The Proteome of Extracellular Vesicles Released from Pulmonary Microvascular Endothelium Reveals Impact of Oxygen Conditions on Biotrauma.

The lung can experience different oxygen concentrations, low as in hypoxia, high as under supplemental oxygen therapy, or oscillating during intermittent hypoxia as in obstructive sleep apnea or intermittent hypoxia/hyperoxia due to cyclic atelectasis in the ventilated patient. This study aimed to characterize the oxygen-condition-specific protein composition of extracellular vesicles (EVs) released from human pulmonary microvascular endothelial cells in vitro to decipher their potential role in biotrauma using quantitative proteomics with bioinformatic evaluation, transmission electron microscopy, flow cytometry, and non-activated thromboelastometry (NATEM). The release of vesicles enriched in markers CD9/CD63/CD81 was enhanced under intermittent hypoxia, strong hyperoxia and intermittent hypoxia/hyperoxia. Particles with exposed phosphatidylserine were increased under intermittent hypoxia. A small portion of vesicles were tissue factor-positive, which was enhanced under intermittent hypoxia and intermittent hypoxia/hyperoxia. EVs from treatment with intermittent hypoxia induced a significant reduction of Clotting Time in NATEM analysis compared to EVs isolated after normoxic exposure, while after intermittent hypoxia/hyperoxia, tissue factor in EVs seems to be inactive. Gene set enrichment analysis of differentially expressed genes revealed that EVs from individual oxygen conditions potentially induce different biological processes such as an inflammatory response under strong hyperoxia and intermittent hypoxia/hyperoxia and enhancement of tumor invasiveness under intermittent hypoxia.

Combined angiotensin-converting enzyme and aminopeptidase inhibition for treatment of experimental ventilator-induced lung injury in mice.

Introduction: Ventilator-induced lung injury (VILI) may aggravate critical illness. Although angiotensin-converting enzyme (ACE) inhibition has beneficial effects in ventilator-induced lung injury, its clinical application is impeded by concomitant hypotension. We hypothesized that the aminopeptidase inhibitor ALT-00 may oppose the hypotension induced by an angiotensin-converting enzyme inhibitor, and that this combination would activate the alternative renin-angiotensin system (RAS) axis to counteract ventilator-induced lung injury. Methods: In separate experiments, C57BL/6 mice were mechanically ventilated with low (LVT, 6 mL/kg) and high tidal volumes (HVT, 30 mL/kg) for 4 h or remained unventilated (sham). High tidal volume-ventilated mice were treated with lisinopril (0.15 µg/kg/min) ± ALT-00 at 2.7, 10 or 100 µg/kg/min. Blood pressure was recorded at baseline and after 4 h. Lung histology was evaluated for ventilator-induced lung injury and the angiotensin (Ang) metabolite profile in plasma (equilibrium levels of Ang I, Ang II, Ang III, Ang IV, Ang 1-7, and Ang 1-5) was measured with liquid chromatography tandem mass spectrometry at the end of the experiment. Angiotensin concentration-based markers for renin, angiotensin-converting enzyme and alternative renin-angiotensin system activities were calculated. Results: High tidal volume-ventilated mice treated with lisinopril showed a significant drop in the mean arterial pressure at 4 h compared to baseline, which was prevented by adding ALT-00 at 10 and 100 µg/kg/min. Ang I, Ang II and Ang 1-7 plasma equilibrium levels were elevated in the high tidal volumes group versus the sham group. Lisinopril reduced Ang II and slightly increased Ang I and Ang 1-7 levels versus the untreated high tidal volumes group. Adding ALT-00 at 10 and 100 µg/kg/min increased Ang I and Ang 1-7 levels versus the high tidal volume group, and partly prevented the downregulation of Ang II levels caused by lisinopril. The histological lung injury score was higher in the high tidal volume group versus the sham and low tidal volume groups, and was attenuated by lisinopril ± ALT-00 at all dose levels. Conclusion: Combined angiotensin-converting enzyme plus aminopeptidase inhibition prevented systemic hypotension and maintained the protective effect of lisinopril. In this study, a combination of lisinopril and ALT-00 at 10 µg/kg/min appeared to be the optimal approach, which may represent a promising strategy to counteract ventilator-induced lung injury that merits further exploration.

Usefulness and limitations of the acute respiratory distress syndrome definitions in non-intubated patients. A narrative review.

