IGF2-enhanced invasion of EPCs is mediated by an increase in MMP9 expression. (A) Invasion assay using vertical collagen gel chamber with IGF2 at the bottom for the chemotactic gradient. Dil-acLDL–labeled EPCs were stimulated with IGF2 (1, 10, 50, and 100 ng/mL). (B) Dil-acLDL–labeled EPCs were preincubated for 6 hours with or without 50 ng/mL PTX or preincubated for 30 minutes with or without 5 μM U73122 or 5 μM BAPTA-AM, and then stimulated with 50 ng/mL IGF2. After 48 hours, invasion was quantified by counting the cells. Scale bar represents 1 mm. (C,D) Total mRNA was isolated from EPCs treated with IGF2 (1, 10, 50, and 100 ng/mL) or untreated, and MMP9 gene expression was assessed by real-time PCR analysis. EPCs were incubated in 0.5% FBS-conditioned medium for 24 hours in the absence or presence of IGF2 (10, 50, and 100 ng/mL) (E) or for the indicated times in the absence or presence of 50 ng/mL IGF2 (F). (G) EPCs were preincubated for 6 hours with or without 50 ng/mL PTX or preincubated for 30 minutes with or without 5 μM U73122 or 5 μM BAPTA-AM, and then incubated in 0.5% FBS-conditioned medium for 24 hours in the absence or presence of 50 ng/mL IGF2. The culture medium was collected and subjected to gelatin zymography. Data are the mean plus or minus SE. **P < .01 versus IGF2 alone.