High Expression of the Pi-Transporter SLC20A1/Pit1 in
Calcific Aortic Valve Disease Promotes Mineralization
through Regulation of Akt-1
Diala El Husseini1, Marie-Chloe ´ Boulanger1, Dominique Fournier1, Ablajan Mahmut1, Yohan Bosse ´2,
Philippe Pibarot2, Patrick Mathieu1*
1Laboratoire d’E´tudes Mole ´culaires des Valvulopathies (LEMV), Groupe de Recherche en Valvulopathies (GRV), Quebec Heart and Lung Institute/Research Center,
Department of Surgery, Laval University, Quebec, Canada, 2Institut universitaire de cardiologie et de pneumologie de Que ´bec, Quebec, Canada
The regulation of phosphate (Pi) handling is crucial during calcification of the aortic valve. Gene profiling of Pi transporters
revealed that VIC culture expresses SLC201A1/Pit1 and SLC20A2/Pit2. On exposure to a mineralizing medium (2 mM Pi), the
expression of Pi transporters in VIC culture is increased several folds, with the highest magnitude for SLC20A1. By using
siRNAs, we established that silencing SLC20A1 significantly reduced Pi-induced mineralization of VICs. In human
pathological specimens, we found that the expression of SCL20A1 was increased in CAVD tissues compared to control non-
mineralized aortic valves. Treatment of VIC culture with Pi promoted the loss of mitochondrial membrane potential (DYm)
and cytochrome c release within the cytosol, leading to apoptosis. Inhibition of Pi transporters with phosphonoformic acid
(PFA) prevented Pi-mediated apoptosis of VICs. Moreover, we discovered that the level of the Akt-1 transcript is diminished
in CAVD tissues compared with control valves. Accordingly, treatment with Pi caused a reduction of the Akt-1 transcript in
VIC culture, and treatment with PFA or siRNA against SLC20A1 restored the level of Akt-1. Overexpression of Akt-1
(pCMVAkt-1) prevented both Pi-induced apoptosis and mineralization of VIC culture. These results strongly suggest that
overexpression of SLC20A1 promotes apoptosis and mineralization by altering the level of Akt-1.
Citation: El Husseini D, Boulanger M-C, Fournier D, Mahmut A, Bosse ´ Y, et al. (2013) High Expression of the Pi-Transporter SLC20A1/Pit1 in Calcific Aortic Valve
Disease Promotes Mineralization through Regulation of Akt-1. PLoS ONE 8(1): e53393. doi:10.1371/journal.pone.0053393
Editor: Ferenc Gallyas, University of Pecs Medical School, Hungary
Received September 7, 2012; Accepted November 28, 2012; Published January 4, 2013
Copyright: ? 2013 El Husseini et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by HSFC grant and CIHR grants MOP114893. D.E.H. is supported by studentship grants from Fonds de Recherche en Sante ´ du
Que ´bec (FRSQ). Y.B. is a research scholar from the Heart and Stroke Foundation of Canada, Ottawa, Ontario, Canada. P.P. holds the Canada Research Chair in
Valvular Heart Diseases, Ottawa, Ontario, Canada. P.M. is a research scholar from the Fonds de Recherche en Sante ´ du Que ´bec, Montreal, Que ´bec, Canada. The
funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
Calcific aortic valve disease (CAVD) is a common disorder of
the aging population . Despite the high prevalence of this
condition, there is so far no medical treatment for CAVD. To this
effect, randomized trials with statins have indicated that a lipid-
lowering strategy in patients with CAVD is no more efficient than
the placebo [2,3,4]. Different risk factors, such as age, male
gender, diabetes, and hypertension, have been identified in CAVD
. Mineralization of the aortic valve is the major culprit in the
development of CAVD. The key molecular processes leading to
the mineralization of the aortic valve are just beginning to be
understood, and this understanding is of utmost importance in
devising novel medical therapies.
Local metabolism and phosphate handling (Pi) are an important
pathway in the control of pathological tissue mineralization .
