scispace - formally typeset

Journal ArticleDOI

In vascular smooth muscle cells paricalcitol prevents phosphate-induced Wnt/β-catenin activation

15 Oct 2012-American Journal of Physiology-renal Physiology (American Physiological Society Bethesda, MD)-Vol. 303, Iss: 8

TL;DR: The differential effect of calcitriol and paricalcitol on vascular calcification appears to be mediated by a distinct regulation of the BMP and Wnt/β-catenin signaling pathways.
Abstract: The present study investigates the differential effect of two vitamin D receptor agonists, calcitriol and paricalcitol, on human aortic smooth muscle cells calcification in vitro. Human vascular sm...
Topics: Paricalcitol (66%), Vascular smooth muscle (63%), Myocyte (56%), Calcitriol receptor (55%), Calcitriol (54%)

Content maybe subject to copyright    Report

In vascular smooth muscle cells paricalcitol prevents phosphate-induced
Wnt/-catenin activation
Julio M. Martínez-Moreno,
1
Juan R. Muñoz-Castañeda,
1
Carmen Herencia,
1
Addy Montes de Oca,
2
Jose C. Estepa,
2
Rocio Canalejo,
1
Maria E. Rodríguez-Ortiz,
1
Pablo Perez-Martinez,
3
Escolástico Aguilera-Tejero,
2
Antonio Canalejo,
4
Mariano Rodríguez,
1
and Yolanda Almadén
3
1
Servicio de Nefrologia, Red in Ren, Instituto Maimónides de Investigación Biomédica de Córdoba, (IMIBIC) Hospital
Universitario Reina Sofia, Cordoba, Spain;
2
Department of Medicina y Cirugia Animal, Universidad de Cordoba, Cordoba,
Spain;
3
Lipid and Atherosclerosis Unit, Instituto Maimónides de Investigación Biomédica de Córdoba (IMIBIC), Reina Sofia
University Hospital/University of Cordoba, and Centro de Investigación Biomédica en Red de la Fisiopatología de la
Obesidad y Nutrición (CIBERobn), Instituto de Salud Carlos III, Cordoba, Spain; and
4
Department of Biologia Ambiental y
Salud Publica, Universidad de Huelva, Huelva, Spain
Submitted 20 December 2011; accepted in final form 2 August 2012
Martínez-Moreno JM, Muñoz-Castañeda JR, Herencia C,
Montes de Oca A, Estepa JC, Canalejo R, Rodríguez-Ortiz ME,
Perez-Martinez P, Aguilera-Tejero E, Canalejo A, Rodríguez M,
Almadén Y. In vascular smooth muscle cells paricalcitol prevents
phosphate-induced Wnt/-catenin activation. Am J Physiol Renal
Physiol 303: F1136 –F1144, 2012. First published August 8, 2012;
doi:10.1152/ajprenal.00684.2011.—The present study investigates the
differential effect of two vitamin D receptor agonists, calcitriol and
paricalcitol, on human aortic smooth muscle cells calcification in
vitro. Human vascular smooth muscle cells were incubated in a high
phosphate (HP) medium alone or supplemented with either calcitriol
10
8
M (HP CTR) or paricalcitol 3·10
8
M (HP PC). HP
medium induced calcification, which was associated with the upregu-
lation of mRNA expression of osteogenic factors such as bone
morphogenetic protein 2 (BMP2), Runx2/Cbfa1, Msx2, and osteocal-
cin. In these cells, activation of Wnt/-catenin signaling was evi-
denced by the translocation of -catenin into the nucleus and the
increase in the expression of direct target genes as cyclin D1, axin 2,
and VCAN/versican. Addition of calcitriol to HP medium (HP
CTR) further increased calcification and also enhanced the expression
of osteogenic factors together with a significant elevation of nuclear
-catenin levels and the expression of cyclin D1, axin 2, and VCAN.
By contrast, the addition of paricalcitol (HP PC) not only reduced
calcification but also downregulated the expression of BMP2 and
other osteoblastic phenotype markers as well as the levels of nuclear
-catenin and the expression of its target genes. The role of Wnt/-
catenin on phosphate- and calcitriol-induced calcification was further
demonstrated by the inhibition of calcification after addition of Dick-
