Abstract Community acquired pneumonia, mainly caused by Streptococcus pneumoniae ( S.pn.), is a common cause of death worldwide. Despite adequate antibiotic therapy, pneumococcal pneumonia can induce pulmonary endothelial hyperpermeability leading to acute lung injury, which often requires mechanical ventilation (MV) causing ventilator-induced lung injury (VILI). Endothelial stabilization is mediated by angiopoietin-1 induced Tie2 activation. PEGylated (polyethylene glycol) Tie2-agonist Vasculotide (VT) mimics Angiopietin-1 effects. Recently, VT has been shown to reduce pulmonary hyperpermeability in murine pneumococcal pneumonia. The aim of this study was to determine whether VT reduces lung damage in S.pn. infected and mechanically ventilated mice. Pulmonary hyperpermeability, immune response and bacterial load were quantified in S.pn. infected mice treated with Ampicillin + /−VT and undergoing six hours of MV 24 h post infection. Histopathological lung changes, Tie2-expression and -phosphorylation were evaluated. VT did not alter immune response or bacterial burden, but interestingly combination treatment with ampicillin significantly reduced pulmonary hyperpermeability, histological lung damage and edema formation. Tie2-mRNA expression was reduced by S.pn. infection and/or MV but not restored by VT. Moreover, Tie2 phosphorylation was not affected by VT. These findings indicate that VT may be a promising adjunctive treatment option for prevention of VILI in severe pneumococcal pneumonia.
Abstract Inflammation and airway remodeling are features associated with chronic asthmatic lungs. Previous reports have shown the benefits of the angiopoietin-1/Tie2 signaling axis on acute experimentally-induced asthma in mice. However, the more chronic effects as well as the interaction between the inflammatory and vascular components is poorly. Thus, we evaluated the impact of vasculotide, a synthetic Tie2 agonist in a chronic model of asthma. During the chronic allergic response in mice, we show that in addition to endothelial Tie2 receptor expression, there is an increased proportion of dendritic cells and monocytes expressing Tie2. Treatment of experimentally-induced chronic airway inflammation in mice with vasculotide correlated with a skewed CD4+ TH subsets and cytokine profile in the bronchoalveolar lavage of animals. This modified cytokine milieu promoted an altered endothelial cell response characterized by reduced expression of CD62E, CD54 and CD106 on endothelial cells, decreased cellular migration of inflammatory cells to the lung as well as reduced mucus accumulation. Analysis of lung tissues of animals treated with vasculotide revealed a decrease in collagen deposition and thinning of the smooth muscle cell layer in the airways of mice with chronic allergic airway inflammation. Our results demonstrate that vasculotide can impact the inflammatory and remodeling components in chronic asthma through its action on both cells of the immune response and on endothelial cells.
Earlier studies by our group have demonstrated that a transgenic animal engineered to express Tie2 under the control of the Tie2 promoter produced animals with a scaly skin phenotype that recapitulated many of the hallmarks of atopic dermatitis (AT-Derm). To test the hypothesis that this model of AT-Derm is driven by dysregulated Tie2-signalling, we have bred AT-Derm transgenic (TG) animals with TG-animals engineered to overexpress Angiopoietin-1 or -2, the cognate Tie2 ligands. These two ligands act to antagonize one another in a context-dependent manner. To further evaluate the role of Ang1-driven-Tie2 signalling, we examined the ability of Vasculotide, an Ang1-mimetic, to modulate the AT-Derm phenotype. AT-Derm+Ang2 animals exhibited an accentuated phenotype, whereas AT-Derm+Ang1 presented with a markedly reduced skin disease, similarly VT-treated AT-Derm animals present with a clear decrease in the skin phenotype. Moreover, a decrease in several important inflammatory cytokines and a decrease in the number of eosinophils was noted in VT-treated animals. Bone marrow differentiation in the presence of VT produced fewer CFU-G colonies, further supporting a role for Tie2-signalling in eosinophil development. Importantly, we demonstrate activation of Tie2, the VT-target, in lung tissue from naïve animals treated with increasing amounts of VT. The AT-Derm phenotype in these animals is driven through dysregulation of Tie2 receptor signalling and is augmented by supplemental Ang2-dependent stimulation. Overexpression of Ang1 or treatment with VT produced a similar amelioration of the phenotype supporting the contention that VT and Ang1 have a similar mechanism of action on the Tie2 receptor and can both counteract the signalling driven by Ang2. Our results also support a possible role for Tie2-signalling in the development of eosinophilic diseases and that activation of Tie2 may directly or indirectly modulate the differentiation of eosinophils, which express Tie2. In summary, these data support the hypothesis that this AT-Derm mouse model is driven by dysregulation of the Tie2 signalling pathway and increased Ang2 levels can aggravate it, whereas it can be reversed by either Ang1-overexpression or VT treatment. Moreover, our data supports the contention that VT acts as an Angiopoietin-1 mimetic and may provide a novel entry point for Tie2-agonist-based therapies for atopic diseases.
