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Plenary paper
Hypoxia-inducible factors in human pulmonary arterial hypertension: a link to the
intrinsic myeloid abnormalities
Samar Farha,1 Kewal Asosingh,2 Weiling Xu,2 Jacqueline Sharp,2 Deepa George,2 Suzy Comhair,2 Margaret Park,3
W. H. Wilson Tang,3 James E. Loyd,4 Karl Theil,5 Raymond Tubbs,5 Eric Hsi,5 Alan Lichtin,6 and Serpil C. Erzurum1,2
1Respiratory
Institute, 2Lerner Research Institute, and 3Heart and Vascular Institute, Cleveland Clinic, Cleveland, OH; 4Allergy, Pulmonary, and Critical Care,
Vanderbilt University, Nashville, TN; and 5Pathology and Laboratory Medicine Institute and 6Taussig Cancer Institute, Cleveland Clinic, Cleveland, OH
Pulmonary arterial hypertension (PAH) is
a proliferative vasculopathy characterized by high circulating CD34ⴙCD133ⴙ
proangiogenic progenitors, and endothelial cells that have pathologic expression
of hypoxia-inducible factor 1 ␣ (HIF-1␣).
Here, CD34ⴙCD133ⴙ progenitor cell numbers are shown to be higher in PAH bone
marrow, blood, and pulmonary arteries
than in healthy controls. The HIF-inducible
myeloid-activating factors erythropoietin,
stem cell factor (SCF), and hepatocyte
growth factor (HGF) are also present at
higher than normal levels in PAH blood, and
related to disease severity. Primary endothelial cells harvested from human PAH lungs
produce greater HGF and progenitor recruitment factor stromal-derived factor 1 ␣
(SDF-1␣) than control lung endothelial cells,
and thus may contribute to bone marrow
activation. Even though PAH patients had
normal numbers of circulating blood elements, hematopoietic alterations in myeloid
and erythroid lineages and reticulin fibrosis
identified a subclinical myeloproliferative
process. Unexpectedly, evaluation of bone
marrow progenitors and reticulin in nonaffected family members of patients with familial PAH revealed similar myeloid abnormalities. Altogether, the results show that PAH is
linked to myeloid abnormalities, some of
which may be related to increased production of HIF-inducible factors by diseased
pulmonary vasculature, but findings in nonaffected family suggest myeloid abnormalities may be intrinsic to the disease process. (Blood. 2011;117(13):3485-3493)
Introduction
Pulmonary arterial hypertension (PAH) is a vasculopathy of the
pulmonary circulation characterized by arterial obliteration secondary to unchecked pathologic angiogenic processes.1-3 An abundance of studies over the past decade provide evidence for the
paradigm of lifelong interdependence between angiogenesis and
hematopoiesis.4-6 The concept of a common hematopoieticendothelial stem cell, that is, hemangioblast, with bidirectional,
reversible gene transcription and persistence is well established in
developmental biology.7 In postnatal life to adulthood, hemangioblasts are readily identifiable in the bone marrow by the CD133selective expression on a small subpopulation of CD34-positive
hematopoietic stem cells.8 Hemangioblasts give rise to all blood
cellular components, but whether these cells give rise to endothelium during postnatal neovascularization is uncertain.9,10 In contrast, studies clearly substantiate that CD34⫹CD133⫹ progenitors
are vital contributors to angiogenesis via proangiogenic effects on
endothelial cells in vessels.11-18 Our and other studies identify that
CD34⫹CD133⫹ progenitors are present at higher than normal
levels in the circulation of PAH patients and are more proliferative
than circulating progenitors of healthy controls.19,20 The relationship of numbers of circulating CD34⫹CD133⫹ cells to severity of
PAH suggest that these cells may promote the angioproliferative
vascular remodeling.20 However, whether the source of greater
numbers of circulating CD34⫹CD133⫹ cells in PAH patients is
related to a greater number of stem cells in the bone marrow and/or
more mobilization of progenitors to the circulation—and/or because of higher levels of the effectors that promote either of these
processes—is unknown.
In general, proliferation and mobilization of bone marrow
progenitors is under the control of growth factors that are transcriptionally regulated by hypoxia-inducible factors (HIF). Classically,
HIF-inducible factors that affect bone marrow progenitors include
erythropoietin (Epo), hepatocyte growth factor (HGF) and stem
cell factor (SCF, also known as Steel Factor), and vascular
endothelial growth factor (VEGF).21-24 In addition to inductive
effects on progenitors, Epo and other HIF-inducible growth factors
also act on pulmonary artery endothelial cells in the vascular bed to
induce a proliferative, promigratory, and antiapoptotic endothelial
cell.6,25,26 Effects on the bone marrow stem cells and endothelial
cells are mediated via activation of signal transducers and activators of transcription factors (STAT).25,27-33 Once mobilized from the
bone marrow, circulating CD34⫹CD133⫹ cells are recruited to
activated tissue sites where vascular repair or growth is needed;
recruitment to specific vascular sites is regulated via local production of HIF-inducible factors, such as stromal-derived factor-1␣
(SDF-1␣).11,16,31,32,34 Thus, bone marrow events and local vascular
changes are coordinately regulated by HIF activation and lead to
robust angiogenic responses. Given the recent discovery of HIF
activation in PAH lung endothelial cells, we hypothesized that
HIF-inducible factors may be higher than normal in PAH patients
and cause both the proliferation of multipotent hemangioblasts in
Submitted September 8, 2010; accepted December 22, 2010. Prepublished
online as Blood First Edition paper, January 21, 2011; DOI 10.1182/blood2010-09-306357.
The online version of this article contains a data supplement.
An Inside Blood analysis of this article appears at the front of this issue.
© 2011 by The American Society of Hematology
BLOOD, 31 MARCH 2011 䡠 VOLUME 117, NUMBER 13
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
3485
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FARHA et al
the bone marrow and increased mobilization of progenitors from
the bone marrow. To test this, numbers of CD34⫹CD133⫹ hemangioblasts in the bone marrow and CD34⫹CD133⫹ progenitors in
the circulation and within the pulmonary arteries were quantitated,
and the serum levels of HIF-inducible factors were determined in
individuals with PAH in comparison to healthy controls. Because
the proliferation, mobilization, and recruitment of progenitors to
the pulmonary vasculature contribute to proliferative vasculopathy,
we also assessed the relationship of HIF-inducible factors to
clinical parameters of PAH severity, such as 6-minute walk
distance and cardiac function. To evaluate the mechanisms contributing to proliferation and mobilization of proangiogenic cells,
numbers of progenitor cells and the activation of tyrosine kinases
that are central to myeloproliferative processes, that is, Janus
Kinase 2 (JAK2) and STAT3 and STAT5, were evaluated in bone
marrow of patients with PAH in comparison to healthy controls.
