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Rationale: Extracellular vesicles (EVs) contain bioactive cargo including microRNAs (miRNAs) and proteins that are released by cells as a form of cell-cell communication. Endothelial cells (ECs) form the innermost lining of all blood vessels and thereby interface with cells in the circulation as well as cells residing in the vascular wall. It is unknown whether ECs have the capacity to release EVs capable of governing recipient cells within two separate compartments, and how this is affected by endothelial activation commonly seen in atheroprone regions.Objective: Given their boundary location, we propose that ECs utilize bidirectional release of distinct EV cargo in quiescent and activated states to communicate with cells within the circulation and blood vessel wall.Methods and Results: EVs were isolated from primary human aortic endothelial cells (ECs) (+/- IL-1β activation), quantified, and analysed by miRNA transcriptomics and proteomics. Compared to quiescent ECs, activated ECs increased EV release, with miRNA and protein cargo that were related to atherosclerosis. RNA sequencing of EV-treated monocytes and smooth muscle cells (SMCs) revealed that EVs from activated ECs altered pathways that were pro-inflammatory and atherogenic. Apical and basolateral EV release was assessed using ECs on transwells. ECs released more EVs apically, which increased with activation. Apical and basolateral EV cargo contained distinct transcriptomes and proteomes that were altered by EC activation. Notably, basolateral EC-EVs displayed greater changes in the EV secretome, with pathways specific to atherosclerosis.In silicoanalysis determined that compartment-specific cargo released by the apical and basolateral surfaces of ECs can reprogram monocytes and SMCs, respectively.Conclusions: The demonstration that ECs are capable of polarized EV cargo loading and directional EV secretion reveals a novel paradigm for endothelial communication, which may ultimately enhance our ability to design endothelial-based therapeutics for cardiovascular diseases such as atherosclerosis where ECs are persistently activated.Non-standard Abbreviations and Acronymscryo-EMcryogenic electron microscopyECendothelial cellEVextracellular vesicleGOgene ontologyHAEChuman aortic endothelial cellSMChuman aortic vascular smooth muscle cellIL-1βinterleukin 1 betaKEGGKyoto encyclopedia of genes and genomesLC-MSlabel-free liquid-chromatography mass spectrometryMVBmultivesicular bodymiRNAmicroRNARNAseqRNA sequencingTEMtransmission electron microscopyTIRFtotal interal reflection fluorescence microscopymiRNAmicroRNAGraphical abstract: Polarized endothelial extracellular vesicle communication with luminal and abluminal vascular cellsEndothelial cell small extracellular vesicle (EC-EV) release from apical (luminal) and basolateral (abluminal) surfaces in quiescence and after endothelial activation. Quiescent EC-EVs are depicted in blue (bright blue=apical, light blue=basolateral), while activated EC-EVs are depicted in red (bright red=apical, light red=basolateral). Luminal monocyte is represented in purple with upregulation of pro-inflammatory transcripts (bright purple) after uptake of activated EC-EVs from the apical surface, compared to uptake of quiescent apical EC-EVs (light purple). Basolateral EC-EVs are taken up by an abluminal resident smooth muscle cell depicted in yellow. Smooth muscle cell uptake of activated basolateral EC-EVs with upregulation of pro-inflammatory/pro-atherogenic transcripts (bright yellow), as compared to uptake of quiescent EC-EVs (light yellow).

Aim: Abdominal aortic aneurysm (AAA) contributes to significant post-rupture mortality in aging populations. AAA management with watchful waiting and surgical repair is based on our limited understanding of disease processes, and current research foci including extracellular vesicles (EVs; nano-sized packages of proteins, RNAs, and lipids that facilitate intercellular communication) may aid in developing novel AAA therapies. To characterize this regulatory cargo, we isolated EVs from human AAA tissue or control aortic punch biopsies and profiled EV content with microRNA (miRNA) sequencing to identify dysregulated pathways. Methods: The study was approved by the University Health Network Research Ethics Board. EVs were isolated from human AAA or aortic punch tissue and enriched using size exclusion chromatography (SEC; qEVoriginal columns 70 nm, Izon Science Ltd.) (n=3). EV size and concentration were determined using nanoparticle tracking analysis (NTA; NanoSight NS300, Malvern Panalytical Ltd.). EV-miRNA sequencing was performed with Illumina NextSeq (HTG Molecular Diagnostics Inc.) and analyzed using Partek Genomics Suite (v.10) and MIENTURNET (19-11-25). Results: Patients were selected for infrarenal AAA requiring surgical repair (AAA) or coronary artery disease requiring bypass graft surgery (control). EV size and concentration were confirmed with NTA. Principal components and gene set analyses revealed distinct clustering of tissue types with 901 and 687 miRNAs enriched in AAA and control samples, respectively. Pathway prediction using established AAA miRNAs (e.g., miR-122, miR-146a, and miR-503) identified significant interactions with proaneurysmal signaling pathways (e.g., PI3K-AKT, JAK-STAT, and HIF-1) as well as cell senescence and adhesion processes (FDR < 0.05). Conclusion: EV-derived miRNAs from patients with AAA prominently associate with cell signaling, senescence, and adhesion pathways in aneurysm pathogenesis. To our knowledge, this is the first study to profile the EV-miRNA landscape in human AAA tissue. Further investigation will explore EV-miRNAs as mediators of communication between distinct vascular cell populations that contribute to AAA development.

