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

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

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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)

slide-1
SLIDE 1

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

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SLIDE 2

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
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SLIDE 3

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

a-SMA

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SLIDE 4

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
  • Second-Hit Hypothesis
  • Not all people with a mutation in BMPR-II have disease
  • Second hit e.g. inflammatory challenge

a-SMA

slide-5
SLIDE 5

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
  • Second-Hit Hypothesis
  • Not all people with a mutation in BMPR-2 have disease
  • Second hit e.g. inflammatory challenge

a-SMA

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SLIDE 6

The Bone Marrow (BM)

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SLIDE 7

The Bone Marrow (BM)

  • The bone marrow is responsible for haematopoiesis
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SLIDE 8

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

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SLIDE 9

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

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SLIDE 10

bmpr2 expression in mouse haematopoietic system

BloodExpress – Courtesy of Emily Groves

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SLIDE 11

MK MK bmpr2

Mouse MK cells express bmpr2

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SLIDE 12

MK MK bmpr2

Lung homogenate Mouse MK RNA

0.0 0.5 1.0 1.5

Fold change in bmpr2 in MK compared with lung homogonate

Mouse MK cells express bmpr2

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SLIDE 13

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)

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SLIDE 14

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)

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SLIDE 15

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)

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SLIDE 16

Evidence for bone marrow derived cells in PAH pathobiology

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SLIDE 17

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)

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SLIDE 18

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)

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SLIDE 19

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)

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SLIDE 20

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)

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SLIDE 21

Evidence for the role of inflammation in PAH

  • Increased IL-1 and 6 in IPAH (Humbert,1995)
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SLIDE 22

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)

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SLIDE 23

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)

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SLIDE 24

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)

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SLIDE 25

A novel mouse model of PAH-second hit

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SLIDE 26

LPS 0.5mg/Kg WT + BMPR-II +/- (MUT) 6 Weeks RHC/Tissue

Soon and Crosby, AJRCCM 2015

A novel mouse model of PAH-second hit

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SLIDE 27

LPS 0.5mg/Kg WT + BMPR-II +/- (MUT) 6 Weeks RHC/Tissue

Soon and Crosby, AJRCCM 2015

A novel mouse model of PAH-second hit

WT BASELINE MUT BASELINE WT LPS MUT LPS 10 20 30 40 50

* #

RVSP (mmHg)

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SLIDE 28

LPS 0.5mg/Kg WT + BMPR-II +/- (MUT) 6 Weeks RHC/Tissue

Soon and Crosby, AJRCCM 2015

A novel mouse model of PAH-second hit

WT BASELINE MUT BASELINE WT LPS MUT LPS 10 20 30 40 50

* #

RVSP (mmHg)

WT BASELINE MUT BASELINE WT LPS MUT LPS 5 10 15

** * *

% Wall thickness Lung vessels

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SLIDE 29

Soon and Crosby, AJRCCM 2015

A novel mouse model of PAH-second hit

WT BASELINE MUT BASELINE WT LPS MUT LPS 0.000 0.005 0.010 0.015

*

Spleen Weight/BW

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SLIDE 30

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

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SLIDE 31

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

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SLIDE 32

1000 rad g irradiation - 137Cs source 1X106 cells

+/+ +/- +/+

  • r
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SLIDE 33

1000 rad g irradiation - 137Cs source 1X106 cells

Mice 7 wks old g Irradiation + BM Reconstitution 1X106 cells/mouse 4 wks bleed 16 wks bleed Lung tissue RVSP RV/LV+S Blood Spleen Bone-marrow LPS challenge 0.5mg/kg – 3X/week

+/+ +/- +/+

  • r

6 weeks

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SLIDE 34

1000 rad g irradiation - 137Cs source 1X106 cells

Mice 7 wks old g Irradiation + BM Reconstitution 1X106 cells/mouse 4 wks bleed 16 wks bleed Lung tissue RVSP RV/LV+S Blood Spleen Bone-marrow LPS challenge 0.5mg/kg – 3X/week

+/+ +/- +/+

  • r

6 weeks

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SLIDE 35

+ + t

  • +

+ L P S +

  • t
  • +

+ L P S 10 20 30

*

RVSP (mmHg)

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SLIDE 36

+ + t

  • +

+ L P S +

  • t
  • +

+ L P S 10 20 30

*

RVSP (mmHg) ++ to ++ LPS +- to ++ LPS 0.0 0.1 0.2 0.3 0.4 RV/LV+S

slide-37
SLIDE 37

++ to ++ LPS +- to ++ LPS 0.00 0.05 0.10 0.15 0.20 0.25

*

Spleen Weight (g)

