Tie YF, Liu H, Zhang T, Meng TW, Liang Q. Dual role and clinical application of extracellular vesicles in acute respiratory distress syndrome: Mechanism analysis and translational challenges. World J Stem Cells 2025; 17(9): 108657 [DOI: 10.4252/wjsc.v17.i9.108657]
Corresponding Author of This Article
Qun Liang, MD, PhD, Chief Physician, Full Professor, Professor, Department of Critical Care Medicine, First Affiliated Hospital of Heilongjiang University of Chinese Medicine, No. 24 Heping Road, Xiangfang District, Harbin 150000, Heilongjiang Province, China. liangqun1@sina.com
Research Domain of This Article
Cell Biology
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This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Yi-Fu Tie, Tong Zhang, Tian-Wei Meng, Heilongjiang University of Chinese Medicine, Harbin 150000, Heilongjiang Province, China
Yi-Fu Tie, Ordos Hospital of Traditional Chinese Medicine, Ordos 017000, Inner Mongolia Autonomous Region, China
Han Liu, Institute for Global Health, University College London, London WC1H 0AW, United Kingdom
Qun Liang, Department of Critical Care Medicine, First Affiliated Hospital of Heilongjiang University of Chinese Medicine, Harbin 150000, Heilongjiang Province, China
Author contributions: Tie YF, Liu H, Zhang T, Meng TW, and Liang Q contributed substantially to data interpretation and critically reviewed the manuscript; Tie YF wrote the manuscript; Liu H, Zhang T, and Meng TW revised the manuscript and validated its key intellectual elements; Liang Q provided critical oversight of the study design. All authors have read and approved the final version.
Supported by National Natural Science Foundation of China, No. 82374400; and Heilongjiang Province “Double First-Class” New Round of Construction Disciplines Collaborative Innovation Achievements Construction Project, No. LJGXCG2022-097.
Conflict-of-interest statement: All the authors report no relevant conflicts of interest for this article.
Open Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: https://creativecommons.org/Licenses/by-nc/4.0/
Corresponding author: Qun Liang, MD, PhD, Chief Physician, Full Professor, Professor, Department of Critical Care Medicine, First Affiliated Hospital of Heilongjiang University of Chinese Medicine, No. 24 Heping Road, Xiangfang District, Harbin 150000, Heilongjiang Province, China. liangqun1@sina.com
Received: April 21, 2025 Revised: May 14, 2025 Accepted: August 21, 2025 Published online: September 26, 2025 Processing time: 157 Days and 17.2 Hours
Abstract
Acute respiratory distress syndrome (ARDS) is a severe and life-threatening manifestation of acute lung injury, characterized by widespread pulmonary inflammation and edema, ultimately resulting in acute respiratory failure. Despite advancements in mechanical ventilation and lung-protective strategies, targeted therapies aimed at modulating dysregulated inflammation and promoting tissue repair remain elusive. Extracellular vesicles (EVs), critical mediators of intercellular communication, have emerged as a promising research focus due to their dual regulatory roles in ARDS pathogenesis. Pro-inflammatory EVs, derived from pathogens or injury-stressed cells, exacerbate alveolar macrophage activation and increase endothelial permeability, thereby aggravating pulmonary damage. In contrast, anti-inflammatory EVs originating from mesenchymal stem cells facilitate alveolar barrier restoration and tissue repair by delivering reparative molecular cargo. This review systematically evaluates the dualistic functions of EVs in ARDS from three key perspectives: Molecular mechanisms, clinical translation, and technical challenges. We further discuss the complexities associated with EV heterogeneity, pathogen interactions, and standardization in EV production. Additionally, we propose future directions that integrate engineered EV modifications and multi-omics approaches to address current therapeutic limitations and enhance ARDS management.
Core Tip: Acute respiratory distress syndrome, a critical illness with a mortality of over 35%, is driven by dysregulated pulmonary inflammation and alveolar injury. Extracellular vesicles (EVs) demonstrate dual regulatory functions - pathogen-derived pro-inflammatory EVs exacerbate macrophage-driven injury, while mesenchymal stem cell EVs suppress inflammation and enhance tissue repair. Current research focuses on EV heterogeneity, spatiotemporal functional dynamics, and clinical translation barriers in therapeutic development. This synthesis integrates mechanistic insights, biomarker advancements, and engineered EV strategies, emphasizing standardization challenges and biosafety considerations. Emerging interdisciplinary approaches, combining multi-omics profiling, nanotechnology, and machine learning, aim to refine EV-based diagnostics and therapeutics, offering transformative potential for acute respiratory distress syndrome management.
