Tag: extracellular vesicles

Extracellular vesicle proteomics uncovers energy metabolism, complement system, and endoplasmic reticulum stress response dysregulation postexercise in males with myalgic encephalomyelitis/chronic fatigue syndrome

Abstract:

Background: Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a debilitating illness characterized by post-exertional malaise (PEM), a worsening of symptoms following exertion. The biological mechanisms underlying PEM remain unclear. Extracellular vesicles (EVs) play a key role in cell-cell communication and may provide insight into ME/CFS pathophysiology post-exertion. Emerging evidence suggests similarities between ME/CFS and Long COVID, including PEM and overlapping immune and metabolic dysfunctions, highlighting the need for deeper mechanistic understanding.

Methods: This study explores the EV proteome response to exercise in 10 males with ME/CFS and 12 well-matched sedentary male controls. Participants underwent a maximal cardiopulmonary exercise test, and plasma samples were collected at baseline, 15 min, and 24 h postexercise. EVs were isolated from plasma using size-exclusion chromatography and characterized with nanoparticle tracking analysis. EV protein abundance was quantified with untargeted proteomics (nanoLC-MS/MS). Comprehensive analyses included differential abundance, pathway enrichment, protein-protein interaction networks, and correlations between EV protein dynamics and clinical or exercise physiology data.

Results: ME/CFS patients exhibited many significantly altered EV proteomic responses compared with controls, including downregulation of TCA cycle-related proteins and upregulation of complement system proteins at 15 min postexercise. Changes in proteins involved in protein folding and the endoplasmic reticulum (ER) stress response during recovery were highly correlated with PEM severity, highlighting their potential as therapeutic targets. EV protein changes postexercise were also associated with disease severity and unrefreshing sleep. Correlations between EV protein levels and the exercise parameters VO₂ peak and ventilatory anaerobic threshold were observed in controls but were absent in ME/CFS patients, suggesting disrupted EV-mediated physiological processes.

Conclusions: ME/CFS patients exhibit a maladaptive EV proteomic response to exercise, characterized by metabolic impairments, immune overactivation, and ER stress response dysregulation. These findings provide insight into the molecular basis of PEM and suggest promising targets for improving recovery and energy metabolism in ME/CFS.

Source: Glass KA, Giloteaux L, Zhang S, Hanson MR. Extracellular vesicle proteomics uncovers energy metabolism, complement system, and endoplasmic reticulum stress response dysregulation postexercise in males with myalgic encephalomyelitis/chronic fatigue syndrome. Clin Transl Med. 2025 May;15(5):e70346. doi: 10.1002/ctm2.70346. PMID: 40465195; PMCID: PMC12135887. https://pmc.ncbi.nlm.nih.gov/articles/PMC12135887/ (Full text)

MicroRNA Profiling of Blood Extracellular Vesicles in ME/CFS

Abstract:

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a chronic debilitating neuroimmune disease affecting many organs and systems which, in the absence of validated biomarkers, remains diagnosed by clinical criteria. Extracellular vesicles (EV) in blood come from practically all cells in our body and therefore may carry the disease-specific biomarkers needed for the diagnosis of ME.

This chapter presents the methodology used on a single pilot study performed to evaluate this possibility to describe a workflow for EV isolation and the analysis of the miRNAs within, which could serve to interrogate additional cohorts of ME/CFS. Among the diverse nature of EV contents miRNAs may constitute a prominent regulatory layer in the development and progress of complex diseases such as ME/CFS, and therefore their study should be further pursued.

Source:Ljungström M, Nathanson L, Oltra E. MicroRNA Profiling of Blood Extracellular Vesicles in ME/CFS. Methods Mol Biol. 2025;2920:39-55. doi: 10.1007/978-1-0716-4498-0_4. PMID: 40372677. https://link.springer.com/protocol/10.1007/978-1-0716-4498-0_4

Blood Markers Show Neural Consequences of LongCOVID-19

Abstract:

