Surgical intervention can result in endothelial denudation, driving growth factor-stimulated vascular smooth muscle cell (VSMC) migration towards the intima, leading to luminal narrowing and restenosis. Clinically approved PGI₂ analogues, including beraprost, activate the cyclic adenosine monophosphate (cAMP) signaling pathway to inhibit VSMC migration in vitro. This pathway is a potential therapeutic target, however the downstream proteins involved in the inhibitory effects of cAMP on migration remain unknown. The aims of this study were to determine the signalling pathways involved in inhibiting VSMC migration through cAMP downstream mediators, protein kinase A (PKA) and the more recently characterised exchange protein activated by cAMP (Epac), and delineate the mechanisms involved. In human saphenous vein VSMCs, Epac activation using an Epac analogue inhibited VSMC migration. Therapeutic concentrations of beraprost (1 nM) also resulted in an inhibition of VSMC migration. The use of fluorescence resonance energy transfer (FRET) confirmed 1 nM beraprost activated Epac, but not PKA. Epac is a guanine nucleotide exchange factor (GEF) for Rap1 thus Rap1 siRNA was used to inhibit the Epac pathway. This blocked the inhibitory effects of beraprost on VSMC migration. Epac1 was localised to the leading edge of migrating VSMCs. Another G-protein, RhoA, was investigated since it is essential for cell migration and is involved in several processes including actin regulation. Epac signaling inhibited PDGF-induced RhoA activation and disassembled F-actin at the leading edge, where Epac1 was previously located. This indicates that beraprost activated the Epac pathway, which inhibited RhoA to decrease VSMC migration. The clinical relevance of this study has discovered the mechanisms of Epac's inhibitory action on VSMC migration and this pathway could be targeted therapeutically to reduce restenosis. In the future the potential use of beraprost on a drug eluting stent might be beneficial to prevent restenosis formation following surgical intervention.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:659069 |
| Date | January 2015 |
| Creators | McKean, Jenny Susan |
| Publisher | University of Aberdeen |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=227109 |
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