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COX-2 suppresses tissue factor expression via endocannabinoid-directed PPARdelta activation.

Ghosh M, Wang H, Ai Y, Romeo E, Luyendyk JP, Peters JM, Mackman N, Dey SK, Hla T - J. Exp. Med. (2007)

Bottom Line: Importantly, PPARdelta agonists suppress coxib-induced TF expression and decrease circulating TF activity.We provide evidence that COX-2-dependent attenuation of TF expression is abrogated by coxibs, which may explain the prothrombotic side-effects for this class of drugs.Furthermore, PPARdelta agonists may be used therapeutically to suppress coxib-induced cardiovascular side effects.

View Article: PubMed Central - PubMed

Affiliation: Center for Vascular Biology, Department of Cell Biology, University of Connecticut Health Center, Farmington, CT 06030, USA.

ABSTRACT
Although cyclooxygenase (COX)-2 inhibitors (coxibs) are effective in controlling inflammation, pain, and tumorigenesis, their use is limited by the recent revelation of increased adverse cardiovascular events. The mechanistic basis of this side effect is not well understood. We show that the metabolism of endocannabinoids by the endothelial cell COX-2 coupled to the prostacyclin (PGI(2)) synthase (PGIS) activates the nuclear receptor peroxisomal proliferator-activated receptor (PPAR) delta, which negatively regulates the expression of tissue factor (TF), the primary initiator of blood coagulation. Coxibs suppress PPARdelta activity and induce TF expression in vascular endothelium and elevate circulating TF activity in vivo. Importantly, PPARdelta agonists suppress coxib-induced TF expression and decrease circulating TF activity. We provide evidence that COX-2-dependent attenuation of TF expression is abrogated by coxibs, which may explain the prothrombotic side-effects for this class of drugs. Furthermore, PPARdelta agonists may be used therapeutically to suppress coxib-induced cardiovascular side effects.

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Endocannabinoids induce PPARδ-dependent transcription in HUVECs. (A) HUVECs were transiently transfected with PPRE-luciferase reporter plasmid pACO-gLuc, and after 24 h cells were incubated with vehicle (DMSO), fatty acids (AA, DHA, and OA), or endocannabinoids (2G, AEA, and NE). Subsequently, luciferase activity was measured as described in Materials and methods. (*, P < 0.05; **, P < 0.01 compared with AA). Structures of the endocannabinoids are shown in Fig. S4. (B) Endocannabinoid activation of endogenous PPARδ-dependent transcription. A chimeric receptor containing the ligand-binding domain of PPARα,γ,δ fused with the DNA-binding domain of yeast Gal4 transcription factor was used. Transactivation was detected by cotransfection with reporter gene (Gal4 response element [UAS-tk-luc]) in the presence of 2-AG (*, P < 0.05; **, P < 0.01, compared with vehicle). (C) Mouse lung ECs were isolated from wild-type (yellow), PPARδ−/− (green), or CB1−/−/CB2−/− (blue) mice, and the Gal4-UAS system of transcriptional activation was assayed in the presence of vehicle (DMSO), 1 or 10 μM 2-AG, or 10 μM PPARδ agonist cPGI as control (*, P < 0.05; **, P < 0.01 compared with vehicle). Fig. S4 is available at http://www.jem.org/cgi/content/full/jem.20070828/DC1.
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fig1: Endocannabinoids induce PPARδ-dependent transcription in HUVECs. (A) HUVECs were transiently transfected with PPRE-luciferase reporter plasmid pACO-gLuc, and after 24 h cells were incubated with vehicle (DMSO), fatty acids (AA, DHA, and OA), or endocannabinoids (2G, AEA, and NE). Subsequently, luciferase activity was measured as described in Materials and methods. (*, P < 0.05; **, P < 0.01 compared with AA). Structures of the endocannabinoids are shown in Fig. S4. (B) Endocannabinoid activation of endogenous PPARδ-dependent transcription. A chimeric receptor containing the ligand-binding domain of PPARα,γ,δ fused with the DNA-binding domain of yeast Gal4 transcription factor was used. Transactivation was detected by cotransfection with reporter gene (Gal4 response element [UAS-tk-luc]) in the presence of 2-AG (*, P < 0.05; **, P < 0.01, compared with vehicle). (C) Mouse lung ECs were isolated from wild-type (yellow), PPARδ−/− (green), or CB1−/−/CB2−/− (blue) mice, and the Gal4-UAS system of transcriptional activation was assayed in the presence of vehicle (DMSO), 1 or 10 μM 2-AG, or 10 μM PPARδ agonist cPGI as control (*, P < 0.05; **, P < 0.01 compared with vehicle). Fig. S4 is available at http://www.jem.org/cgi/content/full/jem.20070828/DC1.

