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Sphingosine 1-phosphate promotes endothelial cell barrier integrity by Edg-dependent cytoskeletal rearrangement.

ABSTRACT: Substances released by platelets during blood clotting are essential participants in events that link hemostasis and angiogenesis and ensure adequate wound healing and tissue injury repair. We assessed the participation of sphingosine 1-phosphate (Sph-1-P), a biologically active phosphorylated lipid growth factor released from activated platelets, in the regulation of endothelial monolayer barrier integrity, which is key to both angiogenesis and vascular homeostasis. Sph-1-P produced rapid, sustained, and dose-dependent increases in transmonolayer electrical resistance (TER) across both human and bovine pulmonary artery and lung microvascular endothelial cells. This substance also reversed barrier dysfunction elicited by the edemagenic agent thrombin. Sph-1-P-mediated barrier enhancement was dependent upon G(ialpha)-receptor coupling to specific members of the endothelial differentiation gene (Edg) family of receptors (Edg-1 and Edg-3), Rho kinase and tyrosine kinase-dependent activation, and actin filament rearrangement. Sph-1-P-enhanced TER occurred in conjunction with Rac GTPase- and p21-associated kinase-dependent endothelial cortical actin assembly with recruitment of the actin filament regulatory protein, cofilin. Platelet-released Sph-1-P, linked to Rac- and Rho-dependent cytoskeletal rearrangement, may act late in angiogenesis to stabilize newly formed vessels, which often display abnormally increased vascular permeability.

SUBMITTER: Garcia JG

PROVIDER: S-EPMC209379 | biostudies-literature | 2001 Sep

REPOSITORIES: biostudies-literature

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Sphingosine 1-phosphate promotes endothelial cell barrier integrity by Edg-dependent cytoskeletal rearrangement.

Garcia J G JG Liu F F Verin A D AD Birukova A A Dechert M A MA Gerthoffer W T WT Bamberg J R JR English D D

The Journal of clinical investigation 20010901 5

Substances released by platelets during blood clotting are essential participants in events that link hemostasis and angiogenesis and ensure adequate wound healing and tissue injury repair. We assessed the participation of sphingosine 1-phosphate (Sph-1-P), a biologically active phosphorylated lipid growth factor released from activated platelets, in the regulation of endothelial monolayer barrier integrity, which is key to both angiogenesis and vascular homeostasis. Sph-1-P produced rapid, sust ...[more]

PMID: 11544274

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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: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.

Dataset Information

Sphingosine 1-phosphate promotes endothelial cell barrier integrity by Edg-dependent cytoskeletal rearrangement.

Publications

Sphingosine 1-phosphate promotes endothelial cell barrier integrity by Edg-dependent cytoskeletal rearrangement.

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