Rationale Simvastatin, an HMG-CoA reductase inhibitor, has protective effects on mechanically stressed human lung endothelial cells (EC) that are mediated by the attenuation of agonist-induced integrin β4 (ITGB4) tyrosine phosphorylation. In addition, overexpression of ITGB4 constructs harboring mutations in tyrosine phosphorylation sites within the cytoplasmic tail results in decreased mechanical stress-induced inflammatory cytokine release by EC. However, the mechanisms by which ITGB4 phosphorylation is regulated is unknown. A molecule of interest in this context is syndecan-1, a cell-surface proteoglycan that binds extraceullar matrix components but also binds the ITGB4 cytoplasmic domain, is expressed by EC, and has been implicated as a mediator of acute lung injury (ALI). Thus, we hypothesized that syndecan-1 is an effector of lung endothelial cell inflammatory responses mediated by ITGB4 in response to mechanical stress.
Methods To investigate the effects of simvastatin on ITGB4 and syndecan-1 expression human pulmonary artery EC lysates treated with simvastatin (5 µM, 16 h) were subjected to Western blotting for ITGB4 and syndecan-1. Simvastatin-treated EC were then used for immunoprecipitation (IP) of syndecan-1 followed by Western blotting for ITGB4. To study the role of syndecan-1 in EC inflammatory responses to mechanical stress mediated by ITGB4, EC were transfected with syndecan-1 siRNA prior to cyclic stretch (18% CS) and lysates were collected for immunoprecipitation of ITGB4 followed by Western blotting with a phospho-tyrosine antibody. Finally, syndecan-1-silenced EC were subjected to 18% CS and the media was collected for measurement of IL-6 and IL-8 levels.
Results Simvastatin treatment of EC resulted in both a dramatic increase in ITGB4 and a marked decreased in syndecan-1 expression levels. In addition, syndecan-1 association with ITGB4 was markedly decreased in EC treated with simvastatin. In EC subjected to 18% CS, ITGB4 phosphorylation was significantly decreased after syndecan-1 knockdown. Finally, silencing of syndecan-1 was associated with a significant decrease in CS-induced EC IL-6 and IL-8 expression (76% and 66%, respectively, p<0.05 for both).
Conclusion Our results implicate syndecan-1 as an important mediator of EC ITGB4 tyrosine phosphorylation affected by both simvastatin and mechanical-stress. These findings represent a novel area of investigation that may ultimately yield new therapeutic targets and strategies for patients with ventilator-induced lung injury, a form of ALI precipitated by excessive lung stretch.
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