Hedgehog Pathway
Targeted by Vitamin E
Therapy in NASH
AASLD
Washington, DC
November 03, 2013
DukeUniversityMedicalCenter
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
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.
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Hypothesis
Response to Vitamin E therapy
would be associated with
reduced Hedgehog pathway activity
in patients with NASH
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Design/Methods
Vitamin E
(n = 30)
Rx
Response (+)
(-)
Placebo
(n = 29)
(+)
(-)
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+)
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Results
Baseline clinical and histological characteristics of
the study population
Placebo
(N=29)
46±11
58.6%
82.8%
6.9%
35.5±7.1
58.6%
48.3%
74.9±37.0
49.4±25.4
4.9±3.9
Age, years
Gender, female %
Race, White %
Ethnicity, Hispanic %
BMI, kg/m2
HTN, %
IGT, %
Serum ALT, IU/L
Serum AST, IU/L
HOMA-IR
Histological scores
Hepatocyte ballooning, 17.2%; 34.5%; 48.3%
grade 0, 1, 2
Fibrosis, stage 0, 1, 2, 3
13.8%; 31.0%;
34.5%; 20.7%
Histological improvement, %**
27.6%
Vitamin E
(N=30)
47±10
60.0%
80.0%
20.0%
35.0±7.5
66.7%
56.7%
87.6±47.3
57.0±29.5
5.7±4.5
P-value#
0.60
0.91
0.79
0.13
0.80
0.52
0.52
0.26
0.30
0.46*
16.7%; 36.7%; 46.7%
0.98
6.7%; 40.0%;
30.0%; 23.3%
43.3%
0.75
0.21
#: 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
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Results
Baseline Immunohistochemistry Data
Shh-positive ballooned hepatocytes,
cells/HPF
K8/18-negative or ubiquitin-positive foci,
cells/HPF
α-SMA-positive stain score, 0 to 4
gli-2+/sox-9+ stain score, 0 to 4
Placebo
(N=29)
Vitamin E
(N=30)
P-value#
4.5 ± 9.1
4.4 ± 8.0
0.60
2.8 ± 5.6
3.4 ± 6.3
0.73
2(1, 2.8)
1(1, 2)
2(2, 3)
2(1, 2)
0.34
0.23
#: p-values were from Wilcoxon Rank Sum tests.
Data were presented as mean ± SD for numeric variables and median (IQR) for ordinal variables.
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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)
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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)
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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)
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Results
 Regardless of the treatment arm, a
response to therapy (as defined by the
PIVENS trial design) was associated
with greater decrease in Shh+
hepatocytes than non-response
(P=0.007)
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A
B
Shh (brown) pretreatment VitE responder
(patient “X”) (400x)
Shh post-treatment VitE responder
(patient “X”) (400x)
C
K8/18 (brown)/Ub (red) pretreatment VitE
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responder
(patient “X”) (400x)
D
K8/18 (brown)/Ub (red) post-treatment VitE
responder (patient “X”) (400x)
IHC: gli-2 (red), sox-9 (blue)
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α-SMA (brown)
A
IHC: gli-2 (red), sox-9 (blue)
α-SMA (brown)
gli-2/sox-9/α-SMA pretreatment VitE responder
(patient “X”) portal tract (400x)
gli-2/sox-9/α-SMA pretreatment VitE responder
(patient “X) zone 3 (400x)
C
D
gli-2/sox-9/α-SMA post-treatment VitE
responder (patient “X”) portal tract (400x)
gli-2/sox-9/α-SMA post-treatment VitE
responder (patient “X”) zone 3 (400x)
B
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
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Illustrative Model
Ballooning
Dying hepatocyte
Progenitors
Lipotoxicity
Quiescent
HSC
Hedgehog
Ligands
Myofibroblasts
Ductular
Reaction
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Speculation
 Inhibiting Hedgehog and/or its effectors might
be a new treatment approach for NASH
- Smoothened antagonist
Mouse
improves NASH*
models
- 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.
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NASH CRN Credits
Principal Investigators
Data Coordinating Center (JHU)
Pathologists
• Patricia Belt
Adult Centers
• Elizabeth Brunt (Wash U)
• Jeanne Clark
• CCF/CWRU: Arthur McCullough • David Kleiner (NCI)
• Michele Donithan
• CU: Joel Lavine
• Cynthia Behling (UCSD)
• Erin Hallinan
• DUKE: Anna Mae Diehl
• Melissa Contos (VCU)
• Milana Isaacson
• IU: Naga Chalasani
• Oscar Cummings (IU)
• SLU: Brent Tetri
• Linda Ferrell, Ryann Gill (UCSF) • Patrick May
• Laura Miriel
• UCSD: Rohit Loomba
• Cynthia Guy (DUKE)
• James Tonascia
• UCSF: Norah Terrault
• Rish Pai (CWRU)
• Aynur Ünalp-Arida
• VMMC: Kris Kowdley
• Matthew Yeh (UW)
• Mark Van Natta
• VCU: Arun Sanyal
• Ivana Vaughn
• Laura Wilson
Pediatric Centers
• Katherine Yates
• BCM: Sarah Barlow
NIDDK Program Official
• CINC: Stavra Xanthakos
• Averell Sherker
• CU: Joel Lavine
NIDDK Project Scientist
• IU: Jean Molleston
• Edward Doo
• JHU: Ann Scheimann
• NWU: Peter Whitington
• SLU: Ajay Jain
• UCSD: Jeffrey Schwimmer
• UCSF: Philip Rosenthal
• UW: Karen Murray
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Thank you
 Anna Mae Diehl’s Lab
Chief, 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
DukeUniversityMedicalCenter
Anna Mae Diehl
Ayako Suzuki
Manal Abdelmalek
Jim Burchette
Additional slides
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Results
Changes in serum AST
150
100
50
0
-50
y = 1.3852x - 9.8841
R=0.16, p=0.002
-100
-150
-40
-20
0
20
Changes in Shh+ BH
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40
Partial residual of changes
in serum AST
Correlation between changes in Shh+
ballooned hepatocytes and changes in serum AST.
50
40
30
20
10
0
-10
-20
-100
y = 0.248x + 1.8887
R=0.75, p<0.0001
0
100
200
Partial residual of changes in
Shh+ BH
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.
Placebo
(N=29)
Vitamin
E
(N=30)
pvalue
Shh-positive ballooned
hepatocytes, cells/HPF
1.2±10.7
-3.0±8.3
0.048
-4.4±2.1
0.037
K8/18 -negative cells or
ubiquitin-positive foci/HPF
0.2±1.4
-2.9±1.3
0.082
-2.6±1.2
0.037
α-SMA-positive stain score,
Grade 0 to 4
0 (0, 0)
0 (-1, 0)
0.097
1.8[0.6,
5.6]
0.34
gli-2/sox-9-positive stain score,
Grade 0 to 4
0 (0, 0)
0 (-1, 0)
0.068
1.7[0.5,
5.5]
0.36
Changes in:
β±SE/
OR[95%C
pI]
value
Data were presented as mean ± SD for numeric variables and median (IQR) for ordinal variables.
#: Wilcoxon Rank-Sum tests
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