Identification of microRNAs involved in early vein graft remodelling

Abstract Background The saphenous vein (SV) is the most commonly-used conduit for coronary artery bypass grafting (CABG). However, these grafts undergo adverse remodelling and suffer a failure rate of ~50% within 10 years. Endothelial cell (EC) dysfunction and vascular smooth muscle cell (VSMC) proliferation after SV graft implantation, drives graft remodelling and subsequent neointimal hyperplasia. Given that microRNAs are key regulators of EC and VSMC function and interaction, they may contribute to vein graft remodelling. Purpose This study aimed to characterise the pathological changes in a porcine SV graft model and identify differentially expressed microRNAs to investigate their relevant gene targets and potential role in graft remodelling, using down-stream bioinformatics. Methods A porcine arterio-venous grafting model was used. SV graft samples were collected after 1, 2, 4 and 12 weeks following grafting into the carotid artery (n=4-7) and compared with control SV samples. EC coverage was assessed through immunohistochemistry for CD31 and VE-cadherin. VSMC proliferation was assessed through immunohistochemistry for cyclin D1 (CCND1). Next generation sequencing was performed on control SVs, week 1 and week 4 SV grafts to identify differentially expressed microRNAs. Bioinformatic platforms, miRNet and STRING, were used to analyse the microRNAs and identify common gene targets. Results Week 2 SV grafts showed a significant reduction in EC coverage compared to SV (41% for CD31, p<0.001; 44% for VE-cadherin, p<0.05). VSMC proliferation was significantly increased by week 4 compared to SV (43.0% vs 2.4%, p<0.01). Using a fold change of ±2.0, we identified 39 microRNAs that were specifically and significantly regulated in week 2 SV grafts, from which 40 genes were predicted to be targets for multiple microRNAs. KEGG pathway analysis revealed features of graft remodelling (p53 signalling, TGF-beta signalling, and cell apoptosis). There were 19 microRNAs specifically and significantly regulated in week 4 SV grafts, from which 38 genes were predicted to be targets for multiple microRNAs. However, there were no relevant enriched pathways. Conclusion The predicted gene targets of the differentially expressed microRNAs 2-weeks post-grafting demonstrated stronger enrichment within pathological pathway changes following SV grafting than at 4-weeks, highlighting an important window of opportunity for therapeutic intervention. Moreover, the identification of several microRNAs predicted to target multiple key genes emphasises their importance as potential targets for modulation to improve CABG patency rates.