Dietary n-3 polyunsaturated fatty acids alter the number, fatty acid profile and coagulatory activity of circulating and platelet-derived extracellular vesicles: a randomized, controlled crossover trial.

BACKGROUND: Extracellular vesicles (EVs) are proposed to play a role in the development of cardiovascular diseases (CVDs) and are considered emerging markers of CVDs. n-3 PUFAs are abundant in oily fish and fish oil and are reported to reduce CVD risk, but there has been little research to date examining the effects of n-3 PUFAs on the generation and function of EVs. OBJECTIVES: We aimed to investigate the effects of fish oil supplementation on the number, generation, and function of EVs in subjects with moderate risk of CVDs. METHODS: A total of 40 participants with moderate risk of CVDs were supplemented with capsules containing either fish oil (1.9 g/d n-3 PUFAs) or control oil (high-oleic safflower oil) for 12 wk in a randomized, double-blind, placebo-controlled crossover intervention study. The effects of fish oil supplementation on conventional CVD and thrombogenic risk markers were measured, along with the number and fatty acid composition of circulating and platelet-derived EVs (PDEVs). PDEV proteome profiles were evaluated, and their impact on coagulation was assessed using assays including fibrin clot formation, thrombin generation, fibrinolysis, and ex vivo thrombus formation. RESULTS: n-3 PUFAs decreased the numbers of circulating EVs by 27%, doubled their n-3 PUFA content, and reduced their capacity to support thrombin generation by >20% in subjects at moderate risk of CVDs. EVs derived from n-3 PUFA-enriched platelets in vitro also resulted in lower thrombin generation, but did not alter thrombus formation in a whole blood ex vivo assay. CONCLUSIONS: Dietary n-3 PUFAs alter the number, composition, and function of EVs, reducing their coagulatory activity. This study provides clear evidence that EVs support thrombin generation and that this EV-dependent thrombin generation is reduced by n-3 PUFAs, which has implications for prevention and treatment of thrombosis. CLINICAL TRIAL REGISTRY: This trial was registered at clinicaltrials.gov as NCT03203512.

Characterization of Nanohybridosomes from Lipids and Spruce Homogenate Containing Extracellular Vesicles.

Introduction: Lipid nanovesicles associated with bioactive phytochemicals from spruce needle homogenate (here called nano-sized hybridosomes or nanohybridosomes, NSHs) were considered. Methods: We formed NSHs by mixing appropriate amounts of lecithin, glycerol and supernatant of isolation of extracellular vesicles from spruce needle homogenate. We visualized NSHs by light microscopy and cryogenic transmission electron microscopy and assessed them by flow cytometry, dynamic light scattering, ultraviolet-visual spectroscopy, interferometric light microscopy and liquid chromatography-mass spectrometry. Results: We found that the particles consisted of a bilayer membrane and a fluid-like interior. Flow cytometry and interferometric light microscopy measurements showed that the majority of the particles were nano-sized. Dynamic light scattering and interferometric light microscopy measurements agreed well on the average hydrodynamic radius of the particles Rh (between 140 and 180 nm), while the concentrations of the particles were in the range between 1013 and 1014/mL indicating that NSHs present a considerable (more than 25%) of the sample which is much more than the yield of natural extracellular vesicles (EVs) from spruce needle homogenate (estimated less than 1%). Spruce specific lipids and proteins were found in hybridosomes. Discussion: Simple and low-cost preparation method, non-demanding saving process and efficient formation procedure suggest that large-scale production of NSHs from lipids and spruce needle homogenate is feasible.

Naringenin regulates cigarette smoke extract-induced extracellular vesicles from alveolar macrophage to attenuate the mouse lung epithelial ferroptosis through activating EV miR-23a-3p/ACSL4 axis.

