Circulating extracellular vesicles derived from tumor endothelial cells hijack the local and systemic anti-tumor immune response: Role of mTOR/G-CSF pathway.

Circulating tumour-derived extracellular vesicles are supposed to contribute to the spreading of distant metastasis. In this study, we investigated the impact of circulating extracellular vesicles derived from tumour-endothelial cells (TEVs) in the expansion of the metastatic bulk. We focus on the role of immune cells in controlling this process using the 4T1 triple negative breast cancer (TNBC) syngeneic model. 4T1 cells were intravenously injected and exposed to circulating TEVs from day 7. The lung, spleen, and bone marrow (BM) were recovered and analysed. We demonstrated that circulating TEVs boost lung metastasis and angiogenesis. FACS and immunohistochemically analyses revealed a significant enrichment of Ly6G+/F4/80+/CD11b+ cells and Ly6G+/F4/80-/CD11b+ in the lung and in the spleen, while Ly6G+/F4/80-/CD11b+ in the BM, indicating the occurrence of a systemic and local immune suppression. TEV immune suppressive properties were further supported by the increased expression of PD-L1, PD-1, and iNOS in the tumour mass. In addition, in vitro experiments demonstrated an increase of CD11+ cells, PD-L1+ myeloid and cancer cells, upregulation of LAG3, CTLA4 and PD-1 in T-cells, release of ROS and NOS, and impaired T-cell-mediated cytotoxic effect in co-culture of TEVs-preconditioned PBMCs and cancer cells. Granulocyte-colony stimulating factor (G-CSF) level was increased in vivo, and was involved in reshaping the immune response. Mechanistically, we also found that mTOR enriched TEVs support G-CSF release and trigger the phosphorylation of the S6 (Ser235/236) mTOR downstream target. Overall, we provided evidence that circulating TEVs enriched in mTOR supported G-CSF release thereby granting tumour immune suppression and metastasis outgrowth.

First pilot case-control interventional study using autologous extracellular vesicles to treat chronic venous ulcers unresponsive to conventional treatments.

Current therapeutic approaches for chronic venous ulcers (CVUs) still require evidence of effectiveness. Diverse sources of extracellular vesicles (EVs) have been proposed for tissue regeneration, however the lack of potency tests, to predict in-vivo effectiveness, and a reliable scalability have delayed their clinical application. This study aimed to investigate whether autologous serum-derived EVs (s-EVs), recovered from patients with CVUs, may be a proper therapeutic approach to improve the healing process. A pilot case-control interventional study (CS2/1095/0090491) has been designed and s-EVs recovered from patients. Patient eligibility included two or more distinct chronic lesions in the same limb with 11 months as median persistence of active ulcer before enrollment. Patients were treated three times a week, for 2 weeks. Qualitative CVU analysis demonstrated that s-EVs-treated lesions displayed a higher percentage of granulation tissue compared to the control group (Sham) (s-EVs 3 out of 5: 75-100 % vs Sham: none), further confirmed at day 30. s-EVs-treated lesions also displayed higher sloughy tissue reduction at the end of treatment even increased at day 30. Additionally, s-EV treatment led to a median surface reduction of 151 mm2 compared to 84 mm2 in the Sham group, difference even more evident at day 30 (s-EVs 385 mm2vs Sham 106 mm2p = 0.004). Consistent with the enrichment of transforming growth factor-ß1 in s-EVs, histological analyses showed a regenerative tissue with an increase in microvascular proliferation areas. This study first demonstrates the clinical effectiveness of autologous s-EVs in promoting the healing process of CVUs unresponsive to conventional treatments.

IL-3 signalling in the tumour microenvironment shapes the immune response via tumour endothelial cell-derived extracellular vesicles.

