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

Abstract

BackgroundIn addition to possessing intracellular vesicles, eukaryotic cells also produce extracellular microvesicles, ranging from 50 to 1000 nm in diameter that are released or shed into the microenvironment under physiological and pathological conditions. These membranous extracellular organelles include both exosomes (originating from internal vesicles of endosomes) and ectosomes (originating from direct budding/shedding of plasma membranes). Extracellular microvesicles contain cell-specific collections of proteins, glycoproteins, lipids, nucleic acids and other molecules. These vesicles play important roles in intercellular communication by acting as carrier for essential cell-specific information to target cells. Endothelial cells in the brain form the blood–brain barrier, a specialized interface between the blood and the brain that tightly controls traffic of nutrients and macromolecules between two compartments and interacts closely with other cells forming the neurovascular unit. Therefore, brain endothelial cell extracellular microvesicles could potentially play important roles in ‘externalizing’ brain-specific biomarkers into the blood stream during pathological conditions, in transcytosis of blood-borne molecules into the brain, and in cell-cell communication within the neurovascular unit.MethodsTo 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.ResultsA 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.ConclusionsThe 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.

Exosomes in stroke pathogenesis and therapy

“…In the brain, exosomes from cultured glioblastoma cells induce angiogenesis by delivering their contents of proangiogenic proteins, mRNAs, and miRNAs into cerebral endothelial cells (68). Additionally, immortalized human brain microvascular endothelial cells secrete exosomes (69). Proteomic analysis has demonstrated that exosomes released by human cerebral endothelial cells contain 1,179 proteins, including several receptors that carry macromolecules across the BBB, such as transferrin receptor and insulin receptor (69).…”

“…Additionally, immortalized human brain microvascular endothelial cells secrete exosomes (69). Proteomic analysis has demonstrated that exosomes released by human cerebral endothelial cells contain 1,179 proteins, including several receptors that carry macromolecules across the BBB, such as transferrin receptor and insulin receptor (69). The role of these exosomal proteins has not been investigated, but interactions between cerebral endothelial exosomes and pericytes have been studied (70).…”

Extracellular-vesicle type of volume transmission and tunnelling-nanotube type of wiring transmission add a new dimension to brain neuro-glial networks

“…A possible fundamental role could be played by extracellular vesicles called endothelial microparticles (EMPs) released from the brain endothelial cells lining capillaries and forming part of the blood -brain barrier for the bidirectional brain-body connections [83]. Brain endothelial cell extracellular vesicles can externalize brain-specific biomarkers into the blood stream and, via transcytosis, allow the migration of blood-borne molecules into the brain [86,87]. They are present in the circulation and are increased in infectious and thrombotic states indicating an involvement in such states [87,88].…”

In vitro evaluation of endothelial exosomes as carriers for small interfering ribonucleic acid delivery

“…[9][10][11][12] However, only a few were used for drug or short-nucleic-acid delivery. [13][14][15][16][17] We hereby presented the isolation and function characterization of exosomes from endothelial cells to deliver siRNA to endothelial cells.…”