Alternatively, to evaluate whether the pulmonary vascular cells
within the diseased lungs nurture the bone marrow progenitor
response, pulmonary artery endothelial cells of PAH patients were
compared with normal pulmonary artery endothelial cells for
production of HIF-inducible factors.
Methods
Study population
Volunteers with familial PAH (FPAH), idiopathic PAH (IPAH), and
associated PAH (APAH) were enrolled in the study as well as nonaffected
family members. All PAH subjects underwent blood draw for quantification
of circulating proangiogenic progenitors and mobilizing factors. A subgroup agreed to have bone marrow biopsy and aspirate. For those subjects,
blood was sent for a complete cell count and differential, reticulocyte count,
and peripheral smear evaluation by the clinical pathologist; and on the same
visit, they underwent bone marrow biopsy and aspirate. Healthy controls
with no known lung diseases provided bone marrow aspirates and blood.
The Institutional Review Board (IRB) at the Cleveland Clinic approved this
study and all subjects gave written informed consent in accordance with the
Declaration of Helsinki. Explanted lungs from PAH subjects undergoing
transplant and donor lungs not used in transplantation were harvested to
obtain pulmonary artery endothelial cells under an IRB-approved protocol.
Flow cytometric evaluation of progenitor cells and
colony-forming assays
CD34⫹CD133⫹ progenitors in peripheral blood and bone marrow were
analyzed by flow cytometry as described in detail previously.20 Colony
formation was performed as described by Hill et al35 using Endocult Liquid
Medium (StemCell Technologies) as reported by our group.20 To analyze
progenitor homing to the pulmonary artery vascular bed, the pulmonary
artery was dissected to terminal branches as far as visible to the human eye
from lungs explanted at transplantation or from donor lungs not used in
transplantation. The blood vessels were excised longitudinally and endothelium was dispersed by collagenase type II solution (2 mg/mL in phosphatebuffered saline [PBS]; Worthington Biochemical) on the inner surface,
followed by brief incubation at 37°C. Cells were collected and washed in
PBS/1% bovine serum albumin (BSA)/0.02% sodium azide. After counting, 100 ⫻ 103 to 200 ⫻ 103 cells were stained with Red LIVE/DEAD dye
(Invitrogen) followed by cell-surface staining for CD34 and CD133.20 Cells
(50 ⫻ 103 to 100 ⫻ 103) were acquired on a FACScan (Becton Dickinson)
flow cytometer and the percentage of CD34⫹CD133⫹ within the live cell
fraction was quantified using CellQuest 3.3 software (Becton Dickinson).
performed as described in “Flow cytometric evaluation of progeuitor cells
and colony-forming assays.” Smears from the bone marrow aspirates were
collected for cell counts and differential performed by experienced
pathologists. Bone marrow biopsies were fixed overnight in 10% formalin
followed by overnight decalcification in decalcifying solution (Fischer) and
processing and embedding in paraffin. Five-micrometer sections were cut
and stained with hematoxylin and eosin (H&E). Reticulin (type III
collagen) staining was performed in the clinical laboratory using standard
silver-impregnation methods. Reticulin was quantified by an experienced
pathologist blinded to the subjects’ diagnosis using the Bauermeister scale:
0, no demonstrable reticulin fibers; 1, occasional fine individual fibers and
foci of a fine fiber network; 2, fine fiber network throughout most of the
section, but no coarse fibers; 3, diffuse fiber network with scattered thick
coarse fibers, but no mature collagen; and 4, diffuse, often coarse fiber
network with areas of collagen reticulin stained.36 JAK2 V617F mutation
that is commonly found in myeloproliferative disorders was evaluated in
bone marrow tissues as described previously.37 Immunohistologic analysis
for pSTAT3 and pSTAT5 was carried out by using the rabbit polyclonal
antihuman phosphoSTAT3 (Cell Signaling), and the mouse monoclonal
anti–human phosphoSTAT5a/b (AdvantexBio) after antigen retrieval with
Antigen Retrieval Citra Plus (Bio Genex). Diaminobenzidine (DAB) and
hydrogen peroxide (H2O2) were applied to develop color. Negative control
of secondary antibody alone was performed on each section of tissue
studied. All slides were counterstained with hematoxylin. The pSTAT3 and
pSTAT5 was evaluated by counting numbers cells with nuclear positivity. A
minimum of 5 fields was examined at ⫻400 magnification and at least
500 cells were counted.
ELISA for HIF-inducible factors
Epo, HGF, SCF, SDF-1␣, and VEGF were measured in plasma using a
quantikine enzyme-linked immunosorbent assay (ELISA; R&D Systems).
Epo, HGF, and SDF-1␣ levels measured in the supernatant of cultured
primary human pulmonary artery endothelial cells were normalized to
protein concentration of cells in culture.
EpoR expression
Epo receptor (EpoR) expression was analyzed by staining of day-5
colony-forming units–endothelial cells (CFUs-ECs) with fluorescein isothiocyanate (FITC)–conjugated anti–human EpoR monoclonal antibodies (R&D
Systems). Isotype-matched control antibody was used as control. Cells
were preincubated with Fc-block (eBioscience) before staining to block
nonspecific Fc-receptor–mediated binding.
CFU-EC:electrophoretic mobility shift assay
Whole-cell extracts (WCEs) from CFU-ECs were prepared as previously
described.1 The duplex oligonucleotide (5⬘-AGATTTCTAGGAATTCAATCC-3⬘) specific for STAT5 binding was purchased from Santa Cruz
Biotechnology. To specifically identify DNA-binding factor in binding
complexes, rabbit polyclonal anti–STAT-5 antibody (Ab; Santa Cruz
Biotechnology) was added to the binding reaction mix.
