MicroRNA profiling of royal jelly extracellular vesicles and their potential role in cell viability and reversing cell apoptosis.

MiRNAs are small non-coding RNA molecules that play important regulatory roles in diverse biological processes. Royal jelly, a milky-white substance produced by nurse honeybees (Apis mellifera), is the primary food of queen bees and plays a crucial role in their development. However, little is known about the microRNA (miRNAs) content of royal jelly and their potential functions. In this study, we isolated extracellular vesicles from the royal jelly of 36 samples through sequential centrifugation and targeted nanofiltration and performed high-throughput sequencing to identify and quantify the miRNA content of honeybee royal jelly extracellular vesicles (RJEVs). We found a total of 29 known mature miRNAs and 17 novel miRNAs. Through bioinformatic analysis, we identified several potential target genes of the miRNAs present in royal jelly, including those involved in developmental processes and cell differentiation. To investigate the potential roles of RJEVs in cell viability, RJEVs were supplemented to apoptotic porcine kidney fibroblasts induced by ethanol 6% exposure for 30 min. TUNEL assay showed a significant reduction in the apoptosis percentage after RJEV supplementation when compared with the non-supplemented control group. Moreover, the wound healing assay performed on the apoptotic cells showed a rapid healing capacity of RJEV-supplemented cells compared to the control group. We observed a significant reduction in the expression of the miRNA target genes such as FAM131B, ZEB1, COL5A1, TRIB2, YBX3, MAP2, CTNNA1, and ADAMTS9 suggesting that RJEVs may regulate the target gene expression associated with cellular motility and cell viability. Moreover, RJEVs reduced the expression of apoptotic genes (CASP3, TP53, BAX, and BAK), while significantly increasing the expression of anti-apoptotic genes (BCL2 and BCL-XL). Our findings provide the first comprehensive analysis of the miRNA content of RJEVs and suggest a potential role for these vesicles in the regulation of gene expression and cell survival as well as augmenting cell resurrection or anastasis.

Extracellular Vesicle-Derived circITGB1 Regulates Dendritic Cell Maturation and Cardiac Inflammation via miR-342-3p/NFAM1.

Acute myocardial infarction (AMI) is a complication of atherosclerosis-related cardiovascular illness that is caused by prolonged ischemia. Circular RNAs (circRNAs) are concentrated in extracellular vesicles (EVs) and have been linked to cardiovascular disease. However, additional research is needed into the expression and function of circRNAs in AMI. In this study, circITGB1 (has_circRNA_0018146), derived from exon 1 of the ITGB1 gene localized on chromosome 10, was shown to be considerably increased in plasma from patients with AMI compared to healthy controls, as demonstrated by the comparison of EV-circRNA expression patterns. Using a luciferase screening assay and a biotin-labeled circITGB1 probe to identify microRNA(s) complementary to circITGB1 sequences, we discovered that circITGB1 competitively binds to miR-342-3p and inhibits its expression, which in turn increase the expression of NFAT activating molecule 1 (NFAM1). Based on western blotting and immunological studies, circITGB1 controls dendritic cell maturation by targeting miR-342-3p and NFAM1. circITGB1 also exacerbated cardiac damage and regulated miR-342-3p and NFAM1 expression in a mouse AMI model. This implies that EV-circITGB1 is involved in dendritic cell maturation and cardiac damage via miR-342-3p/NFAM1, and that is linked to AMI-associated pathogenic processes.

Mindfulness intervention improves cognitive function in older adults by enhancing the level of miRNA-29c in neuron-derived extracellular vesicles.

Although mindfulness-based stress reduction (MBSR) improves cognitive function, the mechanism is not clear. In this study, people aged 65 years and older were recruited from elderly communities in Chitose City, Japan, and assigned to a non-MBSR group or a MBSR group. Before and after the intervention, the Japanese version of the Montreal Cognitive Assessment (MoCA-J) was administered, and blood samples were collected. Then, neuron-derived extracellular vesicles (NDEVs) were isolated from blood samples, and microRNAs, as well as the target mRNAs, were evaluated in NDEVs. A linear mixed model analysis showed significant effects of the MBSR x time interaction on the MoCA-J scores, the expression of miRNA(miR)-29c, DNA methyltransferase 3 alpha (DNMT3A), and DNMT3B in NDEVs. These results indicate that MBSR can improve cognitive function by increasing the expression of miR-29c and decreasing the expression of DNMT3A, as well as DNMT3B, in neurons. It was also found that intracerebroventricular injection of miR-29c mimic into 5xFAD mice prevented cognitive decline, as well as neuronal loss in the subiculum area, by down-regulating Dnmt3a  and Dnmt3b  in the hippocampus. The present study suggests that MBSR can prevent neuronal loss and cognitive impairment by increasing the neuronal expression of miR-29c.

