Monocyte derived large extracellular vesicles in polytrauma.

Despite significant progress in the medical field, there is still a pressing need for minimal-invasive tools to assist with decision-making, especially in cases of polytrauma. Our team explored the potential of serum-derived large extracellular vesicles, so called microparticles/microvesicles/ectosomes, to serve as a supportive tool in decision-making in polytrauma situations. We focused on whether monocyte derived large EVs may differentiate between polytrauma patients with internal organ injury (ISS > 15) and those without. Thus, we compared our EV data to soluble biomarkers such as tumour necrosis factor alpha (TNF alpha) and Interleukin-8 (IL-8). From the blood of 25 healthy and 26 patients with polytrauma large EVs were isolated, purified, and characterized. TNF alpha and IL-8 levels were quantified. We found that levels of these monocyte derived large EVs were significantly higher in polytrauma patients with internal organ damage and correlated with the ISS. Interestingly, we also observed a decline in AnnV+CD14+ large EVs during normal recovery after trauma. Thus, inflammatory serological markers as TNF alpha and as IL-8 demonstrated an inability to discriminate between polytrauma patients with or without internal organ damage, such as spleen, kidney, or liver lacerations/ruptures. However, TNF and IL-8 levels were elevated in polytrauma cases overall when contrasted with healthy non-traumatic controls. These findings suggest that delving deeper into the potential of AnnV+ large EVs derived from monocytes could highly beneficial in the managment of polytrauma, potentially surpassing the efficacy of commonly used serum markers.

Microvesicles derived from mesenchymal stem cells inhibit acute respiratory distress syndrome-related pulmonary fibrosis in mouse partly through hepatocyte growth factor.

BACKGROUND: Pulmonary fibrosis is one of the main reasons for the high mortality rate among acute respiratory distress syndrome (ARDS) patients. Mesenchymal stromal cell-derived microvesicles (MSC-MVs) have been shown to exert antifibrotic effects in lung diseases. AIM: To investigate the effects and mechanisms of MSC-MVs on pulmonary fibrosis in ARDS mouse models. METHODS: MSC-MVs with low hepatocyte growth factor (HGF) expression (siHGF-MSC-MVs) were obtained via lentivirus transfection and used to establish the ARDS pulmonary fibrosis mouse model. Following intubation, respiratory mechanics-related indicators were measured via an experimental small animal lung function tester. Homing of MSC-MVs in lung tissues was investigated by near-infrared live imaging. Immunohistochemical, western blotting, ELISA and other methods were used to detect expression of pulmonary fibrosis-related proteins and to compare effects on pulmonary fibrosis and fibrosis-related indicators. RESULTS: The MSC-MVs gradually migrated and homed to damaged lung tissues in the ARDS model mice. Treatment with MSC-MVs significantly reduced lung injury and pulmonary fibrosis scores. However, low expression of HGF (siHGF-MSC-MVs) significantly inhibited the effects of MSC-MVs (P < 0.05). Compared with the ARDS pulmonary fibrosis group, the MSC-MVs group exhibited suppressed expression of type I collagen antigen, type III collagen antigen, and the proteins transforming growth factor-ß and α-smooth muscle actin, whereas the siHGF-MVs group exhibited significantly increased expression of these proteins. In addition, pulmonary compliance and the pressure of oxygen/oxygen inhalation ratio were significantly lower in the MSC-MVs group, and the effects of the MSC-MVs were significantly inhibited by low HGF expression (all P < 0.05). CONCLUSION: MSC-MVs improved lung ventilation functions and inhibited pulmonary fibrosis in ARDS mice partly via HGF mRNA transfer.

Evaluation of circulating microvesicles and their procoagulant activity in patients with COVID-19.

