The role of Angiopoietin-2 in the development of acute lung failure in severe pneumococcal pneumonia

Schwere bakterielle Pneumonien führen häufig zu einer Schädigung der Lunge. Ein bedeutender Erreger einer schweren Pneumonie ist das grampositive Bakterium Streptococcus pneumoniae. Im Verlauf einer Pneumokokkenpneumonie kann es durch direkten Einfluss von Pathogenitätsfaktoren sowie der körpereigenen Abwehrreaktion zu einer erhöhten endothelialen Permeabilität und einem pulmonalen Barriereversagen kommen, welches in schweren Fällen in ein akutes Lungenversagen münden kann. Angiopoietin-2 ist ein körpereigener Ligand des endothelial vorkommenden Tyrosinkinase-Rezeptors Tie-2. Das Protein hat einen bekannten proinflammatorischen und destabilisierenden Einfluss auf das Gefäßsystem und wird nach einer Stimulation mit Pneumolysin, einem Virulenzfaktor von Streptococcus pneumoniae, vermehrt vom pulmonalen Endothel ausgeschüttet. Die Bedeutung von Angiopoietin-2 in der schweren Pneumokokkenpneumonie ist noch unbekannt. Ziel der vorliegenden Doktorarbeit war es, den Einfluss von Angiopoietin-2 auf das inflammatorische Geschehen und die pulmonale Barrierestabilität in der schweren Pneumokokkenpneumonie mit einhergehendem akuten Lungenversagen näher zu untersuchen. Hierzu wurde ein konditionelles Angiopoietin-2-Knockout-Mausmodell verwendet. Der Knockout der gezüchteten Angiopoietin-2-loxP/loxP-Cre-ERT2 Mauslinie wurde in vivo mit Tamoxifen und in vitro mit 4 -Hydroxytamoxifen induziert. In anschließenden Versuchen wurde die Effizienz des induzierten Angiopoietin-2-Knockout untersucht. Hierbei zeigten die Angiopoietin-2-Knockout-Mäuse eine lückenhafte Gendeletion. Die induzierten Mäuse wiesen im Vergleich zu ihren nicht induzierbaren Geschwistertieren, welche als Kontrolle dienten, zwar eine deutlich verringerte Expression von Angiopoietin-2 auf, eine vollständige Deletion des Proteins konnte jedoch nicht erzielt werden. In vitro bestätigten die erhobenen Daten die bereits bekannte destabilisierende Wirkung von Angiopoietin-2 auf das pulmonale Endothel. Primäre murine pulmonale Endothelzellen von Angiopoietin-2-Knockout-Mäusen wiesen nach einer Stimulation mit Pneumolysin im Vergleich zu pulmonalen Endothelzellen von Kontrollmäusen einen geringeren Integritätsverlust des Zellmonolayers auf. In ergänzenden in vivo Studien zeigten die mit Streptococcus pneumoniae infizierten Angiopoietin-2-Knockout-Mäuse einen verringerten Angiopoietin-2-Plasmaspiegel als die Kontrollmäuse. Ein Unterschied im inflammatorischen Geschehen sowie der alveolokapillären Barierrefunktion war zwischen den Gruppen jedoch nicht ersichtlich, so dass die protektive Wirkung eines Angiopoietin-2-Knockout in der Pneumokokkenpneumonie in diesem Versuchssetting nicht bestätigt werden konnte. Gründe für die abweichenden Ergebnisse der in vitro und in vivo Studie könnten in der verwendeten Mauslinie, deren unvollständigen Induzierbarkeit, sowie im Versuchsdesign liegen. Die Untersuchung des Proteins auch im Hinblick auf die Notwendigkeit neuer innovativer Therapiestrategien bei einem Pneumonie-induzierten akuten Lungenversagen bleibt eine bedeutende Fragestellung. Neben der Erprobung weiterer Tamoxifen-Induktionsprotokolle zum Erreichen einer effektiveren Ang-2 Deletion, oder der Verwendung eines anderen in vivo Versuchdesigns (früherer