kopf-related protein 1 (DKK-1), a specific natural antagonist of the
Wnt/-catenin signaling pathway. In conclusion, the differential ef-
fect of calcitriol and paricalcitol on vascular calcification appears to
be mediated by a distinct regulation of the BMP and Wnt/-catenin
signaling pathways.
vascular calcification; calcitriol; paricalcitol; VSMCs; Wnt/-catenin
UREMIC PATIENTS FREQUENTLY present vascular calcifications
(VC), which contributes to the high rate of cardiovascular
morbidity and mortality observed in these patients (61). The
generation of VC in uremic patients is multifactorial, and the
mechanisms are partially understood (35). Abnormal mineral
metabolism, and particularly the accumulation of phosphate,
plays a central role in the generation of VC (3, 25). Chronic
inflammation has also been proposed as an important factor in
the process of VC (34, 50).
Calcitriol [1,25(OH)
2
D
3
], the most active metabolite of
vitamin D, is a key regulator of mineral metabolism through its
direct effect on intestine, kidney, bone, and parathyroid glands.
Chronic kidney disease (CKD) patients, often develop second-
ary hyperparathyroidism (2°HPT) because of the retention of
phosphate and the deficiency of calcitriol synthesis, both due to
the reduction of functional renal mass, which eventually lead to
hypocalcemia. Thus, in the last decades, therapeutic strategies
to control 2°HPT incorporated high doses of vitamin D, mainly
as calcitriol, that may contribute to the development of VCs
(13). The vitamin D analog 19-nor-1,25(OH)
2
D
2
(paricalcitol)
is now commonly used to treat 2°HPT in CKD patients
because it effectively suppress parathyroid hormone but is less
calcemic and phosphatemic than calcitriol (29, 53).
The administration of calcitriol to CKD patients may cause
VC by increasing the serum levels of calcium and phosphate.
Other studies (45) have suggested that calcitriol and other
vitamin D receptor (VDR) activators such as paricalcitol may
have a direct effect on VC that is independent of calcium and
phosphorus. Paricalcitol seems to produce less calcification
than calcitriol (4, 28, 33). However, the cellular mechanisms
driving the differential effects of calcitriol and paricalcitol on
VC have not been elucidated. We hypothesized that factors
regulating osteogenic differentiation of vascular smooth mus-
cle cells (VSMCs) may respond differently to calcitriol and
paricalcitol.
Bone morphogenetic proteins (BMPs) and proteins of the
Wnt family are extremely potent anabolic regulators of bone
formation, and both have been implicated in the regulation of
VC (7, 17, 31, 49, 51, 57). BMP2 upregulates transcription
factors as the runt-related transcript factor 2 (Runx2/Cbfa1)
and the Msh homeobox 2 (Msx2), master regulators of osteo-
genesis (30). Furthermore, Wnt signaling, which is essential
for the commitment of pluripotent mesenchymal cells, has also
been shown to be activated during the development of VC in
vivo and in vitro (5, 8, 23, 31, 51). Wnt proteins (revised in 26)
are a large family of secreted signaling molecules that signal
through binding to a coreceptor complex formed by the pro-
teins of the frizzled (Fzd) family and the lipoprotein receptor-
related 5/6 proteins (Lrp5/6). The activation of the canonical
Address for reprint requests and other correspondence: Y. A. Peña, Unidad
de Investigacion, IMIBIC, Hospital Reina Sofía, Avda. Menéndez Pidal s/n,
14004 Córdoba, Spain (e-mail: yolandaalmaden@yahoo.es).
Am J Physiol Renal Physiol 303: F1136–F1144, 2012.
First published August 8, 2012; doi:10.1152/ajprenal.00684.2011.
1931-857X/12 Copyright
©
2012 the American Physiological Society http://www.ajprenal.orgF1136
by 10.220.32.246 on November 21, 2017http://ajprenal.physiology.org/Downloaded from

