GM-CSF treated F4/80+ BMCs improve murine hind limb ischemia similar to M-CSF differentiated macrophages

Abstract

Novel cell therapy is required to treat critical limb ischemia (CLI) as many current approaches require repeated aspiration of bone marrow cells (BMCs). The use of cultured BMCs can reduce the total number of injections required and were shown to induce therapeutic angiogenesis in a murine model of hind limb ischemia. Blood flow recovery was significantly improved in mice treated with granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent BMCs that secreted inflammatory cytokines. Angiogenesis, lymphangiogenesis, and blood flow recovery ratio were significantly higher in the GM-CSF-cultured F4/80+ macrophage (GM-Mø)-treated group compared with controls. Furthermore, Foxp3+ cell numbers and tissue IL-10 concentrations were significantly increased compared with controls. There was no significant difference in blood flow recovery between GM-Mø and M-CSF-cultured F4/80+ macrophages (M-Mø). Thus, GM-Mø were associated with improved blood flow in hind limb ischemia similar to M-Mø. The selective methods of culturing and treating GM-Mø cells similar to M-Mø cells could be used clinically to help resolve the large number of cells required for BMC treatment of CLI. This study demonstrates a novel cell therapy for CLI that can be used in conjunction with conventional therapy including percutaneous intervention and surgical bypass.

Figure 1. Expression of cytokine mRNA, and IL-10 protein levels in M-CSF- or GM-CSF-cultured BMCs.

(A) The expression of proinflammatory cytokines (TNF-alpha, IL-6, IL-1beta, IL-12a, and IL12b) and anti-inflammatory cytokine (IL-10) mRNA in M-CSF- or GM-CSF-cultured BMCs. (B) IL-10 concentration in the supernatant of M-CSF- or GM-CSF-cultured BMCs. Data are expressed as mean ± SEM, ***P<0.001, **P<0.01, *P<0.05.

Figure 2. Recovery of blood flow in ischemic limbs treated with M-CSF-cultured BMCs and GM-CSF-cultured BMCs.

Representative laser Doppler perfusion images taken at indicated intervals for the PBS control group, M-CSF-cultured BMCs group and GM-CSF-cultured BMC group. Data are expressed as mean ± SEM, n = 5–7/group, **P<0.01, *P<0.05.

Figure 3. Flow cytometric analysis of F4/80⁺ macrophages from M-CSF- or GM-CSF- cultured BMCs.

Data is representative of three independent experiments.

Figure 4. Recovery of blood flow in ischemic limbs treated with M-Mø and GM-Mø.

Representative laser Doppler perfusion images were taken at indicated intervals for the PBS control group, M-Mø group, and GM-Mø group. Data are expressed as mean ± SEM, n = 5 group, **P<0.01, *P<0.05.

Figure 5. vWF and LYVE-1 staining in M-Mø and GM-Mø treated ischemic hind limbs.

Representative photomicrographs (original magnification ×400). vWF positive and LYVE-1 positive cells are stained green and red, respectively (***P<0.001, **P<0.01, *P<0.05).

Figure 6. Foxp3 expression in M-Mø or GM-Mø treated ischemic hind limbs and the recovery of blood flow in ischemic limbs treated with IL-10.

(A) Photomicrographs show representative DAB staining for Foxp3 (scale bar = 50 µm). The number of Foxp3 positive cells was counted in five random images for each mouse. The thigh muscle of each group was isolated 7 days after the induction of ischemia. (B) IL-10 levels in thigh muscle after limb ischemia at 7 days after intramuscular injection of indicated cells (data are expressed as mean ± SEM, n = 3 group, ***P<0.001, **P<0.01, *P<0.05). (C) Recovery of blood flow in ischemic limbs treated IL-10. Representative laser doppler perfusion images were taken at indicated intervals for the PBS control groups and IL-10 treated groups. Data are expressed as mean ± SEM, n = 5–7/group.