Cellular and molecular mechanisms in Prostaglandin E2 mediated lymphangiogenesis

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Background: We discovered that COX-2 expression in breast cancer promotes lymphangiogenesis due to PGE2-stimulated VEGF-C/D production by cancer cells and macrophages (mf), following activation of the PGE receptor EP4 on these cells and lymphatic endothelial cells (LEC).****Hypothesis: PGE2 is a physiological promoter of lymphangiogenesis caused by activation of EP4 on LECs and mf to upregulate VEGF-C/D production in situ.****Objectives: (1) Dissect cellular/molecular processes in PGE2-mediated lymphangiogenesis using LECs in vitro; (2) Identify the role of EP4 on mf in stimulating lymphangiogenesis in vitro; (3) Identify cellular/molecular partners in PGE2-mediated lymphangiogenesis in vivo. ****Experimental plans: ***Aim 1.Lymphatic sprouting in vivo involves proliferation, migration and tube formation by the micro-vascular LEC that can be measured in vitro. We shall use LEC to measure VEGF-R-3 expression (mRNA with qPCR, protein with western blot); VEGF-C/D production (qPCR for mRNA, ELISA for protein); Proliferation (BrdU uptake , 8-16 h); Migration (24 h) through micro-porous (8 µm pores) membranes; Capillary-like tube formation (12 -24 h) on growth factor-reduced matrigel. Each function will employ 12 hr serum-starved LEC +/- PGE2 or EP4 agonists or VEGF-C (+ve control). EP4 dependence of all functions will be tested with an EP4 antagonist or EP4 knockdown in the LEC. Dependence of the functions on EP4 signaling (PI3k/AKT, MAPK phosphorylation) will be tested with EP4 agonists and specific pathway inhibitors. ****Aim 2. Measure (a) tubulogenesis by the LEC in the presence of a mf cell line or its cell-free supernatants, and (b) the effects of pre-treating mf with EP4 antagonists or EP4 knockdown of mf on VEGF-C/D production by mf and tube formation by the LEC. ****Aim 3. Employ Directed In Vivo Lymphangiogenesis Assay: implant angio-reactors containing matrigel with or without PGE2, or an EP4 agonist or VEGF-C (+ve control) under the dorsal skin in nude mice and 9d later measure lymphangiogenesis by 3 approaches: (a) immunoflourescence of labeled LYVE-1 protein, (b) qPCR for LYVE-1 mRNA and (c) direct counting of lymphatic vessels in cryo-sections dual immuno-labeled for VEGF-C/D and LEC markers LYVE-1 or PROX-1 or podoplanin. We shall also quantify VEGF-C/D-producing mf in situ by double-labeling for F4/80(mf marker) & VEGF-C/D. Treating mice with an EP4 antagonist will test EP4 dependence of the cells contributing to lymphangiogenesis in vivo.***Significance: Understanding basic mechanisms in lymphatic development by a physiological regulator PGE2**