PGE2 cigarette smoke-induced modulates endothelial tissue factor: role of EP1 receptor and SIRT1

Background: Cigarette smoke exposure increases the risk of atherothrombosis, induces the expression of cyclooxygenase-2 (COX-2) and the release COX-2 derived eicosanoids, including Prostaglandin E2 (PGE2). Moreover, cigarette smoke up-regulates the expression and activity of Tissue Factor (TF), the main initiator of the coagulation cascade. However, the relationship between PGE2 and TF has not yet been elucidated. Aims: In this study, we analysed the link between PGE2 and TF, and the impact of their regulation on cigarette smoke-induced atherothrombosis. Methods: The levels of PGE2 and TF expression and activity in plasma were measured in 46 healthy active smokers (AS; 30 men and 16 women) and 19 non-smokers (NS; eight men and 11 women). In addition, PGE2 and TF were also measured in aorta tissue of mice and in mouse cardiac endothelial cells (MCEC) treated with aqueous extracts of cigarette smoke (TS) plus IL-1b (TS/IL-1b) by EIA assay, real-time PCR and procoagulant activity, using one stage-clotting assay, and/or by Zymuphen MP-TF. Arachidonic acid-induced thrombosis was used to study the effect of TS/IL-1b in mice. The expression of SIRT1, the NAD+-dependent protein deacetylase, was measured by Western blotting and by Immunofluorescence. Results: Higher levels of both PGE2 and TF were detected in plasma of AS compared to NS. Similar results were obtained in mice and in MCEC treated with TS/IL-1b. A highly significant correlation between PGE2 and TF activity was observed in both human plasma and mouse tissue. Inhibition of PGE synthase (PGES) activity by CAY10526, or by specific PGES siRNA, markedly diminished both in vitro and in vivo the amount of TF and the mouse carotid artery thrombosis induced by TS/IL-1b. In contrast, treatment with exogenous PGE2 increased both TF and arterial mouse thrombosis. MCEC express three PGE2 receptors: EP1, EP2 and EP4. In these cells, EP1 antagonists (AH6809 and SC51089) prevented the TF induced by TS/IL-1b. In contrast, an EP1 agonist (17-phenyl-trinor- PGE2,) increased TF. We excluded the involvement of other EP receptors because an EP4 antagonist (GW627368X) and EP2/EP4 agonists (misoprostol, butaprost) did not modify TF. The role of SIRT1 in the regulation of TF was also analysed. Both Sirtinol, an SIRT1 deactivator, and the specific SIRT1 siRNA increased TF expression and activity. In contrast, the SIRT-1 activators (resveratrol and CAY10591) reduced TF induced by both TS/IL-1b and EP1 agonist. Finally, EP1 agonist or the selective PGES-1 siRNA prevented the inhibition of SIRT1 mediated by PGE2 or byTS/IL-1b. Summary/Conclusion: We conclude that PGE2 increases TF expression and activity in both mouse carotid arteries and endothelial cells. The phenomenon involves the regulation of the EP1/SIRT-1 pathway. These findings suggest that EP1 may represent a target to prevent thrombotic events