Introduction: Community-acquired pneumonia (CAP) is a significant cause of morbidity and mortality worldwide. Streptococcus pneumoniae (S.p.) is the most frequent causal pathogen identified in CAP.Despite efficient antimicrobial therapy, CAP can induce pulmonary endothelial hyperpermeability resulting in life-threatening lung failure due to an exaggerated host-pathogen interaction. Angiopoietin- (Ang-)1 mediated Tie2-activation reduces and the Ang-1 antagonist Ang-2 increases inflammation and endothelial permeability in sepsis. "Vasculotide" (VT) is a PEGylated (polyethylene glycol) Tie2-agonist mimicking Ang-1. The aim of our study was to investigate experimentally whether VT could diminish pneumonia-induced lung injury. Methods: Human and murine lung endothelial cells were investigated by electrical cell-substrate impedance sensing (ECIS) and immunofluorescence staining. Pulmonary hyperpermeability was quantified in VT-pretreated isolated perfused and ventilated mouse lungs stimulated with the pneumococcal exotoxin pneumolysin (PLY). Also, S.p. infected mice were therapeutically treated with VT. Results: Pretreatment with VT protected lung endothelial cell-monolayers from PLY-induced disruption. Also, decreased PLY-induced pulmonary permeability of isolated mouse lungs was observed in the VT-treated group in comparison to the untreated group. Likewise, therapeutic treatment with VT of S.p. infected mice significantly reduced pneumonia-induced hyperpermeability. Conclusion: VT protected pulmonary endothelial integrity and reduced lung permeability in different models of pneumococcal pneumonia. Thus, VT may provide a novel perspective as adjunctive therapy in addition to antibotics. Support: DFG (SFB/TR84,C6).
Background Most cancer patients are treated with radiotherapy, but the treatment can also damage the surrounding normal tissue. Acute skin damage from cancer radiotherapy diminishes patients’ quality of life, yet effective biological interventions for this damage are lacking. Protecting microvascular endothelial cells from irradiation-induced perturbations is emerging as a targeted damage-reduction strategy. Since Angiopoetin-1 signaling through the Tie2 receptor on endothelial cells opposes microvascular perturbations in other disease contexts, we used a preclinical Angiopoietin-1 mimic called Vasculotide to investigate its effect on skin radiation toxicity using a preclinical model. Methods Athymic mice were treated intraperitoneally with saline or Vasculotide and their flank skin was irradiated with a single large dose of ionizing radiation. Acute cutaneous damage and wound healing were evaluated by clinical skin grading, histology and immunostaining. Diffuse reflectance optical spectroscopy, myeloperoxidase-dependent bioluminescence imaging of neutrophils and a serum cytokine array were used to assess inflammation. Microvascular endothelial cell response to radiation was tested with in vitro clonogenic and Matrigel tubule formation assays. Tumour xenograft growth delay experiments were also performed. Appreciable differences between treatment groups were assessed mainly using parametric and non-parametric statistical tests comparing areas under curves, followed by post-hoc comparisons. Results In vivo, different schedules of Vasculotide treatment reduced the size of the irradiation-induced wound. Although skin damage scores remained similar on individual days, Vasculotide administered post irradiation resulted in less skin damage overall. Vasculotide alleviated irradiation-induced inflammation in the form of reduced levels of oxygenated hemoglobin, myeloperoxidase bioluminescence and chemokine MIP-2. Surprisingly, Vasculotide-treated animals also had higher microvascular endothelial cell density in wound granulation tissue. In vitro, Vasculotide enhanced the survival and function of irradiated endothelial cells. Conclusions Vasculotide administration reduces acute skin radiation damage in mice, and may do so by affecting several biological processes. This radiation protection approach may have clinical impact for cancer radiotherapy patients by reducing the severity of their acute skin radiation damage.
