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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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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Figure 8

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Role of Rac GTPase in Sph-1-P–mediated endothelial actin rearrangement a...
Role of Rac GTPase in Sph-1-P–mediated endothelial actin rearrangement and barrier regulation. (a) Bovine pulmonary artery endothelium were incubated with vehicle (v) Sph-1-P (1 μM), for indicated periods of time, or pretreated with PTX (1 μg/ml, 2 hours) and subsequently incubated with Sph-1-P for 1 minute. Cells were lysed, centrifuged, and supernatants were collected. The activated GTP-bound Rac was precipitated by agarose-conjugated human PAK-1 p21-binding domain and immunoblotted by anti-Rac mAb as described in Methods. Total Rac content was detected using cell lysates. These results indicate rapid Sph-1-P–induced Rac activation, which was completely abolished by PTX. (b–g) Bovine endothelium was transfected with either an empty vector (b and e) or a constitutively active HA-tagged Rac construct (Rac V12) (c, d, f, and g) as described in Methods. Shown are subsequent immunofluorescence images (×100) of endothelial cells stained with either Texas red phalloidin for F-actin (b–d) or anti-HA tag Ab for identification of transfected cells (e–g). b and e, c and f, d and g represent matched images. Overexpression of constitutively active Rac V12, but not control vector, significantly enhances polymerized actin staining (arrows) within the cortical ring with the degree of enhancement dependent upon the level of Rac V-12 overexpression.