There are growing evidences that myelo-monocytic cells play a key role in mediating tissue repair and stimulating angiogenesis in response to ischemia. However, the subpopulations of myelo-monocytic cells participating in vascularization and their underlying mechanisms have not been well identified. We previously demonstrated that CD11b+CX3CR1+ cells, a type of proangiogenic myelo-monocytic cells, promotes angiogenesis in a C57BL/6 hind-limb ischemic mouse model. We now provide insight into mechanisms of CD11b+CX3CR1+ cells during recovery of blood perfusion after ischemia.

Ischemic injuries were induced by dissecting the femoral artery of C57BL/6 mice. Single-cell suspensions were made from ischemic muscles, and ischemic muscle-derived CD11b+CX3CR1+ cells were isolated with a FACS Sorter. Extracted proteins (50ug) from the cells were loaded on nitrocellulose membranes, containing 53 different capture antibodies in proteome profiler mouse angiogenesis Kits (R&D systems, ARY105). For aortic ring sprouting assay, thoracic aortas were embedded in Matrigel.

Proangiogenic effects of CD11b+CX3CR1+ cells were confirmed by enhanced blood perfusion upon injection of additional CD11b+CX3CR1+ cells into ischemic tissues after femoral artery dissection in C57BL/6 mice. Sprouting effects were specifically confirmed through purified CD11b+CX3CR1+ cells co-cultured with aortic ring sprouting assay. Also, medium conditioned by CD11b+CX3CR1+ cells was as efficient as CD11b+CX3CR1+ cells in supporting sprouting, suggesting that vascular endothelial cells (ECs) are likely activated by the proteins secreted by CD11b+CX3CR1+ cells in a paracrine fashion. To investigate whether muscle-derived CD11b+CX3CR1+ cells could secrete angiogenic proteins, we examined the angiogenic proteins in CD11b+CX3CR1+ cells isolated from ischemic muscles after 4 days of surgery. Contrary to our expectations, VEGF proteins were not detected from CD11b+CX3CR1+ cells. However, MCP-1, MIP-1α, platelet factor-4 (PF-4) and MMP-9 were expressed at a higher level in CD11b+CX3CR1+ cells than those of CD11b+CX3CR1- and CD11b-CX3CR1- cells. MCP-1, MIP-1α and MMP-9 have been reported to promote angiogenesis, while PF-4 is recognized as an angiostatic factor. However, it has recently been reported that PF-4 combined with VEGF is essential for the initial separation of ECs from vessels prior to angiogenic sprouting. It is postulated that, after ischemia, collaboration of VEGF with PF-4 secreted from CD11b+CX3CR1+ cells dissociates the ECs from vessels and promotes vascular branching processes.

In summary, CD11b+CX3CR1+ cells are a newly identified myelo-monocytic subset for vessel formation, capable of vascular sprouting via secreting PF-4.


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