microRNA Cargo Research Articles

906: Characterization of microRNA cargo of Wharton’s jelly-derived extracellular vesicles and its future role in neuroregeneration

906: Characterization of microRNA cargo of Wharton’s jelly-derived extracellular vesicles and its future role in neuroregeneration

Dietary Depletion of Milk Exosomes and Their MicroRNA Cargos Elicits a Depletion of miR-200a-3p and Elevated Intestinal Inflammation and Chemokine (C-X-C Motif) Ligand 9 Expression in Mdr1a−/− Mice

ABSTRACTBackgroundExosomes transfer regulatory microRNAs (miRs) from donor cells to recipient cells. Exosomes and miRs originate from both endogenous synthesis and dietary sources such as milk. miR-200a-3p is a negative regulator of the proinflammatory chemokine (C-X-C motif) ligand 9 (CXCL9). Male Mdr1a−/− mice spontaneously develop clinical signs of inflammatory bowel disease (IBD).ObjectivesWe assessed whether dietary depletion of exosomes and miRs alters the severity of IBD in Mdr1a−/− mice owing to aberrant regulation of proinflammatory cytokines.MethodsStarting at 5 wk of age, 16 male Mdr1a−/− mice were fed either milk exosome– and RNA-sufficient (ERS) or milk exosome– and RNA-depleted (ERD) diets. The ERD diet is characterized by a near-complete depletion of miRs and a 60% loss of exosome bioavailability compared with ERS. Mice were killed when their weight loss exceeded 15% of peak body weight. Severity of IBD was assessed by histopathological evaluation of cecum. Serum cytokine and chemokine concentrations and mRNA and miR tissue expression were analyzed by multiplex ELISAs, RNA-sequencing analysis, and qRT-PCR, respectively.ResultsStromal collapse, gland hyperplasia, and additive microscopic disease scores were (mean ± SD) 56.7% ± 23.3%, 23.5% ± 11.8%, and 29.6% ± 8.2% lower, respectively, in ceca of ERS mice than of ERD mice (P < 0.05). The serum concentration of CXCL9 was 35.0% ± 31.0% lower in ERS mice than in ERD mice (P < 0.05). Eighty-seven mRNAs were differentially expressed in the ceca from ERS and ERD mice; 16 of these mRNAs are implicated in immune function. The concentrations of 4 and 1 out of 5 miRs assessed (including miR-200a-3p) were ≤63% lower in livers and ceca, respectively, from ERD mice than from ERS mice.ConclusionsMilk exosome and miR depletion exacerbates cecal inflammation in Mdr1a−/− mice.

Exosomal secretion of a psychosis-altered miRNA that regulates glutamate receptor expression is affected by antipsychotics

The ability of small secretory microvesicles known as exosomes to influence neuronal and glial function via their microRNA (miRNA) cargo has positioned them as a novel and effective method of cell-to-cell communication. However, little is known about the role of exosome-secreted miRNAs in the regulation of glutamate receptor gene expression and their relevance for schizophrenia (SCZ) and bipolar disorder (BD). Using mature miRNA profiling and quantitative real-time PCR (qRT-PCR) in the orbitofrontal cortex (OFC) of SCZ (N = 29; 20 male and 9 female), BD (N = 26; 12 male and 14 female), and unaffected control (N = 25; 21 male and 4 female) subjects, we uncovered that miR-223, an exosome-secreted miRNA that targets glutamate receptors, was increased at the mature miRNA level in the OFC of SCZ and BD patients with positive history of psychosis at the time of death and was inversely associated with deficits in the expression of its targets glutamate ionotropic receptor NMDA-type subunit 2B (GRIN2B) and glutamate ionotropic receptor AMPA-type subunit 2 (GRIA2). Furthermore, changes in miR-223 levels in the OFC were positively and negatively correlated with inflammatory and GABAergic gene expression, respectively. Moreover, miR-223 was found to be enriched in astrocytes and secreted via exosomes, and antipsychotics were shown to control its cellular and exosomal localization in a cell-specific manner. Furthermore, addition of astrocytic exosomes in neuronal cultures resulted in a significant increase in miR-223 expression and a notable reduction in Grin2b and Gria2 mRNA levels, which was strongly inversely associated with miR-223 expression. Lastly, inhibition of astrocytic miR-223 abrogated the exosomal-mediated reduction in neuronal Grin2b expression. Taken together, our results demonstrate that the exosomal secretion of a psychosis-altered and glial-enriched miRNA that controls neuronal gene expression is regulated by antipsychotics.

