Alk1 regulation of embryonic angiogenesis

Alk1 调控胚胎血管生成

• 批准号: 7305240
• 负责人: BETH L ROMAN
• 金额: $ 36.25万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-04-01 至 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7305240
• 关键词: angiogenesis; biological signal transduction; blood circulation; confocal scanning microscopy; embryology; gene expression; gene expression profiling; growth factor receptors; immunocytochemistry; shear stress; telangiectasia; tissue /cell culture; transforming growth factors; zebrafish

DESCRIPTION (provided by applicant): Homozygous inactivation of the endothelial-specific TGFB type I receptor, Alk1, results in embryonic lethality stemming from vascular malformations, and heterozygous inactivation results in a potentially lethal human vascular dysplasia, hereditary hemorrhagic telangiectasia type 2 (HHT2). Clearly, AIk1 plays a necessary role in building and maintaining the vertebrate vasculature; however, the nature of that role is currently unclear. The objective of this proposal is to use the accessible zebrafish embryo, which has a well described, stereotypical vertebrate vasculature, to define the effects of Alkl activation on endothelial cell functional state and transcript expression. Specific Aim 1 comprises experiments designed to test the hypothesis that TGFB signaling through Alk1 is required for angiogenic resolution. Studies in support of this Aim will include time lapse confocal analysis of endothelial sprouting and proliferation in violet beauregarde (vbg) mutants, which harbor a mutation in alk1; and assessment of endothelial migration, proliferation, sprouting, and basement membrane formation in zebrafish embryos overexpressing dominant negative and/or constitutively active forms of alk1, alk5 (the canonical TGFB type I receptor that reportedly competes with Alk1 in determining endothelial state), and tgfB within the endothelium. Specific Aim 2 comprises experiments designed to test the hypothesis that Alk1 is induced by shear stress and plays a role in vascular remodeling. The effect of blood flow on alk1 expression and remodeling of cranial vessels in zebrafish embryos will be determined, and the effect of shear stress on Alk1expression in cultured mammalian cells will be assessed. Further experiments will identify molecular components of the pathway that translates mechanical shear stress into Alk1 induction. Specific Aim 3 comprises experiments designed to identify downstream components of Alk1 signaling. Expression levels of genes reportedly regulated by constitutively active Alk1 in mammalian cell culture will be compared in vbg mutants and wild type siblings, and novel alkl-regulated genes will be sought by comparing the endothelial transcriptome in vbg mutants and wild type siblings. Taken together, the proposed experiments will determine whether Alk1 plays a role in angiogenic activation, resolution, and/or remodeling, and, with the definition of downstream targets, will promote a better understanding of the molecular pathways of normal vessel development and HHT2 pathogenesis.

描述（由申请人提供）：内皮特异性TGFB I型受体Alk1的纯合失活导致血管畸形引起的胚胎致死，杂合失活导致潜在致命的人类血管发育不良，遗传性出血性毛细血管扩张2型（ht2）。显然，AIk1在脊椎动物血管系统的建立和维持中起着必要的作用；然而，这一角色的性质目前尚不清楚。本提案的目的是利用具有良好描述的典型脊椎动物脉管系统的可接近的斑马鱼胚胎来确定Alkl激活对内皮细胞功能状态和转录物表达的影响。特异性目的1包括旨在验证通过Alk1的TGFB信号传导是血管生成分辨率所必需的假设的实验。支持这一目标的研究将包括对含有alk1突变的紫beauregarde （vbg）突变体内皮细胞发芽和增殖的时间推移共聚焦分析；以及评估内皮迁移、增殖、发芽和基底膜形成在斑马鱼胚胎中过度表达显性阴性和/或构成活性形式的alk1、alk5（典型的TGFB I型受体，据报道与alk1竞争决定内皮状态）和内皮内的TGFB。具体目标2包括实验，旨在验证Alk1由剪切应力诱导并在血管重塑中发挥作用的假设。研究将确定血流对斑马鱼胚胎中alk1表达和颅血管重构的影响，并评估剪切应力对培养的哺乳动物细胞中alk1表达的影响。进一步的实验将确定将机械剪切应力转化为Alk1诱导的途径的分子成分。具体目标3包括旨在识别Alk1信号传导下游组分的实验。据报道，在哺乳动物细胞培养中，由组成型活性Alk1调节的基因的表达水平将在vbg突变体和野生型兄弟姐妹中进行比较，并且将通过比较vbg突变体和野生型兄弟姐妹的内皮转录组来寻找新的Alk1调节基因。综上所述，这些实验将确定Alk1是否在血管生成激活、分解和/或重塑中发挥作用，并随着下游靶点的定义，将促进对正常血管发育和HHT2发病机制的分子途径的更好理解。