Cellular and molecular mechanisms in Prostaglandin E2 mediated lymphangiogenesis

背景：我们发现乳腺癌中COX-2的表达促进淋巴管生成，这是由于pge2刺激癌细胞和巨噬细胞（mf）产生VEGF-C/D，在这些细胞和淋巴内皮细胞（LEC）上激活PGE受体EP4。****假设：PGE2是一种生理性的淋巴管生成启动子，通过激活lec和mf上的EP4来原位上调VEGF-C/D的生成。****目的：(1)利用LECs体外解剖pge2介导的淋巴管生成的细胞/分子过程；(2)确定EP4对mf在体外刺激淋巴管生成中的作用；(3)在体内鉴定pge2介导的淋巴管生成的细胞/分子伙伴。****实验计划：***目的：体内淋巴发芽包括增殖、迁移和试管形成的微血管LEC，可以在体外测量。我们将使用LEC检测VEGF-R-3的表达（mRNA用qPCR，蛋白用western blot）；VEGF-C/D生成（qPCR检测mRNA， ELISA检测蛋白）；增殖（BrdU摄取，8-16 h）；通过微孔（8µm孔）膜迁移（24 h）；生长因子还原基质上的毛细血管样管形成（12 -24小时）。每个功能将使用12小时血清饥饿LEC +/- PGE2或EP4激动剂或VEGF-C （+ve对照）。EP4对所有功能的依赖性将在LEC中使用EP4拮抗剂或EP4敲低进行测试。EP4受体激动剂和特异性通路抑制剂将测试EP4信号通路（PI3k/AKT， MAPK磷酸化）的功能依赖性。* * * *的目标2。测量(a)在mf细胞系或其无细胞上清存在的情况下，通过LEC进行小管形成，以及(b)用EP4拮抗剂预处理mf或抑制mf的EP4对mf产生VEGF-C/D和LEC形成小管的影响。* * * *的目标3。采用定向体内淋巴管生成实验：在裸鼠背皮肤下植入含有基质（含或不含PGE2）、EP4激动剂或VEGF-C （+ve对照）的血管反应器，9d后通过3种方法测量淋巴管生成：(a)标记LYVE-1蛋白的免疫荧光，(b) LYVE-1 mRNA的qPCR和(c)冷冻切片中直接计数淋巴管，双重免疫标记VEGF-C/D和LEC标记LYVE-1或PROX-1或podoplanin。我们还将通过F4/80（mf标记）和VEGF-C/D的双重标记来定量原位产生VEGF-C/D的mf。用EP4拮抗剂治疗小鼠将测试EP4对体内淋巴管生成细胞的依赖性。意义：通过生理调节因子PGE2了解淋巴发育的基本机制**

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**