Necrotizing enterocolitis (NEC) is a devastating neonatal disease characterized by acute intestinal injury. Intestinal stem cell (ISC) renewal is required for gut regeneration in response to acute injury. The Wnt/β-catenin pathway is essential for intestinal renewal and ISC maintenance. We found that ISC expression, Wnt activity and intestinal regeneration were all decreased in both mice with experimental NEC and in infants with acute active NEC. Moreover, intestinal organoids derived from NEC-injured intestine of both mice and humans failed to maintain proliferation and presented more differentiation. Administration of Wnt7b reversed these changes and promoted growth of intestinal organoids. Additionally, administration of exogenous Wnt7b rescued intestinal injury, restored ISC, and reestablished intestinal epithelial homeostasis in mice with NEC. Our findings demonstrate that during NEC, Wnt/β-catenin signaling is decreased, ISC activity is impaired, and intestinal regeneration is defective. Administration of Wnt resulted in the maintenance of intestinal epithelial homeostasis and avoidance of NEC intestinal injury. ispartof: Cell Death & Disease vol:10 issue:10 ispartof: location:England status: published

Aims: The transcription factor ERG has emerged as an important regulator of vascular function through its ability to repress inflammation in endothelial cells (ECs) and ERG dysregulation is associated with chronic inflammation in atherosclerosis and aortic aneurysms. To characterize the cellular mechanisms that underlie this regulation, we deleted ERG in human aortic ECs and assessed genome-wide chromatin accessibility and gene expression to identify dysregulated pathways. Furthermore, we have begun to investigate ERG expression in mouse atherosclerotic plaque. Methods: CRISPR/Cas9 was used to delete ERG exon 6 in immortalized human aortic ECs. Wildtype or Δ ERG ECs were profiled with ATAC-seq and RNA-seq (n=2-4). GREAT and clusterProfiler were used to identify enriched pathways (log 2 FC>|1|; FDRLdlr −/− mice were fed a 12-week high-cholesterol diet (1.25%) before sacrifice. Ascending aortic sections were stained with anti-CD68 and anti-ERG (n=2). Images were obtained using a Leica SP8 confocal microscope. Results: Pathway analysis of >21,000 differentially accessible chromatin regions identified 67 enriched processes in Δ ERG ECs. Of these, the top 20% included disrupted wound healing, abnormal aorta morphology, and aneurysm, which were driven by association with proatherogenic and proaneurysmal genes including PDGFRB and TGFBR1 . Gene expression analysis revealed that loss of ERG contributes to endothelial to mesenchymal transition (increased KLF4 and SNAI2 ), inflammation (increased IL18 ), and dysregulated extracellular matrix and cell adhesion (increased FBLN2 and VCAN ). In the mouse aorta, preliminary data suggest that ERG expression may be elevated in EC nuclei overlying plaque compared to non-atherosclerotic regions. Conclusions: The loss of ERG has a broad impact on chromatin accessibility and gene expression in aortic ECs, with enrichment of vascular dysfunction pathways. Further investigation will establish how altered ERG expression contributes to in vivo models of atherosclerosis and aortic aneurysms.

