GLI1+ Cells Contribute to Vascular Remodeling in Pulmonary Hypertension.

BACKGROUND: The precise origin of newly formed ACTA2+ (alpha smooth muscle actin-positive) cells appearing in nonmuscularized vessels in the context of pulmonary hypertension is still debatable although it is believed that they predominantly derive from preexisting vascular smooth muscle cells (VSMCs). METHODS: Gli1Cre-ERT2; tdTomatoflox mice were used to lineage trace GLI1+ (glioma-associated oncogene homolog 1-positive) cells in the context of pulmonary hypertension using 2 independent models of vascular remodeling and reverse remodeling: hypoxia and cigarette smoke exposure. Hemodynamic measurements, right ventricular hypertrophy assessment, flow cytometry, and histological analysis of thick lung sections followed by state-of-the-art 3-dimensional reconstruction and quantification using Imaris software were used to investigate the contribution of GLI1+ cells to neomuscularization of the pulmonary vasculature. RESULTS: The data show that GLI1+ cells are abundant around distal, nonmuscularized vessels during steady state, and this lineage contributes to around 50% of newly formed ACTA2+ cells around these normally nonmuscularized vessels. During reverse remodeling, cells derived from the GLI1+ lineage are largely cleared in parallel to the reversal of muscularization. Partial ablation of GLI1+ cells greatly prevented vascular remodeling in response to hypoxia and attenuated the increase in right ventricular systolic pressure and right heart hypertrophy. Single-cell RNA sequencing on sorted lineage-labeled GLI1+ cells revealed an Acta2high fraction of cells with pathways in cancer and MAPK (mitogen-activated protein kinase) signaling as potential players in reprogramming these cells during vascular remodeling. Analysis of human lung-derived material suggests that GLI1 signaling is overactivated in both group 1 and group 3 pulmonary hypertension and can promote proliferation and myogenic differentiation. CONCLUSIONS: Our data highlight GLI1+ cells as an alternative cellular source of VSMCs in pulmonary hypertension and suggest that these cells and the associated signaling pathways represent an important therapeutic target for further studies.

CEACAM6 as a Novel Therapeutic Target to Boost HO-1-mediated Antioxidant Defense in COPD.

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

SPARC, a Novel Regulator of Vascular Cell Function in Pulmonary Hypertension.

BACKGROUND: Pulmonary hypertension (PH) is a life-threatening disease, characterized by excessive pulmonary vascular remodeling, leading to elevated pulmonary arterial pressure and right heart hypertrophy. PH can be caused by chronic hypoxia, leading to hyper-proliferation of pulmonary arterial smooth muscle cells (PASMCs) and apoptosis-resistant pulmonary microvascular endothelial cells (PMVECs). On reexposure to normoxia, chronic hypoxia-induced PH in mice is reversible. In this study, the authors aim to identify novel candidate genes involved in pulmonary vascular remodeling specifically in the pulmonary vasculature. METHODS: After microarray analysis, the authors assessed the role of SPARC (secreted protein acidic and rich in cysteine) in PH using lung tissue from idiopathic pulmonary arterial hypertension (IPAH) patients, as well as from chronically hypoxic mice. In vitro studies were conducted in primary human PASMCs and PMVECs. In vivo function of SPARC was proven in chronic hypoxia-induced PH in mice by using an adeno-associated virus-mediated Sparc knockdown approach. RESULTS: C57BL/6J mice were exposed to normoxia, chronic hypoxia, or chronic hypoxia with subsequent reexposure to normoxia for different time points. Microarray analysis of the pulmonary vascular compartment after laser microdissection identified Sparc as one of the genes downregulated at all reoxygenation time points investigated. Intriguingly, SPARC was vice versa upregulated in lungs during development of hypoxia-induced PH in mice as well as in IPAH, although SPARC plasma levels were not elevated in PH. TGF-ß1 (transforming growth factor ß1) or HIF2A (hypoxia-inducible factor 2A) signaling pathways induced SPARC expression in human PASMCs. In loss of function studies, SPARC silencing enhanced apoptosis and reduced proliferation. In gain of function studies, elevated SPARC levels induced PASMCs, but not PMVECs, proliferation. Coculture and conditioned medium experiments revealed that PMVECs-secreted SPARC acts as a paracrine factor triggering PASMCs proliferation. Contrary to the authors' expectations, in vivo congenital Sparc knockout mice were not protected from hypoxia-induced PH, most probably because of counter-regulatory proproliferative signaling. However, adeno-associated virus-mediated Sparc knockdown in adult mice significantly improved hemodynamic and cardiac function in PH mice. CONCLUSIONS: This study provides evidence for the involvement of SPARC in the pathogenesis of human PH and chronic hypoxia-induced PH in mice, most likely by affecting vascular cell function.

