Stem cells and Cardioprotection

Myocardial infarction (MI), resulting from the interruption of blood supply to the heart, is a primary target for experimental stem/progenitor cell-based therapies. Although the therapeutic effect of mesenchymal stem cells (MSC) has been attributed to their differentiation into reparative or replacement cell types, the therapeutic importance of cardiovascular lineage remains to be elucidated.

Cell-based therapy has emerged as a promising strategy for the treatment of ischemic heart diseases via myocardial repair [1].

Exploration of stem cells as potential therapeutic agents has increased markedly in the past decade as a novel approach in both animal models and human subjects to reverse myocardial remodeling by reducing infarct size and thereby restoring cardiac pump function. Bone marrow-derived mesenchymal stem cells (MSCs) are among the most promising cell types for the treatment of ischemic heart disease [2]. Compelling evidence has suggested that the adult heart contains cardiac stem cells (CSCs), which are able to differentiate into cardiomyocytes, smooth muscle cells, and endothelial cells in vivo and in vitro [3].

Moreover, over the past years, mesenchymal stem cells (MSCs) originating from either bone marrow or adipose tissue have been consistently shown effective at reducing mortality and improving myocardial function in endotoxin-treated. The useful and beneficial role of MSCs in these studies was believed to be primarily attributed to the interaction of MSCs with host macrophages in circulation and tissues, resulting in a reduced secretion of pro-inflammatory cytokines (i.e., TNF- α, IL-1 β, and IL-6) from macrophages. Hence, MSC-induced cardiac benefits may not be related to their local actions but their systemic effects. Nonetheless, the mechanisms underlying MSC-mediated cardio-protection are still obscure [4].

In addition, the therapeutic effects of MSCs may not only be mediated by their ability to differentiate into cardiac phenotypes to replace damaged cardiac tissues, but may also be related to their salutary paracrine effects. The soluble factors secreted from stem cells include a variety of growth factors, cytokines, and chemokines that orchestrate interactions within the microenvironment to inhibit apoptosis, stimulate proliferation, promote vascularization, and mobilize endogenous cardiac stem cells (CSCs). Subsequent work has affirmed that the paracrine effect of stem cell is also mediated by secreted extracellular vesicles (EVs). Extracellular vesicles secreted from MSC are small, spherical membrane fragments and can be divided into exosomes and shedding vesicles which include microvesicles and apoptotic bodies. In particular, exosomes have been identified as a cardioprotective component in MSC paracrine secretion and have been demonstrated to reduce myocardial ischemia/reperfusion (I/R) injury [2].

The naturally occurring membrane-bound exosomes nanovesicles (50–100 nm) which play a role in the selective release of membrane or cytosolic proteins, RNAs, and/or microRNAs (miRNAs), mediating some aspects of cell-to-cell signaling. While, generally speaking these exosomes are not technically “responsible for the selective release” of signaling molecules, as they do not select the molecular cargo themselves; they just act as transporters.

Previous investigations indicated that exosomes released from progenitor cells contain secreting paracrine factors that stimulate neovascularization, thereby mediating cardiac protection during myocardial ischemia/reperfusion injury [1].

Mostly exosomes have an evolutionarily conserved set of binding proteins that have an affinity to other ligands on cell membranes or within the extracellular matrix. These proteins include members of the tetraspanin family (e.g., CD9, CD63 and CD81) and other proteins unique to the tissue/cell type. Hence etraspanin membrane proteins, CD9 and CD81, could facilitate the cellular uptake of exosomes by specific cell types, including CM, which contain the co-immunoprecipitating complexes of CD81 and CD9. Evidences suggest that exosomes serve as vectors for miR communication between different cell types. miRs are evolutionarily conserved and consist of 18- to 25-nucleotides non-protein coding transcripts that repress mRNA translation or modulate mRNA degradation by binding to the 3′- untranslated region of target mRNAs. Exosome-delivered miRs can regulate target protein expression in recipient cells, which is an important signaling transfer mechanism among neighboring cells. In an earlier study, it was confirmed that exosomes were rapidly internalized by CM, resulting in a transfer of miRs and regulation of protein synthesis in CM.  Exosomes were directly and systematically investigated whether derived from MSC^GATA-4 could deliver anti-apoptotic miRs into cultured CM in vitro and into the ischemic myocardium in vivo to produce a greater cardioprotective effect [2].

