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  • 99
    Thermo Fisher vegfr 2
    VEGF-induced activation of c-Src, Akt, but not <t>VEGFR-2,</t> PLCγ-1 and ERK1/2 requires NADPH oxidase-derived ROS. (A) Protein extracts from HCAEC transfected with control siRNA (Scram-si) or si-p47 phox were subject to Western blots as described in Materials and methods . Serum-starved HCAEC were treated with VEGF (50 ng/ml) for the times indicated. Membranes were sequentially blotted, stripped and re-probed with the phospho-specific antibodies as shown. Blots shown are representative of three independent experiments. (B) Same as in (A) except, the membranes were probed for phosphorylation of Y1175 VEGFR-2, Y783 PLCγ-1 and p42/44 ERK1/2. Anti-β-actin antibody was used as loading control.
    Vegfr 2, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 364 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Millipore vegfr 2
    UVA promoted tyrosine phosphorylation of VEGFR-1 and <t>VEGFR-2</t> in normal human keratinocytes. (a) The time-dependent phosphorylation of VEGFR-1 and VEGFR-2 in keratinocytes induced by 10 J/cm 2 UVA. Cells were harvested 0, 2, 4, 8, 12, 24 h after irradiation. (b) The densitometric analysis of (a). (c) Top panel: The phosphorylation of VEGFR-1 in keratinocytes 12 h after treatmet of 10 J/cm 2 UVA with or without pre-incubation of VEGFR-1 neutralizing antibody (A-VEGFR-1, 5 µg/ml) for 1 h. Bottom panel: The phosphorylation of VEGFR-2 in keratinocytes 12 h after treatmet of 10 J/cm 2 UVA with or without pre-incubation of VEGFR-2 neutralizing antibody (A-VEGFR-2, 5 µg/ml) for 1 h. (d) The densitometric analysis of P-VEGFR-1 and P-VEGFR-2 in (c). The relative protein expression was normalized to the endogenous control GAPDH. NA, neutralizing antibody; P-VEGFR-1, phospho-VEGFR-1 (Y1213); P-VEGFR-2, phospho-VEGFR-2 (Tyr1175); UVA: UVA; * P
    Vegfr 2, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 135 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Cell Signaling Technology Inc vegfr2
    Knocked down <t>VEGFR2</t> in retinal endothelial cells reduces IVNV and AVA and increases retinal thickness measurements. a, b Representative flat mount images of L-LUCshRNA- or L-VEGFR2shRNA-treated OIR eyes stained with lectin. c IVNV was significantly decreased in L-VEGFR2shRNA compared to control, L-LUCshRNA (L-LUCshRNA vs. L-VEGFR2shRNA; 2.62 ± 0.28 vs. 1.79 ± 0.36%; p = 0.03; n = 36 or 16 retinas, respectively). d AVA was significantly decreased in L-VEGFR2shRNA compared to control, L-LUCshRNA (L-LUCshRNA vs. L-VEGFR2shRNA; 39.46 ± 3.29 vs. 32.37 ± 3.89%; p = 0.03; n = 36 or n = 16 retinas, respectively). e, f Representative retinal cross section images of L-LUCshRNA- or L-VEGFR2shRNA-treated OIR eyes stained with DAPI. g Retinal thicknesses were significantly increased in L-VEGFR2shRNA compared to control, L-LUCshRNA for all measurements (L-LUCshRNA vs. L-VEGFR2shRNA; GCL-IPL: 43.53 ± 4.51 vs. 58.34 ± 4.45 µm, p = 0.02; INL: 29.81 ± 2.05 vs. 38.09 ± 2.00 µm, p = 0.004; ONL: 51.57 ± 3.40 vs. 61.96 ± 3.33 µm, p = 0.03; Total: 129.19 ± 7.19 vs. 162.82 ± 7.07 µm, p = 0.001; n = 27 or n = 30 measurements per layer, respectively). L-LUCshRNA images and data were also used as controls in Figs. 2 and 7 . Scale bar in b = 500 µm; f = 100 µm. *Denotes significantly different from L-LUCshRNA ( p
    Vegfr2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 92/100, based on 2332 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Santa Cruz Biotechnology vegfr2
    TSP-1 inhibits VEGF induced <t>VEGFR2</t> phosphorylation through CD47. (A) Late EPCs were treated with VEGF (25 ng/ml) for 0, 5, 10, 15 min respectively. Total protein was extracted and the expression of FLK-1, phospho-VEGFR2 (Tyr1175) was determined by Western blot. (B)Seventy-two hrs after transfection of CD47-specific siRNA or control siRNA, the expression of CD47 in late EPCs was determined by western blotting analysis in three independent experiments (#p
    Vegfr2, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 869 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Abcam vegfr2
    ENZ and CCX771 combination treatment suppresses EGFR, AKT and <t>VEGFR2</t> signaling. Western blotting analysis revealed that CCX771 and ENZ markedly reduced phosphorylation levels of EGFR, AKT and VEGFR2 in VCaP ( a ) and C4‐2B ( c ) cells. Combination treatment (ENZ + CCX771) showed increased suppression of EGFR/AKT signaling compared to both single agents. The quantified ratio of the band intensity for the phosphorylated protein normalized to the total protein and DMSO control, and the VEGFR2 normalized to GAPDH and DMSO control for VCaP ( b ) and C4‐2B ( d ) cells.
    Vegfr2, supplied by Abcam, used in various techniques. Bioz Stars score: 92/100, based on 586 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Cell Signaling Technology Inc anti vegfr2
    Overexpression of miR-150 decreased <t>VEGFR2</t> protein level in endothelial cells. The HUVE cells were transfected with miR-150 (has-miR-150) or a scramble microRNA (Scramble) and cultured for an additional 60 hr. The protein levels of c-Myb and VEGFR2 are significantly lower in HUVE cells with overexpression of miR-150 compared to the scramble (student’s t -test; * p
    Anti Vegfr2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 508 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Cell Signaling Technology Inc phospho vegfr2
    Itraconazole induces a change in the migration pattern of <t>VEGFR2.</t> A , HUVEC were treated for 24 h with the indicated doses of itraconazole or vehicle (DMSO), and VEGFR2 was analyzed by Western blot. B , HUVEC were treated as in A with three inhibitors of
    Phospho Vegfr2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 90/100, based on 201 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    R&D Systems mouse vegf r2 kdr flk 1 antibody
    Itraconazole induces a change in the migration pattern of <t>VEGFR2.</t> A , HUVEC were treated for 24 h with the indicated doses of itraconazole or vehicle (DMSO), and VEGFR2 was analyzed by Western blot. B , HUVEC were treated as in A with three inhibitors of
    Mouse Vegf R2 Kdr Flk 1 Antibody, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 294 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Cell Signaling Technology Inc rabbit anti vegfr2
    The role of VEGFR3 in the mouse aorta. (A) VEGFR3 expression in arterial endothelium. Total RNA isolated from the endothelial layer was analyzed by qPCR for the indicated genes. VEcad and VEGFR3 expression are represented as mean fold enrichment of the endothelial preparation over the remaining media ± SEM (error bars) from four aortas. The relative abundance of the medial layer markers SMA and SM22 indicate the purity of endothelial preparations. (B) VEGFR3 reporter. Aortas from adult VEGFR3-driven YFP gene reporter mice were sectioned longitudinally and stained for the YFP reporter and for nuclei using Hoechst staining. IC, inner curvature. Images are representative of five mice from several litters. (C) <t>VEGFR2</t> iΔEC. Endothelial-specific, inducible VEGFR3 knockout (iΔEC) and WT control mice were treated with tamoxifen, and aortas were removed after 1 wk. Tissue lysates were collected and immunoblotted with the indicated antibodies. IB, immunoblotting. (D and E) Inflammatory markers. VEGFR3 iΔEC and WT control mice were treated with tamoxifen and examined at 3 wk. Aortas were sectioned longitudinally and stained for fibronectin (D) or VCAM-1 (E). Images are representative of 6 mice from two independent experiments. Bars, 100 µm. The ratio of mean fluorescence intensity between the inner and outer curvature was then quantified. Values are means ± SEM (error bars). *, P
    Rabbit Anti Vegfr2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 91/100, based on 282 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology anti vegfr2
    Effects of hypericin on complex formations between <t>VEGFR2,</t> Hsp90 and on HOP-hsp70 intermediate complexes. [A]. Western blot analyses of VEGFR2 protein levels in cytosolic extracts from untreated control cells (C), or cells treated with 30 µM hypericin for 72 hrs (HYP). [B]. Staining of U87-MG cells with Phalloidin-FITC. (1) Untreated cells and (2) cells treated with hypericin 30 µM for 72 hrs. Orange arrows show F-actin filaments; yellow arrows depict collapsed actin globules following exposure to hypericin. [C]. VEGFR2-hsp90 complex formation following treatment with hypericin 30 µM for 72 hrs. Results of immunoprecipitation with anti-Hsp90 antibody and development of Western blots with anti-VEGFR2 antibody. Hypericin diminished VEGFR2-Hsp90 complex formation. [D]. Induction of forced hsp90 poly-ubiquitination by hypericin (30 µM for 72 hrs) in human cancerous cell lines. Top panel – immunoprecipitation with anti-hsp90 and Western blot with anti-hsp90 antibody (control); middle panel - immunoprecipitation with anti-hsp90 and Western blot with anti-ubiquitin, and lower panel immunoprecipitation with anti-ubiquitin and Western blot with anti-hsp90. (I.P – immunoprecipitation; W.B. – Western blots).
    Anti Vegfr2, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 189 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Cell Signaling Technology Inc anti phospho vegfr2
    Reversal effect of cholesterol on the inhibition of <t>VEGFR2</t> and mTOR activities by SERM. (A) and (B) HUVEC were treated with tamoxifen (TMX) or toremifene (TRM) with or without cholesterol (5 μg/ml)/cyclodextrin (0.1%) complex (Chol/CD) for 24
    Anti Phospho Vegfr2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 88/100, based on 60 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Abcam anti vegfr2
    Reversal effect of cholesterol on the inhibition of <t>VEGFR2</t> and mTOR activities by SERM. (A) and (B) HUVEC were treated with tamoxifen (TMX) or toremifene (TRM) with or without cholesterol (5 μg/ml)/cyclodextrin (0.1%) complex (Chol/CD) for 24
    Anti Vegfr2, supplied by Abcam, used in various techniques. Bioz Stars score: 92/100, based on 186 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Angiocrine vegfr2
    Transplantation of wild type PCECs restores the defective alveolar regeneration in the mice deficient in endothelial <t>Vegfr2</t> and Fgfr1
    Vegfr2, supplied by Angiocrine, used in various techniques. Bioz Stars score: 92/100, based on 44 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Becton Dickinson vegfr2
    Correlation between (A) <t>pVEGFR2/VEGFR2</t> (p=0.007) and (B) EC pEGFR/EGFR (p=0.008) ratios and maximal reduction in tumor size (n=11). Complete responses were scored as −100% in tumor size change.
    Vegfr2, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 118 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Santa Cruz Biotechnology p vegfr2
    NOX4 regulates vascular endothelial growth factor–induced STAT3 activation through interacting with phosphorylated vascular endothelial growth factor receptor 2 in human retinal microvascular endothelial cells. Western blots of phosphorylated vascular endothelial growth factor receptor 2 <t>(p-VEGFR2)</t> and total VEGFR2 ( A ) and phosphorylated STAT3 (p-STAT3) and total STAT3 ( B ) in human retinal microvascular endothelial cells (hRMVECs) transfected with ConsiRNA or NOX4siRNA and treated with PBS or VEGF (20 ng/ml); ( C ) coimmunoprecipitation of NOX4 with VEGFR2 and western blots of p-VEGFR2 and total VEGFR2 in hRMVECs treated with VEGF with or without pretreatment of SU5416 (Con, control; SU, SU5416; V, VEGF). D : Western blots of p-STAT3 and total STAT3 were performed in hRMVECs treated with VEGF and pretreated with AG490, apocynin or respective controls (C, control; AG, AG490; APO, apocynin; **p
    P Vegfr2, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 44 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    VEGF-induced activation of c-Src, Akt, but not VEGFR-2, PLCγ-1 and ERK1/2 requires NADPH oxidase-derived ROS. (A) Protein extracts from HCAEC transfected with control siRNA (Scram-si) or si-p47 phox were subject to Western blots as described in Materials and methods . Serum-starved HCAEC were treated with VEGF (50 ng/ml) for the times indicated. Membranes were sequentially blotted, stripped and re-probed with the phospho-specific antibodies as shown. Blots shown are representative of three independent experiments. (B) Same as in (A) except, the membranes were probed for phosphorylation of Y1175 VEGFR-2, Y783 PLCγ-1 and p42/44 ERK1/2. Anti-β-actin antibody was used as loading control.

    Journal: PLoS ONE

    Article Title: Direct Sensing of Endothelial Oxidants by Vascular Endothelial Growth Factor Receptor-2 and c-Src

    doi: 10.1371/journal.pone.0028454

    Figure Lengend Snippet: VEGF-induced activation of c-Src, Akt, but not VEGFR-2, PLCγ-1 and ERK1/2 requires NADPH oxidase-derived ROS. (A) Protein extracts from HCAEC transfected with control siRNA (Scram-si) or si-p47 phox were subject to Western blots as described in Materials and methods . Serum-starved HCAEC were treated with VEGF (50 ng/ml) for the times indicated. Membranes were sequentially blotted, stripped and re-probed with the phospho-specific antibodies as shown. Blots shown are representative of three independent experiments. (B) Same as in (A) except, the membranes were probed for phosphorylation of Y1175 VEGFR-2, Y783 PLCγ-1 and p42/44 ERK1/2. Anti-β-actin antibody was used as loading control.

    Article Snippet: Colocalization of VEGFR-2 with c-Src or EEA1-positive endosome by immunofluorescence HCAEC were plated on fibronectin coated glass-bottom chamber slides (Lab-Tek II, Thermo Scientific) and starved overnight in 0.2% FBS containing EBM-2.

