Abstract
Ischemic stroke causes brain endothelial cell (BEC) death and damages tight junction integrity of the blood-brain barrier (BBB). We harnessed the innate mitochondrial load of endothelial cell-derived extracellular vesicles (EVs) and utilized mixtures of EV/exogenous heat shock protein 27 (HSP27) as a one-two punch strategy to increase BEC survival (via EV mitochondria) and preserve their tight junction integrity (via HSP27 effects). We demonstrated that the medium-to-large (m/lEV) but not small EVs (sEV) transferred their mitochondrial load, which subsequently colocalized with the mitochondrial network of the recipient primary human BECs. BECs treated with m/lEVs increased relative ATP levels and displayed superior mitochondrial function. Importantly, m/lEVs isolated from oligomycin (mitochondrial complex V inhibitor) or rotenone (mitochondrial complex I inhibitor)-exposed BECs (RTN-m/lEVs or OGM-m/lEVs) did not increase BECs ATP levels compared to naïve m/lEVs. In contrast, RTN-sEV and OGM-sEV functionality in increasing cellular ATP levels was minimally impacted in comparison to naïve sEVs. Intravenously administered m/lEVs showed a reduction in brain infarct sizes compared to vehicle-injected mice in a mouse middle cerebral artery occlusion model of ischemic stroke. We formulated binary mixtures of human recombinant HSP27 protein with EVs: EV/HSP27 and ternary mixtures of HSP27 and EV with cationic polymer poly (ethylene glycol)-b-poly (diethyltriamine): (PEG-DET/HSP27)/EV. (PEG-DET/HSP27)/EV and EV/HSP27 mixtures decreased the paracellular permeability of small and large molecular mass fluorescent tracers in oxygen glucose-deprived primary human BECs. This one-two-punch approach to increase BEC metabolic function and tight junction integrity is a promising strategy for BBB protection and prevention of long-term neurological dysfunction post-ischemic stroke.
Highlights
Medium-to-large extracellular vesicles (m/lEVs), not small EVs contain mitochondria
m/lEVs increased ATP and mitochondrial function in brain endothelial cells (BECs)
m/lEVs from oligomycin-exposed BECs did not increase recipient BEC ATP levels
Intravenously injected m/lEVs reduced brain infarct sizes in a mouse stroke model
EV/HSP27 mixtures reduced small and large dextran molecule permeability across BECs
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
We have added new in vivo data demonstrating the potential neuroprotective effects of mitochondria-containing EVs in a mouse model of stroke. Multiple new TEM images show mitochondria in sectioned EVs. Inhibition of mitochondrial complex I and V in the donor cells results in loss of EV mitochondrial function in the recipient cells.
Abbreviations
- BBB
- Blood-brain barrier
- BECs
- Brain endothelial cells
- Calcein AM
- Calcein acetoxymethyl
- ECAR
- Extracellular acidification rate
- EVs
- Extracellular vesicles
- EXOs
- Exosomes
- FT cycle
- Freeze/Thaw cycle
- GAPDH
- Glyceraldehyde 3-phosphate dehydrogenase
- HSP27
- Heat shock protein 27
- hCMEC/D3
- human cerebral microvascular endothelial cell line
- HBMEC
- primary human brain microvascular endothelial cells
- MVs
- Microvesicles
- m/lEV
- medium-to-large EVs
- MitoT-red-EV
- Mitotracker deep red-labeled extracellular vesicles
- OCR
- Oxygen consumption rate
- OGD
- Oxygen-glucose deprivation
- OGD/RP
- Oxygen-glucose deprivation/reperfusion
- PEI
- Polyethylenimine
- PEG-DET
- poly (ethylene glycol)-b-poly (diethyl triamine)
- ROS
- Reactive oxygen species
- sEV
- small EVs
- TRITC
- Tetramethyl rhodamine iso-thiocyanate