Tie Tek Modulation of Cardiac Development

心脏发育的 Tie Tek 调节

基本信息

批准号：
9066778
负责人：
H Scott Baldwin
金额：
$ 45.26万
依托单位：
VANDERBILT UNIVERSITY MEDICAL CENTER
依托单位国家：
美国
项目类别：
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/9066778
关键词：
Affect Alanine Angiopoietin-2 Angiopoietins Animals Aorta Atherosclerosis Attenuated Bioinformatics Cardiac Cardiac development 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 Endothelial Growth Factors Receptor Event Extracellular Domain Foundations Growth Health 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 Factors angiogenesis aortic valve attenuation cardiogenesis congenital heart disorder genetic analysis hemodynamics in utero in vivo migration mutant novel novel therapeutic intervention receptor recombinase-mediated cassette exchange research study response to injury semilunar valve shear stress transcriptome sequencing

项目摘要

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（RTKS）。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Panendotheilal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清楚地描述。此外，尚未确定TIE1的配体或TIE1激活的靶标。我们假设在心血管系统中，Tie1不仅是抑制性tie2激活的抑制性共受体，而且能够自主的TIE2发出信号。 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在整个子宫内心脏发展的连续性中。形态学和功能分析将用于表征由心脏特异性RTK衰减引起的独特表型。 2）定义在体内心脏形成的早期和晚期阶段正常心脏形态发生所需的特定TIE1-TIE2相互作用。重组酶介导的盒式磁带交换（RMCE）将用于评估TIE1-TIE2相互作用的潜在关键域心内膜。我们将产生一系列缺乏细胞外结构域（ECD），细胞内结构域（ICD）以及临界酪氨酸激酶结构域的点突变的cDNA“敲门”小鼠（酪氨酸至苯丙氨酸/Y1113F，Y1113F和裂解至丙氨酸/K8666a）的临界酪氨酸激酶结构域中的点突变。这些动物将越过心内膜特异性CRE系，并评估心脏发育中的特定缺陷。 3）描述心血管系统中TIE1激活的下游靶标。在没有已知的TIE1激活配体的情况下，将使用TIE1信号的血液动力学激活来评估TIE1的定义缺失 /突变的效果。永生化的心内膜细胞系将暴露于剪切应力，湍流和拉伸的改变，并评估了先前描述的信号转导途径的变化，归因于绑带激活。最有用的TIE1突变动物将用于RNA-seq和生物信息学分析，用于构建信号网络，该网络将定义TIE1的独特单签名，并有助于划定心脏中心脏中的内皮细胞自主和非自主信号传导途径。