Decreased NO production in endothelial cells exposed to plasma from ME/CFS patients, Bertinat et al (2022)

[Hutan]

From the presentation by Dr Francisco Westermeier - some brief and selective notes Part 1
Up to the 12 minutes mark
He's a researcher in Austria, although he's from Chile. (pretty impressive language skills)

Nitric oxide (NO) is a signalling molecule and a reactive gas - it only lasts about 10 seconds. It has lots of roles.

Francisco gives the example of a radio signal, sent out and reaching the antennae (receptor) of a radio, the transducer converts the radio waves to sound waves, an amplifier increases the strength of the signal etc. The point is, a signal might not have the same effect on different cells, or the same cell under different conditions.

eNOS is endothelial NO synthase; nNOS is neuronal NO synthase, produced by the vagus nerve and sympathetic nerves
As 'synthase' suggests, these molecules make NO.
NO affects heart rate and mitochondrial respiration in cardiac muscle cells - involved in the muscle relaxation
NO inhibits platelet aggregation and thrombosis (Francisco at this point notes the recent evidence of increased coagulation in people with ME/CFS - although I think the jury is still out on that evidence)
NO promotes vascular dilation, including in response to hypoxia

Increasing evidence of endothelial dysfunction in ME/CFS over the last 4 years - two papers by Francisco and his colleagues, and at least three by other teams.


Paper 1: 2021: Investigation of microRNA linked to eNOS, 2021, Blauensteiner et al
Used plasma from UK ME/CFS Biobank
Evaluate expression of 5 microRNAs known to regulate eNOS (4 down-regulate; 1 up-regulates)
They found statistically significant expression of 4 out of the 5 microRNAs (not sure how that works with both the down-regulating and up-regulating microRNA having increased expression).
But they didn't find any difference in the expression with disease severity


Green is controls; pink is moderately affected patients; red is severely affected patients. Data for the 5 microRNAs. The fourth one is the one that up regulates.




I thought this PCA was quite a good image. Some ME/CFS patients (including ones severely affected) looked much the same as controls in terms of the microRNA. But some, maybe half, clearly look different. However, there's no separation of moderately and severely affected patients. So, either not all the patients actually have ME/CFS, or patients only show this different expression under certain circumstances (PEM?), or endothelial dysfunction is not a core aspect of ME/CFS or...

(So, if I was to go back to that 2021 paper, I'd be wanting to find out more about what they thought was going on with both the down-regulating and up-regulating miRNA having increased expression.)

 

[Hutan]

Part 2 from the 12 minute mark
Paper 2: 2022 Bertinat et al (the paper that is the subject of this thread)
Used same plasma samples
Looking to see the effect of plasma samples (from healthy controls and ME/CFS patients) on healthy endothelial cell NO production in the presence of 4 molecules known to increase the release of NO

insulin
bradykinin
histamine
acetylcholine​

There are two ways to cause the eNOS to make NO:

• Tyrosine kinase receptors - activated by growth factor and hormones (presumably insulin is one) (involves PKA or Akt phosphorylation)
• G-protein coupled receptors - activated by acetylcholine, histamine, bradykinin (involves calcium)

Here's the results of the endothelial cells incubated with either healthy or ME/CFS plasma. They got a bigger decrease in NO production by the ME/CFS plasma compared to the healthy plasma with the three molecules acting on the G protein Coupled Receptors (i.e. GPCR agonists).


To evaluate the activation of eNOS, they measured the phosphorylation of three sites on the eNOS molecule - two (Ser1177 and Ser635) related to activation of eNOS and one (Thr495) related to inhibition of eNOS. (Remember, eNOS is the endothelial NO synthesising molecule).
I'm not completely sure about this but I think they didn't find any difference in the amount of expression of eNOS between the cells incubated with healthy plasma versus cells incubated with ME/CFS plasma. But they did find that there there was significantly more down regulating phosphorylation (i.e. more at the Thr495 site) with ME/CFS plasma. See the picture below. There is some overlap.



up to the 15 minute mark (nearly there as the talk only takes about 20 minutes, the rest was questions and from memory, I didn't take away a lot of information from the questions, although I was trying to paint skirting boards at the time, so I might be wrong).

 

[Hutan]

Part 3, from the 15 minute mark
Still on Paper 2

So, did the ME/CFS plasma actually affect NO production? Yes!