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) persists throughout the world with over 65 million registered cases of survivors with post-COVID-19 sequelae, also known as LongCOVID-19 (LongC). LongC survivors exhibit various symptoms that span multiple organ systems, including the nervous system.
To search for neurological markers of LongC, we investigated the soluble biomolecules present in the plasma and the proteins associated with plasma neuronal-enriched extracellular vesicles (nEVs) in 33 LongC patients with neurological impairment (nLongC), 12 COVID-19 survivors without any LongC symptoms (Cov), and 28 pre-COVID-19 healthy controls (HC). COVID-19 positive participants were infected between 2020 and 2022, not hospitalized, and were vaccinated or unvaccinated before infection.
IL-1β was significantly increased in both nLongC and Cov and IL-8 was elevated in only nLongC. Both brain-derived neurotrophic factor and cortisol were significantly elevated in nLongC and Cov compared to HC. nEVs from people with nLongC had significantly elevated protein markers of neuronal dysfunction, including amyloid beta 42, pTau181 and TDP-43.
This study shows chronic peripheral inflammation with increased stress after COVID-19 infection. Additionally, differentially expressed nEV neurodegenerative proteins were identified in people recovering from COVID-19 regardless of persistent symptoms.
Source: Tang N, Kido T, Shi J, McCafferty E, Ford JM, Dal Bon K, Pulliam L. Blood Markers Show Neural Consequences of LongCOVID-19. Cells. 2024; 13(6):478. https://doi.org/10.3390/cells13060478 https://www.mdpi.com/2073-4409/13/6/478 (Full text)

Genomic communication via circulating extracellular vesicles and long-term health consequences of COVID-19

Abstract:

COVID-19 continues to affect an unprecedented number of people with the emergence of new variants posing a serious challenge to global health. There is an expansion of knowledge in understanding the pathogenesis of Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the impact of the acute disease on multiple organs. In addition, growing evidence reports that the impact of COVID-19 on different organs persists long after the recovery phase of the disease, leading to long-term consequences of COVID-19.

These long-term consequences involve pulmonary as well as extra-pulmonary sequelae of the disease. Noteably, recent research has shown a potential association between COVID-19 and change in the molecular cargo of extracellular vesicles (EVs). EVs are vesicles released by cells and play an important role in cell communication by transfer of bioactive molecules between cells. Emerging evidence shows a strong link between EVs and their molecular cargo, and regulation of metabolism in health and disease.

This review focuses on current knowledge about EVs and their potential role in COVID-19 pathogenesis, their current and future implications as tools for biomarker and therapeutic development and their possible effects on long-term impact of COVID-19.

Source: Nair, S., Nova-Lamperti, E., Labarca, G. et al. Genomic communication via circulating extracellular vesicles and long-term health consequences of COVID-19. J Transl Med 21, 709 (2023). https://doi.org/10.1186/s12967-023-04552-2 https://link.springer.com/article/10.1186/s12967-023-04552-2 (Full text)

 

Investigating the potential role of circulatory extracellular vesicles in myalgic encephalomyelitis/ chronic fatigue syndrome

Abstract:

ME/CFS is a debilitating disease thought to affect millions of individuals. Still, the etiology of ME/CFS is unknown, and there are no standard treatments or established biomarkers. The current symptom-based diagnosis is extensive, and the use of different diagnostic criteria contributes to heterogeneity among patients and may problematize the comparison of findings. Thus, the discovery of a biomarker for ME/CFS is urgent and would benefit both patients and the ME/CFS research field.

Extracellular vesicles (EVs) are membrane limited vesicles secreted by all cells to the extracellular environment and can be collected through biofluids. EVs serve many functions, including transferring functional proteins, lipids, and nucleic acids between cells, thus mediating cell-to-cell communication. EV secretion and cargo may reflect disease state and EVs thus pose great potential as source of minimally invasive biomarkers.

The primary aim of this project was to study EVs in plasma from ME/CFS patients and assess the potential of EVs as source of biomarkers for the disease.

Using size exclusion chromatography, EVs were enriched from plasma from ME/CFS patients (n = 20) and healthy controls (n=20). Success of EV isolation was determined in representative patient- and control EV pools (n=5) using western blotting and transmission electron microscopy. Western blot experiments for detection of EV markers CD9, CD63 and TSG101, and albumin, were optimized and confirmed enrichment of EVs and presence of non-EV eluates in the isolated samples.

