T3 - Mitochondrial Cargo Quality Determines the Paracrine Effects by Endothelial Cell-Derived Extracellular Vesicles

Saturday, October 11, 2025

10:00 AM - 11:00 AM PDT

Location: Exhibit Hall F, G & H

Co-Author(s)

Zahid Manzar, MEng

Graduate Student
SUNY University at Buffalo
Buffalo, New York, United States

Presenting Author(s)

LD

Lucas Davis

Undergraduate Student
SUNY University at Buffalo, United States

Co-Author(s)

KS

Karl Swanson

Undergraduate Student
SUNY University at Buffalo, United States
Erik Munoz

PhD candidate
University at Buffalo -, United States
HS

Hazel Szeto

Professor
Social Profit Network, United States
HM

Hans Minderman

Co-Director, Flow and Immune Analysis Shared Resource Division of Medicine
Roswell Park Comprehensive Cancer Center
Buffalo, New York, United States

Last Author(s)

B. Rita Alevriadou

Empire Innovation Professor in BME
University at Buffalo - SUNY
Buffalo, New York, United States

Primary Investigator(s)

B. Rita Alevriadou

Empire Innovation Professor in BME
University at Buffalo - SUNY
Buffalo, New York, United States

Introduction: : Endothelial cell (EC) dysfunction, the critical step in atherosclerosis development, is known to trigger the release EC-derived extracellular vesicles (EC-EV). Plasma levels of EC-EV are elevated in cardiovascular disease (CVD) patients suggesting that EC-EV may be prognostic and diagnostic biomarkers of atherosclerosis and CVD. While EV are known vectors for intercellular transfer of proteins and nucleic acids, increasing evidence suggests they also shuttle whole mitochondria or fragments (mitoEV). There is a dichotomy in the literature regarding the end-target effects of EV-mediated mitochondrial transfer. Some studies show EV inducing regenerative effects in target/recipient cell whereas other studies implicate EV in initiating proinflammatory, and even proapoptotic, cascades in recipient/target cell. The present study aims to better understand the mechanisms by which mitoEV, via their mitochondrial cargo transfer, regulate target EC (dys)function. We hypothesized that low quality mitoEV mitochondrial cargo, assessed by a diminished mitochondrial membrane potential ΔΨm, may be a key determinant of the mitoEV capacity to induce inflammation in target naïve EC. By utilizing MitoTracker fluorescent probes and advanced imaging flow cytometry, we discovered that EV from EC treated with the cytokine tumor necrosis factor (TNF)-α (TNF-EV) had both a lower mitochondrial cargo quality and an increased ability to induce inflammation compared to EV from control donor EC (C-EV). In addition, C-EV treated with the protonophore FCCP (to specifically depolarize their mitochondrial cargo) gained newfound capability of inducing paracrine inflammation. Our findings suggest the mitoEV mitochondrial cargo ΔΨm as a new biomarker of EC dysfunction, atherosclerosis initiation, and CVD development.

Materials and

Methods: : Human umbilical vein EC (HUVEC) were cultured in growth medium until confluency and then treated, or not, with TNF-α (10 ng/ml, 24h). Some EC monolayers were preincubated with MitoTEMPO (MT; 1 μM, 30 min), and remained during the 24h EC exposure to TNF-α. Following treatments, EV were isolated from conditioned media using differential ultracentrifugation. Amnis MkII-401 ImageStream imaging flow cytometer was utilized for EV and mitoEV characterization. Appropriate controls included use of Triton X-100 for EV, EDTA for Annexin V, and RBC-EV for MitoTracker Green measurements (Fig. 1A). The polarization state of mitoEV cargo was determined by incubating EC-EV with FCCP (2.5 μM, 5 min) and gating for the top 5% of MitoTracker Red intensity. EV-associated DNA was isolated and quantified by qPCR using primers for nuclear gene (B2M) and mitochondrial genes (CYTB, MT-TL1, and ND1). Donor EC were incubated with MitoTracker Green to generate mitoEV carrying green-labeled mitochondrial cargo. EV were incubated with recipient EC (virally transduced to express RFP-labeled mitochondria) and internalization was assessed by flow cytometry and fluorescent microscopy. The EV concentration required for paracrine effects was determined using a dose-response of inflammatory gene expression in naive target EC following their incubation with TNF-EV. Paracrine effects were studied by incubating naïve target EC with EC-EV for 24 h. At the end of 24 h, target EC expression of inflammatory genes ICAM-1, VCAM-1, MCP-1, and IL-8 was measured using RT-qPCR and normalized with respect to untreated ECs.

Results, Conclusions, and Discussions:: Flow cytometric analysis of EC-EV identified two subpopulations: mitoEV (Annexin V+/MitoTracker Green+) as EV with mitochondrial cargo (~50% of EC-EV), and dEV (Annexin V+/MitoTracker Green-) as EV devoid of mitochondrial cargo (Fig. 1B). To confirm presence of mitochondrial cargo, total DNA was extracted from lysed EC-EV and the relative abundance of nucDNA (β2M) vs. mtDNA (CYTB, MT-TL1, ND1) was compared. Every mtDNA gene was significantly more abundant suggesting that EV are selectively enriched in mtDNA (Fig. 1C). Treating C-EV with FCCP (2.5 μM, 5 min) completely abolished the MitoTracker Red signal and was used to threshold and define polarized mitoEV. Mitochondria donated from mitoEV colocalized with target EC mitochondria (Fig. 2A). The maximum projection and orthogonal views of z-stacks showed incorporation of donated mitochondria within the 3D space of the target EC mitochondrial network (Fig. 2A). Flow cytometry quantified the EV-mediated mitochondrial transfer: EC incubated with C-EV (ratio of 300 EV:EC) for 24h showed over 80% of recipient EC becoming MitoTracker Green+ (Fig. 2B). Donor EC were treated to alter their redox state (Fig. 3A) and the released EV cargo quality was determined. While TNF-EV contained mostly depolarized mitochondria, TNF/MT-EV cargo quality was restored to that of C-EV (Fig. 3B). EC incubation with either C- or C/MT-EV had no significant effect on ICAM-1, VCAM-1, MCP-1, and IL-8 transcript levels, whereas TNF-EV significantly upregulated all genes confirming the TNF-EV ability to induce inflammation in recipient EC (Fig. 3C). In contrast, TNF/MT-EV failed to induce inflammatory gene expression suggesting a potential link between the mitoEV mitochondrial cargo quality and their proinflammatory ability (Fig. 3C). To verify the role of mitochondrial cargo in conferring pro-inflammatory paracrine effects, C-EV were treated with FCCP (2.5 μM; overnight) and only C-EV/FCCP, but not C-EV, caused a significant upregulation of inflammatory gene expression in target EC compared to vehicle-treated EC (Fig. 3D). Our study highlights the key role of EC-EV mitochondrial cargo membrane potential (ΔΨm), a surrogate for mitochondrial cargo quality, as a key driver and potential therapeutic target in CVD. Pharmacological interventions that prevent mitoEV depolarization could open new avenues for preventing vascular inflammation/dysfunction and CVD.

Acknowledgements and/or References (Optional): : MD/PhD Program at UB-SUNY (Manzar); NIH F30 HL172394 Predoctoral Fellowship (Manzar); NIH R01 HL142673 (Alevriadou); MitoCure Foundation grant award (Alevriadou).