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Genetic ablation of pannexin1 counteracts liver fibrosis in a chemical, but not in a surgical mouse model

  • Sara Crespo Yanguas
  • Tereza C. da Silva
  • Isabel V. A. Pereira
  • Michaël Maes
  • Joost Willebrords
  • Valery I. Shestopalov
  • Bruna M. Goes
  • Marina Sayuri Nogueira
  • Inar Alves de Castro
  • Guilherme R. Romualdo
  • Luís F. Barbisan
  • Eva Gijbels
  • Mathieu Vinken
  • Bruno Cogliati
Organ Toxicity and Mechanisms

Abstract

Liver fibrosis is the final common pathway for almost all causes of chronic liver injury. This chronic disease is characterized by excessive deposition of extracellular matrix components mainly due to transdifferentiation of quiescent hepatic stellate cell into myofibroblasts-like cells, which in turn is driven by cell death and inflammation. In the last few years, paracrine signaling through pannexin1 channels has emerged as a key player in the latter processes. The current study was set up to investigate the role of pannexin1 signaling in liver fibrosis. Wild-type and whole body pannexin1 knock-out mice were treated with carbon tetrachloride or subjected to bile duct ligation. Evaluation of the effects of pannexin1 deletion was based on a number of clinically relevant read-outs, including markers of liver damage, histopathological analysis, oxidative stress, inflammation and regenerative capacity. In parallel, to elucidate the molecular pathways affected by pannexin1 deletion as well as to mechanistically anchor the clinical observations, whole transcriptome analysis of liver tissue was performed. While pannexin1 knock-out mice treated with carbon tetrachloride displayed reduced collagen content, hepatic stellate cell activation, inflammation and hepatic regeneration, bile duct ligated counterparts showed increased hepatocellular injury and antioxidant enzyme activity with a predominant immune response. Gene expression profiling revealed a downregulation of fibrotic and immune responses in pannexin1 knock-out mice treated with carbon tetrachloride, whereas bile duct ligated pannexin1-deficient animals showed a pronounced inflammatory profile. This study shows for the first time an etiology-dependent role for pannexin1 signaling in experimental liver fibrosis.

Keywords

Pannexin1 Liver fibrosis Inflammation Stellate cells 

Abbreviations

α-SMA

Alpha smooth muscle actin

ALT

Alanine aminotransferase

ALP

Alkaline phosphatase

ANOVA

Analysis of variance

ASC

Apoptosis-associated speck-like protein containing a C-terminal caspase-recruitment domain

AST

Aspartate aminotransferase

ATP

Adenosine-5′-triphosphate

BDL

Bile duct ligation

Casp1

Caspase 1

CCl4

Carbon tetrachloride

Clec7a

Dectin-1

Col1a1

Collagen type 1 alpha 1

Cxcl14

Chemokine ligand 14

GPx

Glutathione peroxidase

GR

Glutathione reductase

HSCs

Hepatic stellate cells

IL

Interleukin

ip

Intraperitoneally

Lbp

Lipopolysaccharide-binding protein

Loxl2

Lysyl oxidase-like 2

Ly96

Lymphocyte antigen 96

MCP-1

Monocyte chemoattractant protein 1

MIP1γ

Macrophage protein 1 gamma

Nalp3

NACHT, LRR, and pyrin domain-containing protein 3

n

Number or repeats

NF-κB

Nuclear factor kappa B

Panx

Pannexin

p

Probability

RT-qPCR

Reverse transcription quantitative real-time polymerase chain reaction

S100a9

S100 calcium-binding protein A9

SEM

Standard error of mean

SOD

Superoxide dismutase

sTNF-RI/-RII

Soluble TNF receptor 1/2

TCA-3

T-cell activation protein 3

TECK

Thymus-expressed chemokine

TLR

Toll-like receptor

TNFα

Tumor necrosis factor alpha

WT

Wild-type

Notes

Acknowledgements

This work was supported by the grants of the “Fundação de Auxílio à Pesquisa do Estado de São Paulo” (FAPESP Grants 14/23890-4; 14/23887-3 and SPEC 13/50420-6), the European Research Council (ERC Starting Grant 335476), the Fund for Scientific Research-Flanders (FWO Grants G009514N and G010214N) and the University Hospital of the Vrije Universiteit Brussel-Belgium (“Willy Gepts Fonds” UZ-VUB). The authors thank Miss Tineke Vanhalewyn, Miss Dinja De Win and Mr Steven Branson for their dedicated technical assistance.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

Supplementary material

204_2018_2255_MOESM1_ESM.pdf (231 kb)
Western blots from p-NF-κBp65 S536 protein expression in liver fibrosis. WT and Panx1-/- mice (n = 10-15) were administered 20% CCl4 ip at a gradually increased dose for 8 weeks or subjected to BDL for 20 days. Representative blot membranes expressing p-NF-κBp65 S536 proteins, total protein loading and β-actin proteins from a the CCl4 model and b the BDL model (PDF 230 KB)
204_2018_2255_MOESM2_ESM.docx (22 kb)
Gene modulation by CCl4 treatment in WT genetic background (DOCX 21 KB)
204_2018_2255_MOESM3_ESM.docx (25 kb)
Gene modulation by the CCl4 treatment independent of background: comparison between WT CCl4 vs WT-oil and Panx1-/- CCl4 vs WT-oil (DOCX 25 KB)
204_2018_2255_MOESM4_ESM.docx (24 kb)
Gene modulation by the BDL procedure in WT genetic background (DOCX 23 KB)
204_2018_2255_MOESM5_ESM.docx (46 kb)
Gene modulation by the BDL procedure independent of background: comparison between WT BDL vs WT-sham and Panx1-/- BDL vs WT-sham (DOCX 45 KB)

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Sara Crespo Yanguas
    • 1
  • Tereza C. da Silva
    • 2
  • Isabel V. A. Pereira
    • 2
  • Michaël Maes
    • 1
  • Joost Willebrords
    • 1
  • Valery I. Shestopalov
    • 3
    • 4
  • Bruna M. Goes
    • 2
  • Marina Sayuri Nogueira
    • 5
  • Inar Alves de Castro
    • 5
  • Guilherme R. Romualdo
    • 6
  • Luís F. Barbisan
    • 6
  • Eva Gijbels
    • 1
  • Mathieu Vinken
    • 1
  • Bruno Cogliati
    • 2
  1. 1.Department of In Vitro Toxicology and Dermato-CosmetologyVrije Universiteit BrusselBrusselsBelgium
  2. 2.Department of Pathology, School of Veterinary Medicine and Animal ScienceUniversity of São PauloSão PauloBrazil
  3. 3.Bascom Palmer Eye Institute, Department of OphthalmologyUniversity of Miami Miller School of MedicineMiamiUSA
  4. 4.Department of Cell Biology and AnatomyUniversity of Miami Miller School of MedicineMiamiUSA
  5. 5.Department of Food and Experimental Nutrition, Faculty of Pharmaceutical SciencesUniversity of São PauloSão PauloBrazil
  6. 6.Department of Pathology, Botucatu Medical SchoolUNESP-São Paulo State UniversityBotucatuBrazil

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