angiopoietin-2 Search Results


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  • 93
    Millipore angiopoietin 2
    VEGF correlates with cancer stem cell increase induced by malignant PE and ascites. The -fold increase in cancer stem cells was plotted against the concentration of VEGF, IGF-I, TGF-β, β2-microglobumin, IL-6, IL-8, TNF-α, osteopontin, <t>angiopoietin-2,</t> endoglin, HGF and HB-EGF. Linear regressions were traced according to the distribution of points.
    Angiopoietin 2, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems angiopoietin 2
    Midtrimester maternal plasma concentrations of angiopoietin 1 (A), <t>angiopoietin</t> 2 (B), placental growth factor (C), and angiopoietin 1/angiopoietin 2 ratio (D) in patients who subsequently developed preeclampsia and controls. In pregnant women who subsequently developed preeclampsia, midtrimester maternal plasma concentrations of angiopoietin 1 and angiopoietin 2 were significantly higher and placental growth factor concentrations were significantly lower than those who did not develop preeclampsia. NS, non-significant.
    Angiopoietin 2, supplied by R&D Systems, used in various techniques. Bioz Stars score: 97/100, based on 351 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Regeneron angiopoietin 2
    Correlation of foveal thickness with aqueous angiopoietin concentrations after pars plana vitrectomy. Increased foveal thickness (A) corresponded with increased <t>angiopoietin</t> 2 and reduced angiopoietin 1 concentration (B).
    Angiopoietin 2, supplied by Regeneron, used in various techniques. Bioz Stars score: 92/100, based on 34 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Reiss Manufacturing reiss y angiopoietin 2
    Correlation of foveal thickness with aqueous angiopoietin concentrations after pars plana vitrectomy. Increased foveal thickness (A) corresponded with increased <t>angiopoietin</t> 2 and reduced angiopoietin 1 concentration (B).
    Reiss Y Angiopoietin 2, supplied by Reiss Manufacturing, used in various techniques. Bioz Stars score: 90/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    R&D Systems human angiopoietin 2 antibody
    Flunarizine reduces <t>Angiopoietin-2</t> (Angpt-2) synthesis in vitro . ( A ) Cropped Angpt-2 immunoblot from human umbilical vein endothelial cell (HUVEC) lysates 15 hrs after 10 μM Flunarizine (FLU) (+) or vehicle (−) treatment. (n = 4) ( B ) After stimulation with 10 μM FLU or vehicle for 1 h, 10 ng/mL Tumor necrosis factor α (TNFα) or control was applied to HUVECs for 24 hrs. Angpt-2 concentration in cell lysates was measured by enzyme-linked immunosorbent assay (ELISA) and is shown relative to whole protein (n = 6). ( C ) Fluorescent immunocytochemistry for Angpt-2 (red) and nuclear staining (4′,6-diamidino-2-phenylindole, blue) in HUVECs after treatment with 10 μM Flunarizine, vehicle or Angpt-2 siRNA as a negative control for 24 hrs (n = 4) (D ) Real-time polymerase chain reaction (RT-qPCR) for Angpt-2 in HUVECs stimulated with 10 ng/mL TNFα or control for 15 hrs after pretreatment with either FLU or vehicle (n = 6) for 1 h. Columns are presented as mean ± SEM.
    Human Angiopoietin 2 Antibody, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 158 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GeneTex angiopoietin 2
    Flunarizine reduces <t>Angiopoietin-2</t> (Angpt-2) synthesis in vitro . ( A ) Cropped Angpt-2 immunoblot from human umbilical vein endothelial cell (HUVEC) lysates 15 hrs after 10 μM Flunarizine (FLU) (+) or vehicle (−) treatment. (n = 4) ( B ) After stimulation with 10 μM FLU or vehicle for 1 h, 10 ng/mL Tumor necrosis factor α (TNFα) or control was applied to HUVECs for 24 hrs. Angpt-2 concentration in cell lysates was measured by enzyme-linked immunosorbent assay (ELISA) and is shown relative to whole protein (n = 6). ( C ) Fluorescent immunocytochemistry for Angpt-2 (red) and nuclear staining (4′,6-diamidino-2-phenylindole, blue) in HUVECs after treatment with 10 μM Flunarizine, vehicle or Angpt-2 siRNA as a negative control for 24 hrs (n = 4) (D ) Real-time polymerase chain reaction (RT-qPCR) for Angpt-2 in HUVECs stimulated with 10 ng/mL TNFα or control for 15 hrs after pretreatment with either FLU or vehicle (n = 6) for 1 h. Columns are presented as mean ± SEM.
    Angiopoietin 2, supplied by GeneTex, used in various techniques. Bioz Stars score: 92/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam angiopoietin 2
    Silibinin enhances <t>angiopoietin-2</t> and Tie-2 levels in urethane-induced lung tumors. Three lung tumor samples were randomly taken from each group and analyzed for pTie-2(Tyr992), Tie-2 and angiopoietin-2 protein levels by immunoblotting. Bands were visualized by enhanced chemiluminescence detection. Membranes were stripped and reprobed with β-actin as a loading control. Densitometric analysis of band intensity for each protein was adjusted with β-actin. Columns , mean intensity of three bands; error bars , SEM for each group. SB, silibinin; Ang-2, angiopoietin-2.
    Angiopoietin 2, supplied by Abcam, used in various techniques. Bioz Stars score: 92/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti angiopoietin 2 antibody
    The potential mechanisms for the effects of PAR2. ( A ) Expression of VEGFA and <t>Angiopoietin</t> 2 (Ang2) were estimated by western blotting. Production of tissue factor (TF) ( B ) and IL-8 ( C ) were analyzed by ELISA. *** P
    Anti Angiopoietin 2 Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 37 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology angiopoietin 2
    CXCL-12 expression is thrombin and <t>angiopoietin-2-dependent.</t> (A) Expression of CXCL-12 at baseline and after incubation with thrombin or angiopoietin-2 for 5 days, expressed as the percentage of cells staining positive in immunocytofluorescence analysis. Anti-TIE-2 antibody used at 50 ng/ml. (B) Secretion of CXCL-12 into supernatant after 5 day incubation with thrombin at the indicated concentrations, analyzed by ELISA. (C) Secretion of CXCL-12 into supernatant after 5 day incubation with angiopoietin-2 at the indicated concentrations, analyzed by ELISA. All data shown from WT CD34+ cells. Abbreviations: Csi , control siRNA. Ang-2si , siRNA specific for angiopoietin-2. Experiments repeated at least twice.
    Angiopoietin 2, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 45 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Abcam anti angiopoietin 2 antibody epr2891 2
    Puerarin upregulates key proangiogenic factors VEGFA, Ang-1 and <t>Ang-2</t> in response to LAD ligation stress. A: Slightly increase of VEGFA, Ang-1 and Ang-2 protein in the MI and MI+S groups in response to MI was detected by western blot. However, protein
    Anti Angiopoietin 2 Antibody Epr2891 2, supplied by Abcam, used in various techniques. Bioz Stars score: 96/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Millipore human angiopoietin 2
    Working model of the roles of MMP-2 and MMP-9 in retinoblastoma cells. Y79 and Weri-1 cells represent the metastatic and the non-metastatic model for Rb, respectively. Our work shows differences in viability, migration and angiogenic-associated responses in Rb cells after inhibition of MMP-2 and MMP-9. a Y79 cells showed a profound defect in migration and invasion along with and a significant reduction in <t>Angiopoietin-2</t> and TGF-β1 proteins. These results highlight Y79’s migratory and invasive potential, which may be dependent upon MMPs. b Analyses of Weri-1 cells show MMP-2 and MMP-9 are involved in multiple processes, including viability of cells and VEGF, as well as TGF-β1 production
    Human Angiopoietin 2, supplied by Millipore, used in various techniques. Bioz Stars score: 91/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    R&D Systems recombinant human angiopoietin 2 protein
    HG modulates <t>angiopoietin-2</t> in diabetic bone marrow
    Recombinant Human Angiopoietin 2 Protein, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Santa Cruz Biotechnology goat anti angiopoietin 2
    CXCL-12 expression is thrombin and <t>angiopoietin-2-dependent.</t> (A) Expression of CXCL-12 at baseline and after incubation with thrombin or angiopoietin-2 for 5 days, expressed as the percentage of cells staining positive in immunocytofluorescence analysis. Anti-TIE-2 antibody used at 50 ng/ml. (B) Secretion of CXCL-12 into supernatant after 5 day incubation with thrombin at the indicated concentrations, analyzed by ELISA. (C) Secretion of CXCL-12 into supernatant after 5 day incubation with angiopoietin-2 at the indicated concentrations, analyzed by ELISA. All data shown from WT CD34+ cells. Abbreviations: Csi , control siRNA. Ang-2si , siRNA specific for angiopoietin-2. Experiments repeated at least twice.
    Goat Anti Angiopoietin 2, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology angiopoietin 2 antibodies
    ( A ) Representative western blots of the angiogenic proteins VEGF-A and <t>angiopoietin-2,</t> with the corresponding densitometric analysis ( B ). ( C ) A representative histological microphotograph of capillary density at 20 weeks in sham ( a ), AB + HA ( b ), and
    Angiopoietin 2 Antibodies, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Abfrontier angiopoietin 2
    Depletion of RRAD decreases angiogenesis-related factors. HUVEC cell was seeded on Matrigel and incubated for 18 h in siControl or siRRAD#1-transfected MKN1 cells ( A ) and DLD1 cells medium ( B ). Tube formation was determined by assessment of the total length of tube in three randomly selected fields. Data represent mean ± SD of three independent experiments. Angiogenesis-related factors including VEGF and <t>angiopoietin</t> 2 were also decreased by siRRAD with immunoblotting ( C ) and ELISA analysis ( D ). Full-length blots are presented in Supplementary Fig. S8 . *P
    Angiopoietin 2, supplied by Abfrontier, used in various techniques. Bioz Stars score: 93/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti angiopoietin 2
    Depletion of RRAD decreases angiogenesis-related factors. HUVEC cell was seeded on Matrigel and incubated for 18 h in siControl or siRRAD#1-transfected MKN1 cells ( A ) and DLD1 cells medium ( B ). Tube formation was determined by assessment of the total length of tube in three randomly selected fields. Data represent mean ± SD of three independent experiments. Angiogenesis-related factors including VEGF and <t>angiopoietin</t> 2 were also decreased by siRRAD with immunoblotting ( C ) and ELISA analysis ( D ). Full-length blots are presented in Supplementary Fig. S8 . *P
    Anti Angiopoietin 2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology anti angiopoietin2
    EGFR activation promotes invasive/non-angiogenic tumor growth in GBM patient biopsies. Tissue microarray (TMA) of GBM biopsies. a EGFR -amplified GBM biopsies as verified by FISH with an EGFR /chromosome 7 probe in red and green, respectively. H E sections and <t>angiopoietin2</t> stainings indicate non-angiogenic ( upper panel ) versus angiogenic areas ( lower panel ) in EGFR -amplified tumors. High pEGFR expression is only found in non-angiogenic areas ( upper panel ). b Immunohistochemical staining of pEGFR positive biopsies selected from the TMA with antibodies against pEGFR and vWF. pEGFR positive tumor areas are non-/less angiogenic compared to angiogenic, pEGFR negative areas within the same biopsies. Scale bars 100 μm. c Area fraction of vascular elements immunostained with vWF from pEGFR positive versus angiogenic, pEGFR negative areas from five different patients. Quantification was performed at ×200 magnification. P
    Anti Angiopoietin2, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    VEGF correlates with cancer stem cell increase induced by malignant PE and ascites. The -fold increase in cancer stem cells was plotted against the concentration of VEGF, IGF-I, TGF-β, β2-microglobumin, IL-6, IL-8, TNF-α, osteopontin, angiopoietin-2, endoglin, HGF and HB-EGF. Linear regressions were traced according to the distribution of points.

    Journal: The Journal of Biological Chemistry

    Article Title: Malignant Pleural Effusion and ascites Induce Epithelial-Mesenchymal Transition and Cancer Stem-like Cell Properties via the Vascular Endothelial Growth Factor (VEGF)/Phosphatidylinositol 3-Kinase (PI3K)/Akt/Mechanistic Target of Rapamycin (mTOR) Pathway *

    doi: 10.1074/jbc.M116.753236

    Figure Lengend Snippet: VEGF correlates with cancer stem cell increase induced by malignant PE and ascites. The -fold increase in cancer stem cells was plotted against the concentration of VEGF, IGF-I, TGF-β, β2-microglobumin, IL-6, IL-8, TNF-α, osteopontin, angiopoietin-2, endoglin, HGF and HB-EGF. Linear regressions were traced according to the distribution of points.

    Article Snippet: The luminex-based multiplex bead array system for IL-8, osteopontin, HGF, angiopoietin-2, endoglin, HB-EGF, IL-1β, IL-13, IL-17, PDGF, EGF, RANTES, FGF-2, Endothelin-1, and BMP-9 were from Millipore Corp.

    Techniques: Concentration Assay

    Midtrimester maternal plasma concentrations of angiopoietin 1 (A), angiopoietin 2 (B), placental growth factor (C), and angiopoietin 1/angiopoietin 2 ratio (D) in patients who subsequently developed preeclampsia and controls. In pregnant women who subsequently developed preeclampsia, midtrimester maternal plasma concentrations of angiopoietin 1 and angiopoietin 2 were significantly higher and placental growth factor concentrations were significantly lower than those who did not develop preeclampsia. NS, non-significant.

    Journal: Obstetrics & Gynecology Science

    Article Title: Midtrimester maternal plasma concentrations of angiopoietin 1, angiopoietin 2, and placental growth factor in pregnant women who subsequently develop preeclampsia

    doi: 10.5468/ogs.2015.58.1.10

    Figure Lengend Snippet: Midtrimester maternal plasma concentrations of angiopoietin 1 (A), angiopoietin 2 (B), placental growth factor (C), and angiopoietin 1/angiopoietin 2 ratio (D) in patients who subsequently developed preeclampsia and controls. In pregnant women who subsequently developed preeclampsia, midtrimester maternal plasma concentrations of angiopoietin 1 and angiopoietin 2 were significantly higher and placental growth factor concentrations were significantly lower than those who did not develop preeclampsia. NS, non-significant.

    Article Snippet: Measurement of angiopoietin 1, angiopoietin 2, and placental growth factor Concentrations of angiopoietin 1, angiopoietin 2, and placental growth factor were measured by commercially available enzyme-linked immunosorbent assay kits (R & D Systems, Minneapolis, MN, USA).

    Techniques:

    Correlation of foveal thickness with aqueous angiopoietin concentrations after pars plana vitrectomy. Increased foveal thickness (A) corresponded with increased angiopoietin 2 and reduced angiopoietin 1 concentration (B).

    Journal: The British Journal of Ophthalmology

    Article Title: Angiopoietin concentrations in diabetic retinopathy

    doi: 10.1136/bjo.2004.049940

    Figure Lengend Snippet: Correlation of foveal thickness with aqueous angiopoietin concentrations after pars plana vitrectomy. Increased foveal thickness (A) corresponded with increased angiopoietin 2 and reduced angiopoietin 1 concentration (B).

    Article Snippet: Then either 30 μl of either angiopoietin 1 (R and D Systems, Minneapolis MN, USA) or angiopoietin 2 (Regeneron Pharmaceuticals Inc, USA) standards, or test samples were added to the wells for 2 hours at room temperature (samples were diluted 1:4 with phosphate buffered saline, pH 7.4).

    Techniques: Concentration Assay

    Flunarizine reduces Angiopoietin-2 (Angpt-2) synthesis in vitro . ( A ) Cropped Angpt-2 immunoblot from human umbilical vein endothelial cell (HUVEC) lysates 15 hrs after 10 μM Flunarizine (FLU) (+) or vehicle (−) treatment. (n = 4) ( B ) After stimulation with 10 μM FLU or vehicle for 1 h, 10 ng/mL Tumor necrosis factor α (TNFα) or control was applied to HUVECs for 24 hrs. Angpt-2 concentration in cell lysates was measured by enzyme-linked immunosorbent assay (ELISA) and is shown relative to whole protein (n = 6). ( C ) Fluorescent immunocytochemistry for Angpt-2 (red) and nuclear staining (4′,6-diamidino-2-phenylindole, blue) in HUVECs after treatment with 10 μM Flunarizine, vehicle or Angpt-2 siRNA as a negative control for 24 hrs (n = 4) (D ) Real-time polymerase chain reaction (RT-qPCR) for Angpt-2 in HUVECs stimulated with 10 ng/mL TNFα or control for 15 hrs after pretreatment with either FLU or vehicle (n = 6) for 1 h. Columns are presented as mean ± SEM.

