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1.
FIGURE 4.

FIGURE 4. From: Heparan Sulfate Regulates VEGF165- and VEGF121-mediated Vascular Hyperpermeability.

Loss of heparan sulfate attenuates eNOS phosphorylation in response to both VEGF165 and VEGF121. Immunoblot analysis of eNOS phosphorylation in mBMEC cells after treatment with heparan lyases I, II, and III (Hep I–III) for 15 min and stimulation with 20 ng/ml VEGF165 or 50 ng/ml VEGF121 was performed. Representative immunoblots are shown. The bar graphs represent the normalized relative signal intensity. n = 3. Error bars indicate S.E. pS1177, phospho-Ser-1177.

Ding Xu, et al. J Biol Chem. 2011 Jan 7;286(1):737-745.
2.
FIGURE 5.

FIGURE 5. From: Heparan Sulfate Regulates VEGF165- and VEGF121-mediated Vascular Hyperpermeability.

Protamine and surfen inhibit VEGFR2 phosphorylation in vitro and dermal vascular permeability in vivo. A, immunoblot analysis of VEGFR2 phosphorylation in mBMEC stimulated with VEGF165 (50 ng/ml) or VEGF121 (100 ng/ml) for 5 min. Cells were treated with protamine at the indicated concentration prior to application of VEGF. The immunoblots are representative of three similar experiments. B, immunoblot analysis of VEGFR2 phosphorylation was performed as in A, except that cells were treated with surfen. C, mice were injected intradermally with 200 ng of VEGF165 alone (filled squares) or with 12.5 μg of protamine (open squares), and the amount of Evan's Blue leakage was measured; n = 9. D, mice were injected intradermally with 200 ng of VEGF165 alone (filled squares) or with 54 μm surfen (open squares); n = 9. E, mice were injected with 200 ng of VEGF121 alone (filled squares) or with 54 μm surfen (open squares); n = 11. Experimental and control treatments of the same mouse in C–E are linked with a line. PBS with 0.1% BSA was used as negative control, which showed an average leakage of 0.9 ± 0.1 μg (C), 0.8 ± 0.1 μg (D), and 0.7 ± 0.1 μg (E). pY1175, phospho-Tyr-1175.

Ding Xu, et al. J Biol Chem. 2011 Jan 7;286(1):737-745.
3.
FIGURE 1.

FIGURE 1. From: Heparan Sulfate Regulates VEGF165- and VEGF121-mediated Vascular Hyperpermeability.

Dermal vascular hyperpermeability induced by VEGF is reduced in Ndst1-deficient mice. A, dermal vascular permeability was measured by Miles assay, in which Evan's Blue was injected intravenously. The amount of dye that leaked into the injection site was measured by absorbance after extraction. Ndst1f/fTie2Cre (WT) and Ndst1f/fTie2Cre+ (labeled as Ndst1 −/−) mice were injected with 200 ng of VEGF165 (n = 20) and VEGF121 (n = 10). Histamine (1 μg, n = 12) was injected as positive control, and PBS with 0.1% BSA (n = 20) was injected as negative control. Error bars indicate S.E. B, dermal vascular hyperpermeability of Ext1f/+Tie2Cre+ (labeled as Ext1+/−) and Ext1f/+Tie2Cre (WT) mice was measured as in A. n = 6–9. C, binding of VEGF165 and VEGF121 to primary lung endothelial cells measured by flow cytometry. VEGF binding to untreated cells is shown as a black line, binding to heparin lyase I-treated cells is shown as a gray line, and the control sample, incubated only with biotinylated antibody and streptavidin-phycoerythrin, is shown as the filled gray histogram. % of Max, percentage of maximum.

Ding Xu, et al. J Biol Chem. 2011 Jan 7;286(1):737-745.
4.
FIGURE 2.

FIGURE 2. From: Heparan Sulfate Regulates VEGF165- and VEGF121-mediated Vascular Hyperpermeability.

