MASSIMO PINZANI, M.D., Ph.D.

DIPARTIMENTO DI MEDICINA INTERNA – CENTER FOR RESEARCH, TRANSFER AND HIGH EDUCATION “DENOTHE” –

UNIVERSITA’ DI FIRENZE, FLORENCE, ITALY

CORSO UNIGASTRO

RIVA DEGLI ETRUSCHI 4-6 MAGGIO 2008

OBESITY

DIABETES

METABOLIC SYNDROME

NAFLDNASH

FIBROSIS

NASH, A FIBROGENIC CHRONIC LIVER DISEASE

15-50% FIBROSIS OR CIRRHOSIS AT PRESENTATION

DIABETES

METABOLIC SYNDROME

NAFLDNASH

FIBROSIS

NASH, A FIBROGENIC CHRONIC LIVER DISEASE

OBESITY

A B

C D

Pinzani M, and Rombouts K., Dig Liv Dis 2004; 36:231-242Patterns of Hepatic Fibrosis Development

Biliary TypePost-necrotic

Vascular Type

Pericellular “Chicken Wire”

ASH/NASH

A B

C D

Pinzani M, and Rombouts K., Dig Liv Dis 2004; 36:231-242Patterns of Hepatic Fibrosis Development

Biliary TypePost-necrotic

Vascular Type

Pericellular “Chicken Wire”

ASH/NASH

FIBROSIS PATTERN

EARLY PORTAL TO CENTRAL

SEPTA

NEO ANGIOGENESIS

REVERSIBILITY

POST-NECROTIC ++++ ++++ +

BILIARY + ++ ++++

CENTROLOBULAR - + ++++

PERICELLULAR -PERISINUSOIDAL

- + +++

REVERSIBILITY OF LIVER FIBROSIS ACCORDING TO THE PATTERN

EXPANDED FAT MASS

INSULIN RESISTANCE

FATTY LIVER NASH

FFA INFLUX

HYPERINSULINEMIA

(HEPATIC IRON ?)

PRODUCTS OF FFA OXIDATION

MITOCHONDRIAL DYSFUNCTION

LIPID PEROXIDATION

OXIDATIVE STRESS

INFLAMMATION

FIBROGENESIS & ANGIOGENESIS

ACTIVATION IKK-β/NF-κB

TNF-α

HEPATOCELLULAR DAMAGE/APOPTOSIS

CB1-R

ANGIOTENSIN II (INCREASE) LEPTIN/RESISTIN (INCREASE) VEGF (INCREASE) ADIPONECTIN (DECREASE) NOREPINEPHRINE (DECREASE)

TWO HITS OR JUSTPREDISPOSITION

(GENERAL/GENETIC?)

Prevalent Mechanism of Fibrogenesis in Different CLD

Chronic direct/indirect damage (Viral, Autoimmune)

Chronic activation of the wound healing process

Chronic Toxic Damage and/or metabolic overload (ETOH Abuse, metals, steatohepatitis)

Oxidative stress

Bile duct proliferation / chronic cholestasis

Epithelial/mesenchymal interaction and Oxidative Stress

Reactive Aldehydes Induce Procollagen Type I mRNA Expression in Human HSC

C HOE HUE

- 5.7- 4.7

α1(I)

- 1.4 36B4

C 3h 6h

HNE

- 5.7- 4.7

α1(I)

- 1.4 36B4

Parola M. et al. Biochem.Biophys.Res.Comm. 1996; 222:261-264

Reactive Aldehydes Induce Procollagen Type I mRNA Expression in Human HSC

C HOE HUE

- 5.7- 4.7

α1(I)

- 1.4 36B4

C 3h 6h

HNE

- 5.7- 4.7

α1(I)

- 1.4 36B4

Parola M. et al. Biochem.Biophys.Res.Comm. 1996; 222:261-264

ROI, Reactive Aldehydes, Acetaldehyde (ETOH)