The role of salivary vesicles as a potential inflammatory biomarker to detect traumatic brain injury in mixed martial artists.

Traumatic brain injury (TBI) is of significant concern in the realm of high impact contact sports, including mixed martial arts (MMA). Extracellular vesicles (EVs) travel between the brain and oral cavity and may be isolated from salivary samples as a noninvasive biomarker of TBI. Salivary EVs may highlight acute neurocognitive or neuropathological changes, which may be particularly useful as a biomarker in high impact sports. Pre and post-fight samples of saliva were isolated from 8 MMA fighters and 7 from controls. Real-time PCR of salivary EVs was done using the TaqMan Human Inflammatory array. Gene expression profiles were compared pre-fight to post-fight as well as pre-fight to controls. Largest signals were noted for fighters sustaining a loss by technical knockout (higher impact mechanism of injury) or a full match culminating in referee decision (longer length of fight), while smaller signals were noted for fighters winning by joint or choke submission (lower impact mechanism as well as less time). A correlation was observed between absolute gene information signals and fight related markers of head injury severity. Gene expression was also significantly different in MMA fighters pre-fight compared to controls. Our findings suggest that salivary EVs as a potential biomarker in the acute period following head injury to identify injury severity and can help elucidate pathophysiological processes involved in TBI.

Pharmacokinetics of Ginsenoside Compound K From a Compound K Fermentation Product, CK-30, and From Red Ginseng Extract in Healthy Korean Subjects.

Natural protopanaxadiol ginsenosides exhibit low absorption in the human intestine. However, ginsenoside compound K (CK) with 1 conjugated glucose molecule exhibits favorable absorption. The purpose of this study was to compare the pharmacokinetics of ginsenoside CK from a CK fermentation product, CK-30, and from a red ginseng extract. A randomized, open-label, 2-treatment, 2×2 crossover study was conducted. The volunteers were randomly divided into 2 groups. One group received CK-30, and the other group received 2.94 g of a red ginseng extract. After a 7-day washout period, the subjects received an alternative treatment for a single dose. The pharmacokinetic parameters, including the maximum plasma concentration (Cmax ) and area under the plasma concentration-time curve from time 0 to time of last measurable concentration, were calculated. The median time to reach Cmax of ginsenoside CK after administration of CK-30 was 3.0 hours, whereas the corresponding value of the red ginseng extract was 10.0 hours. Compared with the red ginseng extract, CK-30 resulted in a higher systemic exposure to ginsenoside CK, with a 118.3-fold increase in Cmax and a 135.1-fold increase in area under the plasma concentration-time curve from time 0 to time of last measurable concentration. The systemic exposure to ginsenoside CK was significantly higher after administration of CK-30 than red ginseng extract.

Morphine-mediated release of miR-138 in astrocyte-derived extracellular vesicles promotes microglial activation.

Opioids, such as morphine, are the mainstay for the management of postsurgical pain. Over the last decade there has been a dramatic increase in deaths related to opioid overdose. While opioid abuse has been shown to result in increased neuroinflammation, mechanism(s) underlying this process, remain less understood. In recent years, microRNAs have emerged as key mediators of gene expression regulating both paracrine signaling and cellular crosstalk. MiRNAs constitute the extracellular vesicle (EV) cargo and can shuttle from the donor to the recipient cells. Exposure of human primary astrocytes to morphine resulted in induction and release of miR-138 in the EVs isolated from conditioned media of cultured astrocytes. Released EVs were, in turn, taken up by the microglia, leading to activation of these latter cells. Interestingly, activation of microglia involved binding of the GUUGUGU motif of miR138 to the endosomal toll like receptor (TLR)7, leading, in turn, to cellular activation. These findings were further corroborated in vivo in wildtype mice wherein morphine administration resulted in increased microglial activation in the thalamus. In TLR7-/- mice on the other hand, morphine failed to induce microglial activation. These findings have ramifications for the development of EV-loaded anti-miRNAs as therapeutics for alleviating neuroinflammation in opioids abusers.

