Quantitative Assessment of Angiogenic Responses by the Directed in Vivo Angiogenesis Assay

Abstract

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 micro l 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 >/=50 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. DIVAA allows accurate dose-response analysis and identification of effective doses of angiogenesis-modulating factors in vivo. TNP-470 potently inhibits angiogenesis (EC(50) = 88 pmol/L) induced by 500 ng/ml of fibroblast growth factor-2. This inhibition correlates with decreased endothelial cell invasion. DIVAA efficiently detects differences in anti-angiogenic potencies of thrombospondin-1 peptides (25 micro mol/L) and demonstrates a partial inhibition of angiogenesis ( approximately 40%) 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 DIVAA as an assay to compare potencies of angiogenic factors or inhibitors, and for profiling molecular markers of angiogenesis in vivo.

A practical and sensitive method of quantitating lymphangiogenesis in vivo

“…DIVAA is an excellent well-studied model to measure angiogenic factor-induced angiogenesis or its inhibition with angiostatic agents after implanting angioreactors in the mouse dorsal flank. 23 This is an improvement on the matrigel plug assay as reported by us for tumor-associated angiogenesis, 29 by containing the BME (or matrigel) in a silicon cylinder to direct the ingrowth of newly formed capillaries into the open end of the cylinder. DIVAA has been effectively applied to studies of both proangiogenic 30 and antiangiogenic 24 functions of various agents.…”

“…Inclusion of tumor cells in the BME within the angioreactor makes its application highly suited for studies of tumor biology and translational cancer research, as has been exploited with the DIVAA model. 22,23 The role of tumor-associated lymphangiogenesis in lymphatic metastasis has been suggested by an association between tumoral expression of the lymphangiogenic factors VEGF-C or -D and lymphatic metastasis in many epithelial cancers including cancers of the prostate, 31 stomach 32 and the colon. 33 This has been validated by a direct association between intra-or peritumoral lymphangiogenesis with lymphatic invasion and metastasis in cancers of the breast, 4,5 pharynx and larynx.…”

“…We significantly modified a directed in vivo angiogenesis assay 23 to measure lymphangiogenesis in addition to angiogenesis. Briefly, sterile angioreactors (Cat.…”

“…To overcome these limitations, we have developed a quick, practical and quantitative in vivo assay for measuring lymphangiogenesis by implanting angioreactors in the mouse dorsal flanks. This method, an adaptation of the directed in vivo angiogenesis assay (DIVAA), [22][23][24] is highly reproducible, providing an opportunity to quantify accurately lymphangiogenesis as early as 10 days. It is applicable to many objectives including quick preclinical screening of drugs in vivo.…”

Improved growth factor directed vascularization into fibrin constructs through inclusion of additional extracellular molecules

“…Another innovative technique is that developed by Guedez et al where ECM-filled semiclosed silicon cylinders are implanted subcutaneously in rats and directed vascular ingrowth into the silicon tube was visualized and quantitated using fluorescein-based fluorescent dyes (Guedez et al, 2003). This technique is quantitative but requires the use of silicon tubing implantation, which is not amenable to the CAM assay.…”