Journal: bioRxiv

Article Title: CD44 Controls Endothelial Proliferation and Functions as Endogenous Inhibitor of Angiogenesis

doi: 10.1101/049494

Figure Lengend Snippet: Increased angiogenesis in CD44-null mice. ( A ) Angiogenesis was analysed in C3H, C57BL/6, and in wild-type and Cd44 −/− mice of mixed genetic backgrounds (WT mix and Cd44KO mix, respectively). Basement membrane extract–filled angioreactors containing premixed FGF-2, VEGF and heparin or PBS for uninduced controls were implanted SC into the flanks of mice. Each mouse received 2 angioreactors, 1 per flank. 14 days after implantation, angioreactors were resected and the population of ECs within the angioreactor matrix was assessed by FITC-lectin staining. The number of fluorescent cells was quantitated by microplate reader. Raw readings from independent experiments were scaled by dividing by their quadratic mean. N = 2–5 mice per condition from 2 independent experiments. P value is from Student’s t-test. ( B ) Angiogenesis in Cd44 −/− mice and their heterozygous ( Cd44 +/− ) and wild-type ( Cd44 +/+ ) littermate that had been backcrossed six generations to the C57BL/6 background. The data are represented as the mean ± SEM. Each dot represents the mean of two angioreactors for an individual mouse. N = 8 mice per condition from 2 independent experiments. P values are from ANOVA post hoc comparisons using the Tukey HSD test.

Article Snippet: After blocking, samples and standards (Recombinant Mouse CD44 Fc Chimera; R&D Systems) were incubated in wells for 30 min at 37°C.

Techniques: Membrane, Staining

Journal: bioRxiv

Article Title: CD44 Controls Endothelial Proliferation and Functions as Endogenous Inhibitor of Angiogenesis

doi: 10.1101/049494

Figure Lengend Snippet: Recombinant CD44-3MUT Fc fusion protein inhibits angiogenesis in vivo . First, the assay and quantitation were performed similarly to those described in , except that each mouse received 4 angioreactors, 2 per flank. The day after implantation, the mice started to receive CD44-3MUT-Fc, control (rhIgG1-Fc) or vehicle (PBS) every second day via IP injections for 14 days. ( A ) Schematic presentation of experimental design. ( B ) Relative blood vessel invasion into matrix–filled angioreactors. The data are represented as the mean ± SEM. Datapoints show the mean of 4 angioreactors for individual mice. N – the number of independent experiments. GF – growth factors (FGF-2/VEGF). ( C ) Effect size with 95% confidence intervals (upper row) and P values (lower row) of pairwise comparisons of the data shown in ( B ). Effect sizes were calculated by Cohen’s d formula. Confidence intervals were derived using bootstrap resampling. P values are from t tests using pooled SD.

Article Snippet: After blocking, samples and standards (Recombinant Mouse CD44 Fc Chimera; R&D Systems) were incubated in wells for 30 min at 37°C.

Techniques: Recombinant, In Vivo, Quantitation Assay, Control, Derivative Assay

Journal: bioRxiv

Article Title: CD44 Controls Endothelial Proliferation and Functions as Endogenous Inhibitor of Angiogenesis

doi: 10.1101/049494

Figure Lengend Snippet: Soluble CD44 concentrations in mouse serum. Blood was collected from mice of different genetic backgrounds and the mice used in the angiogenesis experiments shown in and . ( A ) Serum levels of soluble CD44 in mice from different strains. Each dot represents an individual mouse. Cross indicates the mean. P values are from ANOVA post hoc comparisons using the Tukey HSD test. ( B ) The correlation between relative blood vessel invasion and post-experiment serum sCD44 in wild-type mice. Cd44 -null mice and nude mice were excluded from the dataset. Pearson’s r and the associated P values are shown. ( C ) Post-experiment serum levels of sCD44 in nude mice from different treatment groups. Treatments where more than five mice were analysed are shown. Each dot represents an individual mouse. Cross indicates the mean. GF – growth factors (FGF-2/VEGF). ( D ) The correlation between relative blood vessel invasion and post-experiment serum sCD44 in nude mice. Pearson’s r and the associated P values are shown. In ( B and D ), dashed line is the linear model fit, gray shading is the standard error interval of fitted values.

Article Snippet: After blocking, samples and standards (Recombinant Mouse CD44 Fc Chimera; R&D Systems) were incubated in wells for 30 min at 37°C.

