Extracellular Vesicles Research Articles

GRP75 triggers white adipose tissue browning to promote cancer-associated cachexia

Cachexia, which affects 50–80% of cancer patients, is a debilitating syndrome that leads to 20% of cancer-related deaths. A key feature of cachexia is adipose tissue atrophy, but how it contributes to the development of cachexia is poorly understood. Here, we demonstrate in mouse models of cancer cachexia that white adipose tissue browning, which can be a characteristic early-onset manifestation, occurs prior to the loss of body weight and skeletal muscle wasting. By analysing the proteins differentially expressed in extracellular vesicles derived from cachexia-inducing tumours, we identified a molecular chaperone, Glucose-regulated protein 75 (GRP75), as a critical mediator of adipocyte browning. Mechanistically, GRP75 binds adenine nucleotide translocase 2 (ANT2) to form a GRP75–ANT2 complex. Strikingly, stabilized ANT2 enhances its interaction with uncoupling protein 1, leading to elevated expression of the latter, which, in turn, promotes adipocyte browning. Treatment with withanone, a GRP75 inhibitor, can reverse this browning and alleviate cachectic phenotypes in vivo. Overall, our findings reveal a novel mechanism by which tumour-derived GRP75 regulates white adipose tissue browning during cachexia development and suggest a potential white adipose tissue-centred targeting approach for early cachexia intervention.

Exosome-based drug delivery systems for enhanced neurological therapeutics.

Exosomes are small extracellular vesicles naturally secreted by cells into body fluids, enriched with bioactive molecules such as RNAs, proteins, and lipids. These nanosized vesicles play a crucial role in physiological and pathological processes by facilitating intercellular communication and modulating cellular responses, particularly within the central nervous system (CNS). Their ability to cross the blood-brain barrier and reflect the characteristics of their parent cells makes exosomal cargo a promising candidate for biomarkers in the early diagnosis and clinical assessment of neurological conditions. This review offers a comprehensive overview of current knowledge on the characterization of mammalian-derived exosomes, their application as drug delivery systems for neurological disorders, and ongoing clinical trials involving exosome-loaded cargo. Despite their promising attributes, a significant challenge remains the lack of standardized isolation methods, as current techniques are often complex, costly, and require sophisticated equipment, affecting the scalability and affordability of exosome-based therapies. The review highlights the engineering potential of exosomes, emphasizing their ability to be customized for targeted therapeutic delivery through surface modification or conjugation. Future advancements in addressing these challenges and leveraging the unique properties of exosomes could lead to innovative and effective therapeutic strategies in neurology.

Photobiomodulation associated with alginate-based engineered tissue on promoting chondrocytes-derived biological responses for cartilage regeneration

Articular cartilage is a connective tissue with limited self-healing potential, frequently affected by trauma and degenerative changes, leading to osteoarthritis. Photobiomodulation paired with engineered tissue can improve cartilage's poor intrinsic healing and overcome its restricted self-regeneration. In this study, alginate-based scaffolds were fabricated with varying concentrations of CaCl₂ to achieve optimal mechanical, biocompatible, and biodegradable properties. The fluence-dependence of near-infrared (NIR) laser irradiation (830 nm) on chondrocyte viability and proliferation was investigated in a 2D environment across fluences (2.5–10 J/cm2). Optimal conditions of 3 % w/v CaCl₂ and 5 J/cm2 were identified to construct alginate scaffolds and promote chondrocyte growth in 2D and 3D cultures. Single PBM (830 nm, 5 J/cm2) further exhibited a significant relative intensity of collagen type II immunostaining and stimulation of Col2a1 expression in 2D culture. Multiple PBM sessions (830 nm, 5 J/cm2) significantly enhanced chondrocyte proliferation and glycosaminoglycan production in alginate scaffolds, with a protocol of one session every four days being the most effective. Scanning electron microscopy revealed PBM-induced secretory granule formation, corresponding to a significant increase in extracellular vesicle release. Consequently, integrating PBM and alginate-based scaffolds is a promising technique for accelerating and optimizing cartilage regeneration, with potential application in tissue engineering.

