Role of microRNA-21 in the regulation of human saphenous vein smooth muscle cell function and vascular remodelling.

Cardiovascular diseases including coronary artery disease are the major cause of death worldwide. Saphenous vein (SV) bypass grafting is widely used to revascularise atherosclerotic coronary arteries, although failure rates are problematic. Adverse remodelling and intimal hyperplasia (IH) underlie graft failure primarily driven by aberrant smooth muscle cell (SMC) function. Whilst altered SMC function is pivotal to the adaptation of SV grafts, in the longer term excessive cell proliferation and migration lead to intimal hyperplasia that compromises patency rates. Whilst there are no pharmacological agents to effectively prevent IH, accumulating evidence has implicated microRNAs (miRs), short non-coding RNAs that negatively regulate gene expression, in SMC phenotypic changes and vascular remodelling. In particular, miR-21 is highly expressed in the cardiovascular system and is reportedly dysregulated in pathological conditions. This thesis confirmed a distinct phenotype in SV-SMC from T2DM patients, relative to those from patients without diabetes (ND), although this was not associated with differential miR-21 expression. Artificial overexpression of miR-21 in ND SV-SMC increased total cell area and cell proliferation and its knockdown was able to reverse the increase in cell area. In addition, treating SV-SMC with PDGF-BB, commonly associated with vascular remodelling, led to increased miR-21 expression through activation of both ERK and Akt; inhibition of either pathway abrogated miR-21 expression. Exploring the downstream target genes of miR-21 using a vascular remodelling specific array and follow-up RT-PCR yielded two genes affected by miR-21 overexpression; increased MMP-1 mRNA and decreased IL-1A mRNA. Surprisingly, SV-SMC migration in cells overexpressing miR-21 did not, however, modulate migration through collagen-1 or -3 coated membranes. Potential downstream target genes of miR-21 mediating functional changes related to SMC proliferation and migration were tested. Whilst there were no clear effects on “classical” targets (PDCD4, PTEN and Spry), a significant downregulation of RECK mRNA and protein in miR-21 overexpressing SV-SMC was apparent. Further studies are required to explore in detail how RECK is involved in miR-21 mediated cellular functions and whether targeting miR-21 in SV-SMC would provide a promising therapeutic strategy to improve outcomes in patients following SV grafting