    Techniques: Activation Assay, Derivative Assay, Transfection, Western Blot

    VEGF-induced interaction between VEGFR-2 and c-Src requires NADPH oxidase-derived ROS. (A) Co-immunoprecipitation (co-IP) assay using 1.2 mg protein lysates of HCAEC that were transfected with Scram-si or si-p47 phox and treated without or with VEGF (50 ng/ml for 5 min). IP was carried out using anti-VEGFR-2 antibody followed by immunoblotting using anti-c-Src (upper panel) and anti-VEGFR-2 (lower panel) antibodies. (B) Quantitative analyses of VEGFR-2-bound c-Src. Bar graphs show quantitative densitometric analysis of three independent experiments using NIH image J (-fold change expressed in mean ± S.E.M.). * p

    Journal: PLoS ONE

    Article Title: Direct Sensing of Endothelial Oxidants by Vascular Endothelial Growth Factor Receptor-2 and c-Src

    doi: 10.1371/journal.pone.0028454

    Figure Lengend Snippet: VEGF-induced interaction between VEGFR-2 and c-Src requires NADPH oxidase-derived ROS. (A) Co-immunoprecipitation (co-IP) assay using 1.2 mg protein lysates of HCAEC that were transfected with Scram-si or si-p47 phox and treated without or with VEGF (50 ng/ml for 5 min). IP was carried out using anti-VEGFR-2 antibody followed by immunoblotting using anti-c-Src (upper panel) and anti-VEGFR-2 (lower panel) antibodies. (B) Quantitative analyses of VEGFR-2-bound c-Src. Bar graphs show quantitative densitometric analysis of three independent experiments using NIH image J (-fold change expressed in mean ± S.E.M.). * p

    Article Snippet: Colocalization of VEGFR-2 with c-Src or EEA1-positive endosome by immunofluorescence HCAEC were plated on fibronectin coated glass-bottom chamber slides (Lab-Tek II, Thermo Scientific) and starved overnight in 0.2% FBS containing EBM-2.

    Techniques: Derivative Assay, Co-Immunoprecipitation Assay, Transfection

    c-Src and VEGFR-2 are oxidized in VEGF-treated HCAEC in the presence of NADPH oxidase-derived ROS. (A) Schematic presentation depicting cysteinyl-labeling assay to determine oxidative modification in intracellular proteins. Non-oxidized protein thiols are alkyalated by IAA, oxidized thiols are reduced back to SH-moiety by DTT and subsequently biotinylated by IAP. Biotinylated proteins are then pulled-down by streptavidin-agarose followed by Western blots. (B) Upper panel: cysteinyl labeling assay to Identify thiol oxidation of proteins in VEGF-treated (50 ng/ml for 2 mins) HCAEC lysates using biotinylated IAP probe. HCAEC were transfected with Scram-si or si-p47 phox as indicated. After cell lysis in the presence of IAA followed by DTT treatment and IAP labeling, 1.5 mg biotinylated protein lysates were subject to immunoprecipitation using Streptavidin-agarose beads and immunoblotted using anti-VEGFR-2 and anti-c-Src antibodies. Lower panel: Western blot for VEGFR-2 using 50 µg of parallel HCAEC lysates as loading control. (B) Quantitative analyses of oxidized VEGFR-2 (upper panel) and c-Src (lower panel). Bar graph shows quantitative densitometric analysis of three independent cysteinyl labeling assays (as in A) using NIH J image (-fold change expressed in mean ± S.E.M.). * p

    Journal: PLoS ONE

    Article Title: Direct Sensing of Endothelial Oxidants by Vascular Endothelial Growth Factor Receptor-2 and c-Src

    doi: 10.1371/journal.pone.0028454

    Figure Lengend Snippet: c-Src and VEGFR-2 are oxidized in VEGF-treated HCAEC in the presence of NADPH oxidase-derived ROS. (A) Schematic presentation depicting cysteinyl-labeling assay to determine oxidative modification in intracellular proteins. Non-oxidized protein thiols are alkyalated by IAA, oxidized thiols are reduced back to SH-moiety by DTT and subsequently biotinylated by IAP. Biotinylated proteins are then pulled-down by streptavidin-agarose followed by Western blots. (B) Upper panel: cysteinyl labeling assay to Identify thiol oxidation of proteins in VEGF-treated (50 ng/ml for 2 mins) HCAEC lysates using biotinylated IAP probe. HCAEC were transfected with Scram-si or si-p47 phox as indicated. After cell lysis in the presence of IAA followed by DTT treatment and IAP labeling, 1.5 mg biotinylated protein lysates were subject to immunoprecipitation using Streptavidin-agarose beads and immunoblotted using anti-VEGFR-2 and anti-c-Src antibodies. Lower panel: Western blot for VEGFR-2 using 50 µg of parallel HCAEC lysates as loading control. (B) Quantitative analyses of oxidized VEGFR-2 (upper panel) and c-Src (lower panel). Bar graph shows quantitative densitometric analysis of three independent cysteinyl labeling assays (as in A) using NIH J image (-fold change expressed in mean ± S.E.M.). * p

    Article Snippet: Colocalization of VEGFR-2 with c-Src or EEA1-positive endosome by immunofluorescence HCAEC were plated on fibronectin coated glass-bottom chamber slides (Lab-Tek II, Thermo Scientific) and starved overnight in 0.2% FBS containing EBM-2.

    Techniques: Derivative Assay, Labeling, Modification, Western Blot, Transfection, Lysis, Immunoprecipitation

    Proposed model: thiol oxidation may help propagate signal transduction from VEGFR-2 to downstream c-Src. (A) VEGF activation of VEGFR-2 and downstream PLCγ-1-ERK1/2 signaling pathway appears to be independent of ROS levels in ECs. (B) VEGF induces NADPH oxidase-derived ROS, which in turn oxidizes VEGFR-2 and c-Src. Thiol oxidation of these two tyrosine kinases appears to correlate with VEGF-induced activation of c-Src, and also with the sub-cellular colocalization and interaction between VEGFR-2 and c-Src. Dependence of VEGF-induced thiol oxidation and activation of c-Src on NADPH oxidase-derived ROS render downstream activation of PI3K-Akt redox-sensitive in HCAEC. In this model, VEGFR-2 and/or c-Src act as endothelial redox-sensors that determine whether downstream PI3K-Akt signaling pathway should be activated or not.

    Journal: PLoS ONE

    Article Title: Direct Sensing of Endothelial Oxidants by Vascular Endothelial Growth Factor Receptor-2 and c-Src

    doi: 10.1371/journal.pone.0028454

    Figure Lengend Snippet: Proposed model: thiol oxidation may help propagate signal transduction from VEGFR-2 to downstream c-Src. (A) VEGF activation of VEGFR-2 and downstream PLCγ-1-ERK1/2 signaling pathway appears to be independent of ROS levels in ECs. (B) VEGF induces NADPH oxidase-derived ROS, which in turn oxidizes VEGFR-2 and c-Src. Thiol oxidation of these two tyrosine kinases appears to correlate with VEGF-induced activation of c-Src, and also with the sub-cellular colocalization and interaction between VEGFR-2 and c-Src. Dependence of VEGF-induced thiol oxidation and activation of c-Src on NADPH oxidase-derived ROS render downstream activation of PI3K-Akt redox-sensitive in HCAEC. In this model, VEGFR-2 and/or c-Src act as endothelial redox-sensors that determine whether downstream PI3K-Akt signaling pathway should be activated or not.

    Article Snippet: Colocalization of VEGFR-2 with c-Src or EEA1-positive endosome by immunofluorescence HCAEC were plated on fibronectin coated glass-bottom chamber slides (Lab-Tek II, Thermo Scientific) and starved overnight in 0.2% FBS containing EBM-2.

    Techniques: Transduction, Activation Assay, Derivative Assay, Activated Clotting Time Assay

    VEGF induces subcellular co-localization of c-Src and internalized VEGFR-2 in an ROS-dependent manner. HCAEC transfected with control (Scram-si) (A) or si-p47 phox (B) were immunofluorescently double labeled for internalized VEGFR-2 (green) and c-Src (red). VEGFR-2 on HCAEC was labeled with single chain E-tagged antibody (scFvA7, Fitzerald) as described in Materials and methods . After incubation with VEGF (50 ng/ml for 10 min), in order to remove the antibody from the cell surface, cells were placed on ice and acid washed. In permeabilized and fixed HCAEC, VEGFR-2 was detected with an AlexaFluor488-conjugated secondary antibody and is shown in green. c-Src was labeled with AlexaFluor647-conjugated secondary antibody (red) and nuclei with DAPI (blue). (B) Bar graphs show image analysis for colocalization events using the NIH Image J plugin (as described in Materials and methods ). The graphs present the number of colocalization events normalized for the number of total VEGFR-2–positive immunofluorescence signals. Values are the mean of three experiments ± S.E.M., each containing numbers obtained from five random fields. * p

    Journal: PLoS ONE

    Article Title: Direct Sensing of Endothelial Oxidants by Vascular Endothelial Growth Factor Receptor-2 and c-Src

    doi: 10.1371/journal.pone.0028454

    Figure Lengend Snippet: VEGF induces subcellular co-localization of c-Src and internalized VEGFR-2 in an ROS-dependent manner. HCAEC transfected with control (Scram-si) (A) or si-p47 phox (B) were immunofluorescently double labeled for internalized VEGFR-2 (green) and c-Src (red). VEGFR-2 on HCAEC was labeled with single chain E-tagged antibody (scFvA7, Fitzerald) as described in Materials and methods . After incubation with VEGF (50 ng/ml for 10 min), in order to remove the antibody from the cell surface, cells were placed on ice and acid washed. In permeabilized and fixed HCAEC, VEGFR-2 was detected with an AlexaFluor488-conjugated secondary antibody and is shown in green. c-Src was labeled with AlexaFluor647-conjugated secondary antibody (red) and nuclei with DAPI (blue). (B) Bar graphs show image analysis for colocalization events using the NIH Image J plugin (as described in Materials and methods ). The graphs present the number of colocalization events normalized for the number of total VEGFR-2–positive immunofluorescence signals. Values are the mean of three experiments ± S.E.M., each containing numbers obtained from five random fields. * p

    Article Snippet: Colocalization of VEGFR-2 with c-Src or EEA1-positive endosome by immunofluorescence HCAEC were plated on fibronectin coated glass-bottom chamber slides (Lab-Tek II, Thermo Scientific) and starved overnight in 0.2% FBS containing EBM-2.

    Techniques: Transfection, Labeling, Incubation, Immunofluorescence

    UVA promoted tyrosine phosphorylation of VEGFR-1 and VEGFR-2 in normal human keratinocytes. (a) The time-dependent phosphorylation of VEGFR-1 and VEGFR-2 in keratinocytes induced by 10 J/cm 2 UVA. Cells were harvested 0, 2, 4, 8, 12, 24 h after irradiation. (b) The densitometric analysis of (a). (c) Top panel: The phosphorylation of VEGFR-1 in keratinocytes 12 h after treatmet of 10 J/cm 2 UVA with or without pre-incubation of VEGFR-1 neutralizing antibody (A-VEGFR-1, 5 µg/ml) for 1 h. Bottom panel: The phosphorylation of VEGFR-2 in keratinocytes 12 h after treatmet of 10 J/cm 2 UVA with or without pre-incubation of VEGFR-2 neutralizing antibody (A-VEGFR-2, 5 µg/ml) for 1 h. (d) The densitometric analysis of P-VEGFR-1 and P-VEGFR-2 in (c). The relative protein expression was normalized to the endogenous control GAPDH. NA, neutralizing antibody; P-VEGFR-1, phospho-VEGFR-1 (Y1213); P-VEGFR-2, phospho-VEGFR-2 (Tyr1175); UVA: UVA; * P

    Journal: PLoS ONE

    Article Title: Role of VEGF Receptors in Normal and Psoriatic Human Keratinocytes: Evidence from Irradiation with Different UV Sources

    doi: 10.1371/journal.pone.0055463

    Figure Lengend Snippet: UVA promoted tyrosine phosphorylation of VEGFR-1 and VEGFR-2 in normal human keratinocytes. (a) The time-dependent phosphorylation of VEGFR-1 and VEGFR-2 in keratinocytes induced by 10 J/cm 2 UVA. Cells were harvested 0, 2, 4, 8, 12, 24 h after irradiation. (b) The densitometric analysis of (a). (c) Top panel: The phosphorylation of VEGFR-1 in keratinocytes 12 h after treatmet of 10 J/cm 2 UVA with or without pre-incubation of VEGFR-1 neutralizing antibody (A-VEGFR-1, 5 µg/ml) for 1 h. Bottom panel: The phosphorylation of VEGFR-2 in keratinocytes 12 h after treatmet of 10 J/cm 2 UVA with or without pre-incubation of VEGFR-2 neutralizing antibody (A-VEGFR-2, 5 µg/ml) for 1 h. (d) The densitometric analysis of P-VEGFR-1 and P-VEGFR-2 in (c). The relative protein expression was normalized to the endogenous control GAPDH. NA, neutralizing antibody; P-VEGFR-1, phospho-VEGFR-1 (Y1213); P-VEGFR-2, phospho-VEGFR-2 (Tyr1175); UVA: UVA; * P

    Article Snippet: After treatment with or without 10 J/cm2 UVA, cells were cultured by adding neutralizing antibodies against VEGFR-1 or VEGFR-2 for another 24 h. Then, 20 µl of 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich, USA) at a final concentration of 5 mg/ml in PBS was incubated with the analyzed cells for 4 h at 37°C.

    Techniques: Irradiation, Incubation, Expressing

    Activation of VEGFR-1 and VEGFR-2 by UVA involves activation of PKC and SFK. (a) Western blotting detection of P-VEGFR-1 and P-VEGFR-2 in keratinocytes 12 h after treatment of 10 J/cm 2 UVA with or without pre-incubation of Go6976 (0.5 µM), or rottlerin (5.0 µM) for 1 h. (b) The densitometric analysis of P-VEGFR-1 and P-VEGFR-2 in UVA-treated groups in (a). (c) Western blotting detection of P-VEGFR-1 and P-VEGFR-2 in keratinocytes 12 h after treatment of 0 or 10 J/cm 2 UVA with or without 1 h pre-incubation of GF109203X (3 µM). (d) The densitometric analysis of P-VEGFR-1 and P-VEGFR-2 in UVA-treated groups in (c). (e) Western blotting detection of P-VEGFR-1 and P-VEGFR-2 in keratinocytes 12 h after treatment of 0 or 10 J/cm 2 UVA with or without 1 h pre-incubation of PP2 (10 µM). (f) The densitometric analysis of P-VEGFR-1 and P-VEGFR-2 in UVA-treated groups in (e). The relative expression was normalized to the endogenous control GAPDH. P-VEGFR-1, phospho-VEGFR-1 (Y1213); P-VEGFR-2, phospho-VEGFR-2 (Tyr1175); UVA: UVA; * P

    Journal: PLoS ONE

    Article Title: Role of VEGF Receptors in Normal and Psoriatic Human Keratinocytes: Evidence from Irradiation with Different UV Sources

    doi: 10.1371/journal.pone.0055463

    Figure Lengend Snippet: Activation of VEGFR-1 and VEGFR-2 by UVA involves activation of PKC and SFK. (a) Western blotting detection of P-VEGFR-1 and P-VEGFR-2 in keratinocytes 12 h after treatment of 10 J/cm 2 UVA with or without pre-incubation of Go6976 (0.5 µM), or rottlerin (5.0 µM) for 1 h. (b) The densitometric analysis of P-VEGFR-1 and P-VEGFR-2 in UVA-treated groups in (a). (c) Western blotting detection of P-VEGFR-1 and P-VEGFR-2 in keratinocytes 12 h after treatment of 0 or 10 J/cm 2 UVA with or without 1 h pre-incubation of GF109203X (3 µM). (d) The densitometric analysis of P-VEGFR-1 and P-VEGFR-2 in UVA-treated groups in (c). (e) Western blotting detection of P-VEGFR-1 and P-VEGFR-2 in keratinocytes 12 h after treatment of 0 or 10 J/cm 2 UVA with or without 1 h pre-incubation of PP2 (10 µM). (f) The densitometric analysis of P-VEGFR-1 and P-VEGFR-2 in UVA-treated groups in (e). The relative expression was normalized to the endogenous control GAPDH. P-VEGFR-1, phospho-VEGFR-1 (Y1213); P-VEGFR-2, phospho-VEGFR-2 (Tyr1175); UVA: UVA; * P

    Article Snippet: After treatment with or without 10 J/cm2 UVA, cells were cultured by adding neutralizing antibodies against VEGFR-1 or VEGFR-2 for another 24 h. Then, 20 µl of 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich, USA) at a final concentration of 5 mg/ml in PBS was incubated with the analyzed cells for 4 h at 37°C.

