Endoplasmic Reticulum Stress Participates in Aortic Valve
Calcification in Hypercholesterolemic Animals
Zhejun Cai,* Fei Li,* Wei Gong,* Wanjun Liu, Quanlu Duan, Chen Chen, Li Ni, Yong Xia,
Katherine Cianflone, Nianguo Dong, Dao Wen Wang
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Objectives—Aortic valve (AV) calcification occurs via a pathophysiological process that includes lipoprotein deposition,
inflammation, and osteoblastic differentiation of valvular interstitial cells. Here, we investigated the association between
endoplasmic reticulum (ER) stress and AV calcification.
Approach and Results—We identified ER stress activation in AV of patients with calcified AV stenosis. We generated
an AV calcification model in hypercholesterolemic rabbits and mice, respectively, and found marked AV ER stress
induction. Classical ER stress inhibitor, tauroursodeoxycholic acid, administration markedly prevented AV calcification,
and attenuated AV osteoblastic differentiation and inflammation in both rabbit and mouse models of AV calcification
via inhibition of ER stress. In cultured valvular interstitial cells (VICs), we found that oxidized low density lipoprotein
(oxLDL) caused ER stress in a cytosolic [Ca]2+i-dependent manner. OxLDL promoted osteoblastic differentiation via
ER stress–mediated protein kinase-like ER kinase/activating transcription factor 4/osteocalcin and inositol-requiring
transmembrane kinase and endonuclease-1α (IRE1α)/spliced X-box–binding protein 1/Runx2 pathway, and induced
inflammatory responses through IRE1α/c-Jun N-terminal kinase and IRE1α/nuclear factor kappa-light-chain-enhancer
of activated B cells signaling in VICs. Inhibition of ER stress by either tauroursodeoxycholic acid or 4-phenyl butyric acid
could both suppress oxLDL–induced osteoblastic differentiation and inflammatory responses in VICs.
Conclusions—These data provide novel evidence that ER stress participates in AV calcification development, and suggest
that ER stress may be a novel target for AV calcification prevention and treatment. (Arterioscler Thromb Vasc Biol.
2013;33:2345-2354.)
Key Words: aortic valve, calcification of ◼ endoplasmic reticulum stress ◼ inflammation ◼ osteogenesis
◼ oxidized low density lipoprotein ◼ tauroursodeoxycholic acid
A
chronic inflammation, and osteoblastic differentiation of valvular interstitial cells (VICs).4,5
The endoplasmic reticulum (ER) is a crucial luminal
network for protein synthesis, folding, and transportation.
Conditions disrupting the ER homeostasis cause accumulation of unfolded and misfolded proteins in the ER lumen,
create a state defined as ER stress, and activate a complex signaling network termed the unfolded protein response, which
consists of 3 branches, protein kinase-like ER kinase (PERK),
inositol-requiring transmembrane kinase and endonuclease1α (IRE1α), and activating transcription factor 6 (ATF6).6 So
far, numerous studies have shown that ER stress is linked well
with a variety of pathophysiologic conditions, such as diabetes mellitus, obesity, cancer, atherosclerosis, myocardial injuries, and inflammatory conditions.7–10 However, whether ER
ortic valve (AV) calcification and its subsequent calcified AV stenosis (CAVS) are common clinical problems. Approximately 25% of adults >65 years old have some
degree of AV sclerosis (defined as valvular thickening or
calcification without significant obstruction),1 and CAVS is
present in >4% of very elderly patients,1 which has become
the most common indication for valve replacement and the
second most common indication for cardiac surgery in the
United States and Europe.2,3 Although the disease is associated with increased cardiovascular events and mortality,
there currently is no effective therapy other than surgical or
interventional AV replacement.4 In the past decade, increasing evidence suggests that AV calcification is not simply a
passive degenerative process, but an active disease progress
similar with atherosclerosis, including lipoprotein deposition,
Received on: July 29, 2012; final version accepted on: July 24, 2013.
From the Institute of Hypertension and Department of Internal Medicine, Tongji Hospital (Z.C., W.G., Q.D., C.C., L.N., D.W.W.), and Department of
Cardiovascular Surgery, Union Hospital (F.L., N.D.), Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China; Department
of Cardiology, Second Affiliated Hospital, Medical College, Zhejiang University, Hangzhou, China (Z.C.); Davis Heart and Lung Research Institute,
Division of Cardiovascular Medicine, Department of Molecular and Cellular Biochemistry, Ohio State University College of Medicine, Columbus, OH
(Y.X.); and Centre de Recherche de l’Institut Universitaire de Cardiologie et de Pneumologie de Québec, Université Laval, Québec, Canada (K.C.).
*These authors contributed equally.
The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA.112.300226/-/DC1.
Correspondence to Dao Wen Wang, Department of Internal Medicine and Institute of Hypertension, Tongji Hospital, 1095 Jiefang Ave, Wuhan 430030,
China (e-mail [email protected]); or Nianguo Dong, Department of Cardiovascular Surgery, Union Hospital, 1277 Jiefang Ave, Wuhan 430022,
China (e-mail [email protected]).
© 2013 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org
2345
DOI: 10.1161/ATVBAHA.112.300226
2346 Arterioscler Thromb Vasc Biol October 2013
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stress is also involved in the pathogenesis of AV calcification
remains unknown.
Histological data demonstrate the presence of oxidized
low-density lipoprotein (oxLDL) in calcified AV, indicating
its pathophysiological role in the development of AV calcification.11 Furthermore, several groups have recently demonstrated that oxLDL could trigger ER stress in endothelial
cells, thus playing an important role in the development of
atherosclerosis.12,13 These studies suggest that ER stress may
be induced during AV calcification.
Recent studies reported that ER stress–mediated activation
of activating transcription factor 4 (ATF4) and spliced X-box–
binding protein 1 (XBP1s) are transcriptional activators that
control the important osteoblastic differentiation factors osteocalcin and Runx2.14-16 Moreover, an emerging function of ER
is to regulate inflammation because studies have indicated that
unfolded protein response causes activation of c-Jun N-terminal
kinase (JNK) and nuclear factor kappa-light-chain-enhancer of
activated B cells (NF-κB) pathways.17,18 Notably, activation
of the inflammatory response is also a key contributor to AV
calcification development.5,19,20 We therefore hypothesized that
ER stress may contribute to osteoblastic differentiation and
inflammation in VICs to promote AV calcification.
To test this hypothesis, we investigated the presence of ER
stress in calcified AV from patients with CAVS and experimental AV calcification in animal models. Because oxLDL plays an
important role in AV calcification development and may potentially cause ER stress, we examined the association between
oxLDL and ER stress–mediated inflammation and osteoblastic
differentiation in cultured VICs. Furthermore, we determined
whether a classical ER stress inhibitor, tauroursodeoxycholic
acid (TUDCA), could attenuate diet-induced AV calcification
via alleviation of ER stress in AV calcification animal models.
Materials and Methods
Materials and Methods are available in the online-only Supplement.
Results
ER Stress Is Activated in AV Leaflets
of Patients With CAVS
To determine whether ER stress is induced in patients with
AV calcification, we used AV tissues from 10 patients with
CAVS, as well as 4 traffic accident victims. In parallel with
the increased expression of Runx2 and osteocalcin, we found
marked activation of ER stress in calcified AV compared with
the control subjects as assessed by PERK and IRE1α phosphorylation, and C/EBP homologous protein (CHOP) expression (Figure 1).
High-Cholesterol Diet Induced Significant
AV Calcification in Animals
We used 2 different classical animal models of AV calcification, rabbits and mice, which are similar to human clinical condition.19,21–24 After 12 weeks of high-cholesterol (HC)+vitamin
D2 (vitD2) diet, increased echogenicity occurred in AV of
HC+vitD2-treated rabbits (Figure II in the online-only Data
Supplement). AV area (AVA) and AVA indexed by body surface area in HC+vitD2-treated rabbits decreased markedly
Figure 1. Induction of endoplasmic reticulum (ER) stress in aortic
valve (AV) leaflets of patients with calcified AV stenosis (CAVS).
