Bone Morphogenetic Proteins Protect Pulmonary
Microvascular Endothelial Cells From Apoptosis by
Upregulating α-B-Crystallin
Mariana Ciumas,* Mélanie Eyries,* Odette Poirier,* Svetlana Maugenre, France Dierick,
Natalia Gambaryan, Kevin Montagne, Sophie Nadaud, Florent Soubrier
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Objective—To investigate the role of bone morphogenetic proteins (BMPs) on α-B-crystallin (CRYAB) expression and its
physiological consequences on endothelial cells (ECs).
Approach and Results—We report that the gene encoding for the small heat shock protein, CRYAB, is a transcriptional
target of the BMP signaling pathway. We demonstrate that CRYAB expression is upregulated strongly by BMPs in an
EC line and in human lung microvascular ECs and human umbilical vein ECs. We show that BMP signals through the
BMPR2-ALK1 pathway to upregulate CRYAB expression through a transcriptional indirect mechanism involving Id1. We
observed that the known antiapoptotic effect of the BMPs is, in part, because of the upregulation of CRYAB expression in
EC. We also show that cryab is downregulated in vivo, in a mouse model of pulmonary arterial hypertension induced by
chronic hypoxia where the BMP pathway is downregulated.
Conclusions—We demonstrate a cross-talk between BMPs and CRYAB and a major effect of this regulatory interaction on
resistance to apoptosis. (Arterioscler Thromb Vasc Biol. 2013;33:2577-2584.)
Key Words: ACVRL1 protein, human ◼ anoxia ◼ BMPR2 protein, human ◼ Id1 protein, human
◼ RNA, small interfering ◼ Smad proteins
B
one morphogenetic proteins (BMPs) belong to the transforming growth factor-β superfamily of extracellular
signaling proteins and play crucial roles in embryonic development, adult tissue homeostasis, and in the pathogenesis of
a variety of diseases.1 The highly conserved BMP signaling
pathway comprises the BMP ligands, 2 types of receptors
(type I: ALK1, 2, 3, and 6 and type II: BMPR2, ACTR2A, and
ACTR2B), and signal transducers, the Smad proteins. Once
activated, the receptor complex phosphorylates the carboxy
terminus of the receptor-regulated Smad proteins, Smad1, 5,
and 8. Activated receptor-regulated Smad proteins interact
with the common partner Smad, Smad4, and translocate to the
nucleus, where the Smad complex directly binds defined DNA
sequences and regulates target gene expression.2
BMPs were reported to promote survival of human pulmonary arterial endothelial cells (ECs) and circulating endothelial progenitor cells isolated from normal subjects. This
antiapoptotic effect induced by the BMP signaling pathway
on vascular EC is, in part, because of the decrease in caspase-3 activity.3
CRYAB is a soluble cytoplasmic protein and a member
of the small heat shock protein (sHSP) family. CRYAB was
identified initially as an abundant structural eye lens protein
but seemed later to be expressed in the heart, skeletal muscle, central nervous system, kidney, and lung of mice.4 It is
induced by heat shock, proinflammatory cytokines, and oxidative stress and plays a role in cellular growth, migration, differentiation, and development.5–7 CRYAB has chaperone-like
properties, binds to both desmin and cytoplasmic actin, and
helps to maintain cytoskeletal integrity.8 As in the case of other
chaperones and sHSPs, CRYAB can bind to unfolded proteins
and can prevent their denaturation and aggregation.9 CRYAB
has been shown to protect cells against apoptosis induced by
different factors, such as DNA-damaging agents, tumor necrosis factor-α, tumor-necrosis-factor related apoptosis inducing
ligand, hydrogen peroxide, etc.10–12 CRYAB is known to protect
vascular EC from apoptosis, in part, by binding to caspase-3
and blocking its autoproteolytic maturation.10,13,14 CRYAB is
also considered as an oncogenic protein, by protecting cancer
cells against apoptosis induced by chemotherapeutic agents,
Received on: May 11, 2011; final version accepted on: September 5, 2013.
From the UMR_S 956; Univ Paris 06 (UPMC); Institut National de la Santé et de la Recherche Médicale (INSERM), F-75013, Paris (M.C., M.E., O.P.,
S.M., F.D., K.M., S.N., F.S.); ICAN Institute for Cardiometabolism and Nutrition, Paris, France (M.E., O.P., S.M., F.D., S.N., F.S.); and UMR_S 999;
INSERM; Univ Paris-Sud; LabEx LERMIT, Centre Chirurgical Marie Lannelongue, Le Plessis Robinson, France (N.G.).
*These authors contributed equally to this article.
The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi: 10.1161/ATVBAHA.113.301976/-/DC1.
Correspondence to Florent Soubrier, MD, PhD, INSERM UMR_S 956, University Pierre and Marie Curie, 91 Blvd de l’Hôpital, 75634 Paris Cedex 13,
France. E-mail [email protected]
© 2013 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org
2577
DOI: 10.1161/ATVBAHA.113.301976
2578 Arterioscler Thromb Vasc Biol November 2013
Nonstandard Abbreviations and Acronyms
BMP
EC
HLMEC
HMEC
HUVEC
sHSP
bone morphogenetic protein
endothelial cell
human lung microvascular EC
human microvascular EC
human umbilical vein EC
small heat shock protein
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by interacting with proapoptotic proteins, or inhibiting caspase-3 activation.10,12,15 In cardiomyocytes, it was shown that
CRYAB silencing promoted cell death after exposure to H2O2,
and that CRYAB has a protective role against apoptosis, by
a mechanism involving translocation of CRYAB into mitochondria and by interacting with different proteins, including
voltage-dependent anion channels and caspase-3.16
Here, we show that BMPs induce CRYAB expression by a
transcriptional, indirect molecular mechanism, which requires
the BMPR2 and ALK1 receptors and the transcription factor
Id1. BMPs protect EC from serum starvation and hypoxiainduced apoptosis and the protecting effect is, in part, because
of a strong upregulation of the antiapoptotic protein CRYAB.
cryab is downregulated in the lung of a mouse model of pulmonary hypertension induced by hypoxia, a condition in
which the BMP signaling pathway has decreased activity.
