Extracellular vesicle encapsulated nicotinamide delivered via a trans-scleral route provides retinal ganglion cell neuroprotection.

The progressive and irreversible degeneration of retinal ganglion cells (RGCs) and their axons is the major characteristic of glaucoma, a leading cause of irreversible blindness worldwide. Nicotinamide adenine dinucleotide (NAD) is a cofactor and metabolite of redox reaction critical for neuronal survival. Supplementation with nicotinamide (NAM), a precursor of NAD, can confer neuroprotective effects against glaucomatous damage caused by an age-related decline of NAD or mitochondrial dysfunction, reflecting the high metabolic activity of RGCs. However, oral supplementation of drug is relatively less efficient in terms of transmissibility to RGCs compared to direct delivery methods such as intraocular injection or delivery using subconjunctival depots. Neither method is ideal, given the risks of infection and subconjunctival scarring without novel techniques. By contrast, extracellular vesicles (EVs) have advantages as a drug delivery system with low immunogeneity and tissue interactions. We have evaluated the EV delivery of NAM as an RGC protective agent using a quantitative assessment of dendritic integrity using DiOlistics, which is confirmed to be a more sensitive measure of neuronal health in our mouse glaucoma model than the evaluation of somatic loss via the immunostaining method. NAM or NAM-loaded EVs showed a significant neuroprotective effect in the mouse retinal explant model. Furthermore, NAM-loaded EVs can penetrate the sclera once deployed in the subconjunctival space. These results confirm the feasibility of using subconjunctival injection of EVs to deliver NAM to intraocular targets.

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.

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.

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.

Identification of a Novel Small Molecule That Enhances the Release of Extracellular Vesicles with Immunostimulatory Potency via Induction of Calcium Influx.

Extracellular vesicles (EVs) transfer antigens and immunomodulatory molecules in immunologic synapses as a part of intracellular communication, and EVs equipped with immunostimulatory functions have been utilized for vaccine formulation. Hence, we sought small-molecule compounds that increase immunostimulatory EVs released by antigen-presenting dendritic cells (DCs) for enhancement of vaccine immunogenicity. We previously performed high-throughput screening on a 28K compound library using three THP-1 reporter cell lines with CD63 Turbo-Luciferase, NF-κB, and interferon-sensitive response element (ISRE) reporter constructs, respectively. Because intracellular Ca2+ elevation enhances EV release, we screened 80 hit compounds and identified compound 634 as a Ca2+ influx inducer. 634 enhanced EV release in murine bone marrow-derived dendritic cells (mBMDCs) and increased costimulatory molecule expression on the surface of EVs and the parent cells. EVs isolated from 634-treated mBMDCs induced T cell proliferation in the presence of antigenic peptides. To assess the roles of intracellular Ca2+ elevation in immunostimulatory EV release, we performed structure-activity relationship (SAR) studies of 634. The analogues that retained the ability to induce Ca2+ influx induced more EVs with immunostimulatory properties from mBMDCs than did those that lacked the ability to induce Ca2+ influx. The levels of Ca2+ induction of synthesized analogues correlated with the numbers of EVs released and costimulatory molecule expression on the parent cells. Collectively, our study presents that a small molecule, 634, enhances the release of EVs with immunostimulatory potency via induction of Ca2+ influx. This agent is a novel tool for EV-based immune studies and vaccine development.

Evidence for Effects of Extracellular Vesicles on Physical, Inflammatory, Transcriptome and Reward Behaviour Status in Mice.

Immune-inflammatory activation impacts extracellular vesicles (EVs), including their miRNA cargo. There is evidence for changes in the EV miRNome in inflammation-associated neuropsychiatric disorders. This mouse study investigated: (1) effects of systemic lipopolysaccharide (LPS) and chronic social stress (CSS) on plasma EV miRNome; and (2) physiological, transcriptional, and behavioural effects of peripheral or central delivered LPS-activated EVs in recipient mice. LPS or CSS effects on the plasma EV miRNome were assessed by using microRNA sequencing. Recipient mice received plasma EVs isolated from LPS-treated or SAL-treated donor mice or vehicle only, either intravenously or into the nucleus accumbens (NAc), on three consecutive days. Bodyweight, spleen or NAc transcriptome and reward (sucrose) motivation were assessed. LPS and CSS increased the expression of 122 and decreased expression of 20 plasma EV miRNAs, respectively. Peripheral LPS-EVs reduced bodyweight, and both LPS-EVs and SAL-EVs increased spleen expression of immune-relevant genes. NAc-infused LPS-EVs increased the expression of 10 immune-inflammatory genes. Whereas motivation increased similarly across test days in all groups, the effect of test days was more pronounced in mice that received peripheral or central LPS-EVs compared with other groups. This study provides causal evidence that increased EV levels impact physiological and behavioural processes and are of potential relevance to neuropsychiatric disorders.

