and postnatally. Using these mice, we hereby demonstrate the presence of two waves of Fgf10 expression during embryonic lung development: the first wave, comprising Fgf10-positive cells residing in the submesothelial mesenchyme at early pseudoglandular stage (as well as their descendants); and the second wave, comprising Fgf10-positive cells from late pseudoglandular stage (as well as their descendants). Our lineage-tracing data reveal that the first wave contributes to the formation of parabronchial and vascular smooth muscle cells as well as lipofibroblasts at later developmental stages, whereas the second wave does not give rise to smooth muscle cells but to lipofibroblasts as well as an Nkx2.1(-) E-Cad(-) Epcam(+) Pro-Spc(+) lineage that requires further in-depth analysis. During alveologenesis, Fgf10-positive cells give rise to lipofibroblasts rather than alveolar myofibroblasts, and during adult life, a subpopulation of Fgf10-expressing cells represents a pool of resident mesenchymal stromal (stem) cells (MSCs) (Cd45(-) Cd31(-) Sca-1(+)). Taken together, we show for the first time that Fgf10-expressing cells represent a pool of mesenchymal progenitors in the embryonic and postnatal lung. Our findings suggest that Fgf10-positive cells could be useful for developing stem cell-based therapies for treating interstitial lung diseases.

The development of novel formulations for controlled pulmonary drug delivery purposes has gained remarkable interest in medicine. Although nanomedicine represents attractive concepts for the treatment of numerous systemic diseases, scant information is available on the controlled drug release characteristics of colloidal formulations following lung administration, which might be attributed to the lack of methods to follow their absorption and distribution behavior in the pulmonary environment.In this chapter, we describe the methods of preparation and characterization of drug-loaded polymeric nanoparticles prepared from biodegradable charge-modified branched polyesters, aerosolization of the nanosuspensions using a vibrating-mesh nebulizer, and evaluation of the pulmonary pharmacokinetics (i.e., absorption and distribution characteristics) of the nanoscale drug delivery vehicles following aerosol delivery to the airspace of an isolated lung model. The disclosed methodology may contribute to the design of advanced colloids for the treatment of respiratory disorders.

Acute and sustained hypoxic pulmonary vasoconstriction (HPV), as well as chronic pulmonary hypertension (PH), is modulated by nitric oxide (NO). NO synthesis can be decreased by asymmetric dimethylarginine (ADMA), which is degraded by dimethylarginine dimethylaminohydrolase-1 (DDAH1). We investigated the effects of DDAH1 overexpression (DDAH1(tg)) on HPV and chronic hypoxia-induced PH. HPV was measured during acute (10 min) and sustained (3 h) hypoxia in isolated mouse lungs. Chronic PH was induced by the exposure of mice to 4 weeks of hypoxia. ADMA and cyclic 3',5'-guanosine monophosphate (cGMP) were determined by ELISA, and NO generation was determined by chemiluminescence. DDAH1 overexpression exerted no effects on acute HPV. However, DDAH1(tg) mice showed decreased sustained HPV compared with wild-type (WT) mice. Concomitantly, ADMA was decreased, and concentrations of NO and cGMP were significantly increased in DDAH1(tg). The administration of either Nω-nitro-l-arginine or 1H-[1,2,4]oxadiazolo [4,3-a]quinoxalin-1-one potentiated sustained HPV and partly abolished the differences in sustained HPV between WT and DDAH1(tg) mice. The overexpression of DDAH1 exerted no effect on the development of chronic hypoxia-induced PH. DDAH1 overexpression selectively decreased the sustained phase of HPV, partly via activation of the NO-cGMP pathway. Thus, increased ADMA concentrations modulate sustained HPV, but not acute HPV or chronic hypoxia-induced PH.