According to the Berlin Definition of acute respiratory distress syndrome (ARDS), a positive end-expiratory pressure (PEEP) of at least 5 cmH2O is required to diagnose and grade ARDS. While the Berlin consensus statement specifically acknowledges the role of non-invasive ventilation (NIV) in mild ARDS, this stratification has traditionally presumed a mechanically ventilated patient in the context of moderate to severe ARDS. This may not accurately reflect today's reality of clinical respiratory care. NIV and high-flow nasal cannula oxygen therapy (HFNO) have been used for managing of severe forms of acute hypoxemic respiratory failure with growing frequency, including in patients showing pathophysiological signs of ARDS. This became especially relevant during the COVID-19 pandemic. The levels of PEEP achieved with HFNO have been particularly controversial, and the exact FiO2 it achieves is subject to variability. Pinpointing the presence of ARDS in patients receiving HNFO and the severity in those receiving NIV therefore remains methodically problematic. This narrative review highlights the evolution of the ARDS definition in the context of non-invasive ventilatory support and provides an overview of the parallel development of definitions and ventilatory management of ARDS. It summarizes the methodology applied in clinical trials to classify ARDS in non-intubated patients and the respective consequences on treatment. As ARDS severity has significant therapeutic and prognostic consequences, and earlier treatment in non-intubated patients may be beneficial, closing this knowledge gap may ultimately be a relevant step to improve comparability in clinical trial design and outcomes.

Profiling endogenous adrenal function during veno-venous ECMO support in COVID-19 ARDS: a descriptive analysis.

Background: Prolonged critical illness is often accompanied by an impairment of adrenal function, which has been frequently related to conditions complicating patient management. The presumed connection between hypoxia and the pathogenesis of this critical- illness- related corticosteroid insufficiency (CIRCI) might play an important role in patients with severe acute respiratory distress syndrome (ARDS). Since extracorporeal membrane oxygenation (ECMO) is frequently used in ARDS, but data on CIRCI during this condition are scarce, this study reports the behaviour of adrenal function parameters during oxygenation support with veno-venous (vv)ECMO in coronavirus disease 2019 (COVID-19) ARDS. Methods: A total of 11 patients undergoing vvECMO due to COVID-19 ARDS at the Medical University of Vienna, who received no concurrent corticosteroid therapy, were retrospectively included in this study. We analysed the concentrations of cortisol, aldosterone, and angiotensin (Ang) metabolites (Ang I-IV, Ang 1-7, and Ang 1-5) in serum via liquid chromatography/tandem mass spectrometry before, after 1 day, 1 week, and 2 weeks during vvECMO support and conducted correlation analyses between cortisol and parameters of disease severity. Results: Cortisol concentrations appeared to be lowest after initiation of ECMO and progressively increased throughout the study period. Higher concentrations were related to disease severity and correlated markedly with interleukin-6, procalcitonin, pH, base excess, and albumin during the first day of ECMO. Fair correlations during the first day could be observed with calcium, duration of critical illness, and ECMO gas flow. Angiotensin metabolite concentrations were available in a subset of patients and indicated a more homogenous aldosterone response to plasma renin activity after 1 week of ECMO support. Conclusion: Oxygenation support through vvECMO may lead to a partial recovery of adrenal function over time. In homogenous patient collectives, this novel approach might help to further determine the importance of adrenal stress response in ECMO and the influence of oxygenation support on CIRCI.

The systemic renin-angiotensin system in COVID-19.

SARS-CoV-2 gains cell entry via angiotensin-converting enzyme (ACE) 2, a membrane-bound enzyme of the "alternative" (alt) renin-angiotensin system (RAS). ACE2 counteracts angiotensin II by converting it to potentially protective angiotensin 1-7. Using mass spectrometry, we assessed key metabolites of the classical RAS (angiotensins I-II) and alt-RAS (angiotensins 1-7 and 1-5) pathways as well as ACE and ACE2 concentrations in 159 patients hospitalized with COVID-19, stratified by disease severity (severe, n = 76; non-severe: n = 83). Plasma renin activity (PRA-S) was calculated as the sum of RAS metabolites. We estimated ACE activity using the angiotensin II:I ratio (ACE-S) and estimated systemic alt-RAS activation using the ratio of alt-RAS axis metabolites to PRA-S (ALT-S). We applied mixed linear models to assess how PRA-S and ACE/ACE2 concentrations affected ALT-S, ACE-S, and angiotensins II and 1-7. Median angiotensin I and II levels were higher with severe versus non-severe COVID-19 (angiotensin I: 86 versus 30 pmol/L, p < 0.01; angiotensin II: 114 versus 58 pmol/L, p < 0.05), demonstrating activation of classical RAS. The difference disappeared with analysis limited to patients not taking a RAS inhibitor (angiotensin I: 40 versus 31 pmol/L, p = 0.251; angiotensin II: 76 versus 99 pmol/L, p = 0.833). ALT-S in severe COVID-19 increased with time (days 1-6: 0.12; days 11-16: 0.22) and correlated with ACE2 concentration (r = 0.831). ACE-S was lower in severe versus non-severe COVID-19 (1.6 versus 2.6; p < 0.001), but ACE concentrations were similar between groups and correlated weakly with ACE-S (r = 0.232). ACE2 and ACE-S trajectories in severe COVID-19, however, did not differ between survivors and non-survivors. Overall RAS alteration in severe COVID-19 resembled severity of disease-matched patients with influenza. In mixed linear models, renin activity most strongly predicted angiotensin II and 1-7 levels. ACE2 also predicted angiotensin 1-7 levels and ALT-S. No single factor or the combined model, however, could fully explain ACE-S. ACE2 and ACE-S trajectories in severe COVID-19 did not differ between survivors and non-survivors. In conclusion, angiotensin II was elevated in severe COVID-19 but was markedly influenced by RAS inhibitors and driven by overall RAS activation. ACE-S was significantly lower with severe COVID-19 and did not correlate with ACE concentrations. A shift to the alt-RAS axis because of increased ACE2 could partially explain the relative reduction in angiotensin II levels.

