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. 2020 Mar;19(3):e13103.
doi: 10.1111/acel.13103. Epub 2020 Jan 21.

Cellular senescence contributes to age-dependent changes in circulating extracellular vesicle cargo and function

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Cellular senescence contributes to age-dependent changes in circulating extracellular vesicle cargo and function

Faisal J Alibhai et al. Aging Cell. 2020 Mar.

Abstract

Extracellular vesicles (EVs) have emerged as important regulators of inter-cellular and inter-organ communication, in part via the transfer of their cargo to recipient cells. Although circulating EVs have been previously studied as biomarkers of aging, how circulating EVs change with age and the underlying mechanisms that contribute to these changes are poorly understood. Here, we demonstrate that aging has a profound effect on the circulating EV pool, as evidenced by changes in concentration, size, and cargo. Aging also alters particle function; treatment of cells with EV fractions isolated from old plasma reduces macrophage responses to lipopolysaccharide, increases phagocytosis, and reduces endothelial cell responses to vascular endothelial growth factor compared to cells treated with young EV fractions. Depletion studies indicate that CD63+ particles mediate these effects. Treatment of macrophages with EV-like particles revealed that old particles increased the expression of EV miRNAs in recipient cells. Transfection of cells with microRNA mimics recapitulated some of the effects seen with old EV-like particles. Investigation into the underlying mechanisms using bone marrow transplant studies revealed circulating cell age does not substantially affect the expression of aging-associated circulating EV miRNAs in old mice. Instead, we show that cellular senescence contributes to changes in particle cargo and function. Notably, senolytic treatment of old mice shifted plasma particle cargo and function toward that of a younger phenotype. Collectively, these results demonstrate that senescent cells contribute to changes in plasma EVs with age and suggest a new mechanism by which senescent cells can affect cellular functions throughout the body.

Keywords: aging; extracellular vesicles; microRNA; plasma; senescence; senolytic.

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Conflict of interest statement

None declared.