OXIDATIVE STRESS AND MEMBRANE

LIPID PEROXIDATION

Up-regulation of pro-fibrogenic genes

Accumulation of fibrillar ECM in the space of Disse independent of

cell damage and inflammation

EMERGING MECHANISMSOF HEPATIC FIBROGENESIS

1.- Sympathetic neurotransmitters2.- Viral proteins3.- Renin-angiotensin-aldosterone system4.- Apoptosis products5.- Adipokines

ADIPOKINES LINKED TO INFLAMMATION AND TO THE INFLAMMATORY RESPONSE

resistin

FAT- DERIVED FACTORS REGULATE HEPATIC INFLAMMATORY

RESPONSE AND FIBROGENESIS

LEPTIN

RESISTIN

Uygun A. et al.,Am J Gastroenterol 2000; 95:3584

LEPTIN LEVELS IN NASH

LEPTIN UPREGULATES LIVER DAMAGE AND REPAIR

Ikejima K et al., Hepatology 2001;34:288-297

LEPTIN DEFICIENCY BLOCKS THEDEVELOPMENT OF FIBROSIS

Ikejima K et al., Gastroenterology. 2002;122:1399-1410

Proliferation

Apoptosis

Collagen synthesis

TIMP-1 expression

Oxidative stress

Norepinephrine

Pro-inflammatory cytokines (via NF-kB)

Angiogenic cytokines (via HIF-1α)

LEPTIN AND HEPATIC STELLATE CELLS

Leptin Induces the Expression of HIF1αand VEGF in human HSC

Aleffi S et al., Hepatology 2005;42:1339-1348

Normoxia Hypoxia

HIF-1HIF-2 HIF-3

α β

O2

Hypoxia Responsive Element (HRE)

VEGF

Angiogenesis

Oxygen Sensors PHD 1,2,3

OHHIF-1 HIF-2 HIF-3

α β

OH

Vascular Stability and Quiescence

HRE

Angiogenesis is a Biological Response to Hypoxia

LEPTIN

EXPRESSION OF RESISTIN IN HUMAN CLDS

AAE

NHL

HCV

AAE NASH

RESISTIN

CD43

α−SMA

COLLAGEN I

Bertolani C., et al., Am J Pathol 2006

RESISTIN CD43 MERGE

RESISTIN ENHANCES THE PRO-INFLAMMATORY ACTION OF HUMAN HSC

Bertolani C., et al., Am J Pathol 2006

Brun P., et al., Am J Physiol Gastrointest Liver Physiol 2005; 289:G571-G578

EXPOSURE TO BACTERIAL CELL WALL PRODUCTS TRIGGERS AN INFLAMMATOTY PHENOTYPE IN HEPATIC STELLATE CELLS

Brun P., et al., Am J Physiol Gastrointest Liver Physiol 2007; 292:G518-G525

INCREASED INTESTINAL PERMEABILITY IN OBESE MICE: NEW EVIDENCE IN THE PATHOGENESIS ON NONALCOHOLIC

STEATOHEPATITIS

Ob/Ob (ipoleptin)& Db/Db (iperleptin: lacking leptin receptor)

Altered intestinal mucosal barrier function (Ussing chamber)

Profoundly modified distribution of occludin and zonula occludens-1 in the intestinal mucosa

Higher circulating levels of inflammatory cytokines and portal endotoxemia

HSCs with higher membrane CD14 mRNA levels and more pronounced lipopolysaccharide-induced proinflammatory and fibrogenic responses

Histological Scoring of NAFLDsBrunt E.M., Sem Liv Dis 2001; 21:3-16

Steatosis1. 0-33% fatty inflitration2. 34-66% fatty inflitration3. 67-100% fatty inflitration

Necroinflammation0. Absent1. Occasional balooning and no or very mild inflammation2. Balooning and mild-to-moderate portal inflammation3. Intra-acinar and moderate portal inflammation

Fibrosis0. Absent1. Perisinusoidal/pericellular fibrosis2. Periportal fibrosis3. Bridging fibrosis4. Cirrhosis