Practice of oxygen use in anesthesiology - a survey of the European Society of Anaesthesiology and Intensive Care.

BACKGROUND: Oxygen is one of the most commonly used drugs by anesthesiologists. The World Health Organization (WHO) gave recommendations regarding perioperative oxygen administration, but the practice of oxygen use in anesthesia, critical emergency, and intensive care medicine remains unclear. METHODS: We conducted an online survey among members of the European Society of Anaesthesiology and Intensive Care (ESAIC). The questionnaire consisted of 46 queries appraising the perioperative period, emergency medicine and in the intensive care, knowledge about current recommendations by the WHO, oxygen toxicity, and devices for supplemental oxygen therapy. RESULTS: Seven hundred ninety-eight ESAIC members (2.1% of all ESAIC members) completed the survey. Most respondents were board-certified and worked in hospitals with > 500 beds. The majority affirmed that they do not use specific protocols for oxygen administration. WHO recommendations are unknown to 42% of respondents, known but not followed by 14%, and known and followed by 24% of them. Respondents prefer inspiratory oxygen fraction (FiO2) ≥80% during induction and emergence from anesthesia, but intraoperatively < 60% for maintenance, and higher FiO2 in patients with diseased than non-diseased lungs. Postoperative oxygen therapy is prescribed more commonly according to peripheral oxygen saturation (SpO2), but shortage of devices still limits monitoring. When monitoring is used, SpO2 ≤ 95% is often targeted. In critical emergency medicine, oxygen is used frequently in patients aged ≥80 years, or presenting with respiratory distress, chronic obstructive pulmonary disease, myocardial infarction, and stroke. In the intensive care unit, oxygen is mostly targeted at 96%, especially in patients with pulmonary diseases. CONCLUSIONS: The current practice of perioperative oxygen therapy among respondents does not follow WHO recommendations or current evidence, and access to postoperative monitoring devices impairs the individualization of oxygen therapy. Further research and additional teaching about use of oxygen are necessary.

Tidal Volume-Dependent Activation of the Renin-Angiotensin System in Experimental Ventilator-Induced Lung Injury.

OBJECTIVES: Ventilator-induced lung injury (VILI) is a major contributor to morbidity and mortality in critically ill patients. Mechanical damage to the lungs is potentially aggravated by the activation of the renin-angiotensin system (RAS). This article describes RAS activation profiles in VILI and discusses the effects of angiotensin (Ang) 1-7 supplementation or angiotensin-converting enzyme (ACE) inhibition with captopril as protective strategies. DESIGN: Animal study. SETTING: University research laboratory. SUBJECTS: C57BL/6 mice. INTERVENTIONS: Anesthetized mice ( n = 12-18 per group) were mechanically ventilated with low tidal volume (LV T , 6 mL/kg), high tidal volume (HV T , 15 mL/kg), or very high tidal volume (VHV T , 30 mL/kg) for 4 hours, or killed after 3 minutes (sham). Additional VHV T groups received infusions of 60 µg/kg/hr Ang 1-7 or a single dose of 100 mg/kg captopril. MEASUREMENTS AND MAIN RESULTS: VILI was characterized by increased bronchoalveolar lavage fluid levels of interleukin (IL)-6, keratinocyte-derived cytokine, and macrophage inflammatory protein-2 (MIP2). The Ang metabolites in plasma measured with liquid chromatography tandem mass spectrometry showed a strong activation of the classical (Ang I, Ang II) and alternative RAS (Ang 1-7, Ang 1-5), with highest concentrations found in the HV T group. Although the lung-tissue ACE messenger RNA expression was unchanged, its protein expression showed a dose-dependent increase under mechanical ventilation. The ACE2 messenger RNA expression decreased in all ventilated groups, whereas ACE2 protein levels remained unchanged. Both captopril and Ang 1-7 led to markedly increased Ang 1-7 plasma levels, decreased Ang II levels, and ACE activity (Ang II/Ang I ratio), and effectively prevented VILI. CONCLUSIONS: VILI is accompanied by a strong activation of the RAS. Based on circulating Ang metabolite levels and tissue expression of RAS enzymes, classical ACE-dependent and alternative RAS cascades were activated in the HV T group, whereas classical RAS activation prevailed with VHV T ventilation. Ang 1-7 or captopril protected from VILI primarily by modifying the systemic RAS profile.

