Telocytes‐derived extracellular vesicles alleviate aortic valve calcification by carrying miR‐30b
AIMS: Calcific aortic valve disease (CAVD) is frequent in the elderly. Telocytes (TCs) are implicated in intercellular communication by releasing extracellular vesicles (EVs). This study investigated the role of TC-EVs in aortic valve calcification. METHODS AND RESULTS: TCs were obtained and identified using enzymolysis method and flow cytometry. EVs were isolated from TCs using differential high-speed centrifugation method and identified using transmission electron microscope, western blot, and qNano analysis. The mouse model of CAVD was established. The changes of aortic valve activity-related indicators were analysed by ultrasound, and the expressions of TC markers CD34 and vimentin in mouse valve tissues were detected using RT-qPCR and western blot. The model mice were injected with TC-derived EVs. The expressions of Runx2, osteocalcin, and caspase-3 were detected using RT-qPCR and western blot. The calcification model of valvular interstitial cells (VICs) was established. TC-EVs were co-cultured with calcified VICs, and calcium deposition was detected using alizarin red S staining. miR-30b expression in calcified valvular tissues and cells was detected after EV treatment. miR-30b expression in TCs was knocked down and then EVs were extracted and co-cultured with calcified VICs. The target of miR-30b was predicted through bioinformatics website and verified using dual-luciferase assay. The levels of Wnt/β-catenin pathway-related proteins were detected. ApoE-/- mice fed with a high-fat diet showed decreased aortic valve orifice area, increased aortic transvalvular pressure difference and velocity, reduced left ventricular ejection fraction, decreased CD34 and vimentin, and increased caspase-3, Runx2, and osteocalcin. The levels of apoptosis- and osteogenesis- related proteins were inhibited after EV treatment. TC-EVs reduced calcium deposition and osteogenic proteins in calcified VICs. EVs could be absorbed by VICs. miR-30b expression was promoted in calcified valvular tissues and cells after EV treatment. Knockdown of miR-30b weakened the inhibitory effects of TC-EVs on calcium deposition and osteogenic proteins. miR-30b targeted Runx2. EV treatment inhibited the Wnt/β-catenin pathway, and knockdown of miR-30b in TCs attenuated the inhibitory effect of TC-EVs on the Wnt/β-catenin pathway. CONCLUSION: TC-EVs played a protective role in aortic valve calcification via the miR-30b/Runx2/Wnt/β-catenin axis.
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.