Identification of a common Wnt-associated genetic signature across multiple cell types in pulmonary arterial hypertension.

doi: 10.1152/ajpcell.00057.2014
307:C415-C430, 2014. First published 28 May 2014;Am J Physiol Cell Physiol
Majka
Dwight J. Klemm, Pampee P. Young, W. David Merryman, Darrell Kotton and Susan M.
Charles C. Hong, Barbara Meyrick, James E. Loyd, Aaron B. Bowman, Kevin C. Ess,
Joshua P. Fessel, Johnathan A. Kropski, David Irwin, Lorraine B. Ware, Lisa Wheeler,
Bilousova, Jyh-Chang Jean, Anna R. Hemnes, Swapna Menon, Nathaniel C. Bloodworth,
James D. West, Eric D. Austin, Christa Gaskill, Shennea Marriott, Rubin Baskir, Ganna
hypertension
signature across multiple cell types in pulmonary arterial
Identification of a common Wnt-associated genetic
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CALL FOR PAPERS Stem Cell Physiology and Pathophysiology
Identification of a common Wnt-associated genetic signature across multiple
cell types in pulmonary arterial hypertension
James D. West,1,8
Eric D. Austin,2
Christa Gaskill,1
Shennea Marriott,1
Rubin Baskir,1,3
Ganna Bilousova,10
Jyh-Chang Jean,12
Anna R. Hemnes,1,8
Swapna Menon,1
Nathaniel C. Bloodworth,9
Joshua P. Fessel,1,8
Johnathan A. Kropski,1
David Irwin,10
Lorraine B. Ware,1,5
Lisa Wheeler,1
Charles C. Hong,3,4
Barbara Meyrick,1,5
James E. Loyd,1
Aaron B. Bowman,6,7
Kevin C. Ess,2,6,7
Dwight J. Klemm,10
Pampee P. Young,5,7
W. David Merryman,9
Darrell Kotton,12
and Susan M. Majka1,3,5,7,8,11
1
Division of Allergy, Pulmonary, and Critical Care Medicine, Department of Medicine, Vanderbilt University, Nashville,
Tennessee; 2
Department of Pediatrics, Vanderbilt University, Nashville, Tennessee; 3
Department of Cell and Developmental
Biology, Vanderbilt University, Nashville, Tennessee; 4
Veterans Administration Hospital, Nashville, Tennessee; 5
Department
of Pathology, Microbiology, and Immunology, Vanderbilt University, Nashville, Tennessee; 6
Department of Neurology,
Vanderbilt Brain Institute, Nashville, Tennessee; 7
Vanderbilt Center for Stem Cell Biology, Nashville, Tennessee; 8
Vanderbilt
Vascular Biology Center, Nashville, Tennessee; 9
Department of Biomedical Engineering, Vanderbilt University, Nashville,
Tennessee; 10
Gates Center for Regenerative Medicine and Stem Cell Biology, University of Colorado, Aurora, Colorado;
11
Pulmonary Vascular Research Institute, Kochi, and AnalyzeDat Consulting Services, Kerala, India; and 12
Boston
University, Boston, Massachusetts
Submitted 19 February 2014; accepted in final form 23 May 2014
West JD, Austin ED, Gaskill C, Marriott S, Baskir R, Bilousova
G, Jean JC, Hemnes AR, Menon S, Bloodworth NC, Fessel JP,
Kropski JA, Irwin D, Ware LB, Wheeler L, Hong CC, Meyrick B,
Loyd JE, Bowman AB, Ess KC, Klemm DJ, Young PP, Merry-
man WD, Kotton D, Majka SM. Identification of a common Wnt-
associated genetic signature across multiple cell types in pulmonary arterial
hypertension. Am J Physiol Cell Physiol 307: C415–C430, 2014. First
published May 28, 2014; doi:10.1152/ajpcell.00057.2014.—Understanding
differences in gene expression that increase risk for pulmonary arterial
hypertension (PAH) is essential to understanding the molecular basis
for disease. Previous studies on patient samples were limited by
end-stage disease effects or by use of nonadherent cells, which are not
ideal to model vascular cells in vivo. These studies addressed the
hypothesis that pathological processes associated with PAH may be
identified via a genetic signature common across multiple cell types.
Expression array experiments were initially conducted to analyze cell
types at different stages of vascular differentiation (mesenchymal
stromal and endothelial) derived from PAH patient-specific induced
pluripotent stem (iPS) cells. Molecular pathways that were altered in
the PAH cell lines were then compared with those in fibroblasts from
21 patients, including those with idiopathic and heritable PAH. Wnt
was identified as a target pathway and was validated in vitro using
primary patient mesenchymal and endothelial cells. Taken together,
our data suggest that the molecular lesions that cause PAH are present
in all cell types evaluated, regardless of origin, and that stimulation of
the Wnt signaling pathway was a common molecular defect in both
heritable and idiopathic PAH.
pulmonary arterial hypertension; gene array; induced pluripotent stem cell;
mesenchymal stromal cell; endothelial cell; heritable pulmonary arterial
hypertension; idiopathic pulmonary arterial hypertension; Wnt signaling
PULMONARY ARTERIAL HYPERTENSION (PAH) is characterized
by vascular remodeling, including endothelial cell (EC)
dysfunction and occlusion or rarefaction of the peripheral
pulmonary microvasculature. More recently, the contribu-
tion of multipotent mesenchymal stromal cells (MSC) to
muscularization of microvessels has been described (13).
The interactions between the lung microenvironment, vas-
cular EC, and MSC during remodeling in PAH remain
unclear. All forms of PAH have a high mortality rate,
despite current therapeutic options.
Deregulated bone morphogenetic protein (BMP) receptor
type II (BMPR2) signaling is strongly associated with the
development of PAH in both heritable (BMPR2mut
and
Cav1mut
) and idiopathic cases, although the molecular mecha-
nisms through which BMPR2 derangement promotes PAH are
unknown. Unfortunately, most rodent models of PAH do not
precisely recapitulate the disease pathology; these models dis-
play less substantial pulmonary vascular remodeling in both
proximal arteries and distal microvasculature, significantly
slowing drug discovery efforts. Current in vitro models over-
expressing mutant BMPR2 in cell types of interest are com-
plicated by persistent retention of wild-type (WT) signaling.
Moreover, human PAH tissue is limited in quantity, and
specimens are typically obtained posttransplant or at au-
topsy, which limits conclusions about disease initiation and
propagation. Previous global gene expression analyses using
patient samples to identify risk factors for PAH have had
two fundamental caveats. First, samples isolated from end-
stage disease tissue were likely compromised by effects of
end-stage stress and drugs. We have overcome this difficulty
by analyzing cultured lymphocytes, based on the rationale
that culturing removes the cells from the disease and drug
environment. This strategy has been quite successful and
has identified a number of pathways as risk factors. How-
Address for reprint requests and other correspondence: S. M. Majka, Division of
Allergy, Pulmonary and Critical Care Medicine, Vanderbilt Univ., 1161 21st Ave. S,
T1218 MCN, Nashville, TN 37232 (e-mail: susan.m.majka@vanderbilt.edu.).
Am J Physiol Cell Physiol 307: C415–C430, 2014.
First published May 28, 2014; doi:10.1152/ajpcell.00057.2014.
http://www.ajpcell.org C415
byguestonJuly17,2016http://ajpcell.physiology.org/Downloadedfrom

