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The ability to sense changes in oxygen availability is fundamentally important for the survival of all aerobic organisms. However, cellular oxygen sensing mechanisms and pathologies remain incompletely understood and studies of acute oxygen sensing, in particular, have produced inconsistent results. Current methods cannot simultaneously measure the key cellular events in acute hypoxia (i.e., changes in redox state, electrophysiological properties, and mechanical responses) at controlled partial pressures of oxygen (pO2). The lack of such a comprehensive method essentially contributes to the discrepancies in the field. A sealed microfluidic system that combines i) Raman spectroscopy, ii) patch‐clamp electrophysiology, and iii) live‐cell imaging under precisely controlled pO2 have therefore been developed. Merging these modalities allows label‐free and simultaneous observation of oxygen‐dependent alterations in multiple cellular redox couples, membrane potential, and cellular contraction. This technique is adaptable to any cell type and allows in‐depth insight into acute oxygen sensing processes underlying various physiologic and pathologic conditions. [ABSTRACT FROM AUTHOR]

Therapeutic options for pulmonary arterial hypertension (PAH) have increased over the last decades. The advent of pharmacological therapies targeting the prostacyclin, endothelin, and NO pathways has significantly improved outcomes. However, for the vast majority of patients, PAH remains a life‐limiting illness with no prospect of cure. PAH is characterised by pulmonary vascular remodelling. Current research focusses on targeting the underlying pathways of aberrant proliferation, migration, and apoptosis. Despite success in preclinical models, using a plethora of novel approaches targeting cellular GPCRs, ion channels, metabolism, epigenetics, growth factor receptors, transcription factors, and inflammation, successful transfer to human disease with positive outcomes in clinical trials is limited. This review provides an overview of novel targets addressed by clinical trials and gives an outlook on novel preclinical perspectives in PAH. LINKED ARTICLES: This article is part of a themed issue on Risk factors, comorbidities, and comedications in cardioprotection. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.1/issuetoc [ABSTRACT FROM AUTHOR]

Background and Purpose: Chronic obstructive pulmonary disease, encompassing chronic airway obstruction and lung emphysema, is a major worldwide health problem and a severe socio-economic burden. Evidence previously provided by our group has shown that inhibition of inducible NOS (iNOS) prevents development of mild emphysema in a mouse model of chronic tobacco smoke exposure and can even trigger lung regeneration. Moreover, we could demonstrate that pulmonary hypertension is not only abolished in cigarette smoke-exposed iNOS-/- mice but also precedes emphysema development. Possible regenerative effects of pharmacological iNOS inhibition in more severe models of emphysema not dependent on tobacco smoke, however, are hitherto unknown.Experimental Approach: We have established a mouse model using a single dose of porcine pancreatic elastase or saline, intratracheally instilled in C57BL/6J mice. Emphysema, as well as pulmonary hypertension development was determined by both structural and functional measurements.Key Results: Our data revealed that (i) emphysema is fully established after 21 days, with the same degree of emphysema after 21 and 28 days post instillation, (ii) emphysema is stable for at least 12 weeks and (iii) pulmonary hypertension is evident, in contrast to smoke models, only after emphysema development. Oral treatment with the iNOS inhibitor N(6)-(1-iminoethyl)-l-lysine (L-NIL) was started after emphysema establishment and continued for 12 weeks. This resulted in significant lung regeneration, evident in the improvement of emphysema and reversal of pulmonary hypertension.Conclusion and Implications: Our data indicate that iNOS is a potential new therapeutic target to treat severe emphysema and associated pulmonary hypertension.Linked Articles: This article is part of a themed issue on Risk factors, comorbidities, and comedications in cardioprotection. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.1/issuetoc. [ABSTRACT FROM AUTHOR]

