Effects Of Extracellular Vesicles Research Articles

Effects of extracellular vesicles (EVs) from uterine fluid during estrus and diestrus on porcine embryonic development

Effects of extracellular vesicles (EVs) from uterine fluid during estrus and diestrus on porcine embryonic development

Extracellular Vesicles: A Crucial Player in the Intestinal Microenvironment and Beyond.

The intestinal ecological environment plays a crucial role in nutrient absorption and overall well-being. In recent years, research has focused on the effects of extracellular vesicles (EVs) in both physiological and pathological conditions of the intestine. The intestine does not only consume EVs from exogenous foods, but also those from other endogenous tissues and cells, and even from the gut microbiota. The alteration of conditions in the intestine and the intestinal microbiota subsequently gives rise to changes in other organs and systems, including the central nervous system (CNS), namely the microbiome-gut-brain axis, which also exhibits a significant involvement of EVs. This review first gives an overview of the generation and isolation techniques of EVs, and then mainly focuses on elucidating the functions of EVs derived from various origins on the intestine and the intestinal microenvironment, as well as the impacts of an altered intestinal microenvironment on other physiological systems. Lastly, we discuss the role of microbial and cellular EVs in the microbiome-gut-brain axis. This review enhances the understanding of the specific roles of EVs in the gut microenvironment and the central nervous system, thereby promoting more effective treatment strategies for certain associated diseases.

Effects of extracellular vesicles from adipose-derived stem cells on human keloid fibroblasts via the SOCS1/JAK2/STAT3 pathway.

Keloid represents a benign skin tumor with many cancer-like features. Extracellular vesicles (EVs) derived from human adipose-derived stem cells (hADSCs) play a role in cell migration of multiple diseases. This study aimed to investigate the impact of hADSC-EVs on human keloid fibroblasts (HKFs). hADSCs were cultured to the 3rd generation, and subsequently assessed for their osteogenic, adipogenic, and chondrogenic differentiative abilities using flow cytometry, alizarin red, oil red O, and alcian blue staining techniques. hADSC-EVs were isolated through ultracentrifugation and subsequently identified. HKFs at the 3rd generation were subjected to treatment with hADSC-EVs to observe their endocytosis of EVs by immunofluorescence. CCK-8, wound healing, and Transwell assays were performed to test HKF proliferation and migration. The levels of autophagy proteins, collagens, and Janus kinase 2 (JAK2) and Signal Transducer and Activator of Transcription 3 (STAT3) were determined through Western blot analysis. Suppressor of cytokine signaling 1 (SOCS1) expression was determined by RT-qPCR and Western blot. hADSC-EVs were successfully isolated from hADSCs. PKH67-labeled hADSC-EVs were observed to be endocytosed by HKFs, resulting the inhibition of HKF proliferation, migration, as well as a reduction in collagen deposition. hADSC-EVs carried SOCS1 into HKFs to suppress HKF autophagy. SOCS1 downregulation in hADSC-EVs partially nullified the inhibitory effect of hADSC-EVs on HKFs. hADSC-EV-carried SOCS1 inhibited the activation of the JAK2/STAT3 pathway. JAK2/STAT3 pathway activation partially abrogated the suppression of hADSC-EVs on the proliferation, migration, and collagen deposition of HKF. hADSC-EVs carried SOCS1 into HKFs and suppressed HKF autophagy, proliferation, migration, and collagen deposition by inactivating the JAK2/STAT3 pathway.

Immediate administration of hTERT-MSCs-IDO1-EVs reduces hypoalbuminemia after spinal cord injury.