OBJECTIVE: Several pathological conditions trigger the formation of microvesicles (MVs), including infectious diseases such as COVID-19. The shedding of MVs increases the levels of inflammatory factors (e.g., interleukin-6; IL-6) and ultimately leads to an inflammatory cascade response, while also increasing the procoagulant response. The current study aimed to evaluate the level of circulating MVs and their procoagulant activity as well as the serum level of IL-6 in patients with COVID-19 and healthy controls. In this case-control study, 65 patients with COVID-19 and 30 healthy individuals were sampled after obtaining written informed consent. MVs counting was measured using conjugated CD61, CD45, CD235a, and Annexin-V antibodies. Additionally, the procoagulant activity of MVs and the IL-6 level were estimated using enzyme-linked immunosorbent assay (ELISA). RESULTS: The majority of MVs were platelet-derived MVs (PMVs). Patients with COVID-19 had significantly higher levels of MVs, procoagulant MVs, and IL-6 compared to healthy controls (p < 0.001). MVs were significantly correlated with procoagulant MVs, D-Dimer levels, fibrinogen, and IL-6, but not with platelet, lymphocyte, and neutrophil counts. CONCLUSION: Elevated levels of procoagulant MVs and their association with inflammatory and coagulation markers in patients with COVID-19 are suggested as a novel circulatory biomarker to evaluate and predict the procoagulant activity and severity of COVID-19.

Selective Enrichment of Angiomirs in Extracellular Vesicles Released from Ischemic Skeletal Muscles: Potential Role in Angiogenesis and Neovascularization.

MicroRNAs (miRs) regulate physiological and pathological processes, including ischemia-induced angiogenesis and neovascularization. They can be transferred between cells by extracellular vesicles (EVs). However, the specific miRs that are packaged in EVs released from skeletal muscles, and how this process is modulated by ischemia, remain to be determined. We used a mouse model of hindlimb ischemia and next generation sequencing (NGS) to perform a complete profiling of miR expression and determine the effect of ischemia in skeletal muscles, and in EVs of different sizes (microvesicles (MVs) and exosomes) released from these muscles. Ischemia significantly modulated miR expression in whole muscles and EVs, increasing the levels of several miRs that can have pro-angiogenic effects (angiomiRs). We found that specific angiomiRs are selectively enriched in MVs and/or exosomes in response to ischemia. In silico approaches indicate that these miRs modulate pathways that play key roles in angiogenesis and neovascularization, including HIF1/VEGF signaling, regulation of actin cytoskeleton and focal adhesion, NOTCH, PI3K/AKT, RAS/MAPK, JAK/STAT, TGFb/SMAD signaling and the NO/cGMP/PKG pathway. Thus, we show for the first time that angiomiRs are selectively enriched in MVs and exosomes released from ischemic muscles. These angiomiRs could be targeted in order to improve the angiogenic function of EVs for potential novel therapeutic applications in patients with severe ischemic vascular diseases.

Exploring Salivary Biomarkers for Tumor Diagnosis: A Narrative Review.

A promising method for non-invasive cancer diagnosis and prognosis is through salivary biomarkers. By utilizing the distinct characteristics of saliva and the progress made in biomarker studies, these markers provide more accurate diagnoses for a wider range of malignancies. An attempt was made to thoroughly investigate the field of salivary biomarkers for tumor prognosis and diagnosis, with an emphasis on their use in various cancer forms. Predetermined search criteria were utilized for a systematic search across numerous databases for peer-reviewed papers from 2009 to 2021. Studies concentrating on the detection, validation, and clinical use of salivary biomarkers for different types of cancers were included in the inclusion criteria. Initially, 238 articles were found, of which 15 relevant articles satisfied the inclusion requirements. Information on study aims, methodology, findings, and conclusions were gathered for data extraction. We identified recurrent themes, patterns, and contradictions by a thematic analysis. We also assessed state-of-the-art salivary biomarkers for tumor diagnosis and prognosis. One major finding is the identification of biomolecules in saliva linked to several cancer forms, including pancreatic, oral, breast, lung, and stomach cancers. There is an increasing amount of evidence demonstrating the value of saliva-based diagnostics in oncology. This is due to new detection methods and developments in salivary proteomics and genomics. Identification of exosomes and microvesicles as salivary biomarker profiles offered molecular understandings of the etiology and evolution of cancer, thereby opening new avenues for diagnosis and treatment. Salivary biomarkers are a non-invasive approach for the early detection and prediction of cancer, thanks to the unique properties of saliva and advancements in biomarker research. This potential revolution could enhance patient outcomes and reduce cancer-related deaths.