EV enrichment was further validated through observation of intact EVs on transmission electron micrographs, however few CD63-positive EVs were observed. Through analysis of nanoparticle tracking analysis data, the isolated EV population primarily consisted of small EVs (< 200 nm). Within this EV population, meanand mode EV size was similar between cohorts, but the EV concentration was significantly elevated in samples from patients compared to controls (p = 0.006). However, statistical tests may have been influenced by high variation within the ME/CFS cohort.

Early-stage analysis of tandem mass spectrometry data identified 663 EV associated proteins. The majority of detected proteins overlapped with registered EV proteins, but only few differences could be observed between patient- and control derived samples. However, differential expression was not analyzed.

A biomarker for ME/CFS could not be suggested at this stage of the study, however increased EV concentration suggests abnormality in EV secretion in patients which strengthens their potential as source of biomarkers and further motivates EV research in ME/CFS and related diseases.

Source: Elena Støvring Yran. Investigating the potential role of circulatory extracellular vesicles in myalgic encephalomyelitis/ chronic fatigue syndrome. Master Thesis [University of Oslo] https://www.duo.uio.no/bitstream/handle/10852/103812/1/Masterthesis_ElenaYran_May15th2023.pdf  (Full text)

Dysregulation of extracellular vesicle protein cargo in female ME/CFS cases and sedentary controls in response to maximal exercise

Abstract:

In healthy individuals, physical exercise improves cardiovascular health and muscle stre ngth, alleviates fatigue, and reduces risk of chronic diseases. Although exercise is suggested as a lifestyle intervention to manage various chronic illnesses, it negatively affects people with myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), who suffer from exercise intolerance. We hypothesized that altered extracellular vesicle (EV) signaling in ME/CFS patients after an exercise challenge may contribute to their prolonged and exacerbated negative response to exertion (post-exertional malaise).

EVs were isolated by size exclusion chromatography from the plasma of 18 female ME/CFS patients and 17 age- and BMI-matched female sedentary controls at three time points: before, 15 minutes, and 24 hours after a maximal cardiopulmonary exercise test. EVs were characterized using nanoparticle tracking analysis and their protein cargo was quantified using Tandem Mass Tag-based (TMT) proteomics.

The results show that exercise affects the EV proteome in ME/CFS patients differently than in healthy individuals and that changes in EV proteins after exercise are strongly correlated with symptom severity in ME/CFS. Differentially abundant proteins in ME/CFS patients vs. controls were involved in many pathways and systems, including coagulation processes, muscle contraction (both smooth and skeletal muscle), cytoskeletal proteins, the immune system, and brain signaling.

Source: Ludovic Giloteaux, Katherine A. Glass, Arnaud Germain, Sheng Zhang, Maureen R. Hanson. Dysregulation of extracellular vesicle protein cargo in female ME/CFS cases and sedentary controls in response to maximal exercise. bioRxiv 2023.08.28.555033; doi: https://doi.org/10.1101/2023.08.28.555033 https://www.biorxiv.org/content/10.1101/2023.08.28.555033v1.full (Full text)

Intrinsic factors behind long-COVID: I. Prevalence of the extracellular vesicles

Abstract:

It can be argued that the severity of COVID-19 has decreased in many countries. This could be a result of the broad coverage of the population by vaccination campaigns, which often reached an almost compulsory status in many places. Furthermore, significant roles were played by the multiple mutations in the body of the virus, which led to the emergence of several new SARS-CoV-2 variants with enhanced infectivity but dramatically reduced pathogenicity.

However, the challenges associated with the development of various side effects and their persistence for long periods exceeding 20 months as a result of the SARS-CoV-2 infection, or taking available vaccines against it, are spreading horizontally and vertically in number and repercussions. For example, the World Health Organization announced that there are more than 17 million registered cases of long-COVID (also known as post-COVID syndrome) in the European Union countries alone. Furthermore, by using the PubMed search engine, one can find that more than 10 000 articles have been published focusing exclusively on long-COVID.

In light of these enormous and ever-increasing numbers of cases and published articles, most of which are descriptive of the various long-COVID symptoms, the need to know the reasons behind this phenomenon raises several important questions. Is long-COVID caused by the continued presence of the virus or one/several of its components in the recovering individual body for long periods of time, which urges the body to respond in a way that leads to long-COVID development? Or are there some latent and limited reasons related to the recovering patients themselves? Or is it a sum of both?

