Cardiac cell therapy results in increased capillary density, however only a small percentage of cells successfully engraft the myocardium. This suggests a role for paracrine factors to be involved. These paracrine factors can include soluble proteins, growth factors and exosomes. Exosomes are small membrane vesicles which can facilitate communication between cells. Here, we focused on the effect of cardiomyocytes progenitor cell (CMPC) and mesenchymal stem cell (MSC) exosomes on the angiogenic capacity of endothelial cells (ECs). In addition, we studied the role of extracellular matrix metalloproteinase inducer (EMMPRIN) on exosomes.Methods and Results
Exosomes were isolated from conditioned medium (CM) by centrifugation at 100.000 x g or separated on a sucrose gradient. These exosomes were used in scratch wound, spheroid, and matrigel assays. In addition, the exosomes were or were not treated with an EMMPRIN antibody. CMPCs and MSCs are able to produce exosomes as was observed via EM and western blot analysis for exosomal marker flotillin-1. Both types of exosomes are approximately 100 nm in size and have a density between 1.10 and 1.12 g.ml-1. In the scratch wound assay, CMPC and MSC exosome stimulation of ECs resulted in a closure of the wound of 52% ( + /- 10% SEM) and 39% ( + /- 4% SEM) respectively compared to 4% ( + /- 4% SEM, p < 0.05) when no exosomes were added. The angiogenic capacity of endothelial cells is enhanced in the presence of CMPC and MSC exosomes. Spheroids displayed a 44% ( + /- 16% SEM) and 62% ( + /- 18% SEM) reduction in sprouting length for CMPC and MSC CM depleted for exosomes (p < 0.05). The number of junctions in a matrigel assay was increased when ECs are stimulated with exosomes. CMPC and MSC exosomes express EMMPRIN, a protein known to facilitate cellular migration. Exosomes treated with an EMMPRIN antibody resulted in a diminished closure of the wound in the scratch assay and resulted in reduced number of junctions in the spheroid assay.Conclusion
CMPC and MSCs release exosomes, which can enhance the angiogenic in vitro capacity of ECs via an EMMPRIN mediated mechanism. This phenomenon could be a novel mechanism in which transplanted cells might trigger or enhance migration and organization of endogenous ECs upon cardiac cell therapy leading to increased capillary formation.