Type 2 diabetes mellitus (T2DM) is increasing worldwide, and it is associated with increased risk of coronary artery disease (CAD). For T2DM patients, the main surgical intervention for CAD is autologous saphenous vein grafting. However, T2DM patients have increased risk of saphenous vein graft failure (SVGF). While the mechanisms underlying increased risk of vascular disease in T2DM are not fully understood, hyperglycaemia, insulin resistance, and hyperinsulinaemia have been shown to contribute to microvascular damage, whereas clinical trials have reported limited effects of intensive glycaemic control in the management of macrovascular complications. This suggests that factors other than glucose exposure may be responsible for the macrovascular complications observed in T2DM. SVGF is characterised by neointimal hyperplasia (NIH) arising from endothelial cell (EC) dysfunction and uncontrolled migration and proliferation of vascular smooth muscle cells (SMCs). This is driven in part by proinflammatory cytokines released from the activated ECs and SMCs, particularly interleukin 6 (IL-6). IL-6 stimulation of the Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT) pathway is a key mechanism through which EC inflammation, SMC migration, and proliferation are controlled and whose activation might therefore be enhanced in patients with T2DM. In this review, we investigate how proinflammatory cytokines, particularly IL-6, contribute to vascular damage resulting in SVGF and how suppression of proinflammatory cytokine responses via targeting the JAK/STAT pathway could be exploited as a potential therapeutic strategy. These include the targeting of suppressor of cytokine signalling (SOCS3), which appears to play a key role in suppressing unwanted vascular inflammation, SMC migration, and proliferation.

Aims: Glucose-driven mitochondrial dysfunction has been proposed to promote vascular cell proliferation and migration events responsible for saphenous vein graft failure (VGF) following bypass surgery. However, it is unclear how type 2 diabetes mellitus (T2DM) impacts mitochondrial function in human saphenous vein smooth muscle cells (HSVSMCs) responsible for the maladaptive remodelling responsible for VGF. Therefore, identifying and targeting the signalling pathways involved could offer new therapeutic options to limit VGF. Our aim was to identify signalling pathways that mediate any mitochondrial dysfunction in HSVSMCs in vitro and assess the impact of T2DM. Methods and results: HSVSMCs explanted from surplus HSV tissues from consenting T2DM and non-diabetic patients undergoing coronary artery bypass graft surgery were treated with known activators and inhibitors of the JAK/STAT and MAPK/ERK pathways. Following this, real-time oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) measures of mitochondrial function were then determined. Our findings revealed that both IL-6/sIL-6Rα trans-signalling complexes and PDGF-BB significantly increased OCR in HSVSMCs from T2DM patients but not non-diabetic controls. Meanwhile, only PDGF-BB increased ECAR in HSVSMCs from T2DM patients but not non-diabetic controls. The observed increases in OCR and ECAR were abolished by JAK1/2-selective inhibitor ruxolitinib. Furthermore, thrombin caused a significant increase in OCR specifically in HSVSMCs from T2DM patients, and this was abolished by MEK1/2-selective inhibitor trametinib. Both ruxolitinib and trametinib significantly reduced basal OCR and ECAR in HSVSMCs from both T2DM and non-diabetic patients. Conclusion: Together, these findings demonstrate a JAK/STAT- and MAPK/ERK-mediated regulation of mitochondrial function in HSVSMCs. As such, they represent potential targets for regulation of HSVSMC function that can be explored for drug development to limit saphenous VGF in T2DM.

