Vascular endothelial growth factor-A signaling in bone marrow-derived endothelial progenitor cells exposed to hypoxic stress

Physiol Genomics. 2013 Nov 1;45(21):1021-34. doi: 10.1152/physiolgenomics.00070.2013. Epub 2013 Sep 10.


Bone marrow-derived endothelial progenitor cells (BM-EPCs) are stimulated by vascular endothelial growth factor-A (VEGF-A) and other potent proangiogenic factors. During angiogenesis, an increase in VEGF-A expression stimulates BM-EPCs to enhance endothelial tube formation and contribute to an increase in microvessel density. Hypoxia is known to produce an enhanced angiogenic response and heightened levels of VEGF-A have been seen in oxygen deprived epithelial and endothelial cells, yet the pathways for VEGF-A signaling in BM-EPCs have not been described. This study explores the influence of hypoxia on VEGF-A signaling in rat BM-EPCs utilizing a novel proteomic strategy to directly identify interacting downstream components of the combined VEGF receptor(s) signaling pathways, gene expression analysis, and functional phenotyping. VEGF-A signaling network analysis following liquid chromatographic separation and tandem mass spectrometry revealed proteins related to inositol/calcium signaling, nitric oxide signaling, cell survival, cell migration, and inflammatory responses. Alterations in BM-EPC expression of common angiogenic genes and tube formation in response to VEGF-A during hypoxia were measured and combined with the proteomic analysis to enhance and support the signaling pathways detected. BM-EPC tube formation assays in response to VEGF-A exhibited little tube formation; however, a cell projection/migratory phenotype supported the signaling data. Additionally, a novel assay measuring BM-EPC incorporation into preformed endothelial cell tubes indicated a significant increase of incorporated BM-EPCs after pretreatment with VEGF-A during hypoxia. This study verifies known VEGF-A pathway components and reveals several unidentified mechanisms of VEGF-A signaling in BM-EPCs during hypoxia that may be important for migration to sites of vascular regeneration.

Keywords: endothelial progenitor cells; hypoxia; mass spectrometry; signaling; vascular endothelial growth factor.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Bone Marrow Cells / cytology
  • Bone Marrow Cells / metabolism*
  • Cell Hypoxia
  • Cell Movement / genetics
  • Cell Survival / genetics
  • Cells, Cultured
  • Chromatography, High Pressure Liquid
  • Endothelial Cells / cytology
  • Endothelial Cells / metabolism*
  • Gene Expression Regulation / drug effects
  • Mass Spectrometry / methods
  • Models, Genetic
  • Neovascularization, Physiologic / genetics
  • Peptide Fragments / pharmacology
  • Proteomics / methods
  • Rats
  • Rats, Sprague-Dawley
  • Reverse Transcriptase Polymerase Chain Reaction
  • Signal Transduction / drug effects
  • Signal Transduction / genetics*
  • Stem Cells / cytology
  • Stem Cells / metabolism*
  • Stress, Physiological / genetics
  • Vascular Endothelial Growth Factor A / genetics*
  • Vascular Endothelial Growth Factor A / metabolism
  • Vascular Endothelial Growth Factor A / pharmacology
  • Vascular Endothelial Growth Factor Receptor-2 / genetics
  • Vascular Endothelial Growth Factor Receptor-2 / metabolism


  • Peptide Fragments
  • Vascular Endothelial Growth Factor A
  • vascular endothelial growth factor A (138-165)
  • Vascular Endothelial Growth Factor Receptor-2