Fibroblast growth factor 10 reverses cigarette smoke- and elastase-induced emphysema and pulmonary hypertension in mice.

BACKGROUND: COPD is an incurable disease and a leading cause of death worldwide. In mice, fibroblast growth factor (FGF)10 is essential for lung morphogenesis, and in humans, polymorphisms in the human FGF10 gene correlate with an increased susceptibility to develop COPD. METHODS: We analysed FGF10 signalling in human lung sections and isolated cells from healthy donor, smoker and COPD lungs. The development of emphysema and PH was investigated in Fgf10+/- and Fgfr2b+/- (FGF receptor 2b) mice upon chronic exposure to cigarette smoke. In addition, we overexpressed FGF10 in mice following elastase- or cigarette smoke-induced emphysema and pulmonary hypertension (PH). RESULTS: We found impaired FGF10 expression in human lung alveolar walls and in primary interstitial COPD lung fibroblasts. In contrast, FGF10 expression was increased in large pulmonary vessels in COPD lungs. Consequently, we identified impaired FGF10 signalling in alveolar walls as an integral part of the pathomechanism that leads to emphysema and PH development: mice with impaired FGF10 signalling (Fgf10+/- and Fgfr2b+/- ) spontaneously developed lung emphysema, PH and other typical pathomechanistic features that generally arise in response to cigarette smoke exposure. CONCLUSION: In a therapeutic approach, FGF10 overexpression successfully restored lung alveolar and vascular structure in mice with established cigarette smoke- and elastase-induced emphysema and PH. FGF10 treatment triggered an initial increase in the number of alveolar type 2 cells that gradually returned to the basal level when the FGF10-mediated repair process progressed. Therefore, the application of recombinant FGF10 or stimulation of the downstream signalling cascade might represent a novel therapeutic strategy in the future.

Nicotine promotes e-cigarette vapour-induced lung inflammation and structural alterations.

BACKGROUND: Electronic cigarette (e-cigarette) vapour is gaining popularity as an alternative to tobacco smoking and can induce acute lung injury. However, the specific role of nicotine in e-cigarette vapour and its long-term effects on the airways, lung parenchyma and vasculature remain unclear. RESULTS: In vitro exposure to nicotine-containing e-cigarette vapour extract (ECVE) or to nicotine-free e-cigarette vapour extract (NF ECVE) induced changes in gene expression of epithelial cells and pulmonary arterial smooth muscle cells (PASMCs), but ECVE in particular caused functional alterations (e.g. a decrease in human and mouse PASMC proliferation by 29.3±5.3% and 44.3±8.4%, respectively). Additionally, acute inhalation of nicotine-containing e-cigarette vapour (ECV) but not nicotine-free e-cigarette vapour (NF ECV) increased pulmonary endothelial permeability in isolated lungs. Long-term in vivo exposure of mice to ECV for 8 months significantly increased the number of inflammatory cells, in particular lymphocytes, compared to control and NF ECV in the bronchoalveolar fluid (BALF) (ECV: 853.4±150.8 cells·mL-1; control: 37.0±21.1 cells·mL-1; NF ECV: 198.6±94.9 cells·mL-1) and in lung tissue (ECV: 25.7±3.3 cells·mm-3; control: 4.8±1.1 cells·mm-3; NF ECV: 14.1±2.2 cells·mm-3). BALF cytokines were predominantly increased by ECV. Moreover, ECV caused significant changes in lung structure and function (e.g. increase in airspace by 17.5±1.4% compared to control), similar to mild tobacco smoke-induced alterations, which also could be detected in the NF ECV group, albeit to a lesser degree. In contrast, the pulmonary vasculature was not significantly affected by ECV or NF ECV. CONCLUSIONS: NF ECV components induce cell type-specific effects and mild pulmonary alterations, while inclusion of nicotine induces significant endothelial damage, inflammation and parenchymal alterations.