The epsilon toxin from Clostridium perfringens stimulates calcium-activated chloride channels, generating extracellular vesicles in Xenopus oocytes.

The epsilon toxin (Etx) from Clostridium perfringens has been identified as a potential trigger of multiple sclerosis, functioning as a pore-forming toxin that selectively targets cells expressing the plasma membrane (PM) myelin and lymphocyte protein (MAL). Previously, we observed that Etx induces the release of intracellular ATP in sensitive cell lines. Here, we aimed to re-examine the mechanism of action of the toxin and investigate the connection between pore formation and ATP release. We examined the impact of Etx on Xenopus laevis oocytes expressing human MAL. Extracellular ATP was assessed using the luciferin-luciferase reaction. Activation of calcium-activated chloride channels (CaCCs) and a decrease in the PM surface were recorded using the two-electrode voltage-clamp technique. To evaluate intracellular Ca2+ levels and scramblase activity, fluorescent dyes were employed. Extracellular vesicles were imaged using light and electron microscopy, while toxin oligomers were identified through western blots. Etx triggered intracellular Ca2+ mobilization in the Xenopus oocytes expressing hMAL, leading to the activation of CaCCs, ATP release, and a reduction in PM capacitance. The toxin induced the activation of scramblase and, thus, translocated phospholipids from the inner to the outer leaflet of the PM, exposing phosphatidylserine outside in Xenopus oocytes and in an Etx-sensitive cell line. Moreover, Etx caused the formation of extracellular vesicles, not derived from apoptotic bodies, through PM fission. These vesicles carried toxin heptamers and doughnut-like structures in the nanometer size range. In conclusion, ATP release was not directly attributed to the formation of pores in the PM, but to scramblase activity and the formation of extracellular vesicles.

Development of human amniotic epithelial cell-derived extracellular vesicles as cell-free therapy for dry eye disease

PurposeThis study aimed to investigate the therapeutic potential of extracellular vesicles (EVs) derived from human amniotic epithelial cells (hAEC-EVs) for Dry Eye Disease (DED) treatment. MethodsHighly purified EVs were isolated from the culture supernatants of hAECs, which obtained from term placenta and characterized. Proteomic contents were analyzed for assessing its biological function related to the therapeutic potentials for DED. Subsequently, we examined the therapeutic efficacy of hAEC-EVs on human corneal epithelial cells exposed to hyperosmotic stress and in an experimental DED mouse model induced by desiccation stress. ResultsProteomic analysis of hAEC-EVs revealed proteins linked to cell proliferation and anti-inflammatory responses. We demonstrated efficient uptake of hAEC-EVs by ocular surface cells. Under DED conditions, EV treatment increased corneal epithelial cell proliferation and migration, and concurrently reducing inflammatory cytokines. In the DED mouse model, hAEC-EVs showed significant improvements in corneal staining score, tear secretion, corneal irregularity, and conjunctival goblet cell density. Additionally, hAEC-EVs exhibited a mitigating effect on ocular surface inflammation induced by desiccation. ConclusionsThese findings suggest that hAEC-EVs hold potential as a cell-free therapy for corneal epithelial defects and ocular surface diseases, presenting a promising treatment option for DED.

Innovative Strategies for Liver Transplantation: The Role of Mesenchymal Stem Cells and Their Cell-Free Derivatives.

