Primary Pulmonary Hypertension: Recent Insights into Molecular Pathways Underlying Pathogenesis

1
Primary Pulmonary HypertensionRecent Insights
into Molecular Pathways Underlying Pathogenesis

• Jonathan Alexander MD PhD
• 10 February 2004

2
Outline

• Background
• Pathology
• Genetic analyses of Familial PPH
• Mutations in BMPRII cause FPPH
• Mechanisms by which mutations in BMPRII may cause
  PPH
• Other potential contributors to PPH

3
Outline

• Background
• Pathology
• Genetic analyses of Familial PPH
• Mutations in BMPRII cause FPPH
• Mechanisms by which mutations in BMPRII may cause
  PPH
• Other potential contributors to PPH

4
Pulmonary Hypertension

• Defined by presence of an elevated pulmonary
  arterial pressure (gt 25 mm Hg at rest or gt 30 mm
  Hg during exercise)
• Previously patients were categorized as having
  primary (ie PPH) or secondary (everything else)
  pulmonary hypertension
• Current classification scheme divides patients by
  principal site or etiology of pathologic process

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WHO Classification of Pulmonary Hypertension

• Arterial
• PPH, CVD, L-to-R shunt, portal HTN, HIV,
  drugs/toxins
• Venous
• Left-sided heart disease, valve disease, VOD
• Associated with respiratory system disease
• COPD, ILD, OSA, chronic hypoventilation
• Related to chronic thromboembolic disease
• Disorders of the pulmonary vasculature
• sarcoidosis, Schistosomiasis

Rich (1998) www.who.int/ned/cv/pph.html
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PPH Epidemiology

• Annual incidence of approximately 1-2 cases per
  million people annually in Western populations
• Female predominance (femalemale ratio of
  approximately 21)
• Median age at diagnosis is 36 years but children
  and older people can also be affected
• Approximately 6-10 of cases are inherited

Peacock (1999) Thorax 54 1107 Rudarakanchana et
al (2001) Thorax 56 888
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PPH Presentation and Diagnosis

• Insidious onset and non-specific nature of
  symptoms usually lead to delay in diagnosis
• Symptoms often absent until PAP gt 30 mm Hg
• Demonstration of an elevated pulmonary arterial
  pressure
• Echocardiogram
• Right heart catheterization
• Exclusion of associated conditions or exposures
• Pathological lesions seen in PPH are not specific

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PPH Prognosis

• Mean time of survival after diagnosis is 2.8
  years
• Progression appears to be inevitable
• Available medical therapies (calcium-channel
  blockers, prostacyclin analogues, endothelin
  antagonsists) only delay or slow worsening of the
  disease
• Death occurs due to right heart failure
• Lung transplantation represents the only
  currently available curative therapy

Peacock (1999) Thorax 54 1107 Rudarakanchana et
al (2001) Thorax 56 888
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PPH Prognosis
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Outline

• Background
• Pathology
• Genetic analyses of Familial PPH
• Mutations in BMPRII cause FPPH
• Mechanisms by which mutations in BMPRII may cause
  PPH
• Other potential contributors to PPH

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PPH Pathology

• Lesions are characterized by intimal fibrosis,
  smooth muscle hypertrophy and lumenal
  obliteration
• Different pathological subtypes recognised
• Plexogenic arteriopathy disorganised mass of
  endothelial cells, vascular smooth muscle cells,
  and myofibroblasts that occlude distal pulmonary
  arterioles
• Thrombotic arteriopathy microthrombi present in
  distal pulmonary arterioles
• Both types of lesion may co-exist within the same
  family and even the same patient

Peacock (1999) Thorax 54 1107
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Plexiform Lesion in PPH
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Outline

• Background
• Pathology
• Genetic analyses of Familial PPH
• Mutations in BMPRII cause FPPH
• Mechanisms by which mutations in BMPRII may cause
  PPH
• Other potential contributors to PPH

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Familial PPH

• Represents approximately 6-10 of PPH cases
• Apparent autosomal dominant inheritance
• Vertical transmission in up to five generations
• Male-to-male transmission documented
• Incomplete penetrance and variable expressivity
• Skipping of generations
• Variable age of onset
• Genetic anticipation suggested
• Characteristic of triplet-repeat diseases (eg
  Huntingtons Chorea and Myotonic Dystrophy)

