Dataset Information

FTY720-induced human pulmonary endothelial barrier enhancement is mediated by c-Abl.

ABSTRACT: Strategies to improve pulmonary endothelial barrier function are needed to reverse the devastating effects of vascular leak in acute respiratory distress syndrome. FTY720 is a pharmaceutical analogue of the potent barrier-enhancing phospholipid sphingosine 1-phosphate (S1P). FTY720 decreases vascular permeability by an incompletely characterised mechanism that differs from S1P. Here, we describe its barrier-promoting effects on intracellular signalling and junctional assembly formation in human pulmonary endothelium. Permeability of cultured human pulmonary endothelial cells was assessed using transendothelial electrical resistance and dextran transwell assays. Junctional complex formation was assessed using membrane fractionation and immunofluorescence. Pharmacological inhibitors and small interfering (si)RNA were utilised to determine the effects of individual components on permeability. Unlike S1P, FTY720 failed to induce membrane translocation of adherens junction or tight junction proteins. ?-catenin, occludin, claudin-5 or zona occludens protein (ZO)-1/ZO-2 siRNAs did not alter FTY720-induced barrier enhancement. FTY720 induced focal adhesion kinase (FAK) phosphorylation and focal adhesion formation, with FAK siRNA partially attenuating the prolonged phase of barrier enhancement. Inhibition of Src, protein kinase (PK)A, PKG, PKC or protein phosphatase 2A failed to alter FTY720-induced barrier enhancement. FTY720 increased c-Abl tyrosine kinase activity and c-Abl siRNA attenuated peak barrier enhancement after FTY720. FTY720 enhances endothelial barrier function by a novel pathway involving c-Abl signalling.

SUBMITTER: Wang L

PROVIDER: S-EPMC3758235 | biostudies-literature | 2011 Jul

REPOSITORIES: biostudies-literature

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FTY720-induced human pulmonary endothelial barrier enhancement is mediated by c-Abl.

Wang L L Chiang E T ET Simmons J T JT Garcia J G N JG Dudek S M SM

The European respiratory journal 20101111 1

Strategies to improve pulmonary endothelial barrier function are needed to reverse the devastating effects of vascular leak in acute respiratory distress syndrome. FTY720 is a pharmaceutical analogue of the potent barrier-enhancing phospholipid sphingosine 1-phosphate (S1P). FTY720 decreases vascular permeability by an incompletely characterised mechanism that differs from S1P. Here, we describe its barrier-promoting effects on intracellular signalling and junctional assembly formation in human ...[more]

PMID: 21071472

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Project description:Endothelial cell (EC) barrier dysfunction induced by inflammatory agonists is a frequent pathophysiologic event in multiple diseases. The platelet-derived phospholipid sphingosine-1 phosphate (S1P) reverses this dysfunction by potently enhancing the EC barrier through a process involving Rac GTPase-dependent cortical actin rearrangement as an integral step. In this study we explored the role of the ezrin, radixin, and moesin (ERM) family of actin-binding linker protein in modulating S1P-induced human pulmonary EC barrier enhancement. S1P induces ERM translocation to the EC periphery and promotes ERM phosphorylation on a critical threonine residue (Ezrin-567, Radixin-564, Moesin-558). This phosphorylation is dependent on activation of PKC isoforms and Rac1. The majority of ERM phosphorylation on these critical threonine residues after S1P occurs in moesin and ezrin. Baseline radixin phosphorylation is higher than in the other two ERM proteins but does not increase after S1P. S1P-induced moesin and ezrin threonine phosphorylation is not mediated by the barrier enhancing receptor S1PR1 because siRNA downregulation of S1PR1 fails to inhibit these phosphorylation events, while stimulation of EC with the S1PR1-specific agonist SEW2871 fails to induce these phosphorylation events. Silencing of either all ERM proteins or radixin alone (but not moesin alone) reduced S1P-induced Rac1 activation and phosphorylation of the downstream Rac1 effector PAK1. Radixin siRNA alone, or combined siRNA for all three ERM proteins, dramatically attenuates S1P-induced EC barrier enhancement (measured by transendothelial electrical resistance (TER), peripheral accumulation of di-phospho-MLC, and cortical cytoskeletal rearrangement. In contrast, moesin depletion has the opposite effects on these parameters. Ezrin silencing partially attenuates S1P-induced EC barrier enhancement and cytoskeletal changes. Thus, despite structural similarities and reported functional redundancy, the ERM proteins differentially modulate S1P-induced alterations in lung EC cytoskeleton and permeability. These results suggest that ERM activation is an important regulatory event in EC barrier responses to S1P.

