In vitro and in vivo Signaling Mechanisms during Endothelial Tube Formation

描述（由申请人提供）：我们的长期目标是了解内皮管形成过程中调节囊泡运输的细胞机制。我们的目标是确定参与顶膜形成的关键基因，以及内皮管形成过程中囊泡运输和细胞连接重塑之间的协调。我们的主要假设是内皮管的形成与粘附和紧密连接的重塑紧密耦合，这些顶端连接在体外和体内的顶端膜形成过程中指导囊泡运输。为了支持这一假设，我们已经确定了一些参与细胞连接和囊泡运输的蛋白质，并证明这些基因是培养的人脐静脉内皮细胞和斑马鱼胚胎血管生成过程中内皮管形成所必需的。我们选择用斑马鱼进行研究，是因为它具有与人类基因组相关的定义明确的基因组、有效敲除基因和产生转基因动物的能力，以及胚胎的光学清晰度，可以在体内单细胞水平上详细检查管的形成。作为这一方法的补充，我们在体外遵循同样的生化过程。我们提供的初步证据表明，囊泡运输和根尖交界处重塑密切相关。我们的团队在使用转座子介导的转基因技术，注射反义morpholino寡核苷酸，以及在体外检测蛋白质-蛋白质相互作用方面拥有丰富的经验。我们通过结合体外和体内实验，采取了创新和比较的方法来解决这个问题。我们使用表达荧光标记蛋白的转基因斑马鱼和人内皮细胞来回答有关内皮管形成的基本细胞生物学问题。在批准期结束时，我们将确定内皮管形成过程中起作用的新机制。这种理解对于开发新的治疗策略来调节内皮管的形成和直径是至关重要的。这种能力将对心脏病、缺血和癌症的治疗产生直接影响。

DESCRIPTION (provided by applicant): Our long-term goal is to understand the cellular mechanisms that regulate vesicle trafficking during endothelial tube formation. Our objective is to define critical genes involved in the formation of the apical membrane and the coordination between vesicle trafficking and the remodeling of cellular junctions during endothelial tube formation. Our principle hypothesis is that endothelial tube formation is tightly coupled to adherence and tight junction remodeling and that these apical junctions direct vesicle trafficking during formation of the apical membrane in vitro and in vivo. In support of this hypothesis, we have identified a number of proteins involved in cellular junctions and vesicle trafficking and demonstrated that these genes are required for endothelial tube formation in human umbilical vein endothelial cells in culture and during embryonic angiogenesis in zebrafish. We have chosen to work with zebrafish because of the combined advantages of a well-defined genome that relates to the human genome, the ability to efficiently knockdown genes and produce transgenic animals, and the optical clarity of the embryo that allows a detailed examination of tube formation on the single cell level in vivo. Complementary to this approach, we follow the same processes biochemically in vitro. We provide preliminary evidence suggesting a close coupling of vesicle trafficking and apical junctional remodeling. Our team has extensive experience with the generation of transgenic zebrafish using transposon-mediated transgenesis techniques, the injection of antisense morpholino oligonucleotides, and the examination of protein-protein interactions in vitro. We have taken an innovative and comparative approach to this problem by combining in vitro and in vivo experiments. We use transgenic zebrafish and human endothelial cells that express fluorescently tagged proteins to answer fundamental cell biological questions regarding endothelial tube formation. At the end of the granting period, we will have defined novel mechanisms that function during endothelial tube formation. This understanding is critical to the development of new therapeutic strategies to modulate of endothelial tube formation and diameter. This ability would have direct implications for the treatment of heart disease, ischemia, and cancer. PUBLIC HEALTH RELEVANCE: The creation of tubes by endothelial cells during angiogenesis and vasculogenesis is a critical step in the formation of functional blood vessels. Acquiring a more detailed understanding of the cellular and molecular pathways guiding this process has direct implications for the development of therapeutic interventions for the treatment of heart disease, ischemia, and cancer.