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. 

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More functional studies that address the process of migration use the so-called Boyden chamber [48]. In this assay the migratory capacity of endothelial cells after activation with chemoattractants or pro-angiogenic stimuli is studied. Velocity of migration and the percentage of cells that are capable of migrating through an ECM-coated membrane into another compartment can be determined. 

It is often possible to address function more specifically in in vitro assays, where functional parameters are usually very sensitive readouts of adverse effects. For example trans-epithelial electrical resistance (TEER) is a very sensitive marker of epithelial disturbances. TEER measures the barrier function of the entire mono-layer and is utilized to study functional disturbances of many epithelial/endothelial cell types including blood-brain barrier, pulmonary, renal, and gastrointestinal cells. Its sensitivity lies in the fact that only a small proportion of cell death has a very large impact on barrier function. Additionally, cell stress can interfere with the arrangement and population of tight junction proteins [16] thus, TEER can in certain conditions measure functional disturbances in the absence of cell death [13]. Also since TEER can be measured noninvasively, it is nondestructive and can be used to monitor the effects of treatment over days and weeks [13, 17]. For excitable cells, electrical activity has also been proven to be an extremely sensitive parameter of adverse drug reactions and microelectrode arrays have been employed successfully to monitor neurotoxicity in vitro [18]. Also, for contractile cells, such as cardiomyocytes, the use of impedance measurements to measure the effects of compounds on spontaneous contraction has been demonstrated to be a very sensitive functional monitoring parameter in vitro [19, 20], Admittedly, none of the aforementioned techniques are true biomarkers per se however, such measurements illustrate the fact that in vitro techniques allow certain possibilities that are not practically tenable in the whole body. 

The development of a new coculture-based model of human BBB that enables the prediction of passive and active transport of molecules into the CNS has recently been reported (Josserand et al., 2006). This new model consists of primary cultures of human brain capillary endothelial cells cocultured with primary human glial cells (Megard et al., 2002 Josserand et al., 2006). The advantage of this systan includes the use of human primary cells, avoiding species, age, and interindi-vidual differences since the two cell types are removed from the same human donor and because of the danonstrated expression of functional efflux transporters such as P-gp, MRP-1, MRP-4, MRP-5, and BCRP. Such models have potential for the assessment of permeability of drug and specific transport mechanisms, which is not possible in artificial membrane assays (e.g., PAMPA) or other cell models due to incomplete expression of active transporters. 

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