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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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Role of PG synthases in endocannabinoid-induced PPARδ transactivation. (A) Expression profile of PG synthases in HUVECs was determined by real-time PCR. Expression level (Ro) for each gene was normalized to glyceraldehyde-3-phosphate dehydrogenase. (B) Expression of PGIS or PGFS mRNA in HUVECs after treatment with 100 nM of PGFS or PGIS siRNA (*, P < 0.05, compared with control siRNA). (C) Effects of silencing PGFS or PGIS on PPARδ transactivation in response to 2-AG. Cells were transfected with PGFS or PGIS siRNA with Oligofectamine for 4 h, and were allowed to recover in complete growth medium. After 16 h, cells were transiently transfected with PPARδ-GAL4, UAS-tk-luc, and pβ-gal with Lipofectamine in OptiMEM, followed by addition of 2-AG. Cells were harvested for luciferase activity after 24 h (*, P < 0.05, compared with vehicle).
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fig3: Role of PG synthases in endocannabinoid-induced PPARδ transactivation. (A) Expression profile of PG synthases in HUVECs was determined by real-time PCR. Expression level (Ro) for each gene was normalized to glyceraldehyde-3-phosphate dehydrogenase. (B) Expression of PGIS or PGFS mRNA in HUVECs after treatment with 100 nM of PGFS or PGIS siRNA (*, P < 0.05, compared with control siRNA). (C) Effects of silencing PGFS or PGIS on PPARδ transactivation in response to 2-AG. Cells were transfected with PGFS or PGIS siRNA with Oligofectamine for 4 h, and were allowed to recover in complete growth medium. After 16 h, cells were transiently transfected with PPARδ-GAL4, UAS-tk-luc, and pβ-gal with Lipofectamine in OptiMEM, followed by addition of 2-AG. Cells were harvested for luciferase activity after 24 h (*, P < 0.05, compared with vehicle).

Mentions: Once fatty acid substrates are metabolized by COX-2 into endoperoxides, terminal synthases catalyze the formation of active prostanoids (1). To determine which of the prostanoids are involved in PPARδ transcription, we silenced the expression of genes encoding prostanoid synthases by siRNA in HUVECs and assayed the 2-AG–stimulated transcriptional response. As shown in Fig. 3 A, quantitative (q) RT-PCR analysis indicates that PGF2α synthase (PGFS) and PGI2 synthase (PGIS) mRNAs are highly expressed in HUVECs, although lower levels of cytosolic PGE2 synthase mRNA are also expressed. The inducible microsomal PGE2 synthase mRNA was not detected under basal conditions. This is consistent with our previous observation of abundant synthesis of 6-keto-PGF1α (a stable breakdown product of PGI2), PGF2α, and PGE2 in HUVECs (22). PGIS or PGFS siRNA significantly suppressed the expression of PGIS or PGFS mRNAs in HUVECs, respectively (Fig. 3 B). As shown in Fig. 3 C, 2-AG–induced PPARδ transcription was inhibited by the PGIS, but not by the PGFS siRNA. These data suggest that conversion of 2-AG by COX-2 and PGIS into PGI2 glycerol ester is critical for PPARδ activation in ECs. Attempts to purify the PGI2 glycerol ester or 6-keto-PGF1α glycerol ester from EC extracts were not successful, which is presumably caused by high hydrolytic conversion of prostaglandin glycerol esters by cytosolic esterases (23).


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)

Role of PG synthases in endocannabinoid-induced PPARδ transactivation. (A) Expression profile of PG synthases in HUVECs was determined by real-time PCR. Expression level (Ro) for each gene was normalized to glyceraldehyde-3-phosphate dehydrogenase. (B) Expression of PGIS or PGFS mRNA in HUVECs after treatment with 100 nM of PGFS or PGIS siRNA (*, P < 0.05, compared with control siRNA). (C) Effects of silencing PGFS or PGIS on PPARδ transactivation in response to 2-AG. Cells were transfected with PGFS or PGIS siRNA with Oligofectamine for 4 h, and were allowed to recover in complete growth medium. After 16 h, cells were transiently transfected with PPARδ-GAL4, UAS-tk-luc, and pβ-gal with Lipofectamine in OptiMEM, followed by addition of 2-AG. Cells were harvested for luciferase activity after 24 h (*, P < 0.05, compared with vehicle).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2118704&req=5

fig3: Role of PG synthases in endocannabinoid-induced PPARδ transactivation. (A) Expression profile of PG synthases in HUVECs was determined by real-time PCR. Expression level (Ro) for each gene was normalized to glyceraldehyde-3-phosphate dehydrogenase. (B) Expression of PGIS or PGFS mRNA in HUVECs after treatment with 100 nM of PGFS or PGIS siRNA (*, P < 0.05, compared with control siRNA). (C) Effects of silencing PGFS or PGIS on PPARδ transactivation in response to 2-AG. Cells were transfected with PGFS or PGIS siRNA with Oligofectamine for 4 h, and were allowed to recover in complete growth medium. After 16 h, cells were transiently transfected with PPARδ-GAL4, UAS-tk-luc, and pβ-gal with Lipofectamine in OptiMEM, followed by addition of 2-AG. Cells were harvested for luciferase activity after 24 h (*, P < 0.05, compared with vehicle).
Mentions: Once fatty acid substrates are metabolized by COX-2 into endoperoxides, terminal synthases catalyze the formation of active prostanoids (1). To determine which of the prostanoids are involved in PPARδ transcription, we silenced the expression of genes encoding prostanoid synthases by siRNA in HUVECs and assayed the 2-AG–stimulated transcriptional response. As shown in Fig. 3 A, quantitative (q) RT-PCR analysis indicates that PGF2α synthase (PGFS) and PGI2 synthase (PGIS) mRNAs are highly expressed in HUVECs, although lower levels of cytosolic PGE2 synthase mRNA are also expressed. The inducible microsomal PGE2 synthase mRNA was not detected under basal conditions. This is consistent with our previous observation of abundant synthesis of 6-keto-PGF1α (a stable breakdown product of PGI2), PGF2α, and PGE2 in HUVECs (22). PGIS or PGFS siRNA significantly suppressed the expression of PGIS or PGFS mRNAs in HUVECs, respectively (Fig. 3 B). As shown in Fig. 3 C, 2-AG–induced PPARδ transcription was inhibited by the PGIS, but not by the PGFS siRNA. These data suggest that conversion of 2-AG by COX-2 and PGIS into PGI2 glycerol ester is critical for PPARδ activation in ECs. Attempts to purify the PGI2 glycerol ester or 6-keto-PGF1α glycerol ester from EC extracts were not successful, which is presumably caused by high hydrolytic conversion of prostaglandin glycerol esters by cytosolic esterases (23).

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