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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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COX-2 inhibitors enhance, whereas PPARδ activators suppress, TF gene expression in ECs. (A) qRT-PCR analysis of total RNA extracted from HUVECs upon treatment with 10 μM celecoxib, 10 μM NS398, 10 μM GW501516, 10 μM 2-AG, or DMSO (vehicle) for 3 h. Celecoxib and NS-398 were added 30 min before stimulation with 2-AG or GW501516 for 6 h. Human TF mRNA levels were normalized by the internal control gene GAPDH (*, P < 0.05; **, P <0.01, compared with vehicle). (B) Northern blot analysis of HUVECs treated with LPS for 3–6 h in the presence or absence of COX-2 inhibitors, GW501516, or 2-AG, as described in A. (C) Western blot analysis of TF expression in HUVECs. (D) TF expression in HUVEC extracts was assayed by Western blot analysis as described in Materials and methods. Cells were treated with indicated concentrations (micromolar) of various compounds (D and E) in the presence of 10 μg/ml LPS. (E) TF activity assay in LPS-stimulated HUVEC lysates treated as outlined above. TF concentration of test samples was directly calculated from the standard curve. *, P < 0.05; **, P < 0.01, compared with vehicle.
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fig4: COX-2 inhibitors enhance, whereas PPARδ activators suppress, TF gene expression in ECs. (A) qRT-PCR analysis of total RNA extracted from HUVECs upon treatment with 10 μM celecoxib, 10 μM NS398, 10 μM GW501516, 10 μM 2-AG, or DMSO (vehicle) for 3 h. Celecoxib and NS-398 were added 30 min before stimulation with 2-AG or GW501516 for 6 h. Human TF mRNA levels were normalized by the internal control gene GAPDH (*, P < 0.05; **, P <0.01, compared with vehicle). (B) Northern blot analysis of HUVECs treated with LPS for 3–6 h in the presence or absence of COX-2 inhibitors, GW501516, or 2-AG, as described in A. (C) Western blot analysis of TF expression in HUVECs. (D) TF expression in HUVEC extracts was assayed by Western blot analysis as described in Materials and methods. Cells were treated with indicated concentrations (micromolar) of various compounds (D and E) in the presence of 10 μg/ml LPS. (E) TF activity assay in LPS-stimulated HUVEC lysates treated as outlined above. TF concentration of test samples was directly calculated from the standard curve. *, P < 0.05; **, P < 0.01, compared with vehicle.

Mentions: Because of the prothrombotic effects of coxibs observed in some patients (15, 24), we analyzed the effect of modulating the COX-2–PPARδ pathway on TF expression in ECs. TF is the primary cellular initiator of blood coagulation, and its expression within the vasculature is associated with thrombosis (25). We found that the COX-2 inhibitors celecoxib and NS398 induced TF mRNA expression, as determined by qRT-PCR (Fig. 4 A). In contrast, activation of the COX-2–PPARδ pathway with either 2-AG or GW501516 decreased basal TF mRNA expression (Fig. 4 A). Furthermore, 2-AG reduced celecoxib induction of TF mRNA expression (Fig. 4 A). These data suggest that inhibition of COX-2 induces TF expression, whereas activation of the COX-2–PPARδ pathway suppresses TF expression. We isolated lung ECs from PPARδ−/− mice and tested the expression of TF by qRT-PCR (Fig. S2, available at http://www.jem.org/cgi/content/full/jem.20070828/DC1). Unexpectedly, TF expression was lower than the wild-type counterparts. This phenomenon is analogous to the regulation of inflammatory gene expression by PPARδ in macrophages (26). These authors showed that although PPARδ agonists suppressed inflammatory genes, PPARδ- macrophages had low expression of such genes. They further showed that release of the transcriptional repressor Bcl6 from the apo-PPARδ is responsible for suppression of inflammatory gene expression. We believe that an analogous phenomenon is operative in our PPARδ- ECs. Although molecular details of such mechanisms need to be further elucidated, our data suggest that COX-2–dependent PPARδ activation maintains the antithrombotic phenotype of the normal endothelium by suppressing the TF gene.


