progression of chronic hepatitis: from inflammation to cirrhosis

PROGRESSION OF CHRONIC HEPATITIS: FROM
INFLAMMATION TO CIRRHOSIS
MASSIMO PINZANI, M.D., Ph.D.
DIPARTIMENTO DI MEDICINA INTERNA
CENTER FOR RESEARCH, HIGH EDUCATION AND
TRANSFER “DENOThe”
UNIVERSITA’ DI FIRENZE, FIRENZE, ITALY
FROM INFLAMMATION TO CIRRHOSIS
FIBROGENESIS (AND
INFLAMMATION) IS NOT A
HOMOGENEOS PROCESS
Key Concepts (1)
1. – Hepatic fibrosis develops with different
morphological and spatial patterns.
2. – Different pro-fibrogenic cells contribute
to fibrogenesis.
3. – Hepatic fibrosis is due to different
mechanisms in different clinical settings.
Patterns of Hepatic Fibrosis Development
Pinzani M, and Rombouts K., Dig Liv Dis 2004; 36:231-242
A
B
Post-necrotic
Biliary Type
C
D
ASH/NASH
Pericellular
“Chicken Wire”
Vascular Type
DIFFERENT MECHANISMS AND
DIFFERENT CELLULAR EFFECTORS
Capillarization of Sinusoids
Normal Liver
CLDs
Chronic
Hepatocyte
Injury
Hepatocytes
Loss of
Intercellular
Spaces and
Fenestrations
Space of Disse
Quiescent
HSC
Kupffer
Cell
Sinusoidal
Endothelial
Cell
Hepatic Sinusoid
Activated
Deposition of
HSC
Fibrillar
ECM
Kupffer Cell
Activation
From Friedman SL, J Biol Chem 2000; 275: 22472247-2250
The Multifunctional Pro-fibrogenic Role of HSC
Proliferation
Migration
Fibrillar ECM
Cross-talk with
immune system
Cross-talk with
cancer cells
Cross-talk with
HPC/stem cells
Contraction
Chemoattraction
RESISTANCE TO APOPTOTIC
STIMULI (human HSC)
HYPOTHESIS:
“Fibrogenic
” Cell Types
1.- Differentiation of HSC upon activation
2.- Bone marrow-derived MFs (mesenchymal stem cells)
3.- Both
INTERFACE MFs:
distinct expression
profile and
unknown origin
Portal MFs
SEPTAL MFs:
expression
profile identical
to Portal MFs
HCV - CLD
Hepatic Stellate Cells
IS THERE AN INVOLVEMENT OF
BONE-MARROW-DERIVED STEM
CELLS IN CHRONIC LIVER DISEASE?
α SMA
Vimentin
Fibulin-2
Female patient, BMT at age 10 from Male
donor. Developed HCV cirrhosis requiring
OLT at age 20. No pregnancies.
Commitment of Bone Marrow Cells to
Hepatic Stellate Cells in the Mouse
Baba S. et al, J Hepatol 2004; 40:255-260
GFP
GFAP
2d
desmin
GFP + αSMA
αSMA
7d
HSC from mice transplanted with
GFP-BM
CCl4 in mice transplanted with GFPBM
Prevalent Mechanism of Fibrogenesis in
Different CLD
Chronic direct/indirect
damage (Viral, Autoimmune)
Chronic Toxic Damage and/or
metabolic overload
(ETOH Abuse, metals,
steatohepatitis)
Bile duct proliferation /
chronic cholestasis
Chronic activation of the
wound healing process
Oxidative stress
Epithelial/mesenchymal
interaction and Oxidative
Stress
Key Concepts (2)
1. – Pro-fibrogenic cells are also proinflammatory
2. – Pro-fibrogenic cells are resistant to
apoptosis (human vs. rodent HSC)
3. – Pro-fibrogenic cells are also proangiogenic
HSC ARE ACTIVE PRO-INFLAMMATORY CELLS
Leukocyte recruitment and activation
Leukocyte adhesion
Chemokines
PAF
Complement
Osteopontin
ICAM-1
VCAM-1
NCAM
Fractalkine
PAF
Leukocyte maturation
and survival
M-CSF
SCF
Acute phase
protein synthesis
IL-6
Inhibition of inflammation and fibrogenesis
IL-10
Mast cell recruitment
and activation
SCF
REVERSIBILITY OF FIBROSIS AND CIRRHOSIS
Experimental studies
Animal models Æ Reversion to normal histology after cessation of the
injury
Iredale JP et al. (1998) J C I 102:538-49
Issa R et al. (2001) Gut 48:548-57
associated with:
-
collagenolytic activity
-
HSC apoptosis
Tunel + α-SMA IHC
Culture of rat HSC Æ “in vitro” induction of apoptosis in activated HSC
For a review: Elsharkawy AM, Oakley F, Mann D (2005) Apoptosis 10:927-39.
