reduces susceptibility to pulmonary arterial hypertension in bmpr2 - PowerPoint PPT Presentation

Bone marrow transplantation reduces susceptibility to pulmonary arterial hypertension in bmpr2 deficient mice A Crosby, E Soon, M Southwood, M Toshner, BJ Dunmore, NW Morrell

Pulmonary arterial hypertension • PAH (PAP >25 mmHg) • Characterised by smooth muscle cell and endothelial cell proliferation • Right-sided heart failure • >70% patients with heritable PAH have a mutation in BMPR-II • TGF- b superfamily • Cell proliferation and differentiation • Second-Hit Hypothesis • Not all people with a mutation in BMPR-II have disease • Second hit e.g. inflammatory challenge

Pulmonary arterial hypertension • PAH (PAP >25 mmHg) • Characterised by smooth muscle cell and endothelial cell proliferation • Right-sided heart failure >70% patients with heritable PAH have a mutation in BMPR-II • TGF- b superfamily • Cell proliferation and differentiation a -SMA • Second-Hit Hypothesis • Not all people with a mutation in BMPR-II have disease • Second hit e.g. inflammatory challenge

Pulmonary arterial hypertension • PAH (PAP >25 mmHg) • Characterised by smooth muscle cell and endothelial cell proliferation • Right-sided heart failure • >70% patients with heritable PAH have a mutation in BMPR-2 • TGF- b superfamily • Cell proliferation and differentiation a -SMA • Second-Hit Hypothesis • Not all people with a mutation in BMPR-II have disease • Second hit e.g. inflammatory challenge

Pulmonary arterial hypertension • PAH (PAP >25 mmHg) • Characterised by smooth muscle cell and endothelial cell proliferation • Right-sided heart failure • >70% patients with heritable PAH have a mutation in BMPR-2 • TGF- b superfamily • Cell proliferation and differentiation a -SMA • Second-Hit Hypothesis • Not all people with a mutation in BMPR-2 have disease • Second hit e.g. inflammatory challenge

The Bone Marrow (BM)

The Bone Marrow (BM) • The bone marrow is responsible for haematopoiesis

The Bone Marrow (BM) • The bone marrow is responsible for haematopoiesis • Haematopoietic stem cells (HSC) proliferate throughout life, giving rise to a variety of cell types

The Bone Marrow (BM) • The bone marrow is responsible for haematopoiesis • Haematopoietic stem cells (HSC) proliferate throughout life, giving rise to a variety of cell types

bmpr2 expression in mouse haematopoietic system BloodExpress – Courtesy of Emily Groves

Mouse MK cells express bmpr2 MK MK bmpr2

Mouse MK cells express bmpr2 compared with lung homogonate Fold change in bmpr2 in MK 1.5 MK MK 1.0 bmpr2 0.5 0.0 Lung homogenate Mouse MK RNA

Evidence for bone marrow dysfunction in PAH • High frequency iron-deficiency in PAH patients 43-63% - especially in patients with BMPR-II mutation (Soon et al. Thorax, 2011; Rhodes et al. JACC, 2011)

Evidence for bone marrow dysfunction in PAH • Association between PAH and myeloproliferative disease (MPD) – 13-48% patients with MPD had PAH ( Cortelezzi et al. Leukemia, 2008) – 40-100% patients PAH had Myelodysplasia on bone marrow biopsy (Guilpain et al. Respiration, 2008; Asosingh et al. Blood, 2012) • High frequency iron-deficiency in PAH patients 43-63% - especially in patients with BMPR-II mutation (Soon et al. Thorax, 2011; Rhodes et al. JACC, 2011)

Evidence for bone marrow dysfunction in PAH • Association between PAH and myeloproliferative disease (MPD) – 13-48% patients with MPD had PAH ( Cortelezzi et al. Leukemia, 2008) – 40-100% patients PAH had Myelodysplasia on bone marrow biopsy (Guilpain et al. Respiration, 2008; Asosingh et al. Blood, 2012) • There was increased expression of myeloid-erythroid specific transcription factors in haematopoietic progenitor cells from PAH patients compared with controls ( Asosingh et al. Blood, 2012) • High frequency iron-deficiency in PAH patients 43-63% - especially in patients with BMPR-II mutation (Soon et al. Thorax, 2011; Rhodes et al. JACC, 2011)

Evidence for bone marrow derived cells in PAH pathobiology

Evidence for bone marrow derived cells in PAH pathobiology • PAH patients have higher than normal circulating CD133+ (stem cell) and CD34+ (Haematopoeitic/endothelial cell progenitor) cells (Farha et al. Blood, 2011)

Evidence for bone marrow derived cells in PAH pathobiology • PAH patients have higher than normal circulating CD133+ (stem cell) and CD34+ (Haematopoeitic/endothelial cell progenitor) cells (Farha et al. Blood, 2011) • In the CD133+ fraction from PAH patients there were more multipotent progenitors and they showed greater myeloid commitment ( Asosingh et al. Blood, 2012)

Evidence for bone marrow derived cells in PAH pathobiology • PAH patients have higher than normal circulating CD133+ (stem cell) and CD34+ (Haematopoeitic/endothelial cell progenitor) cells (Farha et al. Blood, 2011) • In the CD133+ fraction from PAH patients there were more multipotent progenitors and they showed greater myeloid commitment ( Asosingh et al. Blood, 2012) • Mice transplanted with CD133+ cells from PAH patients developed vascular injury, thromboses and right ventricular hypertrophy, whereas mice transplanted with CD133+ cells from controls did not ( Asosingh et al. Blood, 2012)