Urinary Extracellular Vesicles Carrying Klotho Improve the Recovery of Renal Function in an Acute Tubular Injury Model.

Acute kidney injury, defined by a rapid deterioration of renal function, is a common complication in hospitalized patients. Among the recent therapeutic options, the use of extracellular vesicles (EVs) is considered a promising strategy. Here we propose a possible therapeutic use of renal-derived EVs isolated from normal urine (urine-derived EVs [uEVs]) in a murine model of acute injury generated by glycerol injection. uEVs accelerated renal recovery, stimulating tubular cell proliferation, reducing the expression of inflammatory and injury markers, and restoring endogenous Klotho loss. When intravenously injected, labeled uEVs localized within injured kidneys and transferred their microRNA cargo. Moreover, uEVs contained the reno-protective Klotho molecule. Murine uEVs derived from Klotho null mice lost the reno-protective effect observed using murine EVs from wild-type mice. This was regained when Klotho-negative murine uEVs were reconstituted with recombinant Klotho. Similarly, ineffective fibroblast EVs acquired reno-protection when engineered with human recombinant Klotho. Our results reveal a novel potential use of uEVs as a new therapeutic strategy for acute kidney injury, highlighting the presence and role of the reno-protective factor Klotho.

ATIM-43. PLASMA EXTRACELLULAR VESICLE MIRNA SIGNATURES IN GBM PATIENTS RECEIVING AN EXPERIMENTAL IMMUNOTHERAPY

Abstract Patients with glioblastoma (GBM) have a median survival of 15 months despite aggressive treatment. Immunotherapies such as dendritic cell (DC) vaccines have modest clinical efficacy in small clinical trials. Treatment-related pseudo-progression confounds outcome assessment by MRI, particularly in patients receiving immunotherapy. Thus, there is a need for additional non-invasive methods to monitor treatment response. Extracellular vesicles (EVs), especially plasma exosomes, contain tumor-specific microRNA (miRNA) cargo that could serve as a liquid biopsy to distinguish true progression from treatment-related pseudo-progression. Plasma exosomes were isolated by serial density gradient ultracentrifugation from 20 newly diagnosed GBM patients enrolled in a clinical trial of allogeneic tumor lysate-pulsed autologous DC vaccination. Short non-coding RNA sequencing and bioinformatics analysis was performed for each patient at three time points (TP): pre-vaccine (TP1), post-initial vaccine (TP2), and at end of treatment (TP3). miRNA expression analysis revealed a total of 14 upregulated and 12 downregulated miRNAs across time points (p-value &lt; 0.05, |logFC| &gt;1), few of which have been previously reported to be differentially expressed in GBM. Interestingly, patients’ miRNA profile expression differed more at the beginning of treatment (e.g. TP1-vs-TP2) and at subsequent time points (e.g. TP2-vs-TP3). Ingenuity Pathway Analysis is in progress to identify pathways associated with immunotherapy treatment response in malignant gliomas. In conclusion, miRNA sequencing from GBM patients’ plasma exosomes enrolled in our DC clinical trial shows marked differential miRNA expression between time points. These results suggest that as patients progress through treatment, consistent differences in plasma exosomal miRNA expression profile can be identified that could be utilized as predictors of treatment response. Thus, plasma EVs may serve as a robust platform to monitor treatment outcome.

Extracellular vesicles, deiminated protein cargo and microRNAs are novel serum biomarkers for environmental rearing temperature in Atlantic cod (Gadus morhua L.)