Myeloid-cell-specific deletion of inducible nitric oxide synthase protects against smoke-induced pulmonary hypertension in mice.

BACKGROUND: Pulmonary hypertension (PH) is a common complication of COPD, associated with increased mortality and morbidity. Intriguingly, pulmonary vascular alterations have been suggested to drive emphysema development. Previously, we identified inducible nitric oxide synthase (iNOS) as an essential enzyme for development and reversal of smoke-induced PH and emphysema, and showed that iNOS expression in bone-marrow-derived cells drives pulmonary vascular remodelling, but not parenchymal destruction. In this study, we aimed to identify the iNOS-expressing cell type driving smoke-induced PH and to decipher pro-proliferative pathways involved. METHODS: To address this question we used 1) myeloid-cell-specific iNOS knockout mice in chronic smoke exposure and 2) co-cultures of macrophages and pulmonary artery smooth muscle cells (PASMCs) to decipher underlying signalling pathways. RESULTS: Myeloid-cell-specific iNOS knockout prevented smoke-induced PH but not emphysema in mice. Moreover, iNOS deletion in myeloid cells ameliorated the increase in expression of CD206, a marker of M2 polarisation, on interstitial macrophages. Importantly, the observed effects on lung macrophages were hypoxia-independent, as these mice developed hypoxia-induced PH. In vitro, smoke-induced PASMC proliferation in co-cultures with M2-polarised macrophages could be abolished by iNOS deletion in phagocytic cells, as well as by extracellular signal-regulated kinase inhibition in PASMCs. Crucially, CD206-positive and iNOS-positive macrophages accumulated in proximity of remodelled vessels in the lungs of COPD patients, as shown by immunohistochemistry. CONCLUSION: In summary, our results demonstrate that iNOS deletion in myeloid cells confers protection against PH in smoke-exposed mice and provide evidence for an iNOS-dependent communication between M2-like macrophages and PASMCs in underlying pulmonary vascular remodelling.

Mitochondrien als universelle Sensoren der akuten Hypoxie?

Adaptation to acute hypoxia through cardiorespiratory responses is mediated by specialized cells in the carotid body and pulmonary vasculature to optimize systemic arterial oxygenation. Acute oxygen sensing thus is a fundamental pre-requisite for aerobic life. Recent studies unravelled basic oxygen sensing mechanisms involving the mitochondrial cytochrome c oxidase subunit 4 isoform 2 that regulates the release of mitochondrial reactive oxygen species and subsequent acute hypoxic responses.

Sex-specific differences in plasma levels of FXII, HK, and FXIIa-C1-esterase inhibitor complexes in community-acquired pneumonia.

Sex-dependent differences in immunity and coagulation play an active role in the outcome of community-acquired pneumonia (CAP). Contact phase proteins act at the crossroads between inflammation and coagulation thus representing a point of convergence in host defense against infection. Here, we measured the levels of factor XII (FXII), FXIIa-C1 esterase inhibitor (C1INH) complexes, and high-molecular-weight kininogen (HK) in plasma of patients with CAP and correlated them to clinical disease severity. Levels of FXIIa-C1INH/albumin ratio were elevated, irrespective of sex, in plasma of patients with CAP (n = 139) as compared with age-matched donors (n = 58). No simultaneous decrease in FXII levels, indicating its consumption, was observed. Stratification by sex revealed augmented FXII levels in plasma of women with CAP as compared with sex-matched donors yet no apparent differences in men. This sex-specific effect was, however, attributable to lower FXII levels in female donors relative to men donors. Plasma estradiol levels mirrored those for FXII. Levels of HK/albumin ratio were decreased in CAP plasma as compared with donors, however, after stratification by sex, this difference was only observed in women and was related to higher HK/albumin values in female donors as opposed to male donors. Finally, strong negative correlation between plasma levels of HK/albumin ratio and CAP severity, as assessed by CRB65 score, in males and females was observed. Our study identifies sex-dependent differences in plasma levels of the contact phase proteins in elderly subjects that may contribute to specific clinical outcomes in CAP between men and women.

