Journal of extracellular biology最新文献

Plant-derived substances are widely used as cosmeceutical and food materials owing to their beneficial properties that promote human health, such as antioxidant, nutritional supply and regenerative potential. In particular, nanovesicles (NVs) from plants contain various biomolecules, including signal proteins, nucleic acids, and metabolites, that participate in cross-kingdom communication. In this study, we isolated NVs from Artemisia princeps (APNVs) based on differential centrifugation and further purification via tangential flow filtration (TFF). Evaluation of the effects of these NVs on the cellular proliferation of fibroblasts clearly indicated their anti-ageing potential for the skin. Specifically, exposure of human dermal fibroblast cells to low concentrations of APNVs (100–200 ng/mL) accelerated cell proliferation over a 7-day period. Treatment with APNVs decreased the senescence level of dermal fibroblast cells, as evidenced by senescence-associated β-galactosidase activity connected with cellular ageing. In the anti-ageing efficacy assessment, inhibition of MMP-1 activity in nanovesicle-treated cells was higher than that induced by the positive control epigallocatechin-3-gallate (EGCG). To validate the inhibitory effect of APNVs on anti-ageing in human skin, three-dimensional, reconstituted human keratinocytes and dermal fibroblasts were cultured with 1000 ng/mL APNVs. Notably, procollagen type I expression was increased in the culture medium following APNVs treatment. Our collective results suggest that APNVs accelerate type I procollagen production through inhibition of MMP-1. In view of the significant anti-ageing potential of APNVs, we recommend their implementation as an active substance in pharmaceutical and functional cosmeceutical products.

Neuronal circuitry remodelling, which comprises excitatory and inhibitory neurons, is critical for improving neurological outcomes after a stroke. Preclinical studies have shown that small extracellular vesicles (sEVs) have a therapeutic effect on stroke recovery. However, it is highly challenging to use sEVs to specifically target individual neuronal populations to enhance neuronal circuitry remodelling after stroke. In the present study, using a chemogenetic approach to specifically activate peri-infarct cortical interneurons in combination with the administration of sEVs derived from cerebral endothelial cells (CEC-sEVs), we showed that the CEC-sEVs were preferentially taken up by the activated neurons, leading to significant improvement of functional outcome after stroke, which was associated with augmentation of peri-infarct cortical axonal/dendritic outgrowth and of axonal remodelling of the corticospinal tract. The ultrastructural and Western blot analyses revealed that neurons with internalization of CEC-sEVs exhibited significantly reduced numbers of damaged mitochondria and proteins that mediate dysfunctional mitochondria, respectively. Together, these data indicate that the augmented uptake of CEC-sEVs by activated peri-infarct cortical interneurons facilitates neuronal circuitry remodelling and functional recovery after stroke, which has the potential to be a novel therapy for improving stroke recovery.

Mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) have shown significant therapeutic potential across a wide range of clinical conditions, complementing the progress of MSC-based therapies, some of which have already received regulatory approval. However, the high cost of these therapies has limited their accessibility, creating an urgent need to explore manufacturing strategies that reduce the cost of goods and selling prices. This study presents the design and simulation of a scalable manufacturing platform for the co-production of clinical-grade MSC and MSC-EVs using SuperPro Designer. Various production scenarios were evaluated to maximise manufacturing capacity while analysing their impact on economic performance. Our findings demonstrate that for MSC-EVs doses containing 10¹⁰ and 10¹¹ particles, selling prices range from 166 to 309€ and from 1659 to 3082€, respectively. For clinical doses of MSC, selling prices vary between 965 and 42,673€ depending on dose size and production scale. Importantly, the co-production approach enables cost-sharing between products, contributing to significantly lower prices compared to individual production. Overall, the proposed platform achieved an attractive payback time of 3 years and a return on investment of 36%. By increasing the number of staggered production units, further price reductions and improved economic metrics could be attained. In conclusion, this study highlights the potential of the proposed manufacturing platform to deliver cost-effective, clinical-grade MSC and MSC-EVs products, advancing the field of regenerative medicine and enhancing the accessibility of these innovative treatments.

