BMP type II receptor as a therapeutic target in pulmonary arterial hypertension

Abstract

Pulmonary arterial hypertension (PAH) is a chronic disease characterized by a progressive elevation in mean pulmonary arterial pressure. This occurs due to abnormal remodeling of small peripheral lung vasculature resulting in progressive occlusion of the artery lumen that eventually causes right heart failure and death. The most common cause of PAH is inactivating mutations in the gene encoding a bone morphogenetic protein type II receptor (BMPRII). Current therapeutic options for PAH are limited and focused mainly on reversal of pulmonary vasoconstriction and proliferation of vascular cells. Although these treatments can relieve disease symptoms, PAH remains a progressive lethal disease. Emerging data suggest that restoration of BMPRII signaling in PAH is a promising alternative that could prevent and reverse pulmonary vascular remodeling. Here we will focus on recent advances in rescuing BMPRII expression, function or signaling to prevent and reverse pulmonary vascular remodeling in PAH and its feasibility for clinical translation. Furthermore, we summarize the role of described miRNAs that directly target the BMPR2 gene in blood vessels. We discuss the therapeutic potential and the limitations of promising new approaches to restore BMPRII signaling in PAH patients. Different mutations in BMPR2 and environmental/genetic factors make PAH a heterogeneous disease and it is thus likely that the best approach will be patient-tailored therapies.

Full text

Vol.:(0123456789)
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Cell. Mol. Life Sci. (2017) 74:2979–2995
DOI 10.1007/s00018-017-2510-4
REVIEW
BMP type II receptor asatherapeutic target inpulmonary
arterial hypertension
MarOrriols1· MariaCatalinaGomez‑Puerto1· PetertenDijke1
Received: 22 December 2016 / Revised: 9 March 2017 / Accepted: 17 March 2017 / Published online: 26 April 2017
© The Author(s) 2017. This article is an open access publication
Keywords Endothelial cell· Vascular smooth muscle
cell· Signal transduction· Inflammation· Vascular
remodeling and autophagy
Abbreviations
ACE Angiotensin-converting enzyme
ACVRII Activin type II receptor type
ACVRL1 Gene encoding activin receptor-like kinase
ALK Activin receptor-like kinase
BMP Bone morphogenetic protein
BMPRI Bone morphogenetic protein type I receptor
BMPRII Bone morphogenetic protein type II receptor
BMPR2 Gene encoding bone morphogenetic protein
receptor II
Cav1 Caveolin-1
cGMP Cyclic guanosine monophosphate
DN Dominant negative
EC Endothelial cell
EndMT Endothelial-to-mesenchymal transition
FKBP12 FK-binding protein-12
GDF Growth and differentiation factor
GDF-2 Gene encoding BMP9
HMGA1 High-mobility group protein
iTOP Induced transduction by osmocytosis and
propanebetaine
MCT Monocrotaline
mPAP Mean pulmonary arterial pressure
miRNA MicroRNA
NMD Non-sense mediated decay
PAEC Pulmonary arterial endothelial cells
PAH Pulmonary arterial hypertension
PASMC Pulmonary arterial smooth muscle cells
PGI2 Prostacyclin
PKG Protein kinase G
PTC Premature termination codon
Abstract Pulmonary arterial hypertension (PAH) is a
chronic disease characterized by a progressive elevation
in mean pulmonary arterial pressure. This occurs due to
abnormal remodeling of small peripheral lung vasculature
resulting in progressive occlusion of the artery lumen that
eventually causes right heart failure and death. The most
common cause of PAH is inactivating mutations in the
gene encoding a bone morphogenetic protein type II recep-
tor (BMPRII). Current therapeutic options for PAH are lim-
ited and focused mainly on reversal of pulmonary vasocon-
striction and proliferation of vascular cells. Although these
treatments can relieve disease symptoms, PAH remains a
progressive lethal disease. Emerging data suggest that res-
toration of BMPRII signaling in PAH is a promising alter-
native that could prevent and reverse pulmonary vascular
remodeling. Here we will focus on recent advances in res-
cuing BMPRII expression, function or signaling to prevent
and reverse pulmonary vascular remodeling in PAH and its
feasibility for clinical translation. Furthermore, we summa-
rize the role of described miRNAs that directly target the
BMPR2 gene in blood vessels. We discuss the therapeutic
potential and the limitations of promising new approaches
to restore BMPRII signaling in PAH patients. Different
mutations in BMPR2 and environmental/genetic factors
make PAH a heterogeneous disease and it is thus likely that
the best approach will be patient-tailored therapies.
Cellular and Molecular LifeSciences
Mar Orriols and Maria Catalina Gomez-Puerto the authors have
contributed equally to this work.
* Peter ten Dijke
p.ten_dijke@lumc.nl
1 Department ofMolecular Cell Biology andCancer Genomics
Center Netherlands, Leiden University Medical Center,
Leiden, TheNetherlands
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2980 M.Orriols et al.
1 3
RV Right ventricle
SMC Smooth muscle cell
TGF-β Transforming growth factor-beta
TNFα Tumor necrosis factor-alfa
Introduction
Pulmonary arterial hypertension (PAH) is a chronic dis-
ease characterized by a progressive elevation in mean pul-
monary arterial pressure (mPAP >25 mmHg) leading to
right heart failure and death [1]. PAH is characterized by
abnormal remodeling of small peripheral lung vascula-
ture resulting in progressive occlusion of the artery lumen.
In addition, at late stages, so-called plexiform lesions are
found, which are complex vascular formations originating
from abnormal endothelial cell (EC) proliferation and vas-
cular smooth muscle cell (SMC) hypertrophy [2]. The basic
pathogenic mechanisms underlying this disease include
vasoconstriction, intimal proliferation, and medial hyper-
trophy. These processes are accompanied by illicit recruit-
ment of inflammatory cells which release factors enhanc-
ing cell proliferation and elastin fibers degradation [3, 4]
(Fig.1).
