Ras signaling directs endothelial specification of VEGFR2+ vascular progenitor cells

Abstract

Vascular endothelial growth factor receptor 2 (VEGFR2) transmits signals of crucial importance to vasculogenesis, including proliferation, migration, and differentiation of vascular progenitor cells. Embryonic stem cell–derived VEGFR2+ mesodermal cells differentiate into mural lineage in the presence of platelet derived growth factor (PDGF)–BB or serum but into endothelial lineage in response to VEGF-A. We found that inhibition of H-Ras function by a farnesyltransferase inhibitor or a knockdown technique results in selective suppression of VEGF-A–induced endothelial specification. Experiments with ex vivo whole-embryo culture as well as analysis of H-ras −/− mice also supported this conclusion. Furthermore, expression of a constitutively active H-Ras[G12V] in VEGFR2+ progenitor cells resulted in endothelial differentiation through the extracellular signal-related kinase (Erk) pathway. Both VEGF-A and PDGF-BB activated Ras in VEGFR2+ progenitor cells 5 min after treatment. However, VEGF-A, but not PDGF-BB, activated Ras 6–9 h after treatment, preceding the induction of endothelial markers. VEGF-A thus activates temporally distinct Ras–Erk signaling to direct endothelial specification of VEGFR2+ vascular progenitor cells.

Dissecting the Signal Transduction Pathway that Directs Endothelial Differentiation Using Embryonic Stem Cell-Derived Vascular Progenitor Cells

“…Vascular anomalies were found in the brain of 73% of H-ras -/-embryos examined at E9.5 although they contained similar numbers of somites as their WT littermates ( Fig. 10; Kawasaki et al, 2008). We next double-stained the cephalic region of the embryos for PECAM1 and VEGFR2, the earliest marker of endothelial cell differentiation.…”

“…In addition to the chimeric receptor approach described above, we also used various pharmacological inhibitors to study endothelial differentiation (Kawasaki et al, 2008), and we found that temporally regulated Ras signaling plays a crucial role in endothelial specification downstream of PLC1.…”

“…We used this system to perform gene silencing studies because we had difficulty introducing siRNA oligonucleotides to VEGFR2 + vascular progenitor cells; we were able to easily introduce them into ES cells or differentiated endothelial cells, however (Kawasaki et al, 2008). It is likely that differentiating ES cells may be resistant to transfection at certain stages of development.…”

“…We next performed an ex vivo whole-embryo culture assay to confirm the effects of FTI-277 on vascular development in a mouse embryo ( Fig. 9; Kawasaki et al, 2008). Concepti at embryonic day 6.75 (E 6.75) were picked out from the uteri of pregnant mice and cultured in vitro for three days, and, in the absence of further manipulation, PECAM1 + blood vessels formed in the yolk sac during this time.…”

“…The molecular basis for the observed signaling specificity remains unclear. To better understand this important process, we focused on signaling downstream of VEGFR2, a functional receptor for VEGF-A, using embryonic stem cellderived VEGFR2 + vascular progenitor cells (Suzuki et al, 2005;Kawasaki et al, 2008;Sase et al, 2009). VEGFR2-null mice demonstrate severe defects in vasculogenesis, and VEGFR2 signaling is thought to play crucial roles in embryonic blood vessel formation (Shalaby et al, 1995;Shalaby et al, 1997; see below for details).…”