Steatosis (0-3)Lobular inflammation (0-2)Hepatocellular ballooning (0-2)Fibrosis (0-4)

NAFLD activity score (NAS) = steatosis + lobular inflammation + hepatocellular ballooning

NASH if NAS≥5‘NO NASH’ if NAS<3

Steatosis (0-3)Lobular inflammation (0-2)Hepatocellular ballooning (0-2)Fibrosis (0-4)

NAFLD activity score (NAS) = steatosis + lobular inflammation + hepatocellular ballooning

NASH if NAS≥5‘NO NASH’ if NAS<3

Consensus means that a lot of people saycollectively what no one believes individually

Abba Eban

THE NAS SCORE IS BASED ON THE ASSUMPTION THAT THE EXTENT FIBROSIS IS AN EXCLUSIVE CONSEQUENCE OF HEPATOCELLULAR DAMAGE AND INFLAMMATION

THIS ASSUMPTION IS NOT PROVEN IN ANY CLD AND PARTICULARLY IN NASH

A. OXIDATIVE STRESS DIRECTLY AFFECTS COLLAGEN GENE EXPRESSION AND SYNTHESIS

B. INDIVIDUAL DIFFERENCES IN THE ABILITY OF DEGRADING FIBRILLAR MATRIX IN CAPILLARIZED SINUSOIDS

DIAGNOSIS OF NASH AND RELATED ISSUES

1. – DIFFERENTIATE FATTY LIVER FROM NASH

2. – DEFINE THE SEVERITY OF NASH

3. – PREDICT AND MONITOR THE FIBROGENIC EVOLUTION OF NASH TOWARDS CIRRHOSIS

4. – DIFFERENTIATE NASH FROM ASH

TO BIOPSY OR NOT TO BIOPSY (AND WHOM) ?

Reasons to perform a liver biopsy

Unknown disease

DIAGNOSIS

Patients with unexplained

alterations of liver enzymes

Known disease

SEVERITY

Patients with a “bright liver”

pattern at the US scan

1.- Reliability of the fibrosis rate (stages treated as linear variables)

2.- Sampling error

3.- Adequacy of biopsy sample

4.- Time between biopsies

LIVER BIOPSY IN NAFLD/NASH

Non-invasive tests may help to identify patients with steatosis in large population studies.

A test differentiating bland steatosis from NASH would be the most useful in this setting.

Several algorythms identify advanced fibrosis in NASH with a good performance.

We need DISCRIMINANT and PROGNOSTIC INDEXES

DIAGNOSIS OF NASH AND RELATED ISSUES

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

Dixon et al., 2001(26 severely obese pts.)

Predictors of advanced fibrosis:Insulin resistanceHypertensionALT levels

Ratziu et al., 2000(93 overweight pts.)

Predictors of septal (F≥2) fibrosis:BMI AgeALTTriglycerides

Bugianesi et al., 2004(167 NAFLD pts. with no

diabetes)

Predictors of septal fibrosis after adjustment for age, and BMI:

Insulin resistanceFerritin (NOT iron overload)

Angulo et al., 1999(144 NASH pts.)

Predictors of severe fibrosis:Older ageObesityDiabetes mellitusAST/ALT ratio >1

How to Predict Fibrosis: Cross-sectional studies

Adams L et al., J Hepatol 2005;42:132

Predictors of fibrosis rate bymultivariate analysis

BMIDiabetesEarlier fibrosis stage

103 patients

Biopsy interval

<24 Months 24–48 Months >48 Months

Progressors 11 (34%) 18 (36%) 9 (43%)

Stable 13 (41%) 13 (26%) 9 (43%)

Regressors 8 (25%) 19 (38%) 3 (14%)

No correlation with changes in ALT or AST

Improvement in ALT correlateswith improvement in steatosis or inflammation but NOT fibrosis

How to Predict Fibrosis: Longitudinal studies

1.- In at least 1/3 of NASH patients fibrosis progresses

2.- No clear parameters at baseline allow to identify patients that will progress

