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1.
FIGURE 3.

FIGURE 3. From: Lymphatic Endothelial Heparan Sulfate Deficiency Results in Altered Growth Responses to Vascular Endothelial Growth Factor-C (VEGF-C).

Heparinase-mediated ablation of lymphatic endothelial cell-surface heparan sulfate inhibits growth signaling and receptor activation in response to VEGF-C. A, phosphorylation of Erk1/2 (p44/42) in serum-starved primary human lung lymphatic endothelial cells was measured before/after growth factor stimulation. Responses to stimulation with recombinant human VEGF-C were measured for cells pretreated with or without heparinase (used to destroy cell-surface heparan sulfate). Total Erk for each panel is shown below respective phospho-Erk panels. Ratios of phospho/total Erk band intensities for each condition were quantified by densitometry and normalized to the respective base-line phospho/total Erk ratio (shown in parentheses below each panel). B, responses to stimulation by FGF-2 (left panels) as well as the non-heparin-binding factor EGF (right panels) pretreated with or without heparinase were also examined. C, VEGFR-3 receptor phosphorylation in response to VEGF-C was examined in the absence/presence of heparan sulfate elimination by heparinase. Receptor was immunoprecipitated from lysates of either serum-starved LEC (baseline) or the same cells preincubated in the absence/presence of heparinase and stimulated with VEGF-C. Immunoblotting with anti-phosphotyrosine was carried out and revealed a phosphorylated product in response to stimulation with VEGF-C (WB: P-Tyr; middle lane), consistent with phosphorylated VEGFR-3. The response of heparinase-treated cells is shown in the right lane. Total receptor in the original lysates (TL) is shown below (WB: VEGFR-3). Graph (right) shows mean signal intensities normalized to base-line signal for n = 3 experiments (±S.E.). *, p = 0.001 for difference relative to normalized base-line signal; **, p = 0.009 for difference between mean heparanase and mean VEGF-C values.

Xin Yin, et al. J Biol Chem. 2011 Apr 29;286(17):14952-14962.
2.
FIGURE 5.

FIGURE 5. From: Lymphatic Endothelial Heparan Sulfate Deficiency Results in Altered Growth Responses to Vascular Endothelial Growth Factor-C (VEGF-C).

Targeting lymphatic endothelial heparan sulfate chain biosynthesis abrogates VEGF-C-dependent gap filling and growth responses in cultured cell systems. Monolayers of serum-starved human lymphatic endothelia transfected with siRNA targeting XylT2 (siXylT2) or with scrambled duplex RNA (siDS; control) were scratched to create a gap in the center at time 0. The ability of cells to fill the gap in response to VEGF-C was examined. A, graphs show quantitative data for percentage of gap filling over time in the presence/absence of VEGF-C by control cells (left panel) and by siXylT2-targeted cells (right panel). Photomicrographs below show gap (with dotted gap boundaries) within confluent cell layers at time 0, and degree of gap filling at 48 h in the presence of VEGF-C (bar, 0.5 mm). *, p = 0.009 and **, p = 0.003 for difference in mean responses by VEGF-C-stimulated versus un-stimulated control (siDS) cells at 24 h and 48 h, respectively. (Graph, mean of n = 3 trials.). B, cell viability assays demonstrate growth responses of control (siDS), siXylT2-, and siNdst1-targeted lymphatic endothelia in response to 24 h of exposure to VEGF-C. *, p < 0.001 for the difference in mean growth responses by control (siDS) cells stimulated ± VEGF-C for 24 h. Other differences were not significant.

Xin Yin, et al. J Biol Chem. 2011 Apr 29;286(17):14952-14962.
3.
FIGURE 2.

FIGURE 2. From: Lymphatic Endothelial Heparan Sulfate Deficiency Results in Altered Growth Responses to Vascular Endothelial Growth Factor-C (VEGF-C).

