Cyclic Strain–Mediated Regulation of Vascular Endothelial Occludin and ZO-1

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Abstract
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. The objective of this study was to investigate the effects of cyclic strain on the expression, modification, and function of 2 pivotal endothelial tight junction components: occludin and ZO-1. Our findings indicate that cyclic strain modulates the expression and phosphorylation of both proteins with consequences for their association and subcellular localization at the cell-cell border and, ultimately, for endothelial barrier integrity.