Materials and Methods
Materials and Methods are available in the online-only Supplement.
Results
BMPs Upregulate CRYAB in
Human Microvascular ECs
Microarray RNA profiling experiments were performed to
identify BMP target genes in human microvascular EC-1
(HMEC-1) stimulated for 7 or 24 hours by 10 ng/mL of
BMP4. Among the modulated genes, including ID1, ID2,
and ID3, which were markedly upregulated (data not shown),
we observed a strong upregulation of CRYAB expression by
BMP4 at both 7 and 24 hours of stimulation.
We confirmed, by real-time polymerase chain reaction
(PCR), the upregulation of CRYAB expression in HMEC-1
treated by 10 ng/mL of BMP4 for 24, 48, and 72 hours
(4.3±4.1, 8.6±0.7, and 7.5±1, respectively; Figure 1A).
CRYAB mRNA was also increased by other members of the
BMP growth factors family, BMP7 (fold increase at 4.8±0.9
at 24 hours, 4.8±1.8 at 48 hours, and 3.4±0.06 at 72 hours)
and BMP9 (fold increase at 86.5±6.3 at 24 hours, 25.1±0.8
at 48 hours, and 15.1±1.5 at 72 hours), similar to BMP4
(Figure 1A). In human lung microvascular ECs (HLMEC),
BMP7 and BMP9 increased CRYAB expression, whereas
BMP4 had no effect on CRYAB expression (Figure I in the
online-only Data Supplement). BMP9 also induced CRYAB
expression in human umbilical vein ECs (HUVEC; Figure
I in the online-only Data Supplement). By Western blot,
we observed that CRYAB levels were increased strongly in
HMEC-1 treated with BMP4, BMP7, and BMP9 compared
with control cells (Figure 1B).
CRYAB Induction by BMPs Is
Transcriptional Indirect
To characterize the molecular mechanism involved in the
BMP-induced CRYAB upregulation, we inhibited DNA
transcription or mRNA translation by pretreating cells with
actinomycin D or cycloheximide, respectively, before BMP
stimulation. When protein translation was stopped by cycloheximide, CRYAB expression was not upregulated in response
to BMP (Figure 2A), showing that this regulation depends
on the induction of the expression of a protein and is therefore indirect. Blocking mRNA transcription by actinomycin
D treatment also abolished the induction of CRYAB expression by BMP (Figure 2A), showing that this regulation
depends on mRNA transcription. However, this actinomycin
D experiment does not differentiate between CRYAB mRNA
transcription and the transcription of an intermediate protein
mRNA. To directly study the transcription of CRYAB mRNA,
we analyzed the transcriptional level of the CRYAB gene by
measuring its pre-mRNA expression by real-time PCR using
primers that hybridize to intronic sequences of the gene and,
therefore, specifically recognize pre-mRNA. After 5 hours of
A
B
Figure 1. Bone morphogenetic proteins (BMPs)
upregulate CRYAB in human microvascular endothelial cell-1 (HMEC-1). Serum-restricted HMEC-1
was stimulated with 10 ng/mL of BMP4, BMP7,
or BMP9 for 24, 48, or 72 hours. A, Expression of
CRYAB was quantified by real-time reverse transcription polymerase chain reaction analysis. The
relative CRYAB mRNA level was normalized to
RPL32 mRNA level and expressed as a fold change
relative to untreated cells. Results are mean±SD
values of 3 independent experiments. *P<0.05,
**P<0.01, and ***P<0.001 relative to untreated cells.
B, Immunoblotting for CRYAB. Blot was reprobed
for β-actin to control for differences in protein
loading.
Ciumas et al A
B
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Figure 2. CRYAB induction by bone morphogenetic proteins
(BMPs) is transcriptional indirect. A, Serum-restricted human
microvascular endothelial cell-1 (HMEC-1) was pretreated or
not with cycloheximide (CHX) or actinomycin D (ActD) for 15
minutes before being stimulated with BMP4, BMP7, or BMP9
(10 ng/mL) for 5 hours. CRYAB expression was quantified
by real-time reverse transcription polymerase chain reaction
(PCR) analysis. The relative CRYAB mRNA level was normalized to RPL32 levels and expressed as a fold change relative to
untreated cells. Results are mean±SD values of 4 independent
experiments. *P<0.05 relative to untreated cells. B, Serumrestricted HMEC-1 was treated with 10 ng/mL of BMP4 or
BMP7 for 5 hours. Nuclear RNAs were isolated and analyzed
by real-time PCR using primers that hybridize to intron 1 and
exon 2 allowing only the detection of the CRYAB pre-mRNA.
CRYAB pre-mRNA expression was normalized to GAPDH mRNA
expression and expressed as a fold change relative to untreated
cells. Results are mean±SD values of 3 independent experiments. *P<0.05 relative to untreated cells.
α-B-Crystallin Induction by BMPs 2579
siRNA value) compared with those transfected with nonspecific siRNA (Figure 3A). In contrast, inhibiting ALK3 by
siRNA did not significantly decrease CRYAB mRNA upregulation (Figure 3A). Altogether, these results demonstrate that
the upregulation of CRYAB expression by BMPs is mediated
by BMPR2 and ALK1, but not by ALK3. ALK6 was not tested
because it is not expressed in EC (data not shown).
