High-throughput surface epitope immunoaffinity isolation of extracellular vesicles and downstream analysis.

Extracellular vesicles (EVs), including exosomes, have significant potential for diagnostic and therapeutic applications. The lack of standardized methods for efficient and high-throughput isolation and analysis of EVs, however, has limited their widespread use in clinical practice. Surface epitope immunoaffinity (SEI) isolation utilizes affinity ligands, including antibodies, aptamers, or lectins, that target specific surface proteins present on EVs. Paramagnetic bead-SEI isolation represents a fit-for-purpose solution for the reproducible, high-throughput isolation of EVs from biofluids and downstream analysis of RNA, protein, and lipid biomarkers that is compatible with clinical laboratory workflows. This study evaluates a new SEI isolation method for enriching subpopulations of EVs. EVs were isolated from human plasma using a bead-based SEI method designed for on-bead and downstream analysis of EV-associated RNA and protein biomarkers. Western blot analysis confirmed the presence of EV markers in the captured nanoparticles. Mass spectrometry analysis of the SEI lysate identified over 1500 proteins, with the top 100 including known EV-associated proteins. microRNA (miRNA) sequencing followed by RT-qPCR analysis identified EV-associated miRNA transcripts. Using SEI, EVs were isolated using automated high-throughput particle moving instruments, demonstrating equal or higher protein and miRNA yield and recovery compared to manual processing. SEI is a rapid, efficient, and high-throughput method for isolating enriched populations of EVs; effectively reducing contamination and enabling the isolation of a specific subpopulation of EVs. In this study, high-throughput EV isolation and RNA extraction have been successfully implemented. This technology holds great promise for advancing the field of EV research and facilitating their application for biomarker discovery and clinical research.

Differential response of placental cells to high D-glucose and its impact on extracellular vesicle biogenesis and trafficking via small GTPase Ras-related protein RAB-7A.

Placental extracellular vesicles (EVs) can be found in the maternal circulation throughout gestation, and their concentration, content and bioactivity are associated with pregnancy outcomes, including gestational diabetes mellitus (GDM). However, the effect of changes in the maternal microenvironment on the mechanisms associated with the secretion of EVs from placental cells remains to be fully established. Here, we evaluated the effect of high glucose on proteins associated with the trafficking and release of different populations of EVs from placental cells. BeWo and HTR8/SVneo cells were used as placental models and cultured under 5-mM D-glucose (i.e. control) or 25-mM D-glucose (high glucose). Cell-conditioned media (CCM) and cell lysate were collected after 48 h. Different populations of EVs were isolated from CCM by ultracentrifugation (i.e. pellet 2K-g, pellet 10K-g, and pellet 100K-g) and characterised by Nanoparticle Tracking Analysis. Quantitative proteomic analysis (IDA/SWATH) and multiple reaction monitoring protocols at high resolution (MRMHR) were developed to quantify 37 proteins related to biogenesis, trafficking/release and recognition/uptake of EVs. High glucose increased the secretion of total EVs across the pellets from BeWo cells, an effect driven mainly by changes in the small EVs concentration in the CCM. Interestingly, no effect of high glucose on HTR8/SVneo cells EVs secretion was observed. High glucose induces changes in proteins associated with vesicle trafficking in BeWo cells, including Heat Shock Protein Family A (Hsp70) Member 9 (HSPA9) and Member 8 (HSPA8). For HTR8/SVneo, altered proteins including prostaglandin F2α receptor regulatory protein (FPRP), RAB5A, RAB35, RAB5B, and RB11B, STAM1 and TSG101. These proteins are associated with the secretion and trafficking of EVs, which could explain in part, changes in the levels of circulating EVs in diabetic pregnancies. Further, we identified that proteins RAB11B, PDCD6IP, STAM, HSPA9, HSPA8, SDCBP, RAB5B, RAB5A, RAB7A and ERAP1 regulate EV release in response to high and low glucose when overexpressed in cells. Interestingly, immunohistochemistry analysis of RAB7A revealed distinct changes in placental tissues obtained from women with normal glucose tolerance (NGT, n = 6) and those with GDM (n = 6), influenced by diet or insulin treatment. High glucose regulation of proteins involved in intercellular dynamics and the trafficking of multivesicular bodies to the plasma membrane in placental cells is relevant in the context of GDM pregnancies.