In vitro and animal
models may lead to
HYPERTROPHIC
conclusions
Discrepancies Between Cell/Animal Models and
the Fibrogenic Evolution of Human CLD
Stellate cells/myofibroblasts become activated by
culture on plastic 48-72 hrs after plating. Their
biology is studied on artificial bidimensional
support.
CCl4
BDL
In most models, cirrhosis develops within 4-8 weeks. No model is identical or
even similar to chronic liver diseases in humans. In general, cirrhosis is rapidly
reversible.
REVERSIBILITY OF FIBROSIS AND CIRRHOSIS
Human CLDs Æ evidence of histological improvement in patients
responding to anti-viral therapy (HCV, HBV)
Kweon YO et al (2001) J Hepatol 35:749 418
Arthur MJP (2002) GE 122:1525
Poynard T et al (2002) GE 122:1303
IHC x αSMA: HBV before (A) and after(B) lamivudine
Kweon YO et al (2001) J Hepatol 35:749-55.
Reversibility of cirrhosis in humans ?
“ … the conclusion is that all studies until now have demonstrated variable
reversal of fibrosis in cirrhosis, and not complete reversal of cirrhosis …”
Roskams T & Desmet VJ (2004) J Hepatol 40:860
ACTIVATED HUMAN HSC DEVELOP RESISTANCE TO APOPTOSIS
HSC 32
HSC 32
freshly activated
isolated passage 7
In vitro:
freshly
isolated
activated
passage 7
Bcl-2
29 kDa
23 kDa
Bax
p-Akt
56 kDa
32 kDa
β actin
Akt
56 kDa
44 kDa
42 kDa
p-ERK
β actin
32 kDa
44 kDa
42 kDa
ERK
In vivo: HCV-cirrhosis
Novo E et al (2006) Gut on line first
doi 10.1136/gut.2005.082791
FIBROGENESIS AND ANGIOGENESIS
IN CHRONIC LIVER DISEASES
CIRRHOSIS
ANGIOGENESIS
FIBROGENESIS
Hepatic Microvasculature in Normal and
Cirrhotic Liver
Onori P. et al., J. Hepatol. 2000; 33:555-563
A
B
Three-Dimensional Reconstruction of Hepatic Bridging
Fibrosis in Chronic HCV Infection
Hoofring A., Boitnott J., and Torbenson M., J Hepatol 2003; 39:738-741
FIBROGENESIS HAS A
CLOSE SPATIAL
RELATIONSHIP WITH THE
PORTAL SYSTEM
PRE-SINUSOIDAL
PORTAL FLOW IS
PROGRESSIVELY
REDUCED
Fibrosis Progression and Its Relationship with Hepatic
Angioarchitecture (Chronic Wound Healing)
Portal tract
Portal-portal
Bridging
CL Vein
Portal-central
Bridging
Portal tract
NEOANGIOGENESIS
and Porto-central shunting
Neo-Angiogenesis in CLDs
1. – Angiogenesis is part of the chronic woundhealing response: VEGF, VEGF-R, bFGF, PDGFBB, TGF-β, MMP-2 activity.
2. – Angiogenesis is a biological response to
hypoxia (capillarization of sinusoids).
3. – Angiogenesis is crucial for the neoplastic
degeneration in CLDs.
INDUCERS
INHIBITORS
CYTOKINES
CD40
CD154
ENDOSTATIN
FGF
CYTOKINES &
CHEMOKINES
CHRONIC
INFLAMMATION
ENDOGENOUS
ANGIOSTATIN
Hypoxia >VEGF
EXOGENOUS
Angiopoietin-1
Anti-VEGF
MMP INHIBITORS
DRUGS (THALIDOMIDE)
ANGIOGENESIS
Lai W.K, Adams D.H., J. Hepatol. 2005; 42: 7-11
Cytokines and Other Soluble Factors Involved
in Hepatic Chronic Wound Healing
Sinusoidal
Endothelium
T- Lymphocytes
Injured Hepatocytes
ROI,
Reactive
Aldehydes,
IGF-1, VEGF
TNF-α ,
IFN-γ
Mononuclear/Kupffer
PDGF-AB ,
bFGF, TGF-b,
TNF-α, IL-1,
PGs, ROI
MCP-1
Thrombin
Angiotensin-II
PDGF-BB ,
bFGF, IL-1,
TGF-β, IGF-1,
PGs, NO,ETs,
ROI, VEGF
PDGF
Platelets
PDGF-AB,
EGF/TGF-α,
TGF-β, TX,
IGF-1, VEGF
VEGF
TGF-β
ET-1
ANG-1
Angiogenesis in DEN-Induced Fibrogenesis in the Rat
Corpechot et al., Hepatology 2002; 35:1010-1021
Hypoxia-Induced VEGF Expression in Rat Liver
Corpechot et al., Hepatology 2002; 35:1010-1021
VEGF
Pimonidazole (Hypoxia)
Why Progressive Hepatic Fibrogenesis is
Associated with Tissue Hypoxia?
