Background: Breast milk has abundant extracellular vesicles (EVs) containing various biological molecules (cargo), including miRNAs. EVs are not degraded in the gastrointestinal system and circulation; thus, breast milk EVs (bEVs) are expected to interact with other organs in breastfed infants and modify the gene expression of recipient cells using miRNAs. Maternal pre-pregnancy BMI is a critical factor influencing the composition of breast milk. Thus, in mothers with obesity, miRNAs in bEVs can be altered, which might be associated with adverse health outcomes in infants. In this study, we examined 798 miRNAs to determine which miRNAs are altered in the bEVs of mothers with obesity and their potential impact on breastfed infants.Methods: We recruited healthy nursing mothers who were either of normal weight (BMI < 25) or with obesity (BMI ≥ 30) based on their pre-pregnancy BMI, and delivered a singleton baby in the prior 6 months. EVs were isolated from breast milk with ultracentrifugation. bEV characteristics were examined by flow cytometry and fluorescence imaging of EV markers. A total of 798 miRNAs were screened using a NanoString human miRNA panel to find differentially expressed miRNAs in bEVs of mothers with obesity compared to mothers of normal weight.Results: We included 65 nursing mothers: 47 of normal weight and 18 with obesity based on pre-pregnancy BMI. After bEV isolation, we confirmed the expression of various EV markers. Out of 37 EV markers, CD326 (EpCaM) was the most highly expressed in bEVs. The most abundant miRNAs in bEVs include miR-30b-5p, miR-4454, miR-494-3p, and let-7 miRNAs. Target genes of the top 10 miRNAs were associated with cancer, prolactin pathway, EGFR, ErbB, and FoxO signaling pathway. In bEVs of mothers with obesity, 19 miRNAs were differentially expressed (adjusted p < 0.05 cut-off), which include miR-575, miR-630, miR-642a-3p, and miR-652-5p. These miRNAs and their target genes were associated with neurological diseases and psychological disorders.Conclusion: In this study, we characterized bEVs and demonstrated altered miRNAs in bEVs of mothers with obesity and identified the pathways of their potential target genes. Our findings will provide insight for future studies investigating the role of bEVs in breastfed infants.

Introduction: Patients who had a mild ischaemic stroke who present with subtle or resolving symptoms sometimes go undiagnosed, are excluded from treatment and in some cases clinically worsen. Circulating immune cells are potential biomarkers that can assist with diagnosis in ischaemic stroke. Understanding the transcriptomic changes of each cell population caused by ischaemic stroke is critical because they work closely in a complicated relationship. In this study, we investigated peripheral blood mononuclear cells (PBMCs) transcriptomics of patients who had a stroke using a single-cell RNA sequencing to understand peripheral immune response after mild stroke based on the gene expression in an unbiased way. Methods: Transcriptomes of PBMCsfrom 10 patients who had an acute ischaemic stroke within 24 hours after stroke onset were compared with 9 race-matched/age-matched/gender-matched controls. Individual PBMCs were prepared with ddSeq^TM (Illumina-BioRad) and sequenced on the Illumina NovaSeq 6000 platform. Results: Notable population changes were observed in patients who had a stroke, especially in NK cells and CD14+ monocytes. The number of NK cells was increased, which was further confirmed by flow cytometry. Functional analysis implied that the activity of NK cells also is enhanced in patients who had a stroke. CD14+ monocytes were clustered into two groups; dendritic cell-related CD14+ monocytes and NK cell-related CD14+ monocytes. We found CD14+ monocyte subclusters were dramatically reduced in patients who had a stroke. Discussion: This is the first study demonstrating the increased number of NK cells and new monocyte subclusters of mild ischaemic stroke based on the transcriptomic analysis. Our findings provide the dynamics of circulating immune response that could assist diagnosis and potential therapeutic development of mild ischaemic stroke.