Antibody-based anti-cancer therapy is considered a successful approach to impair tumour progression. This study aimed to investigate the clinical impact of targeting the IL-3 signalling in the microenvironment of solid tumours. We intended to investigate whether the IL-3Rα blockade on tumour-derived endothelial cells (TEC) can modulate PD-L1 expression in tumour cells and peripheral blood mononuclear cells (PBMC) to reshape the anti-tumour immune response. Extracellular vesicles released by TEC after IL-3Rα blockade (aTEV) were used as the ultimate effectors of the antibody-based approach, while naive TEC-derived extracellular vesicles (nTEV) served as control. Firstly, we demonstrated that, either directly or indirectly via nTEV, IL-3 controls the expression of its receptor on TEC and PBMC respectively. Moreover, we found that nTEV, moulded by the autocrine secretion of IL-3, increased PD-L1 expression in myeloid cells both in vitro and in vivo. In addition, we found that nTEV-primed PBMC favour tumour cell growth (TEC and MDA-MB-231 cells), whereas PBMC-primed with aTEV still retain their anti-tumour properties. Isolated T-cells pre-conditioned with nTEV or aTEV and co-cultured with TEC or MDA-MB-231 cells have no effects, thereby sustaining the key role of myeloid cells in tumour immune editing. In vivo nTEV, but not aTEV, increased the expression of PD-L1 in primary tumours, lung and liver metastases. Finally, we demonstrated that the enrichment of miR-214 in aTEV impacts on PD-L1 expression in vivo. Overall, these data indicate that an approach based on IL-3Rα blockade in TEC rearranges EV cargo and may reshape the anti-tumour immune response.

Differential Therapeutic Effect of Extracellular Vesicles Derived by Bone Marrow and Adipose Mesenchymal Stem Cells on Wound Healing of Diabetic Ulcers and Correlation to Their Cargoes.

Extracellular vesicles (EVs) derived from mesenchymal stem cells isolated from both bone marrow (BMSCs) and adipose tissue (ADSCs) show potential therapeutic effects. These vesicles often show a similar beneficial effect on tissue regeneration, but in some contexts, they exert different biological properties. To date, a comparison of their molecular cargo that could explain the different biological effect is not available. Here, we demonstrated that ADSC-EVs, and not BMSC-EVs, promote wound healing on a murine model of diabetic wounds. Besides a general similarity, the bioinformatic analysis of their protein and miRNA cargo highlighted important differences between these two types of EVs. Molecules present exclusively in ADSC-EVs were highly correlated to angiogenesis, whereas those expressed in BMSC-EVs were preferentially involved in cellular proliferation. Finally, in vitro analysis confirmed that both ADSC and BMSC-EVs exploited beneficial effect on cells involved in skin wound healing such as fibroblasts, keratinocytes and endothelial cells, but through different cellular processes. Consistent with the bioinformatic analyses, BMSC-EVs were shown to mainly promote proliferation, whereas ADSC-EVs demonstrated a major effect on angiogenesis. Taken together, these results provide deeper comparative information on the cargo of ADSC-EVs and BMSC-EVs and the impact on regenerative processes essential for diabetic wound healing.

Extracellular vesicles from human liver stem cells inhibit tumor angiogenesis.

Human liver stem-like cells (HLSC) and derived extracellular vesicles (EVs) were previously shown to exhibit anti-tumor activity. In our study, we investigated whether HLSC-derived EVs (HLSC-EVs) were able to inhibit tumor angiogenesis in vitro and in vivo, in comparison with EVs derived from mesenchymal stem cells (MSC-EVs). The results obtained indicated that HLSC-EVs, but not MSC-EVs, inhibited the angiogenic properties of tumor-derived endothelial cells (TEC) both in vitro and in vivo in a model of subcutaneous implantation in Matrigel. Treatment of TEC with HLSC-EVs led to the down-regulation of pro-angiogenic genes. Since HLSC-EVs carry a specific set of microRNAs (miRNAs) that could target these genes, we investigated their potential role by transfecting TEC with HLSC-EV specific miRNAs. We observed that four miRNAs, namely miR-15a, miR-181b, miR-320c and miR-874, significantly inhibited the angiogenic properties of TEC in vitro, and decreased the expression of some predicted target genes (ITGB3, FGF1, EPHB4 and PLAU). In parallel, TEC treated with HLSC-EVs significantly enhanced expression of miR-15a, miR-181b, miR-320c and miR-874 associated with the down-regulation of FGF1 and PLAU. In summary, HLSC-EVs possess an anti-tumorigenic effect, based on their ability to inhibit tumor angiogenesis.

