basic research
http://www.kidney-international.org
& 2013 International Society of Nephrology
see commentary on page 984
Phosphate-induced autophagy counteracts vascular
calcification by reducing matrix vesicle release
Xiao-Yan Dai1,4, Ming-Ming Zhao1,4, Yan Cai1, Qing-Cong Guan2, Ying Zhao3, Youfei Guan1, Wei Kong1,
Wei-Guo Zhu3, Ming-Jiang Xu1 and Xian Wang1
1
Department of Physiology and Pathophysiology, School of Basic Medical Science, Peking University Health Science Center,
Key Laboratory of Molecular Cardiovascular Science, Ministry of Education, Beijing, China; 2Renal Division, Taizhou Municicpal Hospital,
Taizhou, China and 3Department of Biochemistry and Molecular Biology, School of Basic Medical Science, Peking University Health
Science Center, Beijing, China
Autophagy is a dynamic and highly regulated process of
self-digestion responsible for cell survival and reaction to
oxidative stress. As oxidative stress is increased in uremia and
is associated with vascular calcification, we studied the role of
autophagy in vascular calcification induced by phosphate.
In an in vitro phosphate-induced calcification model of
vascular smooth muscle cells (VSMCs) and in an in vivo model
of chronic renal failure, autophagy was inhibited by the
superoxide dismutase mimic MnTMPyP, superoxide
dismutase-2 overexpression, and by knockdown of the
sodium-dependent phosphate cotransporter Pit1. Although
phosphate-induced VSMC apoptosis was reduced by an
inhibitor of autophagy (3-methyladenine) and knockdown
of autophagy protein 5, calcium deposition in VSMCs was
increased during inhibition of autophagy, even with the
apoptosis inhibitor Z-VAD-FMK. An inducer of autophagy,
valproic acid, decreased calcification. Furthermore,
3-methyladenine significantly promoted phosphate-induced
matrix vesicle release with increased alkaline phosphatase
activity. Thus, autophagy may be an endogenous protective
mechanism counteracting phosphate-induced vascular
calcification by reducing matrix vesicle release. Therapeutic
agents influencing the autophagic response may be of
benefit to treat aging or disease-related vascular calcification
and osteoporosis.
Kidney International (2013) 83, 1042–1051; doi:10.1038/ki.2012.482;
published online 30 January 2013
KEYWORDS: apoptosis; autophagy; chronic renal failure;
hyperphosphatemia; oxidative stress
Correspondence: Ming-Jiang Xu, Department of Physiology and Pathophysiology, School of Basic Medical Science, Peking University Health Science
Center, Key Laboratory of Molecular Cardiovascular Science, Ministry of
Education, Beijing 100191, China. E-mail: mingjiangxu@bjmu.edu.cn
4
These authors contributed equally to this work.
Received 7 January 2012; revised 2 November 2012; accepted 9
November 2012; published online 30 January 2013
1042
Macroautophagy (hereafter referred to as autophagy) is a
dynamic and highly regulated process of self-digestion. It is a
highly conserved cellular process responsible for removal or
recycling of long-lived proteins and organelles, and provides
cells with an alternative source of nutrients from the
reuse of cellular proteins and organelles.1,2 This lysosomal
degradation pathway is essential for cell survival, differentiation, and development, as well as the cellular response to
stress, and thus is associated with neurodegenerative diseases,
cancer, heart disease,3,4 and arteriosclerosis.5,6 Limited
autophagy in response to nutrient starvation has a survival
function, and specific removal of damaged mitochondria by
autophagy can prevent the activation of apoptotic pathways.7
However, in some systems, induced autophagy can
contribute to or enhance the apoptotic response.8
Vascular calcification is a major risk factor of cardiovascular mortality, particularly in patients with end-stage
renal disease.9 Hyperphosphatemia, manifested during
chronic renal failure (CRF) and subsequent dialysis, is
highly associated with the extent of vascular calcification
and contributes directly to high morbidity and mortality in
vascular disease.10 Phosphate (Pi) level was found to be
important in vascular calcification in clinical trials10 and
in vitro11 and in vivo12 experimental models. The potential
mechanisms mediating Pi-induced matrix calcification of
vascular smooth muscle cells (VSMCs) include osteogenic/
chondrogenic conversion, apoptosis, matrix vesicle (MV)
release, and matrix remodeling.13,14 Recently, we found that a
high Pi level induced the production of mitochondrial
O2 and activated NF-kB to induce vascular calcification in
both bovine aortic smooth muscle cells (BASMCs) and a rat
model of CRF.15,16
Many studies have highlighted the important contribution
of mitochondrial reactive oxygen species (ROS), especially
superoxide (O2 ), to inducing autophagy.17–19 ROSmediated basal autophagy has an important role in
maintaining chondrocyte and osteoblast/osteocyte survival
and terminal differentiation, as well as regulating bone
growth.20 However, whether autophagy is involved in
Pi-induced vascular calcification remains to be elucidated.
Kidney International (2013) 83, 1042–1051
X-Y Dai et al.: Autophagy counteracts vascular calcification
Here, we examined the induction of autophagy by Piincreased ROS levels, and the role of autophagy in Pi-induced
calcification in VSMCs and a rat model of CRF.
