VEGI inhibition of EPC differentiation. (A) Cell signaling changes caused by VEGI treatment in early EPCs. Western blotting analysis of the phosphorylation of Erk, Akt, or p38 in freshly isolated Sca-1⁺ mononuclear cells in response to various concentrations of VEGI, as indicated, for 15 minutes. (B) Histograms of flow cytometric analysis of EPC markers on EPCs cultured for day 3, day 7, and day 10 in the absence or presence of VEGI. White areas represent untreated; shaded areas, VEGI-treated. (C) Confocal microscopic images of immunofluorescent-stained endothelial cell marker flk-1 on EPCs cultured for 10 days in the absence or presence of VEGI. Green indicates flk-1; red, actin; blue, cell nuclei (20× objective lens). (D) Phase-contrast images showing morphologic differences between EPCs cultured for 10 days in the absence or presence of VEGI. (E) Analysis of fluorescence intensity of EPC markers. A total of 3000 cells per well were analyzed. Experiments were conducted in triplicate. Data represent mean ± SE of 3 independent experiments. *P < .05.