Fig 2: Trametinib treatment of GNAQQ209L mutant mice rescues vascular phenotype, hyperproliferation, permeability and coagulopathy.a Preventative study scheme. 6 weeks-old iCdh5-GNAQQ209L mice induced with 75 mg/kg tamoxifen and concomitantly treated with Trametinib (2 mg/kg/day) or vehicle. b Macroscopic photographs of subcutaneous tissue from vehicle and Trametinib -treated mice. CD31 (dark brown) stained tissue sections. Scale bars: 2 mm and 50µm. c Vessel size distribution in subcutaneous tissue as percentage of small (<100 µm2) and large (>100 µm2) vessels. d Vascular density and area in vehicle or Trametinib -treated mice. e Representative photograph of intestine from vehicle and Trametinib -treated iCdh5-GNAQQ209L mice. Whole-mount intestinal muscularis stained for CD31 (red), z-stack maximum intensity projection. Scale bars: 2 mm and 100 µm. f Quantification of intestinal muscularis vessel diameter, vascular area, tuft number and tuft area. g Intestinal whole-mounts labeled for EdU (green) and CD31 (red). Scale bar: 20µm. h EdU positive EC (EdU+/CD31+) as percentage over total EC number (CD31+). i Representative z-stack maximum intensity projection of intestinal muscularis stained for TER119 (white) and CD31 (red). Yellow arrows indicate extravasated erythrocytes. Scale bar: 100µm. j Quantification of extravasated erythrocytes normalized to tissue area. k Platelet counts and D-Dimer levels in vehicle or Trametinib -treated iCdh5-GNAQQ209L mice. In d, f, h, j and k: vehicle (n = 7), Trametinib (n = 8), mean±SD, unpaired two-tailed Welch’s t-test. Black dotted lines indicate reference values in untreated control mice. l Representative immunoblot of iEC GNAQ-Q209L treated with DMSO or Trametinib and probed for GNAQ, phosphor-ERK (pERK, Thr202/Tyr204), total ERK (tERK), and GAPDH (n = 3 independent experiments). m Venn diagram of genes upregulated in iEC GNAQ-Q209L and downregulated with Trametinib treatment (6 h). Expression levels of 73 genes were normalized by Trametinib (adjusted P-value of <0.05 and a log2 (fold change) >1). Heatmap of selected genes clustered by GO-terms. Wald test for hypothesis testing. n Therapeutic study scheme. 6 weeks-old iCdh5-GNAQQ209L mice induced with 40 mg/kg tamoxifen and after 8 days treated daily with Trametinib (2 mg/kg) or vehicle. Kaplan-Meier survival curve of vehicle (n = 8, red) and Trametinib -treated (n = 9, turquoise) iCdh5-GNAQQ209L mice. Gehan-Breslow-Wilcoxon test. Trametinib-treated control mice (iCdh5 mice, n = 3, blue). Source data for (c,d,f,h,j,k,l,m,n) are provided as a Source Data file. Schematic in (b,e) created with Biorender.com.

Fig 3: Early-onset vascular abnormalities and vascular tufts in GNAQQ209L expressing mice.a Schematic describing breeding scheme for generating models of GNAQ-related vascular anomalies. The Rosa26-floxed stop-GNAQQ209L mouse was crossed with Cdh5-iCreERT2 to generate EC specific GNAQQ209L expression (mutant mice are thereafter called iCdh5-GNAQQ209L). b Schematic describing Tamoxifen (tam) regimen for postnatal (P1) induction before experimental analysis of vasculature in the subcutaneous and intestinal tissue at P4. Kaplan-Meier curve comparing the survival percentage of iCdh5-GNAQQ209L (n = 12, red) to iCdh5 (n = 8, black) pups. Gehan–Breslow–Wilcoxon test (p < 0.0001). c Left panel: Representative low- and high-power photographs of subcutaneous tissue from tamoxifen-treated iCdh5-GNAQQ209L and control pup (iCdh5). Scale bar: 2 mm. Right panel: Representative confocal z-stack images (max. intensity projection) of whole-mount staining for CD31 (red). The boxed regions (white dashed lines) are enlarged, and 3D reconstructed. Scale bar: 100µm. d Quantification of vessel diameter, vascular area as percentage of total area, tuft number as well as tuft area normalized to vascular area. iCdh5-GNAQQ209L (n = 11) and controls (n = 14), mean±SD, unpaired two-tailed Welch’s t-test. Mouse genotypes are indicated. e Left panel: Representative low- and high-power photographs of intestinal tissue from tamoxifen-treated iCdh5-GNAQQ209L and control pup (iCdh5). Scale bar: 2 mm. Right panel: Representative confocal z-stack images (max. intensity projection) of whole-mount intestinal muscularis stained for CD31 (red). The boxed regions (white dashed lines) are enlarged, and 3D reconstructed. Scale bar: 100µm. f Quantification of vessel diameter, vascular area as percentage of total area, tuft number, as well as tuft area normalized to vascular area. iCdh5-GNAQQ209L (n = 4) and controls (n = 3), mean±SD, unpaired two-tailed Welch’s t-test. Mouse genotypes are indicated. Source data for (b,d,f) are provided as a Source Data file. Schematic in (a,c,e) created with Biorender.com.

