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 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.

The Role of the Redox Enzyme p66Shc in Biological Aging of the Lung.

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.

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.

Pulmonary Hypertension: Unveiling Molecular Mechanisms, Diagnosis, and Therapeutic Targets.

Pulmonary hypertension (PH) is a progressive and life-threatening disease characterized by increased pulmonary arterial pressure, which leads to right heart hypertrophy and eventually right heart failure [...].

Decoding the role of fatty acids and their metabolites in lung fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive and life­threatening interstitial lung disease of familial or sporadic onset. The incidence and prevalence of IPF range from 0.09 to 1.3 and from 0.33 to 4.51 per 10 000 people, respectively. IPF has a poor prognosis, and death usually occurs within 2 to 5 years following the diagnosis due to secondary respiratory failure. Currently, there are 2 drugs available to treat IPF, pirfenidone and nintedanib. Both only slow the disease progression and, in addition, have unfavorable safety profiles. IPF bears the histology of usual interstitial pneumonia, which is characterized by bronchiolization of distal airspaces, honeycombing, fibroblastic foci, and abnormal epithelial hyperplasia. In the last years, alterations in metabolic pathways, in particular those associated with fatty acid (FA) metabolism have been linked with the pathogenesis of lung fibrosis. Changes in FA profiles have been reported in lung tissue, plasma, and bronchoalveolar lavage fluid of IPF patients, and have been found to correlate with the disease progression and outcome. In addition, they have been associated with the development of a profibrotic phenotype of epithelial cells, macrophages, and fibroblasts / myofibroblasts contributing to their (trans)differentiation and production of the disease­relevant mediators. Furthermore, strategies focusing on the correction of FA profiles in experimental models of lung fibrosis brought advances in understanding tissue scarring processes and contributed to the transition of new molecules into clinical development. This review highlights the role of FAs and their metabolites in IPF and provides evidence for therapeutic potential of lipidome manipulations in the treatment of this disease.

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.

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.

TRAIL protects the immature lung from hyperoxic injury.

The hyperoxia-induced pro-inflammatory response and tissue damage constitute pivotal steps leading to bronchopulmonary dysplasia (BPD) in the immature lung. The pro-inflammatory cytokines are considered attractive candidates for a directed intervention but the complex interplay between inflammatory and developmental signaling pathways requires a comprehensive evaluation before introduction into clinical trials as studied here for the death inducing ligand TRAIL. At birth and during prolonged exposure to oxygen and mechanical ventilation, levels of TRAIL were lower in tracheal aspirates of preterm infants <29 weeks of gestation which developed moderate/severe BPD. These findings were reproduced in the newborn mouse model of hyperoxic injury. The loss of TRAIL was associated with increased inflammation, apoptosis induction and more pronounced lung structural simplification after hyperoxia exposure for 7 days while activation of NFκB signaling during exposure to hyperoxia was abrogated. Pretreatment with recombinant TRAIL rescued the developmental distortions in precision cut lung slices of both wildtype and TRAIL-/- mice exposed to hyperoxia. Of importance, TRAIL preserved alveolar type II cells, mesenchymal progenitor cells and vascular endothelial cells. In the situation of TRAIL depletion, our data ascribe oxygen toxicity a more injurious impact on structural lung development. These data are not surprising taking into account the diverse functions of TRAIL and its stimulatory effects on NFκB signaling as central driver of survival and development. TRAIL exerts a protective role in the immature lung as observed for the death inducing ligand TNF-α before.

Association of Clonal Hematopoiesis of Indeterminate Potential with Inflammatory Gene Expression in Patients with COPD.

Chronic obstructive pulmonary disease (COPD) is a disease with an inflammatory phenotype with increasing prevalence in the elderly. Expanded population of mutant blood cells carrying somatic mutations is termed clonal hematopoiesis of indeterminate potential (CHIP). The association between CHIP and COPD and its relevant effects on DNA methylation in aging are mainly unknown. Analyzing the deep-targeted amplicon sequencing from 125 COPD patients, we found enhanced incidence of CHIP mutations (~20%) with a predominance of DNMT3A CHIP-mediated hypomethylation of Phospholipase D Family Member 5 (PLD5), which in turn is positively correlated with increased levels of glycerol phosphocholine, pro-inflammatory cytokines, and deteriorating lung function.

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.

Genetic deletion of p66shc and/or cyclophilin D results in decreased pulmonary vascular tone.

