Cardio-Pulmonary Institute

Clinical and translational research has played a major role in advancing our understanding of pulmonary hypertension (PH), including pulmonary arterial hypertension and other forms of PH with severe vascular remodelling ( e.g. chronic thromboembolic PH and pulmonary veno-occlusive disease). However, PH remains an incurable condition with a high mortality rate, underscoring the need for a better transfer of novel scientific knowledge into healthcare interventions. Herein, we review recent findings in pathology (with the questioning of the strict morphological categorisation of various forms of PH into pre- or post-capillary involvement of pulmonary vessels) and cellular mechanisms contributing to the onset and progression of pulmonary vascular remodelling associated with various forms of PH. We also discuss ways to improve management and to support and optimise drug development in this research field.

Idiopathic pulmonary fibrosis (IPF) is a devastating lung disease with poor prognosis and limited therapeutic options. The incidence of IPF increases with age, and ageing-related mechanisms such as cellular senescence have been proposed as pathogenic drivers. The lung alveolar epithelium represents a major site of tissue injury in IPF and senescence of this cell population is probably detrimental to lung repair. However, the potential pathomechanisms of alveolar epithelial cell senescence and the impact of senolytic drugs on senescent lung cells and fibrosis remain unknown. Here we demonstrate that lung epithelial cells exhibit increased P16 and P21 expression as well as senescence-associated β-galactosidase activity in experimental and human lung fibrosis tissue and primary cells. Primary fibrotic mouse alveolar epithelial type (AT)II cells secreted increased amounts of senescence-associated secretory phenotype (SASP) factors in vitro , as analysed using quantitative PCR, mass spectrometry and ELISA. Importantly, pharmacological clearance of senescent cells by induction of apoptosis in fibrotic ATII cells or ex vivo three-dimensional lung tissue cultures reduced SASP factors and extracellular matrix markers, while increasing alveolar epithelial markers . These data indicate that alveolar epithelial cell senescence contributes to lung fibrosis development and that senolytic drugs may be a viable therapeutic option for IPF.

Endothelial cells play a critical role in the adaptation of tissues to injury. Tissue ischemia induced by infarction leads to profound changes in endothelial cell functions and can induce transition to a mesenchymal state. Here we explore the kinetics and individual cellular responses of endothelial cells after myocardial infarction by using single cell RNA sequencing. This study demonstrates a time dependent switch in endothelial cell proliferation and inflammation associated with transient changes in metabolic gene signatures. Trajectory analysis reveals that the majority of endothelial cells 3 to 7 days after myocardial infarction acquire a transient state, characterized by mesenchymal gene expression, which returns to baseline 14 days after injury. Lineage tracing, using the Cdh5-CreERT2;mT/mG mice followed by single cell RNA sequencing, confirms the transient mesenchymal transition and reveals additional hypoxic and inflammatory signatures of endothelial cells during early and late states after injury. These data suggest that endothelial cells undergo a transient mes-enchymal activation concomitant with a metabolic adaptation within the first days after myocardial infarction but do not acquire a long-term mesenchymal fate. This mesenchymal activation may facilitate endothelial cell migration and clonal expansion to regenerate the vascular network.

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.

AIMS: Coronavirus disease 2019 is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and has emerged as a global pandemic. SARS-CoV-2 infection can lead to elevated markers of cardiac injury associated with higher risk of mortality. It is unclear whether cardiac injury is caused by direct infection of cardiomyocytes or is mainly secondary to lung injury and inflammation. Here, we investigate whether cardiomyocytes are permissive for SARS-CoV-2 infection. METHODS AND RESULTS: Two strains of SARS-CoV-2 infected human induced pluripotent stem cell-derived cardiomyocytes as demonstrated by detection of intracellular double-stranded viral RNA and viral spike glycoprotein expression. Increasing concentrations of viral RNA are detected in supernatants of infected cardiomyocytes, which induced infections in Caco-2 cell lines, documenting productive infections. SARS-CoV-2 infection and induced cytotoxic and proapoptotic effects associated with it abolished cardiomyocyte beating. RNA sequencing confirmed a transcriptional response to viral infection as demonstrated by the up-regulation of genes associated with pathways related to viral response and interferon signalling, apoptosis, and reactive oxygen stress. SARS-CoV-2 infection and cardiotoxicity was confirmed in a 3D cardiosphere tissue model. Importantly, viral spike protein and viral particles were detected in living human heart slices after infection with SARS-CoV-2. Coronavirus particles were further observed in cardiomyocytes of a patient with coronavirus disease 2019. Infection of induced pluripotent stem cell-derived cardiomyocytes was dependent on cathepsins and angiotensin-converting enzyme 2, and was blocked by remdesivir. CONCLUSION: This study demonstrates that SARS-CoV-2 infects cardiomyocytes in vitro in an angiotensin-converting enzyme 2- and cathepsin-dependent manner. SARS-CoV-2 infection of cardiomyocytes is inhibited by the antiviral drug remdesivir.