The main intracellular signaling pathway activated by BMPs
is the Smad pathway. We observed an induction of Smad 1/5/8
phosphorylation after BMP treatment of HMEC-1 in a timedependent manner concomitantly with BMP-induced CRYAB
expression (Figure III in the online-only Data Supplement).
We tested the implication of the transcription factors Id1, Id2,
and Id3, which are strongly induced by BMPs. After silencing these mRNA targets using specific siRNAs (Figure II
in the online-only Data Supplement), we observed that ID1
expression knockdown reduces CRYAB mRNA induction in
HMEC-1 by 55±14%, whereas ID2 and ID3 knockdown did
not modify significantly this induction (Figure 3B). These
data corroborate the actinomycin D and cycloheximide experiments, which suggested the transcription and translation of
an inducer factor.
Role of CRYAB in the Antiapoptotic Effect
of BMPs on Serum-Starved HLMEC
BMP treatment, pre-mRNA levels were strongly induced by
BMP4 or BMP7 stimulation (6.5±2- and 6.1±1-fold respectively; Figure 2B). These data show that CRYAB is induced
at the transcriptional level by BMPs. Altogether, these results
indicate that CRYAB induction by BMPs is dependent on a
transcriptional indirect mechanism and does require de novo
synthesis of proteins.
To determine whether the upregulation of CRYAB mRNA by
BMPs plays a role in the antiapoptotic effect induced by BMPs
on serum-starved HLMEC, we inhibited CRYAB expression
using a specific siRNA targeting CRYAB mRNA (Figure II in
the online-only Data Supplement). In cells transfected with
a nonspecific siRNA, we observed an antiapoptotic effect of
BMP7 in serum-free conditions. This effect was abolished in
cells whose CRYAB expression is knocked down by a specific
siRNA (Figure 4A). This observation was confirmed by flow
cytometry because the percentage of annexin V–positive cells
is higher when CRYAB-targeting siRNA is transfected in cells
treated with BMP7 (6.9±2.2% in siCT-transfected cells versus 13.5±4.52% in siCRYAB-transfected cells) and BMP9
(7.2±2.4% in siCT-transfected cells versus 13.7±4.7% in
siCRYAB-transfected cells) compared with cells transfected
with a control siRNA (Figure 4B and 4C).
These results suggest that BMPs protect EC from apoptosis,
in part, by upregulating the antiapoptotic factor CRYAB.
CRYAB Upregulation by BMPs Is Mediated
by the BMPR2–ALK1–Id1 Pathway
Role of BMP and CRYAB in HypoxiaInduced Apoptosis in HUVEC
We knocked down the expression of BMPR2 using a
BMPR2-targeting siRNA (Figure II in the online-only Data
Supplement). CRYAB upregulation at the mRNA level by
BMPs was nearly abolished in HMEC-1 transfected with
BMPR2-directed siRNA compared with those transfected
with nonspecific siRNA (19.4±8.9% of control siRNA value)
(Figure 3A), demonstrating that the upregulation of CRYAB
expression by BMPs is mediated by BMPR2. We also investigated the involvement of type 1 receptors, ALK1 and ALK3,
using specific targeting siRNAs. BMP upregulation of CRYAB
mRNA was nearly completely abolished in HMEC-1 transfected with ALK1-directed siRNA (24±10.5% of control
To corroborate results obtained on HLMEC by serum-starvation–induced apoptosis, we used hypoxia as another inducer of
apoptosis in EC. HUVEC were cultured in hypoxic conditions
(1% O2). Apoptosis was measured by Hoechst 33342 staining
to quantify condensed pyknotic nuclei in apoptotic cells. In
hypoxic conditions, BMP9 induced a significant decrease of
apoptotic cells in the presence of a control siRNA (41±7%;
Figure 5). This antiapoptotic effect of BMP9 was no more
detectable in cells transfected with a siRNA targeted on the
CRYAB mRNA. Therefore, these results confirm in a different
model of apoptosis (hypoxia-induced apoptosis) that CRYAB
participates in the antiapoptotic effects of BMPs in EC.
2580 Arterioscler Thromb Vasc Biol November 2013
A
B
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Figure 3. CRYAB upregulation by bone morphogenetic proteins (BMPs) in human microvascular endothelial cell-1 (HMEC-1) is mediated
by BMPR2 via the SMAD pathway. HMEC-1 was transfected with siRNA. After transfection, cells were starved overnight and treated or
not with BMP9 (10 ng/mL) for 24 hours. Expression of CRYAB was quantified by real-time reverse transcription polymerase chain reaction analysis. The relative CRYAB mRNA level was normalized to RPL32 levels and expressed as a fold change relative to untreated cells
transfected with siCT or 2xsi CT. A, HMEC-1 was transfected with a nonspecific siRNA (control siRNA [siCT], 50 nmol/L or 2xsi CT, 100
nmol/L), or with siRNA specifically targeting alk1, alk3, or BMPR2 (siAlk1, siAlk3, or siBMPR2, respectively, 50 nmol/L), or alk1 and alk3
combined (siAlk1+siAlk3, 50 nmol/L each). Results are mean±SD values of 4 independent experiments. *P<0.05 relative untreated cells
transfected with siCT or 2xsi CT. B, HMEC-1 was transfected with a nonspecific siRNA (siCT), or with siRNA specifically targeting Id1,
Id2, or Id3 (siId1, siId2, or siId3). Results are mean±SD values of 4 independent experiments. ***P<0.001 relative to BMP9-treated cells
transfected with siCT.
Decreased BMP Activity Correlates With the
Downregulation of CRYAB Expression In Vivo
BMP signaling is known to be altered in pulmonary hypertension. Thus, we studied the BMP signaling pathway and
cryab expression in the mouse hypoxia model of pulmonary
arterial hypertension (PAH). Mice were exposed to chronic
hypoxia for 35 days leading to the development of pulmonary
hypertension as assessed by hemodynamic parameter measurements (Figure IV in the online-only Data Supplement).
