Effects of mesenchymal stromal cell-derived extracellular vesicles in acute respiratory distress syndrome (ARDS): Current understanding and future perspectives

Abstract Acute respiratory distress syndrome (ARDS) is a devastating and life-threatening syndrome that results in high morbidity and mortality. Current pharmacologic treatments and mechanical ventilation have limited value in targeting the underlying pathophysiology of ARDS. Mesenchymal stromal cells (MSCs) have shown potent therapeutic advantages in experimental and clinical trials through direct cell-to-cell interaction and paracrine signaling. However, safety concerns and the indeterminate effects of MSCs have resulted in the investigation of MSC-derived extracellular vesicles (MSC-EVs) due to their low immunogenicity and tumorigenicity. Over the past decades, soluble proteins, microRNAs, and organelles packaged in EVs have been identified as efficacious molecules to orchestrate nearby immune responses, which attenuate acute lung injury by facilitating pulmonary epithelium repair, reducing acute inflammation, and restoring pulmonary vascular leakage. Even though MSC-EVs possess similar bio-functional effects to their parental cells, there remains existing barriers to employing this alternative from bench to bedside. Here, we summarize the current established research in respect of molecular mechanisms of MSC-EV effects in ARDS and highlight the future challenges of MSC-EVs for clinical application.

from 35% to 46% in hospitals. 3 Additionally, another study has estimated that annually ARDS affects more than 3 million patients worldwide and leads to 75,000 deaths in the United States. 4 Nevertheless, low efficacy of the existing pharmacologic interventions of ARDS contributes to new potential therapeutic options being sought by investigators.

Multipotent mesenchymal stromal cells (MSCs) have been shown
to possess the properties of immunomodulation and tissue repair in both experimental acute lung injury (ALI) and sepsis models through the secretion of several growth factors (TNF-α-stimulated gene-6), KGF (keratinocyte growth factor), prostaglandin E2, et al.), and antiinflammatory cytokines (IL-10). [5][6][7] Moreover, in an ex vivo model of perfused human lungs injured with live E. coli bacteria, MSCs were also capable of decreasing lung inflammation, clearing alveolar fluid, and enhancing alveolar macrophage phagocytosis. 8 Despite the existence of data that have shown the benefit of MSCs for ARDS, a growing body of evidence is emerging querying the actual effects of MSCs in clinical practice in terms of the outcome of several completed randomized phase I/II clinical trials [9][10][11][12] (Table 1). Although these early clinical trials have shown that MSCs did not induce prespecified infusion-related adverse events, there was no difference in 28 d mortality between the groups of MSCs and placebo, and the MSC group had higher mean scores of Acute Physiology and Chronic Health Evaluation III, which can provide initial risk stratification and risk estimates for critically ill patients, and higher scores correspond to more severe illness and a higher risk of death. 10,11 Additionally, it has been proven that the efficacy of MSC engraftment and differentiation is limited 13 and MSCs are short-lived cells, which are no longer viable after 24 h in the damaged lung by i.v. infusion. [14][15][16] Of more concern is the fact that MSCs exhibit a high risk of carcinogenesis, and the possibility of tumorigenicity increases when MSCs are expanded in culture, whereas the longterm follow-up of MSC administration is absent in most preclinical experiments and clinical trials. 17,18 Recently, attention has been drawn to MSC-derived extracellular vesicles (MSC-EVs) as a new frontier in the cell-free treatment regime for ARDS. There are increasing data to suggest that the properties of MSC-EVs are similar to their parental cells in anti-inflammation and tissue homeostasis in damaged cells or diseased organs. Furthermore, the characteristics of no risk of tumorigenicity, and a lower possibility of immunologic rejection and self-replication, make them a promising candidate for the treatment of ARDS. 19