Among the idiopathic interstitial pneumonias (IIP), the two entities IPF and NSIP seem to be clinically related, but NSIP has a better outcome. The proteomic signatures which distinguish NSIP from IPF remain still elusive. We therefore performed comparative proteomic analysis of peripheral lung tissue from patients with sporadic IPF (n=14) and fibrotic NSIP (fNSIP, n=8) and organ donors (Controls, n=10), by using the 2-dimensional DIGE technique and MALDI-TOF-MS. The study revealed that the proteomic profiles of IPF and fNSIP were quite similar. Among the upregulated proteins in IPF and fNSIP were stress-induced genes involved in the ER stress-pathway, whereas downregulated proteins in IPF and fNSIP included antiapoptotic factors and antifibrotic molecules. The comparison fNSIP versus IPF indicated upregulation of subunits of the proteasome activator complex and antioxidant enzymes of the peroxiredoxin family. We conclude, that only few protein expression changes exist between IPF and fNSIP, and that epithelial ER- and oxidative stress play a major role in the pathogenesis of both diseases. In contrast to IPF, intracellular clearance of ROS and misfolded protein carbonyls seem to be enhanced in fNSIP due to enhanced expression of antioxidant acting proteins, and may explain the better outcome and survival in patients with fNSIP., Biological Significance: IPF and fibrotic NSIP (fNSIP) belong to the idiopathic interstitial pneumonias and are usually fatal, but fNSIP has a better outcome. In order to identify molecular mechanisms and differences between IPF and fNSIP, we herein present results of a comparative proteome analysis of IPF, fNSIP and control lung tissue. Our data including validation experiments suggest that ER stress and a general stress-response as well as the decline of antioxidant capacity in alveolar epithelium is key in the pathogenesis of IPF and fNSIP. In addition, we could observe a signature of an increased alveolar epithelial protection against oxidative and ER-stress in fNSIP as compared to IPF, which could help to explain the better outcome of fNSIP patients., (Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.)

Liposomes have attracted extensive attention as inhalative drug delivery vehicles. The preparation of tailored liposomal formulations (i.e. nebulization stability and controlled drug release profiles) would facilitate new perspectives for the treatment of pulmonary diseases. 5(6)-Carboxyfluorescein (CF)-loaded submicron liposomal formulations with varying phase transition temperatures were prepared from lipid blends in different molar ratios. Their physicochemical properties, in vitro dye release, stability to nebulization (Aeroneb Pro) and ex vivo pulmonary dye absorption and distribution characteristics were investigated. Phase transitions of liposomes were adjusted below and above body temperature (32.9-55.2 °C). The amount of CF released from liposomes in vitro correlated well with their membrane fluidity. An increase in phase transition temperature resulted in an extended dye release profile. All formulations revealed aerodynamic particle sizes of ∼4 μm with remarkable stability when nebulized by vibrating-mesh technology (percentage of encapsulated model drug ∼80%). Analogous to the release results observed in vitro, liposomal formulations revealing phase transitions above body temperature displayed an increased pulmonary CF retention in an ex vivo lung model. Consequently, an in vitro-ex vivo correlation was established, which demonstrated an excellent agreement of the dye release results with the absorption profiles observed in the biological system (R(2) ≥ 0.91). Overall, the concept of liposomal "phase transition release" is promising for controlled pulmonary drug delivery applications. The ex vivo technique enables a reliable determination of lung-specific pharmacokinetics of drug delivery vehicles, which enhances tailored carrier preparation and testing during early formulation development., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Chronic alveolar hypoxia induces vascular remodeling processes in the lung resulting in pulmonary hypertension (PH). However, the mechanisms underlying pulmonary remodeling processes are not fully resolved yet. To investigate functional changes occurring during hypoxia exposure we applied 2DE to compare protein expression in lungs from