Involvement of Foxo transcription factors in angiogenesis and postnatal neovascularization
Michael Potente, … , Andreas M. Zeiher, Stefanie Dimmeler
Michael Potente, … , Andreas M. Zeiher, Stefanie Dimmeler
Published September 1, 2005
Citation Information: J Clin Invest. 2005;115(9):2382-2392. https://doi.org/10.1172/JCI23126.
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Research Article Angiogenesis

Involvement of Foxo transcription factors in angiogenesis and postnatal neovascularization

Abstract

Forkhead box O (Foxo) transcription factors are emerging as critical transcriptional integrators among pathways regulating differentiation, proliferation, and survival, yet the role of the distinct Foxo family members in angiogenic activity of endothelial cells and postnatal vessel formation has not been studied. Here, we show that Foxo1 and Foxo3a are the most abundant Foxo isoforms in mature endothelial cells and that overexpression of constitutively active Foxo1 or Foxo3a, but not Foxo4, significantly inhibits endothelial cell migration and tube formation in vitro. Silencing of either Foxo1 or Foxo3a gene expression led to a profound increase in the migratory and sprout-forming capacity of endothelial cells. Gene expression profiling showed that Foxo1 and Foxo3a specifically regulate a nonredundant but overlapping set of angiogenesis- and vascular remodeling–related genes. Whereas angiopoietin 2 (Ang2) was exclusively regulated by Foxo1, eNOS, which is essential for postnatal neovascularization, was regulated by Foxo1 and Foxo3a. Consistent with these findings, constitutively active Foxo1 and Foxo3a repressed eNOS protein expression and bound to the eNOS promoter. In vivo, Foxo3a deficiency increased eNOS expression and enhanced postnatal vessel formation and maturation. Thus, our data suggest an important role for Foxo transcription factors in the regulation of vessel formation in the adult.

Authors

Michael Potente, Carmen Urbich, Ken-ichiro Sasaki, Wolf K. Hofmann, Christopher Heeschen, Alexandra Aicher, Ramya Kollipara, Ronald A. DePinho, Andreas M. Zeiher, Stefanie Dimmeler

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Silencing Foxo1 or Foxo3a activity enhances the angiogenic activity of e...
Silencing Foxo1 or Foxo3a activity enhances the angiogenic activity of endothelial cells. (A) HUVECs were transfected with 2 different siRNAs targeted against either Foxo1 (Foxo1 I and Foxo1 II) or Foxo3a (Foxo3a I and Foxo3a II). A nonrelated scrambled siRNA was used a control. Cell lysates were subjected to Western blotting using antibodies against Foxo1 and Foxo3a. Tubulin was used a loading control. (B and C) HUVECs were transfected with 2 different siRNAs targeted against Foxo1, Foxo3a, or a scrambled oligonucleotide. After 18 hours, cells were seeded on a Growth Factor Reduced Matrigel in the presence of VEGF (50 ng/ml). Cumulative sprout length of capillary-like structures was measured by light microscopy after 24 hours. Representative micrographs and statistical summary are shown. Data are presented as mean ± SEM; n = 4 (Foxo1), n = 6 (Foxo3a). *P < 0.001 versus control. (D) Representative micrographs and statistical summary of endothelial cells transfected with siRNAs targeted against Foxo1, Foxo3a, or scrambled control. After 18 hours, cells were seeded in the upper chamber of modified Boyden chamber. Endothelial cell migration was assessed using VEGF (50 ng/ml) as chemoattractant. After 24 hours, nonmigrating cells on the upper side of the chamber were mechanically removed, and the remaining cells on the lower side were fixed and stained with DAPI. Data are presented as mean ± SEM; n = 5. *P < 0.001 versus control. (E) Three-dimensional in vitro angiogenesis with collagen gel–embedded spheroids of Foxo1, Foxo3a, or scrambled siRNA–transfected endothelial cells. Cumulative length of all sprouts originating from an individual spheroid was quantified after 24 hours. Statistical summary represents the mean ± SEM; n = 3. **P < 0.05 versus control. Magnification: ×50 (B); ×200 (D); ×100 (E).