Loyd (2002) Chest 122 284S
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Familial PPH
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Mapping the Familial PPH Locus

• Genome-wide scan for autosomal markers linked to
  the PPH phenotype undertaken by two independent
  groups in different PPH families
• Both groups reported evidence for linkage of a
  putative PPH locus, designated PPH1, to markers
  on chromosome 2q
• Fine mapping narrowed the candidate region to a 3
  cM interval (5.8 Mb) on chromosome 2q33

Nichols et al (1997) Nat Genetics 15 277 Morse
et al (1997) Circulation 952603 Deng et al
(2000) AJRCCM 161 1055
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Linkage of PPH1 to Chr 2q33
Nichols et al (1997) Nat Genetics 15 277
18
Outline

• Background
• Pathology
• Genetic analyses of Familial PPH
• Mutations in BMPRII cause FPPH
• Mechanisms by which mutations in BMPRII may cause
  PPH
• Other potential contributors to PPH

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Molecular Identification of PPH1

• 81 potential transcriptional units identified
  within the candidate region of 2q33
• 17 partially or completely characterized genes
• No mutations identified by sequencing of several
  candidate genes (Casp10, CTLA4, CD28)
• 12 distinct mutations identified in the BMPR2
  gene in different FPPH kindreds

International PPH Consortium (2000) Nat Gen 26
81 Deng et al (2000) Am J Hum Gen 67 737
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BPMRII Mutations in PPH

• Subsequent studies have demonstrated the presence
  of BMPRII mutations in 40/73 families studied
• Several families in which no BMPR2 mutations were
  identified demonstrate linkage to 2q33
• Germ-line mutations in BMPRII are present in
  nearly one third of PPH patients with no family
  history (ie sporadic PPH)
• BMPRII mutation present in a patient with
  apparent hereditary pulmonary VOD

Thomson et al (2000) J Med Genet 37 74 Runo et
al (2002) AJRCCM 167 889
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BPMRII Mutations in PPH
Trembath and Harrison (2003) Ped Res 6 883
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BMPRII

• Member of the type II TGF-b receptor superfamily
• Transmembrane serine/threonine kinase
• Binds ligands of the bone morphogenetic protein
  subgroup of TGF-b ligands (BMP-2,-4,-7)
• Important roles in regulation of development,
  growth and differentiation
• Expressed in pulmonary endothelium and to a
  lesser degree in pulmonary arterial smooth muscle
  cells (as are multiple BMPs)
• Ligand binding promotes formation of heterodimers
  with type I BMP receptors and activation of
  BMPRI/Smad signaling pathways
• Long cytoplasmic tail not present in other type
  II TGFb receptors

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BMP Signaling Pathway
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Outline

• Background
• Pathology
• Genetic analyses of Familial PPH
• Mutations in BMPRII cause FPPH
• Mechanisms by which mutations in BMPRII may cause
  PPH
• Other potential contributors to PPH

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How Do Mutations in BMPRII Cause PPH?

• At the cellular level
• Imbalance between proliferation and apoptosis of
  endothelial cells and/or smooth muscle cells

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Effect of BMPs on Proliferation of PASMCs

• BMP-treatment inhibits serum-stimulated
  proliferation of cultured control and SPH PASMCs
  by approximately 50
• BMP-treatment inhibits serum-stimulated
  proliferation of PPH PASMCs by much less

Morrell et al (2001) Circulation 104 790
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BMP-induced Apoptosisof PASMCs

• BMP treatment increases apoptosis of cultured
  normal and SPH PASMCs by 5-10 fold
• Apoptosis of PPH PASMCs increases by only 2-3
  fold in response to BMP treatment

Zhang et al (2003) Am J Physiol Lung Cell Mol
Physiol 285 L740
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How Do Mutations in BMPRII Cause PPH?

• At the molecular level
• Haploinsufficiency of BMP signaling
• Dominant negative inhibition of BMP signaling
• Predisposition to second hit that results in
  homozygous loss of BMPRII function
  (tumor-suppressor model)
• Gain-of-function mutations lead to high-level,
  constitutive or altered BMP signaling

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How Do Mutations in BMPRII Cause PPH?

