M2 Macrophage-Derived Extracellular Vesicles Encapsulated in Hyaluronic Acid Alleviate Osteoarthritis by Modulating Macrophage Polarization.

An imbalance between M1 and M2 macrophage polarization is critical in osteoarthritis (OA) development. We investigated the effect of M2 macrophage-derived extracellular vesicles (M2-EVs) to reprogramme macrophages from the M1 to M2 phenotype for OA treatment. M1 macrophages and mouse OA models were treated with M2-EVs. Proteomic analysis was performed to evaluate macrophage polarization in vitro. The OA models were as follows: destabilization of the medial meniscus (DMM) surgery-induced OA and collagenase-induced OA (CIOA). Hyaluronic acid (HA) was used to deliver M2-EVs. M2-EVs decreased macrophage accumulation, repolarized macrophages from the M1 to M2 phenotype, mitigated synovitis, reduced cartilage degradation, alleviated subchondral bone damage, and improved gait abnormalities in the CIOA and DMM models. Moreover, HA increased the retention time of M2-EVs and enhanced the efficiency of M2-EVs in OA treatment. Furthermore, proteomic analysis demonstrated that M2-EVs exhibited a macrophage reprogramming ability similar to IL-4, and the pathways might be the NOD-like receptor (NLR), TNF, NF-κB, and Toll-like receptor (TLR) signaling pathways. M2-EVs reprogrammed macrophages from the M1 to M2 phenotype, which resulted in beneficial effects on cartilage and attenuation of OA severity. In summary, our study indicated that M2-EV-guided reprogramming of macrophages is a promising treatment strategy for OA.

Aflatoxin B1-exposed hepatocyte-derived extracellular vesicles: Initiating hepatic stellate cell-mediated liver fibrosis through a p53-Parkin-dependent mitophagy pathway.

Environmental aflatoxin B1 (AFB1) exposure has been proposed to contribute to hepatocellular carcinoma by promoting liver fibrosis, but the potential mechanisms remain to be further elucidated. Extracellular vesicles (EVs) were recognized as crucial traffickers for hepatic intercellular communication and play a vital role in the pathological process of liver fibrosis. The AFB1-exposed hepatocyte-derived EVs (AFB1-EVs) were extracted, and the functional effects of AFB1-EVs on the activation of hepatic stellate cells (HSCs) were explored to investigate the molecular mechanism of AFB1 exposure-induced liver fibrogenesis. Our results revealed that an environment-level AFB1 exposure induced liver fibrosis via HSCs activation in mice, while the AFB1-EVs mediated hepatotoxicity and liver fibrogenesis in vitro and in vivo. AFB1 exposure in vitro increased PINK1/Parkin-dependent mitophagy in hepatocytes, where upregulated transcription of the PARK2 gene via p53 nuclear translocation and mitochondrial recruitment of Parkin, and promoted AFB1-EVs-mediated mitochondria-trafficking communication between hepatocytes and HSCs. The knockdown of Parkin in HepaRG cells reversed HSCs activation by blocking the mitophagy-related AFB1-EVs trafficking. This study further revealed that the hepatic fibrogenesis of AFB1 exposure was rescued by genetic intervention with siPARK2 or p53's Pifithrin-α (PFTα) inhibitors. Furthermore, AFB1-EVs-induced HSCs activation was relieved by GW4869 pharmaceutic inhibition of EVs secretion. These results revealed a novel mechanism that AFB1 exposure-induced p53-Parkin signal axis regulated mitophagy-dependent hepatocyte-derived EVs to mediate the mitochondria-trafficking intercellular communication between hepatocytes and HSCs in the local hepatotoxic microenvironment to promote the activated HSCs-associated liver fibrogenesis. Our study provided insight into p53-Parkin-dependent pathway regulation and promised an advanced strategy targeting intervention to EVs-mediated mitochondria trafficking for preventing xenobiotics-induced liver fibrosis.

Targeting cancer-derived extracellular vesicles by combining CD147 inhibition with tissue factor pathway inhibitor for the management of urothelial cancer cells.

