Molecular basis of the signaling networks that regulate vasculogenesis

VEGFR 2被认为在内皮细胞从血管祖细胞分化中起重要作用：它在血管发生的早期阶段在成血管细胞中表达，并且VEGFR 2敲除小鼠是胚胎致死的，具有缺乏内皮细胞以及造血细胞的表型。小鼠胚胎干细胞（ESC）来源的VEGFR 2 ^+细胞能够分化为αSMA^+壁细胞和PECAM 1 ^+内皮细胞。VEGF-A刺激诱导ESC来源的VEGFR 2 ^+细胞分化为内皮细胞。我们首先使用该体外系统寻找特异性抑制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）可抑制PECAM 1 ^+内皮细胞对VEGF-A的反应。在单细胞来源的集落形成试验中，用FTI-27处理 关于我们 7不影响菌落总数，但降低PECAM 1 ^+菌落的比例。这些发现表明FTI-277特异性抑制ESC衍生的VEGFR 2 ^+细胞向内皮细胞的分化。我们接下来建立了ES细胞系，其中H-Ras使用Tet-off系统被诱导敲低，因为H-Ras是FTI-277的主要靶标之一。当H-Ras被敲低时，VEGF-A诱导的内皮分化被显著抑制。我们还建立了H-Ras[G12 V]，一种组成型活性形式的H-Ras，可以诱导表达的ES细胞系。当H-Ras[G12 V]在ESC来源的VEGFR 2 ^+细胞中表达时，即使在不存在VEGF^A的情况下，它们也分化为PECAM 1 ^+细胞。H-Ras[G12 V]诱导的PECAM 1 ^+细胞表达CD 34、endoglin和乙酰化低密度脂蛋白（acetyl-LDL），并在I型胶原凝胶中三维培养形成管状结构。在集落形成试验中，H-Ras[G12 V]诱导PECAM 1 ^+内皮集落形成，而αSMA^+壁集落形成减少。我们的研究结果表明，Ras信号在血管祖细胞的内皮细胞系的规格的重要作用。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