Tinghuai Wu

Oxidative stress contributes to lung injury and barrier dysfunction via microtubule destabilization.

Oxidative stress is an important part of host innate immune response to foreign pathogens, such as bacterial LPS, but excessive activation of redox signaling may lead to pathologic endothelial cell (EC) activation and barrier dysfunction. Microtubules (MTs) play an important role in agonist-induced regulation of vascular endothelial permeability, but their impact in modulation of inflammation and EC barrier has not been yet investigated. This study examined the effects of LPS-induced oxidative stress on MT dynamics and the involvement of MTs in the LPS-induced mechanisms of Rho activation, EC permeability, and lung injury. LPS treatment of pulmonary vascular EC induced elevation of reactive oxygen species (ROS) and caused oxidative stress associated with EC hyperpermeability, cytoskeletal remodeling, and formation of paracellular gaps, as well as activation of Rho, p38 stress kinase, and NF-κB signaling, the hallmarks of endothelial barrier dysfunction. LPS also triggered ROS-dependent disassembly of the MT network, leading to activation of MT-dependent signaling. Stabilization of MTs with epothilone B, or inhibition of MT-associated guanine nucleotide exchange factor (GEF)-H1 activity by silencing RNA-mediated knockdown, suppressed LPS-induced EC barrier dysfunction in vitro, and attenuated vascular leak and lung inflammation in vivo. LPS disruptive effects were linked to activation of Rho signaling caused by LPS-induced MT disassembly and release of Rho-specific GEF-H1 from MTs. These studies demonstrate, for the first time, the mechanism of ROS-induced Rho activation via destabilization of MTs and GEF-H1-dependent activation of Rho signaling, leading to pulmonary EC barrier dysfunction and exacerbation of LPS-induced inflammation.

Stimulation of Rho signaling by pathologic mechanical stretch is a "second hit" to Rho-independent lung injury induced by IL-6

Most patients with acute lung injury (ALI) and acute respiratory distress syndrome of septic and nonseptic nature require assisted ventilation with positive pressure, which at suboptimal range may further exacerbate lung dysfunction. Previous studies described enhancement of agonist-induced Rho GTPase signaling and endothelial cell (EC) permeability in EC cultures exposed to pathologically relevant cyclic stretch (CS) magnitudes. This study examined a role of pathologic CS in modulation of pulmonary EC permeability caused by IL-6, a cytokine increased in sepsis and acting in a Rho-independent manner. IL-6 increased EC permeability, which was associated with activation of Jak/signal transducers and activators of transcription, p38 MAP kinase, and NF-κB signaling and was augmented by EC exposure to 18% CS. Rho kinase inhibitor Y-27632 suppressed the synergistic effect of 18% CS on IL-6-induced EC monolayer disruption but did not alter the IL-6 effects on static EC culture. 18% CS also increased IL-6-induced ICAM-1 expression by pulmonary EC and neutrophil adhesion, which was attenuated by Y-27632. Intratracheal IL-6 administration in C57BL/6J mice increased protein content and cell count in bronchoalveolar lavage fluid. These changes were augmented by high tidal volume mechanical ventilation (HTV; 30 ml/kg, 4 h). Intravenous injection of Y-27632 suppressed IL6/HTV-induced lung injury. In conclusion, this study proposes a novel mechanism contributing to two-hit model of ALI: in addition to synergistic effects on Rho-dependent endothelial hyper-permeability triggered by thrombin, TNFα, LPS, or other agonists, ventilator-induced lung injury-relevant CS may also exacerbate Rho-independent mechanisms of EC permeability induced by other inflammatory mediators such as IL-6 via mechanisms involving Rho activity.

p190RhoGAP mediates protective effects of oxidized phospholipids in the models of ventilator-induced lung injury.