Wnt pathway results in the inactivation of a destruction com-
plex that phosphorylates -catenin and targets it for ubiquitin-
proteasome-mediated degradation; thus -catenin is able to
translocate to the nucleus and regulates the expression of target
genes.
The aim of the present study was to investigate possible
differences between the effect of calcitriol and paricalcitol on
osteogenic signals, mainly the Wnt/-catenin signaling, in
human aortic smooth muscle cells (HASMCs) in vitro.
MATERIALS AND METHODS
Cell culture. HASMCs were obtained from Clonetics (Lonza,
Walkersville, MD). Cells were cultured in DMEM supplemented with
15% heat-inactivated FBS (BioWhittaker), Na piruvate (1 mmol/l),
glutamine (4.5 g/l), penicillin (100 U/ml), streptomycin (100 mg/ml),
and HEPES (20 mmol/l) at 37°C in a humidified atmosphere with 5%
CO
2
. HASMCs of passage 5– 8 were used in the experiments. To
induce calcification, after reaching 80% confluence, HASMCs were
incubated for 9 days in a high phosphate medium (calcification
medium) that contained Na
2
HPO
4
3
and NaH
2
PO
4
3
salts in 1:2
proportion (Sigma Aldrich) to obtain a final phosphate concentration
of 3.3 mmol/l. The medium was replaced with fresh medium every
2–3 days. Depending on the experiments, calcification medium was
supplemented with calcitriol 10
8
M, paricalcitol 3·10
8
M, or other
drugs such as Dickkopf-related protein 1 (DKK-1; ref 5439-DK-010;
R&D Systems; 100 ng/ml; a secreted, endogenous extracellular Wnt/
-catenin inhibitory gene product that is commercially available).
Cells that were incubated in normal phosphate (0.9 mmol/l) medium
were used as controls.
Assessment of calcium deposition. After 9 days of incubation,
calcification was quantified. Cells were decalcified with HCl (0.6
mol/l) for 24 h. The calcium content of the supernatants was deter-
mined by spectrophotometer at 612 nm by a kit containing phenol-
sulphonephthalein dye (no. DICA008, QuantiChrom calcium assay
kit; BioAssay Systems). Then, the cells were washed three times with
PBS (Sigma Aldrich) and solubilized in 0.1 mol/l NaOH/0.1% SDS.
The protein content was measured using the Bio-Rad protein assay
(Bio-Rad Laboratories, Munich, Germany), and the calcium content
was normalized for total protein.
Real-time RT-PCR. Total RNA was isolated from each sample of
HASMCs using 500 l Trizol (Sigma) by processing according to the
manufacturer’s recommendation. Real-time RT-PCR was performed
in duplicate with QuantiTect SYBR Green one-step RT-PCR Kit (ref.
no. 204243; Qiagen) in a final volume of 20 ul from 30 ng of total
RNA. All PCR amplifications were carried out using Lightcycler 480
(Roche Molecular Biochemicals, Indianapolis, IN). The expression of
target genes was normalized to the expression of GAPDH. The
primers for PCR amplification are indicated in Table 1.
Protein extracts and Western blot. Proteins were isolated from
HASMCs using lysis buffer containing HEPES (10 mmol/l), KCl (10
mmol/), EDTA (0.1 mmol/l), EGTA (0.1 mmol/l), dithiothreitol (1
mmol/l), PMSF (0.5 mmol/l), protease inhibitor cocktail (70 mg/ml;
Sigma Aldrich), and Igepal CA-630 (0.6%) at pH 7.9. The suspension
was centrifuged, and the supernatant (cytosolic extract) was stored.
Nuclear extracts were obtained by incubating the pellet separated
from the cytosolic extract in a lysis buffer containing HEPES (20
mmol/l), NaCl (0.4 mmol/l), EDTA (1 mmol/l), EGTA (1 mmol/l),
dithiothreitol (1 mmol/l), PMSF (1 mmol/l), and protease inhibitor
cocktail (46 mg/ml) at pH 7.9. Protein concentration was determined
with the Bradford method (Bio-Rad Laboratories, Munich, Germany).
