2 - Meccanismi fibrogenetici e implicazioni cliniche
Fabio MarraDipartimento di Medicina Interna
Università di [email protected]
What is fibrosis?
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.
A dynamic, multicellular, integrated, (partially) reversible chronic wound healing process
What is the meaning of hepatic fibrosis?
Vascular
Viral Biliary
ASH/NASH
Patterns of fibrosis development
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
Key introductory concepts
How does fibrosis develop?
Cohen-Naftaly and Friedman, Ther Adv Gastroenterol 2011
Fibrosis progression
SL Friedman, 2008
JP Iredale, J Clin Invest 2007; 117:539
The cell biology of hepatic fibrogenesis
Activated HSC
Deposition of fibrillar extracellular matrix
Inhibition of matrix degradation
Increased proliferation and survival
Cell migration
Quiescent HSC
Cell contraction
Inflammatory cell recruitment
Angiogenesis
Mann & Marra, J Hepatol 2010
All Roads lead to Rome!Fibrosis
ASH NASH
HCV
HBV
Iron
Biliary
Vascular
AIH
HIV coinfection
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
Kim & Chung, Gastroenterology 2009;137:795
Effects of HIV-gp120 on hepatic stellate cells
HSC migration Cytokine expression
Bruno, Galastri et al., Gut 2010
HIV-infected cellsHSC recruitment
via migrationgp120-expressing virions
MCP-1(CCL2) secretion
Further recruitment of fibrogenic and inflammatory cells.
All Roads lead to Rome!Fibrosis
ASH NASH
HCV
HBV
Iron
Biliary
Vascular
AIH
HIV coinfection
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
Treg
M1M2
↑M1
ApoptosisHypoxia
CCL2CCL2
↓Treg↓M2
Weight gain
Weight gain
TNF-IL-1CCL2OPNiNOS
TNF-IL-1CCL2OPNiNOS
↑FFA
Adipose tissue IRLipolysis
↑ Leptin
↓ Adiponectin
↑ Leptin
↓ Adiponectin
Adipose tissue changes after weight gain
Marra & Lotersztajn, Curr Pharm Des 2012; in revision
Metabolic control
Leptin
Adiponectin
Resistin (rodents)
Visfatin
Retinol binding protein 4
Apelin
Vaspin
Omentin
Chemerin
Acylation stimulating protein
Agouti signaling protein
Tissue repair
Angiotensinogen
Renin
Plasminogen-activator inhibitor-1 (PAI-1)
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
Inflammation
Resistin (humans)
Tumor necrosis factor
IL-6
IL-1
IL-10
IL-1 receptor antagonist
Monocyte chemoattractant protein-1 (CCL2)
RANTES (CCL5)
IL-8 (CXCL8)
Interferon.inducible protein-10 (CXCL10)
Migration inhibitory factor (MIF)
Hepcidin
Adipsin
Serum amyloid protein A
Marra & Bertolani, Hepatology 2009;50:957
ADIPOKINES: cytokines of the Adipose tissue
ResistinAdiponectin
Leptin
Deposition of fibrillar matrix
Inhibition of matrix degradation
Proliferation/survival
Cell migration Chemokine secretion
Angiogenesis
Phagocytosis of apoptotic bodies
Leptin
NADPH oxidase activation
Jak-2ERK-1/2PI3K/Akt
ROS
Modified from Bertolani & MarraCurr Pharm Des 2010;16:1929
ADIPONECTIN
Abu-Shanab & Quigley, Nat Rev Gastroenterol Hepatol.2010; 7;691
Based on: Henao-Mejia et al., Nature 2012; 482:179
Innate immune deficiency
Altered microbiota
Activation of TLR4 & TLR9
Proinflammatory signals
NAFLD
Inflammasome deficiency worsens NASH interacting with the microbiome
Transmissible by co-housing
Gut
TLR4
Cytokines (TNF, IL1)
Chemokines(CCL2)
Hepatic stellate cells
Chemokines
Hepatocyte injury
Activated Kupffer cells
ROS
LPS
Fibrogenesis
Hepatocyte steatosis
Cytokines
Cytokines
IL1, danger signals
Monocyte-derived
macrophagerecruitment
Excess free fatty acids
Toxic lipidsTLR9
Bacterial DNA
Inflammatory cell recruitment
Inflammatory cell recruitment
Marra & Lotersztajn,Curr Pharm Des 2012; in revision
Tilg & Moschen, Hepatology 2010;52:1836
The ‘multiple parallel hits’ hypothesis
Genetic predisposition to fibrosis
Zimmer & Lammert, Best Pract Res Clin Gatsroenterol 2011
Zimmer & Lammert, Best Pract Res Clin Gatsroenterol 2011
Valenti et al., Hepatology 2010;51:1209
Adiponutrin (PNPLA3) genotype is associated with the severity of damage and fibrosis
NASH Fibrosis
Patin et al., Gastroenterology 2012
GWAS of susceptibility to fibrosis in HepC
Several susceptibility loci for HCV-induced liver fibrosis, linked to genes that regulate apoptosis.
