RSV restores proliferation and angiogenesis of PT-ECFC in vitro in a SIRT1-dependent manner. ECFCs were used at passage 3. (A) Proliferation rate determined by BrdU incorporation assay in 7500 ECFCs after 72 hours of treatment with 1µM RSV. Results were expressed as arbitrary units of spectrophotometric measurements. Data are means ± SEM of 12 independent samples in experiments performed in triplicate. *P < .05, **P < .01. (B) Representative experiment of capillary tube formation by ECFCs from CT or PT-neonates treated with 1µM RSV, in the presence or not of 1mM NAM. A total of 20 000 ECFCs per well were seeded on growth factor-reduced Matrigel. Tube formation was analyzed 6 hours later on a phase-contrast microscope (original magnification ×4). (C) Quantitative analysis of total closed tubes (top panel) and branches (bottom panel) after 6 hours of treatment. Data are means ± SEM of 12 independent samples in experiments performed in triplicate. *P < .05, **P < .01 . (D) Representative experiment of three-dimensional in vitro angiogenesis assay with collagen gel-embedded spheroids of PT-ECFCs treated with RSV (1µM), in the presence or not of 1mM NAM and CT-ECFCs (original magnification ×20; scale bars, 100µm). (E) Quantification of the number of sprouts per spheroid. (F) Quantification of the number of branch points per spheroid. (G) Quantification of the cumulative sprout length per spheroid. (E-G) For each experiment, sprouts from 20 spheroids were counted. Data are means ± SEM of 12 independent samples from experiments performed in duplicate. *P < .05; **P < .01; ***P < .001.