Project description:Vascular permeability is a hallmark of acute respiratory distress syndrome (ARDS) and ventilator-induced lung injury pathobiology; however, the mechanisms underlying this vascular dysregulation remain unclear, thereby impairing the development of desperately needed effective therapeutics. We have shown that sphingosine-1-phosphate (S1P) and 2-amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol (FTY720) analogues are useful tools for exploring vascular barrier regulation mechanisms. To experimentally define the effects of FTY720 regioisomers on lung endothelial cell barrier regulation. Specific barrier-regulatory receptor and kinase inhibitors were utilized to probe signaling mechanisms involved in FTY720 regioisomer-mediated human lung endothelial cell barrier responses (trans-endothelial electrical resistance, TER). Docking simulations with the S1P1 receptor were performed to further evaluate FTY720 regioisomer signaling. FTY720 regioisomers produced potent endothelial cell barrier disruption reflected by declines in TER alterations. Pharmacologic inhibition of Gi-coupled S1P receptors (S1P1, S1P2, S1P3) failed to alter FTY720 regioisomer-mediated barrier disruption; findings that were corroborated by docking simulations demonstrating FTY720 regiosomers were repelled from S1P1 docking, in contrast to strong S1P1 binding elicited by S1P. Inhibition of either the barrier-disrupting PAR-1 receptor, the VEGF receptor, Rho-kinase, MAPK, NFkB, or PI3K failed to alter FTY720 regioisomer-induced endothelial cell barrier disruption. While FTY720 regioisomers significantly increased protein phosphatase 2 (PP2A) activity, PP2A inhibitors failed to alter FTY720 regioisomer-induced endothelial cell barrier disruption. Together, these results imply a vexing model of pulmonary vascular barrier dysregulation in response to FTY720-related compounds and highlight the need for further insights into mechanisms of vascular integrity required to promote the development of novel therapeutic tools to prevent or reverse the pulmonary vascular leak central to ARDS outcomes.

Project description:ObjectivesAcute respiratory distress syndrome represents the devastating result of acute lung injury, with high mortality. Limited methods are available for rehabilitation of lungs affected by acute respiratory distress syndrome. Our laboratory has demonstrated rehabilitation of sepsis-injured lungs via normothermic ex vivo and in vivo perfusion with Steen solution (Steen). However, mechanisms responsible for the protective effects of Steen remain unclear. This study tests the hypothesis that Steen directly attenuates pulmonary endothelial barrier dysfunction and inflammation induced by lipopolysaccharide.MethodsPrimary pulmonary microvascular endothelial cells were exposed to lipopolysaccharide for 4 hours and then recovered for 8 hours in complete media (Media), Steen, or Steen followed by complete media (Steen/Media). Oxidative stress, chemokines, permeability, interendothelial junction proteins, and toll-like receptor 4-mediated pathways were assessed in pulmonary microvascular endothelial cells using standard methods.ResultsLipopolysaccharide treatment of pulmonary microvascular endothelial cells and recovery in Media significantly induced reactive oxygen species, lipid peroxidation, expression of chemokines (eg, chemokine [C-X-C motif] ligand 1 and C-C motif chemokine ligand 2) and cell adhesion molecules (P-selectin, E-selectin, and vascular cell adhesion molecule 1), permeability, neutrophil transmigration, p38 mitogen-activated protein kinase and nuclear factor kappa B signaling, and decreased expression of tight and adherens junction proteins (zonula occludens-1, zonula occludens-2, and vascular endothelial-cadherin). All of these inflammatory pathways were significantly attenuated after recovery of pulmonary microvascular endothelial cells in Steen or Steen/Media.ConclusionsSteen solution preserves pulmonary endothelial barrier function after lipopolysaccharide exposure by promoting an anti-inflammatory environment via attenuation of oxidative stress, toll-like receptor 4-mediated signaling, and conservation of interendothelial junctions. These protective mechanisms offer insight into the advancement of methods for in vivo lung perfusion with Steen for the treatment of severe acute respiratory distress syndrome.

