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Alzheimer's disease and cerebral amyloid angiopathy are characterized by accumulation of amyloid-b (Ab) at the cerebrovasculature due to decreased clearance at the blood-brain barrier (BBB). However, the exact mechanism of Ab clearance across this barrier has not been fully elucidated. The hCMEC/D3 cell line has been characterized as a valid model for the BBB. In this study we evaluated the use of this model to study Ab clearance across the BBB, with an emphasis on brain-to-blood directional permeability. Barrier integrity of hCMEC/D3 monolayers was confirmed for large molecules in both the apical to basolateral and the reverse direction. However, permeability for smaller molecules was substantially higher, especially in basolateral to apical direction, and barrier formation for Ab was completely absent in this direction. In addition, hCMEC/D3 cells failed to develop a high TEER, possibly caused by incomplete formation of tight junctions. We conclude that the hCMEC/D3 model has several limitations to study the cerebral clearance of Ab. Therefore, the model needs further characterization before this cell system can be generally applied as a model to study cerebral Ab clearance.

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The amyloid-β 43 (Aβ43) peptide has been shown to be abundantly expressed in Alzheimer's disease plaques, whereas only relatively low levels have been demonstrated in cerebral amyloid angiopathy (CAA). To better understand this discrepant distribution, we studied various biochemical properties of Aβ43, in comparison with Aβ40 and Aβ42. We assessed the interaction of Aβ43 with the three apoE isoforms (apoE2, apoE3, and apoE4) using SDS-PAGE/Western blotting and ELISA, aggregation propensity using thioflavin T assays, and cytotoxicity towards cerebrovascular cells using MTT assays. We found that Aβ43 did not differ from Aβ42 in its interaction with apoE, whereas Aβ40 had a significantly lower degree of interaction with apoE. At a molar ratio of 1:100 (apoE:Aβ), all apoE isoforms were comparably capable of inhibiting aggregation of Aβ40 and Aβ42, but not Aβ43. All Aβ variants had a concentration-dependent negative effect on metabolic activity of cerebrovascular cells. However, the degree of this effect differed for the three Aβ isoforms (Aβ40 > Aβ42 > Aβ43), with Aβ43 being the least cytotoxic peptide towards cerebrovascular cells. We conclude that Aβ43 has different biochemical characteristics compared with Aβ40 and Aβ42. Aggregation of Aβ43 is not inhibited by apoE, in contrast to the aggregation of Aβ40 and Aβ42. Furthermore, cerebrovascular cells are less sensitive towards Aβ43, compared with Aβ40 and Aβ42. In contrast, Aβ43 neither differed from Aβ42 in its aggregation propensity (in the absence of apoE) nor in its apoE-binding capacity. Altogether, our findings may provide an explanation for the lower levels of Aβ43 accumulation in cerebral vessel walls.

No abstract