Environmental water monitoring is an important responsibility of municipal governments. In this study, we partnered with several municipalities in an extensive sampling program to investigate the effects of spatiotemporal and environmental factors on bacterial diversity in a complex watershed ecosystem containing specific environments including creeks, a river, canals, stormwater outfalls and freshwater lakes of the Niagara Peninsula. Samples were collected using standard municipal protocols and bacterial DNA extracted from these samples was sequenced using high-throughput DNA sequencing targeting the V3–V4 regions of the 16S rRNA gene. Average taxonomic richness and alpha diversity differed significantly between samples collected from lakes and creeks (P

Background Stem cell therapy has been proven to rescue intestinal injury and stimulate intestinal regeneration in necrotizing enterocolitis (NEC). Specifically, stem cells derived from amniotic fluid (AFSCs) and mesenchymal stem cells (MSCs) derived from bone marrow have shown promising results in the treatment of experimental NEC. This study aims to examine the effects of AFSCs and MSCs on the prevention of intestinal injury during experimental NEC. Methods Supernatants from AFSC and MSC cultures were collected to perform proteomic analysis. Prior to NEC induction, mice received intraperitoneal injections of phosphate-buffered saline (PBS), 2 × 106 AFSCs, or 2 × 106 MSCs. Results We found that AFSCs grew faster than MSCs. Proteomic analysis indicated that AFSCs are primarily involved in cell development and growth, while MSCs are involved in immune regulation. Administering AFSCs before NEC induction decreased NEC severity and mucosal inflammation. Intestinal proliferation and endogenous stem cell activation were increased after AFSC administration. However, administering MSCs before NEC induction had no beneficial effects. Conclusions This study demonstrated that AFSCs and MSCs have different protein release profiles. AFSCs can potentially be used as a preventative strategy for neonates at risk of NEC, while MSCs cannot be used. Impact AFSCs and MSCs have distinct protein secretory profiles, and AFSCs are primarily involved in cell development and growth, while MSCs are involved in immune regulation. AFSCs are unique in transiently enhancing healthy intestinal epithelial cell growth, which offers protection against the development of experimental NEC. The prevention of NEC via the administration of AFSCs should be evaluated in infants at great risk of developing NEC or in infants with early signs of NEC.

Necrotizing enterocolitis (NEC) is a devastating intestinal disease primarily affecting preterm neonates and causing high morbidity, high mortality, and huge costs for the family and society. The treatment and the outcome of the disease have not changed in recent decades. Emerging evidence has shown that stimulating the Wnt/β-catenin pathway and enhancing intestinal regeneration are beneficial in experimental NEC, and that they could potentially be used as a novel treatment. Amniotic fluid stem cells (AFSC) and AFSC-derived extracellular vesicles (EV) can be used to improve intestinal injury in experimental NEC. However, the mechanisms by which they affect the Wnt/β-catenin pathway and intestinal regeneration are unknown. In our current study, we demonstrated that AFSC and EV attenuate NEC intestinal injury by activating the Wnt signaling pathway. AFSC and EV stimulate intestinal recovery from NEC by increasing cellular proliferation, reducing inflammation and ultimately regenerating a normal intestinal epithelium. EV administration has a rescuing effect on intestinal injury when given during NEC induction; however, it failed to prevent injury when given prior to NEC induction. AFSC-derived EV administration is thus a potential emergent novel treatment strategy for NEC. ispartof: CELL DEATH & DISEASE vol:11 issue:9 ispartof: location:England status: published

Necrotizing enterocolitis (NEC) is characterized by intestinal injury and impaired mucin synthesis. We recently showed that breast milk exosomes from rodents promote intestinal cell viability, epithelial proliferation, and stem cell activity, but whether they also affect mucus production is unknown. Therefore, the aim of this study was to investigate the effects of bovine milk-derived exosomes on goblet cell expression in experimental NEC and delineate potential underlying mechanisms of action. Exosomes were isolated from bovine milk by ultracentrifugation and confirmed by Nanoparticle Tracking Analysis and through the detection of exosome membrane markers. To study the effect on mucin production, human colonic LS174T cells were cultured and exposed to exosomes. Compared to control, exosomes promoted goblet cell expression, as demonstrated by increased mucin production and relative expression levels of goblet cell expression markers trefoil factor 3 (TFF3) and mucin 2 (MUC2). In addition, exosome treatment enhanced the expression of glucose-regulated protein 94 (GRP94), the most abundant intraluminal endoplasmic reticulum (ER) chaperone protein that aids in protein synthesis. Furthermore, experimental NEC was induced in mouse pups by hyperosmolar formula feeding, lipopolysaccharide administration and hypoxia exposure on postnatal days 5-9. Milk exosomes were given with each gavage feed. NEC was associated with ileal morphological injury and reduction in MUC2+ goblet cells and GRP94+ cells per villus. Exosome administration to NEC pups prevented these changes. This research highlights the potential novel application of milk-derived exosomes in preventing the development of NEC in high-risk infants when breast milk is not available.

