Nicolene Sarich

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.

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.

VE-cadherin trans-interactions modulate Rac activation and enhancement of lung endothelial barrier by iloprost.

Small GTPase Rac is important regulator of endothelial cell (EC) barrier enhancement by prostacyclin characterized by increased peripheral actin cytoskeleton and increased interactions between VE‐cadherin and other adherens junction (AJ) proteins. This study utilized complementary approaches including siRNA knockdown, culturing in Ca2+‐free medium, and VE‐cadherin blocking antibody to alter VE‐cadherin extracellular interactions to investigate the role of VE‐cadherin outside‐in signaling in modulation of Rac activation and EC barrier regulation by prostacyclin analog iloprost. Spatial analysis of Rac activation in pulmonary EC by FRET revealed additional spike in iloprost‐induced Rac activity at the sites of newly formed cell–cell junctions. In contrast, disruption of VE‐cadherin extracellular trans‐interactions suppressed iloprost‐activated Rac signaling and attenuated EC barrier enhancement and cytoskeletal remodeling. These inhibitory effects were associated with decreased membrane accumulation and activation of Rac‐specific guanine nucleotide exchange factors (GEFs) Tiam1 and Vav2. Conversely, plating of pulmonary EC on surfaces coated with extracellular VE‐cadherin domain further promoted iloprost‐induced Rac signaling. In the model of thrombin‐induced EC barrier recovery, blocking of VE‐cadherin trans‐interactions attenuated activation of Rac pathway during recovery phase and delayed suppression of Rho signaling and restoration of EC barrier properties. These results suggest that VE‐cadherin outside‐in signaling controls locally Rac activity stimulated by barrier protective agonists. This control is essential for maximal EC barrier enhancement and accelerated barrier recovery. J. Cell. Physiol. 227: 3405–3416, 2012.

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.

Oxidized phospholipids protect against lung injury and endothelial barrier dysfunction caused by heat-inactivated Staphylococcus aureus

Increased endothelial cell (EC) permeability and vascular inflammation along with alveolar epithelial damage are key features of acute lung injury (ALI). Products of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine oxidation (OxPAPC) showed protective effects against inflammatory signaling and vascular EC barrier dysfunction induced by gram-negative bacterial wall lipopolysaccharide (LPS). We explored the more general protective effects of OxPAPC and investigated whether delayed posttreatment with OxPAPC boosts the recovery of lung inflammatory injury and EC barrier dysfunction triggered by intratracheal injection of heat-killed gram-positive Staphylococcus aureus (HKSA) bacteria. HKSA-induced pulmonary EC permeability, activation of p38 MAP kinase and NF-κB inflammatory cascades, secretion of IL-8 and soluble ICAM1, fibronectin deposition, and expression of adhesion molecules ICAM1 and VCAM1 by activated EC were significantly attenuated by cotreatment as well as posttreatment with OxPAPC up to 16 h after HKSA addition. Remarkably, posttreatment with OxPAPC up to 24 h post-HKSA challenge dramatically accelerated lung recovery by restoring lung barrier properties monitored by Evans blue extravasation and protein content in bronchoalveolar lavage (BAL) fluid and reducing inflammation reflected by decreased MIP-1, KC, TNF-α, IL-13 levels and neutrophil count in BAL samples. These studies demonstrate potent in vivo and in vitro protective effects of posttreatment with anti-inflammatory oxidized phospholipids in the model of ALI caused by HKSA. These results warrant further investigations into the potential use of OxPAPC compounds combined with antibiotic therapies as a treatment of sepsis and ALI induced by gram-positive bacterial pathogens.

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

Prostacyclin post-treatment improves LPS-induced acute lung injury and endothelial barrier recovery via Rap1☆

Protective effects of prostacyclin (PC) or its stable analog beraprost against agonist-induced lung vascular inflammation have been associated with elevation of intracellular cAMP and Rac GTPase signaling which inhibited the RhoA GTPase-dependent pathway of endothelial barrier dysfunction. This study investigated a distinct mechanism of PC-stimulated lung vascular endothelial (EC) barrier recovery and resolution of LPS-induced inflammation mediated by small GTPase Rap1. Efficient barrier recovery was observed in LPS-challenged pulmonary EC after prostacyclin administration even after 15 h of initial inflammatory insult and was accompanied by the significant attenuation of p38 MAP kinase and NFκB signaling and decreased production of IL-8 and soluble ICAM1. These effects were reproduced in cells post-treated with 8CPT, a small molecule activator of Rap1-specific nucleotide exchange factor Epac. By contrast, pharmacologic Epac inhibitor, Rap1 knockdown, or knockdown of cell junction-associated Rap1 effector afadin attenuated EC recovery caused by PC or 8CPT post-treatment. The key role of Rap1 in lung barrier restoration was further confirmed in the murine model of LPS-induced acute lung injury. Lung injury was monitored by measurements of bronchoalveolar lavage protein content, cell count, and Evans blue extravasation and live imaging of vascular leak over 6 days using a fluorescent tracer. The data showed significant acceleration of lung recovery by PC and 8CPT post-treatment, which was abrogated in Rap1a(-/-) mice. These results suggest that post-treatment with PC triggers the Epac/Rap1/afadin-dependent mechanism of endothelial barrier restoration and downregulation of p38MAPK and NFκB inflammatory cascades, altogether leading to accelerated lung recovery.