MECHANISM DISSECTION OF MSC-EV EFFECTS IN ARDS
MSC-EVs have been demonstrated to have therapeutic benefits for ARDS and severe pneumonia in preclinical studies. The main biofunctionalized benefits of MSC-EVs have been presented in the aspect of attenuating acute inflammation, promoting alveolar epithelial regeneration, and enhancing pulmonary endothelial repair. 23 In this context, accumulating evidence aiming at dissecting molecular mechanisms has shown that MSC-EVs as a shuttle of bio-active messengers is capable of wrapping mRNAs, proteins, microRNA (miRNAs), and mitochondria to modulate immune responses and to repair widespread lung damage in the exudative, proliferative, and fibrotic phases of ARDS, which assist in decreasing proinflammatory cytokine production and improving alveolar fluid clearance 12 (Fig. 2).

Role of mRNAs in MSC-EVs in ARDS
The mRNA is a single-stranded molecule that carries genetic information copied from DNA for protein synthesis. 24 Batagov and colleagues showed that most exosomal mRNAs that are enriched with specific 3′-untranslated regions that may present as a competing RNA in targeted cells to modulate the physiologic processes. 25  (1) mRNA: MSC-EVs deliver mRNA fragments of keratinocyte growth factor (KGF) and angiopoietin 1 (Ang-1) have shown great therapeutic effects in decreasing neutrophil infiltration, increasing anti-inflammatory cytokine production, down-regulating proinflammatory cytokine secretion, total protein, and vascular endothelial permeability. (2) miRNA: MSC-EVs are capable of transferring miRNA21, miRNA27a-3p, miR145, and miR146 to alveolar macrophages, contributing to M2 macrophage polarization, STAT3 signaling activation, macrophage phagocytosis promotion, and down-regulate IL-6 secretion. Furthermore, miR100 can be delivered to fibroblast cells by MSC-EVs, resulting in PI3K/protein kinase B (AKT)/NF-kB signaling activation, down-regulation of total protein content, neutrophil counts, and proinflammatory levels. in MSC-MVs fails to decrease the production of proinflammatory cytokine (TNF-α) and to raise IL-10 production in macrophages. 32 Moreover, Ang-1 mRNA deficient MVs lose the effect of protecting the integrity of LPS-induced microvascular endothelial cells and of decreasing neutrophil influx. These robust data elucidate that MSCs can transfer Ang-1 mRNA to endotheliocytes and macrophages to regulate wound repair and immune responses.
Even the results have shown ALI can be restored by MSC-EV wrapped KGF and Ang-1 mRNA, the standardization, quantification, and characterization of MSC-EVs are absent in these two studies.
Moreover, the potential targets and molecular mechanisms for KGF and Ang-1 mRNA in the alveoli remain uninvestigated.

Effects of MSC-EVs transferred miRNAs in ARDS
The miRNAs are a class of small noncoding RNAs that are transcribed from DNA sequences, normally 22 nucleotides in length, 33 which exert a critical impact on various biologic activities. Mechanically, miRNAs secreted into EVs or fluids are able to interact with targeted mRNAs, thereby suppressing protein translation. However, in the context of several specific microenvironments, miRNAs also promote or modulate mRNA translation. Accumulating evidence has shown that MSC-EVs transfer miR-21p, miR-27p-3a, miR30b-3p, miR100, miR145, and miR146 to injured or inflamed lung tissue for regulating their biogenesis and homeostasis.

miR21
MiR21 is one of most investigated and responsive miRNAs in the context of distinct pathologic processes, including acute or chronic lung inflammation, diabetic complications, and myocardial ischemia. It has been demonstrated that miR21 is able to down-regulate STAT3 signaling to suppress LPS-induced ALI by decreasing the IL-6 secretion in murine RAW264.7 cells. 34 In what could be another mechanism for miR21 biologic function in lung disease, miR21 facilitates the pro-

miR100
Most studies into miR100 in lung diseases concern its antitumor effects and its role as a potential molecular prognostic marker in nonsmall lung cancer. 44,45 Recently, miR100 has been demonstrated to modulate Additionally, miR100 overexpressed MSC-EVs attenuate the total protein content, neutrophil counts, and the proinflammatory levels in the BALF, and decrease cell apoptosis in the BLM-induced ALI rat model. 47

miR145
MiR145 is engaged in normal blood cell development and appears to play a fundamental role in the biogenesis and division of megakaryocytes. 48 Regarding lung diseases, miR145 has been reported to contribute to the pathogenesis of hypoxia-induced pulmonary artery hypertension, and miR145 deficiency has also modulated myofibroblast differentiation to protect BLM-treated lung fibrosis. 49

