Limitations of the hCMEC/D3 cell line as a model for Aβ clearance by the human blood‐brain barrier

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Alzheimer's disease (AD) is the most common neuropathological disease among elderly. Pathologically, AD is characterized by accumulation of the amyloid‐beta (Aβ) protein and Aβ‐associated proteins in extracellular plaques, hyperphosphorylated tau protein in the form of intracellular neurofibrillary tangles and wide‐spread neuronal loss (LaFerla and Oddo, 2005; Selkoe, 1991; Timmer et al., 2010a). In addition, in approximately 80 percent of AD patients, accumulation of Aβ is also seen in the cerebral blood vessels (Kumar‐Singh, 2008; Rensink et al., 2003). This cerebral amyloid angiopathy (CAA) of the Aβ type can severely affect the integrity of blood vessel walls, which often results in small or larger intracerebral bleedings and eventually may lead to hemorrhagic stroke.
Brain levels of Aβ are determined by the balance between local cerebral production, possibly in combination with influx from the peripheral circulation, and clearance of the protein from the brain. Whereas in familial AD production levels of Aβ are clearly increased due to mutations in genes involved in Aβ production, this is not the case for patients with the sporadic form of AD (Bali et al., 2012). It is conceivable that a disturbance of the balance between production and clearance of the Aβ protein towards decreased clearance, is the cause of development of sporadic AD (Mawuenyega et al., 2010).
Clearance of Aβ from the brain can take place via multiple pathways (reviewed by (Miners et al., 2011; Sagare et al., 2012)). One of these pathways is receptor mediated transport of Aβ across the blood‐brain barrier (BBB) into the systemic circulation. The accumulation of Aβ in CAA is likely a result of impaired clearance across the BBB, emphasizing the role of receptor mediated clearance of Aβ. At the capillary level the BBB is composed of highly specialized endothelial cells supported by pericytes and astrocytes (Zlokovic, 2011). The specialized endothelial cells form tight junctions with neighboring endothelial cells. By forming these tight junctions, passive transcytosis, as occurs in systemic blood vessels, is almost absent at the BBB. With the exception of small lipid‐soluble compounds which can passively cross the BBB, other compounds can only pass the intact BBB by active transport. Several receptors on the BBB have been implicated in Aβ clearance, the best known are low‐density lipoprotein receptor related protein‐1 (LRP1) for the transport from brain to blood and the receptor for advanced glycation end products (RAGE) for transport from blood to brain (Candela et al., 2010; Deane et al., 2003; Deane et al., 2004; Wilhelmus et al., 2007). Several other receptors, such as megalin, P‐glycoprotein (P‐gp) and other members of the ATP‐binding cassette (ABC) transporter family may also be involved in this bidirectional transport of Aβ (Cirrito et al., 2005; Elali and Rivest, 2013; Zlokovic et al., 1996).
We aimed to validate an in vitro transport model for the human BBB to study the transport mechanisms of Aβ across the BBB. The hCMEC/D3 cell line has previously been developed to serve as a model for the human BBB (Weksler et al., 2005). This model is most frequently used for transport studies in the apical to basolateral direction (blood‐to‐brain) and has been applied to Aβ transport as well (Andras et al., 2010; Andras et al., 2008; Tai et al., 2009). However, to study cerebral Aβ clearance, the basolateral to apical (or brain‐to‐blood) transport is more relevant. Therefore, we evaluated the use of this hCMEC/D3 cell line as a model to characterize the transport of Aβ across the BBB in the brain‐to‐blood direction.
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