Enhancing precision targeting of ovarian cancer tumor cells in vivo through extracellular vesicle engineering.

Extracellular vesicles (EVs) function as natural mediators of intercellular communication, secreted by cells to facilitate cell-cell signaling. Due to their low toxicity, immunogenicity, biodegradability, and potential to encapsulate therapeutic drugs, EVs hold significant therapeutic promise. Nevertheless, their limited targeting ability often diminishes their therapeutic impact. Therefore, enhancing EVs by incorporating targeting units onto their membranes could bolster their targeting capabilities, enabling them to accumulate in specific cells and tissues. In this study, we engineered EVs to fuse ephrin-B2 with the EV membrane protein LAMP2b. This modification aimed to direct the engineered EVs toward the ephrin-B4 receptor expressed on the surface of ovarian cancer cells. The engineered EVs retained their inherent properties, including size, expression of EV membrane proteins, and morphology, upon isolation. In vitro experiments using real-time imaging revealed that EVs engineered with the ephrin-B2 ligand exhibited substantial internalization and uptake by ovarian cancer cells, in stark contrast to native EVs. In vivo, the engineered EVs carrying the ephrin-B2 ligand effectively targeted ovarian cancer cells, surpassing the targeting efficiency of control EVs. This innovative approach establishes a novel targeting system, enhancing the uptake of EVs by ovarian cancer cells. Our findings underscore the potential of using EVs to target cancer cells, thereby enhancing the effectiveness of anti-cancer therapies while minimizing off-target effects and toxicity in normal cells and organs.

Unveiling clinical applications of bacterial extracellular vesicles as natural nanomaterials in disease diagnosis and therapeutics.

Bacterial extracellular vesicles (BEVs) are naturally occurring bioactive membrane-bound nanoparticles released by both gram-negative and gram-positive bacterial species, exhibiting a multifaceted role in mediating host-microbe interactions across various physiological conditions. Increasing evidence supports BEVs as essential mediators of cell-to-cell communicaiton, influencing bacterial pathogenicity, disease mechanisms, and modulating the host immune response. However, the extent to which these BEV-mediated actions can be leveraged to predict disease onset, guide treatment strategies, and determine clinical outcomes remains uncertain, particularly in terms of their clinical translation potentials. This review briefly describes BEV biogenesis and their internalisation by recipient cells and summarises methods for isolation and characterization, essential for understanding their composition and cargo. Further, it discusses the potential of biofluid-associated BEVs as biomarkers for various diseases, spanning both cancer and non-cancerous conditions. Following this, we outline the ongoing human clinical trials of using BEVs for vaccine development. In addition to disease diagnostics, this review explores the emerging research of using natural or engineered BEVs as smart nanomaterials for applications in anti-cancer therapy and bone regeneration. This discussion extends to key factors for unlocking the clinical potential of BEVs, such as standardization of BEV isolation and characterisation, as well as other hurdles in translating these findings to the clinical setting. We propose that addressing these hurdles through collaborative research efforts and well-designed clinical trials holds the key to fully harnessing the clinical potential of BEVs. As this field advances, this review suggests that BEV-based nanomedicine has the potential to revolutionize disease management, paving the way for innovative diagnosis, therapeutics, and personalized medicine approaches. STATEMENT OF SIGNIFICANCE: Extracellular vesicles (EVs) from both host cells and bacteria serve as multifunctional biomaterials and are emerging in the fields of biomedicine, bioengineering, and biomaterials. However, the majority of current studies focus on host-derived EVs, leaving a gap in comprehensive research on bacteria-derived EVs (BEVs). Although BEVs offer an attractive option as nanomaterials for drug delivery systems, their unique nanostructure and easy-to-modify functions make them a potential method for disease diagnosis and treatment as well as vaccine development. Our work among the pioneering studies investigating the potential of BEVs as natural nanobiomaterials plays a crucial role in both understanding the development of diseases and therapeutic interventions.

