CCN1 Secreted by Tonsil-Derived Mesenchymal Stem Cells Promotes Endothelial Cell Angiogenesis via Integrin αvβ3 and AMPK

CCN1 is highly expressed in cancer cells and has been identified in the secretome of bone marrow‐derived mesenchymal stem cells (BM‐MSC). Although secreted CCN1 is known to promote angiogenesis, its underlying mechanism remains unclear. Here, we examined whether our recently‐established tonsil‐derived MSC (T‐MSC) secrete CCN1 and, if any, how CCN1 promotes the angiogenesis of human umbilical vein endothelial cells (HUVEC). Compared with untreated control T‐MSC, a higher level of CCN1 was secreted by T‐MSC treated with activin A and sonic hedgehog, drugs known to induce endodermal differentiation. Expectedly, conditioned medium collected from differentiated T‐MSC (DCM) significantly increased HUVEC migration and tube formation compared with that from control T‐MSC (CCM), and these stimulatory effects were reversed by neutralization with anti‐CCN1 antibody. Treatment with recombinant human CCN1 (rh‐CCN1) alone also mimicked the stimulatory effects of DCM. Furthermore, treatment with either DCM or rh‐CCN1 increased the phosphorylation of AMP kinase (AMPK), and ectopic expression of siRNA of the AMPK gene inhibited all observed effects of both DCM and rh‐CCN1. However, no alteration of intracellular ATP levels or phosphorylation of LKB1, a well‐known upstream factor of AMPK activation, was observed under our conditions. Finally, the neutralization of integrin αvβ3 with anti‐integrin αvβ3 antibody almost completely reversed the effects of CCN1 on AMPK phosphorylation, and EC migration and tube formation. Taken together, we demonstrated that T‐MSC increase the secretion of CCN1 in response to endodermal differentiation and that integrin αvβ3 and AMPK mediate CCN1‐induced EC migration and tube formation independent of intracellular ATP levels alteration. J. Cell. Physiol. 230: 140–149, 2015. © 2014 Wiley Periodicals, Inc.