Immunohistochemical analyses
For SDF-1␣ and HGF immunostaining, control lungs were collected from
donor lungs not used in transplantation (n ⫽ 3). IPAH lung tissues were
obtained from explanted IPAH lung or postmortem tissues (n ⫽ 3). Rabbit
polyclonal anti–SDF-1␣ Ab (Abcam) and anti-HGF Ab (MyBioSource)
were used after antigen retrieval. Positive control for SDF-1␣ was consisted
of tissue section of a lung carcinoma and mouse liver tissue section was
used as positive control for HGF. Negative control of secondary Ab alone
was performed on each section of tissue studied.
Pulmonary artery endothelial cells
Bone marrow biopsy/aspirate processing
Bone marrow aspirate and biopsy were performed under local anesthesia.
Aspirate was collected in an EDTA tube and flow cytometric analysis was
Primary pulmonary artery endothelial cells (PAECs) were isolated from
explanted PAH lungs or healthy control lungs not used for transplantation as
previously described.3 Briefly, cells were cultured in endothelial cell growth
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BLOOD, 31 MARCH 2011 䡠 VOLUME 117, NUMBER 13
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Table 1. Characteristics of subjects
Age, years
Sex,
F/M
BMPR2
mutation, ⴙ/ⴚ
Mean PAP,
mm Hg
Cardiac output,
L/min
PVR,
wood unit
RVSP,
mm Hg
ERA,
ⴙ/ⴚ
Prostacyclin,
ⴙ/ⴚ
Sildenafil,
ⴙ/ⴚ
IPAH
43 ⫾ 3
20/4
3/21
52 ⫾ 3
5.5 ⫾ 0.5
9.6 ⫾ 1.8
70 ⫾ 5
14/24
21/3
8/16
FPAH
41 ⫾ 4
10/3
4/9
58 ⫾ 7
3.4 ⫾ 0.3
14.6 ⫾ 3.0
66 ⫾ 7
7/13
6/7
7/6
0/11
50 ⫾ 5
5.3 ⫾ 0.6
8⫾2
77 ⫾ 8
9/15
7/15
7/8
.06
.6
⬍ .01
.1
.6
.6
.2
.4
APAH
56 ⫾ 3
11/4
Controls
35 ⫾ 1
46/16
P
⬍ .01
.8
BMPR2 indicates bone morphogenetic protein receptor; F/M, female/male; PAP, pulmonary arterial pressure; PVR, pulmonary vascular resistance; RVSP, right ventricular
systolic pressure; ERA, endothelin receptor antagonist; IPAH, idiopathic pulmonary arterial hypertension; FPAH, familial pulmonary arterial hypertension; and APAH,
associated pulmonary arterial hypertension.
Results
6.2 ⫾ 2.3; FPAH 6.0 ⫾ 0.7; APAH 7.5 ⫾ 0.8, ANOVA P ⫽ .7).
The CD133⫹CD34⫺ cells were similar between PAH and controls
and among the PAH groups (% cells: PAH 2.3 ⫾ 0.3; controls
3.1 ⫾ 0.8, P ⫽ .4). However, numbers of the more mature
CD133⫹CD34⫹ cells in bone marrow of PAH patients were higher
than healthy controls (P ⫽ .008), but similar among PAH groups
(% CD34⫹CD133⫹ cells: IPAH 1.9 ⫾ 0.6; FPAH 2.9 ⫾ 0.4; APAH
2.0 ⫾ 0.8, ANOVA P ⫽ .3; Figure 1). Consistent with an expansion of progenitors at the CD34 differentiation level, the numbers
of CD34⫹CD133⫺ cells in the bone marrow were also higher in
PAH compared with controls without significant difference among
FPAH, IPAH, and APAH (% CD34⫹CD133⫺ cells: PAH 1.53 ⫾ 1.9;
controls 0.63 ⫾ 0.7, P ⬍ .0005). The percentage of CD34⫹CD133⫹
cells in the bone marrow was related to pulmonary vascular
resistance (R ⫽ 0.7, P ⫽ .04) and tended to correlate with pulmonary artery pressures (R ⫽ 0.6, P ⫽ .06).
Study population
Greater numbers of circulating CD34ⴙCD133ⴙ progenitors
Patients with PAH (13 familial [FPAH: class 1.2], 24 idiopathic
[IPAH: 1.1], and 15 associated [APAH: 1.3]) and healthy volunteers (N ⫽ 62) were enrolled in the study (Table 1). Because of
sample limitations, not all samples were available for all assays.
Nine nonaffected family members of FPAH subjects were also
recruited for the study. The hematologic profiles of PAH subjects
were within the normal range of values although there were
significant differences among the groups (Table 2). A subgroup
agreed to undergo bone marrow aspirate and biopsy (14 PAH,
7 healthy controls, 6 nonaffected family members). The differential
cell count in bone marrow aspirates varied among PAH and
controls (Table 3). IPAH patients had increased myelocyte numbers, and FPAH and APAH patients had higher numbers of
erythroid precursors compared with controls.
Similar to previous report,20 circulating CD34⫹CD133⫹ cells were
higher in individuals with PAH compared with controls (P ⫽ .01;
Figure 1). Here, new data are provided to show that FPAH patients
have the highest numbers of circulating CD34⫹CD133⫹ cells
(% CD34⫹CD133⫹ cells: FPAH 0.2 ⫾ 0.04; IPAH 0.09 ⫾ 0.01;
APAH 0.1 ⫾ 0.07; controls 0.06 ⫾ 0.008, ANOVA P ⬍ .01); FPAH
was higher compared with IPAH or controls (all comparisons,
Tukey P ⬍ .05). The numbers of CD34⫹CD133⫺ cells were also
higher in PAH compared with controls, but without significant
difference between FPAH, IPAH, and APAH (% CD34⫹CD133⫺
cells: PAH 0.06 ⫾ 0.00; controls 0.04 ⫾ 0.00, P ⫽ .01). The
numbers of CD133⫹CD34⫺ cells were elevated in subjects with
PAH compared with controls (% CD133⫹CD34⫺ cells: IPAH
1.7 ⫾ 0.3; FPAH 0.8 ⫾ 0.1; APAH 1.3 ⫾ 0.2; controls 0.3 ⫾ 0.05,
ANOVA P ⬍ .001) with IPAH subjects having higher numbers
than FPAH or controls (all comparisons Tukey P ⬍ .05) and APAH
having higher numbers than controls (Tukey P ⬍ .05). The total
circulating progenitors including CD34⫹CD133⫺, CD133⫹CD34⫺,
and CD133⫹CD34⫹ cells were higher in PAH compared with
controls (% total progenitor cells: PAH 1.5 ⫾ 0.2; controls
0.3 ⫾ 0.05, P ⬍ .001) and similar among the different PAH
medium (EGM-2; Cambrex) on plates precoated with coating media
containing fibronectin and passaged at 70%-80% confluence by dissociation from plates with trypsin-EDTA (Invitrogen). Primary cultures of
passages 5-8 were used in experiments. Human umbilical vein endothelial
cells (HUVECs; Lonza) were cultured in EGM-2 (Cambrex).