Spinal cord injury (SCI) presents challenging and unpredictable neurological recovery. During inflammatory conditions, the amount of serum albumin and nutrition consumption decreases. Currently, it is proposed to measure serum albumin and glucose content in human or animal subjects to predict the recovery rate and the efficiency of treatments following SCI. In this study, the effect of extra-cellular vesicles (EVs) from immortalized human adipose tissue-derived mesenchymal stem cells (hTERT-MSCs) equipped with the ectopic expression of the human indoleamine 2,3-dioxygenase-1 (IDO1) gene on serum albumin and glucose levels was investigated. After pre-clearing steps of 72-hr conditioned media, small EVs (sEVs) were isolated based on the ultra-filtration method. They were encapsulated with a chitosan-based hydrogel. Five experimental groups (female rats, N = 30, ~ 230 g) were considered, including SCI, sham, hydrogel, control green fluorescent protein (GFP)-EVs and IDO1-EVs. The 60.00 µL of hydrogel or hydrogels containing 100 µg sEVs from GFP or IDO1-EVs were locally injected immediately after SCI (laminectomy of the T10 vertebra and clip compression). After 8 weeks, non-fasting serum glucose and albumin levels were measured. The results indicated that the level of serum albumin in the animals received IDO1-EVs (3.52 ± 0.04) was increased in comparison with the SCI group (3.00 ± 0.94). Also, these animals indicated higher glucose levels in their serum (250.17 ± 69.61) in comparison with SCI ones (214 ± 45.34). Although these changes were not statistically significant, they could be considered as evidence for the beneficial effects of IDO1-EVs administration in the context of SCI to reduce hypoalbuminemia and improve energy consumption. More detailed experiments are required to confirm these results.

The effects of extracellular vesicles and their cargo on metabolism and its adaptation to physical exercise in insulin resistance and type 2 diabetes.

Lifestyle modification represents the first-line strategy for the prevention and treatment of type 2 diabetes mellitus (T2DM), which is frequently associated with obesity and characterized by defective pancreatic insulin secretion and/or insulin resistance. Exercise training is an essential component of lifestyle modification and has been shown to ameliorate insulin resistance by reducing body fat mass and by enhancing skeletal muscle mitochondrial biogenesis and insulin-independent glucose uptake. Additionally, exercising stimulates the release of exerkines such as metabolites or cytokines, but also long non-coding RNA, microRNAs, cell-free DNA (cf-DNA), and extracellular vesicles (EVs), which contribute to inter-tissue communication. There is emerging evidence that EV number and content are altered in obesity and T2DM and may be involved in several metabolic processes, specifically either worsening or improving insulin resistance. This review summarizes the current knowledge on the metabolic effects of exercise training and on the potential role of humoral factors and EV as new biomarkers for early diagnosis and tailored treatment of T2DM.

STAM transports STING oligomers into extracellular vesicles, down-regulating the innate immune response.

Stimulator of interferon genes (STING) mediates the innate immune response against damaged endogenous double-strand DNA and exogenous virus infection. The location of STING is critical to the accurate control of defence signalling pathways. Recently, the effects of extracellular vesicles (EVs) in the regulation of innate immune signalling have been reported. Nevertheless, the particular roles played by STING in EVs and the related mechanisms have remained largely unknown. Herein, we report that when STING was activated in cells, EVs derived from these cells carried STING oligomers. Signal transducing adapter molecule 1 (STAM) was found to be a STING transporter that directly interacted with STING and facilitated STING transport into EVs. Importantly, the translocation of STING into EVs was a mechanism by which STING was degraded, suppressing the innate immune response. In summary, we elucidated the mechanism and function of the translocation of STING into EVs, adding to the understanding of STING activity regulation.

ACE2-containing defensosomes serve as decoys to inhibit SARS-CoV-2 infection.

