Abstract
Both AD and CAA are characterized by accumulation of A β, which is attributed to a decreased clearance of Aβ, partly due to reduced receptor-mediated transport across the BBB. However, the exact role of receptors involved in A β transport across this barrier has not been determined. Recently the hCMEC/D3 cell line was characterized as a valid model for the BBB. In this study we evaluated the use of these cells as a model to characterize the transport of A β across the BBB. Endothelial hCMEC/D3 cells were cultured in transwell inserts to form cellular monolayers with BBB characteristics. Barrier integrity was determined by 1) measuring trans-endothelial electrical resistance (TEER) and 2) measuring paracellular passage of Dextran-FitC (4 kDa and 70 kDa) in the basolateral to apical direction. Transport of Aβ40 and Aβ42 in the basolateral to apical direction was determined by culturing hCMEC/D3 cells in the presence of fluorescently labeled Aβ, fluorescence was determined in apical samples. To exclude cytotoxic effects of Aβ on barrier formation; transport of Dextran-FitC was also measured in the presence of non-labeled Aβ. ZO-1 expression was determined by immunocytochemistry. hCMEC/D3 cells failed to develop a high TEER. Nevertheless, passage of 70 kDa Dextran was effectively restricted by hCMEC/D3 monolayers in the transwell system. However, this was not seen for 4 kDa Dextran. In addition, no difference in transport of Aβ42 and Aβ40 was seen when comparing empty membranes with membranes containing cells. Presence of Aβ did not alter Dextran permeability, excluding a cytotoxic effect of Aβ. However, failure of tight junction formation in the used culture conditions was confirmed by ZO-1 staining of confluent hCMEC/D3 monolayers grown on transwell membranes. Barrier formation has been shown for larger molecules (e.g. 70 kDa) but not for smaller molecules (e.g. 4 kDa). Therefore, the hCMEC/D3 cell line is not suitable as a model to analyze transport of Aβ across the BBB in AD and CAA.
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