BACKGROUND: Extracellular vesicles (EVs), including microvesicles, hold promise for the management of bladder urothelial carcinoma (BLCA), particularly because of their utility in identifying therapeutic targets and their diagnostic potential using easily accessible urine samples. Among the transmembrane glycoproteins highly enriched in cancer-derived EVs, tissue factor (TF) and CD147 have been implicated in promoting tumor progression. In this in vitro study, we explored a novel approach to impede cancer cell migration and metastasis by simultaneously targeting these molecules on urothelial cancer-derived EVs. METHODS: Cell culture supernatants from invasive and non-invasive bladder cancer cell lines and urine samples from patients with BLCA were collected. Large, microvesicle-like EVs were isolated using sequential centrifugation and characterized by electron microscopy, nanoparticle tracking analysis, and flow cytometry. The impact of urinary or cell supernatant-derived EVs on cellular phenotypes was evaluated using cell-based assays following combined treatment with a specific CD147 inhibitor alone or in combination with a tissue factor pathway inhibitor (TFPI), an endogenous anticoagulant protein that can be released by low-molecular-weight heparins. RESULTS: We observed that EVs obtained from the urine samples of patients with muscle-invasive BLCA and from the aggressive bladder cancer cell line J82 exhibited higher TF activity and CD147 expression levels than did their non-invasive counterparts. The shedding of GFP-tagged CD147 into isolated vesicles demonstrated that the vesicles originated from plasma cell membranes. EVs originating from invasive cancer cells were found to trigger migration, secretion of matrix metalloproteinases (MMPs), and invasion. The same induction of MMP activity was replicated using EVs obtained from urine samples of patients with invasive BLCA. EVs derived from cancer cell clones overexpressing TF and CD147 were produced in higher quantities and exhibited a higher invasive potential than those from control cancer cells. TFPI interfered with the effect when used in conjunction with the CD147 inhibitor, further suppressing homotypic EV-induced migration, MMP production, and invasion. CONCLUSIONS: Our findings suggest that combining a CD147 inhibitor with low molecular weight heparins to induce TFPI release may be a promising therapeutic approach for urothelial cancer management. This combination can potentially suppress the tumor-promoting actions of cancer-derived microvesicle-like EVs, including collective matrix invasion.

Small particles or vesicles released by cancer cells into their surroundings have the potential to stimulate the spread and growth of cancer cells. In this study, we focused on two specific molecules presented by these cancer cell-derived vesicles that could play a role in promoting the dissemination of cancer cells: a protein related to blood clotting and a protein on the cell surface.We found that large vesicles from bladder cancer cells that have the ability to spread had higher levels of these proteins than vesicles from nonspreading cancer cells. We also found that the former could make cancer cells move about more, produce more of a substance that helps cancer cells spread, and invade other tissues.To counteract the cancer-promoting actions of these vesicles, we examined the impact of combining a naturally occurring anticlotting protein that can be released by medications derived from heparin with an inhibitor targeting the cancer cell surface protein. We found that this combination stopped the vesicles from helping cancer cells move about more, produce more of the spreading substance, and invade other tissues.This approach of simultaneously targeting the two protein molecules present on cancer cell-derived vesicles might be a new way to treat bladder cancer.

Mycobacterium abscessus extracellular vesicles increase mycobacterial resistance to clarithromycin in vitro.

Nontuberculous Mycobacteria (NTM) are a group of emerging bacterial pathogens that have been identified in cystic fibrosis (CF) patients with microbial lung infections. The treatment of NTM infection in CF patients is challenging due to the natural resistance of NTM species to many antibiotics. Mycobacterium abscessus is one of the most common NTM species found in the airways of CF patients. In this study, we characterized the extracellular vesicles (EVs) released by drug-sensitive M. abscessus untreated or treated with clarithromycin (CLR), one of the frontline anti-NTM drugs. Our data show that exposure to CLR increases mycobacterial protein trafficking into EVs as well as the secretion of EVs in culture. Additionally, EVs released by CLR-treated M. abscessus increase M. abscessus resistance to CLR when compared to EVs from untreated M. abscessus. Proteomic analysis further indicates that EVs released by CLR-treated M. abscessus carry an increased level of 50S ribosomal subunits, the target of CLR. Taken together, our results suggest that EVs play an important role in M. abscessus resistance to CLR treatment.

Leveraging Human Plasma-Derived Small Extracellular Vesicles as Liquid Biopsy to Study the Induction of Cytochrome P450 3A4 by Modafinil.

