Tiam1 and βPIX mediate Rac‐dependent endothelial barrier protective response to oxidized phospholipids

Am J Respir Cell Mol Biol

Cokic²,

Moldobaeva³

et al. 2009

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Circulating levels of hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF) are increased during acute lung injury; however, combined effects of HGF and VEGF on pulmonary endothelial cell (EC) permeability remain to be elucidated. We have previously shown differential remodeling of focal adhesions (FA) caused by barrier-protective and barrier-disruptive mechanical and chemical stimuli. This study examined a role of FA protein paxillin in the pulmonary EC barrier responses induced by HGF and VEGF. VEGF increased, but HGF decreased, pulmonary EC permeability. These effects were accompanied by differential patterns of site-specific phosphorylation of focal adhesion kinase (FAK) and paxillin and FA redistribution. HGF antagonized random FA formation caused by VEGF challenge and promoted FA accumulation at the cell periphery. HGF attenuated VEGF-induced paxillin redistribution, FA remodeling, and endothelial permeability. SiRNA-based paxillin knockdown attenuated VEGF-induced EC permeability, myosin light chain phosphorylation, and stress fiber and paracellular gap formation. Paxillin knockdown also decreased HGF-induced EC barrier enhancement and suppressed activation of Rac and its effector PAK1. Expression of paxillin-S 273 deficient on PAK1 phosphorylation site prevented HGF-induced cytoskeletal remodeling. These data show a dual role of paxillin in the HGF-and VEGF-mediated endothelial barrier regulation and suggest essential paxillin role in the modulation of Rac-Rho crosstalk. Our results also support a model of pulmonary EC barrier recovery during resolution of ALI via switch from VEGF to HGF signaling.

Section: Discussionmentioning

confidence: 99%

Paxillin Is Involved in the Differential Regulation of Endothelial Barrier by HGF and VEGF

Am J Respir Cell Mol Biol

Cokic²,

Moldobaeva³

et al. 2009

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“…To reduce the content of endogenous GEF-H1, cells were treated with gene-specific siRNA duplexes. Predesigned standard purity siRNA sets (Homo sapiens) were ordered from Dharmacon (Lafayette, CO), and transfection of EC with siRNA was performed as previously described (5,16). After 48 h of transfection, cells were used for experiments or harvested for Western blot verification of specific protein depletion.…”

Section: Methodsmentioning

confidence: 99%

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

American Journal of Physiology-Lung Cellular and Molecular Phys

Xing

et al. 2010

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129

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...

“…To deplete endogenous paxillin or Rac, we treated HPAECs with gene-specific small interfering (siRNA) duplexes, as described elsewhere (3,11,12). Predesigned siRNAs of standard purity were ordered from Ambion (Austin, TX), and transfection of ECs with siRNA was performed as described previously (3,7).…”

Section: Methodsmentioning

confidence: 99%

“…Barrier-protective effects of OxPAPC had been linked to the activation of small GTPases, Rac and Cdc42, which mediated enhancement of the peripheral actin cytoskeleton and increased interactions between the FAs and adherens junction complexes essential for endothelial barrier maintenance (3,11,12). Importantly, protective effects of OxPLs on the EC monolayers exposed to barrier-disruptive agents have been linked to Racmediated downregulation of the Rho-dependent pathways (10).…”

mentioning

confidence: 99%

Cross talk between paxillin and Rac is critical for mediation of barrier-protective effects by oxidized phospholipids

American Journal of Physiology-Lung Cellular and Molecular Phys

Алексеева²,

Cokic³

et al. 2008

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