BACKGROUND: Alveolar macrophages are one of the momentous regulators in pulmonary inflammatory responses, which can secrete extracellular vesicles (EVs) packing miRNAs. Ferroptosis, an iron-dependent cell death, is associated with cigarette smoke-induced lung injury, and EVs have been reported to regulate ferroptosis by transporting intracellular iron. However, the regulatory mechanism of alveolar macrophage-derived EVs has not been clearly illuminated in smoking-related pulmonary ferroptosis. Despite the known anti-ferroptosis effects of naringenin in lung injury, whether naringenin controls EVs-mediated ferroptosis has not yet been explored. PURPOSE: We explore the effects of EVs from cigarette smoke-stimulated alveolar macrophages in lung epithelial ferroptosis, and elucidate the EV miRNA-mediated pharmacological mechanism of naringenin. STUDY DESIGN AND METHODS: Differential and ultracentrifugation were conducted to extract EVs from different alveolar macrophages treatment groups in vitro. Both intratracheal instilled mice and treated epithelial cells were used to investigate the roles of EVs from alveolar macrophages involved in ferroptosis. Small RNA sequencing analysis was performed to distinguish altered miRNAs in EVs. The ferroptotic effects of EV miRNAs were examined by applying dual-Luciferase reporter assay and miRNA inhibitor transfection experiment. RESULTS: Here, we firstly reported that EVs from cigarette smoke extract-induced alveolar macrophages (CSE-EVs) provoked pulmonary epithelial ferroptosis. The ferroptosis inhibitor ferrostatin-1 treatment reversed these changes in vitro. Moreover, EVs from naringenin and CSE co-treated alveolar macrophages (CSE+Naringenin-EVs) markedly attenuated the lung epithelial ferroptosis compared with CSE-EVs. Notably, we identified miR-23a-3p as the most dramatically changed miRNA among Normal-EVs, CSE-EVs, and CSE+Naringenin-EVs. Further experimental investigation showed that ACSL4, a pro-ferroptotic gene leading to lipid peroxidation, was negatively regulated by miR-23a-3p. The inhibition of miR-23a-3p diminished the efficacy of CSE+Naringenin-EVs. CONCLUSION: Our findings firstly provided evidence that naringenin elevated the EV miR-23a-3p level from CSE-induced alveolar macrophages, thereby inhibiting the mouse lung epithelial ferroptosis via targeting ACSL4, and further complemented the mechanism of cigarette-induced lung injury and the protection of naringenin in a paracrine manner. The administration of miR-23a-3p-enriched EVs has the potential to ameliorate pulmonary ferroptosis.

Bioactive compounds and biological functions of medicinal plant-derived extracellular vesicles.

Extracellular vesicles (EVs) are tiny lipid bilayer-enclosed membrane particles released from a variety of cell types into the surrounding environment. These EVs have massive participated in cell-to-cell communication and interspecies communication. In recent years, plant-derived extracellular vesicles (PDEVs) and "exosome-like" EVs populations found in distinct plants have attracted widespread attention. Especially, research on medicinal plant-derived extracellular vesicles (MPDEVs) are increasing, which are considered a kind of promising natural compound. This review summarizes current knowledge on MPDEVs in terms of bioactive compounds, including small RNA, protein, lipid, and metabolite, have been found on the surface and/or in the lumen of MPDEVs. Moreover, both in vitro and in vivo experiments have shown that MPDEVs exert broad biomedical functions, such as anti-inflammatory, anticancer, antioxidant, modulate microbiota, etc. MPDEVs may be a better substitute than animal-derived extracellular vesicles (ADEVs) because of safety and biocompatibility in the future.

Extracellular vesicles in degenerative retinal diseases: A new therapeutic paradigm.