ever, the use of lymphocytes exemplifies a second funda-
mental problem: since lymphocytes are nonadherent, any
pathway relevant to cell-cell contact, matrix interactions,
and polarity is not represented. Taken together, the under-
lying mechanisms of vascular dysfunction remain unclear,
despite known genetic mutations that affect the BMPR2
signaling pathway.
The evaluation of early molecular events in the cells of
PAH patients has been limited, because vascular-specific
cells can only be studied at a late stage in the disease process
(at lung transplant or postmortem evaluation), at the time of
severe pulmonary vascular abnormality. However, the use
of induced pluripotent stem (iPS) cells derived from PAH
patients confers the ability to study early, initiating cellular
events in the pathogenesis of PAH in relevant cell types.
With embryonic stem cells and developmental differentia-
tion used as a road map, these iPS cells may be differenti-
ated to specific affected cell lineages (44, 53). Therefore, to
address the aforementioned limitations and to investigate
molecular pathways affected by dysregulated BMPR2 sig-
naling, we engineered iPS cells and derived vascular mul-
tipotent mesenchymal stromal cells (MSC) and endothe-
lium. We used this approach to study altered gene expres-
sion profiles during differentiation across mesenchymal
cells (MC) and endothelial cells (EC), as well as skin
fibroblasts from control, heritable PAH (HPAH), and idio-
pathic PAH (IPAH) patients. These findings were validated
in vitro using primary patient cell lines. Here we address the
hypothesis that altered human BMPR2 signaling results in a
genetic signature common across multiple cell types, cul-
minating in the pathological processes recognized as PAH.
Taken together, our results suggest that altered Wnt signal-
ing is inherent to the cells of PAH patients and is likely due
to decreased BMPR2 signaling.1
METHODS
Isolation of patient skin fibroblasts and identification and charac-
terization of the BMPR2 mutation. The subjects were recruited via
the Vanderbilt Pulmonary Hypertension Center. The Vanderbilt
University Medical Center Institutional Review Board approved all
study protocols (Vanderbilt University Institutional Review Board
Protocol 9401). All participants gave informed written consent to
participate in genetic and clinical studies and underwent genetic
counseling in accordance with the guidelines of the American
College of Chest Physicians (47). The PAH phenotype was defined
according to accepted international standards of diagnosis. Specif-
ically, PAH was defined diagnostically by autopsy results showing
plexogenic pulmonary arteriopathy in the absence of alternative
causes, such as congenital heart disease, or by clinical and cardiac
catheterization criteria. These criteria included a mean pulmonary
arterial pressure of Ͼ25 mmHg with a pulmonary capillary or left
atrial pressure of Ͻ15 mmHg and exclusion of other causes of
pulmonary hypertension, in accordance with accepted international
diagnostic criteria (45, 59).
Skin biopsy specimens were obtained via a sterile 3-mm punch skin
biopsy. Primary skin fibroblasts were cultured using standardized
measures. All cell lines were grown in the same manner using DMEM
(with 4.5 g/l glucose, L-glutamine, and sodium pyruvate) (Mediatech,
Manassas, VA) with 20% FBS (Invitrogen, Carlsbad, CA). For
identification of BMPR2 gene mutations, genomic DNA was isolated
from whole blood using Puregene DNA Purification Kits (Gentra,
Minneapolis, MN) according to the manufacturer’s protocol. BMPR2
gene mutation was detected by sequencing exons and exon-intron
boundaries of genomic DNA and reverse transcriptase-polymerase
chain reaction (RT-PCR) analysis (14).
Summary of cell lines. PAH iPS and control cell lines (2 each) were
differentiated into MC and subsequent EC-like (ECL) cells (twice
independently). Three pulmonary artery EC (PAEC) control and three
PAEC IPAH primary cell lines were obtained through the Pulmonary
Hypertension Breakthrough Initiative (PHBI).
Karyotyping of skin fibroblasts and iPS cells. Karyotyping was
performed prior to reprogramming on skin fibroblast lines and fol-
lowing transgene removal to confirm normal chromosome comple-
ment and banding. Cultured cells were incubated for 4 h with
colcemid (0.05 ␮g/ml) to enhance mitotic index, trypsinized, and
collected into a centrifuge tube. This process was followed by a
12-min incubation in hypotonic solution (0.075 M KCl) at 37°C. Cells
were then fixed using 3:1 methanol-acetic acid. Slides of metaphase
cells were prepared using a standard air-dry procedure. GTL banding
was performed in 5-day-old slides. Briefly, cells were digested in
trypsin for 30–40 s and incubated for 5 min in Leishman’s stain.
Karyotyping was carried out using BandView software (Applied
Spectral Imaging). A total of 100 metaphases were screened per
specimen for calculation of ploidy, and karyotyping was performed in
Ն10 metaphases. A characterization of patient skin fibroblast samples
is presented in Table 1. Western blot analysis was performed to
evaluate the levels of BMPR2 protein expression in cells exposed to
the primary antibody AF811 (R & D Systems, Minneapolis, MN) for
1 h and the secondary antibody 111-035-003 (Jackson ImmunoRe-
search, West Grove, PA).
iPS cell reprogramming and characterization. To study the pri-
mary effects of BMPR2 mutation without concern for the pressure-
or flow-mediated changes in vascular cell function (or other in vivo
milieu variables), transgene-free iPS cells were generated from a
control patient with no known BMPR2 mutation (WT) and a
HPAH patient with a known BMPR2 mutation (BMPR2mut iPS)
using the excisable polycistronic lentiviral vector (EF1a-hSTEM-
CCA-loxP) encoding the four reprogramming factors (Oct3/4,
Sox2, Klf4, and c-Myc), as described elsewhere (60). Briefly, iPS
cell clones containing a single integrated copy of the vector were
exposed to transient Cre recombinase to excise the floxed STEM-
CCA vector to produce iPS cell lines free of exogenous reprogram-
ming transgenes (60). Karyotyping was performed prior to repro-
gramming and following transgene removal (not shown). Sequenc-
ing of genomic DNA confirmed the retention of mutation in the
BMP3ACr1 iPS cell line. iPS cells exhibited an embryonic stem
cell-like morphology and displayed functional pluripotency in
standard in vivo teratoma assays in nude mice. iPS cells (1 ϫ 106
)
were mixed with 100 ␮l of Matrigel (catalog no. 356237, Becton
Dickinson, San Jose, CA) and injected subcutaneously into the
flank of 6-wk-old severe combined immunodeficiency (SCID) mice
(Jackson Laboratory, Bar Harbor, ME). The animals were moni-
tored for 2 mo for tumor formation. All procedures and protocols
were approved by the Institutional Animal Care and Use Commit-
tee at Vanderbilt University. Principal component analysis (PCA)
was performed to compare two independent patient control iPS cell
clones with two BMPR2mut iPS clones and confirmed that the
clones segregated based on the presence of BMPR2 mutation (not
shown). This segregation illustrates that the mutant clones are
more similar to each other than to controls.
Passage and expansion of iPS cells. Human dermal fibroblasts
(Invitrogen) were expanded and mitomycin C was inactivated in the
medium, as described elsewhere (9, 60). iPS colonies were grown on
this feeder layer in 5% CO2 and routinely passaged every 5–6 days
after disaggregation with collagenase type IV (Invitrogen) at a ratio of
1:4–1:6, depending on colony density, onto a fresh feeder layer with
1
This article is the topic of an Editorial Focus by Katherine A. Cottrill and
Stephen Y. Chan (16).
C416 COMMON GENETIC SIGNATURES IN PAH
AJP-Cell Physiol • doi:10.1152/ajpcell.00057.2014 • www.ajpcell.org

the Rho-associated protein kinase inhibitor Y-27632 (10 ␮M) (60).
Medium was replaced every other day. To separate iPS cell colonies
from the feeder layer, plates were digested using collagenase type IV,
transferred to a 0.1% gelatin (Sigma, St. Louis, MO)-coated tissue
culture plate, and incubated for 1 h, allowing for fibroblast adherence.
The cells and supernatant were then collected and plated on Matrigel-
coated plates with Rho-associated protein kinase inhibitor (10 ␮M).
iPS cells were cultured in this feeder-free system by switching to
MTeSR1 medium.
Directed differentiation of iPS cells and phenotyping. MSC differ-
entiation of iPS cells was performed using defined medium (knockout
DMEM supplemented with 10% serum replacement medium, 10
ng/ml basic fibroblast growth factor, 10 ng/ml platelet-derived growth
factor AB, and 10 ng/ml epidermal growth factor), as previously
described by Lian et al. (43). iPS cells cultured in a feeder-free system
using Matrigel were exposed to the mesenchymal medium until they
became confluent for passage at ϳ14 days (Fig. 1). Upon passage,
cells were cultured on plastic using ␣-MEM supplemented with 20%
fetal calf serum. Differentiation of WT and BMPR2mut iPS cells into
iPS-MSC was demonstrated by surface marker expression of CD73,
Stro-1, CD29, and CD105 and lack of the hematopoietic markers
CD45, CD14, CD3, and c-kit. Multilineage differentiation to mesen-
chymal lineages was also performed (not shown), meeting the estab-
lished criteria for MSC (20) (Fig. 2, A and B). Differentiation to MSC
was performed twice per clone, and characterization was performed
on each line.
MSC at passage 2 were plated onto collagen type I, and
differentiation to EC was performed using the EGM-2 Bullet kit
(Lonza/Clonetics, San Diego, CA). When cells reached confluence
(2 wk), they were incubated with acetylated DiLDL labeled with
Alexa 488 (10 ␮g/ml; Invitrogen) in culture medium for 2 h. Cells
were photographed and RNA was collected for array analysis, or
cells were trypsinized to form a single cell suspension for sorting
by flow cytometry using a MOFlow sorter (Dako Cytomation, Ft.
Collins, CO) and Cell Quest software. DiLDL-enriched iPS-ECL
cells were expanded and, after up to two passages continuing EC
differentiation conditions, trypsinized to form a single cell suspen-
sion and analyzed for the expression of platelet-endothelial cell
adhesion molecule 1 (CD31), CD34, CD45, and vascular endothe-
lial cadherin (CD144) by flow cytometry or cultured in chamber
slides to stain for Flt-1 (Fig. 2, C–E). Differentiation to EC was
performed twice independently. Cell surface determinant expres-
Table 1. Characterization of patient samples
Patient Phenotype Detected Sex
Age at
Biopsy, yr
PAP,
mmHg
6-Minute
Walk, m
PVR, Wood
unit Patient No. Therapy at Time of Biopsy
IPAH Female 50 51 360 10 SPH748EL5025 Bosentan
IPAH Female 30 49 368 9.88 SPH709QK3095 Abrisentan
IPAH Female 64 54 384 14.8 SPH46SH695 Intravenous prostanoid to PDE5
inhibitor
IPAH Female 48 36 365 6.8 SPH137SB868 Intravenous prostanoid ϩ bosentan
IPAH Male 54 51 430 9.05 SPH433TA2148 Oral prostanoid ϩ sildenafil ϩ
bosentan
IPAH Female 50 45 NR 5.9 SPH135KW867 Bosentan
IPAH Female 43 54 325 19.2 SPH400LP1959 Ambrisentan ϩ sildenafil ϩ
intravenous prostanoid
HPAH BMPR2 c.354TϾG Male 41 83 NR 16 PPH14WT266 Transplant
HPAH BMPR2 c.354TϾG Female 34 80 318 29.63 PPH14AR2572 Intravenous prostanoid
HPAH BMPR2 c.354TϾG Male 17 38 350 10.86 PPH14ZR2608 Tadalafil
HPAH BMPR2 c.2504delC Female 40 52 511 21.4 PPH150KW773 Intravenous prostanoid ϩ sildenifil
prior to transplant
HPAH BMPR2 c.G350A Female 32 47 408 10.35 PPH16LW1444 Intravenous prostanoid ϩ
sildenafil
HPAH 57 67 310 NR PPH163RM2621 Intravenous prostanoid ϩ bosentan
Healthy BMPR2
mutation BMPR2 c.G350A Male 58 NA NA NA PPH16LF1447 NA
Healthy WT
control Male 35 NA NA NA SPH497EA2737 NA
Healthy WT
control Female 26 NA NA NA SPH676BN3024 NA
Healthy WT
control Male 64 NA NA NA SPH785JL5104 NA
Control Deceased
fetus
NA NA NA PPH14BR2576 NA
Control NA NA NA VA-005 NA
Control NA NA NA IPF237KM NA
HPAH NR NR NR PPH173TH5026 None/died prior to RHC
IPAH 32 47 311 10 VA011 Intravenous prostanoid
Control Male NA NA NA AH-002 NA
Control Female NA NA NA AH-006 NA
IPAH Female 52 357 5.74 BA-005 Unknown
IPAH Female 45 390 8.86 BA-010 Unknown
Control Male NA NA NA VA-006 NA
IPAH Female See above PAEC clone 3 va011 See above
HPAH BMPR2 See above PPH150KW773 See above
Control Male NA NA NA
Control Female NA NA NA
PAP, pulmonary arterial pressure; PVR, pulmonary vascular resistance; HPAH, heritable pulmonary arterial hypertension; IPAH, idiopathic pulmonary arterial
hypertension; WT, wild-type; BMPR2, bone morphogenetic protein receptor type II; PAEC, pulmonary artery endothelial cell; PDE5, phosphodiesterase 5; RHC,
right heart catheterization; NA, not available; NR, not reported.
C417COMMON GENETIC SIGNATURES IN PAH

