Project description:We assessed the impact of Endothelial Focal Adhesion kinase (ECFAK) knockout, using primary lung endothelial cells isolated (at P10-P14) from a FAK flox/flox mice crossed with mice expressing a tamoxifen-inducible Cre(iCreERT2) that is driven by endothelial cell (EC)-selective platelet-derived growth factor subunit B (Pdgfb) promoter (Pdgfb-iCreERT2) (Tavora et al., 2010). To induce EC restricted FAK deletion, we injected 4-hydroxytamoxifen (4-OHT) from postnatal day 1 (P1) to P2. Molecular signaling events modulated by ECFAK loss, with or without ex vivo VEGF stimulation, were then examined by phospho and total quantitative proteomics analysis using 6plex isobaric tandem mass tagging multiplexing (TMT). In total, three experiments were performed (a an initial pilot experiment not included in this study, followed by two main biological replicates included - i.e. experiments 2, and 3). The following 4 TMT channels were utilised in each experiment: TMT-126: WT-mock(PBS) stimulated TMT-127: WT-Vegf stimulated TMT128: FAK KO-mock (PBS)stimulated TMT 129:FAK KO-Vegf stimulated
Project description:The purpose is to obtain samples for mRNA analysis in primary human lung microvascular endothelial cells treated with universal interferon alpha beta or interferon gamma.
Project description:The purpose of this study was to examine the role of MAVS, ZBP1 and RIPK3 in the phenotype that develops when ADAR1 activity is impaired, in particular when the Za domain of ADAR1 is mutated. Mice homozygous for a Za domain-mutant allele of Adar1 (Adar1mZa/mZa mice) and mice carrying one mZa and one null Adar1 allele (Adar1-/mZa mice) were compared with control mice that were either wild type or heterozygous for the Adar1 mZa allele (Adar1wt/mZa mice). The effects of MAVS deficiency, RIPK3 deficiency, ZBP1 deficiency or ZBP1 Za domain mutations were assessed by analysing compound mutant mice. Given the early postnatal lethal phenotype that develops in Adar1-/mZa mice, comparisons were made in RNA isolated from lung tissue from newborn mice of each genotype (5 mice per genotype). As Adar1-/mZa mice additionally lacking Mavs or Zbp1 are viable, adult mice (15-20 weeks of age) were also used for several compound mutations as donors of lung tissue.
Project description:To investigate the role of a TIE1-function blocking antibody on gene expression of lung endothelial cells. Lung endothelial cells were FACS-isolated from primary tumor-bearing mice. Mice were treated either with IgG or TIE1-blocking antibody during primary tumor growth. Total RNA were used for microarray analysis using Clariom S mouse assays (Applied Biosystems).
Project description:Disruption of pulmonary vascular homeostasis is a central feature of viral pneumonia, wherein endothelial cell (EC) death and subsequent angiogenic responses represent critical determinants of the outcome of severe lung injury. A more granular understanding of the fundamental mechanisms driving reconstitution of the lung endothelium is necessary to facilitate therapeutic targeting of vascular repair. Here, we applied single-cell RNA sequencing (scRNA-seq) to profile lung ECs from mice on D0, D20 and D30 post influenza infection. Our data revealed the dynamics of endothelial subsets during influenza injury.
Project description:Radiation-induced lung injury (RILI) initiates radiation pneumonitis and progresses to fibrosis as the main side effect of lung cancer patients treated with radiotherapy. There is no effective drug for RILI. Sustained vascular activation is a major contributor to the establishment of chronic disease. Here, using a whole thoracic irradiation (WTI) mouse model, we investigated the mechanisms and effectiveness of thrombopoietin mimetic (TPOm) for preventing RILI. We demonstrated that administering TPOm 24 hours before irradiation decreased histologic lung injury score, apoptosis, vascular permeability, expression of pro-inflammatory cytokines, and neutrophil infiltration in the lung of mice 2 weeks after WTI. We described the expression of c-MPL, a TPO receptor, in mouse primary pulmonary microvascular endothelial cells, showing TPOm reduced endothelial cell-neutrophil adhesion by inhibiting ICAM-1 expression. Seven months after WTI, TPOm-treated lung exhibited less collagen deposition, expression of MMP-9, TIMP-1, IL-6, TGF-b, and p21. Moreover, TPOm improved lung vascular structure, lung density, and respiration rate, leading to a prolonged survival time after WTI. Single-cell RNA sequencing analysis of lungs 2 weeks after WTI revealed that TPOm shifted populations of capillary endothelial cells towards a less activated and more homeostatic phenotype. Taken together, TPOm is protective for RILI by inhibiting endothelial cell activation.