Fig. 7

Endothelial cell cycle arrest, per se, enables arterial gene expression. a Treatment of HUVEC with 10 µM clotrimazole or 2 µM palbociclib reduced RB1, phosphorylated RB1 (pRb1), and E2F1, suggestive of G1 arrest. Clotrimazole tended to upregulate CDKN1B expression and preserve GJA4 expression, and CDK4 expression was lost only with CDK4/6i treatment. Uncropped blots presented in Supplementary Fig. 16. b Using FACS to assess cell cycle distribution, clotrimazole was found to arrest HUVEC in G1, even when CDKN1B was knocked down (via si-CDKN1B) (mean difference in cell cycle % ± SEM vs. DMSO; n = 3 (Clotrimazole), n = 5 (DMSO), n = 6 (Clotrimazole+ si-CDKN1B); individual values plotted where n < 5; one-way ANOVA: p = 0.007 (G1), p = 0.03 (S/G2/M); asterisks indicate p < 0.05 in post hoc t-tests). c Clotrimazole upregulated EFNB2 and GJA5 regardless of CDKN1B expression (mean relative mRNA expression ± SEM vs. DMSO; n = 4 (Clotrimazole, p27), n = 5 (Clotrimazole, EFNB2; Clotrimazole, GJA5), n = 7 (Clotrimazole+ si-CDKN1B), n = 8 (DMSO); individual values plotted where n < 5; one-way ANOVA: p = 0.03 (EFNB2, GJA5), p = 0.0007 (CDKN1B)). d Palbociclib treatment also arrested endothelial cells in G1 (mean difference in cell cycle % ± SEM vs. DMSO; n = 3 for all groups; Students’ t-test: p = 0.002 (G1), p < 0.0001 (S/G2/M)), e upregulated EFNB2 and GJA5 (Cx40) mRNA levels, abolished CDK4 expression, and had no effect on GJA4 (Cx37) and CDKN1B (p27), (mean relative mRNA expression ± SEM vs DMSO; n = 9 (GJA4), n = 12 (EFNB2), n = 14 (GJA5), n = 16 (CDKN1B; CDK4); Students’ t-test: p = 0.02 (EFNB2, GJA5), p < 0.0001 (CDK4)). f Endothelial cells within arteries and surrounding plexi of P6 retinas from Cdt1-mOrange+ reporter mice were predominantly in G1 phase, whereas endothelial cells in adjacent veins were not. (Colors: IB4 (red), ERG (blue), pH3 (magenta), CDT1 (green); scale bar: 100 µm). g We hypothesize that in remodeling vessels, arterial shear activates a novel Notch-Cx37-p27 signaling pathway that promotes endothelial cell cycle arrest to enable arterial gene expression