Evidence for the role of inflammation in PAH • Increased IL-1 & 6 in IPAH (Humbert,1995) • IL-6 KO mice resistant to hypoxia-induced increase in pulmonary artery pressures (Savale, 2007) • Increase in pulmonary artery pressures and pulmonary vascular remodelling in IL-6 over-expressing mice ( Steiner, 2009) • Bone Morphogenetic Protein Receptor Type II Deficiency and Increased Inflammatory Cytokine Production. A Gateway to Pulmonary Arterial Hypertension (Soon, 2015)

Evidence for the role of inflammation in PAH • Increased IL-1 and 6 in IPAH (Humbert,1995)

Evidence for the role of inflammation in PAH • Increased IL-1 and 6 in IPAH (Humbert,1995) • IL-6 KO mice resistant to hypoxia-induced increase in pulmonary artery pressures (Savale, 2007)

Evidence for the role of inflammation in PAH • Increased IL-1 and 6 in IPAH (Humbert,1995) • IL-6 KO mice resistant to hypoxia-induced increase in pulmonary artery pressures (Savale, 2007) • There is an increase in pulmonary artery pressures and pulmonary vascular remodelling in IL-6 over-expressing mice ( Steiner, 2009)

Evidence for the role of inflammation in PAH • Increased IL-1 and 6 in IPAH (Humbert,1995) • IL-6 KO mice resistant to hypoxia-induced increase in pulmonary artery pressures (Savale, 2007) • There is an increase in pulmonary artery pressures and pulmonary vascular remodelling in IL-6 over-expressing mice ( Steiner, 2009) • Bone Morphogenetic Protein Receptor Type II Deficiency and Increased Inflammatory Cytokine Production. A Gateway to Pulmonary Arterial Hypertension (Soon, Crosby, 2015)

A novel mouse model of PAH-second hit

A novel mouse model of PAH-second hit WT + BMPR-II +/- (MUT) 6 Weeks LPS 0.5mg/Kg RHC/Tissue Soon and Crosby, AJRCCM 2015

A novel mouse model of PAH-second hit WT + BMPR-II +/- (MUT) 6 Weeks LPS 0.5mg/Kg RHC/Tissue * # 50 RVSP (mmHg) 40 30 20 10 0 WT BASELINE MUT BASELINE WT LPS MUT LPS Soon and Crosby, AJRCCM 2015

A novel mouse model of PAH-second hit WT + BMPR-II +/- (MUT) 6 Weeks LPS 0.5mg/Kg RHC/Tissue * ** 15 # 50 * % Wall thickness Lung vessels RVSP (mmHg) 40 10 * 30 20 5 10 0 0 WT BASELINE MUT BASELINE WT LPS MUT LPS WT BASELINE MUT BASELINE WT LPS MUT LPS Soon and Crosby, AJRCCM 2015

A novel mouse model of PAH-second hit Spleen Weight/BW 0.015 * 0.010 0.005 0.000 WT BASELINE MUT BASELINE WT LPS MUT LPS Soon and Crosby, AJRCCM 2015

bmpr2 heterozygous bone marrow derived cells increase susceptibility to PAH in a mouse model

Can we replicate PAH in a mouse model by replacing wild-type bone marrow with bmpr2+/- bone marrow?

1000 rad g irradiation - 137Cs source +/+ 1X10 6 cells +/+ or +/-

1000 rad g irradiation - 137Cs source +/+ 1X10 6 cells +/+ or +/- LPS challenge Mice 7 16 wks 4 wks 0.5mg/kg – 3X/week wks old bleed bleed RVSP g Irradiation 6 weeks RV/LV+S + BM Lung tissue Reconstitution Blood 1X10 6 cells/mouse Spleen Bone-marrow

1000 rad g irradiation - 137Cs source +/+ 1X10 6 cells +/+ or +/- LPS challenge Mice 7 16 wks 4 wks 0.5mg/kg – 3X/week wks old bleed bleed RVSP g Irradiation 6 weeks RV/LV+S + BM Lung tissue Reconstitution Blood 1X10 6 cells/mouse Spleen Bone-marrow

* 30 RVSP (mmHg) 20 10 0 S S P P L L + + + + o o t t + - + +

* 0.4 30 RVSP (mmHg) 0.3 RV/LV+S 20 0.2 10 0.1 0.0 0 ++ to ++ LPS +- to ++ LPS S S P P L L + + + + o o t t + - + +

0.25 Spleen Weight (g) * 0.20 0.15 0.10 0.05 0.00 ++ to ++ LPS +- to ++ LPS

Can we prevent PAH in a mouse model by replacing bmpr2+/- bone marrow with wild-type bone marrow?

1000 rad g irradiation - 137Cs source +/+ 1X10 6 cells +/- or +/-

1000 rad g irradiation - 137Cs source +/+ 1X10 6 cells +/- or +/- LPS challenge Mice 7 16 wks 4 wks 0.5mg/kg – 3X/week wks old bleed bleed RVSP g Irradiation 6 weeks RV/LV+S + BM Lung tissue Reconstitution Blood 1X10 6 cells/mouse Spleen Bone-marrow