Endothelial cell characterization assays

There are most likely a number of diverse molecular pathways by which NO can alter gene expression, but in the case of MCP-1 this pathway has been partially elucidated. In human endothelial cells inhibition of NO synthesis has been shown to activate proteins capable of binding to oligonucleotides containing the NF-kB binding site (Zeiher etal., 1995), suggesting a molecular link between an oxidant-sensitive transcriptional regulatory mechanism and NO synthesis. However, it cannot be excluded that NO also influences the activation of other transcription factors, such as AP-1, which has recently been characterized as an antioxidant-responsive factor (Meyer et al., 1993). Indeed, in in vitro gel-mobility assays the NO donor sodium nitroprusside, but no free NO, has been reported to S-nitrosylate the AP-1 moiety and inhibit its activity (Tabuchi et al., 1994). 

Since Intestinal absorption and Intestinal and hepatic first-pass metabolism are the major processes limiting bloavallablllty, the majority of the methods described below will focus on appropriate models for these processes. Nevertheless, It should be emphasized that other barriers, notably the blood-brain barrier (BBB) and virtually all organ-related membrane barriers, also need to be considered when Investigating the distribution of a compound throughout the body and ultimately Its tissue bloavallablllty. A recent study by the European Centre for the Validation of Alternative Methods (ECVAM) compared In vitro BBB methods and characterized several models In relation to In vivo studies Immortalized BBB-derlved endothelial cell lines (SV-ARBEC, MBEC4), non-BBB-derlved cell lines (MDCK, Caco-2, ECV-C6), and primary cells derived from BBB. In all cases, the correlations between In vitro and In vivo assays were low [36], and, moreover, most In vitro methods lacked at least some of the features of the In vivo barrier [37, 38]. Therefore, animal studies will continue to play a major role In studies of the BBB. 

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