Fig 2: Angptl2-KD mice exhibit AoV defects at embryonic stage.a Angptl2 gene expression measured by quantitative RT-qPCR in AoV leaflets from adult male WT and Angptl2-KD mice. Data are mean ± SEM of n = 3 for WT and n = 6 for Angptl2-KD mice; *: p < 0.05 determined with Mann–Whitney U test. b, c Front and side views of representative hearts from E10.5 and E11.5 embryos from WT and Angptl2-KD mice. Images are representative of n = 3 independent experiments. d Representative heart sections from E14.5 embryos from WT and Angptl2-KD mice stained with Hematoxylin and Eosin. Higher magnification of AoV at E14.5 is shown in the bottom panel; the 3 leaflets of AoV are indicated by yellow stars. Images are representative of n = 3 independent experiments. e, f Representative AoV sections from E18 embryos from WT and Angptl2-KD mice stained with Hematoxylin and Eosin. The yellow dotted lines represent the regions used for quantification. Quantification of valve thickness shows the enlargement of both (g) the leaflets and (h) the hinges of AoV in E18 embryos from Angptl2-KD mice compared to WT. Data are means ± SEM of n = 8 for WT and n = 9 for Angptl2-KD mice; up to 3 leaflets (n = 21 for WT and n = 23 for Angptl2-KD mice) and 6 hinges (n = 34 for WT and n = 42 for Angptl2-KD mice) were quantified per section; *: p < 0.05 and **: p < 0.01 determined with unpaired t-test.

Fig 3: ANGPTL2 is enriched in human fetal semilunar valves and associated with cell cycle and senescence pathways.a Heatmap of genes expressed in human fetal semilunar valves and adult AoV leaflets. b Close-up view of the heatmap showing significant enrichment of ANGPTL2, HEY1 and NOTCH1 in fetal semilunar valves; *significantly expressed genes (FDR < 0.05 and log2 FC > 1.5). c Pathway enrichment for the turquoise heart valve gene module obtained from WGCNA.

Fig 4: AoV defects in Angptl2-KD mice are associated with reduced Notch1 signalling.a, b Representative images (of n = 3–4 independent experiments per genotype) of Notch1 and CD31 protein expression by immunofluorescence in cardiac sections showing both aortic valve (AoV) and pulmonary valve (PuV); Higher magnification (×40; right panels) showing AoV from E14.5 embryos of WT and Angptl2-KD mice. c The total Notch1 positive area/ROI was quantified. Data are means ± SEM of n = 3 WT and n = 4 Angptl2-KD embryos (1 to 2 sections analyzed/embryo); **: p < 0.01 determined with Mann–Whitney U test. d Representative images (of n = 6 independent experiments) of activated Notch1 (Act-Notch1) protein expression by immunofluorescence in cardiac histological sections from adult 2-month old WT and Angptl2-KD male and female mice. The total Act-Notch1 positive area/ROI was quantified. Data are means ± SEM; n = 6 mice per genotype, males and females were pooled; **: p < 0.01 determined with Mann–Whitney U test. e Representative images (of n = 3 independent experiments) of ANGPTL2 and Notch1 protein expression by immunofluorescence in AoV from E14.5 embryos and 2-month old WT mice. Cells co-expressing ANGPTL2 and Notch1 are indicated by white arrows in AoV leaflets of WT mice. f Gene expression by quantitative RT-qPCR in AoV leaflets from 4-month old males and females Angptl2-KD mice compared to their WT littermates. Data are means ± SEM of n = 9 WT and n = 8–10 Angptl2-KD mice; *: p < 0.05 and **: p < 0.01 determined with t-test or Mann–Whitney U test according to the normality of distribution. g, h Linear correlations between Notch1, Hey1 and Hey2 mRNA levels in AoV leaflets and AoV area (g) and leaflet thickness (h) in male and female 4-month old WT and Angptl2-KD mice. Data are means ± SEM of n = 17–19 mice; *: p < 0.05 determined with Spearman or Pearson correlation tests according to the normality of distribution. i Representative images (of n = 3 independent experiments) of Integrin a5ß1 receptor, ANGPTL2 and Notch1 protein expression by immunofluorescence in AoV from E14.5 embryo from WT mice. Higher magnification shows that Integrin a5ß1, ANGPTL2 and Notch1 are expressed in the border of the leaflet. j Representative images (of n = 3 independent experiments) of PirB receptor and ANGPTL2 protein expression by immunofluorescence in AoV from 2-month old WT mice. Higher magnification shows that PirB and ANGPTL2 are co-expressed in some cells in the border of the leaflet (indicated by white arrows). DAPI staining was used to visualize cell nuclei. k Proposed mechanism for AVS pathogenesis in adult Angptl2-KD mice; At E14.5 during semilunar valve remodelling, the KD of Angptl2 induces a decrease in Notch1 signalling via Integrin a5ß1 receptors in AoV leaflets, which in turn deregulates the fine balance between cell apoptosis, senescence and proliferation. Lower apoptosis and senescence combined with higher cellular proliferation induce inadequate AoV maturation, and lead to thickened AoV leaflets at E18. These structural, cellular and molecular dysfunctions observed at embryonic stage progress to AVS in adult Angptl2-KD mice, in both male and female mice. k Was adapted from Lin et al.87.

Fig 5: Summarized evidence for a role of ANGPTL2 as a potentially new essential player in valvulogenesis in mice and human.At E14.5 during semilunar valve remodelling in mice, the KD of Angptl2 induces a decrease in Notch1 signalling via Integrin a5ß1 receptors in AoV leaflets, which in turn deregulates the fine balance between cell apoptosis, senescence and proliferation. Lower apoptosis and senescence combined with higher cellular proliferation induce inadequate AoV maturation, and lead to thickened AoV leaflets at E18. The thickened AoV leaflets during embryogenesis are associated with the development of AVS in adult mice. ANGPTL2 is also enriched in human fetal semilunar valves compared to adult AoV and is associated with pathways related to cell cycle and senescence. Parts of the figure were drawn by using pictures from Servier Medical Art. Servier Medical Art by Servier is licensed under a Creative Commons Attribution 3.0 Unported License (https://creativecommons.org/licenses/by/3.0/). The upper panel was adapted from Lin et al.87.