Target Discovery and Validation Reviews and Protocols

258 Skovseth, Küchler, and Haraldsen
inhibitor endostatin as an example. To this end, we subcutaneously implanted
microosmotic pumps that released rhendostatin at a constant rate of 2 µg/h
throughout the experimental period (see Note 1). To allow activation of the
osmotic pumps, Matrigel injections were performed 2 d after pump implantation.
In the presence of endostatin, single HUVECs almost completely
failed to migrate or accumulate into vessels. The fraction of cells with a
migratory phenotype (cells expressing F-actin and extending pseudopods)
was at day 10 strongly reduced from 50% in untreated animals to 13% in the
presence of endostatin. Furthermore, the number of human vessels was
reduced by 95% at day 20 (Fig. 1H) and by 99.5% at days 30 and 40 (Fig. 1I)
after treatment with endostatin based on a vessel:single cell ratio. We also
assessed the maturation of the vessels and found that the ratio of mature
vessels (vessels that were covered by α-smooth muscle actin-expressing
cells) was reduced from 64.3% in untreated to 28.6% in endostatin-treated
animals at day 30. To investigate the possible mechanisms that may have
reduced the ratio of vessels covered with α-smooth muscle actin-expressing
cells because of endostatin treatment, we analyzed endothelial cell-expressed
platelet-derived growth factor (PDGF)B (currently considered the most important
recruiter of pericytes during angiogenesis; [31]) by means of in situ
hybridization with a probe to human (h)PDGFB in costaining with Ulex lectin.
Whereas PDGFB transcripts were found in most vascular constructs in both
groups, the remaining single cells that dominate the gels of endostatin-treated
mice were negative. To more accurately quantify the mRNA levels of hPDGFB
at days 10 and 20, we also performed quantitative PCR based on mRNA
from immunopurified human endothelial cells of enzyme-digested Matrigel
plugs and human transcript-specific primers. In line with our in situ observations,
we found that endostatin reduced the copy numbers of hPDGFB by
more than 90 and 70% at days 10 and 20, respectively, compared with
untreated littermates.
The in vivo HUVEC/Matrigel assay is a convenient and a powerful tool for
the study of vascular morphogenesis, physiological and pathological angiogenic
responses, and functional studies on mature vessels such as inflammatory
responses. It offers several possibilities in terms of experimental design,
i.e., a growth factor or inhibitor could be administered to the endothelium
at different stages of vascular development. For example, serial injections of
3 × 300 U of recombinant human tumor necrosis factor-α over 24 h at the edge
of day 60 Matrigel plugs revealed extravasation of mouse leukocytes through
the human vessels (Skovseth and Haraldsen, unpublished data). Furthermore,
incorporation of growth-arrested, transfected cell lines or tumor cell lines into
the Matrigel plug is another possibility for manipulating endothelial cell
phenotype regulation.