Statistical analysis
Descriptive measures for quantitative variables consist of means with
appropriately derived standard errors in the form mean ⫾ SE. Comparisons
of PAH, unaffected family members, and healthy subjects were performed
using analysis of variance (ANOVA) or t test when 2 means were compared.
When ANOVA was significant, Tukey was performed for pairwise comparison. Spearman correlation coefficients were used to describe relationships
among pairs of quantitative variables in a manner free of the normality
assumption.
Greater numbers of hematopoietic progenitors in PAH
Bone marrow aspirates were evaluated for numbers of CD34⫹ and
CD133⫹ progenitors. The total progenitors, including
CD133⫹CD34⫺, CD133⫹CD34⫹, and CD133⫺CD34⫹ cells, were
similar between PAH and controls (% total progenitors: PAH
[N ⫽ 14], 6.3 ⫾ 0.6; controls [N ⫽ 3], 5.1 ⫾ 0.9, P ⫽ .3) and
among the different PAH subgroups (% total progenitor cells: IPAH
Table 2. Baseline hematologic profile
RBC, 106/␮L
Hgb, g/dL
WBC, 103/␮L
Platelets, 103/␮L
ANC, 103/␮L
ALC, 103/␮L
AEC, 103/␮L
AMC, 103/␮L
ABC, 103/␮L
IPAH
4.8 ⫾ 0.1
13.3 ⫾ 0.3
5.9 ⫾ 0.5
213 ⫾ 18
3.7 ⫾ 0.4
1.6 ⫾ 0.1
0.1 ⫾ 0.02
0.7 ⫾ 0.1
0.02 ⫾ 0.01
FPAH
5.1 ⫾ 0.2
14.1 ⫾ 0.6
8.2 ⫾ 0.6
199 ⫾ 24
5.1 ⫾ 0.5
2.2 ⫾ 0.2
0.2 ⫾ 0.03
0.4 ⫾ 0.1
0.04 ⫾ 0.01
APAH
4.4 ⫾ 0.2
12.5 ⫾ 0.5
5.1 ⫾ 0.5
208 ⫾ 47
2.5 ⫾ 0.3
1.7 ⫾ 0.2
0.2 ⫾ 0.1
0.6 ⫾ 0.1
0.04 ⫾ 0.01
.02
.1
⬍ .01
.0009
.002
.01
.2
.2
.6
P
RBC indicates red blood cell; Hgb, hemoglobin; WBC, white blood cell; ALC, absolute lymphocyte count; ANC, absolute neutrophil count; AEC, absolute eosinophil count;
AMC, absolute monocyte count; ABC, absolute basophil count; IPAH, idiopathic pulmonary arterial hypertension; FPAH, familial pulmonary arterial hypertension; and APAH,
associated pulmonary arterial hypertension.
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FARHA et al
Table 3. Bone marrow aspirate differential
% Blasts
% Promyel
% M/M/B/S
IPAH
0.2 ⫾ 0.2
0
72 ⫾ 2
FPAH
1⫾0
0
55 ⫾ 6
APAH
0.5 ⫾ 0.3
0
62 ⫾ 3
2.7 ⫾ 1.1
5.7 ⫾ 1.8
0
22 ⫾ 3
7⫾2
0.7 ⫾ 0.5
Controls
0.5 ⫾ 0.2
0
64 ⫾ 3
4.7 ⫾ 1.1
2.7 ⫾ 0.6
0.5 ⫾ 0.2
13 ⫾ 3
15 ⫾ 3
0.2 ⫾ 0.2
.02
.2
.2
.08
.02
.3
.2
P
.4
% Mono
% Eosino
% Baso
% Erythroids
% Lymphocytes
% Plasma cells
1.8 ⫾ 0.7
4.0 ⫾ 0.8
0.2 ⫾ 0.2
11 ⫾ 3
9⫾4
1.0 ⫾ 0.3
2.5 ⫾ 0.5
5.5 ⫾ 2.5
0
29 ⫾ 6
5⫾2
1.0 ⫾ 0
Promyel indicates promyelocytes; M/M/B/S, myelocytes/metamyelocytes/bands/segs; Mono, monocytes; Eosino, eosinophils; and Baso, basophils.
subgroups (% total progenitor cells: IPAH 1.8 ⫾ 0.3; FPAH
1.1 ⫾ 0.2; APAH 1.5 ⫾ 0.3, ANOVA P ⫽ .3).
Proliferation of CD34⫹CD133⫹ cells as determined by colonyforming assays was significantly higher in individuals with PAH
compared with controls (CFU-EC colonies/106 mononuclear cells:
PAH [N ⫽ 28], 187 ⫾ 31; controls [N ⫽ 10], 48 ⫾ 5, P ⬍ .01).
Patients with IPAH had the most proliferative progenitors compared controls (Tukey P ⬍ .05) but not significantly different from
FPAH or APAH (CFU-EC colonies/106 mononuclear cells: IPAH
251 ⫾ 56; FPAH 134 ⫾ 19; APAH 158 ⫾ 69; controls 48 ⫾ 5,
ANOVA P ⫽ .02).
Hematopoietic CFU assays
CFU-ECs have been shown to be enriched in myeloid CFU–
granulocyte monocyte (CFU-GM) colony forming progenitors.38
Here, hematopoietic CFU assays were performed using sorted bone
marrow hematopoietic progenitors (CD34⫹CD133⫹ cells) from healthy
controls and patients with PAH. The CD34⫹CD133⫹ cells gave rise to
all hematopoietic lineages (erythroid, myeloid, monocyte/macrophage,
and megakaryocytes), but the CFU-GM colony formation was
increased among PAH patients (number of CFU-GM colonies:
PAH 112 ⫾ 23; controls 38 ⫾ 8, P ⫽ .04). This suggested that the
Figure 1. Greater CD34ⴙCD133ⴙ progenitors in PAH.
