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 two-hit 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.

Fig. 1.

Involvement of microtubule (MT) in cyclic stretch (CS)-mediated endothelial cell (EC) remodeling. Pulmonary EC grown to confluence on Flexcell plates were exposed to 18% CS or left under static conditions. At 2 h, cells were stimulated with nocodazole (0.5 μM, 30 min) with or without taxol pretreatment (2 μM, 30 min). A: double immunofluorescence staining was performed with Texas red phalloidin to detect actin filaments and with β-catenin antibodies to visualize adherens junctions. Paracellular gaps are marked by arrows. B: quantitative analysis of gap formation on static and stretched human pulmonary artery endothelial cells (HPAEC). Data are expressed as means ± SD of 5 independent experiments; *P < 0.05.

Fig. 2.

Effect of MT stabilization on CS-induced cytoskeletal remodeling. Human pulmonary EC were pretreated with vehicle or taxol (2 μM, 30 min) followed by exposure to 18% CS for various periods of time. A: actin cytoskeletal remodeling was examined by immunofluorescence staining with Texas red-conjugated phalloidin. Paracellular gaps are marked by arrows. B: levels of myosin light chain (MLC) phosphorylation in the total lysates were determined by Western blot analysis. C: MYPT phosphorylation was analyzed by Western blot in static and CS-challenged EC with or without the presence of taxol.

Fig. 3.

Effects of pathological acute CS on MT dynamics. HPAEC grown on Flexcell plates were subjected to 18% CS for various periods of time. A: MT structure was analyzed by immunofluorescence staining for β-tubulin. Arrows indicate main direction of CS vector. B: quantitative analysis of assembled MT. Data are expressed as means ± SD of 5 independent experiments; *P < 0.05. C: pool of stable MT was determined in stretched EC by Western blot analysis with antibodies against acetylated tubulin. D: effect of taxol (2 μM, 30 min) treatment on CS-induced alteration of stable MT was evaluated by Western blot with antibodies against acetylated tubulin.

Fig. 4.

Role of GEF-H1 in CS-induced cytoskeletal rearrangement. Human pulmonary EC were transfected with GEF-H1-specific or nonspecific siRNA. After 72 h of transfection, cells were subjected to 18% CS for various periods of time. A: cytoskeletal remodeling in control and stretched EC monolayers was analyzed by immunofluorescence staining for F-actin. Inset: Western blot analysis of GEF-H1 depletion in HPAEC. B and C: phosphorylation status of MYPT (B) or MLC (C) was determined by Western blot analysis in static and CS-exposed EC transfected with nonspecific or GEF-H1-specific siRNA.

Fig. 5.

Involvement of GEF-H1 in CS-mediated alterations of MT network. Pulmonary EC grown on Flexcell plates were treated with GEF-H1-specific or nonspecific siRNA for 72 h, followed by 18% CS exposure for 0, 30, or 120 min. A: MT structure was analyzed by immunofluorescence staining for β-tubulin. Arrows indicate main direction of CS vector. B: morphometric analysis of assembled MT. Data are expressed as means ± SD of 3 independent experiments; *P < 0.05. C: pools of stable MT were detected by Western blot for acetylated tubulin in samples from static and stretched EC transfected with nonspecific or GEF-H1-specific siRNA.

Fig. 6.

Role of taxol in the development of ventilator-induced lung injury (VILI). Mice were treated with vehicle or taxol (3.75 × 10⁻⁷ mol/kg iv) before TRAP6 instillation (1.5 × 10⁻⁵ mol/kg it) and mechanical ventilation at HTV (30 ml/kg, 4 h). BAL cell count and protein concentration were determined as described in materials and methods. Data are expressed as means ± SD of 4 independent experiments; *P < 0.05.

Fig. 7.

Role of GEF-H1 in the development of VILI. Mice were transfected with nonspecific or GEF-H1-specific siRNA for 72 h followed by mechanical ventilation at HTV (30 ml/kg, 4 h) with or without TRAP6 instillation (1.5 × 10⁻⁵ mol/mouse, it). A: BAL cell count and protein concentration were determined as described in materials and methods. Data are expressed as means ± SD of 4 independent experiments; *P < 0.01. B: downregulation of GEF-H1 expression was confirmed by qRT-PCR and by Western blot analysis of lung tissue samples. C: whole lungs were fixed, embedded in paraffin, and used for histological evaluation by hematoxylin and eosin staining (n = 3–5 per group). D and E: effect of GEF-H1 knockdown on HTV-induced vascular leak was analyzed by Evans blue-labeled albumin extravasation into the lung tissue (D). The quantitative analysis of Evans blue-labeled albumin extravasation was performed by spectrophotometric analysis of Evans blue extracted from the lung tissue samples (E) (n = 4 per group; *P < 0.05).