Engineering of “humanized” blood vessels using pure isolates of human endothelial cells is a potential alternative to grafting of human skin for propagating human blood vessels in immunocompromised rodents (13
). The need of in vivo
model systems that utilize functional human blood-perfused vessels is dictated by the needs of anti-angiogenic therapies development as well as the needs of tissue engineering in regenerative medicine. Vascular non-invasive imaging could serve as an important adjunct to monitoring of the fate of cell adoption and eventual acceptance in vivo
. Imaging offers a strategy for testing and selecting potential imaging agents directed at the human-specific endothelial target molecules. In particular, imaging of fluorescence in the near-infrared range is especially useful for detecting receptor–ligand or enzyme–substrate interactions in subcutaneous implants due to the fact that near-infrared light has lower scattering and is less absorbed by tissues and blood in vivo
). Moreover, recent progress in development of quantitative methods in optical imaging, resulted in feasibility of fluorescence tomography in vivo
, which enables three-dimensional reconstruction of fluorescence sources and allows measurements of fluorochrome concentrations (23
) and others (19
) previously reported the formation of blood vessels in VEGF and FGF-2 supplemented Matrigel extracellular matrix mixed with human endothelial cells. Matrigel has an important advantage of a natural cell adhesion matrix (collagen I and laminin-based extracellular matrix) in that it exists in a fluid state at lower temperatures and rapidly solidifies at mouse body temperatures in vivo
. It has been previously reported that Matrigel supports sprouting and stabilization of microvessels (25
). Human endothelial vessels generated in mice using collagen implants were found to be immature, transient and having a tendency to regress (13
). However, 4–6 week survival time span of HUVEC-lined vessels was sufficient for our experiments, limited only by continuing degradation and absorption of Matrigel matrix.
We initially tested lectins with well-established endothelial specificity to determine whether they could assist in differentiating between human and mouse endothelial cells. For example, the ability of N
-acetylglucosamine-specific tomato lectin to bind to the apical surface of mouse endothelial cells is well known; and the injection of mice with relatively low amounts of this protein (20–50 μg/animal) enables visualization of tumor blood vessels (27
). We compared tomato lectin with lectin from U. europaeus
which has specificity for fucosylated molecules on human endothelium (29
). Both lectins showed high sensitivity to endothelial cell surface glycoproteins. However, binding experiments in endothelial cell culture demonstrated that both tomato and Ulex
lectins did not exhibit sufficiently high specificity and showed cross-reactivity with human and mouse endothelial cells, respectively. Therefore, we chose to investigate whether human endothelial cells in vivo
could be imaged by using mouse monoclonal antibodies generated against human CD31 chimeric protein (15
). These antibodies demonstrated high levels of binding to constitutively expressed CD31 and resulted in specific association with human endothelial cells (). Unlike human-E-selectin, which is rapidly internalized and degraded (30
), CD31 adhesion molecule surface expression is not triggered by pro-inflammatory stimuli and is less extensively internalized by endothelial cells after specific antibody binding (). This suggests human CD31 as an attractive target for in vivo
imaging. We tested this by using in vivo
imaging () and ex vivo
histology (). The presence of high imaging signal in HUVEC-seeded Matrigel implants and bright staining of vessel-lining cells suggested the presence of intact and viable human endothelial cells in animals at 30 days after the implantation. The observed high target/background imaging signal contrast suggested that the anti-human CD31 antibodies showed no cross-reactivity with mouse endothelial lining cells and suggested no leakage from mouse neovessels formed in Matrigel in vivo
. The observed uptake of antibodies in the spleen of mice was supposedly a result of labeled antibody aggregation with potential opsonization in plasma, and could be viewed as a limitation of the method. However, the use of antibody fragments instead of the whole antibody and the removal of aggregates using sedimentation of size-exclusion methods are expected to decrease the non-specific uptake in non-target organs. Histology suggested that mouse and human vascular networks co-exist predominantly as parallel structures. However, several points of human blood vessel branching off mouse vessels were present and visible by using labeling histology sections with anti-human and anti-mouse CD31 antibodies ().
It is remarkable that human CD31-positive cells () appear to be surrounded by many mouse cells (CD31 negative, DAPI stain positive). We are currently investigating whether this is a result of the recruitment of mouse supporting (mural) cells in response to FGF-2. This could explain an unusual longevity of the human blood vessels in mice that survive for 6 weeks without any additional exogenous supporting cell implantation.
In conclusion, we determined that near-infrared fluorescent anti-human CD31 antibodies show promise in vivo as a potential imaging agent that could be applied for imaging of adoptive transfer of human endothelial cells in mice. The use of these antibodies could assist in determining the fate of human cells for bioengineering purposes and in drug development.