Correction: Soda et al. Electrochemical Detection of Global DNA Methylation Using Biologically Assembled Polymer Beads. Cancers 2021, 13, 3787.

In the original publication [...].

IFPA Joan Hunt Senior Award in Placentology lecture: Extracellular vesicle signalling and pregnancy.

The field of extracellular vesicle (EV) signalling has the potential to transform our understanding of maternal-fetal communication and affords new opportunities for non-invasive prenatal testing and therapeutic intervention. EVs have been implicated in implantation, placentation, maternal adaptation to pregnancy and complications of pregnancy, being detectable in maternal circulation as early as 6 weeks of pregnancy. EVs of differing biogenic origin, composition and bioactivity are released by cells to maintain homoeostasis. Induction of EV signalling is associated with aberrant cellular metabolism and manifests as changes in EV concentrations and/or composition. Characterizing such changes affords opportunity to develop more informative diagnostics and efficacious interventions. To develop accurate and reliable EV-based diagnostics requires: identification of disease-associated biomarkers in specific EV subpopulations; and rapid, reproducible and scalable sample processing. Conventional isolation methods face challenges due to co-isolation of particles with similar physicochemical properties. Methods targeting specific vesicle-surface epitopes and compatible with automated platforms show promise. Effective EV therapeutics require precise targeting, achieved through genetic engineering to release EVs expressing cell-targeting ligands and carrying therapeutic payloads. Unlike cell-based therapies, this approach offers advantages including: low immunogenicity; stability; and long-term storage. Although EV diagnostics and therapeutics in reproductive biology are nascent, available technologies can enhance our understanding of EV signalling between mother and fetus, its role in pregnancies and improve outcomes.

Whole transcriptome profiling of placental pathobiology in SARS-CoV-2 pregnancies identifies placental dysfunction signatures.

Objectives: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) virus infection in pregnancy is associated with higher incidence of placental dysfunction, referred to by a few studies as a 'preeclampsia-like syndrome'. However, the mechanisms underpinning SARS-CoV-2-induced placental malfunction are still unclear. Here, we investigated whether the transcriptional architecture of the placenta is altered in response to SARS-CoV-2 infection. Methods: We utilised whole-transcriptome, digital spatial profiling, to examine gene expression patterns in placental tissues from participants who contracted SARS-CoV-2 in the third trimester of their pregnancy (n = 7) and those collected prior to the start of the coronavirus disease 2019 (COVID-19) pandemic (n = 9). Results: Through comprehensive spatial transcriptomic analyses of the trophoblast and villous core stromal cell subpopulations in the placenta, we identified SARS-CoV-2 to promote signatures associated with hypoxia and placental dysfunction. Notably, genes associated with vasodilation (NOS3), oxidative stress (GDF15, CRH) and preeclampsia (FLT1, EGFR, KISS1, PAPPA2) were enriched with SARS-CoV-2. Pathways related to increased nutrient uptake, vascular tension, hypertension and inflammation were also enriched in SARS-CoV-2 samples compared to uninfected controls. Conclusions: Our findings demonstrate the utility of spatially resolved transcriptomic analysis in defining the underlying pathogenic mechanisms of SARS-CoV-2 in pregnancy, particularly its role in placental dysfunction. Furthermore, this study highlights the significance of digital spatial profiling in mapping the intricate crosstalk between trophoblasts and villous core stromal cells, thus shedding light on pathways associated with placental dysfunction in pregnancies with SARS-CoV-2 infection.