Extracellular vesicles of endosomal origin, exosomes, mediate intercellular communication by transporting substrates with a variety of functions related to tissue homeostasis and disease. Their diagnostic and therapeutic potential has been recognized for diseases such as cancer in which signaling defects are prominent. However, it is unclear to what extent exosomes and their cargo inform the progression of infectious diseases. We recently defined a subset of exosomes termed defensosomes that are mobilized during bacterial infection in a manner dependent on autophagy proteins. Through incorporating protein receptors on their surface, defensosomes mediated host defense by binding and inhibiting pore-forming toxins secreted by bacterial pathogens. Given this capacity to serve as decoys that interfere with surface protein interactions, we investigated the role of defensosomes during infection by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the etiological agent of Coronavirus Disease 2019 (COVID-19). Consistent with a protective function, exosomes containing high levels of the viral receptor ACE2 in bronchoalveolar lavage fluid (BALF) from critically ill COVID-19 patients was associated with reduced intensive care unit (ICU) and hospitalization times. We found ACE2+ exosomes were induced by SARS-CoV-2 infection and activation of viral sensors in cell culture, which required the autophagy protein ATG16L1, defining these as defensosomes. We further demonstrate that ACE2+ defensosomes directly bind and block viral entry. These findings suggest that defensosomes may contribute to the antiviral response against SARS-CoV-2 and expand our knowledge on the regulation and effects of extracellular vesicles during infection.

Research progress of extracellular vesicles in type 2 diabetes and its complications.

Type 2 diabetes is one of the most common chronic diseases in modern society. However, there is still insufficient research on the pathogenesis, diagnosis and treatment of type 2 diabetes and its complications. Extracellular vesicles are small bilayer vesicles secreted by cells. In recent years, the effect of extracellular vesicles in type 2 diabetes and its complications has aroused extensive attention. The research on the influence of protein and nucleic acids carried by extracellular vesicles secreted by stem cells and inflammatory cells on the pathogenesis of type 2 diabetes and its complications provides new ideas for its diagnosis and treatment. This review focuses on the influence of extracellular vesicles on insulin resistance by regulating inflammation and glucose transporter 4 expression. The second part mainly discusses the research progress and limitations of extracellular vesicles use in treating and diagnosing type 2 diabetes and its complications. This review introduces the current research status of type 2 diabetes and its complications, illustrates the biogenesis of extracellular vesicles, their effect on type 2 diabetes pathogenesis and its complications and their potential as therapeutic tools and diagnostic markers in type 2 diabetes and its complications.

Effects of Extracellular Vesicles on the Drug Resistance of Lung Adenocarcinoma Cells by Modulating the ATP Binding Cassette Transporter G2

To investigate the regulatory role of extracellular vesicles (EVs) carrying ATP binding cassette transporter G2 (ABCG2) on the drug resistance of lung adenocarcinoma cells and the relevant molecular mechanisms. A549 cells, human lung adenocarcinoma cells, were used to form cisplatin (or cis-Diaminedichloroplatinum, CDDP)-resistant lung adenocarcinoma cells, i.e., A549/CDDP cells. EVs from A549 and A549/CDDP cells were extracted by gradient centrifugation method and were hence named EVs 1 and EVs 2, respectively. The A549 cells were treated with EVs 1 and EVs 2 for 48 hours, and the cells were named A549-EVs 1 and A549-EVs 2 cells, respectively. A549/ ABCG2 cells were established by transfecting A549 cells with pCDNA3.1- ABCG2 recombinant plasmids. On the other hand, A549 cells transfected with empty vectors were named A549/pCDNA3.1 cells. MTT assay was conducted to calculate the 24-hour cell drug resistance index for CDDP. The ABCG2 gene expression in cells and EVs were assessed with real-time PCR. A549 and A549-EVs 2 cells were transplanted subcutaneously into nude mice, which were labeled the control group and the experimental group accordingly. After tumor formation, 3 mg/kg CDDP was intraperitoneally injected once a week for two times. The ABCG2 gene expression of subcutaneous transplanted tumor cells was examined by real-time PCR. The cell apoptosis rate of subcutaneous transplanted tumor cells was examined by flow cytometry. Using the parental A549 cells as reference, the 24-h CDDP-resistance indexes of 549/CDDP, A549/ ABCG 2, A549/pCDNA3.1, A549-EVs 1, A549-EVs 2 cells were 7.17, 10.06, 1.02, 1.19 and 5.40, respectively. When comparing the ABCG2 gene expression levels in all cells and EVs, the findings were higher in A549/CDDP cells than those inA549 cells, higher in A549/ ABCG2 cells than those in A549/pCDNA3.1 or A549 cells, higher in EVs 2 than those in EVs 1, and higher in A549-EVs 2 than those in A549-EVs 1 cells ( P<0.01) . The volume of transplanted tumor and the ABCG2 gene expression level in the experimental group were higher than those in the control group, while the apoptosis rate was lower than that in the control group ( P<0.01). EVs carrying ABCG2 gene can regulate the drug resistance of lung adenocarcinoma cells.