Despite being the standard treatment for end-stage liver disease, liver transplantation has limitations like donor scarcity, high surgical costs, and immune rejection risks. Mesenchymal stem cells (MSCs) and their derivatives offer potential for liver regeneration and transplantation. MSCs, known for their multipotency, low immunogenicity, and ease of obtainability, can differentiate into hepatocyte-like cells and secrete bioactive factors that promote liver repair and reduce immune rejection. However, the clinical application of MSCs is limited by risks such as aberrant differentiation and low engraftment rates. As a safer alternative, MSC-derived secretomes and extracellular vesicles (EVs) offer promising therapeutic benefits, including enhanced graft survival, immunomodulation, and reduced ischemia-reperfusion injury. Current research highlights the efficacy of MSC-derived therapies in improving liver transplant outcomes, but further studies are necessary to standardize clinical applications. This review highlights the potential of MSCs and EVs to address key challenges in liver transplantation, paving the way for innovative therapeutic strategies.

Research progress on the therapeutic application of extracellular vesicles in erectile dysfunction.

Erectile dysfunction (ED) is one of the most common male sexual dysfunctions and is related to many pathogenic factors. However, first-line treatment, represented by phosphodiesterase 5 inhibitors, is unable to maintain long-term efficacy. Extracellular vesicles (EVs) have recently attracted the attention of researchers in the fields of cardiovascular disease, neurologic disease, and regenerative medicine and may become a treatment for ED. This article reviews recent applications of EVs in the treatment of ED from the aspects of the source, the therapeutic mechanism, and the strategies to enhance therapeutic efficacy. These research advances lay the foundation for further research and provide references for in-depth understanding of the therapeutic mechanism and possible clinical application of EVs in ED.

Therapeutic Efficacy of Extracellular Vesicles Derived from Stem Cell for Alzheimer's Disease: A Meta-Analysis Study.

Alzheimer's disease (AD) poses a significant public health challenge, increasingly affecting patients' finances, mental health, and functional abilities as the global population ages. Stem cell-derived extracellular vesicles (SC-EVs) have emerged as a promising cell-free therapeutic approach for AD, although their precise mechanisms remain unclear. This meta-analysis aims to evaluate the effectiveness of SC-EVs in treating AD. We systematically searched PubMed, EMBASE, and Web of Science databases up to December 31, 2023, identifying studies investigating SC-EVs therapy in AD rodent models. Outcome measures included Morris water maze and Y maze tests, β-amyloid pathology, and inflammatory markers. Statistical analyses utilized Stata 15.1 and R software. This meta-analysis of 16 studies (2017-2023, 314 animals) demonstrates significant efficacy of SC-EVs therapy in AD models. Pooled analyses demonstrated that SC-EVs therapy significantly increased the learning function as measured by Morris water maze tests (MWM) by -1.83 (95% CI = -2.51 to -1.15, p < 0.0001), Y maze test by 1.66 (95% CI = 1.03 to 2.28, p < 0.0001), decreased Aβ plaques in the hippocampal by -2.10 (95% CI = -2.96 to -1.23, p < 0.0001), and proinflammatory cytokines Tumor necrosis factor alpha (TNFα) by -2.61 (95% CI = -4.87 to -0.35, p < 0.05), Interleukin-1 beta (IL-1β) by -2.37 (95% CI = -3.68 to -1.05, p < 0.001). SC-EVs therapy shows promise in enhancing cognitive function and mitigating AD progression in preclinical models. Future research should focus on standardizing methodologies and comparing SC-EVs isolation techniques and dosing strategies to facilitate clinical translation.

Tumor Microenvironment-Derived Exosomes: A Double-Edged Sword for Advanced T Cell-Based Immunotherapy.

The tumor microenvironment (TME) plays a crucial role in cancer progression and immune evasion, partially mediated by the activity of the TME-derived exosomes. These extracellular vesicles are pivotal in shaping immune responses through the transfer of proteins, lipids, and nucleic acids between cells, facilitating a complex interplay that promotes tumor growth and metastasis. This review delves into the dual roles of exosomes in the TME, highlighting both their immunosuppressive functions and their emerging therapeutic potential. Exosomes can inhibit T cell function and promote tumor immune escape by carrying immune-modulatory molecules, such as PD-L1, yet they also hold promise for cancer therapy as vehicles for delivering tumor antigens and costimulatory signals. Additionally, the review discusses the intricate crosstalk mediated by exosomes among various cell types within the TME, influencing both cancer progression and responses to immunotherapies. Moreover, this highlights current challenges and future directions. Collectively, elucidating the detailed mechanisms by which TME-derived exosomes mediate T cell function offers a promising avenue for revolutionizing cancer treatment. Understanding these interactions allows for the development of targeted therapies that manipulate exosomal pathways to enhance the immune system's response to tumors.

