Drug Targeting Organ-Specific Strategies

238 9 Tumour Vasculature Targeting
loss of growth potential. Addition of low concentrations of recombinant or purified growth
factors such as FGF-2 will increase the number of possible passages to 10–12. These limited
subculture possibilities make repeated isolations necessary, thereby introducing significant
variation in the endothelial cell source. To circumvent these drawbacks, it is possible to immortalize
endothelial cells with viral oncogenes such as simian virus-40 large T antigen.
Transfection of endothelial cells with a DNA construct containing the gene for this molecule
can result in cell lines that can be subcultured for over 60 passages. Examples of such cell
lines are HMEC-1 [37], EA.hy926 [38], ECL4n [39], and EvL [40]. The major problem with
these immortalized endothelial cells is, however, that they are not genetically stable, resulting
in loss of phenotypic and functional characteristics and functional resemblance to their in
vivo counterparts. Of note, many endothelial cell lines can be cultured on plastic tissue-culture
material, without being dependent on integrin signalling via ECM molecules for growth
and survival. Careful interpretation of results obtained with these cell lines is necessary.
Endothelial cells can also be prepared from tissue from other species. Capillary endothelial
cells of bovine origin are used quite frequently, because these cells are rather sensitive to
treatment with angiogenesis inhibitors such as angiostatin and endostatin (Griffioen et al.,
unpublished results). It should be noted however, that species-dependent responses to drugs
can occur.
9.2.2 Functional Assays with Endothelial Cells
Angiogenesis is a multi-step process, depending on activation, migration, proliferation and
differentiation of endothelial cells, and all of these stages in the cascade can be studied independently.
9.2.2.1 Cell Growth Assays
Proliferation of endothelial cells can be studied using various assays. Since the cell cycle potential
of endothelial cells is low, with cell doubling times of sometimes over 35 h, cell counting
is a time-consuming and insensitive method of assessing cell growth. A much better way
to determine proliferation is by the measurement of [ 3 H]-thymidine incorporation [41,42].
Alternative assays for the study of endothelial cell proliferation and other mechanisms of cell
activation that do not involve the S-phase of the cell cycle, are based on colorimetric systems
which measure mitochondrial activity. Furthermore, proliferation of endothelial cells can be
analysed by DNA profiling, for example by flow cytometric analysis of cells in G0/G1 phase
(2n DNA), cells in G2/M phase (4n DNA) and cells in S phase (2 < n < 4) after permeabilization
and staining with propidium iodide. In addition, proliferation can be quantified by
determination of cell cycle-dependent expression of molecules such as proliferating cell nuclear
antigen (PCNA) [43] or Ki-67 [44].
The number of cells present depends on the level of cell growth and cell death. Therefore,
detection of cell death is a commonly used approach to average cell growth. Apoptosis induction
can be studied most easily by detection of subdiploid cells or analysis of DNA degradation
profiles on the flow cytometer after DNA extraction and propidium iodide staining.