c-Kit/mbKitL interaction mediates EPC adhesion. (A) The indicated markers were analyzed on EPCs by FACS. Left curves indicate preimmune mouse IgG used as negative control; right curves, CD146 or VE-cadherin expression. (B) EPC extracts were subjected to SDS-PAGE and the filter was immunoblotted (IB) with an anti–c-Kit (K213) antiserum. M07e cells were used as positive control. (C) A representative scanning electron microscope micrograph of EPCs adherent to untreated or TNFα-treated CDC-HMEC-1 cells is reported. K44 was used as indicated (× 1000 magnification). Adherent cells to untreated or TNFα-treated CDC-HMEC-1 cells were also counted for statistical analysis (left panel; data are the mean ± SD; *P < .05 untreated vs TNFα-treated cells). (D) EPCs, treated or not with K44 or K45, were let to adhere to activated CDC-HMEC-1 cells. The anti-KitL antibody was used to pretreat CDC-HMEC-1 cells before the adhesion assay. Adherent cells were counted for statistical analysis and data reported are the mean ± SD (*P < .05 control vs experimental groups). (E) EPCs were added to rhKitL-coated plates and adhesion was evaluated after 45 minutes of incubation. K44 or K45 was added where indicated. Adherent cells were counted and statistical analysis was performed (data are the mean ± SD; *P < .05 BSA vs experimental groups; §P < .05 rhKitL + K44 vs rhKitL). (F) Adhesion was performed on activated CDC-HMEC-1 cells preincubated with an anti–ICAM 1, an anti–E-selectin, or an anti–VCAM 1 antibody. K44 was added to EPCs where indicated. (*P < .05 control vs experimental groups; anti–ICAM 1 + K44 vs anti–ICAM 1; anti–E-selectin + K44 vs anti–E-selectin; anti–VCAM 1 + K44 vs anti–VCAM 1). Four different experiments were performed with similar results.