Group 3 Pulmonary Hypertension FGF2 and FGFR expression is elevated - - PowerPoint PPT Presentation

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Group 3 Pulmonary Hypertension FGF2 and FGFR expression is elevated - - PowerPoint PPT Presentation

Oct 25, 2022 17 likes •98 views

4/21/2018 Fibroblast Growth Factors Protective Role of Fibroblast Growth Factor Signaling in Hypoxia-Induced Pulmonary Hypertension Kel Vin Woo, M.D., Ph.D. Pediatric Cardiology, Fellow Ornitz Lab UCSF 11 th International Conference FGF2

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Protective Role of Fibroblast Growth Factor Signaling in Hypoxia-Induced Pulmonary Hypertension

Kel Vin Woo, M.D., Ph.D. Pediatric Cardiology, Fellow Ornitz Lab UCSF 11th International Conference Neonatal & Childhood Pulmonary Vascular Disease April 21st, 2018

Fibroblast Growth Factors

Ornitz and Itoh.Dev. 2015

  • FGF2 and its receptors FGFR1 and 2 have been studied in many disease

models, including: skin wound healing, retinal microvascular injury, cardiac ischemia/reperfusion injury.

  • FGF2, FGFR1 and 2 are not required for homeostasis but play important roles

in disease.

Control PH

FGF2 and FGFR expression is elevated in patients with PH

1. Izziki et al. JCI 119. 2009 2. Tu et al. Am J Respir Cell Mol Biol 45. 2011 3. Kim et al. Nat Med 19(1). 2013 Pulmonary Hypertension Control

Lung tissue of PH patients

  • FGF signaling is pathologic in Group 1 pulmonary hypertension.

Group 3 Pulmonary Hypertension

  • Group 3 (2nd most common): Alveolar hypoxia due to lung diseases

including bronchopulmonary dysplasia (BPD), interstitial lung disease, COPD, OSA, alveolar hypoventilation disorders, chronic exposure to high altitude.

1.Osterman. Pediatrics 135(6). 2015. 2.Stoll. Pediatrics 126(3). 2010. 3.Mourani. AJRCCM (191). 2015.

  • 4. Khemani. Pediatrics 120. 2007.
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Group 3 Pulmonary Hypertension

  • Group 3 (2nd most common): Alveolar hypoxia due to lung diseases

including bronchopulmonary dysplasia (BPD), interstitial lung disease, COPD, OSA, alveolar hypoventilation disorders, chronic exposure to high altitude.

  • 550,000 premature infants are born in the United States annually.1
  • Up to 60% of premature infants develop BPD.2
  • 29-42% of infants with BPD go on to develop PH.3
  • Infants with BPD and PH have a 2-year mortality of 33-48%.4

1.Osterman. Pediatrics 135(6). 2015. 2.Stoll. Pediatrics 126(3). 2010. 3.Mourani. AJRCCM (191). 2015.

  • 4. Khemani. Pediatrics 120. 2007.
  • Majority of studies to date have focused on Group 1 PH.
  • All 3 classes of PH drugs are targeted toward Group 1.
  • Randomized clinical trials show failure of current PH

therapies in Group 3 PH patients:

1)

  • Bayer. 2016: RCT, Riociguat, increased adverse events and mortality

2) Raghu et al. 2013: RCT, Ambrisentan, patients with PH had no change in time to disease progression 3) Krowka et al. 2007: inh. Iloprost, no difference in 6MWT, NYHA class, dyspnea score, exercise O2sat 4) Jackson et al. 2010: Sildenafil, no difference in 6MWT 5) Zisman et al. 2010: Sildenafil, no improvement in 6MWT

Why we need to study Group 3 PH

adapted from Nathan R. ACC 2017.

  • Increased FGF2 expression in premature infants BPD+PH

Increased FGF2 expression in Group 3 PH

Control BPD + PH

  • 100

100 200 300 400 FGF2 Concentration (pg/ml)

  • Increased FGF2 expression in premature infants BPD+PH
  • Hypoxia-induced PH mouse model:1,2

Increased FGF2 expression in Group 3 PH

  • 1. Chang. Cardiovasc Res. 2015

2.Gore. PLOS ONE. 2015.

