cAng-1 increases the recruitment and the incorporation of BMPCs into endothelial cells in vitro and in vivo ischemia. (A) DiI-labeled human BMPCs (1 × 10⁵; red) were added on the HUVEC monolayer (∼ 90% confluence), incubated under either static or shear-stress conditions, and gently washed. Bar graph shows the count of adhering DiI-labeled BMPCs (n = 3, *P < .05, **P < .01). Hy indicates hypoxia; nSDF-1, neutralizing antibody to SDF-1; and nIgG, neutralizing antibody to IgG. (B) In vitro incorporation assay with a Matrigel tube formation. DiI-labeled HUVECs (red) were seeded onto a Matrigel-coated dish and incubated for 1 hour. CSFE-labeled BMPCs (green) were added, incubated, fixed with 2% PFA, and analyzed by confocal microscopy. Magnification ×200 with immersion oil. Representative images are shown (n = 3). (C-D) In vitro migration assay. DiI-labeled HUVECs (red) were seeded on the bottom of gelatin-coated coverglass dish. The next day, 150 μL of collagen-gel matrix solution (ratio of type 1 collagen:Matrigel:EBM = 2:0.5:1.5) was added onto the HUVECs and incubated for 1 hour at 37°C to form a matrix. cAng-1 was contained in a collagen-gel matrix solution. CFSE-labeled BMPCs (green) were put on top of the collagen-gel matrix and incubated for 24 hours. The migration of BMPCs toward the HUVECs was observed by confocal microscopy, images were obtained by stacking along the z-axis, and the migrated green cells were counted (n = 3). (E) Schematic timetable of BALB/c-nude mice hindlimb ischemia and mouse BM-cell transplantation. (F) LDPI images. BM indicates GFP⁺ bone marrow (BM) cells. (G) LDPI was sequentially evaluated after transplantation of GFP⁺ BM cells (1 × 10⁶) into hindlimb ischemic BALB/c-nude mice (n = 5 each, *P < .001). (H) cAng-1 stimulated the incorporation of BM cells into ischemic muscle. The number of mouse GFP⁺ BM cells (green) that were incorporated into region of SDF-1 expression (red) was greater in the cAng-1/BM group than in the PBS/BM group. The number of the incorporated cells in ischemic tissue of adductor muscle was remarkably lower in the neutralizing anti-CXCR4 pretreatment group (nCXCR4-BM) than the untreated BM-cells or anti-IgG pretreatment group (nIgG-BM). Confocal microscopic image shows conclusively the colocalization of SDF-1 and GFP ⁺BM-cells. Arrows, GFP⁺ cells recruited to the ischemic limb muscle. Representative confocal microscopic photographs are shown and no fluorescence signal in isotype IgG control. Magnification ×100; scale bar, 50 μm. (I) Enlargement views of GFP⁺ cells recruited to the ischemic limb muscle. Magnification ×630 with immersion oil; scale bar, 10 μm. (J) Number of GFP⁺ cells incorporating into ischemic limb muscle. Counting cells in 10 different microscopic fields (n = 5 each, *P < .005). (K) Mechanisms of SDF-1 stimulation by cAng-1 in hypoxic endothelium.