Purified Gr1+CD11b+ Cells Induce Neovascularization in An Ischemic Hind Limb Mouse Model.

Neovascularization in ischemic tissues is enhanced by circulating endothelial precursor cells, some of which have been identified as bone marrow (BM)-derived (BMEPC). However the identity of BM-derived populations that participate in the process of neovascularization is not clear; and indeed several subsets have been proposed as important to this process. Gr1+CD11b+ cells have been recently described as myeloidderived suppressor cells that may be involved in tumor angiogenesis. However, there has been little investigation of the role of Gr1+CD11b+ cells in non-tumor angiogenesis. To study the relationship of Gr1+CD11b+ cells to neovascularization in an ischemic hindlimb model of C57BL/6 mice, we tested the following hypotheses:

1. The number of tissue-resident Gr1+CD11b+ cells increases in BM and ischemic muscle after the ischemic injury;

2. prospectively isolated Gr1+CD11b+ cells can manifest characteristics of endothelial differentiation; and

3. Gr1+CD11b+ cells modulate neovascularization in ischemic hind-limbs of C57BL/6 mice upon direct injection into ischemic muscle.

Results demonstrated that the number of Gr1+CD11b+ cells markedly increased in the ischemic muscle at 4 days post-surgery (n=5 each; 1.08±0.65x10⁵/g tissue in non-surgically treated mice vs 13.43± 7.52x10⁵/g tissue in the ischemic muscle, p<0.05); then returned to basal level at day 10. The number of Gr1+CD11b+ cells from BM in the ischemic group at day 4 trended towards an increase when compared with cells from BM of non-surgically treated C57BL/6 mice (n=5 each; 1.05±0.31x10⁷/2 femur+ 2 tibia in ischemic group vs 0.70±0.32x10⁷/2 femur+ 2 tibia in non-surgically treated C57BL/6 mice). Following culture under endothelial cell growth conditions, purified Gr1+CD11b+ cells from BM exhibited cobblestone and spindle-shaped morphology. FACS analysis demonstrated that cobblestone-shaped cells exhibited development of a CD144+ phenotype, a marker for mature endothelium; while spindle-shaped cells expressed desmin. Following direct injection of purified Gr1+CD11b+ cells from BM or medium control, laser Doppler imaging revealed an increase in blood flow recovery by 14 days post-femoral artery dissection in the cell injected group compared to the control group (n=7 each, 41.14±1.28% in Gr1+CD11b+ cell injected group vs 35.83±2.57% in control group). The mobilization of Gr1+CD11b+ to regions of muscle ischemia, taken together with the novel finding of endothelial differentiation from this BM-derived population, and enhanced flow recovery following local provision of exogenous Gr1+CD11b+ cells, support the hypothesis that this cell population plays an important role in the systemic and local response to ischemic injury.