ABSTRACT: Rationale: Oxidative stress is an important part of host innate immune response to foreign pathogens such as bacterial lypopolysaccharide (LPS), but excessive activation of redox signaling may lead to pathologic endothelial cell (EC) activation and barrier dysfunction. Microtubules (MT) play an important role in agonist-induced regulation of vascular endothelial permeability, but their impact in modulation of inflammation and EC barrier has not been yet investigated. Objective: This study examined effects of LPS-induced oxidative stress on MT dynamics and involvement of MT in the LPS-induced mechanisms of Rho activation, EC permeability and lung injury. Methods and Results: LPS treatment of pulmonary vascular EC induced elevation of reactive oxygen species (ROS) and caused oxidative stress associated with EC hyper-permeability, cytoskeletal remodeling and formation of paracellular gaps, as well as activation of Rho, p38 stress kinase, and NFκB signaling, the hallmarks of endothelial barrier dysfunction. LPS also triggered ROS-dependent disassembly of the microtubule network leading to activation of MT-dependent signaling. Stabilization of MT with epothilone B, or inhibition of MT-associated GEF-H1 activity by siRNA-mediated knockdown suppressed LPS-induced EC barrier dysfunction in vitro and attenuated vascular leak and lung inflammation in vivo. LPS disruptive effects were linked to activation of Rho signaling caused by LPS-induced MT disassembly and release of Rho-specific GEF-H1 from MT. Conclusions: These studies demonstrate for the first time the mechanism of ROS-induced Rho activation via destabilization of MT and GEF-H1-dependent activation of Rho signaling leading to pulmonary EC barrier dysfunction and exacerbation of LPS-induced inflammation.
American Journal of Respiratory Cell and Molecular Biology 07/2012; · 5.13 Impact Factor