Circulating cardiomyocyte-derived extracellular vesicles reflect cardiac injury during systemic inflammatory response syndrome in mice.

The release of extracellular vesicles (EVs) is increased under cellular stress and cardiomyocyte damaging conditions. However, whether the cardiomyocyte-derived EVs eventually reach the systemic circulation and whether their number in the bloodstream reflects cardiac injury, remains unknown. Wild type C57B/6 and conditional transgenic mice expressing green fluorescent protein (GFP) by cardiomyocytes were studied in lipopolysaccharide (LPS)-induced systemic inflammatory response syndrome (SIRS). EVs were separated both from platelet-free plasma and from the conditioned medium of isolated cardiomyocytes of the left ventricular wall. Size distribution and concentration of the released particles were determined by Nanoparticle Tracking Analysis. The presence of GFP + cardiomyocyte-derived circulating EVs was monitored by flow cytometry and cardiac function was assessed by echocardiography. In LPS-treated mice, systemic inflammation and the consequent cardiomyopathy were verified by elevated plasma levels of TNFα, GDF-15, and cardiac troponin I, and by a decrease in the ejection fraction. Furthermore, we demonstrated elevated levels of circulating small- and medium-sized EVs in the LPS-injected mice. Importantly, we detected GFP+ cardiomyocyte-derived EVs in the circulation of control mice, and the number of these circulating GFP+ vesicles increased significantly upon intraperitoneal LPS administration (P = 0.029). The cardiomyocyte-derived GFP+ EVs were also positive for intravesicular troponin I (cTnI) and muscle-associated glycogen phosphorylase (PYGM). This is the first direct demonstration that cardiomyocyte-derived EVs are present in the circulation and that the increased number of cardiac-derived EVs in the blood reflects cardiac injury in LPS-induced systemic inflammation (SIRS).

Extracellular vesicles derived from PM2.5-exposed alveolar epithelial cells mediate endothelial adhesion and atherosclerosis in ApoE-/- mice.

Epidemiological studies suggested that PM2.5 (particle matters with an aerodynamic diameter ≤2.5 µm) exposure is associated with atherosclerosis. Extracellular vesicles (EVs) are messengers between intracellular communications which are important in diseases procession. At present, whether EVs derived from PM2.5-exposed alveolar epithelial cells (P-EVs) involve in atherosclerosis has not been clearly understood. This study is performed to investigate the effects of P-EVs on the development of endothelium adhesion and atherosclerosis. Here, ApoE-/- mice were randomized into different groups receiving one of the following treatments, filtered air (FA), PM2.5, PBS, PBS-treated alveolar epithelial cells-derived EVs (EVs), or P-EVs. Then the atherosclerosis level in aortas or aorta sections was evaluated by oil red O staining. The results indicated that ApoE-/- mice treated with P-EVs or PM2.5 showed more obvious atherosclerosis plaques in aortas and aortic arches than those treated with EVs or PBS. Endothelial cells (ECs) were treated with PBS, EVs, P-EVs, or PM2.5. The adhesion property, miRNAs level and expressions of IκBα, phosphorylated IκBα, NF-κB p65, phosphorylated NF-κB p65, and VCAM1 in ECs were determined. It was found that P-EVs activated IκBα-NF-κB-VCAM1 signaling and increased adhesion of ECs, and such effects could be reversed by adalimumab (the TNF-α inhibitor) or miR-326-3p inhibitor. Further study suggested that P-EVs induced upregulation of TNF-α and miR-326-3p in recipient ECs and contributed to the phosphorylation of NF-κB p65. Collectively, EVs derived from PM2.5-exposed alveolar epithelial cells played an important role in the development of atherosclerosis via activating IκBα-NF-κB-VCAM1 signaling.

MicroRNA-mediated vascular intercellular communication is altered in chronic kidney disease.

