Sphingosine 1-phosphate promotes endothelial cell barrier integrity by Edg-dependent cytoskeletal rearrangement
Joe G.N. Garcia, … , James R. Bamberg, Denis English
Joe G.N. Garcia, … , James R. Bamberg, Denis English
Published September 1, 2001
Citation Information: J Clin Invest. 2001;108(5):689-701. https://doi.org/10.1172/JCI12450.
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Article

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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Signaling events involved in Sph-1-P–mediated endothelial cell barrier e...
Signaling events involved in Sph-1-P–mediated endothelial cell barrier enhancement. (a) Bovine endothelial cells were incubated with tyrosine kinase inhibitors genistein (Gen; 100 μM, 30 minutes), herbimycin A (Herb; 5 μM, 8 hours), or erbstatin analog (Erb; 20 μM, 1 hour); the PI-3′ kinase inhibitor LY294002 (LY; 25 μM, 30 minutes), p42/p44 MAP kinase inhibitor UO126 (UO; 10 μM, 30 minutes), the p38 MAP kinase inhibitor SB203580 (SB; 20 μM, 30 minutes), or p60 src inhibitor PP2 (10 μM, 30 minutes), followed by stimulation with Sph-1-P (1 μM). TER was continuously measured for 3 hours and the maximal Sph-1-P response (10–15 minutes after stimulation) were employed for determining the effect of the inhibitors on Sph-1-P–induced barrier enhancement. Data are presented as mean ± SE, n = 3, P < 0.05. Genistein, herbimycin A, and erbstatin analogues did not significantly alter the basal TER, but inhibited Sph-1-P–induced increases in TER, indicating the involvement of tyrosine kinases in Sph-1-P–mediated modulation of endothelial cell barrier integrity. (b) Endothelial cells were incubated with Sph-1-P (1 μM) for indicated times, followed by Western analysis using an phosphotyrosine Ab. Sph-1-P stimulated tyrosine phosphorylation of multiple proteins as indicated by arrows.