MCP-1–induced angiogenesis in vivo is dependent on VEGF-A and RhoA activation. To chorioallantoic membranes (CAMs) of 10-day-old chick embryos, MCP-1 (10 ng/mL) and, where indicated, Flt2-11 (1 mg/mL; Calbiochem, San Diego, CA), C difficile toxin (20 ng/mL), C3 transferase (100 μg/mL), or 10 μM PD98059 (p42/44 MAPK inhibitor) was applied as described in “Study design.” Bovine serum albumin (BSA; 20 ng/mL) and VEGF (10 μg/mL) were used as negative and positive controls, respectively. (A) After 72 hours of incubation, newly formed blood vessels were photographed. (B) The number of newly formed vessels radiating from the applied spot was counted by 2 independent observers. ▪ indicates VEGF-A165 or MCP-1 alone; □, with Flt2-11; ▦, with C difficile; ▨, with C3 transferase; and ▤, with PD98059. Data are shown as means ± SE. *P < .05, as determined by Mann-Whitney U test.
