Binding of Dickkopf-3 to CXCR7 Enhances Vascular Progenitor Cell Migration and Degradable Graft Regeneration [Original Research]

Rationale: Vascular progenitor cells (VPCs) play key roles in physiological and pathological vascular remodelling, a process that is crucial for the regeneration of acellular biodegradable scaffolds engineered as vital strategies against the limited availability of healthy autologous vessels for bypass grafting. Therefore, understanding the mechanisms driving VPCs recruitment and differentiation could help the development of new strategies to improve Tissue-Engineered Vessel Grafts (TEVGs) and design drug-targeted therapy for vessel regeneration. Objective: In this study we sought to investigate the role of Dickkopf-3 (Dkk3), recently identified as a cytokine promotor of endothelial repair and smooth muscle cell differentiation, on VPC cell migration and vascular regeneration and to identify its functional receptor that remains unknown.
Methods and Results: Vascular stem/progenitor cells were isolated from murine aortic adventitia and selected for the Stem cell antigen-1 (Sca-1) marker. Dkk3 induced the chemotaxis of Sca-1+ cells in vitro in transwell and wound healing assays, and ex vivo in the aortic ring assay. Functional studies to identify Dkk3 receptor revealed that overexpression or knockdown of chemokine receptor CXCR7 in Sca1+ cells resulted in alterations in cell migration. Co-Immunoprecipitation experiments using Sca1+ cells extracts treated with Dkk3 showed the physical interaction between DKK3 and CXCR7, and specific saturation binding assays identified a high affinity Dkk3-CXCR7 binding with a dissociation constant (Kd) of 14.14 nM. Binding of CXCR7 by Dkk3 triggered the subsequent activation of ERK1/2, PI3K/AKT, Rac1 and RhoA signalling pathways involved in Sca1+ cell migration. TEVGs were fabricated with or without Dkk3 and implanted to replace the rat abdominal aorta. Dkk3-loaded TEVGs showed efficient endothelization and recruitment of VPCs, which had acquired characteristics of mature smooth muscle cells. CXCR7 blocking using specific antibodies in this vessel graft model hampered stem/progenitor cell recruitment into the vessel wall, thus Conclusions: We provide a novel and solid evidence that CXCR7 serves as Dkk3 receptor, which mediates Dkk3-induced vascular progenitor migration in vitro and in tissue-engineered vessels, hence harnessing patent grafts resembling native blood vessels.