Alteration of miRNAs in Small Neuron-Derived Extracellular Vesicles of Alzheimer's Disease Patients and the Effect of Extracellular Vesicles on Microglial Immune Responses.

Alzheimer's disease (AD) is one of the most severe neurodegenerative diseases observed in the elderly population. Although the hallmarks of AD have been identified, the methods for its definitive diagnosis and treatment are still lacking. Extracellular vesicles (EVs) have become a promising source for biomarkers since the identification of their content. EVs are released from multiple cell types and, when released from neurons, they pass from the brain to the blood with their cargo molecules. Hence, neuron-specific EV-resident microRNAs (miRNAs) are promising biomarkers for diagnosis of AD. This study aimed to identify altered miRNA content in small neuron-derived extracellular vesicles (sNDEVs) isolated from AD patients and healthy individuals. Furthermore, we examined the role of sNDEV-resident miRNAs in neuron-glia cellular interaction to understand their role in AD propagation. We identified 10 differentially expressed miRNAs in the sNDEVs of patients via next-generation sequencing and validated the most dysregulated miRNA, let-7e, with qRT-PCR. Let-7e was significantly increased in the sNDEVs of AD patients compared with those of healthy controls in a larger cohort. First, we evaluated the diagnostic utility of let-7e via ROC curve analysis, which revealed an AUC value of 0.9214. We found that IL-6 gene expression was increased in human microglia after treatment with sNDEVs of AD patients with a high amount of let-7e. Our study suggests that sNDEV-resident let-7e is a potential biomarker for AD diagnosis, and that AD patient-derived sNDEVs induce a neuroinflammatory response in microglia.

Hypoxic Cell-Derived Extracellular Vesicles Aggravate Rectal Injury Following Radiotherapy via MiR-122-5p.

Radiation-induced rectal injury is a common side effect of radiotherapy. Hypoxia often occurs after radiotherapy. This study aimed to explore the bystander effect of hypoxia on radiation-induced rectal injury. In vivo, apoptosis increased nearby the highly hypoxic area in the rectal tissues in the mouse models of radiation-induced rectal injury, indicating the potential involvement of hypoxia. In vitro, flow cytometry and Western blotting showed that both hypoxia and hypoxic human intestinal epithelial crypt (HIEC) cell supernatant promoted apoptosis in normoxic HIEC cells. The pro-apoptotic effect of extracellular vesicles (EVs) derived from hypoxic HIEC cell to normoxic HIEC cells was then determined. MiR-122-5p was chosen for further studies through a microRNA (miRNA) microarray assay and apoptosis was alleviated in cells receiving miR-122-5p inhibiting hypoxic EVs. Together, our study demonstrated that the miR-122-5p containing-EVs derived from hypoxic HIEC cells promoted apoptosis in normoxic HIEC cells. Hypoxic EV-derived miR-122-5p plays a critical pathologic role in radiation-induced rectal injury and may be a potential therapeutic target.