sion by MSC and EC was analyzed by incubation of primary
antibodies directly conjugated to phycoerythrin (PE), FITC, or
allophycocyanin (APC) (see Supplemental Table S2 in Supplemen-
tal Material for this article available online at the Journal website)
with 1 ϫ 105
cells for 10 min on ice. The cells were then washed
and resuspended for analysis in cold Hanks’ solution containing
2% fetal calf serum with DAPI to exclude dead cells.
Flow cytometry to detect staining was performed using a Beckman
Coulter Cyan analyzer and Cell Quest software. Gates were set
using a known positive and negative for each color. Patient PAEC
T=0
iPS
T=24
Early MSC
T=14 days
Late MSC
T=28 days
EC-like
T=45+ days
EC-like
+ Matrigel
+bFGF
+PDGFAB
+EGF
+ Collagen Type I
+EGF
+FGF B
Msx2
FrzB
CD45-
CD73+
Stro1+
CD29+
CD105+
AcDiLDL
uptake &
enrichment
VE-cadherin (CD144)+
Flt-1
VEGFA
Flt
Flk/KDR
Tie2/TEK
Endoglin (ENG)
Cyp1B1
Fzd4
+IGF-1
+ascorbic acid
+VEGF
A
B CWT iPS-MSC BMPR2 mut iPS-MSC
D E
F G
I J
K L
N
H
CellCounts
100
0
WT iPS ECL
79.3%
mutBMPR2 iPS
ECL 69.8%
M
WT iPS-ECL BMPR2 mut iPS
-ECL
FLT1 FLT1
BMPR2 mut iPS
AcDiLDL
WT iPS
100 105 100 105

were obtained from Ͻ1-mm-diameter vessels through the PHBI
network. Patient characteristics are summarized in Table 1.
BMPR2 expression levels were analyzed by Western blotting (not
shown).
Phenotyping assays. Cells were plated in triplicate at a density of
5 ϫ 104
cells per well in a six-well plate and harvested at 0, 24, 48,
and 72 h. Cell numbers and viability were analyzed using a
Countess counter (Invitrogen). Apoptosis was determined by
flow cytometry using the YoPro propidium iodide kit (Invitrogen).
Flow cytometry to detect staining was performed using a Beckman
Coulter Cyan analyzer and Cell Quest software. Oxidative stress
was measured using the GSH-to-GSSG ratio in cell extracts ac-
cording to the suggested manufacturer’s protocol for the Glutathi-
one Fluorescent Detection Kit (catalog no. K006-F1, Arbor As-
says, Ann Arbor, MI). All assays were performed twice indepen-
dently. For Flexcell tension assays, cells were plated on collagen I
plates (Flexcell International, Hillsboro, NC) and harvested at 0,
24, and 72 h. Cells were exposed to 10% elongation/deformation at
1 Hz to represent approximately one heartbeat and moderate
distension. Control groups included unstretched cells. Two inde-
pendent biological replicates were performed for each sample, and
PCR analysis was performed in triplicate. iPS cell-derived MC
from two control and two PAH patients were analyzed. Tube-
forming ability was determined by using tissue culture-treated
24-well plates that were prechilled and coated with 200 ␮l of
Matrigel, which was allowed to harden at 37°C for 45 min.
Concurrently, cells from both lines were trypsinized, filtered, and
centrifuged to be resuspended at 2 ϫ 105
cells/ml in growth
medium. A volume of 0.5 ml was added to each well. Plates were
incubated at 37°C in 5% CO2, and tube formation was documented
at 2–10 h after plating.
Isolation of primary human lung MSC. Human lung fibroblasts
were isolated from human lung tissue postautopsy or at transplant
by collagenase digestion of lung tissue explants. After expansion in
culture under ambient conditions in ␣-MEM with 20% FBS, the
cells were digested to form a single cell suspension. The cells were
stained with antibody to detect and sort CD45neg
ABCG2pos
cells
(lung MSC). The compensation controls were established as cells
only, cells ϩ DAPI, cells ϩ APC-CD45 antibody, and cells ϩ
PE-ABCG2/ABCG2 antibody, and the sort sample consisted of
cells ϩ DAPI ϩ APC-CD45 antibody ϩ PE-ABCG2 antibody.
Each sample was mixed well and incubated for 20 min at room
temperature. DAPI was used to exclude dead cells. After expan-
sion, cells were analyzed by flow cytometry to confirm the pres-
ence of CD105, CD106, CD73, and ScaI, as well as the absence of
c-kit, CD14, and CD45.
Western blot analysis. Protein extracts were made by scraping
cells in RIPA buffer (catalog no. 9806S, Cell Signaling, Boston,
MA) containing protease and phosphatase inhibitors (catalog no.
78444, ThermoFisher Scientific, Waltham, MA). After determina-
tion of protein concentrations and standardization, cell lysates
were mixed with an equal volume of Laemmli-SDS loading buffer,
resolved on 10% polyacrylamide-SDS gels, and transferred to
PVDF membranes. The blots were blocked with phosphate-buff-
ered saline containing 5% dry milk and 0.1% Tween 20 and then
treated with antibodies that detect the target proteins overnight at
4°C (see Supplemental Table S2). The blots were washed and
subsequently treated with appropriate secondary antibodies conju-
gated to horseradish peroxidase. After the blots are washed, spe-
cific immune complexes were visualized with SuperSignal West
Pico Chemiluminescent Substrate. Secreted frizzled-related protein
2 (Sfrp-2) band size is ϳ33 kDa.
Transcriptome analysis. Total RNA was prepared with RNA Iso-
lation Kit reagents (Qiagen, Valencia, CA). RNA was isolated from
20 patient skin fibroblast cultures, 2 independently isolated control
iPS cell cultures, and 2 independent HPAH iPS cell clones. For
developmental stage analyses, RNA was collected from two indepen-
dently generated clones, and chips were run in duplicate. cDNA
generated from amplified RNA was hybridized to duplicate Af-
fymetrix (Santa Clara, CA) human gene 1.1 or 1.0 ST chips. Gene
ontology groups were analyzed and compiled using Webgestalt (Van-
derbilt University), heat maps using JMP 9, and correlation plots
using Microsoft Excel, with statistics performed using JMP 9. Array
analysis and quantitative RT-PCR validation was performed as de-
scribed elsewhere (13, 37). Quantitative RT-PCR assays were per-
formed in triplicate, and levels of analyzed genes were normalized to
hypoxanthine phosphoribosyltransferase abundance (see primer list in
Supplemental Table S1).
Dual luciferase assay to detect Wnt signaling activity. Cells (5 ϫ
104
per well) were plated on a 12-well plate. Five microliters of
TCF/LEF reporter plasmid (100 ng/␮l stock; Cignal Reporter Assay,
Qiagen) were diluted in 50 ␮l of Opti-MEM (Invitrogen), and 1 ml of
Lipofectamine 2000 (1 mg/ml stock; Invitrogen) was diluted in 50
␮g/ml Opti-MEM (Invitrogen) according to the manufacturers’ in-
structions. The TCF/LEF-responsive construct encodes the firefly
luciferase reporter gene under the control of a minimal CMV pro-
moter and tandem repeats of the TCF/LEF transcriptional response
element. Diluted reporter plasmid and Lipofectamine were combined,
and the cells were incubated at room temperature for 30 min. Cells
were then rinsed with Opti-MEM, and transfection reagents were
added. After 10 h of transfection, the medium was replaced with
culture medium. LiCl (10 mM), a positive regulator of Wnt signaling,
was added to the wells as a positive assay control (not shown). Cells
were harvested at 24 h, 48 h (inhibitor studies), or 72 h using the Dual
Luciferase Reporter Kit (Promega, Madison, WI), and dual luciferase
activity was quantitated using a luminometer. These experiments were
repeated twice independently. Transfection efficiency was standard-
ized to Renilla luciferase.
Detection of Sfrp-2 in human PAH specimens. Human tissue was
obtained from postautopsy specimens from PAH patients (2 con-
trol and 3 PAH with different mutations) after approval from the
Vanderbilt University Institutional Review Boards. Sections of
patient lung tissue were evaluated by antibody staining for the
presence of the secreted Wnt inhibitor Sfrp-2 (catalog no. 92667,
Abcam) using diaminobenzidine detection. Images were captured
using a Nikon Eclipse 90i/DSFi-1 microscope with NIS Elements
Fig. 1. Schematic mapping of inducible pluripotent stem (iPS) cell-derived mesenchymal stromal cell (MSC) and endothelial cell (EC)-like (ECL) cell
differentiation. A: summary of matrix, growth factors, and markers employed to characterize differentiating iPS cell-derived lineages. bFGF, basic fibroblast
growth factor; PDGF AB, platelet-derived growth factor AB; AcDiLDL, acetylated DiLDL. B–G: directed differentiation of heritable pulmonary arterial
hypertension (HPAH) bone morphogenetic protein (BMP) receptor type II (BMPR2) mutant (BMPR2mut) iPS cells to MSC (iPS-MSC). Representative
phase-contrast (B–E) and bright-field (F and G) micrographs of wild-type (WT) and BMPR2mut iPS-MSC are shown. Scale bars, 100 ␮m. H–N: HPAH
BMPR2mut iPS cell-derived cells exhibit endothelial differentiation potential. Successful ECL cell differentiation of WT and BMPR2mut iPS cells was
demonstrated by the appearance of a characteristic phenotype and function. H: iPS-ECL cells were enriched using functional uptake of acetylated DiLDL labeled
with Alexa 488 dye. Overlay of phase-contrast and fluorescent images localizes DiLDL (green) in BMPR2mut iPS-ECL cells; histogram shows results from
analysis of WT and BMPR2mut iPS-ECL cells stained with DiLDL. Fluorescently labeled iPS-ECL cells were analyzed, and the 488 high positives (gate) were
collected by flow cytometry. I and J: phase-contrast micrographs depict a cobblestone morphology. K and L: WT and BMPR2mut iPS-ECL cell expression of
VEGFR1/Flt-1 was detected by immunofluorescent staining. M and N: both populations of enriched ECL cells formed tubes in an in vitro angiogenesis assay.
Scale bars, 50 ␮m. Representative analyses from 2 independent studies per clone are presented.