ENaCγ in Urinary Extracellular Vesicles as an Indicator of MR Signaling in PA.

Aldosterone and the MR (mineralocorticoid receptor) are important therapeutic targets for hypertension and cardiovascular diseases. However, biomarkers of tissue MR signaling are not fully established. Extracellular vesicles released from eukaryotic cells can provide information on tissue signaling. Using samples from patients with primary aldosteronism (PA), we explored the potential of urinary extracellular vesicles (uEVs) as a noninvasive indicator of MR signaling to guide treatment. We analyzed proteins contained in PA uEVs by liquid chromatography tandem mass spectrometry. We narrowed down candidate biomarkers by referring to an existing database of urinary exosomes. The results were validated through Western blot analysis involving 63 patients with PA and 11 healthy volunteers. We identified a total of 1940 proteins in PA uEVs. Comparative analysis with the existing database narrowed down the pathways enriched in PA uEVs, which were related to diabetic complications, Rac1 signaling, and aldosterone-regulated sodium reabsorption. A closer look at the identified proteins revealed ENaCγ (epithelial Na+ channel γ) peptides near the proteolytic cleavage sites, and Western blot analysis confirmed the predominant presence of cleaved ENaCγ, a marker of aldosterone signaling in renal tubules. In PA uEVs, cleaved ENaCγ showed a 4.8-fold increase compared with healthy volunteers and was significantly correlated with the aldosterone-to-renin ratio, aldosterone levels, and fractional excretion of K+. Targeted treatment in PA reduced the abundance of cleaved ENaCγ, suggesting a causal role for MR in its induction. This study provides a list of proteins contained in PA uEVs and suggests that ENaCγ in uEVs is a promising biomarker for renal MR signaling.

Heterogeneity in extracellular vesicles: Same same - but different?

Heterogeneity in extracellular vesicles: Same same - but different?

Milk-Derived Exosomes: Innovative Nanocarriers for Enhanced Anticancer Drug Delivery

This in-depth review examines the recently emerging field of using exosomes from milk as anticancer medication nanocarriers. It gives a summary of the most current developments, difficulties, and opportunities in this cutting-edge therapeutic strategy. Exosomes are found in many different body fluids and are considered a promising option for precision medicine due to their biocompatibility and innate cell-targeting abilities. These extracellular vesicles are nanoscale. The review commences with a comprehensive synopsis of the composition and biogenesis of exosomes derived from milk, highlighting their distinct membrane properties and capacity to transport cargo. Interestingly, these naturally occurring nanocarriers hold a variety of bioactive substances that can be precisely delivered as anticancer medications. These substances include proteins, lipids, and nucleic acids. Because dietary(poly) phenols are rapidly metabolized, their ability to prevent cancer is limited. Extracellular vesicles called exosomes may shield polyphenols from metabolism. Our objective was to evaluate the anticancer effects of free curcumin and resveratrol in breast cancer cell lines compared to their encapsulation in extracellular matrix derived from milk. Breast tissue was disposed of kinetically using rats. Curcumin and resveratrol were assessed using UPLC-QTOF-MS and GV-MS, respectively. Dietary polyphenols have a limited capacity to prevent cancer due to their rapid metabolism. Extracellular vesicles called exosomes may shield polyphenols from metabolism. Our goal was to assess in breast tissue. UPLC-QTOF-MS and GV-MS were used to evaluate curcumin and resveratrol, respectively. Curcumin and Resveratrol anticancer activity and bioavailability were improved by milk extracellular urea, which served as Trojan horses to get around the ABC-mediated chemoresistance of cancer cells. Exosomes derived from milk are being studied for their potential as carriers of therapeutic and diagnostic agents, emphasizing the potential benefits of personalized and precision medicine approaches to cancer treatment. The review also covers the challenges that the clinical translation of milk-derived exosome-based drug delivery systems currently faces, including scalability, standardization and safety profiles. The article's conclusion presents an optimistic view of how milk-derived exosomes will develop in the future in terms of anticancer medication delivery. The review highlights the revolutionary potential of using milk-derived exosomes, nature's nanocarriers, to advance the field toward more precise and effective cancer treatments, anticipating future advancements and emerging trends.