Immunoblotting for osteoblast differentiation markers Runx2
and osteocalcin, and ER stress markers in AV leaflet samples
from patients with CAVS as well as normal leaflets. Runx2 and
osteocalcin expressions were strongly induced, and the expressions of phosphorylated-protein kinase-like ER kinase (p-PERK),
phosphorylated-inositol-requiring transmembrane kinase and
endonuclease-1α (p-IRE1α), and C/EBP homologous protein
(CHOP) were markedly increased in calcified AV compared with
normal AV (**P<0.01 vs normal).
compared with the normal controls (Table 1). We also observed
a significant increase in transvalvular peak jet velocity and a
marked decrease in AVA and AVA index after 24 weeks of HC
diet treatment in ApoE−/− mice (Table 1).
We next assessed the degree of calcification. Alizarin red
staining revealed increased calcium deposit in HC+vitD2 rabbits compared with normal controls (Figure 2A and 2B). The
transition from VICs to myofibroblasts is also a critical change
in valvular pathology.4 We examined myofibroblasts in AV by
immunohistochemical staining using α-smooth muscle actin
(α-SMA) antibody. AV in control animals exhibited sparse αSMA positive myofibroblasts, whereas HC+vitD2 treatment
markedly increased the α-SMA positive staining area (Figure
III in the online-only Data Supplement).Similarly, we also
observed significant calcium deposits in AV of HC diet–treated
ApoE−/− mice by alizarin red staining (Figure 2A and 2C). The
expression of α-SMA was markedly increased in HC-treated
ApoE−/− mice (Figure IV in the online-only Data Supplement).
Activation of ER Stress in Calcified AV
of Hypercholesterolemic Animals
We examined whether ER stress was induced in AV leaflets of
AV calcification animals. Results showed that both HC+vitD2
treatment in rabbits and HC treatment in ApoE−/− mice markedly increased the AV ER stress response compared with normal controls, as evaluated by staining of ER stress markers
Cai et al ER Stress and Aortic Valve Calcification 2347
Table 1. Hemodynamic Parameters of Animals After Different Interventions
Rabbits
ApoE−/− Mice
Control
n=6
HC+vitD2
n=7
HC+vitD2+TUDCA
n=7
Control
n=15
HC
n=15
HC+TUDCA
n=15
Transvalvular peak jet velocity,
cm/s
68.43±8.32
73.07±10.54
71.88±11.60
0.92±0.18
1.57±0.29**
1.23±0.22##
AVA, mm2
23.32±1.44
21.26±1.28*
22.50±1.69
0.97±0.05
0.73±0.07**
0.81±0.05##
1.01±0.03
0.68±0.05**
0.75±0.04##
AVA index, AU
1.17±0.04
0.92±0.04**
0.99±0.06#
Data are presented as mean±SD. AVA indicates aortic valve area; HC, high cholesterol; TUDCA, tauroursodeoxycholic acid; and VitD2, vitamin D2.
*P<0.05 vs control.
**P<0.01 vs control.
#P<0.05 vs HC+vitD2 in rabbits.
##P<0.01 vs HC in ApoE−/− mice.
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KDEL and CHOP (Figure 3A). Moreover, immunoblotting
showed that important ER stress markers, such as p-PERK and
p-IRE1α, and CHOP expression were significantly induced
in AV of HC+vitD2-treated rabbits compared with controls
(Figure VA and VB in the online-only Data Supplement).
Prevention of ER Stress by TUDCA Attenuated
Diet-Induced AV Calcification in Animals
TUDCA is a chemical chaperone that has been well demonstrated to be a classical inhibitor against ER stress by
improving ER folding capacity both in vitro and in vivo.25–27
We examined whether relieving ER stress by TUDCA could
rescue AV calcification. We treated HC+vitD2-fed rabbits
with TUDCA (50 mg/kg per day) by oral gavage. In parallel with the attenuated ER stress in HC+vitD2+TUDCA group
(Figure 3A), we found TUDCA supplementation also
reduced AV echogenicity (Figure II in the online-only
Data Supplement) and restored AVA index compared with
HC+vitD2 group (Table 1). Furthermore, TUDCA significantly suppressed calcium deposit (Figure 2A and 2B) and αSMA staining (Figure III in the online-only Data Supplement)
induced by HC+vitD2 administration.
We also evaluated whether TUDCA administration could
prevent HC diet–induced AV calcification in ApoE−/− mice.
Twenty-four consecutive weeks of TUDCA (0.5 g/kg per
day) oral gavage significantly reduced levels of AV ER stress
markers KDEL and CHOP staining (Figure 3B), prevented
transvalvular peak jet velocity increases, restored AVA index
(Table 1), and reduced calcium deposits (Figure 2A and 2C)
and myofibroblast activation (Figure IV in the online-only
Data Supplement) induced by HC diet.
TUDCA Administration Prevented HC Treatment–
Induced Osteoblastic Differentiation in AV Leaflets
We determined whether ER stress links AV osteoblastic differentiation in AV calcification animals. As shown in Figures
IV and V in the online-only Data Supplement, HC+vitD2 diet
markedly increased Runx2 staining as well as protein and
mRNA expression of important osteoblastic differentiation
markers, osterix, Runx2, and osteocalcin, in AV leaflets in rabbits. Consistent with the results in AV calcification rabbits, AV
staining of osterix, Runx2, and osteocalcin were all markedly
increased after 24 weeks of HC diet treatment in ApoE−/− mice
(Figure 4A–4D). As expected, ER stress inhibitor TUDCA
Figure 2. Aortic valve (AV) calcification of animals
with different intervention. A, Alizarin red staining for calcified nodules in AV leaflets of animals
with indicated intervention. Scale bars, 200 μm in
rabbits or 100 μm in ApoE−/− mice. B and C, Highcholesterol (HC)+vitamin D2 (vitD2) diet led to significant AV calcification in rabbits, and HC diet caused
marked calcification in AV leaflets of ApoE−/− mice
as well. Tauroursodeoxycholic acid (TUDCA) treatment markedly reduced these effects (in rabbits:
control, n=6; HC+vitD2, n=7; HC+vitD2+TUDCA,
n=7; in ApoE−/− mice: control, n=15; HC, n=15; HC
+TUDCA, n=15; **P<0.01 vs control; ##P<0.01 vs
HC+vitD2 in rabbits or HC in ApoE−/− mice).
2348 Arterioscler Thromb Vasc Biol October 2013
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Figure 3. Aortic valve (AV) endoplasmic
reticulum (ER) stress induction in animals
with different intervention. Representative
staining of ER stress markers, KDEL and
C/EBP homologous protein (CHOP), in AV
leaflets of rabbits (A) and ApoE−/− mice (B)
with indicated intervention. Scale bars,
200 μm in rabbits or 100 μm in ApoE−/−
mice. High-cholesterol (HC)+vitamin D2
(vitD2) or HC diet led to significant KDEL
and CHOP induction in AV leaflets of
indicated animals. Tauroursodeoxycholic
acid (TUDCA) treatment markedly relieved
these effects (in rabbits: control, n=6;
HC+vitD2, n=7; HC+vitD2+TUDCA, n=7;
in ApoE−/− mice: control, n=15; HC, n=15;
HC +TUDCA, n=15; **P<0.01 vs control;
##P<0.01 vs HC+vitD2 in rabbits or HC in
ApoE−/− mice).
administration significantly inhibited these effects in both
rabbit and mouse models of AV calcification (Figure 4A–4D;
Figures V and VI in the online-only Data Supplement).
TUDCA Protected Against Leaflet Inflammation
in Diet-Induced AV Calcification Animals
Direct involvement of the ER stress–mediated pathway in the
inflammatory response has been described in some reports,28
but not in a model of AV calcification. We evaluated the effect
of in vivo TUDCA administration on the level of inflammation in AV leaflets. Results showed that hypercholesterolemic animal models of AV calcification markedly increased
macrophage infiltration in AV leaflets as indicated by macrophage markers RAM11 and F4/80, respectively (Figure
4A and 4D; Figure VII in the online-only Data Supplement).
However, TUDCA treatment markedly attenuated AV macrophage infiltration in hypercholesterolemic animals (Figure
4A and 4D; Figure VII in the online-only Data Supplement).