RESULTS
High Pi promotes autophagy in VSMCs
To determine whether autophagy is involved in Pi-induced
vascular calcification, we first examined the effect of Pi on
autophagosome formation in VSMCs. Pi dose dependently
(1.5–3 mmol/l) increased the level of the lipid-conjugated
form of the autophagosome marker light-chain 3-II (LC3II)
in BASMCs (Figure 1a). With lysosome-dependent degradation of LC3II blocked by chlorquinol (25 mmol/l), Pi further
increased the LC3II level (Figure 1b). Increased accumulation
of LC3 puncta tagged with green fluorescent protein (GFP)
indicated Pi induction of autophagosomes (Figure 1c), and
the proportion of cells with GFP–LC3 puncta was sustained
from 0.5 to 7 days with Pi treatment (Figure 1d). Electron
microscopy of typical autophagic structures in BASMCs gave
direct evidence of the autophagy activation, and increased
autophagic structures were observed in cells exposed to Pi for
12 h (Figure 1e). Increased autophagy was observed in
parallel in human aortic VSMCs (HA-VSMCs; Figure 1f
and Supplementary Figure S1 online). In addition, immunofluorescence analysis of LC3 puncta revealed autophagosome
formation in renal artery walls of a patient with CRF
(Figure 1g). Thus, high Pi promoted autophagy in both
animal and HA-VSMCs.
ROS mediate Pi-induced autophagy in VSMCs
Mitochondrial ROS production contributes to autophagosome formation in many conditions, and the superoxide
dismutase mimic MnTMPyP reduces Pi-induced calcification
by eliminating mitochondrial ROS.15 Here, Pi significantly
increased mitochondrial ROS level in BASMCs (Figure 2a).
MnTMPyP (25 mmol/l), as well as SOD2 overexpression,
significantly decreased Pi-increased LC3II level in BASMCs
(Figure 2b and c). These results were further confirmed
by MnTMPyP, decreasing the Pi-increased formation of
GFP–LC3 puncta from 47.2 to 20.3% in BASMCs (Figure 2d).
Therefore, ROS generation mediated Pi-induced autophagy
in vitro. In addition, MnTMPyP reduced LC3 level
(Figure 2e) and mitochondrial ROS level (Figure 2f) in
abdominal aortas of CRF rats with high serum Pi level
(2.73±0.10 mmol/l in control group vs. 5.50±0.36 mmol/l
in CRF group, Po0.05). Indirect immunofluorescence
analysis of LC3 puncta in both calcified and non-calcified
regions of the aorta (Supplementary Figure S2 online) further
showed that MnTMPyP decreased autophagosome formation
in the abdominal aortic wall of CRF rats (Figure 2f).
Type III sodium-dependent Pi cotransporter Pit1 mediates
Pi-induced autophagy
HA-VSMCs were transfected with scramble small interfering
RNA (siRNA) or Pit1 siRNA for 48 h; knockdown of Pit1
expression was confirmed by reverse transcriptase PCR
Kidney International (2013) 83, 1042–1051
basic research
(Figure 3a) and western blot analyses (Figure 3b). Pi
increased LC3II level in scramble siRNA–treated HA-VSMCs,
and Pit1 knockdown significantly reduced Pi-increased LC3II
level (Figure 3c). Similarly, Pit1 knockdown significantly
decreased Pi-induced calcium deposition (Figure 3d). Therefore, Pit1 mediated Pi-induced autophagy and calcification in
VSMCs.
Inhibition of Pi-induced autophagy aggravates calcification
To address the potential role of autophagy in Pi-induced
calcification in VSMCs, we first used 3-MA (5 mmol/l), a
pharmacological inhibitor of autophagy. 3-MA attenuated
the Pi-increased LC3II level (Figure 4a) and formation of
LC3 puncta (43% reduction) (Figure 4b). To confirm the
pharmacological results of 3-MA, siRNA knockdown of
autophagy protein 5 (Atg5) expression in rat VSMCs
(Figure 4c and Supplementary Figure S3 online) resulted in
a significant reduction of LC3 puncta in Pi-treated rat
VSMCs (Figure 4d).
Alizarin red S staining (Figure 5a) and calcium content
assay (Figure 5b) revealed that 3-MA increased calcium
deposition in BASMCs pretreated with Pi (3 mmol/l) at 3, 7,
and 10 days. Similarly, in HA-VSMCs (Figure 5c) and rat
VSMCs (Figure 5d), 3-MA significantly increased Pi-induced
calcium deposition. These findings were further confirmed by
siRNA knockdown of Atg5, which significantly augmented
Pi-induced calcium deposition in both primary rat VSMCs
(Figure 5e) and the rat SMC cell line A7r5 (Figure 5f).
Furthermore, ex vivo experiments of rat aortic rings showed
that 3-MA aggravated Pi-induced calcification (Figure 5g). In
contrast, valproic acid (1 mmol/l), a pharmacological inducer
of autophagy, significantly ameliorated Pi-induced calcium
deposition in both cultured rat aortic rings (Figure 6a) and
BASMCs (Figure 6b). Therefore, autophagy ameliorated
Pi-induced vascular calcification.
The pro-calcification effect of autophagy inhibition does not
involve ROS-induced apoptosis
Autophagy induction under cellular stress may contribute to
cell apoptosis or be a mechanism for cell survival, and VSMC
apoptosis is one of the mechanisms of Pi-promoted vascular
calcification.21,22 As expected, Pi increased the activity of
apoptosis marker–cleaved caspase-3 (Figure 7a) and caspase3/7 (Figure 7b). These increases were reversed by siRNA
knockdown of Atg5 and MnTMPyP in rat VSMCs.
