Dysregulation of extracellular vesicle protein cargo in female ME/CFS cases & sedentary controls in response to maximal exercise, 2023 Giloteaux et al

[Andy]

Preprint. Now published - see post #18

This is from Maureen Hanson's group.

Abstract

In healthy individuals, physical exercise improves cardiovascular health and muscle strength, 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.

https://www.biorxiv.org/content/10.1101/2023.08.28.555033v1

 

[mariovitali]

I really hate that I have to do this.

Network Analysis from 2017, LXR can be clearly seen : https://www.s4me.info/threads/machine-learning-assisted-research-on-me-cfs.5015/page-9#post-456267

Snapshot from Hanson's study :

 

[Ash]

I really hate that I have to do this.

Network Analysis from 2017, LXR can be clearly seen : https://www.s4me.info/threads/machine-learning-assisted-research-on-me-cfs.5015/page-9#post-456267

Snapshot from Hanson's study :

View attachment 20271

can you translate for people who don’t speak lab?

 

[mariovitali]

I am describing one more example on how advanced analytical techniques (available since 2015) have been outperforming findings from conventional researchers. LXR (Liver X receptor) is the latest example which was identified as being important in 2017 by a method called Network Analysis in the link I provided.

Also you can see on the second snapshot that LXR/RXR activation were significantly inhibited 24 hours post-exercise.

 

[Ash]

Thanks @mariovitali!

I wasn’t able to read the paper at top of thread due to exhaustion and was trying to work out if it’s findings were promising or not from the comments section.

 

[mariovitali]

For the record, I believe that what Dr hanson found is related to impaired apoptotic cell clearance. I submit 2 slides from a presentation I made in 2018. Observe where LXR/RXR is situated in the graphs :

 

[rvallee]

This is far above my meager understanding of biology. Which I like. It's so unimpressive looking at BPS "research" and it's basically high school level, both the study and the paper.

Took a few screenshots of some key results:

 

[Fizzlou]

https://britishlivertrust.org.uk/information-and-support/love-your-liver/free-liver-scan/

Don’t know if this is the best place to post but I know @mariovitali you have mentioned Fibro Scan before.

In UK this roadshow by British Liver Trust is offering free Liver Fibro Scan. Only a few locations on map at the moment.

My mother, also pwME has diagnosed fatty liver found after flu induced hospital stay.

 

[DMissa]

I am describing one more example on how advanced analytical techniques (available since 2015) have been outperforming findings from conventional researchers.

A text mining model once pointed at one thing (amongst many things), that also popped up in this study (as one of many things) and this coincidence strengthens your belief in besmirching biologists?

 

[Hutan]

There's a nice summary of work done on EVs in ME/CFS in the paper:

To date, few studies on EVs in ME/CFS have been published (Almenar-Pérez et al., 2020; Bonilla et al., 2022; Castro-Marrero et al., 2018; Eguchi et al., 2020; Giloteaux et al., 2023; Giloteaux et al., 2020; González-Cebrián et al., 2022). In 2018, Castro-Marrero and colleagues were the first to analyze EVs isolated from ME/CFS patients and they found a higher EV concentration and smaller sized EVs in ten ME/CFS patients in comparison to five healthy controls (Castro-Marrero et al., 2018). Almenar-Pérez et al. replicated these results and found dysregulated neuroimmune pathways via analysis of EV miRNA content in a study with 15 severely affected ME/CFS patients compared to 15 healthy subjects (Almenar-Pérez et al., 2020). Our group published two studies measuring the cytokine content of EVs isolated from plasma of 35 and 49 ME/CFS patients and matched healthy controls, respectively (Giloteaux et al., 2023; Giloteaux et al., 2020). Although we did not replicate EV size differences between groups, all four studies found significantly higher concentration of EVs in the ME/CFS group. The cytokine analysis revealed few significant differences compared to controls but significant correlations with patient symptom severity. Eguchi and colleagues also replicated the finding of increased concentration of circulating EVs in a larger cohort of 99 ME/CFS patients compared to 53 controls (Eguchi et al., 2020). However, one study in which EVs were directly quantified without prior isolation showed no difference in EV concentration between 20 ME/CFS patients and 20 controls (Bonilla et al., 2022).

Further proteomic analysis of EV cargo in addition to cytokines could help identify a signature biomarker for ME/CFS. Quantitative untargeted proteomics of isolated EVs from three ME/CFS patients and three controls from the larger Eguchi et al. cohort revealed a protein cargo specific to the ME/CFS group including actin network proteins and 14–3-3 family proteins (Eguchi et al., 2020), but these results need to be validated in a larger study.

Eguchi study link: Identification of actin network proteins, talin-1 and filamin-A, in circulating extracellular vesicles as blood biomarkers...ME/CFS 2019, Eguchi et al. Edit - having just read through the thread for that study, the conclusions on EV proteins in the Eguchi study were based on very small samples.

I think work on difference in the numbers and size of EVs is less interesting than what is actually in the EVs - possibly numbers and size are affected by exercise. It sounds from this as though only one study so far has looked at proteins in the EVs (the Eguchi study). This Giloteaux study looks at the effect of an exercise challenge, which is great. Also great to have a replication of the Eguchi study.

 

[SNT Gatchaman]

I wasn’t able to read the paper at top of thread due to exhaustion and was trying to work out if it’s findings were promising or not from the comments section.