Functional analysis of miR-21-3p, miR-30b-5p and miR-150-5p shuttled by extracellular vesicles from diabetic subjects reveals their association with diabetic retinopathy.

Microvascular dysfunctions due to altered interactions between endothelial cells (ECs) and pericytes are key-events in the pathogenesis of diabetic retinopathy. Extracellular vesicles (EVs) derived from mesenchymal stem cells cultured in diabetic-like conditions enter pericytes, cause their detachment and migration, and stimulate angiogenesis. We recently showed that EVs from diabetic patients with retinopathy have different miRNA profiling patterns from healthy controls, and determine features of retinopathy in in vitro models of retinal microvasculature. In particular, a role for intra-vesicle miR-150-5p, miR-21-3p and miR-30b-5p was hypothesized. In this work, we further characterized EVs from subjects with diabetic retinopathy and investigated miR-150-5p, miR-21-3p and miR-30b-5p functions inside microvascular cells. Human retinal pericytes and ECs were transfected with mimics or inhibitors, as appropriate, of miR-21-3p, miR-30b-5p and miR-150-5p, to evaluate their ability in promoting cell migration and tube formation. mRNA and protein profiling of EVs extracted from diabetic subjects with (DR group) or without retinopathy (noDR group), and healthy controls (CTR group) were also performed. Modulation of miR-150-5p, miR-21-3p and miR-30b-5p inside microvascular cells confirmed their involvement in abnormal angiogenesis. mRNA analysis revealed differing expression of 7 genes involved in angiogenesis, while subsequent protein analysis confirmed increased expression of HIF-1α in DR group. Since all these molecules are involved in the hypoxia-induced retinal damage characteristic of the disease, our data reinforce the hypothesis of a potential use of miR-150-5p, miR-21-3p and miR-30b-5p extracted from circulating EVs as prognostic biomarkers for diabetic retinopathy.

High Resistance of Resting Eggs of Cladoceran Moina macrocopa to the Effect of Heavy Metals.

The research aimed to determine critical concentrations of heavy metals at which survival of resting eggs of the cladoceran Moina macrocopa is negatively affected. Resting eggs' viability was not affected over a 30-days exposure towards copper, cadmium, zinc or nickel at concentrations up to 60-70 g/L. When resting eggs were exposed to sediment contaminated with heavy metals for 8 months, the hatching success was affected at 30 g copper/kg. Thus, resting eggs of Cladocera can tolerate heavy metals at concentrations that far exceed lethal concentrations of heavy metals to active life stage and exceed low or moderate levels of environmental pollution. Follow up investigation of life table parameters of hatchlings from resting eggs exposed to heavy metals demonstrated that neither lifespan nor fecundity of hatchlings differ from control animals. These results demonstrate that zooplankton may rapidly recover from resting egg bank once aquatic habitat becomes unpolluted.

Molecular and functional characterization of circulating extracellular vesicles from diabetic patients with and without retinopathy and healthy subjects.

Diabetic retinopathy is a sight-threatening complication of diabetes, characterized by loss of retinal pericytes and abnormal angiogenesis. We previously demonstrated that extracellular vesicles (EVs) derived from mesenchymal stem cells cultured in diabetic-like conditions are able to enter the pericytes, causing their detachment and migration, and stimulating angiogenesis in vitro. The purpose of this work was the molecular and functional characterization of EVs derived from diabetic subjects with or without diabetic retinopathy, compared with healthy controls. Characterization of EVs extracted from serum/plasma of diabetic patients with or without retinopathy, and healthy controls, was performed by FACS and microarray analysis of microRNA (miRNA) content. Relevant miRNA expression was validated through qRT-PCR. EV influence on pericyte detachment, angiogenesis and permeability of the blood-retinal barrier was also investigated. Diabetic subjects had a 2.5 fold higher EV concentration than controls, while expression of surface molecules was unchanged. Microarray analysis revealed 11 differentially expressed miRNAs. Three of them (miR-150-5p, miR-21-3p and miR-30b-5p) were confirmed by qRT-PCR. Plasma EVs from subjects with diabetic retinopathy induced pericyte detachment and pericyte/endothelial cell migration, increased the permeability of pericyte/endothelial cell bilayers and the formation of vessel-like structures, when compared with EVs from controls. In conclusion, circulating EVs show differences between diabetic patients and healthy subjects. EVs extracted from plasma of diabetic retinopathy patients are able to induce features of retinopathy in in vitro models of retinal microvasculature. Our data suggest a role for miR-150-5p, miR-21-3p and miR-30b-5p as potential biomarkers of the onset of diabetic retinopathy.