Aims: The aim of the study was to investigate the role of cytochrome P450 (CYP) epoxygenase‐derived epoxyeicosatrienoic acids (EETs) in sustained hypoxic pulmonary vasoconstriction (HPV). Methods: Vasomotor responses of isolated mouse intrapulmonary arteries (IPAs) were assessed using wire myography. Key findings were verified by haemodynamic measurements in isolated perfused and ventilated mouse lungs. Results: Pharmacological inhibition of EET synthesis with MS‐PPOH, application of the EET antagonist 14,15‐EEZE or deficiency of CYP2J isoforms suppressed sustained HPV. In contrast, knockdown of EET‐degrading soluble epoxide hydrolase or its inhibition with TPPU augmented sustained HPV almost twofold. All EET regioisomers elicited relaxation in IPAs pre‐contracted with thromboxane mimetic U46619. However, in the presence of KCl‐induced depolarization, 5,6‐EET caused biphasic contraction in IPAs and elevation of pulmonary vascular tone in isolated lungs, whereas other regioisomers had no effect. In patch‐clamp experiments, hypoxia elicited depolarization in pulmonary artery smooth muscle cells (PASMCs), and 5,6‐EET evoked inward whole cell currents in PASMCs depolarized to the hypoxic level, but not at their resting membrane potential. Conclusions: The EET pathway substantially contributes to sustained HPV in mouse pulmonary arteries. 5,6‐EET specifically appears to be involved in HPV, as it is the only EET regioisomer able to elicit not only relaxation, but also sustained contraction in these vessels. 5,6‐EET‐induced pulmonary vasoconstriction is enabled by PASMC depolarization, which occurs in hypoxia. The discovery of the dual role of 5,6‐EET in the regulation of pulmonary vascular tone may provide a basis for the development of novel therapeutic strategies for treatment of HPV‐related diseases. [ABSTRACT FROM AUTHOR]

Severe inflammatory disorders such as sepsis are a major cause of morbidity and mortality worldwide. Mitochondrial dysfunction is regarded as a key feature involved in inflammation pathogenesis. In the present study, we investigated the impact of the omega‐3 fatty acid‐derived lipid mediator Resolvin E1 (RvE1) on mitochondrial function in experimental pulmonary inflammation. RvE1 was found to exert anti‐inflammatory properties in human alveolar epithelial cells during severe inflammation. RvE1 is capable of restoring inflammation‐induced mitochondrial dysfunction and the impaired imbalance of mitochondrial fission and fusion. Experimental inhibition of mitochondrial fission with Mdivi‐1 in our model is associated with a significantly reduced inflammatory response and improved mitochondrial function. These findings suggest a novel functional mechanism for the beneficial effects of RvE1 in experimental pulmonary inflammatory reactions. [ABSTRACT FROM AUTHOR]

New Findings: What is the central question of this study? Hypoxic pulmonary vasoconstriction has never been characterized in isolated mouse pulmonary arteries of different generations in detail. What is the main finding and its importance? We found that only small intrapulmonary arteries (80–200 μm in diameter) exhibit hypoxic pulmonary vasoconstriction. The observed response was sustained, significantly potentiated by depolarization‐induced preconstriction and not dependent on the endothelium or TRPC6 channels. Abstract: Hypoxic pulmonary vasoconstriction (HPV) is a physiological response of pulmonary arteries, which adapts lung perfusion to regional ventilation. The properties of HPV vary significantly between animal species. Despite extensive use of mouse models in studies of HPV, this physiological response has never been characterized in isolated mouse pulmonary arteries in detail. Using wire myography, we investigated the effect of 80 min exposure to hypoxia on the tone in mouse pulmonary arteries of different generations in the presence and absence of preconstriction. Hypoxia induced a sustained relaxation in non‐preconstricted extrapulmonary arteries (500–700 μm in diameter), but not in the presence of KCl‐induced preconstriction. Large intrapulmonary arteries (450–650 μm in diameter) did not exhibit a significant response to the hypoxic challenge. In contrast, in small intrapulmonary arteries (80–200 μm in diameter), hypoxia elicited a slowly developing sustained constriction, which was independent of the endothelium. The response was significantly potentiated in arteries preconstricted with KCl, but not with U46619. Hypoxic pulmonary vasoconstriction was not altered in pulmonary arteries of TRPC6‐deficient mice, which suggests that this response corresponds to the sustained phase of biphasic HPV observed earlier in isolated, buffer‐perfused and ventilated mouse lungs. In conclusion, we have established a protocol that allows the study of sustained HPV in isolated mouse pulmonary arteries. The data obtained might be useful for future studies of the mechanisms of HPV in mice. [ABSTRACT FROM AUTHOR]

