2 - Meccanismi fibrogenetici e implicazioni cliniche

Fabio MarraDipartimento di Medicina Interna

Università di Firenzefabio.marra@unifi.it

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