Citation: Tie YF, Liu H, Zhang T, Meng TW, Liang Q. Dual role and clinical application of extracellular vesicles in acute respiratory distress syndrome: Mechanism analysis and translational challenges. World J Stem Cells 2025; 17(9): 108657
Acute respiratory distress syndrome (ARDS) arises from diverse direct or indirect insults, including infection, trauma, or aspiration, which compromise the integrity of the alveolar-capillary barrier, resulting in non-cardiogenic pulmonary edema, neutrophilic infiltration, and excessive pro-inflammatory cytokine release. Clinically, ARDS is characterized by refractory hypoxemia and respiratory failure[1]. Although the 2012 Berlin Definition refined ARDS diagnostic criteria[2,3], treatment strategies remain predominantly supportive, emphasizing mechanical ventilation and prone positioning rather than targeted therapies that directly modulate inflammatory pathways or promote tissue regeneration. The RECOVERY-RS trial (2021) demonstrated only a modest 6.5% reduction in 28-day mortality in moderate-to-severe ARDS patients undergoing prone positioning[4], thus, highlighting the limitations of current management approaches. The persistently high mortality rate (35%-46%) reflects the intricate pathophysiology of ARDS, which involves a complex interplay of systemic inflammatory response syndrome, neutrophil activation, dysregulated inflammatory mediator release, and multi-organ dysfunction syndrome[5,6].
Recent advances underscore the pivotal role of extracellular vesicles (EVs) as dual regulators in both infectious and non-infectious ARDS[7], offering novel insights into pathophysiology and potential therapeutic interventions[8,9]. EVs are membrane-bound nanoparticles (30-1000 nm) that mediate cellular communication through the transfer of proteins, microRNAs (miRNAs), mRNAs, and lipids. Biomedical research has increasingly focused on leveraging EVs as “smart” drug delivery systems due to their superior targeting capabilities, biocompatibility, and favorable pharmacokinetic profiles compared to synthetic nanoparticles[10,11] (Figure 1). Clinical studies have revealed that plasma-derived exosomes from patients with sepsis-induced ARDS exhibit pro-inflammatory, pro-coagulant, and endothelial permeability-enhancing properties, underscoring their potential as disease biomarkers[9,12]. However, the functionality of EVs is highly context-dependent: Host-derived EVs carrying miR-210d-3p have been shown to drive macrophage M1 polarization and promote alveolar epithelial injury[13], whereas mesenchymal stem cell (MSC)-derived EVs (MSC-EVs), enriched with angiopoietin-1 (Ang-1) mRNA and miR-146a, exert anti-inflammatory effects and enhance endothelial repair[14]. Pathogen-derived EVs, particularly those from Gram-negative bacteria, carry virulence factors such as lipopolysaccharide (LPS), which can exacerbate pulmonary edema through toll-like receptor 4 (TLR4)-mediated signaling. Conversely, these EVs may also contribute to protective immunity by facilitating antigen presentation[15]. The delicate balance between these opposing functions remains poorly understood, presenting a significant challenge for therapeutic EV design.
Figure 1 Biogenetic mechanism of extracellular vesicles.
The diagram depicts cellular processes including endocytosis, vesicle trafficking, and lysosomal degradation in donor cells. Recipient cells internalize exosomes via endocytosis or micropinocytosis, while phagocytosis mediates clearance of apoptotic bodies or apoptotic cells. ER: Endoplasmic reticulum.
This review aims to address the following fundamental questions in EV research related to ARDS: How do EVs regulate macrophage polarization and endothelial barrier integrity in a spatiotemporal manner? How can EV heterogeneity be addressed to facilitate biomarker discovery and therapeutic applications? Are existing EV isolation and in vivo tracking methodologies sufficient for clinical translation? By critically analyzing the current evidence, we explore the dual potential of EVs as both biomarkers and therapeutic agents while addressing key challenges associated with EV heterogeneity, standardization, and translational bottlenecks. In addition, we provide novel perspectives on harnessing EVs for ARDS diagnosis and management.
PRO-INFLAMMATORY, ANTI-INFLAMMATORY MECHANISMS, AND THE SPATIOTEMPORAL DYNAMICS AND MOLECULAR BASIS OF PHENOTYPIC TRANSITION OF EVS
To systematically elucidate the dualistic roles of EVs in ARDS pathogenesis, we present a tripartite analysis encompassing pro-inflammatory mechanisms, anti-inflammatory networks, and spatiotemporal dynamics (Figure 2, Table 1). Figure 2 illustrates the opposing roles of EV subtypes in modulating alveolar inflammation and repair, while Table 1 provides a comprehensive summary of molecular drivers, mechanistic pathways, and clinical correlations. This integrated framework establishes a foundation for understanding the dynamic interplay between EV-mediated injury and regeneration across distinct disease stages.
Alveolar epithelial cell-derived EVs: MiR-155, highly expressed in alveolar macrophages and pulmonary epithelial cells, plays a crucial role in amplifying inflammatory responses during acute lung injury (ALI) by suppressing cytokine signaling suppressor 1, a key negative regulator of inflammation[16]. In a hyperoxia-induced ALI murine model, alveolar macrophages exposed to EVs derived from lung epithelial cells exhibit increased secretion of pro-inflammatory cytokines, including interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and macrophage inflammatory protein-2, further exacerbating lung injury[17] (Figure 2A). Host-derived EVs also contribute to the polarization of macrophages toward a pro-inflammatory M1 phenotype via the transfer of miR-320a and miR-221, both of which are implicated in the regulation of innate immune responses[18]. Advanced single-cell RNA sequencing analyses have revealed that alveolar type 1 cell-derived EVs upregulate nucleotide-binding oligomerization domain-, leucine-rich repeat-, and pyrin domain- containing receptor 3 (NLRP3) inflammasome-associated genes in macrophages, thereby promoting the maturation and release of IL-1β and triggering pyroptotic cell death in alveolar epithelial cells. Notably, EVs isolated from bronchoalveolar lavage fluid (BALF) of Escherichia coli-challenged lungs have been shown to induce ALI when administered to naïve ventilated lungs via both intravenous and bronchial routes, demonstrating their potent inflammatory potential. Interestingly, hyaluronic acid administration has been observed to mitigate lung injury and inflammation, likely through its direct interaction with EVs, which may neutralize their pathogenic effects[19]. These findings highlight the complexity of EV-mediated inflammatory signaling and underscore the need for further research to delineate their precise roles in ARDS pathogenesis.
