Abstract 450: Increased O-GlcNAc Modification Induces Vascular Calcification by Increasing Function of Runx2 and AKT
Objective— Vascular calcification is prevalent in patients with atherosclerosis, diabetes mellitus, and end stage renal disease, and it increases morbidity and mortality of these patients. Dramatic vascular calcification has been demonstrated in several experimental diabetic models, including the streptozotocin-induced mouse model of diabetes. Using low-dose STZ, we demonstrated that STZ-induced hyperglycemia was associated with increased protein O-link glycosylation (O-GlcNAc) modification in the mouse vasculature. The present studies sought to determine the role of O-GlcNAc modifications in regulating vascular calcification.
Methods and Results— We found that Thiamet-G, a specific inhibitor of O-GlcNAcase (OGA), the enzyme that removes O-GlcNAc, dramatically increased O-GlcNAc modification in primary mouse vascular smooth muscle cells (VSMC). Non-toxic concentrations of Thiamet-G (10nM - 10μM) induced calcification of VSMC in a dose-dependent manner. Thiamet-G-induced VSMC calcification was associated with increased expression of osteogenic genes, including the key regulator for VSMC calcification Runx2, osteocalcin, alkaline phosphatase, and collagen I (Runx2=2.97±0.31, OC=4.85±0.10, ALP=2.35±0.33, ColIa=1.86±0.20, p<0.05). In addition, Thiamet-G induced transactivity of Runx2 (fold=1.52±0.09, n=3, p<0.05) and O-GlcNAc modification of Runx2.
Furthermore, OGA shRNA markedly increased O-GlcNAc modification in VSMC. A 3.7-fold increase in calcification, as indicated by calcium content, was demonstrated in OGA knockdown VSMC compared with control VSMC. Increased calcification was associated with a 4.8-fold increase in expression of osteocalcin, a Runx2-regulated osteogenic gene. In addition, OGA knockdown enhanced and sustained phosphorylation/activation of AKT, an upstream signal that we have reported to regulate Runx2 activity.
Conclusion— We have found that increased O-GlcNAc modification in VSMC enhances calcification of VSMC, which may be attributed to increased activation of AKT and Runx2 signals. These results provide novel mechanistic insights into the role of protein O-GlcNAc modification in regulating vascular calcification in diabetes.
- © 2012 by American Heart Association, Inc.