The effect of long-term doxycycline treatment in a mouse model of cigarette smoke-induced emphysema and pulmonary hypertension.

Chronic obstructive pulmonary disease (COPD) is a major cause of death and a still incurable disease, comprising emphysema and chronic bronchitis. In addition to airflow limitation, patients with COPD can suffer from pulmonary hypertension (PH). Doxycycline, an antibiotic from the tetracycline family, in addition to its pronounced antimicrobial activity, acts as a matrix metalloproteinase (MMP) inhibitor and has anti-inflammatory properties. Furthermore, doxycycline treatment exhibited a beneficial effect in several preclinical cardiovascular disease models. In preclinical research, doxycycline is frequently employed for gene expression modulation in Tet-On/Tet-Off transgenic animal models. Therefore, it is crucial to know whether doxycycline treatment in Tet-On/Tet-Off systems has effects independent of gene expression modulation by such systems. Against this background, we assessed the possible curative effects of long-term doxycycline administration in a mouse model of chronic CS exposure. Animals were exposed to cigarette smoke (CS) for 8 mo and then subsequently treated with doxycycline for additional 3 mo in room air conditions. Doxycycline decreased the expression of MMPs and general pro-inflammatory markers in the lungs from CS-exposed mice. This downregulation was, however, insufficient to ameliorate CS-induced emphysema or PH. Tet-On/Tet-Off induction by doxycycline in such models is a feasible genetic approach to study curative effects at least in established CS-induced emphysema and PH. However, we report several parameters that are influenced by doxycycline and use of a Tet-On/Tet-Off system when evaluating those parameters should be interpreted with caution.

Genetic Deficiency and Pharmacological Stabilization of Mast Cells Ameliorate Pressure Overload-Induced Maladaptive Right Ventricular Remodeling in Mice.

Although the response of the right ventricle (RV) to the increased afterload is an important determinant of the patient outcome, very little is known about the underlying mechanisms. Mast cells have been implicated in the pathogenesis of left ventricular maladaptive remodeling and failure. However, the role of mast cells in RV remodeling remains unexplored. We subjected mast cell-deficient WBB6F1-KitW/W-v (KitW/KitW-v) mice and their mast cell-sufficient littermate controls (MC+/+) to pulmonary artery banding (PAB). PAB led to RV dilatation, extensive myocardial fibrosis, and RV dysfunction in MC+/+ mice. In PAB KitW/KitW-v mice, RV remodeling was characterized by minimal RV chamber dilatation and preserved RV function. We further administered to C57Bl/6J mice either placebo or cromolyn treatment starting from day 1 or 7 days after PAB surgery to test whether mast cells stabilizing drugs can prevent or reverse maladaptive RV remodeling. Both preventive and therapeutic cromolyn applications significantly attenuated RV dilatation and improved RV function. Our study establishes a previously undescribed role of mast cells in pressure overload-induced adverse RV remodeling. Mast cells may thus represent an interesting target for the development of a new therapeutic approach directed specifically at the heart.

Riociguat for treatment of pulmonary hypertension in COPD: a translational study.

Chronic obstructive pulmonary disease (COPD), which comprises the phenotypes of chronic bronchitis and emphysema, is often associated with pulmonary hypertension (PH). However, currently, no approved therapy exists for PH-COPD. Signalling of the nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) axis plays an important role in PH and COPD.We investigated the treatment effect of riociguat, which promotes the NO-cGMP pathway, in the mouse model of smoke-induced PH and emphysema in a curative approach, and retrospectively analysed the effect of riociguat treatment on PH in single patients with PH-COPD.In mice with established PH and emphysema (after 8 months of cigarette smoke exposure), riociguat treatment for another 3 months fully reversed PH. Moreover, histological hallmarks of emphysema were decreased. Microarray analysis revealed involvement of different signalling pathways, e.g. related to matrix metalloproteinases (MMPs). MMP activity was decreased in vivo by riociguat. In PH-COPD patients treated with riociguat (n=7), the pulmonary vascular resistance, airway resistance and circulating MMP levels decreased, while oxygenation at rest was not significantly changed.Riociguat may be beneficial for treatment of PH-COPD. Further long-term prospective studies are necessary to investigate the tolerability, efficacy on functional parameters and effect specifically on pulmonary emphysema in COPD patients.

