Transplantation of tPA-treated CD11b⁺ and VEGFR-1⁺ myeloid cells improve neoangiogenesis. (A-F) tPA- or BSA-PBS–mobilized PBMCs from GFP mice or carrier (no cells) were injected intravenously into HL ischemia–induced recipients. (A) Blood flow was determined. (B-F) Ischemic tissues of lower limbs isolated 15 days after injection and HL ischemia induction were stained with (B) H&E and (C) antibodies against CD31 for capillary density evaluation (n = 3 per group). Images were acquired with 20×/0.50 NA and a Carl Zeiss KS400 camera, and analyzed using Axio Vision (Carl Zeiss). (D-E) Within ischemic muscle tissues GFP⁺ donor cells (arrows) were quantified. Inserts depict a magnified view. Images were acquired with a 20×/0.50 NA and a 63×/1.3 oil objective using a Carl Zeiss KS400 camera. (E) Quantification of the number of GFP⁺ cells and GFP⁺ cells coexpressing CD11b in ischemic tissues (n = 8 per group). (F) CD11b (Mac-1) staining of lower limb ischemic tissue from mice receiving carrier injections, from mice that received a transplant with PBMCs, or from BSA-PBS–treated or tPA-treated donor GFP mice. Arrows indicate transplanted GFP⁺CD11b⁺ cells in ischemic tissues (bar = 20 μm). Images were acquired with 40×/0.75 NA and a Carl Zeiss KS400 camera, and analyzed using Axio Vision (Carl Zeiss). (G-H) PB-derived CD11b⁺ or CD11b⁻ cells isolated from tPA- or BSA-PBS–treated donors were transplanted into HL ischemia–induced recipients for 3 days (n = 4 per group). (G) Blood flow was determined. (H) Capillary density was evaluated. (I-J) HL ischemic C57BL/6 mice were treated with tPA and coinjected with anti-CD11b or control monoclonal antibodies. (I) Blood flow was analyzed by laser Doppler analysis. (J) Capillary density was evaluated. (K) PB-derived VEGFR-1⁺ or VEGFR-1⁻ cells, isolated from tPA- or BSA-PBS–treated donors, were injected into HL ischemia–induced recipients, and blood flow was examined (n = 4 per group). Single asterisk (P < .05) and double asterisk (P < .001) indicate a significant difference between the groups. Values represent mean ± SEM.