CD14 CD3
100
0
50
CD45
CD105
95.4%
CD29
99.7%
0 101 102 103 104 0 101 102 103 1040 101 102 103 1040 101 102 103 104 0 101 102 103 1040 101 102 103 1040 101 102 103 1040 101 102 103 104
0
50
100
CD73
100%
Stro-1
65.2%
A
CountsCounts
50
100
0
CD29
99.9%
CD105
97.2%
50
100
CD73
100%
Stro-1
98.3%
0
B
CD45 CD14 CD3
WTiPSMSC
BMPR2mutiPSMSC
CD14 CD34CD144
89.7%
CD105
95.5%
CD106
83.5%
50
100
0
50
100
0
WTiPSECL
C D
BMPR2mutiPSECL
50
100
0
50
100
0
CD144
60.7%
CD14 CD34
CD105
98.9%
CD106
21.0%
0
0.6
1
VEGF-A
0
0.6
1
Tie-2/TEK
NormalizedtoGAPDH
1
2 2
3
KDR/Flk-1 (VEGFR2)
0
0.6
1
Flt-1 (VEGFR1)
0
0.6
1
BMP-4
0
1
3
THBS2
0
WT BMPR2mut WT BMPR2mut WT BMPR2mut WT BMPR2mut WT BMPR2mut WT BMPR2mut
E
WT BMPR2mut
MeanRatioGSH:GSSG
**
**
RA RA HYHY
F
iPS-ECL
iPS MSC
0
1
2
0
800000
0 24 48 72
HPAH
Time (hrs)
MeanCell#
IPAH
Control 1/2 0
24 hours
48 hours
72 hours
0
500000
HPAH IPAH
I Primary Lung MSC
Mean%Apoptosis72hr
BMPR2mut WT
25
15
0
**
MeanCell#
Control
G H
0 102 104 0 102 104
0 102 104 0 102 104 0 102 104 0 102 104

software. ELISAs to detect protein levels in conditioned medium
from iPS and primary cells in culture and plasma were performed
according to the manufacturer’s instructions (MyBioSource, San
Diego, CA).
Statistical analysis. Data were analyzed by one-way ANOVA
followed by Tukey’s honestly significant difference post hoc test
using JMP 9. Significance was defined as P Ͻ 0.05.
RESULTS
iPS cell-derived PAH cell lineages show subtle, but signif-
icant, differences in morphology and differentiation potential.
We employed iPS cell technology to study vascular-associated
MSC and ECL cell lineages that may actively participate in the
cell-based pathology of PAH. This allows us to avoid the
complication of consequences, rather than causes, of disease
found in cells directly obtained from patient explants. It also
allowed the derivation of multiple cell lineages from a single
patient, which allows examination of differentiation state-
dependent effects of dysregulated BMPR2 due to mutation.
Transgene-free iPS cells were generated from WT skin fibro-
blasts or skin fibroblasts with known BMPR2 mutation and
directed to differentiate toward multipotent mesenchymal (20,
43) (iPS-MSC) and, subsequently, ECL (iPS-ECL) cell lin-
eages (Figs. 1 and 2). This direction for differentiation and cell
types to study was selected, because, developmentally, distal
pulmonary microvasculature is thought to be of mesenchymal
origin (3). iPS-MSC exhibited characteristic phenotypes (Fig.
1, B–G) and formed mesenchymal structures in vitro (27).
Visible differences in cell organization, including a more
elongated morphology, were noted. However, both WT and
BMPR2mut iPS-MSC formed characteristic two-dimensional
organizational patterns and ridges of high density (Fig. 1, F and
G) (15, 27). Acetylated DiLDL uptake was employed to enrich
putative ECL cells and also demonstrated that BMPR2 muta-
tion decreased the efficiency of differentiation: 79.3% for WT
and 69.8% for BMPR2mut (Fig. 1, H–K, and Fig. 2D). ECL
cell differentiation was demonstrated by the appearance of a
characteristic morphology and expression of Flt-1 [VEGF
receptor 1 (VEGFR1)], as well as angiogenic tube-forming
ability (Fig. 1, K–N). At the level of gene expression, the
BMPR2mut iPS-ECL cells demonstrated decreased expression
of the differentiated markers VEGF-A, Tie-2/TEK, and Flk1/
KDR (Fig. 2E). The mutant MC exhibited lower rates of
apoptosis and increased oxidant stress in response to hypoxia
(Fig. 2, F and G). The mutant iPS-derived MC and primary
lung MSC did not demonstrate a basal difference in prolifer-
ation rate (Fig. 2, H and I), similar to previous observations of
pulmonary artery smooth muscle (51). We have shown that
BMPR2mut iPS cells can be differentiated to MSC and ECL
cell lineages in vitro. These lineages exhibit characteristics of
PAH cells, and they are therefore an effective model to under-
stand the molecular consequences of altered BMPR2 signaling
in cell function in the context of PAH. Taken together, these
results confirm that reprogramming of the primary patient cells
did not affect their ability to demonstrate a “PAH phenotype.”
BMPR2 mutation alters gene expression dependent on, and
independent of, cell lineage differentiation state. We next
performed global gene expression analysis of these validated
iPS-derived cells to determine how decreased BMPR2 signal-
ing affects gene expression at various stages of differentiation.
We used two arrays per differentiation stage, with four differ-
entiation stages, and both WT and BMPR2 mutant cells, for a
total of 16 arrays. As shown in Fig. 1, differentiation stages
included early MSC, late MSC, and ECL cells at passages 4
and 5.
For initial analysis of the resulting data, we used PCA. PCA
is a powerful approach to circumvent the dimensionality prob-
lem in array data; tens of thousands of probe sets can be
projected onto a small number of principal components that
accurately reflect the variability in the data set (8). After
preprocessing to remove control probe sets and probe sets
below the noise threshold, the remaining 13,062 probe sets
were subjected to PCA. PCA found that differentiation state
accounted for 42% and mutation status for 15% of the vari-
ability between the 16 arrays (Fig. 3A). Gene expression in
passage 4 and 5 ECL cells was very similar within genotype,
suggesting stable molecular phenotype. Progress along the
differentiation axis involved similar gene expression changes
in WT and BMPR2mut cells. Between early MSC and ECL
cells, 826 probe sets changed more than fourfold; 200 of these
probe sets, which are depicted in the heat map in Fig. 3B,
consisted of waves of upregulation of developmental, cell
cycle, and angiogenesis-related genes (see Supplemental Ta-
bles S3–S5), ending in upregulation of cell adhesion molecules
associated with endothelial differentiation (P ϭ 4.8 ϫ 10Ϫ2
for
overrepresentation), including VCAM1, ICAM1, CERCAM,
and ITGBL1, which correlate with the flow cytometry data
(Fig. 3C and Fig. 2, C–E).
In addition to the above-mentioned genes, which are devel-
opmentally regulated but not different between control and
mutant cells, several categories of genes distinguished the
PAH-derived cells from WT cells. These include genes that are
over- or underexpressed in BMPR2 mutant cells at every
differentiation state (Fig. 3, D and E) and genes that are only
differentially expressed in differentiated cell types (Fig. 4).
There were 271 probe sets at least 50% more strongly ex-
pressed in HPAH than WT at every stage (Fig. 3D; see
Supplemental Table S6). These map to 220 unique genes, of
which 85 are developmental (P ϭ 5.0 ϫ 10Ϫ4
for overrepre-
Fig. 2. Directed differentiation of BMPR2mut iPS cells to mesenchymal and ECL cells. A–D: representative analyses of flow cytometric characterization of cell
surface determinants on iPS-derived MSC and ECL cells. Differentiation of multiple lineages was performed twice per clone. A and B: WT and HPAH
BMPR2mut iPS-MSC (blue) were positive for mesenchymal markers (CD73, Stro-1, CD29, and CD105) and lacked hematopoietic (CD45, CD14, and CD3) and
EC (CD144) markers. C and D: WT and HPAH BMPR2mut iPS-ECL cells (blue) were positive for the endothelial markers CD144 (VE-cadherin), CD105
(endoglin), and CD106 (VCAM) and lacked hematopoietic markers (CD14 and CD34). Gates were set to fluorescence Ϫ 1 (FMO) negative controls (red).
E: quantitative PCR analysis was performed to compare WT and BMPR2mut iPS-derived ECL cell expression of characteristic EC markers VEGF-A, Tie2/TEK,
VEGFR1/Flt, VEGFR2/KDR/Flk-1, BMP-4, and thrombospondin 2 (THBS2). F: WT and BMPR2mut iPS-MSC were cultured under normal conditions for 72
h and analyzed by flow cytometry to detect apoptosis. G: WT and BMPR2mut iPS-MSC were exposed to ambient culture oxygen [21% O2, i.e., room air (RA)]
or hypoxia [6% O2 (HY)] for 72 h and analyzed spectrophotometrically to quantitate the GSH-to-GSSG ratio as an indicator of intracellular oxidative stress.
**P Ͻ 0.01. H and I: number of WT and PAH iPS-MSC or primary patient MSC at 0–72 h.