mice subjected to 3 h of alveolar hypoxia and those kept under normoxic conditions. Already after this short-time period several proteins were significantly regulated. Subsequent analysis by MALDI-MS identified cofilin as one of the most prominently upregulated proteins. The regulation was confirmed by western blotting and its cellular localization was determined by immunohisto- and immunocytochemistry. Interestingly, enhanced cofilin serine 3 phosphorylation was observed after short-term and after chronic hypoxia-induced PH in mice, in pulmonary arterial smooth muscle cells (PASMC) from monocrotaline-induced PH in rats, in lungs of idiopathic pulmonary arterial hypertension patients and in hypoxic or platelet-derived growth factor BB-treated human PASMC. Furthermore, elevated cofilin phosphorylation was attenuated by curative treatment of monocrotaline-induced PH in rats and hypoxia-induced PH in mice with the PDGF-BB receptor antagonist imatinib. In conclusion, short-term hypoxic exposure induced prominent changes in lung protein regulation. These very early changes allowed us to identify potential triggers of PH. Thus, respective 2DE analysis can lead to the identification of new target proteins for the possible treatment of PH., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Nanomedicine comprises a significant potential to approach the therapy of severe diseases. Knowledge of nanoparticle behavior at the target site would contribute to the development of specialized tools for respiratory medicine. Here, we were interested in the potential of micro-computed tomography (μCT) imaging to monitor the pulmonary distribution of polymeric nanoparticles. Composite nanoparticles were analyzed for physicochemical properties, morphology and composition. μCT was employed to visualize the pulmonary distribution of composite nanoparticles in an ex vivo lung model. Employed composite nanoparticles were composed of poly(styrene) cores coated by a thin shell of colloidal iron oxide. Particles were mainly located in the interstitial space and associated with pulmonary cells, as observed by light microscopy. μCT detected enhanced X-ray opacities in the conducting (linear pattern) and respiratory airways (aciniform X-ray attenuations). In conclusion, multifunctional nanoparticles will prompt the development of novel therapeutic and diagnostic tools in respiratory medicine., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The alveolo-capillary barrier is effectively impermeable to large solutes such as proteins. A hallmark of acute lung injury/acute respiratory distress syndrome is the accumulation of protein-rich oedema fluid in the distal airspaces. Excess protein must be cleared from the alveolar space for recovery; however, the mechanisms of protein clearance remain incompletely understood. In intact rabbit lungs 29.8 ± 2.2% of the radio-labelled alveolar albumin was transported to the vascular compartment at 37°C within 120 min, as assessed by real-time measurement of 125I-albumin clearance from the alveolar space. At 4°C or 22°C significantly lower albumin clearance (3.7 ± 0.4 or 16.2 ± 1.1%, respectively) was observed. Deposition of a 1000-fold molar excess of unlabelled albumin into the alveolar space or inhibition of cytoskeletal rearrangement or clathrin-dependent endocytosis largely inhibited the transport of 125I-albumin to the vasculature, while administration of unlabelled albumin to the vascular space had no effect on albumin clearance. Furthermore, albumin uptake capacity was measured as about 0.37 mg ml−1 in cultured rat lung epithelial monolayers, further highlighting the (patho)physiological relevance of active alveolar epithelial protein transport. Moreover, gene silencing and pharmacological inhibition of the multi-ligand receptor megalin resulted in significantly decreased albumin binding and uptake in monolayers of primary alveolar type II and type I-like and cultured lung epithelial cells. Our data indicate that clearance of albumin from the distal air spaces is facilitated by an active, high-capacity, megalin-mediated transport process across the alveolar epithelium. Further understanding of this mechanism is of clinical importance, since an inability to clear excess protein from the alveolar space is associated with poor outcome in patients with acute lung injury/acute respiratory distress syndrome.

