Abstract 10076: Angiotensin II Downregulates Micro-RNA 22 to Promote CYR61-Mediated Vascular Smooth Muscle Cell Calcification
Angiotensin II (AngII) has been linked to vascular calcification; however, the molecular mechanism(s) by which this occurs remain unknown. We hypothesized that AngII increases vascular smooth muscle cell (SMC) calcification by regulating microRNAs (miR) to increase the expression of cysteine-rich angiogenic protein 61 (CYR61), which induces osteoblastic differentiation of mesenchymal stem cells. We treated human coronary artery SMC with AngII (10-9 mol/l) and confirmed a 21.6-fold increase in CYR61 mRNA at 1 h (p<0.05) and a 2.1-fold increase in CYR61 protein at 2 h (p<0.05); this increased expression was associated with a 1.7-fold increase in Runx2 protein expression at 8 h (p<0.05). Coincubation with the AngII type I receptor blocker losartan (1 μmol/l) prevented the increase in CYR61 and Runx2 expression. Bioinformatic analysis identified miR22 as a candidate miR to regulate CYR61 expression and real-time PCR demonstrated that AngII decreased miR22 by 52% at 1 h (p<0.05). To confirm that miR22 regulates CYR61 expression, we transfected SMC with an anti-miR22 and demonstrated a 22.6% increase in CYR61 expression. Forced expression of miR22 by transfection with pre-miR22 inhibited AngII-induced CYR61 expression by 49% (p<0.05) and Runx2 protein expression by 41% (p<0.01). Next, COS7 cells were cotransfected with a luciferase reporter plasmid containing the 3'-untranslated region (UTR) of CYR61 and pre-miR22. In this assay, forced expression of miR22 decreased luminosity by 49% (p<0.05) indicating that miR22 binds to the CYR61 3'-UTR to modulate protein expression. Von Kossa staining demonstrated that AngII increased SMC calcification by 146.1 ± 10.6 % (p<0.01) and losartan blocked this effect. Similarly, SMC calcification was increased by downregulation of miR22 (139.0 ± 8.6 %, p<0.01) or adenovirus-mediated overexpression of CYR61 (to increase CYR61 expression 1.8-fold) (136.0 ± 5.7 %, p<0.01). In contrast, a 20% downregulation of CYR61 by transfection with antisense-CYR61 inhibited Ang II-induced calcification by 32.0 ± 4.3 % (p<0.01). These findings demonstrate that AngII downregulates miR22 to increase expression of CYR61 and, thereby, SMC calcification. Therapies that target this signaling pathway may limit vascular calcification.
- © 2011 by American Heart Association, Inc.