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

Background: Pulmonary arterial hypertension pathogenesis Pulmonary vascular remodelling Cardiac functional and structural changes Normal Pulmonary arterial hypertension

Background: An imbalance between ROS and antioxidants results in oxidative stress OXIDATIVE STRESS Glutathione peroxidase Superoxide Dismutase Decreased Antioxidants Hydrogen peroxide (H2O2) Thioredoxin Increased ROS Superoxide anion ( ● O2 – ) Catalase Redox Hydroxyl moiety ( ● OH) Hypochlorite (ClO − ) Balance Peroxynitrite (ONOO − ) Malondialdehyde Glutathione Pulmonary ( µ mol/gP) ( µ mol/gP) hypertension (MDA) Reis et al. Pulm Circ. 3, 2013

Background: Apoptosis-Signal Regulating Kinase1 (ASK1) mediates oxidative stress-induced changes • ASK1 is activated in response to Oxidative Stress increased oxidative stress P ASK1 ASK1 Trx Oxidized • ASK1 promotes cell death, fibrosis, and Trx Reduced inflammation via activation of JNK and MKK3/6 MKK4/7 p38 MAPK p38 JNK • ASK1 is increased in patient PAH fibroblasts Inflammation Apoptosis Fibrosis Necrosis • p38 MAPK inhibition reverse PH

Rationale for ASK1 inhibition as a treatment in PAH Oxidative Stress ASK1 ASK1 inhibitor Pulmonary vascular remodelling Cardiac functional and structural changes Hypothesis: ASK1 inhibition may attenuate oxidative-stress induced maladaptive remodeling and reverse PAH

Previous work: ASK1 inhibition in an in vitro PAH model Rat pulmonary artery fibroblasts • (RPAF) • Acute hypoxia with a small 6 0 0 molecule inhibitor of ASK1 ** N o rm o x ia H y p o x ia counts / m inute -GS444217 4 0 0 • ASK1 inhibition alters downstream 2 0 0 MAPK signalling 0 + A S K 1 in h ib ito r • ASK1 inhibition prevents hypoxia induced RPAF proliferation

Previous work: ASK1 inhibition in an in vitro PAH model ASK1 inhibition prevents hypoxia • induced RPAF migration s cra tch c lo se r (% re d u c tio n fro m 0 h ) 1 0 0 • ASK1 inhibition alters cytokine 8 0 ** expression by RPAF 6 0 N o rm o xia n o rm a lis e d m e a n p ix e l d e n s ity 4 0 1 .5 N o rm o x ia + G S 4 4 4 2 1 7 H yp o xia Normoxia H y p o x ia + G S 4 4 4 2 1 7 2 0 1 .0 0 0 .5 N o rm N o rm H yp H yp Hypoxia + i + i 0 .0 s IC A M 1 L IX IL -6 T IM P -1 C IN C -1 C IN C -2 a /ß M IP -3 a T N F a Hypoxia +GS444217

Previous work: ASK1 inhibition in an in vitro PAH model Interim summary Oxidative Stress ASK1 ASK1 inhibitor ↓ Proliferation ↑ Proliferation ↓ Migration ↑ Migration ↓ Cytokine expression ↑ Cytokine expression ASK1 inhibition prevents cellular changes in RPAF PAH model

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) + 3 weeks normoxia 2 weeks hypoxia RATS Haemodynamic 3 week old Week 2 Week 5 Week 0 and cardiac Male measurements Sprague Dawley 5 weeks normoxia Week 0 Week 5

Current study: ASK1 inhibition in an in vivo PAH model R V S P a t 5 w e e ks R V /(L V + S e p tu m ) a t 5 w e e ks 0 .4 6 0 * * R V /(L V + S e p tu m ) 0 .3 R V S P (m m H g ) 4 0 RV LV 0 .2 2 0 0 .1 0 .0 0 N o rm S u H x Norm N o rm S u H x S A P a t 5 w e e ks H e a rt ra te a t 5 w e e ks 1 5 0 5 0 0 4 0 0 h e a rt ra te (b p m ) S A P (m m H g ) 1 0 0 RV LV 3 0 0 2 0 0 5 0 1 0 0 0 0 N o rm S u H x SuHx N o rm S u H x

Current study: ASK1 inhibition in an in vivo PAH model Reversal treatment arm PAH arm + 3 weeks vehicle Week 8 SU5415 (20mg/kg, S.C) + 3 weeks ASK1 inhibitor + 3 weeks normoxia 2 weeks hypoxia RATS Haemodynamic 3 week old Week 8 Week 2 Week 5 Week 0 and cardiac Male Sprague Dawley measurements + 3 weeks vehicle 5 weeks normoxia Week 8 Week 0 Week 5 + 3 weeks ASK1 inhibitor Week 8

Current study: ASK1 inhibition in an in vivo PAH model R V S P a t 8 w e e ks R V /(L V + s e p tu m ) a t 8 w e e ks 6 0 0 .4 * * * * m R V S P (m m H g ) R V /(L V + S e p tu m ) 0 .3 4 0 0 .2 RV LV 2 0 RV LV 0 .1 0 .0 0 N orm N orm S uH x S uH x N o rm N o rm S u H x S u H x + i + i + i + i Norm Norm + i H e a rt ra te a t 8 w e e ks S A P a t 8 w e e ks 5 0 0 1 5 0 4 0 0 h e a rt ra te (b p m ) RV LV S A P (m m H g ) RV LV 1 0 0 3 0 0 2 0 0 5 0 1 0 0 SuHx SuHx + i 0 0 N o rm N o rm S u H x S u H x N o rm N o rm S u H x S u H x + i + i + i + i

Current study: ASK1 inhibition in an in vivo PAH model Norm SuHx + i SuHx 50µM 50µM 50µM α smooth muscle actin P u lm o n a ry va scu la r re m o d e llin g 1 0 0 * % re m o d e lle d ve sse ls 8 0 6 0 4 0 2 0 0 N o rm S u H x S u H x + i

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 David Welsh Grant Budas Geeshath Jayasekera John Liles Andrew Peacock Colin Church Monika Setiwan Hanna Buist Colin Hughes Margaret Nilsen Mandy MacLean We acknowledge the support of the PVRI who has funded the travel expenses and registration fees for this meeting