Characterization of Serum Extracellular Vesicles with Human Age

National Institute On Aging

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Abstract

We characterized EV attributes across selected age groups of participants from the population based, longitudinal study, Healthy Aging in Neighborhoods of Diversity across the Life Span (HANDLS) in LEPS. We analyzed circulating plasma EVs from the HANDLS study using both a cross-sectional and longitudinal approach to address age-related changes in community-dwelling individuals. This sub-cohort consisted of 30 young individuals, 30 middle-aged individuals, and 14 old individuals, who had contributed plasma at two different time points (Visit 1 and Visit 2) approximately 5 years apart. The participants in each age group were matched by race and sex. We isolated EVs from plasma using a precipitation method and analyzed size and concentration using Nanoparticle Tracking Analysis and linear mixed-model regression. We found that EV concentration decreases with advancing age. We also analyzed whether EV concentration was related to demographic and anthropometric measures. BMI and smoking status were significantly associated with EV concentration only at visit 2. There were no significant changes in EV concentration with sex or race. The major finding that EV concentration decreases with advancing age was further explored by examining uptake of EVs by immune cells. EVs from older individuals were more readily internalized by B cells and increased MHC-II expression on monocytes compared with EVs from younger individuals, indicating that the decreased concentration of EVs with age may be due in part to increased internalization. EVs activated both monocytes and B cells, and activation of B cells by LPS enhanced EV internalization. We found a relative stability of EV concentration and protein amount in individual subjects over time. Our data provide information towards establishing a profile of EVs with human age, which will further aid in the development of EV-based diagnostics for aging and age-related diseases. We found that several immune-related antigens like MUCIN16, MUC1, NY-ESO, CD14 and PDL increase with age. The reduction in EV concentration may be a consequence of aging-related phenotypic changes like cellular senescence or part of altered intercellular signaling; both are important hallmarks of aging. EVs may be part of the aging mechanism and may change because of aging-related mechanisms and thus serve as biological aging indicators. While the current study shows that EVs change with age, further research is required to clarify their role in aging. As a follow-on study we examine the relationship between EVs and the presence of diabetes and pre-diabetes. To address this, we have designed a longitudinal sub-cohort of HANDLS that consists of 58 participants who have donated plasma at two time points approximately 5 years apart. Of these 58 participants, 19 were euglycemic at both time points, 19 participants were euglycemic at time point 1 and developed type 2 diabetes mellitus by the second time point and 20 were pre-diabetic at time point 1 and diagnosed with diabetes mellitus at time point 2. Individuals in this sub-cohort have been matched on obesity status. we designed cross-sectional and longitudinal cohorts of euglycemic, pre-diabetic and diabetic participants. In our recent publication in Diabetes (Freeman DW, Noren Hooten N, Eitan E et al 2018) diabetic individuals had significantly higher levels of EVs in their circulation than euglycemic controls. Using a cell-specific EV assay, we identified that erythrocyte-derived EVs are higher with diabetes. We found that insulin resistance increases EV secretion. Furthermore, the levels insulin signaling proteins were altered in EVs from individuals with high levels of insulin resistance and -cell dysfunction. Moreover, EVs from diabetic individuals were preferentially internalized by circulating leukocytes. Cytokine levels in the media and in EVs were higher from monocytes incubated with diabetic EVs. Microarray of these leukocytes revealed altered gene expression pathways related to cell survival, oxidative stress and immune function. Collectively, these results suggest that insulin resistance increases the secretion of EVs, which are preferential internalized by leukocytes and alters leukocyte function. Future work will focus on the influence of race and mortality on EV cargo and the biologic effects of EV cargo on cellular function in the setting of chronic disease and life span. Inflammation-related atherosclerotic peripheral vascular disease is a major end organ complication of diabetes mellitus that results in devastating morbidity and mortality. We examined EV protein cargo from diabetic individuals and whether these EVs cause functional changes in endothelial cells. We quantified inflammatory protein levels in plasma-derived EVs from a longitudinal cohort of euglycemic and diabetic individuals and used in vitro endothelial cell biological assays to assess the functional effects of these EVs with samples from a cross-sectional cohort. We found several significant associations between EV inflammatory protein levels and diabetes status. The angiogenic factor, vascular endothelial growth factor A (VEGF-A), was associated with diabetes status in our longitudinal cohort. Those with diabetes mellitus had higher EV VEGF-A levels compared to euglycemic individuals. Additionally, EV levels of VEGF-A were significantly associated with homeostatic model assessment of insulin resistance (HOMA-IR) and -cell function (HOMA-B). To test whether EVs with different inflammatory cargo can demonstrate different effects on endothelial cells, we performed cell migration and immunofluorescence assays. We observed that EVs from diabetic individuals increased cell lamellipodia formation and migration when compared to EVs from euglycemic individuals. Higher levels of inflammatory proteins were found in EVs from diabetic individuals. Our data implicate EVs as playing important roles in peripheral vascular disease that occur in individuals with diabetes mellitus and suggest that EVs may serve as an informative diagnostic tool for the disease. We also investigated the mitochondrial free radical theory of aging which suggests that accumulating oxidative damage to mitochondria and mitochondrial DNA (mtDNA) plays a central role in aging. Circulating cell-free mtDNA (ccf-mtDNA) isolated from blood may be a biomarker of disease. Extracellular vesicles (EVs) are small (30-400 nm), lipid-bound vesicles capable of shuttling proteins, nucleic acids, and lipids as part of intercellular communication systems. Here, we report that a portion of ccf-mtDNA in plasma is encapsulated in EVs. To address whether EV mtDNA levels change with human age, we analyzed mtDNA in EVs from individuals aged 30-64 years cross-sectionally and longitudinally. EV mtDNA levels decreased with age. Furthermore, the maximal mitochondrial respiration of cultured cells was differentially affected by EVs from old and young donors. Our results suggest that plasma mtDNA is present in EVs, that the level of EV-derived mtDNA is associated with age, and that EVs affect mitochondrial energetics in an EV age-dependent manner.