Targeting miRNAs to Enhance Cell-Based Therapies
The studies of cell-based therapies using bone marrow-derived cells in patients with acute myocardial infarction (Assmus et al. 2002) triggered a vast number of experimental and clinical investigations using bone marrow-, mesenchymal-, or cardiac- derived stem and progenitor cells (Jakob and Landmesser 2013). Recently, cardiac-derived stem cells were examined in clinical phase I/II trials (Bolli et al. 2011; Makkar et al. 2012). However, while some clinical trials using cell-based therapies after myocardial infarction observed an improvement of left ventricular function or clinical symptoms, others failed to show beneficial effects (Jeevanantham et al. 2012; Fisher et al. 2015; Gyongyosi et al. 2015). Importantly, as the majority of conducted clinical trials included only small number of patients and was mostly underpowered for mortality as an endpoint, a Pan-European clinical phase III trial investigating the effect of cell-based treatment in patients with acute myocardial infarction (MI) (http://www.bami-fp7.eu/) is now being conducted.
The modest effects observed in cell-based therapies may in part be attributable to isolation procedures (Assmus et al. 2010; Seeger et al. 2012), low survival (Li et al.
2009), limited homing (Chavakis and Dimmeler 2011), and highly limited differentiation into contractility contributing cardiomyocytes of the applied cell product (Tongers et al. 2011). Of note, cells derived from patients with cardiovascular diseases show an impaired cardiac repair capacity when compared to cells from healthy subjects (Jakob et al. 2012a; Heeschen et al. 2004). Mechanistic studies have shown that miRNAs are crucially involved in these processes. As a potential mechanism, deregulation of miRNAs in cells with repair capacity was observed (Mocharla et al. 2013; Jakob et al. 2012a; Xu et al. 2012). We and others observed deregulation of miRNA expression in early angiogenic outgrowth cells (EOCs) derived from patients with chronic heart failure (CHF) (Jakob et al. 2012b). Notably, overexpression of miR-126 that was reduced in EOCs from patients with CHF enhanced EOC- mediated cardiac repair capacity in vivo (Jakob et al. 2012b). MiR-126 was previously shown to stimulate angiogenesis (Wang et al. 2008) and is also involved in the prevention of experimental atherosclerosis (Schober et al. 2014). In addition, miR-21, that is upregulated in EOCs from patients with coronary artery disease, impairs their migratory potential through an increase in reactive oxygen species (Fleissner et al. 2010). Another approach is to prevent apoptosis of transplanted cells, a process thought to substantially decrease cardiac repair capacity after cell transplantation due to low survival of transplanted cells. Expression of miR-34a, a pro-apoptotic miRNA, was increased in bone marrow mononuclear cells (BMC) from patients with myocardial infarction (Xu et al. 2012). Pretreatment of BMCs with miR-34a inhibitors improved their capacity to restore cardiac function in a murine infarct model (Xu et al. 2012). Of note, miR-34a is also increased during ageing in the heart (Boon et al. 2013). Hu et al. applied a cocktail consisting of miR-21, miR-24, and miR-221 to cardiac progenitor cells, which increased their survival after cardiac transplantation in an experimental myocardial infarct model and resulted in a better preserved cardiac function (Hu et al. 2011). Bim, an inducer of apoptosis, was repressed by these three miRNAs (Hu et al. 2011), demonstrating that multiple miRNAs can synergistically repress one target. Hence, miRNAs have the potential to improve impaired cardiac repair capacity of adult stem/progenitor cells, and miRNA modulation of adult stem/progenitor cells may serve as a strategy to enhance cardiac repair processes in cell-based therapies (Fig. 4.1).
As cell isolation procedures are labor intensive and expensive and improvement in cardiac function is mostly related to paracrine mechanisms (Gnecchi et al. 2008; Murry et al. 2004), recent studies focused on compounds released from progenitor/ stem cells. In this context, exosomes, small secreted membrane-bound vesicles released from cells, evolve as a potential cell-free therapy for cardioprotection (Vicencio et al. 2015; Chen et al. 2013b). Interestingly, miRNA transferred via exo- somes contributes to this intercellular communication system. Intramyocardial delivery of exosomes derived from mouse embryonic stem cells improved LV function after induction of myocardial infarction in mice (Khan et al. 2015). This was
Fig. 4.1 Experimental (and clinical) strategies to improve cardiac function using cells with miRNA-mediated cardiac repair potential, cell-derived miRNA-containing components, or chemically modified synthetic miRNAs. Chemically modified synthetic miRNAs or viral constructs can be delivered directly (local or systemic) for therapeutic manipulation of miRNAs (a). Systemic or intramyocardial delivered cells enhance cardiac repair by the release of miRNAs (and other growth factors) to host cells through exosomes and gap junctions (b). MiRNA pretreatment of cells may enhance their cardiac repair potential and survival (c). MiRNA-containing exosomes of stem/pro- genitor cells can be isolated and delivered to improve cardiac repair capacity. This cell-free strategy avoids potential side effects that may arise after transplantation of stem cells (d). CM cardiomyocytes, EC endothelial cells, miR microRNA related to an increase in proliferative myocytes and number of cardiac progenitor cells (CPCs, c-kit + cells) in vivo. Of note, miRNA profiling of exosomes revealed an enhanced expression of the cell cycle regulator cluster miR-290. Overexpression of one of the members, miR-294, in CPCs increased proliferation and decreased apoptosis in vitro (Khan et al. 2015). Similarly, hypoxia-induced release of exo- somes in CPCs improved LV function in an experimental ischemia-reperfusion model, which is related to an increased exosome content of miRNAs involved in fibrosis pathways (Gray et al. 2015). In addition, exosomes from host tissue after cell therapy may alter function of the applied cell product. Ong et al. showed that CPCs co-delivered with a minicircle plasmid containing hypoxia-inducible factor 1 (HIF-1)-induced endothelial cells to secrete exosomes enriched of miR-126 and miR-210. Uptake of these miRs in CPCs leads to a higher tolerance against hypoxic stress in vitro which in turn enhances survival of CPCs after intramyocardial delivery (Ong et al. 2014). Interestingly, Hosoda et al. (2011) showed that miR-499 may also be transferred via gap junctions from myocytes to cardiac stem cells, thereby promoting differentiation via suppression of differentiation modulators Sox6 and Rod1. MiR-499 is highly expressed in differentiated cardiomyocytes and markedly reduces proliferation rates of cardiomyocyte progenitor cells (Sluijter et al. 2010).