Abstract

The blood-brain barrier (BBB) is responsible for homeostasis of the brain interstitial fluid (ISF) by separating the central nervous system (CNS) from systemic blood circulation. The BBB exhibits low permeability to hydrophilic solutes and high transendothelial electrical resistance arising from a continuous series of tight junctions (TJ) connecting adjacent microvascular endothelial cells of brain capillaries. The basolateral side of these microvascular endothelial cells are encompassed by astrocyte end feet and bathed in ISF, which is completely devoid of serum. Moreover, the vascular endothelium is continuously exposed to hemodynamic forces, namely shear stress and cyclic circumferential strain, both of which can induce profound changes in endothelial gene expression and cell fate. Disruption of BBB integrity leading to vascular leakage is a central pathophysiologic mechanism of many diseases whilst, the “tightness” of the BBB also proves problematic in drug delivery to the brain. Thus, elucidating the etiology of tight junction formation and the factors effecting BBB permeability may lead to the development of novel strategies to modulate barrier properties and thus have profound clinical impact on several neurological diseases. The focus of this PhD project, therefore, was to elucidate the biochemical events affecting tight junction formation and barrier function caused by serum, astrocyte co-culture and biomechanical shear stress. In order to assess TJ formation in Bovine Brain Microvascular Cells (BBMvECs), the expression, localization and association of occludin and ZO -1, two pivotal TJ proteins was examined, concomitant with measurement of sucrose permeability across the EC monolayer and TEER to assess EC barrier function. Briefly, this research has shown that the increased association of occludin with ZO -1, in parallel with increased membrane localization of the two proteins, is central to tight junction formation, and correlates directly with increased barrier function. In-vivo tight junctions forming the BBB prevent serum entering the ISF. This indicates that the effects of serum on tight junction formation and barrier function are highly polar-specific. Our data subsequently indicated that in the absence of basolateral serum, occludin and ZO-1 expression increased, as did their association and the concomitant redistribution of the two proteins to the plasma membrane. These biochemical events were accompanied by an increase in TEER and decrease in sucrose permeability. Furthermore, our findings indicate that co-culture of BBMvEC with basolaterally applied C6 glioma increases tight junction formation and barrier function and that these changes were enhanced, at least in part, by the removal of serum from the basolateral compartment of the coculture model. In the brain micro vasculature, shear stress is typically in the region of 4 - 20 dynes/cm2. Exposure of BBMvECs to physiological laminar shear stress (pulsatile and non-pulsatile) significantly increases occludin and ZO-1 expression and association, concomitant with translocation of both occludin and ZO-1 to the cell membrane and reduced transendothelial permeability. In summary, this body of work addresses three of the major factors impacting upon the BBB and assesses their individual impacts on tight junction formation and barrier function. Further studies are required to fully elucidate the exact signaling mechanisms by which these events occur.