The efficacy of extracellular vesicles for acute lung injury in preclinical animal models: a meta-analysis

Background With the increasing research on extracellular vesicles (EVs), EVs have received widespread attention as biodiagnostic markers and therapeutic agents for a variety of diseases. Stem cell-derived EVs have also been recognized as a new viable therapy for acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). To assess their efficacy, we conducted a meta-analysis of existing preclinical experimental animal models of EVs for ALI treatment. Methods The database was systematically interrogated for pertinent data encompassing the period from January 2010 to April 2022 concerning interventions involving extracellular vesicles (EVs) in animal models of acute lung injury (ALI). The lung injury score was selected as the primary outcome measure for statistical analysis. Meta-analyses were executed utilizing RevMan 5.3 and State15.1 software tools. Results The meta-analyses comprised 31 studies, exclusively involving animal models of acute lung injury (ALI), categorized into two cohorts based on the presence or absence of extracellular vesicle (EV) intervention. The statistical outcomes from these two study groups revealed a significant reduction in lung injury scores with the administration of stem and progenitor cell-derived EVs (SMD = -3.63, 95% CI [-4.97, -2.30], P < 0.05). Conversely, non-stem cell-derived EVs were associated with an elevation in lung injury scores (SMD = -4.34, 95% CI [3.04, 5.63], P < 0.05). EVs originating from stem and progenitor cells demonstrated mitigating effects on alveolar neutrophil infiltration, white blood cell counts, total cell counts in bronchoalveolar lavage fluid (BALF), lung wet-to-dry weight ratios (W/D), and total protein in BALF. Furthermore, pro-inflammatory mediators exhibited down-regulation, while anti-inflammatory mediators demonstrated up-regulation. Conversely, non-stem cell-derived EVs exacerbated lung injury. Conclusion In preclinical animal models of acute lung injury (ALI), the administration of extracellular vesicles (EVs) originating from stem and progenitor cells demonstrably enhances pulmonary function. This ameliorative effect is attributed to the mitigation of pulmonary vascular permeability and the modulation of immune homeostasis, collectively impeding the progression of inflammation. In stark contrast, the utilization of EVs derived from non-stem progenitor cells exacerbates the extent of lung injury. These findings substantiate the potential utility of EVs as a novel therapeutic avenue for addressing acute lung injury.


Introduction
ALI is a severe disease characterised by an excessive inflammatory response.ARDS is an acute inflammatory lung disease with more severe clinical manifestations than ALI, usually accompanied by increased alveolar permeability, causing severe alveolar oedema and further exacerbation of hypoxia, with high annual morbidity and mortality rates worldwide [1].A worldwide observational investigation carried out from 2016 to 2017 across 145 Pediatric Intensive Care Units (PICUs) in 27 nations delineated a morbidity rate of 17% and a mortality rate of 3.2% among 23,280 patients [2].Notwithstanding strides in therapeutic approaches, the absence of efficacious pharmaceutical interventions capable of substantially mitigating mortality and enhancing patient prognoses persists as a critical challenge [3,4].Currently, the diagnosis and treatment of a number of diseases, including stem cells and their related derivatives, have attracted the attention of researchers [5].For example, a study has shown that serum EV-derived ASS1 levels predicted the progression of HEV-ALF and were strongly correlated with the severity of patients with HEV infection.In addition, serum levels of exosome-derived CPS1 have been reported as a diagnostic and prognostic biomarker for patients with HEV-ALF [6,7].Among various cell therapies, mesenchymal stem cells (MSCs) have been considered as a potential therapy for ALI/ARDS, and over the past decades, MSCs have played an important role in modulating immunity, anti-inflammation, cancer, angiogenesis, and tissue repair [8].Numerous preclinical and medical studies have demonstrated the therapeutic possibilities of MSCs in ARDS [9][10][11][12][13][14][15][16].However, MSCs MSC therapies also have risks, such as tumour induction, and their safety is questionable [17].EVs are derived from cells, secreted by almost all types of cells, and play an important role in cellular communication [18][19][20].EVs include lipids, proteins, and nucleic acids [18].According to the different sources, sizes, and surface marks of EVs, they can be divided into apoptotic bodies (ABs), microvesicles (MVs), and exosomes.ABS is generally considered vesicles containing intracellular contents during apoptosis (1000-5000 nm) [21]; while MVs are thought to be secretory vesicles from the plasma membrane by the outward budding (100-1000 nm) [22].Exosomes are formed from the maturation of intraluminal vesicles as multivesicular bodies before their fusion with the plasma membrane for secretion (30-100 nm) [20][21][22].However, due to the overlapping sizes and lack of specific surface markers in this classification, the International Society for Extracellular Vesicles (ISEV) introduced new guidelines in 2018 to provide a more precise categorization of EVs.According to the updated guidelines, EVs are now divided into small EVs (Small extracellular vesicles,sEVs) and large EVs (Large extracellular vesicles,LEVs).sEVs refer to small vesicles originating from intracellular compartments such as endosomes, multivesicular bodies, or the endoplasmic reticulum, typically ranging in diameter from 30 to 150 nm, and they can be isolated using techniques like ultracentrifugation.On the other hand, LEVs are large vesicles derived from cell membrane budding or extracellular matrix, with diameters generally exceeding 200 nm, and they can be separated through filtration methods [23].This novel classification approach based on the different cellular origins of EVs enables a more accurate description of their distinct characteristics and functions.
We conducted a meta-analysis based on data collected from preclinical animal models of ALI to systematically assess the efficacy of EVs as a potential treatment.