Consistently, both 3-MA and MnTMPyP reduced the Piincreased apoptosis (indicated by terminal transferase dUTP
nick-end labeling staining) (Figure 7c and Supplementary
Figure S4 online) and caspase-3/7 activity (Figure 7d) in
BASMCs. Interestingly, MnTMPyP and autophagy inhibition
by Atg5 siRNA knockdown or 3-MA had a synergistic
effect on reducing caspase 3/7 activation and apoptosis
(Figure 7b–d). However, the apoptosis inhibitor Z-VAD-FMK
(20 mmol/l) significantly ameliorated Pi-induced calcification; 3-MA still significantly aggravated Pi-induced calcification, even in the presence of Z-VAD-FMK (Figure 7e).
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X-Y Dai et al.: Autophagy counteracts vascular calcification
a
b
BASMCs
Pi (mM)
LC3I
LC3II
0
1.5
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β-Actin
LC3II protein level
*
3
*
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1
d
BASMCs
Ctrl
Pi (3 mM)
GFP–LC3
Control
*
8
*
BASMCs
Pi (3 mM)
% Cells with GFP–LC3 puncta
LC3II protein level
4
e
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LC3I
LC3II
β-Actin
c
BASMCs
Pi
Chq
Ctrl
3
6
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*
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80
Control
*
Pi (3 mM)
*
60
*
*
0.5 Days
1 Day
40
20
f
Control
3 Days
HA-VSMCs
7 Days
Pi (3 mM)
GFP-LC3
1 μm
1 μm
g
2 μm
Human renal artery wall
Donor
CKD
2 μm
Figure 1 | Phosphate (Pi) induces autophagy in vascular smooth muscle cells (VSMCs). (a) Western blot analysis of light-chain 3-II (LC3II)
levels in bovine aortic smooth muscle cells (BASMCs) treated with Pi at the indicated concentrations for 12 h and (b) in BASMCs pretreated with
chloroquine (Chq, 25 mmol/l) for 30 min, then with Pi for 12 h. (c) Confocal microscopy of green fluorescent protein (GFP) in BASMCs transfected
with GFP–LC3 plasmids, cultured for 36 h, and then treated with Pi for 12 h. Autophagosomes are indicated by fluorescence puncta.
(d) Percentage of BAMSCs with GFP–LC3 puncta after Pi treatment for the indicated durations. *Po0.05 versus corresponding control.
(e) Electron microscopy of autophagic structures (arrows) in cells exposed to Pi for 12 h. n ¼ 3–4, one representative image is shown.
(f) Confocal microscopy of GFP–LC3 puncta in human aortic VSMCs (HA-VSMCs) transfected with GFP–LC3 plasmids and treated with Pi
(3 mmol/l) for 12 h. (g) Immunofluorescence analysis of LC3 puncta in renal artery wall from human organ donor and recipient with
chronic kidney disease during renal transplantation. CKD, chronic kidney disease.
Thus, the pro-calcification effect of autophagy inhibition
may not be associated with ROS-induced apoptosis.
Autophagy inhibition increases Pi-induced MV release
MVs were isolated from cell lysates by collagenase digestion.
Pi significantly increased MV release, from 0.9 to 5.5 mg, and
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inhibition of autophagy by 3-MA further promoted
Pi-induced MV release, from 5.5 to 9.6 mg, in BASMCs
(Figure 8a). Annexin II protein content was significantly
increased in MVs from calcified BASMCs, and 3-MA further
increased the content (Figure 8b). Furthermore, assay
of alkaline phosphatase (ALP) activity as shown by ELF 97
Kidney International (2013) 83, 1042–1051
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X-Y Dai et al.: Autophagy counteracts vascular calcification
a
b
BASMCs
*
0
3
MnTMPyP
LC3I
0
0
15
3 mM
25 μM
LC3II
Pi (3 mM)
LC3I
LC3II
5
Control
Pi
d
*
*
2.0
1.5
1.0
0.5
% Cells with GFP–LC3
puncta
BASMCs
Control
Pi+MnTMPyP
Pi
–
Ad-SOD2
+
–
+
β-Actin
2.5
LC3II protein level
10
BASMCs
Ad-GFP
β-Actin
LC3II protein level
Fluorescence intensity (a.u.)
20
c
BASMCs
Pi
*
1.5
*
1.0
0.5
60
*
*
40
20
GFP–LC3
Control
e
*
CRF
CRF+MnTMPyP
LC3
β-Actin
LC3 protein level
3
Control
*
2
1
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f
CRF
Anti-LC3
CRF+
MnTMPyP
% Cells with LC3 puncta
*
Mitochondrial ROS
CRF
*
30
20
10
Control
Control
Pi+
MnTMPyP
Pi
CRF
CRF+
MnTMPyP
CRF+MnTMPyP
Figure 2 | Reactive oxygen species (ROS) mediate phosphate (Pi)-induced autophagy in vascular smooth muscle cells (VSMCs).
