Iloprost transcriptionally regulates protease-activated receptor-3 (PAR-3) in human vascular smooth muscle cells

Dr. Rosenkranz, Anke, Dr. Rauch, Bernhard, Prof. Dr. Schrör, Karsten. Universitätsklinikum der Heinrich-Heine Universität Düsseldorf Institut für Pharmakologie & Klinische Pharmakologie, Universitätsstraße 1, 40225 Düsseldorf, Germany.

Background and purpose: Proteolytic cleavage of PAR-3 by the coagulation factor thrombin induces vascular smooth muscle cell (SMC) mitogenesis. Unlike the classical thrombin receptor PAR-1, PAR-3 transcription is dynamically regulated by thrombin and may represent a unique link between vessel injury and remodeling. Prostacyclin (PGI2) is a functional antagonist of thrombin. We examined if iloprost, a PGI2-mimetic, counteracts the vascular effects of thrombin via transcriptional control of PAR-3. Experimental approach: Human saphenous vein SMC were serum-deprived (48h) prior to stimulation ± study drugs. Gene and protein-expression levels were quantified by realtime PCR and western blotting. Mitogenesis was determined by [3H]-thymidine incorporation. Key results: Iloprost significantly attenuated PAR-3 mRNA and protein expression (by 43±0.2 and 66±0.1%) and blunted the ability of thrombin or PAR-3 activating peptide TFRGAP to stimulate SMC mitogenesis (from 156±11 to 112±11% of control) and IL-6 expression (from 5.1±1.6 to 1.2±0.6 fold of control). The inhibitory effect of iloprost on PAR-3 expression was mimicked by agents elevating intracellular cyclic AMP (forskolin, isobutylmethylxanthine, dibutyryl-cAMP) and was prevented by a cell-permeant inhibitor of protein kinase A (myristoyl-PKI). Iloprost, acting via protein kinase A, also significantly reduced nuclear localisation (by 48±0.3%) of nuclear factor of activated T-cells (NFAT). Inhibition of NFAT with cyclosporin A or siRNA-mediated knock-down attenuated PAR-3 expression and the induction of IL-6 by thrombin or TFRGAP to a similar degree as iloprost. Conclusions and implications: Iloprost counteracts the mitogenic and inflammatory actions of thrombin in human vascular SMC in part via transcriptional control of PAR-3, a dynamically regulated thrombin receptor. This mechanism is mediated via protein kinase and inhibition of NFAT. These findings raise the exciting possibility that iloprost applied for example to bypass vessels ex vivo might limit thrombin-mediated hyperplasia and inflammation post revascularisation.