Drug Targeting Organ-Specific Strategies

188 7 Vascular Endothelium in Inflamed Tissue as a Target for Site Selective Delivery of Drugs
be semi-quantitatively assessed. Drugs intervening in this process can thus be tested. Some
drugs, however, inhibit NFκB-mediated gene expression at levels downstream of NFκB binding
to its DNA consensus sequence. For these drugs the EMSA technique is not suitable. Furthermore,
antibodies specifically recognizing the NFκB nuclear localization sequence can be
applied in immunohistochemical analysis to determine the activation and nuclear localization
of this transcription factor [132].
EMSA assays can also be exploited to measure STAT nuclear localization, which is, similar
to NFκB localization, a measure of STAT activity. Determination of JAK phosphorylation
is carried out by immunoprecipitation of the JAK proteins from cell lysates, followed by
SDS-PAGE electrophoresis, immunoblotting with antiphosphotyrosine antibody and JAKspecific
antibody re-probing [99].
Another molecular biological approach to the measurement of (inhibition of) NFκB activity
exploits transfection of endothelial cells with artificial reporter genes.These genes contain
a κB-promotor region preceding a gene encoding a reporter protein such as Green Fluorescent
Protein (GFP) or luciferase. If NFκB becomes activated it will activate the promotor
of the gene construct, resulting in the production of the reporter protein. The expression
level of this protein can subsequently be determined by flow cytometry or enzymatically and
is a direct measure of NFκB activity. The same principle can be applied using, for instance, a
reporter construct with a promotor containing a Glucocorticoid Responsive Element (GRE)
to study the cellular delivery of targeted glucocorticoids such dexamethasone. These transfection
systems can be useful tools in the investigation of the basic principles and characteristics
of drug targeting to endothelial cells.
The general aim in inhibiting endothelial cell function by the targeted delivery of anti-inflammatory
drugs is to inhibit local leucocyte recruitment. In vitro, interactions between leucocytes
and endothelium, and pharmacological intervention can be analysed using quantitative
flow cytometry. In such an assay, endothelium is cultured on collagen gels. After incubation
of leucocytes with endothelium under different experimental conditions, adherent leucocytes
are released by trypsin treatment. Migrated leucocytes are retrieved by digestion of
the collagen using collagenase. For quantitation purposes, known amounts of fluorescent
beads are added to the cell samples prior to flow cytometry analysis. In combination with cell
identification based on forward scatter/side scatter characteristics, accurate determination of
the numbers of leucocytes which adhered to or migrated through the endothelium can be obtained
[133].
The various steps of angiogenesis can be investigated in vitro in assays studying endothelial
cell migration, proliferation or tube formation. These types of assays will be described in
detail in Chapter 9, as most of them have been applied in cancer research. Because angiogenesis
in vivo does not occur with only one cell type present, cell co-culture models were developed.
In these models endothelial cells were grown in the presence of other cells, e.g. tumour
cells, keratinocytes, astroglia cells or fibroblasts, which can induce angiogenesis by producing
soluble factors or by inducing cell–cell contact. Villaschi and Nicosia have examined
the communication between endothelial cells and fibroblasts in a rat aorta explant model. In
these cultures, fibroblasts stabilized microvessel sprouts and hence allowed the study of
processes such as proliferation, migration, tube formation, and later events such as pericyte
migration and extracellular matrix deposition [35,134].