Nanoscale extracellular vesicles (EVs), consisting of exomers, exosomes and microvesicles/ectosomes, have been extensively investigated in the last 20 years, although their biological role is still something of a mystery. EVs are involved in the transfer of lipids, nucleic acids and proteins from donor to recipient cells or distant organs as well as regulating cell-cell communication and signaling. Thus, EVs are important in intercellular communication and this is not limited to sister cells, but may also mediate the crosstalk between different cell types even over long distances. EVs play crucial functions in both cellular homeostasis and the pathogenesis of diseases, and since their contents reflect the status of the donor cell, they represent an additional valuable source of information for characterizing complex biological processes. Recent advances in isolation and analytical methods have led to substantial improvements in both characterizing and engineering EVs, leading to their use either as novel biomarkers for disease diagnosis/prognosis or even as novel therapies. Due to their capacity to carry biomolecules, various EV-based therapeutic applications have been devised for several pathological conditions, including eye diseases. In the eye, EVs have been detected in the retina, aqueous humor, vitreous body and also in tears. Experiences with other forms of intraocular drug applications have opened new ways to use EVs in the treatment of retinal diseases. We here provide a comprehensive summary of the main in vitro, in vivo, and ex vivo literature-based studies on EVs' role in ocular physiological and pathological conditions. We have focused on age-related macular degeneration, diabetic retinopathy, glaucoma, which are common eye diseases leading to permanent blindness, if not treated properly. In addition, the putative use of EVs in retinitis pigmentosa and other retinopathies is discussed. Finally, we have reviewed the potential of EVs as therapeutic tools and/or biomarkers in the above-mentioned retinal disorders. Evidence emerging from experimental disease models and human material strongly suggests future diagnostic and/or therapeutic exploitation of these biological agents in various ocular disorders with a good possibility to improve the patient's quality of life.

Mesenchymal stromal cells secretome restores bioenergetic and redox homeostasis in human proximal tubule cells after ischemic injury.

BACKGROUND: Ischemia/reperfusion injury is the leading cause of acute kidney injury (AKI). The current standard of care focuses on supporting kidney function, stating the need for more efficient and targeted therapies to enhance repair. Mesenchymal stromal cells (MSCs) and their secretome, either as conditioned medium (CM) or extracellular vesicles (EVs), have emerged as promising options for regenerative therapy; however, their full potential in treating AKI remains unknown. METHODS: In this study, we employed an in vitro model of chemically induced ischemia using antimycin A combined with 2-deoxy-D-glucose to induce ischemic injury in proximal tubule epithelial cells. Afterwards we evaluated the effects of MSC secretome, CM or EVs obtained from adipose tissue, bone marrow, and umbilical cord, on ameliorating the detrimental effects of ischemia. To assess the damage and treatment outcomes, we analyzed cell morphology, mitochondrial health parameters (mitochondrial activity, ATP production, mass and membrane potential), and overall cell metabolism by metabolomics. RESULTS: Our findings show that ischemic injury caused cytoskeletal changes confirmed by disruption of the F-actin network, energetic imbalance as revealed by a 50% decrease in the oxygen consumption rate, increased oxidative stress, mitochondrial dysfunction, and reduced cell metabolism. Upon treatment with MSC secretome, the morphological derangements were partly restored and ATP production increased by 40-50%, with umbilical cord-derived EVs being most effective. Furthermore, MSC treatment led to phenotype restoration as indicated by an increase in cell bioenergetics, including increased levels of glycolysis intermediates, as well as an accumulation of antioxidant metabolites. CONCLUSION: Our in vitro model effectively replicated the in vivo-like morphological and molecular changes observed during ischemic injury. Additionally, treatment with MSC secretome ameliorated proximal tubule damage, highlighting its potential as a viable therapeutic option for targeting AKI.

Mesenchymal stem cell-derived extracellular vesicles for metabolic syndrome therapy: Assessing efficacy with nuclear magnetic resonance spectroscopy.

Metabolic syndrome (MetS) represents a cluster of metabolic abnormalities. The prevalence of MetS has surged, transforming it into a pressing public health concern that could potentially affect around 20%-25% of the global population. As MetS continues its ascent, diverse interventions, pharmacological, nonpharmacological and combined have been deployed. Yet, a comprehensive remedy that fully eradicates MetS symptoms remains elusive, compounded by the risks of polypharmacy's emergence. Acknowledging the imperative to grasp MetS's intricate pathologies, deeper insights for future research and therapy optimisation become paramount. Conventional treatments often target specific syndrome elements. However, a novel approach emerges in mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) therapy, promising a holistic shift. MSC-EVs, tiny membranous vesicles secreted by mesenchymal stem cells, have garnered immense attention for their multifaceted bioactivity and regenerative potential. Their ability to modulate inflammation, enhance tissue repair and regulate metabolic pathways has prompted researchers to explore their therapeutic application in MetS. This review primarily aims to provide an overview of how MSC-EVs therapy can improve metabolic parameters in subjects with MetS disease and also introduce the usefulness of NMR spectroscopy in assessing the efficacy of MSC-EVs therapy for treating MetS.