Effects of TUDCA Are Independent of
Serum Lipid or Blood Glucose Levels
We compared the metabolic parameters among groups and
found that there were no differences observed in levels of
plasma glucose, total cholesterol, LDL, and triglycerides
Figure 4. TUDCA administration reduces
osteoblastic differentiation and macrophage infiltration in aortic valve (AV)
leaflets induced by high-cholesterol (HC)
diet in ApoE−/− mice. A, Osteoblastic differentiation markers, osterix, Runx2, and
osteocalcin, and macrophage marker
F4/80 staining in AV leaflets of indicated
groups. Scale bars, 100 μm. HC diet significantly induced AV osterix (B), Runx2
(C), and osteocalcin (D) staining, and
increased AV macrophage infiltration (E)
in ApoE−/− mice. Tauroursodeoxycholic
acid (TUDCA) effectively suppressed
these effects (control, n=15; HC, n=15;
HC +TUDCA, n=15; **P<0.01 vs control;
##P<0.01 vs HC).
Cai et al ER Stress and Aortic Valve Calcification 2349
among groups at baseline (Table II in the online-only Data
Supplement). Significant increases in serum cholesterol
and LDL levels were observed in both animal models of
AV calcification after the indicated diet treatments, whereas
TUDCA treatment did not alter either the lipid profiles or
glucose levels compared with the HC+vitD2 or HC diet–fed
animals (Table 2).
ER Stress Is Involved in OxLDL-Activated
Inflammation Through IRE1α-Mediated
JNK and NF-κB Signaling in VICs
OxLDL presents in calcified AV,11 and therefore we investigated whether oxLDL could trigger ER stress in cultured
VICs. Results showed that oxLDL induced marked ER stress
in VICs in a dose-dependent manner as evaluated by expression of ER stress markers (Figure VIIIA and VIIIB in the
online-only Data Supplement).
It has been reported that oxLDL could increase cytosolic [Ca]2+i in various cell types.29,30 We found incubation of oxLDL led to a significant increase in cytosolic
[Ca]2+i (Figure VIIIC in the online-only Data Supplement).
Moreover, as with oxLDL, incubation of VICs with a calcium ionophore A23187 markedly enhanced ER stress, and,
by contrast, pretreatment with calcium chelator BAPTA-AM
effectively protected VICs from ER stress induced by both
oxLDL and A23187 (Figure VIIID and VIIIE in the onlineonly Data Supplement).
We further explored whether oxLDL could lead to inflammation via the ER stress–mediated IRE1α pathway.31 Results
showed that small interfering RNA silencing of IRE1α
markedly reduced JNK phosphorylation triggered by oxLDL
(Figure 5E). Meanwhile, as expected, IRE1α silencing also
reduced oxLDL-induced nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα)
degradation and NF-κB p65 nuclear translocation (Figure
5F and 5G).
Furthermore, IRE1α inhibition was associated with
reduced expression of inflammatory cytokines induced by
oxLDL in VICs, including interleukin (IL)-6, IL-8, and
monocyte chemotactic protein-1 (MCP-1) (Figure X in the
online-only Data Supplement). The proinflammatory roles of
JNK and NF-κB in VICs were supported by the effect of the
JNK inhibitor SP600125 and NF-κB inhibitor BAY 11-7082,
which, in part, inhibited the oxLDL-induced expression of
IL-6, IL-8, and MCP-1 (Figure XIA–XIC in the online-only
Data Supplement). Moreover, BAY 11-7082 incubation also
significantly suppressed osteocalcin and Runx2 expression
induced by oxLDL (Figure XID in the online-only Data
Supplement), suggesting a role of NF-κB signaling in osteoblastic differentiation in VICs.
ER Stress Promoted OxLDL-Induced Osteoblastic
Differentiation Via PERK/ATF4/Osteocalcin
and IRE1α/XBP1s/Runx2 Signaling in VICs
Inhibition of ER Stress Reduced OxLDLInduced Osteoblastic Differentiation
and Inflammation in VICs
OxLDL Induced ER Stress in VICs in a
Cytosolic [Ca]2+i-Dependent Manner
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It has been reported that the important osteoblastic factors
osteocalcin and Runx2 are the downstream targets of ATF4
and XBP1s, respectively, which are activated during ER
stress.14–16 We therefore examined whether oxLDLs trigger
osteoblastic differentiation in VICs via ER stress activation. As
Figure 5A and 5B depicted, small interfering RNA silencing
of PERK (Figure IXA in the online-only Data Supplement),
or its downstream target ATF4 (Figure IXB in the online-only
Data Supplement), significantly suppressed the expression
of osteocalcin induced by oxLDLs. Moreover, silencing of
IRE1α (Figure IXC in the online-only Data Supplement), or
its downstream target XBP1 (Figure IXD in the online-only
Data Supplement), could markedly inhibit oxLDL-induced
expression of Runx2 (Figure 5C and 5D).
We further investigated whether inhibition of ER stress
would protect VICs from oxLDL-induced osteoblastic differentiation and inflammation. We found that TUDCA and
4-phenyl butyric acid (4-PBA), 2 classical ER stress inhibitors with distinct chemical structures, markedly attenuated
ER stress triggered by oxLDL in VICs (Figure 6A and 6B;
Figure XII in the online-only Data Supplement). We assayed
the effects of TUDCA and 4-PBA, and found that they both
markedly reduced Runx2 and osteocalcin expression induced
by oxLDL incubation in VICs (Figure 6C; Figure XIII in
the online-only Data Supplement). We next explored the
effects of TUDCA and 4-PBA on JNK and NF-κB signaling. Results showed that TUDCA and 4-PBA pretreatments
both effectively inhibited the activation of JNK caused by
Table 2. Serum Lipid Profiles and Glucose Levels in Animals After Different Intervention
Rabbits
Control
n=6
HC+vitD2
n=7
ApoE−/− Mice
HC+vitD2+TUDCA
n=7
Control
n=15
HC
n=15
HC+TUDCA
n=15
Glucose, mmol/L
5.61±0.89
5.73±0.84
5.68±0.85
10.56±0.97
11.86±1.44
11.37±1.65
TC, mmol/L
1.30±0.21
25.17±3.88**
23.34±4.14†
14.92±0.54
42.07±2.15**
40.97±3.19†
LDL-c, mmol/L
0.72±0.18
19.43±3.42**
18.39±3.52†
7.85±0.26
23.97±2.03**
22.08±2.75†
TG, mmol/L
0.85±0.23
1.25±0.33
1.18±0.28
2.26±0.37
4.85±1.08**
4.56±1.13†
Data are presented as mean±SD. HC indicates high cholesterol; LDL-c, LDL cholesterol; TC, total cholesterol; TG, triglyceride; TUDCA, tauroursodeoxycholic acid;
and VitD2, vitamin D2.
**P<0.01 vs control.
†Not significant vs HC+vitD2 in rabbits or HC in ApoE−/− mice.
2350 Arterioscler Thromb Vasc Biol October 2013
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Figure 5. Endoplasmic reticulum stress is involved in oxidized low-density lipoprotein (oxLDL)–mediated osteoblastic differentiation and
inflammation in valvular interstitial cells. A, OxLDL induced abundant osteocalcin expression, whereas small interfering RNA (siRNA) silencing of protein kinase-like ER kinase (PERK) significantly inhibited its downstream activating transcription factor 4 (ATF4) induction as well as
osteocalcin expression stimulated by oxLDL. B, Silencing of ATF4 markedly prevented oxLDL-induced osteocalcin expression. C, OxLDL
incubation led to significant induction of Runx2, whereas siRNA silencing of inositol-requiring transmembrane kinase and endonuclease-1α
(IRE1α) significantly prevented downstream X-box–binding protein 1 (XBP1) splicing and Runx2 expression induced by oxLDL. D, Silencing
of XBP1 markedly reduced Runx2 expression stimulated by oxLDL incubation. E, IRE1α silencing markedly attenuated c-Jun N-terminal
kinase (JNK) phosphorylation induced by oxLDL. F, OxLDL caused marked degradation of nuclear factor of kappa light polypeptide gene
enhancer in B-cells inhibitor, alpha (IκBα), whereas IRE1α silencing blocked this effect. G, IRE1α silencing inhibited nuclear factor kappalight-chain-enhancer of activated B cells (NF-κB) p65 nuclear translocation induced by oxLDL (n=3 for each experiment; *P<0.05 vs scrambled siRNA served as control [Scr siRNA]; **P<0.01 vs Scr siRNA; #P<0.05 vs oxLDL+Scr siRNA; ##P<0.01 vs oxLDL+Scr siRNA).
exposure to oxLDL (Figure 6D; Figure XIVA in the onlineonly Data Supplement) and simultaneously prevented the
NF-kB p65 activation induced by oxLDL (Figure 6E and 6F;
Figures XIVB and XV in the online-only Data Supplement).
