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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. 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. 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.

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.

IntroductionSaphenous 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.MethodsHSVSMCs 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 (O2·) 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.ResultsThere 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.ConclusionData 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 interestNone

IntroductionSuppressor of cytokine signalling 3 (SOCS3) limits multiple signalling pathways involved in vascular inflammation and remodelling responsible for neointimal hyperplasia and vein graft failure. However, SOCS3 function is limited by its short biological half-life, suggesting that SOCS3 stabilisation might prove an effective therapeutic strategy. Through identification of SOCS3 ubiquitination sites, we have engineered a novel SOCS3 transgene resistant to proteasomal degradation and assessed its ability to limit signalling pathways and processes responsible for neointimal hyperplasia.MethodsFlag-tagged SOCS3 transiently expressed in HEK293 cells was immunoprecipitated with anti-Flag antibody and fractionated by SDS-PAGE for trypsin digestion. Tryptic peptides were analysed by liquid chromatography and tandem mass spectrometry, and ubiquitination sites identified from di-Gly-modified SOCS3 peptides. Smooth muscle cells (SMCs) and endothelial cells (ECs) from human saphenous vein (HSV) were transduced with recombinant lentiviruses (LVs) with MOIs ranging from 3.6-22.2 tfu/cell. Ubiquitination was assessed by immunoprecipitation of soluble cell lysates and immunoblotting. Half-lives of SOCS3 transgenes were determined by immunoblotting following HSVSMC incubation ± protein synthesis inhibitor emetine. HSVSMC proliferation was assessed by cell counting. Finally, effects of WT and Lys-less SOCS3 gene delivery on cell signalling was determined by assessing phosphorylation of STAT3 (Tyr705) and ERK1/2 (Thr202/Tyr204) by immunoblotting.ResultsAnalysis of recombinant SOCS3 immunoprecipitated from HEK293 cells revealed that 8 of the 9 Lys residues in human SOCS3 were ubiquitinated. A mutated SOCS3 in which all 9 Lys residues were mutated to Arg was resistant to ubiquitination compared to wild type (WT) SOCS3. LV transduction of WT and Lys-less SOCS3 in HSVSMCs and ECs was highly efficient with >90% of cells expressing SOCS3 transgene after 48 hours. Lys-less SOCS3 was significantly more stable, displaying a biological half-life >4 hours versus <4 hours for WT (n=6, p<0.001). Despite these differences, WT and Lys-less SOCS3 were functionally equivalent in their ability to selectively inhibit STAT3 phosphorylation in response to either sIL-6Rα/IL-6 (74±6% and 80±7% inhibition respectively; n=4, p<0.05 versus controls) or PDGF-BB (67±17% and 72±18% inhibition respectively; n=5) but not ERK1/2 phosphorylation. However, while WT and Lys-less SOCS3 each inhibited cell proliferation in response to sIL-6Rα/IL-6 (83±29% and 89±22% inhibition respectively; n=4, p<0.05 versus controls) only Lys-less SOCS3 significantly inhibited PDGF-BB-stimulated proliferation (67±11% inhibition; n=4, p<0.05 versus controls).ConclusionsThese results provide evidence for possible therapeutic targeting of SOCS3 stabilisation to limit HSVSMC dysfunction responsible for vein graft failure.Conflict of InterestNone

Background Herein, we report the development of a novel nanogels (NG) system loaded with Azadirachta indica (A. indica) Adrien-Henri de Jussieu (A. Juss.), commonly known as neem, for possible topical treatment of wound infections. Methods To develop A. indica extract-loaded NG, first, extract-loaded nanoparticles (NPs) were produced using poly- ε -caprolactone (PCL) as the nanocarrier polymer. Secondly, the NPs were entwined in chitosan (CS) hydrogel loaded with the extract of A. indica to prepare the loaded NG system. Blank NG was produced without the extract. The developed NG was characterized, and its antibacterial effect was evaluated. Results Phytochemical screening of ethanolic extract of A. indica leaves indicated the presence of saponins, flavonoids, glycosides, tannins, alkaloids, steroids, terpenoids, and anthraquinones. The characterization data revealed that the developed NG formulations are nanosized in the ranges of 140–440 nm and 190–610 nm for blank NG and A. indica extract-loaded NG, respectively, and have mostly spherical structures. The developed NG formulation displayed pH-dependent swelling and erosion that are in direct proportion to the change in pH. Fourier transform infrared spectroscopy (FTIR) showed various characteristic bands of A. indica and formulation excipients, confirming the encapsulation of the extract. The minimum inhibitory concentration (MIC) of the loaded NG was found to be 0.250 ± 0.05 mg/ml, 0.625 ± 0.15 mg/mL, and 0.250 ± 0.07 mg/mL for Staphylococcus aureus (S. aureus ), Escherichia coli ( E. coli), and Salmonella typhi (S. typhi ) strains of bacteria, respectively. The NG formulation exhibited significant bacterial inhibition zones which were recorded as 8 ± 2.0 mm ( p  < 0.05), 16 ± 3.0 mm ( p  < 0.05), and 6 ± 1.0 mm ( p  < 0.05) for S. typhi, E. coli, and S. aureus , respectively, as compared with that produced by the crude extract. Conclusions An A. indica extract-loaded NG was successfully developed, and it demonstrated good formulation features, stability under refrigerated and room temperature conditions, as well as useful antibacterial activity that could be used for potential wound infection treatment. Graphical abstract

IntroductionSuppressor 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).MethodsSmooth 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.ResultsLentiviral 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.ConclusionWT 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 interestNone

Vascular inflammation emerge as a key event in development of macrovascular complications of type 2 diabetes mellitus (T2DM). Suppressor of cytokine signalling 3 (SOCS3) is a potent inhibitor of inflammatory pathways (JAK/STAT) involved in the propagation of vascular endothelial cell (EC) inflammation, smooth muscle cell (SMC) migration and proliferation all of which are key events in development of coronary artery bypass graft re-stenosis. However, SOCS3 is limited by its short biological half-life. Therefore a mutated SOCS3 transgene that is resistant to ubiquitination and subsequent proteasome-dependent degradation (‘Lys-less’ SOCS3) has been developed.Hypothesis being tested is that JAK-STAT signalling pathway is enhanced in HSV-SMCs as a consequence of T2DM. In addition, ‘Lys-less’ SOCS3 may have greater therapeutic potential versus wild type (WT) SOCS3 in limiting JAK-STAT mediated processes responsible for neo-intimal hyperplasia and vein graft failure in T2DM. Immuno-fluorescence and confocal imaging have shown that transduction of WT and Lys-less SOCS3 in HSV-SMCs and ECs by lenti-virus can be highly efficient after 48 hours, importantly, there was prolonged and stable SOCS3 protein expression for up to 2 weeks as assessed by immunoblotting. Lys-less SOCS3 was found to be resistant to ubiquitination and has a biological half-life of at least 4 hours versus 45–60 mins for WT SOCS3 as determined from emetine chase experiments in transduced cells. Overexpressed Lys-less SOCS3 and WT SOCS3 inhibited sIL-6Rα/IL-6 mediated STAT3 activation in HSV-SMCs by 83% ±7% and 79% ±7 respectively (p<0.05 versus control response, set at 100%). Furthermore, immunoprecipitation experiments have shown that overexpressed Lys-less SOCS3 precipitated along with Elongin B/C as compared to negative control in SV ECs.In summary, current in-vitro results suggest a mechanism by which overexpressed SOCS3 in SMCs and ECs may supress vascular inflammation causing graft restenosis in T2DM.Abstract 133 Figure 1