Extracellular vesicles (EVs) are released from cells and carry protein and genetic cargo involved in cell communication. EVs isolated from bodyfluids, including plasma and serum, can also be used as markers of pathophysiological changes. Peptidylarginine deiminases (PADs) are phylogenetically conserved enzymes with physiological and pathophysiological roles and cause post-translational protein deimination. This can affect function of target proteins and deimination is also linked to EV release. Atlantic cod (Gadus morhua L.) reared at 4 °C and 9 °C respectively for 18 months, were here assessed for changes in serum derived EVs, including analysis of deiminated protein and micro-RNA cargo markers related to stress and growth. We found that cod reared at 9 °C showed significantly reduced numbers of EVs in serum, compared to cod reared at 4 °C. Some deiminated protein targets, including complement component C3, were found to be considerably higher in EVs of cod reared at 4 °C. Proteomic analysis revealed further differences in deiminated protein targets in EVs isolated from sera of the two temperature groups. Whole cod sera from the two temperature groups furthermore showed differences in deiminated protein targets, including C3, CRP and histone H3, which is a marker of neutrophil extracellular trap formation. MicroRNAs related to inflammation (miRNA-21) and stress (miRNA-155) were elevated in both total cod serum and serum-derived EVs from the 9 °C group, while the growth-related miRNA-206 was higher in the 4 °C group. Our findings highlight EVs as novel biomarkers to assess fish health in response to environmental rearing temperature.

Small RNA Species and microRNA Profiles are Altered in Severe Asthma Nanovesicles from Broncho Alveolar Lavage and Associate with Impaired Lung Function and Inflammation.

MicroRNAs are known to regulate important pathways in asthma pathology including the IL-6 and IFN pathways. MicroRNAs have been found not only within cells but also within extracellular vesicles such as exosomes. In this study, we particularly focused on microRNA cargo of nanovesicles in bronchoalveolar lavage of severe asthmatic patients. We extracted nanovesicle RNA using a serial filtration method. RNA content was analyzed with small RNA sequencing and mapped to pathways affected using WebGestalt 2017 Software. We report that severe asthma patients have deficient loading of microRNAs into their airway luminal nanovesicles and an altered profile of small RNA nanovesicle content (i.e., ribosomal RNA and broken transcripts, etc.). This decrease in microRNA cargo is predicted to increase the expression of genes by promoting inflammation and remodeling. Consistently, a network of microRNAs was associated with decreased FEV1 and increased eosinophilic and neutrophilic inflammation in severe asthma. MicroRNAs in airway nanovesicles may, thus, be valid biomarkers to define abnormal biological disease processes in severe asthma and monitor the impact of interventional therapies.

From the Literature

From the Literature

Abstract 130: Development of Bioinspired Synthetic Exosomes With Proangiogenic Potential

Exosomes with variable microRNA cargos are released from different cell types and stimulate angiogenesis in animal models. Therefore, exosomes are currently considered for their potential to represent a safer and easier biological-based alternative to stem cell therapies. However, the limited amount of exosomes that can be reproducibly prepared from cultured stem cells and the complexity of the cargo of endogenous exosomes make them difficult to develop as off-the-shelf “pharmaceutical products” and limit their clinical potential. These issues could be surpassed by the use of artificial (i.e. nanoparticles mimicking the natural product) exosomes carrying a therapeutic cargo. In consideration of the possibility that not all cargo components of exosomes are required for their therapeutic functions, we reasoned that bioinspired artificial exosomes (AEs) incorporating key therapeutically relevant molecules and devoid of potentially negative or indifferent factors could further improve their therapeutic potential. Therefore, we developed and tested AEs containing exosomal proangiogenic miRNAs as a “proof-of-concept” for therapeutic angiogenesis. In order to achieve this, we: (1) characterized the common microRNA cargo of endogenous proangiogenic exosomes (from stem cells, pericytes and pericardial fluid) using bioinformatics, (2) exploited this knowledge to develop off-the-shelf artificial exosomes (AEs) with proangiogenic capacities, (3) validated the angiogenic potential of the bioinspired AEs. Bioinformatics analyses integrating data of miRNA arrays and panels of proangiogenic exosomes confirmed the enrichment of let-7 family in these exosomes. After testing the angiogenic potential of different members of let-7 family, we produced AEs containing let-7b (the most potent in the family) by microfluidic micromixing. The AEs were uptaken by human ECs cultured under hypoxic conditions, without causing toxicity. let-7b-AEs transferred functional let-7b, thus decreased the expression of validated targets of let-7b in recipient cells. let-7b-AEs improved EC survival, proliferation and angiogenesis in vitro and in vivo . These data suggest the therapeutic potential of bioinspired AEs containing let-7b.