AIMS: The pulmonary vascular tone and hypoxia-induced alterations of the pulmonary vasculature may be regulated by the mitochondrial membrane permeability transition pore (mPTP) that controls mitochondrial calcium load and apoptosis. We thus investigated, if the mitochondrial proteins p66shc and cyclophilin D (CypD) that regulate mPTP opening affect the pulmonary vascular tone. METHODS AND RESULTS: Mice deficient for p66shc (p66shc-/-), CypD (CypD-/-), or both proteins (p66shc/CypD-/-) exhibited decreased pulmonary vascular resistance (PVR) compared to wild-type mice determined in isolated lungs and in vivo. In contrast, systemic arterial pressure was only lower in CypD-/- mice. As cardiac function and pulmonary vascular remodelling did not differ between genotypes, we determined alterations of vascular contractility in isolated lungs and calcium handling in pulmonary arterial smooth muscle cells (PASMC) as underlying reason for decreased PVR. Potassium chloride (KCl)-induced pulmonary vasoconstriction and KCl-induced cytosolic calcium increase determined by Fura-2 were attenuated in all gene-deficient mice. In contrast, KCl-induced mitochondrial calcium increase determined by the genetically encoded Mito-Car-GECO and calcium retention capacity were increased only in CypD-/- and p66shc/CypD-/- mitochondria indicating that decreased mPTP opening affected KCl-induced intracellular calcium peaks in these cells. All mouse strains showed a similar pulmonary vascular response to chronic hypoxia, while acute hypoxic pulmonary vasoconstriction was decreased in gene-deficient mice indicating that CypD and p66shc regulate vascular contractility but not remodelling. CONCLUSIONS: We conclude that p66shc specifically regulates the pulmonary vascular tone, while CypD also affects systemic pressure. However, only CypD acts via regulation of mPTP opening and mitochondrial calcium regulation.

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.

iNOS Deletion in Alveolar Epithelium Cannot Reverse the Elastase-Induced Emphysema in Mice.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is the third leading cause of death worldwide. In addition to chronic bronchitis and emphysema, patients often develop at least mild pulmonary hypertension (PH). We previously demonstrated that inhibition of inducible nitric oxide synthase (iNOS) prevents and reverses emphysema and PH in mice. Interestingly, strong iNOS upregulation was found in alveolar epithelial type II cells (AECII) in emphysematous murine lungs, and peroxynitrite, which can be formed from iNOS-derived NO, was shown to induce AECII apoptosis in vitro. However, the specific cell type(s) that drive(s) iNOS-dependent lung regeneration in emphysema/PH has (have) not been identified yet. AIM: we tested whether iNOS knockout in AECII affects established elastase-induced emphysema in mice. METHODS: four weeks after a single intratracheal instillation of porcine pancreatic elastase for the induction of emphysema and PH, we induced iNOS knockout in AECII in mice, and gave an additional twelve weeks for the potential recovery. RESULTS: iNOS knockout in AECII did not reduce elastase-induced functional and structural lung changes such as increased lung compliance, decreased mean linear intercept and increased airspace, decreased right ventricular function, increased right ventricular systolic pressure and increased pulmonary vascular muscularization. In vitro, iNOS inhibition did not reduce apoptosis of AECII following exposure to a noxious stimulus. CONCLUSION: taken together, our data demonstrate that iNOS deletion in AECII is not sufficient for the regeneration of emphysematous murine lungs, and suggest that iNOS expression in pulmonary vascular or stromal cells might be critically important in this regard.

Retinal tissue develops an inflammatory reaction to tobacco smoke and electronic cigarette vapor in mice.

Cigarette smoke has been identified as a major risk factor for the development of age-related macular degeneration (AMD). As an alternative to conventional cigarettes (C-cigarette), electronic cigarettes (E-cigarette) have been globally promoted and are currently widely used. The increasing usage of E-cigarettes raises concerns with regard to short- (2 weeks), medium- (3 months), and long- (8 months) term consequences related to retinal tissue. In this report, a controlled study in mouse models was conducted to probe the comprehensive effects of E-cigarette vapor on retina, retinal pigmented epithelium (RPE), and choroidal tissues by (1) comparing the effects of C-cigarette smoke and E-cigarette vapor on retina separately and (2) determining the effects of E-cigarette vapor on the RPE and analyzing the changes with regard to inflammatory (IL-1ß, TNFα, iNOS) and angiogenic (VEGF, PEDF) mediators in retina/RPE/choroid by ELISA assays. The data showed that C-cigarette smoke exposure promoted an inflammatory reaction in the retina in vivo. Mice exposed to E-cigarette (nicotine-free) vapor developed inflammatory and angiogenic reactions more pronounced in RPE and choroid as compared to retinal tissue, while nicotine-containing E-cigarette vapor caused even a more serious reaction. Both inflammatory and pro-angiogenic reactions increased with the extension of exposure time. These results demonstrate that exposure to C-cigarette smoke is harmful to the retina. Likewise, the exposure to E-cigarette vapor (with or without nicotine) increases the occurrence and progression of inflammatory and angiogenic stimuli in the retina, which might also be related to the onset of wet AMD in humans. KEY MESSAGES: C-cigarette smoke exposure promotes an inflammatory reaction in the retina in vivo. Mice exposed to E-cigarette (nicotine-free) vapor develop inflammatory and angiogenic reactions more pronounced in RPE and choroid compared to retinal tissue, while nicotine-containing E-cigarette vapor causes even a more serious reaction. Both inflammatory and pro-angiogenic reactions increase with the extension of E-cigarette vapor exposure time.

Epigenetic Regulation by Suv4-20h1 in Cardiopulmonary Progenitor Cells Is Required to Prevent Pulmonary Hypertension and Chronic Obstructive Pulmonary Disease.

