Extracellular vesicles in pulmonary hypertension: lessons from mesenchymal stromal cell-derived exosomes

Hogan et al. (6) investigated the central hypothesis that the smallest of the extracellular vesicle family, the exosome, could repair injured pulmonary vessels and pulmonary artery smooth muscle cells (PASMCs). Treatment of hypoxic PASMCs with exosomes isolated from primary human bone marrow-derived mesenchymal stem cells (MSC) improved amino acid metabolism and prevented the shift to glycolysis. Furthermore, intravenous injection of the isolated exosomes prevented hypoxic pulmonary hypertension (PH) in the mouse model and partially reversed it in the semaxinib/hypoxia rat model (6). This paper contributes to a wider body of literature on the nature of MSC-derived exosomes and their ability to repair vascular injury. In 2016, Aliotta et al. (1) described the ability of MSC-derived exosomes to prevent and even rescue the mouse monocrotaline model of PH. Further, Lee et al. (9) revealed that early intervention in hypoxia-induced PH in mice with MSC-derived exosomes prevented the influx of macrophages and repressed the activation of signal transducer and activator of transcription 3. These examples highlight the varied mechanisms by which MSC-derived exosomes might protect or heal vessels. However, several questions remain: 1) what is the nature of the exosomes and how do they relate to extracellular vesicles, in general; 2) what is the mechanism of action of exosomes and extracellular vesicles; and 3) are extracellular vesicles (including exosomes) truly reparative or injurious?

Since the first descriptions of the effects of “platelet dust” on coagulation as early as 1967, there has been little consensus over how to define or characterize extracellular vesicles; however, great strides have been made in recent years (18). The minimal requirements for defining extracellular vesicles and their primary classes such as exosomes, microvesicles/microparticles, and apoptotic bodies were recently published by the International Society of Extracellular Vesicles (16). In less than a decade since the society was formed, in essence to tackle this issue of defining vesicles, there has been an explosion of literature and interest. Current dogma is that the defining characteristics of an exosome include having a diameter less than 1 μm, generally in the 100- to 200-nm range, and expression of protein markers such as CD63, CD81, TSG01, and ALIX. The work described by Hogan et al. (6) to characterize their functional vesicle is excellent. They provided the reader with a comprehensive view including the marker subsets, transmission electron microscopy, and the concentration and size of vesicles via nanoparticle tracking analysis. This is currently the expectation for publication; however, as new data emerge, more refinement of the definitions of extracellular vesicles may be on the horizon. Each of the protein markers defining exosomes was originally thought to delineate a vesicle, small in size, that was formed in the multivesicular body and released extracellularly. However, in 2016, Kowal et al. (8) identified CD63 and CD9 on the membrane of microvesicles (MVs). MVs are larger in diameter than exosomes and thought to be derived from the cellular plasma membrane. Thus, many publications are using the more encompassing title of extracellular vesicles (7, 15). This highlights an important area in the field of extracellular vesicle research, biogenesis. While some of the mechanisms for extracellular vesicle generation are well-defined, e.g., exosome formation through multivesicular body transport, others are not and include intracellular calcium regulation of the cytoskeleton, alterations in membrane symmetry, and mechanical perturbation (5, 10, 11). Each of these mechanisms will no doubt influence the intravesicle and membrane contents including proteins and RNA. Thus, this area of investigation could yield valuable insight into how we can further manipulate extracellular vesicles, and their tiniest counterpart the exosome, into biological therapeutics and, importantly, how we might also prevent the release of detrimental extracellular vesicles.