Tumor vascular permeability is reduced in Ndst1-deficient mice. A, LLC (4 × 105) were injected into the hindquarters of Ndst1f/fTie2Cre (WT) and Ndst1f/fTie2Cre+ (labeled as Ndst1 −/−) mice. Mice with comparably sized tumors were injected i.v. with 1 mg of Evan's Blue (EB) and sacrificed 1 h later. The amount of dye extravasation into the tumor was measured spectrophotometrically and normalized to the amount tumor mass that was analyzed (wet weight). B, mass (wet weight) of the tumors used in the experiment shown in panel A. C, multiple sections from tumors (n = 4) were stained with antibody to CD31. The density of tumor microvasculature was estimated by counting the number of CD31+ microvessels per 400× microscopic field. D, VEGF expression in tumors was assessed by Western blotting after separation of 20 μg of tumor lysate by SDS-PAGE. Each lane represents a separate tumor. E, sections from tumors (n = 4) were stained with antibody to CD45. The number of CD45+ cells per 400× microscopic field was determined. Error bars indicate S.E. HPF, high power field.

Ding Xu, et al. J Biol Chem. 2011 Jan 7;286(1):737-745.
5.
FIGURE 6.

FIGURE 6. From: Heparan Sulfate Regulates VEGF165- and VEGF121-mediated Vascular Hyperpermeability.

Heparan sulfate-VEGFR2 complexes show a transient increase when stimulated by VEGF165 or VEGF121. A, representative images were obtained by proximity ligation assay. Cells were fixed and incubated with antibodies to heparan sulfate (mAb 10E4) and to the extracellular domain of mouse VEGFR2 (goat IgG) followed by proximity ligation assay reagents. Each red dot indicates an interaction between heparan sulfate and VEGFR2. Nuclei are shown in blue. The Control panel represents cells incubated with 10E4 and nonspecific goat IgG. B, mBMEC were stimulated with 50 ng/ml VEGF121 (striped bars) or 20 ng/ml VEGF165 (white bars) for the indicated time. The data are presented as red dots (Duolink signals)/cell. As a negative control, cells were incubated with mAb 10E4 and nonspecific goat IgG, which gave rise to a background of 3.9 ± 0.3 Duolink signals/cell. The error bar represents S.E., n = 4 separate experiments. C, mBMEC derived from Ndst1f/fTie2Cre (WT) and Ndst1f/fTie2Cre+ (labeled as Ndst1−/−) mice were stimulated with VEGF as indicated and analyzed by proximity ligation assay. The error bars represent S.E., n = 3 separate experiments. All images were taken with a DeltaVision deconvolution microscope (200× magnification), deconvolved with SoftWoRx software and processed with NIH Image J.

Ding Xu, et al. J Biol Chem. 2011 Jan 7;286(1):737-745.
6.
FIGURE 3.

FIGURE 3. From: Heparan Sulfate Regulates VEGF165- and VEGF121-mediated Vascular Hyperpermeability.

Loss of heparan sulfate attenuates VEGFR2 phosphorylation in response to both VEGF165 and VEGF121. A, dermal vascular hyperpermeability induced by VEGF165 was blocked in Vegfr2+/− mice. Wild-type or Vegfr2+/− mice were injected intradermally with 200 ng of VEGF165 (n = 6) or PBS with 0.1% BSA (n = 6) as negative control. B, chemical analysis of heparan sulfate derived from the mBMEC. Heparan sulfate was isolated by anion-exchange chromatography and digested with heparin lyases I, II, and III, and the resulting disaccharides were analyzed by liquid chromatography/mass spectrometry (). The number of sulfate groups at each position was determined and expressed per 100 disaccharides. Ndst1−/−, Ndst1f/fTie2Cre+. C, immunoblot analysis of VEGFR2 phosphorylation in mBMEC isolated from mutant and wild-type mice after stimulation with 20 ng/ml VEGF165 for 2 and 15 min (n = 3). D and E, wild-type mBMEC were treated with heparan lyases I, II, and III (Hep I–III) for 15 min and then stimulated with 20 ng/ml VEGF165 (n = 4) or 50 ng/ml VEGF121 (n = 3). Representative immunoblots are shown. The bar graphs represent the relative signal intensity after correction for the background and normalization to the loading control. The average values ± S.E. were determined from the indicated number of replicates. pY1175, phospho-Tyr-1175.

Ding Xu, et al. J Biol Chem. 2011 Jan 7;286(1):737-745.

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