The effect of hypoxic mediated pro-mitogenic signalling pro-mitogenic signalling associated with vein graft failure and vascular remodelling

Huq, Molly and Currie, Susan and Coats, Paul (2017) The effect of hypoxic mediated pro-mitogenic signalling pro-mitogenic signalling associated with vein graft failure and vascular remodelling. Heart, 103 (S2). A7. 15. ISSN 1468-201X

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Abstract

Tissue hypoxia is known to contribute to the hyperproliferative pathogenesis underlying vein graft stenosis with loss of luminal patency. Hypoxia has been reported to modulate PLC-χ leading to the activation of PKC dependent-cell proliferation through mitogen activated protein kinases (MAPK) signalling pathways. Additionally, hypoxia is positively correlated with the pro-mitogenic HIF-1α derived signalling pathway. This project aims to investigate the potential interaction of HIF-1α and PLC-χ in hypoxic mediated pro-mitogenic signalling associated with vein graft remodelling. Vascular smooth muscle cells (VSMC) were cultured in hypoxic conditions (3%–10%) and compared with normoxic culture. VSMC proliferation was assessed through 3 hour thymidine incorporation assays, mRNA expression levels were measured using RT-PCR and MAPK signalling was evaluated by immunoblotting. Conditioned media experiments from VMSC and endothelial cells were performed along with exosome isolations from both cell types under experimental conditions. Results confirmed a positive correlation between hypoxia and cell proliferation; cells cultured under 3% and 10% oxygen increased cell proliferation by 2.1 and 1.4 fold respectively. The PLC-χ inhibitor U73122 (1 µM) significantly reduced VSMC proliferation under normoxia however had no inhibitory effect in hypoxic culture. Experiments also showed hypoxia to downregulate mRNA expression levels of PLC-χ, by 49.9%. Conditioned media from 3% hypoxic cultured endothelial cells increased VSMC proliferation by 5.8 fold at 24 hours. Initial experiments have confirmed a hypoxic-dependent-increase in exosomal trafficking where the measured exosomal yield from VMSC cultured under 3% hypoxia increased by 30% when compared with normoxic cultures. Moderate hypoxia significantly drives VSMC proliferation in comparison to mild hypoxia. These results raise questions over the association with PLC-χ and hypoxia-dependent pro-mitogenesis of VSMCs. This observation is backed up by the absence of hypoxic-dependent phosphorylation of the MAPKs. Of note, initial experiments have identified hypoxic culture modulates a pro-mitogenic endothelial-derived transferable factor(s) and increase in exosomal trafficking.