Materials and methods
We use the PRISMA statement to conduct this metaanalysis [24].The study protocol was registered on the International Prospective Register of Systematic Reviews (PROSPERO): (CRD42022368159).

Study selection
Two investigators (ZXF and CZY) searched and screened the applicable literature independently and then assessed the titles and abstracts of every retrieved article to decide which required further assessment.Disagreements between the investigators were resolved with a discussion or adjudicated by another reviewer (ZMH).
The inclusion criteria were as follows: (1) any animal models with ALI; (2) any studies intervened with various cell-derived EVs; (3) negative control (treatment without EVs).The primary outcome was the lung injury score.Secondary outcomes included inflammatory factors IL-1β, IL-6 and TNF-α, anti-inflammatory factor IL-10, W/D ratio of the lung, total protein in BALF, white blood cell counts in BALF, and neutrophil counts in BALF.

Data extraction
The data were extracted with the aid of two unbiased reviewers (ZXF and CZY) in a standardized way.For discrepancies, a third reviewer (ZMH) extracted them again.
The following data were collected: first author, country or region, type of ALI model, species, treatment time, measurement time and EV cell origins, diameter, and dose.We extracted data from graphics based on Engauge Digitizer version 4.1 software [25,26].
To extract data from the study, we saved all relevant screenshots from the results as images and uploaded these images to the program.The first step was to determine what type of graph we were analyzing.Secondly, three known values were assigned to three points on the axis to calibrate it.Then, we directly clicked on each point on the graph to get its exact coordinates and used those coordinates to calculate its mean and standard deviation.

Quality assessment
Two independent authors (ZXF and ZMH) evaluated each study's methodological quality with a Collaborative Approach to Meta-Analysis and a Review of Animal Data from Experimental Studies (CAMARADES) 10-item checklist [27].(Table 2).

Statistical analysis
All statistical analyses were conducted using RevMan version 5.3 and State 15.1 statistical software.It was considered statistically significant when the P < 0.05 (two-tailed).Continuous outcomes were expressed as standardized mean differences (SMD) with a 95% confidence interval (95%CI).Using the I 2 statistic, heterogeneity was assessed among studies.An I 2 > 50% indicates significant heterogeneity [28].In order to inspect conceivable between-study heterogeneity and to discover different viable confounding factors, subgroup, sensitivity, and meta-regression analyses were carried out.The publication bias was detected through funnel plots and Egger's test.If publication bias was indicated, we recalculated pooled risk estimates by including those missing studies from the Trimfill method.

Search results and study characteristics
Figure 1 is the diagram of the literature search process; 31 studies conform to the inclusion criteria [14,.
The main animal models were mice and rats; in one study the animal model was a pig.ALI induced by intratracheal injection of lipopolysaccharide or E.coli was the most common method in these studies.At the same time, there are other research methods, such as intratracheal injection of Bleomycin, H1N1 virus, pseudomonas Aeruginosa, cecal ligation and perforation, and trauma.Different studies had different administration methods, intervention times, and intervention doses.In addition, there was a significant difference in the time point at which the results were assessed, with most studies completed within two days and a few lasted one week.All the included studies were peer-reviewed publications.All the experimental animals were randomly assigned to the intervention group and the control.The quality assessment of all studies is shown in Table 2.

