P582Aberrant regulation of RhoA and cytoskeletal derangement in saphenous vein smooth muscle cells derived from patients with Type 2 Diabetes: a potential role for microRNAs?

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Abstract

Purpose

Cardiovascular disease is the leading cause of morbidity and mortality in patients with Type 2 Diabetes Mellitus (T2DM). Not only are they at an increased risk of coronary heart disease but also saphenous vein (SV) bypass graft failure. It has previously been shown that SV smooth muscle cells (SMC) from T2DM patients display altered phenotype and function which may impact their efficacy as bypass grafts. MicroRNAs (miRs) are short, non-coding RNAs that modulate expression of target genes via post-transcriptional inhibition. MiRs are known to modulate SMC function however their influence on the macrovascular complications of T2DM are unknown.

Methods

SV-SMC were cultured from fragments of SV from T2DM and non-diabetic (ND) patients undergoing CABG (n=15 of each). Local ethical committee and informed patient consent were obtained. Cell morphology was analysed using phase contrast imaging and rhodamine phalloidin immunocytochemistry for the f-actin cytoskeleton. RhoA activity, protein and mRNA expression were analysed using G-LISA, western blotting and real-time RT-PCR respectively. Expression of ‘candidate’ microRNAs was determined using TaqMan Assays. MiR-125b and miR-133a have previously been associated with diabetes in animal models and SMC-enriched miR-143 and miR-145 are known to be associated with vascular disease.

Results

T2DM SV-SMC exhibited a ~50% increase in cell area (p < 0.001) and a marked disruption of the f-actin cytoskeleton when compared to their ND counterparts. The activity of RhoA was decreased by ~30% in T2DM SV-SMC (p < 0.05), with a similar fall in protein expression (p < 0.05). However, RhoA mRNA levels were comparable between the two groups (n=15) indicating that miRs may be involved. Although expression of miR-125b and miR-133a was similar in both groups, the SMC-enriched miR-143 and miR-145 were both significantly increased by ~60% in T2DM SV-SMC (p < 0.05).

Conclusion

T2DM patients exhibit increased cardiovascular risk and inferior graft patency. Our study shows that SV-SMC from T2DM patients exhibit disrupted morphology, larger spread area and disorganised cytoskeleton with an impairment of basal RhoA signalling. We saw no changes in miR-125b or miR-133a, however there was significant specific up regulation of miR-143 and miR-145. These miRs may therefore provide attractive therapeutic targets for reducing the vascular complications observed in T2DM.

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