sentation). These include transforming growth factor-␤ (TGF-␤)
pathway genes such as endoglin (ENG) and the repressor latent
TGF-␤-binding protein 2 (LTBP2); numerous homeobox genes,
including DLX1/2, MEIS2, MSX2, PBX1, and SIX2; three sema-
phorins (SEMA3C, SEMA3F, and SEMA7A); and the Wnt path-
way decoy receptor FRZB (see examples in Fig. 3E). The pres-
ence of CYP1B1 (Fig. 3E) in this group sounds a cautionary note
about this approach. We and others previously showed that
CYP1B1 is a powerful modifier gene; expression levels as mea-
sured in lymphoblastoid cells and functionally in patient urine
correlate with disease penetrance, rather than BMPR2 expression
levels (4, 65).
The final group of genes examined were those that were only
differentially regulated by BMPR2 in the context of differen-
tiated cells. Using criteria of no significant difference in early
MC, but a raw P Ͻ 0.05 of a 1.5-fold difference in ECL cells,
we found 190 probe sets representing 164 unique Entrez IDs
that fit this category (Fig. 4A; see Supplemental Table S7).
Overrepresented gene ontology groups [Benjamini and Hoch-
berg (6) multiple test adjusted P Ͻ 0.01] included cell adhesion
(22 genes), cell death (36 genes), proliferation (31 genes),
stimulus response (85 genes), cell surface receptor signaling
(41 genes), and developmental (54 genes). The largest set
consisted of 93 probes representing 72 genes that were specif-
ically upregulated in BMPR2 mutants, but not controls, during
cell differentiation (group I in Fig. 4A). These included 33
developmental genes and 18 genes related to cell death. Of
particular note, these included a large number of cell surface
and secreted Wnt receptors and Wnt pathway target genes (Fig.
4B). Upregulation of the Wnt receptors Fzd4 and Fzd5 and
secreted modulators Srfp1 and Sfrp2, as well as Msx2, Tie2/
TEK, Cyp1B1, and Tsp2, was confirmed by quantitative RT-
PCR, which correlated strongly to array results (Fig. 4, C and
D, and results not shown).
In summary, we have shown that there are genes that are
changed by differentiation state, but not by mutation (Fig. 3, B
and C), genes that are always changed by mutation, regardless
of differentiation state (Fig. 3, D and E), and genes that are
changed by mutation only in differentiated cells (Fig. 4A). One
of the largest groups of genes that are changed by mutation
Fig. 3. BMPR2 mutation causes persistent gene expression differences across cell types but does not interfere with endothelial differentiation. Expression analysis
of WT and BMPR2mut iPS-MSC following 24 h of culture in MSC differentiation medium (Early), differentiated MSC (Late), and ECL cells on subsequent
passages [passage 4 (p4, very confluent) and passage 5 (p5, subconfluent)]. A: principal component analysis segregated WT and BMPR2 mutant early MSC
(Early MC), late MSC (Late MC), and ECL cells into distinct clusters based on BMPR2 mutation and expression of genes involved in cell differentiation. B: heat
map analysis of gene segregation showing high (red) and low (blue) levels of expression. ϩ, Mutant samples. In this set of genes, changes associated with
differentiation are not affected by BMPR2 mutation [compare labeled (ϩ) rows with nonlabeled rows in the same differentiation stage]. C: strong regulation of
markers indicative of differentiation for iPS cells to MSC and ECL cells [ITGBL1 (integrin-␤-like 1), VCAM1 (CD106), CCNA2 (cyclin A2), and CD24].
D: genes that are always more strongly expressed in BMPR2 mutants than WT and are downregulated (I) or upregulated (II) with differentiation and genes that
are always more weakly expressed in BMPR2 mutants than WT and do not undergo change (III) or are downregulated (IV) with differentiation. E: genes that
are always upregulated in BMPR2 mutants compared with WT control at the same time point. Open circles, early MSC; shaded circles, differentiated MSC; solid
circles, ECL cells. Error bars, SE.

included Wnt signaling molecules (Fig. 4B). Confirmation by
RT-PCR correlates well with array results (Fig. 4, C and D, and
results not shown).
PAH-dependent changes in the Wnt pathway are a function
of differentiation, per se, and not a particular somatic lineage.
The comparison of differentiation states of vascular cells de-
rived from iPS cells highlighted genetic signatures conserved
across differentiation state. However, because the iPS cell lines
were derived from outbred individuals, it is not possible a
priori to say which differences between them result from
deregulated BMPR2 signaling and which derive from other
individual-to-individual differences. The most feasible ap-
proach to resolving this limitation was to determine the uni-
versality of the specific differences identified across additional
PAH patients. Therefore, using 21 fibroblast lines derived from
healthy control, HPAH, or IPAH patient skin (see clinical
characteristics in Table 1), we performed global gene expres-
sion profiling. The HPAH patients included both BMPR2 and
caveolin-1 mutations. Hierarchical clustering of samples
showed that, in general, HPAH samples clustered together,
IPAH samples clustered together, and controls clustered to-
gether (Fig. 5A). We found 409 probe sets representing 279
unique Entrez IDs with average differences of at least twofold
between controls and either HPAH or IPAH samples (see
Supplemental Table S8). Analysis of statistically overrepre-
sented gene ontology groups showed that pathways differen-
tially regulated in fibroblast lines were, for the most part,
similar to pathways we previously reported to be dysregulated
in cultured patient lymphocytes (5). These included 134 of the
279 genes related to altered metabolism, 25 cell adhesion genes
[P ϭ 0.013 for overrepresentation of gene ontology group, by
hypergeometric test, with Benjamini and Hochberg (6) multi-
ple comparisons adjustment], 16 circulatory system process
genes (P ϭ 0.0002), and 34 chemical stimulus response genes
(P ϭ 0.022), including 10 oxygen-level response genes (P ϭ
0.008).
To explicitly test the hypothesis that genes identified in the
iPS cells were common to other PAH patients, we examined
expression levels of all 164 unique genes identified in Fig. 4A.
Of these, 154 were also expressed in fibroblasts and 117
showed concordant differential regulation between iPS cell-
derived ECL cells and fibroblasts (e.g., genes upregulated in
ECL cells were also upregulated in fibroblasts, P ϭ 0.0013
by ␹2
test). Correlation in fold change between BMPR2 mutant
and controls and iPS cell-derived ECL cells and fibroblasts was
0.50, with correlation z-test P Ͻ 0.0001 (Fig. 5B). These
results indicate that while cell type-specific changes do exist,
the changes identified in our iPS cell-derived ECL cells are
broadly conserved across differentiated cell types and across
patients. HPAH and IPAH patients also had upregulation of
Wnt pathway genes. Eight of 10 HPAH patients had upregu-
lation of the secreted Wnt receptor SFRP1 compared with
controls and 10 of 10 had upregulation of SFRP2 and the Wnt
target genes PRICKLE2 and WISP2 (Fig. 5C). These differen-
tially regulated Wnt genes were also detected in IPAH patients
(Fig. 5D). That Wnt pathway genes are upregulated in skin
fibroblasts from every patient, not just on average, demon-
strates that our finding of upregulated Wnt genes in iPS-
derived cells is correlated to disease status, rather than indi-
vidual variation. Taken together, these data show that gene
expression changes in both HPAH and IPAH are detectable in
multiple differentiated cell types, are true across individuals,
Fig. 4. BMPR2 mutation causes increased Wnt pathway gene expression only in differentiated cell types. A: heat map analysis of gene segregation showing high
(red) and low (blue) levels of expression. ϩ, Mutant samples. In this set of genes, BMPR2 mutants and control cells are identical in undifferentiated early
mesenchymal cells (top 4 rows). With differentiation, BMPR2 mutants display aberrant induction (I), failure of inhibition (II), aberrant inhibition (III), or failure
of induction (IV). B: Wnt pathway genes show aberrant induction in BMPR2 mutants. Open circles, early MSC; shaded circles, differentiated MSC; solid circles,
ECL cells. Error bars, SE. C: quantitative RT-PCR (qRT-PCR) measurement of secreted Wnt receptor secreted frizzled-related protein (Sfrp-1 and Sfrp-2)
expression shows strong correlation between PCR and array measurements at every developmental stage. Open symbols, early MSC; shaded symbols,
differentiated MSC; solid symbols, ECL cells; circles, Sfrp-1; triangles, Sfrp-2. Horizontal and vertical axes measure expression in BMPR2 mutants divided by
expression in controls. D: qRT-PCR analysis validated gene trends identified in array analysis for FrzB, Fzd4, Msx2, FOXO1, and CYB1B1 in ECL cells.
Correlation between qRT-PCR and array is 0.94.