Mitochondrial Complex IV Subunit 4 Isoform 2 Is Essential for Acute Pulmonary Oxygen Sensing.

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.

Low density lipoprotein receptor-related protein 1 couples ß1 integrin activation to degradation.

Low density lipoprotein receptor-related protein (LRP) 1 modulates cell adhesion and motility under normal and pathological conditions. Previous studies documented that LRP1 binds several integrin receptors and mediates their trafficking to the cell surface and endocytosis. However, the mechanism by which LRP1 may regulate integrin activation remains unknown. Here we report that LRP1 promotes the activation and subsequent degradation of ß1 integrin and thus supports cell adhesion, spreading, migration and integrin signaling on fibronectin. LRP1 interacts with surface ß1 integrin, binds the integrin activator kindlin2 and stimulates ß1 integrin-kindlin2 complex formation. Specifically, serine 76 in the LRP1 cytoplasmic tail is crucial for the interaction with kindlin2, ß1 integrin activation and cell adhesion. Interestingly, a loss of LRP1 induces the accumulation of several integrin receptors on the cell surface. Following internalization, intracellular trafficking of integrins is driven by LRP1 in a protein kinase C- and class II myosin-dependent manner. Ultimately, LRP1 dictates the fate of endocytosed ß1 integrin by directing it down the pathway of lysosomal and proteasomal degradation. We propose that LRP1 mediates cell adhesion by orchestrating a multi-protein pathway to activate, traffic and degrade integrins. Thus, LRP1 may serve as a focal point in the integrin quality control system to ensure a firm connection to the extracellular matrix.

Impact of the mitochondria-targeted antioxidant MitoQ on hypoxia-induced pulmonary hypertension.

Increased mitochondrial reactive oxygen species (ROS), particularly superoxide have been suggested to mediate hypoxic pulmonary vasoconstriction (HPV), chronic hypoxia-induced pulmonary hypertension (PH) and right ventricular (RV) remodelling.We determined ROS in acute, chronic hypoxia and investigated the effect of the mitochondria-targeted antioxidant MitoQ under these conditions.The effect of MitoQ or its inactive carrier substance, decyltriphenylphosphonium (TPP+), on acute HPV (1% O2 for 10 minutes) was investigated in isolated blood-free perfused mouse lungs. Mice exposed for 4 weeks to chronic hypoxia (10% O2) or after banding of the main pulmonary artery (PAB) were treated with MitoQ or TPP+ (50 mg/kg/day).Total cellular superoxide and mitochondrial ROS levels were increased in pulmonary artery smooth muscle cells (PASMC), but decreased in pulmonary fibroblasts in acute hypoxia. MitoQ significantly inhibited HPV and acute hypoxia-induced rise in superoxide concentration. ROS was decreased in PASMC, while it increased in the RV after chronic hypoxia. Correspondingly, MitoQ did not affect the development of chronic hypoxia-induced PH, but attenuated RV remodelling after chronic hypoxia as well as after PAB.Increased mitochondrial ROS of PASMC mediate acute HPV, but not chronic hypoxia-induced PH. MitoQ may be beneficial under conditions of exaggerated acute HPV.

Lung Ischaemia-Reperfusion Injury: The Role of Reactive Oxygen Species.

Lung ischaemia-reperfusion injury (LIRI) occurs in many lung diseases and during surgical procedures such as lung transplantation. The re-establishment of blood flow and oxygen delivery into the previously ischaemic lung exacerbates the ischaemic injury and leads to increased microvascular permeability and pulmonary vascular resistance as well as to vigorous activation of the immune response. These events initiate the irreversible damage of the lung with subsequent oedema formation that can result in systemic hypoxaemia and multi-organ failure. Alterations in the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) have been suggested as crucial mediators of such responses during ischaemia-reperfusion in the lung. Among numerous potential sources of ROS/RNS within cells, nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, xanthine oxidases, nitric oxide synthases and mitochondria have been investigated during LIRI. Against this background, we aim to review here the extensive literature about the ROS-mediated cellular signalling during LIRI, as well as the effectiveness of antioxidants as treatment option for LIRI.