Therapeutic potential of plant-derived extracellular vesicles as nanocarriers for exogenous miRNAs.

Cell-to-cell communication strategies include extracellular vesicles (EVs) in plants and animals. The bioactive molecules in a diet rich in vegetables and fruits are associated with disease-preventive effects. Plant-derived EVs (PDEVs) are biogenetically and morphologically comparable to mammalian EVs and transport bioactive molecules, including miRNAs. However, the biological functions of PDEVs are not fully understood, and standard isolation protocols are lacking. Here, PDEVs were isolated from four foods with a combination of ultracentrifugation and size exclusion chromatography, and evaluated as vehicles for enhanced transport of synthetic miRNAs. In addition, the role of food-derived EVs as carriers of dietary (poly)phenols and other secondary metabolites was investigated. EVs from broccoli, pomegranate, apple, and orange were efficiently isolated and characterized. In all four sources, 4 miRNA families were present in tissues and EVs. miRNAs present in broccoli and fruit-derived EVs showed a reduced RNase degradation and were ferried inside exposed cells. EVs transfected with a combination of ath-miR159a, ath-miR162a-3p, ath-miR166b-3p, and ath-miR396b-5p showed toxic effects on human cells, as did natural broccoli EVs alone. PDEVs transport trace amounts of phytochemicals, including flavonoids, anthocyanidins, phenolic acids, or glucosinolates. Thus, PDEVs can act as nanocarriers for functional miRNAs that could be used in RNA-based therapy.

Replenishing decoy extracellular vesicles inhibits phenotype remodeling of tissue-resident cells in inflammation-driven arthritis.

The interleukin 6 (IL6) signaling pathway plays pleiotropic roles in regulating the inflammatory milieu that contributes to arthritis development. Here, we show that activation of IL6 trans-signaling induces phenotypic transitions in tissue-resident cells toward an inflammatory state. The establishment of arthritis increases the serum number of extracellular vesicles (EVs), while these EVs express more IL6 signal transducer (IL6ST, also known as gp130) on their surface. Transferring these EVs can block IL6 trans-signaling in vitro by acting as decoys that trap hyper IL6 and prevent inflammatory amplification in recipient arthritic mice. By genetically fusing EV-sorting domains with extracellular domains of receptors, we engineered EVs that harbor a higher quantity of signaling-incompetent decoy receptors. These exogenous decoy EVs exhibit significant potential in eliciting efficient anti-inflammatory effects in vivo. Our findings suggest an inherent resistance of decoy EVs against inflammation, highlighting the therapeutic potential of efficient decoy EVs in treating inflammatory diseases.

Immunomodulatory Potential of Fungal Extracellular Vesicles: Insights for Therapeutic Applications.

Extracellular vesicles (EVs) are membranous vesicular organelles that perform a variety of biological functions including cell communication across different biological kingdoms. EVs of mammals and, to a lesser extent, bacteria have been deeply studied over the years, whereas investigations of fungal EVs are still in their infancy. Fungi, encompassing both yeast and filamentous forms, are increasingly recognized for their production of extracellular vesicles (EVs) containing a wealth of proteins, lipids, and nucleic acids. These EVs play pivotal roles in orchestrating fungal communities, bolstering pathogenicity, and mediating interactions with the environment. Fungal EVs have emerged as promising candidates for innovative applications, not only in the management of mycoses but also as carriers for therapeutic molecules. Yet, numerous questions persist regarding fungal EVs, including their mechanisms of generation, release, cargo regulation, and discharge. This comprehensive review delves into the present state of knowledge regarding fungal EVs and provides fresh insights into the most recent hypotheses on the mechanisms driving their immunomodulatory properties. Furthermore, we explore the considerable potential of fungal EVs in the realms of medicine and biotechnology. In the foreseeable future, engineered fungal cells may serve as vehicles for tailoring cargo- and antigen-specific EVs, positioning them as invaluable biotechnological tools for diverse medical applications, such as vaccines and drug delivery.

