261Decisive role of microRNA-494 in smooth muscle cell proliferation and vascular remodeling

Abstract

Abstract Background Proliferation of vascular smooth muscle cells triggered by cytokines and growth factors is a main driver in the development of vascular proliferative diseases such as atherosclerosis and in-stent-restenosis after angioplasty. MicroRNAs (miR) are small noncoding RNAs that can inhibit the expression of multiple genes simultaneously. However, the contribution of microRNAs to the differential gene regulation that triggers vascular remodeling processes is not well understood. Methods and results Neointima formation was induced by a wire-mediated injury of the femoral artery in C57BL/6 mice. Microarray analysis of the developing neointimal lesion showed a strong reduction of miR-494 (0.411±0.04; p<0.05) at 7 days after injury. In order to investigate the expression levels of miRs in vascular cells, human coronary artery smooth muscle cells (SMC), human coronary artery endothelial cells and human monocytes were analyzed via microarray analysis. Intriguingly, miR-494 was found to be predominantly expressed in SMC via microarray and qPCR analysis. The regulation of miR-494 expression was further analyzed after stimulation of SMC with 10%FCS. Following this mitogenic stimulation, mir-494 expression dropped robustly and significantly in a time-dependent manner at 6, 9, and 24 hours. To investigate the functional impact of miR-494 on SMC proliferation, miR-494 was overexpressed using miR-494-mimics (20μM). Overexpression of miR-494 significantly reduced the FCS-induced proliferation of SMC as assessed by BrdU-incorporation. In silico analyses of potential target genes for miR-494 identified ROCK1 and Survivin, both important molecules in the mitogenic response of SMC to cytokines and growth factors, as potential targets of miR-494. Indeed, ROCK1 and Survivin were found down-regulated on the mRNA and protein level after transfection of SMC with miR-494 mimics and both mulecules could be identified as direct targets using luciferase reporter assays. Following the specific inhibition of miR-494 by local application (in a perivascular thermos-responsive, self-degrading pluronic gel) of an in vivo stabilized Pre-miR-494 after wire-induced injury of the mouse femoral artery, SMC proliferation was significantly reduced, as assessed by Ki67 immunofluorescence (26.3% vs 11.2%; p<0.05). Consistently, local application of Pre-miR-494 significantly reduced neointima formation (neointima/media ratio 2.31 in control vs 1.01 in treated animals; p<0.01). Conclusion Our results show that mir-494 is strongly down-regulated in proliferating SMC in vitro as well as during neointimal lesion formation in vivo. Moreover, overexpression/ reconstitution of miR-494 levels effectively prevented SMC proliferation and neointima formation, indicating an important functional role of miR-494 in these processes. Hence, miR-494 may represent an attractive SMC-specific target for future therapeutic interventions for the treatment of vascular proliferative diseases.