Expression of sphingosine kinase-1 is enhanced by the activated coagulation factor-X in human vascular smooth muscle cells

Ermler, Swen , Böhm, Andreas , Dr. Rosenkranz, Anke C. , Prof. Dr. Fischer, Jens W. , Prof. Dr. Schrör, Karsten , Dr. Rauch, Bernhard H. Universitätsklinikum Düsseldorf Institut für Pharmakologie und Klinische Pharmakologie, Universitätsstr. 1, 40225 Düsseldorf, Germany.

Sphingosine kinase-1 (SK1) generates the lipid signaling molecule sphingosine-1-phosphate (S1P), which regulates endothelial permeability and SMC proliferation. Here we demonstrate that the activated coagulation factor-X (FXa) induces expression of SK1 in human vascular smooth muscle cells (SMC) and enhances S1P synthesis. This mechanism enables FXa to antagonize thrombin-induced endothelial cell (EC) layer damage and is required for SMC mitogenesis.

Human aortic SMC and saphenous vein EC were cultured separately or in co-culture. EC permeability was determined by passage of FITC-labelled bovine serum albumin (BSA). SK1 expression was determined by RT-PCR/qPCR, Western blotting and immunostaining. S1P was measured by thin-layer chromatography and HPLC.

FXa induced a significant time (3h to 24h) and concentration-dependent (10-100 nM) upregulation of SK1 mRNA (3-fold) and protein (2-fold) expression in human SMC. This was accompanied by an increased production of S1P by human SMC. In co-culture experiments, EC permeability was increased by thrombin (1-10 U/ml) within 2 hours of incubation. This thrombin-induced EC permeability was attenuated by 80% when SMC were pre-treated with FXa for 24 hours. This effect of FXa was abolished by co-incubation of SMC with a selective SK1 inhibitor. In addition, SK1 inhibition inhibited the FXa-induced mitogenic response in human SMC. In atherosclerotic plaques from 16-weeks old ApoE-deficient mice, either on high-fed or regular diet, intraplaque FX/FXa was observed and co-localized with expression of SK1. This may suggest transendothelial diffusion of FX and activation to FXa which leads to increased SK1 expression in atherosclerotic plaques.

In conclusion, FXa induced SK1 expression and S1P production in human SMC, and thereby antagonizes thrombin-enhanced EC permeability and is required for SMC mitogenesis. This activity may be relevant for endothelial barrier protection in atherosclerotic tissues and the response to injury in vivo.