    Journal: Scientific Reports

    Article Title: Flunarizine suppresses endothelial Angiopoietin-2 in a calcium - dependent fashion in sepsis

    doi: 10.1038/srep44113

    Figure Lengend Snippet: Flunarizine reduces Angiopoietin-2 (Angpt-2) synthesis in vitro . ( A ) Cropped Angpt-2 immunoblot from human umbilical vein endothelial cell (HUVEC) lysates 15 hrs after 10 μM Flunarizine (FLU) (+) or vehicle (−) treatment. (n = 4) ( B ) After stimulation with 10 μM FLU or vehicle for 1 h, 10 ng/mL Tumor necrosis factor α (TNFα) or control was applied to HUVECs for 24 hrs. Angpt-2 concentration in cell lysates was measured by enzyme-linked immunosorbent assay (ELISA) and is shown relative to whole protein (n = 6). ( C ) Fluorescent immunocytochemistry for Angpt-2 (red) and nuclear staining (4′,6-diamidino-2-phenylindole, blue) in HUVECs after treatment with 10 μM Flunarizine, vehicle or Angpt-2 siRNA as a negative control for 24 hrs (n = 4) (D ) Real-time polymerase chain reaction (RT-qPCR) for Angpt-2 in HUVECs stimulated with 10 ng/mL TNFα or control for 15 hrs after pretreatment with either FLU or vehicle (n = 6) for 1 h. Columns are presented as mean ± SEM.

    Article Snippet: Antibodies against Angpt-2 (AF623) (R & D Systems, Minneapolis, MN), mAngpt-2 (Clone #748246) (R & D Systems, Minneapolis, MN), Tie2 (C-20) (sc-324, Santa Cruz Biotechnology, CA), pTie2 (Y1102/Y1100) (R & D systems, Minneapolis, MN), Gr-1 (AbD Serotec), Lcyopersicon esculentum agglutinin (LEA, tomato lectin) (Vector Laboratories, Burlingame, CA), Akt (11E7) (Cell Signaling Technology, Cambridge, UK), pAkt (Ser 473) (Cell Signaling Technology, Cambridge, UK), pJNK (G-7) (sc-6254, Santa Cruz Biotechnology, CA), JNK (Cell Signaling Technology, Cambridge, UK) and GAPDH (FL-335) (Santa Cruz Biotechnology, CA) were utilized.

    Techniques: In Vitro, Concentration Assay, Enzyme-linked Immunosorbent Assay, Immunocytochemistry, Staining, Negative Control, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

    Flunarizine reduces Angiopoietin-2 (Angpt-2) after stimulation in vitro . Human umbilical vein endothelial cells (HUVECs) were stimulated with 10 ng/mL tumor necrosis factor α (TNFα) or control after 1 h pretreatment with either 10 μM Flunarizine (FLU) or vehicle and the concentration of Angpt-2 in the supernatant was determined by enzyme-linked immunosorbent assay (ELISA) ( A ) after six hrs of stimulation (n = 6) ( B ) after 12 hrs of stimulation (n = 6) and ( C ) after 24 hrs of stimulation (n = 6) ( D ) Real-time transendothelial electrical resistance (TER) from HUVECs, who were pretreated for 1 h with 10 μM Flunarizine (FLU) or vehicle and stimulated with 10 ng/mL TNFα, was recorded with an electric cell-substrate impedance sensing (ECIS) device (ibidi). Columns are presented as mean ± SEM.

    Journal: Scientific Reports

    Article Title: Flunarizine suppresses endothelial Angiopoietin-2 in a calcium - dependent fashion in sepsis

    doi: 10.1038/srep44113

    Figure Lengend Snippet: Flunarizine reduces Angiopoietin-2 (Angpt-2) after stimulation in vitro . Human umbilical vein endothelial cells (HUVECs) were stimulated with 10 ng/mL tumor necrosis factor α (TNFα) or control after 1 h pretreatment with either 10 μM Flunarizine (FLU) or vehicle and the concentration of Angpt-2 in the supernatant was determined by enzyme-linked immunosorbent assay (ELISA) ( A ) after six hrs of stimulation (n = 6) ( B ) after 12 hrs of stimulation (n = 6) and ( C ) after 24 hrs of stimulation (n = 6) ( D ) Real-time transendothelial electrical resistance (TER) from HUVECs, who were pretreated for 1 h with 10 μM Flunarizine (FLU) or vehicle and stimulated with 10 ng/mL TNFα, was recorded with an electric cell-substrate impedance sensing (ECIS) device (ibidi). Columns are presented as mean ± SEM.

    Article Snippet: Antibodies against Angpt-2 (AF623) (R & D Systems, Minneapolis, MN), mAngpt-2 (Clone #748246) (R & D Systems, Minneapolis, MN), Tie2 (C-20) (sc-324, Santa Cruz Biotechnology, CA), pTie2 (Y1102/Y1100) (R & D systems, Minneapolis, MN), Gr-1 (AbD Serotec), Lcyopersicon esculentum agglutinin (LEA, tomato lectin) (Vector Laboratories, Burlingame, CA), Akt (11E7) (Cell Signaling Technology, Cambridge, UK), pAkt (Ser 473) (Cell Signaling Technology, Cambridge, UK), pJNK (G-7) (sc-6254, Santa Cruz Biotechnology, CA), JNK (Cell Signaling Technology, Cambridge, UK) and GAPDH (FL-335) (Santa Cruz Biotechnology, CA) were utilized.

    Techniques: In Vitro, Concentration Assay, Enzyme-linked Immunosorbent Assay, Electric Cell-substrate Impedance Sensing

    Multiple structurally distinct putative T-type (but not L-type) calcium channel blockers suppress Angiopoietin-2 (Angpt-2). 10 μM Flunarizine (FLU) or vehicle was applied for 24 hrs after pretreatment with ( A ) 10 μM Mibefradil or control for 1 h, ( B ) 50 μM TTA-A2 (a specific pharmacological inhibitor of t-type CCs) or control for 1 h, (C) 10 μM Amlodipine or control for 1 h. Angpt-2 concentration in the supernatant of human umbilical vein endothelial cells (HUVECs) was measured by enzyme-linked immunosorbent assay (ELISA) (n = 6–10). (D) Before application of 10 μM FLU or vehicle for 24 hrs HUVECs were pretreated with 1 μM Thapsigargin or control for 0.5 hrs. Angpt-2 concentration in the supernatant was measured by ELISA and is shown relative to whole intracellular protein (n = 6–10). ( E ) 10 μM FLU or control was applied for 8 hrs after pretreatment with 10 μM EDTA for 1 h and the concentration of Angpt-2 in the supernatant was determined by ELISA (n = 6). Columns are presented as mean ± SEM.

    Journal: Scientific Reports

    Article Title: Flunarizine suppresses endothelial Angiopoietin-2 in a calcium - dependent fashion in sepsis

    doi: 10.1038/srep44113

    Figure Lengend Snippet: Multiple structurally distinct putative T-type (but not L-type) calcium channel blockers suppress Angiopoietin-2 (Angpt-2). 10 μM Flunarizine (FLU) or vehicle was applied for 24 hrs after pretreatment with ( A ) 10 μM Mibefradil or control for 1 h, ( B ) 50 μM TTA-A2 (a specific pharmacological inhibitor of t-type CCs) or control for 1 h, (C) 10 μM Amlodipine or control for 1 h. Angpt-2 concentration in the supernatant of human umbilical vein endothelial cells (HUVECs) was measured by enzyme-linked immunosorbent assay (ELISA) (n = 6–10). (D) Before application of 10 μM FLU or vehicle for 24 hrs HUVECs were pretreated with 1 μM Thapsigargin or control for 0.5 hrs. Angpt-2 concentration in the supernatant was measured by ELISA and is shown relative to whole intracellular protein (n = 6–10). ( E ) 10 μM FLU or control was applied for 8 hrs after pretreatment with 10 μM EDTA for 1 h and the concentration of Angpt-2 in the supernatant was determined by ELISA (n = 6). Columns are presented as mean ± SEM.

    Article Snippet: Antibodies against Angpt-2 (AF623) (R & D Systems, Minneapolis, MN), mAngpt-2 (Clone #748246) (R & D Systems, Minneapolis, MN), Tie2 (C-20) (sc-324, Santa Cruz Biotechnology, CA), pTie2 (Y1102/Y1100) (R & D systems, Minneapolis, MN), Gr-1 (AbD Serotec), Lcyopersicon esculentum agglutinin (LEA, tomato lectin) (Vector Laboratories, Burlingame, CA), Akt (11E7) (Cell Signaling Technology, Cambridge, UK), pAkt (Ser 473) (Cell Signaling Technology, Cambridge, UK), pJNK (G-7) (sc-6254, Santa Cruz Biotechnology, CA), JNK (Cell Signaling Technology, Cambridge, UK) and GAPDH (FL-335) (Santa Cruz Biotechnology, CA) were utilized.

    Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay

    Flunarizine does not require Tie2 or downstream signalling to reduce Angpt-2. ( A ) Cropped immunoblot for Angiopoietin-2 (Angpt-2), tTie2 and GAPDH as a loading control from HUVECs transfected with control siRNA or Tie2 siRNA and stimulated with 10 μM Flunarizine (FLU) or vehicle for 24 hrs (n = 4). ( B ) HUVECs were treated with 10 μM FLU (+) or vehicle (−) for 24 hrs after transfection with Tie2 siRNA (+) or control siRNA (−) and the concentration of Angpt-2 in the supernatant was determined by ELISA (n = 5–6). Angpt-2 concentration in the supernatant of cells pretreated with ( C ) 1 μM Wortmannin or control for 1 h or with ( D ) 50 μM LY294002 for 1 h and stimulated with 10 μM FLU or vehicle for 12 hrs was measured by enzyme-linked immunosorbent assay (ELISA) (n = 6). ( E ) Real-time transendothelial electrical impedance from HUVECs 24 hrs after transfection with Tie2 siRNA or control siRNA, who were pretreated with 10 μM Flunarizine (FLU) or vehicle for 1 h and stimulated with 10 ng/mL TNFα, was recorded with an electric cell-substrate impedance sensing (ECIS) device (ibidi). Bar graphs represent n = 8 measurements per conditions after 5 hrs of TNF challenge. Columns are presented as mean ± SEM.

    Journal: Scientific Reports

    Article Title: Flunarizine suppresses endothelial Angiopoietin-2 in a calcium - dependent fashion in sepsis

    doi: 10.1038/srep44113

    Figure Lengend Snippet: Flunarizine does not require Tie2 or downstream signalling to reduce Angpt-2. ( A ) Cropped immunoblot for Angiopoietin-2 (Angpt-2), tTie2 and GAPDH as a loading control from HUVECs transfected with control siRNA or Tie2 siRNA and stimulated with 10 μM Flunarizine (FLU) or vehicle for 24 hrs (n = 4). ( B ) HUVECs were treated with 10 μM FLU (+) or vehicle (−) for 24 hrs after transfection with Tie2 siRNA (+) or control siRNA (−) and the concentration of Angpt-2 in the supernatant was determined by ELISA (n = 5–6). Angpt-2 concentration in the supernatant of cells pretreated with ( C ) 1 μM Wortmannin or control for 1 h or with ( D ) 50 μM LY294002 for 1 h and stimulated with 10 μM FLU or vehicle for 12 hrs was measured by enzyme-linked immunosorbent assay (ELISA) (n = 6). ( E ) Real-time transendothelial electrical impedance from HUVECs 24 hrs after transfection with Tie2 siRNA or control siRNA, who were pretreated with 10 μM Flunarizine (FLU) or vehicle for 1 h and stimulated with 10 ng/mL TNFα, was recorded with an electric cell-substrate impedance sensing (ECIS) device (ibidi). Bar graphs represent n = 8 measurements per conditions after 5 hrs of TNF challenge. Columns are presented as mean ± SEM.

    Article Snippet: Antibodies against Angpt-2 (AF623) (R & D Systems, Minneapolis, MN), mAngpt-2 (Clone #748246) (R & D Systems, Minneapolis, MN), Tie2 (C-20) (sc-324, Santa Cruz Biotechnology, CA), pTie2 (Y1102/Y1100) (R & D systems, Minneapolis, MN), Gr-1 (AbD Serotec), Lcyopersicon esculentum agglutinin (LEA, tomato lectin) (Vector Laboratories, Burlingame, CA), Akt (11E7) (Cell Signaling Technology, Cambridge, UK), pAkt (Ser 473) (Cell Signaling Technology, Cambridge, UK), pJNK (G-7) (sc-6254, Santa Cruz Biotechnology, CA), JNK (Cell Signaling Technology, Cambridge, UK) and GAPDH (FL-335) (Santa Cruz Biotechnology, CA) were utilized.

    Techniques: Transfection, Concentration Assay, Enzyme-linked Immunosorbent Assay, Electric Cell-substrate Impedance Sensing

    Flunarizine lowers Angiopoietin-2 (Angpt-2) and vascular inflammation in vivo. Adult male C57BL/6 J mice were pretreated daily with Flunarizine (25 mg/kg body weight (bw) p.o.) or vehicle for three days before the injection with 17.5 mg/kg bw gram-negative endotoxin (lipopolysaccharides [LPS] from Escherichia coli i.p.) or control. Expression of pulmonary mRNA and Angpt-2 serum levels were measured 12 h after LPS (n = 3–9). ( A ) Real-time polymerase chain reaction (RT-PCR) from lung homogenates for tumor necrosis factor α (TNFα). ( B ) RT-PCR from lung homogenates for Interleukin-6 (IL-6). (C) RT-PCR from lung homogenates for Angpt-2. ( D ) Angpt-2 serum levels were quantified by enzyme-linked immunosorbent assay (ELISA). ( E ) RT-PCR from lung homogenates for intercellular adhesion molecule-1 (ICAM-1). ( F ) Immunohistochemistry from murine lungs for Gr-1 (red), endothelial-specific lectin (green) and nuclear staining (4′,6-diamidino-2-phenylindole, blue) ( G ) Kaplan Meier Survival after LPS administration (20 mg/kg bw) in mice that were pretreated with 25 mg/kg bw FLU or vehicle for three days once a day (n = 8–10, p = 0.408). Columns are presented as mean ± SEM.

    Journal: Scientific Reports

    Article Title: Flunarizine suppresses endothelial Angiopoietin-2 in a calcium - dependent fashion in sepsis

    doi: 10.1038/srep44113

    Figure Lengend Snippet: Flunarizine lowers Angiopoietin-2 (Angpt-2) and vascular inflammation in vivo. Adult male C57BL/6 J mice were pretreated daily with Flunarizine (25 mg/kg body weight (bw) p.o.) or vehicle for three days before the injection with 17.5 mg/kg bw gram-negative endotoxin (lipopolysaccharides [LPS] from Escherichia coli i.p.) or control. Expression of pulmonary mRNA and Angpt-2 serum levels were measured 12 h after LPS (n = 3–9). ( A ) Real-time polymerase chain reaction (RT-PCR) from lung homogenates for tumor necrosis factor α (TNFα). ( B ) RT-PCR from lung homogenates for Interleukin-6 (IL-6). (C) RT-PCR from lung homogenates for Angpt-2. ( D ) Angpt-2 serum levels were quantified by enzyme-linked immunosorbent assay (ELISA). ( E ) RT-PCR from lung homogenates for intercellular adhesion molecule-1 (ICAM-1). ( F ) Immunohistochemistry from murine lungs for Gr-1 (red), endothelial-specific lectin (green) and nuclear staining (4′,6-diamidino-2-phenylindole, blue) ( G ) Kaplan Meier Survival after LPS administration (20 mg/kg bw) in mice that were pretreated with 25 mg/kg bw FLU or vehicle for three days once a day (n = 8–10, p = 0.408). Columns are presented as mean ± SEM.

    Article Snippet: Antibodies against Angpt-2 (AF623) (R & D Systems, Minneapolis, MN), mAngpt-2 (Clone #748246) (R & D Systems, Minneapolis, MN), Tie2 (C-20) (sc-324, Santa Cruz Biotechnology, CA), pTie2 (Y1102/Y1100) (R & D systems, Minneapolis, MN), Gr-1 (AbD Serotec), Lcyopersicon esculentum agglutinin (LEA, tomato lectin) (Vector Laboratories, Burlingame, CA), Akt (11E7) (Cell Signaling Technology, Cambridge, UK), pAkt (Ser 473) (Cell Signaling Technology, Cambridge, UK), pJNK (G-7) (sc-6254, Santa Cruz Biotechnology, CA), JNK (Cell Signaling Technology, Cambridge, UK) and GAPDH (FL-335) (Santa Cruz Biotechnology, CA) were utilized.

