Extracellular vesicles from [LC] patients promote RUNX2-mediated cellular stress via dysregulated miR-204 and p53 pathway activation, 2025, Dalle+

[forestglip]

Extracellular vesicles from long COVID patients promote RUNX2-mediated cellular stress via dysregulated miR-204 and p53 pathway activation

Spoiler: Authors

Dalle Carbonare, Luca; Minoia, Arianna; Zouari, Sharazed; Braggio, Michele; Cominacini, Mattia; Gaglio, Salvatore Calogero; Piritore, Francesca Cristiana; Lorenzi, Pamela; Meneghel, Mirko; Dervishi, Kevin; Corsi, Andrea; Pedrinolla, Anna; Giuriato, Gaia; Fiore, Alessandra; Celesia, Adriana; Guerricchio, Laura; Venturelli, Massimo; Schena, Federico; Donadelli, Massimo; Mottes, Monica; Romanelli, Maria Grazia; Perduca, Massimiliano; Guardavaccaro, Daniele; Crisafulli, Ernesto; Zipeto, Donato; Barile, Lucio; Valenti, Maria Teresa

[Line breaks added]

Background:
Subjects with Long COVID, also known as post-acute sequelae of SARS-CoV-2 infection (PASC), experience a wide range of symptoms, including fatigue and respiratory disturbances, affecting their quality of life. Despite the increasing prevalence of Long COVID, the underlying pathogenic mechanisms remain poorly understood. Extracellular vesicles (EVs) are known to be involved in various processes, such as tissue repair and the transmission of viral particles. However, the specific characteristics and functional roles of EVs derived- from patients with Long COVID (LC-EVs) are poorly characterized.

Methods:
To uncover systemic mechanisms underlying Long COVID, we performed a comprehensive characterization of patient-derived extracellular vesicles (EVs) via Nanoparticle Tracking analysis (NTA), Atomic Force Microscopy (AFM), Transmission Electron Microscope (TEM) and flow cytometry. These EVs were applied to lung cells, Mesenchymal Stem Cell (MSCs), Human Umbilical Vein Endothelial Cells (HUVECs) and Aortic Smooth Muscle Cells (ASMCs), revealing stress responses through SESN1, SESN2, and p53 activation. We further assessed mitochondrial respiration to evaluate metabolic dysfunction, and conducted targeted transfection experiments to dissect the molecular pathways involved, shedding light on EV-driven cellular reprogramming.

Results:
Thus, we observed that Long COVID (LC) patients experienced breathlessness and leg discomfort during exertion.

Our data highlighted that LC-EVs induce aberrant RUNX2 expression and activate the p53/p21 pathway in lung cells as well stress responses. Additionally, LC-EVs impair mitochondrial function and cellular adaptability under metabolic stress, reducing maximal respiration and ATP production at high cell densities.

Protein interaction analysis showed RUNX2 involvement in key biological processes and post-transcriptional regulation by hsa-miR-204-5p was identified. Finally, LC-EVs also activated stress pathways and increased RUNX2, SESN, p53, and p21 levels in endothelial cells, aortic smooth muscle cells, and mesenchymal stem cells.

Conclusions:
In conclusions, these findings provide new insights into the role of extracellular vesicles in Long COVID, revealing their involvement in cellular stress and impaired mitochondrial function.

Web | DOI | PDF | Cell Communication and Signaling | Open Access

 

[Kitty]

Thus, we observed that Long COVID (LC) patients experienced breathlessness and leg discomfort during exertion.

I guess that raises the question, "What do you mean by long Covid?"

 

[SNT Gatchaman]

Despite looking at small numbers of hospitalised patients going on to LC vs recovered controls, this could be of interest. p53, mitochondria, EVs and platelets feature.

Apart from the mitochondrial data, the increased RUNX2 finding replicates that of Upregulation of olfactory receptors and neuronal-associated genes highlights complex immune and neuronal dysregulation in Long COVID patients (2025) which among other things highlighted Reelin (note JE's previous comment). They also highlighted RUNX2 —

Another interesting upregulated pathway in LC patients was RUNX2 expression and activity, which is a transcription factor involved in osteogenesis and immune regulation. Its dysregulation might hint at tissue remodeling and skeletal muscle issues or systemic effects beyond the immune response in LC, potentially explaining skeletal muscle complications such as wide spread pain and fatigue in LC patients.

 

[SNT Gatchaman]

Summary quotes from intro and discussion —

this study was designed as a hypothesis-driven investigation aimed at exploring whether cell-to-cell communication mediated by extracellular vesicles (EVs) can modulate cellular stress and thereby contribute to the persistence of Long COVID symptoms.

Our data strongly suggest a key pathogenic mechanism of long COVID mediated by vesicles (EVs). Using cytofluorimetric analysis, we identified a significant overexpression of markers associated with platelet hyperactivation in LC-EVs. Specifically, we observed elevated levels of CD29, CD41b, CD42a, and CD69, which have been reported in the literature as key players in platelet aggregation.

we observed that these vesicles effectively crossed the cellular membrane and dispersed within the cell’s cytoplasm. This observation is consistent across both control and LC-EVs, suggesting a fundamental ability of these vesicles to enter cells regardless of their origin.

we observed that both p53 and its phosphorylated form, as well as p21, were increased in cells exposed to LC-EVs. These findings indicate activation of the p53 pathway, which is commonly involved in cellular responses to DNA damage, oxidative stress, and other forms of cellular stress

Notably, we observed that LC-EVs have a dual effect on mitochondrial function. LC-EVs-treated cells analyzed under oxygen-favorable conditions exhibited higher oxygen consumption […] indicates an increase in mitochondrial respiration, which could be due to enhanced metabolic activity or a compensatory response to stress induced by LC-EVs. However, when metabolic stress is exacerbated by higher cell density, the ability of LC-EV treated cells to sustain maximal respiration significantly declines […] suggests that while LC-EVs may initially stimulate mitochondrial activity, they ultimately impair the cells’ adaptive metabolic flexibility, making them more vulnerable under conditions where resources such as oxygen and nutrients are limited.

A key consequence of this impaired adaptability is the reduction in ATP production and basal respiration at higher cell densities. Since maximal respiration reflects the full capacity of the electron transport chain, its decline in LC-EVs-treated cells implies that mitochondria become less efficient in generating ATP under stress. In contrast, cells treated with control vesicles maintain stable respiration and ATP production their mitochondrial function remains more resilient.

Overall, these findings suggest that LC-EVs disrupt the metabolic homeostasis of recipient cells, particularly in conditions of high metabolic demand. The effects of LCEVs on mitochondrial respiration may reflect a context dependent cellular response. In hyperoxic environments, LC-EVs might promote mitochondrial activity as part of a compensatory mechanism aimed at sustaining energy production and cellular homeostasis. Conversely, under stress conditions the LC-EVs-mediated signaling could contribute to mitochondrial dysfunction, acting as a maladaptive or pathological response. This duality suggests that EVs may play a pivotal role in the balance between adaptation and degeneration, particularly in the context of Long COVID

it has been demonstrated that hsa-miR-204-5p is a well-known direct negative regulator of RUNX2, targeting its 3’UTR in various cell types. We observed that the levels of hsa-miR-204-5p were lower in the cargo of LC-EVs, which was consistent with the increased levels of RUNX2 seen in LC-EVs and in cells treated with LC-EVs.

Thus, our data suggest that this RUNX2 upregulation is, at least in part, due to reduced levels of hsa-miR-204-5p in LC-EVs.

 

[DMissa]

mitochondria

Tbh I’ve come to realise that 99% of the time this ends up being a red flag