Rat Mesentery Angiogenesis Assay

Abstract

The adult rat mesentery window angiogenesis assay is biologically appropriate and is exceptionally well suited to the study of sprouting angiogenesis in vivo [see review papers], which is the dominating form of angiogenesis in human tumors and non-tumor tissues, as discussed in invited review papers ^1,2. Angiogenesis induced in the membranous mesenteric parts by intraperitoneal (i.p.) injection of a pro-angiogenic factor can be modulated by subcutaneous (s.c.), intravenous (i.v.) or oral (p.o.) treatment with modifying agents of choice. Each membranous part of the mesentery is translucent and framed by fatty tissue, giving it a window-like appearance.

The assay has the following advantageous features: (i) the test tissue is natively vascularized, albeit sparsely, and since it is extremely thin, the microvessel network is virtually two-dimensional, which allows the entire network to be assessed microscopically in situ; (ii) in adult rats the test tissue lacks significant physiologic angiogenesis, which characterizes most normal adult mammalian tissues; the degree of native vascularization is, however, correlated with age, as discussed in ¹; (iii) the negligible level of trauma-induced angiogenesis ensures high sensitivity; (iv) the assay replicates the clinical situation, as the angiogenesis-modulating test drugs are administered systemically and the responses observed reflect the net effect of all the metabolic, cellular, and molecular alterations induced by the treatment; (v) the assay allows assessments of objective, quantitative, unbiased variables of microvascular spatial extension, density, and network pattern formation, as well as of capillary sprouting, thereby enabling robust statistical analyses of the dose-effect and molecular structure-activity relationships; and (vi) the assay reveals with high sensitivity the toxic or harmful effects of treatments in terms of decreased rate of physiologic body-weight gain, as adult rats grow robustly.

Mast-cell-mediated angiogenesis was first demonstrated using this assay ^3,4. The model demonstrates a high level of discrimination regarding dosage-effect relationships and the measured effects of systemically administered chemically or functionally closely related drugs and proteins, including: (i) low-dosage, metronomically administered standard chemotherapeutics that yield diverse, drug-specific effects ( i.e., angiogenesis-suppressive, neutral or angiogenesis-stimulating activities ⁵); (ii) natural iron-unsaturated human lactoferrin, which stimulates VEGF-A-mediated angiogenesis ⁶, and natural iron-unsaturated bovine lactoferrin, which inhibits VEGF-A-mediated angiogenesis ⁷; and (iii) low-molecular-weight heparin fractions produced by various means ^8,9. Moreover, the assay is highly suited to studies of the combined effects on angiogenesis of agents that are administered systemically in a concurrent or sequential fashion.

The idea of making this video originated from the late Dr. Judah Folkman when he visited our laboratory and witnessed the methodology being demonstrated.

Review papers (invited) discussing and appraising the assay

Norrby, K. In vivo models of angiogenesis. J. Cell. Mol. Med. 10, 588-612 (2006).

Norrby, K. Drug testing with angiogenesis models. Expert Opin. Drug. Discov. 3, 533-549 (2008).