    Techniques: In Vivo, Mouse Assay, Injection, Expressing, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Staining

    Flunarizine reduces baseline Angiopoietin-2 (Angpt-2) in vitro. ( A ) Human umbilical vein endothelial cells (HUVECs) grown in a 96-well format were treated with a Food and Drug Administration (FDA)-drug library for 24 hrs, and Angpt-2 protein in the supernatant was measured by enzyme-linked immunosorbent assay (ELISA). Results were analyzed as the fold-change relative to the median value and ordered from strongest inhibitors (left, blue bars) to strongest inducers (right, blue bars). The cutoff was set to tenfold reduction. ( B ) HUVECs were treated with different concentrations of Flunarizine for 24 hrs and Angpt-2 in the supernatant was quantified by ELISA (n = 6–12). ( C ) 10 μM Flunarizine (FLU) or vehicle was applied for indicated time points. Angpt-2 in the supernatant was measured by ELISA (n = 5–6). Columns are presented as mean ± SEM.

    Journal: Scientific Reports

    Article Title: Flunarizine suppresses endothelial Angiopoietin-2 in a calcium - dependent fashion in sepsis

    doi: 10.1038/srep44113

    Figure Lengend Snippet: Flunarizine reduces baseline Angiopoietin-2 (Angpt-2) in vitro. ( A ) Human umbilical vein endothelial cells (HUVECs) grown in a 96-well format were treated with a Food and Drug Administration (FDA)-drug library for 24 hrs, and Angpt-2 protein in the supernatant was measured by enzyme-linked immunosorbent assay (ELISA). Results were analyzed as the fold-change relative to the median value and ordered from strongest inhibitors (left, blue bars) to strongest inducers (right, blue bars). The cutoff was set to tenfold reduction. ( B ) HUVECs were treated with different concentrations of Flunarizine for 24 hrs and Angpt-2 in the supernatant was quantified by ELISA (n = 6–12). ( C ) 10 μM Flunarizine (FLU) or vehicle was applied for indicated time points. Angpt-2 in the supernatant was measured by ELISA (n = 5–6). Columns are presented as mean ± SEM.

    Article Snippet: Antibodies against Angpt-2 (AF623) (R & D Systems, Minneapolis, MN), mAngpt-2 (Clone #748246) (R & D Systems, Minneapolis, MN), Tie2 (C-20) (sc-324, Santa Cruz Biotechnology, CA), pTie2 (Y1102/Y1100) (R & D systems, Minneapolis, MN), Gr-1 (AbD Serotec), Lcyopersicon esculentum agglutinin (LEA, tomato lectin) (Vector Laboratories, Burlingame, CA), Akt (11E7) (Cell Signaling Technology, Cambridge, UK), pAkt (Ser 473) (Cell Signaling Technology, Cambridge, UK), pJNK (G-7) (sc-6254, Santa Cruz Biotechnology, CA), JNK (Cell Signaling Technology, Cambridge, UK) and GAPDH (FL-335) (Santa Cruz Biotechnology, CA) were utilized.

    Techniques: In Vitro, Enzyme-linked Immunosorbent Assay

    Silibinin enhances angiopoietin-2 and Tie-2 levels in urethane-induced lung tumors. Three lung tumor samples were randomly taken from each group and analyzed for pTie-2(Tyr992), Tie-2 and angiopoietin-2 protein levels by immunoblotting. Bands were visualized by enhanced chemiluminescence detection. Membranes were stripped and reprobed with β-actin as a loading control. Densitometric analysis of band intensity for each protein was adjusted with β-actin. Columns , mean intensity of three bands; error bars , SEM for each group. SB, silibinin; Ang-2, angiopoietin-2.

    Journal: Cancer prevention research (Philadelphia, Pa.)

    Article Title: Growth Inhibition and Regression of Lung Tumors by Silibinin: Modulation of Angiogenesis by Macrophage-associated Cytokines, and NF-?B and STAT3

    doi: 10.1158/1940-6207.CAPR-08-0095

    Figure Lengend Snippet: Silibinin enhances angiopoietin-2 and Tie-2 levels in urethane-induced lung tumors. Three lung tumor samples were randomly taken from each group and analyzed for pTie-2(Tyr992), Tie-2 and angiopoietin-2 protein levels by immunoblotting. Bands were visualized by enhanced chemiluminescence detection. Membranes were stripped and reprobed with β-actin as a loading control. Densitometric analysis of band intensity for each protein was adjusted with β-actin. Columns , mean intensity of three bands; error bars , SEM for each group. SB, silibinin; Ang-2, angiopoietin-2.

    Article Snippet: Angiopoietin-2 (Ang-2) is the ligand for the Tie-2 (tyrosine kinase) receptor in endothelial as well as immune cells.

    Techniques:

    The potential mechanisms for the effects of PAR2. ( A ) Expression of VEGFA and Angiopoietin 2 (Ang2) were estimated by western blotting. Production of tissue factor (TF) ( B ) and IL-8 ( C ) were analyzed by ELISA. *** P

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    Article Title: Upregulation of Protease-Activated Receptor 2 Promotes Proliferation and Migration of Human Vascular Smooth Muscle Cells (VSMCs)

    doi: 10.12659/MSM.917865

    Figure Lengend Snippet: The potential mechanisms for the effects of PAR2. ( A ) Expression of VEGFA and Angiopoietin 2 (Ang2) were estimated by western blotting. Production of tissue factor (TF) ( B ) and IL-8 ( C ) were analyzed by ELISA. *** P

    Article Snippet: After proteins were well separated, they were transferred to polyvinylidene fluoride membranes (Millipore, USA) for 2 h and blocked with 5% (v/v TBST) skimmed milk for 1 h. The membranes were incubated with primary antibody at 4°C overnight: rabbit anti-PAR2 (#6976), MMP9 (#13667), MMP14 (#13130), and GAPDH (#5174) were purchased from Cell Signaling Technology (USA), and VEGFA (ab52917) and Angiopoietin 2 (ab8452) were purchased from Abcam (USA).

    Techniques: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay

    Differential regulation of VEGF and IL-6 mediated Notch ligands and Ang2 may explain defective pericyte coverage 2×10 5 MLEC cells were plated and treated with control (PBS), VEGF (30ng/ml) or mIL-6 (30ng/ml) for 24 hours. A. Western blot analysis of Jagged1 and DLL4 expression on MLEC cells. B. The induction of Jagged1 by IL-6 and DLL4 by VEGF is further confirmed in the protein analysis of aortas treated with IL-6 or VEGF. C. Hey upregulation in the VEGF treated MLEC confirms the positive VEGF induced Notch-DLL4 interaction in the MLEC. D. Western blot analysis of Ang2 in the MLEC treated with VEGF or IL-6 shows similar upregulation of Ang2 in the MLEC treated with IL-6 compared to VEGF E. Ang2 expression in protein isolated from three aortas per group from wild-type C57BL/6 mice (8–12 weeks) and treated with either VEGF (30ng/ml) or mouse IL-6 (30ng/ml) for 24hours F. Model for mechanism of action of VEGF or IL-6 on endothelial cells. VEGF binding to VEGF receptor leads to upregulation of DLL4 resulting in angiogenesis and maturation of vessels. However, IL-6 forms a complex with IL-6 receptors to activate Jagged1 and Ang2 resulting in angiogenesis with weak or defective pericyte coverage.

    Journal: Cancer research

    Article Title: Interleukin-6 stimulates defective angiogenesis

    doi: 10.1158/0008-5472.CAN-15-1227

    Figure Lengend Snippet: Differential regulation of VEGF and IL-6 mediated Notch ligands and Ang2 may explain defective pericyte coverage 2×10 5 MLEC cells were plated and treated with control (PBS), VEGF (30ng/ml) or mIL-6 (30ng/ml) for 24 hours. A. Western blot analysis of Jagged1 and DLL4 expression on MLEC cells. B. The induction of Jagged1 by IL-6 and DLL4 by VEGF is further confirmed in the protein analysis of aortas treated with IL-6 or VEGF. C. Hey upregulation in the VEGF treated MLEC confirms the positive VEGF induced Notch-DLL4 interaction in the MLEC. D. Western blot analysis of Ang2 in the MLEC treated with VEGF or IL-6 shows similar upregulation of Ang2 in the MLEC treated with IL-6 compared to VEGF E. Ang2 expression in protein isolated from three aortas per group from wild-type C57BL/6 mice (8–12 weeks) and treated with either VEGF (30ng/ml) or mouse IL-6 (30ng/ml) for 24hours F. Model for mechanism of action of VEGF or IL-6 on endothelial cells. VEGF binding to VEGF receptor leads to upregulation of DLL4 resulting in angiogenesis and maturation of vessels. However, IL-6 forms a complex with IL-6 receptors to activate Jagged1 and Ang2 resulting in angiogenesis with weak or defective pericyte coverage.

    Article Snippet: The membrane was blocked overnight (4°C in PBS with 0.1% Tween and 5% milk powder) and probed using the following antibodies: Jagged1 (1:1000, Abcam ab7771), DLL4 (1:1000, Abcam ab7280), Ang2 (1:500, Abcam ab8452), Hey1 (1:500, Abcam ab22614), Phospho-Stat3 (Tyr705) (1:1000, Cell Signaling 9145), Stat3 (1:1000, Cell Signaling 4904), p-ERK (1:1000, Santa Cruz sc-7383), ERK (1:1000, Cell Signaling 9102), β-Actin (1:5000, Sigma A5316/).

    Techniques: Western Blot, Expressing, Isolation, Mouse Assay, Binding Assay

    Relevance of these findings to malignant disease Gene expression data from 285 ovarian cancer biopsies from the AOCS (Australian Ovarian Cancer Study) dataset along with 245 samples obtained by merging two other publicly available datasets were ranked for expression of IL-6 pathway genes. Then 50 samples with the highest (high IL-6) and 50 samples with the lowest (low IL-6) levels of expression were selected and from that a list was generated of the differentially expressed genes between the high and low IL-6 samples. These differentially expressed genes were then associated with various pathways and processes. Significant associations were found between the high IL-6 pathway gene expression and with various processes including angiogenesis. A. The high IL-6 pathway expression correlated positively with Jagged1 and Ang2. RNAseq analysis of omental metastases from patients with stage 3-4 high-grade serous ovarian cancer. The 27 tumor samples were divided into three groups based on histological analysis; uninvolved omental tissue, established tumors and stroma with low tumor burden post chemotherapy. B. Significant positive correlation was observed between expression levels for IL-6, Jagged 1 and Ang2 mRNA, and the best-fit correlation (Spearman r) was found in samples that had a high burden of tumor.

    Journal: Cancer research

    Article Title: Interleukin-6 stimulates defective angiogenesis

    doi: 10.1158/0008-5472.CAN-15-1227

    Figure Lengend Snippet: Relevance of these findings to malignant disease Gene expression data from 285 ovarian cancer biopsies from the AOCS (Australian Ovarian Cancer Study) dataset along with 245 samples obtained by merging two other publicly available datasets were ranked for expression of IL-6 pathway genes. Then 50 samples with the highest (high IL-6) and 50 samples with the lowest (low IL-6) levels of expression were selected and from that a list was generated of the differentially expressed genes between the high and low IL-6 samples. These differentially expressed genes were then associated with various pathways and processes. Significant associations were found between the high IL-6 pathway gene expression and with various processes including angiogenesis. A. The high IL-6 pathway expression correlated positively with Jagged1 and Ang2. RNAseq analysis of omental metastases from patients with stage 3-4 high-grade serous ovarian cancer. The 27 tumor samples were divided into three groups based on histological analysis; uninvolved omental tissue, established tumors and stroma with low tumor burden post chemotherapy. B. Significant positive correlation was observed between expression levels for IL-6, Jagged 1 and Ang2 mRNA, and the best-fit correlation (Spearman r) was found in samples that had a high burden of tumor.

    Article Snippet: The membrane was blocked overnight (4°C in PBS with 0.1% Tween and 5% milk powder) and probed using the following antibodies: Jagged1 (1:1000, Abcam ab7771), DLL4 (1:1000, Abcam ab7280), Ang2 (1:500, Abcam ab8452), Hey1 (1:500, Abcam ab22614), Phospho-Stat3 (Tyr705) (1:1000, Cell Signaling 9145), Stat3 (1:1000, Cell Signaling 4904), p-ERK (1:1000, Santa Cruz sc-7383), ERK (1:1000, Cell Signaling 9102), β-Actin (1:5000, Sigma A5316/).

    Techniques: Expressing, Generated

    CXCL-12 expression is thrombin and angiopoietin-2-dependent. (A) Expression of CXCL-12 at baseline and after incubation with thrombin or angiopoietin-2 for 5 days, expressed as the percentage of cells staining positive in immunocytofluorescence analysis. Anti-TIE-2 antibody used at 50 ng/ml. (B) Secretion of CXCL-12 into supernatant after 5 day incubation with thrombin at the indicated concentrations, analyzed by ELISA. (C) Secretion of CXCL-12 into supernatant after 5 day incubation with angiopoietin-2 at the indicated concentrations, analyzed by ELISA. All data shown from WT CD34+ cells. Abbreviations: Csi , control siRNA. Ang-2si , siRNA specific for angiopoietin-2. Experiments repeated at least twice.

    Journal: Frontiers in Immunology

    Article Title: Inhibition of Angiopoietin-2 Production by Myofibrocytes Inhibits Neointimal Hyperplasia After Endoluminal Injury in Mice

    doi: 10.3389/fimmu.2018.01517

    Figure Lengend Snippet: CXCL-12 expression is thrombin and angiopoietin-2-dependent. (A) Expression of CXCL-12 at baseline and after incubation with thrombin or angiopoietin-2 for 5 days, expressed as the percentage of cells staining positive in immunocytofluorescence analysis. Anti-TIE-2 antibody used at 50 ng/ml. (B) Secretion of CXCL-12 into supernatant after 5 day incubation with thrombin at the indicated concentrations, analyzed by ELISA. (C) Secretion of CXCL-12 into supernatant after 5 day incubation with angiopoietin-2 at the indicated concentrations, analyzed by ELISA. All data shown from WT CD34+ cells. Abbreviations: Csi , control siRNA. Ang-2si , siRNA specific for angiopoietin-2. Experiments repeated at least twice.

    Article Snippet: siRNA Transfection siRNA for angiopoietin 2 (sc-39294), a pool of 3 target-specific 19-25 nt siRNAs designed to knockdown gene expression, and the fluorescein conjugated control siRNAs (sc-36869) were purchased from Santa Cruz biotechnology Inc. Texas 75220, USA.

    Techniques: Expressing, Incubation, Staining, Enzyme-linked Immunosorbent Assay

    Association between intimal hyperplasia and plasma levels of angiopoietin-2 after wire-induced endoluminal injury. (A) Plasma angiopoietin-2 levels in wild-type (WT) mice. Levels in unninjured mice represented by the horizontal bar day 0 only; (squares)—WT CD34+ cells; (circles)—unfractionated CD31-TFPI-Tg CD34+ cells; (diamonds) human tissue factor pathway inhibitor (hTFPI)+ fraction of CD31-TFPI-Tg CD34+ cells; (triangles)—hTFPI-negative fraction of CD31-TFPI-Tg CD34+ cells. *Day 7 p

    Journal: Frontiers in Immunology

    Article Title: Inhibition of Angiopoietin-2 Production by Myofibrocytes Inhibits Neointimal Hyperplasia After Endoluminal Injury in Mice

    doi: 10.3389/fimmu.2018.01517

    Figure Lengend Snippet: Association between intimal hyperplasia and plasma levels of angiopoietin-2 after wire-induced endoluminal injury. (A) Plasma angiopoietin-2 levels in wild-type (WT) mice. Levels in unninjured mice represented by the horizontal bar day 0 only; (squares)—WT CD34+ cells; (circles)—unfractionated CD31-TFPI-Tg CD34+ cells; (diamonds) human tissue factor pathway inhibitor (hTFPI)+ fraction of CD31-TFPI-Tg CD34+ cells; (triangles)—hTFPI-negative fraction of CD31-TFPI-Tg CD34+ cells. *Day 7 p

    Article Snippet: siRNA Transfection siRNA for angiopoietin 2 (sc-39294), a pool of 3 target-specific 19-25 nt siRNAs designed to knockdown gene expression, and the fluorescein conjugated control siRNAs (sc-36869) were purchased from Santa Cruz biotechnology Inc. Texas 75220, USA.