(A-D) CD34⫹CD133⫹ progenitors in PAH determined by
flow cytometry. Box plots indicate median values, upper
and lower quartiles. There are increased hemangioblasts
in PAH bone marrow, and higher levels of CD34⫹CD133⫹
progenitors in circulation and within the pulmonary artery
endothelium than in controls. (E-F) STAT3 and STAT5
activation and localization in bone marrow biopsies.
Cellular localization of phosphoSTAT3 (pSTAT3) by immunohistochemical staining in bone marrow biopsy (arrowheads) from PAH patient and healthy control (E). Immunohistochemical staining for phosphoSTAT5 (pSTAT5) in
bone marrow biopsy (arrowheads) from PAH patient and
healthy control (F). (G) Reticulin increase in bone marrow
of PAH patients. Reticulin staining as a measure of
myelofibrosis, was increased in PAH bone marrows.
Arrowheads identify reticulin staining in bone marrow
biopsies from PAH subject. The reticulin stain around a
blood vessel is normally present, and is shown as internal
positive in the control tissue. (H) EpoR expression by flow
cytometry. Dashed lined histogram indicates background
staining with isotype control antibodies, solid histogram
indicates EpoR staining. CFU-EC have low levels of
EpoR, while lymphocytes are shown as negative control
have no EpoR. (I) STAT5 activation in CFU-EC. Electrophoretic mobility shift assay for STAT5 DNA binding
activation in whole cell extract of CFU-EC from PAH
patient (lane 4) and healthy control (lane 3) is shown.
STAT5 DNA-binding activation is similar among PAH and
control CFU-EC. A549 cells stimulated with Epidermal
Growth Factor (EGF) is a positive control, and the
supershift using antibody to STAT5 identifies the STAT5DNA complex (arrow). Scale bars: 6 ␮m (E); 10 ␮m (F);
40 ␮m (G).
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BLOOD, 31 MARCH 2011 䡠 VOLUME 117, NUMBER 13
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Figure 2. Elevated circulating HIF-inducible factors in PAH. Factors are grouped according to their biologic activity on stem cells/progenitors. Box plots indicate median
values, upper and lower quartiles. Levels of erythropoietin are strongly related to HGF in PAH subjects.
greater numbers of CFU-EC derived from PAH bone marrow and
circulation may be related to alteration of progenitor lineage
commitment.
Recruitment of CD34ⴙCD133ⴙ progenitors into the pulmonary
vascular bed
Lungs of PAH patients undergoing transplantation and donor lungs
not used in transplantation were harvested for the study of
progenitor cell recruitment into the pulmonary vasculature. To
assess whether the increased mobilization of progenitors is paralleled by greater recruitment into the pulmonary arteries, the
percentage of CD34⫹CD133⫹ cells was measured in single-cell
suspensions of the pulmonary artery endothelium obtained from
subjects undergoing lung transplantations or in donor lungs not
used in transplantation. PAH subjects had ⬃ 10-fold higher
CD34⫹CD133⫹ progenitors recruited into the pulmonary artery
wall compared with controls (P ⫽ .01; Figure 1).
JAK/STAT activation in PAH bone marrow
The circulating progenitor cell counts are increased in patients with
myeloproliferative disorders, and related to constitutive activation
of the JAK-STAT signal transduction pathway, most commonly
because of JAK2 1849G⬎T mutation (valine-to-phenylalanine
change at amino acid 617 [JAK 2 V617F]).39,40 Hence, we
evaluated JAK2 V617F mutation in genomic DNA derived from
circulating mononuclear cells, as well as activation of pSTAT3 and
pSTAT5 in bone marrow aspirates and biopsies. JAK2 mutation
was not detected in samples of PAH subjects (N ⫽ 18). Immunohistochemical staining of bone marrow showed that pSTAT3 and
pSTAT5 were present and localized to nuclei of erythroid and
granulocytic precursor cells and megakaryocytes (Figure 1E-F).
Nuclear positive staining of phosphorylated STAT5 (pSTAT5) was
easily detectable in a small subpopulation of mononuclear cells in
bone marrow biopsies of patients and controls (Figure 1F). In
contrast, pSTAT3 immunoreactivity in bone marrow aspirates was
present in more cells, but was less intense (Figure 1E). Because a
prior report identified myelofibrosis in patients with PAH, we
evaluated bone marrows for argyrophilic (⫹) reticulin as a measure
of fibrosis. Among hematologically healthy individuals, reticulin is
usually absent, or present at low levels, that is, Bauermeister grade
0 in 69%, grade 1 in 27%, and grade 2 in 4%.36,41 Here, reticulin in
all healthy human bone marrow was grade 0 (N ⫽ 4). Reticulin
was present at greater than normal levels in PAH bone marrows
(PAH bone marrow: Bauermeister grade 0 [14%], 1 [72%], 2 [14%];
Figure 1G). The high numbers of hematopoietic progenitors and
reticulin fibrosis is consistent with a myeloproliferative process in
PAH patients. Together, these findings and the greater numbers of
circulating progenitors prompted us to analyze soluble factors that
may promote myeloproliferation and progenitor mobilization.
Circulating levels of HIF-regulated bone marrow–activating
factors
Because HIF plays a central role in the regulation of stem/
progenitor cells and is activated in PAH endothelial cells, we
evaluated the circulating levels of HIF-regulated factors. Epo was
higher in PAH compared with controls (P ⫽ .005) with no difference among the PAH subgroups (Epo, mIU/mL: IPAH 25 ⫾ 5;
FPAH 45 ⫾ 13; APAH 57 ⫾ 32, ANOVA P ⫽ .4; all comparisons
Tukey P ⬎ .05). Other progenitor cell mobilization and proliferation factors measured were SCF and HGF; both were higher in
serum of patients with PAH compared with controls (all P ⬍ .05;
Figure 2). VEGF tended to be higher in PAH (P ⫽ .09) but SDF-1␣
was not different between the groups (Figure 2). Factors were
unrelated to circulating or bone marrow percentage of
CD34⫹CD133⫹ cells, percentage of CD34⫹ cells, and percentage
of CD133⫹ cells (all P ⬎ .1). However, Epo levels were related to
progenitor cell colony formation (CFU-EC), an indicator of
proliferation potential (R ⫽ 0.4, P ⫽ .01).