For Western blot, equal amounts of protein were electrophoresed in
10% SDS-polyacrylamide gel (Invitrogen, Carlsbad, CA) and subse-
quently transferred to a nitrocellulose membrane (Invitrogen). The
membranes were blocked with 5% nonfat dried milk for1hatroom
temperature and then incubated with primary antibody for2hatroom
temperature. Primary antibodies used included rabbit polyclonal
-catenin antibody (ref. no. L9562; Cell Signaling) and rabbit poly-
clonal TFIIB antibody (ref. no. SC-225; Santa Cruz Biotechnology,
Santa Cruz, CA). Blots were immunolabeled using a horseradish
peroxidase-conjugated secondary antibody and developed on autora-
diographic film using the ECL Plus Western blotting detection system
(Amersham Biosciences, Little Chalfont, England). Specific bands
were quantified by densitometric analyses with Quantity One 4.4.0
software (Bio-Rad Laboratories) and were normalized to TFIIB levels.
Confocal microscopy. Cells were seeded on coverslips, and after
reaching 90% confluence, they received the different treatments for 24
h. Then, they were rinsed in PBS and fixed and permeated in cold 50%
methanol for 2 min, cold 100% methanol for 20 min, and cold 50%
methanol for 2 min. The specimens were subsequently washed once in
PBS (3 5 min) and incubated for 2 h with anti--catenin antibody
(1:50; BD Pharmigen, Franklin Lakes, NJ) in blocking solution (1%
BSA) at room temperature. After being washed with PBS (3 5 min),
specimens were incubated for 1 h with Alexa Fluor 488 F(ab=)
2
fragment of rabbit anti-mouse IgG (1:500; ref. no. A-21204; Invitro-
gen) in PBS containing 1% BSA. After a final wash with PBS (3
5 min), the specimens were counterstained with DAPI for nuclear
stain. Cells were mounted on slides to examine fluorescence using a
LSM 5 Exciter Carl Zeiss confocal microscope. Pictures were ob-
tained at 40 in Axio Observer Z1 inverted confocal microscope
(LSM5 Exciter Zeiss). ImageJ software (National Institutes of Health)
was used to analyze confocal immunofluorescence staining. Mander’s
coefficient M2 plugin (DAPI vs. green) was used to analyze nuclear
translocation of -catenin. Mander’s coefficient M2 is the percentage
of above-background pixels in blue color (DAPI) that overlap above-
background pixels in green color (-catenin).
Statistical analysis. Results are expressed as means SE. The
difference between means for three or more groups was assessed by
one-way ANOVA followed by post hoc Duncan analysis. The difference
between means for two different groups was determined by t-test. A P
value 0.05 was considered significant. These analyses were performed
with the assistance of a computer program (SPSS 15.0, Chicago, IL).
RESULTS
Effect of calcitriol and paricalcitol on VSMC calcification.
Incubation of HASMCs in a high phosphate (3.3 mmol/l)
medium for 9 days (HP) induced calcification compared with
Table 1. Primers used for quantitative real-time RT-PCR
Gene Sense Primer Antisense Primer
GAPDH 5=-TGATGACATCAAGAAGGTGGTGAAG-3= 5=-TCCTTGGAGGCCATGTGGGCCAT-3=
Cyclin D1 5=-CCGAGGAGCTGCTGCAAATGGA-3= 5=-ATGGAGGGCGGATTGGAAATGAAC-3=
BMP2 5=-AGGAGGCAAAGAAAAGGAACGGAC-3= 5=-GGAAGCAGCAACGCTAGAAGACAG-3=
Msx2 5=-AAATTCAGAAGATGGAGCGGCGTG-3= 5=-CTGGGATGTGGTAAAGGGCGTGCG-3=
Runx2 5=-CCGGAGTGGACGAGGCAAGAGTT-3= 5=-AGCTTCTGTCTGTGCCTTCTGGG-3=
Osteocalcin 5=-GCAGAGTCCAGCAAAGGTGCAGCC-3= 5=-GCCTCCTGAAAGCCGATGTGGTCA-3=
BMP2, bone morphogenetic protein 2.
F1137PARICALCITOL AND Wnt/-CATENIN
AJP-Renal Physiol doi:10.1152/ajprenal.00684.2011 www.ajprenal.org
by 10.220.32.246 on November 21, 2017http://ajprenal.physiology.org/Downloaded from