Lung ischaemia-reperfusion-induced oedema (LIRE) is a life-threatening condition that causes pulmonary oedema induced by endothelial dysfunction. Here we show that lungs from mice lacking nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox2(y/-)) or the classical transient receptor potential channel 6 (TRPC6(-/-)) are protected from LIR-induced oedema (LIRE). Generation of chimeric mice by bone marrow cell transplantation and endothelial-specific Nox2 deletion showed that endothelial Nox2, but not leukocytic Nox2 or TRPC6, are responsible for LIRE. Lung endothelial cells from Nox2- or TRPC6-deficient mice showed attenuated ischaemia-induced Ca(2+) influx, cellular shape changes and impaired barrier function. Production of reactive oxygen species was completely abolished in Nox2(y/-) cells. A novel mechanistic model comprising endothelial Nox2-derived production of superoxide, activation of phospholipase C-γ, inhibition of diacylglycerol (DAG) kinase, DAG-mediated activation of TRPC6 and ensuing LIRE is supported by pharmacological and molecular evidence. This mechanism highlights novel pharmacological targets for the treatment of LIRE.

Background: Acute respiratory disorders may lead to sustained alveolar hypoxia with hypercapnia resulting in impaired pulmonary gas exchange. Hypoxic pulmonary vasoconstriction (HPV) optimizes gas exchange during local acute (0-30 min), as well as sustained (> 30 min) hypoxia by matching blood perfusion to alveolar ventilation. Hypercapnia with acidosis improves pulmonary gas exchange in repetitive conditions of acute hypoxia by potentiating HPV and preventing pulmonary endothelial dysfunction. This study investigated, if the beneficial effects of hypercapnia with acidosis are preserved during sustained hypoxia as it occurs, e.g in permissive hypercapnic ventilation in intensive care units. Furthermore, the effects of NO synthase inhibitors under such conditions were examined., Method: We employed isolated perfused and ventilated rabbit lungs to determine the influence of hypercapnia with or without acidosis (pH corrected with sodium bicarbonate), and inhibitors of endothelial as well as inducible NO synthase on acute or sustained HPV (180 min) and endothelial permeability., Results: In hypercapnic acidosis, HPV was intensified in sustained hypoxia, in contrast to hypercapnia without acidosis when HPV was amplified during both phases. L-NG-Nitroarginine (L-NNA), a non-selective NO synthase inhibitor, enhanced acute as well as sustained HPV under all conditions, however, the amplification of sustained HPV induced by hypercapnia with or without acidosis compared to normocapnia disappeared. In contrast 1400 W, a selective inhibitor of inducible NO synthase (iNOS), decreased HPV in normocapnia and hypercapnia without acidosis at late time points of sustained HPV and selectively reversed the amplification of sustained HPV during hypercapnia without acidosis. Hypoxic hypercapnia without acidosis increased capillary filtration coefficient (Kfc). This increase disappeared after administration of 1400 W., Conclusion: Hypercapnia with and without acidosis increased HPV during conditions of sustained hypoxia. The increase of sustained HPV and endothelial permeability in hypoxic hypercapnia without acidosis was iNOS dependent.

Chronic obstructive pulmonary disease (COPD) is one of the most common causes of death worldwide. We report in an emphysema model of mice chronically exposed to tobacco smoke that pulmonary vascular dysfunction, vascular remodeling, and pulmonary hypertension (PH) precede development of alveolar destruction. We provide evidence for a causative role of inducible nitric oxide synthase (iNOS) and peroxynitrite in this context. Mice lacking iNOS were protected against emphysema and PH. Treatment of wild-type mice with the iNOS inhibitor N(6)-(1-iminoethyl)-L-lysine (L-NIL) prevented structural and functional alterations of both the lung vasculature and alveoli and also reversed established disease. In chimeric mice lacking iNOS in bone marrow (BM)-derived cells, PH was dependent on iNOS from BM-derived cells, whereas emphysema development was dependent on iNOS from non-BM-derived cells. Similar regulatory and structural alterations as seen in mouse lungs were found in lung tissue from humans with end-stage COPD., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The neurotrophins (NTs) are emerging as exciting new participants in normal lung physiology, as well as in several pathological processes in diseased lungs. In this study, the increased expression of NT4/5 and of its cognate receptor, the neurotrophic tyrosine kinase receptor Type 2 (TrkB), was observed in human lungs explanted from patients with idiopathic pulmonary fibrosis (IPF), and in lungs from mice with bleomycin-induced pulmonary fibrosis. The expression of NT4/5 and TrkB localized to hyperplastic alveolar Type II cells (ATII) and fibroblastic foci in affected lungs. Increased concentrations of NT4/5 and TrkB were evident in ATII isolated from the lungs of bleomycin-treated mice. Primary ATII were shown to secrete NT4/5 into the cell culture medium. The profibrotic cytokine transforming growth factor-β1, stimulated TrkB, but not NT4/5 gene expression, suggesting that perturbed profibrotic growth factor signaling in affected lungs may drive the expression of TrkB. NT4/5 enhanced the proliferation of ATII through a TrkB/extracellular-regulated kinase/protein kinase B pathway, and could also drive the proliferation of primary human and murine lung fibroblasts, through TrkB-dependent and protein kinase B-dependent pathways. Taken together, these data suggest that a dysregulated TrkB/NT4/5 axis may contribute to several of the pathological lesions associated with pulmonary fibrosis, including ATII hyperplasia and the proliferation of fibroblasts, and we would add IPF to the list of disorders, such as pain and cancer, for which therapeutic targeting of the TrkB/neurotrophin axis has been proposed for further investigation.