Preparations of plasma-derived small extracellular vesicles (sEVs) were deployed as liquid biopsy to study cytochrome P450 (CYP) 3A4 (CYP3A4) induction following modafinil 400 mg once daily × 14 days (young healthy volunteers, N = 10 subjects). Induction was confirmed using the 4ß-hydroxycholesterol-to-cholesterol (4ßHC/C) ratio, a plasma CYP3A4/5 biomarker, with a mean 2.1-fold increase (Day 15 vs. Day 1; 90% confidence interval (CI) = 1.8-2.3; P value = 0.0004). Proteomic analysis revealed the induction (mean Day 15 vs. Day 1 fold-increase (90% CI)) of both liver (1.3 (1.1-1.5), P value = 0.014) and nonliver (1.9 (1.6-2.2), P value = 0.04) sEV CYP3A4 protein expression. In CYP3A5 nonexpresser subjects, the baseline (pre-dose) 4ßHC/C plasma ratio was more highly correlated with liver sEVs (r = 0.937, P value = 0.001) than nonliver sEVs (r = 0.619, P value = 0.101) CYP3A4 protein expression. When CYP3A5 expressers (CYP3A5*1/*3) were included, the correlation with liver sEVs (r = 0.761, P value = 0.011) and nonliver sEVs (r = 0.391, P value = 0.264) CYP3A4 protein was weaker. Although modafinil-induced changes in plasma 4ßHC/C ratio did not correlate with sEVs CYP3A4 protein expression, the individual subject sEVs proteomic data were used successfully to predict victim drug (midazolam, triazolam, dextromethorphan, 17α-ethinylestradiol, and abemaciclib) area under the plasma concentration-time curve (AUC) ratios (AUCRs) following modafinil. Based on the AUCR values, modafinil was classified as a weak to moderate CYP3A4 inducer (vs. rifampicin). For the first time, it was possible to deploy plasma-derived sEVs to study CYP3A4 induction beyond rifampicin, a more potent CYP3A4 inducer.

Therapeutic Effects of Hyaluronic Acid Against Cytotoxic Extracellular Vesicles Released During Pseudomonas Aeruginosa Pneumonia.

ABSTRACT: Extracellular vesicles (EVs) have now been recognized as important mediators of cellular communication during injury and repair. We previously found that plasma EVs isolated from ex vivo perfused human lungs injured with Escherichia coli bacterial pneumonia were inflammatory, and exogenous administration of high molecular weight (HMW) hyaluronic acid (HA) as therapy bound to these EVs, decreasing inflammation and injury. In the current study, we studied the role of EVs released during severe Pseudomonas aeruginosa (PA) pneumonia in mice and determined whether intravenous administration of exogenous HMW HA would have therapeutic effects against the bacterial pneumonia. EVs were collected from the bronchoalveolar lavage fluid (BALF) of mice infected with PA103 by ultracentrifugation and analyzed by NanoSight and flow cytometry. In a cytotoxicity assay, administration of EVs released from infected mice (I-EVs) decreased the viability of A549 cells compared to EV isolated from sham control mice (C-EVs). Either exogenous HMW HA or an anti-CD44 antibody, when co-incubated with I-EVs, significantly improved the viability of the A549 cells. In mice with PA103 pneumonia, administration of HMW HA improved pulmonary edema and bacterial count in the lungs and decreased TNF-α and caspase-3 levels in the supernatant of lung homogenates. In conclusion, EVs isolated from BALF of mice with P. aeruginosa pneumonia were cytotoxic and inflammatory, and intravenous HMW HA administration was protective against P. aeruginosa pneumonia.

Profiling of extracellular vesicle-bound miRNA to identify candidate biomarkers of chronic alcohol drinking in nonhuman primates.

