Effect of 8-pCPT-cAMP on in vivo homing and neovascularization capacity of EPC. (A) CM-Dil-labeled EPCs in suspension were stimulated with 8-pCPT-2′-O-Me-cAMP (100 μM) or PBS for 15 minutes. After a washing step, EPCs were resuspended in PBS and injected in the tail vein in nude mice 2 days after the induction of hind limb ischemia. The number of EPCs was assessed in the ischemic muscles by microscopy (* P < .05 vs control, control, n = 4; 8-pCPT-cAMP, n = 5; data are mean ± SEM). (B) Representative images of the homing experiment demonstrated in panel A. Infused EPCs were identified as CM-Dil-labeled cells (red fluorescence); the blue fluorescence indicates nuclear staining (Topro III). (C) EPCs in suspension were stimulated with 8-pCPT-2′-O-Me-cAMP (100 μM) or PBS for 15 minutes at 37°C. After a washing step, 8-pCPT-2′-O-Me-cAMP-stimulated-EPCs, nonstimulated EPCs (control EPCs), or PBS (control, no cells) were injected in the tail vein in nude mice one day after the induction of hind limb ischemia. After 15 days, the ischemic muscles were harvested and the capillary density was assessed by microscopy as described in “Model of hind limb ischemia” (^#P < .05 vs PBS, no cells; *P < .05 vs control EPCa; PBS, no cells: n = 7, control EPCs: n = 13, 8-pCPT-cAMP-stimulated EPCs: n = 12; data are mean ± SEM). (D) Representative images from ischemic muscles from nude mice intravenously injected with 8-pCPT-cAMP-stimulated EPCs or control (nonstimulated) EPCs (experiment in panel C). The laminin staining (green fluorescence) indicates the myocytes; the PECAM-1 staining (red fluorescence) indicates the capillaries. Images were acquired as in Figure 4.