10/1/12 Understanding Root Cause: Pathogenesis of Hepatic Fibrosis Hepatitis C Virus Mild inflammation Inflammation Fibrosis Cirrhosis 1
10/1/12 Non-alcoholic Fatty Liver Disease Steatosis Steatohepatitis Fibrosis Cirrhosis Fibrotic Liver Diseases Patients Mice HCV, HBV (Post necrotic cirrhosis) Carbon tetrachloride (CCl4) Thioacetamide (TAA) Mouse with humanized liver with hepatitis infection Secondary biliary fibrosis Bile duct ligation Alcoholic liver disease Intragastric ethanol Autoimmune hepatitis Heterologous serum Non-alcoholic steatohepatitis (NASH) Diet induced obesity Choline deficient diet (CDAA) 2
10/1/12 J Clin Invest. 115: 209, 2005 3
Hepatic Stellate Cell (HSC) activation in culture quiescent HSC activated HSC ACTIVATION Vit-A autofluorescence ECM a-sma Modifiers of hepatic fibrosis using knock out mice Susceptibility to fibrosis Knock out: Increased Protective IL-6, IL-10 inos Plasminogen Interferon g Adiponectin Telomerase Bcl-xL SOCS1 (+/-) Keratin Decreased Fibrogenic TNFR1, TLR4, CD14 Angiotensin R1a NADPH oxidase TGFb1, Smad3, MMP13 p21 FGF1/2, FGFR4 Leptin, OB-R Dopamine bhydroxylase FasL, Cathepsin B C5 Hepatology 37: 653, 2003 4
Oxidative stress induces liver fibrosis Ethanol/acetaldehyde AngII, Bile salts HCV proteins P450IIE1 Apoptosis ROS Activation Proliferation Activation NAD(P)H-oxidase NAD(P)H-oxidase Fibrosis Examples of kos NADPH oxidase, angiotensin R1a Renin Angiotensin Pathway in HSCs Induces ROS and Hepatic Fibrosis Angiotensinogen Renin Angiotensin-I ACE-inh ARB ACE Angiotensin-II AT1 NADPH oxidase gp91 phox p67 phox p22 phox. p47 phox p O 2 O 2 Ca +2 flux HSC activation ECM production AT2 Hepatic fibrosis 5
NOXs in hepatic stellate cells NOX1 Non-phagocytic form of NOX Expressed in VSMCs and fibroblasts Induced by Ang II, Leptin, PDGF NOX2 Classic phagocytic form of NOX Expressed in macrophages Inducible NOX4 Regulated at level of transcription Increased in inflammation and fibrosis Induced by TGFb Liver fibrosis is blocked by NOX 1/4 inhibitor GKT137831 Vehicle NOX1/4 inhibitor WT SOD1mu B Sirius red positive area (%) 15 10 5 0 NI WT SOD1mu * * * - - + + - - + + C Vehicle NOX1/4 inhibitor SOD1mu oil WT D Vehicle NOX1/4 inhibitor WT SOD1mu WT SOD1mu oil oil oil oil -SMA -actin E collagen 1(I) mrna (fold) 120 * * * 80 40 0 NI - - + + - - + + TIMP-1 mrna (fold) 100 50 0 NI - - + + - - + + TGF- mrna (fold) 15 10 WT SOD1mu * * * 5 0 NI - - + + - - + + oil oil oil * * * 6
TGF 1 induces liver fibrogenesis Activates HSCs Stimulates synthesis of ECM Collagens Glycoproteins Proteoglycans Inhibits ECM degradation Inhibits MMPs Stimulates protease inhibitors Increases TGF 1 mrna Examples of ko mice TGFb1, Smad3, MMP13,TLR4 Integrin αvβ6 activates TGFβ TGFb RDG LTBP1 b6 RDG a v TGFbR Sheppard D. Integrin-mediated activation of latent transforming growth factor beta. Cancer Metastasis Rev. 2005 Sep;24(3):395-402. 7