MicroRNA profiling of royal jelly extracellular vesicles and their potential role in cell viability and reversing cell apoptosis.

MiRNAs are small non-coding RNA molecules that play important regulatory roles in diverse biological processes. Royal jelly, a milky-white substance produced by nurse honeybees (Apis mellifera), is the primary food of queen bees and plays a crucial role in their development. However, little is known about the microRNA (miRNAs) content of royal jelly and their potential functions. In this study, we isolated extracellular vesicles from the royal jelly of 36 samples through sequential centrifugation and targeted nanofiltration and performed high-throughput sequencing to identify and quantify the miRNA content of honeybee royal jelly extracellular vesicles (RJEVs). We found a total of 29 known mature miRNAs and 17 novel miRNAs. Through bioinformatic analysis, we identified several potential target genes of the miRNAs present in royal jelly, including those involved in developmental processes and cell differentiation. To investigate the potential roles of RJEVs in cell viability, RJEVs were supplemented to apoptotic porcine kidney fibroblasts induced by ethanol 6% exposure for 30 min. TUNEL assay showed a significant reduction in the apoptosis percentage after RJEV supplementation when compared with the non-supplemented control group. Moreover, the wound healing assay performed on the apoptotic cells showed a rapid healing capacity of RJEV-supplemented cells compared to the control group. We observed a significant reduction in the expression of the miRNA target genes such as FAM131B, ZEB1, COL5A1, TRIB2, YBX3, MAP2, CTNNA1, and ADAMTS9 suggesting that RJEVs may regulate the target gene expression associated with cellular motility and cell viability. Moreover, RJEVs reduced the expression of apoptotic genes (CASP3, TP53, BAX, and BAK), while significantly increasing the expression of anti-apoptotic genes (BCL2 and BCL-XL). Our findings provide the first comprehensive analysis of the miRNA content of RJEVs and suggest a potential role for these vesicles in the regulation of gene expression and cell survival as well as augmenting cell resurrection or anastasis.

Extracellular vesicles: A new communication paradigm of complement in neurological diseases.

The complement system is crucial to the innate immune system. It has the function of destroying pathogens by activating the classical, alternative, and lectin pathways. The complement system is important in nervous system diseases such as cerebrovascular and neurodegenerative diseases. Activation of the complement system involves a series of intercellular signaling and cascade reactions. However, research on the source and transport mechanisms of the complement system in neurological diseases is still in its infancy. Studies have increasingly found that extracellular vesicles (EVs), a classic intercellular communication paradigm, may play a role in complement signaling disorders. Here, we systematically review the EV-mediated activation of complement pathways in different neurological diseases. We also discuss the prospect of EVs as future immunotherapy targets.

Selenium Biofortification Enhanced miR167a Expression in Broccoli Extracellular Vesicles Inducing Apoptosis in Human Pancreatic Cancer Cells by Targeting IRS1.

Purpose: Pancreatic adenocarcinoma (PAAD) presents an extremely high morbidity and mortality rate. Broccoli has excellent anti-cancer properties. However, the dosage and serious side effects still limit the application of broccoli and its derivatives for cancer therapy. Recently, extracellular vesicles (EVs) derived from plants are emerging as novel therapeutic agents. Thus, we conducted this study to determine the effectiveness of EVs isolated from Se-riched broccoli (Se-BDEVs) and conventional broccoli (cBDEVs) for the treatment of PAAD. Methods: In this study, we first isolated Se-BDEVs and cBDEVs by a differential centrifugation method, and characterized them by using nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). Then, miRNA-seq was combined with target genes prediction, and functional enrichment analysis to reveal the potential function of Se-BDEVs and cBDEVs. Finally, the functional verification was conducted in PANC-1 cells. Results: Se-BDEVs and cBDEVs exhibited similar characteristics in size and morphology. Subsequent miRNA-seq revealed the expression of miRNAs in Se-BDEVs and cBDEVs. Using a combination of miRNA target prediction and KEGG functional analysis, we found miRNAs in Se-BDEVs and cBDEVs may play an important role in treating pancreatic cancer. Indeed, our in vitro study showed that Se-BDEVs had greater anti-PAAD potency than cBDEVs due to increased bna-miR167a_R-2 (miR167a) expression. Transfection with miR167a mimics significantly induced apoptosis of PANC-1 cells. Mechanistically, further bioinformatics analysis showed that IRS1, which is involved in the PI3K-AKT pathway, is the key target gene of miR167a. Conclusion: This study highlights the role of miR167a transported by Se-BDEVs which could be a new tool for counteracting tumorigenesis.

