FIGURE 3  Changes in concentrations of circulating cells, RNA expression and bone marrow B-cell staining after therapy.

Figure 3.  Changes in concentrations of circulating cells, RNA expression and bone marrow B-cell staining after therapy.

Each group contains 18–21 animals. Values in panels A and B are expressed as mean SEM. (A) Representative immunostaining patterns of ischemic muscle for CD34⁺ cells in PTH, G-CSF, and PTH + G-CSF groups (upper panel). The lower panel indicates number of CD34⁺ cells in ischemic muscle fiber at 21 days. (B) Upper panel: Ki67⁺ cells in cryostat sections of ischemic muscle counterstained with CD34; arrows indicate Ki67⁺/CD34⁺ cells. Lower panel: differences in numbers of SCA1/KDR-VEGF⁺ circulating cells. (C) Reverse-transcriptase polymerase chain reaction analysis of VEGF and angiopoietin-1 expression in nonischemic and ischemic muscle exposed to different treatments. (D) Immunofluorescence staining and image analysis by confocal laser scanning microscopy of frozen bone marrow sections from ischemic hindlimbs indicated increases in B cells compared with controls. The difference was least with PTH alone, greater with G-CSF alone, and greatest with combined therapy. Images of B-cell staining (red) are merged with DAPI (blue) or fibronectin (green); bar, 50 m. ^aP <0.01 versus control; ^bP <0.002 versus control; ^cP <0.05 versus G-CSF; ^dP <0.001 versus control; ^eP <0.0001 versus control; ^fP <0.01 versus G-CSF; ^gP <0.05 versus nonischemic muscle; ^hP <0.05 versus control. Abbreviations: G-CSF, granulocyte-colony stimulating factor; PTH; parathyroid hormone; SCA1/KDR-VEGF⁺, stem cell antigen 1 or kinase insert domain receptor–vascular endothelial growth factor positive.

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