Safety and preliminary efficacy of sequential multiple ascending doses of solnatide to treat pulmonary permeability edema in patients with moderate-to-severe ARDS-a randomized, placebo-controlled, double-blind trial.

BACKGROUND: Acute respiratory distress syndrome (ARDS) is a complex clinical diagnosis with various possible etiologies. One common feature, however, is pulmonary permeability edema, which leads to an increased alveolar diffusion pathway and, subsequently, impaired oxygenation and decarboxylation. A novel inhaled peptide agent (AP301, solnatide) was shown to markedly reduce pulmonary edema in animal models of ARDS and to be safe to administer to healthy humans in a Phase I clinical trial. Here, we present the protocol for a Phase IIB clinical trial investigating the safety and possible future efficacy endpoints in ARDS patients. METHODS: This is a randomized, placebo-controlled, double-blind intervention study. Patients with moderate to severe ARDS in need of mechanical ventilation will be randomized to parallel groups receiving escalating doses of solnatide or placebo, respectively. Before advancing to a higher dose, a data safety monitoring board will investigate the data from previous patients for any indication of patient safety violations. The intervention (application of the investigational drug) takes places twice daily over the course of 7 days, ensued by a follow-up period of another 21 days. DISCUSSION: The patients to be included in this trial will be severely sick and in need of mechanical ventilation. The amount of data to be collected upon screening and during the course of the intervention phase is substantial and the potential timeframe for inclusion of any given patient is short. However, when prepared properly, adherence to this protocol will make for the acquisition of reliable data. Particular diligence needs to be exercised with respect to informed consent, because eligible patients will most likely be comatose and/or deeply sedated at the time of inclusion. TRIAL REGISTRATION: This trial was prospectively registered with the EU Clinical trials register (clinicaltrialsregister.eu). EudraCT Number: 2017-003855-47 .

Oxygen conditions oscillating between hypoxia and hyperoxia induce different effects in the pulmonary endothelium compared to constant oxygen conditions.

The pulmonary endothelium is an immediate recipient of high oxygen concentrations upon oxygen therapy and mediates down-stream responses. Cyclic collapse and reopening of atelectatic lung areas during mechanical ventilation with high fractions of inspired oxygen result in the propagation of oxygen oscillations in the hypoxic/hyperoxic range. We used primary murine lung endothelial cell cultures to investigate cell responses to constant and oscillating oxygen conditions in the hypoxic to hyperoxic range. Severe constant hyperoxia had pro-inflammatory and cytotoxic effects including an increase in expression of ICAM1, E-selectin, and RAGE at 24 hr exposure. The coagulative/fibrinolytic system responded by upregulation of uPA, tPA, and vWF and PAI1 under constant severe hyperoxia. Among antioxidant enzymes, the upregulation of SOD2, TXN1, TXNRD3, GPX1, and Gstp1 at 24 hr, but downregulation of SOD3 at 72 hr constant hyperoxia was evident. Hypoxic/hyperoxic oscillating oxygen conditions induced pro-inflammatory cytokine release to a lesser extent and later than constant hyperoxia. Gene expression analyses showed upregulation of NFKB p65 mRNA at 72 hr. More evident was a biphasic response of NOS3 and ACE1 gene expression (downregulation until 24 hr and upregulation at 72 hr). ACE2 mRNA was upregulated until 72 hr, but shedding of the mature protein from the cell surface favored ACE1. Oscillations resulted in severe production of peroxynitrite, but apart from upregulation of Gstp1 at 24 hr responses of antioxidative proteins were less pronounced than under constant hyperoxia. Oscillating oxygen in the hypoxic/hyperoxic range has a characteristical impact on vasoactive mediators like NOS3 and on the activation of the renin-angiotensin system in the lung endothelium.

The Renin-Angiotensin System as a Component of Biotrauma in Acute Respiratory Distress Syndrome.