Blood Products and Stem Cells in Osteoarthritis Therapy

The principle of regenerative medicine in the treatment of osteoarthritis pursues a functional restoration of cartilage tissue instead of just repairing cartilage defects. The use of blood products is intended to inhibit chronic inflammatory processes and promote tissue regeneration. Intraarticular injection of autologous platelet-rich plasma (PRP) is a prominent procedure. Clinical evidence supports PRP injection over hyaluronic acid or glucocorticoid injection. Comparability of studies is difficult due to missing standardisation of production procedures, dosing and donor variability. In particular, whether presence of residual leukocytes is required or should be avoided is an open debate. In contrast, stem cell therapies in osteoarthritis therapy are often based on mesenchymal stem cells (MSC) from adipose tissue or bone marrow aspirate. Different sources of MSC might render the cells more suitable for application in a given context. Nevertheless, it became evident that their secretome rather than the cells themselves are responsible for observed regenerative processes. Research on the mechanisms of action have focused on growth factors. However, an overlooked component of blood products called extracellular vesicles (EV) came to the center of attention, which are also released by MSC as intercellular signal carriers. EV cargo molecules such as miRNAs open up new dimensions in the investigation and explanation of clinically observed anti-inflammatory and regenerative effects.

SARS-CoV-2 ORF3a Protein Impairs Syncytiotrophoblast Maturation, Alters ZO-1 Localization, and Shifts Autophagic Pathways in Trophoblast Cells and 3D Organoids.

SARS-CoV-2 infection poses a significant risk to placental physiology, but its impact on placental homeostasis is not well understood. We and others have previously shown that SARS-CoV-2 can colonize maternal and fetal placental cells, yet the specific mechanisms remain unclear. In this study, we investigate ORF3a, a key accessory protein of SARS-CoV-2 that exhibits continuous mutations. Our findings reveal that ORF3a is present in placental tissue from pregnant women infected with SARS-CoV-2 and disrupts autophagic flux in placental cell lines and 3D stem-cell-derived trophoblast organoids (SCTOs), impairing syncytiotrophoblast differentiation and trophoblast invasion. This disruption leads to protein aggregation in cytotrophoblasts (CTB) and activates secretory autophagy, increasing CD63+ extracellular vesicle secretion, along with ORF3a itself. ORF3a also compromises CTB barrier integrity by disrupting tight junctions via interaction with ZO-1, mediated by its PDZ-binding motif, SVPL. Colocalization of ORF3a and ZO-1 in SARS-CoV-2-infected human placental tissue supports our in vitro findings. Deleting the PDZ binding motif in the ORF3a protein (ORF3a-noPBM mutant) restored proper ZO-1 localization at the cell junctions in an autophagy-independent manner. Lastly, we demonstrate that constitutive ORF3a expression induces SC-TOs to transition towards a secretory autophagy pathway likely via the PBM motif, as the ORF3a-NoPBM mutants showed a significant lack of CD63 expression. This study demonstrates the functional impact of ORF3a on placental autophagy and reveals a new mechanism for the activation of secretory autophagy, which may lead to increased extracellular vesicle secretion. These findings provide a foundation for exploring therapeutic approaches targeting ORF3a, specifically focusing on its PBM region to block its interactions with host cellular proteins and limiting placental impact.

