Figure 5. Endothelial NAD⁺ sensitizes ECs to VEGF by suppressing Notch.

(A) Number of migrated MLECs (n = 12).

(B) Sprout length of MLEC spheroids (n = 8).

(C) Sprout length of HAEC spheroids transfected with non-targeting (NT) or SIRT1 (T1) siRNAs (n = 8).

(D) Number of branch points and length of HAEC tube networks (n = 13).

(E) Number and total area of sprouts originating from aortic rings (18-month old, n = 8).

(F) Relative mRNA levels of Notch target genes (HEY1, HES1 and NRARP) and NOTCH1 in HAECs stimulated with VEGF for 1 hr (n = 4).

(G) Notch Intracellular Domain (NICD) protein and relative abundance in HAECs stimulated with VEGF for 5 hrs (n = 3).

(H) Sprout length of VEGF-stimulated HAEC spheroids transduced with NT or T1 siRNAs (n = 8).

(I) Number of sprouts in VEGF-stimulated aortic rings (18-month old, n = 8).

(J) VEGF stimulation during sprouting angiogenesis upregulates expression of Dll4 ligand in the tip cells, activating Notch in the stalk cells, which triggers proteolytic cleavage of Notch receptor by γ-secretase complex to release NICD from the cell membrane so it translocates to the nucleus and induces transcription of target genes. Activation of SIRT1 by the NAD booster NMN promotes migration, proliferation and survival in VEGF-stimulated ECs. In stalk cells, NMN suppresses NICD during VEGF/Dll4 stimulation and Notch target gene activation, thereby promoting sprouting. VEGF receptor inhibitors SU5416 or axitinib block the effects of NMN on angiogenesis.

Data expressed as mean ± SEM. *p < 0.05, **p < 0.005, ***p < 0.0005, ^δp < 0.00005 by Student’s t test (G), one-way (H and I) or two-way (A–F) ANOVA with Bonferroni’s corrections. See also Figure S5, Movie 3 and Movie 4