More than 70% of patients with familial PAH and 20%
of idiopathic PAH show heterozygous mutations in the
bone morphogenetic protein type II receptor (BMPRII)
[5–8]. BMPRII is a transmembrane serine/threonine kinase
receptor of the bone morphogenetic protein (BMP) pathway
which is essential for embryogenesis, development, and
adult tissue homeostasis. Upon BMP-induced heteromeric
complex formation of BMPRII with BMP type I receptor
(BMPRI), BMPRII activates BMPRI by phosphorylation.
Thereafter, the activated BMPRI propagates the signal into
the cell through phosphorylation of the SMAD1/5/8 tran-
scription factors.
In PAH, over 300 mutations have been found in the
BMPR2 gene. These mutations target sequences that
encode the ligand binding and kinase domain and the
long cytoplasmic tail; the mutations compromise BMPRII
function [9]. Although the BMPRII pathway is essential
for vascular homeostasis and there is a strong correlation
between BMPR2 mutations and PAH, the incomplete pen-
etrance of BMPRII mutations (20–30%) suggests that other
genetic and environmental factors contribute to the disease.
Among them, BMPR2 alternative splicing plays a role in
PAH penetrance. One BMPR2 splice variant lacks exon 12,
which is the largest exon of the gene and encodes the cyto-
plasmic tail. It has been shown that carriers of this variant
are more prone to develop PAH through a dominant-neg-
ative effect (DN) effect on wild-type BMPRII [10]. Fur-
thermore, there are mutations in other genes in the BMP
pathway, which further strengthens the notion of a causal
role for this pathway in PAH [11]. Moreover, the co-exist-
ence of modifier genes, infections, toxic exposure, inflam-
mation, or alterations in estrogen metabolism has been
described [11–14] and some of them were found to down-
regulate BMPRII expression. For example, pro-inflam-
matory cytokines such as tumor necrosis factor α (TNFα)
and Interleukin 6 induce the expression of miRNAs that
inhibit BMPRII expression [15]. Furthermore, BMPRII is
essential for maintaining the barrier function of the pulmo-
nary artery endothelial cell lining and BMPRII deficiency
increases endothelial inflammatory responses thereby con-
tributing to adverse vascular remodeling [16–18].
Current therapeutic options for PAH are limited and
focused mainly on reversal of pulmonary vasoconstric-
tion and proliferation of vascular cells through targeting of
Fig. 1 Physiopathological mechanisms of pulmonary arterial hyper-
tension development. Presence of genetic risk factors such as BMPR2
mutations together with exposure to deleterious environmental or
biological stimuli in the lung promotes PAH. PAH development is
characterized by a disturbance on the signaling pathways that con-
trol pulmonary vascular homeostasis. It results in pulmonary vascu-
lar thickening and occlusion compromising lung and heart function.
EndMT endothelial-to-mesenchymal transition
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2981BMP type II receptor asatherapeutic target inpulmonary arterial hypertension
1 3
prostacyclin (PGI2), endothelin, or nitric oxide pathways
[19]. Although these treatments can relieve disease symp-
toms and slow down its progression, PAH remains a pro-
gressive lethal disease. Abundant research over the past
decade has improved our understanding of the molecular
mechanisms underlying PAH progression revealing novel
potential therapeutic interventions [20–22]. Among them
there are several anti-proliferative strategies including cell
cycle inhibitors (e.g., mTOR inhibitor rapamycin) and
anti-apoptotic drugs (e.g., surviving inhibitors) [23]. Fur-
thermore, based on the fact that Rho and ROCK mediate
smooth muscle cell proliferation in a serotonin-BMPR-
dependent pathway, Rho-kinase inhibitors have been also
considered [23, 24]. Although several drugs with possible
benefit in PAH have been identified, only very few have
been approved for use in the clinic due to toxicity or lack of
clinical efficacy. This review will focus on recent advances
on the rescue of BMPRII expression, function, or signaling
to prevent and reverse pulmonary vascular remodeling in
PAH. We will discuss data on the invitro efficacy of the
different approaches together with the physiological out-
comes in pre-clinical models and their feasibility for clini-
cal translation.
BMP signaling invascular biology andPAH
BMPs belong to the multifunctional transforming growth
factor-β (TGF-β) family of secreted dimeric cytokines.
The effects of BMPs are highly dependent on cellular
context [25]. In general, BMPs control cellular prolifera-
tion, differentiation, and apoptosis, and play an important
role in embryonic development and maintaining tissue
homeostasis [26]. Therefore, perturbation of BMP signal-
ing may lead to skeletal diseases, vascular diseases, and
cancer [27]. BMPs can be subdivided into four subgroups
based on their sequence similarity and cell surface recep-
tor affinities: BMP2/4, BMP5/6/7/8, BMP9/10, and growth
and differentiation factor (GDF)-5/6/7 [28, 29]. BMPs sig-
nal via hetero-tetrameric combinations of type I receptors
(activin receptor-like kinase (ALK)1, ALK2, ALK3, or
ALK6) and BMP type II receptors (BMPRII) and activin
type II receptor (ACVRII)A or ACVRIIB complexes [30,
31]. Both, type I and type II receptors have a similar struc-
ture encompassing a short extracellular domain, a single
transmembrane domain and an intracellular domain with
intrinsic serine-threonine kinase activity. In the vascular
endothelium, BMP signaling is mainly activated by BMP2,
4, 6, 9, and 10 [32]. BMP2 and BMP4 bind preferentially to
the BMPRII in complex with ALK3 or ALK6. BMP6 binds
to the ACVRIIA-ALK2 complex, while BMP9 and BMP10
bind to BMPRII or ACVRII in combination with ALK1 or
ALK2. Whereas BMPRII is a specific receptor for BMPs,
ACVRIIA and ACVRIIB can also interact functionally
with other ligands, such as activins, myostatin, and nodal.
Interestingly, ALK2 and 6 are widely expressed in various
cell types, while ALK1 has a more selective expression
pattern and is mainly restricted to ECs. After BMP binding
and receptor complex formation, the type II receptor kinase
phosphorylates the type I receptor on serine and threonine
residues in the glycine-serine rich (GS)-domain causing its
activation and subsequent phosphorylation of the recep-
tor-associated R-SMAD1, 5, and 8 effector proteins. The
R-SMADs that are activated by TGF-β type I and activin
type I receptor (i.e., ALK5 and ALK4, respectively) are
SMAD2 and SMAD3 and these are distinct from BMP
R-SMADs. Activated R-SMAD 1, 5, or 8 forms a hetero-
oligomeric complex with common mediator co-SMAD4.
