Mechanotransduction by GEF-H1 as a novel mechanism of ventilator-induced vascular endothelial permeability

Abstract

Pathological lung overdistention associated with mechanical ventilation at high tidal volumes (ventilator-induced lung injury; VILI) compromises endothelial cell (EC) barrier leading to development of pulmonary edema and increased morbidity and mortality. We have previously shown involvement of microtubule (MT)-associated Rho-specific guanine nucleotide exchange factor GEF-H1 in the agonist-induced regulation of EC permeability. Using an in vitro model of human pulmonary EC exposed to VILI-relevant magnitude of cyclic stretch (18% CS) we tested a hypothesis that CS-induced alterations in MT dynamics contribute to the activation of Rho-dependent signaling via GEF-H1 and mediate early EC response to pathological mechanical stretch. Acute CS (30 min) induced disassembly of MT network, cell reorientation, and activation of Rho pathway, which was prevented by MT stabilizer taxol. siRNA-based GEF-H1 knockdown suppressed CS-induced disassembly of MT network, abolished Rho signaling, and attenuated CS-induced stress fiber formation and EC realignment compared with nonspecific RNA controls. Depletion of GEF-H1 in the murine twohit model of VILI attenuated vascular leak induced by lung ventilation at high tidal volume and thrombin-derived peptide TRAP6. These data show for the first time the critical involvement of microtubules and microtubule-associated GEF-H1 in lung vascular endothelial barrier dysfunction induced by pathological mechanical strain. cyclic stretch; actin; microtubules; endothelium; Rho GEF; mechanical forces REORGANIZATION of the endothelial cell (EC) cytoskeleton, which is composed of actin filaments, microtubules (MT), and intermediate filaments, leads to alteration in cell shape and provides a structural basis for an increase in vascular permeability, implicated in the pathogenesis of many diseases including asthma, sepsis, and acute lung injury (ALI) (24,37,41). MT depolymerization by MT inhibitors nocodazole or vinblastin in pulmonary EC results in increased myosin light chain (MLC) phosphorylation, stress fiber formation, contraction, and EC barrier dysfunction (9,10,19,60,66). These effects are linked to the activation of small GTPase Rho and can be attenuated by cell pretreatment with paclitaxel (taxol), which promotes MT stabilization (9,19,25).Rho and its downstream target Rho-associated kinase (Rhokinase) may directly catalyze MLC phosphorylation or act indirectly via inactivation of MLC phosphatase (MYPT1) (65, 67) by phosphorylating Thr695, Ser894, and Thr850 (28), and cause actomyosin-driven cell contraction and EC barrier dysfunction. Guanine nucleotide exchange factor H1 (GEF-H1) has been recently characterized as a Rho-specific GEF, which localizes on microtubules and exhibits Rho-specific activity (54). In MT-bound state, the guanine-exchange activity of GEF-H1 is suppressed, whereas GEF-H1 release caused by MT disassembly stimulates Rho-specific GEF activity (36). We and others have previously shown that barrier-disruptive effects of thrombin, TGF-␤1, and TNF␣ are associated with part...

Control of Vascular Permeability by Atrial Natriuretic Peptide via a GEF-H1-dependent Mechanism

“…Transfection of EC with siRNA was performed as described previously (21). After 72 h of transfection, cells were used for experiments or harvested for Western blot verification of specific protein depletion.…”

“…Nonspecific RNA (Dharmacon, Lafayette, CO) was used as a control treatment. Plasmids encoding human wild type GEF-H1, wild type PAK1, and dominant negative PAK1 (H83L/H86L/K299R) constructs (22) were kindly provided by G. Bokoch; GEF-H1-S885A was kindly provided by K. Szaszi (St. Michael's Hospital, Toronto, Canada); and plasmids encoding Rac1 constitutively active (G12V) and dominant negative (T17N) mutants were purchased from the Missouri University of Science and Technology (Rolla, MO) and used for transient transfections according to protocols described previously (3,21). Control transfections were performed with empty vectors.…”

“…Unbound FITC-avidin was washed out with PBS (pH 7.4, 37°C), and the fluorescence of matrix-bound FITC-avidin was measured on a Victor X5 multilabel plate reader (PerkinElmer Life Sciences). In permeability visualization experiments, cells were fixed with 3.7% formaldehyde in PBS (10 min, room temperature), and immunofluorescence staining of VE-cadherin was performed as described elsewhere (21). Images were acquired using the Nikon video imaging system Eclipse TE 300 (Nikon, Tokyo, Japan) equipped with a digital camera (DKC 5000, Sony, Tokyo, Japan); 10ϫ objective lenses were used.…”

“…Immunofluorescence and Live Cell Imaging-Endothelial monolayers plated on glass coverslips were subjected to immunofluorescence staining with Texas Red phalloidin to visualize F-actin (21,23). For microtubule quantification, cells were fixed with Ϫ20°C methanol, and immunostaining was carried out with ␤-tubulin or EB1 antibodies as described previously (25,26).…”

Vessels With Cingulin Are Leakproof

“…2 Cingulin has been shown to recruit the RhoGEF guanine nucleotide exchange factor H1 (GEFH1) to TJ, thereby downregulating the activity of RhoA and downstream signaling. 6 Although expression of cingulin in ECs is low, the recently identified role of GEFH1 in permeability 7,8 raises the intriguing possibility that cingulin is also important in regulation of barrier function. In this issue of ATVB, a study by Schossleitner et al 9 provides novel insights into the function of cingulin in ECs.…”