Systemic Levels of G-CSF and IL-6 Determine the Angiogenic Potential of Bone Marrow Resident Monocytes

Recent studies have demonstrated the efficacy of hematopoietic cell-based therapies in promoting therapeutic angiogenesis for a wide variety of vascular syndromes, however the cell populations responsible and the mechanisms involved are poorly understood. Using a mouse model of hindlimb ischemia, we previously showed that an adoptive transfer of donor monocytes significantly enhanced revascularization. Monocytes are a widely heterogeneous cell population and differences in the ability of various monocyte subsets to mediate revascularization have not been previously investigated. Using the hindlimb ischemia model we demonstrate that an adoptive transfer of inflammatory: CX3CR loGr-1+), but not resident: CX3CR1hiGr-1-) monocytes, significantly enhances revascularization post-ischemia. Additionally, we show that the inflammatory subset of monocytes is selectively recruited from the bone marrow to the blood and that these cells accumulate at the ischemic lesion. These findings demonstrate that the adoptive transfer of only a small proportion of monocytes from a non-ischemic donor significantly enhances revascularization despite the presence of a far greater proportion of endogenous: ischemia-conditioned) monocytes. Herein, we provide data suggesting that upon induction of distant ischemia, systemic signals are generated which reduce the angiogenic capacity of bone marrow resident monocytes. We provide evidence that granulocyte-colony stimulating factor: G-CSF) and interleukin-6: IL-6) provide these conditioning signals. Systemic levels of G-CSF and IL-6 are significantly increased following induction of hindlimb ischemia, and accordingly, bone marrow resident monocytes from ischemic mice exhibited increased STAT3 phosphorylation and STAT3 target gene expression. Finally, G-CSF receptor-/- and IL-6-/- mice were resistant to the deleterious effects of ischemic conditioning on monocyte angiogenic potential. The mechanism by which this ischemia-driven signals limit the angiogenic potential of monocytes was examined using RNA expression profiling which suggested that ischemia-conditioned monocytes in the bone marrow are polarized towards expression of M2-associated genes. Consistent with this observation, M2-skewed monocytes from SHIP-/- mice also had impaired angiogenic capacity. Lastly we demonstrate that the efficacy of an adoptive transfer of non-ischemic donor monocytes may be due, at least in part, to increased expression of the fractalkine receptor CX3CR1 as well as to increases in local concentrations of the angiogenic factors MCP-1, VEGF, MMP-9 and ApoA1