Abstract 2590: Nano-exosomes with miRNA: A new therapeutic approach

Abstract Access to reliable and effective therapeutics for patients suffering from aggressive triple-negative breast cancer (TNBC) has been clinically challenging to achieve due to lack of targetable mutations and high heterogeneity in the tumor cells. Therapeutic approaches to reduce mortality in the TNBC patient subset requires an innovative and targeted approach to drug delivery, which is presented here in the form of labeled-exosomes. In this study, we have developed a novel method to bioengineer exosomes, where an antibody is covalently bonded to a fatty acid, which is then anchored into the exosomal membrane to create labeled-exosomes. Our data demonstrate that labeled-exosomes can contain up to 700,000-fold higher miRNA concentrations and can deliver miRNA cargo with increased efficiency into MDA-MB-231 TNBC cells. Results further reveal that insertion of a specific antibody-label to the exosomal membrane significantly increases microRNA uptake into MDA-MB-231 cells, and is further enhanced in cells overexpressing that same antibody marker on their surface. The customizable bioengineering of the labeled-exosomes makes it a highly versatile tool to target cells based on surface marker expression and increase miRNA or other therapeutic cargo delivery. Most importantly, the relevancy of this model reaches far beyond its success in just TNBC cells and can also be translated to several different disease models that require specific targeting with an encapsulated miRNA or therapeutic. We believe this is a novel and exciting utilization of exosomes with a high degree of customization based on surface markers on the target cell, as well as microRNA cargo loaded into the exosome to inhibit a specific oncogene. Citation Format: Sudeepti Kuppa, Balazs Rada, Arthur Roberts, Mandi M. Murph. Nano-exosomes with miRNA: A new therapeutic approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2590.

An ARF6–Exportin-5 axis delivers pre-miRNA cargo to tumour microvesicles

Tumor-derived microvesicles (TMVs) comprise a class of extracellular vesicles released from tumor cells that are now understood to facilitate communication between the tumor and the surrounding microenvironment. Despite their significance, the regulatory mechanisms governing the trafficking of bioactive cargos to TMVs at the cell surface remain poorly defined. Here we describe a molecular pathway for the delivery of microRNA (miRNA) cargo to nascent TMVs involving the dissociation of a pre-miRNA/Exportin-5 complex from Ran-GTP following nuclear export, and its subsequent transfer to a cytoplasmic shuttle comprised of ARF6-GTP and GRP1. As such, ARF6 activation increases pre-miRNA cargo contained within TMVs via a process that requires casein kinase 2-mediated phosphorylation of Ran-GAP1. Further, TMVs were found to contain pre-miRNA processing machinery including Dicer and Argonaute 2, which allow for cell-free pre-miRNA processing within shed vesicles. These findings offer cellular targets to block the loading and processing of pre-miRNAs within TMVs.

