Katherine M Malinda 1 Introduction

Angiogenesis, the process of new blood vessels forming from preexisting vessels, is an important feature in developmental processes, wound healing, and pathologic conditions such as cancer and vascular diseases. Owing to the importance of angiogenesis, a relatively simple and rapid in vivo method to determine the angiogenic potential of compounds is desirable to augment in vitro findings.

One such quantitative method is the murine Matrigel plug assay, which can measure both angiogenesis and antiangiogenesis. Matrigel, an extract of the Englebreth-Holm-Swarm tumor composed of basement membrane components, is liquid at 4°C and forms a gel when warmed to 37°C (1). When plated on Matrigel, human umbilical vein endothelial cells undergo differentiation into capillary-like tube structures in vitro (2,3). In vivo, Matrigel is either injected alone or mixed with potential angiogenic compounds and injected subcutaneously into the ventral region of mice, where it solidifies, forming a "Matrigel plug." When known angiogenic factors, such as basic fibroblast growth factor (bFGF), are mixed with the Matrigel and injected, endothelial cells migrate into the plug and form vessels. The level of angio-genesis is typically viewed by embedding and sectioning the plugs in paraffin and staining using Masson's Trichrome, which stains the Matrigel blue and the endothelial cells/vessels red (Fig. 1). These vessels contain erythrocytes, indicating that they form functional capillaries. Additionally, these capillaries stain positively for factor Vlll-related antigen (4,5). In unsupplemented Matrigel, few cells invade the plug. Strongly angiogenic compounds result in

Fig. 1. Sections of Matrigel plugs stained with Masson's trichrome. All sections are oriented with the side underlying the skin at the top of the image. (A,B) Representative fields of plugs containing 5 |g/mL of Tp4. (C) Field showing Matrigel alone. (D) Representative field of a plug with 10 ng/mL of ECGS (positive control). Sections with Tp4 contain many more cells than Matrigel alone and have cells with a morphology similar to those in the ECGS control. Bar = 100 |im. With permission from Malinda, K. M., Goldstein, A. L., and Kleinman, H. K. (1997). Thymosin P4 stimulates directional migration of human umbilical vein endothelial cells. FASEB J. 11, 474-481.

Fig. 1. Sections of Matrigel plugs stained with Masson's trichrome. All sections are oriented with the side underlying the skin at the top of the image. (A,B) Representative fields of plugs containing 5 |g/mL of Tp4. (C) Field showing Matrigel alone. (D) Representative field of a plug with 10 ng/mL of ECGS (positive control). Sections with Tp4 contain many more cells than Matrigel alone and have cells with a morphology similar to those in the ECGS control. Bar = 100 |im. With permission from Malinda, K. M., Goldstein, A. L., and Kleinman, H. K. (1997). Thymosin P4 stimulates directional migration of human umbilical vein endothelial cells. FASEB J. 11, 474-481.

yellowish plugs, so initial indications of activity can be made at the time when plugs are removed from the mice.

The assay also can be utilized when putative antiangiogenic compounds are being tested. In this assay, Matrigel is premixed with bFGF (angiogenic compound), and the test substances are then added. Thus, test antiangiogenic substances inhibit the formation of vessels induced by bFGF in the Matrigel plug. In this case, the plugs removed from the mouse are relatively colorless, and when viewed by Masson's trichrome staining should contain few endothelial cells. Because of the strong vascular response to bFGF, it is also possible to measure hemoglobin levels with the Drabkin assay (4).

One important consideration in using either of the above assays is the degree of variability that will be observed. Differences in the mice and in the basement membrane preparations will affect the background levels of blood vessel formation one observes. Age and gender of the mice selected also can result in different results between experiments. Vessel formation in young mice (6 mo old) is reduced as compared to mice 12-24 mo old (4). Additionally, if

Fig. 2. Diagram of ventral side of a mouse. Arrows show optimal sites for Matrigel injection.

the Matrigel is injected into different sites in the mouse, variability can result. Lower angiogenic response is observed if the material is injected into the dorsal surface of the animal, while one of the best areas in terms of angiogenic response is the ventral surface of the mouse in the groin area close to the ventral midline (Fig. 2). Regardless of these potential problems, this assay is one of the best for the rapid screening of potential angiogenic and antiangiogenic compounds.

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