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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Figure 5

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Foxo1 and Foxo3a are transcriptional repressors of eNOS. (A) eNOS expres...
Foxo1 and Foxo3a are transcriptional repressors of eNOS. (A) eNOS expression in HUVECs that were transfected with constitutively active constructs of Flag–Foxo1 A3 and HA–Foxo3a A3. Cell lysates were prepared at the indicated time points, and expression of eNOS, Flag, HA, and tubulin was determined by Western blotting using the respective antibodies. (B) eNOS expression in HUVECs that were transfected with Foxo1- or Foxo3a-specific siRNA and lysed at the indicated time points. eNOS, Foxo1, and Foxo3a expression was determined by immunoblotting. SCR, scrambled siRNA; FX1, Foxo1 siRNA; FX3a, Foxo3a siRNA. (C) eNOS expression in the aorta of Foxo3a+/+ and Foxo3a–/– mice. After the aortas from the indicated groups of mice were removed, cell lysates were prepared, and expression of eNOS was determined by immunoblotting. Equal protein loading was confirmed with a tubulin antibody. (D) HUVECs were transfected with Foxo1 A3 (Flag-Foxo1 A3), Foxo3a A3 (HA–Foxo3a A3), Foxo4 A3 (HA–Foxo4 A3), or vector control. After 24 hours, chromatin-bound DNA was immunoprecipitated with an antibody against the Flag or HA epitope. Immunoprecipitated DNA was analyzed by PCR using a primer combination that encompassed the forkhead responsive element (FHRE). The pGL3-eNOS-3500 plasmid was used as a positive control for the PCR. eNOS primers for the eNOS coding sequence were used as a negative control to exclude nonspecific precipitated DNA. CDS, coding sequence. (E) HUVECs were cotransfected with pGL3-eNOS-3500 and either Foxo1 A3 or the empty vector pcDNA3 (pcDNA). Luciferase activity was measured 24 hours later. Values are expressed as the level of luciferase activity of Foxo1 A3 relative to that of pcDNA, which was set as 100%. Data are presented as mean ± SEM; n = 6. *P < 0.001.