NADPH oxidases-do they play a role in TRPC regulation under hypoxia?

In the lung, acute alveolar hypoxia causes hypoxic pulmonary vasoconstriction (HPV) to maintain ventilation perfusion matching and thus optimal oxygenation of blood. In contrast, global chronic hypoxia triggers a pathological thickening of pulmonary arterial walls, called pulmonary vascular remodelling, leading to persistence of pulmonary hypertension (PH). Moreover, ischaemia or hypoxia can lead to a damage of pulmonary endothelial cells with subsequent oedema formation. Alterations in reactive oxygen species (ROS) have been suggested as a crucial mediator of such responses. Among the various sources of cellular ROS production, NADPH oxidases (NOXs) have been found to contribute to these physiological and pathophysiological signalling processes. NOXs are the only known examples that generate ROS as the primary function of the enzyme system. However, the downstream targets of NOX-derived ROS signalling in hypoxia are still not known. Canonical transient receptor potential (TRPC) channels recently have been recognised as directly or indirectly ROS-activated channels and have been shown to be essential for hypoxia-dependent vascular regulatory processes in the lung. Against this background, we here summarise the current knowledge on NOX-mediated TRPC channel signalling during hypoxia in the pulmonary circulation.

Oxygen sensing and signal transduction in hypoxic pulmonary vasoconstriction.

Hypoxic pulmonary vasoconstriction (HPV), also known as the von Euler-Liljestrand mechanism, is an essential response of the pulmonary vasculature to acute and sustained alveolar hypoxia. During local alveolar hypoxia, HPV matches perfusion to ventilation to maintain optimal arterial oxygenation. In contrast, during global alveolar hypoxia, HPV leads to pulmonary hypertension. The oxygen sensing and signal transduction machinery is located in the pulmonary arterial smooth muscle cells (PASMCs) of the pre-capillary vessels, albeit the physiological response may be modulated in vivo by the endothelium. While factors such as nitric oxide modulate HPV, reactive oxygen species (ROS) have been suggested to act as essential mediators in HPV. ROS may originate from mitochondria and/or NADPH oxidases but the exact oxygen sensing mechanisms, as well as the question of whether increased or decreased ROS cause HPV, are under debate. ROS may induce intracellular calcium increase and subsequent contraction of PASMCs via direct or indirect interactions with protein kinases, phospholipases, sarcoplasmic calcium channels, transient receptor potential channels, voltage-dependent potassium channels and L-type calcium channels, whose relevance may vary under different experimental conditions. Successful identification of factors regulating HPV may allow development of novel therapeutic approaches for conditions of disturbed HPV.

Oxygen-dependent expression of cytochrome c oxidase subunit 4-2 gene expression is mediated by transcription factors RBPJ, CXXC5 and CHCHD2.

Cytochrome c oxidase (COX) is the terminal enzyme of the electron transport chain, made up of 13 subunits encoded by both mitochondrial and nuclear DNA. Subunit 4 (COX4), a key regulatory subunit, exists as two isoforms, the ubiquitous isoform 1 and the tissue-specific (predominantly lung) isoform 2 (COX4I2). COX4I2 renders lung COX about 2-fold more active compared with liver COX, which lacks COX4I2. We previously identified a highly conserved 13-bp sequence in the proximal promoter of COX4I2 that functions as an oxygen responsive element (ORE), maximally active at a 4% oxygen concentration. Here, we have identified three transcription factors that bind this conserved ORE, namely recombination signal sequence-binding protein Jκ (RBPJ), coiled-coil-helix-coiled-coil-helix domain 2 (CHCHD2) and CXXC finger protein 5 (CXXC5). We demonstrate that RBPJ and CHCHD2 function towards activating the ORE at 4% oxygen, whereas CXXC5 functions as an inhibitor. To validate results derived from cultured cells, we show using RNA interference a similar effect of these transcription factors in the gene regulation of COX4I2 in primary pulmonary arterial smooth muscle cells. Depending on the oxygen tension, a concerted action of the three transcription factors regulates the expression of COX4I2 that, as we discuss, could augment both COX activity and its ability to cope with altered cellular energy requirements.