Neutrophil-derived EVs: Neutrophils are crucial mediators of ARDS pathogenesis, in part through the secretion of EVs that propagate inflammation. Under LPS stimulation, neutrophils release CD66b+ exosomes (50-100 nm in diameter), which suppress the expression of the autophagy-related gene autophagy related 5 in macrophages via miR-30d-5p delivery. This suppression impairs autophagosome formation and concurrently enhances nuclear factor-kappa B (NF-κB) pathway activation, thereby promoting a sustained inflammatory response[20]. Animal studies further demonstrate that intratracheal administration of neutrophil-derived EVs in LPS-induced ARDS models exacerbates pulmonary neutrophil infiltration and significantly elevates TNF-α and IL-6 Levels in BALF, contributing to disease severity[21]. Clinical cohort studies support these findings, revealing a significant negative correlation between BALF exosomal miR-30d-5p levels and PaO2/FiO2 ratios (r = -0.38, P = 0.03) in ARDS patients, suggesting its potential as a prognostic biomarker[22] (Figure 2B). However, inconsistencies in EV subpopulation composition due to variations in isolation methodologies may influence functional interpretations[23]. For instance, studies indicate that gradient filtration enriches bacterial outer membrane vesicles (OMVs) approximately twofold compared to conventional ultracentrifugation, which may inadvertently enhance observed pro-inflammatory effects[24]. Conversely, fluorescence-activated cell sorting-based isolation techniques have identified exosomes with strong CD63 and CD81 expression but relatively low CD9 and tumor susceptibility gene 101 Levels, highlighting potential discrepancies in defining vesicle subtypes[25]. Such variations underscore the necessity of adopting standardized isolation protocols, such as those outlined in the EV-TRACK guidelines, to improve reproducibility and ensure consistency in functional EV studies.
Bacterial OMVs: Bacterial OMVs, ranging from 20 to 250 nm in diameter, are released by Gram-negative bacteria such as Pseudomonas aeruginosa and play a crucial role in modulating host immune responses. These vesicles are enriched with virulence factors, including LPS and flagellin, which activate alveolar macrophages via dual TLR2/4 receptor signaling. This activation leads to a robust inflammatory response characterized by increased secretion of IL-1β, TNF-α, IL-6, and interferon-gamma (IFN-γ), subsequently promoting neutrophil recruitment to sites of lung injury[26] (Figure 2C). In murine models of Streptococcus pneumoniae-induced lung injury, OMV-treated lung tissues exhibit compromised alveolar epithelial barrier integrity, as evidenced by reduced expression of zonula occludens-1 tight junction proteins and decreased transendothelial electrical resistance in human pulmonary microvascular endothelial cells (ECs)[27]. Additionally, OMVs facilitate the transfer of β-lactamase to host cells via OprF outer membrane protein-mediated fusion, thereby promoting bacterial resistance to β-lactam antibiotics[28]. While OMVs are primarily implicated in exacerbating inflammatory responses[29], emerging evidence suggests that they may also have potential as vaccine vectors[30,31]. Studies have demonstrated that Pseudomonas aeruginosa OMVs carrying specific bacterial antigens can induce protective immunoglobulin G antibody production, leading to a 50.9% enhancement in bacterial clearance rates in animal models (P < 0.001)[32,33]. However, the strong immunogenicity of OMV-associated LPS remains a significant concern, as it may provoke hyperinflammatory responses. Notably, intravenous administration of OMVs to healthy mice has been shown to elevate serum levels of IL-6, IL-8, and TNF-α, highlighting the need for careful evaluation of OMV dosage and administration routes in clinical applications[34]. These findings underscore the dual nature of OMVs in ARDS pathophysiology and emphasize the importance of further investigation into their therapeutic potential.