Studies indicate that Pi transporters, such as SLC20A1/Pit1 play
an important role in the mineralization of blood vessels . To this
effect, intracellular channelling of Pi is known to promote
mineralization. However, the cellular pathways that are triggered
following intracellular entry of Pi remain largely unexplored. It is
worth noting that phosphate-generating enzymes are highly
expressed during mineralization and elevate the concentration of
Pi in the extracellular space. One recent study indicates that the
ectonucleotidase enzyme, ectonucleotide pyrophosphatase/phos-
phodiesterase-1 (ENPP-1), is highly expressed in CAVD and that it
contributes to the elevation of extracellular Pi levels in valve
interstitial cells (VICs), which are the main cellular component of
the aortic valve . It follows that Pi may be channelled into the
intracellular space, contributing to the mineralization of the aortic
valve. Although it is known that Pi induces mineralization in
vascular smooth muscle cells and VICs by promoting apoptosis,
the role of Pi transporters in this process, as well as the chain of
events leading to programmed cell death, has not yet been fully
elucidated . In this study, we hypothesized that Pi transporters
play a major role in delivering signals of apoptosis in VICs by
altering the level of Akt, a kinase involved in cell survival.
We examined 50 aortic valves that were explanted from patients
at the time of aortic valve replacement for CAVD. Control non-
calcified aortic valves (n=28) with normal echocardiographic
analyses were obtained during heart transplant procedures.
Patients with a history of rheumatic disease, endocarditis, and
PLOS ONE | www.plosone.org1January 2013 | Volume 8 | Issue 1 | e53393
inflammatory diseases were excluded. Valves with an aortic valve
regurgitation grade .2+ were excluded. Patients with reduced left
ventricular ejection fraction (LVEF) (,40%) were excluded. All
patients underwent a comprehensive Doppler echocardiographic
examination preoperatively. Doppler echocardiographic measure-
ments included the left ventricular stroke volume and transvalvular
gradients using the modified Bernoulli equation. The protocol was
approved by the local ethical committee and informed consent was
obtained from the subjects.
Immunohistologic analysis for SLC20A1 was performed using
the rabbit antibody anti-SLC20A1 (Novus Biologicals, Oakville,
ON, Canada). Slides were then incubated with HRP-conjugated
and AEC substrate (Dako, Mississauga, ON, Canada). Tissue
sections were counterstained with hematoxylin. Rabbit serum was
used as a negative control in immunohistologic experiments.
Valve Interstitial Cells Isolation and in vitro Analyses of
Human VICs were isolated by collagenase digestion. To
promote calcification, cells were incubated for 7 days with a pro-
calcifying medium containing: DMEM +5% FBS, 1027M insulin,
50 mg/ml ascorbic acid and NaH2PO4 at 2 mM. In some
experiments, phosphonoformic acid (PFA) (1 mM) or LY294002
(50 mM) (Sigma, Oakville, ON, Canada) was added as specified.
The calcium content was determined by the Arsenazo III method
(Synermed, Monterey Park, CA, USA), which relies on the specific
reaction of Arsenazo III with calcium to produce a blue complex.
The results were measured at 650 nm on the Roche Diagnostics
Modular P800 Elecsys (Roche Diagnostics, Laval, QC, Canada).
This reaction is specific for calcium. Magnesium is prevented from
forming a complex with the reactive. The results were normalized
for protein contents and reported as percent changes.
RNA was extracted from valves explanted from 78 patients (50
CAVD and 28 controls). RNA was also extracted from cells during
Figure 1. Pi transporters in human valve interstitial cells (VICs). A. In isolated VICs, only transcripts of SLC20A1 and SL20A2 were expressed.
B. Following exposure to Pi, VICs have increased expression of SLC20A1 and SLC20A2 transcripts by several folds, with the highest magnitude for
SLC20A1 (results expressed in function of SLC20A2 as the referent) (SLC20A1 increased by 5.3-folds when compared to SLC20A2) C. Treatment with
PFA, a Pi transporter inhibitor, blocked the rise of SLC20A1 and SLC20A2 transcripts induced by the mineralizing medium (Pi). D. PFA prevented the
mineralization of VIC cultures induced by Pi. E. PFA prevented the Pi-induced rise of osteopontin, osteonectin, osteocalcin, Runx2 and alkaline
phosphatase transcripts. F and G. In isolated VICs, siRNA-mediated knockdown of SLC20A1 (F) resulted in a decreased Pi-induced mineralization (G).