In this model, a significant reduction of bmpr2 expression in
mouse lung was observed (Figure 6A). In these conditions,
we also observed a decrease of Smad 1/5/8 phosphorylation
compared with control conditions (Figure 6C and 6D). These
results confirmed that the BMP signaling pathway is downregulated in the mouse hypoxia model of PAH in our experimental conditions.
We observed a significant downregulation of the cryab
mRNA and protein in the lung of animals exposed to chronic
hypoxia compared with those exposed to normal oxygen conditions (Figure 6B, 6C, and 6D). These results show that cryab
expression is downregulated in vivo during chronic hypoxia,
and this downregulation correlates with the decreased BMP
signaling pathway activity.
We analyzed the expression of cryab in the mouse lung
by immunofluorescence staining. The expression of cryab
in EC of small vessels was shown by double staining with
antibodies against the EC-specific Von Willebrandt factor and
CRYAB (Figure 6E). We observed that cryab is expressed in
the mouse lung in various types of cells but at different levels. In the lung parenchyma, the highest levels of expression
are observed in EC, in smooth muscle cells (from vessels and
airways) and in bronchiolar epithelial cells (Figure 6E; Figure
VI in the online-only Data Supplement). We confirmed the
specificity of the CRYAB antibody by performing a parallel hybridization in similar conditions on mouse kidney. The
results show specific expression of cryab in kidney vessels in
EC and smooth muscle cells (Figure VII in the online-only
Data Supplement).
Discussion
We demonstrate in this study a cross-talk between the BMP
pathway and the sHSP family regulation, namely the strong
induction of the CRYAB gene transcription and expression by
BMPs. Experiments in vitro favor an indirect mechanism of
transcription induction by BMPs because a protein biosynthesis is required, as shown by cycloheximide experiments.
Therefore, a transcription factor upregulated by BMPs is likely
to be involved, and our results confirm this conclusion because
Id1 knockdown strongly, but not totally, inhibits the induction. The absence of total inhibition could be because of either
incomplete mRNA knockdown by siRNAs or the involvement
of other intermediate transcription factors. Knockdown experiments of intracellular intermediates of BMP signaling show
that BMPR2 and ALK1 are required for the induction.
We initially identified the CRYAB transcriptional upregulation by BMPs in an EC line (HMEC-1), and this result was
Ciumas et al A
α-B-Crystallin Induction by BMPs 2581
B
C
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Figure 4. Knockdown of CRYAB expression prevents the inhibition by bone morphogenetic protein (BMP) of serum-depletion–induced
endothelial cell apoptosis. Human lung microvascular endothelial cells were transfected with a nonspecific siRNA (siCT) or a CRYABspecific siRNA (siCRYAB). After transfection, cells were cultured in normal conditions (fetal calf serum [FCS]), in serum-free medium (SF),
and in serum-free medium with 200 ng/mL BMP7 or BMP9 for 24 hours. A, Apoptosis levels were assessed by colorimetric ELISA measuring histone-associated DNA fragments in the cytoplasmic fraction of cell lysates. Results are expressed as a fold change relative to
cells transfected with siCT and cultured in FCS. Results represent mean±SD of 3 independent experiments. *P<0.05 relative to untreated
cells cultured in SF medium transfected with siCT (B). C, Cells were labeled with annexin V-fluorescein isothyocyanate (FITC) and propidium iodide (PI), and apoptosis was assessed by flow cytometry. C, Representative scatter plots of PI (y axis) vs annexin V-FITC (x axis).
Absence of both markers (lower left quadrants) indicates viable cells; PI-positive alone (upper left quadrants) indicates cellular necrosis,
whereas annexin V staining alone or together with PI (upper right and lower right quadrants) is indicative of early and late-stage apoptosis, respectively. B, Graph showing the quantification of annexin V–positive cells. Results are expressed as the percentage of annexin
V–positive cells (PI+ and PI−). Results are mean±SD of 6 independent experiments. *P<0.05 vs serum-free siCT-transfected cells and
§P<0.05 siCT-transfected cells treated with BMP7 and BMP9 vs siCRYAB-transfected cells treated with BMP7 or BMP9.
extended to HLMEC, and HUVEC, 2 types of human primary
culture of EC from different origins. However, we found some
contrasted effects between BMPs in these cell types because
BMP4, which induces CRYAB in HMEC-1, is not efficient in
Figure 5. Knockdown of CRYAB expression prevents the inhibition by bone morphogenetic protein 9 (BMP9) of hypoxiainduced endothelial cell apoptosis. Human umbilical vein
endothelial cells were transfected with 50 nmol/L of siCT or
sicryab, and treated or not with BMP9 (10 ng/mL). After 24 hours,
cells were submitted to 24 hours of hypoxia (1% O2) followed by
labeling with Hoechst 33342. The nuclear chromatin morphological changes of apoptotic cells were analyzed, and the proportion
of apoptotic nuclei was counted by fluorescent microscopy.
Results are mean±SD of 4 independent experiments. **P<0.01
relative to cells transfected with siCT, but not treated with BMP9.
HLMEC, and BMP9 is clearly the most active inducer in all
cell types.
We evaluated the physiological effect of BMPs in EC taking into account the known physiological effect of CRYAB.
