Seng Wen Lin, Eng Kurt, Lee Jenny, McGrath Patricia
Phylonix Pharmaceuticals, Inc., Cambridge, MA 02139, USA.
Angiogenesis. 2004;7(3):243-53. doi: 10.1007/s10456-004-4181-7.
We have recently generated a monoclonal antibody (mAb), Phy-V002, which specifically labels activated vascular endothelial cells (EC) in zebrafish. Here, we show that this mAb labels activated EC in newly formed vessels in vivo without staining mature vessels or other tissues. Using this mAb, drug effects on in vivo EC migration and vessel formation were visually assessed by whole-mount immunochemical staining in the transparent embryo. In addition, we have developed a quantitative microplate-based ELISA that measures EC proliferation in vivo after drug treatment. We have validated the quantitative in vivo ELISA using several antiangiogenic small molecules with different mechanisms of action which were added directly to the fish water. Some of these drugs, including: 2-methoxyestradiol, flavopiridol, paclitaxel, and genistein, are currently in clinical trials. We also injected large molecule drugs, including 3TSR and TSR2+KRFK, recombinant human antiangiogenic peptides of thrombospondin-1, a natural protein. To demonstrate that proangiogenic effects can also be assessed in zebrafish, we assessed effects of penicillamine and simvastatin, two proangiogenic compounds shown to stimulate vessel formation in rodents. Using whole-mount immunochemical staining with Phy-V002, inhibition of EC migration and inhibition or stimulation of vessel formation were visually observed for each compound. Next, using the quantitative in vivo angiogenesis ELISA, we generated dose-response curves for each compound. Compared to conventional assays, advantages of using zebrafish to assess drug effects on angiogenesis include: (1) a short assay time; (2) easy animal maintenance; (3) use of small quantities of drug; (4) single dosing; (5) a quantitative assay format; and (6) use of statistically significant number of animals per test.
我们最近制备了一种单克隆抗体（mAb），即Phy-V002，它能特异性标记斑马鱼中活化的血管内皮细胞（EC）。在此，我们表明该单克隆抗体可在体内新形成的血管中标记活化的内皮细胞，而不会对成熟血管或其他组织进行染色。利用这种单克隆抗体，通过对透明胚胎进行全胚胎免疫化学染色，可直观评估药物对体内内皮细胞迁移和血管形成的影响。此外，我们开发了一种基于微孔板的定量酶联免疫吸附测定法（ELISA），用于测量药物处理后体内内皮细胞的增殖情况。我们使用了几种具有不同作用机制的抗血管生成小分子直接添加到鱼水中，验证了这种体内定量ELISA。其中一些药物，包括：2-甲氧基雌二醇、黄酮哌啶醇、紫杉醇和染料木黄酮，目前正处于临床试验阶段。我们还注射了大分子药物，包括3TSR和TSR2 + KRFK，它们是血小板反应蛋白-1（一种天然蛋白质）的重组人抗血管生成肽。为了证明在斑马鱼中也可以评估促血管生成作用，我们评估了青霉胺和辛伐他汀的作用，这两种促血管生成化合物已被证明可刺激啮齿动物的血管形成。使用Phy-V002进行全胚胎免疫化学染色，可直观观察到每种化合物对内皮细胞迁移的抑制以及对血管形成的抑制或刺激作用。接下来，使用体内血管生成定量ELISA，我们为每种化合物生成了剂量反应曲线。与传统检测方法相比，利用斑马鱼评估药物对血管生成作用的优势包括：（1）检测时间短；（2）动物饲养容易；（3）药物用量少；（4）单次给药；（5）定量检测形式；（6）每次试验使用具有统计学意义数量的动物。
