Abstract 479: Dickkopf-3 Binds to CXCR7 of Vascular Progenitor Cells to Enhance Degradable Graft Regeneration

Abstract

Background: Dickkopf-3 (Dkk3) is a secreted protein that may have a role in vascular diseases by promoting smooth muscle cell (SMC) differentiation and endothelial repair, two processes in which vascular stem/progenitor cells could be involved. However, the effect of Dkk3 on stem/progenitor cell migration and its specific receptor on these cells remain unknown. Methods: Vascular stem/progenitor cells (VPCs) were isolated from murine aortic adventitia and selected for the Sca-1 marker. The chemotactic ability of Dkk3 for Sca-1+ cells was tested in vitro using transwell and wound healing assays and ex vivo by the aortic ring assay. Chemokine receptors identification was carried out using Western blot (WB), siRNA-mediated knockdown, co-Immunoprecipitation (Co-IP) and saturation binding assays. To assess in vivo the role of Dkk3 in recruiting Sca-1+ cells, tissue-engineered vessel grafts, with or without Dkk3, were fabricated and implanted to replace the rat abdominal aorta. Results: We demonstrated that Dkk3 induced the chemotaxis of Sca-1+ cells in vitro , and ex vivo in culture of aortic rings derived from Sca-1-GFP transgenic mice. Flow cytometry and WB analysis revealed that Sca-1+ cells expressed CXCR7, while overexpression or knockdown of CXCR7 resulted in alterations in cell migration. Interestingly, Co-IP experiments showed the physical interaction between DKK3 and CXCR7, and specific saturation binding assays identified a high affinity Dkk3-receptor binding with a dissociation constant of 14.14 nM. Activation of CXCR7 by Dkk3 triggered the ERK1/2, PI3K/AKT, Rac1 and RhoA signalling pathways. Furthermore, when Dkk3-loaded tissue-engineered vessels were grafted to the abdominal artery of rats, the grafts showed efficient endothelization and recruitment of VPCs, which had acquired characteristics of mature SMCs. CXCR7 blocking using specific antibodies in our vessel graft model hampered stem/progenitor cell recruitment into the vessel wall, thus compromising vascular remodelling. Conclusions: We provide novel and solid evidence that CXCR7 serves as Dkk3 receptor, which mediates Dkk3-induced VPCs migration in vitro and in tissue-engineered vessels, hence harnessing patent grafts resembling native blood vessels.