Epo was also related to parameters of clinical disease, including
right ventricular systolic pressure (R ⫽ 0.3, P ⫽ .05), Pulmonary
vascular resistance (PVR; R ⫽ 0.4, P ⫽ .06), and cardiac index
(R ⫽ ⫺0.3, P ⫽ .05) and tended to associate with brain natriuretic
peptide (BNP), a sign of cardiac failure (R ⫽ 0.3, P ⫽ .08).
Although patients were not anemic and had normal platelet counts,
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FARHA et al
BLOOD, 31 MARCH 2011 䡠 VOLUME 117, NUMBER 13
Epo was inversely related to hemoglobin (R ⫽ ⫺0.4, P ⫽ .01) and
platelets (R ⫽ ⫺0.4, P ⫽ .01), but not to white blood cell count or
creatinine (all P ⬎ .1). Interestingly, BNP was inversely related to
platelets (R ⫽ ⫺0.4, P ⫽ .03) and hemoglobin (R ⫽ ⫺0.3,
P ⫽ .06). Platelets were also related to right ventricular systolic
pressure (R ⫽ ⫺0.5, P ⫽ .001), while hemoglobin was modestly
related to 6-minute walk distances (R ⫽ 0.4, P ⫽ .02). HGF, SCF,
SDF-1␣, and VEGF were unrelated to any clinical parameters of
disease severity (all P ⬎ .1), even though Epo was strongly related
to HGF (R ⫽ 0.6, P ⫽ .005) and SCF (R ⫽ 0.3, P ⫽ .03). Given
the higher levels of HIF-regulated soluble factors, we questioned
whether PAH PAECs that have intrinsic activation of HIF-1␣ might
be the source of production of the factors.
Production of HIF-regulated factors by PAH PAECs
Lungs of PAH patients undergoing transplantation (n ⫽ 4) and
donor lungs not used in transplantation (n ⫽ 3) were available for
harvest of primary PAECs. Epo, HGF, and SDF-1␣ were measured
in the supernatant of cultured PAECs that were obtained from PAH
and healthy control donor lungs. Epo was not produced by cells in
culture, whereas HGF was produced by cultured PAH PAECs at
levels higher than in control cells (HGF [pg/mg protein]: controls
49 ⫾ 6; PAH 682 ⫾ 369, P ⫽ .03). HGF production by PAH cells
was also greater than control cells when the cells were cultured
under hypoxia (⬍ 5% oxygen [all P ⬍ .05]; Figure 3). Although
control cells had no significant change in HGF over time of
hypoxia (ANOVA P ⫽ .6), HGF production by PAH PAECs
significantly increased over time of hypoxia (ANOVA P ⫽ .0001;
Figure 3). SDF-1␣ increased in both PAH cells (ANOVA P ⫽ .0001)
and control cells under hypoxia (ANOVA P ⫽ .0007). SDF-1␣
production was greater in PAH cells compared with control cells at
baseline culture conditions and under conditions of hypoxia (all
P ⬍ .05; Figure 3).
To assess the in vivo production of HGF and SDF-1␣ by
vascular endothelium, we performed immunostaining on lung
tissues from controls and PAH (Figure 3B). Similar to the report by
Toshner et al,19 immunostaining of lung tissues from control
samples (n ⫽ 3) and patients with PAH (n ⫽ 3) revealed that
SDF-1␣ expression was plainly detectable in the endothelium of
plexiform lesions and vessels in PAH lungs, but only mild
positivity was observed in vessel endothelium of control lungs.
Likewise, strong HGF immunopositivity was observed in endothelial cells of plexiform lesions in PAH lungs, but HGF was less
prominent or absent in endothelial cells of healthy lung. The
presence of HGF and SDF-1␣ strong immunopositivity in endothelium of PAH lesional tissues, together with greater HGF levels in
PAH blood and greater secretion of factors by PAH endothelial
cells in culture, suggests that the mobilization and/or recruitment of
progenitors to PAH lungs may be related to the production of these
factors by a pulmonary vascular source in vivo.
Epo signaling in CFU-EC
Because CFU-EC were elevated in PAH patients in whom Epo
concentrations were also elevated, Epo receptor (EpoR) and signal
transduction (STAT5 activation) in CFU-EC of PAH patients were
compared with controls. Flow cytometric analysis indicated similarly low expression of EpoR on CFU-EC from PAH or healthy
controls (P ⫽ .5; Figure 1H). DNA-binding activation of STAT5,
one of the downstream mediators of EpoR signaling that mediates
hematopoiesis, was detectable under standard culture conditions
for the CFU-EC, and activation was similar among PAH (n ⫽ 3)
Figure 3. Increased production of HGF and SDF-1␣ by primary human pulmonary arterial endothelial cells (PAECs) from PAH. (A) HGF and SDF-1␣ is
secreted by PAH PAEC in culture at higher levels than by control cells under both
normoxia and hypoxia conditions (*all P ⬍ .05). HGF secretion by PAH PAEC was
significantly increased on exposure to hypoxia (ANOVA P ⫽ .0001). SDF-1␣ levels
also significantly increased with hypoxia in both PAH (ANOVA P ⫽ .0001) and control
PAEC (ANOVA P ⫽ .0007). (B) HGF and SDF-1␣ in lung tissues from controls and
PAH patients. Endothelial cells in plexiform lesions of PAH lungs had strong positive
immunoreactivity for HGF (arrowheads identify endothelial cells lining vascular
lumens). Endothelial cells of control lung expressed HGF, but immunopositivity was
much less prominent (arrowheads). SDF-1␣ expression was present in endothelial
cells of plexiform lesions in PAH lung (arrowheads). Only mild immunopositivity for
SDF-1␣ was found in vessel endothelium of control lung (arrowheads). Scale bars:
40 ␮m.
and control cells (n ⫽ 3; Figure 1I). STAT5 activation increased
slightly after Epo stimulation (30 minutes) of cells in culture (data
not shown). The modest STAT5 activation is likely because of
low-level EpoR on the cells. These results indicate that alterations
in EpoR/STAT5 activation did not account for greater CFU-EC
numbers in PAH.