Comprehensive strategy for identifying extracellular vesicle surface proteins as biomarkers for chronic kidney disease.

Chronic kidney disease (CKD) poses a significant health burden worldwide. Especially, obesity-induced chronic kidney disease (OCKD) is associated with a lack of accuracy in disease diagnostic methods. The identification of reliable biomarkers for the early diagnosis and monitoring of CKD and OCKD is crucial for improving patient outcomes. Extracellular vesicles (EVs) have emerged as potential biomarkers in the context of CKD. In this review, we focused on the role of EVs as potential biomarkers in CKD and OCKD and developed a comprehensive list of EV membrane proteins that could aid in the diagnosis and monitoring of the disease. To assemble our list, we employed a multi-step strategy. Initially, we conducted a thorough review of the literature on EV protein biomarkers in kidney diseases. Additionally, we explored papers investigating circulating proteins as biomarkers in kidney diseases. To further refine our list, we utilized the EV database Vesiclepedia.org to evaluate the qualifications of each identified protein. Furthermore, we consulted the Human Protein Atlas to assess the localization of these candidates, with a particular focus on membrane proteins. By integrating the information from the reviewed literature, Vesiclepedia.org, and the Human Protein Atlas, we compiled a comprehensive list of potential EV membrane protein biomarkers for CKD and OCKD. Overall, our review underscores the potential of EVs as biomarkers in the field of CKD research, providing a foundation for future studies aimed at improving CKD and OCKD diagnosis and treatment.

Advances in extracellular vesicles as mediators of cell-to-cell communication in pregnancy.

Cell-to-cell communication mediated by Extracellular Vesicles (EVs) is a novel and emerging area of research, especially during pregnancy, in which placenta derived EVs can facilitate the feto-maternal communication. EVs comprise a heterogeneous group of vesicle sub-populations with diverse physical and biochemical characteristics and originate by specific biogenesis mechanisms. EVs transfer molecular cargo (including proteins, nucleic acids, and lipids) between cells and are critical mediators of cell communication. There is growing interest among researchers to explore into the molecular cargo of EVs and their functions in a physiological and pathological context. For example, inflammatory mediators such as cytokines are shown to be released in EVs and EVs derived from immune cells play key roles in mediating the immune response as well as immunoregulatory pathways. Pregnancy complications such as gestational diabetes mellitus, preeclampsia, intrauterine growth restriction and preterm birth are associated with altered levels of circulating EVs, with differential EV cargo and bioactivity in target cells. This implicates the intriguing roles of EVs in reprogramming the maternal physiology during pregnancy. Moreover, the capacity of EVs to carry bioactive molecules makes them a promising tool for biomarker development and targeted therapies in pregnancy complications. This review summarizes the physiological and pathological roles played by EVs in pregnancy and pregnancy-related disorders and describes the potential of EVs to be translated into clinical applications in the diagnosis and treatment of pregnancy complications.

Early-Pregnancy Serum Maternal and Placenta-Derived Exosomes miRNAs Vary Based on Pancreatic ß-Cell Function in GDM.