Virus-packaged EVs: EVs secreted by virus-infected host cells play a pivotal role in viral pathogenesis and immune modulation. Exosomes (80-120 nm in diameter) secreted by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected pulmonary epithelial cells have been found to encapsulate viral RNA and spike proteins, enabling the infection of bystander cells via angiotensin-converting enzyme 2 (ACE2)-independent pathways. These exosomes downregulate ACE2 expression in alveolar epithelial cells, resulting in an accumulation of angiotensin II, increased pulmonary vascular permeability, and exacerbated lung injury[35] (Figure 2C). Clinical studies indicate that plasma-derived exosomes in coronavirus disease 2019-associated ARDS patients correlate with disease severity and elevated D-dimer levels, implicating their role in coagulopathy and suggesting their potential utility as biomarkers for disease progression[36]. However, concerns regarding the specificity of SARS-CoV-2 EV-based biomarkers remain unresolved. Cross-reactivity issues, akin to those observed with SARS-CoV-2 accessory proteins (e.g., antibodies targeting 3a or 7b proteins), may arise if EVs contain viral antigens, leading to false-positive diagnostic results[37]. To enhance diagnostic accuracy, future studies should adopt multi-marker detection strategies, such as dual detection of spike protein and ACE2, in conjunction with multi-platform validation techniques. These approaches will be critical in ensuring the reliable application of virus-packaged EVs as biomarkers in coronavirus disease 2019-related ARDS.
Functional networks of anti-inflammatory EVs
Repair mechanisms of MSC-EVs: MSC-EVs play a crucial role in ARDS by exerting both anti-inflammatory and tissue-regenerative effects. These vesicles are enriched with bioactive molecules that contribute to endothelial protection and immune modulation. One key mechanism involves MSC-EV-mediated delivery of Ang-1 mRNA, which activates the Tie2 receptor signaling pathway. This interaction suppresses endothelial apoptosis, enhances the expression of tight junction proteins, and consequently reduces alveolar-capillary permeability[38]. Additionally, MSC-EVs transport miR-146a, which targets the TNF receptor-associated factor 6 (TRAF6)/NF-κB signaling axis. This downregulation significantly decreases the secretion of pro-inflammatory cytokines such as IL-6 and TNF-α in alveolar macrophages while promoting their polarization toward an anti-inflammatory M2 phenotype[39] (Figure 2D). Notably, MSC-EVs also facilitate metabolic restoration in damaged ECs via mitochondrial transfer. Through direct membrane fusion, MSC-EVs deliver functional mitochondria to recipient cells, replenishing ATP levels, reducing reactive oxygen species accumulation, and enhancing alveolar epithelial repair and re-epithelialization[40,41].
Immunomodulatory role of EC-derived EVs: Under physiological conditions, EC-derived EVs (EC-EVs) contribute to immune homeostasis by regulating macrophage polarization through the miR-223/regulator of G-protein signaling 1 (RGS-1) axis. Clinical studies have revealed that ARDS survivors exhibit significantly higher plasma levels of EC-EV-associated miR-223 compared to non-survivors[42]. These levels positively correlate with an increased proportion of CD45+ M2 macrophages, suggesting a protective role for EC-EVs in modulating immune responses. Mechanistically, miR-223 suppresses RGS-1, thereby alleviating its inhibitory effects on the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway. This cascade enhances signal transducer and activator of transcription 6 (STAT6) phosphorylation, driving M2 macrophage polarization and facilitating the resolution of inflammation[42] (Figure 2E).
Apoptotic body clearance and inflammation resolution: Apoptotic bodies (Apo-EVs) released by dying cells serve as critical mediators of immune regulation, promoting the resolution of secondary inflammation through “silent clearance” mechanisms[43]. When macrophage c-mer tyrosine kinase receptors recognize phosphatidylserine on Apo-EVs, they activate the Rac1/Cdc42 signaling pathway. This interaction enhances the phagocytosis of apoptotic vesicles and promotes the secretion of anti-inflammatory cytokines IL-10 and transforming growth factor beta (TGF-β). These processes collectively inhibit NLRP3 inflammasome activation and support the expansion of regulatory T cells, further dampening inflammation[41,44] (Figure 2F). Therapeutic strategies leveraging Apo-EV-mimetic particles have shown promise in preclinical models. For instance, intratracheal administration of these particles has been demonstrated to significantly reduce inflammatory cytokine levels in murine BALF[45]. Interestingly, monocytes exhibit a preferential ability to internalize Apo-EVs via efferocytosis, even in inflammatory environments containing neutrophil extracellular traps[46]. However, neutrophil extracellular trap-associated elastase and citrullinated histones have been implicated in inducing monocyte extracellular trap formation in a proteolysis-dependent manner, which may counteract the anti-inflammatory effects of Apo-EVs[46,47]. Further investigations have uncovered that MSC-derived Apo-EVs suppress stimulator of interferon genes signaling through membrane-bound ENPP1-mediated hydrolysis of intra- and extracellular cyclic GMP-AMP, effectively reducing excessive immune activation[47]. Moreover, blockade of calreticulin has been shown to impair Apo-EV phagocytosis and diminish their anti-inflammatory efficacy, suggesting that enhancing Apo-EV clearance could be a viable therapeutic approach[48]. These findings highlight the potential of engineered Apo-EVs as novel immunotherapeutic agents for chronic inflammatory diseases, although their clinical translation requires further research into their heterogeneity and interactions within complex inflammatory microenvironments (Table 1).