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SLIDE 38

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

slide-39
SLIDE 39

1000 rad g irradiation - 137Cs source 1X106 cells

+/+ +/- +/-

  • r
slide-40
SLIDE 40

1000 rad g irradiation - 137Cs source 1X106 cells

Mice 7 wks old g Irradiation + BM Reconstitution 1X106 cells/mouse 4 wks bleed 16 wks bleed Lung tissue RVSP RV/LV+S Blood Spleen Bone-marrow LPS challenge 0.5mg/kg – 3X/week

+/+ +/- +/-

  • r

6 weeks

slide-41
SLIDE 41

1000 rad g irradiation - 137Cs source 1X106 cells

Mice 7 wks old g Irradiation + BM Reconstitution 1X106 cells/mouse 4 wks bleed 16 wks bleed Lung tissue RVSP RV/LV+S Blood Spleen Bone-marrow LPS challenge 0.5mg/kg – 3X/week

+/+ +/- +/-

  • r

6 weeks

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SLIDE 42

++ to ++ LPS +- to ++ LPS 10 20 30

*

RVSP (mmHg) + + t

  • +

+ L P S +

  • t
  • +

+ L P S 0.0 0.1 0.2 0.3 0.4 RV/LV+S

slide-43
SLIDE 43

++ to ++ LPS +- to ++ LPS ++ to +- LPS 10 20 30

* *

RVSP (mmHg) + + t

  • +

+ L P S +

  • t
  • +

+ L P S + + t

  • +
  • L

P S 0.0 0.1 0.2 0.3 0.4 RV/LV+S

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SLIDE 44

++ to ++ LPS +- to ++ LPS ++ to +- LPS +- to +- LPS 10 20 30

* * *

RVSP (mmHg) + + t

  • +

+ L P S +

  • t
  • +

+ L P S + + t

  • +
  • L

P S +

  • t
  • +
  • L

P S 0.0 0.1 0.2 0.3 0.4 RV/LV+S

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SLIDE 45

+ + t

  • +

+ L P S +

  • t
  • +

+ L P S 0.00 0.05 0.10 0.15 0.20 0.25

*

Spleen Weight (g)

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SLIDE 46

+ + t

  • +

+ L P S +

  • t
  • +

+ L P S + + t

  • +
  • L

P S 0.00 0.05 0.10 0.15 0.20 0.25

* *

Spleen Weight (g)

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SLIDE 47

+ + t

  • +

+ L P S +

  • t
  • +

+ L P S + + t

  • +
  • L

P S +

  • t
  • +
  • L

P S 0.00 0.05 0.10 0.15 0.20 0.25

* *

Spleen Weight (g)

slide-48
SLIDE 48

+ + t

  • +

+ L P S +

  • t
  • +

+ L P S + + t

  • +
  • L

P S +

  • t
  • +
  • L

P S 100 200 300 400

*

p=0.055

MK counts in spleen

slide-49
SLIDE 49

++ to ++ LPS +- to ++ LPS ++ to +- LPS +- to +- LPS

+ + t

  • +

+ L P S +

  • t
  • +

+ L P S + + t

  • +
  • L

P S +

  • t
  • +
  • L

P S 100 200 300 400

*

p=0.055

MK counts in spleen

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SLIDE 50

Do mice with bmpr2+/- bone marrow exhibit pulmonary vascular remodelling?

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SLIDE 51

++ to ++ LPS +- to ++ LPS +- to +- LPS 1 2 3 4 5

* * % Wall thickness

Do mice with bmpr2+/- bone marrow exhibit pulmonary vascular remodelling?

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SLIDE 52

++ to ++ LPS +- to ++ LPS +- to +- LPS 1 2 3 4 5

* * % Wall thickness

++ to ++ LPS +- to ++ LPS +- to +- LPS 20 40 60 80 100 120 140

Non-muscularised Partially muscularised Muscularised

* % Muscularisation

Do mice with bmpr2+/- bone marrow exhibit pulmonary vascular remodelling?

