RESUMEN
Encapsulation of microRNAs in exosomes confers protection against degradation and a vehicle for shuttling of microRNAs between cells and tissues, and cellular uptake by endocytosis. Exosomes can be found in foods including milk. Humans absorb cow's milk exosomes and deliver the microRNA cargo to peripheral tissues, consistent with gene regulation by dietary nucleic acids across species boundaries. Here, we tested the hypothesis that human vascular endothelial cells transport milk exosomes by endocytosis, constituting a step crucial for the delivery of dietary exosomes and their cargo to peripheral tissues. We tested this hypothesis by using human umbilical vein endothelial cells and fluorophore-labeled exosomes isolated from cow's milk. Exosome uptake followed Michaelis-Menten kinetics (Vmax = 0.057 ± 0.004 ng exosome protein × 40,000 cells/h; Km = 17.97 ± 3.84 µg exosomal protein/200 µl media) and decreased by 80% when the incubation temperature was lowered from 37°C to 4°C. When exosome surface proteins were removed by treatment with proteinase K, or transport was measured in the presence of the carbohydrate competitor d-galactose or measured in the presence of excess unlabeled exosomes, transport rates decreased by 45% to 80% compared with controls. Treatment with an inhibitor of endocytosis, cytochalasin D, caused a 50% decrease in transport. When fluorophore-labeled exosomes were administered retro-orbitally, exosomes accumulated in liver, spleen, and lungs in mice. We conclude that human vascular endothelial cells transport bovine exosomes by endocytosis and propose that this is an important step in the delivery of dietary exosomes and their cargo to peripheral tissues.
Asunto(s)
Endocitosis/fisiología , Células Endoteliales/metabolismo , Exosomas/metabolismo , Leche/metabolismo , Animales , Transporte Biológico Activo/fisiología , Bovinos , Células Cultivadas , HumanosRESUMEN
Light-induced retinal degeneration (LIRD) models are used to recapitulate the pathologies of retinal diseases that affect photoreceptors. Current LIRD models use a dark-adaptation period of 7-14 days followed by high-intensity light exposure. The purpose of this study was to determine whether photoreceptor damage and death would occur in pigmented zebrafish using a short period of dark-adaptation. Zebrafish were dark-adapted for 24 h and then exposed to constant high-intensity light for 48 h. Immunohistochemical analysis was performed on vertical retinal sections to assess damage and apoptosis. Photoreceptors exhibited structural damage, apoptosis, and cell loss after 24 and 48 h of light exposure as previously reported in studies using 7-14 day dark-adaption. Also, photoreceptors lost following light damage were regenerated after 28 days. These results suggest that a short period of dark-adaptation is sufficient for a LIRD model in pigmented zebrafish.
Asunto(s)
Modelos Animales de Enfermedad , Luz/efectos adversos , Células Fotorreceptoras de Vertebrados/patología , Degeneración Retiniana/patología , Animales , Adaptación a la Oscuridad , Femenino , Masculino , Regeneración Nerviosa/fisiología , Degeneración Retiniana/etiología , Pez CebraRESUMEN
MicroRNAs (miRs, miRNAs) play central roles in gene regulation. Previously, we reported that miRNAs from pasteurized, store-bought bovine milk have biological activity in humans. Here, we assessed the effects of milk processing, storage, somatic cell content, and handling by consumers on the degradation of miRNAs in milk; we also quantified miRNAs in dairy products. Pasteurization and homogenization caused a 63% loss of miR-200c, whereas a 67% loss observed for miR-29b was statistically significant only in skim milk. Effects of cold storage and somatic cell content were quantitatively minor (<2% loss). Heating in the microwave caused a 40% loss of miR-29b but no loss of miR-200c. The milk fat content had no effect on miRNA stability during storage and microwave heating. The concentrations of miRNAs in dairy products were considerably lower than in store-bought milk. We conclude that processing of milk by dairies and handling by consumers causes a significant loss of miRNAs.
Asunto(s)
Manipulación de Alimentos/métodos , MicroARNs/metabolismo , Leche/química , Animales , Bovinos , Productos Lácteos/análisis , Almacenamiento de Alimentos , MicroARNs/análisis , MicroARNs/genética , Leche/metabolismoRESUMEN
Workers exposed to organic dusts from concentrated animal feeding operations (CAFOs) are at risk for developing airway inflammatory diseases. Available preventative and therapeutic measures for alleviating dust-induced lung disease are inadequate. Because omega-3 fatty acids can mitigate inflammatory processes, we aimed to determine whether nutritional supplementation with the omega-3 fatty acid docosahexaenoic acid (DHA) could reduce the airway inflammatory consequences of exposures to organic dust. Aqueous extracts of organic dusts from swine CAFOs (ODE) were utilized. In DHA-pretreated human bronchial epithelial cells, lung fibroblasts, monocyte cell cultures, and precision-cut murine lung slices, we found that DHA pretreatment dose-dependently decreased ODE-induced inflammatory cytokine production. To determine the in vivo significance of DHA, C57BL/6 mice were orally administered DHA for seven days prior to treatment with intranasal ODE or saline inhalations. Animals treated with 2 mg DHA demonstrated significant reductions in ODE-induced bronchial alveolar lavage neutrophil influx and pro-inflammatory cytokine/chemokine production compared to mice exposed to ODE alone. Collectively, these data demonstrate that DHA affects several lung cells to reduce the airway inflammatory response to organic dust exposures. Dietary supplementation with DHA may be an effective therapeutic strategy to reduce the airway inflammatory consequences in individuals exposed to agriculture dust environments.