Spatiotemporal dynamics and molecular basis of phenotypic transition
The functional roles of EVs in ARDS are not static but instead exhibit dynamic spatiotemporal variations[49]. These variations are observed across two primary dimensions: Temporal evolution, reflecting stage-dependent changes in EV composition and function, and spatial distribution, representing the heterogeneous localization of EVs within distinct pulmonary microenvironments. The molecular basis of these phenotypic transitions is driven by time-dependent modifications in EV cargo and their differential regulation of target cell signaling pathways.
Temporal dimension (stage-dependent evolution of EV function): During the acute phase of ARDS, pro-inflammatory EVs dominate the inflammatory response. EVs secreted by neutrophils and damaged alveolar epithelial cells intensify lung injury by delivering inflammatory mediators. Under LPS stimulation, neutrophils release CD66b+ exosomes containing miR-30d-5p, which suppress macrophage autophagy and activate the NF-κB pathway, leading to increased IL-6 and TNF-α levels[20,21]. Hyperoxia-exposed alveolar type 1 cells release microvesicles enriched with high-mobility group box 1, triggering macrophage pyroptosis through the TLR4/MyD88-NLRP3 axis, further elevating IL-1β concentrations in BALF. Additionally, bacterial OMVs containing LPS and flagellin activate TLR2/4 signaling, disrupting endothelial tight junctions and decreasing transendothelial electrical resistance, thereby exacerbating vascular permeability[26,27].
In the subacute phase, EVs derived from MSCs and ECs promote tissue repair by delivering reparative molecules. Ang-1 mRNA enhances endothelial barrier integrity via Tie2 receptor activation, while miR-146a downregulates the TRAF6/NF-κB pathway, facilitating macrophage polarization toward an anti-inflammatory M2 phenotype[38,39]. MiR-223 further modulates immune balance by targeting RGS-1, thereby relieving PI3K/Akt pathway inhibition, increasing STAT6 phosphorylation, and promoting M2 macrophage differentiation[42]. Additionally, macrophage c-mer tyrosine kinase-mediated efferocytosis of Apo-EVs inhibits NLRP3 inflammasome activation, reduces IL-1β levels, and expands regulatory T cell populations, fostering inflammation resolution[41,44].
In the chronic phase, EVs contribute to fibrotic progression as persistent inflammation induces a transition toward pro-fibrotic EV phenotypes. TGF-β1 and miR-21 drive myofibroblast differentiation via the Smad3 and Wnt/β-catenin pathways, promoting excessive collagen deposition and fibrosis[7]. Chronic hypoxia-mediated downregulation of miR-146a, coupled with upregulation of miR-155 in MSC-EVs, further accelerates epithelial-mesenchymal transition, reinforcing a fibrotic microenvironment[39].
Spatial dimension (microenvironment-specific distribution and function of EVs): The biological activity of EVs is tightly regulated by their anatomical localization, reflecting regional heterogeneity within the lung microenvironment. Within the alveolar lumen, pathogen-derived EVs, such as Pseudomonas aeruginosa OMVs, compete with host-derived EVs, triggering macrophage activation through TLR2/4 signaling and driving neutrophil recruitment. Conversely, MSC-EVs demonstrate a targeted accumulation at injury sites via C-X-C chemokine receptor type 4 (CXCR4)-mediated chemotaxis, where miR-223 expression at air-liquid interfaces facilitates inflammation suppression[26]. In the pulmonary interstitium, EVs contribute to fibrosis by influencing local immune and stromal cell interactions. Interstitial EVs enriched with Ang-1 mRNA and miR-146a dampen the TRAF6/NF-κB pathway in alveolar macrophages, reducing pro-inflammatory cytokine secretion and attenuating fibrosis[39]. At the vascular endothelium, EVs play a crucial role in preserving barrier function by upregulating tight junction proteins through Tie2 receptor activation[38]. In high-shear vascular regions, EVs are efficiently internalized by macrophages, enabling localized repair mechanisms to restore endothelial integrity[41].
The dynamic transition of EV function in ARDS is governed by a multilayered regulatory network. In the acute phase, transcriptional and epigenetic modifications - primarily driven by NF-κB and STAT1 - upregulate pro-inflammatory miRNA synthesis. In contrast, during the subacute phase, STAT6 and peroxisome proliferator-activated receptor gamma signaling promote the expression of anti-inflammatory miRNAs[38]. Environmental stimuli further dictate EV cargo composition and function. Hypoxia, for instance, induces the expression of miR-210 via hypoxia-inducible factor-1 alpha, thereby promoting fibroblast activation. Similarly, endoplasmic reticulum stress enhances the presence of glucose-regulated protein 78/binding immunoglobulin protein within EVs, which in turn facilitates apoptosis in target cells through protein kinase RNA-like endoplasmic reticulum kinase-eukaryotic initiation factor 2α signaling[38]. Intercellular communication networks further reveal that M1 macrophage-derived EVs impair MSC-EV-mediated repair via miR-155, whereas IL-10 secretion by M2 macrophages counteracts this inhibitory effect, establishing a feedback loop that influences the resolution of inflammation. Additionally, gut-derived OMVs have been shown to modulate pulmonary inflammation by upregulating TLR4 expression in lung-derived EVs, thereby promoting systemic inflammatory crosstalk across distant organ systems[26]. These findings highlight the intricate regulatory landscape governing EV-mediated effects in ARDS, underscoring the need for targeted therapeutic interventions aimed at harnessing EVs for disease modulation at different stages of ARDS progression.