Palmitate alters miRNA content of small extracellular vesicles secreted from NPY/AgRP-expressing hypothalamic neurons.

Exosomes (sEVs) are extracellular vesicles involved in the pathogenesis of obesity. Notably, exosomal microRNAs (miRNAs) have emerged as crucial mediators of communication between cells and are involved in the development of obesity. One region of the brain known to be dysregulated in obesity is the hypothalamus. It coordinates whole-body energy homeostasis through stimulation and inhibition of the orexigenic neuropeptide (NPY)/agouti-related peptide (AgRP) neurons and anorexigenic proopiomelanocortin (POMC) neurons. A role for hypothalamic astrocytic exosomes in communication with POMC neurons was previously elucidated. Yet, it was unknown whether NPY/AgRP neurons secreted exosomes. We previously established that the saturated fat palmitate alters the intracellular levels of miRNAs and we now questioned whether palmitate would also alter the miRNA content of exosomal miRNAs. We found that the mHypoE-46 cell line secreted particles consistent with the size of exosomes and that palmitate altered levels of a spectrum of miRNAs associated with exosomes. The predicted KEGG pathways of the collective miRNA predicted targets included fatty acid metabolism and type II diabetes mellitus. Of note, one of these altered secreted miRNAs was miR-2137, which was also altered within the cells. We also found that while sEVs collected from the mHypoE-46 neurons increased Pomc mRNA in the mHypoA-POMC/GFP-2 cells after 48 h, the effect was absent with sEVs isolated following palmitate treatment, indicating another potential route by which palmitate promotes obesity. Hypothalamic neuronal exosomes may therefore play a role in the control of energy homeostasis that may be disrupted in obese conditions.

Phosphatidylserine-positive extracellular vesicles boost effector CD8+ T cell responses during viral infection.

CD8+ T cells are crucial for the clearance of viral infections. During the acute phase, proinflammatory conditions increase the amount of circulating phosphatidylserine+ (PS) extracellular vesicles (EVs). These EVs interact especially with CD8+ T cells; however, it remains unclear whether they can actively modulate CD8+ T cell responses. In this study, we have developed a method to analyze cell-bound PS+ EVs and their target cells in vivo. We show that EV+ cell abundance increases during viral infection and that EVs preferentially bind to activated, but not naive, CD8+ T cells. Superresolution imaging revealed that PS+ EVs attach to clusters of CD8 molecules on the T cell surface. Furthermore, EV-binding induces antigen (Ag)-specific TCR signaling and increased nuclear translocation of the transcription factor Nuclear factor of activated T-cells (NFATc1) in vivo. EV-decorated but not EV-free CD8+ T cells are enriched for gene signatures associated with T-cell receptor signaling, early effector differentiation, and proliferation. Our data thus demonstrate that PS+ EVs provide Ag-specific adjuvant effects to activated CD8+ T cells in vivo.

Agricultural dust derived bacterial extracellular vesicle mediated inflammation is attenuated by DHA.