Project description:The maintenance of endothelial barrier function is essential for normal physiology, and increased vascular permeability is a feature of a wide variety of pathological conditions, leading to complications including edema and tissue damage. Use of the pharmacological inhibitor imatinib, which targets the Abl family of non-receptor tyrosine kinases (Abl and Arg), as well as other tyrosine kinases including the platelet-derived growth factor receptor (PDGFR), Kit, colony stimulating factor 1 receptor (CSF1R), and discoidin domain receptors, has shown protective effects in animal models of inflammation, sepsis, and other pathologies characterized by enhanced vascular permeability. However, the imatinib targets involved in modulation of vascular permeability have not been well-characterized, as imatinib inhibits multiple tyrosine kinases not only in endothelial cells and pericytes but also immune cells important for disorders associated with pathological inflammation and abnormal vascular permeability. In this work we employ endothelial Abl knockout mice to show for the first time a direct role for Abl in the regulation of vascular permeability in vivo. Using both Abl/Arg-specific pharmacological inhibition and endothelial Abl knockout mice, we demonstrate a requirement for Abl kinase activity in the induction of endothelial permeability by vascular endothelial growth factor both in vitro and in vivo. Notably, Abl kinase inhibition also impaired endothelial permeability in response to the inflammatory mediators thrombin and histamine. Mechanistically, we show that loss of Abl kinase activity was accompanied by activation of the barrier-stabilizing GTPases Rac1 and Rap1, as well as inhibition of agonist-induced Ca(2+) mobilization and generation of acto-myosin contractility. In all, these findings suggest that pharmacological targeting of the Abl kinases may be capable of inhibiting endothelial permeability induced by a broad range of agonists and that use of Abl kinase inhibitors may have potential for the treatment of disorders involving pathological vascular leakage.

Project description:Vascular endothelial barrier dysfunction is the most prominent manifestation and important cause of mortality in infectious acute lung injury (ALI). Exogenous apelin is effective in ameliorating lipopolysaccharide (LPS)-induced inflammatory response in ALI lungs, reducing exudation of lung tissue and decreasing mortality. This study set out to investigate the association between apelin and Friend leukemia integration-1 (Fli-1) in the prevention and treatment of ALI, and to elucidate the molecular mechanism by which apelin protects the permeability of the vascular endothelial barrier. At the vivo functional level, lung wet/dry weight ratio was used to detect whole lung permeability, evans blue assay and dual fluorescent protein tracking assay were used to detect lung vascular endothelial permeability, HE staining to observe the inflammatory status of lung tissue, and immunofluorescence staining for VE-cadherin expression levels in blood vessels. The changes in inflammatory factors in bronchoalveolar lavage fluid (BALF) were detected by ELASA. Western blot was used to detect the expression level of proteins. qRT-PCR was performed to detect changes in mRNA expression of Fli-1 and adherent junction-related proteins. The correlation analysis of Fli-1 with vascular endothelial permeability and SRC showed that Fli-1 participated in the process of ALI. After preventive and therapeutic treatment of ALI mice with exogenous apelin, Fli-1, APJ, VE-cadherin, phosphorylated-VE-cadherin (p-VE-cadherin) and β-catenin were up-regulated, while SRC, phosphorylated-SRC (p-SRC), VEGF and VEGF-R were down-regulated, which indicated that the stability of vascular endothelial barrier was enhanced. With the use of Fli-1 inhibitor irinotecan, the protective effect of apelin was weakened in various functional indexes, genes and proteins. The lung was maintained at the level of the injury. Our research shows that Fli-1 is involved in the LPS-induced ALI process. The molecular mechanism for apelin in preventing endothelial barrier dysfunction in ALI is through up-regulating Fli-1, thus regulating adherens junction-related proteins, and finally recovering the endothelial barrier function.

Dataset Information

FTY720-induced human pulmonary endothelial barrier enhancement is mediated by c-Abl.

Publications

FTY720-induced human pulmonary endothelial barrier enhancement is mediated by c-Abl.

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