(a) Confocal microscopy of fluorescence with control and Pi (3 mmol/l) induction in bovine aortic smooth muscle cells (BASMCs) incubated with
MitoTracker Red CM-H2XRos probe for 30 min to indicate mitochondrial ROS level. (b) Western blot analysis of light-chain 3 (LC3)I/II level in
BASMCs pretreated with MnTMPyP for 30 min, or (c) infected with adenovirus-expressing green fluorescent protein (GFP) or superoxide
dismutase 2 (SOD2) for 24 h, and then treated with Pi for 12 h. (d) Immunofluorescence and percentage of cells with GFP–LC3 puncta in BASMCs
treated with Pi (3 mmol/l) and/or MnTMPyP (25 mmol/l) for 12 h. (e) Western blot analysis of LC3 protein level in abdominal aortas of rats with
chronic renal failure (CRF) with control, vehicle (CRF), and CRF plus MnTMPyP (CRF þ MnTMPyP) treatment and (f) immunofluorescence of
LC3 puncta and MitoTracker Red CM-H2XRos probe (200 nmol/l) for mitochondrial ROS in rat abdominal aorta walls. n ¼ 3–5, *Po0.05.
phosphatase substrate activity indicated increased number of
ELF 97–positive MVs in calcified BASMCs, which could be
further increased by 3-MA treatment (Figure 8c). In addition,
ALP activity, considered a marker of MV maturation, was
Kidney International (2013) 83, 1042–1051
further increased by 3-MA treatment in calcified BASMCs
(Figure 8d), and the release of ALP into the MV fraction,
indicated by the ratio of MV ALP to total cellular ALP, was
also increased in 3-MA-treated cells (Supplementary
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si
R
N
A
Pit1/2 protein level
β-Actin
1.5
Scramble
LC3II protein level
3
0
3
Pit1 siRNA
0
0.5
2
1
*
*
*
2
40
20
1
Pi
Control
3-MA+Pi
200
Atg5
100
β-Actin
1.5
Control Pi
Control Pi
Scramble
Pit1 siRNA
Figure S5a and b online). Thus, the pro-calcification effect
of autophagy inhibition may contribute to its promotion of
MV release.
DISCUSSION
In this study, we showed that Pi-increased ROS levels
potentiated autophagy in cultured VSMCs and aortic walls
of a dietary adenine–induced rat CRF model, and the
sodium-dependent Pi cotransporter Pit1 mediated the Piinduced autophagy. Inhibition of autophagy by 3-MA or
siRNA knockdown of the autophage protein Atg5 significantly aggravated Pi-induced calcium deposition in vitro and
ex vivo. However, valproic acid, a pharmacological inducer of
autophagy, significantly ameliorated the increased calcification. The underlying mechanisms of the pro-calcification
effect of autophagy inhibition involved increased MV release
rather than cell apoptosis. Thus, targeting the autophagy
pathway may help to prevent or treat vascular calcification in
patients with end-stage renal disease.
As autophagy is linked to conditions such as neurodegenerative diseases, cancer, and cardiovascular diseases, it is a
prime target for developing therapeutic treatments for these
diseases.23 Evidence of the importance of hyperphosphatemia
as a major inducer of vascular calcification comes from
studies of genetic syndromes and diseases of renal insufficiency;13 increased Pi exposure results in the conversion of
Rat VSMCs
Rat VSMCs
Control Scramble Atg5 siRNA
*
Figure 3 | Type III sodium-dependent phosphate cotransporter
Pit1 mediates phosphate (Pi)-induced autophagy in vascular
smooth muscle cells (VSMCs). (a) Reverse transcriptase PCR analysis
of mRNA levels of Pit1 and Pit2 in human VSMCs (HA-VSMCs)
transfected with scramble or Pit1 siRNA for 48 h. (b) Western blot
analysis of Pit1/2 protein levels in HA-VSMCs transfected with
scramble or Pit1 siRNA for 48 h. (c) Light chain 3 (LC3)I/II protein
levels in HA-VSMCs transfected with scramble or Pit1 siRNA for 48 h,
then treated with Pi (3 mM) for 12 h. (d) Calcium content assay of HAVSMCs transfected with scramble or Pit1 siRNA for 48 h, then treated
with Pi (3 mM) for 10 days. n ¼ 3, *Po0.05.
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*
*
3 mM
300
*
*
3
1.0
0.5
*
50
% Cells with Lc3 puncta
Scramble
Pi
LC3-I
LC3-II
β-Actin
Pit1 siRNA
1.0
Atg5 protein level
Control
% Cells with
GFP–Lc3 puncta
β-Actin
1.0
*
BASMCs
*
60
LC3II
Pit1/2
0.5
BASMCs
Control
Pi 3-MA+Pi
LC3I
Lc3-II protein level
Pit2
Pi
t1
l
tro
C
on
Pit1
Calcium content
mg/g prot
Relative mRNA level
1.5
Sc
ra
m
bl
e
X-Y Dai et al.: Autophagy counteracts vascular calcification
*
*
40
30
20
10
Scramble Scramble Atg5 siRNA
+Pi
+Pi
Figure 4 | Phosphate (Pi)-induced autophagy is abolished by
autophagy inhibition. (a) Western blot analysis of light-chain 3
(LC3)I/II protein levels in bovine aortic smooth muscle cells (BASMCs)
pretreated with autophagy inhibitor 3-methyladenine (3-MA 5 mmol/
l) for 30 min, and then with Pi (3 mmol/l) for 12 h. (b) Percentage of
cells with green fluorescent protein (GFP)–LC3 puncta in BASMCs
treated with Pi (3 mmol/l) or 3-MA þ Pi for 12 h. (c) Western blot
analysis of protein level of autophagy protein 5 (Atg5) in rat vascular
smooth muscle cells (VSMCs) transfected with scramble or Atg5 small
interfering RNA (siRNA) for 48 h. (d) Percentage of GFP–LC3 puncta in
rat VSMCs transfected with scramble or Atg5 siRNA for 48 h, and then
with Pi for 12 h. n ¼ 3, *Po0.05.
contractile VSMCs to osteogenic/chondrogenic cells, cell
apoptosis, MV release, and matrix remodeling, followed by
vascular calcification.14 Here, we showed that autophagy
involved in Pi-induced vascular calcification. Elevated Pi level
could induce autophagy, as shown by the formation of
GFP–LC3 puncta, increased LC3II level, and electron microscopy of typical autophagic structures in cultured VSMCs and a
rat CRF model, as well as the human aortic wall.
