Extended Data Fig. 3: APOE Expression and Functions in VICs During Elastogenesis.
From: APOE–NOTCH axis governs elastogenesis during human cardiac valve remodeling
a. Left: Representative immunofluorescence staining and RNA in situ hybridization of APOE in four human fetal valves. n = 3 different valves. White arrows represent unidirectional flow directions. Right: Violin plots of APOE and ELN expression within Elastin-VICs across each valve, A/P/M/T represent Aortic, Pulmonary, Mitral, Tricuspid valve respectively; b. Violin plots of elastogenesis-related genes (EMILIN1, LOXL1, FBN1, LTBP1) expression within Elastin-VICs across each valve; c. PCA analysis comparing four valves transcriptome in each VIC subtype; d. qPCR detection of VIC-related marker genes in cultured human fetal VICs, VECs, and HUVECs. Expression levels are normalized to HUVECs; e. Flow cytometry analysis of PECAM1 (VECs) and COL1A1 (VICs) expression in culture VICs; f. immunofluorescence staining of α-SMA (VICs) and PECAM1 (VECs) on cultured VICs; g. qPCR analysis APOE in VICs after APOE KD; h. Quantification of total elastin (both tropo-elastin(soluble) and mature elastin (insoluble)) in VICs after APOE KD. The elastin content was normalized to the total protein content; i. qPCR analysis of elastogenesis-related genes in VICs after APOE KD; j. Violin plot and feature plot of ACTA2 expression in VIC subtypes. FDR: Elastin vs. other VIC subclusters; k. Representative immunofluorescence staining of α-SMA and Elastin in four human fetal valves. White arrows represent unidirectional flow directions. Yellow arrowhead: Elastin-VIC positive for α-SMA. White arrowhead: VICs positive for α-SMA but not Elastin. n = 3; l. Contraction assay comparing scramble and APOE KD in VICs; m. qPCR analysis of contraction-related gene expressions in VICs comparing scramble and APOE KD. Data are shown as the mean ± SEM. n = 3 biological repeats. ns p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001. For g-i, l-m, scramble vs. APOE KD; d-e, VICs vs. VECs. Statistics in d-e, g-i, l-m: Unpaired 2-tailed t-test (2 groups). P values in d: COL1A1: p = 0.0121, ACTA2: p = 0.0437; in e: p < 0.0001; in g: p = 0.0023; in i: FBN2: p = 0.0265, LTBP2: p = 0.6162, LTBP4: p = 0.0422, FBLN1: p = 0.0327, FBLN2: p = 0.0526, LOX: p = 0.0003; in l: p = 0.0362; in m: ACTA2: p = 0.0011, MYH11: p = 0.0223, SMTN: p = 0.1664, CNN1: p = 0.0247. For j: Statistics adhered to a two-sided approach. Significance was determined by adjusted p-values (FDRs) < 0.05. FDR for j: FDR = 8.43E-203. FDR: False Discovery Rate. HUVECs: Human Umbilical Vein Endothelial Cells. KD: Knockdown.