Several reports have demonstrated that CD147, an N-glycosylated protein that is exchanged by cells in soluble form or through small extracellular vesicles (sEVs), can promote cancer progression. However, its activity related to EVs in colorectal cancer (CRC) is still not fully understood. Previously, we showed that sEV secretion during CRC stem cell (CR-CSCs) differentiation is partially controlled by CD147, and that CD147-expressing sEVs (sEVs-CD147) activate a signalling cascade in recipient cells, inducing molecular invasive features in CR-CSCs. In the present study, we demonstrated that sEVs-CD147 increase the expression of myofibroblast and activation markers in cancer-associated fibroblasts (CAF). In sEVs-CD147-activated CAF, aerobic glycolysis was also triggered by the β-catenin signalling pathway and induced lactate release. These effects were associated with NFKβ upregulation and NO secretion that caused increased cytokines production and VEGF release, respectively. Furthermore, co-culture with CAF promoted CR-CSC invasivity in vitro and tumour growth in vivo. Spatial proteomics analysis confirmed in vivo the activation of fibroblasts and the modulation of their metabolic features, within their biological context, after their conditioning with CD147-expressing sEVs. Our findings indicate that sEV-packaged CD147 is involved in CAF activation, thus promoting tumour progression via stroma metabolism modification.

Extracellular vesicles (EVs) are secreted nanoparticles composed of a lipid bilayer that carry lipid, protein, and nucleic acid cargo between cells as a mode of intercellular communication. Although EVs can promote tissue repair in mammals, their roles in animals with greater regenerative capacity are not well understood. Planarian flatworms are capable of whole-body regeneration due to pluripotent somatic stem cells called neoblasts that proliferate in response to injury. Here, using transmission electron microscopy, nanoparticle tracking analysis, and protein content examination, we showed that EVs enriched from the tissues of the planarian Schmidtea mediterranea had similar morphology and size as other eukaryotic EVs, and that these EVs carried orthologs of the conserved EV biogenesis regulators ALIX and TSG101. PKH67-labeled EVs were taken up efficiently by planarian cells, including S/G2 neoblasts, G1 neoblasts/early progeny, and differentiated cells. When injected into living planarians, EVs from regenerating tissue fragments enhanced the upregulation of neoblast-enriched and proliferation-related transcripts. In addition, EV injection increased the number of F-ara-EdU-labelled cells by 49% as compared to buffer injection only. Our findings demonstrate that regenerating planarians produce EVs that promote stem cell proliferation, and suggest the planarian as an amenable in vivo model for the study of EV function during regeneration.

As a life history strategy, some semelparous organisms, such as the ayu fish (Plecoglossus altivelis), reproduce only once in their lifetime and then die. They invest heavily in their single reproductive event, producing many offspring. However, the regulatory mechanisms that trigger mortality after reproduction are not well understood. Exosomes serve as an essential pathway for intercellular communication, with internal microRNA (miRNA) playing a crucial role in regulating physiological activities within the organism. This study aimed to elucidate the function of exosomal miRNA during the semelparous reproduction period in P. altivelis. Exosomes were successfully extracted from P. altivelis plasma during reproduction, and abundant miRNA molecules were discovered through small RNA sequencing. The miRNA expression patterns in ayu fish during reproduction exhibited notable differences between females and males. Furthermore, it was observed that key cellular processes and signalling pathways associated with intercellular transmission and intracellular stress are regulated by miRNA expression, and these changes in regulation may be responsible for post-breeding mortality. This study enhances our understanding of the function of exosomal miRNA during semelparous reproduction in ayu fish and provides further insight into the intrinsic mechanisms of ageing and mortality in organisms.

