Vascular endothelial cells respond phenotypically to the hemodynamic environment created by circulating blood, with diverse consequences for vessel wall homeostasis, remodelling, and vascular diseases. Hemodynamic stimuli primarily comprise cyclic circumferential strain and shear stress, blood flow-associated forces that profoundly impact on endothelial gene expression and phenotype. One such impact relates to the barrier integrity of the endothelium monolayer lining the vessel lumen. Barrier function involves the regulated co-interaction of inter-endothelial tight junction and adherens junction protein complexes and is central to vascular endothelium-mediated regulation of vessel homeostasis [1]. By contrast, barrier disruption is characterized by junctional disassembly leading to elevated permeability and vascular leakage, an acknowledged feature of many vascular pathologies, including atherosclerosis, pulmonary diseases, stroke, and CNS disorders [2]. As these pathologies frequently correlate with a dysregulation in normal vessel hemodynamic loading, we hypothesized a dynamic regulatory association between blood flow-associated hemodynamic forces and endothelial barrier function. Our initial investigation of this hypothesis recently examined the effects of physiological cyclic strain on vascular endothelial tight junction expression and function using a bovine aortic endothelial cell (BAEC) model. Equibiaxial cyclic strain (5%, 60 cycles/min, cardiac waveform) was shown to enhance the expression and co-immunoprecipitation of both occludin and zonula occludens type-1 (ZO-1), pivotal components of the tight junction complex, and to enhance localization of both proteins to the cell-cell border. In parallel investigations, strain-dependent modulation of tight junction phosphorylation state, manifested as decreased pTyr-occludin and increased pSer/Thr-ZO-1, was also observed (note: the recent finding by Kago et al. that increased tyrosine phosphorylation of occludin accompanies endothelial barrier dysfunction in a rat brain model of cerebral ischemia clearly highlights the physiological importance of these phosphorylation changes to tight junction assembly [3]). Finally, these events were consistent with a significant strain-dependent reduction in BAEC permeability, as monitored by transwell permeability assay [4]. In a closely related study, our laboratory have also reported similar findings for physiological laminar shear stress (10 dynes/cm²) using a bovine brain microvascular endothelial cell (BBMvEC) model [5]. Using an in vitro laminar shear stress model, our laboratory is currently working to delineate the precise signal transduction mechanisms that mediate vascular endothelial barrier responses to flow-associated mechanical stimulation. In view of their established signalling roles in endothelial mechanotransduction and cytoskeletal remodelling, our investigations have specifically focussed on the roles of VE-Cadherin [6] and Rac1 GTPase [7], respectively. Inhibition studies using Rac1-selective strategies (NSC23766, dominant-negative Rac1-T17N) for example, confirm that Rac1 signalling (but not RhoA/ROCK signalling) can mediate shear-induced pTyr-occludin reduction, tight junction complex localization, and endothelial barrier enhancement. Similarly, both shear-induced Rac1 activation and vascular endothelial barrier enhancement could be fully attenuated following over-expression of VE-Cad(ΔEXD), a “non-junctional” VE-Cadherin mutant in which the extracellular domain has been replaced with a FLAG epitope, thereby preventing both cell-cell VE-Cadherin engagement and proper signalling through the cytoplasmic domain. In summary, our collective data confirm that blood flow-associated mechanical forces can up-regulate vascular endothelial barrier function through modulation of tight junction protein expression and biochemical properties, findings consistent with the established “atheroprotective” influence of physiological hemodynamic challenge in vivo. Moreover, our data also point to a VE-Cadherin/ Rac1 signalling axis in the regulation of tight junction complex assembly and endothelial barrier response to shear stress. In this regard, recent reports indicating that VE-Cadherin likely functions within a larger mechanotransduction protein complex that also includes vascular endothelial growth factor receptor 2 (VEGFR2) and platelet-endothelial cell adhesion molecule 1 (PECAM1) [6], are of obvious relevance to these studies and will form a basis for more elaborate investigations into our proposed role for VE-Cadherin in the mechanoregulation of vascular endothelial barrier integrity.