• At the molecular level
• Haploinsufficiency of BMP signaling
• Dominant negative inhibition of BMP signaling
• Predisposition to second hit that results in
  homozygous loss of BMPRII function
  (tumor-suppressor model)
• Gain-of-function mutations lead to high-level,
  constitutive or altered BMP signaling

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Signaling of VariousBMPRII-PPH Mutants

• PPH mutations in the extracellular and kinase
  domains of BMPRII do not promote normal
  BMP-stimulated transcriptional activation
• PPH mutations in the cytoplasmic tail of BMPRII
  exhibit normal BMP-stimulated transcriptional
  activation

Nishihara et al (2002) Mol Biol Cell 13 3055
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How Do Mutations in BMPRII Cause PPH?

• At the molecular level
• Haploinsufficiency of BMP signaling
• Dominant negative inhibition of BMP signaling
• Predisposition to second hit that results in
  homozygous loss of BMPRII function
  (tumor-suppressor model)
• Gain-of-function mutations lead to high-level,
  constitutive or altered BMP signaling

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Dominant Negative Effects of BMPRII-PPH Mutations

• Transfection experiments suggest that certain EC
  and kinase domain BMPRII-PPH mutations may act as
  dominant negative inhibitors of BMP-stimulated
  transcriptional activation
• PPH mutations in the cytoplasmic tail of BMPRII
  do not exert a dominant negative effect on
  BMP-stimulated transcriptional activation

Rudarakanchana et al (2002) Hum Mol Gen 11 1517
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How Do Mutations in BMPRII Cause PPH?

• At the molecular level
• Haploinsufficiency of BMP signaling
• Dominant negative inhibition of BMP signaling
• Predisposition to second hit that results in
  homozygous loss of BMPRII function
  (tumor-suppressor model)
• Gain-of-function mutations lead to high-level,
  constitutive or altered BMP signaling

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Clonality of Endothelial Cells within Plexiform
Lesions of PPH

• Analysis of X-chromosome inactivation patterns in
  endothelial cell from PPH and SPH plexiform
  lesions using human androgen-receptor gene
  methylation assay
• X-linked
• (CAG)n repeats gt 90 of women are polymorphic at
  this locus
• Evidence of endothelial monoclonality present in
  17/22 PPH lesions analzyed 0/19 SPH lesions
  appeared to be monoclonal
• Different lesions from the same patient showed
  allelic discordance in 3/4 PPH patients

Lee et al (2002) JCI 101 927
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How Do Mutations in BMPRII Cause PPH?

• At the molecular level
• Haploinsufficiency of BMP signaling
• Dominant negative inhibition of BMP signaling
• Predisposition to second hit that results in
  homozygous loss of BMPRII function
  (tumor-suppressor model)
• Gain-of-function mutations lead to high-level,
  constitutive or altered BMP signaling

36
BMPRII-PPH Mutants Do Not Increase Smad-dependent
Signaling

• BMPRII mutations in PPH affect are widely
  distributed throughout the protein
• Mutations present in the EC domain, TM domain,
  kinase domain and cytoplasmic tail
• Frameshift/nonsense mutations in the 5 end of
  the gene predicted to create truncated protein or
  unstable mRNA
• In transfection experiments most BMPRII-PPH
  mutants result in decreased expression of
  BMP-responsive reporter constructs

Machado et al (2001) Am J Hum Gen 68 92
37
BMPRII-PPH Mutants Stimulate Cell Proliferation
and Increase MAP Kinase Phosphorylation

• Stimulation of TGFb receptors activates p38 MAPK
  pathway in Smad-independent manner
• Transfection of BMPRII-PPH mutants results in
  ligand-independent phosphorylation of p38 MAPK
  and enhanced cell proliferation
• Addition of BMPs promotes further increases in
  both level of MAPK phosphorylation and cell
  proliferation
• Pharmacological MAPK inhibition blocks increases
  in cell proliferation in response to BMPs

