sEVs from tonsil-derived mesenchymal stromal cells alleviate activation of hepatic stellate cells and liver fibrosis through miR-486-5p

Abstract

Mesenchymal stromal cells (MSCs) are considered as a promising therapeutic tool for liver fibrosis, a main feature of chronic liver disease. Because small extracellular vesicles (sEVs) harboring a variety of proteins and RNAs are known to have similar functions with their derived cells, MSC-derived sEVs carry out the regenerative capacities of MSCs. Human tonsil-derived MSCs (T-MSCs) are reported as a novel source of MSCs, but their effects on liver fibrosis remain unclear. In the present study, we investigated the effects of T-MSC-derived sEVs on liver fibrosis. The expression of profibrotic genes decreased in human primary hepatic stellate cells (pHSCs) co-cultured with T-MSCs. Treatment of T-MSC-sEVs inactivated human and mouse pHSCs. Administration of T-MSC-sEVs ameliorated hepatic injuries and fibrosis in chronically damaged liver induced by carbon tetrachloride (CCl₄). miR-486-5p highly enriched in T-MSC-sEVs targeting the hedgehog receptor, smoothened (Smo), was upregulated, whereas Smo and Gli2, the hedgehog target gene, were downregulated in pHSCs and liver tissues treated with T-MSC-sEVs or miR-486-5p mimic, indicating that sEV-miR-486 inactivates HSCs by suppressing hedgehog signaling. Our results showed that T-MSCs attenuate HSC activation and liver fibrosis by delivering sEVs, and miR-486 in the sEVs inactivates hedgehog signaling, suggesting that T-MSCs and their sEVs are novel anti-fibrotic therapeutics for treating chronic liver disease.

Keywords: hepatic stellate cells; liver fibrosis; microRNA; small extracellular vesicles; tonsil-derived mesenchymal stromal cells.

Graphical abstract

Figure 1

T-MSCs inactivate human pHSCs (A) qRT-PCR analysis for TGF-β, α-SMA, COL1α1, VIMENTIN, CTGF, TIMP1, and GFAP in human pHSCs cultured alone (pHSC alone) or co-cultured with T-MSCs (pHSC with T-MSC) for 1 day (D1) or 2 days (D2). Data are presented as mean ± SEM of experiments performed in triplicate (unpaired two-sample Student’s t test; ∗p < 0.05, ∗∗p < 0.005 versus own control). (B) Western blot analysis for TGF-β (25 kDa), VIMENTIN (57 kDa), α-SMA (42 kDa), GFAP (50 kDa) and GAPDH (36 kDa) in these cells. GAPDH was used as an internal control. Immunoblots shown represent one of three independent experiments with similar results.

Figure 2

Characterization of sEVs isolated from T-MSCs (A) The size distribution of sEVs isolated from T-MSCs was measured by dynamic light scattering. (B) Representative microscopic images were obtained from TEM analysis for detecting isolated sEVs. (C) Western blot analysis of sEV surface markers CD63 (45 kDa), CD9 (25 kDa), and CD81 (17 kDa) and sEVs negative marker CALRETICULIN (64 kDa) in HepG2, LX2, T-MSCs, and sEVs isolated from T-MSCs (T-MSC-sEV). Immunoblots shown represent one of three independent experiments with similar results.

Figure 3

Treatment of T-MSC-derived sEVs downregulates profibrotic genes in human pHSCs (A) qRT-PCR analysis for TGF-β, α-SMA, VIMENTIN, and CTGF in pHSCs treated with vehicles (PBS) or T-MSC-sEV (1, 10, and 100 μg/mL) for 1 day (D1) or 2 days (D2). Data are presented as mean ± SEM of experiments performed in triplicate (unpaired two-sample Student’s t test; ∗p < 0.05 versus own control). (B) Western blot and cumulative densitometry analyses of TGF-β (25 kDa), α-SMA (42 kDa), VIMENTIN (57 kDa), and GAPDH (36 kDa) in these cells. Immunoblots shown represent one of three independent experiments with similar results. Band densities were normalized to the expression level of GAPDH, which was used as an internal control. Data are presented as mean ± SEM from three identical experiments (unpaired two-sample Student’s t test; ∗p < 0.05, ∗∗p < 0.005). (C) Representative images for PKH67-labeled sEVs (green) in human pHSCs, which were treated with vehicles (PBS) or T-MSC-sEVs (100 μg/mL) for 2 and 4 h. Hoechst 33342 was used for staining the nuclei of cells (original magnification, ×40; scale bars, 20 μm).

Figure 4

Administration of T-MSC-derived sEVs ameliorates CCl₄-induced liver injury in mice (A) qRT-PCR analysis for Tgf-β, α-Sma, Vimentin, Ctgf, and Timp1 in primary HSCs that were isolated from the livers of mice given CCl₄ and treated with vehicle (PBS) or T-MSC-derived sEVs (100 μg/mL) for 1 day (D1) or 2 days (D2). Data are presented as mean ± SEM of experiments performed in triplicate (unpaired two-sample Student’s t test; ∗p < 0.05, ∗∗p < 0.005 versus own control). (B) Representative images of hematoxylin and eosin-stained liver sections from representative CON+PBS, CCl₄, CCl₄+PBS, and CCl₄+sEV groups (original magnification, ×10; scale bars, 100 μm). (C) The serum levels of AST and ALT from each group (CON+PBS, n = 3; CCl₄, n = 4; CCl₄+PBS, n = 4; and CCl₄+sEV, n = 5). Data are presented as mean ± SEM (unpaired two-sample Student’s t test; ∗p < 0.05). (D) qRT-PCR of G6pc in the livers from each group (CON+PBS, n = 3; CCl₄, n = 4; CCl₄+PBS, n = 4; and CCl₄+sEV, n = 5). Data are presented as mean ± SEM (unpaired two-sample Student’s t test; ∗p < 0.05).

