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PPAR ligands induce prostaglandin production in vascular smooth muscle cells: indomethacin acts as a peroxisome proliferator-activated receptor- antagonist¹

Department of Cardiac, Vascular and Inflammation Research, William Harvey Research Institute, Barts and the London, Queen Mary’s School of Medicine and Dentistry, London EC1M 6BQ, UK

²Correspondence: Department of Cardiac, Vascular and Inflammation Research, William Harvey Research Institute, Barts and the London, Queen Mary’s School of Medicine and Dentistry, Charterhouse Square, London EC1M 6BQ, UK. E-mail: d.bishop-bailey{at}qmul.ac.uk

SPECIFIC AIMS

Peroxisome proliferator-activated receptor- (PPAR) and inducible cyclooxygenase-2 (COX-2) are expressed in atherosclerotic lesions, particularly in the intimal monocytic and vascular smooth muscle cells. PPAR ligands, which include endogenous COX products, induce apoptosis of vascular smooth muscle cells in culture.

We therefore studied the interaction between PPAR and inducible cyclo-oxygenase (COX-2), in rat aortic vascular smooth muscle cells (RASMCs).

PRINCIPAL FINDINGS

1. PPAR activation induces prostanoid release in vascular smooth muscle cells
Rosiglitazone (up to 100 µM) induced PG release 2- to 20-fold from RASMCs. In the absence of serum, IL-1ß (10 ng/mL) induced small increases up to fourfold PGD₂ release, with no effect on PGE₂. In combination with rosiglitazone, IL-1ß produced a synergistic increase in the release of PGs 10- to 100-fold. When used at antagonist concentrations (up to 1 µM), the PPAR partial agonist GW0072 inhibited PGE₂ release induced by either rosiglitazone or rosiglitazone in combination with IL-1ß (Fig. 1 ). Similar results were found with the pure PPAR antagonist GW9662.

View larger version (20K): [in this window] [in a new window]   Figure 1. GW0072 inhibits rosiglitazone induced PGE2 release in RASMCs: the effect of PPAR activation on PGE2 release (48 h) from WKY 12-22 RASMCs incubated with rosiglitazone (Rosi 30 µM) and/or IL-1ß (10 ng/mL) (presence indicated by +). The 4 bars in each cluster indicate increasing concentrations of GW0072; untreated vehicle control (0.1% DMSO), 0.01, 0.1, and 1 µM). The data represent the mean ± SE mean of n = 12 from 4 separate experiments. Symbols denote significance P <0.05 by ANOVA followed by Bonferroni post test: *between control and treatment (in absence of GW0072) and between individual treatment in the presence or absence of GW0072.

2. The major effects of PPAR activation on prostanoid release are on arachidonic acid formation, independent of the PPAR targets COX-2 and type II sPLA₂
Rosiglitazone 30 µM, but not 3 or 10 µM, induced increased expression of COX-2 protein in RASMCs, an effect increased by IL-1ß and inhibited by GW9662. IL-1ß alone also significantly increased COX-2 expression to a similar, if not higher, level as 30 µM rosiglitazone. Rosiglitazone alone (up to 30 µM) had no effect on the expression of mRNA for type II sPLA₂ as determined by RT-PCR. In contrast, coincubation of rosiglitazone with IL-1ß led to a synergistic induction of sPLA₂ mRNA expression. Although rosiglitazone can induce COX-2 and synergize with IL-1ß to type II sPLA₂, rosiglitazone alone induces prostanoid production at concentrations that have no effect on either of these pathways.

3. PLA₂ activity, but not endogenous COX products, mediate PPAR ligand-induced smooth muscle cell apoptosis
Prolonged exposure of rosiglitazone induces RASMC death by apoptosis. Free arachidonic acid or COX products can regulate PPAR activation. The PGD₂ dehydration product 15d-PGJ₂ is a PPAR agonist, while PGF₂ can inhibit PPAR activity by inducing phosphorylation mediated by its classical G-protein-linked receptor pathway. Cell death induced by rosiglitazone in the presence or absence of IL-1 was inhibited by a cPLA₂ (arachidonyl trifluoromethyl ketone, 0.1–10 µM) or sPLA₂ (thioetheramide-PC, 0.1–10 µM) inhibitor. In contrast, rosiglitazone-induced cell death was unaffected by high concentrations of the nonselective COX inhibitor piroxicam (10 µM), an NSAID that has no direct effect on PPARs, or the selective COX-2 inhibitor DFP (1 µM).

4. Indomethacin antagonizes PPAR induced cell death and transcriptional activation
Indomethacin, an alternative inhibitor of cyclooxygenase activity that activates PPAR at "supra-pharmacological" concentrations (high µM-mM), inhibited rosiglitazone-induced cell death at low µM concentrations (up to 10 µM) and rosiglitazone-induced PPAR reporter gene activation (Fig. 2 ).