CLINICAL CHALLENGES AND FUTURE DIRECTIONS IN EV TRANSLATION
The spatiotemporal heterogeneity of EVs presents significant clinical implications. Temporal enrichment of Ang-1 mRNA in repair-phase MSC-EVs provides a molecular basis for stage-specific therapeutic interventions. However, several challenges remain, including the functional diversity of EV subsets (pro-inflammatory vs anti-inflammatory), competitive interactions between host-derived and pathogen-derived EVs (e.g., TLR4 antagonism between OMVs and MSC-EVs), and technical limitations in EV isolation and characterization, all of which pose critical barriers to clinical translation.
Diagnostic biomarker development and engineering strategies
The dual role of EVs as both biomarkers and therapeutic agents is transforming ARDS management. In diagnostics, multi-omic EV signatures have demonstrated superior performance over traditional biomarkers. The combined detection of plasma exosomal miR-223 and IL-8 [sensitivity: 84%, area under the curve (AUC) = 0.88] enhances diagnostic specificity, distinguishing infectious from non-infectious ARDS by linking macrophage polarization to neutrophil infiltration[50,51]. Additionally, pathogen-derived EVs (e.g., LPS-enriched bacterial OMVs or viral envelope proteins) offer novel avenues for infection typing. For instance, the detection of OmpA in BALF from Gram-negative ARDS patients (AUC = 0.91) highlights the potential of pathogen-specific EV biomarkers[52,53]. Despite these advances, key challenges persist, particularly in balancing technical sensitivity with biological complexity. While microfluidic chips integrated with surface-enhanced Raman spectroscopy enable single-EV multiparametric analysis (sensitivity: 103 particles/mL), molecular overlap between host and pathogen-derived EVs (e.g., SARS-CoV-2 spike+ EVs vs host pro-inflammatory EVs) may confound diagnostic accuracy[54,55]. Future research should focus on in situ capture methodologies combined with nucleic acid sequencing to improve biomarker resolution in complex biological samples.
Although significant progress has been made in engineering therapeutic EVs, clinical translation remains hindered by both biological and technical barriers. Targeted modifications - such as CXCR4 chemokine receptor expression or CD47-mediated “immune stealth” strategies - have been shown to increase lung tissue accumulation by 3.5-fold and extend circulatory half-life to 24 hours[56,57]. Additionally, co-delivery of therapeutic cargo, such as miR-146a with sirtuin 1 mRNA, has demonstrated synergistic suppression of inflammatory pathways, leading to a 51% reduction in pulmonary fibrosis area[58]. However, a fundamental paradox remains: Engineering modifications can compromise the natural functionality of EVs. For instance, CXCR4 overexpression has been shown to reduce CD81 membrane protein levels by 40%, potentially impairing EV fusion with target cell membranes[56]. Similarly, artificial EV constructs (e.g., lipidosome-cell membrane hybrids) exhibit batch variability (< 8%), raising concerns regarding long-term stability and immunogenicity[59]. To overcome these challenges, future EV engineering efforts should prioritize the development of “dynamic-responsive” systems, such as inflammation-specific enzyme-activated targeting ligands, to maximize therapeutic efficacy while preserving the innate biological properties of EVs.
Translational bottlenecks and development
While EVs demonstrate significant potential, their successful clinical translation remains hindered by multiple challenges spanning technical, standardization, biological, and clinical domains. Addressing these barriers is crucial for advancing EV-based therapeutics from preclinical research to clinical application.
Heterogeneity of EVs: The variability in EV composition arises from donor-specific factors such as age, genetic background, and pathological status, as well as inconsistencies in isolation techniques. The therapeutic efficacy of MSC-EVs, for example, is significantly influenced by donor age. MSC-EVs obtained from younger donors (< 30 years) exhibit 2.1-fold higher levels of miR-146a compared to those from elderly donors (> 60 years) (P = 0.007), resulting in a 40% greater anti-inflammatory effect[58]. Furthermore, the choice of isolation methodology impacts EV purity and functionality. While ultracentrifugation remains a widely used technique, it frequently results in contamination with Apo-EVs and protein aggregates. In contrast, size-exclusion chromatography enhances EV purity but at the expense of lower functional exosome recovery (35% efficiency).
Lack of standardization: The absence of standardized protocols for EV isolation, characterization, and quality control continues to hinder data reproducibility and clinical translation. Variability in exosome marker detection across laboratories has been reported, with CD9 positivity rates fluctuating by up to 50% due to differences in detection techniques[51]. The International Society for Extracellular Vesicles MISEV2023 guidelines recommend validation of at least three transmembrane protein markers; however, clinical-grade EV preparations necessitate even more rigorous quality control measures, including miRNA profiling and membrane integrity assessment. Furthermore, regulatory ambiguities regarding the classification of EV-based therapeutics have contributed to limited industrial investment in large-scale EV production.
