Protective effects of mesenchymal stem cells-derived extracellular vesicles against ischemia-reperfusion injury of hearts donated after circulatory death: Preliminary study in a pig model.

INTRODUCTION: Insufficient supply of cardiac grafts represents a severe obstacle in heart transplantation. Donation after Circulatory Death (DCD), in addition to conventional donation after brain death, is one promising option to overcome the organ shortage. However, DCD organs undergo an inevitable more extended period of warm unprotected ischemia between circulatory arrest and graft procurement. Mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) have shown remarkable protective effects against ischemia-reperfusion injury. Thus, we aimed to enhance grafts preservation from DCD donors, through treatment with MSC-EVs. METHODS: Female pigs were euthanized by barbiturate overdose and after 20 min of a flat EKG, the chest was opened, the heart harvested and subsequently connected to an extracorporeal perfusion machine. MSC-EVs, isolated by ion exchange chromatography, were added to the perfusion solution (1×1011 particles) and the heart was perfused for 2 h. Then, heart tissue biopsies were taken to assess histological changes, mitochondrial morphology, antioxidant enzyme activity and inflammation mediators' expression. Biochemical parameters of myocardial viability were assessed in the perfusate. RESULTS: The treatment with MSC-EVs significantly prevented mitochondria swelling, mitochondrial cristae loss and oxidative stress in cardiac tissue. The protective effect of MSC-EVs was confirmed by the delayed increase of the cardiac-specific enzymes CK and TnC in the perfusate and the reduction of caspase-3+ cells in tissue sections. CONCLUSION: MSC-EVs improve graft quality by preserving the mitochondrial ultrastructure protecting the myocardium against oxidative stress, reducing apoptosis of cardiac cells and preventing the increase of pro-inflammatory cytokines.

Profile of matrix-entrapped extracellular vesicles of microenvironmental and infiltrating cell origin in decellularized colorectal cancer and adjacent mucosa.

Cellular elements that infiltrate and surround tumours and pre-metastatic tissues have a prominent role in tumour invasion and growth. The extracellular vesicles specifically entrapped and stored within the extracellular matrix (ECM-EVs) may reflect the different populations of the tumour microenvironment and their change during tumour progression. However, their profile is at present unknown. To elucidate this aspect, we isolated and characterized EVs from decellularized surgical specimens of colorectal cancer and adjacent colon mucosa and analyzed their surface marker profile. ECM-EVs in tumours and surrounding mucosa mainly expressed markers of lymphocytes, natural killer cells, antigen-presenting cells, and platelets, as well as epithelial cells, representing a multicellular microenvironment. No difference in surface marker expression was observed between tumour and mucosa ECM-EVs in stage II-III tumours. At variance, in the colon mucosa adjacent to stage IV carcinomas, ECM-EV profile showed a significantly increased level of immune, epithelial and platelet markers in comparison to the matrix of the corresponding tumour. The increase of EVs from immune cells and platelets was not observed in the mucosa adjacent to low-stage tumours. In addition, CD25, a T-lymphocyte marker, resulted specifically overexpressed by ECM-EVs from stage IV carcinomas, possibly correlated with the pro-tolerogenic environment found in the corresponding tumour tissue. These results outline the tissue microenvironmental profile of EVs in colorectal carcinoma-derived ECM and unveil a profound change in the healthy mucosa adjacent to high-stage tumours.

Bottlenose dolphin (Tursiops truncatus) immortalized fibroblasts on novel 3D in vitro collagen-free scaffolds.

Dolphins, as apex predators, can be considered relevant sentinels of the health of marine ecosystems. The creation of 3D cell models to assess in vitro cell-to-cell and cell-to-matrix interactions in environmental-mimicking conditions, is of considerable interest. However, to date the establishment of cetacean 3D culture systems has not yet been accomplished. Thus, in this study, different 3D systems of bottlenose dolphin (Tursiops truncatus) skin fibroblasts have been analyzed. Particularly, novel scaffolds based on hyaluronic acid and ionic-complementary self-assembling peptides such as RGD-EAbuK and EAbuK-IKVAV have been compared to Matrigel. Histological and fluorescent staining, electron microscopy (TEM) analyses and viability assays have been performed and RT-PCR has been used to detect extracellular matrix (ECM) components produced by cells. Results showed that Matrigel induced cells to form aggregates with lower viability and no ECM production compared to the novel scaffolds. Moreover, scaffolds allowed dispersed cells to produce a collagenous ECM containing collagen1a1, laminin B1 and elastin. The HA-EAbuK-IKVAV scaffold resulted in the most suitable 3D model in terms of cell quantity and viability. The development of this innovative approach is the first step towards the possibility to create 3D in vitro models for this protected species.

