The Role Of CD11b+CX3CR1+ Cells In Neovascularization

There is solid evidence that myelo-monocytic cells actively contribute to angiogeneic processes. However, the specific subpopulations of myelo-monocytic cells participating in vascularization and their precise underlying mechanisms have not been well identified. Recently it has been reported that CX3CR1, a receptor for fractalkine (CX3CL1; Fkn), is highly induced at injury sites and CX3CR1 positive cells have an active role for angiogenesis in a mouse wound healing model. Therefore, we evaluated neovascularization activities of CD11b⁺CX3CR1⁺ cells during recovery of blood perfusion in a hind-limb ischemic injury mouse model and also identified their activities during the process of angiogenic sprouting in the retina of early postnatal Sprague-Dawley (SD) rat. Ischemic injuries were induced in C57BL/6 mice by femoral artery dissections. Single-cell suspensions were made from ischemic muscles using collagenase and dispase digestion. Isolated CD11b⁺CX3CR1⁺ cells and CD11b^-CX3CR1^- cells from ischemic muscle (5x10⁵ cells/mouse) were injected into ischemic muscles 2 days after femoral artery dissections. Laser Doppler Perfusion (LDP) indexes were assessed as an indicator for blood perfusion. In order to block the functions of CX3CR1 and Fkn, either anti-CX3CR1 or anti-Fkn neutralizing antibody (Ab) were injected into ischemic muscles. To determine whether CX3CR1 has a role in actively growing retinal vessels, intravitreal microinjections were done with anti-CX3CR1 Ab at postnatal 2 days (P2) SD rat. P5 retinas treated with neutralizing anti-CX3CR1 Ab at P2 were used. CD11b⁺CX3CR1⁺ cells were positive for CD45 and F4/80, monocyte/macrophage marker. CD11b⁺CX3CR1⁺ cells accumulated at the injured muscles in C57BL/6 mice. We tested for contribution of CD11b⁺CX3CR1⁺ cells to neovascularization in the injury sites by injecting ischemic muscle-derived CD11b⁺CX3CR1⁺ cells or CD11b^-CX3CR1^- cells into the adductor muscles of C57BL/6 mice. CD11b⁺CX3CR1⁺ cells enhanced blood perfusion when compared to CD11b^-CX3CR1^- cells (78.0±3.2% vs 39.0±0.3% of LDP indexes at day 28, p<0.01). Both neutralizing Abs to CX3CR1 and Fkn significantly reduced blood perfusion resulting in decreased LDP indexes of 25.9±4.9% and 29.4±4.6% compared to those of the non-ischemic limbs (p<0.01). When CX3CR1 function was blocked with anti-CX3CR1-neutralizing Ab, CD11b⁺CX3CR1⁺cells were completely eliminated and any vessel-like structures were not observed in the ischemic muscles. By staining of isolectin for P5 retina, well-organized primitive vascular plexus was observed. However, blocking the function of CX3CR1 resulted in formation of peripheral plexus that was more highly interconnected. Additionally, vessels coalesced to form a syncytium in some areas. Thus, CD11b⁺CX3CR1⁺ cells accumulate at sites of ischemic injury and enhance recovery of blood flow via new vascular structure formation. Furthermore, CX3CR1 acts to prevent overexuberant vascular growth and promotes formation of well-differentiated vascular network.

In summary, CD11b⁺CX3CR1⁺ cells are a newly identified subset of myelomonocytic cells for vessel formation, capable of regulating vascular formation. CD11b⁺CX3CR1⁺ cells may be an attractive clinical tool to accelerate tissue vasculature development in various ischemic diseases.