Exosomes Explosion for Cardiac Resuscitation

Graphical Abstract

Extracellularly secreted membrane vesicles (EMVs) including exosomes were first discovered 30 years ago and were considered to be garbage bags comprised of unwanted cellular components (1). Exosomes are relatively homogenous in size (about 30–100 nm) and are different from other secreted vesicles such as apoptotic bodies and microvesicles in their origin, density and protein and nucleic acid composition. They are shown to mediate local and distant cellular communications, and by doing so, can play a role in dissemination of diseased or fortified materials from the cell of their origin. The recent explosion in exosome research has demonstrated critical roles for these bio-nanovesicles in nearly all aspects of physiology as well as pathology. Although fast-growing understanding of biogenesis and function of exosomes have emerged in immune regulation, tumor progression and inflammation, intensive characterization of exosomes in cardiovascular research remains largely unexplored.

It is now well-established that the human heart is a dynamic organ with limited capability for cell replacement under pathological insult. Several clinical and preclinical studies have demonstrated beneficial effects of intramyocardial delivery of progenitor cells to post-ischemic hearts. Although secreted paracrine factors and cytokines were thought to be the basis of the beneficial effects, accumulating evidence now points towards extracellular vesicles as being responsible for the therapeutic effects observed. In 2011, a study from our laboratory (2) provided the first evidence of exosomes mediating beneficial effects of clinically successful human stem cells for the treatment of ischemic cardiovascular disease. In several recent investigations, the role of exosomes as a mediator of cardiac communication among different types of heart cells has been studied intensively. Two parallel studies have demonstrated that extracellular vesicles from human cardiac progenitor cells (CPCs) (3) or from human cardiospheres (CSp) (4), but not from dermal fibroblasts inhibited apoptosis in cardiomyocytes, improved angiogenic activity of endothelial cells and improved LV function of the post-ischemic heart. These CPC/CSp-vesicles were enriched with several anti-apoptotic and proangiogenic miRNAs such as miR-210, miR-132, miR-146a, which had been implicated to have functions in cardiac biology. Similarly, cardioprotective and anti-apoptotic nature of mouse cardiac progenitor cell-derived exosomes (5), and mouse fibroblast-iPS cell-derived exosomes (6) have been reported earlier. More recently, Khan et al. (7) have shown that cardioprotective exosomes from mouse embryonic stem cells augments the endogenous cardiac progenitor cell-based repair programs in the heart. They tied the underlying basis of the beneficial effects to exosomes-mediated delivery of ESC-specific miR-294.

Collectively, cell therapy approaches have been shown over the last decade to successfully augment cardiac function by multiple means, such as reducing fibrosis, stimulation of vascular angiogenesis, replacing damaged heart tissue with new functionally integrated myocytes and augmenting endogenous repair processes. Nonetheless, in a fresh approach, Tseliou et al. in this issue of the Journal added another novel dimension to further deepen our understanding of the mechanism by which cell therapy can reprogram fibroblasts by stem cell derived EMVs, and positively impact post-ischemic reverse remodeling. Using rigorous experimental methods, the authors have demonstrated that EMVs secreted from human and rat cardiospheres (CSp) alter the fibroblast phenotype and secretome in a salutary positive feedback loop, converting inert fibroblasts into beneficial fibroblasts. The beneficial fibroblasts in turn secreted proangiogenic and cardioprotective VEGF and SDF-1 to initiate a cascade of events amplifying the beneficial effects of the stem cells and improving the function of the post-ischemic myocardium. In a rat model of chronic MI, intramyocardial injection of CSp-EMVs or, CSp-EMV-primed dermal fibroblasts (DFs) improved left ventricular ejection fraction, reduced scar mass and increased microvessel density as compared to injection of unprimed fibroblasts.

To decipher the underlying molecular and cellular events involved in this process, the authors used a series of interesting in vitro experiments. Treatment of CSp-EMVs to dermal fibroblasts induced a loss of fibroblast phenotype as demonstrated by loss of fibroblast surface marker proteins FSP and DDR2, down regulation of TGF-beta pathway, increased secretion of VEGF and SDF-1 and increased expression of smooth muscle actin. This process, the authors claimed, contributed to generating therapeutic fibroblasts with anti-apoptotic and proangiogenic properties, and amplified the beneficial effect of CSp-EMVs. It would be intriguing to determine whether these alterations in fibroblast phenotype in response to uptake of CSp-EMV were transient effects or durable alterations that were retained by the cells.

