Hedgehog Pathway Targeted by Vitamin E Therapy in NASH

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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

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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/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+)

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  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

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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

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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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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

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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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Dying hepatocyte

Progenitors

Myofibroblasts

HedgehogLigands

Quiescent HSC

DuctularReaction

Lipotoxicity

Ballooning

Illustrative Model

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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

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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

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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

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Additional slides

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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).

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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