Protective roles of seminal plasma exosomes and microvesicles during human sperm cryopreservation

Research questionDo seminal plasma microvesicles and exosomes, as two subtypes of extracellular vesicles, exert cryoprotective properties in sperm cryopreservation? DesignMicrovesicles and exosomes isolated from normozoospermic semen samples (n = 10) by serial ultracentrifugation were determined using scanning electron microscopy, dynamic light scattering and western blot analysis. The interactions between extracellular vesicles and spermatozoa were detected using Dil labelling. Purified spermatozoa from different normozoospermic samples (n = 25) were then treated individually with exosomes or microvesicles for 1 h and subsequently cryopreserved. The effects of extracellular vesicles during cryopreservation were investigated by determining post-thaw sperm motility, morphology, viability, reactive oxygen species (ROS) generation, lipid peroxidation, total antioxidant capacity (TAC), mitochondrial membrane potential (MMP), DNA integrity, and apoptosis rate. ResultsMicrovesicles and exosomes displayed a round-shape morphology, with about 70% of exosomes ranging from 43–144 nm, microvesicles ranging from 144.5–486 nm and both expressed tetraspanin markers. Fluorescence microscopy showed that exosomes and microvesicles absorbed mainly in the sperm head and less frequently in the neck and tail. The post-thawing results indicated that the diluent with exosomes or microvesicles had improved sperm motility (P = 0.007), morphology (P < 0.001) and viability (P < 0.001) compared with untreated samples. The ROS levels decreased significantly (P = 0.001), with a consequent decrease in DNA damage (P = 0.001). The TAC activity (P = 0.001) and MMP levels (P = 0.001) were also significantly improved; levels of malondialdehyde (MDA) (P = 0.62) and apoptosis rate (P = 1.000) remained unchanged. ConclusionSeminal plasma microvesicles and exosomes could protect spermatozoa from cryopreservation chilling injuries.

The effects of extracellular vesicles derived from Kr\xfcppel-Like Factor 2 overexpressing endothelial cells on the regulation of cardiac inflammation in the dilated cardiomyopathy

BackgroundDilated cardiomyopathy (DCM) is one of the common causes of heart failure. Myocardial injury triggers an inflammatory response and recruits immune cells into the heart. High expression of Krüppel-like factor 2 (KLF2) in endothelial cells (ECs) potentially exerts an anti-inflammatory effect. However, the role of extracellular vesicles (EVs) from KLF2-overexpressing ECs (KLF2-EVs) in DCM remains unclear.Methods and resultsEVs were separated from the supernatant of KLF2-overexpressing ECs by gradient centrifugation. Mice were repeatedly administered low-dose doxorubicin (DOX) and then received KLF2-EVs through an intravenous injection. Treatment with KLF2-EVs prevented doxorubicin-induced left ventricular dysfunction and reduced the recruitment of Ly6high Mo/Mø in the myocardium. We used flow cytometry to detect Ly6high monocytes in bone marrow and spleen tissues and to elucidate the mechanisms underlying this beneficial effect. KLF2-EVs increased the retention of Ly6Chigh monocytes in the bone marrow but not in the spleen tissue. KLF2-EVs also significantly downregulated C–C chemokine receptor 2 (CCR2) protein expression in cells from the bone marrow.ConclusionsEVs derived from KLF2-overexpressing ECs reduced cardiac inflammation and ameliorated left ventricular dysfunction in DCM mice by targeting the CCR2 protein to inhibit Ly6Chigh monocyte mobilization from the bone marrow.Graphical

Extracellular Vesicles Derived From Hypoxia-Conditioned Adipose-Derived Mesenchymal Stem Cells Enhance Lymphangiogenesis

