Duquesne University Pittsburgh, Pennsylvania, United States
Purpose: Mitochondrial dysfunction is one of the hallmarks of numerous neurodegenerative disorders and brain injuries, including ischemic stroke. Stroke-induced mitochondrial damage in brain endothelial cells (BECs) results in endothelial/neural cell death and contributes to impaired blood-brain barrier integrity. Therefore, restoring mitochondrial function in BECs via exogenous delivery of mitochondrial components is a promising approach to protecting the neurovascular unit in ischemic stroke. Cell-derived extracellular vesicles (EVs), such as small EVs (sEVs, diameter < 200 nm) and medium to large EVs (m/lEVs, diameter >200 nm), hold immense potential as safe and efficient drug carriers due to their lower immunogenicity and inherent homing capabilities to target cells. We have previously demonstrated that the innate EV mitochondria can be transferred to recipient BECs and increase their cellular bioenergetics. Here, we sought to determine if the innate EV mitochondrial load can be further increased by increasing mitochondrial biogenesis in the donor cells. We refer to these mitochondria-enriched EVs as mito-EVs. We hypothesize that mito-EVs could enhance mitochondrial transfer and increase ATP levels of BECs compared to naïve EVs isolated from non-activated cells. Methods: We increased mitochondrial biogenesis in the donor NIH/3T3 fibroblasts and hCMEC/D3 BECs via pharmacological activation of peroxisome proliferator-activated receptor-gamma coactivator (PGC-1α). Naïve sEVs and m/lEVs were isolated from the conditioned medium of non-activated BECs and mito-sEVs and mito-m/lEVs were isolated from activated BECs using a differential centrifugation protocol. We measured particle diameters and particle concentration of mito-EVs and naïve EVs using dynamic light scattering and nanoparticle tracking analysis, respectively. We stained EV mitochondria with MitoTracker deep red (MitoT-red) dye and evaluated EV mitochondria transfer into recipient BECs using fluorescence microscopy and flow cytometry. Naïve EV- and mito-EV-mediated modulation in relative ATP levels of oxygen-glucose deprived BECs was evaluated using luciferase-based ATP assay. Results: Activated NIH/3T3 fibroblast showed a significant increase in cellular PGC-1α expression compared to untreated cells. mito-sEVs (116 nm) did not show a statistically significant change in particle diameter compared to naïve sEVs (115 nm). In contrast, mito-m/lEVs (280 nm) showed a statistically significant (p < 0.0001) increase in particle diameter compared to naïve m/lEVs (205 nm). PGC-1α activation significantly increased sEV and m/lEV concentrations regardless of the donor cell type, and the most increases were noted in the case of mito-m/lEVs. The data likely suggest increased mitochondrial biogenesis due to PGC-1α activation as a result, the secreted mito-m/lEVs have a greater mitochondrial load compared to naïve m/lEVs. m/lEV-mediated mitochondria transfer was considerably higher than sEVs. The greater transfer of m/lEV mitochondria into the recipient BECs cells is likely due to a greater enrichment of functional mitochondria in m/lEVs compared to sEVs. mito-m/lEVs showed considerably greater MitoT-red signals than their naïve m/lEV counterparts, also suggesting incorporation of a greater mitochondrial load in the mito-m/lEVs compared to naïve m/lEVs. mito-sEVs and mito-m/lEVs showed significantly greater mitochondrial transfer at lower EV doses compared to naïve sEVs and m/lEVs. Importantly, BEC-derived naïve and mito-m/lEVs also showed about a three to four-fold increase in relative ATP levels compared to the untreated cells. Interestingly, mito-m/lEV-mediated increase in ATP levels was significantly (p < 0.01) higher than naïve m/lEVs. Conclusion: Our results suggest that the pharmacological modulation of mitochondrial biogenesis in the donor cells can change the mitochondrial load in the secreted m/lEVs. Mito-EVs remained functionally active at levels greater level compared to naïve EVs. In future studies, we will investigate the detailed mitochondrial profile in mito-EVs.
Acknowledgements:A patent application related to this abstract is now under preparation: “D. S Manickam and K. M. Dave, 2022, “Mitochondria-enriched extracellular vesicles”, patent application under preparation by Duquesne University.”