This complex translocates to the nucleus and regulates the
expression of target genes by binding to specific enhanc-
ers/promoters upstream of these target genes [30, 33, 34]
(Fig. 2). Besides canonical BMP receptor/SMAD signal-
ing, activated BMP receptors can initiate non-SMAD sign-
aling pathways such as ERK, JNK, p38 MAP kinases, and
the phosphatidyl inositol 3 kinase (PI3K)/AKT pathways
[35–37]. These non-SMAD pathways are also important for
diversifying and modulating the canonical SMAD signal-
ing pathways that are activated by the BMP receptors [38,
39]. In addition, BMP activity is also regulated by several
extracellular modulators, including BMP binding proteins
NOGGIN, CHORDIN, and FIBULINs. Co-receptors such
as ENDOGLIN, BETAGLYCAN, and DRAGON fam-
ily members may also modulate the interactions between
BMPs and BMP receptors [40, 41]. Moreover, intracellu-
lar kinases/phosphatases and other binding proteins have
been identified as regulators of the trafficking, subcellular
localization, stability, and function of BMP receptors and
SMADs [26].
Genetic depletion of different components of the BMP
signaling cascade leads to embryonic death due to cardio-
vascular malformations and abnormal vascular remodeling.
BMP signaling plays an important role in vasculogenesis
(de novo formation of blood vessels from undifferentiated
mesodermal cells) and angiogenesis (formation of new
blood vessels from the existing vasculature). In this light,
it is not surprising to discover that, besides PAH, dysfunc-
tion of BMP signaling has been found to be associated with
other vascular diseases including hereditary hemorrhagic
telangiectasia, cerebral cavernous malformation, athero-
sclerosis, and vascular calcification among others [42].
Furthermore, BMPRII downregulation has been found to
be involved in pancreatic and lung fibrosis [43, 44].
Blood vessels are composed of three layers: the tunica
adventitia consisting of fibroblasts and associated collagen
fibers; the tunica media composed of SMCs; and the tunica
intima consisting of ECs coating the interior surface [45,
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2982 M.Orriols et al.
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46]. ECs, SMCs, and fibroblasts have been found to play
a role in the pathogenesis of PAH. Abnormal EC prolif-
eration resulting in the formation of plexiform lesions has
been frequently described in many PAH cases [47]. In addi-
tion, pulmonary arterial SMCs show an increased prolifera-
tion and decreased apoptosis leading to vessel wall thicken-
ing and vascular remodeling (Fig.1). The close interaction
between ECs and SMCs has been found to be involved in
vessel formation and maintenance. For instance, endothe-
lial derived factors, like endothelin and angiotensin II,
affect SMCs which increases vascular tone. Similarly, nitric
oxide and PGI2 secreted by ECs modulate the vasodilator
response of SMCs [48]. In particular, PGI2 has been found
to be reduced in PAH patients [49].
BMP signaling is known to control cell migration, pro-
liferation, and apoptosis in ECs and SMCs [45]. BMP9
and BMP10 are present in the circulation and play an
important role in the vasculature. Their associated recep-
tors BMPRII and ALK1 and co-receptor ENDOGLIN are
predominantly expressed on ECs [50, 51] and together
can modulate the ability of ECs to migrate and prolifer-
ate [27]. Furthermore, BMPRII is also expressed in vas-
cular SMCs where it has been shown to be necessary
for the control of proliferation and differentiation [52].
Besides mutations in the BMPR2 gene, mutations in
the genes of other BMP signaling components (such as
GDF-2, ACVRL1, ENDOGLIN, and SMAD8) have also
been linked to PAH development [11, 53–59]. This asso-
ciation reinforces the importance of BMP signaling in
the control of vascular homeostasis, and suggests that
there is a causal link between perturbation of canonical
BMP/SMAD signaling and PAH. In support of this view,
recent, new DNA sequencing techniques helped to iden-
tify new gene mutations associated to PAH [Caveolin-1
(CAV1), KCNK3, and EIF2AK4] [60, 61] (Fig.2).
The abnormal vascular remodeling that characterizes
PAH involves an accumulation of α-smooth muscle,
actin-expressing mesenchymal-like cells indicating that
the endothelial-to-mesenchymal transition (EndMT)
may be involved in the pathogenesis of the disease [62].
In addition, BMPRII reduction in pulmonary artery
endothelial cells (PAECs) has been found to promote the
trans-differentiation of epithelial cells into motile mesen-
chymal cells via the transcription factors high-mobility
group protein (HMGA)1 and its target SLUG [63].
Fig. 2 BMP signaling in
endothelial cells. BMP9 and
BMP10 present in the circu-
lation initiate signaling by
binding and bringing together
BMPRII and ALK1. BMPRII
phosphorylates ALK1 which
then propagate the signal
through phosphorylation of
SMAD1/5/8. Subsequently,
SMAD4 forms a complex with
SMAD1/5/8, which translo-
cates to the nucleus regulating
the expression of target genes
such as ID1 and ID3. Known
gene mutations associated with
PAH are highlighted in red.
It includes mutations in BMP
signaling components (GDF2,
BMPR2, ALK1, SMAD8, and
ENDOGLIN) as well as recently
discovered non-directly related
BMP genes (CAV1, KCNK3,
and EIF2AK4). CAV caveolin,
EFI2AK4 eukaryotic translation
initiation factor 2α kinase 4,
ENG ENDOGLIN, ID inhibi-
tor of DNA binding, KCNK3
potassium channel subfamily K
member 3
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2983BMP type II receptor asatherapeutic target inpulmonary arterial hypertension
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Animal models ofPAH
PAH has a complex etiology and pathobiology with many
factors contributing to its development [64]. A variety of
pre-clinical rodent models have been used to study the
underlying pathophysiological mechanisms and to test
novel therapeutic strategies for PAH. A proper model
should be reproducible, inexpensive, and faithfully repro-
duce (in a defined period) the basic features of PAH such
as complex destructive neointimal lesions and right ventri-
cle (RV) dysfunction and failure. To date, there is no model
that recapitulates all of the pathophysiological mechanisms
and the clinical course of human PAH. For instance, in
the chronic hypoxic exposure or monocrotaline (MCT)-
induced rat models, pulmonary hypertension rarely devel-
ops with the same severity observed in humans perhaps due
to the absence of obstructive intimal lesions in the periph-
eral pulmonary arteries [65, 66]. Furthermore, the chronic
hypoxia model does not lead to RV failure, while MCT
injection causes myocarditis affecting both ventricles and
causing liver and kidney damage [67]. These limitations
may explain why it is difficult to translate the reversal of
PAH in animal models by several experimental compounds
into therapies for PAH patients.
