Abstract
Microtubule (MT) dynamics is involved in a variety of cell functions, including control of the endothelial cell (EC) barrier. Release of Rho-specific nucleotide exchange factor GEF-H1 from microtubules activates the Rho pathway of EC permeability. In turn, pathologic vascular leak can be prevented by treatment with atrial natriuretic peptide (ANP). This study investigated a novel mechanism of vascular barrier protection by ANP via modulation of GEF-H1 function. In pulmonary ECs, ANP suppressed thrombin-induced disassembly of peripheral MT and attenuated Rho signaling and cell retraction. ANP effects were mediated by the Rac1 GTPase effector PAK1. Activation of Rac1-PAK1 promoted PAK1 interaction with the Rho activator GEF-H1, inducing phosphorylation of total and MT-bound GEF-H1 and leading to attenuation of Rho-dependent actin remodeling. In vivo, ANP attenuated lung injury caused by excessive mechanical ventilation and TRAP peptide (TRAP/HTV), which was further exacerbated in ANP(-/-) mice. The protective effects of ANP against TRAP/HTV-induced lung injury were linked to the increased pool of stabilized MT and inactivation of Rho signaling via ANP-induced, PAK1-dependent inhibitory phosphorylation of GEF-H1. This study demonstrates a novel protective mechanism of ANP against pathologic hyperpermeability and suggests a novel pharmacological intervention for the prevention of increased vascular leak via PAK1-dependent modulation of GEF-H1 activity.
Highlights
Regulation of vascular permeability by microtubule (MT)-associated proteins is not well understood
The protective effects of atrial natriuretic peptide (ANP) against TRAP/high tidal volume (HTV)-induced lung injury were linked to the increased pool of stabilized MT and inactivation of Rho signaling via ANP-induced, PAK1-dependent inhibitory phosphorylation of guanine nucleotide exchange factor H1 (GEF-H1)
This study demonstrates a novel mechanism of ANP barrierprotective effects in endothelial cells and a two-hit model of TRAP/HTV induced lung injury
Summary
Regulation of vascular permeability by microtubule (MT)-associated proteins is not well understood. This study demonstrates a novel protective mechanism of ANP against pathologic hyperpermeability and suggests a novel pharmacological intervention for the prevention of increased vascular leak via PAK1-dependent modulation of GEF-H1 activity. 2 The abbreviations used are: ALI, acute lung injury; EC, endothelial cell; MT, microtubule; MLC, myosin light chain; GEF, guanine nucleotide exchange factor; ANP, atrial natriuretic peptide; HPAEC, human pulmonary artery endothelial cell; XPerT, express micromolecule permeability testing; HTV, high tidal volume; EB1, end-binding protein-1; CA, constitutively activated; DN, dominant negative; RDU, relative density units. This study tested a hypothesis that the protective effect of ANP in the pathologic settings of vascular endothelial barrier dysfunction is directed by Rac-PAK1-dependent GEF-H1 phosphorylation and resultant inactivation and down-regulation of barrier-disruptive Rho signaling. Microtubules and Control of Permeability on MT dynamics, GEF-H1 microtubule association, PAK1GEF-H1 functional interactions, Rho signaling, and vascular permeability in vitro and in vivo
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