Abstract Coronavirus disease 2019 (COVID-19) can damage cerebral small vessels and cause neurological symptoms. Here we describe structural changes in cerebral small vessels of patients with COVID-19 and elucidate potential mechanisms underlying the vascular pathology. In brains of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected individuals and animal models, we found an increased number of empty basement membrane tubes, so-called string vessels representing remnants of lost capillaries. We obtained evidence that brain endothelial cells are infected and that the main protease of SARS-CoV-2 (M pro ) cleaves NEMO, the essential modulator of nuclear factor-κB. By ablating NEMO, M pro induces the death of human brain endothelial cells and the occurrence of string vessels in mice. Deletion of receptor-interacting protein kinase (RIPK) 3, a mediator of regulated cell death, blocks the vessel rarefaction and disruption of the blood–brain barrier due to NEMO ablation. Importantly, a pharmacological inhibitor of RIPK signaling prevented the M pro -induced microvascular pathology. Our data suggest RIPK as a potential therapeutic target to treat the neuropathology of COVID-19.

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 ).

Increasing evidence suggests that neurogenesis occurs in the postnatal and adult mammalian hypothalamus. However, the identity and location of the putative progenitor cells is under much debate, and little is known about the dynamics of neurogenesis in unchallenged brain. Previously, we postulated that Fibroblast growth factor 10-expressing (Fgf10(+)) tanycytes constitute a population of progenitor cells in the mouse hypothalamus. Here, we show that Fgf10(+) tanycytes express markers of neural stem/progenitor cells, divide late into postnatal life, and can generate both neurons and astrocytes in vivo. Stage-specific lineage-tracing of Fgf10(+) tanycytes using Fgf10-creERT2 mice, reveals robust neurogenesis at postnatal day 28 (P28), lasting as late as P60. Furthermore, we present evidence for amplification of Fgf10-lineage traced neural cells within the hypothalamic parenchyma itself. The neuronal descendants of Fgf10(+) tanycytes predominantly populate the arcuate nucleus, a subset of which express the orexigenic neuronal marker, Neuropeptide-Y, and respond to fasting and leptin-induced signaling. These studies provide direct evidence in support of hypothalamic neurogenesis during late postnatal and adult life, and identify Fgf10(+) tanycytes as a source of parenchymal neurons with putative roles in appetite and energy balance.

BACKGROUND: The prevention of hypertension with the angiotensin-converting enzyme inhibitor ramipril in patients with high-normal blood pressure study addresses the issue of whether progression to manifest hypertension in patients with high-normal blood pressure can be prevented with treatment. METHODS: A total of 1008 participants with high-normal office blood pressure were randomized to ramipril treatment group (n = 505) and a control group (n = 503). The patients were followed up for 3 years. Primary endpoint was to prevent or delay the progression to manifest hypertension. Secondary endpoints were reduction in the incidence of cerebrovascular and cardiovascular events, as well as the development of hypertension as defined by ambulatory blood pressure monitoring. FINDINGS: One hundred and fifty-five patients (30.7%) in the ramipril group, and 216 (42.9%) in the control group reached the primary endpoint (relative risk reduction 34.4%, P = 0.0001). Ramipril also proved to be more effective in reducing the incidence of manifest office hypertension in patients with baseline ambulatory blood pressure monitoring high-normal blood pressure. The incidence of cerebrovascular and cardiovascular events showed no statistically significant differences between the two groups. Cough was more frequent in the ramipril group (4.8 vs. 0.4%). INTERPRETATION: There is now good clinical evidence that patients with high-normal blood pressure (prehypertension) are more likely to progress to manifest hypertension than patients with optimal or normal blood pressure. Additional ambulatory blood pressure monitoring seems to be essential to achieve correct diagnosis. Treatment of patients with high-normal office blood pressure with the angiotensin-converting enzyme inhibitor was well tolerated, and significantly reduced the risk of progression to manifest hypertension.