In recent years, second-generation animal models have
been established based on the combination of multiple
triggers: MCT plus pneumonectomy, MCT plus chronic
hypoxia, and SU5416 plus chronic hypoxia. To circumvent
the problem of the embryonic lethality of BMPR2 knock-
out mice, switchable rodent models have been developed,
by means of BMPR2 conditional knock-out, whereby the
mutation can be activated after birth [68–70]. Moreo-
ver, genetic rodent models have been developed includ-
ing overexpression of interleukin-6. These new models
closely mimic the features and the severity of human PAH
although not completely [71]).
Restoring BMPRII signaling asatherapeutic
strategy
While hereditary PAH have been linked to heterozygous
mutations in the BMPR2 gene, non-genetic forms of PAH
show a reduction in BMPRII levels and activity [9]. Con-
sistent with this, heterozygous BMPR2 deletion in PAECs
and pulmonary artery smooth muscle cells (PASMCs)
mimics the PAH phenotype [69, 72]. Furthermore, mice
expressing a dominant-negative BMPRII (lacking an intra-
cellular domain) in vascular SMC, develop vascular lesions
in the lungs [68, 72].
There is strong evidence suggesting that BMPRII signal-
ing has a protective role in the vascular wall by promoting
the survival of PAECs, inhibiting PASMCs proliferation
and triggering anti-inflammatory responses [17, 73, 74].
Based on this, modulation of BMPRII signaling is consid-
ered a promising therapeutic approach for PAH. Impor-
tantly, the rescue of BMPRII expression may not exclu-
sively benefit PAH patients but also patients suffering from
pancreatic and lung fibrosis where BMPRII deficiency has
been implicated [43, 44]. BMPRII restoration can be tar-
geted at different levels: genetic-based therapies, transcrip-
tional and translational regulation, protein activity, and
processing as well as SMAD downstream signaling modu-
lation [27, 75, 76] (Fig.3).
Genetic‑based therapies
Exogenous BMPR2 gene delivery
One strategy to treat PAH patients is to rescue BMPRII
expression through gene therapy targeting ECs. In pre-
clinical models, this was explored by Reynolds etal., who
administrated a vector inducing BMPRII expression via
tail-vein injection. The BMPR2 encoding virus targets the
pulmonary endothelium by binding to the highly expressed
pulmonary endothelial angiotensin-converting enzyme
(ACE) using a bi-specific conjugate antibody. This BMPR2
adenoviral vector restored BMPRII protein levels in human
microvascular PAECs and attenuated the PAH phenotype
in a chronic hypoxia model and MCT-treated rats [77,
78]. Furthermore, BMPRII overexpression in lung tissue
was shown to reverse the imbalance between BMPRII and
TGFβ signaling thus restoring normal levels of pSMAD
1/5/8 and the activation of PI3K and p38 MAP kinase [79].
In contrast, BMPRII administration via an aerosol route
targeting PASMCs did not improve the PAH phenotype
when tested in the MCT model [80]. The later result high-
lights the importance of BMPRII signaling in ECs but not
SMCs. However, further investigations will be required to
elucidate precisely how spatio-temporal control BMPRII
overexpression might provide therapeutic benefit in the
context of BMPR2 mutations. It should be noted that ade-
noviral vectors are only capable of transient gene expres-
sion since the delivered gene is not integrated into the host
chromosome. Stable integration can be achieved and lenti-
viral vectors are potentially an attractive vehicle to deliver
longer term transgene expression since they integrate into
the genome and they can infect non-proliferating cells,
when compared to retroviral vectors. An important poten-
tial limitation of this approach is that integrating vectors
may generate gene mutations upon insertion and newer
advances regarding self-inactivating vectors are needed
[81, 82]. Adeno-associated virus and helper dependent ade-
noviral vectors (the latest generation of recombinant adeno-
viral vectors) are a promising alternative since they deliver
longer durations of transgene expression when compared
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2984 M.Orriols et al.
1 3
to the first-generation vectors. Moreover, they show nei-
ther long-term adverse effects in liver nor an immunologi-
cal response [83, 84]. Taken together, exogenous BMPR2
delivery is a possible therapy for PAH, but further improve-
ments in vector technology are required to translate this
approach to the clinic for the treatment of pulmonary vas-
cular disease.
Transcriptional regulation
miRNA targeting BMPRII
In recent years, there has been an increasing interest in the
role of epigenetics in the development of PAH [85, 86].
Epigenetics refers to heritable changes in gene expres-
sion that do not involve alterations in the DNA sequence.
miRNAs are small non-coding RNAs that negatively, post-
transcriptionally regulate the expression of target genes by
interfering with both the stability of the target transcript as
well as its translation. miRNAs have emerged as essential
players in the development (and diseases) of the cardiovas-
cular system. They also play an important role in vascular
remodeling [87, 88]. miRNAs are expressed in the vascu-
lature and are essential for the regulation of vessel func-
tion. Many miRNAs control proliferation, differentiation,
and apoptosis of ECs and SMCs by targeting components
of the TGF-β/BMP signaling pathways. Several miRNAs,
such as miR-145, miR-21 and the miR17/92 cluster, have
been associated with the disrupted BMPRII pathway in
PAH and can explain the incomplete penetrance of BMPR2
mutations [89–91]). Figure4 and Table1 provides an over-
view of currently described miRNAs that target BMPR2
Fig. 3 Rescuing the BMPRII
signaling pathway in pulmonary
arterial hypertension. Modula-
tion of BMPRII signaling is
considered a promising thera-
peutic approach for PAH. This
could be achieved by different
methods aiming to increase the
amounts of BMPRII present
in the cell or to trigger BMP
signaling. These approaches
include exogenous BMPRII
delivery, inhibition of miRNAs
negatively regulating BMPRII
stability and translation, inhibi-
tion of lysosomal degradation,
and delivery of exogenous BMP
ligands or BMP coactivators
among others
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2985BMP type II receptor asatherapeutic target inpulmonary arterial hypertension
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expression in vascular cells. In addition, Table 1 shows a
list of other miRNAs that are predicted to target BMPR2 in
silico.