Extracellular vesicles from adipose-derived mesenchymal stem cells (ADSCs) play an important role in lymphangiogenesis; however, the underlying mechanisms are not fully understood. In this study, we aimed to investigate the function of extracellular vesicles secreted by hypoxia-conditioned ADSCs in lymphangiogenesis and explore the potential molecular mechanisms. Extracellular vesicles were extracted from ADSCs cultured under hypoxia or normoxia conditions. The uptake of extracellular vesicles by lymphatic endothelial cells (LECs) was detected by immunofluorescence staining. The effects of extracellular vesicles on the viability, migration, and tube formation of LECs were determined by CCK-8 assay, migration assay, and tube formation assay, respectively. Molecules and pathway involved in lymphangiogenesis mediated by ADSC-derived extracellular vesicles were analyzed by luciferase reporter assay, qRT-polymerase chain reaction (PCR), and Western blot. Hypoxia ADSC-derived extracellular vesicles (H-ADSC/evs) significantly enhanced the proliferation, migration, and tube formation of LECs. Hypoxia decreased the expression of miR-129 in ADSC-derived extracellular vesicles. Overexpression of miR-129 counteracted the promoting effect of H-ADSC/evs on lymphangiogenesis. In addition, decreased exosomal miR-129 expression resulted in upregulation of HMGB1 in LECs, which led to AKT activation and lymphangiogenesis enhancement. Our data reveal that extracellular vesicles derived from hypoxia-conditioned ADSCs induce lymphangiogenesis, and this effect is mediated by miR-129/HMGB1/AKT signaling. Our findings imply that hypoxia ADSC-isolated extracellular vesicles may represent as a valuable target for the treatment of diseases associated with lymphatic remodeling.

Abstract 10495: Mechanism of Cardiac Repair in Rat Myocardial Infarction Model Treated with Extracellular Vesicles from Differentiating Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Introduction: A few reports showed the therapeutic effects of extracellular vesicles (EVs) secreted from induced pluripotent stem cell-derived cardiomyocytes (iPSCM) on animal ischemic cardiomyopathy models. However, the mechanism of the cardiac function restoration after EVs administration remained unclear. Then, this study aimed to identify the mechanism for the restoration of cardiac function after iPSCM-EVs treatment. Methods: We collected EVs from differentiating iPSCM (TnT positive &amp;gt; 80%) culture medium. Comprehensive miRNA-seq and proteome analysis in the EVs were performed. In vitro myocardial contraction assay and wound healing assay, and ex vivo aortic ring assay were performed. In vivo , we designed a rat model of myocardial infarction by left anterior descending artery (LAD) ligation and divided rats into two groups. Two weeks after LAD ligation, iPSCM-EVs (Group EV) or PBS (Group Ctl) were injected peri-infarct zone. We analyzed the sequential cardiac function, histological analysis, and comprehensive RNA sequence analysis of the border zone before the treatment and 3, 7, 14, and 28 days after the treatment (n=5 at each time point in each group). Results: EVs showed the potential of increased myocyte contraction velocity (11±0.7 vs 6.6±0.6 μm/s, mean±SD, p &amp;lt; 0.01) and enhanced endothelial migration in vitro and facilitated angiogenesis (46±6 vs 13±5 sprouting count, p=0.02) ex vivo . In vivo , Group EVs showed higher LVEF (57±5 vs 38±4 % at day28, p &amp;lt; 0.01) and smaller LVDd (6.8±0.3 vs 8.5±0.8 mm at day28, p &amp;lt; 0.01). Histological analysis showed a lower % fibrosis area in Group EV (22±6 vs 12±5 % at day28, p &amp;lt; 0.01) and significantly higher microvascular (vWF positive: 1.7 at day14 and 2.1 times higher at day28) and arterial (vWF and αSMA positive: 4.3 at day14 and 6.2 times higher at day28) density. RNA expression analysis showed the expression of HGF, SDF-1, and IGF-1 increased in Group EVs on day3 and 7. CTGF and collagen 1 increased in Group Ctl on day14 and 28. Conclusions: Intracardiac injection of iPSCM-EVs improved cardiac function in rat MI model via angiogenesis, increased myocyte contraction, and reduced fibrosis. Comprehensive expression analysis suggested the mechanism of action of EVs in cardiac repair.

Morphology of the Myocardium after Experimental Bone Tissue Trauma and the Use of Extracellular Vesicles Derived from Mesenchymal Multipotent Stromal Cells.