Acute respiratory distress syndrome (ARDS) is a major concern in critical care medicine with a high mortality of over 30%. Injury to the lungs is caused not only by underlying pathological conditions such as pneumonia, sepsis, or trauma, but also by ventilator-induced lung injury (VILI) resulting from high positive pressure levels and a high inspiratory oxygen fraction. Apart from mechanical factors that stress the lungs with a specific physical power and cause volutrauma and barotrauma, it is increasingly recognized that lung injury is further aggravated by biological mediators. The COVID-19 pandemic has led to increased interest in the role of the renin-angiotensin system (RAS) in the context of ARDS, as the RAS enzyme angiotensin-converting enzyme 2 serves as the primary cell entry receptor for severe acute respiratory syndrome (SARS) coronavirus (CoV)-2. Even before this pandemic, studies have documented the involvement of the RAS in VILI and its dysregulation in clinical ARDS. In recent years, analytical tools for RAS investigation have made major advances based on the optimized precision and detail of mass spectrometry. Given that many clinical trials with pharmacological interventions in ARDS were negative, RAS-modifying drugs may represent an interesting starting point for novel therapeutic approaches. Results from animal models have highlighted the potential of RAS-modifying drugs to prevent VILI or treat ARDS. While these drugs have beneficial pulmonary effects, the best targets and application forms for intervention still have to be determined to avoid negative effects on the circulation in clinical settings.

Understanding Cellular Redox Homeostasis: A Challenge for Precision Medicine.

Living organisms use a large repertoire of anabolic and catabolic reactions to maintain their physiological body functions, many of which include oxidation and reduction of substrates. The scientific field of redox biology tries to understand how redox homeostasis is regulated and maintained and which mechanisms are derailed in diverse pathological developments of diseases, where oxidative or reductive stress is an issue. The term "oxidative stress" is defined as an imbalance between the generation of oxidants and the local antioxidative defense. Key mediators of oxidative stress are reactive species derived from oxygen, nitrogen, and sulfur that are signal factors at physiological concentrations but can damage cellular macromolecules when they accumulate. However, therapeutical targeting of oxidative stress in disease has proven more difficult than previously expected. Major reasons for this are the very delicate cellular redox systems that differ in the subcellular compartments with regard to their concentrations and depending on the physiological or pathological status of cells and organelles (i.e., circadian rhythm, cell cycle, metabolic need, disease stadium). As reactive species are used as signaling molecules, non-targeted broad-spectrum antioxidants in many cases will fail their therapeutic aim. Precision medicine is called to remedy the situation.

Treatment of primary graft dysfunction after lung transplantation with orally inhaled AP301: A prospective, randomized pilot study.

BACKGROUND: Primary graft dysfunction (PGD) after lung transplantation (LTx) carries significant morbidity and mortality in the early post-operative period and is associated with the development of chronic lung allograft dysfunction. AP301, an activator of epithelial sodium channel-mediated Na+ uptake represents a new concept for prevention and treatment of pulmonary edema and has shown promising results in the pre-clinical setting. This pilot study investigated the clinical effect of inhaled AP301 on patients with development of PGD > 1 according to International Society of Heart and Lung Transplantation criteria after primary LTx in a high-volume center and was conducted as a randomized, placebo-controlled, single-center pilot-study including 20 patients. All consecutive patients fulfilling inclusion criteria were screened for PGD at arrival on the intensive care unit (ICU) after LTx. After randomization, inhaled AP301 or placebo was administered by nebulizer twice daily for 7 days or until extubation. Otherwise, patients were treated according to routine clinical protocol. Partial pressure of arterial oxygen (Pao2)/fraction of inspired oxygen (Fio2) values were obtained until extubation and assessed as a primary outcome parameter. Patients were monitored for 30 days within the study protocol. RESULTS: From July 2013 to August 2014, 20 patients were randomized 1:1 to AP301 (Group 1) or placebo (Group 2). Both groups were comparable with regard to sex (40% women/60% men vs 50% women/50% men), mean age (55 ± 13 vs 54 ± 6 years), comorbidities, and diagnosis leading to LTx. The Pao2/Fio2 ratio at the time of inclusion was comparable in both groups, with a mean 235.65 ± 90.78 vs 214.2 ± 95.84 (p = 0.405), and there was no significant difference in the extravascular lung water index (13.88 ± 5.28 vs 16 ± 6.29 ml/kg, p = 0.476). The primary end point was mean Pao2/Fio2 ratio values between baseline and Day 3. In the AP301 group, only 1 patient was ventilated at Day 4 and no patients were ventilated after Day 4. In the placebo group, 5 patients were ventilated on Day 4 and 2 patients on Days 5, 6, and 7. The mean increase in the Pao2/Fio2 ratio was significantly higher in Group 1 patients, and the mean between baseline and at 72 hours was 365.6 ± 90.4 in Group 1 vs 335.2 ± 42.3 in Group 2 (p = 0.049). The duration of intubation was shorter in Group 1 than in Group 2 patients (2 ± 0.82 vs 3.7 ± 1.95 days; p = 0.02). ICU stay was 7.5 ± 3.13 days in Group 1 vs 10.8 ± 8.65 days in group 2 (p = 0.57). Survival at 30 days was 100%. No severe adverse events were recorded. CONCLUSIONS: This study was designed as a proof-of-concept pilot study. Although it was not powered to achieve statistical significances, the study demonstrated relevant clinical effects of inhaled AP301 on patients with PGD after primary LTx. The improved gas exchange led to a significantly shorter duration of mechanical ventilation and a trend towards a shorter ICU stay. Further investigation of AP301 for treatment of PGD in larger studies is warranted. TRIAL REGISTRATION: The trial is registered at https://www.clinicaltrialsregister.eu/ctr-search/trial/2013-000716-21/AT.