Our previous report15 showed that elevated Pi level could
increase the production of mitochondrial ROS, and
scavenging intracellular O2 by MnTMPyP and SOD2 overexpression significantly decreased Pi-induced calcification
and autophagy. Thus, increased O2 level has an important
role in Pi-induced autophagy. Chen et al.17 also showed that
O2 is the major ROS-regulating autophagy. Pit1 was
previously found to have an important role in Pi-induced
calcification.24 We also demonstrated that Pit1 was the
dominant Pi cotransporter in VSMCs. SiRNA knockdown of
Pit1 significantly reduced the Pi-induced calcification and
autophagy. Thus, Pit1-mediated Pi transport and ROS
generation have a vital role in Pi-induced autophagy. High
Pi level may be a new inducer of autophagy, in addition to
starvation and H2O2, which may help in further exploring the
mechanisms of Pi-induced vascular diseases.
Autophagy is a multifunctional process involved in
various cellular activities23 and is essential for survival,
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X-Y Dai et al.: Autophagy counteracts vascular calcification
3-MA+Pi
Pi
3-MA
*
*
300
*
200
100
*
300
Calcium content mg/g prot
Pi
400
Calcium content mg/g prot
BASMCs
Calcium content mg/g prot
Control
Rat VSMCs 10 days
HA-VSMCs 10 days
BASMCs 7 days
Alizarin red S staining
*
200
100
Control 3 Days 7 Days 10 Days
Ctrl
Pi
*
300
200
*
100
3-MA 3-MA+Pi
Ctrl
Pi
3-MA 3-MA+Pi
Calcium content mg/g prot
Calcium content mg/g prot
150
A7r5 10 days
*
100
*
50
Control Pi
Scramble
150
*
100
*
50
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Pi
Scramble
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Atg5 siRNA
Control
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Atg5 siRNA
Calcium content mg/g prot
Rat aortic ring
Rat VSMCs 10 days
10
*
8
6
*
4
2
Control
Pi
3-MA 3-MA+Pi
Figure 5 | Blockage of autophagy exacerbates phosphate (Pi)-induced calcification. (a–d) Cells were treated with Pi (3 mmol/l) or Pi þ 3methyladenine (3-MA) (5 mmol/l) for the indicated days, and then alizarin red S staining (a) and calcium content assay were performed (b–d).
(e) Calcium content assay of rat vascular smooth muscle cells (VSMCs) and (f) A7r5 cells transfected with scramble or autophagy protein
5 (Atg5) small interfering RNA (siRNA) for 48 h, and then treated with Pi for 10 days; (g) rat aortic ring explants treated with Pi (3 mmol/l)
and/or 3-MA (5 mmol/l) for 7 days. n ¼ 3–5, *Po0.05. BASMCs, bovine aortic smooth muscle cells; HA-VSMCs, human aortic VSMCs.
BASMCs
*
3
Calcium content mg/g prot
Calcium content mg/g prot
Rat aortic ring
4
*
2
1
Ctrl
Pi
VPA
VPA+Pi
1000
*
*
800
600
400
200
Ctrl
Pi
VPA
VPA+Pi
Figure 6 | Autophagy inducer valproic acid (VPA) reduces
calcification. (a) Calcium content assay of rat aortic ring explants and
(b) bovine aortic smooth muscle cells (BASMCs) treated with phosphate
(Pi) (3 mmol/l) and/or VPA (1 mmol/l) for 7 days. n ¼ 3, *Po0.05.
differentiation and development, as well as the cell response
to stress.2,25 We wondered whether autophagy functionally
contributed to Pi-induced calcification. Autophagy
inhibition by both the pharmacological inhibitor 3-MA and
siRNA knockdown of Atg5 significantly aggravated
Pi-induced calcium deposition in both animal and human
aortic cells, including BASMCs, rat VSMCs, HA-VSMCs, and
the A7r5 cell line. As expected, the pharmacological
autophagy inducer valproic acid had a therapeutic effect on
Pi-induced calcification in BASMCs and rat aortic rings.
Thus, autophagy may be an endogenous protective
mechanism counteracting vascular calcification under
hyperphosphatemia. Further research is needed regarding
therapeutic agents that influence the autophagic response for
treating aging or disease-related vascular calcification.
We wondered how autophagy inhibition augmented
Pi-induced calcium deposition. VSMC apoptosis is an
important mechanism for Pi-promoted vascular calcification, and drugs that inhibit apoptosis can ameliorate
Kidney International (2013) 83, 1042–1051
calcification.21,22 Autophagy induction under cellular stress
may contribute to cell apoptosis or be a mechanism of cell
survival.26 The cytotoxic effects of autophagy may be
explained by the direct self-destructive potential of massive
autophagy (type II cell death)27 or by hardwiring the
autophagic process to pro-apoptotic signals (type I cell
death).28 In both scenarios, caspase-3 activity is increased.