Meta-analysis: ALI with EVs group versus ALI without EVs group (control group) Heterogeneity text
There were 14 articles in this study.After the Heterogeneity test, I 2 = 90% > 50%, and Q test P < 0.1, the articles were considered to have a strong heterogeneity.The reasons for heterogeneity should be investigated.Based on the data of this study, a subgroup analysis was carried out from the aspects of the origin of extracellular vesicles, the species of experimental animals, intervention time, administration methods, and different scoring system of lung injury score.For the overall 14 articles, random effects were selected for meta-analysis, and the results were as follows (Fig. 2).
The results of the meta-analysis showed that the lung injury score of the experimental group was 1.93 lower than that of the control group, and the degree was statistically significant (P < 0.05).

Sensitivity analysis
A sensitivity analysis of 14 articles was carried out, and the results were as follows (Fig. 3).As can be clearly seen from the above figure, none of the studies significantly caused heterogeneity, and the stability of the overall study was high.Further subgroup analysis was carried out.Fig. 2 Main outcome of the meta-analyses of the ALI with EVs group compared with the ALI without EVs control group.Main outcome is lung injury score.The size of each square represents the proportion of information given by each trial.Crossing with the vertical line suggests no diference between the two groups.ALI acute lung injury, IV inverse variance, CI confdence interval, df degree of freedom Fig. 3 Sensitivity analysis of Lung Injury Score 14 studies were included in the sensitivity analysis, and after excluding each study, the combined effects of the remaining studies were within 95% confidence interval of the total effect

Subgroup analysis
The 14 articles were divided into two groups according to the source of extracellular vesicles.The results of the meta-analysis were as follows (Fig. 4).Based on the subgroup analysis above, the heterogeneity between the two groups was extreme, reaching a high degree of heterogeneity.Among them, the efficacy of stem-progenitor cell extracellular vesicles was -3.63, which was significant (Z = 5.34, P < 0.05).This means that stem progenitor cell extracellular vesicles significantly reduce the severity of acute lung injury to a large extent.Secondly, there was no heterogeneity in the group of non-stem progenitor cell extracellular vesicles, and the efficacy amount was 4.34 (Z = 6.55,P < 0.05).These results suggest that the extracellular vesicles of non-stem progenitor cells significantly increase the severity of acute lung injury.However, that the different sources of extracellular vesicles are the cause of heterogeneity cannot be explained.
Then, the subgroup analysis was carried out from the aspects of experimental animal species, intervention time, intervention mode and different scoring system of lung injury score.The results are shown in Figs. 5, 6, 7,  and 8.Each subgroup shows a high degree of heterogeneity, and the combined results also show high heterogeneity.The results of subgroup analysis do not support that the type of experimental animals, intervention time, intervention mode and different scoring system of lung injury score are the causes of heterogeneity.Next, meta-regression was used to investigate the source of heterogeneity.

Multivariable meta-regression analysis
From the above table (Fig. 9), we can see that the independent variable "different sources of extracellular vesicles "and" the species of experimental animals" can significantly affect the efficacy (P < 0.05), while the other independent variables-the mode of administration, the Fig. 4 subgroup analysis The subgroup meta-analysis of lung injury score that compares different origins of extracellular vesicles.The analysis didn't detect any statistically significant difference among the stem-progenitor cell extracellular vesicles(1), non-stem progenitor cell extracellular vesicles (2) time of intervention and the different scoring system of the lung injury score-have no statistical effect.Overall, we believed that the different sources of extracellular vesicles and the species of experimental animals are the cause of greater heterogeneity.Var9 extracellular vesicles are primarily derived from stem and non-stem progenitor cells; var10 experimental animals are mainly divided into mice and rats; var11 administration is divided into intratracheal administration and intravenous administration; var12 intervention time is divided into whether the intervention time is more than 48 h or not; var13 different scoring system of the lung injury score is divided into the semi-quantitative grading system [58] and the lung injury scoring system [59].

Publication bias
The results were as follows (Fig. 10).From the figure, it can be seen clearly that the funnel diagram of this study is basically symmetrical, and the Egger bias test and the Begg bias test were carried out at the same time.The Egger test showed that there was no significant publication bias (P = 0.390 > 0.05).The Begg test results are consistent with the Egger test results (P = 0.08 > 0.05).Therefore, it can be said that there is no publication bias in the literature of this study.