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BLOOD, 31 MARCH 2011 䡠 VOLUME 117, NUMBER 13
BONE MARROW IN PAH
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Table 4. Baseline hematologic profile of nonaffected family members in comparison to FPAH subjects
RBC,
106/␮L
Hgb,
g/dL
WBC,
103/␮L
Platelets,
103/␮L
ANC,
103/␮L
ALC,
103/␮L
AEC,
103/␮L
AMC,
103/␮L
ABC,
103/␮L
Nonaffected family members
4.6 ⫾ 0.1
13.0 ⫾ 0.4
8.2 ⫾ 0.8
275 ⫾ 16
5.0 ⫾ 0.5
2.4 ⫾ 0.2
0.1 ⫾ 0.03
0.5 ⫾ 0.1
0.02 ⫾ 0.01
FPAH
5.1 ⫾ 0.2
14.1 ⫾ 0.6
8.2 ⫾ 0.6
199 ⫾ 24
5.1 ⫾ 0.5
2.2 ⫾ 0.2
0.2 ⫾ 0.03
0.4 ⫾ 0.1
0.04 ⫾ 0.01
.02
.1
.9
.02
.8
.6
.3
.5
.07
P
RBC indicates red blood cell; Hgb, hemoglobin; WBC, white blood cell; ALC, absolute lymphocyte count; ANC, absolute neutrophil count; AEC, absolute eosinophil count;
AMC, absolute monocyte count; and ABC, absolute basophil count.
Nonaffected family members of patients with FPAH
The findings of higher circulating levels of hematopoietic-active
factors in PAH, and the greater production of HGF and SDF-1␣ by
PAH endothelial cells, suggested that the hematopoietic abnormalities and greater progenitor mobilization in PAH occur, in part, in
response to the primary pulmonary angioproliferative disease. On
the other hand, abnormalities in hematopoietic precursors might be
intrinsic to PAH. To test this latter possibility, we reasoned that if
bone marrow abnormalities were intrinsic to PAH, then family
members of patients with FPAH, who do not have pulmonary
hypertension, might manifest hematopoietic abnormalities, for
example, high numbers of circulating and/or bone marrow–resident
CD34⫹CD133⫹ progenitors. Nine nonaffected family members
agreed to participate (age 43 ⫾ 4 years; female/male, 2/7) in the
study. Echocardiography studies of all nonaffected family members
were normal without signs of PAH Right ventricular systolic
pressure (RVSP; mm Hg: 22 ⫾ 3). Circulating blood counts were
similar to FPAH subjects with the exception of higher platelet
count in nonaffected family members (Table 4). Strikingly, all
nonaffected family members had circulating CD133⫹ cells and
CD34⫹ cell numbers similar to their afflicted relatives, and higher
than healthy nonrelated controls (Tukey P ⬍ .05; Figure 4). Bone
marrow–resident CD133⫹ cells, CD34⫹CD133⫹ cells, and
CD34⫹CD133⫺ cells were similar between FPAH and nonaffected
family members and healthy controls (supplemental Table 1,
available on the Blood Web site; see the Supplemental Materials
link at the top of the online article). However, bone marrow of
nonaffected family members had an increase of reticulin (100%
Bauermeister grade 1; N ⫽ 6) compared with healthy unrelated
controls (100% grade 0; N ⫽ 4). In contrast to the findings of high
circulating progenitors and increased reticulin in nonaffected
family members, levels of Epo, a valid indicator of enhanced HIF
activation or responsiveness, were not different from controls and
lower than FPAH (Epo, mIU/mL: nonaffected family members
9 ⫾ 2; FPAH 45 ⫾ 13; controls 11 ⫾ 1, ANOVA P ⬍ .001 and
TUKEY P ⬎ .05 for controls compared with nonaffected family
members and ⬍ 0.05 for FPAH compared with controls or nonaffected family members).
Discussion
Increased bone marrow hemangioblast numbers, alterations in
erythroid/myeloid lineages, increased reticulin, and greater mobilization of bone marrow progenitor cells define hematopoietic
abnormalities as an integral part of PAH disease. Higher levels of
bone marrow–activating factors are present in circulation, some of
which are produced by the PAH-diseased pulmonary vascular
endothelium and are likely instrumental in causing the hematopoietic effects (Figure 5). However, Epo, one of the most potent
mediators of bone marrow activation, is not derived from PAH PAECs,
which favors an underlying disturbance in the myeloid system.
In this report, we confirm prior work that circulating progenitors
are present at higher than normal levels and are more proliferative
in PAH than controls. Here, patients with FPAH are shown to have
the highest circulating progenitors, while patients with IPAH have
the most proliferative circulating progenitors. Likewise, there are
differences in bone marrow progenitors; patients with FPAH and
APAH have nearly double the erythroid progenitors of IPAH
patients, while patients with IPAH have greater differentiation
toward myeloid progenitors. In this study, we extend observations
regarding myelofibrosis that is associated with PAH. Prior study
identified a high incidence of myelofibrosis in PAH patients
selected for study on the basis of a hematologic indication such as
anemia, low platelets or both.42 Here, PAH patients had no overt
hematologic abnormality by measures of circulating blood counts,
yet still manifest abnormalities typical of myeloproliferative processes. However, somatic mutations of JAK2, which are typical of
primary myeloproliferative processes, were not present in PAH
patients. Rather, Epo levels were higher than normal in PAH
Figure 4. Increased circulating progenitor cells in nonaffected family members of patients with FPAH. Values of circulating progenitors in nonaffected family members
were comparable with their afflicted family members. Single- and double-positive CD34 and CD133 cells were measured in circulation using flow cytometry. Box plots indicate
median values, upper and lower quartiles.
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3492
BLOOD, 31 MARCH 2011 䡠 VOLUME 117, NUMBER 13
FARHA et al
Figure 5. Pulmonary vascular disease and myeloid
abnormalities in PAH. This study identifies hemangioblast proliferation and fibrosis as key components in the
pathophysiology of PAH. Increased levels of HIFinducible factors, some of which are produced by diseased pulmonary vascular cells, promote hemangioblast
proliferation and progenitor cell mobilization. Local production of recruitment factors by pulmonary vascular
endothelium attract mobilized progenitor cells into the
pulmonary artery wall where these cells fuel vascular
remodeling. (Illustration by David Schumick, BS, CMI.