The declining birth rates and fertility challenges in Europe have intensified global concerns over rising infertility, particularly among women. This study decisively investigates follicular fluid-related extracellular vesicles (FF-EVs) from infertile patients with polycystic ovary syndrome (PCOS) or diminished ovarian reserve (DOR) undergoing in vitro fertilization (IVF), comparing them to a healthy control group. We have identified significant variations in protein content and polydispersity in crude follicular fluid using UV-Vis absorption and dynamic light scattering (DLS) techniques. Furthermore, the morphology of the extracellular vesicles (EVs) and the patterns of non-coding RNA content, including miRNAs, reveal distinct differences in infertile patients. These findings offer critical insights into the molecular signatures associated with these conditions. This study plays a vital role in advancing reproductive healthcare by pinpointing potential targets that can enhance diagnosis and deepen our understanding of ovarian disorders.

Phoma stem canker disease of oilseed rape (Brassica napus) is caused by the extracellular fungal pathogen Leptosphaeria maculans. Although this pathogen resides exclusively in apoplastic spaces surrounding plant cells, the significance of extracellular vesicles (EVs) has not been assessed. Here, we show a method to collect apoplastic fluids (AFs) from infected leaves or cotyledons for collection of EVs during the process of host colonisation. The 15,000 × g supernatants of AFs were shown to contain ribulose-bisphosphate carboxylase (RuBisCO) at 7 days post-inoculation with L. maculans, a protein that was absent from unchallenged cotyledons. RuBisCO release coincided with the switch from biotrophy to necrotrophy, suggesting the involvement of host cell death. However, RuBisCO release did not differ between compatible and incompatible interactions, suggesting necrotrophic host cell death might not be the only process involved. EVs were also collected from axenic fungal cultures and characterised for their particle size distribution using nanoparticle tracking analysis and transmission electron microscopy. The protein composition of EV-enriched fractions was analysed using SDS-PAGE and proteomics. Enrichment analysis of gene ontology terms provided evidence for involvement of glucan and chitin metabolism as well as catalase and peptidase activities. Most of the proteins identified have previously been found in EV studies and/or EV databases, and for most of the proteins evidence was found for an involvement in pathogenicity/virulence.

Extracellular vesicles (EV) which play critical roles in intercellular communication, have garnered interest as biomarkers with researchers studying brain-related disease processes due to their ability to be isolated from various biofluids. Astrocytes, a type of glial cell, play a critical role in neuronal regulation and function. As such, EV enriched from astrocytes can be used to interrogate cargo and identify mechanisms by which astrocytes communicate with other cells of the central nervous system or shed light on pathophysiological conditions. This manuscript compared five EV isolation methods (differential ultracentrifugation [dUC], precipitation, precipitation + purification, silicon carbon resin and size exclusion chromatography [SEC]) using small volumes of human plasma and serum with a focus on immunocapture of astrocyte-enriched EV (AEEV), with the excitatory amino acid transporter 1, or GLAST. Methods were evaluated on yield, purity, recovery and downstream application to include immunoassays for tetraspanin, immune and astrocyte markers. Results revealed that whilst precipitation-based methods such as ExoQuick yielded higher EV concentrations, size exclusion (SmartSEC, qEV) provided greater purity, emphasizing a trade-off between yield and purity. This study provides a comprehensive resource for researchers in selecting EV isolation methods tailored to small biobanked clinical samples, with the goal of advancing biomarker discovery in Neuroscience.

Proteasomes are essential for protein degradation and maintaining cellular balance, yet their roles in extracellular fluids are not well understood. Our study investigates the freely circulating proteasome in blood, to uncover its unique molecular characteristics, compared to its intracellular counterparts. Using a transgenic mouse model, mass spectrometry, and biochemical tools, we show that the predominant proteasome in serum is the free uncapped 20S particle, which seems to assemble intracellularly before entering the bloodstream. This serum proteasome is composed of constitutive and immuno subunits and exhibits all three catalytic activities. Moreover, the complex displays distinct post-translational modifications, indicating specialization for extracellular roles, as demonstrated by its enhanced caspase-like activity. We also found that physiological stress significantly upregulates serum 20S proteasome levels, paralleling human data. This research highlights the specialized characteristics of circulating proteasomes, offering new insights into protein turnover in the blood with significant implications for understanding proteostasis beyond the intracellular environment.