The interplay of extracellular vesicles in the pathogenesis of metabolic impairment and type 2 diabetes.

The pathogenesis of type 2 diabetes (T2D) involves dysfunction in multiple organs, including the liver, muscle, adipose tissue, and pancreas, leading to insulin resistance and ß cell failure. Recent studies highlight the significant role of extracellular vesicles (EVs) in mediating inter-organ communication in T2D. This review investigates the role of EVs, focusing on their presence and biological significance in human plasma and tissues affected by T2D. We explore specific EV cargo, such as miRNAs and proteins, which affect insulin signaling and glucose metabolism, emphasizing their potential as biomarkers. By highlighting the diagnostic and therapeutic potential of EVs, we aim to provide new insights into their role in early detection, disease monitoring, and innovative treatment strategies for T2D.

Role of Extracellular Vesicles in Crohn's Patients on Adalimumab Who Received COVID-19 Vaccination.

Crohn's disease (CD) is a type of inflammatory bowel disease (IBD) affecting the gastrointestinal tract that can also cause extra-intestinal complications. Following exposure to the mRNA vaccine BNT162b2 (Pfizer-BioNTech) encoding the SARS-CoV-2 Spike (S) protein, some patients experienced a lack of response to the biological drug Adalimumab and a recrudescence of the disease. In CD patients in progression, resistant to considered biological therapy, an abnormal increase in intestinal permeability was observed, more often with a modulated expression of different proteins such as Aquaporin 8 (AQP8) and in tight junctions (e.g., ZO-1, Claudin1, Claudin2, Occludin), especially during disease flares. The aim of this study is to investigate how the SARS-CoV-2 vaccine could interfere with IBD therapy and contribute to disease exacerbation. We investigated the role of the SARS-CoV-2 Spike protein, transported by extracellular vesicles (EVs), and the impact of various EVs components, namely, exosomes (EXOs) and microvesicles (MVs), in modulating the expression of molecules involved in the exacerbation of CD, which remains unknown.

Human mesenchymal stem cells-derived microvesicles increase oligodendrogenesis and neurogenesis of cultured adult neural stem cells.

Mesenchymal stem cells (MSCs) are involved in tissue repair and anti-inflammatory activities and have shown promising therapeutic efficiency in different animal models of neurodegenerative disorders. Microvesicles (MVs), implicated in cellular communication, are secreted from MSCs and play a key role in determining the fate of cell differentiation. Our study examines the effect of human umbilical cord MSC-derived MVs (hUC-MSC MVs) on the proliferation and differentiation potential of adult neural stem cells (NSCs). Results showed that 0.2 µg MSC derived MVs significantly increased the viability of NSCs and their proliferation, as demonstrated by an increase in the number of neurospheres and their derived cells, compared to controls. In addition, all hUC-MSC MVs concentrations (0.1, 0.2 and 0.4 µg) induced the differentiation of NSCs toward precursors (Olig2 + ) and mature oligodendrocytes (MBP+). This increase in mature oligodendrocytes was inversely proportional to the dose of MVs. Moreover, hUC-MSC MVs induced the differentiation of NSCs into neurons (ß-tubulin + ), in a dose-dependent manner, but had no effect on astrocytes (GFAP+). Furthermore, treatment of NSCs with hUC-MSC MVs (0.1 and 0.2 µg) significantly increased the expression levels of the proliferation marker Ki67 gene, compared to controls. Finally, hUC-MSC MVs (0.1 µg) significantly increased the expression level of Sox10 transcripts; but not Pax6 gene, demonstrating an increased NSC ability to differentiate into oligodendrocytes. In conclusion, our study showed that hUC-MSC MVs increased NSC proliferation in vitro and induced NSC differentiation into oligodendrocytes and neurons, but not astrocytes.