Pulmonary hypertension (PH) is characterized by pulmonary artery remodeling that can subsequently culminate in right heart failure and premature death. Emerging evidence suggests that hypoxia-inducible factor (HIF) signaling plays a fundamental and pivotal role in the pathogenesis of PH. This Review summarizes the regulation of HIF isoforms and their impact in various PH subtypes, as well as the elaborate conditional and cell-specific knockout mouse studies that brought the role of this pathway to light. We also discuss the current preclinical status of pan- and isoform-selective HIF inhibitors, and propose new research areas that may facilitate HIF isoform-specific inhibition as a novel therapeutic strategy for PH and right heart failure.

The respective antitumoral and protumoral roles of M1 and M2 tumor-associated macrophages (TAM) typify the complexity of macrophage function in cancer. In lung cancer, density and topology of distinct TAM phenotypes at the tumor center (TC) versus the invasive margin (IM) are largely unknown. Here, we investigated TAM subtype density and distribution between TC and IM in human lung cancer and TAM associations with overall survival. Macrophages isolated from adjacent nontumor tissue (NM), the TC (TC-TAM), and the IM (IM-TAM) were analyzed with RNA-sequencing (RNA-seq). Lung tumor tissue microarrays from 104 patient samples were constructed. M1 and M2 TAMs were identified using multiplex immunofluorescence staining and a tumor cell-TAM proximity analysis was performed. RNA-seq identified marked differences among NM, TC-TAM, and IM-TAM. On the basis of a panel of five selected markers (CD68, IL12, CCR7, CD163, and ALOX15), M2 predominance over M1 and M2 proximity to tumor cells was observed, especially at IM. Tumor cell proximity to TAM was linked with tumor cell survival and hypoxia was associated with accumulation of M2 TAM. Notably, lower density of M1 TC-TAM and higher proximity of tumor cells to M2 IM-TAM or lower proximity to M1 IM-TAM were linked with poor survival. In addition, three novel molecules (UBXN4, MFSD12, and ACTR6) from RNA-seq served as potential prognostic markers for lung cancer, and M2 predominance and juxtaposition of M2 TAM near tumor cells were associated with poor survival. Together, our results reveal the marked heterogeneity of TAM populations in different tumor regions, with M2 TAM predominance, particularly at IM. SIGNIFICANCE: This study underlines the significance of the density, spatial distribution, and gene expression of TAM phenotypes as prognostic factors for overall survival in lung cancer. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/20/4414/F1.large.jpg.

Studies from the USA have reported that sleep apnoea is common in congestive heart failure (CHF), with Cheyne-Stokes respiration (CSR) being the most frequent type of sleep-disordered breathing (SDB) in these patients. Within the present study, the authors sought to assess the prevalence and type of SDB among CHF patients in Germany. A total of 203 CHF patients participated in this prospective multicentre study. All patients were stable in New York Heart Association classes II and III and had a left ventricular ejection fraction (LVEF)<40%. The patients were investigated by polygraphy and all data were centrally analysed. Patient enrolment was irrespective of sleep-related symptoms. The majority of patients were male with a mean age of 65 yrs and hospitalised. Of the 203 patients, 145 (71%) had an apnoea/hypopnoea index>10.h(-1), obstructive sleep apnoea (OSA) occurred in 43% (n=88) and CSR in 28% (n=57) of patients. The prevalence of sleep-disordered breathing is high in patients with stable severe congestive heart failure from a European population. As sleep-disordered breathing may have a negative impact on the prognosis of congestive heart failure, a sleep study should be performed in every patient with congestive heart failure and a left ventricular ejection fraction of <40%. This diagnostic approach should probably be adopted for all of these patients irrespective of the presence of sleep-related symptoms.

