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Introduction Saphenous vein graft failure and restenosis are associated with reactive oxygen species (ROS)-induced vascular dysfunction. People with type 2 diabetes mellitus (T2DM) are more prone to vein graft failure, which arises from specific alterations in human saphenous vein smooth muscle cell (HSVSMC) and endothelial cell phenotype that trigger vascular remodelling. However, our understanding of the mechanisms linking increased ROS with vascular remodelling remains incomplete. Hence, the aim of this study was to investigate the contribution of the MEK1/2-ERK1/2 signalling pathway in mitochondrial-derived ROS (mROS) production in HSVSMCs of T2DM patients versus non-diabetic controls.Methods HSVSMCs were explanted from surplus HSV tissues from consenting T2DM and non-diabetic patients undergoing coronary artery bypass graft (CABG) surgery under HRA ethical approval (NHS REC:15/NE/0138). Confluent HSVSMCs from T2DM and non-diabetic patients were treated with or without 10 nM MEK1/2-selective inhibitor trametinib for 90 min followed by treatment with well-established MEK1/2-ERK1/2 activators Angiotensin ll (AngII, 100 nM) or thrombin (1 U/mL) for 24 hrs. 5 μM MitoSOX dye was then added to assess mROS (O 2 ·) production by flow cytometry, and%PE-A which measures the percentage of the viable cells that responded to MitoSOX stain was used as a marker for mROS production. Statistical analysis was done using Graphpad Prism® 6, San Diego, USA. Data were analysed using one-way analysis of variance followed by Dunnett’s post-hoc test and p<0.05 was considered statistically significant.Results There was no significant difference in mROS production in HSVSMCs from T2DM after stimulation with Ang II and thrombin versus untreated HSVSMCs, however, Ang II and thrombin both produced significant ( p < 0.01, n=4) ERK activation in these cells. mROS production was significantly higher (p < 0.05, n=12) in HSVSMCs from T2DM patients versus non-diabetic controls. Inhibition of MEK1/2-ERK1/2 pathway with trametinib resulted in significant reduction in the generation of mROS in HSVSMCs from both T2DM (p < 0.05, n=6) and non-diabetic patients (p < 0.05, n=6). The degree of reduction in mROS production following inhibition of the MEK1/2-ERK1/2 pathway with trametinib was greater (p < 0.05, n=6) in T2DM patients compared to non-diabetic controls.Conclusion Data from our study demonstrate that inhibition of the MEK1/2-ERK1/2 pathway with trametinib caused a significant reduction in the production of mROS in HSVSMCs-this reduction was greater in T2DM HSVSMCs. This suggests that MAP/ERK signalling mediate mROS production in HSVSMCs, hence a therapeutic target in ROS-mediated vascular dysfunction.Conflicts of interest None