Indeed, CRYAB is a sHSP known to inhibit apoptosis through
inhibition of caspase-3 activation. Because BMPs are known
to inhibit apoptosis in EC,3 an effect which contrasts with the
documented proapoptotic effects of BMPs during embryogenesis17 and also in adult vascular smooth muscle cells,18,19 we
searched for a specific effect of BMPs on EC apoptosis through
CRYAB. We observed a strong apoptotic effect induced by
serum deprivation of HLMEC, and this effect was antagonized by BMP7 and BMP9 as attested by the lower percentage
of annexin V–positive cells measured by flow cytometry after
treatment with these peptides. This effect is dependent on the
induction of CRYAB expression as the knockdown of CRYAB
expression resulted in a nearly complete loss of the BMP antiapoptotic effect. We confirmed these results using hypoxia
as a stress for inducing apoptosis in HUVEC. In this model,
BMP9 also significantly decreased apoptosis, an effect suppressed by inhibiting CRYAB expression. To eliminate an indirect effect mediated by VEGF, we measured VEGF mRNA
induction by BMP9 in HMEC-1, and we did not observe any
induction (Figure V in the online-only Data Supplement).
2582 Arterioscler Thromb Vasc Biol November 2013
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B
C
D
*
β-
E
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Figure 6. CRYAB expression in mouse lung exposed to normoxia or hypoxia. Total RNAs were isolated from lungs of mice exposed to
normoxia or hypoxia (10% O2) for 35 days, and mRNA levels were quantified by real-time reverse transcription polymerase chain reaction
analysis. Results are mean±SD values of 5 animals per group. A, BMPR2 expression was normalized to RPL13a levels and expressed
as a fold change relative to normoxia. *P<0.05 relative to normoxic group. B, CRYAB expression was normalized to RPL13a levels and
expressed as a fold change relative to normoxia. *P<0.05 relative to normoxic group. C, Immunoblotting of pSmad1/5/8 protein extracted
from the lung of mice exposed to normoxia or hypoxia. All blots were reprobed with β-actin to correct for protein loading. D, Graph
showing densitometry of immunoblots of CRYAB and pSMAD1/5/8. Band intensities were normalized to the β-actin blots. Results are
represented as fold change relative to normoxia. Results are mean±SD values of 5 animals per group. *P<0.05 relative to normoxia.
E, Immunofluorescence analysis of (a) endothelial cells (visualized by Von Willebrandt factor [VWF], green); (b) CRYAB expression (red)
in normal mouse lung; (c) 4′,6-diamidino-2-phenylindole nucleus staining; and (d) merged images. Expression of CRYAB in endothelial
cells of small lung vessels is indicated by arrows. Scale bar, 20 µm.
We looked for a correlation between the activation status
of the BMP signaling pathway and bmpr2 expression in pulmonary tissues by investigating the expression and regulation of bmpr2 in the mouse hypoxia model of PAH. In this
model, we observed a downregulation of the BMP pathway, as
shown by the pSmad1/5/8 decrease after chronic exposure to
hypoxia, confirming previously published results.20–22 In this
experimental condition, bmpr2 is also decreased in the mouse
lung as measured by RNA expression and by Western blot.
The expression of CRYAB was detected in pulmonary EC, but
also localized in other cell types. Therefore, these results show
that, in vivo, a BMP signaling decrease is associated with a
decrease in bmpr2 expression, which is consistent with the
BMP-induced CRYAB regulation found in cultured HMEC-1,
HLMEC, and HUVEC.
A few hereditary vascular diseases result from decreased
BMP signaling and are linked to genetic mutations abrogating
a single allele function of a gene belonging to the BMP pathway.23 Hereditary hemorrhagic telangiectasia is mainly caused
by ALK1 (ACVRL1), Endoglin, and SMAD4 genes mutations,24 and heritable PAH is linked to BMPR2 mutations but
can also complicate ACVRL1-linked hereditary hemorrhagic
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telangiectasia.25 These 4 genes are receptors or coreceptors
for the BMP ligands or intermediate signaling molecules. The
cross-talk identified in our study between BMP and CRYAB
might be of importance for understanding the physiopathology
of these diseases because under stress conditions, a deficient
BMP signaling might result in increased endothelial apoptosis, at least in part because of decreased CRYAB expression. It
has been proposed that, after a stress to pulmonary EC in the
lung, the selection of EC resistant to apoptosis and subsequent
vascular smooth muscle cell proliferation could explain the
remodeling observed in PAH.26
The cross-talk between BMP and CRYAB could have
implication beyond resistance to apoptosis because CRYAB
has wide physiological functions as sHSP. Indeed, CRYAB is
known to be involved in resistance to oxidative stress induced
by high glucose concentration in EC and to prevent apoptosis
in mouse neonatal cardiomyocytes exposed to H2O2.14,16 BMP7
was reported recently to protect mesangial cells from oxidative stress, and it can be speculated that it might be through
CRYAB induction.27
The BMP-CRYAB cross-talk might also be of importance
for the abnormal angiogenesis observed when the BMP signaling pathway is decreased by an ACVRL1 or Endoglin
inactivating mutation such as in hereditary hemorrhagic
telangiectasia because angiogenesis is a balance between
vascular cell apoptosis and proliferation.28 BMP signaling
plays a major role on the balance between endothelial tip
cells, present at the tip of growing capillaries, and stalk cells,
which have a proliferative behavior, form tubes and vessel
branching.29 Cryab null mice present with decreased retinal
vessel density and subtle defects in vascular pattern morphogenesis.30 After experimental retinopathy, cryab null mice
show greater EC apoptosis, reduced neovascular tufts in the
retina, and vessel leakage.30 CRYAB also stabilizes vascular
endothelial growth factor and favors neovascularization in
retinopathies.30 Taken together, these results and ours suggest that CRYAB might be one of the BMP targets whose
decreased expression can lead to the abnormal angiogenesis
observed in hereditary hemorrhagic telangiectasia. We, and
others, previously showed a strong inhibition by BMP9 of
Apelin gene expression, encoding a peptide highly expressed
in endothelial tip cells, which binds to its receptor present
on stalk cells, where it promotes cell proliferation and vessel branching through mTOR signaling.29,31 Therefore, the
expected increase in apelin expression when BMP signaling
is decreased might contribute synergistically with CRYAB
decrease to the abnormal angiogenesis observed in mutation
carriers of the BMP pathway genes.
