Blood-brain barrier (BBB) regulation involves the coordinated interaction of intercellular adherens and tight junctions in response to stimuli. One such stimulus, shear stress, has been shown to upregulate brain microvascular endothelial cell (BMvEC) barrier function, although our knowledge of the signaling mechanisms involved is limited. In this article, we examined the hypothesis that VE-cadherin can transmit shear signals to tight junction occludin with consequences for pTyr-occludin and barrier function. In initial studies, chronic shear enhanced membrane localization of ZO-1 and claudin-5, decreased pTyr-occludin (in part via a dephostatin-sensitive mechanism), and reduced BMvEC permeability, with flow reduction in pre-sheared BMvECs having converse effects. In further studies, VE-cadherin inhibition (VE-cad ΔEXD) blocked shear-induced Rac1 activation, pTyr-occludin reduction, and barrier upregulation, consistent with an upstream role for VE-cadherin in transmitting shear signals to tight junctions through Rac1. As VE-cadherin is known to mediate Rac1 activation via Tiam1 recruitment, we subsequently confirmed that Tiam1 inhibition (Tiam1-C580) could elicit effects similar to VE-cad ΔEXD. Finally, the observed attenuation of shear-induced changes in pTyr-occludin level and barrier phenotype following Rac1 inhibition (NSC23766, T17N) establishes a downstream role for Rac1 in this pathway. In summary, we describe for the first time in BMvECs a role for VE-cadherin in the transmission of physiological shear signals to tight junction occludin through engagement of Tiam1/Rac1 leading to barrier stabilization. A downstream role is also strongly indicated for a protein tyrosine phosphatase in pTyr-occludin modulation. Importantly, these findings suggest an important route of inter-junctional signaling cross-talk during BBB response to flow.

Epidemiological and clinical studies provide compelling support for a causal relationship between Helicobacter pylori infection and endothelial dysfunction, leading to vascular diseases. However, clear biochemical evidence for this association is limited. In the present study, we have conducted a comprehensive investigation of endothelial injury in bovine aortic endothelial cells (BAECs) induced by H. pylori-conditioned medium (HPCM) prepared from H. pylori 60190 [vacuolating cytotoxin A (Vac(+))]. BAECs were treated with either unconditioned media, HPCM (0-25% vol/vol), or Escherichia coli-conditioned media for 24 h, and cell functions were monitored. Vac(+) HPCM significantly decreased BAEC proliferation, tube formation, and migration (by up to 44%, 65%, and 28%, respectively). Posttreatment, we also observed sporadic zonnula occludens-1 immunolocalization along the cell-cell border, and increased BAEC permeability to FD40 Dextran, indicating barrier reduction. These effects were blocked by 5-nitro-2-(3-phenylpropylamino)benzoic acid (VacA inhibitor) and were not observed with conditioned media prepared from either VacA-deleted H. pylori or E. coli. The cellular mechanism mediating these events was also considered. Vac(+) HPCM (but not Vac(-)) reduced nitric oxide (NO) by >50%, whereas S-nitroso-N-acetylpenicillamine, an NO donor, recovered all Vac(+) HPCM-dependent effects on cell functions. We further demonstrated that laminar shear stress, an endothelial NO synthase/NO stimulus in vivo, could also recover the Vac(+) HPCM-induced decreases in BAEC functions. This study shows, for the first time, a significant proatherogenic effect of H. pylori-secreted factors on a range of vascular endothelial dysfunction markers. Specifically, the VacA-dependent reduction in endothelial NO is indicated in these events. The atheroprotective impact of laminar shear stress in this context is also evident.

Our findings demonstrate that laminar shear positively regulates endothelial TOP expression. Moreover, a role for ROS production by NADPH oxidase is indicated. Finally, our studies suggest that shear-dependent TOP induction down-regulates MHC1 levels, pointing to a role for TOP in the flow-mediated regulation of endothelial immunogenicity.

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