Abstract 16341: Msx2 Regulates Osteochondrogenic Gene Expression In Bone Morphogenetic Protein-2-Stimulated Vascular Smooth Muscle Cells From Leptin-Deficient ob/ob Mice
Diabetes increases vascular calcification, which is associated with higher cardiovascular mortality. In order to assess calcifying mechanisms in insulin resistance and obesity, we isolated vascular smooth muscle cells (SMC) from leptin-deficient ob/ob (obob) and from C57BL6 (C57) mice, which were cultured without or with bone morphogenetic protein-2 (BMP2) 50ng/mL, a potent mediator of vascular calcification. Results demonstrated herein are statistically significant (Mean±SEM, ANOVA, p<0.05, n=3 to 6). Calcification increased both in untreated obob vs. C57 SMC (1.44±0.06 vs. 1±0.29) and in BMP2-treated obob vs. C57 SMC (2.03±0.06 vs. 1.32±0.06) after 14 days. Although alkaline phosphatase (ALP) mRNA expression increased in obob vs. C57 SMC both without BMP2 (8.35±1.31 vs. 1.01±0.07) and with BMP2 (15.22±3.21 vs. 1.88±0.23), ALP activity decreased in obob vs. C57 SMC without BMP2 (0.13±0.02 vs. 1±0.03) and was not different with BMP2 (2.56±0.05 vs. 3.12±0.22, p=NS). Then, we showed that MSX2 mRNA expression increased in untreated obob vs. C57 SMC (9.01±1.79 vs. 1.03±0.14) and after incubation with BMP2 for 48h (19.26±5.58 vs. 0.71±0.07). Moreover, obob ex-vivo cultured aorta rings showed increased MSX2 mRNA expression vs. C57 (4.85±0.85 vs. 0.97±0.58). To further determine the role of MSX2 in osteochondrogenic differentiation of SMC, we transfected C57 and obob SMC with either MSX2 siRNA (MSX2C57; MSX2obob) or β-catenin-1 siRNA (βCatC57; βCatobob) or scrambled siRNA (ScrC57; Scrobob) and incubated these cells without or with BMP2 for 48h. BMP2-incubated Scrobob increased MSX2, RUNX2 and ALP mRNA expression vs. untreated Scrobob (3.16±0.91 vs. 1.1±0.22; 2.37±0.57 vs. 1.04±0.14 and 4.51±0.79 vs. 1.12±0.35 respectively), which was abrogated in MSX2obob+BMP2 SMC (0.58±0.13; 0.83±0.22; 2.94±0.41). Moreover, BMP2-treated βCatobob decreased RUNX2 mRNA expression vs. Scrobob+BMP2, which did not occur in C57 SMC. In opposition to obob, BMP2-treated MSX2C57 SMC increased RUNX2 mRNA expression vs. untreated ScrC57 and MSX2C57. Still, increased ALP mRNA expression persisted in MSX2C57 vs. ScrC57 SMC after BMP2 stimulation. In conclusion, MSX2 mediates osteochondrogenic gene expression in BMP2-stimulated obob SMC.
- © 2013 by American Heart Association, Inc.