    Techniques: Activation Assay, Western Blot, Incubation, Expressing

    Immunofluorescence detection of VEGF, VEGFRs and P-VEGFR-2 regulated by UVA in normal human epidermis. (a) Expression and localization of VEGF165, VEGFR-1, VEGFR-2, NRP-1 and P-VEGFR-2 by immunofluorescence regulated by UVA in normal human epidermis. (b) The fluorescence density analysis of (a). Skin samples from 5 independent individuals were used for quantification. Biopsies were taken 24 h after treatment of 0, one MED, and three MEDs of UVA respectively. The presence of VEGF165 and VEGFRs was indicated by red fluorescence. The presence of P-VEGFR-2 was indicated by green fluorescence. The cellular nuclei were counterstained with DAPI (blue nuclear signal). P-VEGFR-2, phospho-VEGFR-2 (Tyr1175); UVA: UVA; MED, minimal erythema dose; NC, negative controls, which were incubated with non-immune mouse IgG. Bars: 50 µm; Asterisk, epidermis; Yellow triangle, dermis; * P

    Journal: PLoS ONE

    Article Title: Role of VEGF Receptors in Normal and Psoriatic Human Keratinocytes: Evidence from Irradiation with Different UV Sources

    doi: 10.1371/journal.pone.0055463

    Figure Lengend Snippet: Immunofluorescence detection of VEGF, VEGFRs and P-VEGFR-2 regulated by UVA in normal human epidermis. (a) Expression and localization of VEGF165, VEGFR-1, VEGFR-2, NRP-1 and P-VEGFR-2 by immunofluorescence regulated by UVA in normal human epidermis. (b) The fluorescence density analysis of (a). Skin samples from 5 independent individuals were used for quantification. Biopsies were taken 24 h after treatment of 0, one MED, and three MEDs of UVA respectively. The presence of VEGF165 and VEGFRs was indicated by red fluorescence. The presence of P-VEGFR-2 was indicated by green fluorescence. The cellular nuclei were counterstained with DAPI (blue nuclear signal). P-VEGFR-2, phospho-VEGFR-2 (Tyr1175); UVA: UVA; MED, minimal erythema dose; NC, negative controls, which were incubated with non-immune mouse IgG. Bars: 50 µm; Asterisk, epidermis; Yellow triangle, dermis; * P

    Article Snippet: After treatment with or without 10 J/cm2 UVA, cells were cultured by adding neutralizing antibodies against VEGFR-1 or VEGFR-2 for another 24 h. Then, 20 µl of 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich, USA) at a final concentration of 5 mg/ml in PBS was incubated with the analyzed cells for 4 h at 37°C.

    Techniques: Immunofluorescence, Expressing, Fluorescence, Incubation

    Immunofluorescence detection of VEGF165, VEGFRs and P-VEGFR-2 affected by NB-UVB phototherapy (a, skin samples from 13 patients) or halomethasone therapy (b, skin samples from 3 patients) in human psoriatic epidermis. The times of phototherapy in relation to during and after therapy were 11.2±2.2 and 26.3±3.9, and the days of halomethasone treatment in relation to during and after therapy were 12.7±2.9 and 36.0±8.0. Biopsies were taken before, during, and after phototherapy respectively. The presence of VEGF165 and VEGFRs was indicated red. The presence of P-VEGFR-2 was indicated green. The cellular nuclei were counterstained with DAPI (blue nuclear signal). P-VEGFR-2, phospho-VEGFR-2 (Tyr1175); UVB, narrowband UVB therapy; HMS, topical halomethasone monohydrate 0.05% cream; NC, negative controls, which were incubated with non-immune mouse IgG. Bars: 100 µm; Asterisk, epidermis; Yellow triangle, dermis.

    Journal: PLoS ONE

    Article Title: Role of VEGF Receptors in Normal and Psoriatic Human Keratinocytes: Evidence from Irradiation with Different UV Sources

    doi: 10.1371/journal.pone.0055463

    Figure Lengend Snippet: Immunofluorescence detection of VEGF165, VEGFRs and P-VEGFR-2 affected by NB-UVB phototherapy (a, skin samples from 13 patients) or halomethasone therapy (b, skin samples from 3 patients) in human psoriatic epidermis. The times of phototherapy in relation to during and after therapy were 11.2±2.2 and 26.3±3.9, and the days of halomethasone treatment in relation to during and after therapy were 12.7±2.9 and 36.0±8.0. Biopsies were taken before, during, and after phototherapy respectively. The presence of VEGF165 and VEGFRs was indicated red. The presence of P-VEGFR-2 was indicated green. The cellular nuclei were counterstained with DAPI (blue nuclear signal). P-VEGFR-2, phospho-VEGFR-2 (Tyr1175); UVB, narrowband UVB therapy; HMS, topical halomethasone monohydrate 0.05% cream; NC, negative controls, which were incubated with non-immune mouse IgG. Bars: 100 µm; Asterisk, epidermis; Yellow triangle, dermis.

    Article Snippet: After treatment with or without 10 J/cm2 UVA, cells were cultured by adding neutralizing antibodies against VEGFR-1 or VEGFR-2 for another 24 h. Then, 20 µl of 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich, USA) at a final concentration of 5 mg/ml in PBS was incubated with the analyzed cells for 4 h at 37°C.

    Techniques: Immunofluorescence, Incubation

    Activated VEGFR-1 and VEGFR-2 by UVA both contribute to the survival of keratinocytes via subsequent activation of ERK1/2 and Akt. (a) Keratinocytes were incubated with or without neutralizing antibodies against VEGFR-1 (A-VEGFR-1, 5 µg/ml) and VEGFR-2 (AVEGFR-2, 5 µg/ml) 24 h after treatment of 0 or 10 J/cm 2 UVA, and cell apoptosis rate was examined by flow cytometry. (b) Keratinocytes were incubated with neutralizing antibodies against VEGFR-1 (A-VEGFR-1, 5 µg/ml) and/or VEGFR-2 (A-VEGFR-2, 5 µg/ml) 24 h after treatment of 0 or 10 J/cm 2 UVA, and cell survival was determined by MTT assay. (c) Western blotting detection of cleaved-caspase-3 and Bcl-2 in keratinocytes treated by 10 J/cm2 UVA with or without incubation of VEGFR-1 neutralizing antibody (A-VEGFR-1, 5 µg/ml) or VEGFR-2 neutralizing antibody (A-VEGFR-2, 5 µg/ml) for 24 h. (d) Western blotting detection of phospho-ERK1/2 and phospho-Akt in keratinocytes 12 h after treatment of 10 J/cm 2 UVA with or without pre-incubation of VEGFR-1 neutralizing antibody (A-VEGFR-1, 5 µg/ml) or VEGFR-2 neutralizing antibody (A-VEGFR-2, 5 µg/ml) for 1 h. GAPDH was served as loading control for protein normalization, UVA: UVA; * P

    Journal: PLoS ONE

    Article Title: Role of VEGF Receptors in Normal and Psoriatic Human Keratinocytes: Evidence from Irradiation with Different UV Sources

    doi: 10.1371/journal.pone.0055463

    Figure Lengend Snippet: Activated VEGFR-1 and VEGFR-2 by UVA both contribute to the survival of keratinocytes via subsequent activation of ERK1/2 and Akt. (a) Keratinocytes were incubated with or without neutralizing antibodies against VEGFR-1 (A-VEGFR-1, 5 µg/ml) and VEGFR-2 (AVEGFR-2, 5 µg/ml) 24 h after treatment of 0 or 10 J/cm 2 UVA, and cell apoptosis rate was examined by flow cytometry. (b) Keratinocytes were incubated with neutralizing antibodies against VEGFR-1 (A-VEGFR-1, 5 µg/ml) and/or VEGFR-2 (A-VEGFR-2, 5 µg/ml) 24 h after treatment of 0 or 10 J/cm 2 UVA, and cell survival was determined by MTT assay. (c) Western blotting detection of cleaved-caspase-3 and Bcl-2 in keratinocytes treated by 10 J/cm2 UVA with or without incubation of VEGFR-1 neutralizing antibody (A-VEGFR-1, 5 µg/ml) or VEGFR-2 neutralizing antibody (A-VEGFR-2, 5 µg/ml) for 24 h. (d) Western blotting detection of phospho-ERK1/2 and phospho-Akt in keratinocytes 12 h after treatment of 10 J/cm 2 UVA with or without pre-incubation of VEGFR-1 neutralizing antibody (A-VEGFR-1, 5 µg/ml) or VEGFR-2 neutralizing antibody (A-VEGFR-2, 5 µg/ml) for 1 h. GAPDH was served as loading control for protein normalization, UVA: UVA; * P

    Article Snippet: After treatment with or without 10 J/cm2 UVA, cells were cultured by adding neutralizing antibodies against VEGFR-1 or VEGFR-2 for another 24 h. Then, 20 µl of 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich, USA) at a final concentration of 5 mg/ml in PBS was incubated with the analyzed cells for 4 h at 37°C.

    Techniques: Activation Assay, Incubation, Flow Cytometry, Cytometry, MTT Assay, Western Blot

    SPECT analysis of in vivo accumulation of Tc-99m-HYNIC-VEGF-c. VEGF-c (which targets both VEGFR2 and VEGFR3) was tagged with HYNIC chelators and then labeled with Tc-99m and injected intravenously in melatonin and vehicle treated mice. One hour after injection, SPECT images were obtained using dedicated animal scanner. Vehicle treated mice showed increased accumulation of Tc-99m-HYNIC-VEGF-c in the mammary tumor (A, Intersection of lines indicate the tumor, with a volume of 865.69 mm 3 at the 21th day) compared to that of melatonin treated mammary tumors (B, Intersection of lines indicate the tumor, with a volume of 130.69 mm 3 at the 21th day) C. Semi-quantitative analysis of total radioactivity normalized to contralateral muscles showing the intensity of radioactivity in the vehicle and melatonin treated animals.

    Journal: PLoS ONE

    Article Title: Effect of Melatonin on Tumor Growth and Angiogenesis in Xenograft Model of Breast Cancer

    doi: 10.1371/journal.pone.0085311

    Figure Lengend Snippet: SPECT analysis of in vivo accumulation of Tc-99m-HYNIC-VEGF-c. VEGF-c (which targets both VEGFR2 and VEGFR3) was tagged with HYNIC chelators and then labeled with Tc-99m and injected intravenously in melatonin and vehicle treated mice. One hour after injection, SPECT images were obtained using dedicated animal scanner. Vehicle treated mice showed increased accumulation of Tc-99m-HYNIC-VEGF-c in the mammary tumor (A, Intersection of lines indicate the tumor, with a volume of 865.69 mm 3 at the 21th day) compared to that of melatonin treated mammary tumors (B, Intersection of lines indicate the tumor, with a volume of 130.69 mm 3 at the 21th day) C. Semi-quantitative analysis of total radioactivity normalized to contralateral muscles showing the intensity of radioactivity in the vehicle and melatonin treated animals.

    Article Snippet: Activation of intracellular signaling of VEGFR2 stimulates an angiogenic response, leading to cell proliferation, migration, permeability, survival and ultimately resulting in tumor growth .

    Techniques: Single Photon Emission Computed Tomography, In Vivo, Labeling, Injection, Mouse Assay, Radioactivity

    Immunohistochemistry staining with VEGFR2 (arrows) in vehicle treated and melatonin treated tumors. Images were taken with 40× magnification. A significant decrease was observed at the tumor in melatonin treated tumors compared to vehicle treated tumors (*p

    Journal: PLoS ONE

    Article Title: Effect of Melatonin on Tumor Growth and Angiogenesis in Xenograft Model of Breast Cancer

    doi: 10.1371/journal.pone.0085311

    Figure Lengend Snippet: Immunohistochemistry staining with VEGFR2 (arrows) in vehicle treated and melatonin treated tumors. Images were taken with 40× magnification. A significant decrease was observed at the tumor in melatonin treated tumors compared to vehicle treated tumors (*p

    Article Snippet: Activation of intracellular signaling of VEGFR2 stimulates an angiogenic response, leading to cell proliferation, migration, permeability, survival and ultimately resulting in tumor growth .

