Enhanced prothrombotic and proinflammatory activity of circulating extracellular vesicles in acute exacerbations of chronic obstructive pulmonary disease.

BACKGROUND: Acute exacerbations of chronic obstructive pulmonary disease (AE-COPD) are associated with a high rate of cardiovascular events. Thromboinflammation (the interplay between coagulation and inflammation) is probably involved in these events. Extracellular vesicles (EV) increase during AE-COPD, but their role in thromboinflammation in COPD is still unknown. We investigated EV-associated prothrombotic and proinflammatory activity in COPD. METHODS: Patients with AE-COPD, stable COPD (sCOPD) and age- and sex-matched subjects (controls) were enrolled. AE-COPD patients were evaluated at hospital admission and 8 weeks after discharge (recovery; longitudinal arm). In a cross-sectional arm, AE-COPD were compared with sCOPD and controls. EV-mediated prothrombotic activity was tested by measuring the concentration of EV-associated phosphatidylserine, as assessed by a prothrombinase assay, and tissue factor, as assessed by a modified one-stage clotting assay (EV-PS and EV-TF, respectively). Synthesis of interleukin-8 (IL-8) and C-C motif chemokine ligand-2 (CCL-2) by cells of the human bronchial epithelial cell line 16HBE incubated with patients' EV was used to measure EV-mediated proinflammatory activity. RESULTS: Twenty-five AE-COPD (median age [interquartile range] 74.0 [14.0] years), 31 sCOPD (75.0 [9.5] years) and 12 control (67.0 [3.5] years) subjects were enrolled. In the longitudinal arm, EV-PS, EV-TF, IL-8 and CCL-2 levels were all significantly higher at hospital admission than at recovery. Similarly, in the cross-sectional arm, EV-PS, EV-TF and cytokines synthesis were significantly higher in AE-COPD than in sCOPD and controls. CONCLUSIONS: EV exert prothrombotic and proinflammatory activities during AE-COPD and may therefore be effectors of thromboinflammation, thus contributing to the higher cardiovascular risk in AE-COPD.

Isolation and Characterization of Cetacean Cell-Derived Extracellular Vesicles.

Cetaceans are of scientific interest because they are good candidates as environmental bioindicators. However, in vivo research is arduous and in vitro studies represent a rarely used valid alternative. Extracellular vesicles (EVs) are membrane-bound structures playing roles in cell-to-cell communication. Despite being a promising investigative tool in different fields of science, EVs have been poorly studied in cetaceans. To fill this gap, we describe the preliminary characterization of EVs isolated from a bottlenose dolphin and a Cuvier's beaked whale cell line. EVs have been isolated with ultracentrifugation (UC) or size exclusion chromatography (SEC) and characterized with nanoparticle tracking analysis (NTA), Western blotting (WB), and scanning transmission electron microscopy (STEM). UC and SEC allowed the isolation of mainly small EVs (<200 nm). A higher number of particles were isolated through UC compared to SEC from both cell lines. At WB, all EVs expressed the EV-markers CD9 and integrin-ß. Only EVs isolated with UC were positive for TSG101. In conclusion, we isolated for the first time EVs from a bottlenose dolphin and a Cuvier's beaked whale cell line using two different techniques. Further studies on cell-derived EVs will be useful to deepen our knowledge on cetacean pathophysiology and health status assessment.

Metastatic Dissemination: Role of Tumor-Derived Extracellular Vesicles and Their Use as Clinical Biomarkers.

Cancer is a major cause of mortality in humans; often, rather than the primary tumor, it is the presence of metastases that are the cause of death. Extracellular vesicles (EVs) are small structures released by both normal and cancer cells; regarding the latter, they have been demonstrated to modulate almost all cancer-related processes, such as invasion, angiogenesis induction, drug resistance, and immune evasion. In the last years, it has become clear how EVs are widely involved in metastatic dissemination as well as in pre-metastatic niche (PMN) formation. Indeed, in order to achieve a successful metastatic process, i.e., penetration by cancer cells into distant tissues, the shaping of a favorable environment into those distant tissue, i.e., PMN formation, is mandatory. This process consists of an alteration that takes place in a distant organ and paves the way for the engraftment and growth of circulating tumor cells derived from the tumor primary site. This review focuses on the role of EVs in pre-metastatic niche formation and metastatic dissemination, also reporting the last studies suggesting the EVs role as biomarkers of metastatic diseases, possibly in a liquid biopsy approach.

