Molecular Determinants of Vertebrate Vasculature and Development

背景：脊椎动物血管系统对血液循环、气体和代谢物交换、体液平衡和免疫细胞运输至关重要。胚胎中的血管形成称为血管发生，出生后称为血管生成。血管生成参与组织再生、炎症和肿瘤发病。血管发生的过程和参与血管发生的分子决定因素尚不清楚。微rna （mi）是一种短链非编码rna，其负向调控靶基因的表达，在胚胎发生、组织稳态和发病机制中发挥关键作用。前列腺素E2可促进炎症，通过上调PGE2受体EP4调控干细胞并诱导炎症相关mirna。阻断EP4活性可抑制PGE2介导的功能。PGE2和mirna在脊椎动物发育和血管生成中的生物学作用尚不清楚。***长期目标：我的研究项目将发现脊椎动物系统中负责胚胎生长和血管形成的新分子决定因素。我们将确定PGE2和miRNA在脊椎动物发育中的作用。我将使用人脐静脉内皮细胞（HUVEC）作为体外模型，斑马鱼作为体内模型。***近期目标：***目的1：研究PGE2诱导的mirna在体外血管生成中的作用。用PGE2处理HUVEC细胞以刺激miRNA表达。miRNA将在这种细胞（HUVEC-miR）中过表达，以测试细胞迁移、增殖和管形成，这是血管生成所需的三个功能。***目的2：研究EP4活性是否调控miRNA功能；我们将用EP4激动剂刺激HUVEC细胞的miRNA，用EP4拮抗剂抑制HUVEC- mir细胞的miRNA功能。功能依赖于EP4介导的信号（PI3K/AKT， ERK磷酸化）将建立mirna与EP4之间的联系。***目的3:(a)研究PGE2在斑马鱼胚胎发育和血管发生中的作用；我们将以剂量依赖的方式在鱼胚中注射PGE2，并记录其发育变化。我们将利用胚胎提取物进行微阵列检测miRNA的变化。(b)为了测试miRNA的功能，我们将在斑马鱼胚胎中敲低miRNA并测量生长变化。这项初步研究将确定在血管发生过程中由PGE2调节的新型mirna。***目的4：观察PGE2在斑马鱼体内的表达变化；我们将对载体和PGE2处理过的鱼胚胎提取的mRNA进行微阵列检测，以鉴定可能参与脊椎动物发育的新基因。***意义：因此，通过各种方法，该计划将在脊椎动物发育目录中添加新的决定因素。PGE2和miRNA在血管发生中的生理作用EP4信号调节miRNA功能，因此该程序将改变目前使用的靶向EP4的药物的进程。

Background: Vertebrate vascular system is vital for blood circulation, gas & metabolite exchange, fluid balance & immune cell trafficking. Vessel formation in embryos is called vasculogenesis, and in postnatal life, angiogenesis. Angiogenesis participates in tissue regeneration, inflammation and tumor pathogenesis. The process of vasculogenesis and the molecular determinants involved in vasculogenesis is not clear yet. Micro(mi)RNAs are short noncoding RNAs, which negatively regulate expression of target genes to play key roles in embryogenesis, tissue homeostasis, and pathogenesis. Prostaglandin(PG) E2 promotes inflammation, can regulate stem cells and induce inflammation associated miRNAs by upregulating EP4, a receptor for PGE2. PGE2 mediated functions can be inhibited by blocking EP4 activity. The biology of PGE2 and miRNAs in vertebrate development and angiogenesis are not known.***Long term goal: My research program will find novel molecular determinants responsible for embryonic growth and vasculogenesis in the vertebrate system. We shall determine the roles of PGE2 and miRNA in vertebrate development. I shall use Human Umbilical Vein Endothelial Cells (HUVEC) as in vitro model and Zebrafish as an in vivo model.***Short-term objectives:***Objective 1: To study angiogenic roles of PGE2 induced miRNAs in vitro. HUVEC cells will be treated with PGE2 to stimulate miRNA expression. miRNA will be overexpressed in this cells (HUVEC-miR) to test cell migration, proliferation, and tube formations, three functions require for angiogenesis.***Objective 2: To investigate if EP4 activity regulates miRNA functions; we shall stimulate miRNA in HUVEC cells with EP4 agonist and inhibit miRNA functions in HUVEC-miR cells with EP4 antagonist. Functional dependence on EP4 mediated signaling (PI3K/AKT, ERK phosphorylation) will establish the link between miRNAs and EP4.***Objective 3: (a) To study the role of the PGE2 in embryonic development and vasculogenesis in Zebrafish model; we shall inject PGE2 in fish embryo in a dose-dependent way and record developmental changes. We shall conduct microarrays with embryo extracts to identify miRNA changes. (b) To test miRNA functions, we shall knock down miRNAs in Zebrafish embryo and measure growth change. This pilot study will identify novel miRNAs regulated by PGE2 during vasculogenesis.***Objective 4: To find gene expression changes due to PGE2 treatment in Zebrafish; we shall conduct microarrays in Vehicle and PGE2 treated fish embryo extracted mRNA to identify novel genes might involve in vertebrate development.***Significance: With various approaches, this program will, therefore, add new determinants in the catalog of vertebrate development. Identification of the physiological roles of PGE2 and miRNA in vasculogenesis; EP4 signaling regulating miRNA functions, this program will, therefore, modify the course of currently used drugs targeting EP4.