3.- Fibrosis progression occurs even in presence of a decrease in necro-inflammatory activity and/or steatosis

4.- Improvement of transaminases is NOT a surrogate marker for improvement of fibrosis

Messages from Paired-Biopsy Studies

Fassio et al., Hepatology 2004;40:820 Harrison et al., Am J Gastroenterol 2003;98:2042Adams et al., J Hepatol 2005;42:132

Noninvasive Model for the Prediction of Fibrosis in Patients with NAFLD

1-Specificity

Sens

itivi

ty

0.0 0.2 0.4 0.6 0.8 1.0

0.0

0.2

0.4

0.6

0.8

1.0

Model Building Data

1-Specificity

Sen

sitiv

ity

0.0 0.2 0.4 0.6 0.8 1.0

0.0

0.2

0.4

0.6

0.8

1.0

Validation Data

Angulo P. et al,HEPATOLOGY, in press

Variable OR 95% CI p ValueAge ≥ 45 years 2.3 1.2, 4.3 0.01Obese (BMI ≥ 30) 2.6 1.4, 4.9 0.004AST/ALT ratio > 1 6.7 3.4, 13.2 < 0.0001Low platelet count (< 150x103/dL) 8.3 3.8, 18.3 < 0.0001AST/platelet > 1 2.4 1.3, 4.5 0.007Diabetes mellitus (yes) 4.7 2.5, 8.8 < 0.0001Hypertriglyceridemia (> 150 mg/dL) 0.5 0.3, 0.8 < 0.02

ELF Study: Algorythm Performance in Different Diagnostic Subgroups

Diagnosis Pts (n) Sensitivity Specificity NPV

NAFLD/NASH 60 92.3 91.7

59.6 66.7

96.6 97.0

HCC & Iron Overload

15 100100

84.676.9

100 100

Disease Post OLT

33 100 100

41.7 33.3

100100

HCV or HBV or Both

289 60.060.7

72.0 72.0

70.271.6

PBC or PSC 33 78.978.9

71.4 78.9

71.4 71.4

SCHEUER ISHAK

Detection of Scheuer Stage 0,1,2 versus 3,4

Ancona – Clinica di GastroenterologiaGrosseto – Divisione di Gastroenterologia Modena e Trieste - Amici del Fegato Napoli – Gastroenterologia II UniversitàNovara - Dipartimento Scienze MedichePadova – Clinica di Gastroenterologia Padova - Dipartimento Medicina Clinica e

SperimentaleRoma – Università Cattolica Savona - UO Semplice di

Gastroenterologia-EpatologiaTorino – Gastroenterologia

209 campioni

AISF-FAT STUDY, Validation of the ELF panel in an Italian NAFLD cohort

PERFORMANCE OF ELF SERUM MARKERS IN PREDICTING FIBROSIS STAGE IN CHILDREN WITH NON ALCOHOLIC FATTY LIVER DISEASEValerio Nobili, Julie Parkes, Gianfranco Bottazzo, Matilde Marcellini, Richard Cross, Daniel Newman, Massimo Pinzani, and William Rosenberg (2008 Submitted)

43210

12.

10.

8.

*

*#

#

EL

F S

co

re

Fibrosis Stage (Kleiner)

112 pts with biopsy proven NAFLD (64 M and 48 F, mean age 13.8± 3.3, age range 3-17 yrs)

PERFORMANCE OF ELF SERUM MARKERS IN PREDICTING FIBROSIS STAGE IN CHILDREN WITH NON ALCOHOLIC FATTY LIVER DISEASEValerio Nobili, Julie Parkes, Gianfranco Bottazzo, Matilde Marcellini, Richard Cross, Daniel Newman, Massimo Pinzani, and William Rosenberg (2008 Submitted)

43210

12.

10.

8.

