Tie Tek Modulation of Cardiac Development

心脏发育的 Tie Tek 调节

• 批准号: 8666044
• 负责人: H Scott Baldwin
• 金额: $ 38.22万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-06-01 至 2018-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8666044
• 关键词: Affect; Alanine; Angiopoietin-2; Angiopoietins; Animals; Aorta; Atherosclerosis; Attenuated; Bioinformatics; Cardiac; Cardiovascular system; Cell Line; Cell Proliferation; Cell physiology; Cell surface; Cells; Complementary DNA; Data; Defect; Deletion Mutation; Development; Dorsal; EGF gene; Embryo; Endocardium; Endothelial Cells; Event; Extracellular Domain; Foundations; Growth; Heart; Immunoglobulins; In Vitro; Inflammatory Response; Knock-in Mouse; Knowledge; Ligands; LoxP-flanked allele; Lymphatic; Lysine; Mediating; Morphogenesis; Mus; Phenotype; Phenylalanine; Phosphorylation; Play; Point Mutation; Protein Tyrosine Kinase; RNA; Receptor Protein-Tyrosine Kinases; Role; Series; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Staging; Stream; Stretching; TIE-2 Receptor; Time; Tyrosine; Tyrosine Kinase Domain; Vascular Endothelial Growth Factor Receptor; angiogenesis; aortic valve; attenuation; cardiogenesis; congenital heart disorder; genetic analysis; hemodynamics; in utero; in vivo; migration; mutant; novel; novel therapeutics; public health relevance; receptor; recombinase; research study; response to injury; semilunar valve; shear stress; transcriptome sequencing

DESCRIPTION (provided by applicant): Tie1 and Tek (also known as Tie2) are endothelial protein receptor tyrosine kinases (RTKs). Along with the vascular endothelial growth factor (VEGF) receptor, these are the only known endothelial cell-specific RTKs. Due to embryonic lethality of panendotheilal deletion of either Tie 1 or Tie2 the exact roles and mechanisms of Tie1 or Tie2 in regulating cardiac development has not been clearly delineated. Furthermore the ligand(s) for Tie1 or the targets of Tie1 activation have not been identified. We hypothesize that in the cardiovascular system Tie1 is not only an inhibitory co-receptor for Tie2 activation, but also capable of signaling autonomously of Tie2. We therefore propose to: 1) Determine the unique functions of Tie1 and Tie2 signaling during early and late stages of cardiac development in vivo: Recently generated mice harboring a floxed allele of Tie1 or Tie2 will be used in conjunction with a novel early endocardial specific (Nfatc1Cre) or a later valvular endocardial Cre line (Nfatc1enCre) to produce stage specific cardiac deletion of Tie1 and Tie2 throughout the continuum of heart development in utero. Morphological and functional analysis will be used to characterize the unique phenotype that results from cardiac specific RTK attenuation. 2) Define the specific Tie1-Tie2 interactions that are required for normal cardiac morphogenesis at both early and late stages of heart formation in vivo. Recombinase mediated cassette exchange (RMCE) will be used to evaluate potential critical domains of Tie1-Tie2 interaction specifically in the endocardium. We will generate a series of cDNA "knock-in" mice lacking the extracellular domain (ECD), intracellular domain (ICD) as well as point mutations in critical tyrosine kinase domains in the cytoplasmic region of Tie1 (tyrosine to phenylalanine/Y1113F and lysine to alanine/K866A). These animals will be crossed to endocardial specific Cre lines and evaluated for specific defects in cardiac development. 3) Delineate down-stream targets of Tie1 activation in the cardiovascular system. In the absence of a known ligand for Tie1 activation, hemodynamic activation of Tie1 signaling will be used to evaluate the effect of defined deletion / mutations of Tie1. Immortalized endocardial cell lines derived from the conditional Tie1 mutants will be exposed to altered shear stress, turbulence and stretch and evaluated for alterations in previously described signal transduction pathways attributed to Tie activation. The most informative Tie1 mutant animals will be used for RNA-Seq and bioinformatic analysis for construction of signaling networks that will define a unique singling signature for Tie1 and help delineate endothelial cell autonomous and non-autonomous signaling pathways in the heart.

描述（由申请人提供）：Tie1 和 Tek（也称为 Tie2）是内皮蛋白受体酪氨酸激酶（RTK）。与血管内皮生长因子 (VEGF) 受体一起，这些是唯一已知的内皮细胞特异性 RTK。由于 Tie 1 或 Tie2 的全内皮缺失的胚胎致死性，Tie1 或 Tie2 在调节心脏发育中的确切作用和机制尚未明确描述。此外，Tie1 的配体或 Tie1 激活的靶标尚未确定。我们假设在心血管系统中，Tie1 不仅是 Tie2 激活的抑制性共受体，而且还能够自主发出 Tie2 信号。因此，我们建议：1）确定Tie1和Tie2信号在体内心脏发育早期和晚期阶段的独特功能：最近产生的携带Tie1或Tie2 floxed等位基因的小鼠将与新型早期心内膜特异性（Nfatc1Cre）或晚期瓣膜心内膜Cre线（Nfatc1enCre）结合使用，以产生Tie1和Tie2的阶段特异性心脏缺失 贯穿心脏在子宫内发育的整个过程。形态学和功能分析将用于表征心脏特异性 RTK 衰减产生的独特表型。 2) 定义体内心脏形成早期和晚期正常心脏形态发生所需的特定 Tie1-Tie2 相互作用。重组酶介导的盒式交换 (RMCE) 将用于评估 Tie1-Tie2 相互作用的潜在关键域，特别是在 心内膜。我们将生成一系列 cDNA“敲入”小鼠，它们缺乏胞外结构域 (ECD)、胞内结构域 (ICD) 以及 Tie1 细胞质区域中关键酪氨酸激酶结构域的点突变（酪氨酸变为苯丙氨酸/Y1113F 和赖氨酸变为丙氨酸/K866A）。这些动物将与心内膜特定的 Cre 系杂交，并评估心脏发育的特定缺陷。 3) 描绘心血管系统中 Tie1 激活的下游目标。在缺乏 Tie1 激活的已知配体的情况下，Tie1 信号传导的血流动力学激活将用于评估 Tie1 定义的删除/突变的效果。来自条件 Tie1 突变体的永生化心内膜细胞系将暴露于改变的剪切应力、湍流和拉伸，并评估先前描述的归因于 Tie 激活的信号转导途径的改变。信息最丰富的 Tie1 突变动物将用于 RNA 测序和生物信息学分析，以构建信号网络，该网络将定义 Tie1 独特的单信号签名，并帮助描绘心脏内皮细胞自主和非自主信号通路。