The effect of extracellular vesicles (EVs) of various origins on the heart structures in the time of health and disease has been well studied. At the same time, data on the distribution of EVs throughout the body after introduction into the tissues and the possibility of the influence of these EVs on organs distant from the injection site are practically absent. It is also necessary to note a certain inconsistency in the results of various researchers: from articles on the direct absorption of EVs derived from mesenchymal multipotent stromal cells (MSC EVs) by cardiomyocytes to the data that the heart is inherently immune to drug delivery mediated by nanoparticles. In this regard, the morphological changes in the myocardium of outbred rabbits of both sexes weighing 3–4 kg were studied at various times after experimental trauma of the bone tissue in the proximal condyle of the tibia (PCT) and the use of MSC EVs. As a result of modeling the PCT defect, rabbits develop myocardial edema in the heart muscle by the 3rd day, their lymphatic vessels expand, and then, on the 7th day, the blood vessels become dilated. In the myocardium, the relative and absolute contents of neutrophils, erythrocytes, and macrophages increase, but the percentage of lymphocytes decreases. By day 10, almost all of these changes return to their initial values. The detected transformations of the myocardium are most likely due to the ingress of detritus with the blood flow from the PCT. The use of MSC EVs to influence the regeneration of damaged tissue of PCT promotes earlier dilatation of the blood vessels of the heart with pronounced diapedesis of erythrocytes or even hemorrhages, prolongation of edema, the formation of blood clots in vessels with obliteration of their lumen, sclerotic transformation of vascular walls and paravascular tissues. In the myocardium, the number density of neutrophils, the percentage of lymphocytes, and neutrophils become smaller, with a simultaneous increase in the relative numbers of erythrocytes and macrophages, and changes in the content of macrophages remained until the end of the observation—up to 10 days after the surgery. The discovered effect of MSC EVs is most likely associated with the suppression of the activity of the inflammatory process in the PCT area, which, in turn, was caused by a longer ingress of detritus with blood flow into the myocardium. The absence of statistically significant differences between changes in the myocardium of the left and right ventricles may indicate that both detritus from the surgical site and MSC EVs affect the heart spreading through the coronary artery system.

Effects of Extracellular Vesicles from Blood-Derived Products on Osteoarthritic Chondrocytes within an Inflammation Model.

Osteoarthritis (OA) is hallmarked by a progressive degradation of articular cartilage. One major driver of OA is inflammation, in which cytokines such as IL-6, TNF-α and IL-1β are secreted by activated chondrocytes, as well as synovial cells—including macrophages. Intra-articular injection of blood products—such as citrate-anticoagulated plasma (CPRP), hyperacute serum (hypACT), and extracellular vesicles (EVs) isolated from blood products—is gaining increasing importance in regenerative medicine for the treatment of OA. A co-culture system of primary OA chondrocytes and activated M1 macrophages was developed to model an OA joint in order to observe the effects of EVs in modulating the inflammatory environment. Primary OA chondrocytes were obtained from patients undergoing total knee replacement. Primary monocytes obtained from voluntary healthy donors and the monocytic cell line THP-1 were differentiated and activated into proinflammatory M1 macrophages. EVs were isolated by ultracentrifugation and characterized by nanoparticle tracking analysis and Western blot. Gene expression analysis of chondrocytes by RT-qPCR revealed increased type II collagen expression, while cytokine profiling via ELISA showed lower TNF-α and IL-1β levels associated with EV treatment. In conclusion, the inflammation model provides an accessible tool to investigate the effects of blood products and EVs in the inflammatory context of OA.

The role of extracellular vesicles in regulating local and systemic inflammation in cardiovascular disease

