Target Discovery and Validation Reviews and Protocols

256 Skovseth, Küchler, and Haraldsen
and retaining its shape during the experimental period. Mouse ECs invade and
vascularize the Matrigel plug in response to the level of angiogenic agents dissolved
in the gel (11).
Although these model systems have certainly proven useful for the study of
the angiogenic processes, they rely on the extrapolation of results obtained in
nonhuman systems to the understanding of human disease. Recently, two
experimental models of surgical implantation of human ECs have been published.
In the first bioassay, developed by Nör and co-workers, microvascular
ECs derived from the skin are suspended in a 1:1 mixture of Matrigel and EC
growth medium and absorbed into poly(L-lactic acid) sponges before subcutaneous
implantation to immunodeficient SCID mice (12,13). The ECs organize
into functional vessels that carry mouse blood and are covered by α-smooth
muscle actin-positive cells in the course of 21 d. The fraction of human ECs in
the vessels dropped from 90–100% at day 14 to 50–65% at day 28, perhaps (as
suggested by the authors) caused by mouse ECs replacing apoptotic human
ECs. Furthermore, analysis of the inflammation markers intercellular adhesion
molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, and E-selectin
revealed their upregulation in the human endothelium peaking at day 7, possibly
indicating proinflammatory activation of the microenvironment because of traumatization
of the tissue. The second assay for transplantation of human ECs was
reported by Schechner and colleagues (14,15). In this assay, Bcl-2–transduced
human umbilical vein-derived ECs, HUVECs are suspended in a collagen/
fibronectin gel and incubated for 20 h in vitro to obtain tubular structures before
subcutaneous implantation into immunodeficient SCID/beige mice. Analysis of
day 60 gels revealed functional vessels that had the characteristics of capillaries,
venules, and arterioles, carried mouse blood, and were invested by α-smooth
muscle actin-positive cells. The authors observed that the use of Bcl-2–transduced
ECs enhanced the initial formation of vascular structures fourfold and that the
ECs were dependent on Bcl-2 transduction to recruit vascular smooth muscle
cells. Furthermore, transplantation of polymerized collagen/fibronectin gels
where the 20-h initial tubular formation had not taken place in vitro resulted in
avascular gels in the mice (14,15).
Here, we describe in detail a novel bioassay of human angiogenesis in which
HUVECs suspended in Matrigel are transferred to immunodeficient RAG2
knockout mice by means of a single subcutaneous injection (Fig. 1A) and
develop into mature vessels in the course of 30 days (16). Differentiated ECs
form a functional vascular network by adapting sprouting, migrating, and
proliferating phenotypes to vascularize the Matrigel plug. This process very
closely resembles angiogenesis as it is described in the literature (3,17,18).
Furthermore, we demonstrate the controlled, sustained delivery of endostatin, a
powerful inhibitor of the angiogenic process (19).