Inhaled AP301 for treatment of pulmonary edema in mechanically ventilated patients with acute respiratory distress syndrome: a phase IIa randomized placebo-controlled trial.

BACKGROUND: High-permeability pulmonary edema is a hallmark of acute respiratory distress syndrome (ARDS) and is frequently accompanied by impaired alveolar fluid clearance (AFC). AP301 enhances AFC by activating epithelial sodium channels (ENaCs) on alveolar epithelial cells, and we investigated its effect on extravascular lung water index (EVLWI) in mechanically ventilated patients with ARDS. METHODS: Forty adult mechanically ventilated patients with ARDS were included in a randomized, double-blind, placebo-controlled trial for proof of concept. Patients were treated with inhaled AP301 (n = 20) or placebo (0.9% NaCl; n = 20) twice daily for 7 days. EVLWI was measured by thermodilution (PiCCO®), and treatment groups were compared using the nonparametric Mann-Whitney U test. RESULTS: AP301 inhalation was well tolerated. No differences in mean baseline-adjusted change in EVLWI from screening to day 7 were found between the AP301 and placebo group (p = 0.196). There was no difference in the PaO2/FiO2 ratio, ventilation pressures, Murray lung injury score, or 28-day mortality between the treatment groups. An exploratory subgroup analysis according to severity of illness showed reductions in EVLWI (p = 0.04) and ventilation pressures (p < 0.05) over 7 days in patients with initial sequential organ failure assessment (SOFA) scores ≥11 inhaling AP301 versus placebo, but not in patients with SOFA scores ≤10. CONCLUSIONS: There was no difference in mean baseline-adjusted EVLWI between the AP301 and placebo group. An exploratory post-hoc subgroup analysis indicated reduced EVLWI in patients with SOFA scores ≥11 receiving AP301. These results suggest further confirmation in future clinical trials of inhaled AP301 for treatment of pulmonary edema in patients with ARDS. TRIAL REGISTRATION: The study was prospectively registered at clinicaltrials.gov, NCT01627613 . Registered 20 June 2012.

Effects of azithromycin and tanomastat on experimental bronchiolitis obliterans.

OBJECTIVE: Azithromycin has become a standard of care in therapy of bronchiolitis obliterans following lung transplantation. Matrix metalloprotease-9 broncho-alveolar lavage levels increase in airway neutrophilia and bronchiolitis obliterans. Interleukin-17 may play a role in lung allograft rejection, and interleukin-12 is downregulated in bronchiolitis obliterans. Whether these mechanisms can be targeted by azithromycin remains unclear. METHODS: Bronchiolitis obliterans was induced by transplantation of Fischer F344 rat left lungs to Wistar Kyoto rats. Allografts with azithromycin therapy from day 1 to 28 or 56 and mono- or combination therapy with the broad-spectrum matrix metalloprotease inhibitor tanomastat from day 1 to 56 were compared to control allografts and isografts. Graft histology was assessed, and tissue cytokine expression studied using Western blotting and immunofluorescence. RESULTS: The chronic airway rejection score in the azithromycin group did not change between 4 and 8 weeks after transplantation, whereas it significantly worsened in control allografts (P = .041). Azithromycin+tanomastat prevented complete allograft fibrosis, which occurred in 40% of control allografts. Azithromycin reduced interleukin-17 expression (P = .049) and the number of IL-17(+)/CD8(+) lymphocytes at 4 weeks, and active matrix metalloprotease-9 at 8 weeks (P = .017), and increased interleukin-12 expression (P = .025) at 8 weeks following transplantation versus control allografts. CONCLUSIONS: The expression of interleukin-17 and matrix metalloprotease-9 in bronchiolitis obliterans may be attenuated by azithromycin, and the decrease in interleukin-12 expression was prevented by azithromycin. Combination of azithromycin with a matrix metalloprotease inhibitor is worth studying further because it prevented complete allograft fibrosis in this study.