Many observations support a positive answer to the first question, whereas others back the second question but typically without releasing a fundamental reason/signal behind it. Whatever the answer is, it seems that the real reasons behind this widespread phenomenon remain unclear. This report opens a series of articles, in which we will try to shed light on the underlying causes that could be behind the long-COVID phenomenon.

Source: El-Maradny YA, Rubio-Casillas A, Uversky VN, Redwan EM. Intrinsic factors behind long-COVID: I. Prevalence of the extracellular vesicles. J Cell Biochem. 2023 May;124(5):656-673. doi: 10.1002/jcb.30415. Epub 2023 May 1. PMID: 37126363. https://pubmed.ncbi.nlm.nih.gov/37126363/

Proteomics and cytokine analyses distinguish myalgic encephalomyelitis/chronic fatigue syndrome cases from controls

Abstract:

Background: Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a complex, heterogenous disease characterized by unexplained persistent fatigue and other features including cognitive impairment, myalgias, post-exertional malaise, and immune system dysfunction. Cytokines are present in plasma and encapsulated in extracellular vesicles (EVs), but there have been only a few reports of EV characteristics and cargo in ME/CFS. Several small studies have previously described plasma proteins or protein pathways that are associated with ME/CFS.

Methods: We prepared extracellular vesicles (EVs) from frozen plasma samples from a cohort of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) cases and controls with prior published plasma cytokine and plasma proteomics data. The cytokine content of the plasma-derived extracellular vesicles was determined by a multiplex assay and differences between patients and controls were assessed. We then performed multi-omic statistical analyses that considered not only this new data, but extensive clinical data describing the health of the subjects.

Results: ME/CFS cases exhibited greater size and concentration of EVs in plasma. Assays of cytokine content in EVs revealed IL2 was significantly higher in cases. We observed numerous correlations among EV cytokines, among plasma cytokines, and among plasma proteins from mass spectrometry proteomics. Significant correlations between clinical data and protein levels suggest roles of particular proteins and pathways in the disease. For example, higher levels of the pro-inflammatory cytokines Granulocyte-Monocyte Colony-Stimulating Factor (CSF2) and Tumor Necrosis Factor (TNFα) were correlated with greater physical and fatigue symptoms in ME/CFS cases. Higher serine protease SERPINA5, which is involved in hemostasis, was correlated with higher SF-36 general health scores in ME/CFS. Machine learning classifiers were able to identify a list of 20 proteins that could discriminate between cases and controls, with XGBoost providing the best classification with 86.1% accuracy and a cross-validated AUROC value of 0.947. Random Forest distinguished cases from controls with 79.1% accuracy and an AUROC value of 0.891 using only 7 proteins.

Conclusions: These findings add to the substantial number of objective differences in biomolecules that have been identified in individuals with ME/CFS. The observed correlations of proteins important in immune responses and hemostasis with clinical data further implicates a disturbance of these functions in ME/CFS.

Source: Giloteaux L, Li J, Hornig M, Lipkin WI, Ruppert D, Hanson MR. Proteomics and cytokine analyses distinguish myalgic encephalomyelitis/chronic fatigue syndrome cases from controls. J Transl Med. 2023 May 13;21(1):322. doi: 10.1186/s12967-023-04179-3. PMID: 37179299. https://translational-medicine.biomedcentral.com/articles/10.1186/s12967-023-04179-3 (Full text)

Diagnosis of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome with partial least squares discriminant analysis: Relevance of blood extracellular vesicles

Abstract:

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS), a chronic disease characterized by long-lasting persistent debilitating widespread fatigue and post-exertional malaise, remains diagnosed by clinical criteria. Our group and others have identified differentially expressed miRNA profiles in the blood of patients. However, their diagnostic power individually or in combinations seems limited. A Partial Least Squares-Discriminant Analysis (PLS-DA) model initially based on 817 variables: two demographic, 34 blood analytic, 136 PBMC miRNAs, 639 Extracellular Vesicle (EV) miRNAs, and six EV features, selected an optimal number of five components, and a subset of 32 regressors showing statistically significant discriminant power. The presence of four EV-features (size and z-values of EVs prepared with or without proteinase K treatment) among the 32 regressors, suggested that blood vesicles carry relevant disease information. To further explore the features of ME/CFS EVs, we subjected them to Raman micro-spectroscopic analysis, identifying carotenoid peaks as ME/CFS fingerprints, possibly due to erythrocyte deficiencies. Although PLS-DA analysis showed limited capacity of Raman fingerprints for diagnosis (AUC = 0.7067), Raman data served to refine the number of PBMC miRNAs from our previous model still ensuring a perfect classification of subjects (AUC=1). Further investigations to evaluate model performance in extended cohorts of patients, to identify the precise ME/CFS EV components detected by Raman and to reveal their functional significance in the disease are warranted.

Source: González-Cebrián Alba, Almenar-Pérez Eloy, Xu Jiabao, Yu Tong, Huang Wei E., Giménez-Orenga Karen, Hutchinson Sarah, Lodge Tiffany, Nathanson Lubov, Morten Karl J., Ferrer Alberto, Oltra Elisa. Diagnosis of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome With Partial Least Squares Discriminant Analysis: Relevance of Blood Extracellular Vesicles. Frontiers in Medicine, 9, 2022 , DOI: 10.3389/fmed.2022.842991 https://www.frontiersin.org/article/10.3389/fmed.2022.842991 (Full study)

Cytokine profiling of extracellular vesicles isolated from plasma in myalgic encephalomyelitis/chronic fatigue syndrome: a pilot study

Abstract:

Background: Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a debilitating disease of unknown etiology lasting for a minimum of 6 months but usually for many years, with features including fatigue, cognitive impairment, myalgias, post-exertional malaise, and immune system dysfunction. Dysregulation of cytokine signaling could give rise to many of these symptoms. Cytokines are present in both plasma and extracellular vesicles, but little investigation of EVs in ME/CFS has been reported. Therefore, we aimed to characterize the content of extracellular vesicles (EVs) isolated from plasma (including circulating cytokine/chemokine profiling) from individuals with ME/CFS and healthy controls.

Methods: We included 35 ME/CFS patients and 35 controls matched for age, sex and BMI. EVs were enriched from plasma by using a polymer-based precipitation method and characterized by Nanoparticle Tracking Analysis (NTA), Transmission Electron Microscopy (TEM) and immunoblotting. A 45-plex immunoassay was used to determine cytokine levels in both plasma and isolated EVs from a subset of 19 patients and controls. Linear regression, principal component analysis and inter-cytokine correlations were analyzed.

Results: ME/CFS individuals had significantly higher levels of EVs that ranged from 30 to 130 nm in size as compared to controls, but the mean size for total extracellular vesicles did not differ between groups. The enrichment of typical EV markers CD63, CD81, TSG101 and HSP70 was confirmed by Western blot analysis and the morphology assessed by TEM showed a homogeneous population of vesicles in both groups. Comparison of cytokine concentrations in plasma and isolated EVs of cases and controls yielded no significant differences. Cytokine-cytokine correlations in plasma revealed a significant higher number of interactions in ME/CFS cases along with 13 inverse correlations that were mainly driven by the Interferon gamma-induced protein 10 (IP-10), whereas in the plasma of controls, no inverse relationships were found across any of the cytokines. Network analysis in EVs from controls showed 2.5 times more significant inter-cytokine interactions than in the ME/CFS group, and both groups presented a unique negative association.

Conclusions: Elevated levels of 30-130 nm EVs were found in plasma from ME/CFS patients and inter-cytokine correlations revealed unusual regulatory relationships among cytokines in the ME/CFS group that were different from the control group in both plasma and EVs. These disturbances in cytokine networks are further evidence of immune dysregulation in ME/CFS.

Source: Giloteaux L, O’Neal A, Castro-Marrero J, Levine SM, Hanson MR. Cytokine profiling of extracellular vesicles isolated from plasma in myalgic encephalomyelitis/chronic fatigue syndrome: a pilot study. J Transl Med. 2020 Oct 12;18(1):387. doi: 10.1186/s12967-020-02560-0. PMID: 33046133. https://pubmed.ncbi.nlm.nih.gov/33046133/