miR-92a regulates angiogenic activity of adipose-derived mesenchymal stromal cells.

Mesenchymal stromal cells including those from adipose tissue (MSCs) regulate angiogenesis in adult tissues. MicroRNAs (miRs), small noncoding RNAs that control gene expression by binding to target mRNAs, reducing their stability and/or inhibiting translation, appear to be important regulators of blood vessel growth. In this study, we examined the impact of angio-miRs on paracrine activities of MSCs. Using Illumina microarrays we found that miR-92a is one of the most abundant angio-miRs in human MSCs. We transfected MSC with pre-miR-92a or anti-miR-92a which led to the coordinated changes of known miR-92a target mRNA levels. Then we tested the ability of conditioned medium from transfected cells to stimulate tube formation by HUVECs. MSC overexpressing miR-92a completely lost the ability to stimulate tubes formation by endothelial cells. However, knocking-out miR-92a by transfection with anti-miR-92a did not increase the ability of MSC to stimulate tube formation. Secretion of hepatocyte growth factor (HGF) and angiopoetin-1 was significantly lower in the medium of miR-92a overexpressing MSC, whereas VEGF secretion did not change significantly. The replenishment of HGF but not angiopoietin-1 has restored the ability of conditioned medium from miR-92a overexpressing MSC to stimulate the tube formation. We conclude that overexpression of miR-92a in MSC suppresses angiogenic properties of these cells by down-regulation of HGF secretion.

Platelet-derived growth factor regulates the secretion of extracellular vesicles by adipose mesenchymal stem cells and enhances their angiogenic potential.

BACKGROUND: Several studies demonstrate the role of adipose mesenchymal stem cells (ASCs) in angiogenesis. The angiogenic mechanism has been ascribed to paracrine factors since these cells secrete a plenty of signal molecules and growth factors. Recently it has been suggested that besides soluble factors, extracellular vesicles (EVs) that include exosomes and microvesicles may play a major role in cell-to-cell communication. It has been shown that EVs are implicated in the angiogenic process. RESULTS: Herein we studied whether EVs released by ASCs may mediate the angiogenic activity of these cells. Our results demonstrated that ASC-derived EVs induced in vitro vessel-like structure formation by human microvascular endothelial cells (HMEC). EV-stimulated HMEC when injected subcutaneously within Matrigel in SCID mice formed vessels. Treatment of ASCs with platelet-derived growth factor (PDGF) stimulated the secretion of EVs, changed their protein composition and enhanced the angiogenic potential. At variance of EVs released in basal conditions, PDGF-EVs carried c-kit and SCF that played a role in angiogenesis as specific blocking antibodies inhibited in vitro vessel-like structure formation. The enhanced content of matrix metalloproteinases in PDGF-EVs may also account for their angiogenic activity. CONCLUSIONS: Our findings indicate that EVs released by ASCs may contribute to the ASC-induced angiogenesis and suggest that PDGF may trigger the release of EVs with an enhanced angiogenic potential.

Growth hormone-releasing hormone antagonist MIA-602 inhibits inflammation induced by SARS-CoV-2 spike protein and bacterial lipopolysaccharide synergism in macrophages and human peripheral blood mononuclear cells.

