The hepatic "matrisome" responds dynamically to injury: Characterization of transitional changes to the extracellular matrix in mice

Abstract

The extracellular matrix (ECM) consists of diverse components that work bidirectionally with surrounding cells to create a responsive microenvironment. In some contexts (e.g., hepatic fibrosis), changes to the ECM are well recognized and understood. However, it is becoming increasingly accepted that the hepatic ECM proteome (i.e., matrisome) responds dynamically to stress well before fibrosis. The term "transitional tissue remodeling" describes qualitative and quantitative ECM changes in response to injury that do not alter the overall architecture of the organ; these changes in ECM may contribute to early disease initiation and/or progression. The nature and magnitude of these changes to the ECM in liver injury are poorly understood. The goals of this work were to validate analysis of the ECM proteome and compare the impact of 6 weeks of ethanol diet and/or acute lipopolysaccharide (LPS). Liver sections were processed in a series of increasingly rigorous extraction buffers to separate proteins by solubility. Extracted proteins were identified using liquid chromatography/tandem mass spectrometry (LC-MS/MS). Both ethanol and LPS dramatically increased the number of matrisome proteins ∼25%. The enhancement of LPS-induced liver damage by ethanol preexposure was associated with unique protein changes.

Conclusion: An extraction method to enrich the hepatic ECM was characterized. The results demonstrate that the hepatic matrisome responds dynamically to both acute (LPS) and chronic (ethanol) stresses, long before more-dramatic fibrotic changes to the liver occur. The changes to the mastrisome may contribute, at least in part, to the pathological responses to these stresses. It is also interesting that several ECM proteins responded similarly to both stresses, suggesting a common mechanism in both models. Nevertheless, there were responses that were unique to the individual and combined exposures. (Hepatology 2017;65:969-982).

FIG. 1

Scheme of transitional ECM changes and extraction methodology. Remodeling in response to chronic injury (i.e., fibrosis) is well known; however the hepatic ECM also responses dynamically to acute stress. These acute responses can be viewed as an arm of the wound-healing response and facilitate recovery from damage, which resolves once the damage is repaired. However, under conditions of chronic injury, these changes contribute to activation of a significant remodeling response that leads to scar formation (i.e., fibrosis).

FIG. 2

Validation of extraction technique with CCl₄ model of fibrosis. Animals were administered CCl₄ or vehicle for 4 weeks. Collagen type I accumulation was determined by Sirius Red staining (A) and by dot blot and hydroxyproline content (B). Collagen 1 mRNA expression (B) was determined by real-time rtPCR. Quantitative changes in other collagens were also determined in the ECM extraction fractions (C). Abbreviations: COL, collagen; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; rtPCR, reverse-transcription PCR.

FIG. 3

Liver extracts have unique protein profiles based on fraction type and experimental group. A heatmap depicting quantitative changes to the total ECM proteome in response to ethanol or LPS is shown in (A). The number of proteins unique to, or shared by, all four extractions of pair-fed animals is shown (B). Bubble graphs are used to show presence of plasminogen, Annexin A1, and fibrin(ogen) gamma chain across the four extracts (x-axis) and the four experimental groups (y-axis; C).

FIG. 4

Ethanol and LPS cause dynamic changes in the matrisome. The impact of ethanol diet (left panels) and 24-hour LPS (right panels) on the types of proteins found in the ECM proteome are shown. Proteins are categorized by class (A–D) and organized by extraction fraction (NaCl, SDS, GnHCl, and pellet). Red proteins indicate those that appeared with exposure, whereas green proteins indicate those that were lost with exposure, compared to control.

FIG. 5

Shared and unique changes to the hepatic matrisome. Venn diagrams (left column) show all proteins within an extract and indicate the number that are shared between, or that are unique to, the four experimental groups. Bubble plots (right column) show quantitative changes in protein expression of proteins that were shared by all four experimental groups. The bubble plots show fold change in protein expression caused by LPS (y-axis), ethanol (x-axis), and the combination of ethanol+LPS (bubble size). Each bubble represents a single extracellular protein; bubble color indicates the protein’s class.

FIG. 6

Quantitative changes to the matrisome. Heatmap analysis of the quantitative changes in protein expression of proteins that were shared by all four experimental groups (see Fig. 5) are shown for the NaCl (A), SDS (B), GnHCl (C), and pellet (D) extraction fractions.