Zeybel et al., Nat Med 2012
Paternal epigenetic suppression of hepatic fibrosis in male progeny
SL Friedman, Nat Med 2012
Paternal epigenetic suppression of hepatic fibrosis in male progeny
Zhang & Friedman, Hepatology 2012
Pathways in fibrosis that promote HCC
How can we measure fibrosis in clinical practice?
Progression of chronic liver diseases
F0 F1 F2 F3 F4
Fibrosis without septa
No fibrosis Few septaNumerous Septa W/O
cirrhosis
Numerous Septa WITH CIRRHOSIS
CancerPortal hypertensionDeranged microvascular anatomy
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
Notes from EASL clinical practice guidelines
Chronic liver diseases: Methods to establish disease progression
Liver Biopsy: 1:50,000 of liver tissue
Serum Markers
Imaging: US, CT, MRI
Stiffness
HVPG
Serum markers
Gressner et al., World J Gastroenterol 2009; 28:2433
Currently available serum markers
Indirect markers
Direct markers
Combination markers
Pinzani et al., Nat Clin Pract Gastroenterol Hepatol. 2008;5:95
Pinzani et al., Nat Clin Pract Gastroenterol Hepatol. 2008;5:95
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
Algorithms?
Castera et al., J Hepatol 2010;52:191
SAFE biopsy for significant fibrosis (> F2)
Castera et al., J Hepatol 2010;52:191
The Castera algorithm for significant fibrosis
Genes instead of biochemical markers?
A 7 gene signature identifies the risk of developing cirrhosis during chronic hepatitis C
Huang et al., Hepatology 2007;46:297
Marcolongo et al., Hepatology 2009
Transient elastography
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
Transient elastography (Fibroscan®)
AUC
GRAY AREAGRAY AREA
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
Modified from Vizzutti et al., Gut 2009;58:157
Suspected Fibrogenic Liver Disease
Transient elastography
Intermediate values ≥12 kPa≤6 kPa
F4F3F2F1F0
Advanced fibrosis or cirrhosis
Gray areaNo significant
fibrosis
Treat and/or screen for
varices and HCC
No biopsyNo biopsy
Follow-up
Biopsy if results influence management
Possible implementation with other NIT
Poor classification of intermediate stages by non-invasive tests
Cales et al., Liver Int 2008;28:1352
L. Castera, Gastroenterology 2012
In search for a better standard
Inaccuracy of biopsy affects marker perfomance
Mehta et al., J Hepatol 2009;50:36
When errors in the diagnostic test and the gold standard are independent, the observed
sensitivity and specificity of the diagnostic test will be underestimated
Parkes et al., Gut 2010;59:1245
ELF test can predict clinical outcomes
Parkes et al., Gut 2010;59:1245
Vergniol et al., Gastroenterology 2011;140:1970
Ghany et al., Gastroenterology 2010;138:136
Predicting clinical outcomes with standard laboratory tests in chronic hepatitis C
Ghany et al., Gastroenterology 2010;138:136
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
Chronic HCV-Hepatitis Chronic HCV-Hepatitis HAI Score: 11 HAI Score: 11 METAVIR: F1METAVIR: F1
PDGF-R PDGF-R
Collagen I Collagen I
Fibrosis Vs. Fibrogenesis
PDGFPDGF
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
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
Role of biopsy in the management of chronic hepatitis C
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?
Liver Biopsy
Nagula et al., J Hepatol 2006; 44:111
Histological-hemodynamic correlations in cirrhosis
Septal thickness Nodule size
Goodman et al., Hepatology 2007;45:886
“In advanced chronic hepatitis C, fibrosis increases at a rapid pace that can only be detected by morphometry”
Morphometric analysis of advanced fibrosis
Collagen area better correlates with HVPG than Ishak stage
Calvaruso, Burroughs et al., Hepatology 2009;49:1236
Predictor of HVPG > 6 mm HgIshak grading score 0.138Ishak staging score 0.067Collagen proportionate area <0.001
Predictor of HVPG > 10 mm HgIshak grading score 0.477Ishak staging score 0.05Collagen proportionate area 0.009
Schuppan & Afdhal, Lancet 2008;371:838
Clinical evaluation
Clinical evaluation
ImagingImaging
Serum markersSerum markers
FibroscanFibroscan
BiopsyBiopsyPatient
categorization
Follow-up with noninvasive
markers
Follow-up with noninvasive
markers
StableUnstable
Repeat biopsyRepeat biopsy
F0F0
F1F1
F2F2
F3F3
F4F4
Davis et al., Gastroenterology 2010;138:513
DecompensationDecompensation
HCCHCC
Present and future of HCV infection
Classification of chronic liver disease
Garcia-Tsao et al., Hepatology 2010
Progression of CLD:Key Pathophysiological Points
From Chronic Damage to Significant Fibrosis
Pre-Cirrhosis Compensated CirrhosisDecompensated
Cirrhosis
Chronic Tissue Damage, Inflammation, Fibrogenesis
Tissue Hypoxia, Angiogenesis
Portal Hypertension, Carcinogenesis
Parenchymal Failure
Systemic Disease
Assessment of fibrosis progression and regression in different disease stages
F0 F4
HVPG > 5 mmHg
HVPG < 10 mmHg
Stage at liver biopsy
Liver stiffness
Biochemical markers
HVPG
Biopsy (TJLB?) + morphometry
Liver stiffness
Biochemical markers?