    Techniques: Mouse Assay

    Pre-incubation of enhanced yellow fluorescent protein (EYFP) CD34+ cells with reagents to target angiopoietin-2 prior to adoptive transfer. (A) Neointimal area (left axis, white bars) and neointima:media ratio (right axis, black bars) of vessels taken from wild-type (WT) animals 28 days post-injury after adoptive transfer of 1 × 10 6 EYFP CD34+ cells that were either pre-incubated with isotype control antibody, an anti-TIE-2 antibody, control scrambled siRNA or specific siRNA targeting angiopoietin-2, and administered immediately post-injury. Data derived from examination of three random sections from six different vessels. * p

    Journal: Frontiers in Immunology

    Article Title: Inhibition of Angiopoietin-2 Production by Myofibrocytes Inhibits Neointimal Hyperplasia After Endoluminal Injury in Mice

    doi: 10.3389/fimmu.2018.01517

    Figure Lengend Snippet: Pre-incubation of enhanced yellow fluorescent protein (EYFP) CD34+ cells with reagents to target angiopoietin-2 prior to adoptive transfer. (A) Neointimal area (left axis, white bars) and neointima:media ratio (right axis, black bars) of vessels taken from wild-type (WT) animals 28 days post-injury after adoptive transfer of 1 × 10 6 EYFP CD34+ cells that were either pre-incubated with isotype control antibody, an anti-TIE-2 antibody, control scrambled siRNA or specific siRNA targeting angiopoietin-2, and administered immediately post-injury. Data derived from examination of three random sections from six different vessels. * p

    Article Snippet: siRNA Transfection siRNA for angiopoietin 2 (sc-39294), a pool of 3 target-specific 19-25 nt siRNAs designed to knockdown gene expression, and the fluorescein conjugated control siRNAs (sc-36869) were purchased from Santa Cruz biotechnology Inc. Texas 75220, USA.

    Techniques: Incubation, Adoptive Transfer Assay, Derivative Assay

    Association between plasma CXCL-12 and intimal hyperplasia (IH): CXCL-12 expression is thrombin and angiopoietin-2-dependent. (A) Plasma CXCL-12 (black filled) and migration inhibitory factor (MIF) (white filled) levels post-injury in wild-type (WT) recipients of WT (squares) or CD31-TFPI-Tg (circles) CD34+ cells. *Day 7 and day 28 p

    Journal: Frontiers in Immunology

    Article Title: Inhibition of Angiopoietin-2 Production by Myofibrocytes Inhibits Neointimal Hyperplasia After Endoluminal Injury in Mice

    doi: 10.3389/fimmu.2018.01517

    Figure Lengend Snippet: Association between plasma CXCL-12 and intimal hyperplasia (IH): CXCL-12 expression is thrombin and angiopoietin-2-dependent. (A) Plasma CXCL-12 (black filled) and migration inhibitory factor (MIF) (white filled) levels post-injury in wild-type (WT) recipients of WT (squares) or CD31-TFPI-Tg (circles) CD34+ cells. *Day 7 and day 28 p

    Article Snippet: siRNA Transfection siRNA for angiopoietin 2 (sc-39294), a pool of 3 target-specific 19-25 nt siRNAs designed to knockdown gene expression, and the fluorescein conjugated control siRNAs (sc-36869) were purchased from Santa Cruz biotechnology Inc. Texas 75220, USA.

    Techniques: Expressing, Migration

    Thrombin-induced proliferation and survival are angiopoietin-2-dependent. In all, responses of wild-type (WT) CD34+ cells are shown as white bars, whereas purified CD31+ myofibrocytes from CD31-TFPI-Tg mice are shown as black bars. 0*Indicates incubation with active site inhibited thrombin. Abbreviations: Csi, control scrambled siRNA; Ang-2si, siRNA specific for angiopoietin-2. (A) Expression of angiopoietin-2 at baseline and after incubation with thrombin for 5 days, expressed as the percentage of CD34+cells staining positive in immunocytofluoresence analysis. (B) Secretion of angiopoietin-2 into supernatant after 5-day incubation with thrombin at the indicated concentrations, analyzed by ELISA. (C) Expression of smooth muscle actin (SMA) and joint expression of CD31 with SMA at baseline and after incubation with thombin for 5 days, either alone or with anti-TIE-2 antibody or siRNA against angiopoietin-2 or control. Data expressed as the percentage of CD34+ cells staining positive in immunocytofluoresence analysis. (D) As (C) but cells incubated with angiopoietin-2 ± anti-TIE-2 antibody. (E) Proliferation, assessed by 3 H-thymidine incorporation and expressed as counts per minute (CPM) after incubation with thrombin at the indicated concentrations. (F) Proliferation, assessed by 3 H-thymidine incorporation and expressed as CPM after incubation with angiopoietin-2 at the indicated concentrations. (G) Degree of apoptosis after incubation with thrombin or angiopoietin-2 at the indicated concentrations. Anti-TIE-2 antibody used at 50 ng/ml. All experiments repeated at least twice.

    Journal: Frontiers in Immunology

    Article Title: Inhibition of Angiopoietin-2 Production by Myofibrocytes Inhibits Neointimal Hyperplasia After Endoluminal Injury in Mice

    doi: 10.3389/fimmu.2018.01517

    Figure Lengend Snippet: Thrombin-induced proliferation and survival are angiopoietin-2-dependent. In all, responses of wild-type (WT) CD34+ cells are shown as white bars, whereas purified CD31+ myofibrocytes from CD31-TFPI-Tg mice are shown as black bars. 0*Indicates incubation with active site inhibited thrombin. Abbreviations: Csi, control scrambled siRNA; Ang-2si, siRNA specific for angiopoietin-2. (A) Expression of angiopoietin-2 at baseline and after incubation with thrombin for 5 days, expressed as the percentage of CD34+cells staining positive in immunocytofluoresence analysis. (B) Secretion of angiopoietin-2 into supernatant after 5-day incubation with thrombin at the indicated concentrations, analyzed by ELISA. (C) Expression of smooth muscle actin (SMA) and joint expression of CD31 with SMA at baseline and after incubation with thombin for 5 days, either alone or with anti-TIE-2 antibody or siRNA against angiopoietin-2 or control. Data expressed as the percentage of CD34+ cells staining positive in immunocytofluoresence analysis. (D) As (C) but cells incubated with angiopoietin-2 ± anti-TIE-2 antibody. (E) Proliferation, assessed by 3 H-thymidine incorporation and expressed as counts per minute (CPM) after incubation with thrombin at the indicated concentrations. (F) Proliferation, assessed by 3 H-thymidine incorporation and expressed as CPM after incubation with angiopoietin-2 at the indicated concentrations. (G) Degree of apoptosis after incubation with thrombin or angiopoietin-2 at the indicated concentrations. Anti-TIE-2 antibody used at 50 ng/ml. All experiments repeated at least twice.

    Article Snippet: siRNA Transfection siRNA for angiopoietin 2 (sc-39294), a pool of 3 target-specific 19-25 nt siRNAs designed to knockdown gene expression, and the fluorescein conjugated control siRNAs (sc-36869) were purchased from Santa Cruz biotechnology Inc. Texas 75220, USA.

    Techniques: Purification, Mouse Assay, Incubation, Expressing, Staining, Enzyme-linked Immunosorbent Assay

    Pre-incubation of enhanced yellow fluorescent protein (EYFP) CD34+ cells with reagents to target CXCR4 or angiopoietin-2 prior to adoptive transfer. Panels show immunohistology of consecutive sections through injured mouse carotid arteries harvested on day 28 post-injury. All sections stained with DAPI (4,6 diamidino-2-phenylindole) nuclear stain (blue) and anti-CXCL-12 (red). The green staining is light emitted by the EYFP cells themselves. Yellow indicates co-localization. The annotated white line defines the junction between neointima and media. (A,B) Injured wild-type (WT) mice were adoptively transferred with CD34+ cells on day of injury. In (A) , the cells came from WT mice. In (B) , the cells came from CD31-TFPI-Tg mice. 1 week later, both groups of mice received a second adoptive transfer of EYFP CD34+ cells that were pre-incubated with either isotype control antibody or anti-CXCR4 as indicated. Sections were harvested 1 week later (day 14 post-injury). The annotated white line defines the junction between neointima and media. The anti-CXCR4 prevented late recruitment of adoptively transferred CD34+ cells in (A) , but in (B) , there is no late recruitment of adoptively transferred EYFP cells, even those pre-incubated with control antibody. (C) Adoptive transfer of EYFP CD34+ cells to injured WT mice at the time of injury. Cells first incubated with an anti-TIE-2 or isotype control, or siRNA targeting angiopoietin-2 or scrambled control. Compared to the control cells and those incubated with isotype control antibody, the anti-TIE-2 antibody significantly reduced the neointimal area but did not inhibit angiopoietin-2 expression. Compared to the scrambled siRNA, the specific siRNA targeting angiopoietin-2 significantly reduced the neointimal area in association with abolition of angiopoietin-2 expression cells. Tables besides panels describe summary of staining from all mice ( n = 6), showing the proportion of the neointimal area that is positive for CXCL-12, EYFP, or both on day of examination (% ± SEM). Data derived from three random sections from each of the arteries from each mouse (see Materials and Methods ). *cf isotype for anti-TIE-2 p

    Journal: Frontiers in Immunology

    Article Title: Inhibition of Angiopoietin-2 Production by Myofibrocytes Inhibits Neointimal Hyperplasia After Endoluminal Injury in Mice

    doi: 10.3389/fimmu.2018.01517

    Figure Lengend Snippet: Pre-incubation of enhanced yellow fluorescent protein (EYFP) CD34+ cells with reagents to target CXCR4 or angiopoietin-2 prior to adoptive transfer. Panels show immunohistology of consecutive sections through injured mouse carotid arteries harvested on day 28 post-injury. All sections stained with DAPI (4,6 diamidino-2-phenylindole) nuclear stain (blue) and anti-CXCL-12 (red). The green staining is light emitted by the EYFP cells themselves. Yellow indicates co-localization. The annotated white line defines the junction between neointima and media. (A,B) Injured wild-type (WT) mice were adoptively transferred with CD34+ cells on day of injury. In (A) , the cells came from WT mice. In (B) , the cells came from CD31-TFPI-Tg mice. 1 week later, both groups of mice received a second adoptive transfer of EYFP CD34+ cells that were pre-incubated with either isotype control antibody or anti-CXCR4 as indicated. Sections were harvested 1 week later (day 14 post-injury). The annotated white line defines the junction between neointima and media. The anti-CXCR4 prevented late recruitment of adoptively transferred CD34+ cells in (A) , but in (B) , there is no late recruitment of adoptively transferred EYFP cells, even those pre-incubated with control antibody. (C) Adoptive transfer of EYFP CD34+ cells to injured WT mice at the time of injury. Cells first incubated with an anti-TIE-2 or isotype control, or siRNA targeting angiopoietin-2 or scrambled control. Compared to the control cells and those incubated with isotype control antibody, the anti-TIE-2 antibody significantly reduced the neointimal area but did not inhibit angiopoietin-2 expression. Compared to the scrambled siRNA, the specific siRNA targeting angiopoietin-2 significantly reduced the neointimal area in association with abolition of angiopoietin-2 expression cells. Tables besides panels describe summary of staining from all mice ( n = 6), showing the proportion of the neointimal area that is positive for CXCL-12, EYFP, or both on day of examination (% ± SEM). Data derived from three random sections from each of the arteries from each mouse (see Materials and Methods ). *cf isotype for anti-TIE-2 p

    Article Snippet: siRNA Transfection siRNA for angiopoietin 2 (sc-39294), a pool of 3 target-specific 19-25 nt siRNAs designed to knockdown gene expression, and the fluorescein conjugated control siRNAs (sc-36869) were purchased from Santa Cruz biotechnology Inc. Texas 75220, USA.

    Techniques: Incubation, Adoptive Transfer Assay, Staining, Mouse Assay, Expressing, Derivative Assay

    Adoptive transfer of CD31-TFPI-Tg CD34+ cells to wild-type (WT) mice immediately post-injury. Panels show immunohistology of representative sections through injured mouse carotid arteries harvested on days post-injury as indicated. All sections stained with DAPI (4,6 diamidino-2-phenylindole) nuclear stain (blue) and (red) anti-collagen-1 (A) or human tissue factor pathway inhibitor (B) and (green) anti-angiopoietin-2. Yellow indicates co-localization. The annotated white line defines the junction between neointima and media. *On day 0, sections harvested within an hour of injury. Table besides panel (A) describes summary of staining from all mice ( n = 6), showing the proportion of the neointimal area that is positive for collagen-1, angiopoietin-2, or both on days 0, 7, and 28 (% ± SEM). Data derived from three random sections from each of the arteries from each mouse (see Materials and Methods ). *cf WT cells (see Figure 1 B) at equivalent time point p = 0.03. † cf WT cells (Figure 1 B) at equivalent time point p

    Journal: Frontiers in Immunology

    Article Title: Inhibition of Angiopoietin-2 Production by Myofibrocytes Inhibits Neointimal Hyperplasia After Endoluminal Injury in Mice

    doi: 10.3389/fimmu.2018.01517

    Figure Lengend Snippet: Adoptive transfer of CD31-TFPI-Tg CD34+ cells to wild-type (WT) mice immediately post-injury. Panels show immunohistology of representative sections through injured mouse carotid arteries harvested on days post-injury as indicated. All sections stained with DAPI (4,6 diamidino-2-phenylindole) nuclear stain (blue) and (red) anti-collagen-1 (A) or human tissue factor pathway inhibitor (B) and (green) anti-angiopoietin-2. Yellow indicates co-localization. The annotated white line defines the junction between neointima and media. *On day 0, sections harvested within an hour of injury. Table besides panel (A) describes summary of staining from all mice ( n = 6), showing the proportion of the neointimal area that is positive for collagen-1, angiopoietin-2, or both on days 0, 7, and 28 (% ± SEM). Data derived from three random sections from each of the arteries from each mouse (see Materials and Methods ). *cf WT cells (see Figure 1 B) at equivalent time point p = 0.03. † cf WT cells (Figure 1 B) at equivalent time point p

    Article Snippet: siRNA Transfection siRNA for angiopoietin 2 (sc-39294), a pool of 3 target-specific 19-25 nt siRNAs designed to knockdown gene expression, and the fluorescein conjugated control siRNAs (sc-36869) were purchased from Santa Cruz biotechnology Inc. Texas 75220, USA.

    Techniques: Adoptive Transfer Assay, Mouse Assay, Staining, Derivative Assay

    Impact of coagulation proteases on myofibrocyte phenotype. In (A,B) , responses of wild-type (WT) CD34+ cells are shown as white bars, whereas isolated CD31+ myofibrocytes from CD31-TFPI-Tg mice are shown as black bars. (A) Cells were incubated with FX in presence or absence of FVIIa and FII (prothrombin) plus FVa. Functional tissue factor on WT cells is illustrated by thrombin generation, angiopoietin-2 (Ang-2) secretion, and CXCL-12 secretion. The presence of human tissue factor pathway inhibitor on purified CD31+ myofibrocytes from CD31-TFPI-Tg mice significantly inhibits all three phenotype changes. (B) Proliferation, assessed by 3 H-thymidine incorporation and expressed as counts per minute (CPM) after incubation with FX and FII in presence of FVIIa. (C) Angiopoietin-2 secretion by WT CD34+ cells (3 × 10 4 /well) after 24 h incubation with either PAR-1 antagonist (black bars), PAR-2 antagonist (white bars), or PAR-4 antagonist (gray bars) at the indicated concentrations for 30 min before addition of FVIIa with FX (both 10 nM) with or without prothrombin (4 nM) and FVa (6 nM) as indicated. All conditions performed in triplicate wells. Error bars indicate SEM. In comparison of increasing concentrations of antagonists with FVIIa + FX, p = 0.027 for PAR2, but p = NS for PAR1 and PAR4. In comparison of increasing concentrations of antagonists with FVIIa + FX + FII + FVa, p = 0.05 for PAR1, but p = not significant (NS) for PAR2 and PAR4. Analysis by one-way ANOVA Kruskal–Wallis test. (D) Angiopoietin-2 secretion by WT CD34+ cells (3 × 10 4 /well) after 24 h incubation with PAR-1, -2, or -4 agonists at the indicated concentrations. All conditions performed in triplicate wells. Error bars indicate SEM. p = 0.017 for comparisons of increasing concentrations of PAR1 agonist, p = 0.012 for PAR2, but p = NS for PAR4 agonist. Analysis by one-way ANOVA Kruskal–Wallis test. (E) Dissection of signaling pathways involved in angiopoietin-2 secretion by WT CD34+ cells induced by 24 h incubation with 10 mM PAR-1 or -2 agonists. Cells were incubated with the agonists with or without 50 mM mitogen-activated protein kinase inhibitor PD98059, 10 mM p38-MAPK inhibitor SB203580, 20 mM NF-kB inhibitor SN50, or 1 mM of the S6K1 inhibitor as indicated. All conditions performed in triplicate wells. Error bars indicate SEM. p = 0.05 for PAR-1 agonist without inhibitor vs. +PD98509 and vs. +SB203580. p = > 0.05 all other comparisons. Analysis by Mann–Whitney T test. All experiments repeated at least twice.