Moreover, as depicted in Figure XVI in the online-only Data
Supplement, oxLDL significantly increased the expression of
IL-6, IL-8, and MCP-1, and the effects could be markedly
suppressed by TUDCA and 4-PBA.
Finally, we evaluated whether ER stress inhibition
could prevent mineralization of VICs induced by oxLDL.
TUDCA and 4-PBA treatments significantly attenuated
oxLDL-induced mineralization and alkaline phosphatase
Figure 6. Tauroursodeoxycholic acid (TUDCA) attenuates
endoplasmic reticulum (ER)
stress–mediated osteoblastic differentiation, and c-Jun N-terminal
kinase (JNK) and nuclear factor kappa-light-chain-enhancer
of activated B cells (NF-κB)
signaling activation in valvular
interstitial cells (VICs). A and B,
Immunoblotting showed that
TUDCA pretreatment markedly
inhibited oxidized low-density
lipoprotein (oxLDL)–induced ER
stress in VICs. C, TUDCA suppressed oxLDL-induced Runx2
and osteocalcin expression in
VICs. TUDCA prevented VICs
from JNK phosphorylation (D),
nuclear factor of kappa light polypeptide gene enhancer in B-cells
inhibitor, alpha (IκBα) degradation (E), and NF-κB p65 nuclear
translocation (F) stimulated by
oxLDL (n=3 for each experiment;
**P<0.01 vs control; #P<0.05 vs
oxLDL; ##P<0.01 vs oxLDL).
Cai et al ER Stress and Aortic Valve Calcification 2351
(ALP) activity in VICs (Figure XVIIA and XVIIB in the
online-only Data Supplement). Consistently, Alizarin staining showed that TUDCA and 4-PBA markedly attenuated
VICs mineralization induced by oxLDL (Figure XVIIC in
the online-only Data Supplement).
Discussion
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Despite the clinical importance of heart disease and intense
study in this field, the mechanisms underlying the development of AV calcification remain unclear. The present study
suggests the important role of ER stress in the pathogenesis
of AV calcification. We identified significant ER stress induction in calcified AV of patients with CAVS. In our hypercholesterolemia-induced AV calcification animal models, we also
found ER stress activation in AV leaflets. Inhibition of ER
stress by TUDCA administration was associated with significant benefits on AV calcification and could markedly attenuate AV osteoblastic differentiation and inflammation. Our in
vitro experiments subsequently indicate that oxLDL causes
ER stress in VICs by increasing cytosolic [Ca]2+i. Furthermore,
we provide evidence that oxLDL induces osteoblastic differentiation via ER stress–mediated PERK/ATF4/osteocalcin and
IRE1α/XBP1s/Runx2 pathways, and oxLDL promotes inflammatory responses via ER stress–mediated IRE1α/JNK and
NF-κB signaling. Inhibition of ER stress by either TUDCA or
4-PBA could suppress osteoblastic differentiation, inflammatory response, and mineralization induced by oxLDL in VICs.
AV Calcification and ER Stress Activation
AV calcification development shares some similarities with
atherosclerosis and arterial calcification.32 It has been widely
accepted that ER stress plays an important role in atherosclerotic plaque progression,33,34 and in vitro data indicate the
potential role of ER stress in arterial calcification development.14 However, whether there is a link between ER stress
and AV calcification has not been reported to date. In this present work, we used calcified AV tissue samples from patients
with CAVS, as well as 2 classical hypercholesterolemic AV
calcification animal models to explore their links.
Lipoprotein deposition, osteoblastic differentiation, and
inflammation all markedly contribute to AV calcification and
its subsequent CAVS in humans.4 In this study, we identified
upregulation of ER stress markers in patients with significant
CAVS. Moreover, we observed that ER stress was markedly
induced in AV leaflets in 2 distinct AV calcification models of 2
species, further suggesting that ER stress activation in AV leaflets potentially correlates with AV calcification development.
Although the above data implicate ER stress in induction
of calcified AV, how ER stress is initiated is still unknown.
To date, oxLDL is associated with proinflammatory and mineralization promoting properties35,36 to induce AV calcification.11 Our current results add another dimension to oxLDL
and provide evidence that oxLDL contributes to ER stress in
AV calcification. Here, we showed that oxLDL stimulation
causes strong ER stress in VICs. As previously documented,
increased cytosolic [Ca]2+i hampers ER abilities of protein
folding, causes unfolded or misfolded protein accumulation,
and could finally lead to ER stress.7,8,37 We hypothesized that
oxLDL stimulated ER stress in VICs via increasing cytosolic
[Ca]2+i. Indeed, incubation of oxLDL increased cytosolic
[Ca]2+i in VICs, which is consistent with recent studies in
which oxLDL increases cytosolic [Ca]2+i in smooth muscle
cells and endothelium cells.13,29 Providing additional support
to this hypothesis, we demonstrated that administration of
A23187, a calcium ionophore, could also strongly induce ER
stress in VICs. Moreover, we found that oxLDL or A23187induced ER stress could be attenuated by reducing cytosolic
[Ca]2+i. These findings suggest that oxLDL-induced ER stress
in VICs is mediated in a cytosolic [Ca]2+i-dependent manner.
ER Stress Links Osteoblastic
Differentiation and Inflammation
Activation of osteoblastic differentiation in VICs has been
proven to play a central role during AV calcification development.4 ATF4 and XBP1s are critical transcription factors
downstream of PERK and IRE1α signaling branches of ER
stress, and they play important roles in the recovery of damaged cells exposed to ER stress.6 Recent studies indicate
that ATF4 and XBP1s mediate not only ER stress responses
but also osteoblastic differentiation because they are transcriptional activators of osteocalcin and Runx2, the central
regulators of osteoblastic differentiation.15,16 Using small
interfering RNA silencing strategy, we provided evidence
that ER stress–mediated PERK/ATF4 and IRE1α/XBP1s signaling is responsible for osteocalcin and Runx2 expression,
and thus directly contributes to oxLDL-induced osteoblastic
differentiation in VICs.
Recent studies revealed that chronic inflammation contributes to the development of AV calcification. Accumulating
infiltrated inflammatory cells are major sources of matrix
metalloproteinases and cysteine endoproteases, which degrade
collagen and elastin in the valvular extracellular matrix,
thereby resulting in valvular dysfunction and further activation
of osteoblastic differentiation.38 As a growing body of evidence
suggests that ER stress can lead to inflammation,28,31,39 ER
stress may also contribute to AV calcification via promotion
of inflammatory responses. We explored the role of the IRE1α
pathway, shown to be linked with JNK and NF-κB signaling.17,18 We found that JNK was activated by oxLDL, in agreement with previous reports,40 and that silencing IRE1α blocked
the phosphorylation of JNK. Furthermore, NF-κB nuclear
translocation induced by oxLDL could also be prevented by
IRE1α silencing. Both NF-κB and JNK activation induced the
expression of the inflammatory genes.28 Our data further suggest that IRE1α silencing could, in part, inhibit the oxLDLinduced inflammatory genes expression, in agreement with the
role of IRE1α/JNK and IRE1α/NF-κB, in inflammation.31
The NF-κB pathway regulates expression of a wide variety of genes that are involved in inflammatory responses.41
Moreover, it has been reported that NF-κB signaling mediates
Runx2 and osteocalcin expression, and promotes osteoblastic differentiation.20,42,43 In our experiment, we showed that
NF-κB inhibition significantly suppressed oxLDL-stimulated
inflammatory responses, and the expression of Runx2 and
osteocalcin as well. These results indicate that NF-κB activation mediates oxLDL-induced proinflammatory effects and
osteoblastic differentiation in VICs.