Analysezeitpunkt oder Einsatz einer Antibiotikabehandlung), sollte die Verwendung einer anderen Mauslinie zur Erforschung des Einflusses von Angiopoietin-2 im Verlauf einer Pneumokokkenpneumonie in Betracht gezogen werden.Severe bacterial pneumonia frequently leads to permanent lung damage. A significant pathogen of severe pneumonia is the gram-positive bacterium Streptococcus pneumoniae. In the course of pneumococcal pneumonia, both the pathogen itself and the inflammatory response to the pathogen can lead to increased endothelial permeability and pulmonary barrier failure finally leading to life-threatening respiratory failure. Angiopoietin-2 is an endogenous ligand of the tyrosine kinase receptor Tie-2. Angiopoietin-2 has known pro-inflammatory and destabilizing effects on the vascular system and is increasingly released from the pulmonary endothelium following stimulation with pneumolysin, a virulence factor of Streptococcus pneumoniae. However, the precise role of Angiopoietin-2 in severe pneumococcal pneumonia is still unclear. The goal of this study was to elucidate the influence of Angiopoietin-2 on inflammatory processes and alveolar-capillary barrier integrity in severe pneumococcal pneumonia with acute respiratory failure. Therefore, a conditional Angiopoietin-2-knockout mouse model was used. The knockout was induced in vivo in the Angiopoietin-2-loxP/loxP-Cre-ERT2-line of mice using Tamoxifen and in vitro using 4-Hydroxytamoxifen. In subsequent experiments, the efficiency of the Angiopoietin-2-knockout was determined. The Angiopoietin-2-knockout showed an incomplete gene deletion of Angiopoietin-2. Compared with their non-inducible siblings, which served as controls, the induced mice showed a reduced expression of angiopoietin-2, but a complete deletion of the protein could not be achieved. In vivo data confirmed previously established findings that Angiopoietin-2 has a destabilizing effect on the pulmonary endothelium. Pulmonary endothelial cells from Angiopoietin-2-knockout mice showed reduced loss of integrity in their cell monolayers following stimulation with pneumolysin when compared to controls. Further in vivo studies also showed reduced Angiopoietin-2 plasma levels in Streptococcus pneumoniae infected Angiopoietin-2-knockout mice compared to their controls. No differences were observed between the groups in markers of the inflammatory process or in the state of the alveolar-capillary barrier. Therefore, a protective effect of Angiopoietin-2-knockout in pneumococcal pneumonia could not be demonstrated in this experiment with regard to these parameters. The discrepancies in findings between the in vitro and in vivo data could be due to the inherent variability between the mice themselves, its incomplete inducibility but also to the study design. Further studies to investigate the significance of this protein for the establishment of new, innovative therapeutic strategies for pneumonia-associated lung failure should be considered. In addition to investigating additional Tamoxifen induction protocols to achieve more effective Ang-2 deletion, or designing another in vivo study (e.g., regarding earlier analysis or initiation of antibiotic treatment), the use of another mouse line to investigate the role of Antiopoietin-2 in the course of pneumococcal pneumonia should be considered

