The Role of Endoglin in Hemangioblast Development

内皮糖蛋白在成血管细胞发育中的作用

• 批准号: 7885323
• 负责人: Rita C. R. Perlingeiro
• 金额: $ 37.75万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-05-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7885323
• 关键词: Adopted; Anemia; Blood; Blood flow; Cell Fraction; Cells; Commit; Complement; Development; Embryo; Endoglin; Erythropoiesis; Event; Frequencies; Gene Expression; Grant; Hematological Disease; Hematopoiesis; Hematopoietic; In Vitro; Injection of therapeutic agent; Knock-out; Knowledge; Maps; Mesoderm; Mesoderm Cell; Modeling; Phenotype; Play; Proteins; Regenerative Medicine; Research Personnel; Role; Signal Pathway; Signal Transduction; Surface Antigens; Testing; Transforming Growth Factor beta; base; blastocyst; embryonic stem cell; in vitro Assay; in vivo; member; overexpression; progenitor; programs; receptor; research study; stem

DESCRIPTION (provided by applicant): This grant aims toward an understanding of the events involved in lineage determination as pluripotent cells commit to hematopoietic and endothelial fates. This knowledge is fundamental to the development of regenerative medicine based on embryonic stem (ES) cells for the treatment of diseases of hematologic and endothelial origin. Our current model postulates that the hematopoietic and endothelial lineages arise in mesoderm from a common bipotent progenitor - the hemangioblast. Although an in vitro assay exists for the hemangioblast, very little is known about what enables some mesodermal cells to adopt this fate while others do not, and how the hemangioblast progeny select between endothelial or hematopoietic fates. In our search for hemangioblast regulatory molecules, we have discovered that endoglin (eng), an ancillary receptor for several members of the TGF-beta superfamily, plays a critical role in hemangioblast specification. Our preliminary studies using ES cells differentiated in vitro demonstrate a profound reduction in hemangioblast frequency in the absence of endoglin. Furthermore, we show that endoglin marks the hemangioblast on day 3 of embryoid body (EB) differentiation. To date the only cell surface antigen known to mark the hemangioblast is Flk-1. Our pilot results point to a role for endoglin in hemangioblast specification as well as provide evidence that endoglin is required for hematopoietic commitment but not endothelial. In this study, we propose to dissect the mechanisms by which endoglin regulates hemangioblast specification as well as to understand its role in hematopoietic and endothelial commitment. In Aim 1, we propose to use inducible gene expression to probe the function of endoglin in temporally defined windows to evaluate its role in promoting hemangioblast specification, and hematopoietic or endothelial lineage selection. To elucidate the mechanism associated with endoglin's function in hemangioblast development, in Aim 2, we will investigate biochemically the cascade of components associated with TGF-beta signaling, including Smads and upstream receptors, in cell fractions highly enriched for hemangioblast precursors isolated from eng -/- as well as endoglin overexpressing and control cells from day 3 EBs. These results will be complemented genetically by rescuing the eng -/- phenotype with appropriate activated proteins. In Aim 3, we will validate our in vitro results by studying the role of endoglin in vivo. We hypothesize that the anemia observed in eng -/- embryos is not a secondary effect due to reduced blood flow but a result of defective erythropoiesis, which is associated with improper hemangioblast development.

描述（由申请人提供）：这项资助旨在了解多能细胞决定造血和内皮细胞命运时涉及谱系决定的事件。这些知识对于基于胚胎干 (ES) 细胞治疗血液和内皮源性疾病的再生医学的发展至关重要。我们目前的模型假设造血和内皮谱系在中胚层中起源于共同的双能祖细胞——成血管细胞。尽管存在针对成血管细胞的体外测定，但对于是什么使一些中胚层细胞采取这种命运而其他细胞不采取这种命运，以及成血管细胞后代如何在内皮或造血命运之间进行选择，人们知之甚少。在寻找成血管细胞调节分子的过程中，我们发现内皮糖蛋白 (eng) 是 TGF-β 超家族几个成员的辅助受体，在成血管细胞规范中发挥着关键作用。我们使用体外分化的 ES 细胞进行的初步研究表明，在缺乏内皮糖蛋白的情况下，成血管细胞频率显着降低。此外，我们发现内皮糖蛋白在胚状体（EB）分化的第3天标记成血管细胞。迄今为止，唯一已知标记成血管细胞的细胞表面抗原是 Flk-1。我们的初步结果指出了内皮糖蛋白在成血管细胞规范中的作用，并提供了内皮糖蛋白是造血承诺所必需的证据，但内皮细胞不是必需的。在这项研究中，我们打算剖析内皮糖蛋白调节成血管细胞规范的机制，并了解其在造血和内皮细胞定向中的作用。在目标 1 中，我们建议使用诱导型基因表达来探测内皮糖蛋白在时间定义的窗口中的功能，以评估其在促进成血管细胞规范以及造血或内皮谱系选择中的作用。为了阐明与内皮糖蛋白在成血管细胞发育中的功能相关的机制，在目标 2 中，我们将从生化角度研究与 TGF-β 信号传导相关的成分的级联，包括 Smad 和上游受体，这些成分在高度富集从 eng -/- 分离的成血管细胞前体的细胞级分中，以及来自第 3 天 EB 的内皮糖蛋白过表达和对照细胞。这些结果将通过用适当的活化蛋白拯救 eng -/- 表型得到遗传补充。在目标 3 中，我们将通过研究内皮糖蛋白在体内的作用来验证我们的体外结果。我们假设在 eng -/- 胚胎中观察到的贫血不是由于血流量减少而引起的继发效应，而是红细胞生成缺陷的结果，这与成血成血管细胞发育不当有关。