Rationale: Low-density lipoprotein receptor–related protein-1 (LRP-1) mediates the endocytic clearance of various proteinases, including matrix metalloproteinases (MMPs). The ectodomain of LRP-1 can be shed from the cell surface, releasing a soluble form of this receptor (sLRP-1), which antagonizes ligand endocytosis by cellular LRP-1., Objectives: To assess if increased LRP-1 shedding occurs in the lungs of patients with acute respiratory distress syndrome (ARDS) and may lead to the accumulation of MMPs and subsequent tissue injury., Methods: We determined sLRP-1 levels in bronchoalveolar lavage fluids (BALF) from 46 patients with ARDS and their correlation with MMP concentration and disease severity. In complementary in vitro studies, we investigated the mechanisms underlying the LRP-1 release from the cell surface and its impact on MMP cellular uptake., Measurements and Main Results: sLRP-1 levels were significantly elevated in BALF but not in plasma from patients with ARDS compared with control subjects and further increased in the later course of the disease. Baseline BALF sLRP-1 concentration was positively correlated with disease severity and significantly higher in nonsurvivors compared with survivors. The presence of ARDS BALF enhanced LRP-1 shedding from cultured lung fibroblasts but not from alveolar type II cells or macrophages. This process was blocked when ARDS BALF was supplemented with metalloproteinase inhibitor resulting in enhanced cellular uptake and degradation of MMP-2 and -9. Accordingly, sLRP-1 BALF concentration in patients with ARDS was positively correlated with MMP levels and laminin, a marker of basement membrane disruption., Conclusions: Increased LRP-1 shedding prevents the cellular clearance of MMPs and might thereby contribute to tissue destruction in ARDS lungs.

Idiopathic pulmonary fibrosis (IPF) is a progressive, dysregulated response to alveolar injury that culminates in compromised lung function from excess extracellular matrix production. Associated with high morbidity and mortality, IPF is generally refractory to current pharmacological therapies. We examined fibrotic lungs from mice and from patients with IPF and detected increased expression of dimethylarginine dimethylaminohydrolases (DDAHs)--key enzymes that metabolize asymmetric dimethylarginine (ADMA), which is an endogenous inhibitor of nitric oxide synthase, to form l-citrulline and dimethylamine. DDAHs are up-regulated in primary alveolar epithelial type II cells from these mice and patients where they are colocalized with inducible nitric oxide synthase. In cultured alveolar epithelial type II cells from bleomycin-induced fibrotic mouse lungs, inhibition of DDAH suppressed proliferation and induced apoptosis in an ADMA-dependent manner. In addition, DDAH inhibition reduced collagen production by fibroblasts in an ADMA-independent but transforming growth factor/SMAD-dependent manner. In mice with bleomycin-induced pulmonary fibrosis, the DDAH inhibitor L-291 reduced collagen deposition and normalized lung function. In bleomycin-induced fibrosis, inducible nitric oxide synthase inhibition decreased fibrosis, but an even stronger reduction was observed after inhibition of DDAH. Thus, DDAH inhibition reduces fibroblast-induced collagen deposition in an ADMA-independent manner and reduces abnormal epithelial proliferation in an ADMA-dependent manner, offering a possible therapeutic avenue for attenuation of pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal disease for which no effective therapy exists to date. To identify the molecular mechanisms underlying IPF, we performed comparative proteome analysis of lung tissue from patients with sporadic IPF (n = 14) and human donor lungs (controls, n = 10) using two-dimensional gel electrophoresis and MALDI-TOF-MS. Eighty-nine differentially expressed proteins were identified, from which 51 were up-regulated and 38 down-regulated in IPF. Increased expression of markers for the unfolded protein response (UPR), heat-shock proteins, and DNA damage stress markers indicated a chronic cell stress-response in IPF lungs. By means of immunohistochemistry, induction of UPR markers was encountered in type-II alveolar epithelial cells of IPF but not of control lungs. In contrast, up-regulation of heat-shock protein 27 (Hsp27) was exclusively observed in proliferating bronchiolar basal cells and associated with aberrant re-epithelialization at the bronchiolo-alveolar junctions. Among the down-regulated proteins in IPF were antioxidants, members of the annexin family, and structural epithelial proteins. In summary, our results indicate that IPF is characterized by epithelial cell injury, apoptosis, and aberrant epithelial proliferation.

