Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Patent
1996-10-31
1999-11-02
Lankford, Jr., Leon B.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
435 29, 435325, C12N 500
Patent
active
059767820
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to a method for determining angiogenesis, a method of screening substances for angiogenesis modulation activity, and a method of determining the appropriate treatment regime in the case of tumours and the like.
BACKGROUND OF THE INVENTION
Angiogenesis, or the development of new blood vessels, is an essential feature of tissue development and wound healing (1). Without the appropriate development of a blood supply, tissues cannot survive; the circulatory system being essential for the supply of oxygen and nutrients to tissues and for removal of by products of metabolism.
In adults, angiogenesis is a relatively rare occurrence except during wound healing. However, there are a number of "angiogenesis-dependent diseases" in adults where angiogenesis is of critical importance (1-3). The most important of these is the angiogenesis associated with the growth of solid tumours, proliferative retinopathies and rheumatoid arthritis. The development of angiogenesis inhibitors may provide a means for controlling these diseases but current assays for angiogenesis are cumbersome, time consuming and usually based on in vivo systems. The three most frequently used models are the rabbit corneal pocket, the hamster cheek pouch and the chicken chorioallantoic membrane (CAM) assays (1-4). In each system an angiogenic substances must be implanted in the cornea, cheek pouch or the CAM in order to induce angiogenesis. All three assays suffer from the need to artificially induce angiogenesis, the requirement for a sustained-release polymeric vehicle for the angiogenic substance and inhibitor (5), and the technical complexities associated with setting up the assay, including using live animals, and measuring the outcome. The rabbit corneal assay has the additional disadvantage of being ethically unacceptable in many research institutions.
Because of these disadvantages there is a great need for physiologically relevant, in vitro assays for angiogenesis, particularly human angiogenesis. Previous in vitro assays have usually entailed establishing long term cultures of endothelial cells and inducing formation of microvessels by placing the cells on extracellular matrices (6-8) or exposing the cells to various angiogenic stimuli (9-11). Such assays are highly artificial and may not represent a physiological response, particularly as the endothelial cells are already activated, having been cultured for considerable periods of time in the presence of growth factors before use.
The in vitro assay of the present invention represents an angiogenic response which mirrors a normal physiological response, namely neo-vascularisation following blood vessel damage. In one embodiment, the assay of the present invention provides substantial advantages over a previously described procedure where a large volume of frequently replaced culture medium is necessary (12). This is because, in one embodiment, use of a miniaturised assay makes it feasible to test pro- and antiangiogenic substances. Furthermore, the overall cost of the assay (i.e. labour costs, media and tissue flask expenses) is dramatically reduced and the assay, when conducted in a convenient 24 or 48 well format, allows rapid examination and quantification by light microscopy. In addition the assay in all of its embodiments is ethically acceptable because it avoids the use of live animals. Furthermore it provides direct information about the effects of particular angiogenic modulating substances on a particular species because vascular tissue from that species can be used in the assay. For example the assay can directly determine whether a particular substance has angiogenic modulating ability in humans since human tissue may be used in the assay.
In work leading up to the present invention, the inventors discovered that small fragments of human placental blood vessels embedded in a fibrin clot displayed a complex network of blood vessels growing out of the vessel fragment following 7-14 days culture. The assay was performed in 24 or 48 well culture plates
REFERENCES:
Nicosia, R.F. et al. "Large-vessel endothelium switches to a microvascular phenotype during angiogenesis in collagen gel culture of rat aorta" Atherosclerosis, 95, 1992, pp. 191-199.
Nicosia, R.F. et al. "Growth of Microvessels in Serum-Free Matrix Culture of Rat Aorta"; Laboratory Investigation, 63, (1) 1990, pp. 115-122.
Gajdusek, C.M. et al. "Basic Fibroblast Growth Factor and Transforming Growth Factor Beta-1"Journal of cellular physiology, 157, 1993, pp. 133-144.
Bezos Anna
Brown Kathryn Joanna Isabel
Maynes Susan Faye
Parish Christopher Richard
Lankford , Jr. Leon B.
The Australian University
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