Target Discovery and Validation Reviews and Protocols

254 Skovseth, Küchler, and Haraldsen
In the healthy adult, the vasculature remains quiescent, except in response to
exercise-induced muscle hypertrophy and during the transient neovascularization
of the uterus. However, angiogenesis is a prominent feature of wound
repair and has the potential to become an important therapeutic modality in
ischemic disease (4). Angiogenesis is also an attractive target of neoplastic
tumor growth, in particular because ECs are genetically stable and therefore less
likely to accumulate mutations that allow them to develop drug resistance (5).
Indeed, the antiangiogenic approach to treat cancer has made substantial progress
reflected in the recent approval of Avastin (a humanized antibody to vascular
endothelial growth factor-A) for support therapy of colorectal cancer (6).
However, it should be noted that Avastin’s effect is restricted to prolonging progression-free
survival times with an average of few months (7). Thus, although
this achievement is a milestone toward antiangiogenic treatment of human cancer,
it is still far from copying the full potential that Avastin ® displayed in animal
experiments. In our view, this discrepancy reflects the lack of both
standardized tools and models that reliably mirror the complexity of tumor
angiogenesis mechanisms in humans.
Characterization of candidates for treatment of angiogenesis-dependent diseases
has traditionally been performed using a variety of in vitro and in vivo
models (reviewed in ref. 8). Here, we briefly describe the most commonly used
in vivo models based on host vessel growth. One classical in vivo approach has
been the chick chorioallantoic membrane (CAM) assay (9) where angiogenesis
can be monitored in response to implanted slow-release polymer pellets or
organ grafts onto the developing CAM. Another assay exploits the avascular
cornea in which slow release pellets induce vascularization from the limbus and
offer another elegant way to study and manipulate angiogenesis in vivo (10). A
third, frequently used in vivo assay is based on the injection of Matrigel into the
abdominal wall of mice where it polymerizes at body temperature. Matrigel is a
commercially available, solubilized basement membrane preparation extracted
from the Engelbreth–Holm–Swarm mouse sarcoma-derived cell line. It contains
laminin, collagen IV, heparan sulfate proteoglycans, entactin, and nidogen, and
it gels rapidly at 22–35°C, forming a solid mass upon subcutaneous injection
Fig. 1. (Opposite page) Development of functional human vessels in Matrigel and
inhibition of angiogenesis after endostatin treatment. (A) H&E staining of Matrigel
plug (MP) positioned between the skin (S) and abdominal muscle layer (M). (B–F, H,
and I) Immunofluorescence staining for α-smooth muscle actin (red) and Ulex lectin
staining (green) of Matrigel sections from control and endostatin-treated animals on
days as indicated. (G) Immunofluorescence staining for hCD31 with luconyl blue
inside the vessels indicating functional vessels (phase contrast and immunofluorescent
image of identical fields). Bars = 500 µm (A), 50 µm (B–I). Images (A, E, G, and G).