Prostaglandin (PG) I₂ and mimetics, e.g. treprostinil sodium, are used for the treatment of pulmonary hypertension because of their ability to reduce the accelerated proliferation of pulmonary vascular cells and fibroblasts. PGI₂ is known to act via activation of cell surface IP receptors. However, PGI₂ and some of its mimetics are also ligands for peroxisome proliferator-activated receptor delta (PPAR) (Lim and Dey, 2002). There are currently few pharmacological tools available to study specifically IP or PPAR receptor function. Here, therefore, we have compared the responses to treprostinil sodium (donated by United Therapeutics Corp) and other mimetics of lung fibroblasts cultured from wild-type mice, PPAR-deficient mice, and IP-deficient mice.

Lungs from PPAR^-/-, IP^-/- or wild-type control (BLK6) mice were cultured into supplemented DMEM using standard explant protocols (Stanford et al., 2003). Explanted cells were detached and re-seeded at 40% confluence in 96-well plates. 24 h later the medium was replaced with fresh DMEM containing BSA (0.1%) but no FCS. Cells were then rested for 2 h before the addition of test drugs or vehicle controls. 4 h later proliferation was stimulated by the addition of FCS (3 %) and cells were then incubated (5% CO₂ in air; 37 °C) for a further 72 h. Proliferation was quantified using a fluorescence assay (CYQUANT; Cambridge Bioscience).

Effects of (A) treprostinil sodium and (B) 15 deoxy-^12,14-PGJ₂ on proliferation of lung fibroblasts from wild-type (WT; open circles), PPARd^/- (filled triangles) or IP^-/- (filled squares). Proliferation was calculated as % growth induced by FCS. Data is mean ± s.e.m for n=6 determinations using cells cultured from 2 separate animals for data point, except graph B WT where n=12 (p<0.05; two-way ANOVA).

15d-PGJ₂ inhibited proliferation in cells from all three types of mice and at the highest concentration was found to induce induce cell death. At high concentrations treprostinil sodium inhibited proliferation in all cell types. However at 10^-4M its effects were blunted in cells from either IP-deficient and, significantly, from PPAR-deficient mice.

Here we show that treprostinil sodium inhibits murine lung cell proliferation, partially via the activation of PPAR receptors. The better studied PPAR ligand, 15d-PGJ₂ did not appear to function via PPAR. These observations have implications for our understanding of therapeutic intervention in pulmonary hypertension.

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This work was funded by the JRB St Bartholomews Hospital, London.