Gastrolearning II lezione Meccanismi fibrogenetici e implicazioni cliniche - Prof. F. Marra (Università di Firenze) www.gastrolearning.it

1. 2 - Meccanismi fibrogenetici
e implicazioni cliniche
Fabio Marra
Dipartimento di Medicina Interna
Università di Firenze
fabio.marra@unifi.it

2. What is fibrosis?

3. What is the meaning of hepatic fibrosis?
The formation of excess fibrous
connective tissue in an organ or tissue in
a reparative or reactive process.

4. What is the meaning of hepatic fibrosis?
A dynamic, multicellular,
integrated, (partially) reversible
chronic wound healing process

5. Patterns of fibrosis development
Viral Biliary
Vascular ASH/NASH

6. Key introductory concepts
Hepatic fibrosis develops with different
morphological and spatial patterns
The process involves resident, infiltrating,
and distant cells
Different molecular mechanisms underly
fibrosis development in different settings
Fibrosis is NOT cirrhosis

7. How does fibrosis develop?

8. Fibrosis progression
Cohen-Naftaly and Friedman, Ther Adv Gastroenterol 2011

9. SL Friedman, 2008

10. The cell biology of hepatic fibrogenesis
JP Iredale, J Clin Invest 2007; 117:539

11. Deposition of fibrillar
extracellular matrix
Inhibition of matrix
degradation
Increased proliferation
and survival
Cell migration
Quiescent HSC Activated HSC
Cell contraction
Inflammatory cell
recruitment
Angiogenesis

12. Mann & Marra, J Hepatol 2010

13. All Roads lead toFibros
Rome!
is
ASH AIH NASH
HCV Iron
HIV
Vascular
coinfection
HBV
Biliary

14. Effects of HIV on hepatitis C
 Enhanced HCV replication
 Decreased response rates to HCV treatment
 Faster progression of fibrosis, leading to the earlier
appearance of end-stage liver disease
 More severe inflammation
 HIV treatment (ART) slows down the progression
of liver disease
NEGATIVE IMPACT ON HCV PATHOGENESIS

15. Kim & Chung, Gastroenterology 2009;137:795

16. Effects of HIV-gp120 on hepatic stellate cells
HSC migration Cytokine expression
Bruno, Galastri et al., Gut 2010

17. HSC recruitment
HIV-infected cells
via migration
gp120-expressing virions
MCP-1(CCL2)
secretion
Further recruitment of fibrogenic
and inflammatory cells.

18. All Roads lead toFibros
Rome!
is
ASH AIH NASH
HCV Iron
HIV
Vascular
coinfection
HBV
Biliary

19. Why fat?
1. Severe obesity is associated with a greater
prevalence of NAFLD, NASH, and cirrhosis
2. Alcoholic steatohepatitis is more severe in the
presence of obesity
3. Steatosis accelerates disease progression in
chronic hepatitis C and other chronic liver diseases
4. The response to antiviral therapy in HCV patients is
lower in the presence of fatty liver

20. Adipose tissue changes after weight gain
↑FFA
Adipose tissue IR
Lipolysis
TNF-α
TNF-α ↑ Leptin
↑ Leptin
IL-1β
IL-1β ↓ Adiponectin
CCL2
↓ Adiponectin
CCL2
OPN
OPN
Treg iNOS
iNOS
↑M1 CCL2
CCL2
Weight
Weight
gain
gain
M1
Apoptosis
M2
↓M2 ↓Treg Hypoxia
Marra & Lotersztajn, Curr Pharm Des 2012; in revision

