Mitochondria-containing Extracellular Vesicles (EV) Reduce Mouse Brain Infarct Sizes and EV/HSP27 Protect Ischemic Brain Endothelial Cultures

Abstract

Ischemic stroke causes brain endothelial cell death and damages tight junction integrity of the blood-brain barrier (BBB). We engineered endothelial cell-derived extracellular vesicles (EVs) for the delivery of exogenous heat shock protein 27 (HSP27) and harnessed the innate EV mitochondrial load as a one, two-punch strategy to increase brain endothelial cell survival (via mitochondrial delivery) and preserve their tight junction integrity (via HSP27 delivery). We demonstrated that endothelial microvesicles but not exosomes transferred their mitochondrial load that subsequently underwent fusion with the mitochondrial network of the recipient primary human brain endothelial cells. This mitochondrial transfer increased the relative ATP levels and mitochondrial function in the recipient endothelial cells. EV-mediated HSP27 delivery to primary human brain endothelial cells decreased the paracellular permeability of small and large molecular mass fluorescent tracers in an in vitro model of ischemia/reperfusion injury. This one, two-punch approach to increase the metabolic function and structural integrity of brain endothelial cells is a promising strategy for BBB protection and prevention of long-term neurological dysfunction post-ischemic stroke.Highlights➢Exosomes and microvesicles (EVs) can be engineered for co-delivery of bio-actives➢Microvesicles (MV) but not exosomes contain functional mitochondria➢MV mitochondria fused with the mitochondria in recipient brain endothelial cells➢MVs increase mitochondrial function while EVs increase cellular ATP levels➢EV-mediated HSP27 delivery decreased dextran permeability in brain endothelial cellsGraphical Abstract

Lipidoid nanoparticles increase ATP uptake into hypoxic brain endothelial cells

“…Free ATP has a very short half-life (∼10 minutes). Here, we have formulated ATP-LNPs for ATP uptake by the BECs, whose protection is of central importance in conditions like stroke (14, 66, 67). Our prior works have shown that transfer of mitochondria to hypoxic BECs increased their relative ATP levels and concomitantly, their cell viability (14, 67).…”

“…For studies using cells maintained under normoxic conditions, they were supplemented with the complete growth medium and maintained in a humidified incubator at 37 ºC and 5% CO 2 . For studies under hypoxic conditions, cells were supplemented with the oxygen-glucose deprived (OGD) medium (14) and were maintained in a Billups-Rothenberg chamber pre-flushed with 5% carbon dioxide, 5% hydrogen and 90% nitrogen at maintained at 37 ± 0.5 °C.…”

Effect of homotypicvs. heterotypic interactions on the cellular uptake of extracellular vesicles

“…Both large (m/lEVs) and small (sEVs) EV fractions are collectively referred to as EVs wherever applicable. We used a differential ultracentrifugation method to isolate m/lEVs and sEVs from the EV-conditioned supernatants of BECs and macrophages [11,14,26,27]…”

“…EVs retain membrane signatures (lipids and proteins) reminiscent of their donor/parent cells that may allow increased uptake into recipient cells of the same type [9,10]. Our laboratory has previously demonstrated that m/lEVs derived from brain endothelial cells (BECs) increase mitochondrial function in the recipient BECs and also result in reduced brain tissue infarct volumes in a mouse model of transient ischemic stroke [11][12][13][14]. We have demonstrated that BEC-derived EVs (BEC-EVs) show a greater extent of fluorescent mitochondria signals in the recipient BECs as opposed to EVs derived from a macrophage cell line.…”

“…Ilahibaks et al showed clathrin-dependent endocytosis and macropinocytosis-mediated delivery of human embryonic kidney 293FT (HEK293FT)-derived EVs [24] into T47D stoplight reporter cells. The uptake route of BECderived EVs into recipient BECs is of specific interest to us as we continue to develop these EVs as a unique class of mitochondria carriers [11][12][13][14]25].…”