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)

COX-2 inhibitors enhance, whereas PPARδ activators suppress, TF gene expression in ECs. (A) qRT-PCR analysis of total RNA extracted from HUVECs upon treatment with 10 μM celecoxib, 10 μM NS398, 10 μM GW501516, 10 μM 2-AG, or DMSO (vehicle) for 3 h. Celecoxib and NS-398 were added 30 min before stimulation with 2-AG or GW501516 for 6 h. Human TF mRNA levels were normalized by the internal control gene GAPDH (*, P < 0.05; **, P <0.01, compared with vehicle). (B) Northern blot analysis of HUVECs treated with LPS for 3–6 h in the presence or absence of COX-2 inhibitors, GW501516, or 2-AG, as described in A. (C) Western blot analysis of TF expression in HUVECs. (D) TF expression in HUVEC extracts was assayed by Western blot analysis as described in Materials and methods. Cells were treated with indicated concentrations (micromolar) of various compounds (D and E) in the presence of 10 μg/ml LPS. (E) TF activity assay in LPS-stimulated HUVEC lysates treated as outlined above. TF concentration of test samples was directly calculated from the standard curve. *, P < 0.05; **, P < 0.01, compared with vehicle.
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fig4: COX-2 inhibitors enhance, whereas PPARδ activators suppress, TF gene expression in ECs. (A) qRT-PCR analysis of total RNA extracted from HUVECs upon treatment with 10 μM celecoxib, 10 μM NS398, 10 μM GW501516, 10 μM 2-AG, or DMSO (vehicle) for 3 h. Celecoxib and NS-398 were added 30 min before stimulation with 2-AG or GW501516 for 6 h. Human TF mRNA levels were normalized by the internal control gene GAPDH (*, P < 0.05; **, P <0.01, compared with vehicle). (B) Northern blot analysis of HUVECs treated with LPS for 3–6 h in the presence or absence of COX-2 inhibitors, GW501516, or 2-AG, as described in A. (C) Western blot analysis of TF expression in HUVECs. (D) TF expression in HUVEC extracts was assayed by Western blot analysis as described in Materials and methods. Cells were treated with indicated concentrations (micromolar) of various compounds (D and E) in the presence of 10 μg/ml LPS. (E) TF activity assay in LPS-stimulated HUVEC lysates treated as outlined above. TF concentration of test samples was directly calculated from the standard curve. *, P < 0.05; **, P < 0.01, compared with vehicle.
Mentions: Because of the prothrombotic effects of coxibs observed in some patients (15, 24), we analyzed the effect of modulating the COX-2–PPARδ pathway on TF expression in ECs. TF is the primary cellular initiator of blood coagulation, and its expression within the vasculature is associated with thrombosis (25). We found that the COX-2 inhibitors celecoxib and NS398 induced TF mRNA expression, as determined by qRT-PCR (Fig. 4 A). In contrast, activation of the COX-2–PPARδ pathway with either 2-AG or GW501516 decreased basal TF mRNA expression (Fig. 4 A). Furthermore, 2-AG reduced celecoxib induction of TF mRNA expression (Fig. 4 A). These data suggest that inhibition of COX-2 induces TF expression, whereas activation of the COX-2–PPARδ pathway suppresses TF expression. We isolated lung ECs from PPARδ−/− mice and tested the expression of TF by qRT-PCR (Fig. S2, available at http://www.jem.org/cgi/content/full/jem.20070828/DC1). Unexpectedly, TF expression was lower than the wild-type counterparts. This phenomenon is analogous to the regulation of inflammatory gene expression by PPARδ in macrophages (26). These authors showed that although PPARδ agonists suppressed inflammatory genes, PPARδ- macrophages had low expression of such genes. They further showed that release of the transcriptional repressor Bcl6 from the apo-PPARδ is responsible for suppression of inflammatory gene expression. We believe that an analogous phenomenon is operative in our PPARδ- ECs. Although molecular details of such mechanisms need to be further elucidated, our data suggest that COX-2–dependent PPARδ activation maintains the antithrombotic phenotype of the normal endothelium by suppressing the TF gene.

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