Fractalkine/CX3CR1 Signaling Promotes Angiogenic Potentials in CX3CR1 Expressing Monocytes

Introduction: Myelo-monocytic cells expressing CD11b are involved in angiogenesis, but their specific roles and underlying mechanisms are unclear. CX3CR1 is the only known receptor for fractalkine (Fkn). CD11b⁺CX3CR1⁺ cells isolated from ischemic muscles expressed F4/80, which is a common marker for mouse macrophages (Tissue (T)-CD11b⁺CX3CR1⁺ macrophages). T-CD11b⁺CX3CR1⁺ macrophages exert pro-angiogenic effect by platelet factor-4 (PF-4) production. PF-4 did not promote angiogenesis by itself but magnified the angiogenic activity of VEGF. Although T-CD11b⁺CX3CR1⁺ macrophages show definite pro-angiogenic effects, their clinical implementation is limited because they require muscle excision for cell harvesting. Therefore, we focused on angiogenic potential of more accessible bone marrow (BM)-CD11b⁺CX3CR1⁺ monocytes, because the CD11b⁺CX3CR1⁺monocytes migrate from BM into ischemic muscles via Fkn-mediated chemotaxis and differentiate into macrophages upon tissue damage.

Materialsand methods: CD11b⁺CX3CR1⁺cells were isolated by MoFlo™ XDP Cell Sorter (Beckman Coulter, Brea, CA). Isolated CD11b⁺CX3CR1⁺ cells were fixed and stained with anti-PF-4 Ab with PE-conjugated IgG for immunofluorescence staining. For aortic tissue sprouting assay, thoracic aortas were embedded in Collagen Type-1. Aortic segments were incubated with CD11b⁺CX3CR1⁺ monocytes in the presence or absence of Fkn, and their conditioned medium (CM) at 37℃ in a humidified 5% CO₂ atmosphere with triweekly media replacement. For Fkn treatment, CD11b⁺CX3CR1⁺monocytes were incubated with Fkn (50ng/mL) for 30 minutes. Morphometric analysis of sprouting was performed using Image J software (National Institute Health, Bethesda, MD, USA).

Results: T-CD11b⁺CX3CR1⁺ macrophages greatly increased sprouting from mouse aortic tissue segments, while T-CD11b⁺CX3CR1^- macrophages showed only modest effects (p<0.05). CM harvested from T-CD11b⁺CX3CR1⁺ macrophages showed equivalent effects as T-CD11b⁺CX3CR1⁺ macrophages in inducing sprouting. Their high angiogenic potential largely attributed to increased expression of PF-4, according to angiogenic protein array. Depletion of PF-4 in CM of T-CD11b⁺CX3CR1⁺ macrophages strongly reduced vascular sprouting compared to control (p <0.001). In contrast to T-CD11b⁺CX3CR1⁺ macrophages, PF-4 expression was not detected in BM-CD11b⁺CX3CR1⁺ monocytes. However, activation of Fkn/CX3CR1 interaction by treating the cells with Fkn (50ng/mL for 30 minutes) induces high PF-4 expression. Heightened expression of PF-4 mRNA in Fkn-treated BM-CD11b⁺CX3CR1⁺ monocytes was confirmed by RT-PCR. Aortic tissue sprouting assay confirmed Fkn-treated BM-CD11b⁺CX3CR1⁺ monocytes achieve a similar angiogenic potential to that of T-CD11b⁺CX3CR1⁺ macrophages. CD11b⁺CX3CR1⁺ monocytes were also found in human blood. CM of Fkn-treated human CD11b⁺CX3CR1⁺ monocytes (50ng/mL) greatly increased vascular sprouting, while CM of human CD11b⁺CX3CR1⁺monocytes without Fkn treatment only showed modest effects on inducing sprouting (p<0.05).

Conclusion; Fkn triggers angiogenic potential of CD11b⁺CX3CR1⁺ monocytes by Fkn/CX3CR1 signaling. Thus, Fkn-treated CD11b⁺CX3CR1⁺monocytes may be of potential therapeutic use to accelerate recovery of blood perfusion in ischemic diseases.