Cellular and molecular mechanisms in Prostaglandin E2 mediated lymphangiogenesis

背景：我们发现乳腺癌组织中COX-2的表达是通过激活癌细胞和巨噬细胞(Mf)和淋巴管内皮细胞(LEC)上的PGE受体Ep4，从而促进淋巴管生成，从而促进淋巴管生成。*假设：PGE2是一种生理性的淋巴管生成促进剂，通过激活LECs和MF上的Ep4，原位上调VEGF-C/D的生成。*目的：(1)体外研究PGE2介导的淋巴管生成的细胞/分子过程；(2)明确Ep4在体外刺激淋巴管生成中的作用；(3)在体内确定PGE2介导的淋巴管生成的细胞/分子伙伴。*实验计划：*目的1.体内淋巴萌发涉及微血管LEC的增殖、迁移和管状形成，这是可以在体外测量的。我们将使用LEC检测VEGF-R-3的表达(qPCR检测mRNA，Western印迹检测蛋白质)；VEGF-C/D产量(qPCR检测mRNA，EL ISA检测蛋白质)；增殖(BrdU摄取，8-16 h)；通过微孔(8µm孔)膜迁移(24 H)；在生长因子抑制的基质细胞上形成毛细管(12-24 h)。每个功能将使用12小时血清饥饿的LEC+/-PGE2或EP4激动剂或血管内皮生长因子-C(+VE对照)。所有功能的EP4依赖性将在LEC中用EP4拮抗剂或EP4基因敲除来测试。我们将用EP4激动剂和特定的通路抑制剂来测试这些功能对EP4信号的依赖性(PI3K/AKT，MAPK磷酸化)。目的2.测定(A)在有MF细胞系或其无细胞培养上清液存在的情况下，LEC的小管形成，以及(B)用Ep4拮抗剂或Ep4基因敲除MF对MF产生的VEGF-C/D和LEC的管状形成的影响。*目的3.在活体淋巴管生成试验中的应用：在裸鼠背部皮肤下植入含有Matrigel和PGE2或EP4激动剂或VEGF-C(+Ve)对照的血管反应物，9d后通过三种方法测量淋巴管生成：(A)标记的LYVE-1蛋白的免疫荧光法，(B)LYVE-1mRNA的定量聚合酶链式反应和(C)双重免疫标记的冷冻切片淋巴管计数。我们还将通过F4/80(MF标记)和VEGF-C/D的双重标记来原位定量产生VEGF-C/D的MF。用Ep4拮抗剂治疗小鼠将测试有助于体内淋巴管生成的细胞对Ep4的依赖性。*意义：通过生理调节器PGE2了解淋巴管发育的基本机制**

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**