Portopulmonary hypertension (POPH) is regarded as a subtype of pulmonary arterial hypertension (PAH); however, established PAH therapies have not been evaluated for this condition. The current authors treated 14 patients (four male, 10 female; mean (range) age 55 (39-75) yrs) with moderate (n = 1) or severe (n = 13) POPH caused by alcoholic liver disease (n = 7), chronic viral hepatitis (n = 3), autoimmune hepatitis (n = 3), and hepatic manifestation of hereditary haemorrhagic teleangiectasia (n = 1) with oral sildenafil. Eight patients were newly started on pulmonary vasoactive treatment, while six patients were already on treatment with inhaled prostanoids (iloprost, n = 5; treprostinil, n = 1). During treatment with sildenafil, mean +/- sd 6-min walk distance increased from 312 +/- 111 m to 397 +/- 99 m after 3 months, and 407 +/- 97 m after 12 months. Mean +/- sd pro-brain natriuretic peptide levels decreased from 582 +/- 315 ng x mL(-1) to 230 +/- 278 ng x mL(-1), and to 189 +/- 274 ng x mL(-1) after 3 and 12 months, respectively. Two patients died after 1 and 2 months from liver failure and cardiac failure, respectively. There was a similar response to sildenafil treatment after 3 and 12 months in patients on monotherapy and those on combination therapy. In conclusion, sildenafil might be effective in monotherapy and in combination therapy with inhaled prostanoids in portopulmonary hypertension, leading to significant improvement by 3 months and sustained response over 12 months.

Reactive oxygen species (ROS) are highly reactive oxygen derivatives that include free radicals such as superoxide anion radical (O2•−) and hydroxyl radical (HO•), as well as non-radical molecules hydrogen peroxide (H2O2), peroxynitrite (ONOO−), and hypochlorous acid (HOCl) [...]

Acetylcholine (ACh), a classical transmitter of parasympathetic nerve fibres in the airways, is also synthesized by a large number of non-neuronal cells, including airway surface epithelial cells. Strongest expression of cholinergic traits is observed in neuroendocrine and brush cells but other epithelial cell types--ciliated, basal and secretory--are cholinergic as well. There is cell type-specific expression of the molecular pathways of ACh release, including both the vesicular storage and exocytotic release known from neurons, and transmembrane release from the cytosol via organic cation transporters. The subcellular distribution of the ACh release machineries suggests luminal release from ciliated and secretory cells, and basolateral release from neuroendocrine cells. The scenario as known so far strongly suggests a local auto-/paracrine role of epithelial ACh in regulating various aspects on the innate mucosal defence mechanisms, including mucociliary clearance, regulation of macrophage function and modulation of sensory nerve fibre activity. The proliferative effects of ACh gain importance in recently identified ACh receptor disorders conferring susceptibility to lung cancer. The cell type-specific molecular diversity of the epithelial ACh synthesis and release machinery implies that it is differently regulated than neuronal ACh release and can be specifically targeted by appropriate drugs.

AIMS: Oxidative stress is thought to be a risk for cardiovascular disease and NADPH oxidases of the Nox family are important producers of reactive oxygen species. Within the Nox family, the NADPH oxidase Nox4 has a unique position as it is constitutively active and produces H2O2 rather than [Formula: see text] . Nox4 is therefore incapable of scavenging NO and its low constitutive H2O2 production might even be beneficial. We hypothesized that Nox4 acts as an endogenous anti-atherosclerotic enzyme. METHODS AND RESULTS: Tamoxifen-induced Nox4-knockout mice were crossed with ApoE⁻/⁻ mice and spontaneous atherosclerosis under regular chow as well as accelerated atherosclerosis in response to partial carotid artery ligation under high-fat diet were determined. Deletion of Nox4 resulted in increased atherosclerosis formation in both models. Mechanistically, pro-atherosclerotic and pro-inflammatory changes in gene expression were observed prior to plaque development. Moreover, inhibition of Nox4 or deletion of the enzyme in the endothelium but not in macrophages resulted in increased adhesion of macrophages to the endothelial surface. CONCLUSIONS: The H2O2-producing NADPH oxidase Nox4 is an endogenous anti-atherosclerotic enzyme. Nox4 inhibitors, currently under clinical evaluation, should be carefully monitored for cardiovascular side-effects.