When compared with the knockdown of SLC20A2, the siRNA against SLC20A1 provided a greater reduction of mineralization of VIC cultures (G). (For
in vitro experiments n=3); PFA: Phosphonoformic acid; * p,0.0001 compared to negative control (Ctn); # p,0.005 compared to mineralizing
SLC20A1/Pit1 and Calcific Aortic Valve Disease
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in vitro experiments. Total RNA was isolated with RNeasy micro
kit from Qiagen (Qiagen, Mississauga, ON, Canada). The RNA
extraction protocol was performed according to the manufac-
turer’s instructions using 100 mg of tissue. The quality of total
RNA was monitored by capillary electrophoresis (Experion,
Biorad, Mississauga, ON, Canada). One mg of RNA was reverse
transcribed using the Quantitec Reverse Transcription Kit from
Qiagen. Quantitative real-time PCR (q-PCR) was performed with
Quantitec SYBR Green PCR kit from Qiagen on the Rotor-Gene
6000 system (Corbett Robotics Inc, San Francisco, CA, USA).
Primers for the following transcripts were obtained from Qiagen
(Mississauga, ON, Canada): SLC20A1, SLC20A2, SLC17A1,
SLC34A1, SLC34A2, Akt-1, Akt-2, and Akt-3. The expression of
hypoxanthine guanine phosphoribosyl transferase (HPRT) was
used as a reference gene to normalize the results.
Transfection of Valve Interstitial Cells with pCMVAkt-1
VICs were seeded in 6-well plates (16105cells/well) for RNA
extraction and in 12-well plates (56104cells/well) for analysis of
calcification. After 24 hours, the cells were transfected with 1 mg of
Akt-1 human cDNA ORF clone incorporated into the vector
pCMV6-AC from Origene (Rockville, MD, USA). The trans-
fection was done using the Turbofectin 8 system from Origene.
After 48 hours, cells were harvested for RNA extraction or
exposed to the mineralizing medium.
Transfection of Valve Interstitial Cells with siRNAs
VICs were cultured into 12-well plates, at a density of 66104
cells per well, for determination of calcification, and into 6-well
plates, at a density of 16105cells per well, for real-time PCR
analysis performed in the transfection experiment. VICs were
grown in a volume of 1 ml and allowed to adhere overnight in
serum-containing antimicrobial-DMEM (5% CO2and 37uC). The
next day, VICs were transfected by incubation in a HiPerfect
reagent containing 2700 ng siRNA (either negative control or
SLC20A1, SLC20A2 and Akt-1 sequences) (Qiagen, Mississauga,
ON, Canada). After two days, the medium was changed and
a second transfection was done with either negative control or
SLC20A1, SLC20A2 and Akt-1 siRNAs. After 24 hours, the
transfection medium was replaced by a serum-containing antimi-
2 mM NaH2PO4 (Sigma, Oakville, ON, Canada). VIC cultures
were maintained for seven days in a calcification medium that was
changed every two days. A third transfection was done three days
after the second transfection, and cells were collected on the
seventh day of the mineralization process. The transfection
efficiency was verified by reduction of the target gene measured
by real-time PCR, using SLC20A1, SLC20A2 and Akt-1 primers
(Qiagen, Mississauga, ON, Canada).
Detection of Apoptosis
Apoptosis was documented in human VIC culture by TUNEL
assay using an Apoptag Plus Peroxidase In Situ Apoptosis
Detection Kit (Millipore, Billerica, MA, USA). For the quantitative
analysis, we counted 300 cells of each well and each condition.