Extracellular Vesicles From Mesenchymal Umbilical Cord Cells Exert Protection Against Oxidative Stress and Fibrosis in a Rat Model of Bronchopulmonary Dysplasia.

Oxidative stress and fibrosis are important stress responses that characterize bronchopulmonary dysplasia (BPD), a disease for which only a therapy but not a cure has been developed. In this work, we investigated the effects of mesenchymal stromal cells-derived extracellular vesicles (MSC-EVs) on lung and brain compartment in an animal model of hyperoxia-induced BPD. Rat pups were intratracheally injected with MSC-EVs produced by human umbilical cord-derived MSC, following the Good Manufacturing Practice-grade (GMP-grade). After evaluating biodistribution of labelled MSC-EVs in rat pups left in normoxia and hyperoxia, oxidative stress and fibrosis investigation were performed. Oxidative stress protection by MSC-EVs treatment was proved both in lung and in brain. The lung epithelial compartment ameliorated glycosaminoglycan and surfactant protein expression in MSC-EVs-injected rat pups compared to untreated animals. Pups under hyperoxia exhibited a fibrotic phenotype in lungs shown by increased collagen deposition and also expression of profibrotic genes. Both parameters were reduced by treatment with MSC-EVs. We established an in vitro model of fibrosis and another of oxidative stress, and we proved that MSC-EVs suppressed the induction of αSMA, influencing collagen deposition and protecting from the oxidative stress. In conclusion, intratracheal administration of clinical-grade MSC-EVs protect from oxidative stress, improves pulmonary epithelial function, and counteracts the development of fibrosis. In the future, MSC-EVs could represent a new cure to prevent the development of BPD.

Expression of targets of the RNA-binding protein AUF-1 in human airway epithelium indicates its role in cellular senescence and inflammation.

Introduction: The RNA-binding protein AU-rich-element factor-1 (AUF-1) participates to posttranscriptional regulation of genes involved in inflammation and cellular senescence, two pathogenic mechanisms of chronic obstructive pulmonary disease (COPD). Decreased AUF-1 expression was described in bronchiolar epithelium of COPD patients versus controls and in vitro cytokine- and cigarette smoke-challenged human airway epithelial cells, prompting the identification of epithelial AUF-1-targeted transcripts and function, and investigation on the mechanism of its loss. Results: RNA immunoprecipitation-sequencing (RIP-Seq) identified, in the human airway epithelial cell line BEAS-2B, 494 AUF-1-bound mRNAs enriched in their 3'-untranslated regions for a Guanine-Cytosine (GC)-rich binding motif. AUF-1 association with selected transcripts and with a synthetic GC-rich motif were validated by biotin pulldown. AUF-1-targets' steady-state levels were equally affected by partial or near-total AUF-1 loss induced by cytomix (TNFα/IL1ß/IFNγ/10 nM each) and siRNA, respectively, with differential transcript decay rates. Cytomix-mediated decrease in AUF-1 levels in BEAS-2B and primary human small-airways epithelium (HSAEC) was replicated by treatment with the senescence- inducer compound etoposide and associated with readouts of cell-cycle arrest, increase in lysosomal damage and senescence-associated secretory phenotype (SASP) factors, and with AUF-1 transfer in extracellular vesicles, detected by transmission electron microscopy and immunoblotting. Extensive in-silico and genome ontology analysis found, consistent with AUF-1 functions, enriched RIP-Seq-derived AUF-1-targets in COPD-related pathways involved in inflammation, senescence, gene regulation and also in the public SASP proteome atlas; AUF-1 target signature was also significantly represented in multiple transcriptomic COPD databases generated from primary HSAEC, from lung tissue and from single-cell RNA-sequencing, displaying a predominant downregulation of expression. Discussion: Loss of intracellular AUF-1 may alter posttranscriptional regulation of targets particularly relevant for protection of genomic integrity and gene regulation, thus concurring to airway epithelial inflammatory responses related to oxidative stress and accelerated aging. Exosomal-associated AUF-1 may in turn preserve bound RNA targets and sustain their function, participating to spreading of inflammation and senescence to neighbouring cells.