Rudarakanchana et al (2001) Hum Mol Gen 11 1517
38
Outline

• Background
• Pathology
• Genetic analyses of Familial PPH
• Mutations in BMPRII cause FPPH
• Mechanisms by which mutations in BMPRII may cause
  PPH
• Other potential contributors to FPPH

39
Are There Other Contributors to PPH?

• Incomplete penetrance of BMPRII mutations
• Variable expressivity of familial PPH phenotype
• Absence of BMPRII mutations in many PPH families
  and sporadic PPH patients

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A Second PPH locus

• Familial PPH linked to chromosome 2q31 in three
  different and apparently unrelated families
• No BMPRII mutations detected in PPH patients from
  these families
• Highest LOD score obtained with markers located
  15-19 cM proximal to BMPR2 on chromosome 2q,
  suggesting that a second FPPH locus (FPPH2) may
  exist

Rindermann et al (2001) JACC 4 2237
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Appetite Suppressants and PPH

• Well-documented association between PAH and
  anorexigen use
• Aminorex (1960s)
• Fenfluramine and dexfenfluramine (1980-90s)
• In one series of patients with PPH thought to be
  related to anorexigen use, ten percent also had
  BMPRII mutations

Rich et al (2001) Chest 117 870 Humbert et al
(2002) Eur Resp J 20 518
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Mutations in ALK1 Associated with Pulmonary
Hypertension

• Hereditary hemorrhagic telangiectasia
  (Osler-Weber-Rendu Syndrome) results from
  mutations in the type 1 and type 3 TGF-b
  receptors, respectively, ALK1 and Endoglin
• HHT families identified in which pulmonary
  hypertension also occus (sometimes in the same
  patient)
• ALK1 mutations detected in these patients
• ALK1 mutations have not been found in FPPH
  kindreds or patients with sporadic PPH

Trempath et al (2001) NEJM 345 325
43
Angiopoietin/TIE2 and Serotonin in Pulmonary
Hypertension

• Increased Ang1 mRNA expression and TIE2
  phosphorylation found in lung tissue from
  patients with pulmonary hypertension due to
  various causes
• In rats lung-specific Ang1 overexpression causes
  pulmonary hypertension and increases pulmonary
  5-HT levels
• Anorexigens alter 5-HT metabolism
• Genetic or pharmacologic inhibition of 5-HT
  receptors blocks hypoxia-induced pulmonary
  hypertension in mice

Du et al (2001) NEJM 348 500 Sullivan et al
(2003) PNAS 100 12331 Launay et al (2002) Nat
Med 8 1129
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Absence of BMPRII Mutations in PPH Patients with
CTD

• Two small studies involving 36 total patients
• 33 with systemic sclerosis
• 2 with SLE
• 1 with MCTD
• No BMPRII mutations detected in these patients

Tew et al (2002) Arth Rheum 46 2829 Morse et al
(2002) J Rheum 29 2379
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HHV8 and PPH

• Human herpesvirus 8 has been associated with
  Kaposis sarcoma and Castlemans disease
• Detection of HHV8 in microdissected endothelial
  cells from PPH and SPH plexiform lesions
• HHV8 LANA-1 present in 10/16 PPH specimens but in
  0/14 SPH specimens
• HHV8 DNA detected by PCR in the same 10/16 PPH
  specimens and in only 1/14 SPH specimens
• BMPRII mutations present in 4/16 PPH patients
  (2/4 were positive for HHV8)

Cool et al (2003) NEJM 349 1113
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Conclusions

• Abnormal BMPRII signaling plays an important role
  in the pathogenesis of many cases of PPH, both
  familial and sporadic
• How mutations in BMPRII contribute to PPH remains
  to be elucidated
• Not all cases of PPH are due to BMPRII mutations
• BMPRII mutations may be involved in some but not
  likely most cases of SPH
• Abnormalities in other signaling pathways must be
  important in the pathogenesis of PAH

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Future Directions

• Mouse model of FPPH
• BMPRII -/- homozygous mice die early in
  development
• No report of PAH in heterozygotes
• Downstream components of the BMP signaling
  pathway(s) that contribute to PPH
• Interactions between BMPRII signaling and other
  molecular pathways implicated in PAH