More than Nutrition: Therapeutic Potential of Breast Milk-Derived Exosomes in Cancer.

Human breast milk (HBM) is an irreplaceable source of nutrition for early infant growth and development. Breast-fed children are known to have a low prevalence and reduced risk of various diseases, such as necrotizing enterocolitis, gastroenteritis, acute lymphocytic leukemia, and acute myeloid leukemia. In recent years, HBM has been found to contain a microbiome, extracellular vesicles or exosomes, and microRNAs, as well as nutritional components and non-nutritional proteins, including immunoregulatory proteins, hormones, and growth factors. Especially, the milk-derived exosomes exert various physiological and therapeutic function in cell proliferation, inflammation, immunomodulation, and cancer, which are mainly attributed to their cargo molecules such as proteins and microRNAs. The exosomal miRNAs are protected from enzymatic digestion and acidic conditions, and play a critical role in immune regulation and cancer. In addition, the milk-derived exosomes are developed as drug carriers for delivering small molecules and siRNA to tumor sites. In this review, we examined the various components of HBM and their therapeutic potential, in particular of exosomes and microRNAs, towards cancer.

microRNA-155 Is Decreased During Atherosclerosis Regression and Is Increased in Urinary Extracellular Vesicles During Atherosclerosis Progression.

Background: Atherosclerosis is a chronic inflammatory disease driven by macrophage accumulation in medium and large sized arteries. Macrophage polarization and inflammation are governed by microRNAs (miR) that regulate the expression of inflammatory proteins and cholesterol trafficking. Previous transcriptomic analysis led us to hypothesize that miR-155-5p (miR-155) is regulated by conjugated linoleic acid (CLA), a pro-resolving mediator which induces regression of atherosclerosis in vivo. In parallel, as extracellular vesicles (EVs) and their miR content have potential as biomarkers, we investigated alterations in urinary-derived EVs (uEVs) during the progression of human coronary artery disease (CAD). Methods: miR-155 expression was quantified in aortae from ApoE-/- mice fed a 1% cholesterol diet supplemented with CLA blend (80:20, cis-9,trans-11:trans-10,cis-12 respectively) which had been previously been shown to induce atherosclerosis regression. In parallel, human polarized THP-1 macrophages were used to investigate the effects of CLA blend on miR-155 expression. A miR-155 mimic was used to investigate its inflammatory effects on macrophages and on ex vivo human carotid endarterectomy (CEA) plaque specimens (n = 5). Surface marker expression and miR content were analyzed in urinary extracellular vesicles (uEVs) obtained from patients diagnosed with unstable (n = 12) and stable (n = 12) CAD. Results: Here, we report that the 1% cholesterol diet increased miR-155 expression while CLA blend supplementation decreased miR-155 expression in the aorta during atherosclerosis regression in vivo. CLA blend also decreased miR-155 expression in vitro in human THP-1 polarized macrophages. Furthermore, in THP-1 macrophages, miR-155 mimic decreased the anti-inflammatory signaling proteins, BCL-6 and phosphorylated-STAT-3. In addition, miR-155 mimic downregulated BCL-6 in CEA plaque specimens. uEVs from patients with unstable CAD had increased expression of miR-155 in comparison to patients with stable CAD. While the overall concentration of uEVs was decreased in patients with unstable CAD, levels of CD45+ uEVs were increased. Additionally, patients with unstable CAD had increased CD11b+ uEVs and decreased CD16+ uEVs. Conclusion: miR-155 suppresses anti-inflammatory signaling in macrophages, is decreased during regression of atherosclerosis in vivo and is increased in uEVs from patients with unstable CAD suggesting miR-155 has potential as a prognostic indicator and a therapeutic target.

Macrophages exposed to HIV viral protein disrupt lung epithelial cell integrity and mitochondrial bioenergetics via exosomal microRNA shuttling.