Airspaces-derived exosomes contain disease-relevant protein signatures in a mouse model of cystic fibrosis (CF)-like mucoinflammatory lung disease.

Exosomes, membrane-bound extracellular vesicles, ranging from approximately 30-200nm in diameter, are released by almost all cell types and play critical roles in intercellular communication. In response to the prevailing stress, the exosome-bound protein signatures vary in abundance and composition. To identify the bronchoalveolar lavage fluid (BALF) exosome-bound proteins associated with mucoinflammatory lung disease and to gain insights into their functional implications, we compared BALF exosomes-derived proteins from adult Scnn1b transgenic (Scnn1b-Tg+) and wild type (WT) mice. A total of 3,144 and 3,119 proteins were identified in BALF exosomes from Scnn1b-Tg+ and WT mice, respectively. Using cutoff criteria (Log2 fold-change > 1 and adjusted p-value < 0.05), the comparison of identified proteins revealed 127 and 30 proteins that were significantly upregulated and downregulated, respectively, in Scnn1b-Tg+ versus WT mice. In addition, 52 and 27 proteins were exclusively enriched in Scnn1b-Tg+ and WT mice, respectively. The identified exosome-bound proteins from the homeostatic airspaces of WT mice were mostly relevant to the normal physiological processes. The protein signatures enriched in the BALF exosomes of Scnn1b-Tg+ mice were relevant to macrophage activation and mucoinflammatory processes. Ingenuity pathway analyses revealed that the enriched proteins in Scnn1b-Tg+ mice contributed to the inflammatory responses and antimicrobial defense pathways. Selective proteins including, RETNLA/FIZZ1, LGALS3/Galectin-3, S100A8/MRP8, and CHIL3/YM1 were immunolocalized to specific cell types. The comparative analysis between enriched BALF exosome proteins and previously identified differentially upregulated genes in Scnn1b-Tg+ versus WT mice suggested that the compartment-/cell-specific upregulation in gene expression dictates the enrichment of their respective proteins in the lung airspaces. Taken together, this study demonstrates that the BALF exosome-bound protein signatures reflect disease-relevant disturbances. Our findings suggest that the exosomes carry disease-relevant protein signatures that can be used as a diagnostic as well as predictive biomarkers for mucoinflammatory lung disease.

Serum Extracellular Vesicles Reveal Metabolic Responses to Time-Restricted Feeding in High Fat Diet-Induced Obesity in Male Mice.

Extracellular vesicle (EV) secretion and cargo composition are dysregulated in metabolic diseases. This study aimed to identify changes in the EV size profile and protein cargoes in diet-induced obesity following time-restricted feeding (TRF) and to establish the role of EVs in obesity-related metabolic responses. Mice were fed a high-fat diet (HFD) for 18 weeks prior to being placed either ad libitum or a time-restricted feeding for an additional 10 weeks. Mice on a normal chow ad libitum served as the control. The TRF group had food available for 10 hours and fasted for 14 hours per day. The serum EV size profile and amount displayed sex- and age-dependent changes in HFD-induced obesity, with age reducing EV amounts. HFD decreased small EV populations and increased larger EV populations, while TRF reversed these changes. Quantitative proteomic analysis showed that the abundance and composition of EV proteins changed in response to both acute stimulation with lipopolysaccharides (LPS) and HFD. Gene ontology analysis identified specific sets of EV proteins and their involved biological processes, reflecting the effect of LPS and HFD, as well as the reversal effect of TRF on metabolic and inflammatory pathways. EV proteins altered by HFD and those reversed by TRF had low protein overlap but significant functional overlap in biological processes. TRF activated the PPAR signaling pathway and the AKT-mTOR signaling pathway. The most significant impacts of HFD and TRF were observed on lipoprotein and carbohydrate metabolism, complement system, and neutrophil degranulation. The reversal effect of TRF on the complement system was pathway-specific, significantly activating the lectin complement pathway and restoring neutrophil degranulation. Our data indicate that EVs are involved in diet-induced metabolic and inflammatory responses. Different EV populations may carry distinct sets of proteins involved in specific biological processes, thereby regulating diverse metabolic pathways efficiently.