CD19(+)CD21(low) B cells and patients at risk for NIH-defined chronic graft-versus-host disease with bronchiolitis obliterans syndrome.

Bronchiolitis obliterans syndrome (BOS), pathognomonic for chronic graft-versus-host disease (cGVHD) of the lung, is a progressive and often fatal complication after allogeneic hematopoietic cell transplantation (HCT). Biomarkers for the prediction and diagnosis of BOS are urgently needed to improve patients' prognosis. We prospectively evaluated B-cell subpopulations and B-cell activating factor (BAFF) in 136 patients (46 BOS, 41 no cGVHD, 49 cutaneous cGVHD) to define novel biomarkers for early diagnosis of National Institutes of Health-defined BOS diagnosed a median of 11 mo after HCT. Patients with newly diagnosed BOS had significantly higher percentages of CD19(+)CD21(low) B cells (25.5 versus 6.6%, P < .0001), BAFF (7.3 versus 3.5 ng/mL, P = .02), and BAFF/CD19(+) ratio (0.18 versus 0.02 ng/10(3) CD19(+) B cells, P 5 .007) compared with patients without cGVHD. The area under the receiver operating curve for CD19(+)CD21(low) B cells was 0.97 (95% confidence interval, 0.94-0.99) and a cutoff point >9% was optimal for diagnosing BOS in patients with first drop of pulmonary function tests with a sensitivity of 96% and a negative predictive value of 94%. Thus, elevated levels of CD19(+)CD21(low) B cells are a potential novel biomarker for HCT patients at risk for developing BOS at an early stage and could allow improvement of patient outcome.

Persistent plasminogen activator inhibitor 1 gene expression in cardiac transplant recipients with idiopathic dilated cardiomyopathy.

OBJECTIVES: Plasminogen activator inhibitor 1 is the primary regulator of urokinase plasminogen activator and tissue plasminogen activator. Plasminogen activator inhibitor 1 is essential in the control of the thrombotic/fibrinolytic balance and is a marker of endothelial cell injury. Idiopathic dilated cardiomyopathy is reportedly associated with endothelial cell dysfunction. Whether endothelial cell damage plays a role in patients with dilated cardiomyopathy after cardiac transplantation remains unknown. METHODS: In this study explanted hearts of cardiac transplant recipients with ischemic cardiomyopathy and dilated cardiomyopathy, as well as control myocardial tissue, were investigated for expression of urokinase plasminogen activator, tissue plasminogen activator, urokinase plasminogen activator receptor, and plasminogen activator inhibitor 1 and 2. Furthermore, plasminogen activator inhibitor 1 expression was examined in endomyocardial biopsy specimens and sera of patients with ischemic cardiomyopathy and those with dilated cardiomyopathy during the first posttransplantation year. The effect of the patient's serum on endothelial cells was assessed in vitro to examine the role of circulating endothelial cell damage-related factors. RESULTS: Plasminogen activator inhibitor 1 expression was upregulated in ischemic cardiomyopathy and dilated cardiomyopathy myocardial tissue versus that seen in control tissue. After transplantation, plasminogen activator inhibitor 1 expression returned to control levels in patients with ischemic cardiomyopathy. In patients with dilated cardiomyopathy, plasminogen activator inhibitor 1 expression increased at 24 weeks after transplantation in both biopsy specimens and sera versus that seen in control tissue. Sera of patients with dilated cardiomyopathy, but not that of patients with ischemic cardiomyopathy, inhibited vascular endothelial growth factor A-induced proliferation of endothelial cells, although downstream target gene activation of early growth response factor 1 and NGFI-A binding protein 2 was not affected. CONCLUSIONS: These data suggest for the first time that the endothelial cell damage-related process recurs in patients with dilated cardiomyopathy after transplantation, which, independently of vascular endothelial growth factor, is associated with increased plasminogen activator inhibitor 1 expression, and that this pathology might play a role in allograft remodeling in patients with dilated cardiomyopathy.

Apollon/RNF41 myocardial messenger RNA diagnoses cardiac allograft apoptosis in rejection.