Therapeutic Application of Extracellular Vesicles Derived from Mesenchymal Stem Cells in Domestic Animals.

Recently, the therapeutic potential of extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) has been extensively studied in both human and veterinary medicine. EVs are nano-sized particles containing biological components commonly found in other biological materials. For that reason, EV isolation and characterization are critical to draw precise conclusions during their investigation. Research on EVs within veterinary medicine is still considered in its early phases, yet numerous papers were published in recent years. The conventional adult tissues for deriving MSCs include adipose tissue and bone marrow. Nonetheless, alternative sources such as synovial fluid, endometrium, gingiva, and milk have also been intermittently used. Fetal adnexa are amniotic membrane/fluid, umbilical cord and Wharton's jelly. Cells derived from fetal adnexa exhibit an intermediate state between embryonic and adult cells, demonstrating higher proliferative and differentiative potential and longer telomeres compared to cells from adult tissues. Summarized here are the principal and recent preclinical and clinical studies performed in domestic animals such as horse, cattle, dog and cat. To minimize the use of antibiotics and address the serious issue of antibiotic resistance as a public health concern, they will undoubtedly also be utilized in the future to treat infections in domestic animals. A number of concerns, including large-scale production with standardization of EV separation and characterization techniques, must be resolved for clinical application.

Extracellular vesicles at the crossroad between cancer progression and immunotherapy: focus on dendritic cells.

Extracellular vesicles (EVs) are nanosized heat-stable vesicles released by virtually all cells in the body, including tumor cells and tumor-infiltrating dendritic cells (DCs). By carrying molecules from originating cells, EVs work as cell-to-cell communicators in both homeostasis and cancer but may also represent valuable therapeutic and diagnostic tools. This review focuses on the role of tumor-derived EVs (TEVs) in the modulation of DC functions and on the therapeutic potential of both tumor- and DC-derived EVs in the context of immunotherapy and DC-based vaccine design. TEVs were originally characterized for their capability to transfer tumor antigens to DCs but are currently regarded as mainly immunosuppressive because of the expression of DC-inhibiting molecules such as PD-L1, HLA-G, PGE2 and others. However, TEVs may still represent a privileged system to deliver antigenic material to DCs upon appropriate engineering to reduce their immunosuppressive cargo or increase immunogenicity. DC-derived EVs are more promising than tumor-derived EVs since they expose antigen-loaded MHC, costimulatory molecules and NK cell-activating ligands in the absence of an immunosuppressive cargo. Moreover, DC-derived EVs possess several advantages as compared to cell-based drugs such as a higher antigen/MHC concentration and ease of manipulation and a lower sensitivity to immunosuppressive microenvironments. Preclinical models showed that DC-derived EVs efficiently activate tumor-specific NK and T cell responses either directly or indirectly by transferring antigens to tumor-infiltrating DCs. By contrast, however, phase I and II trials showed a limited clinical efficacy of EV-based anticancer vaccines. We discuss that the future of EV-based therapy depends on our capability to overcome major challenges such as a still incomplete understanding of their biology and pharmacokinetic and the lack of standardized methods for high-throughput isolation and purification. Despite this, EVs remain in the limelight as candidates for cancer immunotherapy which may outmatch cell-based strategies in the fullness of their time.

Mitochondrial DNA-boosted dendritic cell-based nanovaccination triggers antitumor immunity in lung and pancreatic cancers.

