Fig 2: Irradiation causes upregulation of miR-711 and downregulation of its target genes expression. (A) qPCR quantification of miR-711 (F(12,26) = 224.6, p = 0.0264 for 6 h 2Gy; p < 0.0001 for 3 h and 6 h 8Gy, p = 0.0016 for 24 h 8Gy; p < 0.0001 for 30 min, 3 h and 6 h 32Gy, compared to control), Akt mRNA (F(12,52) = 15.04; p = 0.0035 for 3 h 2Gy; p = 0.0264 for 30 min 8Gy; p < 0.0001 for 6 h and 24 h 8Gy and for 32Gy at all time points, compared to control) and Ang-1 mRNA (F(12,52) = 28.39; p = 0.0103 for 6 h 2Gy; p = 0.0042 for 30 min 8Gy, p < 0.0001 for 3 h and 6 h 8 and 32Gy; p = 0.0236 for 30 min 32Gy; p = 0.0085 for 24 h 32Gy, compared to control). n = 3/group, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. control RCNs. (B) Neurons were collected 6 h after 8Gy irradiation and subjected to RIP with Ago2 antibodies; samples were used for qPCR analysis. qPCR quantification of miR-711 (T(4) = 5.078, p = 0.0035), Akt mRNA T(4) = 20.41, p < 0.0001) and Ang-1 mRNA (T(4) = 8.45, p < 0.0001). n = 3/group. Significance assigned by one-tailed t-test, ** p < 0.01, *** p < 0.001 vs. control RCNs. miR-711 inhibition attenuates IR-induced downregulation of Akt and Ang-1. (C) RCNs were transfected with miR-711 inhibitor and miR-ve inhibitor 1 h before exposure to 8Gy. qPCR quantification of Akt mRNA (F(10,43) = 51.96, p < 0.0001 at 1, 3 and 6 h after IR with miR-ve inhibitor, compared to non-irradiated control; for miR-711 inhibitor, compared to miR-ve control, p < 0.0001 at 30 min and 1 h, p = 0.0010 at 3 h, p = 0.0356 at 6). Ang-1 mRNA (F(10,43) = 19.56, p < 0.0001 at 3, 6 and 24 h after IR with miR-ve inhibitor, compared to non-irradiated control; for miR-711 inhibitor, compared to miR-ve inhibitor, p < 0.0001 at 3 and 24 h, p = 0.0003 at 6 h). n = 4 for controls, n = 5/group for treatments, * p < 0.05, *** p< 0.001, vs. control; ^ p < 0.05, ^^^ p < 0.001, ^^^^ p < 0.0001 vs. corresponding 8Gy + miR-ve inhibitor. (D) RCNs were treated with recombinant Ang-1 to final concentrations 50, 100, 200 and 400 ng/mL, or with 200 ng/mL of recombinant Ang-1 and 6.25uM Akt inhibitor or 25nMWortmannin and exposed to 8Gy. LDH release was measured 24 h after irradiation (F(7,40) = 58.08, p < 0.0001 for IR alone vs. control; p < 0.0001 for 8Gy with 200 or 400 ng/mL Ang-1 vs. IR; p < 0.0001 for both inhibitor-containing groups vs. 8Gy with 200ng/mL Ang-1). n = 6/group, **** p < 0.0001 vs. control RCNs, ^^^^ p < 0.0001 vs. IR alone RCNs, &&&& p < 0.0001 vs. 8Gy with 200 ng/mL Ang-1. (E) qPCR quantification of mature miR-711 (F(10,22) = 95.15, p = 0.0035 at 1 h, p < 0.0001 at 3, 6 and 24 h after IR with miR-ve inhibitor, compared to control) and pri-mir-711 (F(10,25) = 54.32, p = 0.0056 at 1 h, p < 0.0001 at 3 and 6 h, p = 0.0300 at 24 h after IR with miR-ve inhibitor, compared to control). n = 3/group, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. control. (F) Neurons were treated as describe above. Cells were collected in 3 h for RIP analysis for levels of miR-711 (F(2,6) = 112.8, p < 0.0001 for both treatments, compared to control), Akt1 mRNA (F(2,6) = 54.17, p = 0.0001 after IR with miR-ve inhibitor, compared to control, p = 0.0034 for miR-711 inhibitor, compared to miR-ve inhibitor) and Ang-1 mRNA (F(2,6) = 788.7, p < 0.0001 for all comparisons). n = 3/group. *** p < 0.001, **** p < 0.0001 vs. control RCNs; ^^ < 0.01, ^^^ p < 0.001, ^^^^ p < 0.0001 vs. corresponding 8Gy + miR-ve inhibitor group.

Fig 3: ANGPT1 expression in human and mouse tissues.(a) Organ-wide average gene expression (normalized transcripts per million, nTPM), of ANGPT1 in different cell types, extracted from Human Protein Atlas scRNA-seq data. ANGPT1 expression in SMCs is highlighted in red and in BECs in blue. (b) Gene expression of ANGPT1 and selected mural cell markers in different skin cell types extracted from Human Protein Atlas scRNA-seq data. (c) Whole mount immunofluorescence of ANGPT1+ SMCs (αSMA) associated with veins (arrows) but not arteries (arrowhead) in the ear skin of a control mouse. Similar results were obtained from 3 mice in 2 independent experiments. Scale bars: 50 μm.