COVID-19 is characterized by an excessive inflammatory response and macrophage hyperactivation, leading, in severe cases, to alveolar epithelial injury and acute respiratory distress syndrome. Recent studies have reported that SARS-CoV-2 spike (S) protein interacts with bacterial lipopolysaccharide (LPS) to boost inflammatory responses in vitro, in macrophages and peripheral blood mononuclear cells (PBMCs), and in vivo. The hypothalamic hormone growth hormone-releasing hormone (GHRH), in addition to promoting pituitary GH release, exerts many peripheral functions, acting as a growth factor in both malignant and non-malignant cells. GHRH antagonists, in turn, display potent antitumor effects and antinflammatory activities in different cell types, including lung and endothelial cells. However, to date, the antinflammatory role of GHRH antagonists in COVID-19 remains unexplored. Here, we examined the ability of GHRH antagonist MIA-602 to reduce inflammation in human THP-1-derived macrophages and PBMCs stimulated with S protein and LPS combination. Western blot and immunofluorescence analysis revealed the presence of GHRH receptor and its splice variant SV1 in both THP-1 cells and PBMCs. Exposure of THP-1 cells to S protein and LPS combination increased the mRNA levels and protein secretion of TNF-α and IL-1ß, as well as IL-8 and MCP-1 gene expression, an effect hampered by MIA-602. Similarly, MIA-602 hindered TNF-α and IL-1ß secretion in PBMCs and reduced MCP-1 mRNA levels. Mechanistically, MIA-602 blunted the S protein and LPS-induced activation of inflammatory pathways in THP-1 cells, such as NF-κB, STAT3, MAPK ERK1/2 and JNK. MIA-602 also attenuated oxidative stress in PBMCs, by decreasing ROS production, iNOS and COX-2 protein levels, and MMP9 activity. Finally, MIA-602 prevented the effect of S protein and LPS synergism on NF-кB nuclear translocation and activity. Overall, these findings demonstrate a novel antinflammatory role for GHRH antagonists of MIA class and suggest their potential development for the treatment of inflammatory diseases, such as COVID-19 and related comorbidities.

Tumour Derived Extracellular Vesicles: Challenging Target to Blunt Tumour Immune Evasion.

Control of the immune response is crucial for tumour onset and progression. Tumour cells handle the immune reaction by means of secreted factors and extracellular vesicles (EV). Tumour-derived extracellular vesicles (TEV) play key roles in immune reprogramming by delivering their cargo to different immune cells. Tumour-surrounding tissues also contribute to tumour immune editing and evasion, tumour progression, and drug resistance via locally released TEV. Moreover, the increase in circulating TEV has suggested their underpinning role in tumour dissemination. This review brings together data referring to TEV-driven immune regulation and antitumour immune suppression. Attention was also dedicated to TEV-mediated drug resistance.

Interleukin-3-Receptor-α in Triple-Negative Breast Cancer (TNBC): An Additional Novel Biomarker of TNBC Aggressiveness and a Therapeutic Target.

Tumour molecular annotation is mandatory for biomarker discovery and personalised approaches, particularly in triple-negative breast cancer (TNBC) lacking effective treatment options. In this study, the interleukin-3 receptor α (IL-3Rα) was investigated as a prognostic biomarker and therapeutic target in TNBC. IL-3Rα expression and patients' clinical and pathological features were retrospectively analysed in 421 TNBC patients. IL-3Rα was expressed in 69% human TNBC samples, and its expression was associated with nodal metastases (p = 0.026) and poor overall survival (hazard ratio = 1.50; 95% CI = 1.01-2.2; p = 0.04). The bioinformatics analysis on the Breast Invasive Carcinoma dataset of The Cancer Genome Atlas (TCGA) proved that IL-3Rα was highly expressed in TNBC compared with luminal breast cancers (p = 0.017, padj = 0.026). Functional studies demonstrated that IL-3Rα activation induced epithelial-to-endothelial and epithelial-to-mesenchymal transition, promoted large blood lacunae and lung metastasis formation, and increased programmed-cell death ligand-1 (PD-L1) in primary tumours and metastases. Based on the TCGA data, IL-3Rα, PD-L1, and EMT coding genes were proposed to discriminate against TNBC aggressiveness (AUC = 0.86 95% CI = 0.82-0.89). Overall, this study identified IL-3Rα as an additional novel biomarker of TNBC aggressiveness and provided the rationale to further investigate its relevance as a therapeutic target.

