Abstract

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.

Disclosures: No relevant conflicts of interest to declare.

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