Atg5 has a dual role in autophagy, and apoptosis is regulated
by proteolysis of Atg5; thus, siRNA knockdown of Atg5 was
found to abolish autophagy and reduce apoptosis.29 In our
study, the Pi-increased caspase-3 activity and terminal
transferase dUTP nick-end labeling-positive cells in VSMCs
was reversed by the ROS scavenger MnTMPyP. In addition,
autophagy inhibition by 3-MA and Atg5 siRNA knockdown
reduced the formation of autophagosomes and Pi-increased
apoptosis. Finally, autophagy inhibition aggravated
Pi-induced calcification even when cell apoptosis was
blocked by Z-VAD-FMK. Thus, ROS mediates Pi-induced
apoptosis and calcification, but the pro-calcification effect of
autophagy inhibition might not involve ROS-mediated
VSMC apoptosis.
Thus, an additional mineralization-specific mechanism
could be involved in autophagy inhibition–aggravated
calcification. Growing evidence suggests that membranebound vesicles from cells are involved in both physiological
and pathological calcification.30 Calcifying membrane-bound
vesicles (here, MVs) released from VSMCs participating in
VSMC mineralization have been reported in both human
SMCs and BASMCs.31,32 MVs are membrane-enclosed
microstructures released from the cell membrane that
have a key role in hydroxyapatite formation and the
mineralization process, and the release of ALP into the MV
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a
b
!
Rat VSMCs
–
–
0
0
–
–
0
3
+
–
0
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+
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3
–
–
25 μM
3 mM
β-Actin
1.0
!
10
!
!
*
15
–
–
0
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–
–
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+
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0
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–
+
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–
–
25 μM
3 mM
#
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#
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5 mM
25 μM
3 mM
#
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#
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Atg5 siRNA
MnTMPyP
Pi
1
2
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4
+
–
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+
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+
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+
+
25
3
e
BASMCs
8
10
#
0.5
*
!
6
#
#
4
#
2
3-MA
MnTMPyP
Pi
*
300
!
1
2
3
4
5
0
0
0
0
0
3
5
0
3
0
25
3
5 mM
25 μM
3 mM
5
–
+
25 μM
3 mM
BASMCs
!
Luminescence
(RLU ×10 5)
%TUNEL-positive cells
#
d
BASMCs
20
3-MA
MnTMPyP
Pi
1.5
Scramble
Atg5 siRNA
MnTMPyP
Pi
c
*
*
*
2.0
*
*
Calcium content
mg/g prot
Scramble
Atg5 siRNA
MnTMPyP
Pi
Cleaved
caspase-3
Rat VSMCs
15
Luminescence
(RLU ×10 4)
Cleaved caspase-3
protein level
2.5
200
100
3-MA
Z-VAD-FMK
Pi
1
2
3
4
5
0
0
0
0
0
3
5
0
3
0
20
3
5 mM
20 μM
3 mM
Figure 7 | The pro-calcification effect of autophagy inhibition does not involve reactive oxygen species (ROS)-mediated vascular
smooth muscle cell (VSMC) apoptosis. (a) Cleaved caspase-3 and (b) caspase-3/7 activity detected in rat VSMCs transfected with scramble or
autophagy protein 5 (Atg5) small interfering RNA (siRNA) for 48 h, and then treated with MnTMPyP and/or phosphate (Pi) as indicated for
10 days. (c) Quantification of terminal transferase dUTP nick-end labeling (TUNEL) staining of apoptotic cells in bovine aortic smooth muscle
cells (BASMCs) treated with Pi and/or 3-methyladenine (3-MA) and MnTMPyP as indicated for 7 days. (d) Caspase-3/7 activity and (e) calcium
content assay in BASMCs treated with 3-MA, MnTMPyP, Z-VAD-FMK, and/or Pi as indicated for 7 days. n ¼ 3, *Po0.05 versus corresponding
group 1 or as indicated; #Po0.05 versus corresponding group 2; !Po0.05 versus corresponding group as indicated.
a
BASMCs
b
*
BASMCs
Pi (3 mM)
–
+
3-MA (5 mM)
–
+
Annexin II
10
*
5
Ctrl
c
*
5
Annexin II
protein level
MV release (μg)
15
Pi
4
3
*
2
1
3-MA 3-MA+Pi
BASMCs
Ctrl
Pi
1 μm
1 μm
Pi+3-MA
3-MA
ALP activity (IU/mg prot)
d
BASMCs
*
0.3
*
0.2
0.1
Ctrl
1 μm
Pi
3-MA 3-MA+Pi
1 μm
Figure 8 | Blocking autophagy promotes matrix vesicle (MV) release. (a–c) Bovine aortic smooth muscle cells (BASMCs) were treated with
phosphate (Pi) (3 mmol/l) and/or 3-methyladenine (3-MA) (5 mmol/l) for 3 days, MVs were isolated by centrifugation, and then (a) MV release
was assessed by total protein content. (b) Western blot analysis of protein level of annexin II (MV marker) in MVs. (c) Alkaline phosphatase (ALP)
activity shown by ELF 97 phosphatase substrate in cultured BASMCs. (d) Assay of ALP activity in MVs. n ¼ 4, *Po0.05.
