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

Chronic Hepatitis C Chronic Hepatitis C

Dr.Amro El-karefDr.Amro El-karefAssistant Professor of PathologyAssistant Professor of Pathology

Department of PathologyDepartment of PathologyMansoura UniversityMansoura University

IntroductionIntroduction* Chronic Hepatitis C is a worldwide liver * Chronic Hepatitis C is a worldwide liver disease that causes liver fibrosis, cirrhosis disease that causes liver fibrosis, cirrhosis & hepatocellular carcinoma. & hepatocellular carcinoma. (Lauer 2001)(Lauer 2001)

* Tenascin-C (TN-C), a hexameric ECM glyco-* Tenascin-C (TN-C), a hexameric ECM glyco- protein, is a part of provisional matrix that protein, is a part of provisional matrix that modulates cellular functions during tissue modulates cellular functions during tissue remodleing & cancer invasion. remodleing & cancer invasion. (Chiquet-Ehrismann 1993)(Chiquet-Ehrismann 1993)

* TN-C has different isoforms due to alternative * TN-C has different isoforms due to alternative splicing of its mRNA. splicing of its mRNA. (Jones 2001)(Jones 2001)

* Serum levels of large TN-C was found to * Serum levels of large TN-C was found to correlate well with the grades of piecmeal correlate well with the grades of piecmeal necrosis. necrosis. (Tanaka 2005 – in press)(Tanaka 2005 – in press)

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

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

11 22 33 44 55 A1A1 A2A2 A3A3 A4A4 BB AD2AD2 AD1AD1 CC DD 66 77 88

FNIII Domains TA EGFL Repeats Alternatively Spliced Domains Fbg

4F10TT 6C4TT 4C8MS

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

Materials and methods

Materials and methods

A) Monoclonal antibodies preparation (4F10TT, 6C4TT & 4C8MS)

B) In vivo study: liver biopsies of chronic HCV patients - H&E & Sirus red To diagnose & grade the activity (Ishak’s HAI) - Immunohistochemistry * TN-C * α-SMA - Combined IHC & In situ hybridization α-SMA & human TN-C mRNA probes

Materials and methods

A) Monoclonal antibodies preparation (4F10TT, 6C4TT & 4C8MS)

B) In vivo study: liver biopsies of chronic HCV patients - H&E & Sirus red To diagnose & grade the activity (Ishak’s HAI) - Immunohistochemistry * TN-C * α-SMA - Combined IHC & In situ hybridization α-SMA & human TN-C mRNA probes

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

4F10TT 6C4TT 4C8MS

Immunostaining of liver biopsies of Chronic HCV Patients with different Anti-TN-C Antibodies

Normal

4F10TT 6C4TT 4C8MS

Immunostaining of liver biopsies of Chronic HCV Patients with different Anti-TN-C Antibodies

Normal

Piecemeal necrosis

4F10TT 6C4TT 4C8MS

Immunostaining of liver biopsies of Chronic HCV Patients with different Anti-TN-C Antibodies

Normal

Piecemeal necrosis

Confluent necrosis

4F10TT 6C4TT 4C8MS

Immunostaining of liver biopsies of Chronic HCV Patients with different Anti-TN-C Antibodies

Normal

Piecemeal necrosis

Confluent necrosis

4F10TT 6C4TT 4C8MS

Immunostaining of liver biopsies of Chronic HCV Patients with different Anti-TN-C Antibodies