Benefits of MSC-EV packaged mitochondria in ARDS
Mitochondria, referred to as the powerhouse of the cell, are believed to exert a vital impact on the pathologic processes of the illness by regulating cell metabolism and homeostasis. Optimal performance of cellular mitochondria relies on the sophisticated and dynamic system that provides a complete electron transport chain (ETC), an efficient citric acid cycle, and intact mitochondria constituents. Conversely, damaged ETC, excessive superoxide-derived reactive oxygen species production, unbalanced cytosolic and mitochondrial calcium levels, and morphologic changes (mitochondria leak and uncoupling) contribute to mitochondrial dysfunction and cell injury. 59,60 Of note, mitochondrial bioenergetic failure also plays a key role in the pathogenesis of ARDS. 61 Ever-increasing evidence has suggested mitochondrial dyshomeostasis is implicated in ARDS development and pathophysiology. 62 A preclinical study of ARDS has revealed that significant ATP decline and low arterial oxygenation have been shown in the LPStreated mouse model, which are considered to be associated with prolyl hydroxylases and hypoxia-inducible factor. 63 The preliminary data from Ten et al. have demonstrated that ADP-phosphorylating respiration in mitochondria isolated from mouse injured lungs that were treated with LPS is lower than the control group. 64 To the best of our knowledge, Spees and coworkers have initially  (Table 2).
Due to there being no precise and specific antiviral medicine for this emerging illness, MSCs were applied in several critically ill COVID-19 patients, contributing to the decline of plasma C reactive protein (CRP), aspartic aminotransferase, creatine kinase activity, and myoglobin. Furthermore, combined with symptomatic and supportive treatment, chest CT manifestation and SARS-CoV-2 nucleic acid detection improved markedly or returned to negative after 2 wk. [78][79][80][81] Although the positive results mentioned earlier supported the administration of MSC-EVs as a safe and efficient alternative for critically ill COVID-19 patients, the un-rigorous experimental design and small sample size of these studies contributed to these unimpressive and suspect data. Especially, only one clinical trial randomly divided subjects into MSC-treatment group and standard treatment group, and no clinical trials have tested the MSC viability before infusion.
Recently, a prospective nonrandomized open-label cohort study was conducted to evaluate the safety and efficacy of allogeneic bone marrow MSC-derived exosomes (ExoFlo) in severe COVID19 patients who suffered from moderate to severe ARDS. 82 To this end, 24 patients were i.v. administrated 15 ml ExoFlo (single dose) that was added into 100 ml normal saline solution. In the 72 h of post-treatment, no adverse events were observed related to the administration. After a 2 wk observation, 17 patients (71%) recovered, 3 patients (13%) remained severely ill, and 4 patients (16%) died. The laboratory results of the patients have shown that the levels of CRP, ferritin, and D-dimer, and neutrophil count decreased, whereas the average CD3+, CD4+, and CD8+ lymphocyte count increased. However, the paper did not mention how the ExoFlo was developed and manufactured, and there appears to be no difference in mortality between this cohort and that of the global survey (Table 3). To date, even though several clinical trials are ongoing to determine the effects of MSC-EVs for COVID19, the International Society of Cell and Gene Therapy and ISEV strongly urge that the decision to apply of MSC-EVs for COVID should be taken carefully due to the limited preclinical and clinical data, and they also suggest any use of EVs should be carefully evaluated through rational clinical trial design. 83

CONCLUSION
The

DISCLOSURES
The authors declare no conflicts of interest.