Figure 5.

The effects of osteocalcin on human aortic smooth muscle cell (HASMC) migration and proliferation. (a) Representative images from a scratch wound assay performed in HASMCs in which the scratch area was monitored for up to 48 hr. (b) Rate of migration/closure of scratch of HASMCs treated with either vehicle or ucOCN (10 ng/ml; n = 23 from four experimental repeats). (c) ucOCN concentration‐dependent effect (0.1–50.0 ng/ml) on cell proliferation as measured by MTS assay after 48 hr (n = 12 from three experimental repeats). (d) Cell proliferation was further assessed using PI3K inhibitor (LY294002 hydrochloride) and MAPK (MKK/MEK) inhibitor (PD98059) (three experimental repeats, total at least n = 9). (e) MMP‐3 secretion over 24–72 hr by HAECs treated with vehicle or ucOCN (10 ng/ml; n = 12 for control and n = 24 for ucOCN, from three experimental repeats). One‐way ANOVAs were used to analyze proliferation data and multiple testing corrected for by Dunnett's statistical test. Data are given as means with error bars representing SEM. *Denotes a significant difference compared to control (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001). ^#Denotes significant difference compared to ucOCN. ANOVA: analysis of variance; SEM: standard error of mean; MMP‐3, matrix metalloproteinase‐3; MTS: Cell Titre 96 AQueous One Solution Cell Proliferation Assay; ucOCN, uncarboxylated osteocalcin