Then the percentage of apoptotic cells over the total counted cells
was calculated. In some of the experiments with VICs, apoptosis
was also confirmed using the Apopercentage apoptosis assay
(Biocolor, Carrickfergus, UK), and the apoptosis levels were
analysed using Image Pro Plus Version 6.1 image analysis software
and expressed as pixel units. In some of the experiments,
cyclosporine A (0.05 mM) or PFA (1 mM) were added to the
growth medium to inhibit the mitochondrial permeability
transition pore (MTP) or Pi transporters respectively.
Release of Cytochrome c in Valve Interstitial Cells
The cells were fixed with paraformaldehyde +0.1% Triton and
washed in phosphate-buffered saline (PBS), before being incubated
with rabbit antibody against cytochrome c (Cell Signaling
Technology, Danvers, MA, USA) diluted in 5% BSA/PBS. After
being washed, the cells were treated with an autofluorescence
eliminator reagent and incubated with a secondary anti-rabbit
antibody (Abcam, Cambridge, MA, USA).They were also treated
with 49,6-diamidino-2-phenylindole (DAPI, 1 mg/ml). Images
were acquired with an Olympus 1X81-UCB microscope using
Image Pro Plus Version 6.1 and processed using ImageJ 1.44p.
Measurement of the Mitochondrial Membrane Potential
(DYm) in Valve Interstitial Cells
Valve interstitial cells were grown in a normal or mineralizing
medium for four days, in the presence of 1 mM PFA where
indicated. On the fourth day, cells were incubated for 20 minutes
with 200 nM of MitoPT TRME (ImmunoChemistry technologies,
Bloomington, MN, USA). The medium was replaced with
a complete medium without phenol red, to remove excess dye.
The cells with intact mitochondrial membrane potential were
counted in comparison to the total population. Epifluorescence
microscopy was performed with an Olympus IX81 inverted
microscope (40X) and an Olympus IEQ camera. Images were
corrected for background, and subjected to fast iteration and a fine
noise filter using Volocity 6.0.1 (Perkin Elmer).
Quantification of Akt and pAkt by ELISA
VICs were collected (1 ml from each well, 1.106per condition)
and centrifuged at 12,000 rpm for 10 minutes to pellet off dead
cells and debris. The quantification of Akt or pAkt was determined
in accordance with the manufacturer’s instructions (EMD
Table 1. Clinical characteristics of patients used for SLC20A1
and Akt-1 quantitative real-time PCR analysis.
Normal Valve CAVDp-value
BMI (kg/m2)25.665.527.864.6 0.03
AVA (cm2)– 0.7960.02–
Aortic mean gradient
TG (mmol/L)1.5660.94 1.3960.63NS
Creatinine (mmol/L) 113.34650.7092.15625.790.02
Creatinine clearance (ml/min) 68.97626.1163.60620.14NS
CAVD: calcific aortic valve disease; BMI: body mass index; LDL: Low-density
lipoprotein; HDL: High-density lipoprotein; TG: Triglycerides; BAV: Bicuspid
SLC20A1/Pit1 and Calcific Aortic Valve Disease
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Millipore, Billerica MA, USA) and normalized with protein
Results were expressed as means 6SEM. For continuous data,
values were compared between groups with Student t-test or a one-
way ANOVA to test the group effect (when more than two groups
were compared). Post hoc Tukey analyses were done when the p
value of the ANOVA was ,0.05. Categorical data were expressed
as a percentage and compared with the chi-square test. A p value
,0.05 was considered as statistically significant. Statistical analysis
was performed with a commercially available software package
(JMP IN 8.1).
Pi Transporters in Human Valve Interstitial Cells
A gene profiling of potential Pi transporters in human primary
VICs, including SLC20A1, SLC20A2, SLC17A1, SLC34A1 and
SLC34A2, revealed that only transcripts of SLC20A1 (Pit1) and
SLC20A2 (Pit2) were detected (Figure 1A). Of note, the levels of
SLC20A1 transcript were 2.5-fold higher than SLC20A2. On
exposure to the mineralizing medium (Pi 2 mM), the transcript
levels of both SLC20A1 and SLC20A2 increased by several-folds,
with the highest magnitude for SLC20A1 (Figure 1B). A treatment
with the mineralizing medium did not induce the expression of
other phosphate transporters (SLC17A1, SLC34A1, SLC34A2).