Products resulting from oxidation of cell membrane phospholipid 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC) exhibit potent protective effects against lung endothelial cell (EC) barrier dysfunction caused by pathologically relevant mechanical forces and inflammatory agents. These effects were linked to enhancement of peripheral cytoskeleton and cell adhesion interactions mediated by small GTPase Rac and inhibition of Rho-mediated barrier-disruptive signaling. However, the mechanism of OxPAPC-induced, Rac-dependent Rho downregulation critical for vascular barrier protection remains unclear. This study tested the hypothesis that Rho negative regulator p190RhoGAP is essential for OxPAPC-induced lung barrier protection against ventilator-induced lung injury (VILI), and investigated potential mechanism of p190RhoGAP targeting to adherens junctions (AJ) via p120-catenin. OxPAPC induced peripheral translocation of p190RhoGAP, which was abolished by knockdown of Rac-specific guanine nucleotide exchange factors Tiam1 and Vav2. OxPAPC also induced Rac-dependent tyrosine phosphorylation and association of p190RhoGAP with AJ protein p120-catenin. siRNA-induced knockdown of p190RhoGAP attenuated protective effects of OxPAPC against EC barrier compromise induced by thrombin and pathologically relevant cyclic stretch (18% CS). In vivo, p190RhoGAP knockdown significantly attenuated protective effects of OxPAPC against ventilator-induced lung vascular leak, as detected by increased cell count and protein content in the bronchoalveolar lavage fluid, and tissue neutrophil accumulation in the lung. These results demonstrate for the first time a key role of p190RhoGAP for the vascular endothelial barrier protection in VILI.

Iloprost improves endothelial barrier function in lipopolysaccharide-induced lung injury.

The protective effects of prostacyclin and its stable analogue iloprost are mediated by elevation of intracellular cyclic AMP (cAMP) leading to enhancement of the peripheral actin cytoskeleton and cell–cell adhesive structures. This study tested the hypothesis that iloprost may exhibit protective effects against lung injury and endothelial barrier dysfunction induced by bacterial wall lipopolysaccharide (LPS). Endothelial barrier dysfunction was assessed by measurements of transendothelial permeability, morphologically and by analysis of LPS-activated inflammatory signalling. In vivo, C57BL/6J mice were challenged with LPS with or without iloprost or 8-bromoadenosine-3′,5′-cyclic monophosphate (Br-cAMP) treatment. Lung injury was monitored by measurements of bronchoalveolar lavage protein content, cell count and Evans blue extravasation. Iloprost and Br-cAMP attenuated the disruption of the endothelial monolayer, and suppressed the activation of p38 mitogen-activated protein kinase (MAPK), the nuclear factor (NF)-&kgr;B pathway, Rho signalling, intercellular adhesion molecular (ICAM)-1 expression and neutrophil migration after LPS challenge. In vivo, iloprost was effective against LPS-induced protein and neutrophil accumulation in bronchoalveolar lavage fluid, and reduced myeloperoxidase activation, ICAM-1 expression and Evans blue extravasation in the lungs. Inhibition of Rac activity abolished the barrier-protective and anti-inflammatory effects of iloprost and Br-cAMP. Iloprost-induced elevation of intracellular cAMP triggers Rac signalling, which attenuates LPS-induced NF-&kgr;B and p38 MAPK inflammatory pathways and the Rho-dependent mechanism of endothelial permeability.

Afadin controls p120-catenin-ZO-1 interactions leading to endothelial barrier enhancement by oxidized phospholipids.

Afadin is a novel regulator of epithelial cell junctions assembly. However, its role in the formation of endothelial cell junctions and the regulation of vascular permeability remains obscure. We previously described protective effects of oxidized 1‐palmitoyl‐2‐arachidonoyl‐sn‐glycero‐3‐phosphorylcholine (OxPAPC) in the in vitro and in vivo models of lung endothelial barrier dysfunction and acute lung injury, which were mediated by Rac GTPase. This study examined a role of afadin in the OxPAPC‐induced enhancement of interactions between adherens junctions and tight junctions as a novel mechanism of endothelial cell (EC) barrier preservation. OxPAPC induced Rap1‐dependent afadin accumulation at the cell periphery and Rap1‐dependent afadin interaction with adherens junction and tight junction proteins p120‐catenin and ZO‐1, respectively. Afadin knockdown using siRNA or ectopic expression of afadin mutant lacking Rap1 GTPase binding domain suppressed OxPAPC‐induced EC barrier enhancement and abolished barrier protective effects of OxPAPC against thrombin‐induced EC permeability. Afadin knockdown also abolished protective effects of OxPAPC against ventilator‐induced lung injury in vivo. These results demonstrate for the first time a critical role of afadin in the regulation of vascular barrier function in vitro and in vivo via coordination of adherens junction–tight junction interactions. J. Cell. Physiol. 227: 1883–1890, 2012.