10/1/12 Hepatocyte apoptosis induces hepatic fibrosis Kupffer cell Phagocytosis Apoptosis MIP-2 KC TGFb1 HSC Apoptotic bodies Hepatocyte Activated HSC F I B R O S I S Hepatocyte Examples of ko mice Fas, Cathepsin B, Bcl-xL, IKKb TLR4-mutant mice display strongly reduced fibrosis after BDL!SMA-staining BDL TLR4WT Sham BDL Sham BDL TLR4mutant Sham BDL Sirius red pos-area (%) Sirius red staining Sham TLR4mutant Hydroxyproline (mg/g liver) TLR4WT 20 Sirius red 15 10 5 0 400 sham BDL Hydroxyproline 300 200 100 0 sham BDL TLR4WT TLR4mutant 8
LPS activates TLR4 to induce fibrosis LPS TGFb-RI TLR4 TGF Chemokines Bambi quiescent HSC Adhesion molecules Kupffer cell HSC activation Collagen deposition Liver fibrosis Studies of modifier genes in patients Modifier genes: Genes that alters the expression of other gene(s) and affects phenotype. Genetic polymorphisms: Stable variations in DNA that are observed in 1% or more of the population (i.e. SNPs). Association studies: epidemiological studies assessing the role of genetic polymorphisms in disease susceptibility and/or progression. 9
Modifiers of hepatic fibrosis in patients HCV HFE, Angiotensinogen, TGFb1, CTLA-4, MnSOD, TNFa, IL10, MTP, MCPI/Z, MMP 1/3/9, RANYES, Kertin 8/18, a AT, C5, DDX5, CPT-1A, IFN8 ALD HFE, TGFb1, CTLA-4, MnSOD, TNFa, IL10, CD14, GSTMI/T1 NASH HFE, Angiotensinogen, TGFb1, MnSOD, TNFa, MTP Problems: Inadequate sample size, different populations, statistical stringency, small genetic effects blue genes that modify multiple liver diseases Hepatology 37:653, 2003 Celera Whole Genome Scan for Cirrhosis in HCV Gastro, 2006 Hepatology, 2007 10
Modifier Genes of Hepatic Fibrosis in HCV (Celera, Hepatol. 2008) Predictors Gene (Chr) Risk of genotype or phenotype Frequency (Caucasian Patients) SNP1b SNP2 SNP3 SNP4 SNP5 SNP6 SNF7 Sex Age Alcohol AZIN1 (Chr 8) TLR4 (Chr 9) TRPM5 (Chr 11) None (Chr 15) None (Chr 1) None (Chr 3) AQP2 (Chr 12) GG CC CT, CC AG, AA GG AG, AA GG Male Older 50 g/day 86.9% 87.3% 63.6% 41.6% 72.3% 18.5% 66.7% 69.3% N/A 32.9% Chronic Inflammation leads to fibrosis WBC recruitment INJURY LPS TLR4 Kupffer cell activation TNF- IL-1 MIP-1 HSC activation Activated Myofibroblast TGF-b Ang II Leptin ROS FIBROSIS Ko mice TNF-, TLR4, CD14, LBP 11
Reversal of CC1 4 -induced Hepatic Fibrosis Collagen Activated HSCs CC1 4 Post 7d Iredale, JCI 102:538, 1998 Fibrosis and Resolution Fibrosis Resolution ECMs Collagenase TIMPs Activated HSCs 12
10/1/12 Removing known stimuli of fibrogenesis reverses fibrosis in patients Inflammation autoimmune hepatitis Ethanol alcoholic liver disease Copper Wilson s disease Iron hemochromatosis Schistosomes schistosomiasis Biliary Obstruction 2ndary biliary cirrhosis Hepatitis B and C viral hepatitis NASH obesity, diabetes Lamivudine decreases activated HSCs a smooth muscle actin staining Pre-LAM Post-LAM J Hepatol 35:749,2001 13
Rates of Regression of HCV cirrhosis with SVR D Ambrosio et al. Hepatology. Volume 56, pages 532, 2012 Gastric Bypass Surgery Decreased hepatic fibrogenesis in NAFLD Decreased steatosis Decreased activated hepatic stellate cells Pre-GBS Decreased mrnas for intermediate biomarkers of fibrosis: collagen α1(i), TGFβ1, TIMP-1, and αsma Post-GBS 14
Chronic Liver Injury Pathogenesis of Hepatic Fibrosis Apoptosis Quiescent HSC Angiotensin PDGF Leptin Fibroblast Fibrogenesis NADPH oxidase Fibrocyte ROS TGFb1 Proliferation SMA+ Myofibroblast 15