Extended Data Fig. 1: Characterization of Pik3ca-driven VM-formation across tissues and their dependency on VEGF.
From: Angiopoietin–TIE2 feedforward circuit promotes PIK3CA-driven venous malformations
(a) Top: Bright field images of ears from 4-OHT-treated Pik3caH1047R;Vegfr1-CreERT2 mice and Cre- littermate control mice (Ctrl) four weeks post-induction, showing macroscopic VM lesions and redness of ears skin in the mutant. Below: Whole-mount immunofluorescence showing overgrowth of EMCN+ veins but unaffected PDPN+ lymphatic vessels in the mutant. Similar results were obtained from 7 mice in 2 independent experiments. (b) Female reproductive tract (FRT) following topical application of 4-OHT (50 μg) to the ear skin of 3-week-old Pik3caH1047R;Vegfr1-CreERT2 mice and Cre- littermate control mice (Ctrl), showing vascular lesions in ovaries, fallopian tube and the uterine horn in the mutant. Boxed regions are magnified below. Similar results were obtained from 10 mice in 6 independent experiments. (c) Immunofluorescence (top) and Hematoxylin and Eosin staining (H&E) (below) of cryo-sections visualizing ECs (PECAM1) and SMCs (αSMA) in the uterus of Pik3caH1047R;Vegfr1-CreERT2 and Cre- littermate control mice (Ctrl). Arrowheads point to enlarged, SMC-covered vasculature in endometrial stroma (e). The boxed regions in both the H&E and immunofluorescence images correspond to the same areas, which are magnified on the right. m, myometrium. Similar results were obtained from 4 mice in 2 independent experiments. (d) Thyroid vasculature four weeks after intraperitoneal injection of AAV vectors encoding VEGF-Grab or a control molecule, and three weeks post-4-OHT induction. EMCN+ veins and αSMA+ SMCs are visualized. Similar results were obtained from 3 mice in 1 experiment. (e) Top: Experimental scheme for the induction of VMs followed by intraperitoneal injection of AAV vectors encoding VEGF-Grab or a control molecule to inhibit VEGF signaling. Below: Whole-mount immunofluorescence images of ear skin from VEGF-Grab or control-Trap-treated Pik3caH1047R;Cdh5-CreERT2 mice. Right: Quantification of vessel growth, represented as a change in EMCN+ vessel area relative to Cre- littermate control (Ctrl), mean ± sd (n = 4 (Ctrl+Ctrl-Trap), n = 7 (Pik3caH1047R+Ctrl-Trap), n = 6 (Ctrl+VEGF-Grab), n = 7 (Pik3caH1047R + VEGF-Grab) mice) from three independent experiments, indicated by symbol. ns, P = 0.8562 (not significant, ns), Unpaired two-tailed Student’s t-test. Scale bars: 1 cm (a, overview), 1 mm (a, b magnification), 500 μm (c, low magnification), 200 μm (d, c high magnification).