In conclusion, we have detected a molecular link between
the BMP signaling pathway and a chaperone protein acting in different cell processes, in particular, in resistance to
apoptosis, and this relationship might play a major role in the
physiopathology of vascular diseases linked to BMP signaling
deficiency.
Acknowledgments
We thank David Tregouet, PhD, for his advice for statistical analysis;
Catherine Blanc, PhD, for help in flow cytometry experiments; and
Mathilde Keck, PhD, for help in experimental data.
α-B-Crystallin Induction by BMPs 2583
Sources of Funding
M. Ciumas was supported by Société de Pneumologie de Langue
Française. M. Eyries was supported by the Fondation LefoulonDelalande. This work was supported by the Fondation de France, the
Legs Poix, and by the Program Hospitalier de Recherche Clinique
(AOM07041).
Disclosures
None.
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Downloaded from http://atvb.ahajournals.org/ by guest on July 31, 2017
Significance
We show the strong induction of CRYAB, a chaperone protein which is a small heat shock protein, by bone morphogenetic proteins. This
induction is transcriptional indirect and is dependent on BMPR2, ALK1, and the transcription factor Id1. We show that, in vitro, bone morphogenetic protein inhibits endothelial cell apoptosis induced by serum starvation and by hypoxia, and that this inhibition is dependent on
CRYAB induction. We also show in the mouse hypoxia model a decrease in the bone morphogenetic protein pathway activity and a decrease
in CRYAB expression. We speculate that the CRYAB decrease observed in this model can favor initial apoptosis and secondary endothelial
cell proliferation.
Downloaded from http://atvb.ahajournals.org/ by guest on July 31, 2017
Bone Morphogenetic Proteins Protect Pulmonary Microvascular Endothelial Cells From
Apoptosis by Upregulating α-B-Crystallin
Mariana Ciumas, Mélanie Eyries, Odette Poirier, Svetlana Maugenre, France Dierick, Natalia
Gambaryan, Kevin Montagne, Sophie Nadaud and Florent Soubrier
Arterioscler Thromb Vasc Biol. 2013;33:2577-2584; originally published online September 26,
2013;
doi: 10.1161/ATVBAHA.113.301976
Arteriosclerosis, Thrombosis, and Vascular Biology is published by the American Heart Association, 7272
Greenville Avenue, Dallas, TX 75231
Copyright © 2013 American Heart Association, Inc. All rights reserved.
Print ISSN: 1079-5642. Online ISSN: 1524-4636
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Data Supplement (unedited) at:
http://atvb.ahajournals.org/content/suppl/2013/09/26/ATVBAHA.113.301976.DC1
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Materials and Methods
BMPs up-regulate -B-crystallin in endothelial cells, and that mediates in part their antiapoptotic effect.
Reagents. Recombinant human BMP4, BMP7 and BMP9 were purchased from R&D
Systems (Minneapolis, MN). Cycloheximide and Actinomycin D were obtained from
Boehringer Ingelheim GmbH (Ingelheim, Germany). Primary antibodies used were:
αB-crystallin: SPA-222, for Western blot and SPA-223 for immmunofluorescence, (both from
Enzo lifescience, Villeurbanne, France), pSmad1/5/8, SMAD5 (Cell Signaling Technology,
Danvers, MA), β-actin (Sigma-Aldrich, St Louis, MO), Von Willebrandt Factor (#ab11713
from Abcam, Cambridge, UK).
Cell culture. The human dermal microvascular endothelial cell line HMEC-1 was obtained
from Thomas J. Lawley (Emory University, School of Medicine, Atlanta, GA) and grown as
previously described1. Human lung microvascular endothelial cells (HLMEC) were obtained
from Clonetics (Baltimore, MD) and cultured in endothelial cell Basal Medium 2 (EBM-2)
supplemented with EGM-2MV Single Quots (Clonetics, Baltimore, MD). HUVEC (Human
Umbilical Vascular Endothelial Cell) were extracted from umbilical cord as previously
described2 and cultured in endothelial cell basal medium 2 (EBM-2) supplemented with
EGM-2MV (Lonza, Basel Switzerland).
siRNA transfection. Synthetic small interfering RNA (siRNA) targeting the human BMPR2
mRNA, CRYAB mRNA, ALK1 mRNA, ALK3 mRNA, Id1 mRNA , Id2 mRNA and Id3 mRNA
were purchased from Dharmacon Research (Lafayette, CO). Non-targeting siRNA 2
(Dharmacon #D-001210-02) was used as a non-specific control. SiRNA were transfected
using Dharmafect-1 transfection reagent according to manufacturer’s recommendation. After
transfection, cells were starved for 24h then treated with BMPs for indicated time before lysis
or staining.
Real-time RT-PCR. Real-time RT-PCR assay was performed as previously described3.