CONTEXT: Pancreatic ß-cell function impairment is a key mechanism for developing gestational diabetes mellitus (GDM). Maternal and placental exosomes regulate maternal and placental responses during hyperglycemia. Studies have associated exosomal micro-RNAs (miRNAs) with GDM development. To date, no studies have been reported that evaluate the profile of miRNAs present in maternal and placental exosomes in the early stages of gestation from pregnancies that develop GDM. OBJECTIVE: We assessed whether early-pregnancy serum maternal and placenta-derived exosomes miRNA profiles vary according to pancreatic ß-cell function in women who will develop GDM. METHODS: A prospective nested case-control study was used to identify exosomal miRNAs that vary in early-pregnancy stages (<18 weeks of gestation) from women with normoglycemia and those who developed GDM based on their pancreatic ß-cell function using the homeostasis model assessment of pancreatic ß-cell function (HOMA-%ß) index. Early-pregnancy serum maternal and placenta-derived exosomes were isolated to obtain miRNA profiles. Potential target and pathway analyses were performed to identify molecular and metabolic pathways associated with the exosomal miRNAs identified. RESULTS: In early-pregnancy stages, serum maternal exosome size and concentration are modified in GDM group and fluctuate according to HOMA-%ß index. Serum maternal exosomal hsa-miR-149-3p and hsa-miR-455-3p in GDM are related to insulin secretion and signaling, lipolysis, and adipocytokine signaling. Early-pregnancy serum placenta-derived exosomes hsa-miR-3665 and hsa-miR-6727-5p in GDM are related to regulating genes involved in response to immunological tolerance of pregnancy and pathways associated with placental dysfunction. CONCLUSION: Early serum exosomal miRNAs differ depending on their origin (maternal or placental) and pancreatic ß-cell function. This research provides insights into the interactions between maternal and placental exosomal miRNAs and may have implications for identifying potential biomarkers or therapeutic targets for GDM.

Chemically-Induced Lipoprotein Breakdown for Improved Extracellular Vesicle Purification.

Extracellular vesicles (EVs) are nanosized biomolecular packages involved in intercellular communication. EVs are released by all cells, making them broadly applicable as therapeutic, diagnostic, and mechanistic components in (patho)physiology. Sample purity is critical for correctly attributing observed effects to EVs and for maximizing therapeutic and diagnostic performance. Lipoprotein contaminants represent a major challenge for sample purity. Lipoproteins are approximately six orders of magnitude more abundant in the blood circulation and overlap in size, shape, and density with EVs. This study represents the first example of an EV purification method based on the chemically-induced breakdown of lipoproteins. Specifically, a styrene-maleic acid (SMA) copolymer is used to selectively breakdown lipoproteins, enabling subsequent size-based separation of the breakdown products from plasma EVs. The use of the polymer followed by tangential flow filtration or size-exclusion chromatography results in improved EV yield, preservation of EV morphology, increased EV markers, and reduced contaminant markers. SMA-based EV purification enables improved fluorescent labeling, reduces interactions with macrophages, and enhances accuracy, sensitivity, and specificity to detect EV biomarkers, indicating benefits for various downstream applications. In conclusion, SMA is a simple and effective method to improve the purity and yield of plasma-derived EVs, which favorably impacts downstream applications.

Microvesicle-eluting nano-engineered implants influence inflammatory response of keratinocytes.

Besides enhancing osseo- and soft tissue integration, modulating inflammation at the implant site is also crucial for dental implant success. Uncontrolled peri-implant inflammation can cause significant loss of surrounding tissue and implant failure. It was recently shown that microvesicles (MVs), a less-studied type of extracellular vesicles, play a crucial role in cell-to-cell communication and may modulate angiogenesis and inflammatory response. The effect of MVs on regulating inflammation at an implant site, however, remains unexplored. In the current study, MVs were isolated and characterised from human primary gingival fibroblasts (hGFs) and loaded within titania nanotubes (TNTs, fabricated via anodisation on 3D Ti wire implants) towards their local release. The modified implants were characterised using SEM and confocal imaging to confirm the loading and local release of MVs from TNTs. In vitro studies demonstrated the internalisation of hGFs-MVs by human gingival keratinocytes (OKF6/TERT2 cell line), which caused a significant reduction in the production of pro-inflammatory cytokines. The results support MVs-releasing TNTs as a promising implant surface modification strategy to reduce inflammation, paving the way for further advancements in therapeutic dental implants.

Extracellular vesicles as a potential delivery platform for CRISPR-Cas based therapy in epithelial ovarian cancer.