The addition of PFA, a Pi transporter inhibitor, prevented the rise
of SLC20A1 and SLC20A2 mRNA brought about by Pi
Figure 2. Expression of SCL20A1 in human calcific aortic valve disease. A. In human calcific aortic valve disease (CAVD) tissues, expression
of SLC20A1 was increased, both in tricuspid and bicuspid aortic valves, when compared to control non-mineralized aortic valves. B, C and D.
Immunostaining for SLC20A1 revealed faint expression in control aortic valves (B), whereas in CAVD tissues we observed a strong immunostaining in
areas of tissue remodelling in the vicinity of calcific nodules (C) (1006) (in panel D magnification 2006of inset in C). * p,0.005 compared to control
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(Figure 1C). Furthermore, PFA also prevented the mineralization
of VIC cultures (Figure 1D) as well as the expression of the bone
associated-proteins, osteopontin, osteonectin, osteocalcin, alkaline
phosphatase and the master bone transcription factor Runx2
(Figure 1E). VICs were transfected with siRNAs against SLC20A1
and SLC20A2 in order to delineate the role of each Pi transporter
on mineralization in vitro. In this experiment, both transcripts were
reduced by more than 80% by using their respective siRNAs
(Figure 1F). While the siRNA for SLC20A2 only slightly reduced
mineralization, the siRNA targeting SLC20A1 reduced mineral-
ization of VIC cultures by 78% (Figure 1G). Hence, considering
the higher expression of SLC20A1 when compared to SLC20A2
in human VICs, it is likely that the former plays a more important
role for Pi transport and mineralization during aortic valve
Expression of SLC20A1 in Human Calcific Aortic Valve
Next, the expression level of SLC20A1 in CAVD tissues was
examined. Clinical criteria of these patients are presented in
Table 1. Compared to control non-calcified aortic valves, the
CAVD tissues had significantly more expression of SLC20A1 in
both the bicuspid and tricuspid aortic valves (Figure 2A). The
expression of the Pi transporter was then confirmed by immuno-
histochemistry studies, which showed faint expression of SLC20A1
in control non-calcified aortic valves (Figure 2B) and a strong
staining in CAVD tissues (Figure 2C and inset in D). In CAVD,
immunostaining for SLC20A1 demonstrated that the protein was
expressed by cells located at the periphery of mineralized nodules,
where the remodelling process is active (Figure 2D).
Figure 3. Pi induces apoptosis of valve interstitial cells through the mitochondrial pathway. A. The percentage of apoptotic cells,
measured by TUNEL assay, increased significantly during VIC mineralization, whereas treatment with PFA blocked this response. B. The mitochondrial
membrane potential (DYm) decreased following treatment with Pi, but addition of PFA protected the DYm. C. Epifluorescence images of VICs with
the MitoPT TRME fluorescent dye in different conditions. In control cells, the mitochondrial uptake of MitoPT TRME gives a clear and distinct
fluorescent pattern, indicating a normal DYm. Following treatment with Pi, however, the fluorescent pattern is diffuse and accompanied by an
abnormal mitochondrial morphology indicating a loss in the DYm. The addition of PFA prevented Pi-mediated loss in the DYm. D. This protection
with PFA was also confirmed with an immunofluorescence assay measuring cytochrome c release in VICs under mineralizing condition. E. Cyclosporin
A which is an inhibitor of MTP, prevented Pi-mediated apoptosis of VIC cultures as detected with TUNEL assay. F. The effect of cyclosporine A on Pi-
mediated apoptosis was confirmed by the APOPercentage assay, which relies on changes in membrane asymmetry during apoptosis. G. Cyclosporin
A prevented Pi-induced mineralization of VIC cultures. (For in vitro experiments n=3); PFA: Phosphonoformic acid; MTP: mitochondrial permeability
transition pore; * p,0.0001 compared to negative control (Ctn); # p,0.0001 compared to mineralizing medium (PO4).