Anti-Inflammatory Effects of OxPAPC Involve Endothelial Cell Mediated Generation of LXA4

Rationale: Oxidation of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC) generates a group of bioactive oxidized phospholipid products with a broad range of biological activities. Barrier-enhancing and anti-inflammatory effects of OxPAPC on pulmonary endothelial cells are critical for prevention of acute lung injury caused by bacterial pathogens or excessive mechanical ventilation. Anti-inflammatory properties of OxPAPC are associated with its antagonistic effects on Toll-like receptors and suppression of RhoA GTPase signaling. Objective: Because OxPAPC exhibits long-lasting anti-inflammatory and lung-protective effects even after single administration in vivo, we tested the hypothesis that these effects may be mediated by additional mechanisms, such as OxPAPC-dependent production of anti-inflammatory and proresolving lipid mediator, lipoxin A4 (LXA4). Methods and Results: Mass spectrometry and ELISA assays detected significant accumulation of LXA4 in the lungs of OxPAPC-treated mice and in conditioned medium of OxPAPC-exposed pulmonary endothelial cells. Administration of LXA4 reproduced anti-inflammatory effect of OxPAPC against tumor necrosis factor-&agr; in vitro and in the animal model of lipopolysaccharide-induced lung injury. The potent barrier-protective and anti-inflammatory effects of OxPAPC against tumor necrosis factor-&agr; and lipopolysaccharide challenge were suppressed in human pulmonary endothelial cells with small interfering RNA–induced knockdown of LXA4 formyl peptide receptor-2 (FPR2/ALX) and in mFPR2−/− (mouse formyl peptide receptor 2) mice lacking the mouse homolog of human FPR2/ALX. Conclusions: This is the first demonstration that inflammation- and injury-associated phospholipid oxidation triggers production of anti-inflammatory and proresolution molecules, such as LXA4. This lipid mediator switch represents a novel mechanism of OxPAPC-assisted recovery of inflamed lung endothelium.

Role of microtubules in attenuation of PepG-induced vascular endothelial dysfunction by atrial natriuretic peptide.

Apart from control of circulating fluid, atrial natriuretic peptide (ANP) exhibits anti-inflammatory effects in the lung. However, molecular mechanisms of ANP anti-inflammatory effects are not well-understood. Peripheral microtubule (MT) dynamics is essential for agonist-induced regulation of vascular endothelial permeability. Here we studied the role of MT-dependent signaling in ANP protective effects against endothelial cell (EC) barrier dysfunction and acute lung injury induced by Staphylococcus aureus-derived peptidoglican-G (PepG). PepG-induced vascular endothelial dysfunction was accompanied by MT destabilization and disruption of MT network. ANP attenuated PepG-induced MT disassembly, NFκB signaling and activity of MT-associated Rho activator GEF-H1 leading to attenuation of EC inflammatory activation reflected by expression of adhesion molecules ICAM1 and VCAM1. ANP-induced EC barrier preservation and MT stabilization were linked to phosphorylation and inactivation of MT-depolymerizing protein stathmin. Expression of stathmin phosphorylation-deficient mutant abolished ANP protective effects against PepG-induced inflammation and EC permeability. In contrast, siRNA-mediated stathmin knockdown prevented PepG-induced peripheral MT disassembly and endothelial barrier dysfunction. ANP protective effects in a murine model of PepG-induced lung injury were associated with increased phosphorylation of stathmin, while exacerbated lung injury in the ANP knockout mice was accompanied by decreased pool of stable MT. Stathmin knockdown in vivo reversed exacerbation of lung injury in the ANP knockout mice. These results show a novel MT-mediated mechanism of endothelial barrier protection by ANP in pulmonary EC and animal model of PepG-induced lung injury via stathmin-dependent control of MT assembly.