If you just look at the y axis, which is units of NO, and look at the green dots (cells in control plasma) and red dots (cells in ME/CFS plasma), there's a decent separation in the NO production.

Taking the x-axis into account, the phosphorylation of Thr495 site did seem to affect production - more phosphorylation of that inhibiting site did reduce NO production.

There's clearly a lot of mechanisms and different things affecting the operation of the mechanisms when it comes to NO.

Moving on the next project - arginine metabolism
The project is funded by Solve ME




So, they are looking at arginine which is taken into endothelial cells and broken down into citrulline and NO. Increased levels of SDMA have been described as reducing the uptake of arginine into the endothelial cell. Increased levels of ADMA and MMA have been described as inhibiting the production of citrulline and NO.

Francisco's team will be working out if the levels of the various molecules are different in ME/CFS plasma.

Francisco closed with some thanks, including to CureME, MERUK, SolveME and Austrian patients and the ME/CFS community who donated funds.

From the talk, it all seemed pretty solid and worthwhile research to me, not hyped.

Finished by 20 minute mark.

Edited to add - I may well have some things wrong - please do point out errors.

 

[Hoopoe]

I've had an arginine infusion as part of a medical test and it made me feel good. Maybe that's got something to do with NO?

 

[Robert 1973]

If reduced NO production is inhibiting vasodilation would it make sense to try vasodilators? This was suggested to me by a consultant a few years ago but I didn’t try them at the time because I’d recently had kidney stones which are a contraindication.

 

[Hutan]

It could be that it's the moment by moment response to particular situations, possibly even in particular parts of the body, that isn't appropriate. That is, the control systems aren't working. So making the body awash with a substance that increases vasodilation in all parts of the body all of the time perhaps wouldn't help, and might even just make things worse.

 

[Ravn]

Thanks for the of the video @Hutan. I don't have the brain power right now to digest the science properly but I do like the way Francisco is very cautious about drawing conclusions, takes the time to really think through his answers and is willing to say he doesn't know. Makes me more confident he's doing solid science.

 

[Ravn]

If reduced NO production is inhibiting vasodilation would it make sense to try vasodilators? This was suggested to me by a consultant a few years ago but I didn’t try them at the time because I’d recently had kidney stones which are a contraindication.

There must be lots of pwME who take drugs that affect vasoconstriction for other conditions, plus lots of pwME take supplements like arginine or citrulline. As usual some report some effect some of the time but if there was a general and significant effect in ME that probably would have shown up by now. Or it could be that the existing drugs and supplements just aren't strong enough.

It could be that it's the moment by moment response to particular situations, possibly even in particular parts of the body, that isn't appropriate. That is, the control systems aren't working. So making the body awash with a substance that increases vasodilation in all parts of the body all of the time perhaps wouldn't help, and might even just make things worse.

Yes, it's probably more complicated than just too much or too little of something. More likely some sort of combination of the wrong amount in the wrong place at the wrong time. Dysregulation rather than deficiency.

Of the more visible vasodilation-vasoconstriction comorbidities, some pwME report Raynaud's, others report Erythromelalgia-like signs, and some of us have both. I can have one foot in Raynaud's mode and the other in EM mode - at the same time, which would suggest some local signalling going awry.

Also, the problem could be - I think Francisco mentions this in the video - that there may be plenty of vasodilating molecules about but the mechanism to use them is broken, so adding extra wouldn't help.

Plus, dysregulation of vasodilation-vasoconstriction could be a secondary adaptation that's actually helpful (as in, the lesser evil). I find my ME symptoms ease when I'm cold and all my extremities are nice shades of white and blue, i.e. lots of vasoconstriction. My entirely speculative hypothesis is that it keeps more blood going to the brain (which inconveniently contradicts my local signalling hypothesis above ).

 

[ahimsa]

I find my ME symptoms ease when I'm cold and all my extremities are nice shades of white and blue, i.e. lots of vasoconstriction.

Same. Cold always helps me a lot.

I always thought it might be due to low blood volume and that extra vasoconstriction pushing more blood to the core (heart/lungs) - maybe also the brain? But that's a complete guess.

 

[Mithriel]

I might have mentioned this on the thread before but I found that clonidine made a difference to me. I went from not managing to go out alone to the odd trip to the shops.

I carried on with the deterioration but it gave me an extra year of independence.