Considerable insight in the molecular and cellular biology of angiogenesis has been obtained by in vitro studies using endothelial cells, isolated from either capillaries or large vessels (42, 64, 120). Most steps in the angiogenic cascade can be analyzed in vitro, including endothelial cell proliferation, migration and differentiation (156). The proliferation studies are based on cell counting, thymidine incorporation, or immuno histochemical staining for cell proliferation (by measurement of PCNA) or cell death (by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling or Tunel assay). Chemotaxis can be examined in a Boyden chamber, which consists of an upper and lower well separated by a membrane filter. Chemotactic solutions are placed in the lower well, cells are added to the top well, and after a period of incubation the cells that have migrated toward the chemotactic stimulus are counted on the lower surface of the membrane. Cell migration can also be studied by making a "wound" in a confluent cell layer and calculating the number of cells that migrate and the distance of migration of the cells from the edge of the wound (64). Finally, differentiation can be induced in vitro by culturing endothelial cells in different ECM components (156), including two- and three-dimensional fibrin clots, collagen gels and matrigel (16). Microvessels have also been shown to grow from rings of rat aorta embedded in a three dimensional fibrin gel (169). Advantages of these in vitro systems include the possibility to control the different parameters (i.e. the spatial and temporal concentration of angiogenic mediators) involved, the ability to study individual steps in the angiogenic process, and the lower costs and efforts, as compared to in vivo experiments. However, the great diversity in the reagents (endothelial cell origin and passage number, content of matrigel substrate, growth media, etc…) that are being used makes comparison between different studies rather difficult. Furthermore, any compound that causes a significant effect on cell proliferation, migration of differentiation in vitro might not have the same response in vivo, where it might be inactivated or activate other molecules with opposite effects. Likewise, substances with no evident chemotactic and/or mitogenic effect in vitro may play a crucial role in angiogenesis in vivo.

To discover and evaluate the potency of new anti-angiogenic compounds, it is crucial to have suitable in vivo models. Classical angiogenesis assays include the chick chorioallantoic membrane, rabbit cornea assay, sponge implant models, matrigel plugs and conventional tumor models (42, 64, 120, 194).
 

The chick chorioallantoic membrane (CAM) assay is perhaps the most widely used assay for screening purposes (196). The early chick embryo lacks a mature immune system and was therefore used to study tumor-induced angiogenesis (77). Tissue grafts were placed on the CAM through a window made in the eggshell. This caused a typical radial rearrangement of vessels towards, and a clear increase of vessels around the graft within four days after implantation. Blood vessels entering the graft were counted under a stereomicroscope (25). To assess the anti-angiogenic or angiogenic activity of test substances, the compounds are either prepared in slow release polymer pellets, absorbed by gelatin sponges or air-dried on plastic discs and then implanted onto the CAM. Several variants of the CAM assay including culturing of shell-less embryos in Petri dishes (8), and different quantification methods (i.e. measuring the rate of basement membrane biosynthesis using radio-labeled proline, counting the number of vessels under a microscope or image analysis) have been described. The CAM assay is relatively simple and inexpensive and thus suitable for large-scale screening. The major disadvantage of this assay is that the CAM contains already a well-developed vascular network, which makes it difficult to discriminate between new capillaries and already existing ones.

The cornea presents an in vivo avascular site. Therefore, any vessels penetrating from the limbus into the corneal stroma can be identified as newly formed. To induce an angiogenic response, slow release polymer pellets [i.e. poly-2-hydroxyethyl-methacrylate (hydron) (99) or ethylene-vinyl acetate copolymer (ELVAX)] (189), containing an angiogenic substance (i.e. FGF-2 of VEGF) are implanted in "pockets" created in the corneal stroma of a rabbit. Also, a wide variety of tissues, cells, cell extracts and conditioned media have been examined for their effect on angiogenesis in the cornea. The vascular response can be quantified by computer image analysis after perfusion of the cornea with India ink. This method is very reliable, but technically more demanding and more expensive than the CAM assay, which makes it not a practical screening assay.

Subcutaneous implantation of various artificial sponges (i.e. polyvinyl alcohol, gelatin) in animals has been used frequently to study angiogenesis in vivo. Compounds to be evaluated are either injected directly into the sponges (105) or incorporated into ELVAX (62) or hydron pellets, which are placed in the center of the sponge. Neovascularization of the sponges is assessed either histologically, morphometrically (vascular density), biochemically (hemoglobin content) or by measuring the blood flow rate in the vasculature of the sponge using a radioactive tracer (105). The differences in sponge materials, shape and size make direct data comparison difficult. Moreover, implantation of these materials is associated with non-specific immune responses, which may cause a significant angiogenic response even in the absence of exogenous growth factors in the sponge.

Matrigel is a matrix of a mouse basement membrane neoplasm known as Engelbreth-Holm-Swarm murine sarcoma. It is a complex mixture of basement membrane proteins including laminin, collagen type IV, heparan sulfate, fibrin and growth factors, including EGF, TGF-b , PDGF and IGF-1 (131). It was originally developed to study endothelial cell differentiation in vitro. However, matrigel-containing FGF-2 can be injected subcutaneously in mice (178). Matrigel is liquid at 4°C but forms a solid gel at 37°C that traps the growth factor to allow its slow release. After 10 days, the matrigel plugs are removed and angiogenesis is quantified histologically or morphometrically in plug sections. Matrigel is expensive but, unlike artificial sponges, it provides a more natural environment to initiate an angiogenic response.
 

Numerous animal tumor models have been developed to test the anti-angiogenic and anti-cancer activity of potential drugs. In many cases, tumor cells are engrafted subcutaneously and tumor size is determined at regular time intervals. Frequently used tumor cells include C6 rat glioma, B16BL6 melanoma, LLC, and Walker 256 carcinoma (64). Transfection of endothelial and tumor cells with angiogenic factors has been carried out to assess the effect of overexpressing a single angiogenic factor on angiogenesis and tumor growth in vivo. Finally, the efficacy of potential anti-angiogenic agents can be evaluated on strongly vascularized tumors and in tumors of vascular origin, including polyomavirus middleT-transformed (256) or chemically induced (209) hemangiosarcomas, hemangioendotheliomas overexpressing FGF-2 (223) or Kaposi’s Sarcoma (60).