USP8 Down-Regulation Promotes Parkin-Independent Mitophagy in the Drosophila Brain and in Human Neurons.

Stress-induced mitophagy, a tightly regulated process that targets dysfunctional mitochondria for autophagy-dependent degradation, mainly relies on two proteins, PINK1 and Parkin, which genes are mutated in some forms of familiar Parkinson's Disease (PD). Upon mitochondrial damage, the protein kinase PINK1 accumulates on the organelle surface where it controls the recruitment of the E3-ubiquitin ligase Parkin. On mitochondria, Parkin ubiquitinates a subset of mitochondrial-resident proteins located on the outer mitochondrial membrane, leading to the recruitment of downstream cytosolic autophagic adaptors and subsequent autophagosome formation. Importantly, PINK1/Parkin-independent mitophagy pathways also exist that can be counteracted by specific deubiquitinating enzymes (DUBs). Down-regulation of these specific DUBs can presumably enhance basal mitophagy and be beneficial in models in which the accumulation of defective mitochondria is implicated. Among these DUBs, USP8 is an interesting target because of its role in the endosomal pathway and autophagy and its beneficial effects, when inhibited, in models of neurodegeneration. Based on this, we evaluated autophagy and mitophagy levels when USP8 activity is altered. We used genetic approaches in D. melanogaster to measure autophagy and mitophagy in vivo and complementary in vitro approaches to investigate the molecular pathway that regulates mitophagy via USP8. We found an inverse correlation between basal mitophagy and USP8 levels, in that down-regulation of USP8 correlates with increased Parkin-independent mitophagy. These results suggest the existence of a yet uncharacterized mitophagic pathway that is inhibited by USP8.

Multiple Forms of Neural Cell Death in the Cyclical Brain Degeneration of A Colonial Chordate.

Human neuronal loss occurs through different cellular mechanisms, mainly studied in vitro. Here, we characterized neuronal death in B. schlosseri, a marine colonial tunicate that shares substantial genomic homology with mammals and has a life history in which controlled neurodegeneration happens simultaneously in the brains of adult zooids during a cyclical phase named takeover. Using an ultrastructural and transcriptomic approach, we described neuronal death forms in adult zooids before and during the takeover phase while comparing adult zooids in takeover with their buds where brains are refining their structure. At takeover, we found in neurons clear morphologic signs of apoptosis (i.e., chromatin condensation, lobed nuclei), necrosis (swollen cytoplasm) and autophagy (autophagosomes, autolysosomes and degradative multilamellar bodies). These results were confirmed by transcriptomic analyses that highlighted the specific genes involved in these cell death pathways. Moreover, the presence of tubulovesicular structures in the brain medulla alongside the over-expression of prion disease genes in late cycle suggested a cell-to-cell, prion-like propagation recalling the conformational disorders typical of some human neurodegenerative diseases. We suggest that improved understanding of how neuronal alterations are regulated in the repeated degeneration-regeneration program of B. schlosseri may yield mechanistic insights relevant to the study of human neurodegenerative diseases.

Fumarate induces vesicular release of mtDNA to drive innate immunity.

Mutations in fumarate hydratase (FH) cause hereditary leiomyomatosis and renal cell carcinoma1. Loss of FH in the kidney elicits several oncogenic signalling cascades through the accumulation of the oncometabolite fumarate2. However, although the long-term consequences of FH loss have been described, the acute response has not so far been investigated. Here we generated an inducible mouse model to study the chronology of FH loss in the kidney. We show that loss of FH leads to early alterations of mitochondrial morphology and the release of mitochondrial DNA (mtDNA) into the cytosol, where it triggers the activation of the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING)-TANK-binding kinase 1 (TBK1) pathway and stimulates an inflammatory response that is also partially dependent on retinoic-acid-inducible gene I (RIG-I). Mechanistically, we show that this phenotype is mediated by fumarate and occurs selectively through mitochondrial-derived vesicles in a manner that depends on sorting nexin 9 (SNX9). These results reveal that increased levels of intracellular fumarate induce a remodelling of the mitochondrial network and the generation of mitochondrial-derived vesicles, which allows the release of mtDNAin the cytosol and subsequent activation of the innate immune response.