Thiamine transporter 2 is involved in high glucose-induced damage and altered thiamine availability in cell models of diabetic retinopathy.

Thiamine prevents high glucose-induced damage in microvasculature, and progression of retinopathy and nephropathy in diabetic animals. Impaired thiamine availability causes renal damage in diabetic patients. Two single-nucleotide polymorphisms in SLC19A3 locus encoding for thiamine transporter 2 are associated with absent/minimal diabetic retinopathy and nephropathy despite long-term type 1 diabetes. We investigated the involvement of thiamine transporter 1 and thiamine transporter 2, and their transcription factor specificity protein 1, in high glucose-induced damage and altered thiamine availability in cells of the inner blood-retinal barrier. Human endothelial cells, pericytes and Müller cells were exposed to hyperglycaemic-like conditions and/or thiamine deficiency/over-supplementation in single/co-cultures. Expression and localization of thiamine transporter 1, thiamine transporter 2 and transcription factor specificity protein 1 were evaluated together with intracellular thiamine concentration, transketolase activity and permeability to thiamine. The effects of thiamine depletion on cell function (viability, apoptosis and migration) were also addressed. Thiamine transporter 2 and transcription factor specificity protein 1 expression were modulated by hyperglycaemic-like conditions. Transketolase activity, intracellular thiamine and permeability to thiamine were decreased in cells cultured in thiamine deficiency, and in pericytes in hyperglycaemic-like conditions. Thiamine depletion reduced cell viability and proliferation, while thiamine over-supplementation compensated for thiamine transporter 2 reduction by restoring thiamine uptake and transketolase activity. High glucose and reduced thiamine determine impairment in thiamine transport inside retinal cells and through the inner blood-retinal barrier. Thiamine transporter 2 modulation in our cell models suggests its major role in thiamine transport in retinal cells and its involvement in high glucose-induced damage and impaired thiamine availability.

Extracellular Vesicles Released by Tumor Endothelial Cells Spread Immunosuppressive and Transforming Signals Through Various Recipient Cells.

Head and neck squamous cell carcinoma (HNSCC) has a high recurrence and metastatic rate with an unknown mechanism of cancer spread. Tumor inflammation is the most critical processes of cancer onset, growth, and metastasis. We hypothesize that the release of extracellular vesicles (EVs) by tumor endothelial cells (TECs) induce reprogramming of immune cells as well as stromal cells to create an immunosuppressive microenvironment that favor tumor spread. We call this mechanism as non-metastatic contagious carcinogenesis. Extracellular vesicles were collected from primary HNSCC-derived endothelial cells (TEC-EV) and were used for stimulation of peripheral blood mononuclear cells (PBMCs) and primary adipose mesenchymal stem cells (ASCs). Regulation of ASC gene expression was investigated by RNA sequencing and protein array. PBMC, stimulated with TEC-EV, were analyzed by enzyme-linked immunosorbent assay and fluorescence-activated cell sorting. We validated in vitro the effects of TEC-EV on ASCs or PBMC by measuring invasion, adhesion, and proliferation. We found and confirmed that TEC-EV were able to change ASC inflammatory gene expression signature within 24-48 h. TEC-EV were also able to enhance the secretion of TGF-ß1 and IL-10 by PBMC and to increase T regulatory cell (Treg) expansion. TEC-EV carry specific proteins and RNAs that are responsible for Treg differentiation and immune suppression. ASCs and PBMC, treated with TEC-EV, enhanced proliferation, adhesion of tumor cells, and their invasion. These data indicate that TEC-EV exhibit a mechanism of non-metastatic contagious carcinogenesis that regulates tumor microenvironment and reprograms immune cells to sustain tumor growth and progression.

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.

Targeting IL-3Rα on tumor-derived endothelial cells blunts metastatic spread of triple-negative breast cancer via extracellular vesicle reprogramming.

