Vitronectin deficiency attenuates hepatic fibrosis in a non-alcoholic steatohepatitis-induced mouse model

Abstract

Vitronectin (VN), an extracellular matrix protein, is a promising immune biomarker of non-alcoholic steatohepatitis (NASH); however, its precise function remains unclear. This study investigated how VN deficiency contributes to the development of NASH. Towards this aim, wild-type (WT) and VN-/- mice were fed with a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) for 6 and 10 weeks to induce NASH, and the livers were isolated. In WT mice fed with CDAHFD for 6 and 10 weeks, the expression of Vn mRNA and protein was up-regulated compared with that in mice fed with the MF control diet, indicating that VN is regulated in NASH condition. VN-/- mice showed decreased picrosirius red staining in the liver area and Col1a2 mRNA expression levels, compared with WT mice, indicating that the severity of hepatic fibrosis is attenuated in the CDAHFD-fed VN-/- mice. In addition, VN deficiency did not affect the area of lipid droplets in haematoxylin-eosin staining and the mRNA expression levels of fatty acid synthases, Srebp, Acc and Fas in the CDAHFD-fed mice. Moreover, VN deficiency decreased the inflammation score and the mRNA expression levels of Cd11b and F4/80, macrophage markers, as well as Tnf-α and Il-1β, inflammatory cytokines in the CDAHFD-fed mice. Furthermore, VN deficiency decreased the protein and mRNA expression levels of α-smooth muscle actin in the CDAHFD-fed mice, suggesting that VN deficiency inhibits the activation of hepatic stellate cells (HSCs). Our findings indicate that VN contributes to the development of fibrosis in the NASH model mice via modulation of the inflammatory reaction and activation of HSCs.

Keywords: hepatic fibrosis; hepatic stellate cell; inflammation; non-alcoholic steatohepatitis; vitronectin.

Figure 1

Expression of VN in the liver of MF (control)‐ and CDAHFD‐fed WT mice. A, Western blot analysis of VN in liver of CDAHFD‐fed mice. Mouse plasma was used as a positive control. B, Relative expression levels of VN protein in the liver of MF‐ and CDAHFD‐6‐ and 10‐wk‐fed mice. The raw values of the expression levels were normalized against GAPDH protein levels, and the mean values were normalized against the VN protein levels in MF‐6‐wk‐fed mice. C,D, Expression levels of Vn and αv integrin mRNA in the liver of MF‐ and CDAHFD‐6‐ and 10‐wk‐fed mice. Transcripts were quantified by real‐time PCR, normalized to Gapdh, and subsequently normalized to the level of MF‐6‐wk‐fed mice. E‐G, Images of immunohistochemistry using anti‐VN in the liver of WT mice fed with MF (E), CDAHFD (F), and VN−/− mice fed with CDAHFD (G) for 10 wks. Higher magnification images are shown in the insets (E’‐G’). Scale bar: 50 μm. The values represent means ± SEM of at least three mice per group at each specified week. *P < 0.05. ^# P < 0.05, ^## P < 0.01 vs MF [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 2

Effects of VN deficiency on hepatic fibrosis in CDAHFD‐fed mice. A, Picrosirius red staining in the liver of WT and VN−/− mice fed with MF (control) and CDAHFD for 6 and 10 wks. Scale bar: 50 μm. B, Picrosirius red‐stained area per field in liver sections of CDAHFD‐fed VN−/− mice. C, mRNA expression levels of Col1a2 were normalized to Gapdh mRNA levels and subsequently normalized to the values of MF‐fed WT mice for 6 wks. D,E, Analysis of MMP‐2 activity using gelatin zymography in the liver of VN−/− mice fed with CDAHFD for 10 wks. The values represent means ± SEM of at least three mice per group at each specified week. *P < 0.05. ^# P < 0.05, ^## P < 0.01 vs MF [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 3

Effects of VN deficiency on lipid accumulation in CDAHFD‐fed mice. A, Liver sections of CDAHFD‐10‐wk‐fed WT and VN−/− mice stained for HE. Scale bar: 200 μm. B, Lipid droplet area per field. C, mRNA levels of Srebp, Acc and Fas in the liver of WT and VN−/− mice fed with MF and CDAHFD for 6 and 10 wks. The values represent means ± SEM of at least three mice per group at each specified week. ^# P < 0.05, ^## P < 0.01, ^### P < 0.001 vs MF. n.s., not significant [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 4

Effects of VN deficiency on hepatic inflammation in CDAHFD‐fed mice. A, HE staining images of the liver in WT and VN−/− mice fed with MF and CDAHFD for 6 and 10 wks. Scale bar: 100 μm. Arrows: inflammatory cell infiltration areas. B, Inflammation score in livers of CDAHFD‐fed VN−/− mice. C‐F, mRNA levels of Cd11b, F4/80, Tnf‐α and Il‐1β in livers of WT and VN−/− mice fed with MF and CDAHFD for 6 and 10 wks. The values represent means ± SEM of at least three mice per group at each specified week. *P < 0.05, **P < 0.01, ***P < 0.001. ^# P < 0.05, ^## P < 0.01, ^### P < 0.001 vs MF [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 5

Effects of VN deficiency on α‐SMA expression in NASH livers. A‐D, Images of enzymatic immunohistochemistry using anti‐α‐SMA antibody in the liver of WT (A,B) and VN−/− (C,D) mice fed with either MF (A,C) or CDAHFD (B,D) for 10 wks. Arrows: α‐SMA positive cells. Scale bar: 50 μm. (E‐H) Images of immunofluorescence staining using anti‐ α‐SMA antibody in the liver of WT (E,F) and VN−/− (G,H) mice fed with either MF (E,G) or CDAHFD (F,H) for 10 wks. V: Vessel. Scale bar: 20 μm. (I,J) Western blot analysis of α‐SMA in the liver of WT and VN−/− mice fed with MF and CDAHFD for 6 and 10 wks. (J) Relative expression levels of α‐SMA protein in the livers. The raw values of the α‐SMA protein levels were normalized against GAPDH protein levels, and the mean values were normalized against α‐SMA protein levels in MF‐fed WT mice for 6 wks. (K,L) Expression levels of α‐Sma and Tgf‐β1 mRNA in the livers of CDAHFD‐fed VN−/− mice. The values represent means ± SEM of at least three mice per group at each specified week. *P < 0.05, **P < 0.01. ^# P < 0.05, ^## P < 0.01, ^### P < 0.001 vs MF. V, vascular lesion [Colour figure can be viewed at wileyonlinelibrary.com]