Biodistribution of Intratracheal, Intranasal, and Intravenous Injections of Human Mesenchymal Stromal Cell-Derived Extracellular Vesicles in a Mouse Model for Drug Delivery Studies.

Mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) are extensively studied as therapeutic tools. Evaluation of their biodistribution is fundamental to understanding MSC-EVs' impact on target organs. In our work, MSC-EVs were initially labeled with DiR, a fluorescent lipophilic dye, and administered to BALB/c mice (2.00 × 1010 EV/mice) through the following routes: intravenous (IV), intratracheal (IT) and intranasal (IN). DiR-labeled MSC-EVs were monitored immediately after injection, and after 3 and 24 hours (h). Whole-body analysis, 3 h after IV injection, showed an accumulation of MSC-EVs in the mice abdominal region, compared to IT and IN, where EVs mainly localized at the levels of the chest and brain region, respectively. After 24 h, EV-injected mice retained a stronger positivity in the same regions identified after 3 h from injection. The analyses of isolated organs confirmed the accumulation of EVs in the spleen and liver after IV administration. Twenty-four hours after the IT injection of MSC-EVs, a stronger positivity was detected selectively in the isolated lungs, while for IN, the signal was confined to the brain. In conclusion, these results show that local administration of EVs can increase their concentration in selective organs, limiting their systemic biodistribution and possibly the extra-organ effects. Biodistribution studies can help in the selection of the most appropriate way of administration of MSC-EVs for the treatment of different diseases.

Long-term proliferation of immature hypoxia-dependent JMML cells supported by a 3D in vitro system.

Juvenile myelomonocytic leukemia (JMML) is a rare clonal stem cell disorder that occurs in early childhood and is characterized by the hyperactivation of the RAS pathway in 95% of the patients. JMML is characterized by a hyperproliferation of granulocytes and monocytes, and little is known about the heterogeneous nature of leukemia-initiating cells, as well as of the cellular hierarchy of the JMML bone marrow. In this study, we report the generation and characterization of a novel patient-derived three-dimensional (3D) in vitro JMML model, called patient-derived JMML Atypical Organoid (pd-JAO), sustaining the long-term proliferation of JMML cells with stem cell features and patient-specific hallmarks. JMML cells brewed in a 3D model under different microenvironmental conditions acquired proliferative and survival advantages when placed under low oxygen tension. Transcriptomic and microscopic analyses revealed the activation of specific metabolic energy pathways and the inactivation of processes leading to cell death. Furthermore, we demonstrated the pd-JAO-derived cells' migratory, propagation, and self-renewal capacities. Our study contributes to the development of a robust JMML 3D in vitro model for studying and defining the impact of microenvironmental stimuli on JMML disease and the molecular mechanisms that regulate JMML initiating and propagating cells. Pd-JAO may become a promising model for compound tests focusing on new therapeutic interventions aimed at eradicating JMML progenitors and controlling JMML disease.

Small intestinal submucosa-derived extracellular matrix as a heterotopic scaffold for cardiovascular applications.