Primers used for real-time RT-PCR are indicated below:
Primer sequences
Gene
Forward
Reverse
hCRYAB
5’-GAAGAGCGCCAGGATGAACAT-3’
5’-TGAGGACCCCATCAGATGACA-3’
RPL32
5’-CCCAAGATCGTCAAAAAGA-3’
5’-TCAATGCCTCTGGGTTT-3’
mCRYAB
5’-TGGATTGACACCGGACTCTCA-3’
5’-CCCCCAGAACCTTGACTTTGA-3’
mBMPR2
5’-GGTCTCACATCGGTGATCCC-3’
5’-GTCAGGGGGTGGAAAGTTCTC-3’
mRPL13a
5’-TCTGGAGGAGAAACGGAAGGA-3’
5’-GGTTCACACCAAGAGTCCATTG-3’
GAPDH
5‘-TTGAGGTCAATGAAGGGGTC-3’
5’-GAAGGTGAAGGTCGGAGTCA-3’
VEGF
5’-CACACAGGATGGCTTGAAGA-3’
5’-AGGGCAGAATCATCACGAAG-3’
Western Blot. Cells were lysed with 100μl of Ripa buffer (25 mM Tris•HCl pH 7.6, 150 mM
NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) supplemented with Protease
Inhibitor Cocktail, Phosphatase Inhibitor Cocktail 1 and 2 (Sigma-Aldrich, St Louis, MO),
snap frozen and stored at -80°C until use. Frozen tissues were homogenized in the same
lysis buffer, centrifugated for 5 minutes at 14000 rpm at 4°C in a chilled micro centrifuge and
the supernatant was stored at -80°C until use. Samples were subjected (20µg of proteins per
lane for cell extracts and 50µg of proteins per lane for tissue extracts) to SDS-polyacrylamide
gel electrophoresis (PAGE) in Tris-Glycine gels. The proteins were transferred to the PVDF
membrane using XCell II Blot Module (Invitrogen, Carlsbad, CA). Membranes were then
incubated overnight with corresponding antibodies (CRYAB 1:1000, pSMAD1/5/8 and
SMAD5 1:1000, β-actin 1: 40000) followed by an anti-rabbit IgG or anti-mouse IgG
secondary antibody conjugated to horse-radish peroxidase (1:40000, 1h; Jackson
ImmunoResearch Laboratories, West Grove, PA). Bands were visualized using an enhanced
chemiluminescence
substrate
system
ECL+
detection
system
(GE
Healthcare,
Buckinghamshire, UK) using either High Performance Chemiluminescence Films or Ettan
DIGE Imager (GE Healthcare, Buckinghamshire, UK). The intensity of each band was
analyzed by densitometry using ImageJ software or using ImageQuant software (GE
Healthcare, Buckinghamshire, UK) respectively.
Transcription assay of the CRYAB gene. Gene transcription was measured by quantifying
pre-mRNA as previously described4. CRYAB pre-mRNA was measured by real-time PCR
using the following intron-exon primers: upstream 5’-TTGGTCTCACCTAAGGGGA-3’ located
in intron 1 and downstream 5’-TGTCATCTGATGGGGTCCTCA-3’ located in exon 2. CRYAB
pre-mRNA expression was normalized to GAPDH expression measured by real-time PCR
using the following primers: upstream 5’-GAAGGTGAAGGTCGGAGT-3’ placed on exon 2
and downstream 5’-GAAGATGGTGATGGGATTTC-3’ placed on exon 4.
Apoptosis assay. HLMEC were plated at a density of 2.5x105 cells/well in 6-well plates for
24h. Cells were then cultured for 48h in complete medium or SF medium, in the presence of
BMP7 (200 ng/ml). Cell lysates were tested for apoptosis by measurement of cytoplasmic
nucleosomes using a Cell Death Detection ELISA kit (Roche Diagnostics GmbH, Roche
Molecular
Biochemicals,
Mannheim,
Germany)
according
to
the
manufacturer’s
recommendation. This assay quantitatively detects histone-associated DNA fragments in the
cytoplasmic fraction of cell lysates.
Annexin assay. Apoptosis was assessed in HLMEC by flow cytometry analysis using the
Annexin V:FITC Apoptosis Detection Kit II (BD Pharmingen, San Diego, CA) under control or
apoptosis-inducing conditions (SF), in the presence or absence of BMPs and siRNA targeting
CRYAB mRNA or a non-targeting siRNA. At the end of the treatment period (72h), cells were
suspended in Enzyme Free Cell Dissociation Buffer (InVitrogen, Carlsbad, CA) and washed
twice with cold PBS. Annexin assay was performed according to the manufacturer’s
recommendation Fluorescence was measured on a Beckman Coulter XL analyzer (Beckman
Coulter, Fullerton, CA).
Nuclear chromatin condensation assay. HUVEC were seeded in 6-well plates at a density
of 2×105 per well, and cultured in normoxia or hypoxia (1%O2) conditions for 24h. Plates
were washed twice with PBS, and cells were fixed using 4% paraformaldehyde for 15 min,
followed by another two washes of PBS and incubated in 500 μl of Hoechst 33342 solution
(5 μg/ml in PBS) for 15 min in the dark. Analysis was performed by fluorescent microscopy.
Blue cells were scored as apoptotic or normal based on morphological criteria: small, round,
condensed cells were scored as apoptotic, and all other cells were scored as healthy. For
each sample, about 1000 cells were scored in a blinded manner.
Animals. Eight weeks-old male C57Bl/6 mice (CERJ, Orléans, France) were used for this
study and were divided into two groups: a control group exposed to normoxic conditions, and
one group exposed to hypoxic conditions (10% O2) in a normobaric hypoxic chamber for 35
days. After 35 days, mice were euthanized immediately after hemodynamic assessment.
Lungs were washed in PBS, immediately frozen in liquid nitrogen and stored at -80° before
RNA and protein extraction, or inflated with diluted OCT (1:2 in PBS, 2ml) and frozen in
isopentane dipped into liquid nitrogen for immunofluorescence analysis. Mice were treated in
accordance with our institutional guidelines (Ministère de l’Agriculture, France; authorization
75-1206) and conformed to the Directive 2010/63/EU of the European Parliament).
Immunofluorescence staining and microscopy. Frozen mouse lung sections (6 µm) were
fixed in 4% PFA for 15 minutes and washed in PBS. Sections were blocked in 3% BSA for 1h
at room temperature followed by incubation with primary antibodies (SPA-223 1:100, VWF
1:1000) in blocking solution over night at 4°C. Sections were washed in PBS and incubated
with Alexa-conjugated secondary antibodies (Invitrogen, Carlsbad, CA) in PBS buffer for 1h,
followed by nuclear staining with DAPI. Sections were mounted using Fluorescent Mounting
Medium (Dako, Denmark A/S). Slices were analyzed using an Olympus XL microscope and
images were then deconvoluted, using MetaMorph softwear (MDS Analytical Technologies,
Downingtown, PA).
