Molecular Determinants of Vertebrate Vasculature and Development

背景：脊椎动物的血管系统对血液循环、气体生成至关重要。前列腺素（PG）E2促进炎症，可以调节干细胞，并通过上调PGE 2的受体EP 4诱导炎症相关的miRNA。PGE 2介导的功能可以通过阻断EP 4活性来抑制。PGE 2和miRNA在脊椎动物发育和血管生成中的生物学尚不清楚。长期目标：我的研究计划将发现负责脊椎动物系统中胚胎生长和血管发生的新分子决定因素。我们将确定PGE 2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：研究前列腺素E2（PGE 2）诱导的miRNAs在体外的血管生成作用。HUVEC细胞将用PGE 2处理以刺激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种功能。目标二：为了研究EP 4活性是否调节miRNA功能;我们将用EP 4激动剂刺激HUVEC细胞中的miRNA，并用EP 4拮抗剂抑制HUVEC-miR细胞中的miRNA功能。对EP 4介导的信号传导（PI 3 K/AKT、ERK磷酸化）的功能依赖性将建立miRNA与EP 4之间的联系。目标三：（a）研究PGE 2在斑马鱼胚胎发育和血管发生中的作用，以剂量依赖的方式将PGE 2注射到斑马鱼胚胎中，并记录其发育变化。我们将使用胚胎提取物进行微阵列以鉴定miRNA的变化。(b)为了测试miRNA的功能，我们将敲除斑马鱼胚胎中的miRNA并测量生长变化。这项初步研究将鉴定在血管发生过程中由PGE 2调节的新型miRNA。目标4：为了发现斑马鱼中由于PGE 2处理引起的基因表达变化;我们将在载体和PGE 2处理的鱼胚胎提取的mRNA中进行微阵列，以鉴定可能涉及脊椎动物发育的新基因。意义：通过各种方法，该计划将在脊椎动物发育的目录中添加新的决定因素。鉴定PGE 2和miRNA在血管发生中的生理作用; EP 4信号调节miRNA功能，因此，该计划将修改目前使用的靶向EP 4的药物的过程。

Background: Vertebrate vascular system is vital for blood circulation, gas ogenesis. 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.