21. Inflammation
ADIPOKINES: cytokines of the Resistin (humans)
Tumor necrosis factor
Adipose tissue IL-6
IL-1
IL-10
IL-1 receptor antagonist
Monocyte chemoattractant protein-1 (CCL2)
RANTES (CCL5)
IL-8 (CXCL8)
Metabolic control
Interferon.inducible protein-10 (CXCL10)
Leptin Migration inhibitory factor (MIF)
Adiponectin Hepcidin
Resistin (rodents) Adipsin
Visfatin Serum amyloid protein A
Retinol binding protein 4
Apelin
Vaspin
Omentin Tissue repair
Chemerin Angiotensinogen
Renin
Acylation stimulating protein
Plasminogen-activator inhibitor-1 (PAI-1)
Agouti signaling protein
Nerve growth factor
Vascular endothelial growth factor
Transforming growth factor-β
Hepatocyte growth factor (HGF)
Heparin-binding, epidermal growth factor-like
growth factor (H-EGF)
Insulin-like growth factor-1
Tissue factor
Marra & Bertolani, Hepatology 2009;50:957

22. Resistin
Adiponectin
Leptin

23. Leptin
Deposition of fibrillar
Proliferation/survival
matrix
Jak-2
ERK-1/2
PI3K/Akt
Phagocytosis of ROS Inhibition of matrix
apoptotic bodies degradation
Cell migration Chemokine
secretion
ADIPONECTIN
NADPH oxidase Angiogenesis
activation Modified from Bertolani & Marra
Curr Pharm Des 2010;16:1929

24. Abu-Shanab & Quigley, Nat Rev
Gastroenterol Hepatol.2010; 7;691

25. Inflammasome deficiency worsens NASH
interacting with the microbiome
Innate immune deficiency

Altered microbiota

Activation of TLR4 & TLR9

Proinflammatory signals

Transmissible by co-housing NAFLD
Based on: Henao-Mejia et al., Nature 2012; 482:179

26. Gut
Excess free LPS
fatty acids Bacterial
Toxic lipids TLR4
DNA
TLR9
IL1, danger signals
ROS
Activated
Kupffer cells
Chemokines Cytokines
(CCL2) (TNF, IL1)
Hepatocyte Hepatocyte
steatosis injury
Monocyte-
derived
macrophage Inflammatory cell
Inflammatory cell
recruitment Cytoki
Cytokirecruitment
recruitment
nes
nes
Chemokines
Hepatic stellate
cells
Fibrogenesis Marra & Lotersztajn,
Curr Pharm Des 2012; in revision

27. The ‘multiple parallel hits’ hypothesis
Tilg & Moschen, Hepatology 2010;52:1836

28. Genetic predisposition to fibrosis

29. Zimmer & Lammert, Best Pract Res Clin Gatsroenterol 2011

30. Zimmer & Lammert, Best Pract Res Clin Gatsroenterol 2011

31. Adiponutrin (PNPLA3) genotype is associated
with the severity of damage and fibrosis
NASH Fibrosis
Valenti et al., Hepatology 2010;51:1209

32. GWAS of susceptibility to fibrosis in HepC
Several susceptibility
loci for HCV-induced
liver fibrosis, linked to
genes that regulate
apoptosis.
Patin et al., Gastroenterology 2012

34. Paternal epigenetic suppression of
hepatic fibrosis in male progeny
Zeybel et al., Nat Med 2012

35. Paternal epigenetic suppression of
hepatic fibrosis in male progeny
SL Friedman, Nat Med 2012

36. Pathways in fibrosis that promote HCC
Zhang & Friedman, Hepatology 2012

37. How can we measure fibrosis in
clinical practice?

38. Progression of chronic liver diseases
F0 F1 F2 F3 F4
Numerous Numerous
Fibrosis
No fibrosis Few septa Septa W/O Septa WITH
without septa
cirrhosis CIRRHOSIS
Deranged microvascular anatomy Portal hypertension Cancer

39. Notes from EASL clinical practice guidelines
 Assessment of the severity of hepatic fibrosis is
important in decision making in chronic hepatitis C
treatment and prognosis
 Liver biopsy is still regarded as the reference
method to assess the grade of inflammation and the
stage of fibrosis
 Serological markers and transient elastography […]
have a performance, when used alone or together,
[which] has been reported to be comparable with liver
biopsy