Figures

Figure 1
Figure 1
Aging alters plasma EV‐like particle concentration and size. (a) Size exclusion chromatography isolation of plasma EV enriched fractions from young (top) and old (bottom) plasma. (b) Representative transmission electron microscopy (TEM) images of particles isolated from young and old plasma. (c) Quantification of particle size distribution in 25 nm bins from TEM images, n = 3/group. (d) Representative Western blots images and (e) quantification, n = 4/group, *p < .05. All values are mean ± SEM
Figure 2
Figure 2
Aging alters plasma EV‐like particle function. (a) Gene expression in young macrophages treated with plasma EVs for 24 hr; data are relative to PBS‐treated cells (dashed line), n = 4–5/group. (b) Gene expression in young macrophages treated with EVs for 24 hr then LPS for 4 hr. Expression is relative to cells treated with PBS for 24 hr then LPS for 4 hr (dashed line), n = 5/group, p < .05 versus PBS‐treated cells, *p < .05 versus all other groups. (c) Representative flow cytometry images of macrophage phagocytosis and (d) quantification, n = 6/group, *p < .05 versus all groups. (e) Quantification of tube formation in HUVECs treated with plasma EVs. (f) Quantification of tube formation of cells treated with plasma EVs and stimulated with VEGF. (g) Representative tube formation images. n = 8/group, *p < .05. (h) Young macrophages treated with old EVs (IgG or CD63 depleted) for 24 hr then LPS for 4 hr, n = 5/group, **p < .05 versus all groups. (i) Phagocytic activity of young macrophages treated with old EVs (IgG or CD63 depleted), n = 3/group, **p < .05 versus all groups. All values are mean ± SEM
Figure 3
Figure 3
Aging alters plasma EV‐like particle Cargo. (a) Venn diagram showing differentially expressed miRNAs between young and old EVs, n = 4 samples per group. (b) Real‐Time PCR validation of differentially expressed miRNAs in an independent sample set, n = 6/group, *p < .05. (c) Bioinformatics analysis of the molecular function of miRNAs upregulated in old EVs and (d) predicted KEGG pathways targeted. (e) MiRNA expression in macrophage treated with either young or old plasma EVs. Expression is relative to PBS‐treated cells (dashed line). n = 6/group, *p < .05 old EV versus young EV. (f) Log2 gene expression in macrophages transfected with miRNA mimics then stimulated with LPS for 4 hr, n = 5/group. (g) Quantification of tube formation in stimulated/unstimulated cells in control, miR‐146a, and miR‐21 transfected cells, n = 5‐7/group. (h) Representative tube formation images. All values are mean ± SEM
Figure 4
Figure 4
Effect of bone marrow (BM) and circulating cell age on EV miRNA expression. (a) Quantification young and old reconstitution in aged mice, n = 8/group. (b) Representative flow cytometry images of donor CD45/GFP+ cells in the blood. (c) Principle component analysis of miRNA expression in plasma EVs from young, old, YO and OO mice. (d) Differentially expressed miRNAs in plasma EVs from YO and OO mice identified by miRNA qPCR array, n = 4/group, *p < .05. (e) Bioinformatics analysis of the molecular function of miRNAs differentially expressed between YO and OO mice and (f) predicted KEGG pathways targeted. (g) Expression of age‐associated miRNAs in YO and OO plasma EVs. Groups are relative to young plasma EV miRNA expression (dashed line), n = 6/group, p < .05 versus young plasma EVs. (h) Plasma EV miRNA expression in YY, OY, YO, and OO mice, n = 4–6/group, *p < .05 versus YO and OO, and †† p < .05 versus OO. All values are mean ± SEM
Figure 5
Figure 5
Induction of cellular senescence alters EV secretion and cargo. Expression of p16 and p21 mRNA in the (a) liver and (b) lung of control, 2 and 4 month post‐TBI mice, n = 4–6/group, *p < .05 versus control, **p < .05 versus all groups. (c) Quantification of plasma particle concentration by NTA, n = 4–7/group, *p < .05 versus control. (d) Representative Western blot image and (e) quantification, n = 4/group, *p < .05. (f) CD63 abundance by flow cytometry, n = 5/group, *p < .05. (g) Expression of miRNAs in plasma EVs in control, 2‐ and 4‐month post‐TBI mice, n = 4/group, *p < .05 4 month TBI versus control. (h) Quantification of EV concentration and size secreted by control and senescent cells by NTA, n = 3–5/group. (i) Expression of miRNAs in EVs isolated from control and senescent cells. n = 5/group. (j) Expression of miRNAs in control and senescent cells, n = 4/group, *p < .05. All values are mean ± SEM
Figure 6
Figure 6
Rejuvenation of old EV‐like particle cargo and function by D + Q treatment. (a) Expression of p16 and p21 in the lung and liver of vehicle and D + Q‐treated mice, n = 5/group. (b) Representative size distribution graph from NTA of plasma particles. (c) CD81 Western blot image and (d) quantification, n = 4/group, *p < .05. (e) CD63 abundance by flow cytometry, n = 5/group, *p < .05. (f) Expression of miRNAs in EVs isolated from vehicle and D + Q plasma, n = 6/group, *p < .05. (g) Expression of miRNAs in spleen CD11b+ cells isolated from young or old mice, n = 6/group. (h) MiRNA expression in CD11b+ cells isolated from vehicle or D + Q‐treated old mice, n = 6/group. (i) Macrophages treated with vehicle or D + Q plasma EVs then stimulated with LPS, n = 5/group. (j) Quantification of tube formation of HUVECs treated with vehicle or D + Q plasma EVs followed by stimulation with VEGF, n = 6 for vehicle EV and n = 5 for D + Q EV, *p < .05. (k) Representative tube formation images. All values are mean ± SEM

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