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SLIDE 53

++ to ++ LPS +- to ++ LPS +- to +- LPS

++ to ++ LPS +- to ++ LPS +- to +- LPS 1 2 3 4 5

* * % Wall thickness

++ to ++ LPS +- to ++ LPS +- to +- LPS 20 40 60 80 100 120 140

Non-muscularised Partially muscularised Muscularised

* % Muscularisation

Do mice with bmpr2+/- bone marrow exhibit pulmonary vascular remodelling?

slide-54
SLIDE 54

Is there a change in circulating blood cells?

slide-55
SLIDE 55

+ + t

  • +

+ L P S +

  • t
  • +

+ L P S + + t

  • +
  • L

P S +

  • t
  • +
  • L

P S 2 4 6

WBC

+ + t

  • +

+ L P S +

  • t
  • +

+ L P S + + t

  • +
  • L

P S +

  • t
  • +
  • L

P S 2 4 6 8 10

RBC

++ to ++ LPS +- to ++ LPS ++ to +- LPS +- to +- LPS 50 100 150

HGB g/l

++ to ++ LPS +- to ++ LPS ++ to +- LPS +- to +- LPS 0.0 0.1 0.2 0.3 0.4 0.5

HCT (l/l)

+ + t

  • +

+ L P S +

  • t
  • +

+ L P S + + t

  • +
  • L

P S +

  • t
  • +
  • L

P S 20 40 60 80

% LYM

++ to ++ LPS +- to ++ LPS ++ to +- LPS +- to +- LPS 2 4 6 8 10

% MON

++ to ++ LPS +- to ++ LPS ++ to +- LPS +- to +- LPS 10 20 30 40

% GRA

+ + t

  • +

+ L P S +

  • t
  • +

+ L P S + + t

  • +
  • L

P S +

  • t
  • +
  • L

P S 2 4 6 8

% EOS

Is there a change in circulating blood cells?

slide-56
SLIDE 56

+ + t

  • +

+ L P S +

  • t
  • +

+ L P S + + t

  • +
  • L

P S +

  • t
  • +
  • L

P S 500 1000 1500

* * *

Circulating Platelets

Is there a change in circulating blood cells?

slide-57
SLIDE 57

What could be responsible for the increase in circulating platelets?

slide-58
SLIDE 58

What could be responsible for the increase in circulating platelets?

++ Baseline +- Baseline ++ to ++ Saline +- to ++ Saline ++ to ++ LPS +- to ++ LPS 10 20 30

** **

No Megakaryoctes/HPF

slide-59
SLIDE 59

Can we identify a defect in the bone marrow using histology?

slide-60
SLIDE 60

Can we identify a defect in the bone marrow using histology?

+- to ++ BMT saline +- to ++ BMT LPS

H+E

slide-61
SLIDE 61

Can we identify a defect in the bone marrow using histology?

++ to ++ LPS +- to ++ LPS 20 40 60 80

  • No. B cells in BM section
slide-62
SLIDE 62

Can we identify a defect in the bone marrow using histology?

++ to ++ LPS +- to ++ LPS 20 40 60 80

  • No. B cells in BM section

++ to ++ LPS +- to ++ LPS 2 4 6

  • No. T cells in BM section
slide-63
SLIDE 63

Working Hypothesis

slide-64
SLIDE 64

bmpr2+/-

Working Hypothesis

slide-65
SLIDE 65

bmpr2+/- Megakaryocyte

Working Hypothesis

slide-66
SLIDE 66

bmpr2+/- Megakaryocyte LPS

Working Hypothesis

slide-67
SLIDE 67

bmpr2+/- Megakaryocyte Platelets LPS

Working Hypothesis

slide-68
SLIDE 68

bmpr2+/- Megakaryocyte Platelets Release of proangiogenic and vasoconstrictor substances LPS

Working Hypothesis

slide-69
SLIDE 69

bmpr2+/- Megakaryocyte Platelets Release of proangiogenic and vasoconstrictor substances

Composite Volume (uL) 33 32 31 30 29 28 Pressure 1 (mmHg) 2 4 6 8 10 12 14 16 18 20 22 24 26 RVSP

LPS

Working Hypothesis

slide-70
SLIDE 70

bmpr2+/- Megakaryocyte Platelets Release of proangiogenic and vasoconstrictor substances

Composite Volume (uL) 33 32 31 30 29 28 Pressure 1 (mmHg) 2 4 6 8 10 12 14 16 18 20 22 24 26 RVSP

LPS Splenomegaly

Working Hypothesis

slide-71
SLIDE 71

bmpr2+/- Megakaryocyte Platelets Release of proangiogenic and vasoconstrictor substances

Composite Volume (uL) 33 32 31 30 29 28 Pressure 1 (mmHg) 2 4 6 8 10 12 14 16 18 20 22 24 26 RVSP

LPS Splenomegaly

Working Hypothesis

?

slide-72
SLIDE 72

Acknowledgements

  • Nick Morrell
  • Mark Toshner
  • Elaine Soon
  • Mark Southwood
  • Ben Dunmore
  • Ian Horan

PVRI Fellowship grant