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X-Y Dai et al.: Autophagy counteracts vascular calcification
fraction is an important process for MV-mediated
mineralization.30–32 Recently, 1,25(OH)2D3 was found to
stimulate mineralization by increasing MV production.33
Here, inhibition of autophagy increased Pi-induced MV
release and ALP activity, which may be the cause of calcification aggravated by autophagy inhibition. Furthermore,
previous works have shown that MV can selectively load ALP
in both VSMCs34 and chondrocytes, and actin
polymerization inhibited by cytochalasin D can increase the
release of ALP into the MV fraction.35,36 In the present study,
the ratio of MV ALP to cell ALP indicated increased release of
ALP into the MV fraction in 3-MA-treated cells without
affecting cellular ALP and osteogenic gene expression, which
may indicate that cellular ALP is interrelated with osteogenic
differentiation, whereas the release of ALP into the MV
fraction involves other mechanisms. The process and
mechanism of MV formation and release are not
completely understood, but some evidence suggests that the
actin cytoskeleton is involved in the release of MVs from
chondrocytes/osteoblast-like cells.36–38 Despite the absence of
direct evidence of the influence of autophagy on the
cytoskeleton, cytoskeletal components contribute to the
formation of autophagy,39 and pro-renin receptor knockout
can alter the cytoskeleton and lead to the accumulation of
LC3-positive vesicles.40 Our results showed that 3-MA
markedly decreased SM-a-actin expression (Supplementary
Figure 6a online) and affected cell shape and actin/SM-aactin distribution (Supplementary Figure 6b online) but
not the expression of Pi-induced osteogenesis genes
(Supplementary Figure 6a online). Therefore, we can not
exclude the fact that autophagy inhibition may contribute to
MV release and load of ALP by regulating the cytoskeleton,
which needs further exploration.
Several epidemiological studies suggested a relationship
between vascular calcification, impaired bone metabolism,
and increased mortality, both in patients with chronic
kidney disease and the general aging population.41–43 Basal
autophagy has an important role in maintaining chondrocyte
and osteoblast/osteocyte survival and terminal differentiation, as well as in regulating bone growth.20 With our
finding that autophagy is an endogenous protective
mechanism counteracting vascular calcification under
hyperphosphatemia, therapeutic agents acting on the
autophagic response and activities of autophagy-regulating
pathways could be developed to treat aging or disease-related
vascular calcification and osteoporosis.
MATERIALS AND METHODS
Animal protocols
Abdominal aortas were from the CRF rat model fed with 0.75%
adenine and 0.9% phosphorus, as we described previously15 and
used for both morphology and western blot analysis. The rat
CRF model was confirmed by increased levels of serum creatinine,
blood urea nitrogen, and blood Pi in blood samples. MnTmPyP
(C44H36Cl5MnN8,
30 nmol/l/day,
Calbiochem,
Darmstadt,
Germany) was given intraperitoneally.
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Cell culture and cell calcification model
BASMCs and rat VSMCs were isolated and cultured as
described.44–46 Briefly, segments of aortas were obtained from
prepubertal bovines and rats. The inner portion of the medium was
removed and cut into B1-mm2 sections, which were placed in a
culture dish with Dulbecco’s modified Eagle’s medium (DMEM)
containing 4.5 g/l glucose supplemented with 20% fetal bovine
serum (FBS) and 10 mmol/l sodium pyruvate. Cells that migrated
from the explants were grown in DMEM plus 15% FBS; cells
between passages 3 and 8 were used in experiments. T/G
HA-VSMCs were from ATCC (Rockville, MD) and cultured in
DMEM plus 10% FBS.
For calcification experiments, cells were seeded at a density of
1104 cells/cm2 (day 0) and maintained in 15% FBS–DMEM until
confluence (day 6), when calcification was induced by adding
10 mmol/L-b-glycerophosphate or 3 mmol/l Pi (Sigma, St Louis,
MO). Calcium deposits were detected by measuring calcium content
or by alizarin red staining, as we described.15,46 For treatment with
antioxidants or inhibitors, MnTMPyP (25 mmol/l), Z-VAD-FMK
(Promega, Madison, WI, 20 mmol/l), autophagy inhibitor 3-MA
(Calbiochem, 5 mmol/l), or chloroquine (Sigma, 25 mmol/l) was
added with Pi every 3 days.
Aortic ring calcification
Aortas (from the thoracic to the iliac arteries) were removed in a
sterile manner from rats. After the adventitia and endothelium were
carefully removed, the vessels were cut into 2- to 3-mm rings and
placed in high-Pi (3 mmol/l) or normal culture medium at 37 1C in
5% CO2 for 7 days, with medium changes every 3 days.
MV isolation
MVs were harvested by following a modified MV isolation
protocol.31,47 Confluent BASMCs were washed twice with PBS and
transferred to control or high-Pi (3 mmol/l) medium for 3 days,
and then the medium was decanted after digestion with collagenase
and centrifuged at 10,000 g to remove cells and apoptotic bodies.
MVs were then harvested from the supernatant by centrifugation at
100,000 g for 30 min at 4 1C in a Hitachi Ultracentrifuge
(Hitachinaka, Ibaraki, Japan). MVs were resuspended with 0.1%
HCl or 1% Triton X-100, and protein and ALP activity were
determined. An increase in protein level indicated an increased
number of MVs.
Western blot analysis
Cell and aortic extracts were collected after treatment. The protein
underwent sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transfer to a nitrocellulose membrane, which was incubated
successively with 3% bovine serum albumin and the primary
antibodies anti-LC3 (1:1000, CST, Danvers, MA), anti-cleaved
caspase 3 (1:1000, CST), anti-Pit1/2, and anti-b-actin (1:500, both
Santa Cruz Biotechnology, Santa Cruz, CA), followed by IRDye700or 800–conjugated secondary antibody (1:20,000, Rockland,
Gilbertsville, PA) for 1 h. Fluorescence signals were detected
by the Odyssey infrared imaging system (LI-COR Biosciences,
Lincoln, NE).