Maternal Dicer Knockout Impairs Growth and Gut Health in Nursing Wild-type Pups in Mice (P11-137-19)

ObjectivesBackground: Dicer catalyzes microRNA biogenesis. Administration of tamoxifen to heterozygous Dicer conditional knockout (KO) mice (Dicer + /-) deletes one Dicer allele (Dicer KO). Nanoparticles (exosomes) transfer microRNAs from donor cells to recipient cells. Humans absorb exosomes and microRNA cargos from milk. MicroRNAs regulate the expression of 60% of mammalian genes.Objectives: Assess whether the loss of microRNA biogenesis in lactating Dicer KO dams impairs growth and gut health in nursing wild-type pups. MethodsNewborn pups from wild-type (WT) C57BL/6 breeders were fostered to synchronized Dicer + /- dams. Dams were injected with tamoxifen or vehicle (control). Additional controls included WT pups fostered to WT dams injected with tamoxifen or vehicle. Body weight gain and milk consumption were measured at timed intervals up to 21 days when pups were euthanized. Body fat accretion was assessed using NMR. Sections of the gastrointestinal tract were evaluated by histology analyses. The unpaired t-test was used for statistical analysis (tamoxifen vs. vehicle); P < 0.05 was considered statistically significant. ResultsBody weight and fat accretion in male and female pups nursed by Dicer KO dams was significantly lower than in vehicle controls at age 3 weeks (Figs. 1 and 2). Histology assessment revealed villous atrophy in the small intestine from pups nursed by Dicer KO dams compared to vehicle controls at age 3 weeks (Figs. 3–5). Tamoxifen administration did not elicit phenotypes in wild-type pups nursed by wild-type dams (not shown). Milk consumption was not significantly different between pups nursed by Dicer KO dams and controls (not shown). ConclusionsLoss of microRNA biogenesis in lactating Dicer KO dams impairs growth and gut health in nursing wild-type pups. The quality of milk secreted by Dicer KO dams remains to be assessed. Funding SourcesNIFA, NIH, Gerber Foundation, Gates Foundation, PureTech, Inc. and USDA Hatch and Multistate. J.Z. is a consultant for PureTech. Supporting Tables, Images and/or Graphs▪▪▪▪

Microbial mRNAs in Bovine Milk Exosomes Are Bioavailable in Humans and Mice and Increase Survival of Mice Challenged with Influenza A (OR12-07-19)

ObjectivesExosomes are natural nanoparticles that facilitate cell-to-cell communication by transferring RNAs from donor to recipient cells. Previous studies focused on microRNA cargos and disregarded mRNAs. Exosomes and RNA cargos may be absorbed from milk. RNAs elicit antiviral responses by activating interferon type I (IFN) and NF-kappa B (NF-kB) signaling (Figure 1A).To assess whether 1) bovine milk exosomes (BME) contain microbial mRNAs, 2) microbial mRNAs are bioavailable in humans and mice, and 3) microbial mRNAs activate antiviral responses in reporter cells and mice. MethodsThe mRNA content in BME was assessed by RNA-sequencing. mRNA bioavailability was assessed by RNA-sequencing analysis of human plasma before and 4 h after consumption of 1 L milk and by administering BME transfected with synthetic, fluorophore (IRDye)-labeled Pseudomonas fluorescens capB mRNA to Balb/c mice by oral gavage. (capB mRNA was abundantly expressed in all BME samples sequenced.) Antiviral responses were assessed using liposomes loaded with capB mRNA and cultured with IFN and NF-kB J774 reporter macrophages, and by survival curves of mice fed BME-free or BME-sufficient diets and challenged with influenza A virus. Unpaired t-test was used for statistical analyses; P < 0.05 was considered significant. ResultsAbout 50% of the mRNA-sequencing reads in BME were non-bovine; after contig assembly, up to two thirds of the non-bovine reads mapped to microbial mRNAs (Figure 1B). IRDye-capB mRNA accumulated in murine intestinal mucosa and liver (and perhaps brain) 6 h and 24 h, respectively, after oral gavage (Figure 2). Microbial mRNAs, including 55 and 36 mRNAs from P. fluorescens and E. coli were detected in human plasma after but not before a milk meal (Tables 1, 2). capB mRNA-loaded liposomes activated NF-kB and IFN signaling in reporter macrophages (Figure 3A). C57BL/6 mice fed a BME-depleted diet died within 11 days of influenza A challenge whereas controls fed a BME-sufficient diet survived (Figure 3B). ConclusionsThis is the first report to suggest a novel interaction between microbiome and hosts: microbial mRNAs in BME are bioavailable, activate antiviral IFN and NF-kB pathways and increase resistance to influenza A. Funding SourcesNIFA, NIH, Gates Foundation, PureTech Health, USDA Hatch & Multistate. J.Z. is a consultant for PureTech. Supporting Tables, Images and/or Graphs▪▪▪▪▪