Reprinted with permission, Cleveland Clinic Center for
Medical Art & Photography, 2010. All rights reserved.)
patients; Epo binding to its receptor results in phosphorylation of
the receptor and activation of JAK2, which then phosphorylates/
activates STAT5 and STAT3. Along with JAK2 abnormalities,
altered activation of STAT pathways are typically present in
myeloproliferative disorders.43 In this context, although bone
marrow cells did not manifest JAK/STAT abnormalities, pulmonary artery endothelial cells in PAH lungs have been shown to have
intrinsically high levels of STAT3 activation,1,3 identifying alterations in the tyrosine kinase signal transduction pathway in PAH
lung endothelial cells. Together with prior studies, the findings
point to an interdependent hematopoietic and angiogenic pathology
in PAH. How might the myeloid abnormalities influence the
disease? In this report, we provide quantitative data showing
CD34⫹CD133⫹ cells recruitment into the pulmonary vascular bed
using immunoquantitative FACS analyses, analogous to prior
report of immunostaining of tissues.44 Recruitment to the pulmonary vasculature is likely related to intrinsic production of chemoattractants HGF and SDF-1␣ by the diseased endothelium. The
production of these factors is consistent with prior report that PAH
endothelial cells have HIF-1␣ and STAT3 activation, leading to a
proangiogenic phenotype.3,45,46 Likewise, the greater response to
hypoxia in the PAH endothelial cells confirms prior findings that
intracellular hypoxia sensing is altered in PAH. Based on the
increases of Epo in this report, hypoxia-sensing abnormalities may
be present systemically and contribute to the myeloproliferative
process. On the other hand, the greater bone marrow resident and
circulating progenitors in nonaffected family members of PAH
patients suggests that hematopoietic abnormalities may precede
and/or predispose to the disease, and are not just a consequence of
pulmonary vasculopathy. In this context, although PAH patients
were not anemic, high Epo levels or hemoglobin levels in the lower
range were related to more severe clinical disease. This suggests
that the condition of anemia or iron deficiency, which activate
HIF-inducible factors including Epo, would lead to worsening
clinical status in PAH patients. In fact, iron deficiency and/or
anemia influences the development and clinical course of PAH. For
example, low hemoglobin is related to decreased survival in all
classes of PAH,47 and the classic pulmonary hypertensive response
to hypoxia is significantly attenuated by intravenous iron infusion.48 Here, we identify that a subclinical myeloproliferative
process is intrinsic to PAH disease, and that the myeloid abnormalities result in processes that promote the pathologic vascular
remodeling in the lung. The fact that PAH patients are highly prone
to develop overt myelofibrosis or thrombocytopenia provides
support for the idea that PAH is coupled to underlying alterations in
early bone marrow progenitors. Likewise, patients with myeloproliferative diseases, such as primary myelofibrosis and myeloid
leukemia, often develop PAH3,45,46,49,50 Reports identifying that
PAH resolves with treatment of the myeloproliferative process,
also support a role for bone marrow progenitors in PAH pathogenesis.51,52 Overall, the findings in this study together with other
reports indicate that myeloid abnormalities exist concurrently with
pulmonary vascular disease, although not manifest clinically.
Myeloid abnormalities appear to predispose to PAH, and PAH
patients are prone to development of myeloid diseases. This
supports the interdependence of hematopoiesis and angiogenesis,
not only in functional repair, but also in pathologic vascular
remodeling, and indicates that treatments targeting the bone
marrow myeloproliferative process may be effective in treating the
proliferative angiopathic processes in PAH.
Acknowledgments
We thank A. Janocha, J. Hanson, L. Mavrakis, and R Steinle for
excellent technical assistance; L. Vargo, Dr J. Drazba, and Dr A. J.
Peterson in the Lerner Research Institute Digital Imaging Core; C.
Shemo and S. O’Bryant in the Lerner Research Institute Flow
Cytometry Core for technical advise and excellent assistant with
instrument operation; and M. Baaklini, M. Cleggett-Mattox, and
M. Koo for patient recruitment.
This work was supported by grants HL60917 and M01
RR018390 from the National Institutes of Health, the Cleveland
Clinic Research Programs Council, American Thoracic Society/
Pulmonary Association Research grant (PH-07-003) to K.A., and
the Hematopoietic Stem Cell Core Facility of the Case Comprehensive Cancer Center (P30 CA43703).
Authorship
Contribution: S.F. conducted the study, performed research, analyzed and interpreted data, and wrote the manuscript; K.A.
performed research, analyzed and interpreted the data, and wrote
the manuscript; W.X. performed research, analyzed and interpreted
the data, and wrote the manuscript; J.S. conducted the study and
collected data; D.G. conducted the study and recruited subjects;
S.C. performed research and provided tissue and cells; M.P.
conducted research and recruited subjects; W.H.W.T. analyzed data
and reviewed the manuscript; J.E.L. recruited subjects and reviewed the manuscript; K.T. performed research and reviewed the
manuscript; R.T. performed research; E.H. performed research and
reviewed the manuscript; A.L. performed research and reviewed
the manuscript; and S.C.E. designed research, analyzed data and
wrote the manuscript.
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BLOOD, 31 MARCH 2011 䡠 VOLUME 117, NUMBER 13
BONE MARROW IN PAH
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Samar Farha, MD, Cleveland Clinic, 9500
3493
Euclid Ave, NC22, Cleveland, OH 44195; e-mail: [email protected];
or Serpil Erzurum, MD, Cleveland Clinic, 9500 Euclid Ave, NC22,
Cleveland, OH 44195; e-mail: [email protected].
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From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
2011 117: 3485-3493
doi:10.1182/blood-2010-09-306357 originally published
online January 21, 2011
Hypoxia-inducible factors in human pulmonary arterial hypertension: a
link to the intrinsic myeloid abnormalities
Samar Farha, Kewal Asosingh, Weiling Xu, Jacqueline Sharp, Deepa George, Suzy Comhair,
Margaret Park, W. H. Wilson Tang, James E. Loyd, Karl Theil, Raymond Tubbs, Eric Hsi, Alan Lichtin
and Serpil C. Erzurum
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