    Techniques: Immunohistochemistry, Staining

    Knocked down VEGFR2 in retinal endothelial cells reduces IVNV and AVA and increases retinal thickness measurements. a, b Representative flat mount images of L-LUCshRNA- or L-VEGFR2shRNA-treated OIR eyes stained with lectin. c IVNV was significantly decreased in L-VEGFR2shRNA compared to control, L-LUCshRNA (L-LUCshRNA vs. L-VEGFR2shRNA; 2.62 ± 0.28 vs. 1.79 ± 0.36%; p = 0.03; n = 36 or 16 retinas, respectively). d AVA was significantly decreased in L-VEGFR2shRNA compared to control, L-LUCshRNA (L-LUCshRNA vs. L-VEGFR2shRNA; 39.46 ± 3.29 vs. 32.37 ± 3.89%; p = 0.03; n = 36 or n = 16 retinas, respectively). e, f Representative retinal cross section images of L-LUCshRNA- or L-VEGFR2shRNA-treated OIR eyes stained with DAPI. g Retinal thicknesses were significantly increased in L-VEGFR2shRNA compared to control, L-LUCshRNA for all measurements (L-LUCshRNA vs. L-VEGFR2shRNA; GCL-IPL: 43.53 ± 4.51 vs. 58.34 ± 4.45 µm, p = 0.02; INL: 29.81 ± 2.05 vs. 38.09 ± 2.00 µm, p = 0.004; ONL: 51.57 ± 3.40 vs. 61.96 ± 3.33 µm, p = 0.03; Total: 129.19 ± 7.19 vs. 162.82 ± 7.07 µm, p = 0.001; n = 27 or n = 30 measurements per layer, respectively). L-LUCshRNA images and data were also used as controls in Figs. 2 and 7 . Scale bar in b = 500 µm; f = 100 µm. *Denotes significantly different from L-LUCshRNA ( p

    Journal: Angiogenesis

    Article Title: Gene therapy knockdown of VEGFR2 in retinal endothelial cells to treat retinopathy

    doi: 10.1007/s10456-018-9618-5

    Figure Lengend Snippet: Knocked down VEGFR2 in retinal endothelial cells reduces IVNV and AVA and increases retinal thickness measurements. a, b Representative flat mount images of L-LUCshRNA- or L-VEGFR2shRNA-treated OIR eyes stained with lectin. c IVNV was significantly decreased in L-VEGFR2shRNA compared to control, L-LUCshRNA (L-LUCshRNA vs. L-VEGFR2shRNA; 2.62 ± 0.28 vs. 1.79 ± 0.36%; p = 0.03; n = 36 or 16 retinas, respectively). d AVA was significantly decreased in L-VEGFR2shRNA compared to control, L-LUCshRNA (L-LUCshRNA vs. L-VEGFR2shRNA; 39.46 ± 3.29 vs. 32.37 ± 3.89%; p = 0.03; n = 36 or n = 16 retinas, respectively). e, f Representative retinal cross section images of L-LUCshRNA- or L-VEGFR2shRNA-treated OIR eyes stained with DAPI. g Retinal thicknesses were significantly increased in L-VEGFR2shRNA compared to control, L-LUCshRNA for all measurements (L-LUCshRNA vs. L-VEGFR2shRNA; GCL-IPL: 43.53 ± 4.51 vs. 58.34 ± 4.45 µm, p = 0.02; INL: 29.81 ± 2.05 vs. 38.09 ± 2.00 µm, p = 0.004; ONL: 51.57 ± 3.40 vs. 61.96 ± 3.33 µm, p = 0.03; Total: 129.19 ± 7.19 vs. 162.82 ± 7.07 µm, p = 0.001; n = 27 or n = 30 measurements per layer, respectively). L-LUCshRNA images and data were also used as controls in Figs. 2 and 7 . Scale bar in b = 500 µm; f = 100 µm. *Denotes significantly different from L-LUCshRNA ( p

    Article Snippet: Knockdown of downstream STAT3 in retinal endothelial cells reduces pathologic IVNV and maintains retinal thickness, but has no effect on physiologic retinal vascular development The transcription factor, signal transducer and activator of transcription 3 (STAT3), is activated downstream of VEGF-induced activation of VEGFR2 and is important for VEGF-VEGFR2 induced rRMVEC proliferation [ ].

    Techniques: Antiviral Assay, Staining

    L-VEGFR2shRNA knocks down VEGFR2 mRNA and protein in rat endothelial cells. a, b rRMVECs transduced by L-LUCshRNA or L-VEGFR2shRNA for 72 h. c RT-qPCR, mRNA expression for VEGFR2 was reduced in rRMVECs transduced by L-VEGFR2shRNA, compared to L-LUCshRNA (L-LUCshRNA vs. L-VEGFR2shRNA; 1.02 ± 0.14 vs. 0.45 ± 0.11; p = 0.03; n = 2–4). d Image of Western blot gel from rRMVECs. e, f Densitometry quantification of total VEGFR2 protein ( e ) and p-VEGFR2 ( f ) relative to β-actin. e VEGFR2 protein was significantly reduced in rRMVECs transduced by L-VEGFR2shRNA, compared to L-LUCshRNA when cells were treated with PBS or VEGF (L-LUCshRNA vs. L-VEGFR2shRNA; p = 3.56 × 10 −4 ; n = 6). f p-VEGFR2 protein was significantly increased in VEGF-treated rRMVECs transduced by L-LUCshRNA compared to PBS treatment (L-LUCshRNA + PBS vs. L-LUCshRNA + VEGF; p = 0.003; n = 3) that was reduced in rRMVECs transduced by L-VEGFR2shRNA (L-LUCshRNA + VEGF vs. L-VEGFR2shRNA + VEGF; p = 0.04; n = 3). Scale bar in b = 50 µm. Data are means ± SEM. GFP green fluorescent protein, KDR kinase insert domain receptor (VEGFR2 gene), p-VEGFR2 phosphylated-VEGFR2

    Journal: Angiogenesis

    Article Title: Gene therapy knockdown of VEGFR2 in retinal endothelial cells to treat retinopathy

    doi: 10.1007/s10456-018-9618-5

    Figure Lengend Snippet: L-VEGFR2shRNA knocks down VEGFR2 mRNA and protein in rat endothelial cells. a, b rRMVECs transduced by L-LUCshRNA or L-VEGFR2shRNA for 72 h. c RT-qPCR, mRNA expression for VEGFR2 was reduced in rRMVECs transduced by L-VEGFR2shRNA, compared to L-LUCshRNA (L-LUCshRNA vs. L-VEGFR2shRNA; 1.02 ± 0.14 vs. 0.45 ± 0.11; p = 0.03; n = 2–4). d Image of Western blot gel from rRMVECs. e, f Densitometry quantification of total VEGFR2 protein ( e ) and p-VEGFR2 ( f ) relative to β-actin. e VEGFR2 protein was significantly reduced in rRMVECs transduced by L-VEGFR2shRNA, compared to L-LUCshRNA when cells were treated with PBS or VEGF (L-LUCshRNA vs. L-VEGFR2shRNA; p = 3.56 × 10 −4 ; n = 6). f p-VEGFR2 protein was significantly increased in VEGF-treated rRMVECs transduced by L-LUCshRNA compared to PBS treatment (L-LUCshRNA + PBS vs. L-LUCshRNA + VEGF; p = 0.003; n = 3) that was reduced in rRMVECs transduced by L-VEGFR2shRNA (L-LUCshRNA + VEGF vs. L-VEGFR2shRNA + VEGF; p = 0.04; n = 3). Scale bar in b = 50 µm. Data are means ± SEM. GFP green fluorescent protein, KDR kinase insert domain receptor (VEGFR2 gene), p-VEGFR2 phosphylated-VEGFR2

    Article Snippet: Knockdown of downstream STAT3 in retinal endothelial cells reduces pathologic IVNV and maintains retinal thickness, but has no effect on physiologic retinal vascular development The transcription factor, signal transducer and activator of transcription 3 (STAT3), is activated downstream of VEGF-induced activation of VEGFR2 and is important for VEGF-VEGFR2 induced rRMVEC proliferation [ ].

    Techniques: Quantitative RT-PCR, Expressing, Western Blot

    SAR131675 decreases high-glucose and palmitate-induced vascular endothelial cell growth factor-C (VEGF-C), vascular endothelial cell growth factor receptor-3 (VEGFR-3), and LYVE-1 expression in human kidney-2 (HK2) cells. To determine whether the addition of SAR131675 might modulate lymphangiogenesis in human proximal tubular epithelial HK2 cells, the cells were stimulated with palmitate (500 μM) and exposed to SAR131675 at 1, 10, or 100 nM in low-glucose (LG; 5 mmol/L d -glucose) or high-glucose (HG; 30 mmol/L d -glucose) medium. Representative western blots for VEGF-C, VEGFR-1, VEGFR-2, VEGFR-3, and β-actin and quantitative data. * P

    Journal: Cell Death & Disease

    Article Title: Inhibition of lymphatic proliferation by the selective VEGFR-3 inhibitor SAR131675 ameliorates diabetic nephropathy in db/db mice

    doi: 10.1038/s41419-019-1436-1

    Figure Lengend Snippet: SAR131675 decreases high-glucose and palmitate-induced vascular endothelial cell growth factor-C (VEGF-C), vascular endothelial cell growth factor receptor-3 (VEGFR-3), and LYVE-1 expression in human kidney-2 (HK2) cells. To determine whether the addition of SAR131675 might modulate lymphangiogenesis in human proximal tubular epithelial HK2 cells, the cells were stimulated with palmitate (500 μM) and exposed to SAR131675 at 1, 10, or 100 nM in low-glucose (LG; 5 mmol/L d -glucose) or high-glucose (HG; 30 mmol/L d -glucose) medium. Representative western blots for VEGF-C, VEGFR-1, VEGFR-2, VEGFR-3, and β-actin and quantitative data. * P

    Article Snippet: VEGF-C, VEGFR-3, and LYVE-1 expression was increased upon exposure to palmitate in HK2 cells, but was normalized to the levels comparable to that of low glucose without palmitate after SAR131675 treatment, whereas the expression of VEGFR-1 and VEGFR-2 was not altered by SAR131675 treatment.

    Techniques: Expressing, Western Blot

    SAR131675 decreases high-glucose and palmitate-induced vascular endothelial cell growth factor-C (VEGF-C), vascular endothelial cell growth factor receptor-3 (VEGFR-3), and LYVE-1 expression and M polarization in RAW264.7 cells. To determine whether SAR131675 might modulate lymphangiogenesis and macrophage polarization in RAW264.7 murine macrophage cells, the cells were stimulated with palmitate (500 μM) in high-glucose medium (HG; 30 mmol/l d -glucose) and exposed to SAR131675 at 1, 10, and 100 nM. a Representative western blots for VEGF-C, VEGFR-1, VEGFR-2, VEGFR-3, and β-actin and quantitative data. b Representative western blots for CD68, arginase I, and arginase II, inducible nitric oxide synthase (iNOS), and β-actin and quantitative data. c Representative confocal microscopy images showing dihydroethidium (DHE) and MitoSOX fluorescence in the human kidney-2 (HK2) and RAW264.7 cells and quantitative data. * P

    Journal: Cell Death & Disease

    Article Title: Inhibition of lymphatic proliferation by the selective VEGFR-3 inhibitor SAR131675 ameliorates diabetic nephropathy in db/db mice

    doi: 10.1038/s41419-019-1436-1

    Figure Lengend Snippet: SAR131675 decreases high-glucose and palmitate-induced vascular endothelial cell growth factor-C (VEGF-C), vascular endothelial cell growth factor receptor-3 (VEGFR-3), and LYVE-1 expression and M polarization in RAW264.7 cells. To determine whether SAR131675 might modulate lymphangiogenesis and macrophage polarization in RAW264.7 murine macrophage cells, the cells were stimulated with palmitate (500 μM) in high-glucose medium (HG; 30 mmol/l d -glucose) and exposed to SAR131675 at 1, 10, and 100 nM. a Representative western blots for VEGF-C, VEGFR-1, VEGFR-2, VEGFR-3, and β-actin and quantitative data. b Representative western blots for CD68, arginase I, and arginase II, inducible nitric oxide synthase (iNOS), and β-actin and quantitative data. c Representative confocal microscopy images showing dihydroethidium (DHE) and MitoSOX fluorescence in the human kidney-2 (HK2) and RAW264.7 cells and quantitative data. * P

    Article Snippet: VEGF-C, VEGFR-3, and LYVE-1 expression was increased upon exposure to palmitate in HK2 cells, but was normalized to the levels comparable to that of low glucose without palmitate after SAR131675 treatment, whereas the expression of VEGFR-1 and VEGFR-2 was not altered by SAR131675 treatment.

    Techniques: Expressing, Western Blot, Confocal Microscopy, Fluorescence

    SAR131675 attenuates transforming growth factor (TGF-β) expression and lymphangiogenesis in the kidneys in db/db mice. Effects of SAR131675 on the expression of TGF-β, vascular endothelial cell growth factor-C (VEGF-C), vascular endothelial cell growth factor receptor-1 (VEGFR-1), VEGFR-2, VEGFR-3, LYVE-1, and podoplanin in the cortex and medulla were determined at 20 weeks in db/m and db/db mice treated with or without SAR131675. a Representative immunohistochemistry for TGF-β and VEGF-C. b Representative western blots for VEGF-C, VEGFR-1, VEGFR-2, VEGFR-3, and β-actin in the cortex and medullar and quantitative data. c Representative images of immunohistochemical staining for LYVE-1 and podoplanin, and d western blots for LYVE-1, podoplanin, and β-actin in the cortex and medullar and quantitative data. * P

    Journal: Cell Death & Disease

    Article Title: Inhibition of lymphatic proliferation by the selective VEGFR-3 inhibitor SAR131675 ameliorates diabetic nephropathy in db/db mice

    doi: 10.1038/s41419-019-1436-1

    Figure Lengend Snippet: SAR131675 attenuates transforming growth factor (TGF-β) expression and lymphangiogenesis in the kidneys in db/db mice. Effects of SAR131675 on the expression of TGF-β, vascular endothelial cell growth factor-C (VEGF-C), vascular endothelial cell growth factor receptor-1 (VEGFR-1), VEGFR-2, VEGFR-3, LYVE-1, and podoplanin in the cortex and medulla were determined at 20 weeks in db/m and db/db mice treated with or without SAR131675. a Representative immunohistochemistry for TGF-β and VEGF-C. b Representative western blots for VEGF-C, VEGFR-1, VEGFR-2, VEGFR-3, and β-actin in the cortex and medullar and quantitative data. c Representative images of immunohistochemical staining for LYVE-1 and podoplanin, and d western blots for LYVE-1, podoplanin, and β-actin in the cortex and medullar and quantitative data. * P

    Article Snippet: VEGF-C, VEGFR-3, and LYVE-1 expression was increased upon exposure to palmitate in HK2 cells, but was normalized to the levels comparable to that of low glucose without palmitate after SAR131675 treatment, whereas the expression of VEGFR-1 and VEGFR-2 was not altered by SAR131675 treatment.