Relationship Between HDL Functional Characteristics and Cardiovascular Health and Potential Impact of Dietary Patterns: A Narrative Review.

Cardiovascular disease is a leading cause of death around the world. Overall diet quality and dietary behaviors are core contributors to metabolic health. While therapeutic targets have traditionally focused on levels of lipoprotein cholesterol when evaluating cardiovascular risk, current perspectives on high-density lipoprotein (HDL) have shifted to evaluating the functionality of this lipoprotein particle. Effects of diet on cardiovascular health are mediated through multiple pathways, but the impact on HDL composition and function deserves greater attention. Potential areas of investigation involve changes in particle characteristics, distribution, microRNA cargo, and other functional changes such as improvements to cholesterol efflux capacity. Various dietary patterns like the Mediterranean diet and Dietary Approaches to Stop Hypertension (DASH) diet have beneficial effects on cardiovascular health and may prevent cardiovascular events. These healthful dietary patterns tend to be rich in plant-based foods, with cardiovascular benefits likely resulting from synergistic effects of the individual dietary components. The purpose of this review is to summarize current perspectives on selected functions of HDL particles and how various dietary patterns affect cardiovascular health biomarkers, with a focus on HDL functionality.

Circulating exosomes derived from transplanted progenitor cells aid the functional recovery of ischemic myocardium.

The stem cell field is hindered by its inability to noninvasively monitor transplanted cells within the target organ in a repeatable, time-sensitive, and condition-specific manner. We hypothesized that quantifying and characterizing transplanted cell-derived exosomes in the recipient plasma would enable reliable, noninvasive surveillance of the conditional activity of the transplanted cells. To test this hypothesis, we used a human-into-rat xenogeneic myocardial infarction model comparing two well-studied progenitor cell types: cardiosphere-derived cells (CDCs) and c-kit+ cardiac progenitor cells (CPCs), both derived from the right atrial appendage of adults undergoing cardiopulmonary bypass. CPCs outperformed the CDCs in cell-based and in vivo regenerative assays. To noninvasively monitor the activity of transplanted CDCs or CPCs in vivo, we purified progenitor cell-specific exosomes from recipient total plasma exosomes. Seven days after transplantation, the concentration of plasma CPC-specific exosomes increased about twofold compared to CDC-specific exosomes. Computational pathway analysis failed to link CPC or CDC cellular messenger RNA (mRNA) with observed myocardial recovery, although recovery was linked to the microRNA (miRNA) cargo of CPC exosomes purified from recipient plasma. We further identified mechanistic pathways governing specific outcomes related to myocardial recovery associated with transplanted CPCs. Collectively, these findings demonstrate the potential of circulating progenitor cell-specific exosomes as a liquid biopsy that provides a noninvasive window into the conditional state of the transplanted cells. These data implicate the surveillance potential of cell-specific exosomes for allogeneic cell therapies.

Muscle-derived miR-34a increases with age in circulating extracellular vesicles and induces senescence of bone marrow stem cells.