SLC20A1/Pit1 and Calcific Aortic Valve Disease
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Pi Induces Apoptosis of Valve Interstitial Cells through
the Mitochondrial Pathway
To further evaluate the process by which SLC20A1 may
promote the mineralization of VIC culture, the level of apoptosis
was measured following treatment with a Pi transporter inhibitor,
PFA . It should be noted that we had already demonstrated
that Pi-induced mineralization of VICs is, to a large extent,
dependent on apoptosis . Apoptosis levels were measured by
using a TUNEL assay and we demonstrated that PFA completely
blocked Pi-induced apoptosis of VICs (Figure 3A). In addition, on
exposure to Pi, there was a loss of the mitochondrial membrane
potential (DYm) in the VIC culture, which was prevented by PFA
(Figure 3B and C). Accordingly, we found that PFA impedes the
Pi-mediated cytochrome c release within the cytosol of VICs
(Figure 3D). Overall, these findings suggest that intracellular
channelling of Pi promotes apoptosis of VIC culture by activating
the mitochondrial pathway and the opening of the mitochondrial
permeability transition pore (MTP). To address the involvement of
the MTP, VICs were then treated with cyclosporine A, an MTP
inhibitor. Significantly, cyclosporin A inhibited Pi-induced apo-
ptosis of VICs (Figure 3E). These results were also corroborated by
the APOPercentage technique, which relies on changes in
membrane asymmetry during programmed cell death (Figure 3F).
Consequently, cyclosporin A also prevented Pi-mediated miner-
alization of VICs (Figure 3G).
Pi-mediated Regulation of mRNA Akt-1 and
Mineralization of Valve Interstitial Cells
Akt is a kinase involved in cell survival and we thought that it
might be involved in apoptosis-mediated mineralization of the
aortic valve. Gene profiling of the three genes encoding for Akt
demonstrated that mRNAs encoding for Akt-1 and Akt-2 were
present in VICs, with Akt-1 being the most highly expressed
(Figure 4A). While Akt-2 is involved in glucose metabolism, Akt-1
has been shown to control apoptosis . Measurements of Akt-1
transcript levels in human CAVD tissues indicated that there was
a substantial reduction of the transcript levels (4-folds) when
compared to control non-mineralized aortic valves (Figure 4B). In
VIC culture, we then determined that the Akt-1 transcript levels
were lower when the cells were grown in the mineralizing medium
(PO4) (Figure 4C). Both PFA and a siRNA targeting SLC20A1 re-
established the Akt-1 mRNA levels (Figure 4 C and D). On that
Figure 4. Pi-mediated regulation of Akt levels. A. In isolated VICs Akt-1 and Akt-2 were expressed. B. The levels of Akt-1 transcript were
reduced significantly in CAVD tissues (n=50) when compared to control non-mineralized aortic valves (n=28). C and D. In isolated VICs, the levels of
Akt-1 transcript were lowered following exposure to Pi, whereas PFA (C) and siRNA targeting SLC20A1 (D) prevented this response. E and F. Both Akt
(E) and pAkt (F) protein levels were reduced by Pi treatment of VICs, whereas in the presence of PFA, levels were maintained. (For in vitro experiments
n=3); PFA: Phosphonoformic acid; CAVD: Calcific Aortic Valve Disease; * p,0.0001 compared to negative control (Ctn); # p,0.005 compared to
mineralizing medium (PO4).
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account, measurement of the Akt protein levels by ELISA
indicated that on exposure to the mineralizing medium, the levels
of Akt and phosphorylated Akt (pAkt) were reduced (Figure 4E
and F). Treatment of VIC culture with PFA prevented the
decrease of Akt and pAkt levels (Figure 4E and F). In addition,
VICs were transfected with pCMVAkt-1 (Figure 5A) and apoptosis
and mineralization were measured. Overexpression of Akt-1
prevented Pi-mediated apoptosis and mineralization (Figure 5B
and C). Conversely, inhibition of PI3K, a kinase acting upstream
of Akt, with LY294002 led to a significant rise in the
mineralization of the VIC cultures (Figure 5D). Similarly, siRNA
against Akt-1 (Figure 5F) increased the mineralization of VIC
cultures by 2.6-folds (Figure 5E). When taken together, these
results indicate that the cellular entry of Pi can modulate the
expression of Akt-1, which constitutes an important target in
apoptosis-mediated mineralization of the aortic valve.
