hedgehog pathway targeted by vitamin e therapy in nash
DESCRIPTION
Hedgehog Pathway Targeted by Vitamin E Therapy in NASH. Cynthia D Guy 1 Ayako Suzuki 2 Manal F Abdelmalek 1 James L Burchette 1 Anna Mae Diehl 1 Duke University Medical Center 1 University of Arkansas for Medical Sciences 2 f or the NASH CRN. AASLD Washington, DC - PowerPoint PPT PresentationTRANSCRIPT
DukeUniversityMedicalCenter
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 CRNAASLDWashington, DC November 03, 2013
DukeUniversityMedicalCenter
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.
DukeUniversityMedicalCenter
Hypothesis
Response to Vitamin E therapy would be associated with
reduced Hedgehog pathway activity
in patients with NASH
DukeUniversityMedicalCenter
Design/MethodsVitamin E(n = 30)
Placebo(n = 29)
RxResponse (+)(+) (-) (-)
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+)
DukeUniversityMedicalCenter
Placebo(N=29)
Vitamin E(N=30)
P-value#
Age, years 46±11 47±10 0.60Gender, female % 58.6% 60.0% 0.91
Race, White % 82.8% 80.0% 0.79Ethnicity, Hispanic % 6.9% 20.0% 0.13
BMI, kg/m2 35.5±7.1 35.0±7.5 0.80HTN, % 58.6% 66.7% 0.52IGT, % 48.3% 56.7% 0.52
Serum ALT, IU/L 74.9±37.0 87.6±47.3 0.26Serum AST, IU/L 49.4±25.4 57.0±29.5 0.30
HOMA-IR 4.9±3.9 5.7±4.5 0.46*Histological scores
Hepatocyte ballooning, grade 0, 1, 2
17.2%; 34.5%; 48.3%
16.7%; 36.7%; 46.7%
0.98
Fibrosis, stage 0, 1, 2, 3 13.8%; 31.0%; 34.5%; 20.7%
6.7%; 40.0%; 30.0%; 23.3%
0.75
Histological improvement, %**
27.6% 43.3% 0.21 #: p-values were from t-test or Chi-square test, except for * (Wilcoxon Rank-sum test). **: defined by the PIVENS study designBMI: body mass index, IGT: impaired glucose tolerance, HTN: systemic hypertension, HOMA-IR: Homeostasis Model of Assessment - Insulin Resistance
ResultsBaseline clinical and histological characteristics of
the study population
DukeUniversityMedicalCenter
Placebo(N=29)
Vitamin E(N=30)
P-value#
Shh-positive ballooned hepatocytes, cells/HPF 4.5 ± 9.1 4.4 ± 8.0 0.60
K8/18-negative or ubiquitin-positive foci, cells/HPF 2.8 ± 5.6 3.4 ± 6.3 0.73
α-SMA-positive stain score, 0 to 4 2(1, 2.8) 2(2, 3) 0.34gli-2+/sox-9+ stain score, 0 to 4 1(1, 2) 2(1, 2) 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.
Results Baseline Immunohistochemistry Data
DukeUniversityMedicalCenter
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)
DukeUniversityMedicalCenter
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)
DukeUniversityMedicalCenter
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)
DukeUniversityMedicalCenter
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)
DukeUniversityMedicalCenter
Shh (brown) pretreatment VitE responder (patient “X”) (400x)
Shh post-treatment VitE responder (patient “X”) (400x)
K8/18 (brown)/Ub (red) pretreatment VitEresponder (patient “X”) (400x)
K8/18 (brown)/Ub (red) post-treatment VitEresponder (patient “X”) (400x)
A B
C D
DukeUniversityMedicalCenterIHC: 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)
gli-2/sox-9/α-SMA post-treatment VitEresponder (patient “X”) portal tract (400x)
gli-2/sox-9/α-SMA post-treatment VitEresponder (patient “X”) zone 3 (400x)
IHC: gli-2 (red), sox-9 (blue) α-SMA (brown)A B
C D
DukeUniversityMedicalCenter
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
DukeUniversityMedicalCenter
Dying hepatocyte
Progenitors
Myofibroblasts
HedgehogLigands
Quiescent HSC
DuctularReaction
Lipotoxicity
Ballooning
Illustrative Model
DukeUniversityMedicalCenter
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.
Mousemodels
DukeUniversityMedicalCenter
NASH CRN Credits
Principal InvestigatorsAdult 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
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
DukeUniversityMedicalCenter
Thank you
Anna Mae Diehl’s LabChief, Division of Gastroenterology and Hepatology, Department of Medicine, Duke University Medical Center
Steve ChoiGregory MichelottiMarzena-Swiderska-SynGuanhua XieGamze F. KaracaLeandi KrugerThiago D PereiraMariana V MachadoKatherine GarmanCynthia MoylanJerome BoussierW. Carl Stone
Anna Mae DiehlAyako SuzukiManal AbdelmalekJim Burchette
DukeUniversityMedicalCenter
Additional slides
DukeUniversityMedicalCenter
ResultsCorrelation between changes in Shh+ ballooned hepatocytes and changes in serum AST.
-40 -30 -20 -10 0 10 20 30 40-150
-100
-50
0
50
100
150
f(x) = 1.38520742096137 x − 9.88410005014861
Changes in Shh+ BH
Chan
ges
in s
erum
AST
-100 -50 0 50 100 150-20-10
01020304050
f(x) = 0.248014418729364 x + 1.88872537962101
Partial residual of changes in Shh+ BH
Part
ial r
esid
ual o
f ch
ange
s in
ser
um A
ST
ResultsCorrelation 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).
ResultsCorrelation 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).
DukeUniversityMedicalCenter
Changes in:Placebo(N=29)
Vitamin E
(N=30)
p-value
β±SE/OR[95%
CI]
p-valu
e
Shh-positive ballooned hepatocytes, cells/HPF
1.2±10.7
-3.0±8.
30.048 -4.4±2.1 0.03
7
K8/18 -negative cells or ubiquitin-positive foci/HPF 0.2±1.4
-2.9±1.
30.082 -2.6±1.2 0.03
7α-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 40 (0, 0) 0 (-1,
0) 0.068 1.7[0.5, 5.5] 0.36
ResultsComparison 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