Influence of basolateral condition on the regulation of brain microvascular endothelial tight junction properties and barrier function.

Basolateral condition of the brain microvascular endothelium is believed to influence blood-brain barrier (BBB) phenotype, although the precise transcriptional and post-translational mechanisms involved are poorly defined. In vivo, the basolateral surface of the blood-brain endothelium is bathed in serum-free interstitial fluid and encompassed by astrocytic end-feet. We hypothesized that these conditions impact on BBB function by directly modulating expression and biochemical properties of tight junctions. To investigate this, an in vitro transwell culture model was employed to selectively modify the basolateral environment of bovine brain microvascular endothelial cells (BBMvECs). In the absence of basolateral (but not apical) serum, we observed higher levels of expression, association and plasma membrane localization for the tight junction proteins, occludin and zonula occludens-1 (ZO-1), in parallel with elevated transendothelial electrical resistance (TEER) and reduced (14)[C]-sucrose permeability of BBMvEC monolayers. We further examined the effects of non-contact co-culture with basolateral astrocytes (C6 glioma) on indices of BBMvEC barrier function in both the presence and absence of serum. Astrocyte co-culture with serum led to enhanced occludin protein expression, occludin/ZO-1 association, and ZO-1 membrane localization, in parallel with increased TEER of BBMvEC monolayers. Astrocyte co-culture in the absence of serum (i.e. basolateral conditions most consistent with in vivo BBB physiology) however, gave the highest increases in BBMvEC barrier indices. Thus, we can conclude that factors influencing condition of the basolateral environment of the brain microvasculature can directly, and independently, modify BBB properties by regulating the expression and biochemical properties of the tight junction proteins, occludin and ZO-1.

Cyclic strain-mediated regulation of vascular endothelial occludin and ZO-1: influence on intercellular tight junction assembly and function.

OBJECTIVE: The vascular endothelium constitutes a highly effective fluid/solute barrier through the regulated apposition of intercellular tight junction complexes. Because endothelium-mediated functions and pathology are driven by hemodynamic forces (cyclic strain and shear stress), we hypothesized a dynamic regulatory link between endothelial tight junction assembly/function and hemodynamic stimuli. We, therefore, examined the effects of cyclic strain on the expression, modification, and function of 2 pivotal endothelial tight junction components, occludin and ZO-1. METHODS AND RESULTS: For these studies, bovine aortic endothelial cells were subjected to physiological levels of equibiaxial cyclic strain (5% strain, 60 cycles/min, 24 hours). In response to strain, both occludin and ZO-1 protein expression increased by 2.3+/-0.1-fold and 2.0+/-0.3-fold, respectively, concomitant with a strain-dependent increase in occludin (but not ZO-1) mRNA levels. These changes were accompanied by reduced occludin tyrosine phosphorylation (75.7+/-8%) and increased ZO-1 serine/threonine phosphorylation (51.7+/-9% and 82.7+/-25%, respectively), modifications that could be completely blocked with tyrosine phosphatase and protein kinase C inhibitors (dephostatin and rottlerin, respectively). In addition, there was a significant strain-dependent increase in endothelial occludin/ZO-1 association (2.0+/-0.1-fold) in parallel with increased localization of both occludin and ZO-1 to the cell-cell border. These events could be completely blocked by dephostatin and rottlerin, and they correlated with a strain-dependent reduction in transendothelial permeability to FITC-dextran. CONCLUSIONS: Overall, these findings indicate that cyclic strain modulates both the expression and phosphorylation state of occludin and ZO-1 in vascular endothelial cells, with putative consequences for endothelial tight junction assembly and barrier integrity.

Regulation of bovine brain microvascular endothelial tight junction assembly and barrier function by laminar shear stress.

Blood-brain barrier (BBB) controls paracellular solute diffusion into the brain microenvironment and is maintained primarily by tight junctions between adjacent microvascular endothelial cells. Studies implicate blood flow-associated shear stress as a pathophysiological mediator of BBB function, although detailed biochemical data are scarce. We hypothesize that shear stress upregulates BBB function via direct modulation of expression and properties of pivotal tight-junction proteins occludin and zonula occludens-1 (ZO-1). Bovine brain microvascular endothelial cells (BBMvECs) were exposed to either steady or pulsatile shear stress (10 and 14 dyn/cm(2), respectively) for 24 h. Sheared BBMvECs were monitored for occludin-ZO-1 expression, association, and subcellular localization, and transendothelial permeability of BBMvECs to FITC-dextran and (14)[C]sucrose was assessed. Actin reorganization and BBMvEC realignment were observed following steady shear stress for 24 h. Substantial increases in occludin mRNA and protein expression (2.73 +/- 0.26- and 1.83 +/- 0.03-fold) and in occludin-ZO-1 association (2.12 +/- 0.15-fold) were also observed. Steady shear stress also induced clear relocalization of both proteins to the cell-cell border in parallel with reduced transendothelial permeability to FITC-dextran (but not sucrose). Following pulsatile shear stress, increased protein levels for both occludin and ZO-1 (2.15 +/- 0.02- and 1.67 +/- 0.21-fold) and increased occludin-ZO-1 association (2.91 +/- 0.14-fold) were observed in parallel with a reduction in transendothelial permeability to (14)[C]sucrose. Shear stress upregulates BBMvEC barrier function at the molecular level via modulation of expression, association, and localization of occludin and ZO-1. The pulsatile shear model appeared to give the most profound biochemical responses.