Mentions: The COX-2 isoenzyme has a larger active site pocket than COX-1 and therefore is capable of oxidizing many polyunsaturated fatty acids in addition to the common substrate arachidonic acid (AA) (16). We tested if metabolism of various substrates of COX-2 would lead to intracellular activation of PPARδ in ECs. Human umbilical vein ECs (HUVECs), which express COX-2 were transfected with a PPAR-responsive transcription reporter (pACO-Luc) (17), incubated with various fatty acid substrates and transcriptional reporter (luciferase) activity was measured. As shown in Fig. 1 A, endocannabinoids, 2-arachidonyl glycerol (2-AG), noladin ether (NE), and anandamide (AEA) stimulated PPAR-dependent transcription. In contrast, the effect of AA was modest, and neither n-3 fatty acids (docosahexaenoic acid or eicosapentaenoic acid) nor non–COX-2 substrates (palmitate or oleate) induced PPAR-dependent transcription. The concentration of endocannabinoids that induced transcription is significantly below the Km of 2-AG for COX-2, which is estimated to be ∼4 μM (16). NE, which is a nonhydrolyzable ether analogue of 2-AG, is more potent, suggesting that hydrolytic pathways are involved in attenuating the 2-AG effect. These data provide evidence that endocannabinoid ligands, which are alternative substrates for COX-2 but not COX-1, are capable of activating the endogenous PPAR system in ECs.


COX-2 suppresses tissue factor expression via endocannabinoid-directed PPARdelta activation.

Ghosh M, Wang H, Ai Y, Romeo E, Luyendyk JP, Peters JM, Mackman N, Dey SK, Hla T - J. Exp. Med. (2007)

