Exosomes in cardiac injury and repair

Stem cell therapy has been proposed as a strategy to regenerate the damaged myocardium after myocardial infarction. The differentiation capacity of many different stem cells to cardiomyocytes and blood vessels and their effect on cardiac function has been studied. Despite low retention and engraftment of transplanted cells, small improvements in cardiac function and the formation of new vessels and cardiomyocytes, originating from transplanted and endogenous cells, could be observed. The activation of endogenous cells by cell transplantation instigated the formulation of the paracrine hypothesis. Stem cells conditioned medium diminished cardiac injury after myocardial infarction and stimulated regeneration. Exosomes being part of the conditioned medium were suggested to be involved. Exosomes are small membrane vesicles produced via the endosomal pathway involved in inter cellular communication. Cardiomyocyte progenitor cell (CMPC) and mesenchymal stem cell (MSC) exosomes are characterized and their role in angiogenesis and immune modulation is determined. Within the conditioned medium different types of vesicles could be detected of which the majority of the vesicles are in the exosomal range (30-100 nm). Analysis of sucrose gradient purified exosomes demonstrated that these vesicles express exosome specific proteins at a density of 1.10 – 1.12 g/ml. Incubation of endothelial cells with fluorescently labeled exosomes demonstrated the uptake of exosomes by endothelial cells. In different cell-based assays, the pre-requisites of angiogenesis were enhanced. In vivo, CMPC and MSC exosomes enhanced the infiltration of angiogenic cells into a matrigel plug, demonstrating the pro-angiogenic potential of these exosomes. Endothelial cells stimulated with strategically manipulated CMPC exosomes, via overexpression of miR-1 in CMPCs led to the horizontal transfer of miR-1 from CMPC exosomes to endothelial cells. Additionally the migration of these endothelial cells in a scratch wound migration assay was enhanced. CMPCs and MSCs were also found to be involved in the suppression of activated T-cell proliferation. The expression of pro-inflammatory cytokines of activated T-cells is suppressed upon co-culture with both types of stem cells. However, CMPCs and MSCs mode of action is not dependent on cell-cell contact as trans-well experiments and exosomes also suppressed T-cell proliferation. Immune suppression of the inflammatory response specifically for T-cells is important to persevere cardiac function and reduce negative cardiac remodeling in long-term heart failure. The effect on other immune cells, which are involved in the acute response after myocardial infarction still need to be investigated. Finally this thesis describes the release of exosomes and other vesicles upon cardiac injury and their role in the activation of endothelial cells. Upon ischemia reperfusion injury, more vesicles are released into the circulation, suggesting that they could play a role as biomarkers. Exosomes released upon ischemia/reperfusion injury enhance the migration of endothelial cells, which could indicate that these vesicles stimulate endogenous repair. Taken together the amount of exosomes released upon cardiac injury to the circulation is increased. These vesicles activate endothelial cells, suggestive of the intrinsic regenerative capacity of these vesicles released upon cardiac injury. Additionally, exosomes from CMPCs and MSCs are a new and promising therapeutic strategy to reduce cardiac injury and enhance cardiac regeneration.