Structural cardiac lesions are often surgically repaired using prosthetic patches, which can be biological or synthetic. In the current clinical scenario, biological patches derived from the decellularization of a xenogeneic scaffold are gaining more interest as they maintain the natural architecture of the extracellular matrix (ECM) after the removal of the native cells and remnants. Once implanted in the host, these patches can induce tissue regeneration and repair, encouraging angiogenesis, migration, proliferation, and host cell differentiation. Lastly, decellularized xenogeneic patches undergo cell repopulation, thus reducing host immuno-mediated response against the graft and preventing device failure. Porcine small intestinal submucosa (pSIS) showed such properties in alternative clinical scenarios. Specifically, the US FDA approved its use in humans for urogenital procedures such as hernia repair, cystoplasties, ureteral reconstructions, stress incontinence, Peyronie's disease, penile chordee, and even urethral reconstruction for hypospadias and strictures. In addition, it has also been successfully used for skeletal muscle tissue reconstruction in young patients. However, for cardiovascular applications, the results are controversial. In this study, we aimed to validate our decellularization protocol for SIS, which is based on the use of Tergitol 15 S 9, by comparing it to our previous and efficient method (Triton X 100), which is not more available in the market. For both treatments, we evaluated the preservation of the ECM ultrastructure, biomechanical features, biocompatibility, and final bioinductive capabilities. The overall analysis shows that the SIS tissue is macroscopically distinguishable into two regions, one smooth and one wrinkle, equivalent to the ultrastructure and biochemical and proteomic profile. Furthermore, Tergitol 15 S 9 treatment does not modify tissue biomechanics, resulting in comparable to the native one and confirming the superior preservation of the collagen fibers. In summary, the present study showed that the SIS decellularized with Tergitol 15 S 9 guarantees higher performances, compared to the Triton X 100 method, in all the explored fields and for both SIS regions: smooth and wrinkle.

Stemness Activity Underlying Whole Brain Regeneration in a Basal Chordate.

Understanding how neurons regenerate following injury remains a central challenge in regenerative medicine. Adult mammals have a very limited ability to regenerate new neurons in the central nervous system (CNS). In contrast, the basal chordate Polycarpa mytiligera can regenerate its entire CNS within seven days of complete removal. Transcriptome sequencing, cellular labeling, and proliferation in vivo essays revealed that CNS regeneration is mediated by a newly formed neural progeny and the activation of neurodevelopmental pathways that are associated with enhanced stem-cell activity. Analyzing the expression of 239 activated pathways enabled a quantitative understanding of gene-set enrichment patterns at key regeneration stages. The molecular and cellular mechanisms controlling the regenerative ability that this study reveals can be used to develop innovative approaches to enhancing neurogenesis in closely-related chordate species, including humans.

Retinoic acid-induced 1 gene haploinsufficiency alters lipid metabolism and causes autophagy defects in Smith-Magenis syndrome.

Smith-Magenis syndrome (SMS) is a neurodevelopmental disorder characterized by cognitive and behavioral symptoms, obesity, and sleep disturbance, and no therapy has been developed to alleviate its symptoms or delay disease onset. SMS occurs due to haploinsufficiency of the retinoic acid-induced-1 (RAI1) gene caused by either chromosomal deletion (SMS-del) or RAI1 missense/nonsense mutation. The molecular mechanisms underlying SMS are unknown. Here, we generated and characterized primary cells derived from four SMS patients (two with SMS-del and two carrying RAI1 point mutations) and four control subjects to investigate the pathogenetic processes underlying SMS. By combining transcriptomic and lipidomic analyses, we found altered expression of lipid and lysosomal genes, deregulation of lipid metabolism, accumulation of lipid droplets, and blocked autophagic flux. We also found that SMS cells exhibited increased cell death associated with the mitochondrial pathology and the production of reactive oxygen species. Treatment with N-acetylcysteine reduced cell death and lipid accumulation, which suggests a causative link between metabolic dyshomeostasis and cell viability. Our results highlight the pathological processes in human SMS cells involving lipid metabolism, autophagy defects and mitochondrial dysfunction and suggest new potential therapeutic targets for patient treatment.

The Flavone Cirsiliol from Salvia x jamensis Binds the F1 Moiety of ATP Synthase, Modulating Free Radical Production.

Several studies have shown that mammalian retinal rod outer segments (OS) are peculiar structures devoid of mitochondria, characterized by ectopic expression of the molecular machinery for oxidative phosphorylation. Such ectopic aerobic metabolism would provide the chemical energy for the phototransduction taking place in the OS. Natural polyphenols include a large variety of molecules having pleiotropic effects, ranging from anti-inflammatory to antioxidant and others. Our goal in the present study was to investigate the potential of the flavonoid cirsiliol, a trihydroxy-6,7-dimethoxyflavone extracted from Salvia x jamensis, in modulating reactive oxygen species production by the ectopic oxidative phosphorylation taking place in the OS. Our molecular docking analysis identified cirsiliol binding sites inside the F1 moiety of the nanomotor F1Fo-ATP synthase. The experimental approach was based on luminometry, spectrophotometry and cytofluorimetry to evaluate ATP synthesis, respiratory chain complex activity and H2O2 production, respectively. The results showed significant dose-dependent inhibition of ATP production by cirsiliol. Moreover, cirsiliol was effective in reducing the free radical production by the OS exposed to ambient light. We report a considerable protective effect of cirsiliol on the structural stability of rod OS, suggesting it may be considered a promising compound against oxidative stress.