Decades of work have shown that MSC infusions are beneficial for pulmonary vasculopathy, but later work determined that merely the conditioned medium, and not the cells themselves, was sufficient for repair. It has now become clear that a factor in that medium, the isolated extracellular vesicle or exosome, is a primary functioning component. Lee et al. (9) elegantly illustrated that MSC-derived exosome-depleted media did not prevent hypoxic PH, nor did fibroblast-derived exosomes. Inflammatory cell infiltration is a hallmark of pulmonary vascular lesion formation, and MSC-derived exosomes blocked macrophage infiltration. Hogan et al. (6) highlight this with their work, indicating that even a single dose of intravenous MSC-derived exosomes prevented hypoxic PH and that repeated dosing reversed the more robust semaxinib/hypoxia rat model of PH. Hogan et al. examined the exosomes directly, using proteomics and RNAseq to guide their investigations and found metabolic programming as a potential mechanism. MSC-derived exosomes in the hands of Hogan et al., clearly inhibit the hypoxic activation of sirtuin 4, releasing glutamate dehydrogenase-1, and pyruvate dehydrogenase to drive metabolism and improve mitochondrial function. However, Aliotta et al. (1) also examined MSC-derived exosomes directly and their data suggest that a miRNA component of the exosome drives repair. Use of MSC-derived exosomes for repair is not limited to the pulmonary vasculature, but also tracheal instillation reduces hyperoxia-induced damage in a rat model of bronchopulmonary dysplasia (14). These data demonstrate the complexity of extracellular vesicle signaling. Whether the exosomes (or other extracellular vesicles) themselves have pleiotropic effects, whether the starting material and method of stimulation alters the exosome contents, or whether the mechanisms used by the recipient cells to make use of extracellular vesicle contents differ are all questions that will drive the field forward.

While there is mounting evidence that MSC-derived exosomes can generally be considered “good”, during disease many extracellular vesicles deliver injurious messages such as alterations in nitric oxide signaling and stimulation of inflammatory molecules on the endothelium (2, 4, 17). Microparticles (MPs) in particular, especially those with phosphatidylserine on their membrane, are frequently reported to induce injury in a variety of lung diseases including PH, acute lung injury, and cigarette smoking-induced damage. So, how might we tip the balance between “good” and “bad” extracellular vesicles? Mohning et al. (12) examined clearance mechanisms of MPs by alveolar macrophages and determine phosphatidylserine-positive MPs were cleared by the phosphatidylserine-binding MerTK receptors expressed on resident macrophages. While inducing clearance of dangerous MPs is an attractive concept, not all injurious extracellular vesicles express phosphatidylserine; therefore, future studies into alternative clearance mechanisms would benefit the field as a whole. In an effort to maximize the potential for “good” exosomes to deliver their message, Zhang et al. (19) have provided a novel methodology to enrich microRNAs (miRNAs) in isolated exosomes for delivery to recipient cells. Prior work to enrich miRNAs in exosomes focused on upregulating the miRNA in parent cells; however, the novelty of Zhang’s method is direct enrichment in the isolated exosome. As we continue to understand the constituents of circulating and cell culture-derived extracellular vesicles, it is intriguing to envision a time when we can manipulate the vesicles to our benefit, either inhibiting the “bad” ones or enriching the “good” ones, to restore homeostasis.

The work by Hogan et al. (6) provides direct evidence of mitochondrial repair in PASMCs, and further, the contents of the MSC-derived exosomes includes metabolism-associated genes and proteins. However, we still do not fully understand how extracellular vesicles deliver their contents to recipient cells, otherwise known as the “endocytic problem”. Many studies have examined the interactions and uptake of labeled extracellular vesicles into recipient cells and tissues. The mechanisms involve the endocytic pathway and a variety of necessary cytoskeletal proteins (3, 5, 13). However, direct evidence of intracellular processing of the intact or even opened vesicle and its contents has not been determined. Future studies into intracellular processing of extracellular vesicles will significantly strengthen their potential as therapeutics and provide mechanisms to inhibit delivery of dangerous content.

Severe PH is a devastating syndrome with no curative therapeutics, and current therapies have minimal effects on mortality. Therefore, the work described by Hogan et al. (6), investigating a novel mechanism of vessel and pulmonary vascular smooth muscle mitochondrial repair utilizing MSC-derived exosomes, is timely and important. While we clearly have many hurdles to overcome in our understanding of extracellular vesicle/exosome signaling, it is reasonable that study of these extracellular vesicles will provide novel insight into the pathology and potential treatment of PH.

GRANTS

This work was supported in part by NIH National Heart, Lung, and Blood Institute Grants R01 HL133066 (N. N. Bauer) and T32 HL076125 (J. L. Hewes).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

N.N.B. drafted manuscript; J.L.H. and N.N.B. edited and revised manuscript; J.L.H. and N.N.B. approved final version of manuscript.