Primary outcome
Lung injury score is considered the primary outcome.Secondary outcomes included IL-1β, IL-6, TNF-α, IL-10, W/D ratio, total protein in BALF, BALF total cell counts, white blood cell counts, and BALF neutrophil counts.
The results showed that compared with the negative control group, stem progenitor cell extracellular vesicle therapy could significantly reduce the lung injury score.The standardized mean difference (SMD) = -3.6395%CI [-4.97, 2.30], p < 0.05 I 2 = 86.6%,while the

Discussion
This meta-analysis of 31 studies provides a comprehensive summary of the impact of EVs on preclinical animal models of ALI.Analysis of the combined data suggests that the attenuation of the inflammatory response and the improvement in lung function depend on the type of EVs involved.
Previous meta-analyses have shown that MSCderived EVs reduce the inflammatory response of inflammatory cells, decrease lung permeability, decrease the activity of inflammatory mediators, and increase the activity of anti-inflammatory mediators in an animal model of ARDS, thereby attenuating lung injury and improving survival in an animal model of ARDS [60].However, previous studies have not considered the effects of other sources of EVs on ALI/ ARDS.In our meta-analysis, we incorporated extracellular vesicles (EVs) from diverse cellular origins to investigate their impact on lung injury scores, cell, and inflammatory factors in bronchoalveolar lavage fluid (BALF).Our findings offer valuable insights for future studies.Multiple regression analyses revealed that the cell source independently influenced the efficacy of EVs.Additionally, the choice of experimental animal introduced notable heterogeneity, while no statistically significant differences were observed among modes of administration, duration of intervention, and lung injury scoring systems.This meta-analysis demonstrates the mitigating effects of stem progenitor cell-derived extracellular vesicles (EVs) on ALI/ARDS severity in an animal model.The Lung Injury Score (LIS), a pivotal pathophysiological metric in clinical trials for assessing lung injury severity, was significantly reduced by stem progenitor cell-derived EVs, indicative of a diminished overall severity of lung injury.In addition, our study found that stem cell-derived EVs Fig. 7 subgroup analysis The subgroup meta-analysis of lung injury score that compares different administrations.The analysis didn't detect any statistically significant difference among the intravenous administration (1), intratracheal administration(2) Fig. 8 subgroup analysis The subgroup meta-analysis of lung injury score that compares different scoring system.The analysis didn't detect any statistically significant difference among the semi-quantitative grading system(1), the lung injury scoring system(2) Fig. 9 multivariable meta-regression analysis P < 0.05 Considering this variable is the cause of heterogeneity.(var9:extracellular vesicles are primarily derived from stem progenitor cells and non-stem progenitor cells; var10: experimental animals are mainly divided into mice and rats; var11: administration is divided into intratracheal administration and intravenous administration; var12: intervention time is divided into whether the intervention time is more than 48 h or not; var13: different scoring system of the lung injury score is divided into the semi-quantitative grading system and the lung injury scoring system) down-regulated the levels of inflammatory factors such as IL-1 β, IL-6, and TNF-a, and up-regulated the levels of IL-10, a traditional anti-inflammatory cytokine.Modulation of the balance of pro-inflammatory and anti-inflammatory cytokines by stem cell-derived EVs may be important for ameliorating lung injury and improving survival.In contrast, EVs from alveolar epithelial cells and macrophages exacerbate the extent of lung injury.The lung W/D ratio is a widely used index to assess pulmonary vascular permeability in animal experiments.In the present meta-analysis, the lung W/D ratio decreased, suggesting that stem cell-derived EVs increase lung water clearance.Also, our metaanalysis showed that neutrophils, leukocytes, and total protein in bronchoalveolar lavage fluid were reduced after intervention with stem cell EVs.This suggests that stem cell-derived EV therapy reduces the effects of lung tissue infection, vascular permeability and lung tissue damage.Numerous research have proven that RNAs carried by means of EVs are integral for their therapeutic characteristics [61,62], and the proteins contained in EVs are also additionally associated with various biological functions in the human body.EVs are membrane-bound vesicles launched by way of all cell types, which are necessary data carriers for controlling angiogenesis, extracellular matrix remodelling, gene expression, inflammation, cell proliferation, cell migration, and morphogenetic elements [63][64][65][66].
As surface molecules, EVs can target receptors, facilitating signal transduction via receptor-ligand interactions.They undergo internalization through endocytosis, phagocytosis, and fusion with target cell membranes.With a composition akin to normal cell membrane and surface proteins, EVs are readily taken up by target cells, delivering their contents to the cytoplasm and thereby modulating the physiological state of recipient cells.This characteristic raises optimism regarding their potential as the next generation of drug transporters [67][68][69][70].EV derived from stem cells reduces immunogenicity compared to stem cells and also reduces the dangers associated with cellular therapies such as cytokine release syndrome [63].Currently, there are two main therapeutic approaches to ALI/ARDS: supportive therapy and pharmacological interventions.Despite increased understanding of the pathophysiology of ARDS, the efficacy of preferred treatments such as lung-protective ventilation, prone positioning, and neuromuscular blockers is often limited [71].Currently, there are no successful pharmacological therapies for ARDS [72].T herefore, there is a need to study the effects of EVs on ALI/ARDS.
Extracellular vesicles are emerging as a promising therapeutic and diagnostic tool, with studies demonstrating their potential role in the treatment and diagnosis of digestive system diseases, cancer, and other areas of research [6,73,74].Additionally, a meta-analysis have shown that stem cell-derived EVs improve The size of each square represents the proportion of information given by each trial.Crossing with the vertical line suggests no diference between the two groups.ALI acute lung injury, IV inverse variance, CI confdence interval, df degree of freedom cardiac function and reduce infarct size in myocardial infarction animals [75].Another meta-analysis have found that MSC-EVs have therapeutic potential for acute and chronic liver diseases [76].Furthermore, several meta-analysis studies have indicated that EVs are involved in the treatment of acute kidney injury and osteoporosis [77,78].However, some studies have suggested that EVs may not only have a therapeutic effect in disease development but may also contribute to disease progression.A study found that the increase of peripheral blood miR-1298-5p in sepsis-associated ALI patients triggered lung inflammation by inhibiting the proliferation of human bronchial epithelial cells and inducing epithelial permeability changes [79].Another study revealed that EVs in the plasma of sepsis patients are rich in miR-210-3p, which can enhance various responses associated with sepsis-induced ALI by regulating autophagy and inflammation.These responses include macrophage inflammatory responses, bronchial epithelial cell apoptosis, and changes in lung microvascular endothelial cell permeability [80].
Recent studies have shown that there may be differences in the membrane lipid composition and capsule contents released by the same cells [81,82].In our meta-analysis, EVs of various sizes were included.the large heterogeneity among EVs is a major obstacle to understanding the composition and functional characteristics of the different secreted components.For further studies, the isolation, identification, and compositional analysis of EVs of different sizes are key determinants of ALI treatment.In addition, more efficient and safer methods of EVs preparation, isolation, characterisation and stockpiling skills are also important factors in determining whether EVs can be used on a large scale in the clinic.