    Journal: Frontiers in Immunology

    Article Title: Inhibition of Angiopoietin-2 Production by Myofibrocytes Inhibits Neointimal Hyperplasia After Endoluminal Injury in Mice

    doi: 10.3389/fimmu.2018.01517

    Figure Lengend Snippet: Impact of coagulation proteases on myofibrocyte phenotype. In (A,B) , responses of wild-type (WT) CD34+ cells are shown as white bars, whereas isolated CD31+ myofibrocytes from CD31-TFPI-Tg mice are shown as black bars. (A) Cells were incubated with FX in presence or absence of FVIIa and FII (prothrombin) plus FVa. Functional tissue factor on WT cells is illustrated by thrombin generation, angiopoietin-2 (Ang-2) secretion, and CXCL-12 secretion. The presence of human tissue factor pathway inhibitor on purified CD31+ myofibrocytes from CD31-TFPI-Tg mice significantly inhibits all three phenotype changes. (B) Proliferation, assessed by 3 H-thymidine incorporation and expressed as counts per minute (CPM) after incubation with FX and FII in presence of FVIIa. (C) Angiopoietin-2 secretion by WT CD34+ cells (3 × 10 4 /well) after 24 h incubation with either PAR-1 antagonist (black bars), PAR-2 antagonist (white bars), or PAR-4 antagonist (gray bars) at the indicated concentrations for 30 min before addition of FVIIa with FX (both 10 nM) with or without prothrombin (4 nM) and FVa (6 nM) as indicated. All conditions performed in triplicate wells. Error bars indicate SEM. In comparison of increasing concentrations of antagonists with FVIIa + FX, p = 0.027 for PAR2, but p = NS for PAR1 and PAR4. In comparison of increasing concentrations of antagonists with FVIIa + FX + FII + FVa, p = 0.05 for PAR1, but p = not significant (NS) for PAR2 and PAR4. Analysis by one-way ANOVA Kruskal–Wallis test. (D) Angiopoietin-2 secretion by WT CD34+ cells (3 × 10 4 /well) after 24 h incubation with PAR-1, -2, or -4 agonists at the indicated concentrations. All conditions performed in triplicate wells. Error bars indicate SEM. p = 0.017 for comparisons of increasing concentrations of PAR1 agonist, p = 0.012 for PAR2, but p = NS for PAR4 agonist. Analysis by one-way ANOVA Kruskal–Wallis test. (E) Dissection of signaling pathways involved in angiopoietin-2 secretion by WT CD34+ cells induced by 24 h incubation with 10 mM PAR-1 or -2 agonists. Cells were incubated with the agonists with or without 50 mM mitogen-activated protein kinase inhibitor PD98059, 10 mM p38-MAPK inhibitor SB203580, 20 mM NF-kB inhibitor SN50, or 1 mM of the S6K1 inhibitor as indicated. All conditions performed in triplicate wells. Error bars indicate SEM. p = 0.05 for PAR-1 agonist without inhibitor vs. +PD98509 and vs. +SB203580. p = > 0.05 all other comparisons. Analysis by Mann–Whitney T test. All experiments repeated at least twice.

    Article Snippet: siRNA Transfection siRNA for angiopoietin 2 (sc-39294), a pool of 3 target-specific 19-25 nt siRNAs designed to knockdown gene expression, and the fluorescein conjugated control siRNAs (sc-36869) were purchased from Santa Cruz biotechnology Inc. Texas 75220, USA.

    Techniques: Coagulation, Isolation, Mouse Assay, Incubation, Functional Assay, Purification, Dissection, MANN-WHITNEY

    Comparison of phenotype of intimal hyperplasia in wild-type (WT) animals vs. WT after adoptive transfer of WT CD34+ cells immediately post-injury. Panels show immunohistology of representative sections through injured mouse carotid arteries harvested on days post-injury as indicated. All sections stained with DAPI (4,6 diamidino-2-phenylindole) nuclear stain (blue) and (red) anti-collagen-1 (A,B) , or angiopoietin-2 (C) , and (green) anti-angiopoietin-2 (A,B) . Yellow indicates co-localization. In (C) , the adoptively transferred cells came from enhanced yellow fluorescent protein (EYFP) mice, which spontaneously fluoresce as shown. The annotated white line defines the junction between neointima and media. (A) Injured mice received no adoptively transferred cells. *On day 0, sections harvested within an hour of injury. (B) Mice injected with cells from WT mice. (C) Mice injected with cells from EYFP mice. Expanded area illustrates that most of the early recruited cells are adoptively transferred, angiopoietin-2+ cells. Tables beside each panel describe summary of staining from all mice ( n = 6), showing the proportion of the neointimal area that is positive for collagen-1, angiopoietin-2 or both or EYFP, angiopoietin-2 or both on days 0, 5/7, and day 28 (% ± SEM). Data derived from three random sections from each of the arteries from each mouse (see Materials and Methods ). Subtracting the proportional area occupied by dual positive cells from the total area occupied by angiopoietin-2+ cells suggests the proportional area occupied by collagen-1-negative cells expressing angiopoietin-2. *cf WT (no adoptive transfer) at equivalent time: p

    Journal: Frontiers in Immunology

    Article Title: Inhibition of Angiopoietin-2 Production by Myofibrocytes Inhibits Neointimal Hyperplasia After Endoluminal Injury in Mice

    doi: 10.3389/fimmu.2018.01517

    Figure Lengend Snippet: Comparison of phenotype of intimal hyperplasia in wild-type (WT) animals vs. WT after adoptive transfer of WT CD34+ cells immediately post-injury. Panels show immunohistology of representative sections through injured mouse carotid arteries harvested on days post-injury as indicated. All sections stained with DAPI (4,6 diamidino-2-phenylindole) nuclear stain (blue) and (red) anti-collagen-1 (A,B) , or angiopoietin-2 (C) , and (green) anti-angiopoietin-2 (A,B) . Yellow indicates co-localization. In (C) , the adoptively transferred cells came from enhanced yellow fluorescent protein (EYFP) mice, which spontaneously fluoresce as shown. The annotated white line defines the junction between neointima and media. (A) Injured mice received no adoptively transferred cells. *On day 0, sections harvested within an hour of injury. (B) Mice injected with cells from WT mice. (C) Mice injected with cells from EYFP mice. Expanded area illustrates that most of the early recruited cells are adoptively transferred, angiopoietin-2+ cells. Tables beside each panel describe summary of staining from all mice ( n = 6), showing the proportion of the neointimal area that is positive for collagen-1, angiopoietin-2 or both or EYFP, angiopoietin-2 or both on days 0, 5/7, and day 28 (% ± SEM). Data derived from three random sections from each of the arteries from each mouse (see Materials and Methods ). Subtracting the proportional area occupied by dual positive cells from the total area occupied by angiopoietin-2+ cells suggests the proportional area occupied by collagen-1-negative cells expressing angiopoietin-2. *cf WT (no adoptive transfer) at equivalent time: p

    Article Snippet: siRNA Transfection siRNA for angiopoietin 2 (sc-39294), a pool of 3 target-specific 19-25 nt siRNAs designed to knockdown gene expression, and the fluorescein conjugated control siRNAs (sc-36869) were purchased from Santa Cruz biotechnology Inc. Texas 75220, USA.

    Techniques: Adoptive Transfer Assay, Staining, Mouse Assay, Injection, Derivative Assay, Expressing

    Interpretation of our findings. (A) Post-endoluminal injury, fibrocytes (green) are mobilized into the circulation, a subpopulation of which express smooth muscle actin (SMA) (“myofibrocytes”-yellow cells) and CD31 and these are recruited to the site of injury, via CXCR4 from CXCL-12 released from luminal platelets (data not shown). These myofibrocytes express tissue factor (TF), which generates FXa and thrombin (FIIa), which can signal through PARs-1 and -2 on the cell surface. These result in the secretion of angiopoietin-2, which through cell surface TIE-2, causes proliferation and secretion of CXCL-12, which can attract more myofibrocytes from the circulation. The myofibrocytes also orchestrate expression of angiopoietin-2 by other cells within the vessel wall, by a mechanism not elucidated. (B) Inhibition of TF on the myofibrocytes, either by cell surface expression of a transgenic human tissue factor pathway inhibitor (hTFPI) fusion protein, or by an anti-TF antibody inhibits angiopoietin-2 secretion and results in a small neointima, consisting of non-proliferating myofibrocytes. An siRNA to angiopoietin-2 or a blocking anti-TIE-2 antibody has the same effect, illustrating the key importance of the angiopoietin-2/TIE axis for the development of intimal hyperplasia. *Treatment of whole CD34+ cells with an anti-TF inhibits intimal hyperplasia (IH) but also prevents significant recruitment of myofibrocytes, in a similar way as expressing hTFPI fusion protein on SMA+ fibrocytes ( 8 ).

    Journal: Frontiers in Immunology

    Article Title: Inhibition of Angiopoietin-2 Production by Myofibrocytes Inhibits Neointimal Hyperplasia After Endoluminal Injury in Mice

    doi: 10.3389/fimmu.2018.01517

    Figure Lengend Snippet: Interpretation of our findings. (A) Post-endoluminal injury, fibrocytes (green) are mobilized into the circulation, a subpopulation of which express smooth muscle actin (SMA) (“myofibrocytes”-yellow cells) and CD31 and these are recruited to the site of injury, via CXCR4 from CXCL-12 released from luminal platelets (data not shown). These myofibrocytes express tissue factor (TF), which generates FXa and thrombin (FIIa), which can signal through PARs-1 and -2 on the cell surface. These result in the secretion of angiopoietin-2, which through cell surface TIE-2, causes proliferation and secretion of CXCL-12, which can attract more myofibrocytes from the circulation. The myofibrocytes also orchestrate expression of angiopoietin-2 by other cells within the vessel wall, by a mechanism not elucidated. (B) Inhibition of TF on the myofibrocytes, either by cell surface expression of a transgenic human tissue factor pathway inhibitor (hTFPI) fusion protein, or by an anti-TF antibody inhibits angiopoietin-2 secretion and results in a small neointima, consisting of non-proliferating myofibrocytes. An siRNA to angiopoietin-2 or a blocking anti-TIE-2 antibody has the same effect, illustrating the key importance of the angiopoietin-2/TIE axis for the development of intimal hyperplasia. *Treatment of whole CD34+ cells with an anti-TF inhibits intimal hyperplasia (IH) but also prevents significant recruitment of myofibrocytes, in a similar way as expressing hTFPI fusion protein on SMA+ fibrocytes ( 8 ).

    Article Snippet: siRNA Transfection siRNA for angiopoietin 2 (sc-39294), a pool of 3 target-specific 19-25 nt siRNAs designed to knockdown gene expression, and the fluorescein conjugated control siRNAs (sc-36869) were purchased from Santa Cruz biotechnology Inc. Texas 75220, USA.

    Techniques: Expressing, Inhibition, Transgenic Assay, Blocking Assay

    Puerarin upregulates key proangiogenic factors VEGFA, Ang-1 and Ang-2 in response to LAD ligation stress. A: Slightly increase of VEGFA, Ang-1 and Ang-2 protein in the MI and MI+S groups in response to MI was detected by western blot. However, protein

    Journal: International Journal of Clinical and Experimental Medicine

    Article Title: Puerarin accelerate scardiac angiogenesis and improves cardiac function of myocardial infarction by upregulating VEGFA, Ang-1 and Ang-2 in rats

    doi:

    Figure Lengend Snippet: Puerarin upregulates key proangiogenic factors VEGFA, Ang-1 and Ang-2 in response to LAD ligation stress. A: Slightly increase of VEGFA, Ang-1 and Ang-2 protein in the MI and MI+S groups in response to MI was detected by western blot. However, protein

    Article Snippet: Primary antibodies against Ang-1 (ab118380) and Ang-2 (ab155106) were purchased from Abcam.

    Techniques: Ligation, Western Blot

    ANGPT2 expression in the uterus on D5, D6, and D8. (A) Immunohistochemistry of ANGPT2 in paraffin sections of the uterus. The right pictures are the higher magnification images of the black boxes on the left. Arrow indicates the positive staining. L, lumen; E, embryo; M, mesometrial; AM, anti-mesometrial; VSF, vascular sinus folding. Original magnification and scale bar were shown in the pictures. (B) Protein levels of ANGPT2 in pregnant uterus (remove embryo) by western blot analysis ( n = 3). (C) Quantification of ANGPT2 protein expression. (D) mRNA levels of ANGPT2 in pregnant uterus (remove embryo) by qRT-PCR ( n = 5). Data represent mean ± SD. β-actin was used as the reference.

    Journal: Frontiers in Pharmacology

    Article Title: Bushen Huoxue Recipe Alleviates Implantation Loss in Mice by Enhancing Estrogen–Progesterone Signals and Promoting Decidual Angiogenesis Through FGF2 During Early Pregnancy

    doi: 10.3389/fphar.2018.00437

    Figure Lengend Snippet: ANGPT2 expression in the uterus on D5, D6, and D8. (A) Immunohistochemistry of ANGPT2 in paraffin sections of the uterus. The right pictures are the higher magnification images of the black boxes on the left. Arrow indicates the positive staining. L, lumen; E, embryo; M, mesometrial; AM, anti-mesometrial; VSF, vascular sinus folding. Original magnification and scale bar were shown in the pictures. (B) Protein levels of ANGPT2 in pregnant uterus (remove embryo) by western blot analysis ( n = 3). (C) Quantification of ANGPT2 protein expression. (D) mRNA levels of ANGPT2 in pregnant uterus (remove embryo) by qRT-PCR ( n = 5). Data represent mean ± SD. β-actin was used as the reference.

    Article Snippet: Anti-ERα (sc-542; 1:1000; Santa Cruz Biotechnology), anti-PR (ab133526; 1:1000; Abcam), anti-VEGFA (AF-493-NA; 1:1000; R & D Systems), anti-ANGPT2 (ab155106; 1:1000; Abcam), anti-FGF2 (sc-79; 1:100; Santa Cruz Biotechnology), and anti-β-actin (GB13001-1; 1:1000; Goodbio Technology Co, Wuhan, China) antibodies were used for incubation overnight at 4°C.

    Techniques: Expressing, Immunohistochemistry, Staining, Western Blot, Quantitative RT-PCR

    Ang-2 expression, gene transcription and shRNA suppression Ang-2 expression and gene transcription in lung Beas-2B cell, lung cancer (SPC-A-1, NCI-1650, A549, and NCI-1975) cell lines were analyzed at protein- by Western blotting or at mRNA-level by qRT-PCR (A) , the Ang-2 expressions in different cells at protein level with β-actin as control; (B) , the ratios from Ang-2 to β-actin in different cells at protein level; (C) , the ratios from Ang-2 to GAPDH expression in different cells at mRNA level; (D) , the phase-contrast image (X100 magnification) with the Ang-2-shRNA1 successfully transfected into A549 cells at 24 h; (E) , the fluorescence image (X100 magnification) with the Ang-2-shRNA1 successfully transfected into A549 cells; (F) , the different alterations of Ang-2 expression with β-actin as loading control at 48 h; (G) , the ratios from Ang-2 to β-actin protein in different cell lines; and (H) , the similar alterations of Ang-2 normalized to GAPDH in different cell lines at mRNA level. Ang-2 , angiopoietin-2; Ang-2 Exp., angiopoietin-2 protein expression; shRNA , the Ang-2-shRNA transfection group; NC , the negative control group; and Control , the blank control group. * P

    Journal: Oncotarget

    Article Title: Ang-2 promotes lung cancer metastasis by increasing epithelial-mesenchymal transition

    doi: 10.18632/oncotarget.24061

    Figure Lengend Snippet: Ang-2 expression, gene transcription and shRNA suppression Ang-2 expression and gene transcription in lung Beas-2B cell, lung cancer (SPC-A-1, NCI-1650, A549, and NCI-1975) cell lines were analyzed at protein- by Western blotting or at mRNA-level by qRT-PCR (A) , the Ang-2 expressions in different cells at protein level with β-actin as control; (B) , the ratios from Ang-2 to β-actin in different cells at protein level; (C) , the ratios from Ang-2 to GAPDH expression in different cells at mRNA level; (D) , the phase-contrast image (X100 magnification) with the Ang-2-shRNA1 successfully transfected into A549 cells at 24 h; (E) , the fluorescence image (X100 magnification) with the Ang-2-shRNA1 successfully transfected into A549 cells; (F) , the different alterations of Ang-2 expression with β-actin as loading control at 48 h; (G) , the ratios from Ang-2 to β-actin protein in different cell lines; and (H) , the similar alterations of Ang-2 normalized to GAPDH in different cell lines at mRNA level. Ang-2 , angiopoietin-2; Ang-2 Exp., angiopoietin-2 protein expression; shRNA , the Ang-2-shRNA transfection group; NC , the negative control group; and Control , the blank control group. * P

    Article Snippet: Equivalent amounts (50 μg/per lane) from each sample was separated using a 10% SDS-polyacrylamide gels at 80 V for 40 min, then 120 V for 1h, and finally the gel was transferred to polyvinylidine difluoride (PVDF) membranes (Millipore Corporation, USA) at 300 mA for 120 min, blocked in 5% bovine serum albumin (BSA) in blocking buffer (Solarbio, China), and incubated with the primary antibodies rabbit anti-human Ang-2 (ab155106, diluted 1:1000; Abcam, UK), or anti-E-Cadherin (ab1416, diluted 1: 200; Abcam, UK), or anti-Vimentin (VIM, ab20346, diluted 1:2000; Abcam, UK), anti-Snail (ab167609, diluted 1:500; Abcam, UK), anti-Twist (ab50887, diluted 1:200; Abcam, UK), and β-actin (1:1000 dilution, CST, USA) overnight at 4°C, followed by incubation with secondary antibody horseradish peroxidase-conjugated (HRP)-conjugated goat anti-rabbit antibody (1:1000, Abbkine, China) at room temperature for 2h.