Control BPD + PH

  • 100

100 200 300 400 FGF2 Concentration (pg/ml)

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Hypothesis

Endothelial FGF signaling is important for the pathogenesis of Group 3 hypoxia-induced pulmonary hypertension.

Method

  • Determine whether endothelial FGFR1 and FGFR2 signaling

contribute to the development of hypoxia-induced pulmonary hypertension

2 weeks, 10% vs 21% FiO2

  • Mouse CV Phenotyping

Core

  • Echocardiogram
  • Cardiac Cath
  • IHC + Quantify
  • PA muscularization
  • RV hypertrophy

2 weeks hypoxia-induced PH

  • Mice challenged with 10% oxygen (hypoxia) or room air

(normoxia).

  • RV systolic pressure measured by cardiac catheterization

– DCKO: Tie2-Cre;FGFR1-flox/flox;FGFR2-flox/flox – DFF: FGFR1-flox/flox;FGFR2-flox/flox

2 weeks hypoxia-induced PH

  • Mice challenged with 10% oxygen (hypoxia) or room air

(normoxia).

  • RV systolic pressure measured by cardiac catheterization

– DCKO: Tie2-Cre;FGFR1-flox/flox;FGFR2-flox/flox – DFF: FGFR1-flox/flox;FGFR2-flox/flox

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Echocardiogram analysis of 2 weeks hypoxia-induced PH

  • Pulmonary Artery Acceleration (PAAT) = time to maximal velocity of RV

ejection.

  • PAAT is an accepted non-invasive measure of pulmonary hypertension in

both patients and mouse models.

Loss of FGFR1,2 worsens smooth muscle hyperplasia

  • Fgfr1 and Fgfr2 knockout (DCKO) + 2 weeks hypoxia causes

increased tunica media/smooth muscle hyperplasia.

H&E

Normoxia Hypoxia DFF DCKO

Loss of FGFR1,2 worsens smooth muscle hyperplasia

  • Fgfr1 and Fgfr2 knockout (DCKO) + 2 weeks hypoxia causes

increased tunica media/smooth muscle hyperplasia.

A B /

  • A

SMA

Normoxia Hypoxia DFF DCKO

DFF DCKO DFF DCKO 0.0 0.2 0.4 0.6 0.8 Media/CSA(20-50um) p<0.05 Hypoxia Normoxia p<0.001

H&E

Normoxia Hypoxia DFF DCKO

Loss of FGFR1,2 increases numbers of muscularized vessels

  • Fgfr1 and Fgfr2 knockout (DCKO) + 2 weeks hypoxia increases proportion
  • f muscularized vessels.

CD31 + SMA

Partially Muscularized Fully Muscularized Non- Muscularized

DFF DCKO DFF DCKO 50 100 %Vessels Muscularized (20-50um) Non-Muscularized Partially Muscularized Fully Muscularized Hypoxia Normoxia

* *

*p<0.001

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  • Induced expression of constitutively active FGFR1
  • Tie2Cre; RosartTA; Tre-caFgfr1

FGFR1 signaling prevents hypoxia-induced PH

WT caFGFR1 WT caFGFR1 20 40 60

RVSP (mmHg) Normoxia Hypoxia p<0.01 p<0.01

  • Global FGF2-knockout1 (FGF2KO) mice

FGF2-KO hypoxia-induced PH

WT FGF2KO WT FGF2KO 20 40 60 RVSP (mmHg) *p<0.05 Hypoxia Normoxia *p<0.05

  • 1. Zhou et al. 1998. Nat Med.
  • Global FGF2-knockout1 (FGF2KO) mice

FGF2-KO hypoxia-induced PH

WT FGF2KO WT FGF2KO 20 40 60 RVSP (mmHg) *p<0.05 Hypoxia Normoxia *p<0.05

  • 1. Zhou et al. 1998. Nat Med.