Extracellular vesicles are heterogeneous structures surrounded by cell membranes and carry complex contents including nucleotides, proteins, and lipids. These proteins include cytokines and chemokines that are important for exaggerating local and systemic inflammation in disease. Extracellular vesicles are mainly categorized as exosomes and micro-vesicles, which are directly shed from the endosomal system or originated from the cell membrane, respectively. By transporting several bioactive molecules to recipient cells and tissues, extracellular vesicles have favorable, neutral, or detrimental impacts on their targets, such as switching cell phenotype, modulating gene expression, and controlling biological pathways such as inflammatory cell recruitment, activation of myeloid cells and cell proliferation. Extracellular vesicles mediate these functions via both autocrine and paracrine signaling. In the cardiovascular system, extracellular vesicles can be secreted by multiple cell types like cardiomyocytes, smooth muscle cells, macrophages, monocytes, fibroblasts, and endothelial cells, and affect functions of cells or tissues in distant organs. These effects involve maintaining homeostasis, regulating inflammation, and triggering pathological process in cardiovascular disease. In this review, we mainly focus on the role of micro-vesicles and exosomes, two important subtypes of extracellular vesicles, in local and systemic inflammation in cardiovascular diseases such as myocardial infarction, atherosclerosis and heart failure. We summarize recent findings and knowledge on the effect of extracellular vesicles in controlling both humoral and cellular immunity, and the therapeutic approaches to harness this knowledge to control exacerbated inflammation in cardiovascular diseases.

Senescence did not alter the chondroprotective effect of extracellular vesicles from mesenchymal stromal cells

Senescence did not alter the chondroprotective effect of extracellular vesicles from mesenchymal stromal cells

Senescence did not alter the chondroprotective effect of extracellular vesicles from adipose mesenchymal stem cells in osteoarthritis

Purpose: Age is the most important risk factor in degenerative osteoarthritis (OA) and is associated with the accumulation of senescent cells that contribute to functional decline of joint. We previously demonstrated that extracellular vesicles (EVs) from mesenchymal stromal cells (MSCs) largely mediate the therapeutic effect of parental cells in OA. Here, we assessed the impact of senescence on the characteristics of EVs from adipose tissue-derived MSCs (ASC-EVs) and their properties in an in vitro model of OA Methods: ASCs were induced to senescence using 25μM etoposide for 24 hours. Senescence was assessed at day 12 by quantifying proliferation rate (Trypan blue counting, BrdU incorporation), SA-βGal activity (immunostaining and dedicated quantitative assay), nuclear γH2AX foci number (immunofluorescence), stress fiber visualization (phalloidin staining) and expression of cyclin dependent kinase inhibitors (CDKI) (RT-qPCR). ASC-EVs were isolated by differential ultracentrifugation and characterized by size, concentration, total protein content, structure (cryo-TEM) and immunophenotype (CD9, CD63, CD81, Alix). In vitro OA model used human chondrocytes isolated from OA patients, which were stimulated with IL1β for 48h before culture with ASCs or ASC-EVs for 7 days. Expression of chondrocytic and inflammatory markers was quantified by RT-qPCR and SASP factors were quantified by ELISA in supernatants Results: After 12 days, senescence-induced ASCs experienced growth arrest and increase of SA-βGal staining, of p21 CDKI expression, of nuclear γH2AX foci, of stress fibers and of several SASP factors (IL6, IL8, MMP3) confirming expression of all main features of senescence. Senescent ASCs produced 4-fold more EVs than healthy ASCs and senescent ASC-EVs were larger and contained slightly less total protein amounts. In the in vitro model of OA chondrocytes, both healthy and senescent ASCs decreased fibrotic markers (type III COLLAGEN), catabolic and hypertrophic markers (MMP3, MMP13, AP) and increased COX2. By contrast, healthy ASCs decreased the expression of IL6 while senescent ASCs highly increased IL6 expression in OA chondrocytes. Looking at the role of ASC-EVs on OA chondrocytes, we found out that both healthy and senescent ASC-EVs were able to increase the expression of AGG and type II COLLAGEN while they decreased the expression of MMP13, AP, type X COLLAGEN, HMOX1 and IL6. Finally, healthy and senescent ASC-EVs decreased the number of SA-βGal positive OA chondrocytes but did not impact the expression of p21 in these IL1β-induced chondrocytes. Conclusions: Our Results indicated a chondroprotective effect of ASC-EVs, independently of the senescent state of parental cells and suggested that EVs might act through different mechanisms than ASCs, which warrants further investigation