The Mediterranean diet (MD) has been reported to have beneficial effects on breast cancer and cardiovascular diseases. Recently, microRNAs (miRNAs) have been suggested as biomarkers for the diagnosis and disease prognosis in cancer and cardiovascular diseases. We evaluated the influence of the MD on the plasma-derived extracellular vesicle miRNA signature of overweight breast cancer survivors. Sixteen participants instructed to adhere to the MD for eight weeks were included in this study. To curate differentially expressed miRNAs after MD intervention, we employed two methods: significance analysis of microarrays and DESeq2. The selected miRNAs were analyzed using ingenuity pathway analysis. After an eight-week intervention, body mass index, waist circumference, fasting glucose, fasting insulin, and homeostatic model assessment for insulin resistance were significantly improved. Expression levels of 798 miRNAs were comprehensively analyzed, and 42 extracellular vesicle miRNAs were significantly differentially regulated after the eight-week MD (36 were up and 6 were down-regulated). We also identified enriched pathways in genes regulated by differentially expressed 42 miRNAs, which include signaling associated with breast cancer, energy metabolism, glucose metabolism, and insulin. Our study indicates that extracellular vesicle miRNAs differentially expressed as a result of the MD might be involved in the mechanisms that relate to cardiometabolic risk factors in overweight breast cancer survivors.

Background: Endothelial cell (EC) dysfunction is an early sign of compromised vascular integrity and is associated with various cardiovascular diseases (CVDs). Activation of Toll-like receptor 4 (TLR4) plays a central role in this process. Extracellular vesicles (EVs) derived from milk have known anti-inflammatory properties, particularly in suppressing TLR4 activation. This study investigates the therapeutic potential of human breast milk-derived EVs (HBM-EVs) in mitigating EC dysfunction related to CVDs. Methods: HBM-EVs were isolated from the breast milk of healthy nursing mothers using ultracentrifugation. HBM-EVs were applied to lipopolysaccharide (LPS)-treated human umbilical vein endothelial cells (HUVECs), and inflammatory marker expression was assessed through qPCR and Western blotting. Mitochondrial oxidative stress was measured using MitoSOX. The effects of HBM-EVs were further evaluated in ex vivo studies using mesenteric arteries from diet-induced obese mice. Additionally, the effect of HBM-EVs on angiogenesis was tested via a wound closure assay. Results: In HUVECs, pre-treatment with HBM-EVs inhibited LPS-induced expression of inflammatory markers, including IL-6 and VCAM-1, as well as the phosphorylation of NFκB. Additionally, HBM-EVs reduced LPS-induced mitochondrial oxidative stress. In animal studies, HBM-EV treatment restored EC-dependent vasorelaxation in mesenteric arteries from diet-induced obese mice. Furthermore, HBM-EVs enhanced EC migration, leading to improved wound closure in HUVECs. Conclusion: This study demonstrates the therapeutic potential of HBM-EVs in alleviating EC dysfunction, offering a promising new approach to the treatment of CVDs. Future research will focus on identifying the specific cargo of HBM-EVs and further exploring their therapeutic mechanisms in endothelial dysfunction.

Background: Middle-aged adults have the highest obesity rates, leading to significant health complications in later years. Obesity triggers the release of altered molecules, including extracellular vesicles (EVs) from excess adipose tissue (AT), contributing to various health complications. In this study, we assessed the effects of age and a high-fat diet on AT-derived EV miRNA profiles to understand their potential roles in aging and obesity. Method: C57BL/6 male mice were subjected to a normal chow diet (NCD) or a high-fat diet (HFD) for either 10–12 weeks (young mice, n = 10) or 50–61 weeks (middle-aged mice, n = 12). After evaluating metabolic characteristics, peri-gonadal white AT was isolated and cultured to obtain EVs. AT-derived EV miRNAs were profiled using a NanoString miRNA panel (n = 599). Results: Middle-aged mice exhibited obesity regardless of diet. Young mice fed an HFD showed similar metabolic traits to middle-aged mice. In the NCD group, 131 differentially expressed miRNAs (DE-miRNAs) emerged in middle-aged mice compared to young mice, including miR-21, miR-148a, and miR-29a, associated with cancer, neuro/psychological disorders, and reproductive diseases. In the HFD group, 55 DE-miRNAs were revealed in middle-aged mice compared to young mice. These miRNAs were associated with significantly suppressed IGF1R activity. Conclusion: This study demonstrates the potential significant impact of miRNAs of AT EVs on aging- and obesity-related diseases.