| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2006: ¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 2005: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Research Abstract |
VEGFR2 is considered to play an essential role in differentiation of endothelial cells from vascular progenitor cells : it is expressed in haemangioblasts at the early stage of vasculogenesis, and VEGFR2-knockout mice are embryonic lethal with a phenotype lacking endothelial as well as haematopoietic cells. Signal transduction pathways that direct endothelial differentiation are, however, not well understood.
Mouse embryonic stem cell (ESC)-derived VEGFR2^+ cells are capable of differentiating into αSMA^+ mural cells and PECAM1^+ endothelial cells. VEGF-A stimulation induces differentiation of ESC-derived VEGFR2^+ cells into endothelial cells. We first searched for low molecular weight compounds that specifically inhibit VEGF-A-induced endothelial differentiation using this in vitro system. We found that FTI-277, a farnesyltransferase inhibitor, supperssed appearance of PECAM1^+ endothelial cells in response to VEGF-A. In single cell-derived colony formation assay, treatment with FTI-27
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7 did not affect total colony number, but reduced the ratio of PECAM1^+ colonies. These findings suggest that FTI-277 specifically inhibit differentiation of ESC-derived VEGFR2^+ cells into endothelial cells. We next established ES cell lines in which H-Ras is inducibly knocked down using Tet-off system, because H-Ras is one of the principal targets of FTI-277. When H-Ras was knocked down, VEGF-A-induced endothelial differentiation was significantly suppressed.
We also established ES cell lines in which H-Ras[G12V], a constitutively active from of H-Ras, can be inducibly expressed. When H-Ras[G12V] was expressed in ESC-derived VEGFR2^+ cells, they differentiated into PECAM1^+ cells even in the absence of VEGF^A. H-Ras[G12V]-induced PECAM1^+ cells were positive for CD34, endoglin, acetyl-LDL incorporation, and formed tubule-like structure in three dimensional culture in type I collagen gel. In colony formation assay, H-Ras[G12V] caused induction of PECAM1^+ endothelial colonies at the expense of αSMA^+ mural colonies. Our findings suggest the important role of Ras signaling in specification of endothelial lineage from vascular progenitor cells. Less
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