Investigation of the role of microRNA-143 and microRNA-145 in acute vascular injury

[摘要] Vascular smooth muscle cell (VSMC) activation leading to proliferation, migration and extracellular matrix (ECM) production is a major cause of neointimal formation after stenting and coronary artery bypass grafting. Drug-eluting stents have reduced clinical incidence of in-stent restenosis by inhibiting this proliferative response but they can also delay vessel re-endothelialisation after injury leading to an increased thrombotic risk. MicroRNAs (miRNAs) are short (~22 nt) non-protein-coding RNAs which act as regulators of gene expression largely through binding to the 3’ untranslated region of target genes and causing degradation or repression of expression. MiR-143 and miR-145 are a miRNA family that are enriched in VSMCs and have been previously shown to influence VSMC phenotype through regulation of their gene targets. Consequently, the aim of this study was to investigate the role that miR-143 and miR-145 play in the neointimal response to stenting.Initial experiments investigated whether modulation of miR-143 or miR-145 expression was capable of significantly altering VSMC phenotype. It was found that modulation of miRNA levels in human saphenous vein (HSV) SMC or endothelial cells (EC) using adenoviruses was not ideal due to transduction and toxicity issues. Use of antimiR miRNA inhibitors and premiR miRNA mimics revealed that modulation of miR-143 or miR-145 levels alone was not sufficient to alter proliferation or migration of HSV SMCs in vitro. Knockdown of miR-143 expression in cells resulted in de-repression of target genes kruppel-like factor 4 (KLF4) and KLF5 but expression levels of other previously identified target genes were unaltered by miRNA modulation.Pre-clinical stenting studies are largely performed in porcine models due to similarities in vessel structure and neointimal formation, however large animal models are not ideal for early investigative studies. In order to examine the role of miR-143 and miR-145 in stent-induced vascular injury we utilised a mouse model where a bare metal stent is deployed in the thoracic aorta of a donor mouse and interposition grafted into the carotid artery of a recipient. This model resulted in the development of a defined neointima over 28 days which consisted largely of VSMCs and ECM, similar to that of the human in-stent restenosis.Vessel expression of miR-143 and miR-145 has been previously shown to be reduced following vascular injury and furthermore overexpression of these miRNA can reduce neointimal formation. MiR-143 and miR-145 knockout (KO) mice have previously been shown to have abnormal VSMC phenotype in their vessel walls including perturbed stress fiber formation, increased presence synthetic machinery and an increased number of synthetic versus contractile VSMCs. MiR-143 and miR-145 KO mice were found to develop significantly less neointimal formation in response to stenting than WT mice indicating that these miRNA are essential for normal vessel response to injury.The reduced neointimal formation following genetic ablation of miR-143 or miR-145 led to the investigation of whether pharmacological knockdown of these miRNA was able to mimic this effect. An antimiR consisting of DNA and locked nucleic acid bases targeted against mature miR-143 expression was used to knockdown miR-143 expression in mice prior to stenting. AntimiR-143-mediated knockdown of miR-143 expression did not significantly alter the degree of neointimal formation seen 28 days following stent deployment when compared to mice that received a control non-targeted antimiR (antimiR-ctl). The neointima of both antimiR-143 and antimiR-ctl mice were comparable and consisted largely of VSMCs and ECM. Re-endothelialisation had occurred by day 28 post-injury in antimiR-143 and antimiR-ctl treated mice indicating that knockdown of miR-143 did not significantly delay EC repopulation in this model.Expression of miRNA and mRNA after vascular injury can be both spatial and temporal. Target de-repression was not detected in the aorta or heart of miRNA KO or antimiR-143 treated mice. This is likely to reflect the complex nature of miRNA gene regulation in vivo which is governed by many different factors including the relative expression of the miRNA and its target, cell stress, and transcriptional activation or inhibition by growth and transcription factors.In summary, the influence of miR-143 and miR-145 on VSMC biology was investigated in vitro using a range of molecular biology techniques and in vivo in a mouse model of in-stent stenosis. Results have extended current knowledge of the degree of influence these miRNA exert over VSMC phenotype and identified that miR-143 and miR-145 are involved in neointimal formation after stenting.

[发布机构] University:University of Glasgow;Department:Institute of Cardiovascular and Medical Sciences

[关键词] microRNA, vascular smooth muscle cell, vascular injury, stent, restenosis