Reversal of pulmonary hypertension in a Sugen/hypoxic rat model - - PowerPoint PPT Presentation

Reversal of pulmonary hypertension in a Sugen/hypoxic rat model using an ASK1 inhibitor

Kathryn Wilson

Background: Pulmonary arterial hypertension pathogenesis

Lumen

Lumen

Fibroblast Proliferation Hypertrophy Immune cell recruitment Collagen and ECM deposition Smooth muscle cells (SMC) Proliferation Hypertrophy Endothelial cells (EC) EC dysfunction SMC infiltration Adventitia Intima Media Normal pulmonary artery Pulmonary hypertensive artery

Background: Pulmonary arterial hypertension pathogenesis

Normal Pulmonary arterial hypertension Pulmonary vascular remodelling Cardiac functional and structural changes

Background: An imbalance between ROS and antioxidants results in oxidative stress

Redox Balance

Hydrogen peroxide (H2O2) Superoxide anion (●O2–) Hydroxyl moiety (●OH) Hypochlorite (ClO−) Peroxynitrite (ONOO−) Glutathione peroxidase Superoxide Dismutase Thioredoxin Catalase

OXIDATIVE STRESS

Glutathione

(µmol/gP)

Decreased Antioxidants

Malondialdehyde (MDA)

(µmol/gP)

Increased ROS

Reis et al. Pulm Circ. 3, 2013

Pulmonary hypertension

ASK1 Oxidative Stress ASK1

TrxReduced TrxOxidized

p38 JNK

MKK3/6 MKK4/7

Background: Apoptosis-Signal Regulating Kinase1 (ASK1) mediates oxidative stress-induced changes

• ASK1 is activated in response to

increased oxidative stress

• ASK1 promotes cell death, fibrosis, and

inflammation via activation of JNK and p38 MAPK

• ASK1 is increased in patient PAH

fibroblasts

• p38 MAPK inhibition reverse PH

P

Inflammation Fibrosis Apoptosis Necrosis

ASK1

Oxidative Stress

ASK1 inhibitor Hypothesis: ASK1 inhibition may attenuate oxidative-stress induced maladaptive remodeling and reverse PAH

Rationale for ASK1 inhibition as a treatment in PAH

Pulmonary vascular remodelling Cardiac functional and structural changes

Previous work: ASK1 inhibition in an in vitro PAH model

• Rat pulmonary artery fibroblasts

(RPAF)

• Acute hypoxia with a small

molecule inhibitor of ASK1

• GS444217
• ASK1 inhibition alters downstream

MAPK signalling

• ASK1 inhibition prevents hypoxia

induced RPAF proliferation

counts / m inute + A S K 1 in h ib ito r 2 0 0 4 0 0 6 0 0

N o rm o x ia H y p o x ia

**

Previous work: ASK1 inhibition in an in vitro PAH model

• ASK1 inhibition prevents hypoxia

induced RPAF migration

• ASK1 inhibition alters cytokine

expression by RPAF

s cra tch c lo se r (% re d u c tio n fro m 0 h ) N o rm N o rm + i H yp H yp + i 2 0 4 0 6 0 8 0 1 0 0

**

Normoxia Hypoxia Hypoxia +GS444217

T IM P -1 C IN C -1 C IN C -2 a /ß s IC A M 1 L IX IL -6 M IP -3 a T N F a 0 .0 0 .5 1 .0 1 .5

n o rm a lis e d m e a n p ix e l d e n s ity N o rm o xia N o rm o x ia + G S 4 4 4 2 1 7 H yp o xia H y p o x ia + G S 4 4 4 2 1 7

Previous work: ASK1 inhibition in an in vitro PAH model

ASK1 inhibition prevents cellular changes in RPAF PAH model

ASK1

Oxidative Stress

↑ Proliferation ↑ Migration ↑ Cytokine expression

ASK1 inhibitor

↓ Proliferation ↓ Migration ↓ Cytokine expression Interim summary

Can ASK1 inhibition reverse PH in an in vivo model?

Current study: ASK1 inhibition in an in vivo PAH model

SU5415 (20mg/kg, S.C) 2 weeks hypoxia + 3 weeks normoxia RATS 3 week old Male Sprague Dawley 5 weeks normoxia Week 0 Week 2 Week 5 Week 0 Week 5

Haemodynamic and cardiac measurements

Current study: ASK1 inhibition in an in vivo PAH model

N o rm S u H x 0 .0 0 .1 0 .2 0 .3 0 .4

R V /(L V + S e p tu m ) a t 5 w e e ks R V /(L V + S e p tu m ) * N o rm S u H x 2 0 4 0 6 0 R V S P a t 5 w e e ks R V S P (m m H g ) *

N o rm S u H x 5 0 1 0 0 1 5 0

S A P a t 5 w e e ks

S A P (m m H g )

Norm SuHx

LV RV LV RV

N o rm S u H x 1 0 0 2 0 0 3 0 0 4 0 0 5 0 0

H e a rt ra te a t 5 w e e ks h e a rt ra te (b p m )

+ 3 weeks vehicle + 3 weeks vehicle + 3 weeks ASK1 inhibitor + 3 weeks ASK1 inhibitor Week 8 Week 8 Week 8 Week 8

Haemodynamic and cardiac measurements

SU5415 (20mg/kg, S.C) 2 weeks hypoxia + 3 weeks normoxia RATS 3 week old Male Sprague Dawley 5 weeks normoxia Week 0 Week 2 Week 5 Week 0 Week 5

Reversal treatment arm PAH arm

Current study: ASK1 inhibition in an in vivo PAH model

N o rm N o rm + i S u H x S u H x + i 2 0 4 0 6 0

R V S P a t 8 w e e ks

m R V S P (m m H g ) * * *

N orm N orm + i S uH x S uH x + i 0 .0 0 .1 0 .2 0 .3 0 .4

R V /(L V + s e p tu m ) a t 8 w e e ks

R V /(L V + S e p tu m ) * N o rm N o rm + i S u H x S u H x + i 5 0 1 0 0 1 5 0

S A P a t 8 w e e ks

S A P (m m H g )

Norm Norm + i SuHx SuHx + i

LV RV LV RV LV RV LV RV

Current study: ASK1 inhibition in an in vivo PAH model

N o rm N o rm + i S u H x S u H x + i 1 0 0 2 0 0 3 0 0 4 0 0 5 0 0

H e a rt ra te a t 8 w e e ks

h e a rt ra te (b p m )

N o rm S u H x S u H x + i 2 0 4 0 6 0 8 0 1 0 0

P u lm o n a ry va scu la r re m o d e llin g

% re m o d e lle d ve sse ls *

Norm SuHx

50µM 50µM 50µM

SuHx + i

Current study: ASK1 inhibition in an in vivo PAH model

α smooth muscle actin

Summary

Previous work

• ASK1 inhibition prevents hypoxia induced cellular changes in vitro

– ↓cellular proliferation – ↓cellular migration – Alters cellular cytokine expression Current work

• ASK1 inhibition reverses phenotype changes in a SuHx rat model of PAH

– ↓ RVSP – ↓ vascular remodelling – Trend to reduction in RV/LV+septum

• ASK1 inhibition reverses pulmonary hypertension in a Sugen/hypoxic model

Acknowledgements

We acknowledge the support of the PVRI who has funded the travel expenses and registration fees for this meeting

David Welsh Geeshath Jayasekera Andrew Peacock Colin Church Monika Setiwan Hanna Buist Colin Hughes Margaret Nilsen Mandy MacLean Grant Budas John Liles