Biomarkers and diagnostic significance of non-coding RNAs in extracellular vesicles of pathologic pregnancy.

Intercellular communication is an important mechanism for the development and maintenance of normal biological processes in all organs, including the female reproductive system. Extracellular vesicles, as important carriers of intercellular communication, contain a variety of biologically active molecules, such as mRNAs, miRNAs, lncRNAs, and circRNAs, which are involved in cell-to-cell exchanges as well as in many physiological and pathological processes in the body. Compared with biomarkers found in tissues or body fluids, extracellular vesicles show better stability due to the presence of their envelope membrane which prevents the degradation of the RNA message in their vesicles. Therefore, the genomic and proteomic information contained in extracellular vesicles can serve as important markers and potential therapeutic targets for female reproductive system-related diseases or placental function. Moreover, changes in the expression of non-coding RNAs (mainly miRNAs, lncRNAs, and circRNAs) in maternal extracellular vesicles can accurately and promptly reflect the progress of female reproductive system diseases. The aim of this review is to collect information on different types of non-coding RNAs with key molecular carriers in female pathologic pregnancies (preeclampsia and recurrent spontaneous abortion), so as to explore the relevant molecular mechanisms in female pathologic pregnancies and provide a theoretical basis for clinical research on the pathogenesis and therapeutic approaches of reproductive system diseases. The current state of the art of exosome isolation and extraction is also summarized.

The Systemic Effect of Ischemia Training and Its Impact on Bone Marrow-Derived Monocytes.

Monocytes are innate immune cells that play a central role in inflammation, an essential component during neovascularization. Our recent publication demonstrated that ischemia training by 24 h unilateral occlusion of the femoral artery (FA) can modify bone marrow-derived monocytes (BM-Mono), allowing them to improve collateral remodeling in a mouse model of hindlimb ischemia. Here, we expand on our previous findings, investigating a potential systemic effect of ischemia training and how this training can impact BM-Mono. BM-Mono from mice exposed to ischemia training (24 h) or Sham (same surgical procedure without femoral artery occlusion-ischemia training) procedures were used as donors in adoptive transfer experiments where recipients were subjected to hindlimb ischemia. Donor cells were divided corresponding to the limb from which they were isolated (left-limb previously subjected to 24 h ischemia and right-contralateral limb). Recipients who received 24 h ischemic-trained monocytes isolated from either limb had remarkable blood flow recovery compared to recipients with Sham monocytes (monocytes isolated from Sham group-no ischemia training). Since these data suggested a systemic effect of ischemic training, circulating extracellular vesicles (EVs) were investigated as potential players. EVs were isolated from both groups, 24 h-trained and Sham, and the former showed increased expression of histone deacetylase 1 (HDAC1), which is known to downregulate 24-dehydrocholesterol reductase (Dhcr24) gene expression. Since we previously revealed that ischemia training downregulates Dhcr24 in BM-Mono, we incubated EVs from 24 h-trained and Sham groups with wild-type (WT) BM-Mono and demonstrated that WT BM-Mono incubated with 24 h-trained EVs had lower gene expression of Dhcr24 and an HDAC1 inhibitor blunted this effect. Next, we repeated the adoptive transfer experiment using Dhcr24 KO mice as donors of BM-Mono for WT mice subjected to hindlimb ischemia. Recipients who received Dhcr24 KO BM-Mono had greater limb perfusion than those who received WT BM-Mono. Further, we focused on the 24 h-trained monocytes (which previously showed downregulation of Dhcr24 gene expression and higher desmosterol) to test the expression of a few genes downstream of the desmosterol pathway, confirm the Dhcr24 protein level and assess its differentiation in M2-like macrophage phenotype. We found that 24 h-trained BM-Mono had greater expression of key genes in the desmosterol pathway, such as liver X receptors (LXRs) and ATP-binding cassette transporter (ABCA1), and we confirmed low protein expression of Dhcr24. Further, we demonstrated that ischemic-trained BM-Mono polarized towards an anti-inflammatory M2 macrophage phenotype. Finally, we demonstrated that 24 h-trained monocytes adhere less to endothelial cells, and the same pattern was shown by WT BM-Mono treated with Dhcr24 inhibitor. Ischemia training leads to a systemic effect that, at least in part, involves circulating EVs and potential epigenetic modification in BM-Mono. These ischemic-trained BM-Mono demonstrated an anti-inflammatory phenotype towards M2 macrophage differentiation and less ability to adhere to endothelial cells, which is associated with the downregulation of Dhcr24 in those cells. These data together suggest that Dhcr24 might be an important target within monocytes to improve the outcomes of hindlimb ischemia.

