Molecular Determinants of Vertebrate Vasculature and Development

脊椎动物脉管系统和发育的分子决定因素

• 批准号: RGPIN-2017-04787
• 负责人: Majumder, Mousumi
• 金额: $ 1.89万
• 依托单位: Brandon University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=658541
• 关键词: Molecular; Determinants; Vertebrate; Vasculature; Development

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.

背景：脊椎动物的血管系统对血液循环、气体和代谢物交换、液体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生成，在出生后的生命中称为血管生成。血管生成参与组织再生、炎症和肿瘤的发病。血管生成的过程和参与血管生成的分子决定因素尚不清楚。Micro(Mi)RNAs是一类非编码短小的RNAs，它负性调节靶基因的表达，在胚胎发育、组织动态平衡和发病机制中发挥关键作用。前列腺素(PG)E2通过上调PGE2受体EP4的表达，促进炎症，调节干细胞，诱导炎症相关的miRNAs。PGE2介导的功能可通过阻断EP4的活性而被抑制。PGE2和miRNAs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：我的研究计划将找到新的分子决定因素，负责脊椎动物系统中的胚胎生长和血管生成。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。*短期目标：*目的1：研究PGE2诱导的miRNAs在体外的血管生成作用。HUVEC细胞将用PGE2刺激miRNA的表达。我们将在该细胞(HUVEC-miR)中过表达miRNA，以检测细胞的迁移、增殖和管状形成，这是血管生成所必需的三个功能。*目的2：探讨Ep4活性是否调节miRNA功能；我们将用Ep4激动剂刺激HUVEC细胞中的miRNA，并用Ep4拮抗剂抑制HUVEC-miR细胞中的miRNA功能。对EP4介导的信号转导的功能依赖(PI3K/AKT，ERK磷酸化)将建立miRNAs和EP4之间的联系。*目的3：(A)研究PGE2在斑马鱼胚胎发育和血管形成中的作用；我们将PGE2以剂量依赖的方式注射到鱼类胚胎中，并记录发育变化。我们将用胚胎提取物进行微阵列来识别miRNA的变化。(二)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。这项初步研究将发现在血管发生过程中受PGE2调控的新的miRNAs。*目标4：发现PGE2处理后斑马鱼基因表达的变化；我们将在载体和PGE2处理的鱼胚胎提取的mRNA中进行微阵列，以寻找可能参与脊椎动物发育的新基因。*意义：因此，该计划将在脊椎动物发育的目录中增加新的决定因素。鉴定PGE2和miRNA在血管生成中的生理作用；Ep4信号调节miRNA功能，因此，该计划将修改目前使用的针对Ep4的药物的进程。