    Techniques: Expressing, shRNA, Western Blot, Quantitative RT-PCR, Transfection, Fluorescence, Negative Control

    Kaplan-Meier survival curves of Ang-2 overexpressionThe survival curves of high Ang-2 expression in patients with lung cancer were made by the Kaplan-Meier method (A) , the overall survival curve of high Ang-2 expression in patients with lung cancer; (B) , the overall survival curve of high Ang-2 expression in patients with lung cancer at TNM stage I; (C) , the overall survival curve of high Ang-2 expression in patients with lung cancer at stage II; and (D) , the overall survival curve of high Ang-2 expression in patinets with lung cancer at stage III-IV, respectively.

    Journal: Oncotarget

    Article Title: Ang-2 promotes lung cancer metastasis by increasing epithelial-mesenchymal transition

    doi: 10.18632/oncotarget.24061

    Figure Lengend Snippet: Kaplan-Meier survival curves of Ang-2 overexpressionThe survival curves of high Ang-2 expression in patients with lung cancer were made by the Kaplan-Meier method (A) , the overall survival curve of high Ang-2 expression in patients with lung cancer; (B) , the overall survival curve of high Ang-2 expression in patients with lung cancer at TNM stage I; (C) , the overall survival curve of high Ang-2 expression in patients with lung cancer at stage II; and (D) , the overall survival curve of high Ang-2 expression in patinets with lung cancer at stage III-IV, respectively.

    Article Snippet: Equivalent amounts (50 μg/per lane) from each sample was separated using a 10% SDS-polyacrylamide gels at 80 V for 40 min, then 120 V for 1h, and finally the gel was transferred to polyvinylidine difluoride (PVDF) membranes (Millipore Corporation, USA) at 300 mA for 120 min, blocked in 5% bovine serum albumin (BSA) in blocking buffer (Solarbio, China), and incubated with the primary antibodies rabbit anti-human Ang-2 (ab155106, diluted 1:1000; Abcam, UK), or anti-E-Cadherin (ab1416, diluted 1: 200; Abcam, UK), or anti-Vimentin (VIM, ab20346, diluted 1:2000; Abcam, UK), anti-Snail (ab167609, diluted 1:500; Abcam, UK), anti-Twist (ab50887, diluted 1:200; Abcam, UK), and β-actin (1:1000 dilution, CST, USA) overnight at 4°C, followed by incubation with secondary antibody horseradish peroxidase-conjugated (HRP)-conjugated goat anti-rabbit antibody (1:1000, Abbkine, China) at room temperature for 2h.

    Techniques: Expressing

    Possibility mechanism of tumor derived Ang-2 up-regulating EMT and facilitating lung cancer metastasis Ang-2 has been implicated in mediating inflammatory processes, and upregulated in multiple inflammation-related tumors or signaling pathway. With lung cancer volume increasing, intratumoral hypoxia in the absence of vascularization resulted in tumor cells to express Ang-2 inducing angiogenesis for tumor growth and increasing vascular permeability. Overexpression of Ang-2 facilitating proliferation, invasion, metastasis, and EMT of lung cancer cells that could be a new mechanism insight into tumor metastasis by inhibiting E-cadherin and upregulating VIM, Twist and Snail signaling. Ang , angiopoietin; Ang-2 , angiopoietin-2; CaMK, calcium/calmodulin-dependent protein kinase; EMT , epithelial-mesenchymal transition; ENO1 , enolase 1; EPO , erythropoietin; ET1 , endothelin-1; GAPDH , glyceraldehyde-3-phosphate dehydrogenase; Glut1 , glucose transporter 1; HIF-1α , hypoxia-inducible factor-1α; HK1 , hexokinase 1; HRE , hypoxia-response element; IGF2 , insulin-like growth factor 2; IP3 , inositol 1,4,5-trisphate; mTOR , mechanistic target of rapamycin kinase; NOS2 , nitric oxide synthase 2; Oncogenes , ras, c-myc etc; PLC-γ , phospholipase C gamma; PI3K, phosphatidylinositol 3-kinase; PKC , protein kinase C; RTK, receptor tyrosine kinase; TGFA , transforming growth factor alpha; tumor suppressor genes , pVHL, P 53 , PTEN, etc. VEGF , vascular endothelial growth factors; VHL , von Hippel-Lidau; VIM , Vimentin.

    Journal: Oncotarget

    Article Title: Ang-2 promotes lung cancer metastasis by increasing epithelial-mesenchymal transition

    doi: 10.18632/oncotarget.24061

    Figure Lengend Snippet: Possibility mechanism of tumor derived Ang-2 up-regulating EMT and facilitating lung cancer metastasis Ang-2 has been implicated in mediating inflammatory processes, and upregulated in multiple inflammation-related tumors or signaling pathway. With lung cancer volume increasing, intratumoral hypoxia in the absence of vascularization resulted in tumor cells to express Ang-2 inducing angiogenesis for tumor growth and increasing vascular permeability. Overexpression of Ang-2 facilitating proliferation, invasion, metastasis, and EMT of lung cancer cells that could be a new mechanism insight into tumor metastasis by inhibiting E-cadherin and upregulating VIM, Twist and Snail signaling. Ang , angiopoietin; Ang-2 , angiopoietin-2; CaMK, calcium/calmodulin-dependent protein kinase; EMT , epithelial-mesenchymal transition; ENO1 , enolase 1; EPO , erythropoietin; ET1 , endothelin-1; GAPDH , glyceraldehyde-3-phosphate dehydrogenase; Glut1 , glucose transporter 1; HIF-1α , hypoxia-inducible factor-1α; HK1 , hexokinase 1; HRE , hypoxia-response element; IGF2 , insulin-like growth factor 2; IP3 , inositol 1,4,5-trisphate; mTOR , mechanistic target of rapamycin kinase; NOS2 , nitric oxide synthase 2; Oncogenes , ras, c-myc etc; PLC-γ , phospholipase C gamma; PI3K, phosphatidylinositol 3-kinase; PKC , protein kinase C; RTK, receptor tyrosine kinase; TGFA , transforming growth factor alpha; tumor suppressor genes , pVHL, P 53 , PTEN, etc. VEGF , vascular endothelial growth factors; VHL , von Hippel-Lidau; VIM , Vimentin.

    Article Snippet: Equivalent amounts (50 μg/per lane) from each sample was separated using a 10% SDS-polyacrylamide gels at 80 V for 40 min, then 120 V for 1h, and finally the gel was transferred to polyvinylidine difluoride (PVDF) membranes (Millipore Corporation, USA) at 300 mA for 120 min, blocked in 5% bovine serum albumin (BSA) in blocking buffer (Solarbio, China), and incubated with the primary antibodies rabbit anti-human Ang-2 (ab155106, diluted 1:1000; Abcam, UK), or anti-E-Cadherin (ab1416, diluted 1: 200; Abcam, UK), or anti-Vimentin (VIM, ab20346, diluted 1:2000; Abcam, UK), anti-Snail (ab167609, diluted 1:500; Abcam, UK), anti-Twist (ab50887, diluted 1:200; Abcam, UK), and β-actin (1:1000 dilution, CST, USA) overnight at 4°C, followed by incubation with secondary antibody horseradish peroxidase-conjugated (HRP)-conjugated goat anti-rabbit antibody (1:1000, Abbkine, China) at room temperature for 2h.

    Techniques: Derivative Assay, Permeability, Over Expression, Planar Chromatography

    Working model of the roles of MMP-2 and MMP-9 in retinoblastoma cells. Y79 and Weri-1 cells represent the metastatic and the non-metastatic model for Rb, respectively. Our work shows differences in viability, migration and angiogenic-associated responses in Rb cells after inhibition of MMP-2 and MMP-9. a Y79 cells showed a profound defect in migration and invasion along with and a significant reduction in Angiopoietin-2 and TGF-β1 proteins. These results highlight Y79’s migratory and invasive potential, which may be dependent upon MMPs. b Analyses of Weri-1 cells show MMP-2 and MMP-9 are involved in multiple processes, including viability of cells and VEGF, as well as TGF-β1 production

    Journal: BMC Cancer

    Article Title: Inhibition of MMP-2 and MMP-9 decreases cellular migration, and angiogenesis in in vitro models of retinoblastoma

    doi: 10.1186/s12885-017-3418-y

    Figure Lengend Snippet: Working model of the roles of MMP-2 and MMP-9 in retinoblastoma cells. Y79 and Weri-1 cells represent the metastatic and the non-metastatic model for Rb, respectively. Our work shows differences in viability, migration and angiogenic-associated responses in Rb cells after inhibition of MMP-2 and MMP-9. a Y79 cells showed a profound defect in migration and invasion along with and a significant reduction in Angiopoietin-2 and TGF-β1 proteins. These results highlight Y79’s migratory and invasive potential, which may be dependent upon MMPs. b Analyses of Weri-1 cells show MMP-2 and MMP-9 are involved in multiple processes, including viability of cells and VEGF, as well as TGF-β1 production

    Article Snippet: Human Angiopoietin-2 was purchased from Sigma-Aldrich (St. Louis, MO).

    Techniques: Migration, Inhibition

    HG modulates angiopoietin-2 in diabetic bone marrow

    Journal: Stem cells (Dayton, Ohio)

    Article Title: Switch from canonical to noncanonical Wnt signaling mediates high glucose-induced adipogenesis

    doi: 10.1002/stem.1659

    Figure Lengend Snippet: HG modulates angiopoietin-2 in diabetic bone marrow

    Article Snippet: Recombinant human Ang2 (623-AN-025; R & D Systems) and Wnt11 in DMEM media were used to create standard curves.

    Techniques:

    CXCL-12 expression is thrombin and angiopoietin-2-dependent. (A) Expression of CXCL-12 at baseline and after incubation with thrombin or angiopoietin-2 for 5 days, expressed as the percentage of cells staining positive in immunocytofluorescence analysis. Anti-TIE-2 antibody used at 50 ng/ml. (B) Secretion of CXCL-12 into supernatant after 5 day incubation with thrombin at the indicated concentrations, analyzed by ELISA. (C) Secretion of CXCL-12 into supernatant after 5 day incubation with angiopoietin-2 at the indicated concentrations, analyzed by ELISA. All data shown from WT CD34+ cells. Abbreviations: Csi , control siRNA. Ang-2si , siRNA specific for angiopoietin-2. Experiments repeated at least twice.

    Journal: Frontiers in Immunology

    Article Title: Inhibition of Angiopoietin-2 Production by Myofibrocytes Inhibits Neointimal Hyperplasia After Endoluminal Injury in Mice

    doi: 10.3389/fimmu.2018.01517

    Figure Lengend Snippet: CXCL-12 expression is thrombin and angiopoietin-2-dependent. (A) Expression of CXCL-12 at baseline and after incubation with thrombin or angiopoietin-2 for 5 days, expressed as the percentage of cells staining positive in immunocytofluorescence analysis. Anti-TIE-2 antibody used at 50 ng/ml. (B) Secretion of CXCL-12 into supernatant after 5 day incubation with thrombin at the indicated concentrations, analyzed by ELISA. (C) Secretion of CXCL-12 into supernatant after 5 day incubation with angiopoietin-2 at the indicated concentrations, analyzed by ELISA. All data shown from WT CD34+ cells. Abbreviations: Csi , control siRNA. Ang-2si , siRNA specific for angiopoietin-2. Experiments repeated at least twice.

    Article Snippet: Frozen sections were immersed in 1% BSA(Sigma)-PBS for 30 min and then incubated overnight at 4°C with the following antibodies: goat anti-angiopoietin-2 (Santa Cruz biotechnology Inc.), mouse monoclonal anti-collagen 1 (as a marker of fibocytes), rabbit anti-CXCR4, rabbit anti-CXCL12 (all from Abcam), mouse anti-human TFPI (Enzyme Research Laboratories, Swansea, UK), and rat anti-mouse CD31(BD).

    Techniques: Expressing, Incubation, Staining, Enzyme-linked Immunosorbent Assay

    Association between intimal hyperplasia and plasma levels of angiopoietin-2 after wire-induced endoluminal injury. (A) Plasma angiopoietin-2 levels in wild-type (WT) mice. Levels in unninjured mice represented by the horizontal bar day 0 only; (squares)—WT CD34+ cells; (circles)—unfractionated CD31-TFPI-Tg CD34+ cells; (diamonds) human tissue factor pathway inhibitor (hTFPI)+ fraction of CD31-TFPI-Tg CD34+ cells; (triangles)—hTFPI-negative fraction of CD31-TFPI-Tg CD34+ cells. *Day 7 p

    Journal: Frontiers in Immunology

    Article Title: Inhibition of Angiopoietin-2 Production by Myofibrocytes Inhibits Neointimal Hyperplasia After Endoluminal Injury in Mice

    doi: 10.3389/fimmu.2018.01517

    Figure Lengend Snippet: Association between intimal hyperplasia and plasma levels of angiopoietin-2 after wire-induced endoluminal injury. (A) Plasma angiopoietin-2 levels in wild-type (WT) mice. Levels in unninjured mice represented by the horizontal bar day 0 only; (squares)—WT CD34+ cells; (circles)—unfractionated CD31-TFPI-Tg CD34+ cells; (diamonds) human tissue factor pathway inhibitor (hTFPI)+ fraction of CD31-TFPI-Tg CD34+ cells; (triangles)—hTFPI-negative fraction of CD31-TFPI-Tg CD34+ cells. *Day 7 p

    Article Snippet: Frozen sections were immersed in 1% BSA(Sigma)-PBS for 30 min and then incubated overnight at 4°C with the following antibodies: goat anti-angiopoietin-2 (Santa Cruz biotechnology Inc.), mouse monoclonal anti-collagen 1 (as a marker of fibocytes), rabbit anti-CXCR4, rabbit anti-CXCL12 (all from Abcam), mouse anti-human TFPI (Enzyme Research Laboratories, Swansea, UK), and rat anti-mouse CD31(BD).

    Techniques: Mouse Assay

    Pre-incubation of enhanced yellow fluorescent protein (EYFP) CD34+ cells with reagents to target angiopoietin-2 prior to adoptive transfer. (A) Neointimal area (left axis, white bars) and neointima:media ratio (right axis, black bars) of vessels taken from wild-type (WT) animals 28 days post-injury after adoptive transfer of 1 × 10 6 EYFP CD34+ cells that were either pre-incubated with isotype control antibody, an anti-TIE-2 antibody, control scrambled siRNA or specific siRNA targeting angiopoietin-2, and administered immediately post-injury. Data derived from examination of three random sections from six different vessels. * p

    Journal: Frontiers in Immunology

    Article Title: Inhibition of Angiopoietin-2 Production by Myofibrocytes Inhibits Neointimal Hyperplasia After Endoluminal Injury in Mice

    doi: 10.3389/fimmu.2018.01517

    Figure Lengend Snippet: Pre-incubation of enhanced yellow fluorescent protein (EYFP) CD34+ cells with reagents to target angiopoietin-2 prior to adoptive transfer. (A) Neointimal area (left axis, white bars) and neointima:media ratio (right axis, black bars) of vessels taken from wild-type (WT) animals 28 days post-injury after adoptive transfer of 1 × 10 6 EYFP CD34+ cells that were either pre-incubated with isotype control antibody, an anti-TIE-2 antibody, control scrambled siRNA or specific siRNA targeting angiopoietin-2, and administered immediately post-injury. Data derived from examination of three random sections from six different vessels. * p

    Article Snippet: Frozen sections were immersed in 1% BSA(Sigma)-PBS for 30 min and then incubated overnight at 4°C with the following antibodies: goat anti-angiopoietin-2 (Santa Cruz biotechnology Inc.), mouse monoclonal anti-collagen 1 (as a marker of fibocytes), rabbit anti-CXCR4, rabbit anti-CXCL12 (all from Abcam), mouse anti-human TFPI (Enzyme Research Laboratories, Swansea, UK), and rat anti-mouse CD31(BD).