Statistical Analysis- Statistical analysis was performed with XLSTAT (Addinsoft, Paris,
France) using a one-way analysis of variance for all data, followed by a Student NewmanKeuls post-hoc analysis for multiple tests. Pairwise comparisons were analysed using the
non parametric Mann-Whitney test. The null hypothesis was rejected for p<0.05. Data are
expressed as mean ± S.D.
References
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umbilical
veins.
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and
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Fold change of CRYAB
mRNA expression
A
**
1.2
***
2
20
1
10
0
0
0.8
0.4
0
-
24h
48h
-
72h
24h
BMP4 200ng/ml
Fold change of CRYAB
mRNA expression
B
48h
BMP7 200ng/ml
72h
-
24h
48h
72h
BMP9 200ng/ml
50
*
40
30
20
10
0
-
BMP9
10 ng/ml
Supplementary Figure I. BMPs up-regulate CRYAB in endothelial cells.(A) Serum-restricted HLMEC were
stimulated with 200ng/ml of BMP7 or BMP9 for 24, 48 or 72h. (B) Serum-restricted HUVEC were stimulated with
10ng/ml of BMP9 for 24h. Expression of CRYAB was quantified by real-time RT-PCR analysis. The relative CRYAB
mRNA level was normalized to RPL32 mRNA level and expressed as a fold change relative to untreated cells. Results
are mean ± S.D. values of 3 independent experiments.* P<0.05 relative to untreated cells.
Specific mRNA /GAPDH mRNA levels
(fold change of levels in untreated cells)
2.0
Si CT
Si specific
1.5
1.0
0.5
0.0
ALK1
ALK3
BMPR2
CRYAB
ID1
ID2
ID3
Supplementary figure II. Knock-down expression of targeted genes by specific siRNA. HMEC-1 were
transfected with a non specific siRNA (Si CT) or specific siRNA targeting ALK1, ALK3, BMPR2, CRYAB, ID1, ID2 or
ID3. Each specific gene expression was quantified by real time RT-PCR analysis. The targeted gene mRNA level was
normalized to GAPDH levels and expressed as a fold change of its expression in untreated cells.
A
BMP4 10ng/ml
BMP9 10ng/ml
BMP7 10ng/ml
0h 24h
0h 24h 48h 72h
48h
0h 24h 48h 72h
72h
pSMAD 1/5/8
SMAD 5
SMAD1/5/8 Activation
(arbitrary units)
B
16
14
12
10
8
6
4
2
0
10
8
6
4
2
0
0h
24h
48h
BMP4 10ng/ml
72h
25
20
15
10
5
0
0h
24h
48h
BMP7 10ng/ml
72h
0h
24h
48h
72h
BMP9 10ng/ml
Supplementary figure III. SMAD pathway is activated by BMPs in HMEC-1. Serum-restricted HMEC-1 were
stimulated with 10ng/ml of BMP4, BMP7 or BMP9 for 24, 48 or 72h. (A) Protein extracts were analyzed by Western
blot using specific phospho- SMAD1/5/8 and SMAD5 antibodies.(B) Densitometric analysis of SMAD1/5/8 activation.
SMAD activation is defined as the ratio between the phospho-SMAD 1/5/8 signal and SMAD5 signal
B
*
60
RVSP (mmHg)
Hematocrit (%)
80
40
20
0
Normoxia
Hypoxia
C
35
30
25
20
15
10
5
0
*
Normoxia
Hypoxia
RV/(LV+S) (Fulton Index)
A
45
40
35
30
25
20
15
10
5
0
*
Normoxia
Hypoxia
Supplementary figure IV. Hemodynamic parameters in mice exposed to normoxia or hypoxia for 35
days.(A) Hematocrit. (B) Right Ventricule Systolic Pressure (RVSP) (C) Fulton index
Fold change of VEGF mRNA expression
2.0
ns
1.5
1.0
0.5
0.0
-
BMP9 10 ng/ml
Supplementary Figure V. Absence of VEGF mRNA induction in HMEC-1 treated with BMP9.
Serum-restricted HMEC-1 were stimulated with 10 ng/ml BMP9 for 24h. Expression of VEGF mRNA
was quantified by real-time RT-PCR analysis. The relative VEGFmRNA level was normalized to GAPDH
mRNA level and expressed as a fold change relative to untreated cells. Results are mean ± S.D.
values of 5 independent experiments. ns, not significant.
E
br
mv
nmv
Supplementary Figure VI CRYAB expression in mouse lung exposed to normoxia or hypoxia. (E) Immunofluorescence
analysis of endothelial cells (visualized by Von Willebrandt factor (VWF) -green) shows CRYAB expression (in red) in endothelial
cells of lung vessels but also in vascular and airway smooth muscle cells and in bronchiolar (br) epithelial cells normal mouse lung;
nuclei are stained with DAPI;. Expression of CRYAB in endothelial cells of small muscularized (mv) and non muscularized (nmv) lung
vessels is indicated by arrows. Scale bar 50µm.
KIDNEY
CRYAB
VWF
MERGE
DAPI
v
v
v
v
Negative Control
Merge
Supplementary Figure VII. CRYAB expression in mouse kidney vessels showing the specificity of the Cryab antibody.
Immunofluorescence analysis of endothelial cells (visualized by Von Willebrandt factor (VWF) -green) shows CRYAB expression (in red) in
endothelial cells of kidney vessels (v) but not in other kidney cells. Scale bar 50µm.