Dietary long-chain omega-3 polyunsaturated fatty acids (n-3 PUFA) and their pro-resolving metabolites are protective against atherosclerotic disease, and ameliorate systemic inflammatory conditions including lupus erythematosus, psoriasis, and bronchial asthma. Organic bioaerosol inhalation is a common and injurious hazard associated with agricultural occupations such as work in swine concentrated animal feeding operations (CAFOs) and is known to increase the risk for developing respiratory conditions such as asthma and COPD. Nearly all cells secrete membrane-bound vesicles (extracellular vesicles, EVs) that have the capacity to transmit protein, nucleic acid, and lipid signaling mediators between cells. Using a polymer-based isolation technique (ExoQuick, PEG) followed by ultracentrifugation, EVs were isolated from CAFO dust extracts, and were quantified and partially characterized. Here, we investigated the role of the n-3 PUFA docosahexaenoic acid (DHA) as a component of n-6 to n-3 PUFA mixtures used to recapitulate physiologically relevant dietary ratios in the resolution of inflammatory injury caused by exposure to EVs carried by agricultural organic dust in vitro. Primary human bronchial epithelial cells, fibroblasts and monocyte-derived macrophages were exposed to EVs isolated from swine CAFO dust. Cells were treated with mixtures of n-6 and n-3 PUFA during recovery from the EV-induced injury. CAFO dust extract (DE) was found to contain EVs that contributed significantly to the overall consequences of exposure to complete DE. DHA-rich PUFA ratios inhibited DE-derived EV-induced proinflammatory cytokine release dose-dependently. DHA-rich PUFA ratios also reversed the damaging effects of EVs on recellularization of lung matrix scaffolds, accelerated wound healing, and stimulated the release of pro-resolution mediators. These results underscore the importance of n-3 PUFA as anti-inflammatory compounds during recovery from EV-laden environmental dust exposure in the context of cellular responses in vitro, warranting future translational studies.

Circulating Extracellular Vesicle-Propagated microRNA Signature as a Vascular Calcification Factor in Chronic Kidney Disease.

BACKGROUND: Chronic kidney disease (CKD) accelerates vascular calcification via phenotypic switching of vascular smooth muscle cells (VSMCs). We investigated the roles of circulating small extracellular vesicles (sEVs) between the kidneys and VSMCs and uncovered relevant sEV-propagated microRNAs (miRNAs) and their biological signaling pathways. METHODS AND RESULTS: We established CKD models in rats and mice by adenine-induced tubulointerstitial fibrosis. Cultures of A10 embryonic rat VSMCs showed increased calcification and transcription of osterix (Sp7), osteocalcin (Bglap), and osteopontin (Spp1) when treated with rat CKD serum. sEVs, but not sEV-depleted serum, accelerated calcification in VSMCs. Intraperitoneal administration of a neutral sphingomyelinase and biogenesis/release inhibitor of sEVs, GW4869 (2.5 mg/kg per 2 days), inhibited thoracic aortic calcification in CKD mice under a high-phosphorus diet. GW4869 induced a nearly full recovery of calcification and transcription of osteogenic marker genes. In CKD, the miRNA transcriptome of sEVs revealed a depletion of 4 miRNAs, miR-16-5p, miR-17~92 cluster-originated miR-17-5p/miR-20a-5p, and miR-106b-5p. Their expression decreased in sEVs from CKD patients as kidney function deteriorated. Transfection of VSMCs with each miRNA-mimic mitigated calcification. In silico analyses revealed VEGFA (vascular endothelial growth factor A) as a convergent target of these miRNAs. We found a 16-fold increase in VEGFA transcription in the thoracic aorta of CKD mice under a high-phosphorus diet, which GW4869 reversed. Inhibition of VEGFA-VEGFR2 signaling with sorafenib, fruquintinib, sunitinib, or VEGFR2-targeted siRNA mitigated calcification in VSMCs. Orally administered fruquintinib (2.5 mg/kg per day) for 4 weeks suppressed the transcription of osteogenic marker genes in the mouse aorta. The area under the curve of miR-16-5p, miR-17-5p, 20a-5p, and miR-106b-5p for the prediction of abdominal aortic calcification was 0.7630, 0.7704, 0.7407, and 0.7704, respectively. CONCLUSIONS: The miRNA transcriptomic signature of circulating sEVs uncovered their pathologic role, devoid of the calcification-protective miRNAs that target VEGFA signaling in CKD-driven vascular calcification. These sEV-propagated miRNAs are potential biomarkers and therapeutic targets for vascular calcification.

Small extracellular vesicle targeting of hypothalamic AMPKα1 promotes weight loss in leptin receptor deficient mice.