    Techniques: Expressing, Mouse Assay, Immunohistochemistry, Western Blot, Staining

    Avascular zones, distal microaneurysms, and lack of deep plexi in Srf iECKO retinae at P10. ( A ) ILB4-stained retinal flat-mounts. Red arrow indicates recessed angiogenic front in the Srf iECKO primary plexus. Images are composites (see Methods). ( B ) Radial outgrowth, expressed as percent of control. n = 19 retinae (control); 9 retinae ( Srf iECKO ). ( C ) Higher magnification of ILB4-stained retinal flat-mounts. White arrows indicate microaneurysms in Srf iECKO retinae. ( D ) EM image of blood vessels near the inner limiting membrane (ILM) to visualize the primary plexus. P, pericyte; L, lumen; BL, basal lamina. ( E and F ) ILB4-stained retinal capillaries of ( E ) the primary plexus and ( F ) deep plexi, which revealed complete absence of deeper capillaries in Srf iECKO retinae. ( G ) EM image visualizing deep plexi. OPL, outer plexiform layer. ( H ) Semiquantitative RT-PCR of mRNA expression in purified ECs of P10 retinae. n = 4 ( Srf ); 3 ( Kdr and Actb ); 5 ( Cdh5 ). mRNA levels were normalized to Gapdh and expressed as percent of control. ( I ) Western blot analysis of 2 representative pairs of control and Srf iECKO P10 whole retinal tissue. ( J ) Quantitation of Western blot. SRF ( n = 5) and VEGF-R2 ( n = 4) levels were normalized to GAPDH and expressed as percent of control. Scale bars: 1 mm ( A ), 100 μm ( C ), 2 μm ( D and G , left), 50 μm ( E and F ), 5 μm ( G , right). * P

    Journal: The Journal of Clinical Investigation

    Article Title: Endothelial SRF/MRTF ablation causes vascular disease phenotypes in murine retinae

    doi: 10.1172/JCI64201

    Figure Lengend Snippet: Avascular zones, distal microaneurysms, and lack of deep plexi in Srf iECKO retinae at P10. ( A ) ILB4-stained retinal flat-mounts. Red arrow indicates recessed angiogenic front in the Srf iECKO primary plexus. Images are composites (see Methods). ( B ) Radial outgrowth, expressed as percent of control. n = 19 retinae (control); 9 retinae ( Srf iECKO ). ( C ) Higher magnification of ILB4-stained retinal flat-mounts. White arrows indicate microaneurysms in Srf iECKO retinae. ( D ) EM image of blood vessels near the inner limiting membrane (ILM) to visualize the primary plexus. P, pericyte; L, lumen; BL, basal lamina. ( E and F ) ILB4-stained retinal capillaries of ( E ) the primary plexus and ( F ) deep plexi, which revealed complete absence of deeper capillaries in Srf iECKO retinae. ( G ) EM image visualizing deep plexi. OPL, outer plexiform layer. ( H ) Semiquantitative RT-PCR of mRNA expression in purified ECs of P10 retinae. n = 4 ( Srf ); 3 ( Kdr and Actb ); 5 ( Cdh5 ). mRNA levels were normalized to Gapdh and expressed as percent of control. ( I ) Western blot analysis of 2 representative pairs of control and Srf iECKO P10 whole retinal tissue. ( J ) Quantitation of Western blot. SRF ( n = 5) and VEGF-R2 ( n = 4) levels were normalized to GAPDH and expressed as percent of control. Scale bars: 1 mm ( A ), 100 μm ( C ), 2 μm ( D and G , left), 50 μm ( E and F ), 5 μm ( G , right). * P

    Article Snippet: Primary antibodies were as follows: GAPDH (1:20,000 dilution; Hytest Ltd.), VEGF-R2 (1:1,000 dilution; Cell Signaling), SRF (2C5, undiluted; ref. ), P-cofilin (1:500 dilution; Cell Signaling).

    Techniques: Staining, Reverse Transcription Polymerase Chain Reaction, Expressing, Purification, Western Blot, Quantitation Assay

    TSP-1 inhibits VEGF induced VEGFR2 phosphorylation through CD47. (A) Late EPCs were treated with VEGF (25 ng/ml) for 0, 5, 10, 15 min respectively. Total protein was extracted and the expression of FLK-1, phospho-VEGFR2 (Tyr1175) was determined by Western blot. (B)Seventy-two hrs after transfection of CD47-specific siRNA or control siRNA, the expression of CD47 in late EPCs was determined by western blotting analysis in three independent experiments (#p

    Journal: PLoS ONE

    Article Title: Effect and Mechanism of Thrombospondin-1 on the Angiogenesis Potential in Human Endothelial Progenitor Cells: An In Vitro Study

    doi: 10.1371/journal.pone.0088213

    Figure Lengend Snippet: TSP-1 inhibits VEGF induced VEGFR2 phosphorylation through CD47. (A) Late EPCs were treated with VEGF (25 ng/ml) for 0, 5, 10, 15 min respectively. Total protein was extracted and the expression of FLK-1, phospho-VEGFR2 (Tyr1175) was determined by Western blot. (B)Seventy-two hrs after transfection of CD47-specific siRNA or control siRNA, the expression of CD47 in late EPCs was determined by western blotting analysis in three independent experiments (#p

    Article Snippet: After 14 days of cultivation, EPCs exhibited a cobblestone morphology, spindle-like shape ( ) and displayed endothelial cell markers including VEGFR2, vWF and CD31( ).

    Techniques: Expressing, Western Blot, Transfection

    Intestinal ischemia reperfusion (IIR)-induced changes of vascular endothelial growth factor receptor (VEGFR)-2 in the lung tissue and immunoblotting of VEGF and its receptors. (a) VEGFR-2 immunostaining (four animals per group). Slides (magnification 1,000×) shown are representatives of indicated groups. Positively stained cells are in brown (examples are indicated with arrowheads). In the IIR group, some of the positive cells appear to be interstitial monocytes with strong staining in the cytoplasm. (b) Quantification of VEGFR-2-positive cells per field. Ten fields were counted from each animal and four animals from each group. (c) Western blotting for VEGF and its receptors. Results from two animals per group are used as examples. The optical density of blot bands were quantified with desitometry and normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as controls. No significant difference was found among these three groups.

    Journal: Critical Care

    Article Title: The early responses of VEGF and its receptors during acute lung injury: implication of VEGF in alveolar epithelial cell survival

    doi: 10.1186/cc5042

    Figure Lengend Snippet: Intestinal ischemia reperfusion (IIR)-induced changes of vascular endothelial growth factor receptor (VEGFR)-2 in the lung tissue and immunoblotting of VEGF and its receptors. (a) VEGFR-2 immunostaining (four animals per group). Slides (magnification 1,000×) shown are representatives of indicated groups. Positively stained cells are in brown (examples are indicated with arrowheads). In the IIR group, some of the positive cells appear to be interstitial monocytes with strong staining in the cytoplasm. (b) Quantification of VEGFR-2-positive cells per field. Ten fields were counted from each animal and four animals from each group. (c) Western blotting for VEGF and its receptors. Results from two animals per group are used as examples. The optical density of blot bands were quantified with desitometry and normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as controls. No significant difference was found among these three groups.

    Article Snippet: VEGFR-2, mainly located on endothelial cells, but also on epithelial type II cells [ ], is responsible for most of the known functions of VEGF in the lung [ ] and, in particular, is involved in the anti-apoptotic properties of VEGF on endothelial cells [ ].

    Techniques: Immunostaining, Staining, Western Blot

    Protein expression and phosphorylated/total protein ratio of mechanosensor complex before and after 14 days of sucrose intervention A , protein expression of VEGF‐R2, PECAM‐1 and VE‐cadherin. B , phosphorylated VEGF‐R2 (tyr1175 ), PECAM‐1 (tyr713 ) and VE‐cadherin (tyr731 ) to total protein ratio in muscle homogenates from musculus vastus lateralis at rest and after 20 min of passive leg movement. C , expression of VE‐CAD, VEGF‐R2 and PECAM‐1 in isolated primary skeletal muscle cells (SkMu Cell) and skeletal muscle tissue homogenate (SkMu Tissue). D , representative blots are shown from one subject in duplicates from rest (1) and after passive leg movement (2) under the control situation and from rest (3) and after passive leg movement (4) after the sucrose intervention. Data are presented as the mean ± SD ( n = 12). * Significantly different compared to before the sucrose intervention during the condition of passive leg movement. ** Overall significant effect of the sucrose intervention.

    Journal: The Journal of Physiology

    Article Title: Endothelial mechanotransduction proteins and vascular function are altered by dietary sucrose supplementation in healthy young male subjects

    doi: 10.1113/JP274623

    Figure Lengend Snippet: Protein expression and phosphorylated/total protein ratio of mechanosensor complex before and after 14 days of sucrose intervention A , protein expression of VEGF‐R2, PECAM‐1 and VE‐cadherin. B , phosphorylated VEGF‐R2 (tyr1175 ), PECAM‐1 (tyr713 ) and VE‐cadherin (tyr731 ) to total protein ratio in muscle homogenates from musculus vastus lateralis at rest and after 20 min of passive leg movement. C , expression of VE‐CAD, VEGF‐R2 and PECAM‐1 in isolated primary skeletal muscle cells (SkMu Cell) and skeletal muscle tissue homogenate (SkMu Tissue). D , representative blots are shown from one subject in duplicates from rest (1) and after passive leg movement (2) under the control situation and from rest (3) and after passive leg movement (4) after the sucrose intervention. Data are presented as the mean ± SD ( n = 12). * Significantly different compared to before the sucrose intervention during the condition of passive leg movement. ** Overall significant effect of the sucrose intervention.

    Article Snippet: This impairment in vascular function was paralleled by an up‐regulation of PECAM‐1, eNOS, NOX and Rac1 protein expression in whole muscle tissue, as well as by an increased phosphorylation status of VEGF‐R2 and a reduced phosphorylation status of PECAM‐1.

    Techniques: Expressing, Isolation

    Skeletal muscle mRNA levels of eNOS, PECAM‐1 and VEGR‐R2 before and after 14 days of sucrose intervention mRNA levels of eNOS, PECAM‐1 and VEGF‐R2 in muscle homogenates from musculus vastus lateralis at rest and after 20 min of passive leg movement. Data are presented as the mean ± SD ( n = 12). * Significantly different compared to before the sucrose intervention under the same condition. There was no significant effect of passive leg movement.

    Journal: The Journal of Physiology

    Article Title: Endothelial mechanotransduction proteins and vascular function are altered by dietary sucrose supplementation in healthy young male subjects

    doi: 10.1113/JP274623

    Figure Lengend Snippet: Skeletal muscle mRNA levels of eNOS, PECAM‐1 and VEGR‐R2 before and after 14 days of sucrose intervention mRNA levels of eNOS, PECAM‐1 and VEGF‐R2 in muscle homogenates from musculus vastus lateralis at rest and after 20 min of passive leg movement. Data are presented as the mean ± SD ( n = 12). * Significantly different compared to before the sucrose intervention under the same condition. There was no significant effect of passive leg movement.

    Article Snippet: This impairment in vascular function was paralleled by an up‐regulation of PECAM‐1, eNOS, NOX and Rac1 protein expression in whole muscle tissue, as well as by an increased phosphorylation status of VEGF‐R2 and a reduced phosphorylation status of PECAM‐1.

    Techniques:

    ENZ and CCX771 combination treatment suppresses EGFR, AKT and VEGFR2 signaling. Western blotting analysis revealed that CCX771 and ENZ markedly reduced phosphorylation levels of EGFR, AKT and VEGFR2 in VCaP ( a ) and C4‐2B ( c ) cells. Combination treatment (ENZ + CCX771) showed increased suppression of EGFR/AKT signaling compared to both single agents. The quantified ratio of the band intensity for the phosphorylated protein normalized to the total protein and DMSO control, and the VEGFR2 normalized to GAPDH and DMSO control for VCaP ( b ) and C4‐2B ( d ) cells.

    Journal: International Journal of Cancer

    Article Title: Enzalutamide and CXCR7 inhibitor combination treatment suppresses cell growth and angiogenic signaling in castration‐resistant prostate cancer models

    doi: 10.1002/ijc.31237

    Figure Lengend Snippet: ENZ and CCX771 combination treatment suppresses EGFR, AKT and VEGFR2 signaling. Western blotting analysis revealed that CCX771 and ENZ markedly reduced phosphorylation levels of EGFR, AKT and VEGFR2 in VCaP ( a ) and C4‐2B ( c ) cells. Combination treatment (ENZ + CCX771) showed increased suppression of EGFR/AKT signaling compared to both single agents. The quantified ratio of the band intensity for the phosphorylated protein normalized to the total protein and DMSO control, and the VEGFR2 normalized to GAPDH and DMSO control for VCaP ( b ) and C4‐2B ( d ) cells.

    Article Snippet: Single agent AMD3100 treatment did not reduce expression of p‐EGFR, VEGFR2 and p‐AKT in either cell line (Fig. ).

    Techniques: Western Blot

    VEGFR-2 is the potential target of pioglitazone. A, 3D molecular docking model of pioglitazone with VEGFR-2; green structure, the conformer of pioglitazone. B, 2D molecular docking model of pioglitazone with VEGFR-2; purple structure, the conformer of pioglitazone. H-bonding interactions between the pioglitazone and VEGFR-2 were indicated with green dashed lines. C and D, Representative western bolotting trace of VEGFR-2 and phospho-VEGFR-2 (Tyr1175) protein levels in rat neonatal cardiomyocytes under hypertrophic stimuli, and treated with 0, 10, 20 (µM) pioglitazone for 24 hours, and intensity of the bands in C normalized to β-actin (n = 12 in each group). All data shown are the mean ± SD. *p

    Journal: Arquivos Brasileiros de Cardiologia

    Article Title: Pioglitazone Induces Cardiomyocyte Apoptosis and Inhibits Cardiomyocyte Hypertrophy Via VEGFR-2 Signaling Pathway

    doi: 10.5935/abc.20180108

    Figure Lengend Snippet: VEGFR-2 is the potential target of pioglitazone. A, 3D molecular docking model of pioglitazone with VEGFR-2; green structure, the conformer of pioglitazone. B, 2D molecular docking model of pioglitazone with VEGFR-2; purple structure, the conformer of pioglitazone. H-bonding interactions between the pioglitazone and VEGFR-2 were indicated with green dashed lines. C and D, Representative western bolotting trace of VEGFR-2 and phospho-VEGFR-2 (Tyr1175) protein levels in rat neonatal cardiomyocytes under hypertrophic stimuli, and treated with 0, 10, 20 (µM) pioglitazone for 24 hours, and intensity of the bands in C normalized to β-actin (n = 12 in each group). All data shown are the mean ± SD. *p

    Article Snippet: - VEGFR-2 is a tyrosine kinase receptor that dimerizes upon ligand binding and is activated by trans-phosphorylation.

    Techniques: Western Blot

    Pioglitazone and Apatinib regulate VEGFR-2 signaling in neonatal rat cardiomyocytes under hypertrophy induced by Angiotensin II (n = 12 in each group). A and B, Representative western bolotting trace of phospho-VEGFR-2, VEGFR-2, phospho-mTOR, mTOR, phospho-Akt, Akt, Bax, phospho-P53 and P53 protein levels in rat neonatal cardiomyocytes under hypertrophic stimuli, and treated with pioglitazone (20 µM) or apatinib (2 µM) for 24 hours, and intensity of the above bands in A normalized to GAPDH. All data represent the means ± SD. *p

    Journal: Arquivos Brasileiros de Cardiologia

    Article Title: Pioglitazone Induces Cardiomyocyte Apoptosis and Inhibits Cardiomyocyte Hypertrophy Via VEGFR-2 Signaling Pathway

    doi: 10.5935/abc.20180108

    Figure Lengend Snippet: Pioglitazone and Apatinib regulate VEGFR-2 signaling in neonatal rat cardiomyocytes under hypertrophy induced by Angiotensin II (n = 12 in each group). A and B, Representative western bolotting trace of phospho-VEGFR-2, VEGFR-2, phospho-mTOR, mTOR, phospho-Akt, Akt, Bax, phospho-P53 and P53 protein levels in rat neonatal cardiomyocytes under hypertrophic stimuli, and treated with pioglitazone (20 µM) or apatinib (2 µM) for 24 hours, and intensity of the above bands in A normalized to GAPDH. All data represent the means ± SD. *p

    Article Snippet: - VEGFR-2 is a tyrosine kinase receptor that dimerizes upon ligand binding and is activated by trans-phosphorylation.