Extracellular vesicles (EVs) are known to play important roles in cell-cell communication. Here we investigated the role of muscle-derived EVs and their microRNAs in the loss of bone stem cell populations with age. Aging in male and female C57BL6 mice was associated with a significant increase in expression of the senescence-associated microRNA miR-34a-5p (miR-34a) in skeletal muscle and in serum –derived EVs. Muscle-derived, alpha-sarcoglycan positive, EVs isolated from serum samples also showed a significant increase in miR-34a with age. EVs were isolated from conditioned medium of C2C12 mouse myoblasts and primary human myotubes after cells were treated with hydrogen peroxide to simulate oxidative stress. These EVs were shown to have elevated levels of miR-34a, and these EVs decreased viability of bone marrow mesenchymal (stromal) cells (BMSCs) and increased BMSC senescence. A lentiviral vector system was used to overexpress miR-34a in C2C12 cells, and EVs isolated from these transfected cells were observed to home to bone in vivo and to induce senescence and decrease Sirt1 expression of primary bone marrow cells ex vivo. These findings suggest that aged skeletal muscle is a potential source of circulating, senescence-associated EVs that may directly impact stem cell populations in tissues such as bone via their microRNA cargo.

Extracellular Vesicles From Human Urine-Derived Stem Cells Ameliorate Erectile Dysfunction in a Diabetic Rat Model by Delivering Proangiogenic MicroRNA.

IntroductionStem cell therapies represent a promising new frontier for the treatment of refractory diabetic erectile dysfunction (DED). The use of stem cell-derived extracellular vesicles (EVs) is a novel strategy for cell-free stem cell therapy. We have reported that urine-derived stem cells (USCs) can improve DED; however, the therapeutic effects of EVs secreted by USCs (USC-EVs) remain unknown.AimTo determine the therapeutic effects of USC-EVs on DED in a rat model.MethodsUSC-EVs were isolated from conditioned medium by ultracentrifugation. DED was induced in male Sprague–Dawley rats via an intraperitoneal injection of streptozotocin. Sixteen DED rats were divided into phosphate-buffered saline (PBS) and USC-EV groups. Eight normal rats served as the normal control group. PBS or USC-EVs were transplanted into the corpora cavernosa in the corresponding groups.Main Outcome MeasureIntracavernosal pressure (ICP), mean arterial pressure (MAP), expression of endothelial markers (CD31), endothelial nitric oxide synthase (eNOS), phospho-eNOS, and neural nitric oxide synthase (nNOS) were assessed in each group. Masson’s trichrome staining was used to determine the collagen deposition and ratio of smooth muscle cells to collagen. The microRNA (miRNA) cargo of USC-EVs was characterized by high-throughput RNA sequencing.ResultsRecovery of erectile function was observed in the USC-EV group, as represented by improved ICP and ICP/MAP ratio. CD31, eNOS, phospho-eNOS, and nNOS expression in the penis was significantly improved in the USC-EV group. In addition, the ratio of smooth muscle to collagen was significantly increased in the USC-EV group. RNA sequencing revealed that USC-EVs were enriched for distinct classes of miRNA (miR-21-5p, let-7 family, miR-10 family, miR-30 family, and miR-148a-3p) that promote angiogenesis.ConclusionUSC-EV transplantation can ameliorate DED in rats. Its mechanism may involve the delivery of proangiogenic miRNA.Ouyang B, Xie Y, Zhang C, et al. Extracellular Vesicles From Human Urine-Derived Stem Cells Ameliorate Erectile Dysfunction in a Diabetic Rat Model by Delivering Proangiogenic MicroRNA. Sex Med 2019;7:241–250.

Comparing small urinary extracellular vesicle purification methods with a view to RNA sequencing—Enabling robust and non-invasive biomarker research

