Angiogenesis Research Laboratory, Department of Restorative Sciences, University of Michigan School of Dentistry, Ann Arbor, USA.
Lab Invest. 2012 Jul;92(7):988-98. doi: 10.1038/labinvest.2012.77. Epub 2012 May 7.
Angiogenesis is fundamentally required for the initialization, development and metastatic spread of cancer. A rapidly expanding number of new experimental, chemical modulators of endothelial cell function have been described for the therapeutic inhibition of angiogenesis in cancer. Despite this expansion, there has been very limited parallel growth of in vitro angiogenesis models or experimental tools. Here we present the Responsive Angiogenic Implanted Network (RAIN)-Droplet model and novel angiogenesis assay using an endothelial cell culture model of microvascular endothelial cells encapsulated in a spontaneously self-assembling, toroidal hydrogel droplet uniquely yielding discrete, pre-formed, angiogenic networks that may be embedded in 3D matrices. On embedding, radial growth of capillary-like sprouts and cell invasion was observed. The sprouts formed not only as outgrowths from endothelial cells on the surface of the droplets, but also, uniquely, from the pre-formed network structures within the droplet. We demonstrate proof of principle for the utility of the model showing significant inhibition of sprout formation (P<0.001) in the presence of bevacizumab, an anti-angiogenic antibody. Using the RAIN-Droplet assay, we also demonstrate a novel dose-dependent pro-angiogenic function for the characteristically anti-angiogenic multi-kinase inhibitor sorafenib. Exposure of endothelial cells in 3D culture to low, non-lethal doses (<1 μM) of sorafenib after initiation of sprouting resulted in the formation of significantly (P<0.05) more endothelial sprouts compared with controls over a 48-h period. Higher doses of sorafenib (5 μM) resulted in a significant (P<0.05) reduction of sprouting over the same time period. The RAIN-Droplet model is a highly versatile and simply constructed 3D focal sprouting approach well suited for the study of vascular morphogenesis and for preclinical testing of drugs. Furthermore, the RAIN-Droplet model has facilitated the discovery of a novel pro-angiogenic capacity for sorafenib, which may impact the clinical application and dosing regimen of that drug.
血管生成对于癌症的起始、发展和转移扩散是根本必需的。已经有大量新的实验性、内皮细胞功能的化学调节剂被描述出来，用于治疗性抑制癌症中的血管生成。尽管有了这种扩展，但在体外血管生成模型或实验工具方面的平行发展却非常有限。在这里，我们提出了响应性血管生成植入网络（RAIN）-液滴模型和新的血管生成测定法，该方法使用微血管内皮细胞的内皮细胞培养模型，将内皮细胞包裹在一种自发自组装的、环形水凝胶液滴中，该液滴独特地产生离散的、预先形成的血管生成网络，可以嵌入 3D 基质中。在嵌入后，观察到毛细血管样芽的径向生长和细胞入侵。这些芽不仅从液滴表面的内皮细胞向外生长，而且还从液滴内的预先形成的网络结构中独特地生长。我们证明了该模型的原理验证，表明贝伐单抗（一种抗血管生成抗体）的存在显著抑制了芽的形成（P<0.001）。使用 RAIN-液滴测定法，我们还证明了特征性抗血管生成多激酶抑制剂索拉非尼具有新颖的剂量依赖性促血管生成功能。在起始芽形成后，将内皮细胞暴露于 3D 培养中的低剂量（<1 μM）、非致死剂量的索拉非尼，与对照组相比，在 48 小时内形成了明显（P<0.05）更多的内皮芽。在相同时间内，较高剂量的索拉非尼（5 μM）导致芽的形成显著减少（P<0.05）。RAIN-液滴模型是一种高度通用且构建简单的 3D 焦点发芽方法，非常适合用于研究血管形态发生以及用于药物的临床前测试。此外，RAIN-液滴模型促进了发现索拉非尼的一种新的促血管生成能力，这可能会影响该药物的临床应用和剂量方案。