40. Chronic liver diseases:
Methods to establish disease progression
Imaging: US, CT, MRI
Serum Markers
Stiffness
HVPG
Liver Biopsy: 1:50,000 of liver tissue

41. Serum markers

42. Gressner et al., World J Gastroenterol 2009; 28:2433

43. Currently available serum markers
Indirect markers
Direct markers
Combination markers

44. Pinzani et al., Nat Clin Pract Gastroenterol Hepatol. 2008;5:95

45. Pinzani et al., Nat Clin Pract Gastroenterol Hepatol. 2008;5:95

46. General considerations on
serum markers of fibrosis
 Minimal (F0-F1) vs. significant (≥ F2) fibrosis:

 Detection of advanced (≥F3) fibrosis:

 Detection of cirrhosis:

 Stepwise differentiation of fibrosis stages:

 Fibrogenic process monitoring:

 Selection of patients to be biopsied


47. Algorithms?

48. SAFE biopsy for significant fibrosis (> F2)
Castera et al., J Hepatol 2010;52:191

49. The Castera algorithm for significant fibrosis
Castera et al., J Hepatol 2010;52:191

51. Genes instead of biochemical markers?

52. A 7 gene signature identifies the risk of
developing cirrhosis during chronic hepatitis C
Huang et al., Hepatology 2007;46:297

53. Marcolongo et al., Hepatology 2009

54. Transient elastography

55. Transient elastography (Fibroscan®)
Based on a ultrasound transducer
probe mounted on the axis of a
vibrator.
Vibrations induce an elastic shear
wave that propagates through the
underlying liver tissue.
The velocity of the wave is directly
related to tissue stiffness and to the
amount of fibrotic tissue
Tests approximately 1/500 of the
liver
Not reliable with obesity or ascites

56. AUC
GRAY AREA

57. Multilevel likelihood ratios for the prediction of
significant fibrosis, and cirrhosis
Likelihood ratios above 10 and below 0.1 provide strong evidence to rule in
or rule out diagnoses, respectively.
Arena et al., Gut 2008;57;1288

58. Suspected Fibrogenic Liver Disease
Transient elastography
≤6 kPa Intermediate values ≥12 kPa
No significant Advanced fibrosis or
Gray area
fibrosis cirrhosis
F0 F1 F2 F3 F4
No biopsy
Biopsy if results
No biopsy
influence management
Treat and/or
Possible implementation screen for
Follow-up varices and HCC
with other NIT
Modified from Vizzutti et al., Gut 2009;58:157

59. Poor classification of intermediate
stages by non-invasive tests
Cales et al., Liver Int 2008;28:1352

60. L. Castera, Gastroenterology 2012

61. In search for a better standard

62. Inaccuracy of biopsy affects marker perfomance
When errors in the diagnostic test and the gold
standard are independent, the observed
sensitivity and specificity of the diagnostic test
will be underestimated
Mehta et al., J Hepatol 2009;50:36

63. ELF test can predict clinical outcomes
Parkes et al., Gut 2010;59:1245

64. Parkes et al., Gut 2010;59:1245

65. Vergniol et al., Gastroenterology 2011;140:1970

66. Predicting clinical outcomes with standard
laboratory tests in chronic hepatitis C
Ghany et al., Gastroenterology 2010;138:136

67. Ghany et al., Gastroenterology 2010;138:136

68. What we would like to have from a non-
invasive tool
1. Diagnostic accuracy >0.8 for advanced fibrosis
2. Diagnostic accuracy >0.9 for cirrhosis
3. Ability to detect major changes in fibrosis (e.g. >2
METAVIR stages)
4. Correlate with long-term clinical outcomes
5. Applicability across different types of liver diseases
6. Known profile in control subjects
7. Possibility to be combined with other staging
modalities to build an algorithm