VEGF-C-mediated Erk1/2 activation in lymphatic endothelium is inhibited by the presence of heparin, with unique responses in the presence of sulfate-modified heparinoids. A, phosphorylation of Erk1/2 (p44/42) in serum-starved human primary lung lymphatic endothelial cells in response to treatment with recombinant human VEGF-C was measured in the absence/presence of increasing commercial heparin concentration. Lymphatic endothelial cell purity was confirmed by immunostaining to detect nuclear expression of Prox-1 (right photomicrographs; with IgG isotype-matched control antibody stain to right; bar, 50 μm). Maximum phosphorylation was noted in the absence of heparin (+VEGF-C, heparin = 0). Phospho/total signal intensities were quantified by densitometry and normalized relative to that of maximum stimulation (Signal Relative to Max, below panel). Preliminary testing showed that marked inhibition was achieved at heparin doses >1 μg/ml. B, effects of N-desulfated (N-deS), 2-O-desulfated (2-O-deS), or 6-O-desulfated (6-O-deS) heparin species were examined at the same doses as that used for unmodified commercial heparin in A. C, quantitative assessment for responses in the presence of heparin versus sulfate-modified heparinoids dosed at 10 μg/ml is shown (graph). Values represent the densitometry ratio of phospho/total Erk for VEGF-C-mediated stimulation in the presence of either heparin (+ Hep) or each of the respective heparinoids normalized to the signal for stimulation in the absence of heparin (− Hep). Graph shows mean of four experiments. *, p < 0.001 for + heparin versus − heparin; **, p < 0.001 for 6-O-deS versus − heparin; differences in means for N-deS versus − heparin or 2-O-deS versus − heparin were not significant).

Xin Yin, et al. J Biol Chem. 2011 Apr 29;286(17):14952-14962.
4.
FIGURE 4.

FIGURE 4. From: Lymphatic Endothelial Heparan Sulfate Deficiency Results in Altered Growth Responses to Vascular Endothelial Growth Factor-C (VEGF-C).

Silencing of lymphatic endothelial heparan sulfate chain biosynthesis inhibits VEGF-C-dependent Erk1/2 activation and reduces receptor-dependent binding of VEGF-C. A, cultured primary human lymphatic endothelia were transfected with siRNA targeting XylT2 (siXylT2) or with scrambled duplex RNA (siDS) as a control. Under each condition, phosphorylation of Erk1/2 in serum-starved cells before/after recombinant human VEGF-C exposure was examined by Western blotting. Total Erk for each condition is shown below respective phospho-Erk lanes. Ratios of phospho/total Erk band intensities for each condition were quantified and normalized to the respective base-line ratio (values in parentheses). Graph (below) shows mean signal intensities for n = 4 experiments (±S.E.). B, in separate experiments, exposure of mock-transfected (siDS) control cells to exogenous recombinant human VEGF-C followed by washing resulted in the engagement of numerous cell-surface VEGF-C-VEGFR-3 complexes (red dots, top right photomicrograph; siDS +VEGF-C), as measured through PLA. There was minimal endogenous signal at base line, prior to exogenous VEGF-C application (top left; siDS). Exposure of siXylT2-targeted cells to exogenous VEGF-C resulted in minimal complex formation over base line (bottom photomicrographs; bar, 50 μm). C, quantified results, showing the fold increase in fluorescent signal for each condition indexed to base-line signal for siDS control. Also shown on the graph (right) is the mean signal achieved by cells treated with siRNA targeting VEGFR-3 and exposed to exogenous VEGF-C (labeled siVR3+VEGF-C). *, p values for difference between siDS + VEGF-C and the following: siDS base line (p < 0.001); siXylT2 + VEGF-C (p = 0.001); siVR3 + VEGF-C (p = 0.004). **, p = 0.04 for difference in base-line signals (siDS versus siXylT2).

Xin Yin, et al. J Biol Chem. 2011 Apr 29;286(17):14952-14962.
5.
FIGURE 1.

FIGURE 1. From: Lymphatic Endothelial Heparan Sulfate Deficiency Results in Altered Growth Responses to Vascular Endothelial Growth Factor-C (VEGF-C).