Flaxseed is high in ω-3 polyunsaturated fatty acids, fiber, and lignans known to lower cholesterol levels. However, its use for prevention or treatment of inflammatory bowel diseases has yielded mixed results, perhaps related to dietary interactions. In this study, we evaluated the impact of ground flaxseed supplementation on the severity of Citrobacter rodentium-induced colitis in the setting of either a high-fat (HF, ~36%kcal) or reduced-fat (RF, ~12%kcal) diet. After weaning, C57BL/6 mice ( n = 8–15/treatment) were fed ground flaxseed (7 g/100 g diet) with either HF (HF Flx) or RF (RF Flx) diets for 4 wk before infection with C. rodentium or sham gavage. Weight changes, mucosal inflammation, pathogen burden, gut microbiota composition, tissue polyunsaturated fatty acids, and cecal short-chain fatty acids were compared over a 14-day infection period. The RF diet protected against C. rodentium-induced colitis, whereas the RF Flx diet increased pathogen burden, exacerbated gut inflammation, and promoted gut dysbiosis. When compared with the RF diet, both HF and HF Flx diets resulted in more severe pathology in response to C. rodentium infection. Our findings demonstrate that although an RF diet protected against C. rodentium-induced colitis and associated gut dysbiosis in mice, beneficial effects were diminished with ground flaxseed supplementation. NEW & NOTEWORTHY Our results demonstrate a strong protective effect of a reduced-fat diet against intestinal inflammation, dysbiosis, and pathogen burden during Citrobacter rodentium-induced colitis. However, ground flaxseed supplementation in the setting of a reduced-fat diet exacerbated colitis despite higher levels of intestinal n-3 polyunsaturated fatty acids and cecal short-chain fatty acids.

Background Prebiotics are non-digestible food ingredients that enhance the growth of certain microbes within the gut microbiota. Prebiotic consumption generates immune-modulatory effects that are traditionally thought to reflect microbial interactions within the gut. However, recent evidence suggests they may also impart direct microbe-independent effects on the host, though the mechanisms of which are currently unclear. Methods Kinome arrays were used to profile the host intestinal signaling responses to prebiotic exposures in the absence of microbes. Identified pathways were functionally validated in Caco-2Bbe1 intestinal cell line and in vivo model of murine endotoxemia. Results We found that prebiotics directly regulate host mucosal signaling to alter response to bacterial infection. Intestinal epithelial cells (IECs) exposed to prebiotics are hyporesponsive to pathogen-induced mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) activations, and have a kinome profile distinct from non-treated cells pertaining to multiple innate immune signaling pathways. Consistent with this finding, mice orally gavaged with prebiotics showed dampened inflammatory response to lipopolysaccharide (LPS) without alterations in the gut microbiota. Conclusions These findings provide molecular mechanisms of direct host-prebiotic interactions to support prebiotics as potent modulators of host inflammation. Electronic supplementary material The online version of this article (10.1186/s40168-017-0357-4) contains supplementary material, which is available to authorized users.

Scope Necrotizing enterocolitis (NEC) is a leading cause of morbidity and death in preterm infants, occurring more often in formula-fed than breastfed infants. Studies in both rats and humans show that human milk oligosaccharides (HMOs) lower the incidence of NEC, but the mechanism underlying such protection is currently unclear. Methods and results By extracting HMOs from pooled human breastmilk, the impact of HMOs on the intestinal mucin levels in a murine model of NEC are investigated. To confirm the results, the findings are validated by exposing human intestinal epithelial cells and intestinal organoids to HMOs and evaluated for mucin expression. HMO-gavage to pups increases Muc2 levels and decreases intestinal permeability to macromolecular dextran. HMO-treated cells have increased Muc2 expression, decreased bacterial attachment and dextran permeability during challenge by enteric pathogens. To identify the mediators involved in HMO induction of mucins, it is demonstrated that HMOs directly induce the expression of chaperone proteins including protein disulfide isomerase (PDI). Suppression of PDI activity removes the protective effects of HMOs on barrier function in vitro as well as NEC protection in vivo. Conclusions Taken together, the results provide insights to the possible mechanisms by which HMOs protect the neonatal intestine through upregulation of mucins.