Abstract 15099: Bone Morphogenetic Protein 2 and Leptin Promote Calcification of Vascular Smooth Muscle Cells from Leptin-Deficient ob/ob Mice Independently of Alkaline Phosphatase Activity

Abstract

Vascular calcification and diabetes are associated with coronary artery disease and cardiovascular mortality. However, the impact of obesity, insulin resistance and leptin on vascular calcification is poorly understood. We hypothesized that bone morphogenetic protein 2 (BMP2) and/or leptin would stimulate different calcifying responses in vascular smooth muscle cells (SMC) from ob/ob mice and in SMC from C57BL6 mice. We investigated the effect of BMP2 and/or leptin on mineralization of SMC from ob/ob mice in comparison with calcification of SMC from C57BL6 mice and we assessed osteogenic gene and protein expression, such as RUNX2, MSX2 and alkaline phosphatase (ALP) in these cells. Aortic SMC harvested from the aorta of C57BL6 or ob/ob mice were cultured without BMP2 (C57/Cont or ObOb/Cont) or with BMP2 50ng/mL (C57/BMP2 or ObOb/BMP2). RUNX2 mRNA (48h) and protein expression (72h) increased respectively 1.54±0.12 and 2.62±0.29 in C57/BMP2 versus (vs) C57/Cont (p<0.05, n=3) SMC; 1.77±0.04 and 2.50±0.19 in ObOb/BMP2 vs ObOb/Cont SMC (p<0.05). Moreover, MSX2 mRNA increased 2.75±0.08 only in ObOb/BMP2 vs ObOb/Cont (p<0.05, n=3), but not in C57/BMP2 SMC. ALP mRNA increased 4.05±0.18 in ObOb/Cont vs C57/Cont SMC (p<0.05, n=4) after 72h of culture under baseline condition, and further increased 7.69±0.38 in ObOb/BMP2 vs C57/Cont and 1.9±0.1 in ObOb/BMP2 vs ObOb/Cont (p<0.05, n=3), but it did not enhance in C57/BMP2 SMC. After 14 days, C57/BMP2 and ObOb/BMP2 SMC calcification augmented respectively 1.24±0.03 and 1.36±0.01 vs C57/Cont (p<0.05, n=4). In parallel, C57/BMP2 ALP activity increased 1.82±.22 vs C57/Cont SMC (p<0.05, n=3). Incubation with leptin 10ng/mL alone for 14 days increased ObOb SMC calcification 1.54±0.05 vs C57/Cont (p<0.05, n=3) and co-incubation with leptin and BMP2 potentiated ObOb SMC calcification 1.88±0.02 vs C57/Cont (p<0.05, n=3). Nevertheless, leptin incubation didn’t increase calcification in C57/Cont SMC without or with BMP2. Despite increased calcification of ObOb/BMP2 and ObOb/BMP2/Lep SMC vs C57/Cont, ALP activity decreased 0.22±0.02 and 0.12±0.003 respectively vs C57/Cont (p<0.05, n=3). In conclusion, increased calcification of SMC from ob/ob mice with BMP2, leptin or both occurred independently of ALP activity.