Currently, there are different technologies to inhibit
aberrantly overexpressed miRNAs including the use of
antisense oligonucleotides, masking, sponges, erasers,
or decoys [92, 93]. In addition, administration of miRNA
mimics can enhance the expression of downregulated miR-
NAs [94]. These strategies are still under development
and more research is needed to establish how modulation
of miRNA function could offer therapeutic benefits in a
clinical setting while avoiding off-target effects, especially
in the liver, where systemically administrated miRNAs or
modulating compounds preferentially accumulate [95].
The delivery routes mostly used to target lung dis-
ease are local intranasal and intra-tracheal administration.
These naked miRNAs are directly delivered into the lung
with minimal systemic side effects [96]. Nevertheless, this
method remains ineffective and challenging due to the
complexity of the lung [97]. Recent advances in delivery
strategies, such as the use of liposomes, nanoparticles, or
virus, combined with improvements in chemically modi-
fying miRNAs, represent promising strategies to improve
lung miRNA delivery [98]. More than 20 miRNAs are cur-
rently in clinical trials, several in Phase III stage, highlight-
ing the potential of miRNA therapeutics to restore BMPRII
in PAH [99]. To date, the potential for miRNAs as a thera-
peutic tool is relatively limited. Further studies focusing on
the specificity, safety, efficiency, and stable systemic deliv-
ery of miRNAs into target cells or tissues will improve the
process of translating these findings to the clinic.
Translational regulation
Read-through premature STOP codons
Most BMPR2 mutations (~70%) are non-sense muta-
tions (frame-shift deletions and insertions) generated
by the insertion of a premature termination codon (PTC)
resulting in truncated reading frames which produce non-
functional proteins [7]. To prevent formation of truncated
proteins, mutated transcripts are directly degraded through
non-sense mediated decay (NMD) resulting in insufficient
amounts of the functional protein, which is produced only
by the wild-type allele (haplo-insufficiency) [100, 101].
NMD usually does not completely reduce the levels of
mutated transcripts and as a result truncated proteins per-
sist, and may exert a DN effect [102].
An approach aimed to correct these types of mutations
consists on the induction of PTC read-through. Read-
through of truncated mutations by aminoglycoside anti-
biotics, such as Gentamicin, has been extensively studied
and has reached the clinical trial stage for genetic disorders
such as cystic fibrosis [103] and Duchenne muscular dys-
trophy [104–110]. Aminoglycoside antibiotics bind to the
decoding site of ribosomal RNA and eliminates the PTC
Fig. 4 miRNAs targeting
BMPRII in the vascular wall.
The illustration shows hypoxia
and BMPRII mutations as regu-
lators of miRNAs expression in
endothelial or smooth muscle
cells. These miRNAs negatively
regulate BMPRII expression
resulting in increased cell
proliferation and impaired
apoptosis. Green arrows
indicate activation, red arrows
represent inhibition, and black
arrows correspond to unknown
regulation. EC endothelial cells,
IL interleukin, miR micro RNA,
mut mutant, SMC smooth mus-
cle cell, STAT signal transducer,
and activator of transcription
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2986 M.Orriols et al.
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by incorporating an amino acid to generate full-length pro-
teins [111]. Importantly, this read-through function of ami-
noglycosides does not affect normal translation because
of the presence of upstream and downstream regulatory
sequences around a normal termination codon that ensure
optimal efficiency of termination [112]. Gentamicin treat-
ment has been tested in lymphocytes derived from two
PAH patients with PTC mutations [113, 114]. The results
demonstrated increased amounts of full-length BMPRII
protein, a reduction in the mutated BMPRII product and
enhanced BMPRII downstream signaling. Although amino-
glycosides are commonly used in the clinic to treat infec-
tions and are safe when administered directly to the lung by
inhalation, several side effects of long-term treatments and/
or high concentrations of the drug have been shown.
Recently, a high-throughput screening for compounds
that suppress non-sense mutations identified a new small
molecule named Ataluren (PTC124) which mediates
read-through of premature stop codons without acute
side effects [115]. Aldred et al. have demonstrated that
after Ataluren treatment, BMPRII protein levels were
normalized and BMP-dependent phosphorylation of the
downstream target R-SMADs was increased in PAECs
and PASMCs from PAH patients. In addition, the hyper-
proliferative phenotype of these cells was reversed even
in the presence of significant non-sense mediated mRNA
decay. Although, further studies, including animal mod-
els, are required to explore the relevance of Ataluren
in vivo in a PAH context, the compound has emerged
as a promising therapeutic strategy for a subset of PAH
patients.