BACKGROUND: The pathogenesis of life-threatening cardiopulmonary diseases such as pulmonary hypertension (PH) and chronic obstructive pulmonary disease (COPD) originates from a complex interplay of environmental factors and genetic predispositions that is not fully understood. Likewise, little is known about developmental abnormalities or epigenetic dysregulations that might predispose for PH or COPD in adult individuals. METHODS: To identify pathology-associated epigenetic alteration in diseased lung tissues, we screened a cohort of human patients with PH and COPD for changes of histone modifications by immunofluorescence staining. To analyze the function of H4K20me2/3 in lung pathogenesis, we developed a series of Suv4-20h1 knockout mouse lines targeting cardiopulmonary progenitor cells and different heart and lung cell types, followed by hemodynamic studies and morphometric assessment of tissue samples. Molecular, cellular, and biochemical techniques were applied to analyze the function of Suv4-20h1-dependent epigenetic processes in cardiopulmonary progenitor cells and their derivatives. RESULTS: We discovered a strong reduction of the histone modifications of H4K20me2/3 in human patients with COPD but not patients with PH that depend on the activity of the H4K20 di-methyltransferase SUV4-20H1. Loss of Suv4-20h1 in cardiopulmonary progenitor cells caused a COPD-like/PH phenotype in mice including the formation of perivascular tertiary lymphoid tissue and goblet cell hyperplasia, hyperproliferation of smooth muscle cells/myofibroblasts, impaired alveolarization and maturation defects of the microvasculature leading to massive right ventricular dilatation and premature death. Mechanistically, SUV4-20H1 binds directly to the 5'-upstream regulatory element of the superoxide dismutase 3 (Sod3) gene to repress its expression. Increased levels of the extracellular SOD3 enzyme in Suv4-20h1 mutants increases hydrogen peroxide concentrations, causing vascular defects and impairing alveolarization. CONCLUSIONS: Our findings reveal a pivotal role of the histone modifier SUV4-20H1 in cardiopulmonary codevelopment and uncover the developmental origins of cardiopulmonary diseases. We assume that the study will facilitate the understanding of pathogenic events causing PH and COPD and aid the development of epigenetic drugs for the treatment of cardiopulmonary diseases.

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.

Amelioration of elastase-induced lung emphysema and reversal of pulmonary hypertension by pharmacological iNOS inhibition in mice.

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.

IRAG1 Deficient Mice Develop PKG1ß Dependent Pulmonary Hypertension.

PKGs are serine/threonine kinases. PKG1 has two isoforms-PKG1α and ß. Inositol trisphosphate receptor (IP3R)-associated cGMP-kinase substrate 1 (IRAG1) is a substrate for PKG1ß. IRAG1 is also known to further interact with IP3RI, which mediates intracellular Ca2+ release. However, the role of IRAG1 in PH is not known. Herein, WT and IRAG1 KO mice were kept under normoxic or hypoxic (10% O2) conditions for five weeks. Animals were evaluated for echocardiographic variables and went through right heart catheterization. Animals were further sacrificed to prepare lungs and right ventricular (RV) for immunostaining, western blotting, and pulmonary artery smooth muscle cell (PASMC) isolation. IRAG1 is expressed in PASMCs and downregulated under hypoxic conditions. Genetic deletion of IRAG1 leads to RV hypertrophy, increase in RV systolic pressure, and RV dysfunction in mice. Absence of IRAG1 in lung and RV have direct impacts on PKG1ß expression. Attenuated PKG1ß expression in IRAG1 KO mice further dysregulates other downstream candidates of PKG1ß in RV. IRAG1 KO mice develop PH spontaneously. Our results indicate that PKG1ß signaling via IRAG1 is essential for the homeostasis of PASMCs and RV. Disturbing this signaling complex by deleting IRAG1 can lead to RV dysfunction and development of PH in mice.

NADPH oxidase subunit NOXO1 is a target for emphysema treatment in COPD.

Chronic obstructive pulmonary disease (COPD) is a major cause of morbidity and death worldwide. Peroxynitrite, formed from nitric oxide, which is derived from inducible nitric oxide synthase, and superoxide, has been implicated in the development of emphysema, but the source of the superoxide was hitherto not characterized. Here, we identify the non-phagocytic NADPH oxidase organizer 1 (NOXO1) as the superoxide source and an essential driver of smoke-induced emphysema and pulmonary hypertension development in mice. NOXO1 is consistently upregulated in two models of lung emphysema, Cybb (also known as NADPH oxidase 2, Nox2)-knockout mice and wild-type mice with tobacco-smoke-induced emphysema, and in human COPD. Noxo1-knockout mice are protected against tobacco-smoke-induced pulmonary hypertension and emphysema. Quantification of superoxide, nitrotyrosine and multiple NOXO1-dependent signalling pathways confirm that peroxynitrite formation from nitric oxide and superoxide is a driver of lung emphysema. Our results suggest that NOXO1 may have potential as a therapeutic target in emphysema.