2352 Arterioscler Thromb Vasc Biol October 2013
Inhibition of ER Stress as a Therapeutic
Approach to AV Calcification
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Chemical chaperones, such as TUDCA and 4-PBA, enhance
the ER folding ability to prevent protein aggregation, and
thus have the ability to alleviate ER stress.27 In this study,
we found TUDCA and 4-PBA protected against oxLDLinduced ER stress in VICs. Concomitant with reduced ER
stress, these 2 structurally distinct ER stress inhibitors significantly suppressed osteoblastic differentiation and inflammatory responses induced by oxLDL in VICs, and therefore add
strength to the proposal that the oxLDL-triggered osteoblastic
differentiation and inflammation in VICs is, at least in part,
induced via ER stress.
Altogether, the results provide support to the hypothesis
that ER stress in AV leaflets is associated with AV calcification development. Furthermore, these implications raise a crucial question: whether reducing AV ER stress could be used
as a strategy to alter the progression of AV calcification. The
chemical chaperone TUDCA has been proven to be protective in various diseases, such as diabetes mellitus, obesity, and
atherosclerosis, via prevention of ER stress.25–27 We also found
that TUDCA protected against hypercholesterolemia-induced
AV calcification in animal models. TUDCA administration
significantly reduced leaflet ER stress activation, prevented
osteoblastic differentiation and inflammation, and attenuated
AV calcification development. These protective effects were
independent of serum lipid or glucose metabolism and were
demonstrated to act via inhibition of AV ER stress, further
implicating the involvement of ER stress in AV calcification
development.
The most striking result in the present study was that
TUDCA administration could significantly reduce the development of AV stenosis, which might lead to a novel therapeutic
approach. Several clinical trials indicate that ER stress–inhibitor chemical chaperones could improve liver and muscle
insulin sensitivity in obese subjects, while ER stress plays an
essential role in insulin resistance and diabetes mellitus development.44 Our study provides evidence that ER stress inhibitors might be protective against AV calcification in humans.
Many other clinical conditions other than hypercholesterolemia may contribute to the development of AV calcification.
Studies indicate that chronic kidney disease is closely associated with AV calcification; increased parathyroid hormone and
inflammation might contribute to this.45 Recently, Zhou et al46
reported that chronic kidney disease could lead to vascular ER
stress activation and cause insulin resistance. Although further
studies are required, this may provide additional evidence supporting our findings and suggests that ER stress may also play
an important role during chronic kidney disease–induced AV
calcification. Patients with bicuspid AV malformation develop
AV calcification and stenosis spontaneously at an early age.47
Local blood turbulence and inflammation may be the major
contributor to the development of AV calcification in bicuspid
AV malformation,48 and the role of ER stress in such a condition needs further investigation.
Evidence suggests that AV calcification possesses characteristics of arterial calcification, and both share similar epidemiological risk factors.49 Inflammation and osteoblastic
differentiation play a key role in the development of both
these conditions.50,51 In the absence of in vivo data, Masuda
et al14 and Saito et al16 reported that stearate and bone morphogenetic protein 2 stimulated osteoblastic differentiation in
vascular smooth muscle cells via activating ER stress. This is
similar to our study that shows that oxLDL triggers osteoblastic differentiation via ER stress–mediated PERK/ATF4/osteocalcin and IRE1α/XBP1s/Runx2 signaling in VICs. However,
arterial calcification and AV calcification development are
mediated through differential processes. In AV calcification,
VICs should first be activated by various stimuli to become
α-SMA–containing activated VICs, which are then further
differentiated into osteoblast-like VICs,4 whereas vascular
smooth muscle cells are already α-SMA positive and can be
directly stimulated into osteoblast-like cells.52 With respect to
these important differences between arterial calcification and
AV calcification, future studies are required to verify the links
between ER stress and arterial calcification in vivo.
Although LDL and oxLDL are closely associated with AV
calcification and CAVS development, recent randomized clinical trials of statins on CAVS led to conflicting results, which
did not show protective effects on CAVS development.53–55
In addition, recent studies indicate statin therapy for vascular calcification seemed to paradoxically accelerate vascular
calcification.56,57 In addition to the well-established lipid lowering and anti-inflammatory effects of statins, several reports
suggested that statins could also trigger ER stress.58–60 This
implies that induction of ER stress by statins may be one of
the mechanisms limiting their therapeutic efficacy in CAVS.
Moreover, our study showed that inhibition of ER stress did
not alter lipid profiles, suggesting this as a potential approach
to managing osteogenic differentiation, independent of classical risk factors.
Limitations
One limitation in our study is the limited number of human normal AV samples available. Our previous report demonstrated
that valve tissues of patients with cardiomyopathy receiving
heart transplantation strongly exhibit ER stress activation
because of severe heart failure,9 and therefore these samples
are unfortunately not suitable to serve as a negative control in
our study. Furthermore, we only used small interfering RNAs
against ER stress signaling to support the potential roles of
ER stress–mediated osteoblastic differentiation and inflammation in AV calcification in vitro. Because IRE1α or XBP1
deficiency both cause embryonic lethality in mice, and PERK
or ATF4 deficiency in mice spontaneously develop diabetes
mellitus or skeletal dysplasia, respectively,17 these potential
targets are thus unavailable or not suitable for in vivo study.
In summary, we demonstrate that ER stress is involved in
the pathogenesis of AV calcification. Specifically, oxLDL promotes osteoblastic differentiation and inflammation, at least
in part via activating ER stress, and ultimately promotes AV
calcification. Inhibition of ER stress markedly relieved ER
stress, inhibited osteoblastic differentiation and inflammation in VICs, and attenuated AV calcification development in
hypercholesterolemia animals. The proposed mechanism is
outlined in Figure XVIII in the online-only Data Supplement.
Cai et al ER Stress and Aortic Valve Calcification 2353
These results collectively indicate that ER stress may be a
novel target against AV calcification.
Acknowledgments
We acknowledge the extensive assistance with echocardiography
from Drs Xiaojun Bi, Han Chen, and Jing Zhao. We also thank Dr Jia
Wei for graphic assistance.
Sources of Funding
This work was supported by the National Natural Science
Foundation of China (81000097 and 31130031), the “973” Program
(2012CB518004), and the Zhejiang Provincial Natural Science
Foundation of China (LQ13H020003).
Disclosures
None.
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Significance
Aortic valve (AV) calcification and its subsequent calcified AV stenosis are associated with increased cardiovascular events and mortality
without effective therapy other than surgical or interventional AV replacement. The work presented in this report suggests that endoplasmic
reticulum (ER) stress signaling is involved in the development of AV calcification. The authors demonstrate that ER stress is activated in
calcified AV leaflets from human samples as well as animal models of AV calcification. This work indicates that ER stress directly contributes
to osteoblastic differentiation and inflammatory responses in valvular interstitial cells. Most importantly, ER stress inhibition effectively prevented AV calcification in animal models. ER stress inhibitors have already been approved by Food and Drug Administration, suggesting they
are conceivable treatment for patients with AV calcification. Further work would be needed to assess whether ER stress inhibition is a safe
and effective treatment strategy for patients with AV calcification.