BACKGROUND: Long-term alcohol drinking is associated with numerous health complications including susceptibility to infection, cancer, and organ damage. However, due to the complex nature of human drinking behavior, it has been challenging to identify reliable biomarkers of alcohol drinking behavior prior to signs of overt organ damage. Recently, extracellular vesicle-bound microRNAs (EV-miRNAs) have been found to be consistent biomarkers of conditions that include cancer and liver disease. METHODS: In this study, we profiled the plasma EV-miRNA content by miRNA-Seq from 80 nonhuman primates after 12 months of voluntary alcohol drinking. RESULTS: We identified a list of up- and downregulated EV-miRNA candidate biomarkers of heavy drinking and those positively correlated with ethanol dose. We overexpressed these candidate miRNAs in control primary peripheral immune cells to assess their potential functional mechanisms. We found that overexpression of miR-155, miR-154, miR-34c, miR-450a, and miR-204 led to increased production of the inflammatory cytokines TNFα or IL-6 in peripheral blood mononuclear cells after stimulation. CONCLUSION: This exploratory study identified several EV-miRNAs that could serve as biomarkers of long-term alcohol drinking and provide a mechanism to explain alcohol-induced peripheral inflammation.

Treatment Failure in Acute Myeloid Leukemia: Focus on the Role of Extracellular Vesicles.

Acute myeloblastic leukemia (AML) is one of the most common types of blood malignancies that results in an AML-associated high mortality rate each year. Several causes have been reported as prognostic factors for AML in children and adults, the most important of which are cytogenetic abnormalities and environmental risk factors. Following the discovery of numerous drugs for AML treatment, leukemic cells sought a way to escape from the cytotoxic effects of chemotherapy drugs, leading to treatment failure. Nowadays, comprehensive studies have looked at the role of extracellular vesicles (EVs) secreted by AML blasts and how the microenvironment of the tumor changes in favor of cancer progression and survival to discover the mechanisms of treatment failure to choose the well-advised treatment. Reports show that malignant cells secrete EVs that transmit messages to adjacent cells and the tumor's microenvironment. By secreting EVs, containing immune-inhibiting cytokines, AML cells inactivate the immune system against malignant cells, thus ensuring their survival. Also, increased secretion of EVs in various malignancies indicates an unfavorable prognostic factor and the possibility of drug resistance. In this study, we briefly reviewed the challenges of treating AML with a glance at the EVs' role in this process. It is hoped that with a deeper understanding of EVs, new therapies will be developed to eliminate the relapse of leukemic cells.

Movement of Mitochondria with Mutant DNA through Extracellular Vesicles Helps Cancer Cells Acquire Chemoresistance.

Triple negative breast cancer (TNBC) is one of the most aggressive subtypes of breast cancer with the worst prognosis after chemo- or radiation therapy. This is mainly due to the development of cancer chemoresistance accompanied by tumor recurrence. In this work, we investigated a new mechanism of acquired chemoresistance of TNBC cells. We showed that extracellular vehicles (EVs) of chemoresistant TNBC cells can transfer mitochondria to sensitive cancer cells, thus increasing their chemoresistance. Such transfer, but with less efficiency, can be carried out over short distances using tunneling nanotubes. In addition, we showed that exosome fractions carrying mitochondria from resistant TNBC cells contribute to acquired chemoresistance by increasing mtDNA levels with mutations in the mtND4 gene responsible for tumorigenesis. Blocking mitochondrial transport by exosome inhibitors, including GW4869, reduced acquired TNBC chemoresistance. These results could lead to the identification of new molecular targets necessary for more effective treatment of this type of cancer.

The Pyrazolyl-Urea Gege3 Inhibits the Activity of ANXA1 in the Angiogenesis Induced by the Pancreatic Cancer Derived EVs.

The pyrazolyl-urea Gege3 molecule has shown interesting antiangiogenic effects in the tumor contest. Here, we have studied the role of this compound as interfering with endothelial cells activation in response to the paracrine effects of annexin A1 (ANXA1), known to be involved in promoting tumor progression. ANXA1 has been analyzed in the extracellular environment once secreted through microvesicles (EVs) by pancreatic cancer (PC) cells. Particularly, Gege3 has been able to notably prevent the effects of Ac2-26, the ANXA1 mimetic peptide, and of PC-derived EVs on endothelial cells motility, angiogenesis, and calcium release. Furthermore, this compound also inhibited the translocation of ANXA1 to the plasma membrane, otherwise induced by the same ANXA1-dependent extracellular stimuli. Moreover, these effects have been mediated by the indirect inhibition of protein kinase Cα (PKCα), which generally promotes the phosphorylation of ANXA1 on serine 27. Indeed, by the subtraction of intracellular calcium levels, the pathway triggered by PKCα underwent a strong inhibition leading to the following impediment to the ANXA1 localization at the plasma membrane, as revealed by confocal and cytofluorimetry analysis. Thus, Gege3 appeared an attractive molecule able to prevent the paracrine effects of PC cells deriving ANXA1 in the tumor microenvironment.