BACKGROUND: Endomyocardial biopsy (EMB) remains the gold standard for acute cellular rejection (ACR) diagnosis in cardiac transplantation yet is subject to interobserver variability. A method that could avoid discordant EMB analysis would be desirable. The apoptosis rate in EMB correlates with ACR severity. Apollon inhibits apoptosis, and RNF41 catalyzes its degradation. Whether tissue Apollon/RNF41 could diagnose ACR is not known. This study addressed this issue. METHODS: Apollon/RNF41 messenger RNA (mRNA) was measured by real time reverse-transcriptase polymerase chain reaction and apoptosis was quantified with TUNEL assays in EMBs of 268 transplant recipients. EMBs were obtained at 1, 2, 3, 4, 7, 12, 24, and 52 posttransplant weeks. RESULTS: At all time points posttransplant, Apollon mRNA decreased significantly in EMBs with ACR grades 2R/3R combined (P

Donor serum SMARCAL1 concentrations predict primary graft dysfunction in cardiac transplantation.

BACKGROUND: Primary graft dysfunction (PGD) is a life-threatening complication in cardiac transplantation. A sensitive, specific, and easily measurable predictor in donors could facilitate PGD prevention. METHODS AND RESULTS: SMARCAL1 is a matrix-associated regulator of chromatin with helicase and ATPase activities, and its serum concentrations were significantly increased in a targeted protein array in donors whose grafts developed PGD. Therefore, this study analyzed SMARCAL1 serum concentrations by ELISA in 336 heart donors before and after aortic cross-clamping (ACC) and in recipients at 10, 30, and 60 minutes reperfusion. Demographic and hemodynamic parameters of donors and recipients as well as transplant procedure characteristics were documented. PGD (n=68) was defined as ventricular dilation and hypocontractility associated with systolic blood pressure <90 mm Hg, pulmonary capillary wedge pressure >20 mm Hg, and decreased mixed venous oxygen saturation necessitating mechanical circulatory support. SMARCAL1 serum protein concentration was significantly increased only before and after ACC in donors (P<0.0001) whose grafts developed PGD compared to those who did not. In receiver operating characteristic curve analysis, SMARCAL1 serum concentration at a cut-off level of > or =1.25 ng/mL before ACC in donors predicted PGD (P<0.0001, AUC=0.988, OR=17.050, 95% CI=5.200 to 55.901) with 96% sensitivity and 88% specificity. SMARCAL1 serum concentrations <1.25 ng/mL in donors before ACC resulted in 97% PGD-free outcome and SMARCAL1 concentrations > or =1.25 resulted in 83% PGD occurrence. CONCLUSIONS: Donor serum SMARCAL1 may serve as a specific, sensitive, and noninvasive predictive marker in the assessment of cardiac graft quality.

Vascular endothelial growth factor increases pulmonary vascular permeability in cystic fibrosis patients undergoing lung transplantation.

OBJECTIVE: Vascular endothelial growth factor (VEGF) is the prime regulator of angiogenesis and vascular permeability and its serum levels increase in cystic fibrosis (CF). The mechanisms of VEGF overproduction and its impact on CF lung pathology and pulmonary vascular permeability during lung transplantation are not fully understood. METHODS: The expression of VEGF, its receptors, hypoxia inducible factor (HIF)-1alpha, beta, angiopoietins, and endothelial cell marker CD31 were studied in lung biopsies of CF and COPD patients and controls, using real time reverse transcription (RT)-PCR and Western blotting. DNA binding activity of HIF-1 to VEGF-A promoter was assessed by electrophoretic mobility shift assay (EMSA) and wet-to-dry lung weight ratios as well as microvascular density (MVD) were determined. Serum VEGF-A concentrations in enzyme-linked immunosorbent assay (ELISA) and wet-to-dry weight ratios of donor lungs were monitored during transplantation in CF and COPD patients. Primary graft dysfunction (PGD) was diagnosed and graded according to the guidelines of the International Society for Heart and Lung Transplantation. RESULTS: VEGF-A165 and Flt-1 mRNA expression (P<0.05), VEGF-A (P<0.05), and HIF-1alpha (P<0.05) protein levels, DNA binding activity of HIF-1 to VEGF promoter (P<0.001) and extravascular lung water content (P<0.05) were increased in CF lungs versus controls, whereas MVD was unchanged. Before and during lung transplantation, VEGF-A serum concentrations were higher in CF versus COPD patients (P<0.05) and 60 min following reperfusion donor lungs transplanted to CF patients had higher tissue water contents than in COPD patients (P<0.05). PGD grade 3 occurred more frequently in CF (22.7%) versus COPD patients (4%). PGD grade 3 patients had significantly higher VEGF serum concentrations versus PGD grade 0-2 patients (P<0.001). CONCLUSIONS: These data indicate that upregulated VEGF-A levels are most likely induced by enhanced HIF-1 binding to VEGF-A promoter, possibly contributing to elevated serum VEGF-A levels in CF. Furthermore, CF patients undergoing lung transplantation are possibly more susceptible to PGD because of increased VEGF-A expression that mediates increased lung graft vascular permeability.