Cytochrome c oxidase (COX) is the terminal enzyme of the mitochondrial electron transport chain. The purpose of this study was to analyze the function of lung-specific cytochrome c oxidase subunit 4 isoform 2 (COX4i2) in vitro and in COX4i2-knockout mice in vivo. COX was isolated from cow lung and liver as control and functionally analyzed. COX4i2-knockout mice were generated and the effect of the gene knockout was determined, including COX activity, tissue energy levels, noninvasive and invasive lung function, and lung pathology. These studies were complemented by a comprehensive functional screen performed at the German Mouse Clinic (Neuherberg, Germany). We show that isolated cow lung COX containing COX4i2 is about twice as active (88 and 102% increased activity in the presence of allosteric activator ADP and inhibitor ATP, respectively) as liver COX, which lacks COX4i2. In COX4i2-knockout mice, lung COX activity and cellular ATP levels were significantly reduced (-50 and -29%, respectively). Knockout mice showed decreased airway responsiveness (60% reduced Penh and 58% reduced airway resistance upon challenge with 25 and 100 mg methacholine, respectively), and they developed a lung pathology deteriorating with age that included the appearance of Charcot-Leyden crystals. In addition, there was an interesting sex-specific phenotype, in which the knockout females showed reduced lean mass (-12%), reduced total oxygen consumption rate (-8%), improved glucose tolerance, and reduced grip force (- 14%) compared to wild-type females. Our data suggest that high activity lung COX is a central determinant of airway function and is required for maximal airway responsiveness and healthy lung function. Since airway constriction requires energy, we propose a model in which reduced tissue ATP levels explain protection from airway hyperresponsiveness, i.e., absence of COX4i2 leads to reduced lung COX activity and ATP levels, which results in impaired airway constriction and thus reduced airway responsiveness; long-term lung pathology develops in the knockout mice due to impairment of energy-costly lung maintenance processes; and therefore, we propose mitochondrial oxidative phosphorylation as a novel target for the treatment of respiratory diseases, such as asthma. [ABSTRACT FROM AUTHOR]

Chronic exposure of adult mice to inhalational anesthetic nitrous oxide (N20) results in anesthetic tolerance. N20 is an NMDA (N-methyl-D- aspartate) antagonist. It has been demonstrated that chronic administration of members of the NMDA antagonist class of drugs (e.g., MK801) causes upregulation of NMDA receptors in certain brain regions that could, at least in part, explain the development of tolerance. We sought to determine whether the anesthetic tolerance resulting from chronic exposure to N20 reflects changes in the number and/or distribution of NMDA receptors. We exposed mice to either a 50- or 75-vol% N20 atmosphere continuously for 1 or 2 weeks and performed binding studies with [3H]MK801 and NR1 antibodies. Binding studies revealed a significant (P < 0.05) increase in [3H]MK801 binding in the cerebral cortex after 2 weeks of N20 (50- and 75-vol%) exposure. Immunocytochemical binding of NR1 antibodies in selected brain regions showed no changes in distribution pattern. The timing of this increase in [3H]MK801 binding correlates with the time period required for development of tolerance. [ABSTRACT FROM AUTHOR]

The role of costimulatory molecules other than CD80/CD86 in endothelial cell (EC)-dependent CD8 T cell activation including the generation of a distinct subset of endothelium-specific CTL (EC-CTL) remains unclear. Inducible costimulator (ICOS) and its ligand (ICOSL) are new members of the CD28 family mediating effector T cell differentiation and graft rejection in animal models. In this study endothelial ICOSL expression/regulation and effects on CD8 T cell allo-activation were analyzed. Constitutive expression of ICOSL was found on human EC. IL-1α and TNF-α induced ICOSL in an NF-κB-dependent manner on human umbilical vein endothelial cells (HUVEC). ICOS receptor was not detected on resting CD8 T cells but was induced in co-cultures with HUVEC. ICOSL blockade reduced CD8 T cell proliferation by 70% along with a marked decrease of IL-2 and IFN-γ production in co-cultures with HUVEC. IL-2 supplementation of co-cultures could overcome the effect of ICOSL blockade; similarly the generation of EC-CTL was not impaired by ICOSL blockade in an IL-2-containing system. In vivo, weak constitutive ICOSL expression was found on coronary microvessels, which was significantly up-regulated during acute cardiac allograft rejection ( p=0.04). Our data indicate a distinct role for ICOSL in EC-mediated CD8 T cell costimulation with implications for human cardiac allograft rejection. [ABSTRACT FROM AUTHOR]