Fig 4: RNA-Sequencing of iEC GNAQ-Q209L reveals enriched expression of genes related to angiogenesis, MAPK signaling, inflammatory response, and coagulation.a Principal Component Analysis (PCA) of RNA-sequencing data obtained from iEC GNAQ-WT no Dox, iEC GNAQ-WT + Dox and iEC GNAQ-Q209L + Dox. b iEC GNAQ-WT and to GNAQ-Q209L (n = 4 biological replicates per group) were treated with 1 µg/mL doxycycline for 24 h and total RNA was collected for RNA-seq analysis. Differentially expressed genes (DEG) are defined as log2 (fold change) =1 and adjusted P value of <0.05. Heat maps of differentially expressed transcripts in iEC GNAQ-Q209L compared to GNAQ-WT. Wald test for hypothesis testing. c Volcano-plot of DEGs in iEC GNAQ-Q209L compared to GNAQ-WT. A total of 617 genes showed increased expression while 300 genes showed decreased expression in iEC GNAQ-Q209L compared to iEC GNAQ-WT. d KEGG and GO-BP pathway analyses of DEGs. e A selected group of DEGs were validated using RT-qPCR. n = 4 samples/group (median and quartiles), unpaired two-tailed Welch’s t-test. f Representative immunoblots of GNAQ, phospho-ERK (pERK, Thr202/Tyr204), total ERK (tERK), phospho-AKT (pAKT, Ser473), total AKT (tAKT), ANGPT2, and GAPDH in iEC GNAQ-WT and GNAQ-Q209L 48 h after doxycycline induction (0, 0.25, 0.5, 1 and 2 µg/ml of Dox). The expression of pERK was quantified and normalized to tERK expression (n = 3 independent experiments). Mean±SD, one-way ANOVA with Tukey’s multiple comparisons. P-values for statistical analysis between all groups are listed in the Source data file. Source data for (c,d,e,f) are provided as a Source Data file.

Fig 5: Increased ERK activation in the vasculature of GNAQQ209L expressing mice and patient vascular tumor.a Tamoxifen induction scheme for analysis of subcutaneous tissue. b Representative images of subcutaneous tissue of tamoxifen-treated control and iCdh5-GNAQQ209L mutant mice stained for ERG (white), pERK (red), Ki67 (green), and merge. Scale bar: 50 µm. c ERG staining marks EC nuclei. Quantification of ERG+ cells normalized per subcutaneous tissue area. pERK+ and Ki67+ and double-positive endothelial cells were normalized to total EC number. iCdh5-GNAQQ209L (n = 9) and control littermates (n = 9), mean±SD, unpaired two-tailed Welch’s t-test. Mouse genotypes are indicated. d GNAQQ209L mutant patient-derived vascular tumor tissue was analyzed. e Representative images of neonatal human foreskin and patient-derived tissue (cutaneous vascular tumor) with somatic GNAQ p.Q209L mutation immuo-stained for UEAI (Ulex europaeus agglutinin-I) (brown). Scale bar: 50µm. f Representative images of neonatal human foreskin and patient-derived tissue with somatic GNAQ p.Q209L mutation labeled for UEAI (white), pERK (red), Ki67 (green), and merge with DAPI (blue). Scale bar: 50 µm. g Quantification of the UEAI+ vascular area as percentage of total field area, pERK+, Ki67+ and double-positive EC calculated as events per mm2 of vascular area; human foreskin tissue from n = 5 different donors, and n = 1 patient-derived tumor tissue, mean±SD. Source data for (c,g) are provided as a Source Data file. Schematic in (a,d) created with Biorender.com.