BACKGROUND AND AIMS: Leptin receptor (LEPR) deficiency promotes severe obesity and metabolic disorders. However, the current therapeutic options against this syndrome are scarce. METHODS: db/db mice and their wildtypes were systemically treated with neuronal-targeted small extracellular vesicles (sEVs) harboring a plasmid encoding a dominant negative mutant of AMP-activated protein kinase alpha 1 (AMPKα1-DN) driven by steroidogenic factor 1 (SF1) promoter; this approach allowed to modulate AMPK activity, specifically in SF1 cells of the ventromedial nucleus of the hypothalamus (VMH). Animals were metabolically phenotyped. RESULTS: db/db mice intravenously injected with SF1-AMPKα1-DN loaded sEVs showed a marked feeding-independent weight loss and decreased adiposity, associated with increased sympathetic tone, brown adipose tissue (BAT) thermogenesis and browning of white adipose tissue (WAT). CONCLUSION: Overall, this evidence indicates that specific modulation of hypothalamic AMPK using a sEV-based technology may be a suitable strategy against genetic forms of obesity, such as LEPR deficiency.

Oral nicotinamide riboside raises NAD+ and lowers biomarkers of neurodegenerative pathology in plasma extracellular vesicles enriched for neuronal origin.

Declining nicotinamide adenine dinucleotide (NAD+ ) concentration in the brain during aging contributes to metabolic and cellular dysfunction and is implicated in the pathogenesis of aging-associated neurological disorders. Experimental therapies aimed at boosting brain NAD+ levels normalize several neurodegenerative phenotypes in animal models, motivating their clinical translation. Dietary intake of NAD+ precursors, such as nicotinamide riboside (NR), is a safe and effective avenue for augmenting NAD+ levels in peripheral tissues in humans, yet evidence supporting their ability to raise NAD+ levels in the brain or engage neurodegenerative disease pathways is lacking. Here, we studied biomarkers in plasma extracellular vesicles enriched for neuronal origin (NEVs) from 22 healthy older adults who participated in a randomized, placebo-controlled crossover trial (NCT02921659) of oral NR supplementation (500 mg, 2x /day, 6 weeks). We demonstrate that oral NR supplementation increases NAD+ levels in NEVs and decreases NEV levels of Aß42, pJNK, and pERK1/2 (kinases involved in insulin resistance and neuroinflammatory pathways). In addition, changes in NAD(H) correlated with changes in canonical insulin-Akt signaling proteins and changes in pERK1/2 and pJNK. These findings support the ability of orally administered NR to augment neuronal NAD+ levels and modify biomarkers related to neurodegenerative pathology in humans. Furthermore, NEVs offer a new blood-based window into monitoring the physiologic response of NR in the brain.

ESP-B4 promotes nasal epithelial cell-derived extracellular vesicles containing miR-146a-5p to modulate Smad3/GATA-3 thus relieving allergic rhinitis: ESP-B4/miR-146a-5p in AR.

BACKGROUND: Though generally a mild affliction, allergic rhinitis (AR) is very common and causes considerable discomfort. Ephedra sinica polysaccharide is a candidate cost-effective therapy to relieve AR symptoms. PURPOSE: We explore the molecular mechanism of pure polysaccharide ESP-B4 action in AR. METHODS: RPMI2650 cells were treated with lipopolysaccharide to induce an in vitro sensitization model, and extracellular vesicles (EVs) were isolated. A rat model of AR was established using ovalbumin as the allergen and was treated with Ephedra sinica polysaccharide to observe changes in rhinitis symptoms, nasal mucosa histopathology and molecular pathology. ESP-B4-treated sensitized cells were adopted in vitro to verify effect of Ephedra sinica polysaccharide on miR-146a-5p expression in RPMI2650 cell-derived EVs and helper T cell differentiation. RESULTS: miR-146a-5p inhibited Smad3, impeded the Smad3/GATA-3 interaction, upregulated IFN-γ expression, and promoted CD4+T cell Th1 differentiation. Treatment with ESP-B4 relieved AR in rats, and elevated miR-146a-5p in the EVs from the nasal epithelial cells, apparently in relation to effects on helper T cell Th1/Th2 equilibrium. CONCLUSION: Overall, ESP-B4 can promote miR-146a-5p secretion, affect the Th1/Th2 balance of helper T cells, and relieve AR symptoms through Smad3/GATA-3 interaction, thus presenting a potential strategy for AR treatment.