Aesculetin Accelerates Osteoblast Differentiation and Matrix-Vesicle-Mediated Mineralization.

The imbalance between bone resorption and bone formation in favor of resorption results in bone loss and deterioration of bone architecture. Osteoblast differentiation is a sequential event accompanying biogenesis of matrix vesicles and mineralization of collagen matrix with hydroxyapatite crystals. Considerable efforts have been made in developing naturally-occurring plant compounds, preventing bone pathologies, or enhancing bone regeneration. Coumarin aesculetin inhibits osteoporosis through hampering the ruffled border formation of mature osteoclasts. However, little is known regarding the effects of aesculetin on the impairment of matrix vesicle biogenesis. MC3T3-E1 cells were cultured in differentiation media with 1-10 µM aesculetin for up to 21 days. Aesculetin boosted the bone morphogenetic protein-2 expression, and alkaline phosphatase activation of differentiating MC3T3-E1 cells. The presence of aesculetin strengthened the expression of collagen type 1 and osteoprotegerin and transcription of Runt-related transcription factor 2 in differentiating osteoblasts for 9 days. When ≥1-5 µM aesculetin was added to differentiating cells for 15-18 days, the induction of non-collagenous proteins of bone sialoprotein II, osteopontin, osteocalcin, and osteonectin was markedly enhanced, facilitating the formation of hydroxyapatite crystals and mineralized collagen matrix. The induction of annexin V and PHOSPHO 1 was further augmented in ≥5 µM aesculetin-treated differentiating osteoblasts for 21 days. In addition, the levels of tissue-nonspecific alkaline phosphatase and collagen type 1 were further enhanced within the extracellular space and on matrix vesicles of mature osteoblasts treated with aesculetin, indicating matrix vesicle-mediated bone mineralization. Finally, aesculetin markedly accelerated the production of thrombospondin-1 and tenascin C in mature osteoblasts, leading to their adhesion to preformed collagen matrix. Therefore, aesculetin enhanced osteoblast differentiation, and matrix vesicle biogenesis and mineralization. These findings suggest that aesculetin may be a potential osteo-inductive agent preventing bone pathologies or enhancing bone regeneration.

Different Ability of Multidrug-Resistant and -Sensitive Counterpart Cells to Release and Capture Extracellular Vesicles.

Cancer multidrug resistance (MDR) is one of the main challenges for cancer treatment efficacy. MDR is a phenomenon by which tumor cells become resistant to several unrelated drugs. Some studies have previously described the important role of extracellular vesicles (EVs) in the dissemination of a MDR phenotype. EVs' cargo may include different players of MDR, such as microRNAS and drug-efflux pumps, which may be transferred from donor MDR cells to recipient drug-sensitive counterparts. The present work aimed to: (i) compare the ability of drug-sensitive and their MDR counterpart cells to release and capture EVs and (ii) study and relate those differences with possible distinct fate of the endocytic pathway in these counterpart cells. Our results showed that MDR cells released more EVs than their drug-sensitive counterparts and also that the drug-sensitive cells captured more EVs than their MDR counterparts. This difference in the release and capture of EVs may be associated with differences in the endocytic pathway between drug-sensitive and MDR cells. Importantly, manipulation of the recycling pathway influenced the response of drug-sensitive cells to doxorubicin treatment.

Myofibroblast transdifferentiation is associated with changes in cellular and extracellular vesicle miRNA abundance.