This is the first study to demonstrate that SLC20A1 is highly
expressed in CAVD tissues and that it promotes mineralization of
VICs by altering the level of mRNA encoding for Akt-1.
Specifically, we found that Pi promotes apoptosis of VICs by
altering the DYm, the formation of the MTP and the release of
cytochrome c within the cytosol. In addition, we observed that Pi-
mediated down-regulation of Akt-1 is a key event in the process
leading to programmed cell death and the mineralization of VICs.
These findings may therefore have a significant impact on our
understanding on how local handling of Pi by VICs promotes
mineralization of the aortic valve.
SLC20A1/Pit1 is Highly Expressed in Calcific Aortic Valve
Disease: Functional Relevance
The present findings highlight that SLC20A1 is highly
expressed in CAVD. The localization of SLC20A1 in the vicinity
of calcified nodules suggests that the Pi transporter might play
a role in the control of mineralization in CAVD tissue. Moreover,
by using gene-profiling studies, we established that among the
different Pi transporters, SLC20A1 is the most highly expressed. In
addition, on exposure to Pi, the expression of SLC20A1 transcripts
was increased by several-folds, indicating that there is a positive
feedback loop between the availability of Pi and the transporter
expression. It is worth noting that SLC20A2 was also expressed
but to a lower level compared to SLC20A1. SLC20A1 and
SLC20A2 knockdown demonstrates that the reduction of
Figure 5. Akt-1 a regulator of Pi-induced mineralization. Transfection of VICs with a pCMVAkt-1 resulted in higher expression of Akt-1
transcripts. B. The transfection of Akt-1 (pCMVAkt-1) prevented Pi-induced apoptosis of VICs (measured with the TUNEL assay). C. Transfection of Akt-
1 reduced significantly Pi-induced mineralization of VIC cultures. D. Inhibition of PI3K, a kinase acting upstream of Akt, with Ly294002 increased
mineralization of VIC cultures by several-folds. E and F. Similarly, the knockdown of Akt-1 (F) resulted in higher mineralization of VIC cultures (E). (For
in vitro experiments n=3); pCMV empty is the control (ctn) in panels A–C * p,0.0001 compared to negative control (Ctn); # p,0.0001 compared to
mineralizing medium (PO4).
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SLC20A1 induces a significant decrease in VIC mineralization.
This finding is consistent with the notion that the level of Pi
transporters expressed in a tissue is the main limiting factor that
will modulate the channelling of Pi within the intracellular space
Pi-mediated Apoptosis Relies on the Mitochondrial
The expression of SLC201A is ubiquitous and its role in the
mineralization of the vascular wall and smooth muscle cells has
been previously highlighted . Nevertheless, the precise
mechanism by which cellular entry of Pi mediates the mineral-
ization of VICs has yet to be clearly delineated. Previous
investigations with vascular smooth muscle cells have revealed
that Pi-mediated mineralization is accompanied by the expression
of bone-related proteins . In this regard, the present study also
emphasized that on exposure to Pi, VICs expressed several bone-
related proteins, including Runx2, a master bone transcription
factor. Furthermore, we documented that inhibiting SLC20A1
with PFA prevented the rise of bone-related transcripts induced by
the mineralizing medium. It is worth noting that despite the
expression of bone-related proteins, Pi-mediated mineralization of
both vascular smooth muscle cells and VICs relies mainly on
apoptosis . In this regard, the level of apoptosis in human
pathological CAVD specimens is high, and the inhibition of
caspases prevents Pi-induced mineralization of VIC cultures .
