Method for isolation and molecular characterization of extracellular microvesicles released from brain endothelial cells

To study cell-specific molecular make-up and functions of brain endothelial cell exosomes, methods for isolation of extracellular microvesicles using mass spectrometry-compatible protocols and the characterization of their signature profiles using mass spectrometry -based proteomics were developed.A total of 1179 proteins were identified in the isolated extracellular microvesicles from brain endothelial cells. The microvesicles were validated by identification of almost 60 known markers, including Alix, TSG101 and the tetraspanin proteins CD81 and CD9. The surface proteins on isolated microvesicles could potentially interact with both primary astrocytes and cortical neurons, as cell-cell communication vesicles. Finally, brain endothelial cell extracellular microvesicles were shown to contain several receptors previously shown to carry macromolecules across the blood brain barrier, including transferrin receptor, insulin receptor, LRPs, LDL and TMEM30A.The methods described here permit identification of the molecular signatures for brain endothelial cell-specific extracellular microvesicles under various biological conditions. In addition to being a potential source of useful biomarkers, these vesicles contain potentially novel receptors known for delivering molecules across the blood–brain barrier.Brain endothelial cells (BEC) lining the brain capillaries are sealed by tight junctions and exhibit a specialized molecular and functional phenotype referred to as the blood–brain barrier (BBB). The BBB functions as a physical and enzymatic barrier and employs polarized transport systems to control the exchange of nutrients and macromolecules between the blood and the brain [1]. BECs are tightly integrated with other neighbouring cells, pericytes and astrocytes; astrocytes also communicate with neurons acting as a liaison for endothelial–neuronal coupling (the neurovascular unit; NVU). The luminal, blood-facing surface of BEC is endowed by a thick and dynamic glycocalyx i