Fig 2: PI3K inhibition rescues downstream signalling components.(a,b) Anti phospho-S6 (pS6) and IB4 staining of retinal flat mounts from P5 mice treated or not with BMP9/10 blAB. (c) Quantification of the percentage of pS6+ vascular area per field of view. n=8 Retinas per group. Error bars: s.e.m., ***P<0.001, Mann–Whitney U-test. (d–g) Anti-pS6 and IB4 staining of retinal flat mounts from Ctrl (d,f) or Alk1i?EC (e,g) treated with PBS (d,e) or PI3Ki (f,g). (h) Quantification of the percentage of pS6+ vascular area per field of view. n=6–8 Retinas per group. Error bars: s.e.m., *P<0.05 and **P<0.01, Mann–Whitney U-test. (i,l) Anti-Eng and IB4 staining of retinal flat mounts from Ctrl (i,k) or Alk1i?EC (j,l) treated with PBS (i,j) or PI3Ki (k,l). (m) Quantification of the percentage of Eng+ vascular area per field of view. Yellow arrowheads in b,e,j indicate AV shunts. n=5–12 Retinas per group. Error bars: s.e.m., **P<0.01 and ***P<0.001, Mann–Whitney U-test. Scale bars, 100 µm in a,b,d–g,i–l. a, artery in red; v, vein in blue.

Fig 3: Assessment of LV remodelling induced by PAB upon cardiac depletion of Hand2. (A) Assessment of LV eccentricity index at end-diastole and (B) end-systole; (C) quantification of cardiomyocyte CSA from images of histological sections stained for WGA, n =30 microscopic field/heart, 3 hearts. (D–H) Quantitative real-time PCR analysis was performed to assess the expression levels of several genes known to be related to cardiac hypertrophy in RV tissue from Hand2F/F and MCM-Hand2F/F animals either after sham or PAB surgery: (D) Nppa; (E) Nppb; (F) β-myosin heavy chain; (G) Acta1; (H) regulator of Calcineurin 1 Isoform 4; (I) quantification of collagen deposition from images of histological sections stained for Sirius Red, n =30 microscopic field/heart, 3 hearts. (J–L) Quantitative real-time PCR analysis of (J) collagen type I alpha 1 chain; (K) TGFb and (L) Eng; (M) representative images of western blot analysis of COL1A, PAI-1, ENG, and GAPDH as loading control in LV tissue of Hand2F/F and MCM-Hand2F/F animals subjected to either sham or PAB surgery (N) capillaries in RV sections of Hand2F/F and MCM-Hand2F/F animals subjected to either sham or PAB surgery were identified by isolectin B4 immunohistochemistry combined with WGA and, from the images obtained, we determined the ratio of capillaries per cardiomyocyte ratios; (O) quantitative real-time PCR analysis of Vegfr2 expression levels in the RV of Hand2F/F and MCM-Hand2F/F animals, subjected to either sham or PAB surgery. (P) Representative images of western blot analysis of VEGFR2 and GAPDH as loading control in LV tissue of Hand2F/F and MCM-Hand2F/F animals subjected to either sham or PAB surgery. Data are from 5–10 animals per group, except for the western blots where protein lysates from 3–5 hearts per group were pulled in one sample. Statistical analysis using one-way ANOVA with Tukey’s multiple comparisons test. ∗P<0.05 between indicated groups (error bars are SEM).

Fig 4: MRS1754 reduces tumor blood vessel formation and angiogenesis marker expression in vivo on GBM38. (A) H&E staining of representative sections of tumors from control and MRS treatment groups. Scale bar is 200 µm. (B) Immunofluorescence images of representative areas from control and MRS treatment groups. Stained with DAPI (in blue), CD105 (in red), and Vimentin (in green). The scale bar is 100 µm. (C) Immunofluorescence images of representative areas from control and MRS treatment groups. Stained with DAPI (in blue) and VEGFA (in red). The scale bar is 100 µm. (D) Quantification of CD105 levels in immunofluorescence images. Shown here is the integrated intensity density of CD105 in the control and MRS treated groups. **p value <0.01. (E) Quantification of VEGFA levels in immunofluorescence images. Shown here is the mean intensity of VEGFA in the control and MRS treated groupsis shown here. (F) mRNA expression levels of mCD31, hVEGFA, hVEGFR2, and mVEGFR2 in both control and MRS treatment groups. **p value <0.01. n.s, non-significant.

Fig 5: Assessment of RV remodelling induced by PAB upon cardiac depletion of Hand2. (A) Fulton’s index values (ratio of RV weight to LV plus septal weight) of Hand2F/F and MCM-Hand2F/F animals either after sham or PAB surgery; (B) high-magnification of representative images of histological sections stained for WGA; and (C) quantification of CSA in (B), n =30 microscopic field/heart, 3 hearts. (D–H) Quantitative real-time PCR analysis was performed to assess the expression levels of several genes known to be related to cardiac hypertrophy in RV tissue from Hand2F/F and MCM-Hand2F/F animals either after sham or PAB surgery: (D) Nppa; (E) Nppb; (F) Acta1; (G) ß-myosin heavy chain; (H) regulator of Calcineurin 1 Isoform 4; (I) high-magnification of representative images of histological sections stained for Sirius Red; (J) quantification of collagen deposition in (I), n =30 microscopic field/heart, 3 hearts. Quantitative real-time PCR analysis of (K) collagen type I alpha 1 chain; (L) Tgfb and (M) Eng; (N) representative images of western blot analysis of COL1A, PAI-1, ENG, and GAPDH as loading control in RV tissue of Hand2F/F and MCM-Hand2F/F animals subjected to either sham or PAB surgery (O) Capillaries in RV sections of Hand2F/F and MCM-Hand2F/F mice subjected to either sham or PAB surgery were identified by isolectin B4 immunohistochemistry combined with WGA and, from the images obtained, we determined the ratio of capillaries per cardiomyocyte ratios; (P) quantitative real-time PCR analysis of Vegfr2 expression levels in the RV of Hand2F/F and MCM-Hand2F/F animals, subjected to either sham or PAB surgery. (Q) Representative images of western blot analysis of VEGFR2 and GAPDH as loading control in RV tissue of Hand2F/F and MCM-Hand2F/F animals subjected to either sham or PAB surgery. Data are from 5–10 animals per group, except for the western blots where protein lysates from 3–5 hearts per group were pulled in one sample. Statistical analysis using one-way ANOVA with Tukey’s multiple comparisons test. *P<0.05 between indicated groups (error bars are SEM).