IGF2 increases EPC chemotaxis. (A) Total mRNA was isolated from EPCs and OECs, and gene expression profiles were assessed using an Affymetrix gene chip. Red indicates high expression. IGF2R mRNA and protein levels were measured by RT-PCR (B),Western blotting (C), and flow cytometric analysis (D). (E) EPCs were treated with IGF2 (1, 10, 50, and 100 ng/mL). (F,G) EPCs were preincubated for 30 minutes with or without IGF2RN (1, 2.5, 5, and 10 μg/mL) or M6P (5 mM) and stimulated with IGF2 (50 ng/mL), VEGF (50 ng/mL), or SDF-1 (50 ng/mL). After 24 hours, chemotaxis was quantified by counting the cells that migrated to the lower side of the filter with optical microscopy at 200× magnification. (H,I) EGFP CD34⁺ mouse BMMNCs were isolated using the magnetic-activated cell sorting (MACS) system and then cultured for 7 days. Immunofluorescent staining of Dil-acLDL was performed (H) using an Olympus IX81-ZDC microscope with a UPL SAPO 10×/0.40 NA lens. Mouse IGF2R, KDR, and CXCR4 mRNA expression was measured by RT-PCR (I). (J) EGFP CD34⁺ mBMMNCs were preincubated for 30 minutes with or without 5 mM M6P and stimulated with 50 ng/mL mIGF2 or 50 ng/mL mVEGF. All data are presented as the mean plus or minus SE from 3 different experiments in duplicate. **P < .01 versus IGF2 alone.