Table 1 miRNA targeting BMPRII expression
MicroRNA Cell type Function Model Expression References
miR-17/92 EC Interleukin-6 modulates the expres-
sion of the BMPRII through a novel
STAT3–microRNA Cluster 17/92
pathway
PAEC ∃ Brock etal. [13]
SMC Inhibition of miR-17 enhances
BMPRII expression and improves
heart and lung function in experi-
mental PH
PASMC Hypoxia-induced PH mice
MCT-induced PH rats
? Pullamsetti etal. [164]
miR-20A SMC Treatment with antagomiR-20a
restores functional levels of BMPRII
in pulmonary arteries and prevents
the development of vascular remod-
eling
PASMC Hypoxia-induced PH mice ? Brock etal. [165]
miR-21 EC Hypoxia and BMPRII signaling
independently upregulate miR-21. In
a reciprocal feedback loop, miR-21
downregulates BMP receptor type II
expression
PAEC Several rodent models of PH
miR-21-null mice
# Parikh etal. [166]
SMC BMPRII was downregulated in
PASMCs overexpressing miR-21
PASMC Hypoxia-induced PH mice # Yang etal. [167]
miR-125 EC Inhibition of miR-125a resulted in
upregulated BMPRII expression
accompanied by increased prolifera-
tion of EC
PAEC Hypoxia-induced PH mice
Plasma PAH patients
# Huber etal. [168]
miR-143/145 SMC miR-145 expression is increased in
primary PASMCs cultured from
patients with BMPRII mutations and
in the lungs of BMPRII-deficient
mice
PASMC Hypoxia-induced PH mice
BMPRII R899X knock-In Mice
miR-145 knock-Out Mice Lugn tis-
sue PAH patients
# Caruso etal. 2012 [169]
miR-302 SMC Inhibition of miR-302 by BMP4
increases BMPRII expression
and facilitates the BMP signaling
pathway
PASMC ? Kang etal. 2012 [170]
miR-181c cardiac Increased miR-181c expression
in human cardiac samples from
individuals with ventricular septal
defects (VSD) was correlated with
downregulated BMPRII levels
Human VSD cardiac samples # Li etal. [171]
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2987BMP type II receptor asatherapeutic target inpulmonary arterial hypertension
1 3
Protein processing regulation
Rescue ofBMPRII trafficking
30% of BMPR2 mutations are missense mutations leading
to single amino acid substitutions in a conserved domain
affecting the overall function of the protein [7]. Mutations
resulting in the substitution of cysteine residues in the
ligand binding and kinase domains disrupt protein folding
and trafficking of BMPRII to the cell surface leading to
retention of the mutant receptor in the endoplasmic reticu-
lum (ER) [113, 116]. A potentially promising therapeutic
strategy to increase the expression of BMPRII at the plasma
membrane is to enhance the activity of chaperones which
facilitate protein folding and trafficking. This can be done
by means of chemical chaperones such as sodium phenyl-
butyrate (4-PBA), probenecid, and tauroursodeoxycholic
acid (TUDCA). These have been shown to improve protein
trafficking via several distinct mechanisms [117–126]. Dif-
ferent groups have demonstrated that treatment with chemi-
cal chaperones can partially restore cell surface expression
of BMPRII in ECs. As a result, BMP-induced SMAD 1/5/8
phosphorylation and the expression of the target gene ID1
is restored [127–129]. These agents are showing promise
in clinical trials for other diseases caused by misfolded
proteins, such as cystic fibrosis. Since the currently used
chemical chaperones are federal drug administration (FDA)
approved drugs, there is an immediate translational poten-
tial to treat PAH patients [118, 130–133]. However, fur-
ther invivo studies are required to test the viability of this
approach.
Even though chemical chaperones have the potential to
rescue the BMPRII mutants which are retained in the ER, it
remains to be investigated whether the amount of BMPRII
reaching the plasma membrane is enough to induce a clini-
cally relevant effect. Also, BMPRII with a protein-folding
defect expressed at the cell surface may have a dominant-
negative activity and adverse effects on BMP signaling
[127]. Moreover, patients harboring missense mutations
that affect the activity of the receptor (kinase domain) may
not benefit from this therapeutic strategy. Further research,
taking this mutation variability into account, is required to
determine which patients might benefit from this approach.
Inhibition oflysosomal degradation
The deciphering of mechanisms which regulate cell sur-
face expression levels of BMPRII are of potential clini-
cal importance, particularly those mechanisms that pre-
vent its rapid turnover and thereby restore downstream
BMPRII signaling and function. In this context, several
studies focused on the potential of targeting the degra-
dation of BMPRII by preventing lysosomal degradation
[134]. Durrington et al. have demonstrated that after
Kaposi sarcoma-associated herpesvirus infection,
BMPRII is ubiquitinated by K5 (membrane-associated
RING E3 viral ubiquitin protein ligase) leading to lyso-
somal degradation in primary cultured pulmonary vascu-
lar cells [134]. In addition, cells treated with the lysoso-
mal inhibitor concanamycin A exhibit increase levels of
BMPRII. Furthermore, through siRNA screening of the
NEDD4-like family E3 ubiquitin protein ligases, it was
found that knockdown of ITCH expression resulted in
increased BMPRII protein levels [134]. Whether ITCH
ubiquitinates BMPRII, leading to lysosomal degrada-
tion, has yet to be investigated. Satow etal. have dem-
onstrated that BMPRII is degraded via the proteosomal
pathway in HEK 293T cells, when it is associated with
Dullard phosphatase [135]. This might suggest that more
than one mechanism accounts for BMPRII proteasome-
mediated degradation. It is noteworthy that Satow etal.
used a BMPRII overexpression system, whereas Dur-
rington et al. studied the degradation of endogenous
BMPRII [134]. Different membrane trafficking pathways
such as endocytosis, phagocytosis, micropinocytosis, and
autophagy, use lysosomes for the digestion of diverse
macromolecules [136]. Caveolae-mediated endocytosis
affects multiple cellular signaling pathways by the redis-
tribution of transmembrane receptors and receptor-ligand
complexes [137–139]. BMPRII localization has been
found to be regulated by CAV1 in vascular SMC [137].
Recently, ELAFIN (endogenous serine protease inhibitor)
treatment has been shown to prevent and reverse PAH in
the SU-hypoxia rat model. This occurs via elastase inhi-
bition and by promoting the interaction of BMPRII with
CAV1. Interestingly, when ELAFIN was combined with
BMP9, there was enhanced co-localization of CAV1 and
BMPRII on PAEC surfaces, which led at an increase in
BMP9-dependent SMAD1/5 phosphorylation and induc-
tion of ID1 [137]. Furthermore, transgenic mice overex-
pressing human ELAVIN in the cardiovascular system
(by placing ELAVIN expression under the control of the
pre-proendothelin-1 promoter), exhibited reduced SMC
proliferation and medial/intimal thickening after carotid
artery wire injury [140] and were protected from hypoxic
pulmonary hypertension [141]. In agreement with this,
peptidyl trifluoromethylketone serine elastase inhibitors
such as M249314 or ZD0892, have been used to prevent
and reverse PAH in the MCT rat model [142]. However,
the clinical use of these compounds was not pursued due
to hepatotoxicity. ELAFIN has been shown to inhibit
myocardial ischaemia-reperfusion injury induced during
coronary artery bypass graft surgery [143]. Even though
ELAFIN infusion was safe and resulted in >50% inhi-
bition of elastase activity in the first 24 h, myocardial
injury was not reduced after 48h. Based on the biology
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2988 M.Orriols et al.
