136 Theurapeutic targeting of jak-stat signalling pathways responsible for vascular restenosis in type 2 diabetes mellitus

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Abstract

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.

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