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Endoplasmic Reticulum Stress Participates in Aortic Valve Calcification in
Hypercholesterolemic Animals
Zhejun Cai, Fei Li, Wei Gong, Wanjun Liu, Quanlu Duan, Chen Chen, Li Ni, Yong Xia,
Katherine Cianflone, Nianguo Dong and Dao Wen Wang
Arterioscler Thromb Vasc Biol. 2013;33:2345-2354; originally published online August 8, 2013;
doi: 10.1161/ATVBAHA.112.300226
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
The online version of this article, along with updated information and services, is located on the
World Wide Web at:
http://atvb.ahajournals.org/content/33/10/2345
Data Supplement (unedited) at:
http://atvb.ahajournals.org/content/suppl/2013/08/08/ATVBAHA.112.300226.DC1
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published
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Supplemental Material
Endoplasmic Reticulum Stress Participates in Aortic Valve
Calcification in Hypercholesterolemic Animals
Zhejun Cai, Fei Li, Wei Gong, Wanjun Liu, Quanlu Duan, Chen Chen, Li Ni,
Yong Xia, Katherine Cianflone, Nianguo Dong, Dao Wen Wang
Supplemental Figure I
Identification of porcine valvular interstitial cells (VICs). Cultured VICs were
identified by α-SMA and vimentin.
Supplemental Figure II
Echocardiographic images and echogenicity data of rabbits with different
interventions. (A) Echocardiographic images of control (left panels), HC+vitD2
(middle panel), and HC+vitD2+TUDCA aortic valves (right panels) after 12
weeks of intervention. (B) Echogenicity increased in aortic valves from
HC+vitD2 rabbits compared with the control group. TUDCA greatly attenuated
this effect (Control: n=6; HC+vitD2: n=7; HC+vitD2+TUDCA: n=7; **P<0.01 vs.
control; ##P<0.01 vs. HC+vitD2).
Supplemental Figure III
AV
myofibroblast
transition
of
rabbits
with
different
interventions.
Representative immunohistochemical staining of α-SMA in AV leaflets of
control group (A), HC+vitD2 group (B), and HC+vitD2+TUDCA group (C). Scale
bars: 200 μm. (D) TUDCA treatment markedly reduced number of
myofibroblastsin AV induced by HC+vitD2 diet in rabbits (Control: n=6;
HC+vitD2: n=7; HC+vitD2+TUDCA: n=7; **P<0.01 vs. control; ##P<0.01 vs.
HC+vitD2).
Supplemental Figure IV
AV myofibroblast transition of ApoE-/- mice with different interventions.
Representative immunohistochemical staining of α-SMA in AV leaflets of
control group (A), HC group (B), and HC+TUDCA group (C). Scale bars: 100
μm. (D) TUDCA treatment markedly reduced the number of myofibroblasts in
AV induced by HC diet in ApoE-/- mice (Control: n=15; HC: n=15;
HC+vitD2+TUDCA: n=15; **P<0.01 vs. control; ##P<0.01 vs. HC).
Supplemental Figure V
TUDCA inhibited AV ER stress activation and osteoblastic differentiation in
HC+vitD2 fed rabbits. (A) Representative immunoblotting image of ER stress
and osteoblastic differentiation markers of AV leaflets in rabbits with different
interventions. TUDCA significantly prevented ER stress activation (B), and
osteoblastic differentiation markers Runx2 and osteocalcin protein (C) and
mRNA (D) expression induced by 12 weeks of HC+vitD2 administration in
rabbits (**P<0.01 vs. control; ##P<0.01 vs. HC+vitD2).
Supplemental Figure VI
TUDCA administration reduced Runx2 expression in AV leaflets of HC+vitD2
fed rabbits. Representative immunohistochemical staining of Runx2 in AV
leaflets of control group (A), HC+vitD2 group (B), and HC+vitD2+TUDCA group
(C). Scale bars: 200 μm. (D)HC+vitD2 diet significantly induced AV Runx2
expression in rabbits, while TUDCA treatment markedly reduced the effect
(Control: n=6; HC+vitD2: n=7; HC+vitD2+TUDCA: n=7; **P<0.01 vs. control;
##P<0.01 vs. HC+vitD2).
Supplemental Figure VII
TUDCA administration reduces AV macrophage infiltration induced by
HC+vitD2 diet in rabbits. Macrophage (RAM11) staining in AV of control group
(A), HC+vitD2 group (B), and HC+vitD2+TUDCA group (C). Scale bars: 200 μm.
(D) HC+vitD2 diet significantly increased AV macrophage infiltration, while
TUDCA effectively suppressed this effect (Control: n=6; HC+vitD2: n=7;
HC+vitD2+TUDCA: n=7; **P<0.01 vs. control; ##P<0.01 vs. HC+vitD2).
Supplemental Figure VIII
OxLDL-induced ER stress in VICs is cytosolic [Ca2+]i dependent. (A)
Immunoblotting for ER stress markers of VICs exposed to oxLDL at different
concentrations. (B) Blots were scanned and relative expression levels were
normalized against β-actin. OxLDL-induced ER stress in VICs in a dose
dependent manner. (C) Incubation with oxLDL increased cytosolic [Ca2+]i to
1.73 fold vs. control. (D) Immunoblotting for ER stress markers of VICs
exposed to oxLDL and calcium ionophore A23187, with or without calcium
chelator BAPTA-AM pretreatment. (E) As with oxLDL, A23187 triggered ER in
VICs, while BAPTA-AM abolished the ER stress-stimulating effects of A23187
and oxLDL in VICs (n=3 for each experiment; *P<0.05 vs. control; **P<0.01 vs.
control; ##P<0.01 vs. oxLDL; ††P<0.01 vs. A23187).
Supplemental Figure IX
Silencing efficacy of indicated siRNAs. SiRNAs against PERK (A), ATF4 (B),
IRE1α (C), and XBP1 (D) reduced expression of the indicated proteins.
Supplemental Figure X
IRE1α signaling mediates oxLDL-induced inflammatory gene expression in
VICs. IRE1α silencing suppressed oxLDL-induced IL-6, IL-8 and MCP-1
mRNA expression (n=3 for each experiment; ScrsiRNA indicates scrambled
siRNA which served as control; **P<0.01 vs. ScrsiRNA; ##P<0.01 vs.
oxLDL+ScrsiRNA).
Supplemental Figure XI
JNK and NF-κB signaling mediate oxLDL-induced inflammation and
osteoblastic differentiation in VICs. Pretreatment of VICs with JNK inhibitor
SP600125
or
NF-κB
inhibitor
BAY
11-7082
markedly
suppressed
oxLDL-induced IL-6 (A), IL-8 (B), and MCP-1 (C) mRNA expression. (D)
NF-κB inhibitor BAY 11-7082 significantly inhibited oxLDL-induced Runx2 and
osteocalcin expression in VICs (n=3 for each experiment; **P<0.01 vs. control;
#P<0.05 vs. oxLDL; ##P<0.01 vs. oxLDL).
Supplemental Figure XII
4-PBA attenuates ER stress induced by oxLDL in VICs. Immunoblotting
showed that 4-PBA pretreatment markedly inhibited oxLDL-induced ER stress
in VICs (n=3 for each experiment; **P<0.01 vs. control; ## P<0.01 vs. oxLDL).
Supplemental Figure XIII
4-PBA attenuates osteoblastic differentiation induced by oxLDL in VICs.
Immunoblotting
showed
that
4-PBA
pretreatment
markedly
inhibited
oxLDL-induced Runx2 and osteocalcin expression in VICs (n=3 for each
experiment; **P<0.01 vs. control; ##P<0.01 vs. oxLDL).
Supplemental Figure XIV
4-PBA attenuates oxLDL-induced JNK and NF-κBactivation in VICs. TUDCA
prevented JNK phosphorylation (A) and NF-κB p65 nuclear translocation (B) in
VICs (n=3 for each experiment; **P<0.01 vs. control; ##P<0.01 vs. oxLDL).
Supplemental Figure XV
TUDCA prevents oxLDL-induced NF-κB p65 nuclear translocation in VICs.
Representative immunocytofluorescence staining for NF-κB p65 in VICs.
TUDCA pretreatment markedly attenuated NF-κB p65 nuclear translocation
stimulated by oxLDL.
Supplemental Figure XVI
ER stress inhibition attenuates oxLDL-induced inflammation in VICs. TUDCA
(A) and 4-PBA (B) preincubation could both markedly down-regulate mRNA
expression of IL-6, IL-8, and MCP-1 stimulated by oxLDL (n=3 for each
experiment; **P<0.01 vs. control; ##P<0.01 vs. oxLDL).
