Fig. 5: 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 α5β1 receptor, ANGPTL2 and Notch1 protein expression by immunofluorescence in AoV from E14.5 embryo from WT mice. Higher magnification shows that Integrin α5β1, 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 α5β1 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.