*

*#

#

EL

F S

co

re

Fibrosis Stage (Kleiner)

0%

10%20%

30%40%

50%

60%70%

80%90%

100%

“any” fibrosis

“any”fibrosis

1c-4 2-4 3-4

cannot be allocated

avoid biopsy incorrectly

avoid biopsy correctly

8.84+9.54 9.54 9.54 10.3 10.3

112 pts with biopsy proven NAFLD (64 M and 48 F, mean age 13.8± 3.3, age range 3-17 yrs)

ACCURACY AND REPRODUCIBILITY OF TRANSIENT ELASTOGRAPHY FOR THE DIAGNOSIS OF FIBROSIS IN PEDIATRIC NON-ALCOHOLIC

STEATOHEPATITISValerio Nobili, Francesco Vizzutti, Umberto Arena, Juan G. Abraldes, Fabio Marra, Andrea

Pietrobattista, Rodolfo Fruhwirth, Matilde Marcellini, and Massimo PinzaniHEPATOLOGY 2008 (IN PRESS)

When should intervention begin?

Are study endpoints consistent and complementary to clinical goals and actual outcomes?

1. – EFFECT ON MORTALITY (NOT REALISTIC)

3. – EFFECT ON INSULIN RESISTANCE (NOT NECESSARILY ASSOCIATED WITH LIVER CHANGES IN THE SHORT TERM)

4. – EFFECT ON HISTOLOGY: DOWNSTAGING NAS SCORE.

2. – EFFECT ON LIVER FUNCTION TESTS (NOT RELIABLE)

5.- EFFECT ON HISTOLOGY: DOWNSTAGING FIBROSIS.

How does the stage of NAFLD/NASH influence the option of intervention, therapeutic target, and clinical outcomes and goals?

1. – NO FIBROSIS OR MILD PERISINUSOIDAL FIBROSIS

2. - SIGNIFICANT FIBROSIS WITHOUT BRIDGING AND ANGIOGENESIS

3. ADVANCED FIBROSIS (ALSO PERIPORTAL)

4. HISTOLOGICAL CIRRHOSIS

What features of a NAFLD/NASH study could be required to test efficacy?

1.- Histology

2.- Metabolic parameters

3.- Cardiovascular risk

4.- Adipokine levels (Adiponectin increase)

Potential therapeutic approaches

BW REDUCTIONSIBUTRAMINE

ORLISTATSURGERY

METFORMINTHIAZOLIDINEDIONESGLP1-R AGONISTS

(EXENDIN-4)

BOWEL DECONT & PRE-PROBIOTICS

ANTI-TNF (PTFYLLIN)IKK-β INHIBITORS

SAMeBETAINE

SYLIBININ

HEPATIC ACETYLCoA

CARBOXILASES 1-2 INHIBITORS

ACE INHIB/ATII RBANTI-VEGF?

CB1-RB

CHEMOKINERECEPTOR

BLOCKERS (CCR2)

DIAGNOSIS OF NASH AND RELATED ISSUES

1. – DIFFERENTIATE FATTY LIVER FROM NASH

2. – DEFINE THE SEVERITY OF NASH

3. – PREDICT AND MONITOR THE FIBROGENIC EVOLUTION OF NASH TOWARDS CIRRHOSIS

4. – DIFFERENTIATE NASH FROM ASH

Alcohol Consumption by Country, Adults aged 15 years and above, 2001, Europe

0.0

2.0

4.0

6.0

8.0

10.0

12.0

14.0

16.0

18.0

20.0

litre

s/pe

rson

/yea

r

Under-reported or surreptitious alcohol consumption (the “desperate house wife syndrome”)

The “gourmet business man syndrome”

ASH vs. NASH

How do NASH studies relate to the Country’s average alcohol consumption and drinking habits?

MCV + AST/ALT + BMI + GENDER = ANI (ALD/NAFLD INDEX)

ACCURACY OF ANI IN DISTINGUISHING ALD FROM NAFLD COMPARED TO STANDARD

METHODS

DISTRIBUTION OF ANI IN NAFLD COMPARED TO ABSTINENT OR ACTIVE ALCOHOLICS