Fig 2: FSS-NOTCH-GJA4-CDKN1B axis regulates arterial identity genes. a Disorganized or absent SMA investment was observed in Gja4 -/- and WT + DAPT mice, compared to WT controls. (Colors: PECAM1 (red), SMA (white); scale bar: 100 µm; symbols: a = artery, v = vein, R = remodeling, M = mature). b Radial SMA investment of arteries, as well as c radial distance of GJA5 (Cx40) expression, were reduced in Gja4 -/- and DAPT-treated animals (mean radial distance as % of total vascular outgrowth ± SEM vs WT; n = 3 (WT + DAPT; WT, GJA5), n = 4 (WT, SMA), n = 6 (Gja4 -/-); one-way ANOVA: p = 0.03 (SMA), p = 0.04 (GJA5); asterisks indicate p < 0.05 in post hoc t-tests vs. WT). d DLL4 activation of NOTCH signaling upregulated EFNB2 (EphrinB2) and GJA5, and this effect was abolished with si-GJA4 or si-CDKN1B (mean relative mRNA expression ± SEM vs. PBS; n = 3 (si-GJA4; si-CDKN1B, EFNB2), n = 4 (si-Ctrl, GJA5; si-CDKN1B, GJA5), n = 4 (si-Ctrl, EFNB2); Students’ t-test: p = 0.02 (si-Ctrl, EFNB2), p = 0.04 (si-Ctrl, GJA5), p = 0.0005 (si-GJA4, GJA5), p < 0.0001 (si-CDKN1B, GJA5)). e NOTCH activation was maximal with 1 h exposure to 18 dynes/cm2 shear (mean NICD R.F.U. ± SEM; n = 7000 cells, representative of N = 3). f Expression of GJA4 (Cx37), GJA5 (Cx40) and EFNB2 (EphrinB2) were also maximal at 18 dynes/cm2 (mean relative mRNA expression ± SE; n = 3, representative of 4 experiments). g Knockdown of NOTCH1, GJA4 or CDKN1B reduced basal and 10 h FSS-induced upregulation of EFNB2 and GJA5 (mean relative mRNA expression ± SEM vs. Static; n = 3 technical replicates; representative of two biological replicates)

Fig 3: Connexin 40 (Cx40) expression was reduced in angiotensin II type 1 receptor (AT1R) mice of both age groups but Connexin 43 (Cx43) protein expression was not affected. A, Atrial Gja5 (Cx40) messenger RNA (mRNA) expression was reduced at both ages in AT1R mice (50 days and 6 months, n=6; *P<0.0001). B, Cx40 protein expression was reduced in AT1R mice compared with their respective controls at 50 days and also tended to be reduced at 6 months (50 days, *P=0.0057; 6 months, P=0.0660 [n=3 per group]) (see Figures S1 and S2 for stain-free images). C, Quantitative polymerase chain reaction results showed a reduction in Gja1 (Cx43) mRNA expression in 50-day and 6-month AT1R mice (50 days, *P=0.0068; 6 months, *P=0.0002; n=6 per group). D, However, Cx43 protein expression was not changed by AT1R overexpression (50 days, P=0.8846; 6 months, P=0.8606; n=3 per group). Film-free chemiluminescence protein signal was normalized by the total protein content using stain-free quantification (see Figure S2 for stain-free images). Statistical significance interpretation: *P<0.05; **P<0.01; ***P<0.0001.

Fig 4: LDL reduces connexin-40 expression in HL-1 myocytes.Effect of increasing doses of LDL (0, 50, 100 and 200 µg/mL) on connexin-40 (Cx40) and connexin-43 (Cx43) mRNA (A) or protein (B) levels in HL-1 myocyte cultures C Relationship between intracellular cholesteryl ester (CE) and Cx40 and Cx43 protein levels in HL-1 myocyte cultures. Results are shown as mean±SEM of three independent experiments performed in duplicate. *P<0.05 vs. cells incubated in absence of LDL.

Fig 5: A distal region of mouse Hey1/human HEY1 functions as an endothelial enhancer. A, the p300 ChIP-Seq result using E11.5 endothelial cells and genomic conservation proposed the mouse Hey1 -18 kb region as an endothelial enhancer candidate. B, the BAC-LacZ reporter lacking the -18 kb region (f) did not induce robust ß-gal activity in the vasculature of E9.5 embryos. Both the mouse -18 kb region (g) and the human -16 kb region (h) showed vascular activity in DA, PAAs, and ISV. Summary of the results is shown in A, and the results of other F0 embryos are displayed in Fig. S8. C, Tg mouse lines confirmed endothelial activity of the -18 kb-LacZ reporter in DA and PAAs (numbered). The floor plate of neural tube (NT) also expressed the LacZ reporter. LV, left ventricle; OFT, outflow tract; RV, right ventricle. D, the -18 kb-EGFP reporter Tg embryos recapitulated arterial endothelial expression of the LacZ reporter. CV, cardinal vein; FG, foregut. Scale bars in the whole-mount images and sections of B, C, and D are 500 and 200 µm, respectively. E, EGFPhigh and EGFPlow populations of Pecam1high endothelial cells were sorted from E10.5 embryos. F, real-time PCR analysis revealed that EGFPhigh cells had high expression of Hey1 and arterial markers (Cxcr4, Efnb2, Gja4, Gja5, Dll4, Jagged1, and Hey2), although the expression levels of pan-endothelial (Cdh5 and Pecam1) and venous (Ephb4) genes were equivalent between EGFPhigh and EGFPlow cells. *, p < 0.05; **, p < 0.01; ns, not significant.