Characterisation of Anti-inflammatory and Pro-inflammatory Influences on Blood-brain Barrier Phenotype Using an in Vitro Human Brain Microvascular Endothelial Model PDF Download

Author: Keith D. Rochfort
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Languages : en
Pages : 364

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Book Description
Introduction: Within the central nervous system, the cerebral endothelial cells have highly specialised structural and functional properties. Compared to the endothelium of the periphery, brain endothelial cells are phenotypically unique in that they have enhanced inter-endothelial junction complexes that provide a highly restrictive yet controlled paracellular barrier for the brain from the constituents of the circulation, effectively embodying a blood-brain barrier. Disruption of the proteins that form these junctional complexes (i.e. occludin, claudin-5, VE-cadherin and ZO-1) has therefore been implicated in several CNS disease states. Endothelial functions can be modulated by both local and systemic environmental factors. The ability of the endothelium to sense its humoral and biomechanical environment and modify its functional phenotype accordingly plays a pivotal role in the maintenance of vascular homeostasis or the development of vascular pathology. However, much remains unknown about how anti- and pro- inflammatory stimuli modulate blood-brain barrier phenotype at the molecular level, thus framing the context of this thesis. In the current study, in view of the opposing physiological and pathophysiological actions demonstrated thus far in similar yet distinctly different in vivo and in vitro endothelial models, we propose to investigate how anti-inflammatory laminar shear stress and pro-inflammatory cytokines can differentially modulate BBB phenotype, with functional consequences for endothelial homeostasis, particularly with regards to the coordination and maintenance of the BBB interendothelial junction complex. Results: Exposure of cultured human brain microvascular endothelial cells (HBMvECs) to physiological levels of laminar shear stress (8 dynes cm-2) resulted in a reduction in monolayer permeability. The transcription and translation of occludin, claudin-5, VE-cadherin and ZO-1 were all upregulated, with an enhanced localisation of said proteins at the cell-cell junctions. Moreover, our studies demonstrated laminar shear stress caused a substantial reduction in pTyr and pThr levels on each protein, with functional consequences for barrier integrity as determined using dephostatin and genistein. In addition, laminar shear stress promoted a number of anti-inflammatory mechanisms, which, in the presence of inflammatory cytokines, could partially ameliorate the injurious effects of the latter. Sophisticated co-IP techniques, coupled with mass spectrometry analysis, identified several isoforms of the 14-3-3 family of proteins as intracellular binding partners to the interendothelial junction complex. This family of proteins was implicated in contributing to the protective, barrier- stabilising effect of laminar shear, whilst inhibition of their activity exacerbated cytokine injury. In parallel studies, exposure of cultured HBMvECs to pathophysiological levels of inflammatory cytokines, TNF-alpha and IL-6, resulted in an increase in monolayer permeability, an effect directly attributable to the reduction in the transcription and translation of the aforementioned tight and adherens junction proteins. Moreover, each cytokine caused a substantial increase in pTyr/Thr levels on each protein. Noteworthy, all cytokine effects were dose- and time-dependent. This increase in injury can be possibly attributed to a correlative reduction in anti-inflammatory mechanisms coupled with a correlative increase in the production and release of inflammatory mediators such as ROS and other cytokines such as IL-6 into the local environment. Conclusions: Physiological levels of laminar shear stress and pathophysiological levels of inflammatory cytokines, TNF-alpha and IL-6, modulate the expression and post-translational properties of BBB tight and adherens junction proteins in an opposing manner. Inflammatory cytokines mediate their effects in part through the induced release of injury potentiating agents such as ROS and IL-6. A novel role for 14-3-3 isoforms in the modulation of interendothelial junction assembly is also implicated in these studies.