Extracellular vesicle-encapsulated miR-30c-5p reduces aging-related liver fibrosis

Abstract

Aging is associated with decreased health span, and despite the recent advances made in understanding the mechanisms of aging, no antiaging drug has been approved for therapy. Therefore, strategies to promote a healthy life in aging are desirable. Previous work has shown that chronic treatment with extracellular vesicles (EVs) from young mice prolongs lifespan in old mice, but the mechanism of action of this effect on liver metabolism is not known. Here we investigated the role of treatment with EVs derived from young sedentary (EV-C) or exercised (EV-EX) mice in the metabolism of old mice and aimed to identify key youthful-associated microRNA (miRNA) cargos that could promote healthy liver function. We found that aged mice treated with either EV-C or EV-EX had higher insulin sensitivity, higher locomotor activity resulting in longer distance traveled in the cage, and a lower respiratory exchange ratio compared to mice treated with EVs from aged mice (EV-A). In the liver, treatment with young-derived EVs reduced aging-induced liver fibrosis. We identified miR-30c in the EVs as a possible youth-associated miRNA as its level was higher in circulating EVs of young mice. Treatment of aged mice with EVs transfected with miR-30c mimic reduced stellate cell activation in the liver and reduced fibrosis compared to EV-negative control by targeting Foxo3. Our results suggest that by delivering juvenile EVs to old mice, we can improve their liver health. Moreover, we identified miR-30c as a candidate for antiaging liver therapy.

Keywords: aging; exercise; extracellular vesicles; fibrosis; miR‐30; microRNAs.

FIGURE 1

Treatment of aged mice with extracellular vesicles (EVs) from young mice improved their metabolism and distance traveled. Eighteen‐month‐old male mice were intravenously injected PBS or EVs from aged (EV‐A), young sedentary (EV‐C) or young exercised (EV‐EX) mice weekly for 4 weeks. (a) Fasting glucose levels. (b) Insulin tolerance test (ITT) was performed after 5 h of fasting in the last week of treatment. (c, d) Area under the curve (AUC) and insulin tolerance rate decay (K_ITT) calculated from the ITT curve in (b). (e) Body weight before and after EV treatment. (f–j) Data obtained from indirect calorimetry in the dark/light cycles and total period performed after the last injection. Food consumption (f); energy balance (g); energy expenditure (h); distance traveled in cage (i) and respiratory exchange ratio (RER) (j). (k,l) Fat depots and gastrocnemius muscle collect after euthanasia and weighed. Three experimental cohorts were performed with two or three animals in each group. Each symbol inside the column represents one animal n = 7–8 (PBS), n = 6–7 (EV‐A), n = 8–9 (EV‐C) and n = 5–6 (EV‐EX). *indicates p < 0.05 after performing Tukey's post hoc test (a–e; i–j), ANCOVA (F‐H).

FIGURE 2

Effect of the treatment of aged mice with extracellular vesicles from young (EV‐C), young exercised (EV‐EX), and aged mice (EV‐A) in key insulin‐sensitive tissues‐ skeletal muscle, liver, and adipose tissue. (a) Western blot image of phospho‐Akt (Ser473) (p‐Akt (Ser 473)) and Gapdh in the skeletal muscle of EV‐A (n = 3), EV‐C (n = 4) and EV‐EX (n = 3) groups. PBS samples were not detected and excluded from this panel. (b) Quantification of p‐Akt was performed after normalization with Gapdh. Relative levels to the EV‐A group are shown. (c) Transcript levels of genes related to fatty acid synthesis and oxidation in the inguinal adipose tissue (iWAT). Each symbol inside the column represents one animal n = 4–7 (PBS), n = 5–7 (EV‐A), n = 6–8 (EV‐C), and n = 5–6 (EV‐EX). (d, e) Transcript levels of Irs1 and Irs2 (d) and genes related to fatty acid metabolism (e) measured by RT‐qPCR in the liver of aged mice treated with EVs. (f) Representative image of the western blot of p‐Akt (Ser473) and Akt from liver protein of young (y), aged treated with PBS (P), aged treated with EV‐C (C); aged treated with EV‐EX (E). (g) Quantification of phospho‐Akt/Akt ratio of all samples in the liver. From (d–g) Each symbol inside the column represents one animal n = 6–7 (PBS), n = 5–6 (EV‐A), n = 5–7 (EV‐C) and n = 4–6 (EV‐EX). *indicates p < 0.05 after performing Tukey's post hoc test.

FIGURE 3

Treatment of aged mice with EV from young mice improves aging‐induced liver fibrosis. Eighteen‐month‐old male mice were intravenously injected PBS or extracellular vesicles from aged (EV‐A), young sedentary (EV‐C) or young exercised (EV‐EX) mice weekly for 4 weeks. (a, b) Liver weight and liver triglyceride (TG) content. (c) Representative photomicrographs of liver stained with Masson's Trichrome from aged mice treated with PBS or EVs. (d) Calculated fibrosis grade from each animal, from 0 to 4. No animals were scored 4. (e) Fibrosis area calculated using ImageJ. (f, g) Serum alanine transaminase (ALT) (f) and aspartate transaminase (AST) (g) enzyme activities. Serum with hemolysis were excluded as interferes with the measurement. Each symbol inside the column represents one animal n = 3–8 (PBS), n = 4–7 (EV‐A), n = 3–8 (EV‐C) and n = 3–6 (EV‐EX). *p < 0.05 as indicated by Tukey's post hoc test.