This raises an important question: By which mechanism does Pi
transport within the cell contribute to apoptosis-mediated miner-
alization of the aortic valve? To answer this, we observed, in VIC
culture, that Pi-induced apoptosis was dependent on the
mitochondrial pathway. Specifically, Pi promotes a loss of the
DYm which is associated with cytochrome c release. The release of
cytochrome c from mitochondria within the cytosol is a key process
in the activation of effector caspases during programmed cell
death . It should be pointed out that the inhibition of Pi
transporter with PFA prevented DYm loss, cytochrome c release
and consequently Pi-mediated apoptosis of VIC cultures. These
findings suggest that MTP opening might be involved in the
mineralization of VIC cultures. Accordingly, we found that
inhibiting the MTP with cyclosporine A prevented Pi-mediated
apoptosis and the mineralization of VIC cultures. Overall, these
findings suggest that Pi-induced mineralization of VIC culture is
dependent on the intracellular channelling of Pi, whereby
apoptosis is triggered through the mitochondrial pathway.
Pi-mediated Mineralization of Valve Interstitial Cells is
Dependent on Akt-1
VIC survival is largely dependent on the PI3K/Akt pathway. In
the present study, we established that mRNA levels of Akt-1 were
significantly reduced in VIC culture following treatment with Pi.
Moreover, Akt-1 transcript levels were severely down-regulated in
CAVD tissues when compared to control non-mineralized aortic
valves. In the same line, a recent study documented that Akt
signalling is down-regulated in mineralized aortic valves .
Interestingly, in vitro, both PFA and a siRNA against SLC20A1
prevented Pi-mediated down-regulation of Akt-1 mRNA tran-
script levels, indicating that this transporter is involved in the
regulation of the PI3K/Akt pathway. Reduced level of Akt-1 at
the mRNA level was associated with a decrease in the protein level
of phosphorylated Akt (pAkt). Overall, these findings suggest that
the regulation of VIC apoptosis and mineralization is dependent
on the level of Akt-1. This latter hypothesis was reinforced by the
discovery that the overexpression of Akt-1 in VIC cultures
prevented Pi-mediated apoptosis and mineralization. Although
the mechanism by which Pi modulates Akt-1 mRNA levels is not
yet fully elucidated, it is possible that Pi affects transcription and/
or mRNA stability.
This study examined CAVD tissues with advanced pathological
mineralization, but we cannot necessarily transpose these findings
to early processes involved in the development of aortic sclerosis.
Nevertheless, the present findings highlight the role of Pi
transporters and Akt-1 in the process of aortic valve mineraliza-
tion. In this study, we used PFA as a pharmacological inhibitor of
Pi transport. Although PFA was criticized as being a potentially
weak inhibitor of SLC20A1 in one study , other reports using
a longer incubation time of the inhibitor in cultured cells have
documented that PFA in fact significantly reduces the intracellular
channelling of Pi . Specifically, Jono et al. showed that the
incubation of human vascular smooth muscle cells with PFA
(1 mM) for several days reduced Pi uptake by 75%. Under similar
conditions, we found that the Pi-mediated expression of osteoblast
genes  and the mineralization of VIC cultures were inhibited
by the addition of PFA for 7 days. Nonetheless, in the present
study, we established some important findings by decreasing the
SLC20A1 level. In this regard, we have conclusively shown that
Pi-induced mineralization and the down-regulation of Akt-1 were
abrogated by both PFA and the knockdown of SLC20A1. Hence,
these findings indicate that both the expression level and
functional properties of SLC20A1 are important in regulating
the mineralization of the aortic valve.
This study provides evidence that SLC20A1 is overexpressed in
CAVD tissue and that it contributes to mineralization by
modifying the level of Akt-1. Pathological mineralization of aortic
valve may be, at least in part, dependent on the intracellular
channelling of Pi, whereby the mitochondrial-dependent apoptosis
pathway is triggered. Further research on Pi transport in CAVD
may help to develop novel therapies based on Pi handling.
Conceived and designed the experiments: DEH MCB DF AM YB PP PM.
Performed the experiments: DEH MCB DF AM. Analyzed the data: DEH
PM. Contributed reagents/materials/analysis tools: DEH MCB DF AM.
Wrote the paper: PM DEH MCB.
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