Pentraxin 3, a new biomarker for the diagnosis and management of PJI in primary and revision hip arthroplasty.

Background/Aim of the study: The periprosthetic or superficial site infections are one of the most catastrophic and difficult to manage complications following total hip arthroplasty. Recently, in addition to well know systemic markers of inflammation, the blood and synovial fluid biomarkers are focused to have a possible role in the infection diagnosis. The long Pentraxin 3 (PTX3) seems to be a sensitive biomarker of acute phase inflammation. The objectives of this prospective and multicentre study were (1) to establish the plasma trend effectiveness of PTX3 in patients undergoing primary hip replacement, and (2) to evaluate the diagnostic accuracy of blood and synovial PTX3 in patients undergoing prosthetic revision of infected hip arthroplasty. METHODS: Human PTX3 was measured by ELISA in two cohorts of patients, 10 patients undergoing primary hip replacement for osteoarthritis and 9 patients with infected hip arthroplasty. RESULTS: The Authors were able to demonstrate that PTX3 is a viable biomarker for acute phase inflammation. CONCLUSIONS: An increase in PTX3 protein concentration in the synovial fluid of patients undergoing implant revision has a strong diagnostic capacity for periprosthetic joint infection, showing 97% specificity.

PCSK9 Confers Inflammatory Properties to Extracellular Vesicles Released by Vascular Smooth Muscle Cells.

Vascular smooth muscle cells (VSMCs) are key participants in both early- and late-stage atherosclerosis and influence neighbouring cells possibly by means of bioactive molecules, some of which are packed into extracellular vesicles (EVs). Proprotein convertase subtilisin/kexin type 9 (PCSK9) is expressed and secreted by VSMCs. This study aimed to unravel the role of PCSK9 on VSMCs-derived EVs in terms of content and functionality. EVs were isolated from human VSMCs overexpressing human PCSK9 (VSMCPCSK9-EVs) and tested on endothelial cells, monocytes, macrophages and in a model of zebrafish embryos. Compared to EVs released from wild-type VSMCs, VSMCPCSK9-EVs caused a rise in the expression of adhesion molecules in endothelial cells and of pro-inflammatory cytokines in monocytes. These acquired an increased migratory capacity, a reduced oxidative phosphorylation and secreted proteins involved in immune response and immune effector processes. Concerning macrophages, VSMCPCSK9-EVs enhanced inflammatory milieu and uptake of oxidized low-density lipoproteins, whereas the migratory capacity was reduced. When injected into zebrafish embryos, VSMCPCSK9-EVs favoured the recruitment of macrophages toward the site of injection. The results of the present study provide evidence that PCSK9 plays an inflammatory role by means of EVs, at least by those derived from smooth muscle cells of vascular origin.

Cancer Three-Dimensional Spheroids Mimic In Vivo Tumor Features, Displaying "Inner" Extracellular Vesicles and Vasculogenic Mimicry.

The role of extracellular vesicles (EVs) as mediators of cell-to-cell communication in cancer progression is widely recognized. In vitro studies are routinely performed on 2D culture models, but recent studies suggest that 3D cultures could represent a more valid model. Human ovarian cancer cells CABA I were cultured by the hanging drop method to form tumor spheroids, that were moved to low adhesion supports to observe their morphology by Scanning Electron Microscopy (SEM) and to isolate the EVs. EVs release was verified by SEM and their identity confirmed by morphology (Transmission Electron Microscopy, TEM), size distribution (Nanoparticles Tracking Analysis), and markers (CD63, CD9, TSG-101, Calnexin). CABA I form spheroids with a clinically relevant size, above 400 µm; they release EVs on their external surface and also trap "inner" EVs. They also produce vasculogenic mimicry-like tubules, that bulge from the spheroid and are composed of a hollow lumen delimited by tumor cells. CABA I can be grown as multicellular spheroids to easily isolate EVs. The presence of features typical of in vivo tumors (inner entrapped EVs and vasculogenic mimicry) suggests their use as faithful experimental models to screen therapeutic drugs targeting these pro-tumorigenic processes.