The lack of approved targeted therapies highlights the need for new treatments for triple-negative breast cancer (TNBC) patients. Interleukin-3 (IL-3) acts as an autocrine factor for tumor-endothelial cells (TEC), and exerts pro-angiogenic paracrine action via extracellular vesicles (EVs). IL-3Rα blockade on TEC changes TEC-EV (anti-IL-3R-EV) microRNA (miR) content and promotes the regression of established vessels. As TEC is the doorway for "drug" entry into tumors, we aimed to assess whether IL-3R blockade on TEC impacts tumor progression via its unique EV cargo. First, the expression of IL-3Rα was evaluated in 27 human TNBC samples. It was noticed that, besides TEC and inflammatory cells, tumor cells from 55.5% of the human TNBC samples expressed IL-3Rα. Using human TNBC cell lines for in vitro studies, we found that, unlike native TEC-EVs (nEVs), anti-IL-3R-EVs increase apoptosis and reduced cell viability and migration. In vivo, anti-IL-3R-EV treatment induced vessel regression in established tumors formed of MDA-MB-231 cells, decreased Vimentin, ß-catenin, and TWIST1 expression, almost abolished liver and lung metastases from primary tumors, and reduced lung metastasis generated via the intravenous injection of MDA-MB-231 cells. nEVs depleted of miR-24-3p (antago-miR-24-3p-EVs) were effective as anti-IL-3R-EVs in downregulating TWIST1 and reducing metastatic lesions in vivo. Consistent with network analyses of miR-24-3p gene targeting, anti-IL-3R-EVs and antago-miR-24-3p-EVs upregulate SPRY2 in MDA-MB-231 cells. Finally, SPRY2 silencing prevented anti-IL-3R-EV and antago-miR-24-3p-EV-mediated apoptotic cues.Overall, these data provide the first evidence that IL-3Rα is highly expressed in TNBC cells, TEC, and inflammatory cells, and that IL-3Rα blockade on TEC impacts tumor progression.

Correction: Adipose-Derived Stem Cells Stimulate Regeneration of Peripheral Nerves: BDNF Secreted by These Cells Promotes Nerve Healing and Axon Growth De Novo.

[This corrects the article DOI: 10.1371/journal.pone.0017899.].

Characterization and Gene Expression Analysis of Serum-Derived Extracellular Vesicles in Primary Aldosteronism.

Patients affected by primary aldosteronism (PA) display an increased risk of cardiovascular events compared with essential hypertension (EH). Endothelial dysfunction favors initiation and progression of atherosclerosis and circulating extracellular vesicles (EVs), reflecting endothelial cell activity, could represent one of the mediators of endothelial dysfunction in these patients. The aim of this study was to characterize circulating EVs from patients diagnosed with PA and to explore their functional significance. Serum EVs were isolated from 12 patients with PA and 12 with EH, matched by sex, age, and blood pressure, and compared with 8 normotensive controls. At nanoparticle tracking analysis, EVs concentration was 2.2× higher in patients with PA ( P=0.033) compared with EH and a significant correlation between EV number and serum aldosterone and potassium levels was identified. Fluorescence-activated cell sorting analysis demonstrated that patients with PA presented a higher absolute number of endothelial-derived EVs compared with both patients with EH and normotensive controls. Through EV mRNA profiling, 15 up-regulated and 4 down-regulated genes in patients with PA compared with EH were identified; moreover, EDN1 was expressed only in patients with PA. Microarray platform was validated by quantative real-time polymerase chain reaction on 4 genes ( CASP1, EDN1, F2R, and HMOX1) involved in apoptosis, inflammation, and endothelial dysfunction. After unilateral adrenalectomy, EVs number and expression of CASP1 and EDN1 significantly decreased in patients with PA ( P<0.05). Additionally, the incubation with PA-derived EVs reduced angiogenesis and induced apoptosis in vitro. Circulating EVs might not only represent a marker of endothelial dysfunction but also contribute themselves to vascular dysfunction in patients with PA.