Endocannabinoids induce PPARδ-dependent transcription in HUVECs. (A) HUVECs were transiently transfected with PPRE-luciferase reporter plasmid pACO-gLuc, and after 24 h cells were incubated with vehicle (DMSO), fatty acids (AA, DHA, and OA), or endocannabinoids (2G, AEA, and NE). Subsequently, luciferase activity was measured as described in Materials and methods. (*, P < 0.05; **, P < 0.01 compared with AA). Structures of the endocannabinoids are shown in Fig. S4. (B) Endocannabinoid activation of endogenous PPARδ-dependent transcription. A chimeric receptor containing the ligand-binding domain of PPARα,γ,δ fused with the DNA-binding domain of yeast Gal4 transcription factor was used. Transactivation was detected by cotransfection with reporter gene (Gal4 response element [UAS-tk-luc]) in the presence of 2-AG (*, P < 0.05; **, P < 0.01, compared with vehicle). (C) Mouse lung ECs were isolated from wild-type (yellow), PPARδ−/− (green), or CB1−/−/CB2−/− (blue) mice, and the Gal4-UAS system of transcriptional activation was assayed in the presence of vehicle (DMSO), 1 or 10 μM 2-AG, or 10 μM PPARδ agonist cPGI as control (*, P < 0.05; **, P < 0.01 compared with vehicle). Fig. S4 is available at http://www.jem.org/cgi/content/full/jem.20070828/DC1.
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fig1: Endocannabinoids induce PPARδ-dependent transcription in HUVECs. (A) HUVECs were transiently transfected with PPRE-luciferase reporter plasmid pACO-gLuc, and after 24 h cells were incubated with vehicle (DMSO), fatty acids (AA, DHA, and OA), or endocannabinoids (2G, AEA, and NE). Subsequently, luciferase activity was measured as described in Materials and methods. (*, P < 0.05; **, P < 0.01 compared with AA). Structures of the endocannabinoids are shown in Fig. S4. (B) Endocannabinoid activation of endogenous PPARδ-dependent transcription. A chimeric receptor containing the ligand-binding domain of PPARα,γ,δ fused with the DNA-binding domain of yeast Gal4 transcription factor was used. Transactivation was detected by cotransfection with reporter gene (Gal4 response element [UAS-tk-luc]) in the presence of 2-AG (*, P < 0.05; **, P < 0.01, compared with vehicle). (C) Mouse lung ECs were isolated from wild-type (yellow), PPARδ−/− (green), or CB1−/−/CB2−/− (blue) mice, and the Gal4-UAS system of transcriptional activation was assayed in the presence of vehicle (DMSO), 1 or 10 μM 2-AG, or 10 μM PPARδ agonist cPGI as control (*, P < 0.05; **, P < 0.01 compared with vehicle). Fig. S4 is available at http://www.jem.org/cgi/content/full/jem.20070828/DC1.
Mentions: The COX-2 isoenzyme has a larger active site pocket than COX-1 and therefore is capable of oxidizing many polyunsaturated fatty acids in addition to the common substrate arachidonic acid (AA) (16). We tested if metabolism of various substrates of COX-2 would lead to intracellular activation of PPARδ in ECs. Human umbilical vein ECs (HUVECs), which express COX-2 were transfected with a PPAR-responsive transcription reporter (pACO-Luc) (17), incubated with various fatty acid substrates and transcriptional reporter (luciferase) activity was measured. As shown in Fig. 1 A, endocannabinoids, 2-arachidonyl glycerol (2-AG), noladin ether (NE), and anandamide (AEA) stimulated PPAR-dependent transcription. In contrast, the effect of AA was modest, and neither n-3 fatty acids (docosahexaenoic acid or eicosapentaenoic acid) nor non–COX-2 substrates (palmitate or oleate) induced PPAR-dependent transcription. The concentration of endocannabinoids that induced transcription is significantly below the Km of 2-AG for COX-2, which is estimated to be ∼4 μM (16). NE, which is a nonhydrolyzable ether analogue of 2-AG, is more potent, suggesting that hydrolytic pathways are involved in attenuating the 2-AG effect. These data provide evidence that endocannabinoid ligands, which are alternative substrates for COX-2 but not COX-1, are capable of activating the endogenous PPAR system in ECs.

Bottom Line: Importantly, PPARdelta agonists suppress coxib-induced TF expression and decrease circulating TF activity.We provide evidence that COX-2-dependent attenuation of TF expression is abrogated by coxibs, which may explain the prothrombotic side-effects for this class of drugs.Furthermore, PPARdelta agonists may be used therapeutically to suppress coxib-induced cardiovascular side effects.

View Article: PubMed Central - PubMed

Affiliation: Center for Vascular Biology, Department of Cell Biology, University of Connecticut Health Center, Farmington, CT 06030, USA.

ABSTRACT
Although cyclooxygenase (COX)-2 inhibitors (coxibs) are effective in controlling inflammation, pain, and tumorigenesis, their use is limited by the recent revelation of increased adverse cardiovascular events. The mechanistic basis of this side effect is not well understood. We show that the metabolism of endocannabinoids by the endothelial cell COX-2 coupled to the prostacyclin (PGI(2)) synthase (PGIS) activates the nuclear receptor peroxisomal proliferator-activated receptor (PPAR) delta, which negatively regulates the expression of tissue factor (TF), the primary initiator of blood coagulation. Coxibs suppress PPARdelta activity and induce TF expression in vascular endothelium and elevate circulating TF activity in vivo. Importantly, PPARdelta agonists suppress coxib-induced TF expression and decrease circulating TF activity. We provide evidence that COX-2-dependent attenuation of TF expression is abrogated by coxibs, which may explain the prothrombotic side-effects for this class of drugs. Furthermore, PPARdelta agonists may be used therapeutically to suppress coxib-induced cardiovascular side effects.

Show MeSH
Related in: MedlinePlus