Abstract
Vascular progenitor cells play key roles in physiological and pathological vascular remodeling-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 vascular progenitor cells recruitment and differentiation could help the development of new strategies to improve tissue-engineered vessel grafts and design drug-targeted therapy for vessel regeneration. In this study, we sought to investigate the role of Dkk3 (dickkopf-3), recently identified as a cytokine promotor of endothelial repair and smooth muscle cell differentiation, on vascular progenitor cells cell migration and vascular regeneration and to identify its functional receptor that remains unknown. Vascular stem/progenitor cells were isolated from murine aortic adventitia and selected for the Sca-1 (stem cell antigen-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 (C-X-C chemokine receptor type 7) in Sca-1+ cells resulted in alterations in cell migration. Coimmunoprecipitation experiments using Sca-1+ cell 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 of 14.14 nmol/L. Binding of CXCR7 by Dkk3 triggered the subsequent activation of ERK1/2 (extracellular signal-regulated kinases 1/2)-, PI3K (phosphatidylinositol 3-kinase)/AKT (protein kinase B)-, Rac1 (Ras-related C3 botulinum toxin substrate 1)-, and RhoA (Ras homolog gene family, member A)-signaling pathways involved in Sca-1+ cell migration. Tissue-engineered vessel grafts were fabricated with or without Dkk3 and implanted to replace the rat abdominal aorta. Dkk3-loaded tissue-engineered vessel grafts showed efficient endothelization and recruitment of vascular progenitor cells, 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 compromising vascular remodeling. 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.
Highlights
To further assess the chemotactic effect of Dkk[3] in a setting that better mimics the in vivo 3-dimensional conditions and to study the effect of Dkk[3] on endogenous resident vascular progenitor cells (VPCs) of the vessel wall, the aortic ring assay was performed with aortas of wild-type and ApoE−/− mice
The explanation could be that vascular cells derived from the native atherosclerosis model ApoE−/− mice are potentially more responsive or sensitive to stimuli than the cells derived from wild-type mice
We demonstrated that Dkk[3] induces chemotaxis for Sca1+ stem/progenitor cells via binding to CXCR7 resulting in better regeneration of TEVGs
Summary
The data that support the findings of this study are available from the corresponding author on reasonable request.Details of materials and experimental procedures are available in the Online Data Supplement.Sca-1+ Adventitial Progenitor Cell Isolation and Dkk[3] BindingMouse VPCs were isolated from the outgrowth of aortic adventitial tissues as described previously.[13]. The data that support the findings of this study are available from the corresponding author on reasonable request. Details of materials and experimental procedures are available in the Online Data Supplement. Sca-1+ Adventitial Progenitor Cell Isolation and Dkk[3] Binding. Mouse VPCs were isolated from the outgrowth of aortic adventitial tissues as described previously.[13] Sca-1+ VPCs were treated with 25 ng/mL of Dkk[3] for 3 hours. The eluted immune complexes and input samples were separated on a 4% to 12% Bis-Tris gel, and the immunoblot was probed with Dkk[3] antibody. His-tag pull-down assay was performed according to the instructions provided in MagneHis Protein Purification System (Promega). Receptor affinity binding assay was done as described previously.[22]
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