Following rinsing with PBS, cells were permeabilized with 0.3% Triton-X 100 (Sigma) for 15 min. relative release of histamine and tryptase. A: toluidine blue staining of peritoneal mast cells. (a) control group; (b) compound 48/80-treated cells; (c) Ang-1 100 ng/ml-treated cells; (d) Soluble form of Tie2 (sTie-2)-treated cells and (e) ON-013100 RGD-treated cells. B: Statistical analysis of amplitudes of compound 48/80-induced cell degranulation from all groups. It was performed in a blinded fashion. The data shown is the meanSD of 3 separate experiments. C: Degranulation stimulated by compound 48/80 was determined by measuring the release of histamine through OPT-fluorometric assay as previously reported in duplicates. D: Degranulation stimulated by compound 48/80 was determined by measuring the release of tryptase-2 (mMCP-6) through commercial ELISA kit in duplicates. The data shown are mean SD of 3 separate experiments. *P 0.05.(TIF) pone.0089148.s002.tif (11M) GUID:?94FD76DA-E0A8-4096-9662-8FAE9DE14CC9 Figure S3: ON-013100 Ang-1 suppressed FcRI-mediated mast cells degranulation. Degranulation was determined by staining with dyes and measuring the release of histamine and trptase. A: Mast cell degranulation was observed by microscope 20 min after DNP-BSA 10 g/ml treatment after overnight incubated with 250 ng/ml. Cells were stained with alcian blue (aCe) and toluidine blue (fCj) (100). (a,f) control group, (b,g) IgE-DNP/DNP-BSA-treated cells, (c,h) Ang-1 100 ng/ml-treated cells, (d,i) Soluble form of Tie2 Eptifibatide Acetate (sTie-2)-treated cells and (e,j) RGD-treated cells. B and C: Quantification of P815 mast cells degranulation by IgE-DNP/DNP-BSA. It was performed in a blinded fashion. The data shown is the meanSD of 3 separate experiments. *P 0.05.(TIF) pone.0089148.s003.tif (11M) GUID:?152C1CF9-2846-4475-94D7-56B2292C7038 Abstract Since morbidity and mortality rates of anaphylaxis diseases have been increasing year by year, how to prevent and manage these diseases effectively has become an important issue. Mast cells play a central regulatory role in allergic diseases. Angiopoietin1 (Ang-1) exhibits anti-inflammatory properties by inhibiting vascular permeability, leukocyte migration and cytokine production. However, Ang-1’s function in mast cell activation and anaphylaxis diseases is unknown. The results of our study suggest that Ang-1 decreased lipopolysaccharide (LPS)-induced pro-inflammatory cytokines production of mast cells by suppressing IB phosphorylation and NF-B nuclear translocation. Ang-1 also strongly inhibited compound 48/80 induced and FcRI-mediated mast cells degranulation by decreasing intracellular calcium levels lentivirus-mediated delivery of Ang-1 in mice exhibited alleviated leakage in IgE-dependent passive cutaneous anaphylaxis (PCA). Furthermore, exogenous Ang-1 intervention treatment prevented mice from compound 48/80-induced mesentery mast cell degranulation, attenuated increases in pro-inflammatory cytokines, relieved lung injury, and improved survival in anaphylaxis shock. The results of our study reveal, for the first time, the important role of Ang-1 in the activation of mast cells, and identify a therapeutic effect of Ang-1 on anaphylaxis diseases. Introduction When Angiopoietin1 (Ang-1) was first discovered as a specific ligand of Tie-2 in 1996, people were ON-013100 concerned about its role in promoting angiogenesis . Ang-1 cooperates with vascular endothelial growth factor (VEGF) in the later stages of embryonic angiogenesis to form the mature vascular endothelial barrier . Moreover, in adult microvasculature, binding of Ang-1 to the Tie-2 receptor stabilizes endothelial cell interactions with the extracellular matrix and junctional proteins, and enhances endothelial barrier functions . Transgenic mice over-expressing Ang-1 in dermal micro-vessels were resistant to leakage of albumin-binding Evans blue dye in response to VEGF and other inflammatory agents . Adenoviral-mediated delivery of Ang-1 in adult mouse vascular endothelia markedly reduced vascular leakage . An improved mortality rate in mice with endotoxic shock was seen with an adenoviral construct encoding Ang-1 pretreatment . Local administration of recombinant Ang-1 protects against histological, biochemical, and functional changes observed in an OVA-induced mouse allergic asthma model . These findings raise the possibility that Ang-1 has anti-inflammatory properties. studies have found that Ang-1 directly stimulates migration, and possibly inhibits vascular endothelial growth factor-induced eosinophil and neutrophil chemotaxis , . Moreover, Ang-1 can promote monocyte chemotaxis, endothelial binding, and trans-endothelial migration, which are key events in the progression of atherosclerosis . The Ang-1/Tie-2.