Control of vascular permeability by atrial natriuretic peptide via a GEF-H1-dependent mechanism.

Background: Regulation of vascular permeability by microtubule (MT)-associated proteins is not well understood. Results: ANP promoted MT peripheral growth and protected endothelial barrier via Rac-PAK1-dependent inactivation of GEF-H1 and consequent suppression of Rho signaling. Conclusion: A PAK1-GEF-H1 dependent mechanism mediates endothelial barrier protection by ANP. Significance: Modulation of GEF-H1 activity represents a novel approach in prevention of pathologic vascular leak. 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.

Novel role of stathmin in microtubule-dependent control of endothelial permeability

Microtubule (MT) dynamics in vascular endothelium are modulated by vasoactive mediators and are critically involved in the control of endothelial cell (EC) permeability via Rho GTPase‐dependent crosstalk with the actin cytoskeleton. However, the role of regulators in MT stability in these mechanisms remains unclear. This study investigated the involvement of the MT‐associated protein stathmin in the mediation of agonist‐induced permeability in EC cultures and vascular leak in vivo. Thrombin treatment of human pulmonary ECs induced rapid dephosphorylation and activation of stathmin. Inhibition of stathmin activity by small interfering RNA‐based knockdown or cAMP‐mediated phosphorylation abrogated thrombin‐induced F‐actin remodeling and Rho‐dependent EC hyperpermeability, while expression of a phosphorylation‐deficient stathmin mutant exacerbated thrombininduced EC barrier disruption. Stathmin suppression preserved the MT network against thrombin‐induced MT disassembly and release of Rho‐specific guanine nucleotide exchange factor, GEF‐H1. The protective effects of stathmin knockdown were observed in vivo in the mouse 2‐hit model of ventilator‐induced lung injury and were linked to MT stabilization and down‐regulation of Rho signaling in the lung. These results demonstrate the mechanism of stathmin‐dependent control of MT dynamics, Rho signaling, and permeability and suggest novel potential pharmacological interventions in the prevention of increased vascular leak via modulation of stathmin activity.—Tian, X., Tian, Y., Sarich, N., Wu, T., Birukova, A. A. Novel role of stathmin in microtubule‐dependent control of endothelial permeability. FASEB J. 26, 3862–3874 (2012). www.fasebj.org

Stiffness-Activated GEF-H1 Expression Exacerbates LPS-Induced Lung Inflammation

Acute lung injury (ALI) is accompanied by decreased lung compliance. However, a role of tissue mechanics in modulation of inflammation remains unclear. We hypothesized that bacterial lipopolysacharide (LPS) stimulates extracellular matrix (ECM) production and vascular stiffening leading to stiffness-dependent exacerbation of endothelial cell (EC) inflammatory activation and lung barrier dysfunction. Expression of GEF-H1, ICAM-1, VCAM-1, ECM proteins fibronectin and collagen, lysyl oxidase (LOX) activity, interleukin-8 and activation of Rho signaling were analyzed in lung samples and pulmonary EC grown on soft (1.5 or 2.8 kPa) and stiff (40 kPa) substrates. LPS induced EC inflammatory activation accompanied by expression of ECM proteins, increase in LOX activity, and activation of Rho signaling. These effects were augmented in EC grown on stiff substrate. Stiffness-dependent enhancement of inflammation was associated with increased expression of Rho activator, GEF-H1. Inhibition of ECM crosslinking and stiffening by LOX suppression reduced EC inflammatory activation and GEF-H1 expression in response to LPS. In vivo, LOX inhibition attenuated LPS-induced expression of GEF-H1 and lung dysfunction. These findings present a novel mechanism of stiffness-dependent exacerbation of vascular inflammation and escalation of ALI via stimulation of GEF-H1 - Rho pathway. This pathway represents a fundamental mechanism of positive feedback regulation of inflammation.