orF1 F2 F3
COMPENSATED CIRRHOSIS
Hirooka et al., Radiology 2011
Splenic elasticity is a marker of portalHypertension
Predict and Monitor rate of Fibrosis RegressionPredict and Monitor rate of Fibrosis Regression
From Chronic Damage to From Chronic Damage to Significant FibrosisSignificant Fibrosis Pre-CirrhosisPre-Cirrhosis Compensated CirrhosisCompensated CirrhosisDecompensated Decompensated
CirrhosisCirrhosis
Detect significant fibrosis Detect significant fibrosis Predict rate of progression:Predict rate of progression:
Distinguish Advanced Fibrosis Distinguish Advanced Fibrosis from Cirrhosisfrom Cirrhosis
Monitor the anatomical Monitor the anatomical worsening beyond F4worsening beyond F4
Detect early Detect early predictors of predictors of decompensationdecompensation
Predict the occurrence Predict the occurrence of HCCof HCC
Beginning of Beginning of infection infection difficult to difficult to assessassess
Progression of hepatitis C:clinical needs for patient management
Towards a new classification of cirrhosis
Arvaniti et al., Gastroenterology 2010;139:1246
Does increased knowledge of pathogenesis translate into
antifibrotic therapies?
Damage
Virus, Ethanol, Iron, Autoimmunity,
Fat, Biliary damage
Treat the primary disease:
Interferon/RibavirinNucleos(t)ide analogsAbstinenceVenesectionImmunosuppressionWeight loss & physical activityBiliary decompression
Damage
Inflammation
Oxidative stress
Apoptosis
Cytokine secretion
Virus, Ethanol, Iron, Autoimmunity,
Fat, Biliary damage
CorticosteroidsChemokine antagonists
AntioxidantsHerbal medicines (curcumin)
UDCACaspase inhibitorsHepatic Growth Factor (HGF)
PentoxifyllineDecoy receptorsMAb
Modified from Marra et al., Semin Immunopathol 2009; e-pub Jun 17
Targeting hepatic stellate cell activation
Interferon-PPAR- agonists
Imatinib (PDGF)
Bosentan (ET-1)
NO-donors
CB-1antagonists
ACE-I/ARB
Limitation of matrix deposition
Anti-TGF-Colchicine
Apoptosis of activated stellate cells
SulfasalazineGliotoxin
Promote matrix degradation
HalofuginoneMMP over-expression
Colchicine
Pentoxifylline
Canrenone
Statins
COX inhibitors
N-acetyl-L-cysteine (NAC)
NO donors
Thiazolidinediones
Angiotenin receptor blockers
Antifibrotic drugs already in clinical use
Translational research and the development of an antifibrogenic drug
Mo
lecu
lar
pla
usi
bili
ty
In v
itro
ac
tio
ns
Sa
fety
/to
lera
bili
ty
Eff
ect
s in
in v
ivo
mo
de
ls
Clin
ica
l tri
als
Us
e in
clin
ica
l pra
cti
ce
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
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
Selection of patients for antifibrotic trials
Etiology:HCV or HBV: known natural historyNASH: several components for prognosisAlcohol: dependance of abstinenceBiliary
Fibrosis stage:Avoid advanced cirrhosisConsider grading
Rate of fibrosis progression:Stratification
Progression of autoimmune hepatitis
Hudacko & Thiese Arch Pathol Lab Med 2011
Changes in fibrillar matrix affects degradation
ESTABLISHED FIBROSIS
COLLAGEN CROSS-LINKING
PRESENCE OF ELASTIN
ACELLULAR FIBROSIS
EARLY FIBROSIS
MMPs
Deranged microvascular anatomy in cirrhosis
Onori et al., J Hepatol 2000; 33:555
BB
Cirrhotic liverExtensive FIBROSIS and conversion of normal liver architecture intoSTRUCTURALLY ABNORMAL NODULES
Establishment of INTRAHEPATICVASCULAR SHUNTS between afferentand efferent vessels of the liver
Normal liver
Which is the best drug to test?
1. Easily administered2. Tolerable for a long time3. Safe and devoid of off-target effects4. Potency might not be the real issue
Angiotensin receptor signaling predicts the response to losartan
IKKNEMO)
IKK2IKK1
cytoplasm
nucleus
IB
p50 p65
ATII-Ra
P
P
modified from Marra, Gut 2008;57:570 Oakley et al., Gastroenterology 2009;136:2334
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)
Perspectives for antifibrotic therapies
Cell-based therapies
Gut microbiota/TLRs
Angiogenesis
Weight loss
Herbal medicine