are likely independent of the state of disease progression, and
are consistent with our previously reported findings.
Decreased BMPR2 signaling activity deregulates Wnt sig-
naling in MSC. Dysregulation of Wnt signaling has previously
been noted in group I PAH, but in a context in which it was not
clear whether it was a consequence of end-stage disease (39),
a modifier (18), or a direct consequence of known mutation.
Our identification of Wnt pathway upregulation in our iPS cells
and as one of the most powerful common factors across 21
patient fibroblast lines suggests that it is a direct effect of
deregulated BMPR2 signaling.
We next performed functional analyses to quantify canonical
Wnt signaling activity in BMPR2mut iPS-MSC in vitro. First,
Wnt activity was measured indirectly via TCF/LEF luciferase
activity and normalized to Renilla luciferase activity following
transfection (Fig. 6A). At 72 h, Wnt signaling was significantly
increased in BMPR2mut iPS-MSC compared with WT iPS-
MSC. Second, to show that this result was specific to decreased
BMPR2 signaling, we took advantage of a group of small-
molecule kinase inhibitors with differential effects on BMPR2
and decreased BMP signaling in WT cells (32). BMPR2, a type
II serine/threonine kinase receptor, transduces signals through
heterotrimeric complexes with BMPR type I receptors
(BMPR1). Normal BMPR2 signaling requires a type I receptor.
Dorsomorphin (DM) is a prototype inhibitor that targets
BMPR1 and BMPR2 [IC50 ϭ 74 nM for BMPR2 (31, 34, 67)],
whereas LDN-193189 (LDN) and DM homolog 1 (DMH1) are
selective inhibitors of BMPR1 and are less active against
BMPR2 (IC50 ϭ 3,845 and Ͼ100,000 nM, respectively). We
tested canonical Wnt signaling activity in WT iPS-MSC in the
presence or absence of DM and the BMPR1 selective inhibitors
DMH1 and LDN (Fig. 6B). BMPR2 signaling inhibition by
DM significantly increased Wnt signaling at 24 h. DMH and
LDN treatment slightly increased Wnt signaling in WT iPS-
MSC at 48 h (results not shown).
In both iPS-derived MSC and ECL cells, as well as skin
fibroblasts, Sfrp-2 expression was strongly upregulated. To
validate these findings, we evaluated expression of Sfrp-2 on a
protein level using both iPS cell-derived and primary patient
cells. Using supernatant from iPS-MSC and iPS-ECL cells, we
Fig. 5. Differential regulation of genes by deregulated BMPR2 signaling in iPS-ECL cells is strongly correlated to that in skin fibroblasts from PAH patients.
Gene expression arrays were performed using RNA from 21 fibroblast lines derived from HPAH (n ϭ 10), idiopathic pulmonary arterial hypertension (IPAH,
n ϭ 7), or control (n ϭ 4) patients applied to Affymetrix Human Genome ST 1 chips. From 54,675 initial probe sets, 13,062 had a range of Ͼ0.4 and at least
1 sample with an expression Ͼ7 in log base 2 units. Restriction of analysis to these genes prevents inclusion of noise. A: heat map of 409 probe sets representing
255 unique Entrez IDs shows average changes of Ն2-fold between controls and either HPAH or IPAH. B: differential regulation of genes by deregulated BMPR2
signaling in iPS-ECL cells is strongly correlated to average differential regulation of genes in skin fibroblasts from PAH patients (correlation ϭ 0.50, P Ͻ 0.0001
by correlation z-test). Each circle represents 1 gene. C: upregulation of Wnt pathway and target genes in skin fibroblasts from HPAH patients compared with
controls. Each symbol represents gene expression in 1 patient, normalized to average of controls. D: analyses of developmental pathways with altered gene
expression confirm alterations in Wnt signaling, including secreted modulators Srfp2 and WISP2, and included the Notch pathways in both cells from IPAH and
HPAH patients relative to control.

MeanFoldChangeoverWTControl
vehicle
+DM(2μM)
+DM(5μM)
+DMH(0.5μM)
+LDN(0.5μM)
WT iPS MSCA B
WTiPSMSC
+LiCl(10mM)
BMPR2mutiPSMSC
+LiCl(10mM)
BMPR2mutiPSMSC0
3.5
7
WTiPSMSC
0
5
2.5
***
***
***
MeanFoldChangeoverVehicle
C D
SecretedSfrp-2ng/mlELISA
0
60
120
WTiPSECL
BMPiPSECL
ControlPAEC
PAHPAEC
WTiPSMSC
BMPiPSMSC
0
0.1
0.5
0.9
MeanSfrp-2(NormalizedDensitometry)
**
*
*
*
sfrp-2
β-actin
Ratio 0.6 0.2 0.5 0.5 0.9 0.6
Control
HPAH
(BMPR2)
IPAH
Hu Lung MSC
Non PAH
WTiPSECL
BMPiPSECL
ControlPAEC
PAHPAEC
F
14
0
FoldChangeinGeneExpression
overVehicleControliPSMSC
Veh DM
(5μM)
**
G
0
6
12
MeanChangeinExpressionLevel
ofSfrp-2NormalizedtoHPRT
Control PAH
UT Stretch UT Stretch
***
***
sfrp2
β−actin
WT mut mutWT
iPS ECL PAEC
Control PAEC
PAH PAEC
E
Fig. 6. Deregulated BMPR alters the Wnt signaling pathway and Sfrp-2 expression. A: Wnt activity by WT or BMPR2mut iPS-MSC was measured at 72 h using
a luciferase reporter assay. LiCl was used as a positive control for Wnt activation. B: Wnt activity by WT iPS-MSC in the presence of BMPR2 and BMPR1
signaling inhibitors was measured using the Wnt-luciferase reporter assay. Values represent mean fold change over WT or vehicle control at 24 h. DM,
dorsomorphin; DMH, DM homolog; LDN, LDN-193189. C and D: ELISA and Western blot analysis of secreted and cell-associated expression levels of Sfrp-2
protein by WT and BMPR2mut iPS-MSC and iPS-ECL cells. C: ELISA of secreted Sfrp-2 protein using cell conditioned medium, performed in triplicate. D
and E: representative Western blots of Sfrp-2 and ␤-actin, repeated twice independently and normalized to actin. Primary PAEC: n ϭ 2 control and 5 PAH.
Primary PAEC: n ϭ 2 control and 3 PAH. Primary human lung MSC: n ϭ 1 WT, 3 non-PAH, and 2 PAH. F: qRT-PCR was performed to detect the effect of
decreased BMPR signaling on Sfrp-2 expression. Values are shown as mean fold change compared with vehicle (Veh) controls at 24 h; n ϭ 2 control independent
patient iPS-MSC lines. G: effects of mechanical stretch on Sfrp-2 expression in control vs. PAH iPS-MSC was analyzed by qRT-PCR. Cells were plated on
collagen-coated plates and separated into unstretched (UT) and stretched groups. Values are shown as mean change normalized to hypoxanthine phosphoribo-
syltransferase (HPRT) at 72 h; n ϭ 2 control and 2 PAH independent patient iPS-MSC lines. *P Ͻ 0.05; **P Ͻ 0.01; ***P Ͻ 0.001.