69. Fibrosis Vs. Fibrogenesis
Chronic HCV-Hepatitis
Collagen I
HAI Score: 11
METAVIR: F1
PDGF
PDGF-R β

70. The need for a ‘dynamic’ serum marker to
assess fibrosis in clinical practice
1. Not for cross-sectional staging or diagnosis
2. Sensitive to rapid changes in fibrogenesis and/or
fibrolysis
3. Possibly related to ECM turnover
4. Specific for a given chronic liver disease

71. Role of biopsy in the management of
chronic hepatitis C
Contribution to staging in selected cases
Grading
Assessment of associated lesions (e.g.
NAFLD)
Additional information in the presence of
discrepant non-invasive tests
Masurement of collagen proportionate area

72. Liver Biopsy
1. - Provides clues about etiology
2. - Provides clues about cofactors
3. - Allows immunohistochemical,
biochemical and biomolecular
investigations
4. - Allows assessment of iron content
5. - Allows grading (activity) and staging
(fibrosis): gold standard?

73. Histological-hemodynamic correlations
in cirrhosis
Septal thickness Nodule size
Nagula et al., J Hepatol 2006; 44:111

74. Morphometric analysis of advanced fibrosis
“In advanced chronic hepatitis C, fibrosis increases at a rapid
pace that can only be detected by morphometry”
Goodman et al., Hepatology 2007;45:886

75. Collagen area better correlates with HVPG than
Ishak stage
Predictor of HVPG > 6 mm Hg
Ishak grading score 0.138
Ishak staging score 0.067
Collagen proportionate area <0.001
Predictor of HVPG > 10 mm Hg
Ishak grading score 0.477
Ishak staging score 0.05
Collagen proportionate area 0.009
Calvaruso, Burroughs et al., Hepatology 2009;49:1236

76. Schuppan & Afdhal, Lancet 2008;371:838

77. Biopsy
Patient
Biopsy
categorization
Clinical
Clinical
evaluation
evaluation Follow-up with
Follow-up with
noninvasive
noninvasive
markers
markers
Imaging
Imaging
Serum markers
Serum markers Unstable Stable
Fibroscan
Fibroscan Repeat biopsy
Repeat biopsy

78. Present and future of HCV infection
F0
Decompensation
F1 F4
F2
F3 HCC
Davis et al., Gastroenterology 2010;138:513

79. Classification of chronic liver disease
Garcia-Tsao et al., Hepatology 2010

80. Progression of CLD:
Key Pathophysiological Points
Systemic Disease
Parenchymal Failure
Portal Hypertension, Carcinogenesis
Tissue Hypoxia, Angiogenesis
Chronic Tissue Damage, Inflammation, Fibrogenesis
From Chronic Damage to Decompensated
Pre-Cirrhosis Compensated Cirrhosis
Significant Fibrosis Cirrhosis

81. Assessment of fibrosis progression and
regression in different disease stages
HVPG
Stage at liver biopsy
Biopsy (TJLB?) + morphometry
Liver stiffness
Liver stiffness
Biochemical markers
Biochemical markers?
COMPENSATED CIRRHOSIS
F0 F1 F2 F3 F4
or HVPG < 10 mmHg
HVPG > 5 mmHg

82. Splenic elasticity is a marker of portal
Hypertension
Hirooka et al., Radiology 2011

83. Progression of hepatitis C:
clinical needs for patient management
Beginning of
infection Predict and Monitor rate of Fibrosis Regression
difficult to
assess
Detect early
Detect significant fibrosis Monitor the anatomical
predictors of
Predict rate of progression: worsening beyond F4
decompensation
From Chronic Damage to Decompensated
Pre-Cirrhosis Compensated Cirrhosis
Significant Fibrosis Cirrhosis
Distinguish Advanced Fibrosis Predict the occurrence
from Cirrhosis of HCC

84. Towards a new classification of cirrhosis
Arvaniti et al., Gastroenterology 2010;139:1246

85. Does increased knowledge of
pathogenesis translate into
antifibrotic therapies?

86. Virus, Ethanol, Iron, Treat the primary disease:
Autoimmunity,
Interferon/Ribavirin
Fat, Biliary damage
Nucleos(t)ide analogs
Abstinence
Venesection
Immunosuppression
Weight loss & physical activity
Biliary decompression
Damage