Figure 5

Hepatic fibrosis is alleviated in mice given CCl₄ with T-MSC-derived sEVs (A) qRT-PCR analysis of Tgf-β, α-Sma, Vimentin, and Ctgf in the livers from CON+PBS, CCl₄, CCl₄+PBS, and CCl₄+sEV groups (n = 3 per group). Data are presented as mean ± SEM (unpaired two-sample Student’s t test; ∗p < 0.05, ∗∗p < 0.005). (B and C) Western blot (B) and cumulative densitometric (C) analyses of Tgf-β (25 kDa), α-Sma (42 kDa), Vimentin (57 kDa), Col1α1 (140 kDa), and Gapdh (36 kDa) in the livers from each group (n = 4 per group). Immunoblots shown represent one of three independent experiments with similar results. Band densities were normalized to the expression level of GAPDH, which was used as an internal control. Data are presented as mean ± SEM (unpaired two-sample Student’s t test; ∗p < 0.05, ∗∗p < 0.005). (D) Representative images of Sirius-red-stained (top) and α-Sma-stained (bottom) liver sections from each group (original magnification, ×10; scale bars, 100 μm). (E) Relative levels of hydroxyproline contents in the livers from each group (CON+PBS, n = 4; CCl₄, n = 4; CCl₄+PBS, n = 5; and CCl₄+sEV, n = 6). Data are presented as mean ± SEM (unpaired two-sample Student’s t test; ∗p < 0.05).

Figure 6

miR-486-5p is highly enriched in T-MSC-derived sEVs and directly targets to SMO (A) qRT-PCR analysis of miR-185-5p, miR-486-5p, miR-125b-5p, and miR-130a-3p in human normal liver tissues, LX2, pHSCs, T-MSCs, and T-MSC-derived sEVs. Data are presented as mean ± SEM (unpaired-two sample Student’s t test; ∗∗p < 0.005). (B) Using a miRNA database (miRWalk), the potential binding site (red fonts) of miR-486-5p was predicted in the 3′ UTR of SMO mRNA in mice and humans. The dashed line represents complementary base pairs between miR-486-5p and SMO mRNA, whereas the gray shading indicates the seed sequence of miR-486-5p. (C) A dual-luciferase assay was performed to verify binding interaction between miR-486-5p and SMO mRNA. HepG2 or LX2 was co-transfected with psiCHECK-2 vector containing either wild-type (WT) or mutant (mut) target sites plus either the miR-486-5p mimic or scrambled (Scr-)miR (control). Results of relative luciferase assay activity are shown as mean ± SEM obtained from triplicate experiments (unpaired two-sample Student’s t test; ∗p < 0.05 versus own control).

Figure 7

miR-486-5p decreases expression of HSC activation markers and Hh-activated genes (A) qRT-PCR analysis of miR-486-5p, SMO, GLI2, α-SMA, VIMENTIN, CTGF, and TGF-β in pHSCs transfected with Scr-miR or miR-486-5p mimic for 1 day (D1) or 2 days (D2). All data are presented as mean ± SEM obtained from triplicate experiments (unpaired two-sample Student’s t test; ∗p < 0.05). (B and C) Western blot (B) and cumulative densitometric (C) analyses of SMO (86 kDa), GLI2 (133 kDa), TGF-β (25 kDa), α-SMA (42 kDa), VIMENTIN (57 kDa), and GAPDH (36 kDa) in these cells. GAPDH was used as an internal control. Immunoblots shown represent one of three independent experiments with similar results. Band densities were normalized to the expression level of GAPDH, which was used as an internal control. Data are presented as mean ± SEM from three identical experiments (unpaired two-sample Student’s t test; ∗p < 0.05, ∗∗p < 0.005 versus own control).

Figure 8

miR-486-5p transferred by T-MSC-derived sEVs downregulates Hh-activated genes in pHSCs and damaged livers (A) qRT-PCR analysis of miR-486-5p, SMO, and GLI2 in human pHSCs treated with vehicles (PBS) or T-MSC-derived sEVs (100 μg/mL) for 1 day (D1) or 2 days (D2). Data are presented as mean ± SEM of experiments performed in triplicate (unpaired two-sample Student’s t test; ∗p < 0.05 versus own control). (B) Western blot analysis of SMO (86 kDa) and GLI2 (133 kDa) in these cells. GAPDH (36 kDa) was used as an internal control. Immunoblots shown represent one of three independent experiments with similar results. (C) qRT-PCR analysis of miR-486-5p, Smo, and Gli2 in livers from CON+PBS, CCl₄, CCl₄+PBS, and CCl₄+sEV groups (n = 3 per group). Data are presented as mean ± SEM (unpaired two-sample Student’s t test; ∗p < 0.05, ∗∗p < 0.005). (D) In situ PCR for the human miR-486-5p gene in the livers of representative mice from these groups (original magnification, ×60, magnified images, ×120; scale bars, 20 μm). In the magnified images, black arrows indicate miR-486-5p-positive HSC-like cells colored blue/violet. (E) Western blot and cumulative densitometric analyses of Smo (86 kDa) and Gli2 (133 kDa) in the livers from each group. Immunoblots shown represent one of three independent experiments with similar results Band densities were normalized to the expression level of LAMIN B1 (66 kDa) or GAPDH (36 kDa), which was used as an internal control. Data are presented as mean ± SEM (unpaired two-sample Student’s t test; ∗p < 0.05, ∗∗p < 0.005). (F) Representative images of Gli2-immunostained liver sections from representative mice from each group (original magnification, ×40; scale bars, 20 μm).