Supplemental Figure XVII
ER stress inhibition prevents oxLDL-induced mineralization in VICs. (A)
TUDCA and 4-PBA could both reduced mineralization (A) and ALP (B) activity
induced by 7-day incubation of oxLDL in the presence of 2mmol/l
β-glycerophosphate, 100 nmol/l dexamethasone, and50 μg/ml ascorbic
acid.(C) Alizarin staining for calcification nodule formation in VIC cells with
different interventions (n=3 for each experiment; **P<0.01 vs. control;
##P<0.01 vs. oxLDL).
Supplemental Figure XVIII
The proposed mechanisms of ER stress promotion of AV calcification. OxLDL
increase cytosolic [Ca2+]i to trigger ER stress in VICs. This leads to the
activation of PERK/ATF4 and IRE1α/XBP1s signaling branches of ER stress to
induce osteocalcin and Runx2 expression, and promotes osteoblastic
differentiation. Meanwhile, activation of the IRE1α pathway promotes
expression of inflammatory response markers via JNK and NF-κB signaling.
The enhanced inflammatory responses and osteoblastic differentiation could
then accelerate AV calcification development.
Supplemental Table I
Clinical characteristics of CAVS patients undergoing AV replacement
Patient
1
2
3
4
5
6
7
8
9
10
Age
55
56
62
58
65
53
68
63
72
70
Female
Male
Male
Male
Male
Female
Male
Male
Male
Male
NYHA class
3
2
2
3
3
2
3
2
3
3
Mean pressure gradient(mmHg)
47
25
33
50
52
38
55
49
53
50
Smoking
no
yes
Yes
yes
yes
no
yes
yes
yes
no
Diabetes
no
no
No
no
no
no
yes
no
no
no
Hypertension
no
no
No
yes
yes
no
yes
yes
yes
yes
2.53
2.38
2.79
3.12
2.88
2.19
3.79
3.27
2.97
3.26
Gender
LDL cholesterol (mmol/l)
Supplemental Table II
Serum lipid profiles and glucose levels in animals at baseline
ApoE-/- Mice
Rabbits
Control
HC+vitD2
HC+vitD2+TUDCA
Control
HC
HC+TUDCA
n=6
n=7
n=7
n=15
n=15
n=15
Glucose (mmol/l)
5.67±0.73
5.52±0.89
5.73±0.79
9.02±0.38
8.96±0.48
8.87±0.60
TC (mmol/l)
1.28±0.19
1.35±0.23
1.34±0.28
2.64±0.22
2.58±0.17
2.72±0.23
LDL-c (mmol/l)
0.50±0.21
0.52±0.28
0.48±0.20
1.23±0.17
1.26±0.12
1.24±0.16
TG (mmol/l)
0.75±0.22
0.70±0.19
0.73±0.24
0.46±0.04
0.48±0.05
0.50±0.04
TC: total cholesterol; TG: triglyceride; LDL-c: LDL cholesterol
Data are presented as mean ± SD.
Supplemental Table III
Serum calcium, phosphate, and PTH levels in rabbits after different
interventions
Control
HC+vitD2
HC+vitD2+TUDCA
(n=6)
(n=7)
(n=7)
Calcium (mg/dl)
13.4±0.4
16.7±0.6**
16.5±0.6n.s
Phosphate (mg/dl)
4.6±0.2
8.7±0.4**
8.3±0.4n.s.
PTH (ng/l)
17.8±0.4
15.3±0.5**
15.9±0.3n.s.
Data are presented as mean ± SD. ** P<0.01 vs. control; n.s. not significant vs.
HC+vitD2. PTH: parathyroid hormone.
Supplemental Methods
Human AV samples
This study complied with the Declaration of Helsinki, and was approved by
the Review Board of Tongji Hospital and Tongji Medical College, and the
subjects recruited to the study provided written informed consent. We obtained
calcified AV from ten patients undergoing valve replacement surgery due to
symptomatic, severe CAVS without moderate or severe aortic regurgitation or
mitral valve diseases. In addition, patients with a history of myocardial
infarction, renal insufficiency were excluded (Detailed characteristics were
listed in Supplemental Table I). Four normal AV were from age-matched traffic
accident victims. Tissue samples were obtained and kept frozen in liquid
nitrogen and then stored at -80°C until use.
Experimental animal model
All animal experimental protocols complied with the Guide for the Care and
Use of Laboratory Animals published by the US National Institutes of Health
(NIH Publication No. 85-23, revised 1996). The animal studies were approved
by the Institutional Animal Research Committee of Tongji Medical College.
For experiments in rabbits, male New Zealand White rabbits weighing
2.5-3.0 kg were obtained from the Experimental Animal Center of Tongji
Medical College (Wuhan, China) and were housed at the animal care facility of
Tongji Medical College at 25°C with 12/12 h light/dark cycles. The method to
induce AV calcification was applied as described previously 1. After initial
quarantine, rabbits were randomly assigned into three groups and received
different diet feeding for a 12-week protocol: control group (n=6) receiving
normal diet, HC+vitD2 group (n=7) receiving 0.5% cholesterol plus vitamin D2
50,000 IU/day, and HC+vitD2+TUDCA group (n=7) receiving HC+vitD2 diet
supplemented with 50mg/kg/day TUDCA dissolved in water. Vitamin D2 and
TUDCA were supplied by oral gavage. Animals without TUDCA treatment
received equal amount of vehicle by oral gavage.
For experiments in mice, 8-week old ApoE-/- mice weighing 18-22g were
purchased from Vital River Laboratory Animal Technology Co. Ltd., China, and
were housed at the animal care facility of Tongji Medical College at 25°C with
12/12 h light/dark cycles. AV calcification was generated by a 24-week protocol
as described previously
2
. After initial quarantine, mice were randomly
assigned into three groups: control group (n=15) receiving normal chow, HC
group (n=15) receiving 0.25% cholesterol diet, and HC+TUDCA group (n=15)
receiving HC diet supplemented with 0.5g/kg/day TUDCA by oral gavage.
Animals without TUDCA treatment received equal amount of vehicle (drinking
water) by oral gavage.
Isolation of LDL and preparation of oxLDL
LDL (density=1.019-1.063 g/ml) were isolated from pooled human plasma
by sequential ultracentrifugation as previously described 3. For oxidation, LDL
was incubated with 10 mmol/l CuSO4 for 18 h at 37°C and terminated by
adding 1 mmol/l EDTA. OxLDL was then dialyzed, sterilized by filtering, and
diluted to 1 mg/ml in PBS. The prepared oxLDL was stored at 4°C and used
within 2 weeks.
Cell culture and treatment
VICs were isolated from AV leaflets of adult pigs from a local
slaughterhouse by collagenase digestion as previously described
4, 5
. The
VICs phenotype was identified by immunocytofluorescence with antibodies
against α-SMA (1:50 dilution, Boster, China) and vimentin (1:50 dilution, Boster)
(Supplemental Figure I). All experiments were performed with cells in
passages 2-4. After confluence, cells were incubated with oxLDL at the
concentrations for 12 h. If needed, pharmacological reagents, including 10
μmol/l BAPTA-AM (Sigma-Aldrich, St. Louis, MO), 10 μmol/l SP600125 (Merck,
Germany), 10 μmol/l BAY 11-7082 (Merck), 1 μmol/l TUDCA (Calbiochem,
Germany), and 2.5 mmol/l 4-PBA (Calbiochem) were added 1 h prior to the
addition of 100 μg/ml oxLDL or 5 μmol/l A23187 (Sigma-Aldrich). Cells were
starved in serum-free DMEM for 6 h before experiments. For mineralization
experiments, VICs were seeded in six-well plates. After 70% confluence, cells
were incubated with indicated interventions in DMEM supplemented with 5%
FBS,
2
mmol/l
β-glycerophosphate
(Sigma-Aldrich),
100
nmol/l
dexamethasone (Sigma-Aldrich), and 50 μg/ml ascorbic acid (Sigma-Aldrich)
for 7 days.