Cyclooxygenase-2 Upregulated by Temozolomide in Glioblastoma Cells Is Shuttled In Extracellular Vesicles Modifying Recipient Cell Phenotype.

Temozolomide (TMZ) resistance is frequent in patients with glioblastoma (GBM), a tumor characterized by a marked inflammatory microenvironment. Recently, we reported that cyclooxygenase-2 (COX-2) is upregulated in TMZ-resistant GBM cells treated with high TMZ concentrations. Moreover, COX-2 activity inhibition significantly counteracted TMZ-resistance of GBM cells. Extracellular vesicles (EV) are considered crucial mediators in orchestrating GBM drug resistance by modulating the tumor microenvironment (TME) and affecting the surrounding recipient cell phenotype and behavior. This work aimed to verify whether TMZ, at low and clinically relevant doses (5-20 µM), could induce COX-2 overexpression in GBM cells (T98G and U87MG) and explore if secreted EV shuttled COX-2 to recipient cells. The effect of COX-2 inhibitors (COXIB), Celecoxib (CXB), or NS398, alone or TMZ-combined, was also investigated. Our results indicated that TMZ at clinically relevant doses upregulated COX-2 in GBM cells. COXIB treatment significantly counteracted TMZ-induced COX-2 expression, confirming the crucial role of the COX-2/PGE2 system in TMZ-resistance. The COXIB specificity was verified on U251MG, COX-2 null GBM cells. Western blotting of GBM-EV cells showed the COX-2 presence, with the same intracellular trend, increasing in EV derived from TMZ-treated cells and decreasing in those derived from COXIB+TMZ-treated cells. We then evaluated the effect of EV secreted by TMZ-treated cells on U937 and U251MG, used as recipient cells. In human macrophage cell line U937, the internalization of EV derived by TMZ-T98G cells led to a shift versus a pro-tumor M2-like phenotype. On the other hand, EV from TMZ-T98G induced a significant decrease in TMZ sensitivity in U251MG cells. Overall, our results, in confirming the crucial role played by COX-2 in TMZ-resistance, provide the first evidence of the presence and effective functional transfer of this enzyme through EV derived from GBM cells, with multiple potential consequences at the level of TME.

Extracellular Vesicles-ceRNAs as Ovarian Cancer Biomarkers: Looking into circRNA-miRNA-mRNA Code.

Ovarian cancer (OC) is one of the most lethal gynecologic malignancies in females worldwide. OC is frequently diagnosed at an advanced stage due to a lack of specific symptoms and effective screening tests, resulting in a poor prognosis for patients. Age, genetic alterations, and family history are the major risk factors for OC pathogenesis. Understanding the molecular mechanisms underlying OC progression, identifying new biomarkers for early detection, and discovering potential targets for new drugs are urgent needs. Liquid biopsy (LB), used for cancer detection and management, consists of a minimally invasive approach and practical alternative source to investigate tumor alterations by testing extracellular vesicles (EVs), circulating tumor cells, tumor-educated platelets, and cell-free nucleic acids. EVs are nanosize vesicles shuttling proteins, lipids, and nucleic acids, such as DNA, RNA, and non-coding RNAs (ncRNAs), that can induce phenotypic reprogramming of target cells. EVs are natural intercellular shuttles for ncRNAs, such as microRNAs (miRNAs) and circular-RNAs (circRNAs), known to have regulatory effects in OC. Here we focus on the involvement of circRNAs and miRNAs in OC cancer progression. The circRNA-microRNA-mRNA axis has been investigated with Circbank and miRwalk analysis, unraveling the intricate and detailed regulatory network created by EVs, ncRNAs, and mRNAs in OC.

Tumor-Derived Extracellular Vesicles Activate Normal Human Fibroblasts to a Cancer-Associated Fibroblast-Like Phenotype, Sustaining a Pro-Tumorigenic Microenvironment.