Targeting inefficiency: Following systemic administration, only 0.5%-5% of EVs reach injured lung tissues, with the majority being sequestered in the liver and spleen[59]. Strategies to enhance targeted EV delivery include CXCR4 chemokine receptor engineering, which has been shown to increase MSC-EV accumulation in lung tissue by 3.5-fold[56]. However, the effectiveness of such modifications is often constrained by fluctuating stromal cell derived factor-1alpha concentrations within inflamed tissues. Additionally, engineering modifications can disrupt native EV membrane integrity. For instance, CXCR4 overexpression has been reported to reduce CD81 expression by 40% (P = 0.002), impairing membrane fusion efficiency with target cells. Future strategies should focus on developing dynamic targeting approaches, such as inflammation-specific enzyme-activated “stealth-exposure” modifications, to optimize EV delivery[60].
Long-term safety concerns: Although EV-based therapies hold great promise, concerns regarding long-term safety remain unresolved. MSC-EVs enriched with miR-21 have been shown to activate the PI3K/Akt pathway through phosphatase and tensin homolog suppression, leading to a 1.8-fold increase in lung cancer incidence in animal models (P = 0.03)[57]. Additionally, allogeneic EVs express major histocompatibility complex class I (MHC-I) molecules on their surface, which can activate CD8+ T cells, elevating serum IFN-γ levels by 2.3-fold (P < 0.001) and restricting repeated dosing[61]. To mitigate these risks, pre-therapeutic miRNA screening strategies that exclude oncogenic miRNAs (e.g., miR-21 and miR-155) should be implemented. While autologous EV administration may minimize immunogenicity, its prolonged production timeline (> 2 weeks) is impractical for urgent ARDS treatment. The development of universal, gene-edited EV variants - such as MHC-knockout EVs - represents a promising avenue for overcoming these limitations[58].
Challenges and development of EVs in ARDS
The advancement of EVs in ARDS presents significant challenges.
EV heterogeneity and its impact on mechanistic studies: The intrinsic heterogeneity of EVs poses significant challenges in mechanistic studies and therapeutic applications. Current isolation techniques, such as ultracentrifugation, fail to distinguish exosomes (CD63+/CD81+) from microvesicles (Annexin V+), resulting in functional ambiguity. Studies indicate that EV samples obtained via ultracentrifugation exhibit a 65% overlap in CD9 and CD63 expression profiles, complicating the identification of distinct EV subpopulations involved in alveolar macrophage polarization[62]. While density gradient centrifugation offers improved subpopulation isolation, its time-consuming nature and low recovery rate (< 30%) limit its clinical applicability. Furthermore, clinical samples such as BALF and plasma contain mixed EVs derived from both host cells (e.g., alveolar epithelium, endothelium) and pathogens (e.g., bacterial OMVs, viral envelope EVs). In sepsis-associated ARDS, host-derived EVs (CD63+/CD9+) co-exist with Pseudomonas aeruginosa OMVs (LPS+/OmpA+) in patient plasma, collectively activating inflammation via the TLR4/MyD88 signaling pathway. However, isolating individual contributions to disease progression remains a major challenge[52].
Biosafety concerns in clinical translation: The potential oncogenic and immunogenic risks of EVs must be carefully addressed in clinical translation. MSC-EVs have been shown to carry pro-tumorigenic miRNAs, such as miR-21, which activates the PI3K/Akt pathway by suppressing phosphatase and tensin homolog. Prolonged MSC-EV administration (> 4 weeks) has been associated with a 1.8-fold increase in lung cancer incidence in animal models (P = 0.03), emphasizing the need for pre-therapeutic miRNA profiling using next-generation sequencing to exclude high-risk molecules[57]. Additionally, allogeneic EVs may trigger adaptive immune responses due to MHC-I molecule expression on their surfaces, leading to CD8+ T-cell activation and a 2.3-fold increase in serum IFN-γ levels (P < 0.001), thereby limiting the feasibility of repeated dosing in clinical trials[63]. While autologous EVs mitigate immune rejection, their lengthy production time (> 2 weeks) renders them impractical for urgent ARDS treatment.
Innovative isolation and characterization techniques: To overcome these challenges, novel technological advancements are required. Microfluidic sorting based on surface markers (CD63/CD81) and particle size has demonstrated superior EV subpopulation isolation (> 90% purity) while preserving functional molecular integrity, achieving miR-146a recovery rates exceeding 85%. Wang et al[62] developed an integrated microfluidic system capable of simultaneously isolating EVs, quantifying miRNAs, and detecting protein markers, with a processing capacity of 1000 samples per hour - far surpassing conventional methods. Additionally, single-EV analysis using atomic force microscopy combined with Raman spectroscopy has facilitated spatial mapping of EV membrane components, miRNAs, and proteins[62]. Zhou et al[51] reported that pro-inflammatory EVs exhibit 1.6-fold higher membrane rigidity than anti-inflammatory EVs (P = 0.004), providing a physical criterion for functional sorting.