Limitations
Firstly, the overall sample size we analysed was very small due to the small sample size of the preclinical trial.Secondly, extracting data from drawings using Engauge Digitizer software in the absence of raw data may have altered the raw data and thus affected the results.This is despite the fact that we used a method where data were extracted separately by multiple reviewers.Finally, due to the lack of large animal tests and routine clinical parameters (e.g., respiratory mechanics), small tests may miss important information that is not conducive to guiding clinical applications.

Conclusion
This meta-analysis demonstrated that stem cell-derived EV therapy could improve lung function and inflammatory response in preclinical ALI animal models, while non-stem cell-derived EVs aggravate lung injury.This result provides an essential clue for human clinical trials of EVs.

Fig. 1
Fig. 1 The diagram of the literature search process

Fig. 5
Fig.5subgroup analysis The subgroup meta-analysis of lung injury score that compares different species of animals.The analysis didn't detect any statistically significant difference among the SD rats(1), C57BL/6J mice(2)

Fig. 6
Fig. 6 subgroup analysis The subgroup meta-analysis of lung injury score that compares different intervention time.The analysis didn't detect any statistically significant difference among the intervention time less than 48 h (1), intervention time more than 48 h (2)

Fig. 13
Fig.13 Secondary outcomes of the meta-analyses of the ALI with EVs group compared with the ALI without EVs control group.Secondary outcomes is TNF-a.The size of each square represents the proportion of information given by each trial.Crossing with the vertical line suggests no diference between the two groups.IV inverse variance, CI confdence interval, df degree of freedom

Fig. 14
Fig.14 Secondary outcomes of the meta-analyses of the ALI with EVs group compared with the ALI without EVs control group.Secondary outcomes are total protein in BALF(A), W/D ratio(B).The size of each square represents the proportion of information given by each trial.Crossing with the vertical line suggests no diference between the two groups.ALI acute lung injury, IV inverse variance, CI confdence interval, df degree of freedom

Table 1
Abbreviations: ADMSC adipose derived mesenchymal stromal cells, BMSC bone marrow mesenchymal stromal cells, C control group, CLP caecal ligation and

Table 2
Abbreviation: 1,peer-reviewed journal; 2,temperature control; 3,animals were randomly allocated; 4,blind established model; 5, blinded outcome assessment; 6,use of anesthetic without significant intrinsic vascular protection activity; 7, appropriate animal model (diabetic, advanced age or hypertensive); 8,calculation of sample size; 9,statement of compliance with animal welfare regulations; 10,statement of potential conflict of interests