Sphingosine 1-phosphate promotes endothelial cell barrier integrity by Edg-dependent cytoskeletal rearrangement
Joe G.N. Garcia, Feng Liu, Alexander D. Verin, Anna Birukova, Melissa A. Dechert, William T. Gerthoffer, James R. Bamberg, Denis English
Joe G.N. Garcia, Feng Liu, Alexander D. Verin, Anna Birukova, Melissa A. Dechert, William T. Gerthoffer, James R. Bamberg, Denis English
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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 Giα-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.

Authors

Joe G.N. Garcia, Feng Liu, Alexander D. Verin, Anna Birukova, Melissa A. Dechert, William T. Gerthoffer, James R. Bamberg, Denis English

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Sph-1-P enhances endothelial cell barrier properties. Endothelial cells ...
Sph-1-P enhances endothelial cell barrier properties. Endothelial cells were plated on gold microelectrodes to measure TER and were cultured to confluence. Growth medium was replaced with serum-free M199, and after equilibration and stabilization, cells were challenged with agonist (Sph-1-P or thrombin). Data are representative of multiple independent experiments (minimum of three). Shown are the responses to increasing concentrations of Sph-1-P in bovine pulmonary artery endothelial cells (BPAEC) (a) and in bovine lung microvascular endothelial cells (BLMVEC) (b). (c) The effect of Sph-1-P on thrombin-induced barrier dysfunction in human pulmonary artery endothelial cells (HPAEC). In these experiments, endothelial cells were simultaneously treated with Sph-1-P (1 μM, 30 minutes) and with the potent edemagenic agent thrombin (100 nM) (38). Sph-1-P consistently elicits a significant increase in TER, which returns to baseline control values in the presence of thrombin but does not exhibit the 40% decline below control values produced by thrombin alone. Furthermore, Sph-1-P rapidly and consistently restores human endothelial cell barrier integrity in cells stimulated previously with thrombin (d).