Advanced therapeutic engineering and targeting strategies: The integration of photothermal-responsive materials with EVs represents a promising avenue for controlled drug delivery. For instance, Han et al[60] engineered near-infrared-activated EV “robots” that release IL-10 at inflamed sites upon localized heating (42 °C), reducing lung IL-6 Levels by 68% with minimal (< 5%) off-target release. Furthermore, alveolar-capillary barrier chips that replicate ARDS pathology (e.g., hypoxia, hyperpermeability) offer high-throughput platforms for screening therapeutic EVs[60]. Guo et al[56] demonstrated that CXCR4-modified EVs exhibited a 3.5-fold increase in transmembrane efficiency compared to unmodified EVs (P < 0.01), optimizing dosing to 1 × 109 particles/cm2.
Spatial mapping and machine learning integration: Emerging spatial transcriptomics approaches provide deeper insights into EV interactions within lung tissues. Using 10× Genomics Visium, Sun et al[59] demonstrated that TGF-β1 concentrations at fibrotic lesion margins were 1.8-fold higher than in central regions (P = 0.007), positioning EVs as “sentinels” of fibrotic progression. Machine learning-based analytical platforms further enhance EV-based precision medicine[59]. Li et al[58] developed an artificial intelligence-driven predictive model (AUC = 0.89) integrating EV-miRNA profiles, clinical parameters, and radiomic features to forecast patient responses to MSC-EV therapy within 72 hours, enabling personalized treatment stratification.
In summary, the application of EVs in ARDS diagnostics and therapeutics has evolved from conceptual validation to the complex stage of clinical translation. However, several challenges remain, including the need to balance high-purity EV isolation with cost-effective scalability for urgent care applications, mitigating risks associated with engineering modifications (e.g., unintended disruption of native signaling pathways), and implementing stringent quality control measures that may delay regulatory approval. Future advancements should focus on developing organ-on-chip models for evaluating EVs under ARDS-mimetic pathological conditions, establishing global EV molecular databases for regulatory standardization, and exploring autologous EV biobanks or universal gene-edited EVs (e.g., MHC-knockout variants) to enhance safety and accessibility. By integrating technological innovation with regulatory frameworks, EV-based therapies have the potential to transition from experimental modalities to transformative clinical solutions, reshaping the future landscape of ARDS management.
CONCLUSION
EVs play a dual role in ARDS, acting both as “disease amplifiers” that exacerbate inflammation and as “repair regulators” that facilitate tissue restoration. This study highlights that in the early stages of ARDS, pro-inflammatory EVs derived from neutrophils and pathogens contribute to alveolar-capillary barrier dysfunction by delivering miR-30d-5p, LPS, and other inflammatory mediators, which activate NF-κB and TLR4/5 signaling pathways. In contrast, during the reparative phase, anti-inflammatory EVs secreted by MSCs and ECs drive macrophage M2 polarization and promote endothelial barrier repair through the controlled release of reparative cargo, such as Ang-1 mRNA and miR-146a. Single-cell transcriptomic and spatial multi-omic analyses further reveal that EV functional heterogeneity is influenced not only by cellular origin but also by dynamic microenvironmental cues, including hypoxic gradients and mechanical stresses. These insights establish a crucial theoretical foundation for the development of stage-specific, precision-targeted EV therapies in ARDS.
Despite providing a comprehensive delineation of the EV regulatory network, this study acknowledges key limitations. The mechanisms underlying the gut-lung axis and the role of intestinal EVs in modulating pulmonary inflammation remain poorly understood. Additionally, current animal models fail to fully replicate the heterogeneity of human ARDS, particularly in terms of age-related and immune microenvironmental differences. Furthermore, long-term safety concerns associated with engineered EVs - such as the potential disruption of native homing pathways due to CXCR4 overexpression - have yet to be validated in clinical settings.
Overcoming these challenges requires a multidisciplinary approach. Future research should focus on technological innovations, including the development of organ-on-chip platforms for high-throughput EV screening and the integration of artificial intelligence to optimize spatiotemporal delivery strategies. From a translational perspective, establishing international EV molecular databases in alignment with ISO/TC 276 standards will be essential for harmonizing regulatory frameworks and ensuring data consistency across clinical studies. Clinically, the exploration of autologous EV biobanks and universal gene-edited EVs - such as MHC-knockout variants - could provide a balance between therapeutic safety and large-scale production feasibility. Through continued interdisciplinary collaboration, EV-based therapies have the potential to evolve from adjunctive treatments into targeted mechanistic interventions, ultimately addressing the clinical challenges of ARDS management and redefining therapeutic paradigms in critical care medicine.
Footnotes
Provenance and peer review: Unsolicited article; Externally peer reviewed.
Peer-review model: Single blind
Specialty type: Cell and tissue engineering
Country of origin: China
Peer-review report’s classification
Scientific Quality: Grade B, Grade B, Grade B, Grade B
Novelty: Grade B, Grade B, Grade B, Grade C
Creativity or Innovation: Grade B, Grade B, Grade B, Grade B
Scientific Significance: Grade B, Grade B, Grade B, Grade B
P-Reviewer: Ying GH, Associate Chief Physician, China; Yuan Z, PhD, Assistant Professor, China S-Editor: Wang JJ L-Editor: A P-Editor: Zhang XD
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