    Techniques: Western Blot

    Proposed cascade of events following activation of 12/15-LOX by hyperglycemia. Lipid metabolites of 12/15-LOX (12- and 15-HETE) activate vascular NADPH oxidase leading to overproduction of reactive oxygen species (ROS). Generation of ROS suppresses the activity of protein tyrosine phosphatases with subsequent activation of VEGF-R2 signal pathway and disruption of retinal endothelial cell barrier.

    Journal: PLoS ONE

    Article Title: 12/15-Lipoxygenase-Derived Lipid Metabolites Induce Retinal Endothelial Cell Barrier Dysfunction: Contribution of NADPH Oxidase

    doi: 10.1371/journal.pone.0057254

    Figure Lengend Snippet: Proposed cascade of events following activation of 12/15-LOX by hyperglycemia. Lipid metabolites of 12/15-LOX (12- and 15-HETE) activate vascular NADPH oxidase leading to overproduction of reactive oxygen species (ROS). Generation of ROS suppresses the activity of protein tyrosine phosphatases with subsequent activation of VEGF-R2 signal pathway and disruption of retinal endothelial cell barrier.

    Article Snippet: In particular, NADPH oxidase has been shown to modulate VEGF signaling pathway in endothelial cells via activating VEGF-R2 , .

    Techniques: Activation Assay, Activity Assay

    12-HETE induces phosphorylation of VEGF-R2 and dephosphorylation of protein tyrosine phosphatase (PTP) SHP1 in REC. Western blotting analysis (A) demonstrated significant increase in the level of pVEGF-R2 after 5 and 60 minutes from the beginning of treatment with 12-HETE. *P

    Journal: PLoS ONE

    Article Title: 12/15-Lipoxygenase-Derived Lipid Metabolites Induce Retinal Endothelial Cell Barrier Dysfunction: Contribution of NADPH Oxidase

    doi: 10.1371/journal.pone.0057254

    Figure Lengend Snippet: 12-HETE induces phosphorylation of VEGF-R2 and dephosphorylation of protein tyrosine phosphatase (PTP) SHP1 in REC. Western blotting analysis (A) demonstrated significant increase in the level of pVEGF-R2 after 5 and 60 minutes from the beginning of treatment with 12-HETE. *P

    Article Snippet: In particular, NADPH oxidase has been shown to modulate VEGF signaling pathway in endothelial cells via activating VEGF-R2 , .

    Techniques: De-Phosphorylation Assay, Western Blot

    Effect of VEGF-R2 inhibition on 12-HETE-induced REC hyperpermeability. Retinal endothelial cells were treated with 0.1 µM 12-HETE in the presence or absence of the VEGF-R2 inhibitor, ZM323881 hydrochloride (10 nM) for 12 hrs before adding the FITC-dextran to the upper chamber of the transwell. Four hrs later the fluorescence intensity in the lower chamber was measured by the plate reader and corrected to the intensity in the upper one. The permeability effect of 12-HETE was significantly prevented by the ZM323881 hydrochloride (12-HETE+I). * P

    Journal: PLoS ONE

    Article Title: 12/15-Lipoxygenase-Derived Lipid Metabolites Induce Retinal Endothelial Cell Barrier Dysfunction: Contribution of NADPH Oxidase

    doi: 10.1371/journal.pone.0057254

    Figure Lengend Snippet: Effect of VEGF-R2 inhibition on 12-HETE-induced REC hyperpermeability. Retinal endothelial cells were treated with 0.1 µM 12-HETE in the presence or absence of the VEGF-R2 inhibitor, ZM323881 hydrochloride (10 nM) for 12 hrs before adding the FITC-dextran to the upper chamber of the transwell. Four hrs later the fluorescence intensity in the lower chamber was measured by the plate reader and corrected to the intensity in the upper one. The permeability effect of 12-HETE was significantly prevented by the ZM323881 hydrochloride (12-HETE+I). * P

    Article Snippet: In particular, NADPH oxidase has been shown to modulate VEGF signaling pathway in endothelial cells via activating VEGF-R2 , .

    Techniques: Inhibition, Fluorescence, Permeability

    Overexpression of miR-150 decreased VEGFR2 protein level in endothelial cells. The HUVE cells were transfected with miR-150 (has-miR-150) or a scramble microRNA (Scramble) and cultured for an additional 60 hr. The protein levels of c-Myb and VEGFR2 are significantly lower in HUVE cells with overexpression of miR-150 compared to the scramble (student’s t -test; * p

    Journal: PLoS ONE

    Article Title: Deletion of miR-150 Exacerbates Retinal Vascular Overgrowth in High-Fat-Diet Induced Diabetic Mice

    doi: 10.1371/journal.pone.0157543

    Figure Lengend Snippet: Overexpression of miR-150 decreased VEGFR2 protein level in endothelial cells. The HUVE cells were transfected with miR-150 (has-miR-150) or a scramble microRNA (Scramble) and cultured for an additional 60 hr. The protein levels of c-Myb and VEGFR2 are significantly lower in HUVE cells with overexpression of miR-150 compared to the scramble (student’s t -test; * p

    Article Snippet: The primary antibodies used in this study were anti-VEGFR2 (Cell Signaling Technology, Denver, MA, USA), anti-pan actin (Cell Signaling Technology), and anti-GAPDH (Cell Signaling Technology).

    Techniques: Over Expression, Transfection, Cell Culture

    Electron immunomicroscopy detection of the anti-VEGFR-2 probe in the endothelial plasma membrane/cytoplasm and cell nuclei within GL261 rat gliomas. A. Electron immunomicroscopy of a GL261 glioma-bearing mouse administered the anti-VEGFR2 probe. The biotin

    Journal: American Journal of Nuclear Medicine and Molecular Imaging

    Article Title: OKN-007 decreases VEGFR-2 levels in a preclinical GL261 mouse glioma model

    doi:

    Figure Lengend Snippet: Electron immunomicroscopy detection of the anti-VEGFR-2 probe in the endothelial plasma membrane/cytoplasm and cell nuclei within GL261 rat gliomas. A. Electron immunomicroscopy of a GL261 glioma-bearing mouse administered the anti-VEGFR2 probe. The biotin

    Article Snippet: The membranes were then probed with anti-VEGFR2 antibody (Cell Signaling Technology) visualized by SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific) as described previously [ ].

    Techniques:

    Expression profile of RTKs and the effect of dovitinib on RTK signaling in HCC and endothelial cells. A ) Expression of PDGFR-β, FGFR-1, and VEGFR-2 in HCC and endothelial cell lines as detected by immunoblotting. B ) Expression of FGFR-3, and VEGFR-1 in HCC and endothelial cell lines C ) Phosphorylation of p-PDGFR-β and p-ERK were inhibited by dovitinib at pharmacologically relevant concentrations in MHCC-97H and SMMC7721 cells. D ) Dovitinib inhibited the phosphorylation of FGFG-1, VEGFR-2, and downstream ERK in HMVEC and HUVEC endothelial cells at pharmacologically relevant concentrations.

    Journal: Journal of Translational Medicine

    Article Title: Dovitinib preferentially targets endothelial cells rather than cancer cells for the inhibition of hepatocellular carcinoma growth and metastasis

    doi: 10.1186/1479-5876-10-245

    Figure Lengend Snippet: Expression profile of RTKs and the effect of dovitinib on RTK signaling in HCC and endothelial cells. A ) Expression of PDGFR-β, FGFR-1, and VEGFR-2 in HCC and endothelial cell lines as detected by immunoblotting. B ) Expression of FGFR-3, and VEGFR-1 in HCC and endothelial cell lines C ) Phosphorylation of p-PDGFR-β and p-ERK were inhibited by dovitinib at pharmacologically relevant concentrations in MHCC-97H and SMMC7721 cells. D ) Dovitinib inhibited the phosphorylation of FGFG-1, VEGFR-2, and downstream ERK in HMVEC and HUVEC endothelial cells at pharmacologically relevant concentrations.

    Article Snippet: Protein (1 mg) from each sample was immunoprecipitated overnight at 4°C with an anti-VEGFR-2, anti-PDGFR-β, or anti-FGFR-1 (Cell Signaling Technology) antibody plus protein G/A agarose beads (Pierce).

    Techniques: Expressing

    Itraconazole induces a change in the migration pattern of VEGFR2. A , HUVEC were treated for 24 h with the indicated doses of itraconazole or vehicle (DMSO), and VEGFR2 was analyzed by Western blot. B , HUVEC were treated as in A with three inhibitors of

    Journal: The Journal of Biological Chemistry

    Article Title: The Antifungal Drug Itraconazole Inhibits Vascular Endothelial Growth Factor Receptor 2 (VEGFR2) Glycosylation, Trafficking, and Signaling in Endothelial Cells *

    doi: 10.1074/jbc.M111.278754

    Figure Lengend Snippet: Itraconazole induces a change in the migration pattern of VEGFR2. A , HUVEC were treated for 24 h with the indicated doses of itraconazole or vehicle (DMSO), and VEGFR2 was analyzed by Western blot. B , HUVEC were treated as in A with three inhibitors of

    Article Snippet: After a 24-h treatment, the cells were lysed by the addition of SDS sample buffer and incubated on ice for 10 min followed by boiling for 10 min. For phospho-VEGFR2 (Cell Signaling, catalog no. 2478), VEGFR2 (Cell Signaling, catalog no. 2479), VEGFR1 (Cell Signaling, catalog no. 2893), PLCγ1 (Cell Signaling, catalog no. 2822), and phospho-PLCγ1 (Cell Signaling, catalog no. 2821) blotting, lysates were subjected to 6 or 8% SDS-PAGE.

    Techniques: Migration, Western Blot

    Subcellular localization of VEGFR2 after treatment with itraconazole or other glycosylation inhibitors. HUVEC were treated for 24 h with itraconazole ( Ita ; 800 n m ) or vehicle (DMSO), fixed, and stained with anti-VEGFR2 and anti-GM130 ( A ), a cis- Golgi

    Journal: The Journal of Biological Chemistry

    Article Title: The Antifungal Drug Itraconazole Inhibits Vascular Endothelial Growth Factor Receptor 2 (VEGFR2) Glycosylation, Trafficking, and Signaling in Endothelial Cells *

    doi: 10.1074/jbc.M111.278754

    Figure Lengend Snippet: Subcellular localization of VEGFR2 after treatment with itraconazole or other glycosylation inhibitors. HUVEC were treated for 24 h with itraconazole ( Ita ; 800 n m ) or vehicle (DMSO), fixed, and stained with anti-VEGFR2 and anti-GM130 ( A ), a cis- Golgi

    Article Snippet: After a 24-h treatment, the cells were lysed by the addition of SDS sample buffer and incubated on ice for 10 min followed by boiling for 10 min. For phospho-VEGFR2 (Cell Signaling, catalog no. 2478), VEGFR2 (Cell Signaling, catalog no. 2479), VEGFR1 (Cell Signaling, catalog no. 2893), PLCγ1 (Cell Signaling, catalog no. 2822), and phospho-PLCγ1 (Cell Signaling, catalog no. 2821) blotting, lysates were subjected to 6 or 8% SDS-PAGE.

    Techniques: Staining

    Itraconazole's effects on glycosylation extend beyond VEGFR2 and HUVEC. A , HUVEC were treated with the indicated doses of itraconazole ( Ita ) or DMSO vehicle (0 μ m itraconazole dose), and the migration pattern of VEGFR1 was analyzed by Western

    Journal: The Journal of Biological Chemistry

    Article Title: The Antifungal Drug Itraconazole Inhibits Vascular Endothelial Growth Factor Receptor 2 (VEGFR2) Glycosylation, Trafficking, and Signaling in Endothelial Cells *

    doi: 10.1074/jbc.M111.278754

    Figure Lengend Snippet: Itraconazole's effects on glycosylation extend beyond VEGFR2 and HUVEC. A , HUVEC were treated with the indicated doses of itraconazole ( Ita ) or DMSO vehicle (0 μ m itraconazole dose), and the migration pattern of VEGFR1 was analyzed by Western

    Article Snippet: After a 24-h treatment, the cells were lysed by the addition of SDS sample buffer and incubated on ice for 10 min followed by boiling for 10 min. For phospho-VEGFR2 (Cell Signaling, catalog no. 2478), VEGFR2 (Cell Signaling, catalog no. 2479), VEGFR1 (Cell Signaling, catalog no. 2893), PLCγ1 (Cell Signaling, catalog no. 2822), and phospho-PLCγ1 (Cell Signaling, catalog no. 2821) blotting, lysates were subjected to 6 or 8% SDS-PAGE.

    Techniques: Migration, Western Blot

    Itraconazole blocks VEGF 165 binding to VEGFR2 and inhibits VEGFR2 signaling. A , HUVEC were grown in low serum medium for 24 h in the presence of vehicle (DMSO), itraconazole ( Ita ), or the VEGFR2 inhibitor sunitinib. The cells were then stimulated for

    Journal: The Journal of Biological Chemistry

    Article Title: The Antifungal Drug Itraconazole Inhibits Vascular Endothelial Growth Factor Receptor 2 (VEGFR2) Glycosylation, Trafficking, and Signaling in Endothelial Cells *

    doi: 10.1074/jbc.M111.278754

    Figure Lengend Snippet: Itraconazole blocks VEGF 165 binding to VEGFR2 and inhibits VEGFR2 signaling. A , HUVEC were grown in low serum medium for 24 h in the presence of vehicle (DMSO), itraconazole ( Ita ), or the VEGFR2 inhibitor sunitinib. The cells were then stimulated for

    Article Snippet: After a 24-h treatment, the cells were lysed by the addition of SDS sample buffer and incubated on ice for 10 min followed by boiling for 10 min. For phospho-VEGFR2 (Cell Signaling, catalog no. 2478), VEGFR2 (Cell Signaling, catalog no. 2479), VEGFR1 (Cell Signaling, catalog no. 2893), PLCγ1 (Cell Signaling, catalog no. 2822), and phospho-PLCγ1 (Cell Signaling, catalog no. 2821) blotting, lysates were subjected to 6 or 8% SDS-PAGE.