FIGURE 4

Treatment of aged mice with EV from young mice reduces hepatic stellate cells activation. (a) Livers from aged mice treated with PBS or extracellular vesicles from aged (EV‐A), young sedentary (EV‐C), or young exercised (EV‐EX) were harvested and fixed in 10% formalin for 48 h and subjected to immunofluorescence (IF) staining of alpha‐smooth muscle Actin (α‐Sma) in red and vimentin in green. Hoechst in blue stained the nucleus. (b, d) If quantification of the percentage of the area stained was performed using ImageJ software. (c, e) Transcript levels of Acta2 and Vimentin (Vim) in the liver of treated mice measured by RT‐qPCR. (f) Representative bands for vinculin and Gapdh from protein of young (y), aged treated with PBS (P), aged treated with EV‐C (C); aged treated with EV‐EX (E) by western blotting. (g) Quantification of vinculin. Vinculin content was normalized to Gapdh content and was expressed relative to the PBS group. (h) Transcript levels of genes related to fibrosis measured by RT‐qPCR in the liver of aged mice treated with PBS or EVs. Each symbol inside the column represents one animal n = 5–8 (PBS), n = 5–7 (EV‐A), n = 5–8 (EV‐C), and n = 5–6 (EV‐EX). *p < 0.05 as indicated by Tukey's post hoc test.

FIGURE 5

Treatment of aged mice with EV from young mice promotes fibrosis resolution. Livers from aged mice treated with PBS or extracellular vesicles (EVs) from aged (EV‐A), young sedentary (EV‐C) or young exercised (EV‐EX) were harvested and used for protein or RNA extraction. (a) Representative bands of western blot for inhibitor of NF‐kB (IkB), NF‐kB p65, phospho‐NF‐kB p65 (Ser536), and Gapdh or total ponceau used as references from liver protein of young (Y), aged treated with PBS (P), aged treated with EV‐C (C); aged treated with EV‐EX (E). Gapdh gel bands in A represented under NF‐kB p65 bands were also used in Figure 4f, as the same membrane was used for the detection of both proteins of interest. (b) Quantification of the IkB, NF‐kB p65, and phospho‐NF‐kB p65 blots for all samples (n = 5/group). *p < 0.05 as indicated by Tukey's post hoc test. (c, d) Transcript levels of senescence‐associated secretory phenotype‐related genes in the liver measured by RT‐qPCR; (c) Pro‐inflammatory‐related genes; (d) Cellular senescence markers. Each symbol inside the column represents one animal n = 6 (PBS), n = 7 (EV‐A), n = 7 (EV‐C) and n = 5 (EV‐EX). *p < 0.05 as indicated by Tukey's post hoc test. (e) Profile of mean spot pixel density obtained from cytokine array immunoassay from young and aged mouse livers treated with EV‐A, EV‐C or EV‐EX. Ratio to 2‐m.o.‐mouse or to aged treated with EV‐A are shown in the heatmap (n = 1).

FIGURE 6

MiRNA‐30c is reduced in liver of aged mice with accelerated fibrosis and treatment with extracellular vesicle‐miR‐30c‐5p (EV‐miR‐30c) reduces liver fibrosis. Serum EVs were isolated from young (2 m.o.) sedentary (EV‐C) or acutely exercised (EV‐EX) and aged mice (18 m.o.) and total RNA was extracted for miR‐30c‐5p quantification (a) or used to treat aged mice. Each symbol inside the column represents one animal n = 11 (Aged), n = 8 (Young), n = 6 (Young exercised). (b, c) Mir‐30c‐5p mature and pri‐mir‐30c‐2 expression of aged mice treated with PBS or EVs (EV‐A, EV‐C or EV‐EX) measure by RT‐qPCR. Each symbol inside the column represents one animal n = 6 (PBS), n = 7 (EV‐A), n = 8 (EV‐C) and n = 5 (EV‐EX). (d–n) Data from aged mice treated with EV‐miR‐30c or EV containing a negative control (EV‐Scr). Each symbol represents one mouse (n = 7/group). (d) Liver mass after treatment. (e, f) Serum alanine transaminase (ALT) (e) and aspartate transaminase (AST) (f) enzyme activities. Two serums with hemolysis were excluded as erythrocytes have high ALT and AST activities and interfere with the measurement. (g) Representative photomicrographs of liver stained with Mason's Trichrome (MTS) from aged mice treated with EV‐Scr or EV‐miR‐30c. (h) Fibrosis score calculated from MTS images for each mouse; (i) Expression of mRNA of fibrosis‐related genes measured by RT‐qPCR. Levels were normalized with Hprt1 and are expressed as fold‐change. The PBS group was used as a calibrator. (j) Representative photomicrographs of immunofluorescence for Vimentin (green) and α‐smooth Actin (α‐Sma) in red. Hoescht was used for nuclear staining (blue). (k, l) Quantification of α‐Sma and Vimentin performed using Image J software for each mouse. (m) Representative bands of western blot for Foxo3 and Gapdh. (n) Quantification of the optical density of the bands obtained in (m) using Image Studio. Levels of Foxo3 were normalized with Gapdh. EV‐mir: EV‐miR‐30c‐treated mice and EV‐Scr: EV‐Scrambled‐treated mice. *p < 0.05 as indicated by t test (d–n) or Tukey's post hoc test (a–b).