Cell cycle alterations due to perfluoroalkyl substances PFOS, PFOA, PFBS, PFBA and the new PFAS C6O4 on bottlenose dolphin (Tursiops truncatus) skin cell.

Per- and polyfluoroalkyl substances (PFAS) have become ubiquitous environmental contaminants in aquatic ecosystems worldwide. Marine mammals, as top predators, are constantly exposed to several PFAS compounds that accumulate in different tissues. As a proxy to assess cytotoxicity of PFAS in the bottlenose dolphin (Tursiops truncatus), we generated a new immortalized cell line derived from skin samples of bottlenose dolphin. Using high content imaging, we assessed the effects of increasing concentrations of PFOS, PFOA, PFBS, PFBA and C6O4 on cell viability and cell cycle phases. In particular, we classified all cells based on multiple morphometric differences of the nucleus in three populations, named respectively "Normal" (nuclei in G0, S and M phase); "Large" (nuclei showing characteristics of senescence) and "Small" (nuclei with fragmentation and condensed chromatin). Combining this approach with cell cycle analysis we determined which phases of the cell cycle were influenced by PFAS. The results revealed that the presence of PFOS, PFBS and PFBA could increase the number of cells in G0+G1 phase and decrease the number of those in the S phase. Moreover, PFOS and PFBS lowered the fraction of cells in the M phase. Interestingly PFOS, PFBS and PFBA reduced the prevalence of the senescence phenotype ("large" nuclei), suggesting a potential tumorigenic effect. Besides, the presence of PFOS and PFBS correlated also with a significant decrease in the number of "small" nuclei. The C6O4 exposure did not highlighted morphometric alteration or cell cycle modification bottlenose dolphin skin cell nuclei. While the effects of PFAS on cell cycle was clear, no significant change was detected either in term of cell proliferation or of viability. This study fosters the overall knowledge on the cellular effects of perfluoroalkyl substances in marine mammals.

Two distinct evolutionary conserved neural degeneration pathways characterized in a colonial chordate.

Colonial tunicates are marine organisms that possess multiple brains simultaneously during their colonial phase. While the cyclical processes of neurogenesis and neurodegeneration characterizing their life cycle have been documented previously, the cellular and molecular changes associated with such processes and their relationship with variation in brain morphology and individual (zooid) behavior throughout adult life remains unknown. Here, we introduce Botryllus schlosseri as an invertebrate model for neurogenesis, neural degeneration, and evolutionary neuroscience. Our analysis reveals that during the weekly colony budding (i.e., asexual reproduction), prior to programmed cell death and removal by phagocytes, decreases in the number of neurons in the adult brain are associated with reduced behavioral response and significant change in the expression of 73 mammalian homologous genes associated with neurodegenerative disease. Similarly, when comparing young colonies (1 to 2 y of age) to those reared in a laboratory for ∼20 y, we found that older colonies contained significantly fewer neurons and exhibited reduced behavioral response alongside changes in the expression of 148 such genes (35 of which were differentially expressed across both timescales). The existence of two distinct yet apparently related neurodegenerative pathways represents a novel platform to study the gene products governing the relationship between aging, neural regeneration and degeneration, and loss of nervous system function. Indeed, as a member of an evolutionary clade considered to be a sister group of vertebrates, this organism may be a fundamental resource in understanding how evolution has shaped these processes across phylogeny and obtaining mechanistic insight.

Single extracellular vesicle analysis in human amniotic fluid shows evidence of phenotype alterations in preeclampsia.

