Angiogenesis Assays: A Critical Overview

Abstract

Background: Angiogenesis, the formation of new blood vessels, is an integral part of both normal developmental processes and numerous pathologies, ranging from tumor growth and metastasis to inflammation and ocular disease. Angiogenesis assays are used to test efficacy of both pro-and antiangiogenic agents. Methods: Most studies of angiogenesis inducers and inhibitors rely on various models, both in vitro and in vivo, as indicators of efficacy. In this report we describe the principal methods now in use: the in vivo Matrigel plug and corneal neovascularization assays, the in vivo/in vitro chick chorioallantoic membrane (CAM) assay, and the in vitro cellular (proliferation, migration, tube formation) and organotypic (aortic ring) assays. We include description of two new methods, the chick aortic arch and the Matrigel sponge assays. Conclusions: In vitro tests are valuable, can be carried out expeditiously, and lend themselves to quantification, but must be interpreted with extreme caution. In vitro tests are best viewed as providing initial information, subject to confirmation by in vivo assays. Multiple tests should be used to obtain maximum benefit from in vitro tests. In vivo tests are more difficult and timeconsuming to perform, thereby limiting the number of tests that can run at any one time. Quantification is generally more difficult as well. However, in vivo assays are essential because of the complex nature of vascular responses to test reagents, responses that no in vitro model can fully achieve.

In vitro assays of angiogenesis for assessment of angiogenic and anti-angiogenic agents

“…This can be done by using higher levels of serum in cell culture assays, by including angiogenic factors such as VEGF in the culture media, or by using assays such as the mouse metatarsal assay that provide high baseline levels of angiogenic activity. It should also be noted that in many assays cytotoxic effects will not be easily distinguished from specific anti-angiogenic effects (reviewed in Auerbach et al, 2003). It is therefore important to visually inspect cell cultures to look for signs of necrotic cell death.…”

“…Due to constraints of space, only selected in vitro angiogenesis assays are described; descriptions of additional assays can be found in the following excellent reviews: (Auerbach et al, 2000;Auerbach et al, 2003;Eccles et al, 2005;Entschladen et al, 2005;Huerta et al, 2007;Renvoizé et al, 1998;Staton et al, 2004;Vailhé et al, 2001).…”

A novel imaging‐based high‐throughput screening approach to anti‐angiogenic drug discovery

“…Images were acquired for every third day as 53 5 montages with a 310 objective and quantified with the parameter tube total length (BD Attovision software). Once the network is formed at day 3, this parameter reflects the stability of the network (day [3][4][5][6][7][8][9][10][11][12], and inhibition of network formation due to drug treatment can be measured. (b) Images from a developing network acquired every third day during a 12-day period showing fluorescent EC (gray).…”

Endothelial progenitor cells: Implications for cardiovascular disease

“…In line with the idea that EPCs participate in angiogenesis, it is reasonable to test this ability in vitro. One of the most specific test for angiogenesis is the measurement of endothelial cells or there progenitors to form three-dimensional structures (tube formation) [reviewed in (40,41)]. With the discovery that Matrigel [a matrix-rich product prepared from Engelbreth-Holm-Swarm (EHS) tumor cells whose primary component is laminin (42,43)] can evoke endothelial cell tube formation within 24 h, tube formation assays have achieved a prominent place in the array of angiogenesis measurements (44,45).…”