Quantitative assessment of angiogenic responses by the directed in vivo angiogenesis assay
Quantitative assessment of angiogenic responses by the directed in vivo angiogenesis assay
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DOI：
10.1016/s0002-9440(10)64276-9
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发表时间：
2003-05-01
影响因子：
6
通讯作者：
Stetler-Stevenson, WG
中科院分区：
文献类型：
--
作者：
Guedez, L;Rivera, AM;Stetler-Stevenson, WG
血管生成研究中的主要问题之一仍然是缺乏合适的方法来定量体内血管生成反应。我们描述了定向体内血管生成试验（DIVAA）的发展和应用，并证明它是可重复的和定量的。本试验包括将半封闭硅胶圆柱体（血管反应器）皮下植入裸鼠体内。血管反应器仅填充有18穆尔与或不与血管生成因子预混合的细胞外基质。血管反应器内的血管化通过在其恢复之前静脉内注射异硫氰酸荧光素（FITC）-葡聚糖来定量，然后通过荧光分光光度法。免疫荧光检查显示细胞和入侵的血管生成血管在不同的发展阶段的血管反应。最低限度可检测到的血管生成反应需要植入后9天和大于或等于50 ng/ml（P < 0.01）的成纤维细胞生长因子-2或血管内皮生长因子。该测定系统的表征表明，FITC标记的葡聚糖定量是高度可重复的，并且FITC-葡聚糖的水平不受血管通透性的显著影响。DIVAN允许精确的剂量反应分析和体内血管生成调节因子有效剂量的鉴定。TNP-470有效抑制由500 ng/ml成纤维细胞生长因子-2诱导的血管生成（EC 50 = 88 pmol/L）。这种抑制与内皮细胞侵袭减少相关。DIVAN有效地检测了血小板反应蛋白-1肽（25 mumol/L）抗血管生成效力的差异，并证明与野生型动物相比，基质金属蛋白酶（MMP）-2缺陷小鼠中的血管生成部分抑制（约40%）。MMP缺陷和对照动物的血管反应器的酶谱显示MMP表达的定量变化。这些结果支持DIVAN作为比较血管生成因子或抑制剂的效力的测定，以及用于分析体内血管生成的分子标志物。
One of the major problems in angiogenesis research remains the lack of suitable methods for quantifying the angiogenic response in vivo. We describe the development and application of the directed in vivo angiogenesis assay (DIVAA) and demonstrated that it is reproducible and quantitative. This assay consists of subcutaneous implantation of semiclosed silicone cylinders (angioreactors) into nude mice. Angioreactors are filled with only 18 mul of extracellular matrix premixed with or without angiogenic factors. Vascularization within angioreactors is quantified by the intravenous injection of fluorescein isothiocyanate (FITC)-dextran before their recovery, followed by spectrofluorimetry. Angioreactors examined by immunofluorescence show cells and invading angiogenic vessels at different developmental stages. The minimally detectable angiogenic response requires 9 days after implantation and greater than or equal to50 ng/ml (P < 0.01) of either fibroblast growth factor-2 or vascular endothelial growth factor. Characterization of this assay system demonstrates that the FITC-labeled dextran quantitation is highly reproducible and that levels of FITC-dextran are not significantly influenced by vascular permeability. DIVAN allows accurate dose-response analysis and identification of effective doses of angiogenesis-modulating factors in vivo. TNP-470 potently inhibits angiogenesis (EC50 = 88 pmol/L) induced by 500 ng/ml of fibroblast growth factor-2. This inhibition correlates with decreased endothelial cell invasion. DIVAN efficiently detects differences in anti-angiogenic potencies of thrombospondin-1 peptides (25 mumol/L) and demonstrates a partial inhibition of angiogenesis (similar to40%) in a matrix metalloprotease (MMP)-2-deficient mouse compared with that in wild-type animals. Zymography of angioreactors from MMP-deficient and control animals reveals quantitative changes in MMP expression. These results support DIVAN as an assay to compare potencies of angiogenic factors or inhibitors, and for profiling molecular markers of angiogenesis in vivo.