Perties of the EPIC line, we have described the expression of molecules involved in the active migration of EPDCs like order 61177-45-5 ephrins and their Eph receptors [44] through FACS analysis (Fig. S3). Remarkably, EphA2+ human cardiac stem cells have been described to be dependent on this ephrin receptor to migrate in response to myocardial infarction [45], but it is not known if the stem cells described in this work derive from the epicardium, as suggested for other cardiac stem cells [35]. On the other hand, the segregated expression of ephrins and Eph receptors in epicardial derivatives and other cell types in the cardiac interstitium could be instrumental to build up a functional cardiac niche microenvironment for cardiovascular progenitor/stem cells. EPICs also express a variety of cell surface markers related to EPDC/fibroblastic adhesion to the ECM [46] and cell mobilization/migration [47] like CD106/VCAM [34]. Additional analyses of standard markers for stem-like/progenitor cells indicate that EPICs are Sca1-positive but c-kit- and CD90-negative, an expression Anlotinib chemical information profile previously described for some CSC [48]. EPIC also express other markers associated with mesenchymal stem cells like CD44 [49]. These results are in agreement with a recent report supporting the epicardial origin of a population of cardiac stem cells [35]. As it can be inferred from the previous paragraph, migration of CICs necessarily involves active ECM proteolysis, but also an efficient attachment of cells to the matrix. Our experimentalsetting compares the response of EPICs to regular and engineered fibrin gels (with TG-bound growth factors). Covalent binding of growth factors like BMP2 and VEGF to fibrin enhances cell attachment and spreading (`sprouting’) of EPICs to the matrix without its massive degradation, whereas culturing these same cells on regular fibrin gels rather activates a strong proteolytic response. Then, the addition of soluble, non fibrin-bound Wnt3a and 5a abrogates EPIC degradation of fibrin matrices in our experiments, suggesting a limited migratory ability for these cells. As a matter of fact, it has been reported that Wnts can inhibit the migration of rat cardiac fibroblasts [50]. All these data, taken together, indicate that a fine balance between different signals is necessary to promote an efficient and permissive degradation of the matrix so that cells can initiate migration. Matrix metalloproteinases (MMPs), endogenous tissue inhibitors of metalloproteinases (TIMPs), and disintigrin and metalloproteinase (ADAM) family proteins are known to be the main agents of ECM remodeling [5]. Our EPIC line displays a high expression level of MMP-11, ADAM-15 and TIMP-1, 2 and 3 as compared with E11.5 whole heart tissue. EPICs also express MM-14 and ADAM-10, but at a similar level to that found in embryonic whole heart extracts. This proteolytic profile correlates with an effective degradation of fibrin gels in vitro. Deficiencies in TIMP-2 activity after myocardial infarction accelerates adverse myocardial remodeling due to enhanced MMP-14 activity [51]. These results may suggest that the response of different CICs to heart damage, specially that of CFs, is diverse, and may be linked to endogenous proteolytic programs that, in turn, could relate to the embryonic origin of CFs. In this regard, TIMP-2 and MMP-14 are known to be involved in the activation of pro-MMP-2 via the formation of a trimolecular complex at the cell surface [51], which might play multiple.Perties of the EPIC line, we have described the expression of molecules involved in the active migration of EPDCs like ephrins and their Eph receptors [44] through FACS analysis (Fig. S3). Remarkably, EphA2+ human cardiac stem cells have been described to be dependent on this ephrin receptor to migrate in response to myocardial infarction [45], but it is not known if the stem cells described in this work derive from the epicardium, as suggested for other cardiac stem cells [35]. On the other hand, the segregated expression of ephrins and Eph receptors in epicardial derivatives and other cell types in the cardiac interstitium could be instrumental to build up a functional cardiac niche microenvironment for cardiovascular progenitor/stem cells. EPICs also express a variety of cell surface markers related to EPDC/fibroblastic adhesion to the ECM [46] and cell mobilization/migration [47] like CD106/VCAM [34]. Additional analyses of standard markers for stem-like/progenitor cells indicate that EPICs are Sca1-positive but c-kit- and CD90-negative, an expression profile previously described for some CSC [48]. EPIC also express other markers associated with mesenchymal stem cells like CD44 [49]. These results are in agreement with a recent report supporting the epicardial origin of a population of cardiac stem cells [35]. As it can be inferred from the previous paragraph, migration of CICs necessarily involves active ECM proteolysis, but also an efficient attachment of cells to the matrix. Our experimentalsetting compares the response of EPICs to regular and engineered fibrin gels (with TG-bound growth factors). Covalent binding of growth factors like BMP2 and VEGF to fibrin enhances cell attachment and spreading (`sprouting’) of EPICs to the matrix without its massive degradation, whereas culturing these same cells on regular fibrin gels rather activates a strong proteolytic response. Then, the addition of soluble, non fibrin-bound Wnt3a and 5a abrogates EPIC degradation of fibrin matrices in our experiments, suggesting a limited migratory ability for these cells. As a matter of fact, it has been reported that Wnts can inhibit the migration of rat cardiac fibroblasts [50]. All these data, taken together, indicate that a fine balance between different signals is necessary to promote an efficient and permissive degradation of the matrix so that cells can initiate migration. Matrix metalloproteinases (MMPs), endogenous tissue inhibitors of metalloproteinases (TIMPs), and disintigrin and metalloproteinase (ADAM) family proteins are known to be the main agents of ECM remodeling [5]. Our EPIC line displays a high expression level of MMP-11, ADAM-15 and TIMP-1, 2 and 3 as compared with E11.5 whole heart tissue. EPICs also express MM-14 and ADAM-10, but at a similar level to that found in embryonic whole heart extracts. This proteolytic profile correlates with an effective degradation of fibrin gels in vitro. Deficiencies in TIMP-2 activity after myocardial infarction accelerates adverse myocardial remodeling due to enhanced MMP-14 activity [51]. These results may suggest that the response of different CICs to heart damage, specially that of CFs, is diverse, and may be linked to endogenous proteolytic programs that, in turn, could relate to the embryonic origin of CFs. In this regard, TIMP-2 and MMP-14 are known to be involved in the activation of pro-MMP-2 via the formation of a trimolecular complex at the cell surface [51], which might play multiple.
