Hedgehog Pathway Targeted by Vitamin E Therapy in NASH Cynthia D Guy1 Ayako Suzuki2 Manal F Abdelmalek1 James L Burchette1 Anna Mae Diehl1 Duke University Medical Center1 University of Arkansas for Medical Sciences2 for the NASH CRN AASLD Washington, DC November 03, 2013
Background • Hedgehog signaling promotes fibrogenic repair • Hedgehog pathway is re-activated during NASH • Ballooned hepatocytes Hedgehog ligands NASH progression • PIVENS Trial (NASH CRN) Vitamin E improved NASH ? Choi SS, Omenetti A, Syn WK, Diehl AM. The role of Hedgehog signaling in fibrogenic repair. Int J Biochem Cell Biol. 2011 43(2):238-244. Jung Y, Witek RP, Syn WK, Choi SS, Omenetti A, Premont R, Guy CD, Diehl AM. Signals from dying hepatocytes trigger growth of liver progenitors. Gut. 2010 May;59(5):655-65. doi: 10.1136/gut.2009.204354 Rangwala F, Guy CD, Lu J, Suzuki A, Burchette JL, Abdelmalek MF, Chen W,Diehl AM. Increased production of sonic hedgehog by ballooned hepatocyte. J Pathol. 2011 Jul;224(3):401-10. doi: 10.1002/path.2888. Epub 2011 May 5. Guy CD, Suzuki A, Zdanowicz M, Abdelmalek MF, Burchette J, Unalp A, Diehl AM; NASH CRN. Hedgehog pathway activation parallels histologic severity of injury and fibrosis in human nonalcoholic fatty liver disease. Hepatology. 2012 Jun;55(6):1711-21. doi: 10.1002/hep.25559. Epub 2012 Apr 18.
Hypothesis Response to Vitamin E therapy would be associated with reduced Hedgehog pathway activity in patients with NASH
Design/Methods Vitamin E (n = 30) Placebo (n = 29) Rx Response (+) (-) (+) (-) Immunohistochemistry/Analysis • #’s of Hh ligand producing cells • Shh+ cells • #’s of ballooned hepatocytes • K8/18-negative/Ub+ cells • #’s of Hh-responsive cells • Progenitors (gli-2+/sox-9+) • Myofibroblasts (gli-2+/α-SMA+)
Results • Baseline clinical and histological characteristics of • the study population #: p-values were from t-test or Chi-square test, except for * (Wilcoxon Rank-sum test). **: defined by the PIVENS study design BMI: body mass index, IGT: impaired glucose tolerance, HTN: systemic hypertension, HOMA-IR: Homeostasis Model of Assessment - Insulin Resistance
Results • BaselineImmunohistochemistry Data #: p-values were from Wilcoxon Rank Sum tests. Data were presented as mean ± SD for numeric variables and median (IQR) for ordinal variables.
Results • Regardless of the treatment arm, #’s of Shh+ hepatocytes correlated with - K8/18-negative/Ub+ ballooned hepatocytes (r2=0.47; P<0.001) - AST (r2=0.15; P=0.002)
Results • After adjusting for baseline numbers of Shh+ and K8/18-negative Ub+ cells, by multiple linear regression analysis, - Changes in #’s of Shh+ hepatocytes correlated with changes in: - AST (r2=0.75; P<0.001) - ALT (r2=0.26, P<0.0001) -H&E Ballooning (P=0.004) -Trichrome Fibrosis stage (P=0.02) - Ductular reaction #’s of Shh-responsive progenitors (P=0.03) #’s of α-SMA+ cells (P=0.10)
Results • Vitamin E group: greater reduction in - Shh+ hepatocytes (P<0.05) -K8/18-negative/Ub+ cells, foci (P=0.08) • Multiple linear regression analysis, VitE group had greater decrease in - Shh+ hepatocytes (P<0.04) -K8/18-negative/Ub+ (ballooned) hepatocytes (P< 0.04)
Results • Regardless of the treatment arm, aresponse to therapy (as defined by the PIVENS trial design) was associated with greater decrease in Shh+ hepatocytes than non-response (P=0.007)
A B Shh post-treatment VitE responder (patient “X”) (400x) Shh (brown) pretreatment VitE responder (patient “X”) (400x) D C K8/18 (brown)/Ub (red) post-treatment VitE responder (patient “X”) (400x) K8/18 (brown)/Ub (red) pretreatment VitE responder (patient “X”) (400x)
IHC: gli-2 (red), sox-9 (blue) α-SMA (brown)
IHC: gli-2 (red), sox-9 (blue) α-SMA (brown) A B gli-2/sox-9/α-SMA pretreatment VitE responder (patient “X”) portal tract (400x) gli-2/sox-9/α-SMA pretreatmentVitE responder (patient “X) zone 3 (400x) C D • gli-2/sox-9/α-SMA post-treatment VitE • responder (patient “X”) zone 3 (400x) gli-2/sox-9/α-SMA post-treatment VitE responder (patient “X”) portal tract (400x)