Transforming growth factor-beta 1 (TGF-ß1), a pro-fibrotic tumour-derived factor promotes fibroblast differentiation in the tumour microenvironment and is thought to contribute to the development of pro-tumourigenic cancer-associated fibroblasts (CAFs) by promoting myofibroblast differentiation. miRNA dysregulation has been demonstrated in myofibroblast transdifferentiation and CAF activation, however, their expression varies among cell types and with the method of fibroblast induction. Here, the expression profile of miRNA in human primary oral fibroblasts treated with TGF-ß1, to derive a myofibroblastic, CAF-like phenotype, was determined compared to untreated fibroblasts. Myofibroblast transdifferentiation was determined by the expression of alpha-smooth muscle actin (α-SMA) and fibronectin-1 extra domain A (FN-EDA1) using quantitative real-time PCR (qRT-PCR) and western blot. The formation of stress fibres was assessed by fluorescence microscopy, and associated changes in contractility were assessed using collagen contraction assays. Extracellular vesicles (EVs) were purified by using size exclusion chromatography and ultracentrifugation and their size and concentration were determined by nanoparticle tracking analysis. miRNA expression profiling in oral fibroblasts treated with TGF-ß1 and their extracellular vesicles was carried out using tiling low-density array cards. The Database for Annotation, Visualization, and Integrated Discovery (DAVID) was used to perform functional and pathway enrichment analysis of target genes. In this study, TGF-ß1 induced a myofibroblastic phenotype in normal oral fibroblasts as assessed by expression of molecular markers, the formation of stress fibres and increased contractility. TaqMan Low-Density Array (TLDA) analysis demonstrated that miR-503 and miR-708 were significantly upregulated, while miR-1276 was significantly downregulated in TGF-ß1-treated oral fibroblasts (henceforth termed experimentally-derived CAF, eCAF). The gene functional enrichment analysis showed that the candidate miRNAs have the potential to modulate various pathways; including the Ras associated protein 1 (Rap1), PI3K-Akt, and tumour necrosis factor (TNF) signalling pathways. In addition, altered levels of several miRNAs were detected in eCAF EV, including miR-142 and miR-222. No differences in size or abundance of EV were detected between eCAF and normal oral fibroblast (NOF). Little overlap was observed between changes in cellular and EV miRNA profiles, suggesting the possibility of selective loading of EV miRNA. The study reveals miRNA expression signature could be involved in myofibroblast transdifferentiation and the miRNA cargo of their EV, providing novel insight into the involvement of miRNA in CAF development and function.

Reversing the surface charge of MSC-derived small extracellular vesicles by εPL-PEG-DSPE for enhanced osteoarthritis treatment.

Mesenchymal stem cell-derived small extracellular vesicles (MSC-sEVs) possess a great therapeutical potential for osteoarthritis (OA) treatment. However, the steric and electrostatic hindrance of cartilage matrix leads to very limited distribution of MSC-sEVs in cartilage and low bioavailability of MSC-sEVs after intra-articular injection. To overcome this, a strategy to reverse the surface charge of MSC-sEVs by modifying the MSC-sEVs with a novel cationic amphiphilic macromolecule namely ε-polylysine-polyethylene-distearyl phosphatidylethanolamine (PPD) was developed in this study. Through incubation with 100 µg/ml PPD, positively charged MSC-sEVs (PPD-sEVs) were obtained, and the modification process showed nearly no disturbance to the integrity and contents of sEVs and exhibited good stability under the interference of anionic macromolecules. A more effective cellular uptake and homeostasis modulation ability of PPD-sEVs than unmodified MSC-sEVs to chondrocytes was demonstrated. More importantly, PPD-sEVs demonstrated significantly enhanced cartilage uptake, cartilage penetration, and joint retention capacity as compared to MSC-sEVs. Intra-articular injection of PPD-sEVs into a mouse OA model showed significantly improved bioavailability than MSC-sEVs, which resulted in enhanced therapeutic efficacy with reduced injection frequency. In general, this study provides a facile and effective strategy to improve the intra-articular bioavailability of MSC-sEVs and has a great potential to accelerate the clinical practice of MSC-sEVs based OA therapy.

Extracellular vesicles and immunogenic stress in cancer.

Tumor progression requires bidirectional cell-to-cell communication within a complex tumor microenvironment (TME). Extracellular vesicles (EVs) as carriers have the capacity to shuttle regulatory molecules, including nucleic acids, proteins, and lipids, between cancer cells and multiple stromal cells, inducing remarkable phenotypic alterations in the TME. Recently proposed the concept "immunogenic stress", which means in some stressed microenvironment, cancer cells can release EVs containing specific immunoregulatory mediators, depending on the initiating stress-associated pathway, thereby provoking the changes of immune status in the TME. Considerable evidence has revealed that the intracellular mechanisms underlying the response to diverse stresses are mainly autophagy, endoplasmic reticulum (ER) stress reactions and the DNA damage response (DDR). In addition, the activation of immunogenic stress responses endows hosts with immune surveillance capacity; in contrast, several cargoes in EVs under immunogenic stress trigger a passive immune response by mediating the function of immune cells. This review discusses the current understanding of the immunogenic stress pathways in cancer and describes the interrelation between EVs and immunogenic stress to propose potential treatment strategies and biomarkers.

