Target Discovery and Validation Reviews and Protocols

In Vivo Assay of Human Angiogenesis 257
To follow the fate of injected HUVECs and to relate them to the contribution
of preexisting murine vessels, we double stained tissue sections with
antibodies recognizing either human or mouse CD31, which in both species
is a pan-endothelial cell marker. In another protocol, we detected human ECs
and invading smooth muscle cells by means of Ulex lectin staining and an
antibody to α-smooth muscle actin, respectively. Thus, specimens excised
1 or 3 d after Matrigel injection revealed single HUVECs uniformly distributed
in the gel (Fig. 1B). After 10 d, we observed single ECs with elongated
cytoplasmic protrusions resembling pseudopods and also ECs that were
lining up and in contact with other ECs (Fig. 1C), particularly at the border
of the Matrigel plug and close to preexisting mouse vessels. After 20 d, we
observed HUVECs forming tubular structures in all areas of the gels and
50% of the vessels were covered by α-smooth muscle actin-positive cells
(Fig. 1D). Some of the vessel structures observed after 20 d also contained
erythrocytes, indicative of functional vessels. On day 40, nearly all human
vessels were fully mature, containing erythrocytes and being surrounded
by α-smooth muscle actin-expressing cells (94.5%) (Fig. 1E,F). Mature
vessels can still be detected in the mice 100 d postinjection, and the human
vessels are composed almost exclusively by human ECs and α-smooth
muscle actin-expressing cells. To further investigate the maturity and functionality
of the vessels, we injected intracardially a water-insoluble dye that
does not leave the vascular circulation (luconyl blue) before termination of
the experiment. Because the dye could be observed in the human vessels
together with the erythrocytes, it nicely demonstrated that the vessels were
circulated at the point of sacrifice (Fig. 1G). Furthermore, no signs of infection
or inflammation could be observed after injection and during the experimental
period, as assessed by general histology and the finding that the
proinflammatory markers ICAM-1, VCAM-1, and E-selectin were negative
at all time-points.
Several tumor-derived, circulating angiogenesis inhibitors generated
in vivo by proteolytic degradation have been recently identified (reviewed
in refs. 20 and 21). In particular, a 20-kDa C-terminal proteolytic fragment
of collagen XVIII, termed endostatin, inhibits tumor growth in several
animal models (5,22–26). Thus, it is not only considered a promising anticancer
drug but also has so far failed to reproduce its properties in human
clinical trials (27–29). Although the mechanisms of action are incompletely
understood, endostatin is thought to interact with several endothelial cell
surface receptors that are critically involved in angiogenesis (reviewed in
ref. 30).
In addition to the model itself, we also show how to target adoptively transferred
human endothelial cells by using sustained delivery of the angiogenesis