VEGF-C binds to lymphatic endothelial heparan sulfate. A, oil granuloma/lymphangioma lesions were induced in wild type mice, and cultured lymphatic endothelial cells from lesions were radiolabeled with 35SO4. [35S]Heparan sulfate from the cells was purified and incubated with recombinant mouse VEGF-C (mature, fully processed 125-amino acid sequence, described under “Experimental Procedures”), followed by passage over nitrocellulose membrane filters. Membrane-bound complexes of [35S]heparan sulfate and protein collected on the filters were quantified and plotted relative to that of FGF-2 (open bars, left). Binding was also tested in the presence of heparin as a competitor. Controls included binding to EGF and BSA. The same experiment was carried out using recombinant human VEGF-C (mature, fully processed 125-amino acid sequence, described under “Experimental Procedures”) and [35S]heparan sulfate from primary human lung lymphatic endothelial cells (filled bars, right; p = 0.01 for FGF-2 versus VEGF-C; average of four experiments). B, to assess binding of the same recombinant human VEGF-C protein (designated recomb human #1) to commercial heparin, affinity chromatography using heparin-Sepharose columns was carried out. Fractions collected over the indicated NaCl step concentration range were run on SDS-PAGE with silver staining to reveal the protein elution profile. The level to which native protein migrates on the gel is shown at left (Pre column sample in 1st lane with kDa range as indicated); column flow-through (FT) is shown in 2nd lane; and elution profile (subsequent lanes) is shown to the right. For comparison, heparin affinity chromatography was carried out for another commercial recombinant human VEGF-C preparation (mature, fully processed 125-amino acid sequence, recomb human #2, shown below). C, binding of recombinant human VEGF-A (VEGF165) was also examined.

Xin Yin, et al. J Biol Chem. 2011 Apr 29;286(17):14952-14962.
6.
FIGURE 6.

FIGURE 6. From: Lymphatic Endothelial Heparan Sulfate Deficiency Results in Altered Growth Responses to Vascular Endothelial Growth Factor-C (VEGF-C).

Targeting lymphatic endothelial Ndst1 results in reduced growth and sprouting responses to VEGF-C and FGF-2. A, serum-starved human lymphatic endothelia were transfected with siRNA targeting Ndst1 (siNdst1) or with scrambled duplex RNA (siDS; control). Phosphorylation of Erk1/2 before/after VEGF-C exposure was examined by Western blotting. Total Erk for each condition is shown. Ratios of phospho/total Erk band intensities for each condition were quantified and normalized to the respective base-line ratio (values in parentheses). Graph (right) shows mean signal intensities for n = 4 experiments (±S.E.). B, ability of the siRNA-targeted lymphatic endothelia to fill a gap made between cells in a scratch assay was examined over 48 h in the presence/absence of growth factor. Photomicrographs show gap (with dotted gap boundaries) within confluent cell layers at time 0, and at 48 h in the presence of VEGF-C (bar, 0.5 mm). Control siDS cells partially filled the gap over 48 h (upper photomicrographs), whereas response by siNdst1-targeted cells is shown below. Mean values for responses in the presence/absence of VEGF-C were graphed (top). *, p = 0.01 for VEGF-C versus un-stimulated cells at 48 h. Graphs for assays carried out in the presence of the cell proliferation inhibitor mitomycin-C are also shown (labeled +Mito-C). The scratch assay was also carried out for cell stimulation in the presence/absence of FGF-2 (graphs shown below photomicrographs). *, p = 0.008 for FGF-2 versus unstimulated cells at 24 h; **, p < 0.001 for FGF-2 versus unstimulated cells at 48 h. (Graphs, mean of n = 3 trials.) C, lymphatic endothelia were isolated from oil granuloma/lymphangioma lesions generated in Ndst1f/fTekCre(+) mutant mice and Cre(−) littermates, with podoplanin and LYVE-1 expression shown by FACS. D, sprouting responses in collagen upon stimulation with recombinant VEGF-C or FGF-2 were examined. Significant responses to growth factor were noted for Cre(−) wild type cells (*, p = 0.003, and **, p = 0.008 for respective VEGF-C and FGF-2 responses versus base-line responses in the absence of growth factor, None). Mutant responses (right) were not significantly different from base line. Sulfation of heparan sulfate disaccharides purified from mutant versus wild type cells was determined by liquid chromatography/mass spectrometry and quantified as a percentage of total disaccharides (bottom graph). The detected disaccharide species are listed (x axis) according to published nomenclature (), with D0A0 (left-most bars) indicating the percentages of unsulfated disaccharides.

Xin Yin, et al. J Biol Chem. 2011 Apr 29;286(17):14952-14962.

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