Exploring the Extracellular Vesicle MicroRNA Expression Repertoire in Patients with Rheumatoid Arthritis and Ankylosing Spondylitis Treated with TNF Inhibitors.

Rheumatoid arthritis (RA) and ankylosing spondylitis (AS) belong to the most common inflammatory rheumatic diseases. MicroRNAs (miRNAs) are small 18-22 RNA molecules that function as posttranscriptional regulators. They are abundantly present within extracellular vesicles (EVs), small intercellular communication vesicles that can be found in bodily fluids and that have key functions in pathological and physiological pathways. Recently, EVs have gained much interest because of their diagnostic and therapeutic potential. Using NanoString profiling technology, the miRNA repertoire of serum EVs was determined and compared in RA and AS patients before and after anti-TNF therapy to assess its potential use as a diagnostic and prognostic biomarker. Furthermore, possible functional effects of those miRNAs that were characterized by the most significant expression changes were evaluated using in silico prediction algorithms. The analysis revealed a unique profile of differentially expressed miRNAs in RA and AS patient serum EVs. We identified 12 miRNAs whose expression profiles enabled differentiation between RA and AS patients before induction of anti-TNF treatment, as well as 4 and 14 miRNAs whose repertoires were significantly changed during the treatment in RA and AS patients, respectively. In conclusion, our findings suggest that extracellular vesicle miRNAs could be used as potential biomarkers associated with RA and AS response to biological treatment.

Cytochalasin B-induced membrane vesicles from human mesenchymal stem cells overexpressing TRAIL, PTEN and IFN-ß1 can kill carcinoma cancer cells.

Extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) are of interest as a new vector for the delivery of therapeutic agents into the tumor microenvironment. Cell-free EV-based therapy has a number of advantages over cell-based therapy, since the use of EVs allows avoiding potential undesirable transformation associated with MSCs. MSC-derived EVs can transfer natural proteins with immunomodulatory or antitumor properties. The aim of this study was to produce vesicles from mesenchymal stem cells with simultaneous overexpression of TRAIL, PTEN and IFN-ß1 and analyze its antitumor and immunomodulatory properties. In this work, a stable line of human adipose tissue-derived mesenchymal stem cells (hADSCs) with simultaneous overexpression of TRAIL, PTEN and IFN-ß1 was produced. To obtain this cell line hADSCs were genetically modified with a genetic multicistronic cassette encoding TRAIL, PTEN, and IFN-ß1 genes separated with a self-cleaving P2A peptide nucleotide sequence. Membrane vesicles (CIMVs) were obtained from genetically modified hADSCs using cytochalasin B treatment. Antitumor and immunomodulatory properties of the CIMVs were analyzed in vitro. It was shown that CIMVs isolated from genetically modified hADSCs overexpressing TRAIL, PTEN and IFN-ß1 genes are able to activate human immune cells and induce apoptosis in various types of carcinomas in vitro. Thus, the immunomodulatory and antitumor properties of CIMVs were shown. However, further studies on animal models in vivo are required.

PINK1 drives production of mtDNA-containing extracellular vesicles to promote invasiveness.

The cystine-glutamate antiporter, xCT, supports a glutathione synthesis program enabling cancer cells to cope with metabolically stressful microenvironments. Up-regulated xCT, in combination with glutaminolysis, leads to increased extracellular glutamate, which promotes invasive behavior by activating metabotropic glutamate receptor 3 (mGluR3). Here we show that activation of mGluR3 in breast cancer cells activates Rab27-dependent release of extracellular vesicles (EVs), which can transfer invasive characteristics to "recipient" tumor cells. These EVs contain mitochondrial DNA (mtDNA), which is packaged via a PINK1-dependent mechanism. We highlight mtDNA as a key EV cargo necessary and sufficient for intercellular transfer of invasive behavior by activating Toll-like receptor 9 in recipient cells, and this involves increased endosomal trafficking of pro-invasive receptors. We propose that an EV-mediated mechanism, through which altered cellular metabolism in one cell influences endosomal trafficking in other cells, is key to generation and dissemination of pro-invasive microenvironments during mammary carcinoma progression.