Small extracellular vesicles (EVs) are 50–200 nm sized mediators in intercellular communication that reflect both physiological and pathophysiological changes of their parental cells. Thus, EVs hold great potential for biomarker detection. However, reliable purification methods for the downstream screening of the microRNA (miRNA) cargo carried within urinary EVs by small RNA sequencing have yet to be established. To address this knowledge gap, RNA extracted from human urinary EVs obtained by five different urinary EV purification methods (spin column chromatography, immunoaffinity, membrane affinity, precipitation and ultracentrifugation combined with density gradient) was analyzed by small RNA sequencing. Urinary EVs were further characterized by nanoparticle tracking analysis, Western blot analysis and transmission electron microscopy. Comprehensive EV characterization established significant method-dependent differences in size and concentration as well as variances in protein composition of isolated vesicles. Even though all purification methods captured enough total RNA to allow small RNA sequencing, method-dependent differences were also observed with respect to library sizes, mapping distributions, number of miRNA reads and diversity of transcripts. Whereas EVs obtained by immunoaffinity yielded the purest subset of small EVs, highly comparable with results attained by ultracentrifugation combined with density gradient, precipitation and membrane affinity, sample purification by spin column chromatography indicated a tendency to isolate different subtypes of small EVs, which might also carry a distinct subset of miRNAs. Based on our results, different EV purification methods seem to preferentially isolate different subtypes of EVs with varying efficiencies. As a consequence, sequencing experiments and resulting miRNA profiles were also affected. Hence, the selection of a specific EV isolation method has to satisfy the respective research question and should be well considered. In strict adherence with the MISEV (minimal information for studies of extracellular vesicles) guidelines, the importance of a combined evaluation of biophysical and proteomic EV characteristics alongside transcriptomic results was clearly demonstrated in this present study.

Exosomes - beyond stem cells for restorative therapy in stroke and neurological injury.

Stroke is a leading cause of disability worldwide, and brain injuries devastate patients and their families, but currently no drugs on the market promote neurological recovery. Limited spontaneous recovery of function as a result of brain remodelling after stroke or injury does occur, and cell-based therapies have been used to promote these endogenous processes. Increasing evidence is demonstrating that the positive effects of such cell-based therapy are mediated by exosomes released from the administered cells and that the microRNA cargo in these exosomes is largely responsible for the therapeutic effects. This evidence raises the possibility that isolated exosomes could be used alone as a neurorestorative therapy and that these exosomes could be tailored to maximize clinical benefit. The potential of exosomes as a therapy for brain disorders is therefore being actively investigated. In this Review, we discuss the current knowledge of exosomes and advances in our knowledge of their effects on endogenous neurovascular remodelling events. We also consider the opportunities for exosome-based approaches to therapeutic amplification of brain repair and improvement of recovery after stroke, traumatic brain injury and other diseases in which neurorestoration could be a viable treatment strategy.

Glyoxalase 1 Prevents Chronic Hyperglycemia Induced Heart-Explant Derived Cell Dysfunction

Decades of work have shown that diabetes increases the risk of heart disease and worsens clinical outcomes after myocardial infarction. Because diabetes is an absolute contraindication to heart transplant, cell therapy is increasingly being explored as a means of improving heart function for these patients with very few other options. Given that hyperglycemia promotes the generation of toxic metabolites, the influence of the key detoxification enzyme glyoxalase 1 (Glo1) on chronic hyperglycemia induced heart explant-derived cell (EDC) dysfunction was investigated.Methods: EDCs were cultured from wild type C57Bl/6 or Glo1 over-expressing transgenic mice 2 months after treatment with the pancreatic beta cell toxin streptozotocin or vehicle. The effects of Glo1 overexpression was evaluated using in vitro and in vivo models of myocardial ischemia.Results: Chronic hyperglycemia reduced overall culture yields and increased the reactive dicarbonyl cell burden within EDCs. These intrinsic cell changes reduced the angiogenic potential and production of pro-healing exosomes while promoting senescence and slowing proliferation. Compared to intra-myocardial injection of normoglycemic cells, chronic hyperglycemia attenuated cell-mediated improvements in myocardial function and reduced the ability of transplanted cells to promote new blood vessel and cardiomyocyte growth. In contrast, Glo1 overexpression decreased oxidative damage while restoring both cell culture yields and EDC-mediated repair of ischemic myocardium. The latter was associated with enhanced production of pro-healing extracellular vesicles by Glo1 cells without altering the pro-healing microRNA cargo within.Conclusions: Chronic hyperglycemia decreases the regenerative performance of EDCs. Overexpression of Glo1 reduces dicarbonyl stress and prevents chronic hyperglycemia-induced dysfunction by rejuvenating the production of pro-healing extracellular vesicles.