AIMS: Chronic kidney disease (CKD) is an independent risk factor for the development of coronary artery disease (CAD). For both, CKD and CAD, the intercellular transfer of microRNAs (miRs) through extracellular vesicles (EVs) is an important factor of disease development. Whether the combination of CAD and CKD affects endothelial function through cellular crosstalk of EV-incorporated miRs is still unknown. METHODS AND RESULTS: Out of 172 screened CAD patients, 31 patients with CAD + CKD were identified and matched with 31 CAD patients without CKD. Additionally, 13 controls without CAD and CKD were included. Large EVs from CAD + CKD patients contained significantly lower levels of the vasculo-protective miR-130a-3p and miR-126-3p compared to CAD patients and controls. Flow cytometric analysis of plasma-derived EVs revealed significantly higher numbers of endothelial cell-derived EVs in CAD and CAD + CKD patients compared to controls. EVs from CAD + CKD patients impaired target human coronary artery endothelial cell (HCAEC) proliferation upon incubation in vitro. Consistent with the clinical data, treatment with the uraemia toxin indoxyl sulfate (IS)-reduced miR-130a-3p levels in HCAEC-derived EVs. EVs from IS-treated donor HCAECs-reduced proliferation and re-endothelialization in EV-recipient cells and induced an anti-angiogenic gene expression profile. In a mouse-experiment, intravenous treatment with EVs from IS-treated endothelial cells significantly impaired endothelial regeneration. On the molecular level, we found that IS leads to an up-regulation of the heterogenous nuclear ribonucleoprotein U (hnRNPU), which retains miR-130a-3p in the cell leading to reduced vesicular miR-130a-3p export and impaired EV-recipient cell proliferation. CONCLUSION: Our findings suggest that EV-miR-mediated vascular intercellular communication is altered in patients with CAD and CKD, promoting CKD-induced endothelial dysfunction.

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.

Tempol Alters Urinary Extracellular Vesicle Lipid Content and Release While Reducing Blood Pressure during the Development of Salt-Sensitive Hypertension.

Salt-sensitive hypertension resulting from an increase in blood pressure after high dietary salt intake is associated with an increase in the production of reactive oxygen species (ROS). ROS are known to increase the activity of the epithelial sodium channel (ENaC), and therefore, they have an indirect effect on sodium retention and increasing blood pressure. Extracellular vesicles (EVs) carry various molecules including proteins, microRNAs, and lipids and play a role in intercellular communication and intracellular signaling in health and disease. We investigated changes in EV lipids, urinary electrolytes, osmolality, blood pressure, and expression of renal ENaC and its adaptor protein, MARCKS/MARCKS Like Protein 1 (MLP1) after administration of the antioxidant Tempol in salt-sensitive hypertensive 129Sv mice. Our results show Tempol infusion reduces systolic blood pressure and protein expression of the alpha subunit of ENaC and MARCKS in the kidney cortex of hypertensive 129Sv mice. Our lipidomic data show an enrichment of diacylglycerols and monoacylglycerols and reduction in ceramides, dihydroceramides, and triacylglycerols in urinary EVs from these mice after Tempol treatment. These data will provide insight into our understanding of mechanisms involving strategies aimed to inhibit ROS to alleviate salt-sensitive hypertension.

Drug-Induced Lysosomal Impairment Is Associated with the Release of Extracellular Vesicles Carrying Autophagy Markers.

Amiodarone is a cationic amphiphilic drug used as an antiarrhythmic agent. It induces phospholipidosis, i.e., the accumulation of phospholipids within organelles of the endosomal-lysosomal system. Extracellular vesicles (EVs) are membrane-enclosed structures released by any type of cell and retrieved in every fluid of the body. EVs have been initially identified as a system to dispose cell waste, but they are also considered to be an additional manner to transmit intercellular signals. To understand the role of EVs in drug-induced phospholipidosis, we investigated EVs release in amiodarone-treated HEK-293 cells engineered to produce fluorescently labelled EVs. We observed that amiodarone induces the release of a higher number of EVs, mostly of a large/medium size. EVs released upon amiodarone treatment do not display significant morphological changes or altered size distribution, but they show a dose-dependent increase in autophagy associated markers, indicating a higher release of EVs with an autophagosome-like phenotype. Large/medium EVs also show a higher content of phospholipids. Drugs inducing lysosomal impairment such as chloroquine and bafilomycin A1 similarly prompt a higher release of EVs enriched in autophagy markers. This result suggests a mechanism associated with amiodarone-induced lysosomal impairment more than a connection with the accumulation of specific undigested substrates. Moreover, the implementation of the lysosomal function by overexpressing TFEB, a master gene regulator of lysosomal biogenesis, prevents the amiodarone-induced release of EVs, suggesting that this could be a feasible target to attenuate drug-induced abnormalities.

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.