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三重大学医学部病理 Deficiency of tenascin-C attenuates liver
fibrosis in immune-mediated chronic hepatitis in mice 三重大学医学部病理 El-Karef Amro 303D006
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Progression from Hepatitis to Fibrosis
Chronic liver diseases Viral Hepatitis, Autoimmune Hepatitis etc Inflammatory Response Hepatocyte Injury T Lymphocytes Kupffer Cells Activation and Recruitment of HSCs (Hepatic Stellate Cells) & MFBs (Portal Myofibroblasts) Collagen Synthesis Liver Fibrosis (Cirrhosis)
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Tenascin-C (TN-C) in Chronic Liver Diseases
highly expressed in chronic viral hepatitis, autoimmune hepatitis and bile obstruction highly deposited in interfaces of fibrotic areas and in peri-sinusoidal spaces synthesized by HSCs Possible involvement in liver fibrosis
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Molecular model of the subunit
Tenascin-C (TNC) is an ECM glycoprotein upregulated in remodeling tissues Hexameric Structure FNIII Repeat TA EGFL Repeats Alternatively spliced region FG 1 2 3 4 5 A1 A2 A3 A4 B AD2 AD1 C D 6 7 8 Molecular model of the subunit
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Mice develop normally without Tenascin-C (Saga Y et al. Gene Dev 1992)
Tenascin-C Knockout (TNKO) Mice Mice develop normally without Tenascin-C (Saga Y et al. Gene Dev 1992) Abnormal repair in diseased tissues of adult mice Neuromuscular junction Venom-induced glomerulonephritis Chemically induced dermatitis Corneal injury Granulation tissue after cardiac injury
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Aim of Study Immune-mediated chronic hepatitis model [ Concanavalin A (ConA)-induced ] × WT mice and TNKO mice To obtain direct evidence for promotive roles of TNC in live fibrosis
![Aim of Study Immune-mediated chronic hepatitis model. [ Concanavalin A (ConA)-induced ] × WT mice and TNKO mice.](http://slideplayer.com/slide/8924068/27/images/6/Aim+of+Study+Immune-mediated+chronic+hepatitis+model.+%5B+Concanavalin+A+%28ConA%29-induced+%5D+%C3%97+WT+mice+and+TNKO+mice..jpg "To obtain direct evidence for promotive roles of TNC in live fibrosis.")
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Experimental Protocol of ConA Challenge
8-week-old female BALB/c mice Experimental Groups ConA challenges (20 mg/Kg/ week, i.v.) 12w 0w 13w x 9w sacrifice x 6w x 3w x Saline alone Controls CTL2 x CTL1 x
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Histological and Biochemical Analyses
Histological & immunohitochemical staining: - H&E: Histological analyses for necrosis and inflammation. - Picrosirus red: Deposited collagen fibrils - TNC: TNC protein - a smooth muscle actin (α-SMA ): Activated HSCs/MFBs Liver RNA extraction & RT-PCR: - Quantitative real-time PCR (qPCR) ECM proteins: TNC – Collagen I & III Inflammatory cytokines: INF-γ, IL-4 & TNF-α Fibrogenic cytokine: TGF-β
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Immunohistochemical staining of TNC
Liver tissues of WT mice CTL1 3w 6w 9w TNC deposition is gradually increased after ConA administration. 12w CTL2
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RT- PCR and qPCR for TNC mRNA
6 p<0.01 p<0.001 5 b-actin TNC - 540 - 344 4 p<0.01 p<0.05 TNC mRNA （folds) 3 2 CTL1 3w 6w 9w 12w CTL2 1 Conventional PCR CTL1 3w 6w 9w 12w CTL2 Quantitative real time PCR TNC mRNA levels sequentially increase in WT livers.
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Collagen deposition of WT & TNKO mice livers
CTL2 12w Picrosirius red staining Collagen deposition was less intense in TNKO mice after 12th ConA injection (12w) than WT mice.
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Image analysis of fibrosis
WT TNKO Total liver fibrosis Parenchymal fibrosis 25 7 p<0.001 p<0.001 p<0.05 p<0.001 p<0.05 ns p<0.05 p<0.01 20 6 5 15 Fibrotic area (%) 4 Fibrotic area (%) 10 3 2 5 1 CTL1 3w 6w 9w 12w CTL2 CTL1 3w 6w 9w 12w CTL2 Quantification was performed by NIH image using picrosirius red-stained sections Fibrosis areas in WT liver were more than in TNKO.
