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A novel transduction pathway for the powerful angiogenic factor VEGF has been recently shown in endothelial cells to operate through NAADP-controlled intracellular release of Ca(2+). In the present report the possible involvement of NAADP-controlled Ca(2+) signaling in tumor vascularization, growth and metastatic dissemination was investigated in a murine model of VEGF-secreting melanoma. Mice implanted with B16 melanoma cells were treated with NAADP inhibitor Ned-19 every second day for 4 weeks and tumor growth, vascularization and metastatization were evaluated. Control specimens developed well vascularized tumors and lung metastases, whereas in Ned-19-treated mice tumor growth and vascularization as well as lung metastases were strongly inhibited. In vitro experiments showed that Ned-19 treatment controls the growth of B16 cells in vitro, their migratory ability, adhesive properties and VEGFR2 expression, indicating NAADP involvement in intercellular autocrine signaling. To this regard, Ca(2+) imaging experiments showed that the response of B16 cells to VEGF stimulation is NAADP-dependent. The whole of these observations indicate that NAADP-controlled Ca(2+) signaling can be relevant not only for neoangiogenesis but also for direct control of tumor cells.

The object of this study was to develop an in vitro bioengineered three-dimensional vascularized skeletal muscle tissue, named eX-vivo Muscle Engineered Tissue (X-MET). This new tissue contains cells that exhibit the characteristics of differentiated myotubes, with organized contractile machinery, undifferentiated cells, and vascular cells capable of forming "vessel-like" networks. X-MET showed biomechanical properties comparable with that of adult skeletal muscles; thus it more closely mimics the cellular complexity typical of in vivo muscle tissue than myogenic cells cultured in standard monolayer conditions. Transplanted X-MET was able to mimic the activity of the excided EDL muscle, restoring the functionality of the damaged muscle. Our results suggest that X-MET is an ideal in vitro 3D muscle model that can be employed to repair muscle defects in vivo and to perform in vitro studies, limiting the use of live animals.