Investigation of cell-to-cell transfer of polystyrene microplastics through extracellular vesicle-mediated communication

Plastics are an essential part of human life and their production is increasing every year. Plastics degrade into small particles (<5 mm, microplastics, MPs) in the environment due to various factors. MPs are widely distributed in the environment, and all living organisms are exposed to the effects of MPs. Extracellular vesicles (EVs) are small membrane particles surrounded by a lipid bilayer that are released into the environment by various cell types and are highly involved in inter- and intra-cellular communication through the exchange of proteins, nucleic acids, and lipids between cells. There have been numerous reports of adverse effects associated with the accumulation of MPs in human and animal cells, with recent studies showing that plastic treatment increases the number of EVs released from cells, but the mechanisms by which MPs accumulate and move between cells remain unclear. In this study, we investigated whether polystyrene (PS)-MPs are transferred cell-to-cell via EVs. This study showed that cell-derived EVs can transport plastic particles. Furthermore, we confirmed the accumulation of PS-MPs transported by EVs within cells using a real-time imaging device. This study provides an understanding of potential EVs-mediated effects of PS-MPs on organisms and suggests directions for further research.

Extracellular vesicles from cancer cell lines of different origins drive the phenotype of normal oral fibroblasts in a CAF-like direction.

Normal oral fibroblasts (NOFs) are located in the connective tissue of the oral mucosa. The NOFs play an important role in wound healing, tumor progression, and metastasis. They are subjected to influence by external and internal stimuli, among them extracellular vesicles (EVs), that are considered as important players in cell to cell communication, especially in carcinogenesis and metastatic processes. During tumorigenesis, stromal NOFs may undergo activation into cancer-associated fibroblasts (CAFs) that modify their phenotype to provide pro-oncogenic signals that in turn facilitate tumor initiation, progression, and metastasis. The aim of the study was to reveal the effect of EVs derived from local (oral squamous cell carcinoma - OSCC) and distant (pancreatic adenocarcinoma - PDAC; malignant melanoma brain metastasis - MBM) cancer origin on NOFs and their possible change into a CAF-like direction. The effect of each of the cancer EV types on NOFs proliferation, viability, and migration was tested. Also, changes in gene expression of the well-established CAF biomarkers ACTA2, FAP, PDGFR, and two putative CAF biomarkers, the Ca2+- activated ion channels ANO1 and KCNMA, were studied. Obtained results indicate that NOFs receive and process signals transmitted by EVs originating from both OSCC, PDAC, and MBM. The fibroblast response was dependent on EV origin and concentration, and duration of EV exposure. The present results indicate that the molecular cargo of the EVs direct NOFs towards a pro-tumorigenic phenotype.