Molecular basis of the signaling networks that regulate vasculogenesis

调节血管发生的信号网络的分子基础

基本信息

批准号：
17570106
负责人：
MIYAZAWA Keiji
金额：
$ 2.24万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17570106/
关键词：
endothelial cells mural cells growth factor differentiation

项目摘要

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

VEGFR2被认为在分化内皮细胞与血管祖细胞的分化中起着至关重要的作用：在血管生成的早期阶段，它在血管细胞中表达，而vegfr2 knockout小鼠是胚胎致死的，是缺乏内皮细胞和赤型止血细胞的表型。但是，直接内皮分化的信号转移途径尚不清楚。小鼠胚胎干细胞（ESC）衍生的VEGFR2^+细胞能够区分为αSMA^+壁细胞细胞和PECAM1^+内皮细胞。 VEGF-A模拟诱导ESC衍生的VEGFR2^+细胞分化为内皮细胞。我们首先搜索了使用此体外系统专门抑制VEGF-A诱导的内皮分化的低分子量化合物。我们发现FTI-277是一种Farneylsylansferase抑制剂，响应VEGF-A的PECAM1^+内皮细胞的出现。在单细胞衍生的菌落形成测定中，FTI-27的处理……更多的7不会影响总菌落数，而是降低了PECAM1^+菌落的比率。这些发现表明，FTI-277特别抑制了ESC衍生的VEGFR2^+细胞分化为内皮细胞。接下来，我们建立了ES细胞系，其中H-RAS使用TET-OFF系统击倒H-RAS，因为H-RAS是FTI-277的主要靶标之一。当H-RAS被击倒时，VEGF-A诱导的内皮分化得到了显着抑制。我们还建立了ES细胞系，其中H-RAS [G12V]（G12V]（来自H-RAS的组成型活性）可以诱导地表达。当H-Ras [G12V]在ESC衍生的VEGFR2^+细胞中表达时，即使在没有VEGF^a的情况下，它们也会分化为PECAM1^+细胞。 H-RAS [G12V]诱导的PECAM1^+细胞对CD34，内og，乙酰LDL掺入呈阳性，并在I型胶原蛋白凝胶中形成三维培养的管状结构。在菌落形成测定中，H-RAS [G12V]引起PECAM1^+内皮菌落的诱导，而牺牲了αSMA^+壁画菌落。我们的发现表明，RAS信号传导在血管祖细胞的内皮谱系规范中的重要作用。较少的