Figure 1.
Zoledronate impaired ischemia-induced neovascularization.
(A) Representative results of laser Doppler measurements before operation and 4 weeks after hindlimb ischemia surgery in controls and in low-dose and high-dose zoledronate-treated mice. Color scale illustrates blood flow variations from minimal (dark blue) to maximal (red) values. Arrows indicate ischemic (right) limb after hindlimb ischemia surgery. (B) At 28 days, compared to control group, both low-dose and high-dose zoledronate-treated groups had significantly lower recovery of blood flows measured by laser Doppler perfusion imaging. Results are means ± SEM. (*p<0.01, **p<0.001 compared to control; n = 16–19).
Figure 2.
Zoledronate resulted in a decrease in new vessel formation and an increase in auto-amputation.
(A) Capillary densities were significantly lower in mice treated with low-dose and high-dose zoledronate (*p<0.001, **p<0.001 compared to control). (B) Compared to the control group, the incidence of limb necrosis was also significantly higher in mice treated with low dose zoledronate and in mice treated with high dose zoledronate (*p<0.05, **p<0.005 compared to control).
Figure 3.
Zoledronate treatment resulted in impaired EPC mobilization and reduced bone marrow EPC number.
(A) The number of Sca-1+/Flk-1+ cells in peripheral blood mononuclear cells was examined by an fluorescence-activated cell sorter. Circulating EPCs were quantified by enumerating Sca-1+/Flk-1+ cells, and the number of EPCs was determined by flow cytometry before and after hind limb ischemia surgery (24 hours) in wild-type mice and FHL2−/− mice. Impaired mobilization of EPCs in peripheral blood was observed in mice treated with low-dose zoledronate and in mice treated with high-dose zoledronate after hindlimb ischemia surgery. (*P<0.05 compared to before operation; n = 6 for each group) (B) BM EPCs were determined 2 days after hindlimb ischemic surgery in study groups. (*p<0.05, **p<0.05 compared to control; n = 6 for each group). (C) Zymographic detection of MMP-9 gelatinolytic activities in BM tissues were assessed in untreated wild-type mice and in mice treated with low-dose or high-dose zoledronate (*p<0.001, **p<0.001 compared to control; n = 6 for each group).
Figure 4.
Zoledronate treatment resulted in decreased expression of VEGF and downregulation of eNOS, Akt and MMP-9 activities in ischemic tissue.
(A) Zoledronate decreased the expression of VEGF (*p<0.05, **p<0.005 compared to control; n = 6 for each group). High-dose zolendronate significantly downregulated eNOS activity in ischemic muscle (*p<0.05 compared to control), and decreased Akt activity both in low-dose and high-dose regimens (*p<0.05, **p<0.05 compared to control). (B) Zoledronate inhibited MMP-9 activity in ischemic hindlimbs (*p<0.05, ** p<0.05 compared to control). However MMP-2 activity was not significantly inhibited (*p = 0.17; **p = 0.21, compared to control).
Figure 5.
Morphology and characterization of human EPCs from peripheral blood.
MNCs were plated on a fibronectin-coated culture dish on the first day (A). Four days after plating, adherent early EPCs with a spindle shape were shown (B). Three weeks after plating, endothelial colony-forming cells (ECFCs) with a cobblestone-like morphology were selected, reseeded, and grown to confluence (E). ECFCs were characterized by immunofluorescence detection of CD34 (F), CD31 (G), VE-cadherin (H), KDR(I), and AC133 (J). Early EPCs and ECFCs were shown to simultaneously bind fluorescein isothiocyanate UEA-1 (lectin, green; C, K) and endocytose DiI-acLDL (red; D, L). Cells were counterstained with DAPI for the nuclei (blue).
Figure 6.
Effects of zoledronate on EPCs viability, senescence, and apoptosis.
(A)\The effects of zoledronate on EPC viability were analyzed by the MTT assay. (*p<0.05 compared to control.) (B) To determine the onset of cellular aging, acidic ß-galactosidase was used as a biochemical marker for acidification, typical of EPC senescence. (*p<0.005 compared to control.) (C) Detection of apoptosis of EPCs was performed with a terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay. (*p<0.05, **p<0.001 compared to control group) (n = 4 for each experiment).
Figure 7.
Zoledronate inhibited migration and tube formation of EPCs in vitro.
(A) The migratory function of EPCs was evaluated by a scratch injury model. Zoledronate significantly impaired EPC migration (*p<0.05 compared to control). (B) An in vitro angiogenesis assay for EPCs was carried out using ECMatrix gel. Representative photos of in vitro angiogenesis are shown. Cells were stained with crystal violet, and the averages of the total area of complete tubes formed by cells were counted. (*p<0.001 compared to control).