Amniotic fluid surrounding the developing fetus is a complex biological fluid rich in metabolically active bio-factors. The presence of extracellular vesicles (EVs) in amniotic fluid has been mainly related to foetal urine. We here characterized EVs from term amniotic fluid in terms of surface marker expression using different orthogonal techniques. EVs appeared to be a heterogeneous population expressing markers of renal, placental, epithelial and stem cells. Moreover, we compared amniotic fluid EVs from normal pregnancies with those of preeclampsia, a hypertensive disorder affecting up to 8% of pregnancies worldwide. An increase of CD105 (endoglin) expressing EVs was observed in preeclamptic amniotic fluid by bead-based cytofluorimetric analysis, and further confirmed using a chip-based analysis. HLA-G, a typical placental marker, was not co-expressed by the majority of CD105+ EVs, in analogy with amniotic fluid stromal cell derived-EVs. At a functional level, preeclampsia-derived EVs, but not normal pregnancy EVs, showed an antiangiogenic effect, possibly due to the decoy effect of endoglin. Our results provide a characterization of term amniotic fluid-EVs, supporting their origin from foetal and placental cells. In preeclampsia, the observed antiangiogenic characteristics of amniotic fluid-EVs may reflect the hypoxic and antiangiogenic microenvironment and could possibly impact on the developing fetus or on the surrounding foetal membranes.

Temperature-Related Effects of Myocardial Protection Strategies in Swine Hearts after Prolonged Warm Ischemia.

Insufficient supply of cardiac grafts represents a severe obstacle in heart transplantation. Donation after circulatory death (DCD), in addition to conventional donation after brain death, is one promising option to overcome the organ shortage. However, DCD organs undergo an inevitably longer period of unprotected warm ischemia between circulatory arrest and graft procurement. In this scenario, we aim to improve heart preservation after a warm ischemic period of 20 min by testing different settings of myocardial protective strategies. Pig hearts were collected from a slaughterhouse and assigned to one of the five experimental groups: baseline (BL), cold cardioplegia (CC), cold cardioplegia + adenosine (CC-ADN), normothermic cardioplegia (NtC + CC) or normothermic cardioplegia + cold cardioplegia + adenosine (NtC-ADN + CC). After treatment, tissue biopsies were taken to assess mitochondrial morphology, antioxidant enzyme activity, lipid peroxidation and cytokine and chemokine expressions. NtC + CC treatment significantly prevented mitochondria swelling and mitochondrial cristae loss. Moreover, the antioxidant enzyme activity was lower in this group, as was lipid peroxidation, and the pro-inflammatory chemokine GM-CSF was diminished. Finally, we demonstrated that normothermic cardioplegia preserved mitochondria morphology, thus preventing oxidative stress and the subsequent inflammatory response. Therefore, normothermic cardioplegia is a better approach to preserve the heart after a warm ischemia period, with respect to cold cardioplegia, before transplantation.

Extracellular Vesicles Secreted by Mesenchymal Stromal Cells Exert Opposite Effects to Their Cells of Origin in Murine Sodium Dextran Sulfate-Induced Colitis.

Several reports have described a beneficial effect of Mesenchymal Stromal Cells (MSCs) and of their secreted extracellular vesicles (EVs) in mice with experimental colitis. However, the effects of the two treatments have not been thoroughly compared in this model. Here, we compared the effects of MSCs and of MSC-EV administration in mice with colitis induced by dextran sulfate sodium (DSS). Since cytokine conditioning was reported to enhance the immune modulatory activity of MSCs, the cells were kept either under standard culture conditions (naïve, nMSCs) or primed with a cocktail of pro-inflammatory cytokines, including IL1ß, IL6 and TNFα (induced, iMSCs). In our experimental conditions, nMSCs and iMSCs administration resulted in both clinical and histological worsening and was associated with pro-inflammatory polarization of intestinal macrophages. However, mice treated with iEVs showed clinico-pathological improvement, decreased intestinal fibrosis and angiogenesis and a striking increase in intestinal expression of Mucin 5ac, suggesting improved epithelial function. Moreover, treatment with iEVs resulted in the polarization of intestinal macrophages towards and anti-inflammatory phenotype and in an increased Treg/Teff ratio at the level of the intestinal lymph node. Collectively, these data confirm that MSCs can behave either as anti- or as pro-inflammatory agents depending on the host environment. In contrast, EVs showed a beneficial effect, suggesting a more predictable behavior, a safer therapeutic profile and a higher therapeutic efficacy with respect to their cells of origin.