Cell transfection of siRNAs
SiRNAs against indicated proteins were a pool of three sequences
provided by Ribobio, China, which were designed against Sus scrofa PERK
(XM_003124925.1), Sus scrofa IRE1α (XM_003131282.1), Sus scrofa ATF4
(NM_001123078.1), and Sus scrofa XBP1 (NM_001142836.1). Scrambled
siRNAs (Ribobio) were used as normal control. After 30-40% confluence, VICs
were transfected with scrambled or indicated siRNAs using Lipofectamine
2000 (Invitrogen, Carlsbad, CA) and Opti-MEM (Gibco, Carlsbad, CA)
according to manufacturer’s recommendations, and changed the medium after
6 h.
Immunoblotting
Tissue samples and cells were homogenized and subsequentely
performed for western blotting as previously described
6
. The following
antibodies were used: p-IRE1α (1:2000 dilution, PA1-16927) from Pierce,
Rockford, IL; p-PERK (1:500 dilution, sc-32577), PERK (1:1000 dilution,
sc-13073), IRE1α (1:1000 dilution, sc-20790), C/EBP homologous protein
(1:500 dilution, sc-575) , X-box binding protein 1 (XBP1, 1:500 dilution,
sc-7160), Runx2 (1:1000 dilution, sc-10758), osteocalcin (1:1000 dilution,
sc-30045), p-JNK (1:500 dilution, sc-6254), lamin B1 (1:1000 dilution,
sc-56145) and β-actin (1:1000 dilution, sc-47778) from Santa Cruz
Biotechnology, Santa Cruz, CA; JNK (1:500 dilution, BS1544), Inhibitor of
NF-κB α (IκBα, 1:500 dilution, BS1190) and NF-κB p65 (1:500 dilution, BS1257)
from Bioworld, Atlanta, GA. β-actin was used as normalization for total protein
or cytosolic protein determination, and lamin B1 for nuclear protein
determination. In phosphorylation induction assay, indicated total protein
expression was used as normalization. Bands were quantified by densitometry
using Quantity One Software (Bio-Rad, Hercules, CA).
Measurement of cytosolic [Ca2+]i
Cytosolic [Ca2+]i was determined using Fluo-3/AM (Invitrogen) under a
fluorometer as previously described
7, 8
. Briefly, VICs were incubated with a
loading solution containing HEPES-buffered saline (HBS; 135 mmol/l NaCl,
5.9 mmol/l KCl, 1.2 mmol/l MgCl2, 1.5 mmol/l CaCl2, 11.6 mmol/l HEPES and
11.5 mmol/l glucose, pH=7.3) supplemented with 5 mmol/l Fluo-3/AM, 0.02%
pluronic F-127 (Sigma-Aldrich) and 1 mg/ml BSA for 30 min and then
incubated in the loading solution without Fluo-3/AM for 30 min. The
fluorescence was recorded at 495 nm and 525 nm for excitation and emission
wavelengths, respectively. Results were normalized on protein levels, and
expressed in ratio of control as previously reported 7, 8.
Immunocytofluorescence
Cells grown on cover glass slides were washed with PBS and fixed in 4%
paraformaldehyde in PBS for 10 min and then in 0.25% Triton-X 100 for 10 min.
After blocking with 3% BSA for 30 min, cells were incubated in indicated
antibodies at 4°C overnight. The slides were then revealed with appropriate
FITC or Cy3-conjugated secondary antibodies for 1 h at 37°C and DAPI
(Sigma-Aldrich) for nuclear counter staining, and were subsequently visualized
under a Nikon DXM1200 fluorescence microscope. Image-Pro Plus (Media
Cybernetics, Bethesda, MD) was applied for image merging.
Calcium content determination in VICs
Calcium deposition in the plates was quantified as previously described 9.
Cells were decalcified by 0.6 mol/l HCl solution, and the supernatant was
subsequently
subjected
to
calorimetrical
quantification
using
the
ocresolphthalein method. The result was calibrated by protein amount and
then normalized to the controls.
ALP activity
ALP activity was measured using p-nitrophenyl phosphate as the substrate
using the commercial kit from Wako, Japan.
Quantitative PCR for mRNA analysis
Total RNA was isolated and reverse transcribed to cDNA as previously
described 10. The cDNA was subsequently subjected to quantitative PCR using
SYBR Premix Ex Taq kit (TAKARA, Japan) and specific primers for rabbit
Runx2 (F: 5’- GCA GTT CCC AAG CAT TTC ATC-3’ and R: 5’- GTG TAA GTA
AAG GTG GCT GGA TA-3’); osteocalcin (F: 5’ GCT CAC CCT TCG TGT CCA
AG-3’ and R: 5’- CCG TCG ATC AGT TGG CGC-3’); β-actin (S: 5’- CAC CCT
GAT GCT CAA GTA CC-3’ and R: 5’- CGC AGC TCG TTG TAG AAG G-3’);
porcine IL-6 (F: 5’-ATC AGG AGA CCT GCT TGA TG-3’ and R: 5’-TGG TGG
CTT TGT CTG GAT TC-3’); IL-8 (F: 5’-TCC TGC TTT CTG CAG CTC TC-3’
and R: 5’-GGG TGG AAA GGT GTG GAA TG-3’); MCP-1 (F: 5’-GTC ACC
AGC AGC AAG TGT C-3’ and R: 5’-CCA GGT GGC TTA TGG AGT C-3’); and
β-actin (F: 5’-GAC CTG ACC GAC TAC CTC-3’ and R: 5’-GCT TCT CCT TGA
TGT CCC-3’). Results were normalized to β-actin expression and calculated
by the ΔΔCt method.
Echocardiography
Transthoracic echocardiography was performed at baseline and at 12
weeks of respective treatment. Animals were sedated with intramuscular
injection of 30 mg/kg ketamine and 3.5 mg/kg xylazine. A comprehensive
echocardiographic study, including two-dimensional imaging, Doppler imaging
and M-mode imaging, and subsequent data calculations were performed by
operator blinded to the treatment assignment as described previously using
the Vevo 2100 system
11
. A semi-quantitative index (range 0-3) for estimating
rabbit AV echogenicity, an index of valve thickening and calcification, as
follows: 0-absent, 1-mild; 2-moderate, with preserved motility/opening;
3-severe, with decreased motility/opening, was applied as previously reported
11
. Data were analyzed on a blinded fashion.
Histopathology and immunohistochemistry
After final transthoracic echocardiographic assessment, animals were
euthanized by intravenous injection of a lethal dose of penthobarbital sodium
(100 mg/kg), and AV were then rapidly harvested, rinsed in PBS, fixed in 4%
paraformaldehyde in PBS, and embedded in paraffin for histopathological
analyses. 3 μm sections were subsequently stained with alizarin red.
Immunohistochemical staining were performed using the following antibodies:
KDEL (1:100 dilution, Santa Cruz, sc-33806 and sc-58774 identifying multiple
proteins which indicate ER stress activation), CHOP (1:100 dilution, Santa
Cruz, sc-575 and sc-7351), α-SMA (1:100 dilution, Sigma-Aldrich, sc-58774),
osterix (1:100 dilution, Santa Cruz, sc-22536-R), Runx2 (1:100 dilution, Santa
Cruz, sc-10758 and sc-8566), osteocalcin (1:100 dilution, Santa Cruz,
sc-30045), F4/80 (1:100 dilution, Santa Cruz, sc-25830), RAM11 (1:100
dilution, Dako, Denmark, M0633). Image-Pro Plus (Media Cybernetics) was
applied to determine quantitative results (positive staining area/total AV area)
for 2 sections of each AV leaflet, and at least 2 of the 3 AV leaflets for each
animal were analyzed. Investigators performing the analyses were blinded to
the study groups.
Determination of serum cholesterol, LDLs, triglyceride, and glucose
levels
Animals underwent a 14-15 h fast before blood samples were collected.
Serum cholesterol, LDLs, triglyceride, and glucose levels were determined
using indicated kits from Biosino, China, according to the manufacturer’s
instructions.
Statistical analysis
All values are presented as mean±SD. After confirming that all variables
were normally distributed using the Kolmogorov-Smirnov test, statistical
differences were evaluated by Student t-test or ANOVA followed by
Bonferroni’s multiple comparison test. P<0.05 was accepted as statistically
significant.
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