Abstract 517: The MicroRNA Cluster 99b/let7-e/125a is Downregulated Following Vein Grafting in a Porcine Model, and Upregulation Alters Vascular Cell Phenotype in vitro

Abstract

Introduction: Coronary artery bypass surgery is a common surgical procedure. However, 20-50% of patients present vein graft failure within 10 years. The occlusion of the graft is induced by intimal hyperplasia formation promoted by enhanced vascular smooth muscle cells (SMC) proliferation and migration. MicroRNAs are becoming recognized for their role in SMC proliferation and migration associated with vascular pathologies. Here we investigate a cluster of miRNAs (99b/let-7e/125a) in a model of vein graft failure and assessed the effect of modulating its expression in SMCs. Methods: Global miRNA profiles were analyzed in porcine saphenous vein grafts 7 and 28 days post-engraftment (n=6) by a TaqMan low density array. The three miRNAs encoded by the cluster were further validated by qPCR. SMCs were isolated from human saphenous vein and then transduced with a lentiviral vector (Lv-cluster) carrying entire cluster sequence. The effects on proliferation (BrdU incorporation) and migration (scratch wound) were analyzed. Computational analysis identified AKT-1, a known pro-proliferative factor, as a common previously validated target for 99b/let7-e/125a miRNAs. AKT-1 mRNA (qPCR) and protein expression (western blot) were analyzed in SMCs previously infected with Lv-cluster. Results: All three miRNAs encoded by this cluster were downregulated in graft samples 7 and 28 days post-engraftment, compared to ungrafted saphenous vein (Fold change at 7 days: 99b: 0.5±0.07, let-7e: 0.34±0.03, 125a: 0.6±0.07; p<0.05. Fold 28 days: 99b: 0.32±0.05, let-7e: 0.24±0.05, 125a: 0.35+0.07; p<0.001). Infection of SMCs with this Lv-cluster increased the expression of the mature miRNAs by ~ 5 fold compared to controls. This increase was associated with a significant (p<0.01) reduction in SMCs migration (20%±1) and proliferation (90%±4). These results demonstrate that overexpression of this cluster in SMCs significantly (p<0.05) reduced the mRNA and protein levels of AKT-1 (0.6 and 0.4 fold, respectively). Conclusion: MiRNAs levels of the cluster 99b/let-7e/125a are downregulated in a porcine model of saphenous vein grafting. In vitro results suggest that overexpression of this miRNA cluster reduces migration and proliferation in SMCs by targeting AKT/mTOR pathway.