Figure 4. Patient stromal cells (SCs) and transforming growth factor A (TGFA) induce an angiogenic endothelial cell (EC) phenotype together with VEGF-A.

(A–B) Venous malformation (VM) patient SCs induce sprouting of genotypically normal ECs. HUVECs on collagen-coated beads were embedded into a fibrin gel and patient SCs or control HPF-c cells were put on top. Representative images are presented at d7. ECs are labeled with phalloidin (red). nuclei with DAPI (blue; A) The number of sprouts per bead in each condition is shown (B). Two independent experiments were done in triplicates. Two-tailed Mann-Whitney U test (data not normally distributed). *p<0.05. In all images, scale bar is 100 µm. (C–E) Fibrin bead assay with HUVECs and HPF-c cells shows increased EC sprouting after stimulation with rhVEGF-A and rhTGFA at d6. ECs are labeled with phalloidin (red), and nuclei with DAPI (blue; C). The number of sprouts per bead (D) or sprout area (E) in each condition was determined from confocal images by ImageJ (45 beads/group). 2 independent experiments were done in triplicates. Brown-Forsythe and Welch ANOVA with Dunnet T3 post-hoc test (D) or Kruskal-Wallis test with two-stage step-up method of Benjamini, Krieger and Yekutieli to control FDR (E). ***p<0.001. (F) Co-culture experiments with HPF-c and HUVEC cells showed an increased growth rate in wells with PIK3CA^H1047R-expressing ECs in comparison to wells with ECs expressing PIK3CA^wt. The response was abolished after inhibition of endogenous TGFA in ECs by specific siRNA, demonstrating the involvement of TGFA in PIK3CA^H1047R-induced responses. Wells with siCtrl-transduced ECs (marked to be negative for siTGFA) were used as a control group in the experiments. Cellular growth was monitored using IncuCyte Live-Cell Imaging system. Data are presented as relative growth rate from two experiments done in triplicates. One-way ANOVA with Bonferroni or Sidac post-hoc test. ***p<0.001. In all data, mean and SEM are presented.

Figure 4—figure supplement 1. rhTGFA stimulation induces VEGFA in normal fibroblasts.

rhTGFA stimulation of HPF-c cells increased VEGFA mRNA expression (A, 6 hr) and VEGF-A protein secretion (B, 24 hr). The data is from three independent experiments done in triplicates. Two-tailed Mann-Whitney U test (data not normally distributed). ***p<0.001.

Figure 4—figure supplement 2. Quantification for the proliferation of cells in co-culture experiments combining endothelial cells (ECs) expressing PI3KCA^H1047R or PI3KCAwt ^wt+/−TGFA and genetically normal HPF-c.

Knock-down of transforming growth factor A (TGFA) was performed using siRNA targeting to TGFA and non-targeting control siRNA as a control. Just prior to imaging, HUVECs and HPF-c were mixed at a ratio of 8:1 to induce crosstalk between the cell types in cultures without the external addition of growth factors. Cell growth was monitored using IncuCyte S3 Live-cell Imaging System for 48 hr in 3 hr intervals, four images/well. (A) Growth curves for each condition show fold change in the confluency of cells in relation to time. Data are presented as mean and SEM from two experiments done in triplicates. (B) Simple linear regression for cell growth in each condition. Regression lines were generated between time points 12–48 hr, as the first 12 hr were considered as a time when the cells settle on wells after seeding. Mean and SEM of the slopes of the regression lines, indicating the growth rate of the cells, are shown in the table.