Low migratory dendritic cell (DC) levels pose a challenge in cancer immune surveillance, yet their impact on tumor immune status and immunotherapy responses remains unclear. We present clinical evidence linking reduced migratory DC levels to immune-cold tumor status, resulting in poor patient outcomes. To address this, we develop an autologous DC-based nanovaccination strategy using patient-derived organoid or cancer cell lysate-pulsed cationic nanoparticles (cNPs) to load immunogenic DC-derived microvesicles (cNPcancer cell@MVDC). This approach transforms immune-cold tumors, increases migratory DCs, activates T cells and natural killer cells, reduces tumor growth, and enhances survival in orthotopic pancreatic and lung cancer models, surpassing conventional methods. In vivo imaging reveals superior cNPcancer cell@MVDC accumulation in tumors and lymph nodes, promoting immune cell infiltration. Mechanistically, cNPs enrich mitochondrial DNA, enhancing cGAS-STING-mediated DC activation and migration. Our strategy shifts cold tumors to a hot state, enhancing antitumor immunity for potential personalized cancer treatments.

Efficacy of stem cells versus microvesicles in ameliorating chronic renal injury in rats (histological and biochemical study).

Chronic exposure to heavy metals as aluminum chloride (AlCl3) could result in severe health hazards such as chronic renal injury. The present study aimed to evaluate the therapeutic potential of adipose tissue-derived stem cells (ASCs) in comparison to their microvesicles (MV) in AlCl3-induced chronic renal injury. Forty-eight adult male Wistar rats were divided into four groups: Control group, AlCl3-treated group, AlCl3/ASC-treated group, and AlCl3/MV-treated group. Biochemical studies included estimation of serum urea and creatinine levels, oxidative biomarkers assay, antioxidant biomarkers, serum cytokines (IL-1ß, IL-8, IL-10, and IL-33), real time-PCR analysis of renal tissue MALT1, TNF-α, IL-6, and serum miR-150-5p expression levels. Histopathological studies included light and electron microscopes examination of renal tissue, Mallory trichrome stain for fibrosis, Periodic acid Schiff (PAS) stain for histochemical detection of carbohydrates, and immunohistochemical detection of Caspase-3 as apoptosis marker, IL-1B as a proinflammatory cytokine and CD40 as a marker of MVs. AlCl3 significantly deteriorated kidney function, enhanced renal MDA and TOS, and serum cytokines concentrations while decreased the antioxidant parameters (SOD, GSH, and TAC). Moreover, serum IL-10, TNF-α, miR-150-5p, and renal MALT1 expression values were significantly higher than other groups. Kidney sections showed marked histopathological damage in both renal cortex and medulla in addition to enhanced apoptosis and increased inflammatory cytokines immunoexpression than other groups. Both ASCs and MVs administration ameliorated the previous parameters levels with more improvement was detected in MVs-treated group. In conclusion: ASCs-derived MVs have a promising ameliorating effect on chronic kidney disease.

CD66b+/CD68+ circulating extracellular vesicles, lactate dehydrogenase and neutrophil-to-lymphocyte ratio can differentiate coronavirus disease 2019 severity during and after infection.

Coronavirus disease 2019 (COVID-19) has been a major public health burden. We hypothesised that circulating extracellular vesicles (cEVs), key players in health and disease, could trace the cell changes during COVID-19 infection and recovery. Therefore, we studied the temporal trend of cEV and inflammatory marker levels in plasma samples of COVID-19 patients that were collected within 24 h of patient admission (baseline, n = 80) and after hospital discharge at day-90 post-admission (n = 59). Inflammatory markers were measured by standard biochemical methods. cEVs were quantitatively and phenotypically characterized by high-sensitivity nano flow cytometry. In patients recovered from COVID-19 lower levels of inflammatory markers were detected. cEVs from vascular (endothelial cells) and blood (platelets, distinct immune subsets) cells were significantly reduced at day-90 compared to admission levels, a pattern also observed for cEVs from progenitor, perivascular and epithelial cells. The best discriminatory power for COVID-19 severity was found for inflammatory markers lactate dehydrogenase and neutrophil-to-lymphocyte ratio and for granulocyte/macrophage-released CD66b+/CD68+-cEVs. Albeit inflammatory markers were good indicators of systemic inflammatory response and discriminators of COVID-19 remission, they do not completely reveal cell stress and organ damage states. cEVs reaching baseline pre-infection levels at 90 days post-infection in recovered patients discriminate parental cells affected by disease.