Antiretroviral therapy extends survival but does not eliminate HIV from its cellular reservoirs. Between immune and stromal cells in the tissue microenvironment, a dynamic intercellular communication might influence host viral immune responses via intercellular transfer of extracellular vehicles (EVs) (microvesicles, exosome, or apoptotic bodies). It is increasingly recognized that HIV-infected macrophage-secreted nucleotide-rich exosomes might play a critical role in mediating communication between macrophages and other structural cells; however, molecular mechanisms underlying cell-cell crosstalk remain unknown. Here we show that HIV-1-infected macrophages and HIV-1 proteins Tat or gp120-treated macrophages express high levels of microRNAs, including miR-23a and miR-27a. Identical miRNAs expression patterns were detected in macrophage-secreted exosomes isolated from bronchoalveolar lavage fluid of HIV transgenic rats. Tat-treated macrophage-derived exosomal miR-23a attenuated posttranscriptional modulation of key tight junction protein zonula occludens (ZO-1) 3'-UTR in epithelial cells. In parallel, exosomal miR-27a released from Tat-treated macrophages altered the mitochondrial bioenergetics of recipient lung epithelial cells by targeting peroxisome proliferator-activated receptor gamma (PPARγ), while simultaneously stimulating glycolysis. Together, exosomal miRNAs shuttle from macrophages to epithelial cells and thereby explain in part HIV-mediated lung epithelial barrier dysfunction. These studies suggest that targeting miRNAs may be of therapeutic value to enhance lung health in HIV.

ATP redirects cytokine trafficking and promotes novel membrane TNF signaling via microvesicles.

Cellular stress or injury induces release of endogenous danger signals such as ATP, which plays a central role in activating immune cells. ATP is essential for the release of nonclassically secreted cytokines such as IL-1ß but, paradoxically, has been reported to inhibit the release of classically secreted cytokines such as TNF. Here, we reveal that ATP does switch off soluble TNF (17 kDa) release from LPS-treated macrophages, but rather than inhibiting the entire TNF secretion, ATP packages membrane TNF (26 kDa) within microvesicles (MVs). Secretion of membrane TNF within MVs bypasses the conventional endoplasmic reticulum- and Golgi transport-dependent pathway and is mediated by acid sphingomyelinase. These membrane TNF-carrying MVs are biologically more potent than soluble TNF in vivo, producing significant lung inflammation in mice. Thus, ATP critically alters TNF trafficking and secretion from macrophages, inducing novel unconventional membrane TNF signaling via MVs without direct cell-to-cell contact. These data have crucial implications for this key cytokine, particularly when therapeutically targeting TNF in acute inflammatory diseases.-Soni, S., O'Dea, K. P., Tan, Y. Y., Cho, K., Abe, E., Romano, R., Cui, J., Ma, D., Sarathchandra, P., Wilson, M. R., Takata, M. ATP redirects cytokine trafficking and promotes novel membrane TNF signaling via microvesicles.

Proteomic and Bioinformatic Characterization of Extracellular Vesicles Released from Human Macrophages upon Influenza A Virus Infection.

Influenza A viruses (IAVs) are aggressive pathogens that cause acute respiratory diseases and annual epidemics in humans. Host defense against IAV infection is initiated by macrophages, which are the principal effector cells of the innate immune system. We have previously shown that IAV infection of human macrophages is associated with robust secretion of proteins via conventional and unconventional protein release pathways. Here we have characterized unconventional, extracellular vesicle (EV)-mediated protein secretion in human macrophages during IAV infection using proteomics, bioinformatics, and functional studies. We demonstrate that at 9 h postinfection a robust EV-mediated protein secretion takes place. We identified 2359 human proteins from EVs of IAV-infected macrophages compared with 1448 proteins identified from EVs of control cells. Bioinformatic analysis shows that many proteins involved in translation, like components of spliceosome machinery and the ribosome, are secreted in EVs in response to IAV infection. Our data also shows that EVs derived from IAV-infected macrophages contain fatty acid-binding proteins, antiviral cytokines, copper metabolism Murr-1 domain proteins, and autophagy-related proteins. In addition, our data suggest that secretory autophagy plays a role in activating EV-mediated protein secretion during IAV infection.