Lung Fibrosis Is Improved by Extracellular Vesicles from IFNγ-Primed Mesenchymal Stromal Cells in Murine Systemic Sclerosis.

BACKGROUND: Systemic sclerosis (SSc) is a severe autoimmune disease for which mesenchymal stromal cells (MSCs)-based therapy was reported to reduce SSc-related symptoms in pre-clinical studies. Recently, extracellular vesicles released by MSCs (MSC-EVs) were shown to mediate most of their therapeutic effect. Here, we aimed at improving their efficacy by increasing the MSC-EV dose or by IFNγ-priming of MSCs. METHODS: small size (ssEVs) and large size EVs (lsEVs) were recovered from murine MSCs that were pre-activated using 1 or 20 ng/mL of IFNγ. In the HOCl-induced model of SSc, mice were treated with EVs at day 21 and sacrificed at day 42. Lung and skin samples were collected for histological and molecular analyses. RESULTS: increasing the dose of MSC-EVs did not add benefit to the dose previously reported to be efficient in SSc. By contrast, IFNγ pre-activation improved MSC-EVs-based treatment, essentially in the lungs. Low doses of IFNγ decreased the expression of fibrotic markers, while high doses improved remodeling and anti-inflammatory markers. IFNγ pre-activation upregulated iNos, IL1ra and Il6 in MSCs and ssEVs and the PGE2 protein in lsEVs. CONCLUSION: IFNγ-pre-activation improved the therapeutic effect of MSC-EVs preferentially in the lungs of SSc mice by modulating anti-inflammatory and anti-fibrotic markers.

Cocaine augments neuro-inflammation via modulating extracellular vesicle release in HIV-1 infected immune cells.

BACKGROUND: Extracellular Vesicles (EV) recently have been implicated in the pathogenesis of HIV-1 syndromes, including neuroinflammation and HIV-1 associated neurological disorder (HAND). Cocaine, an illicit stimulant drug used worldwide is known to exacerbate these HIV-1 associated neurological syndromes. However, the effects of cocaine on EV biogenesis and roles of EVs in enhancing HIV-1 pathogenesis are not yet well defined. RESULTS: Here, we investigated the effects of cocaine on EV biogenesis and release in HIV-1 infected immune cells and explored their roles in elicitation of neuroinflammation. We found that cocaine significantly augmented the release of EVs from uninfected and HIV-1 infected T-cells, DCs and macrophages. Further analysis of the molecular components of EVs revealed enhanced expression of adhesion molecules integrin ß1 and LFA-1 in those EVs derived from cocaine treated cells. Intriguingly, in EVs derived from HIV-1 infected cells, cocaine treatment significantly increased the levels of viral genes in EVs released from macrophages and DCs, but not in T-cells. Exploring the molecular mechanism to account for this, we found that DCs and macrophages showed enhanced expression of the cocaine receptor Sigma 1-Receptor compared to T-cells. In addition, we found that cocaine significantly altered the integrity of the RNA-induced silencing complex (RISC) in HIV-1 infected macrophages and DCs compared to untreated HIV-1 infected cells. Characterizing further the molecular mechanisms involved in how cocaine increased EV release, we found that cocaine decreased the expression of the interferon-inducible protein BST-2; this resulted in altered trafficking of intracellular virus containing vesicles and EV biogenesis and release. We also observed EVs released from cocaine treated HIV-1 infected macrophages and DCs enhanced HIV-1 trans-infection to T-cells compared to those from untreated and HIV-1 infected cells. These EVs triggered release of proinflammatory cytokines in human brain microvascular endothelial cells (HBMECs) and altered monolayer integrity. CONCLUSIONS: Taken together, our results provide a novel mechanism which helps to elucidate the enhanced prevalence of neurological disorders in cocaine using HIV-1 infected individuals and offers insights into developing novel therapeutic strategies against HAND in these hosts.