Mitochondrial cargo quality determines the paracrine effects of extracellular vesicles derived from vascular endothelial cells

Cell-derived extracellular vesicles (EV) are mediators of intercellular communication with increased circulating levels of endothelial cell-derived EV (EC-EV) reported in cardiovascular diseases (CVD). The EC-EV ability to elicit either detrimental or restorative effects on target EC is thought to be, in part, due to horizontal transfer of their mitochondrial cargo. To understand the role of mitochondrial cargo in EC-EV paracrine effects, large EV were collected from media of cultured human EC, and the number of mitochondria-carrying EV (mitoEV), EV mitochondrial cargo mass, and mitoEV quality/polarization were quantified. EC activation with tumor necrosis factor (TNF)-α caused an increased release rate of EV (TNF-EV), including mitoEV that carried a larger and more depolarized mitochondrial cargo, compared to EV released from control EC (C-EV). EC co-treatment with TNF-α and the mitochondria-targeted antioxidant MitoTEMPO restored both the mitochondrial cargo quality and the number of mitoEV carrying polarized mitochondria to levels similar to C-EV. TNF-EV, but not C-EV, dose-dependently upregulated inflammatory gene expression in target naïve EC. Fluorescence microscopy showed the EV mitochondrial cargo to transfer and colocalize with the target EC mitochondrial network. Mitochondrial cargo depolarization of C-EV using carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone was sufficient for those EV to trigger inflammation in target naïve EC. In conclusion, the mitochondrial redox state of donor EC regulates mitoEV mitochondrial cargo quality that, at least in part, determines their capacity to cause target EC dysfunction and promote CVD. The mitochondrial membrane potential (ΔΨ_m) in EC-mitoEV may be a new biomarker and therapeutic target in vascular biology and medicine.