Urinary extracellular vesicles as a source of protein‐based biomarkers in feline chronic kidney disease and hypertension
OBJECTIVES: To validate a methodology for isolating feline urinary extracellular vesicles and characterise the urinary extracellular vesicle population and proteome in cats with normal renal function and cats with normotensive or hypertensive chronic kidney disease. METHODS: Feline urinary extracellular vesicles were isolated using three different methods (precipitation alone, precipitation followed by size exclusion chromatography and ultrafiltration followed by size exclusion chromatography, which were compared via transmission electron microscopy and nanoparticle tracking analysis. Cats with normal renal function (n=9), normotensive chronic kidney disease (n=10) and hypertensive chronic kidney disease (n=9) were identified and urinary extracellular vesicles isolated from patient urine samples via ultrafiltration followed by size exclusion chromatography. Extracellular vesicle size and concentration were determined using nanoparticle tracking analysis, and subsequently underwent proteomic analysis using liquid chromatography with tandem mass spectrometry to identify differences in protein expression between categories. RESULTS: Urinary extracellular vesicle preparations contained particles of the expected size and morphology, and those obtained by ultrafiltration + size exclusion chromatography had a significantly higher purity (highest particle: protein ratio). The urinary extracellular vesicle proteomes contained extracellular vesicle markers and proteins originating from all nephron segments. Urinary extracellular vesicle concentration and size were unaffected by renal disease or hypertension. There were no differentially expressed proteins detected when comparing urinary extracellular vesicles derived from cats in the healthy category with the combined chronic kidney disease category, but five differentially expressed proteins were identified between the normotensive chronic kidney disease and hypertensive chronic kidney disease categories. CLINICAL SIGNIFICANCE: Feline urinary extracellular vesicles can be successfully isolated from stored urine samples. Differentially expressed urinary extracellular vesicle proteins were discovered in cats with hypertensive chronic kidney disease, and warrant further investigation into their utility as biomarkers or therapeutic targets.
Phospholipid fatty acid remodeling and carbonylated protein increase in extracellular vesicles released by airway epithelial cells exposed to cigarette smoke extract
Cigarette smoke (CS) represents one of the most relevant environmental risk factors for several chronic pathologies. Tissue damage caused by CS exposure is mediated, at least in part, by oxidative stress induced by its toxic and pro-oxidant components. Evidence demonstrates that extracellular vesicles (EVs) released by various cell types exposed to CS extract (CSE) are characterized by altered biochemical cargo and gained pathological properties. In the present study, we evaluated the content of oxidized proteins and phospholipid fatty acid profiles of EVs released by human bronchial epithelial BEAS-2B cells treated with CSE. This specific molecular characterization has hitherto not been performed. After confirmation that CSE reduces viability of BEAS-2B cells and elevates intracellular ROS levels, in a dose-dependent manner, we demonstrated that 24 h exposure at 1% CSE, a concentration that only slight modifies cell viability but increases ROS levels, was able to increase carbonylated protein levels in cells and released EVs. The release of oxidatively modified proteins via EVs might represent a mechanism used by cells to remove toxic proteins in order to avoid their intracellular overloading. Moreover, 1% CSE induced only few changes in the fatty acid asset in BEAS-2B cell membrane phospholipids, whereas several rearrangements were observed in EVs released by CSE-treated cells. The impact of changes in acyl chain composition of CSE-EVs accounted for the increased saturation levels of phospholipids, a membrane parameter that might influence EV stability, uptake and, at least in part, EV-mediated biological effects. The present in vitro study adds new information concerning the biochemical composition of CSE-related EVs, useful to predict their biological effects on target cells. Furthermore, the information regarding the presence of oxidized proteins and the specific membrane features of CSE-related EVs can be useful to define the utilization of circulating EVs as marker for diagnosing of CS-induced lung damage and/or CS-related diseases.
A portable elliptical dichroism spectrometer targeting secondary structural features of tumorous protein for pancreatic cancer detection
Stereochemical analysis is essential for understanding the complex function of biomolecules. Various direct and indirect approaches can be used to explore the allosteric configuration. However, the size, cost, and delicate nature of these systems limit their biomedical usage. Here, we constructed elliptical dichroism (ED) spectrometer for biomedical applications, whose performance is validated by experiment and theoretical simulation (Jones/Mueller calculus and time-dependent density-functional theory). Instead of complicated control of circular polarization, ED spectrometer adopted the absorbance of left- and right-oriented elliptically polarized light. With a simplified design, we demonstrated the potential of ED spectrometry as an alternative for secondary structural analysis of biomolecules, their conformation and chirality. It not only provides a portable, low-cost alternative to the sophisticated instruments currently used for structural analysis of biomolecules but also provides superior translational features: low sample consumption(200μl), easy operation, and multiple working modes, for noninvasive cancer detection.
Endosomal escape of nucleic acids from extracellular vesicles mediates functional therapeutic delivery
Extracellular vesicles hold great promise as a drug delivery platform for RNA-based therapeutics. However, there is a lack of experimental evidence for the intracellular trafficking of nucleic acid cargos, specifically, whether they are capable of escaping from the endolysosomal confinement in the recipient cells to be released into the cytosol and hence, interact with their cytoplasmic targets. Here, we demonstrated how red blood cell-derived extracellular vesicles (RBCEVs) release their therapeutic RNA/DNA cargos at specific intracellular compartments characteristic of late endosomes and lysosomes. The released cargos were functional and capable of knocking down genes of interest in recipient cells, resulting in tumor suppression in vitro and in an acute myeloid leukemia murine model without causing significant toxicity. Notably, surface functionalization of RBCEVs with an anti-human CXCR4 antibody facilitated their specific uptake by CXCR4+ leukemic cells, leading to enhanced gene silencing efficiency. Our results provide insights into the cellular uptake mechanisms and endosomal escape routes of nucleic acid cargos delivered by RBCEVs which have important implications for further improvements of the RBCEV-based delivery system.