Ovarian Cancer (OC) is the most common gynecological malignancy and the eighth most diagnosed cancer in females worldwide. Presently, it ranks as the fifth leading cause of cancer-related mortality among patients globally. Major factors contributing to the lethality of OC worldwide include delayed diagnosis, chemotherapy resistance, high metastatic rates, and the heterogeneity of subtypes. Despite continuous efforts to develop novel targeted therapies and chemotherapeutic agents, challenges persist in the form of OC resistance and recurrence. In the last decade, CRISPR-Cas-based genome editing has emerged as a powerful tool for modifying genetic and epigenetic mechanisms, holding potential for treating numerous diseases. However, a significant challenge for therapeutic applications of CRISPR-Cas technology is the absence of an optimal vehicle for delivering CRISPR molecular machinery into targeted cells or tissues. Recently, extracellular vesicles (EVs) have gained traction as potential delivery vehicles for various therapeutic agents. These heterogeneous, membrane-derived vesicles are released by nearly all cells into extracellular spaces. They carry a molecular cargo of proteins and nucleic acids within their intraluminal space, encased by a cholesterol-rich phospholipid bilayer membrane. EVs actively engage in cell-to-cell communication by delivering cargo to both neighboring and distant cells. Their inherent ability to shield molecular cargo from degradation and cross biological barriers positions them ideally for delivering CRISPR-Cas ribonucleoproteins (RNP) to target cells. Furthermore, they exhibit higher biocompatibility, lower immunogenicity, and reduced toxicity compared to classical delivery platforms such as adeno-associated virus, lentiviruses, and synthetic nanoparticles. This review explores the potential of employing different CRISPR-Cas systems to target specific genes in OC, while also discussing various methods for engineering EVs to load CRISPR components and enhance their targeting capabilities.

Metabolomics profiling and chemoresistance mechanisms in ovarian cancer cell lines: Implications for targeting glutathione pathway.

Ovarian cancer presents a significant challenge due to its high rate of chemoresistance, which complicates the effectiveness of drug-response therapy. This study provides a comprehensive metabolomic analysis of ovarian cancer cell lines OVCAR-3 and SK-OV-3, characterizing their distinct metabolic landscapes. Metabolomics coupled with chemometric analysis enabled us to discriminate between the metabolic profiles of these two cell lines. The OVCAR-3 cells, which are sensitive to doxorubicin (DOX), exhibited a preference for biosynthetic pathways associated with cell proliferation. Conversely, DOX-resistant SK-OV-3 cells favored fatty acid oxidation for energy maintenance. Notably, a marked difference in glutathione (GSH) metabolism was observed between these cell lines. Our investigations further revealed that GSH depletion led to a profound change in drug sensitivity, inducing a shift from a cytostatic to a cytotoxic response. The results derived from this comprehensive metabolomic analysis offer potential targets for novel therapeutic strategies to overcome drug resistance. Our study suggests that targeting the GSH pathway could potentially enhance chemotherapy's efficacy in treating ovarian cancer.

Genomic communication via circulating extracellular vesicles and long-term health consequences of COVID-19.

COVID-19 continues to affect an unprecedented number of people with the emergence of new variants posing a serious challenge to global health. There is an expansion of knowledge in understanding the pathogenesis of Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the impact of the acute disease on multiple organs. In addition, growing evidence reports that the impact of COVID-19 on different organs persists long after the recovery phase of the disease, leading to long-term consequences of COVID-19. These long-term consequences involve pulmonary as well as extra-pulmonary sequelae of the disease. Noteably, recent research has shown a potential association between COVID-19 and change in the molecular cargo of extracellular vesicles (EVs). EVs are vesicles released by cells and play an important role in cell communication by transfer of bioactive molecules between cells. Emerging evidence shows a strong link between EVs and their molecular cargo, and regulation of metabolism in health and disease. This review focuses on current knowledge about EVs and their potential role in COVID-19 pathogenesis, their current and future implications as tools for biomarker and therapeutic development and their possible effects on long-term impact of COVID-19.

