Molecular Determinants of Vertebrate Vasculature and Development

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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.