Techniques:

Journal: bioRxiv

Article Title: CD44 Controls Endothelial Proliferation and Functions as Endogenous Inhibitor of Angiogenesis

doi: 10.1101/049494

Figure Lengend Snippet: CD44-3MUT-Fc inhibits EC growth. ( A ) Real-time track of cell adhesion and synchronisation of HUVECs seeded onto 96-well electrode arrays. After seeding, the cells were grown for about 24 h. After that the cells were starved overnight in the media supplemented with 1% FBS (gray area). 1 h before the release from serum starvation (vertical dashed line) the cells were preincubated with different concentrations of rhIgG-Fc or CD44-3MUT-Fc in 5% FBS–containing media. ( B ) Growth curves of HUVECs released from serum starvation by supplementing preincubation media with 25 ng/ml VEGF. Facet labels show rhIgG-Fc or CD44-3MUT-Fc concentrations during preincubation. The data are represented as the mean ± SEM. N – the number of independent experiments. ( C–F ) HUVECs were synchronised and pretreated as in panel A. After preincubation, the cells were stimulated either with 25 ng/ml FGF-2 ( C ), 25 ng/ml VEGF ( D ), 63 ng/ml HGF ( E ) or 10 ng/ml GDF-2 ( F ). After 72 h, the number of viable cells was quantitated by measuring the ATP per well. Left: the effect of growth factor stimulation and the effect of 10 nM fumagillin (FUM) as a positive control for inhibition of cell proliferation. Right: the dose-response curves of rhIgG-Fc (filled triangles) and CD44-3MUT-Fc (filled circles). The data are represented as the mean ± SEM. N = 3–4 independent experiments. ( G ) CD44-3MUT-Fc dose-response curves for FGF-2, GDF-2, HGF or VEGF stimulated HUVEC. The data are represented as the mean ± SEM. ( H ) Apoptosis of HUVECs stimulated with different growth factors and treated with 12.64 μM (-4.9 log 10 M) rhIgG-Fc, CD44-3MUT-Fc or left untreated. Apoptosis was quantitated by Annexin V staining. The data are represented as the mean ± SEM. N = 2 independent experiments. P values are from the ANOVA post hoc comparisons using the Tukey HSD test.

Article Snippet: After blocking, samples and standards (Recombinant Mouse CD44 Fc Chimera; R&D Systems) were incubated in wells for 30 min at 37°C.

Techniques: Positive Control, Inhibition, Staining

Journal: bioRxiv

Article Title: CD44 Controls Endothelial Proliferation and Functions as Endogenous Inhibitor of Angiogenesis

doi: 10.1101/049494

Figure Lengend Snippet: CD44 knockdown augments EC growth. siRNA transfected HUVECs were plated onto 96-well electrode arrays. After 24 h, the cells were starved in 1% FBS media overnight. After starving, the cells were released from cell cycle block by the addition of 20% FBS (A) ; 8 ng/ml, 25 ng/ml, or 79 ng/ml FGF-2 or VEGF (C) ; and 2.5 ng/ml or 10 ng/ml GDF-2 (E) . Following stimulation, HUVEC growth was monitored by recording electrode impedance. Raw impedance readings are shown to allow direct comparison to endpoint measurements. N = 2 for 5% and 20% FBS, 25 ng/ml and 79 ng/ml FGF-2, and 8 ng/ml and 25 ng/ml VEGF. (B, D, F) 72 h after release from cell cycle block the viable cell numbers were determined by measuring the ATP per well. Treatments are labeled as shown in ( B ). The data are represented as the mean ± SEM. P values are from the ANOVA post hoc comparisons using the Tukey HSD test. P values ≤ 0.05 of siCD44-siNTP comparisons are shown. siCD44-siNTP comparisons: N = 4 for 2.5 ng/ml and 10 ng/ml GDF-2, and N = 5 for 20% FBS, 25 ng/ml and 79 ng/ml FGF-2, and 8 ng/ml and 25 ng/ml VEGF. (G) Western blot analysis of CD44 silencing in HUVECs transfected with 30 nM siRNAs for 48 h. siNTP – non-targeting siRNA pool, siVIM – vimentin-targeting pool, siCD44 – CD44-targeting pool, UT – non-transfected cells.

Article Snippet: After blocking, samples and standards (Recombinant Mouse CD44 Fc Chimera; R&D Systems) were incubated in wells for 30 min at 37°C.

Techniques: Knockdown, Transfection, Blocking Assay, Comparison, Labeling, Western Blot