1 3
of ischemia–reperfusion injury and PAH, we believe it
is worth testing whether ELAFIN together with BMP9
could reverses PAH in patients.
Interestingly, autophagy has also been found to be
involved in PAH. Autophagy (literally, “self-eating” in
Greek) is a highly regulated catabolic process that involves
sequestration and lysosomal degradation of cytosolic
components such as dysfunctional organelles, misfolded
proteins, lipid droplets, and invading pathogens [144].
Autophagy can be considered to be a general housekeep-
ing mechanism maintaining the integrity of intracellular
organelles and proteins. It is also triggered during devel-
opment, differentiation, infection, and stress conditions.
Thus, autophagy can be activated in the presence of dam-
aged organelles, protein aggregates, intracellular patho-
gens, hypoxia, amino acid starvation, reactive oxygen spe-
cies, and DNA damage [145]. Long etal. have shown that
in rats suffering from PAH induced by MCT treatment,
there is increased autophagy together with a decrease of
BMPRII protein expression [146]. Moreover, inhibition of
autophagic degradation by the lysosomal inhibitors chloro-
quine and hydroxychloroquine [147] prevents the develop-
ment of PAH as well as its progression. The authors dem-
onstrated that chloroquine and ATG5 (an autophagy protein
involved in the elongation and closure of the autophago-
somal membrane) knockdown inhibited proliferation and
increased apoptosis of PASMCs and these effects corre-
lated with increased levels of BMPRII via lysosomal inhi-
bition. Although autophagy seems to be involved in the
degradation of BMPRII, the exact mechanism by which
this takes place has yet to be elucidated. Chloroquine and
hydroxychloroquine have been widely utilized in malaria
prophylaxis [148]. They have also been used to treat rheu-
matoid arthritis and lupus erythematosus (as anti-inflam-
matory agents) [148]. Since inflammation is thought to be
a crucial second hit in PAH [149], these drugs might be
effective at inhibiting PAH progression by impairing the
degradation of BMPRII as well as inhibiting the inflam-
matory response. However, it is important to keep in mind
that since lysosomal degradation is a ubiquitous cellular
mechanism for regulating protein processing, this approach
can lead to widespread and non-specific off-target effects
independent of BMPRII signaling. Therefore, an improved
understanding of the molecular mechanisms underlying
BMPRII turnover is required for the development of more
directed interventions.
BMPRII signaling regulation
Delivery ofexogenous BMP ligand
As mentioned previously, BMP signaling in the vascular
endothelium is mainly activated by BMP2, 4, 6, 9, and 10
[32]. In particular, BMP9 and BMP10 appear to play an
important role in the vasculature due to their presence in
the circulation and based on the fact that they are known
to signal through receptors expressed on the endothelium,
such as ALK1 and BMPRII or ACVRIIB. Therefore, the
stimulation of BMP signaling with exogenous recombi-
nant ligand is an interesting approach for PAH treatment
[11, 150]. Long et al. have shown that BMP9 prevents
apoptosis and enhances the integrity of ECs in PAECs and
blood outgrowth ECs from PAH patients. Furthermore,
therapeutic BMP9 delivery prevents and reverses PAH in
several mouse models [70]. BMP10 is the least studied
BMP ligand; however, it may present a better treatment
than BMP9 since it binds to ALK1 and BMPRII with
higher affinity and because of its lack of osteogenic activ-
ity in vitro [151]. Further studies have to be performed
to evaluate the delivery strategies, efficiency, and poten-
tial side effects of BMP9 and BMP10 invivo. Finally, the
development of a small peptide mimetics of BMP9 or
BMP10, with an increased affinity for the receptor, is a the-
oretical alternative for efficiently activating BMP signaling
and thereby reversing PAH [150].
Enhance downstream SMAD signaling
An additional approach to reverse the effect of mutant
BMPRII is use small molecules to enhance signaling of
the wild-type functional proteins. Sildenafil is a phospho-
diesterase type-5 (PDE5) inhibitor currently used in the
clinic for PAH treatment [152–154]. Its mode of action is to
block the degradation of cyclic guanosine monophosphate
(cGMP) resulting in corrective vasodilatory and anti-pro-
liferative effects in the arterial wall [155]. Furthermore, it
has been described that protein kinase G (PKG) activated
by cGMP, is a modulator of BMP signaling [156] and that
PASMCs expressing a BMPRII mutant, showed an increase
in BMP signaling after Sildenadil treatment via a cGMP/
PKG-dependent mechanism. In addition, in vivo stud-
ies confirmed that Sildenafil treatment enhanced BMP
signaling and partially reversed PAH development in the
MCT rat model [157, 158]. Although Sildenafil therapy
during 12 weeks improves multiple clinical symptoms
in PAH patients, it appears to have no effect on reducing
either mortality or serious adverse events [159]. Further-
more, the long-term efficiency and safety of Sildenafil
therapy in PAH requires further studies based on large and
well-designed clinical trials [159].
