Expression of Large Tenascin-C Splice Variants by Hepatic Stellate Cells in Chronic Hepatitis C

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Expression of Large Tenascin-C Splice Variants by
Hepatic Stellate Cells in Chronic Hepatitis C

• Dr.Amro El-karef
• Assistant Professor of Pathology
• Department of Pathology
• Mansoura University

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Introduction

• Chronic Hepatitis C is a worldwide liver
• disease that causes liver fibrosis, cirrhosis
• hepatocellular carcinoma. (Lauer 2001)
• Tenascin-C (TN-C), a hexameric ECM glyco-
• protein, is a part of provisional matrix that
• modulates cellular functions during tissue
• remodleing cancer invasion.
  (Chiquet-Ehrismann 1993)
• TN-C has different isoforms due to alternative
• splicing of its mRNA. (Jones 2001)
• Serum levels of large TN-C was found to
• correlate well with the grades of piecmeal
• necrosis. (Tanaka 2005 in press)

3
The structure of human TN-C molecule and the
recognition sites of different anti-TN-C
antibodies
4
The structure of human TN-C molecule and the
recognition sites of different anti-TN-C
antibodies
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The structure of human TN-C molecule and the
recognition sites of different anti-TN-C
antibodies

FNIII Domains TA EGFL Repeats
Alternatively Spliced Domains
Fbg
1 2 3 4 5 A1 A2 A3 A4 B AD2 AD1 C D 6 7 8
4F10TT
6C4TT
4C8MS
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Materials and methods
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Materials and methods A) Monoclonal
antibodies preparation (4F10TT, 6C4TT 4C8MS)
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Materials and methods A) Monoclonal
antibodies preparation (4F10TT, 6C4TT 4C8MS)
B) In vivo study liver biopsies of chronic
HCV patients - HE Sirus red To
diagnose grade the activity (Ishaks HAI)
- Immunohistochemistry TN-C
a-SMA - Combined IHC In situ
hybridization a-SMA human TN-C mRNA
probes
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Materials and methods A) Monoclonal
antibodies preparation (4F10TT, 6C4TT 4C8MS)
C) In vitro study - LI90 human HSC line
culture stimulated with PDGF-BB and
TGF-B. - Immunoblotting of culture
medium cell lysate to detect TN-C its
splice variants. - RNA isolation,
RT-PCR Real time PCR to quantify
TN-C mRNA. - Sequencing of transcripts
of TN-C to conform the different splice
variants
B) In vivo study liver biopsies of chronic
HCV patients - HE Sirus red To
diagnose grade the activity (Ishaks HAI)
- Immunohistochemistry TN-C
a-SMA - Combined IHC In situ
hybridization a-SMA human TN-C mRNA
probes
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Immunostaining of liver biopsies of Chronic HCV
Patients with different Anti-TN-C Antibodies
Immunostaining of liver biopsies of Chronic HCV
Patients with different Anti-TN-C Antibodies
4F10TT
6C4TT
4C8MS
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Immunostaining of liver biopsies of Chronic HCV
Patients with different Anti-TN-C Antibodies
Immunostaining of liver biopsies of Chronic HCV
Patients with different Anti-TN-C Antibodies
4F10TT
6C4TT
4C8MS
Normal
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Immunostaining of liver biopsies of Chronic HCV
Patients with different Anti-TN-C Antibodies
Immunostaining of liver biopsies of Chronic HCV
Patients with different Anti-TN-C Antibodies
4F10TT
6C4TT
4C8MS
Normal
Piecemeal necrosis
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Immunostaining of liver biopsies of Chronic HCV
Patients with different Anti-TN-C Antibodies
Immunostaining of liver biopsies of Chronic HCV
Patients with different Anti-TN-C Antibodies
4F10TT
6C4TT
4C8MS
Normal
Piecemeal necrosis
Confluent necrosis
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Immunostaining of liver biopsies of Chronic HCV
Patients with different Anti-TN-C Antibodies
Immunostaining of liver biopsies of Chronic HCV
Patients with different Anti-TN-C Antibodies
4F10TT
6C4TT
4C8MS
Normal
Piecemeal necrosis
Confluent necrosis
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Immunostaining of liver biopsies of Chronic HCV
Patients with different Anti-TN-C Antibodies
4F10TT
6C4TT
4C8MS
Normal
Piecemeal necrosis
Confluent necrosis
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Immunostaining of liver biopsies of Chronic HCV
Patients with different Anti-TN-C Antibodies
4F10TT
6C4TT
4C8MS
Normal
Piecemeal necrosis
Confluent necrosis
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TN-C expression Vs Activity Grades and Fibrosis
Stage
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HSCs are major sources of TN-C in the liver
TN-C
a-SMA
a-SMA TN-C
a-SMA TN-C mRNA
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TN-C expression Vs HSC Count
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Immunoblotting of TN-C Protein and its Large
Variants from LI90 HSC after Induction with
PDGF-BB
Medium
Lysate
Lysate
4F10TT
4F10TT
6C4TT
4C8MS
KDa
KDa
250 -
250 -
TGF-b
TGF-b
Control
PDGF-BB
PDGF-BB stimulated LI90 HSC to produce small and
large TN-C variants more than TGF-ß compared to
control
Most of large TN-C variants expressed by PDGF-BB
contained A1/A4 and B domains as detected by 6C4
and 4C8 respectively
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mRNA levels of Total TN-C and its large variants
in LI90 HSC Line
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mRNA levels of Total TN-C and its large variants
in LI90 HSC Line
Conventional PCR
Real time PCR
Total TN-C mRNA increased with PDGF-BB
stimulation more than TGF-ß or control cells
TN-C
- 344 - 540
b-actin
TGF-b
Control
PDGF-BB
TGF-b
Control
PDGF-BB
bp
- 2000
- 1600


1500 -


Most of TN-C expressed contains alternatively
spliced domains


- 1000
1000 -




- 500
500 -


TGF-b
BamHI-HindIII
Control
PDGF-BB
TGF-b
Control
PDGF-BB
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TN-C variants produced by LI90 HSC
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Conclusion 1- The previously characterized TN-C
is only the small variants. 2- TN-C is
markedly upregulated in active liver lesions
especially the large variants. 3- Large TN-C
variants is strongly well correlated with
piecemeal and confluent necrosis, the most
reliable prognostic lesions of chronic
hepatitis. 4- Hepatic stellate cells are major
sources of large TN-C variants after their
activation by growth factors.
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