Fig. 7. Milk fat globule–epidermal growth factor 8 (MFG-E8) promotes the osteogenic transdifferentiation of vascular smooth muscle cells (VSMCs) and activation of the transforming growth factor-β1 (TGF-β1)-mediated signaling cascade through β1 integrin engagement.

a, b Ligated common carotid arteries (CCAs) of wild-type (WT) and MFG-E8-knockout (KO) mice were treated with phosphate-buffered saline (PBS) and CaCl₂ (0.4 M) by using pluronic gel. a Representative immunohistochemistry (IHC) microphotographs of activated β1 integrin in the CCAs of mice 21 days after ligation. Scale bar: 50 μm. b Quantitative analysis of the IHC intensities of activated β1 integrin in the intimal medial area (WT + PBS: n_mice = 3, KO + PBS: n_mice = 3, WT + CaCl₂: n_mice = 6, KO + CaCl₂: n_mice = 6). Data are presented as mean ± standard error of the mean. Each data point is derived from an assessment of three sections of an individual animal. **P < 0.01, as obtained using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test. c–l A10 cells treated with PBS, rMFG-E8 (100 ng/mL), and rMFG-E8 with β1 inhibitory antibody (50 μg/mL) were cultured in osteogenic medium (OM) for 7 days. Immunoblotting was performed to assess the protein expression of matrix metalloproteinase 2 (MMP2) (c), phosphorylated Smad2 (P-Smad2) (e), runt-related transcription factor 2 (Runx2) (g), and bone morphogenetic protein-2 (BMP-2) (i) in A10 cells. The quantitative analyses of the levels of MMP2, Runx2, and BMP-2 normalized to those of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (d, h, j, respectively), and P-Smad2 to total Smad2/3 (f) were conducted (n = 3). Data are presented as mean ± standard deviation (SD). Three independent experiments were performed. Each data point was derived from each of the three repeated experiments. *P < 0.05 and **P < 0.01, obtained using one-way ANOVA followed by Tukey’s multiple comparison test. k Representative microscopic images of the alizarin red stain (ARS) of 7-day cultured A10 cells. Scale bar: 50 μm. l Quantitative analysis of ARS levels in A10 cells (n = 3). Data are presented as mean ± SD. Three independent experiments were performed, and each experiment was repeated with similar results. The data were derived from one of the representative experiments. ****P < 0.0001, obtained using one-way ANOVA followed by Tukey’s multiple comparison test.