From first read through, I would say the findings are likely valid and useful. Others may have an opinion on methodological weaknesses that I haven't realised though.

I hope the next paper in the series will be looking at the microRNA content of EVs, similar to Assessing diagnostic value of microRNAs from peripheral blood mononuclear cells and extracellular vesicles in ME/CFS (2020)

 

[DMissa]

Okay having read it this is a cool paper. We have EV people in the building where I work, if I find money for it, it could be a fun side project to see how EV proteomes from patient primary cells or cell lines compare to a) whole cell proteomes and b) the published plasma EV proteomes.

 

[mariovitali]

A text mining model once pointed at one thing (amongst many things), that also popped up in this study (as one of many things) and this coincidence strengthens your belief in besmirching biologists?

The following have been identified before conventional research using Artificial Intelligence methods : pyruvate dehydrogenase complex, phospholipid metabolism dysregulation, peroxisomal dysfunction,bile acid metabolism dysregulation, endoplasmic reticulum stress (latest NIH study),LXR (there may be more, I will have to go thhrough the results)

If you consider how many millions of possible concepts exist in medicine and also that there was no help from biologists, I would say that this is quite good for a result.

 

[Hutan]

There's lots to think about in this paper.

One is how variable exosome contents are before, during and after exercise (see Figure 5). It means that any study of them has to really carefully control for exertion. I wonder how long exosomes last and if there is variation diurnally and also with menstrual cycle.

I wonder if cognitive exertion (as opposed to physical exertion) affects exosome composition - I'd love to see a study of that.

It's interesting to look at the pathways that are upregulated or down regulated in ME/CFS compared to the controls - many normalise straight after exercise, but then either revert to the baseline level of abnormality or become even more abnormal. Those ones that become more abnormal might tell us something about PEM, pathways like

LXR/RXR Activation (down)
RHOGDI Signalling (down)
PAK signalling (up)
RAC signalling (up)
Ephrin receptor signalling (up)​

as well as those that become very different immediately after exercise that might cause some downstream impact on cells, pathways like

coagulation system (down)
acute phase response signalling (down)
ERK5 signalling (up)
14-3-3 mediated signalling (up)
actin cytoskeleton signalling (down)
RHOA signalling (down)​

Figure 5

 

[mariovitali]

@Hutan I think we need better enrichment analysis . Phagocytosis should be among the results and also apoptotic cell clearance

 

[SNT Gatchaman]

15 minutes post-CPET, 13 EV proteins had lower abundances and three had higher abundances in the ME/CFS group [...] including the coagulation factors F8 and F13A1, fibronectin 1 (FN1), and heat shock protein family A member 5 (HSPA5) which were decreased and orosomucoid 1 (ORM1) which was increased.

I'm uncertain whether this might relate, but just to note that freely circulating FN1 was high in serum in Prusty's preprint (and IgM-FN1 was low), and that HSPA5 is aka Binding immunoglobulin protein (BiP) aka 78 kDa glucose-regulated protein (GRP-78). BiP was abnormally low in the recent WASF3 paper, when it should be upregulated in response to ER stress.

 

[mariovitali]

I just realised something regarding RHOGDI. RHOGDI is associated according to Wikipedia with RhOA :

https://en.wikipedia.org/wiki/Low-affinity_nerve_growth_factor_receptor#RhoGDI_and_RhoA


Now observe the following slide and you will see RhoA-GTP along with LXR/RXR.Also, next to the red annotation (on the left) you can see TSP-1 , this is Thrombospondin which OMF is looking at :


https://www.omf.ngo/study-of-thrombospondin-1/


We also have the following post where C1Q is mentioned from a spanish study :

https://mecfsskeptic.com/2021-looking-back-at-a-year-of-me-cfs-research/


It gets more interesting : Next to the red annotation we read MFGE8 also known as Lactadherin. Lactadherin could be ibvestigated for its use in order to avoid fibrin formation :

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5794502/


Could all of this be a coincidence?

 

[SNT Gatchaman]

Published as —

Dysregulation of extracellular vesicle protein cargo in female myalgic encephalomyelitis/chronic fatigue syndrome cases and sedentary controls in response to maximal exercise
Ludovic Giloteaux; Katherine A. Glass; Arnaud Germain; Carl J. Franconi; Sheng Zhang; Maureen R. Hanson

In healthy individuals, physical exercise improves cardiovascular health and muscle strength, alleviates fatigue and reduces the 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) signalling 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 min, and 24 h 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 versus 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 signalling.

Link | PDF (Journal of Extracellular Vesicles)

 

[SNT Gatchaman]

Increased clusterin abundance post-exercise positively correlates with pain and fatigue

We found that the clusterin (CLU) 15 min/0 h ratio positively correlated with fatigue the morning of exercise and it was the only protein in which increased abundance 24 h post-exercise positively correlated with both average severity of myalgia and arthralgia. CLU was also one of the few proteins with decreased abundance 15 min post-exercise in controls. CLU is a multifunctional molecular chaperone protein involved in a variety of physiological and pathologic processes, including clearance of cell debris, apoptosis, and assisting in the folding and conformational maturation of newly synthesized proteins as well as stress-denatured proteins.

In our study, elevated CLU in EVs post-exercise is associated with worse myalgia, arthralgia, and fatigue indicating that this protein may have an important role in ME/CFS pathophysiology.

 

[SNT Gatchaman]