DCKO FGF2KO 35 40 45 50 55 Hypoxia RV Pressure (mmHg) *p<0.01

  • FGFs (FGF2) exerts independent effects on ECs and SMCs,

and may influence EC-SMC interaction

Proposed Model

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Endothelial-Smooth Muscle Interaction

  • EC-SMC microfluidics co-culture

– Pulmonary-ECs, SMCs isolated by FACs

Endothelial-Smooth Muscle Interaction

  • EC-SMC microfluidics co-culture

– Pulmonary-ECs, SMCs isolated by FACs

Brightfield EC-GFP SMA-tdTomato 24 hours DFF

Endothelial-Smooth Muscle Interaction

  • EC-SMC microfluidics co-culture

– Pulmonary-ECs, SMCs isolated by FACs

Brightfield EC-GFP SMA-tdTomato 24 hours DFF EC-DCKO

Endothelial-Smooth Muscle Interaction

  • EC-SMC microfluidics co-culture

– Pulmonary-ECs, SMCs isolated by FACs

Brightfield EC-GFP SMA-tdTomato 24 hours 48 hours DFF EC-DCKO DFF EC-DCKO Brightfield EC-GFP SMA-tdTomato

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Summary

  • Loss of Fgfr1 and Fgfr2 in endothelial cells worsens pulmonary

hypertension in vivo and alters smooth muscle cell activity in vitro.

  • Loss of Fgf2 leads to more severe pulmonary hypertension than

endothelial deletion of Fgfr1 and Fgfr2, and overexpression of constitutively active FGFR1 prevents the phenotype, suggesting: 1) a protective role for FGF2-FGFR1/2 signaling axis, or FGFR1 signaling, 2) FGF therapy might be beneficial for Group 3 PH, the

  • pposite effect of previous reports in Group 1 PH.

Future Directions

1. Investigate role of FGFs on neonatal (P0-P14) hypoxia-induced PH. 2. Investigate the applicability of human iPS derived ECs and SMCs in microfluidic devive 3. Evaluate effects of FGF signaling on whole lung vascular distribution with high resolution Xray microscopy.

  • Support:

– Oliver Langenberg Pediatric Physician Scientist Training Program – Children’s Discovery Institute Postdoctoral Fellowship – American College of Cardiology Research Award – Washington University ICTS – Child Health Forum Challenge – NIH (T32) Pediatric Cardiovascular and Pulmonary Research Training Program – Washington University Center for Cellular Imaging Microgrants

Acknowledgements

  • Mentor: David Ornitz

Ornitz Lab members: – Stacey House, Yin Yongjun – Drew Hagan, Lu Yang – Richard Li – Craig Smith, Stefan Traian, Ling Li

  • Collaborators

– Mouse Cardiovascular Phenotyping Core: Attila Kovacs, Carla Weinheimer – Steven George (UC Irvine), Aziz Traore – Dept of Engr: Mark Meacham – Gautam Singh, Phil Levy – WUCCI: James Fitzpatrick, Matt Joens

  • Committee Members

– Mark Grady – Patrick Jay – Steve Brody – Robert Mecham

Tre-caFgfr1

  • Chimeric receptor: FGFR3c(R248C) mutant extracellular and

transmembrane domain fused to the FGFR1 tyrosine kinase domain (caFGFR1).

  • FGFR3c(R248C) mutation confers ligand-independent dimerization

= constitutively active FGFR1 intracellular tyrosine kinase domain.

Cilvik et al. PLOs One. 2013

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Non-Linear Regression Fit Model

Echocardiogram analysis of 2 weeks hypoxia-induced PH

  • Pulmonary Artery Acceleration (PAAT) = time to maximal velocity of RV

ejection

  • PAAT is an accepted non-invasive measure of pulmonary hypertension in

both patients and mouse models.

  • Clinically diagnosed as elevated pulmonary artery pressures (>25mmHg)
  • High pulmonary pressure -> increased right heart workload -> right

heart failure

  • Endothelial dysfunction, excessive vascular remodeling, inflammation ->

increased pulmonary vascular resistance and gradual vessel occlusion

  • 1. Lai et al. Circ Res, 2016.

Pulmonary Hypertension