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. 2015 Aug 28;24(22):6374–6389. doi: 10.1093/hmg/ddv349

Figure 8.

Figure 8.

VM lesion growth in a mouse transplantation assay. (A) VM lesion on the tongue of a patient and (B) vascularized plug from EC-spheroid implanted mouse. Vascular structures in a mouse model macroscopically resemble human VM lesions. (C) Transplanted human-derived mutant ECs (red) were distinguished from mouse host vasculature (green) using species-specific CD31 antibodies. Scale bar 100 μm. (D and E) Immunohistochemical staining of sections from vascularized TIE2-WT and Y897F-R915L plugs using endothelial anti-human CD31 antibodies (hCD31) with nuclear staining with hematoxylin. When compared with the capillary-sized vessels in WT, vascular lesions showed a pathognomonic increase in vessel size (white arrows) and patchy perivascular cells (black arrows indicating nuclei). Scale bar 100 μm. (F) Maximal vessel area was quantified from histological sections. Two biggest vessels/sections were imaged and area measured using ImageJ. Double-mutated TIE2 expressing HUVECs formed notably large structures, displayed in the graph on the right (note different y-axis scaling for single and double-mutants). 25th quartile, median, 75th quartile (box), average (square), 5th and 95th percentile (whiskers) and outliers (dot) are shown. P < 0.001 in Kruskal–Wallis one-way analysis of variance (all groups in both graphs included). Pairwise comparisons were performed with Mann–Whitney U-test and thresholds of significant P-values were adjusted with Bonferroni correction for 36 pairwise comparisons. n = 13–54, each vessel was counted as an observation. Mouse host ECs formed capillary-sized vessels and were smaller than in other groups (¤P < 0.05). R915L, L914F and double-mutants formed statistically significantly larger vessels than TIE-WT (###P < 0.001) and Y897F-R915L showed a clear enlargement in vessel size compared with single constituent mutations (‡‡P < 0.01 versus Y897F, ‡‡‡P < 0.001 versus R915L). Other double-mutations induced similar vessel enlargement (***P < 0.001, *P < 0.05 versus single mutations). NS, statistically non-significant.