performed ELISA to measure levels of secreted Sfrp-2. We
found that, in both cell types, BMPR2 mutant patient-derived
cells displayed significantly higher levels of secreted Sfrp-2
(Fig. 6C). We then investigated Sfrp-2 secretion in primary
PAEC cultured from explanted IPAH patient or failed donor
control lungs (n ϭ 3–4 for each) and found significantly higher
secreted Sfrp-2 in small vessel-derived EC lines (Fig. 6C). By
Western blot analysis of whole cell lysates from all these cell
types, we found that, as we expected, the PAH EC that secreted
higher levels of Sfrp-2 had less cell-associated Sfrp-2 protein
(Fig. 6D) than primary human lung MSC, which had increased
levels of Sfrp-2 protein (Fig. 6E). To directly link decreased
BMPR signaling to regulation of Sfrp-2 expression, we used
DM to inhibit BMPR signaling in control iPS-MSC (Fig. 6F).
Inhibition of BMPR signaling resulted in significantly in-
creased expression of Sfrp-2 message. Because we are study-
ing a simple system in the absence of physiological influence,
we next evaluated the effect mechanical forces might have on
Sfrp-2 gene expression in control vs. PAH iPS-MSC (Fig. 6G).
Cells were exposed to the deformation of approximately one
heartbeat and moderate distension. At 72 h, baseline expression
of Sfrp-2 transcript was greater in PAH iPS-MSC than con-
trols. Interestingly, stretch did not significantly affect the con-
trol iPS-MSC; however, the PAH cells decreased their expres-
sion levels by approximately twofold. The decrease in PAH
expression rendered the levels similar to controls. No signifi-
cant difference in gene expression was observed at 24 h. Taken
together, our data imply that PAH patients produce more
Sfrp-2 and the expression of Sfrp-2 transcript may be regulated
by vascular tone.
To validate these findings in human PAH tissue specimens,
we performed immunohistochemistry to detect Sfrp-2 (Fig. 7).
Interestingly, Sfrp-2 localized to endothelium, parenchyma,
and smooth muscle cells in control and PAH tissue. However,
the intensity of Sfrp-2 staining was significantly greater in
HPAH tissue, in areas of remodeling and in smooth muscle
cells, than in control specimens (Fig. 7, A–F). ELISA was
performed to analyze levels of Sfrp-2 in PAH patient plasma
relative to controls (Fig. 7G). We did not detect a significant
difference between control, HPAH, and IPAH plasma samples,
suggesting that Sfrp-2 is likely retained locally in the lung.
This theory was confirmed by Western blotting to detect levels
of Sfrp-2 protein in vivo using murine lungs, in the absence or
presence of the R899x BMPR2 mutation (Fig. 7H). As antic-
F
EC
EC
EC/SMC
A B
0
30
60
Meanng/mlSfrp-2in
PatientPlasmaSamples
G
C
EC
SMC
SMC
EC
D E
Control Distal Lung
HPAH Distal Lung (ex2 LBD) HPAH Distal Lung (ex9 Cyt)
Control Proximal PA PA FAM 14 HPAH (ex2 LBD) PA FAM 28 HPAH (ex9 Cyt)
EC
SMC
Control HPAH IPAH
Sfrp-2
β-Actin
RatioSfrp-2
toβ-actin
RVSP
23.7
24
24.7
23.5
59.6
67.3
65.7
35.4
0
0.5
0.25
**
Control mutBMPR2
(R899x)
Control Lung
H mutBMPR2
(R899x)
Fig. 7. Increased Sfrp-2 expression and
localization in control and PAH lung tis-
sue. Patient lung tissue was analyzed by
immunostaining of paraffin-embedded lung
sections using diaminobenzidine detection
(black). Sfrp-2 localized to intimal lesions
and areas of remodeling in HPAH patient
lung tissue. Mutation types are noted. A–F:
representative bright-field images of immu-
nohistochemical localization of Sfrp-2.
Sfrp-2 staining localized to airway epithe-
lium, endothelial, smooth muscle, and paren-
chymal cells. Localization was not depen-
dent on the patient-specific BMPR2 mutation
(n ϭ 2 control and 5 HPAH). Scale bars, 20
␮m. C–E: Sfrp-2 was also present with in-
creasing intensity in smooth muscle layers of
PAH tissue relative to control. EC, endothe-
lial cell layer; BV, blood vessel; EC/SMC,
endothelium and smooth muscle. Scale bars,
100 ␮m. G: ELISA of secreted Sfrp-2 pro-
tein using patient plasma, performed in trip-
licate; n ϭ 14 control, 7 HPAH, and 7 IPAH.
H: Western blot analysis of expression levels
of Sfrp-2 protein by control (n ϭ 4) and
BMPR2 mutant (R899x Cyt, n ϭ 4) mouse
lung tissue; results, normalized to ␤-actin,
are from 2 independent experiments. **P Ͻ
0.01.

ipated, Sfrp-2 protein levels were significantly increased in the
mutant mouse lungs. Our results suggest that abnormal
BMPR2 signaling regulates Wnt signaling during PAH and
may be, in part, due to Sfrp-2.
DISCUSSION
The relationship between the BMPR2 signaling pathway and
Wnt signaling pathways has been delineated during develop-
ment with regard to body axis patterning; however, their
relationship during adult PAH is incompletely understood. In
the present study, we used patient tissue samples and primary
patient cell lines (iPS cell-derived, IPAH and HPAH) to
demonstrate that BMPR2 dysfunction alters Wnt signaling and
yields a common molecular phenotype at various stages of
vascular cell development, as well as in adult somatic cells. We
used iPS cell-derived MSC and ECL cell lines to identify
molecular signatures of PAH without concern for confounding
by secondary effects of end-stage disease, drug therapy, or
altered local milieu from elevated pressures. These studies, as
well as previous data from our murine modeling systems,
suggest that decreased BMPR2 signaling increases canonical
Wnt signaling.
iPS cell-derived and primary patient MSC were used in this
study, because mesenchyme is a source of multipotent vascular
precursors during development, as well as in the adult lung,
and their role in PAH has not been delineated (1, 2, 13, 37, 46).
The intimacy of the relationship between mesenchyme and
epithelium/endothelium persists into the adult tissue and is
recapitulated during organ repair and remodeling. Using this
method, we identified cell and developmental stage-specific
signatures through comparative analyses (1, 2). iPS-MSC and
iPS-ECL cells demonstrated consistent expression of BMPR2,
thus providing the opportunity to model deregulated BMPR2
and Wnt signaling in vitro. Here we demonstrate that the iPS
cell-derived cell lineages retain characteristics of PAH, includ-
ing decreased expression of Tie2, VEGF-A, Flk-1/KDR, and
BMP-4. Although characteristic surface markers of MC and
EC were demonstrated, there were significant differences in
differentiation in ECL cells, as well as gene expression profiles
and Wnt signaling between the WT and PAH-derived cells,
including VEGF-A, Tie2/TEK, and Flk1/KDR, all factors that
regulate vascular stability. These genes lack identified SMAD-
binding sites within their promoters, which suggests an indirect
regulation of expression, not direct regulation by BMP SMAD
signaling. It is likely that a derangement of BMPR2 signaling
pathways in the differentiated cells resulted in alteration of
additional signaling pathways, affecting cell self-renewal, cell
proliferation, and cell fate determination (25, 68).
Apoptosis followed by proliferation of apoptosis-resistant
EC is a paradigm to explain vascular remodeling in PAH (21,
41, 61, 62). Our studies expand this paradigm and show that
while PAH iPS cell-derived and primary MC did not have
significantly different rates of proliferation relative to control
lines, they were also less likely to undergo apoptosis. Also
interesting was the finding that intracellular oxidative stress
was not increased in BMPR2 mutant MC under ambient
culture conditions, which is consistent with previous reports
from studies using cells with BMPR2 mutations (38). How-
ever, we showed that when placed in low oxygen over time,
BMPR2 mutant MC had a significant increase in intracellular
accumulation of GSSH, decreasing the ratio of GSH to GSSG,
indicative of oxidative stress. This may be important in areas of
tissue hypoxia that increase with the progression of disease.
We exploited the use of global gene expression analysis to
identify common molecular pathways affected by deregulated
BMPR2 signaling. Analysis of multiple stages of differentia-
tion from MSC to ECL cells, as well as dermal fibroblasts
(including control, HPAH, and IPAH samples), demonstrated
consistent increases or decreases in expression levels of Wnt
signaling pathway members, including modulators or inhibi-
tors, as well as receptors. The Wnt signaling pathway influ-
ences cell-cell communication, adult tissue maintenance, and
gene expression. BMPR2 signaling may regulate both canon-
ical and noncanonical Wnt pathways in EC and MC to influ-
ence proliferation, survival, and motility during angiogenesis
and remodeling of the pulmonary circulation (3, 19, 39). While
the relationship of BMPR2 and Wnt pathways has been defined
during development, the regulatory targets of Wnt signaling,
common across multiple cell types, in BMPR2-associated PAH
are unknown.
On the basis of these data, we evaluated Wnt signaling in
PAH-susceptible MC. In our iPS-MSC model, decreased BMP
signaling via BMPR2 mutation or soluble inhibitor specific to
BMPR2 (31, 34, 67) resulted in increased Wnt signaling
activity. We used this approach, since modulators of the Wnt
canonical and noncanonical planar cell polarity signaling path-
ways were previously shown to have increased expression in
patients with PAH (39) and because proper BMP signaling
regulates both canonical and planar cell polarity pathways in
the endothelium and smooth muscle to influence cell prolifer-
ation, survival, and motility in the pulmonary circulation (19).
Increased Wnt signaling in adult lung MSC has been correlated
with their transition to a contractile cell that participates in
vascular remodeling during PAH (13).
Expression analyses across multiple cell types identified
Sfrp-2 as differentially regulated in PAH cell lines vs. control.
It was not surprising that Akt (protein kinase B), a critical
component of vascular remodeling in PAH (35), is a key
mediator of Sfrp-2 expression (49). Here we directly link
decreased BMP signaling and the mechanical properties of the
vasculature to regulation of Sfrp-2 transcript expression. Fur-
thermore, both PAH patient lung tissue and BMPR2 mutant
mouse lungs expressed higher levels of the protein. Sfrp-2 was
initially identified as a Wnt antagonist, typically expressed
during lung morphogenesis to promote alveolarization (26).
Sfrp proteins are required for Wnt diffusion, activation, canon-
ical signaling, and proper tissue differentiation; their effects on
Wnt signaling are dependent on their concentration or Wnt
ligands present in tissue (23, 26, 42, 63a). For example, Sfrp-2
can enhance activation of the canonical Wnt pathway (17, 64,
66) while inhibiting the noncanonical pathway, resulting in
abnormal cell alignment and shape (12). Sfrp-2 can inhibit
BMP-4 expression and prevent programmed cell death (22).
Interestingly, the BMPR2 mutant iPS EC had decreased levels
of BMP-4 expression relative to WT. Sfrp-2 is also proangio-
genic, inhibits cell apoptosis, and increases migration (17, 49)
and, therefore, has the potential to play a role in the pathology
of disease. Sfrp-2 is also known to decrease bone formation via
regulation of BMP (54). This is likely due to the inhibitory
effect of Sfrp-2 on BMP-1 and other tolloid proteases neces-
sary for cleavage and inactivation of BMP antagonists, includ-