Normal

Piecemeal necrosis

Confluent necrosis

4F10TT 6C4TT 4C8MS

Normal

Piecemeal necrosis

Confluent necrosis

4F10TT 6C4TT 4C8MS

TN-C stainingn=7 n=20 n=12 n=9

Ac

tivi

ty S

co

re

P<0.0001r =0.77

4.9± 0.9

I II III IV

7.8± 2.4

12.3±2.9

12.8± 1.6

Ac

tivi

ty S

co

ren=12 n=20 n=13 n=3

5.9± 1.8 8.9±3.2

12.8± 2.1

13± 0.0

I II III IV

P<0.0001r = 0.71

2

6

10

14

18

n=2 n=24 n=17 n=5 0 I II III

4.5± 0.7 7.8± 3.211.7± 2.9

11.8±2.2

P< 0.0001r = 0.58

Ac

tivi

ty S

co

re

2

6

10

14

18

4F10TT

TN-C staining

6C4TT 4C8MS

TN-C staining

2

6

10

14

18

00 0

Fibrosis stage

F1 F2 F3 F4n=13 n=14 n=8 n=13

TN

-C s

tain

ing IV

III

II

I 3.0± 0.83.3± 0.8

P<0.003r = 0.64

3

9

1

4

7

2

2

4

2

2

6

51

4F10TT 6C4TT 4C8MS

Fibrosis stage

F1 F2 F3 F4n=13 n =14 n=8 n=13

2.8± 0.7

2.8± 0.7

P<0.002r = 0.57

3

1

4

7

4

5

Fibrosis stage

F1 F2 F3 F4n=13 n =14 n=8 n=13

P<0.019r = 0.53

3

1

42

2

2

1

3

5

8 10 9

3

3

8

TN

-C s

tain

ing IV

III

II

IT

N-C

sta

inin

g0

III

II

I

1.8± 0.6 2.0± 0.9 1.7± 0.81.6± 0.5 1.1± 0.5 1.2± 0.6

2.1± 0.81.9± 0.6

6

2

HSCs are major sources of TN-C in the liver

TN-C α-SMA

α-SMA + TN-C α-SMA + TN-C mRNA

TN-C staining

-S

MA

po

siti

ve

cel

ls

TN-C staining

4C8MS

n=2 n=24 n=17 n = 5

P<0.0001r = 0.51

0

20

40

60

80

100

120

140

0 I II IIII II III IVn=12 n=20 n=13 n=3

P<0.0001r = 0.69

0

20

40

60

80

100

120

140

TN-C staining

6C4TT4F10TT

0

20

40

60

80

100

120

140 P<0.0001r = 0.78

n=7 n=20 n =12 n =9 I II III IV

-S

MA

po

siti

ve

cel

ls

-S

MA

po

siti

ve

cells

25.1± 8.8

43.3± 23.4

85.5± 28.9

101.6± 11.1

32.7± 13.0

52.9± 32.6

93.8± 21.7

103.7± 11.0

26.5± 12.0

46.9± 29.1 80.2± 36.4

87.6± 12.6

Medium

250 -

KDa

Lysate

4F10TT 4F10TT

Con

trol

PDG

F-B

B

TGF-

C

ontr

olPD

GF-

BB

TGF-

KDa6C4TT 4C8MS

250 -

Lysate

Con

trol

PDG

F-B

B C

ontr

olPD

GF-

BB

PDGF-BB stimulated LI90 HSC to produce small and large TN-C variants more than TGF-β 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

-actin

TN-C - 344

- 540

Con

trol

PDG

F-B

BTG

F-

500 -

1000 -

1500 -

- 500

- 1000

- 1600- 2000

Con

trol

PDG

F-B

BTG

F-

*

*****

bp

*

*****

BamHI-H

indI

II

**NS

*

6

5

4

3

2

1

0

Rela

tive m

RN

A o

f T

N-C

SV

6

5

4

3

2

1

0Rela

tive m

RN

A o

f to

tal

TN

-C

***NS

**

Total TN-C mRNA increased with PDGF-BB stimulation more than TGF-β or control

cells

Most of TN-C expressed contains alternatively

spliced domains

Conventional PCRReal time PCRC

ontr

ol

PDG

F-B

B

TGF-

Con

trol

PDG

F-B

B

TGF-

-actin

TN-C - 344

- 540

Con

trol

PDG

F-B

BTG

F-

500 -

1000 -

1500 -

- 500

- 1000

- 1600- 2000

Con

trol

PDG

F-B

BTG

F-

*

*****

bp

*

*****

BamHI-HindIII

Control

**NS

PDGF-B

TGF-β

*

6

5

4

3

2

1

0

Rel

ativ

e m

RN

A o

f T

N-C

SV

6

5

4

3

2

1

0Rela

tive m

RN

A o

f to

tal

TN-C

***NS

**Total TN-C mRNA increased with PDGF-BB stimulation

more than TGF-β or control cells

Most of TN-C expressed contains alternatively

spliced domains

Conventional PCRReal time PCR C

ontr

ol

PDG

F-B

B

TGF-

Con

trol

PDG

F-B

B

TGF-

54321 54321 876 876

DD

DD

DD

DD

DD

DD

DD

DD

DD

DD

DD

DD

DD

DD

DD

DD

DD

CC

CC

CC

CC

CC

BB

BB

BB

BB

BB

BB

BB

BB

BB

BB

BBA4A4

A4A4

A4A4

A4A4

A4A4

A4A4

A4A4

A4A4

A4A4

A4A4

A3A3

A3A3

A3A3

A3A3

A3A3

A3A3

A2A2

A2A2

A2A2

A2A2

A2A2

A2A2

A2A2

A2A2

A2A2

A2A2

A2A2

A2A2A1A1

A1A1

A1A1

A1A1

A1A1

A1A1

A1A1

A1A1

A1A1

A1A1

A1A1

A2A2

A1A1

A1A1

A1A1

A1A1

DDA2A2A1A1

BB

No. of FNIII repeats1

2

3

4

5

6

% Clones screened

100 60

40

30

23 43

4

3

44

27

15 41

12

2

55

14

14 22

9

4

4

68

15

9 22

4

4

100 50

TN-C variants

produced by

LI90 HSC

Conclusion1- 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.