Focused ultrasound (FUS), in presence of microbubbles, transiently induces blood-brain barrier (BBB) permeability to allow for minimally invasive delivery of therapeutics, to targeted areas of the brain. Restoration of the BBB following FUS occurs rapidly in healthy models, however, the primary clinical use would be in neurological disorders, such as Alzheimer's disease (AD), where BBB integrity may be compromised. The use of FUS to deliver AD therapeutics could have beneficial clinical implications, however, it is unknown whether the plasticity of the BBB and its capacity for repair is maintained in the presence of AD pathology. Furthermore, we sought to accelerate BBB restoration using Vasculotide (VT), a synthetic angiopoietin-1 mimetic that activates the Tie2 signaling cascade; a pathway known to promote vascular stability and cell survival in peripheral organs. Using a transgenic (Tg) mouse model of amyloidosis and their non-Tg littermates, VT was injected, every 48 hours for 2-3 months. We used FUS to induce transient increases in BBB permeability. The entry of gadolinium in the brain was monitored by MRI and quantified using MATLAB at 6, 12 and 20 hours post-FUS. Lastly, in a separate cohort of mice, Evans Blue dye was injected to evaluate BBB closure 24 hours post-FUS. VT significantly accelerated BBB restoration, with no significant difference between Tg and non-Tg mice. Furthermore, VT reduced the initial enhancement and acoustic pressure required to induce BBB permeability, with no difference between Tg and non-Tg mice. Finally, Evans Blue dye injected 24 hours post-FUS confirmed BBB closure. Our research is the first to examine the effects of VT on BBB permeability and provides a better understanding of the effects of FUS. This research presents a novel method of facilitating BBB closure that could lead to a VT pre-treatment with FUS delivery of AD therapeutics.
AIMTo investigate the therapeutic potential of vasculotide (VT) -a Tie2 activating therapeutic -in kidney transplantation. METHODSWe performed a murine MHC-mismatched renal transplant model (C57Bl/6 male into Balb/c female) with 60 min cold and 30 min warm ischemia time.500 ng VT was administered i.p. to donor mice 1 h before organ removal.In addition, recipients received 500 ng VT i.p. directly and 3 d after surgery.Survival was monitored and remaining animals were sacrificed 28 d after transplantation.In this model, we analyzed: (1) organ function; (2) Kaplan-Meier survival; (3) organ damage (periodic acid Schiff staining) via semi-quantitative scoring [0-4 (0 = no injury/inflammation to 4 = very severe injury/inflammation)]; (4) expression of renal endothelial adhesion molecules (ICAM-1) via
Introduction: In pneumonia, pathogen-host interaction evokes inflammation and disruption of pulmonary endothelial barrier integrity resulting in acute lung injury despite antibiotic therapy. Treatment of acute lung injury is mainly supportive, since key elements of inflammation-induced barrier disruption remain undetermined. The angiopoietins (Ang) -1 and -2 and their receptor Tie2 are involved in the regulation of vascular permeability and inflammation. Tie2-activation by Angiopoietin (Ang)-1 reduced, while Tie2-blockade by Ang-2 increased inflammation and permeability in sepsis. The PEGylated (polyethylene glycol) Tie2-agonist "Vasculotide" (VT) mimics Ang-1 and induces Tie2-activation. The aim of our study was to experimentally investigate whether VT could be a new therapeutic option for the treatment of pneumonia-induced lung injury.