    Techniques: Binding Assay

    Itraconazole-induced VEGFR2 hypoglycosylation and signaling inhibition is rescued by supplementation of cellular cholesterol. A , HUVEC were treated with itraconazole or vehicle alone in the presence of free cholesterol or free β-estradiol, free

    Journal: The Journal of Biological Chemistry

    Article Title: The Antifungal Drug Itraconazole Inhibits Vascular Endothelial Growth Factor Receptor 2 (VEGFR2) Glycosylation, Trafficking, and Signaling in Endothelial Cells *

    doi: 10.1074/jbc.M111.278754

    Figure Lengend Snippet: Itraconazole-induced VEGFR2 hypoglycosylation and signaling inhibition is rescued by supplementation of cellular cholesterol. A , HUVEC were treated with itraconazole or vehicle alone in the presence of free cholesterol or free β-estradiol, free

    Article Snippet: After a 24-h treatment, the cells were lysed by the addition of SDS sample buffer and incubated on ice for 10 min followed by boiling for 10 min. For phospho-VEGFR2 (Cell Signaling, catalog no. 2478), VEGFR2 (Cell Signaling, catalog no. 2479), VEGFR1 (Cell Signaling, catalog no. 2893), PLCγ1 (Cell Signaling, catalog no. 2822), and phospho-PLCγ1 (Cell Signaling, catalog no. 2821) blotting, lysates were subjected to 6 or 8% SDS-PAGE.

    Techniques: Inhibition

    Itraconazole's effects on VEGFR2 glycosylation occur in parallel to other itraconazole-induced effects. A , cholesterol localization was visualized by filipin staining of HUVEC treated with 800 n m itraconazole ( Ita ), 500 μ m castanospermine ( Cast

    Journal: The Journal of Biological Chemistry

    Article Title: The Antifungal Drug Itraconazole Inhibits Vascular Endothelial Growth Factor Receptor 2 (VEGFR2) Glycosylation, Trafficking, and Signaling in Endothelial Cells *

    doi: 10.1074/jbc.M111.278754

    Figure Lengend Snippet: Itraconazole's effects on VEGFR2 glycosylation occur in parallel to other itraconazole-induced effects. A , cholesterol localization was visualized by filipin staining of HUVEC treated with 800 n m itraconazole ( Ita ), 500 μ m castanospermine ( Cast

    Article Snippet: After a 24-h treatment, the cells were lysed by the addition of SDS sample buffer and incubated on ice for 10 min followed by boiling for 10 min. For phospho-VEGFR2 (Cell Signaling, catalog no. 2478), VEGFR2 (Cell Signaling, catalog no. 2479), VEGFR1 (Cell Signaling, catalog no. 2893), PLCγ1 (Cell Signaling, catalog no. 2822), and phospho-PLCγ1 (Cell Signaling, catalog no. 2821) blotting, lysates were subjected to 6 or 8% SDS-PAGE.

    Techniques: Staining

    Itraconazole alters N -linked glycosylation in HUVEC. A , Western blot of VEGFR2 from HUVEC treated for 24 h with itraconazole ( Ita ) or N -linked glycosylation inhibitors Sws, dMM, or castanospermine ( Cast ). HUVEC were treated for the indicated times with

    Journal: The Journal of Biological Chemistry

    Article Title: The Antifungal Drug Itraconazole Inhibits Vascular Endothelial Growth Factor Receptor 2 (VEGFR2) Glycosylation, Trafficking, and Signaling in Endothelial Cells *

    doi: 10.1074/jbc.M111.278754

    Figure Lengend Snippet: Itraconazole alters N -linked glycosylation in HUVEC. A , Western blot of VEGFR2 from HUVEC treated for 24 h with itraconazole ( Ita ) or N -linked glycosylation inhibitors Sws, dMM, or castanospermine ( Cast ). HUVEC were treated for the indicated times with

    Article Snippet: After a 24-h treatment, the cells were lysed by the addition of SDS sample buffer and incubated on ice for 10 min followed by boiling for 10 min. For phospho-VEGFR2 (Cell Signaling, catalog no. 2478), VEGFR2 (Cell Signaling, catalog no. 2479), VEGFR1 (Cell Signaling, catalog no. 2893), PLCγ1 (Cell Signaling, catalog no. 2822), and phospho-PLCγ1 (Cell Signaling, catalog no. 2821) blotting, lysates were subjected to 6 or 8% SDS-PAGE.

    Techniques: Western Blot

    The role of VEGFR3 in the mouse aorta. (A) VEGFR3 expression in arterial endothelium. Total RNA isolated from the endothelial layer was analyzed by qPCR for the indicated genes. VEcad and VEGFR3 expression are represented as mean fold enrichment of the endothelial preparation over the remaining media ± SEM (error bars) from four aortas. The relative abundance of the medial layer markers SMA and SM22 indicate the purity of endothelial preparations. (B) VEGFR3 reporter. Aortas from adult VEGFR3-driven YFP gene reporter mice were sectioned longitudinally and stained for the YFP reporter and for nuclei using Hoechst staining. IC, inner curvature. Images are representative of five mice from several litters. (C) VEGFR2 iΔEC. Endothelial-specific, inducible VEGFR3 knockout (iΔEC) and WT control mice were treated with tamoxifen, and aortas were removed after 1 wk. Tissue lysates were collected and immunoblotted with the indicated antibodies. IB, immunoblotting. (D and E) Inflammatory markers. VEGFR3 iΔEC and WT control mice were treated with tamoxifen and examined at 3 wk. Aortas were sectioned longitudinally and stained for fibronectin (D) or VCAM-1 (E). Images are representative of 6 mice from two independent experiments. Bars, 100 µm. The ratio of mean fluorescence intensity between the inner and outer curvature was then quantified. Values are means ± SEM (error bars). *, P

    Journal: The Journal of Cell Biology

    Article Title: Intramembrane binding of VE-cadherin to VEGFR2 and VEGFR3 assembles the endothelial mechanosensory complex

    doi: 10.1083/jcb.201408103

    Figure Lengend Snippet: The role of VEGFR3 in the mouse aorta. (A) VEGFR3 expression in arterial endothelium. Total RNA isolated from the endothelial layer was analyzed by qPCR for the indicated genes. VEcad and VEGFR3 expression are represented as mean fold enrichment of the endothelial preparation over the remaining media ± SEM (error bars) from four aortas. The relative abundance of the medial layer markers SMA and SM22 indicate the purity of endothelial preparations. (B) VEGFR3 reporter. Aortas from adult VEGFR3-driven YFP gene reporter mice were sectioned longitudinally and stained for the YFP reporter and for nuclei using Hoechst staining. IC, inner curvature. Images are representative of five mice from several litters. (C) VEGFR2 iΔEC. Endothelial-specific, inducible VEGFR3 knockout (iΔEC) and WT control mice were treated with tamoxifen, and aortas were removed after 1 wk. Tissue lysates were collected and immunoblotted with the indicated antibodies. IB, immunoblotting. (D and E) Inflammatory markers. VEGFR3 iΔEC and WT control mice were treated with tamoxifen and examined at 3 wk. Aortas were sectioned longitudinally and stained for fibronectin (D) or VCAM-1 (E). Images are representative of 6 mice from two independent experiments. Bars, 100 µm. The ratio of mean fluorescence intensity between the inner and outer curvature was then quantified. Values are means ± SEM (error bars). *, P

    Article Snippet: Total and phospho-VEGFRs were detected with rabbit anti-VEGFR2 (#2479; Cell Signaling Technology), goat anti-HsVEGFR3 (AF349; R & D Systems), goat anti-MmVEGFR3 (AF743; R & D Systems), rabbit anti-VEGFR2pY1175 (#2478; Cell Signaling Technology), rabbit anti-VEGFRpY1054/9 (44-1047G; Invitrogen), and rabbit anti-VEGFR3pY1230/1 (CY1115; Cell Applications).

    Techniques: Expressing, Isolation, Real-time Polymerase Chain Reaction, Mouse Assay, Staining, Knock-Out, Fluorescence

    VEcad TMD in flow signaling. (A) Requirement for VEcad. HUVECS were infected with scrambled or anti-VEcad shRNA-containing lentiviruses then subjected to 12 dynes/cm 2 laminar shear for 1 min. Activation of SFKs (SFK pY416 , ∼55 kD) and VEGFR2 (VEGFR2 pY1175 , ∼250 and 220 kD) were assayed by immunoblotting, with actin as a loading control. (B) VEcad TMD requirement for VEGFR activation. VEcad −/− cells reconstituted with VEcad WT , Ncad VE-TMD , Ncad WT , and VEcad N-TMD were subjected to laminar shear stress for 1 min, then VEGFR2 activation was assayed as in A. (C) VEcad requirement for PI3K signaling. Cells were subjected to laminar shear stress, then p85 immunoprecipitated and immunoblotted with anti-p85 pY458 antibody. Values beneath each panel indicate phosphorylation relative to cells without flow, quantified by densitometry with total p85 serving as a loading control. For all panels, values are means ± SEM, n = 3. IB, immunoblotting.

    Journal: The Journal of Cell Biology

    Article Title: Intramembrane binding of VE-cadherin to VEGFR2 and VEGFR3 assembles the endothelial mechanosensory complex

    doi: 10.1083/jcb.201408103

    Figure Lengend Snippet: VEcad TMD in flow signaling. (A) Requirement for VEcad. HUVECS were infected with scrambled or anti-VEcad shRNA-containing lentiviruses then subjected to 12 dynes/cm 2 laminar shear for 1 min. Activation of SFKs (SFK pY416 , ∼55 kD) and VEGFR2 (VEGFR2 pY1175 , ∼250 and 220 kD) were assayed by immunoblotting, with actin as a loading control. (B) VEcad TMD requirement for VEGFR activation. VEcad −/− cells reconstituted with VEcad WT , Ncad VE-TMD , Ncad WT , and VEcad N-TMD were subjected to laminar shear stress for 1 min, then VEGFR2 activation was assayed as in A. (C) VEcad requirement for PI3K signaling. Cells were subjected to laminar shear stress, then p85 immunoprecipitated and immunoblotted with anti-p85 pY458 antibody. Values beneath each panel indicate phosphorylation relative to cells without flow, quantified by densitometry with total p85 serving as a loading control. For all panels, values are means ± SEM, n = 3. IB, immunoblotting.

    Article Snippet: Total and phospho-VEGFRs were detected with rabbit anti-VEGFR2 (#2479; Cell Signaling Technology), goat anti-HsVEGFR3 (AF349; R & D Systems), goat anti-MmVEGFR3 (AF743; R & D Systems), rabbit anti-VEGFR2pY1175 (#2478; Cell Signaling Technology), rabbit anti-VEGFRpY1054/9 (44-1047G; Invitrogen), and rabbit anti-VEGFR3pY1230/1 (CY1115; Cell Applications).

    Techniques: Flow Cytometry, Infection, shRNA, Activation Assay, Immunoprecipitation

    VEcad TMD in shear-mediated VEGFR2 and VEGFR3 activation. (A) VEGFR activation. HUVECs exposed to 12 dynes/cm 2 laminar shear for the indicated times were immunoblotted with the indicated antibodies. The upper band on the anti-VEGFR2/3 pY1054/9 blot is active VEGFR2; the lower band is active VEGFR3 (arrowheads). IB, immunoblotting. (B) Dependence on TMD. VEcad −/− , VEcad WT , and VEcad ΔTMD cells were subjected to shear stress, then lysed and immunoblotted as in A. The anti-VEGFR pY1230 blots were quantified by densitometry of the ∼120-kD band with actin serving as a loading control. Values are means ± SEM (error bars), n ≥ 3. Yellow lines mark boundaries between cell types.

    Journal: The Journal of Cell Biology

    Article Title: Intramembrane binding of VE-cadherin to VEGFR2 and VEGFR3 assembles the endothelial mechanosensory complex

    doi: 10.1083/jcb.201408103

    Figure Lengend Snippet: VEcad TMD in shear-mediated VEGFR2 and VEGFR3 activation. (A) VEGFR activation. HUVECs exposed to 12 dynes/cm 2 laminar shear for the indicated times were immunoblotted with the indicated antibodies. The upper band on the anti-VEGFR2/3 pY1054/9 blot is active VEGFR2; the lower band is active VEGFR3 (arrowheads). IB, immunoblotting. (B) Dependence on TMD. VEcad −/− , VEcad WT , and VEcad ΔTMD cells were subjected to shear stress, then lysed and immunoblotted as in A. The anti-VEGFR pY1230 blots were quantified by densitometry of the ∼120-kD band with actin serving as a loading control. Values are means ± SEM (error bars), n ≥ 3. Yellow lines mark boundaries between cell types.

    Article Snippet: Total and phospho-VEGFRs were detected with rabbit anti-VEGFR2 (#2479; Cell Signaling Technology), goat anti-HsVEGFR3 (AF349; R & D Systems), goat anti-MmVEGFR3 (AF743; R & D Systems), rabbit anti-VEGFR2pY1175 (#2478; Cell Signaling Technology), rabbit anti-VEGFRpY1054/9 (44-1047G; Invitrogen), and rabbit anti-VEGFR3pY1230/1 (CY1115; Cell Applications).

    Techniques: Activation Assay

    Effects of hypericin on complex formations between VEGFR2, Hsp90 and on HOP-hsp70 intermediate complexes. [A]. Western blot analyses of VEGFR2 protein levels in cytosolic extracts from untreated control cells (C), or cells treated with 30 µM hypericin for 72 hrs (HYP). [B]. Staining of U87-MG cells with Phalloidin-FITC. (1) Untreated cells and (2) cells treated with hypericin 30 µM for 72 hrs. Orange arrows show F-actin filaments; yellow arrows depict collapsed actin globules following exposure to hypericin. [C]. VEGFR2-hsp90 complex formation following treatment with hypericin 30 µM for 72 hrs. Results of immunoprecipitation with anti-Hsp90 antibody and development of Western blots with anti-VEGFR2 antibody. Hypericin diminished VEGFR2-Hsp90 complex formation. [D]. Induction of forced hsp90 poly-ubiquitination by hypericin (30 µM for 72 hrs) in human cancerous cell lines. Top panel – immunoprecipitation with anti-hsp90 and Western blot with anti-hsp90 antibody (control); middle panel - immunoprecipitation with anti-hsp90 and Western blot with anti-ubiquitin, and lower panel immunoprecipitation with anti-ubiquitin and Western blot with anti-hsp90. (I.P – immunoprecipitation; W.B. – Western blots).