Another promising strategy is to identify compounds in
drug libraries that activate BMP/SMAD signaling. FK506
(Tacrolimus) was identified as the best BMP coactiva-
tor among 3756 FDA-approved drugs and bioactive com-
pounds (using a high-throughput BMP/SMAD-driven tran-
scriptional reporter assay) [160]. FK506 promotes BMP
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2989BMP type II receptor asatherapeutic target inpulmonary arterial hypertension
1 3
signaling and endothelial-specific gene regulation of genes
such as APELIN. This occurs even in the absence of exog-
enous ligand via a dual mechanism of action: acting as an
inhibitor of phosphatase CALCINEURIN and binding FK-
binding protein-12 (FKBP12), a repressor of BMP signal-
ing. FK506 promotes the release of FKBP12 from the type
I receptor which leads to activation of SMAD1/5 down-
stream of BMP as well as MAPK signaling and ID1 gene
regulation [161]. Furthermore, FK506 treatment increases
ALK1 and ENDOGLIN expression in ECs [162]. Recently,
a randomized, double-blind, placebo-controlled phase IIa
trial was performed to investigate the efficacy of FK506
treatment in three patients with end-stage PAH. The results
suggest a potential clinical benefit of low-dose FK506 (the
evidence being that patients demonstrated cardiac function
stabilization and required less intensive hospital care for
RV failure despite the severity of the illness). It was also
found that changes in serologic biomarkers indicated that
BMPRII had been successfully targeted [163]. However,
these results are based on a limited group of patients and
the efficacy of this therapy must be validated in appropri-
ate, well-designed clinical trials. FK506 (also known as
Tacrolimus) is an immunosuppressive drug with a known
pharmacokinetic and toxicity profile. It is widely used in
solid organ transplantations to lower the risk of organ rejec-
tion [164]. High doses of FK506 caused systemic hyper-
tension and transplant vasculopathy in animal models
[165]. Also, organ transplant patients treated with FK506,
have an increased risk of renal injury, which might occur
due to the inhibition of calcineurin expression in the kid-
ney [166–168]. In contrast, low doses of FK506 did not
induce systemic hypertension in animal models, even after
3 weeks of treatment. FK506 has shown significant clinical
benefits, nonetheless long-term use of this agent for treat-
ing PAH still needs to be rigorously monitored for toxicity
effects.
Conclusions andperspectives
Exogenous BMPRII delivery to ECs has been shown to
be an effective means to restore BMPRII expression and
function [77, 78]. An interesting approach, which is yield-
ing promising results in mice, is to deliver BMPRII spe-
cifically to ECs using BMPRII adenoviral vectors carry-
ing a bi-specific conjugate antibody that targets the virus
to ACE, a membrane-bound protease highly expressed on
pulmonary endothelial cells [77, 78]. One of the draw-
backs of this strategy is the use of two components namely,
adenovirus and antibody. Additional restrictions related
to the use of viral transduction such as safety, specificity,
and delivery of sufficient protein to revert the phenotype
must also be taken into consideration. The utilization of
CRISPR/Cas9 may overcome some of these limitations, for
instance by minimizing the risk that the foreign gene will
be integrated in the wrong place in the genome. Further-
more, it will place the gene under the control of its natu-
ral promoter. However, the delivery of CRISPR/Cas9 into
the patient is still challenging and the Cas9 enzyme could
cleave at unwanted locations. Similarly, the use of miRNAs
targeting BMPRII has to be evaluated for off-target effects
and an effective delivery system has to be found in order
to consider this approach as a promising treatment. A solu-
tion for both plasmid DNA and miRNA delivery might be
the use of liposomes [169] or iTOP (induced transduction
by osmocytosis and propanebetaine), which is an active
uptake mechanism in which NaCl-mediated hyperosmo-
larity together with propanebetaine triggers the uptake of
macromolecules [170]. Another therapeutic strategy is the
use of FDA-approved drugs that have been found to be ben-
eficial in PAH mice models or similar diseases. Ataluren,
for example, allows the cellular machinery to read-through
premature stop codons [115]. Although most of the BMPR2
mutations (~70%) are non-sense mutations, not all patients
will benefit from this approach. Nevertheless, further
invivo studies are worth pursuing in the context of PAH.
Likewise, clinical trials using chloroquine have to be per-
formed to test its effectiveness in PAH patients. The use of
chloroquine has to be carefully evaluated because blocking
lysosomal degradation might trigger non-specific off-target
effects when used as a long-term treatment. An alterna-
tive drug showing significant clinical benefits for PAH is
FK506/Tacrolimus. However, it still needs to be monitored
for side effects since it is an immunosuppressive drug (cur-
rently utilized after allogeneic organ transplant). Moreover,
the effectiveness of FK506 at low doses has to be rigor-
ously tested.
It is important to highlight that although several drugs
showed beneficial outcomes in animal models, most of the
drugs have failed in the clinic. In light of this, we should
focus on a more personalized approach which takes into
account the co-existence of modifier genes, infections,
toxic exposure, inflammation, or alterations in estrogen
metabolism. Combining treatments which target not only
BMPRII signaling but also inflammation and hypoxia
should improve outcomes. Lastly, the use of human exvivo
models such as lung or vessel on a chip [171] could be ben-
eficial for drug discovery and efficacy testing in the context
of PAH. We anticipate that such models may improve the
relevance of pre-clinical results by using patient derived
cells, especially since animal models of PAH are frequently
difficult to translate into clinical practice.
Taken together, previously discussed data suggest that
modulation of BMPRII signaling in PAH is a promising
alternative that could prevent and reverse pulmonary vascu-
lar remodeling. However, different therapeutic approaches
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2990 M.Orriols et al.
1 3
aimed at to increasing the levels of BMPRII signaling are
needed, and these approaches will depend on the particular
genetic background of each patient. In addition, for more
efficient treatments, targeting other genetic and environ-
mental factors that contribute to the disease must be taken
into consideration. In this regard, modulators of the inflam-
matory response and estrogen metabolism could be used to
help restore BMPRII signaling.
Acknowledgements We acknowledge the support from the Neth-
erlands CardioVascular Research Initiative: the Dutch Heart Foun-
dation, Dutch Federation of University Medical Centers, the Neth-
erlands Organisation for Health Research and Development, and the
Royal Netherlands Academy of Sciences.
Open Access This article is distributed under the terms of the
Creative Commons Attribution 4.0 International License (http://
creativecommons.org/licenses/by/4.0/), which permits unrestricted
use, distribution, and reproduction in any medium, provided you give
appropriate credit to the original author(s) and the source, provide a
link to the Creative Commons license, and indicate if changes were
made.
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