Fibroblasts in the tumor microenvironment have been proven to actively participate in tumor progression; they can be "educated" by cancer cells acquiring an activated state and, as such, are identified as cancer-associated fibroblasts (CAFs); CAFs, in turn, remodel tumor stroma to be more advantageous for cancer progression by modulating several processes, including angiogenesis, immunosuppression, and drug access, presumably driving the chemoresistance. That is why they are believed to hamper the response to clinical therapeutic options. The communication between cancer cells and fibroblasts can be mediated by extracellular vesicles (EVs), composed of both exosomes (EXOs) and microvesicles (MVs). To verify the role of different subpopulations of EVs in this cross-talk, a nearly pure subpopulation of EXO-like EVs and the second one of mixed EXO- and MV-like EVs were isolated from ovarian cancer cells and administered to fibroblasts. It turned out that EVs can activate fibroblasts to a CAF-like state, supporting their proliferation, motility, invasiveness, and enzyme expression; EXO-like EV subpopulation seems to be more efficient in some of those processes, suggesting different roles for different EV subpopulations. Moreover, the secretome of these "activated" fibroblasts, composed of both soluble and EV-associated molecules, was, in turn, able to modulate the response of bystander cells (fibroblasts, tumor, and endothelial cells), supporting the idea that EVs sustain the mutual cross-talk between tumor cells and CAFs.

Extracellular vesicles mediate the communication between multiple myeloma and bone marrow microenvironment in a NOTCH dependent way.

Multiple myeloma (MM) is an incurable hematologic neoplasm, whose poor prognosis is deeply affected by the propensity of tumor cells to localize in the bone marrow (BM) and induce the protumorigenic activity of normal BM cells, leading to events associated with tumor progression, including tumor angiogenesis, osteoclastogenesis, and the spread of osteolytic bone lesions. The interplay between MM cells and the BM niche does not only rely on direct cell-cell interaction, but a crucial role is also played by MM-derived extracellular vesicles (MM-EV). Here, we demonstrated that the oncogenic NOTCH receptors are part of MM-EV cargo and play a key role in EV protumorigenic ability. We used in vitro and in vivo models to investigate the role of EV-derived NOTCH2 in stimulating the protumorigenic behavior of endothelial cells and osteoclast progenitors. Importantly, MM-EV can transfer NOTCH2 between distant cells and increase NOTCH signaling in target cells. MM-EV stimulation increases endothelial cell angiogenic ability and osteoclast differentiation in a NOTCH2-dependent way. Indeed, interfering with NOTCH2 expression in MM cells may decrease the amount of NOTCH2 also in MM-EV and affect their angiogenic and osteoclastogenic potential. Finally, we demonstrated that the pharmacologic blockade of NOTCH activation by γ-secretase inhibitors may hamper the biological effect of EV derived by MM cell lines and by the BM of MM patients. These results provide the first evidence that targeting the NOTCH pathway may be a valid therapeutic strategy to hamper the protumorigenic role of EV in MM as well as other tumors.

EV Separation: Release of Intact Extracellular Vesicles Immunocaptured on Magnetic Particles.

Extracellular vesicles (EVs) have attracted considerable interest due to their role in cell-cell communication, disease diagnosis, and drug delivery. Despite their potential in the medical field, there is no consensus on the best method for separating micro- and nanovesicles from cell culture supernatant and complex biological fluids. Obtaining a good recovery yield and preserving physical characteristics is critical for the diagnostic and therapeutic use of EVs. The separation of a single class of EVs, such as exosomes, is complex because blood and cell culture media contain many nanoparticles in the same size range. Methods that exploit immunoaffinity capture provide high-purity samples and overcome the issues of currently used separation methods. However, the release of captured nanovesicles usually requires harsh conditions that hinder their use in certain types of downstream analysis. A novel capture and release approach for small extracellular vesicles (sEVs) is presented based on DNA-directed immobilization of antiCD63 antibody. The flexible DNA linker increases the capture efficiency and allows for releasing EVs by exploiting the endonuclease activity of DNAse I. This separation protocol works under mild conditions, enabling the release of vesicles suitable for analysis by imaging techniques. In this study, sEVs recovered from plasma were characterized by established techniques for EV analysis, including nanoparticle tracking and transmission electron microscopy.

The Inflammatory Cytokine IL-3 Hampers Cardioprotection Mediated by Endothelial Cell-Derived Extracellular Vesicles Possibly via Their Protein Cargo.