Immunofluorescence analysis
Treated samples were fixed with 4% paraformaldehyde and then
rendered permeable by incubation in 0.1% Triton X-100 in 5%
bovine serum albumin–phosphate-buffered saline for 15 min; the
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basic research
samples were then treated with the antibody anti-LC3, actin, or
SM-a-actin (1:200) overnight at 4 1C, followed by treatment with
secondary antibody Alexa Fluor R488 donkey anti-rabbit IgG
(1:500) for 1 h at 37 1C. Nuclei were labeled with Hoechst 33342.
Replacing the primary antibody with normal IgG served as a
negative control.
Alizarin red S staining
Cells in 6-well plates were washed three times with PBS and fixed
with 10% formaldehyde for 10 min. After three washes with PBS,
cells were exposed to 1% Alizarin red S for 30 min and washed
with 0.2% acetic acid. Positively stained cells showed a reddish/
purple color.
Apoptosis analysis
VSMC apoptosis was assessed by measuring caspase 3/7 activity with
the use of the Caspase-Glo 3/7 Assay Kit (Promega) and terminal
transferase dUTP nick-end labeling assay (Promega).
Analysis of autophagy by GFP–LC3 redistribution
At 24 h after plating, cells were transfected with GFP–LC3 expression
vector (kindly provided by Dr Wei-Gou Zhu, Peking University) by
the Lipofectamine 2000 method (Invitrogen, Grand Island, NY).
Next, 6 h later, the transfection medium was replaced with DMEM
containing 10% FBS. The next day, cells were treated as described.
The GFP signal was monitored by confocal laser scanning
microscopy (Leica, Bannockburn, IL). The following criteria were
used to determine cells with punctuate GFP–LC3 (positive cells): (1)
uneven, ring-shaped dots in cytoplasm; (2) mean number of
uneven, ring-shaped dots per cell among 500 randomly selected
normal cells; and (3) more dots than the mean number of normal
cells. The percentage of positive cells was recorded for quantification.
RNA interference
siRNA against rat Atg5, human type III sodium-dependent Pi
cotransporters Pit1 (Slc20a1), and control siRNA were synthesized
by Invitrogen. The siRNA sequences for Atg5 were 50 -UUCAGGACC
UGGAAAGCCAUUGUUG-30 (sense) and 50 -CAACAAUGGCUUU
CCAGGUCCUGAA-30 (antisense); Pit1 50 -GCGCAAUAAUAGCUA
UACUTT-30 (sense); and 50 -AGUAUAGCUAUUAUUGCGCTT-30
(antisense). Cells cultured in 6-well plates were transfected with
siRNA at a final concentration of 10 nmol/l by the Lipofectamine
RNAiMAX Reagent method (Invitrogen), and then incubated for 4 h
at 37 1C before the addition of 10% fetal calf serum for 48 h.
At the end of treatment, cells were harvested for experiments. Gene
knockdown was confirmed by real-time PCR and western blot
analyses. The primers for rat Atg5 were 50 -TCAGTGGAGGCAACAG
AACC-30 (sense) and 50 -CAGACACGAACTGGCACATT-30 (antisense); Pit1 50 -CCAACTGTGCAGGCATAGAA-30 (sense) and 50 -TT
CTTCCTGGTTCGTGCATT-30 (antisense).
Electron microscopy
Electron microscopy was performed at our institution’s microscopy
facility. Briefly, cells were prefixed with 3% glutaraldehyde and
postfixed with 2% osmium tetraoxide. After dehydration, cells were
embedded in epoxy resin. Ultrathin sections were cut, contrasted
with uranyl acetate and lead citrate, and examined under a Zeiss
EM-109 electron microscope (Carl Zeiss AG, Carl-Zeiss-Strasse,
Oberkochen, Germany).
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X-Y Dai et al.: Autophagy counteracts vascular calcification
ALP activity
ALP activity was measured colorimetrically as the hydrolysis of
p-nitrophenyl Pi with the use of an ALP assay kit (Jiancheng
Bioengineering, Nanjing, China). Results were normalized to levels
of total protein. Cells or aortic rings were homogenized with 1%
Triton X-100 in 0.9% saline on ice and centrifuged in a microfuge at
8000 g for 5 min. The supernatant was removed for ALP and protein
concentration assay.
Statistical analysis
Data are reported as mean±s.e.m. Unpaired Student’s t-test was
used for analysis of two groups and one-way analysis of variance for
three or more groups, followed by Bonferroni’s multiple comparison
test as applicable by the use of Prism 5. Po0.05 was considered to be
statistically significant.
DISCLOSURE
All the authors declared no conflict of interest.
ACKNOWLEDGMENTS
This study was supported by the National Basic Research Program
of China (2010CB912504 and 2011CB503904), the National Science &
Technology Pillar Program (2012BAI39B03), the National Natural
Science Foundation of China (81270370, 30900566, 81170099,
81121061, and 31230035), and the Beijing Natural Science
Foundation (Mammalian Target of Rapamycin Complex-1 (mTORC1)
Promotes Vascular Smooth Muscle Cell Calcification).
SUPPLEMENTARY MATERIAL
Figure S1. Pi induced autophagy in T/G human aortic VSMCs (HAVSMCs).
Figure S2. LC3 puncta cells were found in both calcified and noncalcified aortic wall in chronic renal failure rats.
Figure S3. Knockdown of Atg5 by siRNA.
Figure S4. TUNEL staining (red) to identify apoptotic cells.
Figure S5. The effects of autophagy inhibition on release of alkaline
phosphatase (ALP) into the matrix vesicle (MV) fraction.
Figure S6. The effects of autophagy inhibition on VSMC phenotypic
transition.
Supplementary material is linked to the online version of the paper at
http://www.nature.com/ki
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