Abstract 18084: MiR-30e Targets IGF2-Regulated Osteogenesis in Bone Marrow Derived Mesenchymal Stem Cells, Aortic Smooth Muscle Cells, and APOE-/- Mice
Objective: In atherosclerosis, activation of an osteogenic transcriptional program in de-differentiated mesenchymal-like smooth muscle cells (SMCs) contributes substantially to the initiation of aortic calcification. We found that aortic miR-30e is downregulated with age and atherosclerosis and inversely proportional to osteogenic markers. To assess the effect of miR-30e downregulation, we tested the hypothesis that miR-30e regulates osteogenic program in bone marrow derived mesenchymal stem cells (MSCs), aortic SMCs, and APOE-/- mice.
Method and Results: In aortas of old (13.5 mo) and young (6 mo) wild type and APOE-/- mice (n=4-15), we found that NFYC gene and hosted miR-30e transcripts are downregulated with age and atherosclerosis while the osteogenic markers Runx2, Opn, and Igf2 are upregulated - p<.05. MiR-30e over-expression reduced the proliferation of MSCs and SMCs while increased adipogenic differentiation of MSCs and smooth muscle differentiation of SMCs, as quantified by expression of Fabp4 and Pparg and Oil Red Staining in MSCs and expression of SM22a, Cnn1, and VCl in SMCs - p<.05. Electron microscopy showed more miR-30e-caused fat droplets in MSCs, and less mitochondria (p<.01) in SMCs. In MSCs and SMCs over-expressing miR-30e reduced Igf2 transcripts - p<.01. At 2 wk osteogenic differentiation, an osteogenic panel was dramatically enhanced in MSCs but reduced by miR-30e, with a decrease in calcium deposition. Inhibiting miR-30e in MSCs showed over-expression of Igf2 transcripts -p<.01. Luciferase and mutagenesis assays showed binding of miR-30e to a novel and essential site at the 3’UTR of Igf2. Treating MSCs with IGF2 protein increased (p<.05) miR-30e transcripts at 8 and 24 hrs, suggesting a miR-30e-Igf2 feedback loop. In 8mo APOE-/- mice (n=6-7), 8 wk injections of antimiR-30e oligos increased Igf2 expression in the aortas and livers as measured by qPCR and Elisa - p<.05, and modestly enhanced calcium deposition in aortic roots as depicted by Alizarin Red and Van Kossa staining.
Conclusion: MiR-30e represses the osteogenic program in MSCs and SMCs by targeting IGF2 and drives their differentiation into different lineages. Downregulation of miR-30e in aortas with age and atherosclerosis may trigger vascular calcification.
Author Disclosures: W. Ding: None. J. Li: None. J. Singh: None. R. Alif: None. R.I. Vazquez-Padron: None. S.A. Gomes: None. J.M. Hare: Employment; Modest; Vestion. Ownership Interest; Modest; Biscayne. Ownership Interest; Significant; Vestion Inc. L.A. Shehadeh: Research Grant; Modest; AHA GIA. Ownership Interest; Modest; Patent.
This research has received full or partial funding support from the American Heart Association
- © 2014 by American Heart Association, Inc.