Fractalkine Treated of CD11b⁺CX3CR1⁺ Monocytes Promotes Angiogenesis through Pigment Epithelium-Derived Factor

Introduction: Myelo-monocytic cells expressing CD11b are involved in angiogenesis, but their specific roles and underlying mechanisms are unclear. We successfully identified a subset of CD11b⁺ monocytes expressing CX3CR1, the only receptor of fractalkine (Fkn; CX3CL1), in G-CSF mobilized peripheral blood stem cell collection (PBSC). We first verified Fkn triggers proangiogenic activities in CD11b⁺CX3CR1⁺ monocytes. Subsequently, we found that Fkn-treated CD11b⁺CX3CR1⁺ monocytes express pigment epithelium-derived factor (PEDF) once Fkn activates Fkn/CX3CR1 signaling. PEDF is a glycoprotein, which belongs to the serpin family and stimulates various physiologic processes such as angiogenesis, cell proliferation, and survival. In this study, we investigated the role of Fkn-treated CD11b⁺CX3CR1⁺ monocytes in angiogenesis and their mechanisms of action involving PEGF expression.

Materialsand methods: To mobilize mononuclear cells (MNCs) including CD11b⁺CX3CR1⁺ monocytes into peripheral blood (PB), heathy donors were subcutaneously injected with G-CSF (10μg/kg) for 5 days. Apheresis MNCs were collected from donor PB using a COBE spectra cell separator (COBE, Lakewood, CO) after G-CSF injections. Then, CD11b⁺CX3CR1⁺cells were isolated by a MoFlo™ XDP Cell Sorter (Beckman Coulter, Brea, CA). HUVECs were isolated from human cords and cultured in either the presence or absence of Fkn-treated CD11b⁺CX3CR1⁺ monocytes, or their conditioned media (CM) on top of collagen at 37 °C in 5% CO2. For Fkn treatment, CD11b⁺CX3CR1⁺ monocytes were incubated with Fkn (50ng/mL) for 30 minutes. All procedures were approved by the Institutional Review Board at St. Vincent's Hospital, the Catholic University of Korea. All clinical samples were obtained from donors with informed consent.

Results: CD11b⁺CX3CR1⁺ monocytes were found at 19.6±3.58% of total MNCs in human G-CSF mobilized PBSC. They expressed CD14 and CD16, which are common markers for human monocytes. The average number of CD11b⁺CX3CR1⁺ monocytes collected from one apheresis was 9.2±1.2X10⁹. Administration of Fkn (50ng/mL for 30 minutes) in vitro greatly enhanced the angiogenic potential of CD11b⁺CX3CR1⁺ monocytes on HUVECs. CM harvested from Fkn-treated CD11b⁺CX3CR1⁺ monocytes showed equivalent effects to that of Fkn-treated CD11b⁺CX3CR1⁺ monocytes. Their high angiogenic potential is largely attributed to increased expression of PEDF according to angiogenic protein array results, and treating Fkn-treated CD11b⁺CX3CR1⁺ monocytes with a specific PEDF inhibitor largely abrogated HUVEC proliferation and vascular structure formation compared to control (p <0.001).

Conclusion: We were able to collect enough CD11b⁺CX3CR1⁺ monocytes from G-CSF mobilized PBSC. High angiogenic potential of Fkn-treated CD11b⁺CX3CR1⁺ monocytes is largely attributed to increased expression of PEDF. Thus, G-CSF mobilization of CD11b⁺CX3CR1⁺ monocytes and subsequent Fkn treatment of the monocytes may be a novel therapeutic approach to translate to clinical utility in order to accelerate recovery of blood perfusion in ischemic diseases.