RATIONALE: MicroRNAs (miRNAs or miRs) are implicated in the pathogenesis of various cardiovascular diseases, including pulmonary arterial hypertension (PAH). OBJECTIVES: We sought to measure changes in plasma levels of miRNAs in patients with PAH and relate them to the severity of the disease. METHODS: A microarray screen was performed on total plasma RNA from eight patients with PAH and eight healthy control subjects. Quantitative polymerase chain reaction confirmed reduced miR-150 concentrations and was then used to measure miR-150 levels in (1) two separate cohorts of patients with PAH, from London (n = 145) and Sheffield (n = 30), respectively; (2) circulating microvesicles and blood cells; and (3) lungs from a monocrotaline rat model. MEASUREMENTS AND MAIN RESULTS: Fifty-eight miRNAs showed differences in plasma concentration and miR-150 the largest down-regulation in PAH. Receiver-operator-characteristic analysis showed both raw and normalized plasma miR-150 levels correlated with 2-year survival (P < 0.01) in patients with PAH. Cox regression analysis confirmed miR-150 levels as a significant predictor of survival. Age, baseline cardiac index, World Health Organization functional class, 6-minute walk distance, disease duration, and red cell distribution width also predicted survival. Entering these covariates in a multivariable model verified plasma miR-150 levels as an independent predictor of survival in PAH (hazard ratio, 0.533; P = 0.010). miR-150 levels also predicted survival in a second, independent PAH cohort. miR-150 levels were significantly reduced in circulating microvesicles from patients with PAH and the lungs of the monocrotaline rat. CONCLUSIONS: Reduced circulating miR-150 levels are associated with poor survival in PAH.

OBJECTIVE: To compare rates and predictors of pregnancy complications in mothers with systemic lupus erythematosus (SLE) with and without previous nephritis (PN). METHODS: Retrospective analysis of 107 pregnancies in 83 women with SLE diagnosed prepregnancy. RESULTS: Mothers with PN had higher rates of preterm delivery (< 37/40, 30% vs 11%, p = 0.029) than those without PN. Women with PN had earlier onset of preeclampsia [median 34.5 weeks (IQR 32-37) vs 37.5 weeks (IQR 35-38, p = 0.047)] that was more frequently complicated by preterm delivery (p = 0.02). Risk factors for preeclampsia in women with PN include 10-13 weeks' gestation diastolic blood pressure > 80 mmHg and proteinuria, and prepregnancy estimated glomerular filtration rate (eGFR) < 90 ml/min/1.73 m(2). In women with PN, midtrimester uterine-artery-Doppler notching had low negative predictive value (47%). After 39 months followup, eGFR was stable in women with or without PN. CONCLUSION: In SLE, preterm deliveries are more frequent and preeclampsia occurs earlier in women with PN, but longterm eGFR is preserved.

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

Skin wounds are characterized by injury to the skin due to trauma, tearing, cuts, or contusions. As such injuries are common to all human groups, they may at times represent a serious socioeconomic burden. Currently, increasing numbers of studies have focused on the role of mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) in skin wound repair. As a cell-free therapy, MSC-derived EVs have shown significant application potential in the field of wound repair as a more stable and safer option than conventional cell therapy. Treatment based on MSC-derived EVs can significantly promote the repair of damaged substructures, including the regeneration of vessels, nerves, and hair follicles. In addition, MSC-derived EVs can inhibit scar formation by affecting angiogenesis-related and antifibrotic pathways in promoting macrophage polarization, wound angiogenesis, cell proliferation, and cell migration, and by inhibiting excessive extracellular matrix production. Additionally, these structures can serve as a scaffold for components used in wound repair, and they can be developed into bioengineered EVs to support trauma repair. Through the formulation of standardized culture, isolation, purification, and drug delivery strategies, exploration of the detailed mechanism of EVs will allow them to be used as clinical treatments for wound repair. In conclusion, MSC-derived EVs-based therapies have important application prospects in wound repair. Here we provide a comprehensive overview of their current status, application potential, and associated drawbacks.