While glucose-driven mitochondrial dysfunction has been proposed to promote vascular dysfunction responsible for saphenous vein graft failure (VGF) following bypass surgery, the impact of type 2 diabetes mellitus (T2DM) on mitochondrial function in human saphenous vein smooth muscle cells (HSVSMCs) responsible for maladaptive remodelling is unknown. Our aim was to identify signalling pathways that mediate any mitochondrial dysfunction in HSVSMCs in vitro and assess the impact of T2DM. HSVSMCs explanted from surplus HSV tissues from consenting T2DM and non-diabetic patients undergoing coronary artery bypass graft surgery were treated with known activators and inhibitors of the JAK/STAT and MAPK/ERK pathways. Following this, real-time oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) measures of mitochondrial function were then determined. Our findings revealed that both IL-6/sIL-6Rα trans-signalling complexes and platelet-derived growth factor-BB (PDGF-BB) significantly increased OCR in HSVSMCs from T2DM patients but not non-diabetic controls. Meanwhile, only PDGF-BB increased ECAR in HSVSMCs from T2DM patients but not in non-diabetic controls. The observed increases in OCR and ECAR were abolished by JAK1/2-selective inhibitor ruxolitinib. Furthermore, thrombin caused a significant increase in OCR, specifically in HSVSMCs from T2DM patients, and this effect was abolished by the MEK1/2-selective inhibitor trametinib. Both ruxolitinib and trametinib significantly reduced basal OCR and ECAR in HSVSMCs from both T2DM and non-diabetic patients. Together, these findings demonstrate a JAK/STAT- and MAPK/ERK-mediated regulation of mitochondrial function in HSVSMCs. As such, they represent potential targets for regulation of HSVSMC function that can be explored for drug development to limit saphenous VGF in T2DM.

Introduction Suppressor of cytokine signalling 3 (SOCS3) limits JAK/STAT pathways involved in vascular inflammation and remodelling responsible for vein graft failure. However, SOCS3 is limited by its short biological half-life. Therefore, mutation of all 9 Lys residues that are potential sites of ubiquitination to Arg should produce a mutated SOCS3 resistant to ubiquitin-mediated proteasomal degradation (“Lys-less” SOCS3). We hypothesise that a stablised “Lys-less” SOCS3 may have greater therapeutic potential versus wild type (WT) SOCS3 in limiting JAK/STAT mediated processes responsible for neointimal hyperplasia and vein graft failure in type 2 diabetes mellitus (T2DM). Methods Smooth muscle cells (SMCs) and endothelial cell (ECs) isolated from human saphenous vein (HSV) were transduced with recombinant lentiviruses, MOI= 3.6 (WT), 22.2 (Lys-less SOCS3) and 5.6 (GFP) tu/cell. Successful transduction was confirmed by immunofluorescence and immunoblotting. Ubiquitylation was tested by immunoprecipitation and immunoblotting and half-life was determined by immunoblotting following incubation ± protein synthesis inhibitor emetine. HSVSMC proliferation (cell counting and CyQuant proliferation assay) and migration (Boyden chamber assay) were assessed in transduced HSVSMCs. Finally, the effect of WT and Lys-less SOCS3 gene delivery on IL-6 and PDGF-BB signalling in HSVSMCs was assessed by phosphorylation of STAT3 (Tyr705) and ERK1/2 (Thr202/Tyr204) by immunoblotting. Results Lentiviral transduction of WT and Lys-less SOCS3 in HSVSMCs and ECs was highly efficient after 48hrs with 97±0.9% (n=4) and was sustained for at-least 2 weeks. Lys-less SOCS3 was resistant to ubiquitylation in HSVECs in contrast to WT (n=3). Lys-less SOCS3 was also more stable (t1/2=4 h) than WT (t1/2<4 h) (n=6, p<0.001). Concomitant with a significant reduction in proliferative response to sIL-6Rα/IL-6 in HSVSMCs treated with WT and Lys-less SOCS3 was a selective inhibition of sIL-6Rα/IL-6-mediated STAT3 activation by 74±6% and 80±6% respectively (n=5, p<0.001 versus sIL-6Rα/IL-6 alone) but not ERK1/2. Time course experiments indicated that PDGF-BB-induced STAT3 and not ERK1/2 activation was blocked by WT SOCS3 in HSVSMCs by 59±4% at 5 minutes and 38±1% at 15 minutes (n=3 p<0.05 versus PDGF-BB/GFP). WT and Lys-less SOCS3 did not affect proliferative responses to 20% foetal bovine serum as well as PDGF-BB-induced migration. Conclusion WT and Lys-less SOCS3 can be successfully transduced into HSVSMCs and ECs with high efficiency using recombinant lentiviruses. Lys-less SOCS3 is more stable than WT yet functionally equivalent in inhibiting HSVSMC proliferation. WT SOCS3 was also capable of inhibiting both IL-6 and PDGF-BB signalling. These results provide evidence for the possible therapeutic targeting of SOCS3 to limit SMC dysfunction responsible for graft failure. Conflict of interest None