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. 2016 Jun 6:6:27398.
doi: 10.1038/srep27398.

The alterations in the extracellular matrix composition guide the repair of damaged liver tissue

Mariliis Klaas  1 , Triin Kangur  2 , Janeli Viil  1 , Kristina Mäemets-Allas  1 , Ave Minajeva  3   4 , Krista Vadi  1 , Mikk Antsov  2 , Natalia Lapidus  4 , Martin Järvekülg  2 , Viljar Jaks  1   5
Affiliations

The alterations in the extracellular matrix composition guide the repair of damaged liver tissue

Mariliis Klaas et al. Sci Rep. .

Abstract

While the cellular mechanisms of liver regeneration have been thoroughly studied, the role of extracellular matrix (ECM) in liver regeneration is still poorly understood. We utilized a proteomics-based approach to identify the shifts in ECM composition after CCl4 or DDC treatment and studied their effect on the proliferation of liver cells by combining biophysical and cell culture methods. We identified notable alterations in the ECM structural components (eg collagens I, IV, V, fibronectin, elastin) as well as in non-structural proteins (eg olfactomedin-4, thrombospondin-4, armadillo repeat-containing x-linked protein 2 (Armcx2)). Comparable alterations in ECM composition were seen in damaged human livers. The increase in collagen content and decrease in elastic fibers resulted in rearrangement and increased stiffness of damaged liver ECM. Interestingly, the alterations in ECM components were nonhomogenous and differed between periportal and pericentral areas and thus our experiments demonstrated the differential ability of selected ECM components to regulate the proliferation of hepatocytes and biliary cells. We define for the first time the alterations in the ECM composition of livers recovering from damage and present functional evidence for a coordinated ECM remodelling that ensures an efficient restoration of liver tissue.

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Figures

Figure 1
Figure 1. Proteomics analysis of liver ECM.
(A) Study scheme. Groups of mice (n = 3) were subjected to intraperitoneal injection of CCl4 (48 h) or DDC diet (2 wk). At specified time points the livers were decellularized followed by proteomics analysis by nano-LC-MS/MS. (B) Summary of the results of proteomics studies. Number of proteins changed in CCl4- and DDC-induced liver damage and number of proteins changed simultaneously in both damages are shown. (C) ECM proteins up- or downregulated in damaged liver samples.
Figure 2
Figure 2. Immunofluorescence microscopy analysis of structural ECM components altered in damaged mouse (A, n = 4) and human (B, n = 5) livers.
Fn-fibronectin. Scale bars 50 μm (A), 200 μm (B).
Figure 3
Figure 3. Alterations in the microstructure of damaged livers.
SEM images of control (A,B), CCl4- (C,D) and DDC-treated (E,F) decellularized livers. E-elastic fibers, C-collagen fibers and R-reticular fibers. Scale bars: 10 μm (top panels), 500 nm (bottom panels), n = 3. (G) The average force-displacement curves of decellularized liver matrices. The median elastic stiffness for control, CCl4- and DDC-treated livers: 0.18 ± 0.02 MPa, 0.9 ± 0.3 MPa and0.35 ± 0.03 MPa, respectively.
Figure 4
Figure 4. Non-hepatocyte cell growth assay on dishes coated with fibronectin (Fn), col1 or col4.
(A) Colonies stained with anti-ck19 and -Ki67 antibodies. Scale bars: 200 μm. (B) Statistical analysis of the average number of cells in a colony on differently coated plates. (C) Percentage of proliferating ck19 + cells. Bars show the average of 3 independent experiments ± SD. Two-way ANOVA tests were used to assess differences between groups, *indicates a statistically significant (P < 0.01) difference.
Figure 5
Figure 5. Hepatocyte fraction cell growth assay on dishes coated with fibronectin (Fn), col1 or col4.
(A) Cells stained with anti-albumin and -Ki67 antibodies. Scale bars are 200 μm. (B) Percentage of proliferating cells on dishes coated with either Fn, col1 or col4. Bars show the average of 3 independent experiments ± SD. *indicates a statistically significant (P < 0.01) difference. (C) Immunofluorescence microscopy analysis of col4 and Ki67 in portal areas of damaged mouse livers (n = 4). Scale bars: 50 μm.
Figure 6
Figure 6. Immunofluorescence microscopy analysis of non-structural ECM components altered in mouse (A, n = 4) and human (B, n = 5) livers.
Scale bars: 50 μm (A), 200 μm (B). Vn-vitronectin. Hepatocyte (C) and NHC (D) growth assays on dishes coated with col1 and added HEK293-conditioned media containing recombinant Olfm4, Thbs4 or Armcx2. Media from sham-transfected cells was used as control. The percentage of proliferating cells ± SD is shown (n = 3). *indicates a statistically significant (P < 0.01) difference.
Figure 7
Figure 7. The alterations in ECM constitution regulate liver repair.
See Discussion for closer explanation.
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