Exosomes that are generated from MSC dramatically increased the resistance of CM to hypoxic injury. Intramyocardial injections of exosomes promoted cardiac functional recovery and reduced infarction size. Exo^GATA-4 enhances cardioprotection, which may be related to Exo^GATA-4 enriched antiapoptotic miRs, e.g., miR-19a. Exo^GATA-4 may provide an attractive safer therapeutic alternative to cell therapy in the treatment of myocardial infarction. MSCs overexpressing GATA-4 can significantly improve ischemic myocardial function. Paracrine effect plays an important role in MSC^GATA-4 mediated cardioprotection, and cardioprotection by MSC^GATA-4 may be regulated in part by a transfer of certain anti-apoptotic miRs contained within extracellular vesicles. In addition to these factors, the paracrine effect depends on the transfer of other proteins, bioactive lipids, and genetic material including mRNAs, microRNAs (miRs), and other noncoding RNAs [3]. The overexpression of GATA-4 not only increases MSC differentiation into cardiac cell phenotypes, but also increases the survival of MSC in ischemic environments. Furthermore, MSCs overexpressing GATA-4 (MSC^GATA-4) increase CM survival, reduce CM apoptosis, and enhance angiogenesis in the ischemic myocardium and consequently improve cardiac function significantly [2].

MSC^GATA-4 produces salutary cellular and regenerative tissue effects in vivo; by secreting exosomes from MSC^GATA-4 which contain higher anti-apoptotic miRs, particularly miR-19a (previously we reported on the role of miR-221), and can be transferred to cardiomyocytes. Exosomes can play a role in delivering a specific miR (miR-19a) to the ischemic myocardium to induce myocardial protection and promote in vivo ischemic myocardium repair. It was also reported that exosomes can rapidly activate multiple cardioprotective pathways, which can lead to infarct size reduction and the prevention of heart function deterioration after myocardial ischemic reperfusion injury. Also it was demonstrated that purified exosomes, in a concentration-dependent manner, directly increased the resistance of CM against hypoxic injury. Intramyocardial injection of exosomes into the ischemic border of the left ventricle significantly improved cardiac function and reduced infarct size [2].

Numerous studies have indicated that miR-223 can negatively regulate the expression of many inflammatory genes (i.e., IL-6 and NLRP3). Prior work also showed that loss of miR- 223 aggravated myocardial depressions and mortality through up-regulation of Sema3A and Stat3, two known inflammation-related genes [4]. Previous study timely provided the first evidence showing that MSCs are able to reprogram macrophages and cardiomyocytes via the exosome-mediated transfer of miR-223. Subsequently MiR-223, it was found to be detectable in multiple tissues (i.e. heart, lung, kidney, liver, and spleen). Increasing evidence points to miR-223 as an important factor in the regulation of inflammatory response [4].

Previously it was shown that WT-MSCs could secret miR-223-enriched exosomes, which were subsequently taken up by recipient cells (i.e., macrophages and cardiomyocytes). As a result, the miR-223 targets (i.e., Sema3A and Stat3) were down-regulated, leading to the inhibition of inflammatory response in macrophages and attenuation of cardiomyocyte death (Fig. 8). Exosomes released from miR-223-absent MSCs contain higher levels of Sema3A and Stat3, which might be delivered to macrophages and cardiomyocytes, leading to promotion of macrophage inflammation and cardiomyocyte death [4].

Sema3A and Stat3 both are consistently validated to be bona fie targets of miR-223 in macrophages and cardiomyocytes. As a matter of fact, recent studies have shown that MSC-exosomes injected into mice via the tail vein can be detectable in the heart, the kidney, the lung, the liver, and the brain, leading to protection from ischemia/reperfusion-induced cardiac injury, chronic kidney disease, liver fibrosis, endotoxin-triggered acute lung injury and stroke [4].

Expression levels of miRs were different in exosomes secreted from MSCs subjected to ischemic preconditioning (ExoIPC). MiR-22 was highly upregulated in ExoIPC. The delivery of miR-22 reduced apoptosis in ischemic cardiomyocytes, ameliorated fibrosis, and improved cardiac function post-myocardial infarction. The treatment of infarcted hearts with ExoIPC resulted in a better cardiac outcome as compared to treatment with the miR-22 mimic or Mecp2 [2].