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

Gawlak

Journal of Biological Chemistry

et al. 2016

Self Cite

Agonist-induced activation of Rho GTPase signaling leads to endothelial cell (EC) permeability and may culminate in pulmonary edema, a devastating complication of acute lung injury. Cingulin is an adaptor protein first discovered in epithelium and is involved in the organization of the tight junctions. This study investigated the role of cingulin in control of agonist-induced lung EC permeability via interaction with RhoA-specific activator GEF-H1. The siRNA-induced cingulin knockdown augmented thrombin-induced EC permeability monitored by measurements of transendothelial electrical resistance and endothelial cell permeability for macromolecules. Increased thrombin-induced permeability in ECs with depleted cingulin was associated with increased activation of GEF-H1 and RhoA detected in pulldown activation assays. Increased GEF-H1 association with cingulin was essential for down-regulation of thrombin-induced RhoA barrier disruptive signaling. Using cingulin-truncated mutants, we determined that GEF-H1 interaction with the rod ؉ tail domain of cingulin was required for inactivation of GEF-H1 and endothelial cell barrier preservation. The results demonstrate the role for association of GEF-H1 with cingulin as the mechanism of RhoA pathway inactivation and rescue of EC barrier after agonist challenge.

Section: Methodsmentioning

confidence: 99%

Role of Cingulin in Agonist-induced Vascular Endothelial Permeability

Gawlak

Journal of Biological Chemistry

et al. 2016

Self Cite

American Journal of Physiology-Lung Cellular and Molecular Phys

“…Actin filaments were stained with Texas red-conjugated phalloidin for 1 h at room temperature. After immunostaining, the glass slides were prepared using mounting medium (Kirkegaard and Perry Laboratories, Gaithersburg, MD) and analyzed using Nikon video-imaging system (Nikon Instech, Tokyo, Japan) as previously described (5,11,12). Quantitative analysis of assembled MT was performed as previously described (10,18,19).…”

Section: Methodsmentioning

confidence: 99%

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

Birukova

Xing

et al. 2010

Self Cite

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...

American Journal of Physiology-Lung Cellular and Molecular Phys

“…Measurement of transendothelial electrical resistance across confluent HPAEC monolayers was performed using an electrical cellsubstrate impedance sensing system (Applied BioPhysics, Troy, NY) as previously described (4,8).…”

Section: Methodsmentioning

confidence: 99%

“…After agonist treatment, EC grown on glass coverslips were subjected to double immunofluorescence staining for VE-cadherin and F-actin as previously described (4,7,8).…”

Section: Methodsmentioning

confidence: 99%

Atrial natriuretic peptide attenuates LPS-induced lung vascular leak: role of PAK1

Birukova

Xing

et al. 2010

Self Cite

(ANP) in the models of sepsis, pulmonary edema, and acute respiratory distress syndrome (ARDS) suggest its potential role in the modulation of acute lung injury. We have recently described ANP-protective effects against thrombininduced barrier dysfunction in pulmonary endothelial cells (EC). The current study examined involvement of the Rac effector p21-activated kinase (PAK1) in ANP-protective effects in the model of lung vascular permeability induced by bacterial wall LPS. C57BL/6J mice or ANP knockout mice (Nppa Ϫ/Ϫ ) were treated with LPS (0.63 mg/kg intratracheal) with or without ANP (2 g/kg iv). Lung injury was monitored by measurements of bronchoalveolar lavage protein content, cell count, Evans blue extravasation, and lung histology. Endothelial barrier properties were assessed by morphological analysis and measurements of transendothelial electrical resistance. ANP treatment stimulated Rac-dependent PAK1 phosphorylation, attenuated endothelial permeability caused by LPS, TNF-␣, and IL-6, decreased LPS-induced cell and protein accumulation in bronchoalveolar lavage fluid, and suppressed Evans blue extravasation in the murine model of acute lung injury. More severe LPS-induced lung injury and vascular leak were observed in ANP knockout mice. In rescue experiments, ANP injection significantly reduced lung injury in Nppa Ϫ/Ϫ mice caused by LPS. Molecular inhibition of PAK1 suppressed the protective effects of ANP treatment against LPS-induced lung injury and endothelial barrier dysfunction. This study shows that the protective effects of ANP against LPS-induced vascular leak are mediated at least in part by PAK1-dependent signaling leading to EC barrier enhancement. Our data suggest a direct role for ANP in endothelial barrier regulation via modulation of small GTPase signaling. pulmonary endothelium; permeability; acute lung injury; cytoskeleton; small interfering RNA in vivo ACUTE LUNG INJURY (ALI) and its more severe form, acute respiratory distress syndrome (ARDS), are associated with high morbidity and mortality in patients. During the progression of ALI, the endothelial cell (EC) barrier of the pulmonary vasculature becomes compromised, leading to pulmonary edema, a characteristic feature of ALI. It is well-established that EC barrier dysfunction is initiated by agonist-induced cytoskeletal remodeling, which leads to disruption of cell-cell contacts and formation of paracellular gaps, allowing penetration of protein-rich fluid and inflammatory cells (14, 33). However, far less is known about the processes that determine EC barrier enhancement or protection in the injured lung.Natriuretic peptides (NP) are a family of three peptide hormones: atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP). Although the major source of ANP is mammalian atrial myocytes, NP are found in other tissues, including aorta, lung, kidney, and brain (2). NP regulate a variety of physiological functions by interacting with receptors at the plasma membrane either to alter le...