Fig 4: Loss of FOXO1-induced ANGPT2 expression and increase in TIE2 activity in Pik3caH1047R-expressing BECs.a, Volcano plot of 7,700 DEGs between Pik3ca-2 and vein clusters. Negative log2 fold changes (x axis) represent downregulated gene expression, while positive log2 fold changes represent upregulated gene expression in the mutant. Differential gene expression was assessed using Seurat’s Wilcoxon rank-sum test. Significant upregulated and downregulated genes are marked in green (P value < 0.00001, log2FC ≥ 1; y axis) and those shared with FOXO1A3 downregulated genes (from c) are highlighted in red. b, Whole-mount immunofluorescence of mouse ear skin showing ANGPT2 in EMCN+ venous vessels (arrowheads), but not in vascular lesions in the Pik3caH1047R mutant mouse (arrow). Single-channel images for ANGPT2 staining are shown on the right. Similar results were obtained from three mice in two independent experiments. c, ANGPT2 transcript levels in HUVECs transduced with AKT-resistant FOXO1A3 and Ctrl, analyzed by RNA-seq at different time points. Data points represent individual biological replicates of ANGPT2 mRNA expression levels (in fold change), mean ± s.d. (n = 3 (Ctrl) and n = 3 (FOXO1A3) at each time point). ***P < 0.001, unpaired two-tailed Student’s t-test: P(16 h) = 0.0006, P(24 h) = 0.0005, P(32 h) = 0.0001. d, ChIP–seq, ATAC-seq and RNA-seq signals at the ANGPT2 genomic locus performed in FOXO1A3-expressing HUVECs. FOXO consensus motifs bound by FOXO1 are indicated in orange. Unbound FOXO motifs are shown in gray. Sequencing signals are represented as reads per kilobase million (RPKMs). e,f, Immunoblot analysis of immunoprecipitated TIE2 (top) or total cell lysates (TCL; below) from Ctrl HUVECs and HUVECs expressing PIK3CAH1047R (e) or FOXO1A3 (f) using the indicated antibodies. AKT, S6, TIE2 and tubulin TCL western blots were used as sample loading controls. Cells were starved of serum and left untreated, or stimulated with ANGPT1 (50 ng ml-1 (e) or 200 ng ml-1 (f)), in the presence or absence of the TIE2 inhibitor BAY-826. Mr(K) indicates protein molecular weight marker (in kDa). IgG isotype control was used as a negative control. Data are representative of two independent experiments. g, Illustration of the TIE2–PI3K–FOXO pathways (left) and effects of FOXO1 (via FOXO1A3 expression, middle) and PI3K activation (via PIK3CAH1047R expression, right) on their pathway effectors. Blue indicates reduced activity; red indicates increased activity. Scale bars, 50 μm (a).Source data

Fig 5: Increased TIE2 phosphorylation and SMC coverage in Pik3ca-driven VM in mice.a, PLA staining of activated TIE2 on ear skin paraffin sections from Pik3caH1047R;Vegfr1-CreERT2 and Cre− littermate Ctrl mice, detected using pTyr and TIE2 antibodies. DAPI marks cell nuclei. b, Quantification of PLA signals within PECAM1+ blood vessels. Data represent the number of PLA dots per μm2 of PECAM1+ vessel area, mean ± s.d. (n = 8 (Ctrl) and n = 26 (Pik3caH1047R) vessels from four mice per genotype, unpaired two-tailed Student’s t-test, ****P = 0.0000054). c, Immunofluorescence of ear skin paraffin sections from Pik3caH1047R;Vegfr1-CreERT2 and Cre− littermate Ctrl mice using phospho-TIE2 antibodies. d, Phospho-TIE2 signal within EMCN+ vessels, represented as corrected total cell fluorescence (CTFC) of EMCN+ vessel area. Data points represent pTIE2 CTFC, mean ± s.d. (n = 20 (Ctrl) and n = 31 (Pik3caH1047R) vessels from two mice per genotype, unpaired two-tailed Student’s t-test, ****P = 0.000096; Extended Data Fig. 8d). e, Whole-mount immunofluorescence of ear skin from Pik3caH1047R;Vegfr1-CreERT2 and Cre− littermate Ctrl mice using αSMA antibodies 6 weeks after 4-OHT induction. f, Quantification of SMC coverage of veins and capillaries, shown as average percentage of EMCN+ area, mean ± s.d. (n = 4 (Ctrl) and n = 8 (Pik3caH1047R) mice, unpaired two-tailed Student’s t-test, P(vein) = 0.173 (NS, not significant) and ****P(capillary) = 0.0000078). g, Whole-mount immunofluorescence of ANGPT1+ cells associated with veins and capillaries one week after 4-OHT induction. Proliferating cells (arrowheads) were labeled in mutant mice with EdU 16 h before analysis. Asterisks indicate vessel-detached ANGPT1+ cells. h,i, Violin plots showing gene expression of Angpt1 and selected marker genes of EC–SMC interaction (h) and heat map showing relative importance of two selected ligand–receptor pairs, generated using CellChat (i), in Ctrl and Pik3ca EC clusters from Fig. 3a as well as in SMCs from the same dataset. Scale bars, 50 μm (a and g), 20 μm (c) and 100 μm (e).Source data