View larger version (18K): [in this window] [in a new window]   Figure 2. Indomethacin inhibits PPAR-mediated RASMC death and transcriptional activation. A) The effects of indomethacin (0.01–10 µM, filled squares) on RASMC viability alone or in the presence of rosiglitazone 30 µM (open triangles) and 100 µM (open circles). Cell viability was measured by the MTT assay and expressed as % of the signal from cells prepared under control culture conditions. Data represent the mean ± SE mean of n = 12 from 4 separate experiments. B) The effect on transcriptional activation of a PPAR reporter gene of indomethacin (Indo, 10 µM) alone or in the presence of rosiglitazone (Rosi, 30 µM). Data represent the mean ± SE mean of n = 8–9 from 3 separate experiments. *P<0.05 (paired t test) between rosiglitazone in the presence and absence of indomethacin.

CONCLUSIONS

Intimal smooth muscle cells represent a novel PPAR target in vascular disease. Here we shown that PPAR activates COX-2 though multiple pathways. Moreover, the potent nonselective COX inhibitor indomethacin, a NSAID commonly used to treat chronic inflammation inhibited PPAR-mediated responses independent of an effect on COX activity.

PPAR ligands can both induce and inhibit the induction of COX-2 in monocytes. In RASMCs, rosiglitazone induced PG release and the expression of COX-2. However, increases in PG production could not be accounted for by an effect on COX-2 alone as 1) lower concentrations of rosiglitazone (10 µM) induced PG release but had no effect COX-2 expression, and 2) IL-1ß alone induced COX-2 expression but not PG release. These results indicate that a major effect of PPAR activation alone was on substrate formation. PPAR ligands induce a protein synthesis-dependent release of arachidonic acid in rat liver cells and induce the expression of type II PLA₂ in RASMC. In our experiments rosiglitazone alone induced the release of prostanoids without the induction of type II sPLA₂. In the presence of IL-1ß, rosiglitazone did act synergistically to induce type II sPLA₂. We can therefore conclude that PPAR activation induces prostanoid production primarily via substrate formation. When combined with a proinflammatory stimuli such as IL-1ß, PPAR activation results in a large increase in prostanoids associated with both AA-generating enzyme induction and COX-2 induction (see Fig. 3 ).

View larger version (28K): [in this window] [in a new window]   Figure 3. PPAR and COX-2 cross-talk in vascular smooth muscle cells. Phospholipase A2 enzymes cytosolic (c) and secretory (s) utilize membrane phospholipids to produce free arachidonic acid. Free arachidonic acid is used by COX-2 to produce PGH2, the precursor or all the prostanoids. Moreover, free arachidonic acid is a stimulus for apoptosis induction. PPAR activation induces substrate formation for COX enzymes and weakly induces COX-2 (dotted line). IL-1ß is a strong inducer of COX-2 (solid line) but a weak inducer of substrate formation (dotted line). NSAID compounds inhibit COX-2. The NSAID indomethacin also inhibits PPAR-mediated transcription and apoptosis.

We tested whether the PGs released following PPAR activation could feedback to PPAR-dependent RASMC apoptosis. Coincubation of the RASMCs with high concentrations of piroxicam or DFP (a selective COX-2 inhibitor) had no effect on rosiglitazone induced cell death. This clearly indicates no significant feedback between COX and PPAR in these cells. Consistent with our findings on prostanoid release, arachidonic acid substrate formation/PLA₂ activity was a critical factor in PPAR-induced RAMSC apoptosis. Surprisingly, indomethacin, an NSAID shown to activate PPAR and PPAR, inhibited rosiglitazone-induced cell death. Furthermore, at the same concentrations, indomethacin inhibited rosiglitazone-induced transcriptional activation of a PPAR reporter gene, supporting the conclusion that this was a direct effect on PPAR and not a COX-mediated event. Indomethacin binds to and activates PPAR at concentrations above 1 µM. However, the maximum effects of indomethacin on binding and activation were reported to occur at drug concentrations of 100 µM–1 mM. Such concentrations would never be achieved in vivo following therapeutic dosages, where 75–100 mg per day gives steady-state plasma concentrations in the human of 1–2 µM. Significantly, these results indicate that indomethacin affects PPAR at concentrations that accord with its therapeutically active levels.

In summary, rosiglitazone increased prostanoid production by influences at the levels of both COX-2 expression and substrate formation. Indomethacin inhibited PPAR activation in a COX-independent manner. These results have implications not only for the cardiovascular system, but also for the processes underlying other chronic proliferative and inflammatory conditions, where NSAIDs are regularly used, and PPAR ligands have promise as novel anti-inflammatory therapies.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-1075fje; doi: 10.1096/fj.02-1075fje

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