Fig. 9

MFG-E8 increases VSMC proliferation but impairs VSMC migration. a–b Photomicrographs of the immunostaining of Ki-67 in cross-sections of ligated aged CCAs either with or without periadventitial delivery of rMFG-E8 (2 μg/mL) at 21 days after ligation; bar, 5 μm. c Quantitative IHC analysis of the proliferation index (Ki-67⁺ cells over total cell number) in the intimal–medial area of the vessel 21 days after ligation (n = 4 for each experimental group). Data are presented as mean ± SEM. **P < 0.01; Student’s t test. d–e VSMCs isolated from the aortas of aged mice were cultured either with or without MFG-E8 (250 ng/mL) for 24 h. Cell lysates were analyzed through immunoblotting with antibodies specific for PCNA. The PCNA level, normalized to that of tubulin, was quantified (n = 3). Data are presented as mean ± standard deviation. *P < 0.05; Student’s t test. f VSMCs derived from aged aortas were incubated either with or without MFG-E8 for 24 h. The cells were trypsinized and then seeded into the upper wells of 24-well Transwell plates with 8-μm pores for 6 h of incubation with PDGF-BB (10 ng/mL) in the lower chamber. The cells that had migrated into the lower chambers were stained with calcein-AM and quantified using the fluorescence intensity. Data are presented as mean ± standard deviation. ****P < 0.0001; Student’s t test