Ceramide-mediated orchestration of oxidative stress response through filopodia-derived small extracellular vesicles.

Extracellular vesicles (EVs) are shed from the plasma membrane, but the regulation and function of these EVs remain unclear. We found that oxidative stress induced by H2O2 in Hela cells stimulated filopodia formation and the secretion of EVs. EVs were small (150 nm) and labeled for CD44, indicating that they were derived from filopodia. Filopodia-derived small EVs (sEVs) were enriched with the sphingolipid ceramide, consistent with increased ceramide in the plasma membrane of filopodia. Ceramide was colocalized with neutral sphingomyelinase 2 (nSMase2) and acid sphingomyelinase (ASM), two sphingomyelinases generating ceramide at the plasma membrane. Inhibition of nSMase2 and ASM prevented oxidative stress-induced sEV shedding but only nSMase2 inhibition prevented filopodia formation. nSMase2 was S-palmitoylated and interacted with ASM in filopodia to generate ceramide for sEV shedding. sEVs contained nSMase2 and ASM and decreased the level of these two enzymes in oxidatively stressed Hela cells. A novel metabolic labeling technique for EVs showed that oxidative stress induced secretion of fluorescent sEVs labeled with NBD-ceramide. NBD-ceramide-labeled sEVs transported ceramide to mitochondria, ultimately inducing cell death in a proportion of neuronal (N2a) cells. In conclusion, using Hela cells we provide evidence that oxidative stress induces interaction of nSMase2 and ASM at filopodia, which leads to shedding of ceramide-rich sEVs that target mitochondria and propagate cell death.

Mitochondria-containing extracellular vesicles from mouse vs. human brain endothelial cells for ischemic stroke therapy.

Ischemic stroke-induced mitochondrial dysfunction in the blood-brain barrier-forming brain endothelial cells (BECs) results in long-term neurological dysfunction post-stroke. We previously reported data from a pilot study where intravenous administration of human BEC (hBEC)-derived mitochondria-containing extracellular vesicles (EVs) showed a potential efficacy signal in a mouse middle cerebral artery occlusion (MCAo) model of stroke. We hypothesized that EVs harvested from donor species homologous to the recipient species (e.g., mouse) may improve therapeutic efficacy, and therefore, use of mouse BEC (mBEC)-derived EVs may improve post-stroke outcomes in MCAo mice. We investigated potential differences in the mitochondria transfer of EVs derived from the same species as the recipient cell (mBEC-EVs and recipient mBECs or hBECs-EVs and recipient hBECs) vs. cross-species EVs and recipient cells (mBEC-EVs and recipient hBECs or vice versa). Our results showed that while both hBEC- and mBEC-EVs transferred EV mitochondria, mBEC-EVs outperformed hBEC-EVs in increasing ATP levels and improved recipient mBEC mitochondrial function via increasing oxygen consumption rates. mBEC-EVs significantly reduced brain infarct volume and neurological deficit scores compared to vehicle-injected MCAo mice. The superior therapeutic efficacy of mBEC-EVs in MCAo mice support the continued use of mBEC-EVs to optimize the therapeutic potential of mitochondria-containing EVs in preclinical mouse models.

Endothelial-derived microvesicles promote pro-migratory cross-talk with smooth muscle cells by a mechanism requiring tissue factor and PAR2 activation.