87. Inflammation
Corticosteroids
Chemokine antagonists
Oxidative stress
Virus, Ethanol, Iron,
Antioxidants
Autoimmunity, Herbal medicines (curcumin)
Fat, Biliary damage
Apoptosis
UDCA
Caspase inhibitors
Hepatic Growth Factor (HGF)
Cytokine secretion
Pentoxifylline
Decoy receptors
Damage MAb

88. Targeting hepatic stellate cell activation
Interferon-γ
PPAR-γ agonists
Imatinib (PDGF)
Bosentan (ET-1)
NO-donors
CB-1antagonists
ACE-I/ARB
Modified from Marra et al., Semin Immunopathol 2009; e-pub Jun 17

89. Limitation of matrix deposition
Anti-TGF-β
Colchicine
Apoptosis of activated stellate cells Promote matrix degradation
Sulfasalazine Halofuginone
Gliotoxin MMP over-expression

90. Antifibrotic drugs already in clinical use
Colchicine
Pentoxifylline
Canrenone
Statins
COX inhibitors
N-acetyl-L-cysteine (NAC)
NO donors
Thiazolidinediones
Angiotenin receptor blockers

91. Molecular plausibility
In vitro actions
Effects in in vivo models
Safety/tolerability
Clinical trials
of an antifibrogenic drug
Use in clinical practice
Translational research and the development

93. Problems with trials of antifibrotic drugs
Clinical benefit requires a long period of time
‘Competition’ with antiviral agents
Requirement for liver biopsy
Efficacy may not be assessed by a simple test
Difficulties in measuring the endpoint
Identification of patients more likely to respond

94. Lessons from antifibrotic trials
Clinical benefits require a long period of time
Need for noninvasive markers
Quantitative analysis of biopsies to assess
fibrogenesis
Possible interference with viral replication
Biomarker identification for personalized
therapy

95. Selection of patients for antifibrotic trials
Etiology:
HCV or HBV: known natural history
NASH: several components for prognosis
Alcohol: dependance of abstinence
Biliary
Fibrosis stage:
Avoid advanced cirrhosis
Consider grading
Rate of fibrosis progression:
Stratification

96. Progression of autoimmune hepatitis
Hudacko & Thiese Arch Pathol Lab Med 2011

97. Changes in fibrillar matrix affects degradation
EARLY
FIBROSIS
MMPs
COLLAGEN CROSS-LINKING
PRESENCE OF ELASTIN
ACELLULAR FIBROSIS
ESTABLISHED FIBROSIS

98. Deranged microvascular anatomy in cirrhosis
Extensive FIBROSIS and conversion
Normal liver of normal liver architecture into Cirrhotic liver
STRUCTURALLY ABNORMAL NODULES
Establishment of INTRAHEPATIC
VASCULAR SHUNTS between afferent B
and efferent vessels of the liver
Onori et al., J Hepatol 2000; 33:555

99. Which is the best drug to test?
1. Easily administered
2. Tolerable for a long time
3. Safe and devoid of off-target effects
4. Potency might not be the real issue

100. Angiotensin receptor signaling predicts the
response to losartan
ATII-Ra
cytoplasm
IKKγ (NEMO)
IKK1 IKK2
P IκB
p50 p65
P
nucleus
modified from Marra, Gut 2008;57:570 Oakley et al., Gastroenterology 2009;136:2334

101. Diagnostic endpoints
Biopsy: scored the right way and assessing
fibrogenesis (e.g. alpha-SMA)
Serum tests
Elastography
Imaging
Portal pressure (advanced fibrosis or early
cirrhosis)

104. Perspectives for antifibrotic therapies
Cell-based therapies
Gut microbiota/TLRs
Angiogenesis
Weight loss
Herbal medicine