Background: Mast cells (MCs) are implicated in inflammation and tissue remodeling. Accumulation of lung MCs is described in pulmonary hypertension (PH); however, whether MC degranulation and c-kit, a tyrosine kinase receptor critically involved in MC biology, contribute to the pathogenesis and progression of PH has not been fully explored., Methods: Pulmonary MCs of idiopathic pulmonary arterial hypertension (IPAH) patients and monocrotaline-injected rats (MCT-rats) were examined by histochemistry and morphometry. Effects of the specific c-kit inhibitor PLX and MC stabilizer cromolyn sodium salt (CSS) were investigated in MCT-rats both by the preventive and therapeutic approaches. Hemodynamic and right ventricular hypertrophy measurements, pulmonary vascular morphometry and analysis of pulmonary MC localization/counts/activation were performed in animal model studies., Results: There was a prevalence of pulmonary MCs in IPAH patients and MCT-rats as compared to the donors and healthy rats, respectively. Notably, the perivascular MCs were increased and a majority of them were degranulated in lungs of IPAH patients and MCT-rats (p < 0.05 versus donor and control, respectively). In MCT-rats, the pharmacological inhibitions of MC degranulation and c-kit with CSS and PLX, respectively by a preventive approach (treatment from day 1 to 21 of MCT-injection) significantly attenuated right ventricular systolic pressure (RVSP) and right ventricular hypertrophy (RVH). Moreover, vascular remodeling, as evident from the significantly decreased muscularization and medial wall thickness of distal pulmonary vessels, was improved. However, treatments with CSS and PLX by a therapeutic approach (from day 21 to 35 of MCT-injection) neither improved hemodynamics and RVH nor vascular remodeling., Conclusions: The accumulation and activation of perivascular MCs in the lungs are the histopathological features present in clinical (IPAH patients) and experimental (MCT-rats) PH. Moreover, the accumulation and activation of MCs in the lungs contribute to the development of PH in MCT-rats. Our findings reveal an important pathophysiological insight into the role of MCs in the pathogenesis of PH in MCT-rats.

Phosphodiesterases (PDEs) modulate the cellular proliferation involved in the pathophysiology of pulmonary hypertension (PH) by hydrolyzing cAMP and cGMP. The present study was designed to determine whether any of the recently identified PDEs (PDE7-PDE11) contribute to progressive pulmonary vascular remodeling in PH. All in vitro experiments were performed with lung tissue or pulmonary arterial smooth muscle cells (PASMCs) obtained from control rats or monocrotaline (MCT)-induced pulmonary hypertensive (MCT-PH) rats, and we examined the effects of the PDE10 inhibitor papaverine (Pap) and specific small interfering RNA (siRNA). In addition, papaverine was administrated to MCT-induced PH rats from day 21 to day 35 by continuous intravenous infusion to examine the in vivo effects of PDE10A inhibition. We found that PDE10A was predominantly present in the lung vasculature, and the mRNA, protein, and activity levels of PDE10A were all significantly increased in MCT PASMCs compared with control PASMCs. Papaverine and PDE10A siRNA induced an accumulation of intracellular cAMP, activated cAMP response element binding protein and attenuated PASMC proliferation. Intravenous infusion of papaverine in MCT-PH rats resulted in a 40%-50% attenuation of the effects on pulmonary hypertensive hemodynamic parameters and pulmonary vascular remodeling. The present study is the first to demonstrate a central role of PDE10A in progressive pulmonary vascular remodeling, and the results suggest a novel therapeutic approach for the treatment of PH.

Background: Hypoxic pulmonary vasoconstriction (HPV) is an essential mechanism of the lung that matches blood perfusion to alveolar ventilation to optimize gas exchange. Recently we have demonstrated that acute but not sustained HPV is critically dependent on the classical transient receptor potential 6 (TRPC6) channel. However, the mechanism of TRPC6 activation during acute HPV remains elusive. We hypothesize that a diacylglycerol (DAG)-dependent activation of TRPC6 regulates acute HPV., Methods: We investigated the effect of the DAG analog 1-oleoyl-2-acetyl-sn-glycerol (OAG) on normoxic vascular tone in isolated perfused and ventilated mouse lungs from TRPC6-deficient and wild-type mice. Moreover, the effects of OAG, the DAG kinase inhibitor R59949 and the phospholipase C inhibitor U73122 on the strength of HPV were investigated compared to those on non-hypoxia-induced vasoconstriction elicited by the thromboxane mimeticum U46619., Results: OAG increased normoxic vascular tone in lungs from wild-type mice, but not in lungs from TRPC6-deficient mice. Under conditions of repetitive hypoxic ventilation, OAG as well as R59949 dose-dependently attenuated the strength of acute HPV whereas U46619-induced vasoconstrictions were not reduced. Like OAG, R59949 mimicked HPV, since it induced a dose-dependent vasoconstriction during normoxic ventilation. In contrast, U73122, a blocker of DAG synthesis, inhibited acute HPV whereas U73343, the inactive form of U73122, had no effect on HPV., Conclusion: These findings support the conclusion that the TRPC6-dependency of acute HPV is induced via DAG.