Extracellular Vesicles Containing Innate Mitochondria Are a Promising Tool for Increasing Cellular ATP Levels in Ischemic Brain Endothelial Cells

Gina Joy, Anisha D’Souza, and Devika S Manickam, Department of Pharmaceutical Sciences, Duquesne University, 600 Forbes Avenue Pittsburgh, Pennsylvania 15219

Extracellular vesicles (EVs) are naturally secreted by different cells in the body and play a key role in intercellular communication. EVs are membrane-bound and contain a variety of lipids, proteins, and nucleic acids. Recent reports have also suggested the presence of functional mitochondria and mitochondrial DNA in EVs. In ischemic stroke, the blood-brain barrier (BBB) suffers damage leading to cell death and is an emerging target for therapies that can increase endothelial cell survival. The endothelial cells lining the BBB are a critical component whose recovery post-stroke can restore its inherent protective functions and limit further damage to brain tissue. Our goal is to utilize endothelial cell-derived EVs to support cell survival in ischemic events.  Our laboratory has demonstrated that mitochondrial load in EVs (referred henceforth as mito-EVs) can be successfully transferred to recipient cells. We labeled mitochondria in the hCMEC/D3 monolayers (a human brain endothelial cell line used as a surrogate model of the human BBB) using Mitotracker Red and isolated labeled EVs using differential centrifugation. The isolated EVs were characterized using protein gel electrophoresis, particle size analysis, and western blotting to confirm characteristic EV markers. Healthy hCMEC/D3 monolayers treated with the mito-EVs demonstrated robust uptake as determined using fluorescence microscopy. We hypothesize that the delivery of mito-EVs to ischemic endothelial cells will increase intracellular ATP levels, thus switching their fate from cell death to survival. We will treat hCMEC/D3 monolayers exposed to oxygen-glucose deprivation (an in vitro model of stroke) using EVs at doses ranging from 12-200 µg total EV protein. Following exposure to mito-EVs, we will determine the effects of the treatment by measuring ATP levels using a Cell-Glo luminescence assay.  Through these findings, we hope to advance the preclinical testing of EVs for endothelial protection to further improve patient outcomes in stroke therapies.

Additional Abstract Information

Presenter: Gina Joy

Institution: Duquesne University

Type: Poster

Subject: Biochemistry

Status: Approved

Time and Location

Session: Poster 2
Date/Time: Mon 3:00pm-4:00pm
Session Number: 2506