Characterization of exosomes from body fluids of dairy cows.

Exosomes are a specific subpopulation of extracellular vesicles that are widely released by cells of different origins with divergent functions that make their way into body fluids that can be conveniently sampled. In the current study, we isolated and evaluated exosomes from concurrently collected samples of milk, plasma, saliva, and urine from a group of 6 pregnant Holstein-Friesian dairy cows (aged 7 mo, 174 to 203 d of gestation). The cows had BCS of 3.5 to 5.25 (on a scale of 1 to 10), and the milk production for the season to the time of sampling ranged between 5,118 and 6,959 kg. The low levels of extracellular vesicles in saliva and urine (more than 86% fewer compared to the extracellular vesicles in milk and plasma) precluded further detailed evaluation since utility for diagnostics was deemed unlikely. In exosomes isolated from milk and plasma, size distribution, morphology, and the presence of exosome markers was confirmed by nanoparticle tracking analysis, electron microscopy, and Western blot. In addition, a targeted proteomic approach using the quadrupole ion trap mass spectrometer was also used in the study to screen for the exosome marker (e.g., Tumor susceptibility gene 101). Following confirmation of the presence of exosomes, the proteomic profiles of milk and plasma exosomes were evaluated using information-dependent acquisition-mediated liquid chromatography-tandem mass spectrometry (LC-MS/MS). The milk exosomes contain proteins that differed greatly from the plasma exosomes, with only 8 similar proteins harbored in both the milk and plasma exosomes. The milk and plasma exosomes were found to contain proteins (e.g., immunoglobulin J chain and α2 macroglobulin) associated with specific biological processes and molecular functions. Hence, the fluid of origin required for exosome analysis will be dependent on the specific information needed. In conclusion, isolated exosomes from milk and plasma samples collected at the same time point from the same dairy cows encapsulated different profiles of proteins associated with different biological processes and molecular functions.

Effect of circulating exosomes from transition cows on Madin-Darby bovine kidney cell function.

The greatest risk of metabolic and infectious disease in dairy cows is during the transition from pregnancy to lactating (i.e., the transition period). The objective of this experiment was to determine the effects of extracellular vesicles (microvesicles involved in cell-to-cell signaling) isolated from transition cows on target cell function. We previously identified differences in the protein profiles of exosomes isolated from cows divergent in metabolic health status. Therefore, we hypothesized that these exosomes would affect target tissues differently. To investigate this, 2 groups of cows (n = 5/group) were selected based on the concentration of ß-hydroxybutyrate and fatty acids in plasma and triacylglycerol concentration in liver at wk 1 and 2 postcalving. Cows with high concentrations of ß-hydroxybutyrate, fatty acids, and triacylglycerol were considered at increased risk of clinical disease during the transition period (high-risk group; n = 5) and were compared with cows that had low concentrations of the selected health indicators (low-risk group; n = 5). At 2 time points during the transition period (postcalving at wk 1 and 4), blood was sampled and plasma exosomes were isolated from the high-risk and low-risk cows. The exosomes were applied at concentrations of 10 and 1 µg/mL to 5 × 103 Madin-Darby bovine kidney cells grown to 50% confluence in 96-well plates. Results indicate a numerical increase in cell proliferation when exosomes from high-risk cows were applied compared with those from low-risk cows. Consistent with an effect on cell proliferation, quantitative reverse transcriptase PCR indicated a trend for upregulation of 3 proinflammatory genes (granulocyte colony-stimulating factor, ciliary neurotrophic factor, and CD27 ligand) with the application of high-risk exosomes, which are involved in cellular growth and survival. Proteomic analysis indicated 2 proteins in the low-risk group that were not identified in the high-risk group (endoplasmin and catalase), which may also be indicative of the metabolic state of origin. It is likely that the metabolic state of the transition cow affects cellular function through exosomal messaging; however, more in-depth research into cross-talk between exosomes and target cells is required to determine whether exosomes influence Madin-Darby bovine kidney cells in this manner.

Review: Eicosanoids in preterm labor and delivery: Potential roles of exosomes in eicosanoid functions.

Preterm delivery is a major obstetric health problem contributing to poor neonatal outcome including low birth weight, respiratory distress syndrome, gastrointestinal, immunologic, central nervous system, hearing, and vision problems. Worldwide, approximately 15 million babies are born prematurely each year. The critical question which remains is how to identify women destined to deliver preterm from those who will achieve a term delivery. Prostaglandins, in all mammals, are important in the parturient process. Increased intrauterine prostaglandin production is associated with labor and in fact prostaglandin E2 (PGE2) or analogs are widely used clinically for cervical ripening and labor induction. Measurements of circulating eicosanoids have been problematic because of the rapid and major clearance by the lungs and then kidneys resulting in very low concentrations in plasma. Moreover, since eicosanoids are produced by all mammalian tissues, the sources of the measured eicosanoids are unknown. Our understanding of how cells communicate has undergone a paradigm shift with the recognition of the role of exosomes in intercellular signaling. Recent publications have identified enzymes and products of arachidonic acid metabolism (eicosanoids) within exosomes. This review will explore the potential roles of exosomes in eicosanoid functions that are critical in preterm labor and delivery.