Carlin, Christopher M.
The effects of statins on hypoxia-induced proliferation and cell signalling pathways in pulmonary artery fibroblasts.
PhD thesis, University of Glasgow.
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Chronic hypoxia, in animals and man, results in remodelling of the pulmonary vasculature with consequent pulmonary hypertension. The pulmonary artery fibroblast (PAF) has been shown to play an early and important role in hypoxia-induced pulmonary vascular remodelling. In acute and chronic hypoxia there is excess proliferation of PAFs. Morevoer, it is likely that cell-cell interactions
between hypoxia-stimulated PAFs and other vascular cells – particularly smooth muscle cells - initiates and progresses the changes that occur in pulmonary vascular
remodelling in the other vessel compartments. Although hypoxic proliferation of PAFs has been shown to be circulation specific and dependant on phosphorylation of
p38 mitogen-activated protein (MAP) kinase, the cell signalling pathway(s)underlying this are incompletely characterised. Hypoxic activation of PAFs is a
potential therapeutic target but, as p38 MAP kinase inhibitors are not established for clinical use, work was proposed to better characterise this pathway and identify
agent(s) which may inhibit p38 MAPK indirectly.
The HMG-CoA reductase inhibitor simvastatin was recently shown to inhibit hypoxic pulmonary vascular remodelling in rats, but the applicability of this finding
to clinical practice is incompletely established and the mechanism of action of the statin is unclear. Statins have been shown to influence MAP kinase pathways in other cell types and, as their modes of action are well established, they can be used to interrogate uncharacterised upstream cell signalling pathways. On this basis, the
aims of this study were firstly to determine whether statins had a therapeutically useful inhibitory effect on hypoxia-induced, p38 MAP kinase-mediated PAF proliferation. A second aim was to exploit the known effects of statins to better characterise hypoxic cell signalling upstream of p38 MAP kinase in PAFs. Lastly,
comparison of the effects of statins with established pulmonary hypertension therapeutics and a preliminary assessment – also using statins as an experimental tool
- of cell-cell interactions between PAFs and pulmonary artery smooth muscle cells (PASMCs) was proposed.
1μM of fluvastatin was found to selectively inhibit acute and chronic hypoxia-induced p38 MAP kinase phosphorylation and proliferation in rat PAFs. At this dose, fluvastatin had no effect on serum-induced proliferation in PAFs, no effect on systemic adventitial fibroblast proliferation, and no effect on the phosphorylation status of other MAP kinases. Selective use of mediators and inhibitors related to the HMG-CoA pathway indicated that a geranylgeranylated protein, probably Rac1, had an obligatory role upstream of p38 MAPK, in this signalling pathway. Co-culture and conditioned media experiments with bovine PAFs and PASMCs demonstrated the release of PASMC mitogens from hypoxic PAFs. 1μM fluvastatin and the p38 MAP kinase inhibitor SB203580 selectively blocked the hypoxic PAF-PASMC
interaction. Results with hypoxic PAF proliferation with the prostacyclin analogue treprostinil, the phosphodiesterase-5 inhibitor sildenafil and the endothelin-1 antagonist bosentan were negative. Bosentan, however, inhibited the hypoxic PAF-PASMC interaction, suggesting endothelin-1 release by hypoxic PAFs, with proproliferative effects on PASMCs.
The results reported in this thesis provide new information on hypoxic signalling,PAF proliferation and PAF cell-cell interactions in hypoxic states. A circulation and stimulus specific anti-proliferative effect of fluvastatin on PAFs was identified and
this may be of clinical relevance in hypoxia-associated pulmonary hypertension.
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