    Techniques: Incubation, Adoptive Transfer Assay, Derivative Assay

    Association between plasma CXCL-12 and intimal hyperplasia (IH): CXCL-12 expression is thrombin and angiopoietin-2-dependent. (A) Plasma CXCL-12 (black filled) and migration inhibitory factor (MIF) (white filled) levels post-injury in wild-type (WT) recipients of WT (squares) or CD31-TFPI-Tg (circles) CD34+ cells. *Day 7 and day 28 p

    Journal: Frontiers in Immunology

    Article Title: Inhibition of Angiopoietin-2 Production by Myofibrocytes Inhibits Neointimal Hyperplasia After Endoluminal Injury in Mice

    doi: 10.3389/fimmu.2018.01517

    Figure Lengend Snippet: Association between plasma CXCL-12 and intimal hyperplasia (IH): CXCL-12 expression is thrombin and angiopoietin-2-dependent. (A) Plasma CXCL-12 (black filled) and migration inhibitory factor (MIF) (white filled) levels post-injury in wild-type (WT) recipients of WT (squares) or CD31-TFPI-Tg (circles) CD34+ cells. *Day 7 and day 28 p

    Article Snippet: Frozen sections were immersed in 1% BSA(Sigma)-PBS for 30 min and then incubated overnight at 4°C with the following antibodies: goat anti-angiopoietin-2 (Santa Cruz biotechnology Inc.), mouse monoclonal anti-collagen 1 (as a marker of fibocytes), rabbit anti-CXCR4, rabbit anti-CXCL12 (all from Abcam), mouse anti-human TFPI (Enzyme Research Laboratories, Swansea, UK), and rat anti-mouse CD31(BD).

    Techniques: Expressing, Migration

    Thrombin-induced proliferation and survival are angiopoietin-2-dependent. In all, responses of wild-type (WT) CD34+ cells are shown as white bars, whereas purified CD31+ myofibrocytes from CD31-TFPI-Tg mice are shown as black bars. 0*Indicates incubation with active site inhibited thrombin. Abbreviations: Csi, control scrambled siRNA; Ang-2si, siRNA specific for angiopoietin-2. (A) Expression of angiopoietin-2 at baseline and after incubation with thrombin for 5 days, expressed as the percentage of CD34+cells staining positive in immunocytofluoresence analysis. (B) Secretion of angiopoietin-2 into supernatant after 5-day incubation with thrombin at the indicated concentrations, analyzed by ELISA. (C) Expression of smooth muscle actin (SMA) and joint expression of CD31 with SMA at baseline and after incubation with thombin for 5 days, either alone or with anti-TIE-2 antibody or siRNA against angiopoietin-2 or control. Data expressed as the percentage of CD34+ cells staining positive in immunocytofluoresence analysis. (D) As (C) but cells incubated with angiopoietin-2 ± anti-TIE-2 antibody. (E) Proliferation, assessed by 3 H-thymidine incorporation and expressed as counts per minute (CPM) after incubation with thrombin at the indicated concentrations. (F) Proliferation, assessed by 3 H-thymidine incorporation and expressed as CPM after incubation with angiopoietin-2 at the indicated concentrations. (G) Degree of apoptosis after incubation with thrombin or angiopoietin-2 at the indicated concentrations. Anti-TIE-2 antibody used at 50 ng/ml. All experiments repeated at least twice.

    Journal: Frontiers in Immunology

    Article Title: Inhibition of Angiopoietin-2 Production by Myofibrocytes Inhibits Neointimal Hyperplasia After Endoluminal Injury in Mice

    doi: 10.3389/fimmu.2018.01517

    Figure Lengend Snippet: Thrombin-induced proliferation and survival are angiopoietin-2-dependent. In all, responses of wild-type (WT) CD34+ cells are shown as white bars, whereas purified CD31+ myofibrocytes from CD31-TFPI-Tg mice are shown as black bars. 0*Indicates incubation with active site inhibited thrombin. Abbreviations: Csi, control scrambled siRNA; Ang-2si, siRNA specific for angiopoietin-2. (A) Expression of angiopoietin-2 at baseline and after incubation with thrombin for 5 days, expressed as the percentage of CD34+cells staining positive in immunocytofluoresence analysis. (B) Secretion of angiopoietin-2 into supernatant after 5-day incubation with thrombin at the indicated concentrations, analyzed by ELISA. (C) Expression of smooth muscle actin (SMA) and joint expression of CD31 with SMA at baseline and after incubation with thombin for 5 days, either alone or with anti-TIE-2 antibody or siRNA against angiopoietin-2 or control. Data expressed as the percentage of CD34+ cells staining positive in immunocytofluoresence analysis. (D) As (C) but cells incubated with angiopoietin-2 ± anti-TIE-2 antibody. (E) Proliferation, assessed by 3 H-thymidine incorporation and expressed as counts per minute (CPM) after incubation with thrombin at the indicated concentrations. (F) Proliferation, assessed by 3 H-thymidine incorporation and expressed as CPM after incubation with angiopoietin-2 at the indicated concentrations. (G) Degree of apoptosis after incubation with thrombin or angiopoietin-2 at the indicated concentrations. Anti-TIE-2 antibody used at 50 ng/ml. All experiments repeated at least twice.

    Article Snippet: Frozen sections were immersed in 1% BSA(Sigma)-PBS for 30 min and then incubated overnight at 4°C with the following antibodies: goat anti-angiopoietin-2 (Santa Cruz biotechnology Inc.), mouse monoclonal anti-collagen 1 (as a marker of fibocytes), rabbit anti-CXCR4, rabbit anti-CXCL12 (all from Abcam), mouse anti-human TFPI (Enzyme Research Laboratories, Swansea, UK), and rat anti-mouse CD31(BD).

    Techniques: Purification, Mouse Assay, Incubation, Expressing, Staining, Enzyme-linked Immunosorbent Assay

    Pre-incubation of enhanced yellow fluorescent protein (EYFP) CD34+ cells with reagents to target CXCR4 or angiopoietin-2 prior to adoptive transfer. Panels show immunohistology of consecutive sections through injured mouse carotid arteries harvested on day 28 post-injury. All sections stained with DAPI (4,6 diamidino-2-phenylindole) nuclear stain (blue) and anti-CXCL-12 (red). The green staining is light emitted by the EYFP cells themselves. Yellow indicates co-localization. The annotated white line defines the junction between neointima and media. (A,B) Injured wild-type (WT) mice were adoptively transferred with CD34+ cells on day of injury. In (A) , the cells came from WT mice. In (B) , the cells came from CD31-TFPI-Tg mice. 1 week later, both groups of mice received a second adoptive transfer of EYFP CD34+ cells that were pre-incubated with either isotype control antibody or anti-CXCR4 as indicated. Sections were harvested 1 week later (day 14 post-injury). The annotated white line defines the junction between neointima and media. The anti-CXCR4 prevented late recruitment of adoptively transferred CD34+ cells in (A) , but in (B) , there is no late recruitment of adoptively transferred EYFP cells, even those pre-incubated with control antibody. (C) Adoptive transfer of EYFP CD34+ cells to injured WT mice at the time of injury. Cells first incubated with an anti-TIE-2 or isotype control, or siRNA targeting angiopoietin-2 or scrambled control. Compared to the control cells and those incubated with isotype control antibody, the anti-TIE-2 antibody significantly reduced the neointimal area but did not inhibit angiopoietin-2 expression. Compared to the scrambled siRNA, the specific siRNA targeting angiopoietin-2 significantly reduced the neointimal area in association with abolition of angiopoietin-2 expression cells. Tables besides panels describe summary of staining from all mice ( n = 6), showing the proportion of the neointimal area that is positive for CXCL-12, EYFP, or both on day of examination (% ± SEM). Data derived from three random sections from each of the arteries from each mouse (see Materials and Methods ). *cf isotype for anti-TIE-2 p

    Journal: Frontiers in Immunology

    Article Title: Inhibition of Angiopoietin-2 Production by Myofibrocytes Inhibits Neointimal Hyperplasia After Endoluminal Injury in Mice

    doi: 10.3389/fimmu.2018.01517

    Figure Lengend Snippet: Pre-incubation of enhanced yellow fluorescent protein (EYFP) CD34+ cells with reagents to target CXCR4 or angiopoietin-2 prior to adoptive transfer. Panels show immunohistology of consecutive sections through injured mouse carotid arteries harvested on day 28 post-injury. All sections stained with DAPI (4,6 diamidino-2-phenylindole) nuclear stain (blue) and anti-CXCL-12 (red). The green staining is light emitted by the EYFP cells themselves. Yellow indicates co-localization. The annotated white line defines the junction between neointima and media. (A,B) Injured wild-type (WT) mice were adoptively transferred with CD34+ cells on day of injury. In (A) , the cells came from WT mice. In (B) , the cells came from CD31-TFPI-Tg mice. 1 week later, both groups of mice received a second adoptive transfer of EYFP CD34+ cells that were pre-incubated with either isotype control antibody or anti-CXCR4 as indicated. Sections were harvested 1 week later (day 14 post-injury). The annotated white line defines the junction between neointima and media. The anti-CXCR4 prevented late recruitment of adoptively transferred CD34+ cells in (A) , but in (B) , there is no late recruitment of adoptively transferred EYFP cells, even those pre-incubated with control antibody. (C) Adoptive transfer of EYFP CD34+ cells to injured WT mice at the time of injury. Cells first incubated with an anti-TIE-2 or isotype control, or siRNA targeting angiopoietin-2 or scrambled control. Compared to the control cells and those incubated with isotype control antibody, the anti-TIE-2 antibody significantly reduced the neointimal area but did not inhibit angiopoietin-2 expression. Compared to the scrambled siRNA, the specific siRNA targeting angiopoietin-2 significantly reduced the neointimal area in association with abolition of angiopoietin-2 expression cells. Tables besides panels describe summary of staining from all mice ( n = 6), showing the proportion of the neointimal area that is positive for CXCL-12, EYFP, or both on day of examination (% ± SEM). Data derived from three random sections from each of the arteries from each mouse (see Materials and Methods ). *cf isotype for anti-TIE-2 p

    Article Snippet: Frozen sections were immersed in 1% BSA(Sigma)-PBS for 30 min and then incubated overnight at 4°C with the following antibodies: goat anti-angiopoietin-2 (Santa Cruz biotechnology Inc.), mouse monoclonal anti-collagen 1 (as a marker of fibocytes), rabbit anti-CXCR4, rabbit anti-CXCL12 (all from Abcam), mouse anti-human TFPI (Enzyme Research Laboratories, Swansea, UK), and rat anti-mouse CD31(BD).

    Techniques: Incubation, Adoptive Transfer Assay, Staining, Mouse Assay, Expressing, Derivative Assay

    Adoptive transfer of CD31-TFPI-Tg CD34+ cells to wild-type (WT) mice immediately post-injury. Panels show immunohistology of representative sections through injured mouse carotid arteries harvested on days post-injury as indicated. All sections stained with DAPI (4,6 diamidino-2-phenylindole) nuclear stain (blue) and (red) anti-collagen-1 (A) or human tissue factor pathway inhibitor (B) and (green) anti-angiopoietin-2. Yellow indicates co-localization. The annotated white line defines the junction between neointima and media. *On day 0, sections harvested within an hour of injury. Table besides panel (A) describes summary of staining from all mice ( n = 6), showing the proportion of the neointimal area that is positive for collagen-1, angiopoietin-2, or both on days 0, 7, and 28 (% ± SEM). Data derived from three random sections from each of the arteries from each mouse (see Materials and Methods ). *cf WT cells (see Figure 1 B) at equivalent time point p = 0.03. † cf WT cells (Figure 1 B) at equivalent time point p

    Journal: Frontiers in Immunology

    Article Title: Inhibition of Angiopoietin-2 Production by Myofibrocytes Inhibits Neointimal Hyperplasia After Endoluminal Injury in Mice

    doi: 10.3389/fimmu.2018.01517

    Figure Lengend Snippet: Adoptive transfer of CD31-TFPI-Tg CD34+ cells to wild-type (WT) mice immediately post-injury. Panels show immunohistology of representative sections through injured mouse carotid arteries harvested on days post-injury as indicated. All sections stained with DAPI (4,6 diamidino-2-phenylindole) nuclear stain (blue) and (red) anti-collagen-1 (A) or human tissue factor pathway inhibitor (B) and (green) anti-angiopoietin-2. Yellow indicates co-localization. The annotated white line defines the junction between neointima and media. *On day 0, sections harvested within an hour of injury. Table besides panel (A) describes summary of staining from all mice ( n = 6), showing the proportion of the neointimal area that is positive for collagen-1, angiopoietin-2, or both on days 0, 7, and 28 (% ± SEM). Data derived from three random sections from each of the arteries from each mouse (see Materials and Methods ). *cf WT cells (see Figure 1 B) at equivalent time point p = 0.03. † cf WT cells (Figure 1 B) at equivalent time point p

    Article Snippet: Frozen sections were immersed in 1% BSA(Sigma)-PBS for 30 min and then incubated overnight at 4°C with the following antibodies: goat anti-angiopoietin-2 (Santa Cruz biotechnology Inc.), mouse monoclonal anti-collagen 1 (as a marker of fibocytes), rabbit anti-CXCR4, rabbit anti-CXCL12 (all from Abcam), mouse anti-human TFPI (Enzyme Research Laboratories, Swansea, UK), and rat anti-mouse CD31(BD).

    Techniques: Adoptive Transfer Assay, Mouse Assay, Staining, Derivative Assay

    Impact of coagulation proteases on myofibrocyte phenotype. In (A,B) , responses of wild-type (WT) CD34+ cells are shown as white bars, whereas isolated CD31+ myofibrocytes from CD31-TFPI-Tg mice are shown as black bars. (A) Cells were incubated with FX in presence or absence of FVIIa and FII (prothrombin) plus FVa. Functional tissue factor on WT cells is illustrated by thrombin generation, angiopoietin-2 (Ang-2) secretion, and CXCL-12 secretion. The presence of human tissue factor pathway inhibitor on purified CD31+ myofibrocytes from CD31-TFPI-Tg mice significantly inhibits all three phenotype changes. (B) Proliferation, assessed by 3 H-thymidine incorporation and expressed as counts per minute (CPM) after incubation with FX and FII in presence of FVIIa. (C) Angiopoietin-2 secretion by WT CD34+ cells (3 × 10 4 /well) after 24 h incubation with either PAR-1 antagonist (black bars), PAR-2 antagonist (white bars), or PAR-4 antagonist (gray bars) at the indicated concentrations for 30 min before addition of FVIIa with FX (both 10 nM) with or without prothrombin (4 nM) and FVa (6 nM) as indicated. All conditions performed in triplicate wells. Error bars indicate SEM. In comparison of increasing concentrations of antagonists with FVIIa + FX, p = 0.027 for PAR2, but p = NS for PAR1 and PAR4. In comparison of increasing concentrations of antagonists with FVIIa + FX + FII + FVa, p = 0.05 for PAR1, but p = not significant (NS) for PAR2 and PAR4. Analysis by one-way ANOVA Kruskal–Wallis test. (D) Angiopoietin-2 secretion by WT CD34+ cells (3 × 10 4 /well) after 24 h incubation with PAR-1, -2, or -4 agonists at the indicated concentrations. All conditions performed in triplicate wells. Error bars indicate SEM. p = 0.017 for comparisons of increasing concentrations of PAR1 agonist, p = 0.012 for PAR2, but p = NS for PAR4 agonist. Analysis by one-way ANOVA Kruskal–Wallis test. (E) Dissection of signaling pathways involved in angiopoietin-2 secretion by WT CD34+ cells induced by 24 h incubation with 10 mM PAR-1 or -2 agonists. Cells were incubated with the agonists with or without 50 mM mitogen-activated protein kinase inhibitor PD98059, 10 mM p38-MAPK inhibitor SB203580, 20 mM NF-kB inhibitor SN50, or 1 mM of the S6K1 inhibitor as indicated. All conditions performed in triplicate wells. Error bars indicate SEM. p = 0.05 for PAR-1 agonist without inhibitor vs. +PD98509 and vs. +SB203580. p = > 0.05 all other comparisons. Analysis by Mann–Whitney T test. All experiments repeated at least twice.

