Molecular basis of the signaling networks that regulate vasculogenesis

VEGFR2被认为在内皮细胞从血管祖细胞分化的过程中起着至关重要的作用：它在血管形成的早期阶段在血管母细胞中表达，VEGFR2敲除小鼠是胚胎致死性的，表型缺乏内皮细胞和造血细胞。然而，直接内皮细胞分化的信号转导通路尚不清楚。小鼠胚胎干细胞（ESC）来源的VEGFR2^+细胞能够分化为αSMA^+壁细胞和PECAM1^+内皮细胞。VEGF-A刺激诱导内皮细胞来源的VEGFR2^+细胞向内皮细胞分化。我们首先在体外系统中寻找特异性抑制vegf - a诱导的内皮分化的低分子量化合物。我们发现FTI-277，一种法尼基转移酶抑制剂，抑制PECAM1^+内皮细胞对VEGF-A的反应。在单细胞衍生集落形成实验中，fti - 27,7 +处理不影响总集落数，但降低了PECAM1^+集落的比例。这些发现表明，FTI-277特异性抑制内皮细胞来源的VEGFR2^+细胞向内皮细胞的分化。由于H-Ras是FTI-277的主要靶点之一，我们接下来建立了使用et-off系统诱导敲除H-Ras的ES细胞系。当H-Ras基因被敲低时，vegf - a诱导的内皮分化明显受到抑制。我们还建立了可诱导表达H-Ras[G12V]的ES细胞系。当H-Ras[G12V]在esc来源的VEGFR2^+细胞中表达时，即使没有VEGF^A，它们也会分化为PECAM1^+细胞。H-Ras[G12V]诱导的PECAM1^+细胞CD34、内啡肽、乙酰- ldl掺入阳性，在I型胶原凝胶中三维培养形成小管样结构。在集落形成实验中，H-Ras[G12V]诱导了PECAM1^+内皮集落，而αSMA^+壁上集落则被破坏。我们的研究结果表明Ras信号在血管祖细胞内皮谱系的指定中起重要作用。少

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 … More 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