MicroRNA expression profiling using microarrays indicated significant difference in miRNA expression profiles of CSp-EMV treated fibroblasts compared with untreated cells. This is not surprising given that in their earlier publication the authors have already established that beneficial miRNAs such as miR-146a play an essential role in the therapeutic activity of CSp-EMV. Consistent with literature, the miRNA expression pattern in CSp—EMV treated fibroblasts partially resembled to the expression in CSp cells and partly to the expression in CSp-EMVs indicating new miRNA synthesis in the target cells. While the microarray experiments indicate that miRNAs may mediate the beneficial changes in fibroblasts, their mode of action in recipient fibroblasts as well as their effects on other cardiac cells including cardiomyocytes and endothelial cells warrants future investigation. Furthermore, miRNAs as the biologically active component in exosomes is likely only a part of the complex puzzle because other active compounds may play additional role in the recipient cells.

Several different therapies including pharmacological and cellular therapies have demonstrated prevention or slowing down ventricular remodeling after MI in both preclinical and clinical studies. However, it is not clear whether cardiac remodeling may be reversed once it has developed in response to a pathological insult. In their new exciting work, the authors presented evidence that injection of CSp-EMV-primed DFs improved remodeling of the myocardium (reduced scar mass, improved wall thickness and ejection fraction as well as left ventricular end-systolic diameter), as compared to unprimed DFs measured after one month of injection. A phenotypically and functionally improved myocardium after any post-ischemic treatment compared to a control may be attained largely by 2 means: i) the treatment agent prevents or protects the cardiac tissue from further deterioration or ii) the treatment agent stimulates myocardial regeneration mechanisms to reverse the remodeling. Here, the authors have concluded that CSp-EMV treatment actually reversed cardiac pathological remodeling. However, in the absence of assessment of the myocardial damage at baseline, i.e., before the injection of the treatment, a possibility exists that this conclusion may prove to be premature. Further, deciphering the time period within which such reverse remodeling can take place in response to a treatment in the ischemic heart will have important clinical implication.

Myocardial injury induces cardiomyocyte death and destroys the vasculature and initiates a cardiac repair mechanism eventually resulting fibrosis. Cellular communication is further highlighted by plasticity of different cardiac cells and their interactions to induce fibrosis by a process called epithelial/endothelial to mesenchymal transition to generate fibroblasts. This process also stimulates quiescent fibroblasts to become secreting myofibroblasts to enhance fibrotic response. An interesting piece of puzzle remains unanswered here is whether CSp-EMVs alter epithelial/endothelial to mesenchymal transition and affect the collagen secretion in the remodeled tissue.

This study clearly represents a significant advance in our understanding of the extracellular vesicle function. Nevertheless, the authors acknowledged an important limitation of using ExoQuick (a polyethylene based precipitation) to isolate CSp-EMVs for majority of their in vitro and in vivo experiments. As ExoQuick precipitation collects all extracellular vesicular plus nonvesicular components, the study could not pinpoint exosomes as the only biological component responsible for the observed effects. Nevertheless, using rigorous characterization methods the authors have demonstrated presence of membrane vesicles in their isolate. In addition, they had isolated EMVs using an ultracentrifugation method that recapitulated equivalent bioactivity in treated fibroblasts in vitro. These results collectively implicated participation of membrane vesicles in the observed beneficial effects.

The study also hints at the complexity of the cargo carried by progenitor cell-derived exosomes suggesting their enormous potential to directly influence the biology of ischemic repair as well as to augment endogenous cardiac repair and regeneration. This impressive work by the authors further established an important role of EMVs in improvement of cardiovascular disease and opened a promising future for EMV-based cell-free therapeutic strategies. However, developing an efficient isolation method that can collect adequate amounts of exosomes for clinical applications may be a major roadblock in translating the therapeutic benefits of EMVs to clinics. ExoQuick and polyethylene-based methods, although allow easy isolations, may not be suitable for clinical applications, where as ultracentrifugation-based methods, although yield high purity, are time consuming and often associated with substantial loss of the starting material. Going forward, we can anticipate that discovery-driven proteomic or transcriptomic analyses will dissect the molecular events that result from vesicles trafficking in ischemic microenvironment and lead to the identification of more suitable therapeutic targets. While the specifics are intriguing, more broadly exosomes research has opened a vista for interrogating a new form of cell-to-cell communication.

Our understanding of the role of exosomes in local and distant microcommunications in the setting of cardiovascular diseases has evolved significantly over the last few years. In the future, the study of exosomes will not only unravel novel mechanisms of cell-to-cell communications and identify novel biomarkers of cardiovascular disease, but provide novel platforms for treatment strategies.