Normal Liver
Hepatocytes
1.- Sinusoidal blood flow
becomes more and more
“arterialized”
CLDs
Chronic
Hepatocyte
Injury
2.- Liver tissue progressively
adapts to a higher PO2
Loss of
Intercellular
Spaces and
Fenestrations
Space of Disse
3.- Capillarization of
sinusoids impairs O2
diffusion Quiescent
HSC
Kupffer
Cell
Sinusoidal
Endothelial
Cell
Hepatic Sinusoid
Activated
Deposition of
HSC
Fibrillar
ECM
Kupffer Cell
Activation
Angiogenesis, Vasculogenesis, and
Collateral Vessel Growth
SUBMITTED 2006
EFFECT OF ANGIOGENIC CITOKINES ON HUMAN HEPATIC
STELLATE CELLS MIGRATION
IN VIVO LOCALIZATION OF HSC/MF, PRO-ANGIOGENIC
CITOKINES AND RELATED RECEPTORS (RAT CCl4-9wks)
IN VIVO LOCALIZATION OF HSC/MF, PRO-ANGIOGENIC
CITOKINES AND RELATED RECEPTORS (RAT CCl4-9wks)
DEVELOPING FIBROGENESIS (ACTIVE
SEPTA): ANGIOGENIC CYTOKINES AND
RECEPTORS PREDOMINANT IN
ACTIVATED HSC/MF
CIRRHOSIS: ANGIOGENIC
CYTOKINES AND RECEPTORS
PREDOMINANT IN
ENDOTHELIAL CELLS
MAIN CAUSES OF NEOANGIOGENESIS IN THE
FIBROGENIC PROCESS
1. – Part of the chronic wound-healing process
2. – Progressive increase of hypoxia
3. – Others?
EMERGING MECHANISMS
OF HEPATIC FIBROGENESIS
1.- Not only Hepatic Stellate Cells
2.- Sympathetic neurotransmitters
3.- Viral proteins
4.- Apoptosis products
5.- Apoptosis of fibrogenic cell types
6.- Fibrogenesis & Angiogenesis
7.- Adipokines
LEPTIN AND HEPATIC STELLATE CELLS
Proliferation
Apoptosis
Collagen synthesis
TIMP-1 expression
Oxidative stress
Norepinephrine
Pro-inflammatory cytokines (via NF-kB)
Angiogenic cytokines (via HIF-1α)
EFFECT OF LEPTIN ON THE EXPRESSION OF
ANGIOGENIC MOLECULES IN HUMAN HSC
Aleffi S. et al., Hepatology 2005
Angiogenesis is a Biological Response to Hypoxia
Normoxia
HIF-1
HIF-2
HIF-3
O2
Hypoxia
OH
α
OH
HRE
Vascular
Stability and
Quiescence
β
Oxygen Sensors
PHD 1,2,3
HIF-1
HIF-2
HIF-3
α
β
Hypoxia Responsive
Element (HRE)
VEGF
Angiogenesis
LEPTIN INCREASES THE EXPRESSION OF
HIF-1α IN HUMAN HSC
Aleffi S. et al., Hepatology 2005
Angiogenesis is a Biological Response to Hypoxia
Normoxia
HIF-1
HIF-2
HIF-3
O2
Hypoxia
OH
α
β
Oxygen Sensors
PHD 1,2,3
OH
HRE
Vascular
Stability and
Quiescence
LEPTIN
HIF-1
HIF-2
HIF-3
α
β
Hypoxia Responsive
Element (HRE)
VEGF
Angiogenesis
FIBROGENESIS AND ANGIOGENESIS IN CLDs
Leptin, HCV
proteins
FIBROGENESIS
(Chronic Wound
Healing)
PERIPORTAL
FIBROSIS
ARTERIALIZATION OF
SINUSOIDS
ANGIOGENESIS
Shear Stress
CAPILLARIZATION OF
SINUSOIDS
TISSUE HYPOXIA
A RATIONAL APPROACH TO
THE TREATMENT OF CLDs
Normal Liver
A
Inflamed/WHR
B
Fibrotic Liver
C
Cirrhotic Liver
D
Resolution
Remodelling
E
F
?
Alessandra CALIGIURI
Raffaella DE FRANCO
Antonio MAZZOCCA
Krista ROMBOUTS
Benedetta LOTTINI
Marco BROGI
Chiara SALI
Fabio MARRA
Maurizio PAROLA
Erica NOVO