ing chordin and noggin (10, 23, 33, 36, 50), described during
developmental processes. Multiple tolloid and proteases can
cleave and inactivate secreted BMP antagonists. However, the
specific mechanisms by which deregulated BMPR2 regulates
Sfrp-2 and Wnt signaling pathways to regulate cell phenotype
and function remain to be determined and is likely cell-
specific.
Prior etiological interpretation of previous expression array
studies using patient-derived samples may have been compli-
cated by the overwhelming signal induced by end-stage disease
and treatment. In one study of PAH lung tissue, of ϳ14,000
genes with measurable expression, 13,889 were altered (57).
Among studies of PAH patient-derived samples, there have
been three studies of lung tissue specimens (28, 39, 57), four
studies of freshly isolated circulating cells (11, 30, 56, 63), and
three studies of cells cultured from PAH patients (5, 24, 65).
While the overall results have been recently reviewed (48), we
reexamined these data to determine whether they also identi-
fied alterations in expression of Wnt signaling molecules as a
disease-associated signaling pathway (Table 2). Wnt pathway
signal, aside from increased TCF expression, was not apparent
in any of the studies relying on fresh or cultured peripheral
blood mononuclear cells (PBMC). On the basis of our own
published arrays, this is likely because cell-specific Wnt path-
way components are not significantly altered in PBMC (5, 65).
Thus PBMC are likely not the ideal candidate cell type relative
to adherent/polar vascular cells in which to assay this pathway.
Of the four remaining studies, the most recent three showed a
strong indication of increased Wnt signaling. In both normo-
tensive and hypertensive pulmonary arterioles from idiopathic
pulmonary fibrosis patients, Patel et al. (55) demonstrated that
gene expression indicative of activated Wnt signaling was
increased, which was characteristic of abnormal proliferation,
apoptosis, and adverse remodeling.
Our studies show that global gene expression data obtained
using multiple adherent somatic cell types from IPAH and
HPAH patients could be utilized to identify common pathways
affected in PAH, specifically the canonical Wnt pathway. We
recognize that while iPS cells and patient primary cells provide
a powerful model to understand PAH at the cellular level,
limitations to this approach remain. 1) On the basis of the low
efficiency of reprogramming observed, a major obstacle to
deriving iPS cells from HPAH patients with BMPR2 mutation
clearly exists when nonintegrating or excisable technology is
used. This limitation is most likely due to the importance of
intact coordinated BMP and Wnt signaling required for the
formation and proper differentiation of the pluripotent epiblast
(7, 58). These newer technologies are preferred over multiple
integrating viruses (29), which may mask the function of the
mutation, induce their own mutant behavior, and obscure true
penetrance via the creation of virus-dependent genetic altera-
tions (52, 60). However, with the rapid simplification of
accessible technology, these limitations are being overcome. 2)
The iPS cell model system lacks the capacity to model complex
in vivo events and alterations in the physiological milieu. 3) It
is possible that the cellular ramifications of a BMPR2 mutation
are not uniform across mutation type. However, in contrast to
in vitro models that ectopically express BMPR2 mutation in
WT cells while retaining WT BMPR2 signaling, the iPS and
primary patient cells have allowed us to preserve the disease-
specific regulation of two key signaling pathways involved in
PAH. We have been successful in identifying the cell-specific
changes in Wnt signaling, including Sfrp-2, that have been
linked in development but have not been studied in the context
of adult disease. Ongoing investigation in our laboratories is
focused on understanding the regulation and role of Sfrp-2
during vascular lesion formation in PAH, as well as correction
of BMPR2 mutation in our HPAH line using CRISPR gene-
editing technology to elucidate the direct effects of BMPR2 on
this system.
Our studies linked deregulated developmental pathways
with adult disease over multiple cell types and differentiation
states. We show that decreased BMPR2 signaling results in a
genetic signature common across multiple cell types, culmi-
nating in the pathological processes recognized as PAH. Taken
together, our results suggest that increased Wnt signaling is
inherent to the cells of PAH patients and is likely due to
decreased BMPR2 signaling. This combination of iPS and
primary patient cell modeling may ultimately enable the iden-
tification of cellular defects that lead to the clinical manifesta-
tions of PAH and provide access to multiple renewable cell
types in which to test potential therapies.
ACKNOWLEDGMENTS
The authors thank T. Blackwell, A. Omari, N. Wickersham, Tim Sullivan,
Heidi Miller, and Lora Hedges for expert technical assistance and the Van-
derbilt Institute of Chemical Biology Synthesis Core for small-molecule BMP
inhibitors. Tissue samples (pulmonary artery endothelial cells) were provided
by the Pulmonary Hypertension Breakthrough Initiative (PHBI).
GRANTS
This work was funded by American Heart Association Grant GIA-
0855953G and National Institutes of Health (NIH) Grants R01 HL-091105,
R01 HL-11659701, and R21 DK-094132–01 (S. M. Majka). Additional
funding was provided by NIH Grants K23 HL-098743 (E. Austin), R01
HL-082694 and R01 HL-095797 (J. D. West), and 1R01 NS-078289 (K. C.
Ess), and ES-016931 (A. B. Bowman), and NIH Grants HL-094707 and
HL-115103, National Science Foundation Grant 1055384, and National Center
for Biotechnology Information Grant 5T32 GM-007347-34 (W. D. Merry-
man). Funding for the PHBI is provided by the Cardiovascular Medical
Research and Education Fund. Experiments were performed using the Univer-
sity of Colorado Cancer Center Flow Cytometry Core (UCCC; NIH Grant
5P30 CA-46934 and Skin Diseases Research Core Grant P30 AR-057212), the
UCCC Microarray Core (NIH Grant P30 CA 46934-14), and the UCCC Skin
Diseases Research Morphology and Phenotyping Core (NIH Grant P30 AR-
Table 2. Global gene expression studies highlight Wnt pathway involvement in PAH
Study Tissue Wnt-Related Results
Fantozzi et al. (24) Cultured PASMC Wnt ligand expression correlates to PAP
Geraci et al. (28) Whole lung Not discussed/no raw data
Laumanns et al. (39) Laser capture of small arteries Increased noncanonical Wnt signaling
Rajkumar et al. (57) Whole lung Increased secreted Wnt modulators
PAH, pulmonary arterial hypertension; PASMC, pulmonary artery smooth muscles cells.

057212). This work was also supported in part by Vanderbilt Clinical and
Translational Science Award 1 UL1 RR-024975 from the National Center for
Research Resources.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
AUTHOR CONTRIBUTIONS
J.D.W., E.D.A., A.R.H., C.C.H., B.O.M., J.E.L., P.P.Y., W.D.M., D.K., and
S.M.M. are responsible for conception and design of the research; J.D.W.,
E.D.A., C.G., R.B., S. Menon, J.P.F., J.A.K., L.B.W., L.A.W., C.C.H., D.J.K.,
D.K., and S.M.M. analyzed the data; J.D.W., S. Marriott, R.B., A.R.H., S.
Menon, J.P.F., L.B.W., C.C.H., B.O.M., J.E.L., D.J.K., P.P.Y., and S.M.M.
interpreted the results of the experiments; J.D.W., E.D.A., C.G., R.B., G.B., S.
Menon, L.A.W., D.K., and S.M.M. prepared the figures; J.D.W., A.R.H., and
S.M.M. drafted the manuscript; J.D.W., E.D.A., C.G., S. Marriott, R.B., G.B.,
A.R.H., S. Menon, J.P.F., L.B.W., C.C.H., B.O.M., J.E.L., D.J.K., P.P.Y.,
D.K., and S.M.M. edited and revised the manuscript; J.D.W., E.D.A., C.G., S.
Marriott, R.B., G.B., J.-C.J., A.R.H., S. Menon, N.C.B., J.P.F., J.A.K., D.C.I.,
L.B.W., L.A.W., C.C.H., B.O.M., J.E.L., A.B.B., D.J.K., P.P.Y., W.D.M.,
D.K., and S.M.M. approved the final version of the manuscript; E.D.A., C.G.,
S. Marriott, R.B., G.B., J.-C.J., N.C.B., D.C.I., L.B.W., L.A.W., A.B.B.,
K.C.E., D.J.K., W.D.M., D.K., and S.M.M. performed the experiments.
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