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qPCR for procollagen I & III mRNA
WT TNKO Procollagen I Procollagen III 6 4 p<0.05 p<0.05 p<0.01 p<0.001 p<0.01 p<0.05 p<0.001 p<0.05 5 Wild 3 KO Wild 4 KO COL1A2 mRNA (folds) COL3A1 mRNA (folds) 3 2 2 1 1 CTL1 3w 6w 9w 12w CTL2 CTL1 3w 6w 9w 12w CTL2 Procollagen I & III expression was upregulated in WT mice livers than in TNKO.
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How could TNC promote liver fibrosis?
TNC expressed in WT mice can contribute to liver fibrosis. How could TNC promote liver fibrosis?
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Inflammatory cell infiltrate in WT & TNKO mice
CV PT PT: portal tract CV: central vein TNKO PT CV Inflammatory infiltrate was more severe in WT mice livers than in TNKO.
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Semi-quantification of the inflammatory infiltrate
WT TNKO * ns ns * * p<0.05 3 3 2 2 2 4 2 Lymphocytic Infiltration 1 3 1 4 3 3 4 2 3 1 2 1 3w w w w modified Knodell’s HAI scoring system Lymphocytic infiltrate scores were significantly higher in WT than in TNKO after Con A administration
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qPCR of inflammatory cytokines mRNA
2 4 6 8 10 12 CTL1 3w 6w 9w 12w CTL2 INF - γ mRNA (fold value) ** * *** IL 4 mRNA TNF a WT TNKO * p<0.05 ** p<0.01 *** p<0.001 Cytokines were significantly upregulated in WT than in TNKO.
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α-SMA immunostaining of WT & TNKO liver
PT: portal tract CV: central vein Arrows: Positive cells (Activated HSCs/MFBs) α-SMA positive cells were more common in WT than in TNKO.
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a-SMA-positive cells ( /mm2 )
α-SMA positive cell counts WT TNKO p<0.001 p<0.05 p<0.01 300 200 a-SMA-positive cells ( /mm2 ) 100 CTL1 3w 6w 9w 12w CTL2 α-SMA positive cells significantly increased to a greater extent in WT than in TNKO.
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qPCR for TGF-β mRNA WT TNKO 5 p<0.01 p<0.001 p<0.001 4 3 TGF-β1 mRNA (folds) 2 1 CTL1 3w 6w 9w 12w CTL2 TGF-β1 mRNA was significantly increased in WT than in TNKO.
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TNC TNC promotes inflammatory response & cytokine upregulation
Con A Rolling ↑ Migration ↑ TNC Liver Activated T lymphocyte Cytokines ↑ INF-γ INF-γ IL- 4 Kupffer cell Liver cell injury TNF-α (IL-1)
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TNC TNC promotes activation of HSC Liver cell injury Liver
Inflammatory Response ↑ Liver cell injury TNF-α Kupffer cell TNF-α (Lipid metabolites) Phenotypic change ↑ (IL-1, NO, O*) Quiescent HSC Activated HSC (a-SMA +)
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TNC TNC promotes recruitment of HSCs/MFBs & TGF-b upregulation Liver
Activated HSC (a-SMA +) Quiescent HSC TGF-b Expression/ Signaling ↑ TNC HSC proliferation TGF-b Activation & Migration ↑ Activated MFBs (a-SMA +) Portal MFBs
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TNC-rich Matrix TNC HSCs/MFBs provides TNC-rich matrix Liver TGF-b
Activated HSC (a-SMA +) Quiescent HSC TGF-b Expression/ Signaling ↑ TNC-rich Matrix TNC HSC proliferation TGF-b Activation & Migration ↑ Activated MFBs (a-SMA +) Portal MFBs
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Procollagen ↑ Fibrosis TNC TNC promotes liver fibrosis Liver TGF-b
Activated HSC (a-SMA +) Quiescent HSC TGF-b Expression/ Signaling ↑ Procollagen ↑ Fibrosis TNC HSC proliferation TGF-b Activation & Migration ↑ Activated MFBs (a-SMA +) Portal MFBs
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Conclusion Tenascin-C actively contributes to hepatic fibrosis through possible mechanisms as follows: Augments the inflammatory response & upregulates inflammatory cytokines as INF-γ, IL-4 & TNF-α. Recruits & activates HSCs & MFBs. Up-regulates TGF-β.
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