Prognostic and Pathogenic Role of Angiopoietin-1 and -2 in Pneumonia

RATIONALE: During pneumonia, pathogen-host interaction evokes inflammation and lung barrier dysfunction. Tie2 activation by angiopoietin-1 reduces, whereas Tie2 blockade by angiopoietin-2 increases, inflammation and permeability during sepsis. The role of angiopoietin-1/-2 in pneumonia remains unidentified. OBJECTIVES: To investigate the prognostic and pathogenic impact of angiopoietins in regulating pulmonary vascular barrier function and inflammation in bacterial pneumonia. METHODS: Serum angiopoietin levels were quantified in pneumonia patients of two independent cohorts (n\u2009=\u2009148, n\u2009=\u2009395). Human postmortem lung tissue, pneumolysin- or angiopoietin-2-stimulated endothelial cells, isolated perfused and ventilated mouse lungs, and mice with pneumococcal pneumonia were investigated. MEASUREMENTS AND MAIN RESULTS: In patients with pneumonia, decreased serum angiopoietin-1 and increased angiopoietin-2 levels were observed as compared with healthy subjects. Higher angiopoietin-2 serum levels were found in patients with community-acquired pneumonia who died within 28 days of diagnosis compared with survivors. Receiver operating characteristic analysis revealed improved prognostic accuracy of CURB-65 for 28-day survival, intensive care treatment, and length of hospital stay if combined with angiopoietin-2 serum levels. In vitro, pneumolysin enhanced endothelial angiopoietin-2 release, angiopoietin-2 increased endothelial permeability, and angiopoietin-1 reduced pneumolysin-evoked endothelial permeability. Ventilated and perfused lungs of mice with angiopoietin-2 knockdown showed reduced permeability on pneumolysin stimulation. Increased pulmonary angiopoietin-2 and reduced angiopoietin-1 mRNA expression were observed in Streptococcus pneumoniae-infected mice. Finally, angiopoietin-1 therapy reduced inflammation and permeability in murine pneumonia. CONCLUSIONS: These data suggest a central role of angiopoietin-1/-2 in pneumonia-evoked inflammation and permeability. Increased angiopoietin-2 serum levels predicted mortality and length of hospital stay, and angiopoietin-1 may provide a therapeutic target for severe pneumonia

Vasculotide reduces pulmonary hyperpermeability in experimental pneumococcal pneumonia

Abstract Background Community-acquired pneumonia (CAP) is a significant cause of morbidity and mortality worldwide. Despite effective antimicrobial therapy, CAP can induce pulmonary endothelial hyperpermeability resulting in life-threatening lung failure due to an exaggerated host-pathogen interaction. Treatment of acute lung injury is mainly supportive because key elements of inflammation-induced barrier disruption remain undetermined. Angiopoietin-1 (Ang-1)-mediated Tie2 activation reduces, and the Ang-1 antagonist Ang-2 increases, inflammation and endothelial permeability in sepsis. Vasculotide (VT) is a polyethylene glycol-clustered Tie2-binding peptide that mimics the actions of Ang-1. The aim of our study was to experimentally test whether VT is capable of diminishing pneumonia-induced lung injury. Methods VT binding and phosphorylation of Tie2 were analyzed using tryptophan fluorescence spectroscopy and phospho-Tie-2 enzyme-linked immunosorbent assay. Human and murine lung endothelial cells were investigated by immunofluorescence staining and electric cell-substrate impedance sensing. Pulmonary hyperpermeability was quantified in VT-pretreated, isolated, perfused, and ventilated mouse lungs stimulated with the pneumococcal exotoxin pneumolysin (PLY). Furthermore, Streptococcus pneumoniae-infected mice were therapeutically treated with VT. Results VT showed dose-dependent binding and phosphorylation of Tie2. Pretreatment with VT protected lung endothelial cell monolayers from PLY-induced disruption. In isolated mouse lungs, VT decreased PLY-induced pulmonary permeability. Likewise, therapeutic treatment with VT of S. pneumoniae-infected mice significantly reduced pneumonia-induced hyperpermeability. However, effects by VT on the pulmonary or systemic inflammatory response were not observed. Conclusions VT promoted pulmonary endothelial stability and reduced lung permeability in different models of pneumococcal pneumonia. Thus, VT may provide a novel therapeutic perspective for reduction of permeability in pneumococcal pneumonia-induced lung injury

Additional file 1: Figures S1–S6. of Vasculotide reduces pulmonary hyperpermeability in experimental pneumococcal pneumonia

Supplementary figures. (PDF 1144 kb