Effects of Mitochondrial Dysfunction on Extracellular Vesicle Release and Subsequent Disruption of the Lung Vascular Endothelial Barrier

Letsiou, Eleftheria, Al Matni, Mohammed Yaman, Meliton, Lucille, Lawrence-Foster, Rachel, Kadioglu, Aras ORCID: 0000-0003-1137-6321 and Dudek, Steven (2024) Effects of Mitochondrial Dysfunction on Extracellular Vesicle Release and Subsequent Disruption of the Lung Vascular Endothelial Barrier CIRCULATION, 150 (Suppl_). a4135700-a4135700. ISSN 0009-7322, 1524-4539

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Abstract

Background: Lung endothelial cell (EC) barrier disruption is a hallmark of acute lung injury (ALI), which is commonly caused by bacterial pneumonia. Alveolar epithelial cells (AEC) are the first line of defence against invading respiratory pathogens in the lungs and can mediate inflammatory signaling to the adjacent EC contributing to barrier disruption. We have previously demonstrated that pneumolysin (PLY), a major Streptococcus pneumoniae virulence factor, induces both mitochondrial dysfunction in lung AEC and release of large extracellular vesicles (EVs). These EVs, which are enriched with mitochondrial cargo, contribute to increased permeability of the lung EC barrier. Here, we investigate strategies to inhibit mitochondrial dysfunction in AEC and examine how these interventions impact EC barrier function. Methods: Human alveolar epithelial cells (A549) or immortalized AEC (Cell Biologics) were pre-treated with mitoTEMPO (mtROS scavenger), P110 (excessive fission inhibitor), or ensifentrine [dual phosphodiesterase (PDE) 3/4 inhibitor] prior to PLY treatment (100-300 ng/ml, 4 hrs). Mitochondrial function was assessed by the JC-1 assay. EVs were isolated from cell media and analyzed for their mitochondrial content (Tom20 and Tim23 expression). To assess their functional role, EVs were added to TNF-α treated human lung microvascular EC, and barrier function was then determined using the ECIS assay to measure transendothelial electrical resistance (TER). Results: PLY-induced mitochondrial dysfunction was reduced in the presence of mitoTempo or P110. EVs from PLY-treated AEC (EV PLY ; enriched mitochondrial content) exacerbated TNF-α-induced EC barrier permeability as indicated by the significant decrease in TER. EV PLY derived from MitoTEMPO or P110-pretreated AEC contained less mitochondrial cargo and resulted in less EC permeability after TNF-α. Compared to mitoTEMPO and P110, ensifentrine exhibited superior protection against mitochondrial dysfunction after PLY exposure. In addition, in the presence of ensifentrine, the quantity of EV release and their extracelllular mitochondrial content were both dramatically reduced. In contrast to control EV PLY , EV PLY from ensifentrine-pretreated AEC demonstrated no barrier disruptive properties. Conclusions: Mitochondrial dysfunction in AEC contributes to vascular endothelial injury in ALI through mechanisms involving the release of barrier-disruptive EVs, which can be effectively targeted by inhibiting PDE3/4.

Item Type:	Article
Uncontrolled Keywords:	32 Biomedical and Clinical Sciences, 3201 Cardiovascular Medicine and Haematology, 3202 Clinical Sciences, Lung, Acute Respiratory Distress Syndrome, Pneumonia & Influenza, Infectious Diseases, Pneumonia, Rare Diseases, 2.1 Biological and endogenous factors
Divisions:	Faculty of Health & Life Sciences Faculty of Health & Life Sciences > Inst. Infection, Vet & Ecological Sciences Faculty of Health & Life Sciences > Inst. Infection, Vet & Ecological Sciences > Inst. Infection, Vet & Ecological Sciences (T&R Staff) Faculty of Health & Life Sciences > Inst. Infection, Vet & Ecological Sciences > Clinical Infection, Microbiology & Immunology
Depositing User:	Symplectic Admin
Date Deposited:	01 Dec 2025 08:33
Last Modified:	27 Jul 2026 10:14
DOI:	10.1161/circ.150.suppl_1.4135700
Open Access URL:	https://www.ahajournals.org/doi/10.1161/circ.150.s...
Related Websites:	Author Publisher
URI:	https://livrepository.liverpool.ac.uk/id/eprint/3195699
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