Introduction: Microvesicles (MV) released by endothelial cells (EC) following injury or inflammation contain tissue factor (TF) and mediate communication with the underlying smooth muscle cells (SMC). Ser253-phosphorylated TF co-localizes with filamin A at the leading edge of migrating SMC. In this study, the influence of endothelial-derived TF-MV, on human coronary artery SMC (HCASMC) migration was examined. Methods and Results: MV derived from human coronary artery EC (HCAEC) expressing TFWt accelerated HCASMC migration, but was lower with cytoplasmic domain-deleted TF. Furthermore, incubation with TFAsp253-MV, or expression of TFAsp253 in HCASMC, reduced cell migration. Blocking TF-factor VIIa (TF-fVIIa) procoagulant/protease activity, or inhibiting PAR2 signaling on HCASMC, abolished the accelerated migration. Incubation with fVIIa alone increased HCASMC migration, but was significantly enhanced on supplementation with TF. Neither recombinant TF alone, factor Xa, nor PAR2-activating peptide (SLIGKV) influenced cell migration. In other experiments, HCASMC were transfected with peptides corresponding to the cytoplasmic domain of TF prior to stimulation with TF-fVIIa. Cell migration was suppressed only when the peptides were phosphorylated at position of Ser253. Expression of mutant forms of filamin A in HCASMC indicated that the enhancement of migration by TF but not by PDGF-BB, was dependent on the presence of repeat-24 within filamin A. Incubation of HCASMC with TFWt-MV significantly reduced the levels of Smoothelin-B protein, and upregulated FAK expression. Discussion: In conclusion, Ser253-phosphorylated TF and fVIIa released as MV-cargo by EC, act in conjunction with PAR2 on SMC to promote migration and may be crucial for normal arterial homeostasis as well as, during development of vascular disease.

Plasma NOTCH3 and the risk of cardiovascular recurrence in patients with ischemic stroke.

BACKGROUND: Ischemic stroke patients are more prone to developing another cardiovascular event. AIM: This study aims to examine potential biological predispositions to cardiovascular recurrence in patients with ischemic stroke. DESIGN: Human and preclinical studies. METHODS: Quantitative proteomic analysis, animal stroke, atherosclerosis models and circulating endothelial cells (CECs) were employed to examine candidate biomarkers derived from an ischemic stroke cohort in Singapore. RESULTS: Proteomic analysis of pooled microvesicles of "Event" (n = 24) and without "Event" (n = 24) samples identified NOTCH3 as a candidate marker; plasma NOTCH3 were shown to be elevated in "Event" patients compared to those without "Events" and age-matched controls. In a validation cohort comprising 431 prospectively recruited ischemic stroke patients (mean age 59.1 years; median follow-up 3.5 years), men with plasma NOTCH3 (>1600pg/ml) harbored increased risk of cardiovascular recurrence (adjusted hazards ratio 2.29, 95% CI 1.10-4.77); no significant association was observed in women. Chronic renal failure, peripheral artery disease and NT-pro-brain natriuretic peptide were significant predictors of plasma NOTCH3 in men without ischemic stroke (adjusted r2=0.43). Following middle cerebral artery occlusion, NOTCH3 expression in mouse sera increased and peaked at 24 hrs, persisting thereafter for at least 72 hours. In Apoe-/- atherosclerotic mice, NOTCH3 stained the endothelium of defective arterial lining and atherosclerotic plaques. Analysis of CECs isolated from stroke patients revealed increased gene expression of NOTCH3, further supporting endothelial damage underpinning NOTCH3-mediated atherosclerosis. CONCLUSION: Findings from this study suggests that NOTCH3 could be important in cardiovascular recurrence following an ischemic stroke.

Deciphering m6A methylation in monocyte-mediated cardiac fibrosis and monocyte-hitchhiked erythrocyte microvesicle biohybrid therapy.