A role for VEGFR2 activation in endothelial responses caused by barrier disruptive OxPAPC concentrations.

Introduction Oxidation products of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphatidylcholine (OxPAPC) differentially modulate endothelial cell (EC) barrier function in a dose-dependent fashion. Vascular endothelial growth factor receptor-2 (VEGFR2) is involved in the OxPAPC-induced EC inflammatory activation. This study examined a role of VEGFR2 in barrier dysfunction caused by high concentrations of OxPAPC and evaluated downstream signaling mechanisms resulting from the effect of OxPAPC in EC from pulmonary and systemic circulation. Methods EC monolayer permeability in human pulmonary artery endothelial cells (HPAEC) and human aortic endothelial cells (HAEC) was monitored by changes in transendothelial electrical resistance (TER) across EC monolayers. Actin cytoskeleton was examined by immunostaining with Texas Red labeled phalloidin. Phosphorylation of myosin light chains (MLC) and VE-Cadherin was examined by Western blot and immunofluorescence techniques. The role of VEGFR2 in OxPAPC-induced permeability and cytoskeletal arrangement were determined using siRNA-induced VEGFR2 knockdown. Results Low OxPAPC concentrations (5–20 µg/ml) induced a barrier protective response in both HPAEC and HAEC, while high OxPAPC concentrations (50–100 µg/ml) caused a rapid increase in permeability ; actin stress fiber formation and increased MLC phosphorylation were observed as early as 30 min after treatment. VEGFR2 knockdown dramatically decreased the amount of MLC phosphorylation and stress fiber formation caused by high OxPAPC concentrations with modest effects on the amount of VE-cadherin phosphorylation at Y731. We present evidence that activation of Rho is involved in the OxPAPC/VEGFR2 mechanism of EC permeability induced by high OxPAPC concentrations. Knockdown of VEGFR2 did not rescue the early drop in TER but prevented further development of OxPAPC-induced barrier dysfunction. Conclusions This study shows that VEGFR2 is involved in the delayed phase of EC barrier dysfunction caused by high OxPAPC concentrations and contributes to stress fiber formation and increased MLC phosphorylation.

Cell-type-specific crosstalk between p38 MAPK and Rho signaling in lung micro- and macrovascular barrier dysfunction induced by Staphylococcus aureus-derived pathogens

Lung inflammation and alterations in endothelial cell (EC) micro- and macrovascular permeability are key events to development of acute lung injury. Using ECs derived from human pulmonary artery and lung microvasculature, we investigated the interplay between p38 stress mitogen-activated protein kinase (MAPK) and Rho guanosine triphosphatase signaling in inflammatory and hyperpermeability responses. Both cell types were treated with Staphylococcus aureus-derived peptidoglycan (PepG) and lipoteichoic acid (LTA) with or without pretreatment with p38 MAPK or Rho kinase inhibitors. LTA and PepG increased permeability markedly in both pulmonary macrovascular and microvascular ECs. Agonist-induced hyperpermeability was accompanied by cytoskeletal remodeling, disruption of cell-cell contacts, formation of paracellular gaps, and activation of p38 MAPK, nuclear factor kappa-B (NFκB), and Rho/Rho kinase signaling. In macrovascular ECs, pharmacologic inhibition of Rho kinase with Y27632 suppressed p38 MAP kinase cascade activation significantly, whereas inhibition of p38 MAPK with SB203580 had no effect on Rho activation. In contrast, inhibition of p38 MAPK in microvascular ECs suppressed LTA/PepG-induced activation of Rho, whereas the Rho inhibitor suppressed activation of p38 MAPK. Inhibition of either p38 MAPK or Rho kinase attenuated activation of NFκB signaling substantially. These results demonstrate cell-type-specific differences in signaling induced by Staphylococcus aureus-derived pathogens in pulmonary endothelium. Thus, although Gram-positive bacterial compounds caused barrier dysfunction in both EC types, it was induced by a different pattern of crosstalk between Rho, p38 MAPK, and NFκB signaling. These observations may have important implications in defining microvasculature-specific therapeutic strategies aimed at the treatment of sepsis and acute lung injury induced by Gram-positive bacterial pathogens.