Overexpression or silencing of FOXO3a affects proliferation of endothelial progenitor cells and expression of cell cycle regulatory proteins

PLoS One. 2014 Aug 5;9(8):e101703. doi: 10.1371/journal.pone.0101703. eCollection 2014.


Endothelial dysfunction is involved in the pathogenesis of many cardiovascular diseases such as atherosclerosis. Endothelial progenitor cells (EPCs) have been considered to be of great significance in therapeutic angiogenesis. Furthermore, the Forkhead box O (FOXO) transcription factors are known to be important regulators of cell cycle. Therefore, we investigated the effects of changes in FOXO3a activity on cell proliferation and cell cycle regulatory proteins in EPCs. The constructed recombinant adenovirus vectors Ad-TM (triple mutant)-FOXO3a, Ad-shRNA-FOXO3a and the control Ad-GFP were transfected into EPCs derived from human umbilical cord blood. Assessment of transfection efficiency using an inverted fluorescence microscope and flow cytometry indicated a successful transfection. Additionally, the expression of FOXO3a was markedly increased in the Ad-TM-FOXO3a group but was inhibited in the Ad-shRNA-FOXO3a group as seen by western blotting. Overexpression of FOXO3a suppressed EPC proliferation and modulated expression of the cell cycle regulatory proteins including upregulation of the cell cycle inhibitor p27(kip1) and downregulation of cyclin-dependent kinase 2 (CDK2), cyclin D1 and proliferating cell nuclear antigen (PCNA). In the Ad-shRNA-FOXO3a group, the results were counter-productive. Furthermore, flow cytometry for cell cycle analysis suggested that the active mutant of FOXO3a caused a noticeable increase in G1- and S-phase frequencies, while a decrease was observed after FOXO3a silencing. In conclusion, these data demonstrated that FOXO3a could possibly inhibit EPC proliferation via cell cycle arrest involving upregulation of p27(kip1) and downregulation of CDK2, cyclin D1 and PCNA.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Cycle Checkpoints / genetics
  • Cell Cycle Proteins / genetics*
  • Cell Cycle Proteins / metabolism
  • Cell Proliferation / genetics*
  • Cells, Cultured
  • Cyclin D1 / metabolism
  • Cyclin-Dependent Kinase 2 / metabolism
  • Cyclin-Dependent Kinase Inhibitor p27 / metabolism
  • Endothelial Progenitor Cells / physiology*
  • Forkhead Box Protein O3
  • Forkhead Transcription Factors / genetics*
  • Gene Expression Regulation / physiology
  • Humans
  • Proliferating Cell Nuclear Antigen / metabolism
  • RNA Interference
  • Transfection
  • Up-Regulation


  • CCND1 protein, human
  • Cell Cycle Proteins
  • FOXO3 protein, human
  • Forkhead Box Protein O3
  • Forkhead Transcription Factors
  • Proliferating Cell Nuclear Antigen
  • Cyclin D1
  • Cyclin-Dependent Kinase Inhibitor p27
  • CDK2 protein, human
  • Cyclin-Dependent Kinase 2

Grant support

This work was supported by National Natural Science Foundation of China (Grant No. 30973152 and 81270205). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.