The biological relevance of extracellular vesicles (EV) released in an ischemia/reperfusion setting is still unclear. We hypothesized that the inflammatory microenvironment prevents cardioprotection mediated by endothelial cell (EC)-derived extracellular vesicles. The effects of naïve EC-derived EV (eEV) or eEV released in response to interleukin-3 (IL-3) (eEV-IL-3) were evaluated in cardiomyoblasts (H9c2) and rat hearts. In transwell assay, eEV protected the H9c2 exposed to hypoxia/reoxygenation (H/R) more efficiently than eEV-IL-3. Conversely, only eEV directly protected H9c2 cells to H/R-induced damage. Consistent with this latter observation, eEV, but not eEV-IL-3, exerted beneficial effects in the whole heart. Protein profiles of eEV and eEV-IL-3, established using label-free mass spectrometry, demonstrated that IL-3 drives changes in eEV-IL-3 protein cargo. Gene ontology analysis revealed that both eEV and eEV-IL-3 were equipped with full cardioprotective machinery, including the Nitric Oxide Signaling in the Cardiovascular System. eEV-IL-3 were also enriched in the endothelial-nitric oxide-synthase (eNOS)-antagonist caveolin-1 and proteins related to the inflammatory response. In vitro and ex vivo experiments demonstrated that a functional Mitogen-Activated Protein Kinase Kinase (MEK1/2)/eNOS/guanylyl-cyclase (GC) pathway is required for eEV-mediated cardioprotection. Consistently, eEV were found enriched in MEK1/2 and able to induce the expression of B-cell-lymphoma-2 (Bcl-2) and the phosphorylation of eNOS in vitro. We conclude that an inflammatory microenvironment containing IL-3 changes the eEV cargo and impairs eEV cardioprotective action.

Biological effects of selective COX-2 inhibitor NS398 on human glioblastoma cell lines.

BACKGROUND: Cyclooxygenase-2 (COX-2), an inflammation-associated enzyme, has been implicated in tumorigenesis and progression of glioblastoma (GBM). The poor survival of GBM was mainly associated with the presence of glioma stem cells (GSC) and the markedly inflammatory microenvironment. To further explore the involvement of COX-2 in glioma biology, the effects of NS398, a selective COX-2 inhibitor, were evaluated on GSC derived from COX-2 expressing GBM cell lines, i.e., U87MG and T98G, in terms of neurospheres' growth, autophagy, and extracellular vesicle (EV) release. METHODS: Neurospheres' growth and morphology were evaluated by optical and scanning electron microscopy. Autophagy was measured by staining acidic vesicular organelles. Extracellular vesicles (EV), released from neurospheres, were analyzed by transmission electron microscopy. The autophagic proteins Beclin-1 and LC3B, as well as the EV markers CD63 and CD81, were analyzed by western blotting. The scratch assay test was used to evaluate the NS398 influence on GBM cell migration. RESULTS: Both cell lines were strongly influenced by NS398 exposure, as showed by morphological changes, reduced growth rate, and appearance of autophagy. Furthermore, the inhibitor led to a functional change of EV released by neurospheres. Indeed, EV secreted by NS398-treated GSC, but not those from control cells, were able to significantly inhibit adherent U87MG and T98G cell migration and induced autophagy in recipient cells, thus leading to effects quite similar to those directly caused by NS398 in the same cells. CONCLUSION: Despite the intrinsic diversity and individual genetic features of U87MG and T98G, comparable effects were exerted by the COX-2 inhibitor NS398 on both GBM cell lines. Overall, our findings support the crucial role of the inflammatory-associated COX-2/PGE2 system in glioma and glioma stem cell biology.

Breast Cancer Derived Extracellular Vesicles in Bone Metastasis Induction and Their Clinical Implications as Biomarkers.

Cancer incidence and mortality are rapidly growing worldwide. The main risk factors for cancer can be associated with aging as well as the growth of the population and socioeconomic condition. Breast cancer, a crucial public health problem, is the second cause of death among women. About 70% of patients with advanced breast cancer have bone metastases. In bone metastasis, cancer cells and osteoclasts form a vicious cycle: cancer cells promote osteoclast differentiation and activation that, in turn, induce cancer cell seeding and proliferation in the bone. Growing evidence shows that extracellular vesicles (EVs) play a key role in carcinogenesis, proliferation, pre-metastatic niche formation, angiogenesis, metastasis, and chemoresistance in several tumors, such as breast, lung, prostate, and liver cancer. Here, we discuss the role of EVs released by breast cancer cells, focusing on bone metastasis induction and their clinical implications as biomarkers.