Clinical and pulmonary function analysis in long-COVID revealed that long-term pulmonary dysfunction is associated with vascular inflammation pathways and metabolic syndrome.

Introduction: Long-term pulmonary dysfunction (L-TPD) is one of the most critical manifestations of long-COVID. This lung affection has been associated with disease severity during the acute phase and the presence of previous comorbidities, however, the clinical manifestations, the concomitant consequences and the molecular pathways supporting this clinical condition remain unknown. The aim of this study was to identify and characterize L-TPD in patients with long-COVID and elucidate the main pathways and long-term consequences attributed to this condition by analyzing clinical parameters and functional tests supported by machine learning and serum proteome profiling. Methods: Patients with L-TPD were classified according to the results of their computer-tomography (CT) scan and diffusing capacity of the lungs for carbon monoxide adjusted for hemoglobin (DLCOc) tests at 4 and 12-months post-infection. Results: Regarding the acute phase, our data showed that L-TPD was favored in elderly patients with hypertension or insulin resistance, supported by pathways associated with vascular inflammation and chemotaxis of phagocytes, according to computer proteomics. Then, at 4-months post-infection, clinical and functional tests revealed that L-TPD patients exhibited a restrictive lung condition, impaired aerobic capacity and reduced muscular strength. At this time point, high circulating levels of platelets and CXCL9, and an inhibited FCgamma-receptor-mediated-phagocytosis due to reduced FcγRIII (CD16) expression in CD14+ monocytes was observed in patients with L-TPD. Finally, 1-year post infection, patients with L-TPD worsened metabolic syndrome and augmented body mass index in comparison with other patient groups. Discussion: Overall, our data demonstrated that CT scan and DLCOc identified patients with L-TPD after COVID-19. This condition was associated with vascular inflammation and impair phagocytosis of virus-antibody immune complexes by reduced FcγRIII expression. In addition, we conclude that COVID-19 survivors required a personalized follow-up and adequate intervention to reduce long-term sequelae and the appearance of further metabolic diseases.

Comprehensive Strategy for Identifying Extracellular Vesicle Surface Proteins as Biomarkers for Non-Alcoholic Fatty Liver Disease.

Non-alcoholic fatty liver disease (NAFLD) is a liver disorder that has become a global health concern due to its increasing prevalence. There is a need for reliable biomarkers to aid in the diagnosis and prognosis of NAFLD. Extracellular vesicles (EVs) are promising candidates in biomarker discovery, as they carry proteins that reflect the pathophysiological state of the liver. In this review, we developed a list of EV proteins that could be used as diagnostic biomarkers for NAFLD. We employed a multi-step strategy that involved reviewing and comparing various sources of information. Firstly, we reviewed papers that have studied EVs proteins as biomarkers in NAFLD and papers that have studied circulating proteins as biomarkers in NAFLD. To further identify potential candidates, we utilized the EV database Vesiclepedia.org to qualify each protein. Finally, we consulted the Human Protein Atlas to search for candidates' localization, focusing on membrane proteins. By integrating these sources of information, we developed a comprehensive list of potential EVs membrane protein biomarkers that could aid in diagnosing and monitoring NAFLD. In conclusion, our multi-step strategy for identifying EV-based protein biomarkers for NAFLD provides a comprehensive approach that can also be applied to other diseases. The protein candidates identified through this approach could have significant implications for the development of non-invasive diagnostic tests for NAFLD and improve the management and treatment of this prevalent liver disorder.

Effects of periodontal cells-derived extracellular vesicles on mesenchymal stromal cell function.