    Journal: PLoS ONE

    Article Title: Degradation of HIF-1alpha under Hypoxia Combined with Induction of Hsp90 Polyubiquitination in Cancer Cells by Hypericin: a Unique Cancer Therapy

    doi: 10.1371/journal.pone.0022849

    Figure Lengend Snippet: Effects of hypericin on complex formations between VEGFR2, Hsp90 and on HOP-hsp70 intermediate complexes. [A]. Western blot analyses of VEGFR2 protein levels in cytosolic extracts from untreated control cells (C), or cells treated with 30 µM hypericin for 72 hrs (HYP). [B]. Staining of U87-MG cells with Phalloidin-FITC. (1) Untreated cells and (2) cells treated with hypericin 30 µM for 72 hrs. Orange arrows show F-actin filaments; yellow arrows depict collapsed actin globules following exposure to hypericin. [C]. VEGFR2-hsp90 complex formation following treatment with hypericin 30 µM for 72 hrs. Results of immunoprecipitation with anti-Hsp90 antibody and development of Western blots with anti-VEGFR2 antibody. Hypericin diminished VEGFR2-Hsp90 complex formation. [D]. Induction of forced hsp90 poly-ubiquitination by hypericin (30 µM for 72 hrs) in human cancerous cell lines. Top panel – immunoprecipitation with anti-hsp90 and Western blot with anti-hsp90 antibody (control); middle panel - immunoprecipitation with anti-hsp90 and Western blot with anti-ubiquitin, and lower panel immunoprecipitation with anti-ubiquitin and Western blot with anti-hsp90. (I.P – immunoprecipitation; W.B. – Western blots).

    Article Snippet: Immunoprecipitates were prepared using rabbit polyclonal anti-ubiquitin, anti-HIF-1α, anti-VEGFR2, anti-Hsp90 and anti-GAPDH antibodies (Santa Cruz Biotechnology, CA).

    Techniques: Western Blot, Staining, Immunoprecipitation

    Reversal effect of cholesterol on the inhibition of VEGFR2 and mTOR activities by SERM. (A) and (B) HUVEC were treated with tamoxifen (TMX) or toremifene (TRM) with or without cholesterol (5 μg/ml)/cyclodextrin (0.1%) complex (Chol/CD) for 24

    Journal: Cancer letters

    Article Title: Inhibition of angiogenesis by selective estrogen receptor modulators through blockade of cholesterol trafficking rather than estrogen receptor antagonism

    doi: 10.1016/j.canlet.2015.03.022

    Figure Lengend Snippet: Reversal effect of cholesterol on the inhibition of VEGFR2 and mTOR activities by SERM. (A) and (B) HUVEC were treated with tamoxifen (TMX) or toremifene (TRM) with or without cholesterol (5 μg/ml)/cyclodextrin (0.1%) complex (Chol/CD) for 24

    Article Snippet: The blots were blocked with 5% non-fat dried milk at room temperature for 1 h, incubated with the primary antibodies including anti-VEGFR2 (Cell Signaling Technology), anti-phospho-VEGFR2 (Tyr1175, Cell Signaling Technology), anti-FGFR1 (Cell Signaling Technology), anti-mTOR (Cell Signaling Technology), anti-phospho-mTOR (Ser2448, Cell Signaling Technology) anti-S6K (Cell Signaling Technology), anti-phospho-S6K (Thr389, Cell Signaling Technology), anti-PDGFRβ (Santa Cruz Biotechnologies), anti-actin (Santa Cruz Biotechnologies) or anti-α-tubulin (Santa Cruz Biotechnologies) antibodies overnight at 4°C and then incubated with HRP-conjugated secondary antibodies at room temperature for 1 h. The immune-complexes were detected using enhanced chemiluminescence (ECL) detection reagent (GE Healthcare, Pittsburgh, PA).

    Techniques: Inhibition

    Effects of SERM and cholesterol on the subcellular localization of VEGFR2 and mTOR in HUVEC. (A) HUVEC were treated with or without 1 μM tamoxifen (TMX) for 24 h and subcellular localization of VEGFR2 was assessed under a confocal microscope.

    Journal: Cancer letters

    Article Title: Inhibition of angiogenesis by selective estrogen receptor modulators through blockade of cholesterol trafficking rather than estrogen receptor antagonism

    doi: 10.1016/j.canlet.2015.03.022

    Figure Lengend Snippet: Effects of SERM and cholesterol on the subcellular localization of VEGFR2 and mTOR in HUVEC. (A) HUVEC were treated with or without 1 μM tamoxifen (TMX) for 24 h and subcellular localization of VEGFR2 was assessed under a confocal microscope.

    Article Snippet: The blots were blocked with 5% non-fat dried milk at room temperature for 1 h, incubated with the primary antibodies including anti-VEGFR2 (Cell Signaling Technology), anti-phospho-VEGFR2 (Tyr1175, Cell Signaling Technology), anti-FGFR1 (Cell Signaling Technology), anti-mTOR (Cell Signaling Technology), anti-phospho-mTOR (Ser2448, Cell Signaling Technology) anti-S6K (Cell Signaling Technology), anti-phospho-S6K (Thr389, Cell Signaling Technology), anti-PDGFRβ (Santa Cruz Biotechnologies), anti-actin (Santa Cruz Biotechnologies) or anti-α-tubulin (Santa Cruz Biotechnologies) antibodies overnight at 4°C and then incubated with HRP-conjugated secondary antibodies at room temperature for 1 h. The immune-complexes were detected using enhanced chemiluminescence (ECL) detection reagent (GE Healthcare, Pittsburgh, PA).

    Techniques: Microscopy

    Effect of SERM on VEGFR2 glycosylation and mTORC1 pathway in HUVEC. (A) HUVEC were treated with SERM including tamoxifen (TMX, 5 μM), toremifene (TRM, 5 μM) and clomifene (CLM, 5 μM) for 24 h and VEGFR2 glycosylation was assessed

    Journal: Cancer letters

    Article Title: Inhibition of angiogenesis by selective estrogen receptor modulators through blockade of cholesterol trafficking rather than estrogen receptor antagonism

    doi: 10.1016/j.canlet.2015.03.022

    Figure Lengend Snippet: Effect of SERM on VEGFR2 glycosylation and mTORC1 pathway in HUVEC. (A) HUVEC were treated with SERM including tamoxifen (TMX, 5 μM), toremifene (TRM, 5 μM) and clomifene (CLM, 5 μM) for 24 h and VEGFR2 glycosylation was assessed

    Article Snippet: The blots were blocked with 5% non-fat dried milk at room temperature for 1 h, incubated with the primary antibodies including anti-VEGFR2 (Cell Signaling Technology), anti-phospho-VEGFR2 (Tyr1175, Cell Signaling Technology), anti-FGFR1 (Cell Signaling Technology), anti-mTOR (Cell Signaling Technology), anti-phospho-mTOR (Ser2448, Cell Signaling Technology) anti-S6K (Cell Signaling Technology), anti-phospho-S6K (Thr389, Cell Signaling Technology), anti-PDGFRβ (Santa Cruz Biotechnologies), anti-actin (Santa Cruz Biotechnologies) or anti-α-tubulin (Santa Cruz Biotechnologies) antibodies overnight at 4°C and then incubated with HRP-conjugated secondary antibodies at room temperature for 1 h. The immune-complexes were detected using enhanced chemiluminescence (ECL) detection reagent (GE Healthcare, Pittsburgh, PA).

    Techniques:

    Transplantation of wild type PCECs restores the defective alveolar regeneration in the mice deficient in endothelial Vegfr2 and Fgfr1

    Journal: Cell

    Article Title: Endothelial-derived inductive angiocrine signals initiate and sustain regenerative lung alveolarization

    doi: 10.1016/j.cell.2011.10.003

    Figure Lengend Snippet: Transplantation of wild type PCECs restores the defective alveolar regeneration in the mice deficient in endothelial Vegfr2 and Fgfr1

    Article Snippet: Tamoxifen treatment selectively deletes Vegfr2 in endothelial cells ( Vegfr2 iΔEC/iΔEC mice).

    Techniques: Transplantation Assay, Mouse Assay

    Inducible deletion of Vegfr2 and partial knockdown of Fgfr1 in endothelial cells (EC) attenuates lung regeneration

    Journal: Cell

    Article Title: Endothelial-derived inductive angiocrine signals initiate and sustain regenerative lung alveolarization

    doi: 10.1016/j.cell.2011.10.003

    Figure Lengend Snippet: Inducible deletion of Vegfr2 and partial knockdown of Fgfr1 in endothelial cells (EC) attenuates lung regeneration

    Article Snippet: Tamoxifen treatment selectively deletes Vegfr2 in endothelial cells ( Vegfr2 iΔEC/iΔEC mice).

    Techniques:

    Correlation between (A) pVEGFR2/VEGFR2 (p=0.007) and (B) EC pEGFR/EGFR (p=0.008) ratios and maximal reduction in tumor size (n=11). Complete responses were scored as −100% in tumor size change.

    Journal: The lancet oncology

    Article Title: A Phase II Study of Erlotinib and Bevacizumab in Patients with Recurrent or Metastatic Squamous Cell Carcinoma of the Head and Neck

    doi: 10.1016/S1470-2045(09)70002-6

    Figure Lengend Snippet: Correlation between (A) pVEGFR2/VEGFR2 (p=0.007) and (B) EC pEGFR/EGFR (p=0.008) ratios and maximal reduction in tumor size (n=11). Complete responses were scored as −100% in tumor size change.

    Article Snippet: Furthermore, VEGF increases proliferation of breast cancer cells and is dependent on VEGFR2( ).

    Techniques:

    (A) Representative laser scanned images show high expression of phosphorylated VEGFR2/total VEGFR2 in CR compared to non-CR patients (pVEGFR2 stained red, VEGFR2 stained green). (B) High expression of phosphorylated EGFR/total EGFR in endothelial cells

    Journal: The lancet oncology

    Article Title: A Phase II Study of Erlotinib and Bevacizumab in Patients with Recurrent or Metastatic Squamous Cell Carcinoma of the Head and Neck

    doi: 10.1016/S1470-2045(09)70002-6

    Figure Lengend Snippet: (A) Representative laser scanned images show high expression of phosphorylated VEGFR2/total VEGFR2 in CR compared to non-CR patients (pVEGFR2 stained red, VEGFR2 stained green). (B) High expression of phosphorylated EGFR/total EGFR in endothelial cells

    Article Snippet: Furthermore, VEGF increases proliferation of breast cancer cells and is dependent on VEGFR2( ).

    Techniques: Expressing, Staining

    Kaplan-Meier curves for the association between progression-free survival and (A) pVEGFR2/VEGFR2 (p=0.32) or (B) EC pEGFR/EGFR (p=0.12) ratios. Solid line represents subjects with a respective ratio at or above the median (n=6). Dotted line represents

    Journal: The lancet oncology

    Article Title: A Phase II Study of Erlotinib and Bevacizumab in Patients with Recurrent or Metastatic Squamous Cell Carcinoma of the Head and Neck

    doi: 10.1016/S1470-2045(09)70002-6

    Figure Lengend Snippet: Kaplan-Meier curves for the association between progression-free survival and (A) pVEGFR2/VEGFR2 (p=0.32) or (B) EC pEGFR/EGFR (p=0.12) ratios. Solid line represents subjects with a respective ratio at or above the median (n=6). Dotted line represents

    Article Snippet: Furthermore, VEGF increases proliferation of breast cancer cells and is dependent on VEGFR2( ).

    Techniques:

    Effect of diabetes on the proportion of cells that co-expressed (A) VEGFR-2 and CD34, or (B) VEGFR-2 and CD133 in cardiac ischemic patients (n=15 in each group, ***p

    Journal: Prostaglandins & other lipid mediators

    Article Title: Elevated level of pro-inflammatory eicosanoids and EPC dysfunction in diabetic patients with cardiac ischemia

    doi: 10.1016/j.prostaglandins.2012.12.002

    Figure Lengend Snippet: Effect of diabetes on the proportion of cells that co-expressed (A) VEGFR-2 and CD34, or (B) VEGFR-2 and CD133 in cardiac ischemic patients (n=15 in each group, ***p

    Article Snippet: Results are presented as the percentage of PMNCs (after selection for CD45+ and exclusion of debris) co-expressing either VEGFR-2 and CD133, or VEGFR-2 and CD34.

    Techniques:

    NOX4 regulates vascular endothelial growth factor–induced STAT3 activation through interacting with phosphorylated vascular endothelial growth factor receptor 2 in human retinal microvascular endothelial cells. Western blots of phosphorylated vascular endothelial growth factor receptor 2 (p-VEGFR2) and total VEGFR2 ( A ) and phosphorylated STAT3 (p-STAT3) and total STAT3 ( B ) in human retinal microvascular endothelial cells (hRMVECs) transfected with ConsiRNA or NOX4siRNA and treated with PBS or VEGF (20 ng/ml); ( C ) coimmunoprecipitation of NOX4 with VEGFR2 and western blots of p-VEGFR2 and total VEGFR2 in hRMVECs treated with VEGF with or without pretreatment of SU5416 (Con, control; SU, SU5416; V, VEGF). D : Western blots of p-STAT3 and total STAT3 were performed in hRMVECs treated with VEGF and pretreated with AG490, apocynin or respective controls (C, control; AG, AG490; APO, apocynin; **p

    Journal: Molecular Vision

    Article Title: Endothelial NADPH oxidase 4 mediates vascular endothelial growth factor receptor 2-induced intravitreal neovascularization in a rat model of retinopathy of prematurity

    doi:

    Figure Lengend Snippet: NOX4 regulates vascular endothelial growth factor–induced STAT3 activation through interacting with phosphorylated vascular endothelial growth factor receptor 2 in human retinal microvascular endothelial cells. Western blots of phosphorylated vascular endothelial growth factor receptor 2 (p-VEGFR2) and total VEGFR2 ( A ) and phosphorylated STAT3 (p-STAT3) and total STAT3 ( B ) in human retinal microvascular endothelial cells (hRMVECs) transfected with ConsiRNA or NOX4siRNA and treated with PBS or VEGF (20 ng/ml); ( C ) coimmunoprecipitation of NOX4 with VEGFR2 and western blots of p-VEGFR2 and total VEGFR2 in hRMVECs treated with VEGF with or without pretreatment of SU5416 (Con, control; SU, SU5416; V, VEGF). D : Western blots of p-STAT3 and total STAT3 were performed in hRMVECs treated with VEGF and pretreated with AG490, apocynin or respective controls (C, control; AG, AG490; APO, apocynin; **p

    Article Snippet: NOX4 silencing had no effect on basal levels of phosphorylated VEGFR2 (p-VEGFR2; ) or STAT3 (p-STAT3; ), but partly reduced p-VEGFR2 ( ) and completely inhibited VEGF-induced p-STAT3 , providing support that STAT3 was a downstream effector of activated NOX4 and that NOX4 involved VEGFR2 signaling.

    Techniques: Activation Assay, Western Blot, Transfection