INSIDE Project: Individual Air Pollution Exposure, Extracellular Vesicles Signaling and Hypertensive Disorder Development in Pregnancy.

Hypertensive disorders are common complications during pregnancy (HDP) with substantial public health impact. Acute and chronic particulate matter (PM) exposure during pregnancy increases the risk of HDP, although the underlying molecular mechanisms remain unclear. Extracellular vesicles (EVs) may be the ideal candidates for mediating the effects of PM exposure in pregnancy as they are released in response to environmental stimuli. The INSIDE project aims to investigate this mechanism in pregnancy outcomes. The study population is enrolled at the Fetal Medicine Unit of Fondazione IRCCS Ca'Granda-Ospedale Maggiore Policlinico at 10-14 weeks of gestation. Exposure to PM10 and PM2.5 is assessed using the flexible air quality regional model (FARM) and Bayesian geostatistical models. Each woman provides a blood sample for EV analysis and circulating biomarker assessment. Moreover, a subgroup of recruited women (n = 85) is asked to participate in a cardiovascular screening program including a standard clinical evaluation, a non-invasive assessment of right ventricular function, and pulmonary circulation at rest and during exercise. These subjects are also asked to wear a personal particulate sampler, to measure PM10, PM2.5, and PM1. The INSIDE study is expected to identify the health impacts of PM exposure on pregnancy outcomes.

Type I Collagen Suspension Induces Neocollagenesis and Myodifferentiation in Fibroblasts In Vitro.

The ability of a collagen-based matrix to support cell proliferation, migration, and infiltration has been reported; however, the direct effect of an aqueous collagen suspension on cell cultures has not been studied yet. In this work, the effects of a high-concentration aqueous suspension of a micronized type I equine collagen (EC-I) have been evaluated on a normal mouse fibroblast cell line. Immunofluorescence analysis showed the ability of EC-I to induce a significant increase of type I and III collagen levels, parallel with overexpression of crucial proteins in collagen biosynthesis, maturation, and secretion, prolyl 4-hydroxylase (P4H) and heat shock protein 47 (HSP47), as demonstrated by western blot experiments. The treatment led, also, to an increase of α-smooth muscle actin (α-SMA) expression, evaluated through western blot analysis, and cytoskeletal reorganization, as assessed by phalloidin staining. Moreover, scanning electron microscopy analysis highlighted the appearance of plasma membrane extensions and blebbing of extracellular vesicles. Altogether, these results strongly suggest that an aqueous collagen type I suspension is able to induce fibroblast myodifferentiation. Moreover, our findings also support in vitro models as a useful tool to evaluate the effects of a collagen suspension and understand the molecular signaling pathways possibly involved in the effects observed following collagen treatment in vivo.

In vitro evidence supporting applications of platelet derivatives in regenerative medicine.

The role of platelets in haemostasis has long been known, but understanding of these cells' involvement in wound healing/tissue repair is more recent and has given rise to a multitude of translational studies. Tissue repair processes consist of complex, regulated interactions between cells modulated by biologically active molecules, most of which are growth factors released by activated platelets: this aspect represents the rationale on which the use of platelet derivatives for clinical purposes is based. In the last years, many in vitro studies have focused on the mechanisms of action by which these growth factors affect the biological activities of cells, thus supporting tissue healing. Although limited by some drawbacks (two-dimensional in vitro monocultures cannot replicate the tissue architecture and organisation of organs or the continuous interplay between different cell types), in vitro studies do have the advantages of giving rapid results and allowing precise control of platelet concentrations and other parameters.This review offers an updated overview of the data obtained from the most recent bench-top studies focused on the effects of platelet derivatives on a wide variety of human cells, highlighting their possible impact for in vivo applications. The heterogeneity of the data obtained so far is very evident. This can be explained by the different experimental settings used in each study, which may be the cause of the variability in clinical outcomes. In fact, in vitro studies suggest that the composition of platelet derivatives and the method used for their production and activation (or not) and the platelet concentration used can have profound effects on the final results.

NOS2 inhibitor 1400W Induces Autophagic Flux and Influences Extracellular Vesicle Profile in Human Glioblastoma U87MG Cell Line.