Rationale: Device implantation frequently triggers cardiac remodeling and fibrosis, with monocyte-driven inflammatory responses precipitating arrhythmias. This study investigates the role of m6A modification enzymes METTL3 and METTL14 in these responses and explores a novel therapeutic strategy targeting these modifications to mitigate cardiac remodeling and fibrosis. Methods: Peripheral blood mononuclear cells (PBMCs) were collected from patients with ventricular septal defects (VSD) who developed conduction blocks post-occluder implantation. The expression of METTL3 and METTL14 in PBMCs was measured. METTL3 and METTL14 deficiencies were induced to evaluate their effect on angiotensin II (Ang II)-induced myocardial inflammation and fibrosis. m6A modifications were analyzed using methylated RNA immunoprecipitation followed by quantitative PCR. NF-κB pathway activity and levels of monocyte migration and fibrogenesis markers (CXCR2 and TGF-ß1) were assessed. An erythrocyte microvesicle-based nanomedicine delivery system was developed to target activated monocytes, utilizing the METTL3 inhibitor STM2457. Cardiac function was evaluated via echocardiography. Results: Significant upregulation of METTL3 and METTL14 was observed in PBMCs from patients with VSD occluder implantation-associated persistent conduction block. Deficiencies in METTL3 and METTL14 significantly reduced Ang II-induced myocardial inflammation and fibrosis by decreasing m6A modification on MyD88 and TGF-ß1 mRNAs. This disruption reduced NF-κB pathway activation, lowered CXCR2 and TGF-ß1 levels, attenuated monocyte migration and fibrogenesis, and alleviated cardiac remodeling. The erythrocyte microvesicle-based nanomedicine delivery system effectively targeted inflamed cardiac tissue, reducing inflammation and fibrosis and improving cardiac function. Conclusion: Inhibiting METTL3 and METTL14 in monocytes disrupts the NF-κB feedback loop, decreases monocyte migration and fibrogenesis, and improves cardiac function. Targeting m6A modifications of monocytes with STM2457, delivered via erythrocyte microvesicles, reduces inflammation and fibrosis, offering a promising therapeutic strategy for cardiac remodeling associated with device implantation.

The diffusion of normal skin wound myofibroblast-derived microvesicles differs according to matrix composition.

Microvesicles (MVs) are a subtype of extracellular vesicles that can transfer biological information over long distances, affecting normal and pathological processes including skin wound healing. However, the diffusion of MVs into tissues can be impeded by the extracellular matrix (ECM). We investigated the diffusion of dermal wound myofibroblast-derived MVs into the ECM by using hydrogels composed of different ECM molecules such as fibrin, type III collagen and type I collagen that are present during the healing process. Fluorescent MVs mixed with hydrogels were employed to detect MV diffusion using fluorometric methods. Our results showed that MVs specifically bound type I collagen and diffused freely out of fibrin and type III collagen. Further analysis using flow cytometry and specific inhibitors revealed that MVs bind to type I collagen via the α2ß1 integrin. These data demonstrate that MV transport depends on the composition of the wound environment.

Microvesicles derived from dermal myofibroblasts modify the integrity of the blood and lymphatic barriers using distinct endocytosis pathways.

Microvesicles (MVs) are a subtype of extracellular vesicles that can transfer biological information from their producer cells to target cells. This communication can in turn affect both normal and pathological processes. Mounting evidence has revealed that dermal wound myofibroblasts (Wmyo) produce MVs, which can transfer biomolecules impacting receptor cells such as human dermal microvascular endothelial cells (HDMECs). While the effects of MVs on HDMECs are generally well described in the literature, little is known about the transport of MVs across the HDMEC barrier, and their potential effect on the barrier integrity remains unknown. Here, we investigated these roles of Wmyo-derived MVs on two sub-populations of HDMECs, blood endothelial cells (BECs) and lymphatic endothelial cells (LECs). Using an in vitro model to mimic the endothelial barrier, we showed that MVs crossed the LEC barrier but not the BEC barrier. In addition, we demonstrated that MVs were able to influence the cell-cell junctions of HDMECs. Specifically, we observed that after internalization via the predominantly caveolin-dependent pathway, MVs induced the opening of junctions in BECs. Conversely, in LECs, MVs mainly use the macropinocytosis pathway and induce closure of these junctions. Moreover, proteins in the MV membrane were responsible for this effect, but not specifically those belonging to the VEGF family. Finally, we found that once the LEC barrier permeability was reduced by MV stimuli, MVs ceased to cross the barrier. Conversely, when the BEC barrier was rendered permeable following stimulation with MVs, they were subsequently able to cross the barrier via the paracellular pathway. Taken together, these results suggest that the study of Wmyo-derived MVs offers valuable insights into their interaction with the HDMEC barrier in the context of wound healing. They highlight the potential significance of these MVs in the overall process.