Conclusions • Reduction of NASH injury with vitamin E Rx decreased: - hepatocyte ballooning - production of Hh ligand - #’s of Hh-responsive progenitors • Inhibition of Hh pathway activity was associated with improved: - liver injury - fibrosis stage - treatment response
Illustrative Model Dying hepatocyte Ballooning Progenitors Lipotoxicity Quiescent HSC Hedgehog Ligands Myofibroblasts Ductular Reaction
Speculation • Inhibiting Hedgehog and/or its effectors might be a new treatment approach for NASH - Smoothened antagonist improves NASH* - Antagonists of Osteopontin (fibrogenic Hedgehog target gene) improve NASH** *Hirsova P, Ibrahim Sh, Bronk SF, Yagita H, Gores GJ. Vismodegib suppresses TRAIL-mediated liver injury in a mouse model of nonalcoholic steatohepatitis. PLoS One. 2013 Jul 22;8(7):e70599doi: 10.1371/journal.pone.0070599. Print 2013. ** Syn WK, Choi SS, Liakou E, Karaca GF, Agboola KM et al. Osteopontin is Induced by Hedgehog Pathway Activation and Promotes Fibrosis Progression in Nonalcoholic Steatohepatitis. Hepatology. 2011;53:106-115. Mouse models
Principal Investigators Adult Centers CCF/CWRU: Arthur McCullough CU: Joel Lavine DUKE: Anna Mae Diehl IU: Naga Chalasani SLU: Brent Tetri UCSD: Rohit Loomba UCSF: Norah Terrault VMMC: Kris Kowdley VCU: Arun Sanyal Pediatric Centers BCM: Sarah Barlow CINC: Stavra Xanthakos CU: Joel Lavine IU: Jean Molleston JHU: Ann Scheimann NWU: Peter Whitington SLU: Ajay Jain UCSD: Jeffrey Schwimmer UCSF: Philip Rosenthal UW: Karen Murray Data Coordinating Center (JHU) Patricia Belt Jeanne Clark Michele Donithan Erin Hallinan Milana Isaacson Patrick May Laura Miriel James Tonascia Aynur Ünalp-Arida Mark Van Natta Ivana Vaughn Laura Wilson Katherine Yates NASH CRN Credits Pathologists • Elizabeth Brunt (Wash U) • David Kleiner (NCI) • Cynthia Behling (UCSD) • Melissa Contos (VCU) • Oscar Cummings (IU) • Linda Ferrell, Ryann Gill (UCSF) • Cynthia Guy (DUKE) • Rish Pai (CWRU) • Matthew Yeh (UW) NIDDK Program Official • Averell Sherker NIDDK Project Scientist • Edward Doo
Thank you Anna Mae Diehl Ayako Suzuki Manal Abdelmalek Jim Burchette • Anna Mae Diehl’s LabChief, Division of Gastroenterology and Hepatology, Department of Medicine, Duke University Medical Center Steve Choi Gregory Michelotti Marzena-Swiderska-Syn Guanhua Xie Gamze F. Karaca Leandi Kruger Thiago D Pereira Mariana V Machado Katherine Garman Cynthia Moylan Jerome Boussier W. Carl Stone
Results Correlation between changes in Shh+ ballooned hepatocytes and changes in serum AST.
Results Correlation between changes in Shh+ ballooned hepatocytes and changes in K8/18-negative hepatocytes/ubiquitin-positive foci. Figure legend: The partial regression plot shows the relationship between the changes in Shh+ ballooned hepatocytes and the changes in K8/18-negative hepatocytes/ubiquitin-positive foci after adjusting for baseline values. The horizontal axis is partial residual of the changes in K8/18-negative/ubiquitin-positive hepatocytes while the vertical axis is partial residual of the changes in Shh+ ballooned hepatocytes. Solid line is a regression line, and dotted curve lines depict 95% confidence curve. There was significant positive correlation (p=0.0039).
Results Correlation between changes in Shh+ ballooned hepatocytes and changes in serum AST. Figure legend: The partial regression plot shows the relationship between the changes in Shh+ ballooned hepatocytes and the changes in serum AST after adjusting for baseline values. The horizontal axis is partial residual of the changes in Shh+ ballooned hepatocytes while the vertical axis is partial residual of the changes in serum AST. Solid line is a regression line, and dotted curve lines depict 95% confidence curve. There was significant positive correlation (p<0.0001).
Results Comparison of changes in immunohistochemical data with and without adjusting for baseline values: Placebo vs. Vitamin E groups. • Data were presented as mean ± SD for numeric variables and median (IQR) for ordinal variables. • #: Wilcoxon Rank-Sum tests