The relevance of nitric oxide synthase 2 (NOS2) as a prognostic factor in Glioblastoma Multiforme (GBM) malignancy is emerging. We analyzed the effect of NOS2 inhibitor 1400W on the autophagic flux and extracellular vesicle (EV) secretion in U87MG glioma cells. The effects of glioma stem cells (GSC)-derived EVs on adherent U87MG were evaluated. Cell proliferation and migration were examined while using Cell Counting Kit-8 assay (CCK-8) and scratch wound healing assay. Cell cycle profile and apoptosis were analyzed by flow cytometry. Autophagy-associated acidic vesicular organelles were detected and quantified by acridine orange staining. The number and size of EVs were assessed by nanoparticle tracking analysis. EV ultrastructure was verified by transmission electron microscopy (TEM). WB was used to analyze protein expression and acid sphingomyelinase was determined through ceramide levels. 1400W induced autophagy and EV secretion in both adherent U87MG and GSCs. EVs secreted by 1400W-treated GSC, but not those from untreated cells, were able to inhibit adherent U87MG cell growth and migration while also inducing a relevant level of autophagy. The hypothesis of NOS2 expression as GBM profile marker or interesting therapeutic target is supported by our findings. Autophagy and EV release following treatment with the NOS2 inhibitor could represent useful elements to better understand the complex biomolecular frame of GBM.

Short exposure to tranexamic acid does not affect, in vitro, the viability of human chondrocytes.

BACKGROUND: Only few studies have investigated the effect of topical application of tranexamic acid (TXA) on "minimally" invasive joint surgical procedures in which articular cartilage is preserved; for this reason, actually many surgeons avoid the use of topical TXA even if the disadvantage related to a blood loss can occur. The aim of this study was to evaluate the cytotoxicity, on human chondrocytes, of TXA at different concentrations and times of exposure and the mechanisms of cell death. METHODS: Experiments were carried out on isolated human chondrocytes harvested from eight patients who underwent total knee replacement. Cell viability was determined using XTT assay and was assessed at 0, 24 and 48 h intervals after a 10-min-long treatment, followed by thorough washes, or at 24 and 48 h of treatment at TXA concentrations of 20, 50, 70 and 100 mg/ml. Cell cycle alterations and occurrence of cell death for apoptosis or necrosis were assessed by cytofluorimetry. Data were analyzed using Proc Mixed Procedure; LSMEANS was used to compare multiple group means with Tukey's honestly significant difference test. RESULTS: A significant correlation between the controlled for factors (type of treatment, time and concentration) was found in the performed experiment. No significant effect on cell viability was observed when the TXA exposure was limited to 10 min, while for increased exposure, 24 and 48 h, a remarkable reduction was found; cell death occurred by apoptosis and was already appreciable after 24 h, reaching a statistical significance after the 48-h-long treatment. CONCLUSION: A prolonged exposure to TXA may cause cartilage damage, thus its topical application can be expanded also to clinical scenarios that include retention of native cartilage chondrocytes, only if it is limited to few minutes and used at concentrations of 70 mg/ml or less.

Ovarian cancer-derived extracellular vesicles affect normal human fibroblast behavior.

It has become clear that non-tumor cells in the microenvironment, especially fibroblasts, actively participate in tumor progression. Fibroblasts conditioned by tumor cells become "activated" and, as such, are identified as CAFs (cancer-associated fibroblasts). These CAFs remodel the tumor stroma to make it more favourable for cancer progression. The aim of this work was to verify whether EVs (extracellular vesicles - whose role as mediators of information between tumor and stromal cells is well known) released from human ovarian cancer cells were able to activate fibroblasts. EVs isolated from SKOV3 (more aggressive) and CABA I (less aggressive) cells were administered to fibroblasts. The consequent activation was supported by morphological and molecular changes in treated fibroblasts; XTT assays, zymographies, wound healing tests and invasion assays also highlighted higher proliferation, motility, invasiveness and enzyme expression. The secretome of these "activated" fibroblasts was, in turn, able to modulate the responses (proliferation, motility and invasion) of fibroblasts, and of tumor and endothelial cells. These findings support the idea that ovarian cancer cells can modulate fibroblast behaviour through the release of EVs, activating them to a CAFs-like state; the latter are able, in turn, to stimulate the surrounding cells. EVs from SKOV3 rather than from CABA I seem to be more efficient in some processes.