| Summary: | Stem/Progenitor cells, such as Sca-1+ cells, are abundant in the vascular adventitia and they can differentiate into SMCs and ECs. These cells participate in the vascular wall remodeling observed in atherosclerosis, injury-induced neointimal hyperplasia and vein graft atherosclerosis. The detailed mechanisms of progenitor cell migration towards the intima have not been fully investigated. We hypothesize that Dkk3 plays an important role in resident Sca-1+ vascular progenitor cell migration. In this work, mouse vascular progenitor cells were isolated from the adventitia and sorted for the Sca-1 marker. Using transwell and wound healing assays, we showed that Dkk3 induced adventitia-derived Sca-1+ vascular progenitor cell (Sca-1+ APCs) migration in vitro. Additionally, the aortic ring assay demonstrated that Dkk3 was also able to induce Sca-1+ cell migration ex vivo. Analysis of the signaling pathways revealed that MAPK kinase signalling cascade, PI3K/AKT pathway and Rho-GTPases family of proteins were involved in the regulation of Dkk3-induced Sca-1+ progenitor cell migration. Our experiments also identified similarities in the migratory response of Sca-1+ cells to Dkk3 and Sdf-1α. Sdf-1α receptors are well established and they include CXCR4 and CXCR7. In contrast, the receptor for Dkk3 has not been identified yet. The current study aimed at identifying the receptor(s) involved in Dkk3-driven migration of Sca-1+ cells. Co-IP analysis and Affinity Binding assay revealed that Dkk3 binds to CXCR7. The downregulation of CXCR7 by SiRNA transfection reduced Dkk3-mediated Sca-1+ APC migration and the activation of the downstream signalling pathways. Overexpression of CXCR7, on the contrary, enhanced cell migration. In order to perform an exhaustive analysis of the potential binding partners of Dkk3 and identify additional receptors, we performed a Yeast Two Hybrid experiment. The results showed that integrin α5 (ITGα5) and integrin β1 (ITGβ1) interacted with Dkk3. Co-IP and Affinity Binding assays confirmed that Dkk3 bound to ITGα5 and ITGβ1. Furthermore, downregulation of these integrins by SiRNA transfection supressed the migration of Sca-1+ APCs triggered by Dkk3. Our work shows for the first time that Dkk3 acts as a chemokine-like protein able to induce the migration of resident Sca-1+ vascular progenitor cells. The pioneer identification of Dkk3 receptors and the elucidation of the signalling pathways involved in Sca-1+ APC migration could be of major interest for the development of novel drug-targeted therapies in vascular diseases.
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