OBJECTIVE: To enrich and compare three extracellular vesicles-EV subtypes (apoptotic bodies, microvesicles and small EV) from three periodontal cells (periodontal ligament cells-PDLCs, alveolar bone-derived osteoblasts-OBs and gingival fibroblasts-GFs), and assess uptake and cell function changes in buccal fat pad-derived mesenchymal stromal cells (BFP-MSCs). BACKGROUND: Periodontal cells such as PDLCs, OBs and GFs have the potential to enhance bone and periodontal regeneration, but face significant challenges, such as the regulatory and cost implications of in vitro cell culture and storage. To address these challenges, it is important to explore alternative 'cell-free' strategies, such as extracellular vesicles which have emerged as promising tools in regenerative medicine, to facilitate osteogenic differentiation and bone regeneration. METHODS AND MATERIALS: Serial centrifuges at 2600 and 16 000 g were used to isolate apoptotic bodies and microvesicles respectively. Small EV-sEV was enriched by our in-house size exclusion chromatography (SEC). The cellular uptake, proliferation, migration and osteogenic/adipogenic differentiation genes were analysed after EVs uptake in BFP-MSCs. RESULTS: Three EV subtypes were enriched and characterised by morphology, particle size and EV-associated protein expression-CD9. Cellular uptake of the three EVs subtypes was observed in BFP-MSCs for up to 7 days. sEV from the three periodontal cells promoted proliferation, migration and osteogenic gene expression. hOBs-sEV showed superior levels of osteogenesis markers compared to that hPDLCs-sEV and hGFs-sEV, while hOBs-16k EV promoted adipogenic gene expression compared to that from hPDLCs and hGFs. CONCLUSIONS: Our proof-of-concept data demonstrate that hOBs-sEV might be an alternative cell-free therapeutic for bone tissue engineering.

Extracellular vesicle-mediated targeting strategies for long-term health benefits in gestational diabetes.

Extracellular vesicles (EVs) are critical mediators of cell communication, playing important roles in regulating molecular cross-talk between different metabolic tissues and influencing insulin sensitivity in both healthy and gestational diabetes mellitus (GDM) pregnancies. The ability of EVs to transfer molecular cargo between cells imbues them with potential as therapeutic agents. During pregnancy, the placenta assumes a vital role in metabolic regulation, with multiple mechanisms of placenta-mediated EV cross-talk serving as central components in GDM pathophysiology. This review focuses on the role of the placenta in the pathophysiology of GDM and explores the possibilities and prospects of targeting the placenta to address insulin resistance and placental dysfunction in GDM. Additionally, we propose the use of EVs as a novel method for targeted therapeutics in treating the dysfunctional placenta. The primary aim of this review is to comprehend the current status of EV targeting approaches and assess the potential application of these strategies in placental therapeutics, thereby delivering molecular cargo and improving maternal and fetal outcomes in GDM. We propose that EVs have the potential to revolutionize GDM management, offering hope for enhanced maternal-fetal health outcomes and more effective treatments.

Clinical Translation of Extracellular Vesicles.

Extracellular vesicles (EVs) occur in a variety of bodily fluids and have gained recent attraction as natural materials due to their bioactive surfaces, internal cargo, and role in intercellular communication. EVs contain various biomolecules, including surface and cytoplasmic proteins; and nucleic acids that are often representative of the originating cells. EVs can transfer content to other cells, a process that is thought to be important for several biological processes, including immune responses, oncogenesis, and angiogenesis. An increased understanding of the underlying mechanisms of EV biogenesis, composition, and function has led to an exponential increase in preclinical and clinical assessment of EVs for biomedical applications, such as diagnostics and drug delivery. Bacterium-derived EV vaccines have been in clinical use for decades and a few EV-based diagnostic assays regulated under Clinical Laboratory Improvement Amendments have been approved for use in single laboratories. Though, EV-based products are yet to receive widespread clinical approval from national regulatory agencies such as the United States Food and Drug Administration (USFDA) and European Medicine Agency (EMA), many are in late-stage clinical trials. This perspective sheds light on the unique characteristics of EVs, highlighting current clinical trends, emerging applications, challenges and future perspectives of EVs in clinical use.