    Journal: Frontiers in Immunology

    Article Title: Inhibition of Angiopoietin-2 Production by Myofibrocytes Inhibits Neointimal Hyperplasia After Endoluminal Injury in Mice

    doi: 10.3389/fimmu.2018.01517

    Figure Lengend Snippet: Impact of coagulation proteases on myofibrocyte phenotype. In (A,B) , responses of wild-type (WT) CD34+ cells are shown as white bars, whereas isolated CD31+ myofibrocytes from CD31-TFPI-Tg mice are shown as black bars. (A) Cells were incubated with FX in presence or absence of FVIIa and FII (prothrombin) plus FVa. Functional tissue factor on WT cells is illustrated by thrombin generation, angiopoietin-2 (Ang-2) secretion, and CXCL-12 secretion. The presence of human tissue factor pathway inhibitor on purified CD31+ myofibrocytes from CD31-TFPI-Tg mice significantly inhibits all three phenotype changes. (B) Proliferation, assessed by 3 H-thymidine incorporation and expressed as counts per minute (CPM) after incubation with FX and FII in presence of FVIIa. (C) Angiopoietin-2 secretion by WT CD34+ cells (3 × 10 4 /well) after 24 h incubation with either PAR-1 antagonist (black bars), PAR-2 antagonist (white bars), or PAR-4 antagonist (gray bars) at the indicated concentrations for 30 min before addition of FVIIa with FX (both 10 nM) with or without prothrombin (4 nM) and FVa (6 nM) as indicated. All conditions performed in triplicate wells. Error bars indicate SEM. In comparison of increasing concentrations of antagonists with FVIIa + FX, p = 0.027 for PAR2, but p = NS for PAR1 and PAR4. In comparison of increasing concentrations of antagonists with FVIIa + FX + FII + FVa, p = 0.05 for PAR1, but p = not significant (NS) for PAR2 and PAR4. Analysis by one-way ANOVA Kruskal–Wallis test. (D) Angiopoietin-2 secretion by WT CD34+ cells (3 × 10 4 /well) after 24 h incubation with PAR-1, -2, or -4 agonists at the indicated concentrations. All conditions performed in triplicate wells. Error bars indicate SEM. p = 0.017 for comparisons of increasing concentrations of PAR1 agonist, p = 0.012 for PAR2, but p = NS for PAR4 agonist. Analysis by one-way ANOVA Kruskal–Wallis test. (E) Dissection of signaling pathways involved in angiopoietin-2 secretion by WT CD34+ cells induced by 24 h incubation with 10 mM PAR-1 or -2 agonists. Cells were incubated with the agonists with or without 50 mM mitogen-activated protein kinase inhibitor PD98059, 10 mM p38-MAPK inhibitor SB203580, 20 mM NF-kB inhibitor SN50, or 1 mM of the S6K1 inhibitor as indicated. All conditions performed in triplicate wells. Error bars indicate SEM. p = 0.05 for PAR-1 agonist without inhibitor vs. +PD98509 and vs. +SB203580. p = > 0.05 all other comparisons. Analysis by Mann–Whitney T test. All experiments repeated at least twice.

    Article Snippet: Frozen sections were immersed in 1% BSA(Sigma)-PBS for 30 min and then incubated overnight at 4°C with the following antibodies: goat anti-angiopoietin-2 (Santa Cruz biotechnology Inc.), mouse monoclonal anti-collagen 1 (as a marker of fibocytes), rabbit anti-CXCR4, rabbit anti-CXCL12 (all from Abcam), mouse anti-human TFPI (Enzyme Research Laboratories, Swansea, UK), and rat anti-mouse CD31(BD).

    Techniques: Coagulation, Isolation, Mouse Assay, Incubation, Functional Assay, Purification, Dissection, MANN-WHITNEY

    Comparison of phenotype of intimal hyperplasia in wild-type (WT) animals vs. WT after adoptive transfer of WT CD34+ cells immediately post-injury. Panels show immunohistology of representative sections through injured mouse carotid arteries harvested on days post-injury as indicated. All sections stained with DAPI (4,6 diamidino-2-phenylindole) nuclear stain (blue) and (red) anti-collagen-1 (A,B) , or angiopoietin-2 (C) , and (green) anti-angiopoietin-2 (A,B) . Yellow indicates co-localization. In (C) , the adoptively transferred cells came from enhanced yellow fluorescent protein (EYFP) mice, which spontaneously fluoresce as shown. The annotated white line defines the junction between neointima and media. (A) Injured mice received no adoptively transferred cells. *On day 0, sections harvested within an hour of injury. (B) Mice injected with cells from WT mice. (C) Mice injected with cells from EYFP mice. Expanded area illustrates that most of the early recruited cells are adoptively transferred, angiopoietin-2+ cells. Tables beside each panel describe summary of staining from all mice ( n = 6), showing the proportion of the neointimal area that is positive for collagen-1, angiopoietin-2 or both or EYFP, angiopoietin-2 or both on days 0, 5/7, and day 28 (% ± SEM). Data derived from three random sections from each of the arteries from each mouse (see Materials and Methods ). Subtracting the proportional area occupied by dual positive cells from the total area occupied by angiopoietin-2+ cells suggests the proportional area occupied by collagen-1-negative cells expressing angiopoietin-2. *cf WT (no adoptive transfer) at equivalent time: p

    Journal: Frontiers in Immunology

    Article Title: Inhibition of Angiopoietin-2 Production by Myofibrocytes Inhibits Neointimal Hyperplasia After Endoluminal Injury in Mice

    doi: 10.3389/fimmu.2018.01517

    Figure Lengend Snippet: Comparison of phenotype of intimal hyperplasia in wild-type (WT) animals vs. WT after adoptive transfer of WT CD34+ cells immediately post-injury. Panels show immunohistology of representative sections through injured mouse carotid arteries harvested on days post-injury as indicated. All sections stained with DAPI (4,6 diamidino-2-phenylindole) nuclear stain (blue) and (red) anti-collagen-1 (A,B) , or angiopoietin-2 (C) , and (green) anti-angiopoietin-2 (A,B) . Yellow indicates co-localization. In (C) , the adoptively transferred cells came from enhanced yellow fluorescent protein (EYFP) mice, which spontaneously fluoresce as shown. The annotated white line defines the junction between neointima and media. (A) Injured mice received no adoptively transferred cells. *On day 0, sections harvested within an hour of injury. (B) Mice injected with cells from WT mice. (C) Mice injected with cells from EYFP mice. Expanded area illustrates that most of the early recruited cells are adoptively transferred, angiopoietin-2+ cells. Tables beside each panel describe summary of staining from all mice ( n = 6), showing the proportion of the neointimal area that is positive for collagen-1, angiopoietin-2 or both or EYFP, angiopoietin-2 or both on days 0, 5/7, and day 28 (% ± SEM). Data derived from three random sections from each of the arteries from each mouse (see Materials and Methods ). Subtracting the proportional area occupied by dual positive cells from the total area occupied by angiopoietin-2+ cells suggests the proportional area occupied by collagen-1-negative cells expressing angiopoietin-2. *cf WT (no adoptive transfer) at equivalent time: p

    Article Snippet: Frozen sections were immersed in 1% BSA(Sigma)-PBS for 30 min and then incubated overnight at 4°C with the following antibodies: goat anti-angiopoietin-2 (Santa Cruz biotechnology Inc.), mouse monoclonal anti-collagen 1 (as a marker of fibocytes), rabbit anti-CXCR4, rabbit anti-CXCL12 (all from Abcam), mouse anti-human TFPI (Enzyme Research Laboratories, Swansea, UK), and rat anti-mouse CD31(BD).

    Techniques: Adoptive Transfer Assay, Staining, Mouse Assay, Injection, Derivative Assay, Expressing

    Interpretation of our findings. (A) Post-endoluminal injury, fibrocytes (green) are mobilized into the circulation, a subpopulation of which express smooth muscle actin (SMA) (“myofibrocytes”-yellow cells) and CD31 and these are recruited to the site of injury, via CXCR4 from CXCL-12 released from luminal platelets (data not shown). These myofibrocytes express tissue factor (TF), which generates FXa and thrombin (FIIa), which can signal through PARs-1 and -2 on the cell surface. These result in the secretion of angiopoietin-2, which through cell surface TIE-2, causes proliferation and secretion of CXCL-12, which can attract more myofibrocytes from the circulation. The myofibrocytes also orchestrate expression of angiopoietin-2 by other cells within the vessel wall, by a mechanism not elucidated. (B) Inhibition of TF on the myofibrocytes, either by cell surface expression of a transgenic human tissue factor pathway inhibitor (hTFPI) fusion protein, or by an anti-TF antibody inhibits angiopoietin-2 secretion and results in a small neointima, consisting of non-proliferating myofibrocytes. An siRNA to angiopoietin-2 or a blocking anti-TIE-2 antibody has the same effect, illustrating the key importance of the angiopoietin-2/TIE axis for the development of intimal hyperplasia. *Treatment of whole CD34+ cells with an anti-TF inhibits intimal hyperplasia (IH) but also prevents significant recruitment of myofibrocytes, in a similar way as expressing hTFPI fusion protein on SMA+ fibrocytes ( 8 ).

    Journal: Frontiers in Immunology

    Article Title: Inhibition of Angiopoietin-2 Production by Myofibrocytes Inhibits Neointimal Hyperplasia After Endoluminal Injury in Mice

    doi: 10.3389/fimmu.2018.01517

    Figure Lengend Snippet: Interpretation of our findings. (A) Post-endoluminal injury, fibrocytes (green) are mobilized into the circulation, a subpopulation of which express smooth muscle actin (SMA) (“myofibrocytes”-yellow cells) and CD31 and these are recruited to the site of injury, via CXCR4 from CXCL-12 released from luminal platelets (data not shown). These myofibrocytes express tissue factor (TF), which generates FXa and thrombin (FIIa), which can signal through PARs-1 and -2 on the cell surface. These result in the secretion of angiopoietin-2, which through cell surface TIE-2, causes proliferation and secretion of CXCL-12, which can attract more myofibrocytes from the circulation. The myofibrocytes also orchestrate expression of angiopoietin-2 by other cells within the vessel wall, by a mechanism not elucidated. (B) Inhibition of TF on the myofibrocytes, either by cell surface expression of a transgenic human tissue factor pathway inhibitor (hTFPI) fusion protein, or by an anti-TF antibody inhibits angiopoietin-2 secretion and results in a small neointima, consisting of non-proliferating myofibrocytes. An siRNA to angiopoietin-2 or a blocking anti-TIE-2 antibody has the same effect, illustrating the key importance of the angiopoietin-2/TIE axis for the development of intimal hyperplasia. *Treatment of whole CD34+ cells with an anti-TF inhibits intimal hyperplasia (IH) but also prevents significant recruitment of myofibrocytes, in a similar way as expressing hTFPI fusion protein on SMA+ fibrocytes ( 8 ).

    Article Snippet: Frozen sections were immersed in 1% BSA(Sigma)-PBS for 30 min and then incubated overnight at 4°C with the following antibodies: goat anti-angiopoietin-2 (Santa Cruz biotechnology Inc.), mouse monoclonal anti-collagen 1 (as a marker of fibocytes), rabbit anti-CXCR4, rabbit anti-CXCL12 (all from Abcam), mouse anti-human TFPI (Enzyme Research Laboratories, Swansea, UK), and rat anti-mouse CD31(BD).

    Techniques: Expressing, Inhibition, Transgenic Assay, Blocking Assay

    ( A ) Representative western blots of the angiogenic proteins VEGF-A and angiopoietin-2, with the corresponding densitometric analysis ( B ). ( C ) A representative histological microphotograph of capillary density at 20 weeks in sham ( a ), AB + HA ( b ), and

    Journal: Cardiovascular Research

    Article Title: SOCS1 gene transfer accelerates the transition to heart failure through the inhibition of the gp130/JAK/STAT pathway

    doi: 10.1093/cvr/cvs261

    Figure Lengend Snippet: ( A ) Representative western blots of the angiogenic proteins VEGF-A and angiopoietin-2, with the corresponding densitometric analysis ( B ). ( C ) A representative histological microphotograph of capillary density at 20 weeks in sham ( a ), AB + HA ( b ), and

    Article Snippet: The membrane was incubated with the STAT3, phospho-STAT3, JAK-1, phosphor-JAK-1, VEGF-A, and angiopoietin-2 antibodies (Santa Cruz Biotechnology).

    Techniques: Western Blot

    Depletion of RRAD decreases angiogenesis-related factors. HUVEC cell was seeded on Matrigel and incubated for 18 h in siControl or siRRAD#1-transfected MKN1 cells ( A ) and DLD1 cells medium ( B ). Tube formation was determined by assessment of the total length of tube in three randomly selected fields. Data represent mean ± SD of three independent experiments. Angiogenesis-related factors including VEGF and angiopoietin 2 were also decreased by siRRAD with immunoblotting ( C ) and ELISA analysis ( D ). Full-length blots are presented in Supplementary Fig. S8 . *P

    Journal: Scientific Reports

    Article Title: RRAD expression in gastric and colorectal cancer with peritoneal carcinomatosis

    doi: 10.1038/s41598-019-55767-7

    Figure Lengend Snippet: Depletion of RRAD decreases angiogenesis-related factors. HUVEC cell was seeded on Matrigel and incubated for 18 h in siControl or siRRAD#1-transfected MKN1 cells ( A ) and DLD1 cells medium ( B ). Tube formation was determined by assessment of the total length of tube in three randomly selected fields. Data represent mean ± SD of three independent experiments. Angiogenesis-related factors including VEGF and angiopoietin 2 were also decreased by siRRAD with immunoblotting ( C ) and ELISA analysis ( D ). Full-length blots are presented in Supplementary Fig. S8 . *P

    Article Snippet: ELISA assay Secreted protein levels of VEGF and angiopoietin 2 were measured on culture media (200 μL) collected from siRRAD transfected cells.

    Techniques: Incubation, Transfection, Enzyme-linked Immunosorbent Assay

    EGFR activation promotes invasive/non-angiogenic tumor growth in GBM patient biopsies. Tissue microarray (TMA) of GBM biopsies. a EGFR -amplified GBM biopsies as verified by FISH with an EGFR /chromosome 7 probe in red and green, respectively. H E sections and angiopoietin2 stainings indicate non-angiogenic ( upper panel ) versus angiogenic areas ( lower panel ) in EGFR -amplified tumors. High pEGFR expression is only found in non-angiogenic areas ( upper panel ). b Immunohistochemical staining of pEGFR positive biopsies selected from the TMA with antibodies against pEGFR and vWF. pEGFR positive tumor areas are non-/less angiogenic compared to angiogenic, pEGFR negative areas within the same biopsies. Scale bars 100 μm. c Area fraction of vascular elements immunostained with vWF from pEGFR positive versus angiogenic, pEGFR negative areas from five different patients. Quantification was performed at ×200 magnification. P

    Journal: Acta Neuropathologica

    Article Title: EGFR wild-type amplification and activation promote invasion and development of glioblastoma independent of angiogenesis

    doi: 10.1007/s00401-013-1101-1

    Figure Lengend Snippet: EGFR activation promotes invasive/non-angiogenic tumor growth in GBM patient biopsies. Tissue microarray (TMA) of GBM biopsies. a EGFR -amplified GBM biopsies as verified by FISH with an EGFR /chromosome 7 probe in red and green, respectively. H E sections and angiopoietin2 stainings indicate non-angiogenic ( upper panel ) versus angiogenic areas ( lower panel ) in EGFR -amplified tumors. High pEGFR expression is only found in non-angiogenic areas ( upper panel ). b Immunohistochemical staining of pEGFR positive biopsies selected from the TMA with antibodies against pEGFR and vWF. pEGFR positive tumor areas are non-/less angiogenic compared to angiogenic, pEGFR negative areas within the same biopsies. Scale bars 100 μm. c Area fraction of vascular elements immunostained with vWF from pEGFR positive versus angiogenic, pEGFR negative areas from five different patients. Quantification was performed at ×200 magnification. P

    Article Snippet: Primary antibodies used were anti-pAkt (Ser-473) diluted 1:500 (Cell Signaling), anti-pStat3 (Tyr-705) diluted 1:2,000 (Cell Signaling), anti-pMAPK (Thr-202/Tyr-204) diluted 1:2,000 (Cell Signaling), anti-EGFR diluted 1:500 [Life Technologies (Biosource)], anti-EGFRvIII diluted 1:1,000 (clone L8A, a gift kindly provided by S. Clayton, Duke University, Durham, NC), anti-VEGF diluted 1:200 (Santa Cruz), anti-HIF-1α diluted 1:500 (Becton–Dickinson, San Jose, CA), anti-angiopoietin1 diluted 1:300 (Santa Cruz), anti-angiopoietin2 diluted 1:500 (Santa Cruz), anti-FGF2 diluted 1:500 (Santa Cruz), anti-CD133/1 clone AC133 diluted 1:100 (Miltenyi, Bergisch-Gladbach, Germany), anti-vimentin diluted 1:500 (DAKO), anti-snail diluted 1:100 (Abgent, San Diego, CA), anti-Twist diluted 1:100 (Santa Cruz), anti-beta-Actin diluted 1:1,000 (Abcam, Cambridge, UK) and anti-GAPDH diluted 1:2,500 (Abcam).

    Techniques: Activation Assay, Microarray, Amplification, Fluorescence In Situ Hybridization, Expressing, Immunohistochemistry, Staining