cells maintained in a medium with 0.9 mmol/l phosphate
(control; P 0.05; Fig. 1). The addition of calcitriol (10
8
M)
to cells in HP medium (HP CTR) further increased the
degree of calcification observed with HP alone (P 0.05).
Conversely, the addition of paricalcitol (3 10
8
M; HP
PC) produced a significant reduction in calcification relative to
the observed in cells in HP medium (P 0.05). Nevertheless,
in the HP PC cells the calcium content remained higher than
in control cells cultured in 0.9 mmol/L P (P 0.05).
Osteoblast phenotype. The levels of BMP2 mRNA were
greater in cells incubated in HP medium than in controls (P
0.05; Fig. 2A). The addition of calcitriol further increased the
expression of BMP2 mRNA compared with the HP cells (P
0.05). In cells treated with paricalcitol, the levels of BMP2
mRNA were not different from control cells.
The gene expression of the osteoblastic-specific marker
Runx2 was significantly increased in cells on HP compared
with controls (P 0.05; Fig. 2B). Again calcitriol caused
additional increase of Runx2 expression (P 0.05 vs. HP
cells), whereas paricalcitol failed to increase the expression of
Runx2 mRNA beyond the values observed in HP cells.
Similarly to that observed with Runx2, the expression of
Msx2 mRNA was increased in HP and further increased by
addition of calcitriol (Fig. 2C). The addition of paricalcitol
decreased the Msx2 mRNA levels to control values. The
expression of Bglap/osteocalcin (OC) was not increased by HP
but it increased when calcitriol was added to the HP medium
(Fig. 2D). By contrast, paricalcitol did not modify the OC
expression.
Role of the canonical Wnt/
-catenin signaling pathway.
Activation of the Wnt/-catenin signaling pathway results in
nuclear translocation of -catenin. The presence of -catenin
in the nucleus was assessed by Western blotting of nuclear
extracts. The incubation of cells in HP induced a significant
increase of the expression of nuclear -catenin compared with
controls (P 0.05). The addition of calcitriol to HP medium
increased the nuclear content of -catenin; however, the addi-
tion of paricalcitol caused a reduction in the levels of nuclear
-catenin to a level similar to that observed in control cells
(Fig. 3A). Intracellular localization of -catenin was visualized
by immunofluorescence using confocal microscopy (Fig. 3B).
Control cells showed immunofluorescence staining of -catenin
only in the cytoplasm, whereas cells cultured in HP showed
marked expression of -catenin at the nuclear level. HP CTR
cells also exhibited marked staining for nuclear -catenin. By
contrast, in HP PC, -catenin expression was mainly re-
stricted to the cytoplasm. Quantification by the Mander’s
3
4
5
6
on
(µg/mg protein)
*
*
#
*
0
1
2
Ca
depositio
Control HP HP+CTR HP+PC
*
#
Fig. 1. Effect of calcitriol and paricalcitol on human aortic smooth muscle cell
(HASMC) calcification. HASMCs are incubated for 9 days in a high (3.3
mmol/l) phosphate (HP) medium (calcification medium) alone or supple-
mented with either calcitriol 10
8
M (HP CTR) or paricalcitol 3·10
8
M
(HP PC). Cells incubated in normal phosphate (0.9 mmol/l) medium are
used as controls. Calcium content is determined with the phenolsulphoneph-
thalein dye. Bars are means SE (5 independent experiments; 6 repetitions in
each experiment). *P 0.05 vs. control. #P 0.05 vs. HP.
3
ntrol)
*
#
8
ntrol)
*
#
BA
0
0.5
1
1.5
2
2.5
BMP2 mRNA levels (vs. Con
Control HP HP+CTR HP+PC
*
#
0
2
4
6
Runx 2 mRNA levels (vs. Con
Control HP HP+CTR HP+PC
**
*
0.5
1
1.5
2
2.5
Msx 2 mRNAlevels (vs. Control)
*
*
#
0.5
1
1.5
2
OC mRNA levels (vs. Control)
*
CD
0
Control HP HP+CTR HP+PC
0
Control HP HP+CTR HP+PC
Fig. 2. Effect of calcitriol and paricalcitol on the expression of markers of osteoblastic phenotype during HASMC calcification. HASMCs are incubated for 9
days in a high (3.3 mmol/l) phosphate (HP) medium (calcification medium) alone or supplemented with either calcitriol 10
8
M (HP CTR) or paricalcitol
3·10
8
M (HP PC). Cells incubated in normal phosphate (0.9 mmol/l) medium are used as controls. mRNA levels are analyzed by real-time RT-PCR technique.
Expression of target genes are normalized to the expression of GAPDH. A: bone morphogenetic protein 2 (BMP2) mRNA expression. B: Runx2/Cbfa1 mRNA
expression. C: Msx2 mRNA expression. D: osteocalcin/Bglap mRNA expression. Bars are means SE (3 independent experiments; 6 repetitions in each
experiment). OC, osteocalcin. *P 0.05 vs. control. #P 0.05 vs. HP.
F1138 PARICALCITOL AND Wnt/-CATENIN
AJP-Renal Physiol doi:10.1152/ajprenal.00684.2011 www.ajprenal.org
by 10.220.32.246 on November 21, 2017http://ajprenal.physiology.org/Downloaded from

coefficient M2 plugin (DAPI vs. green), confirmed the signif-
icance of differences in the levels of -catenin fluorescence in
HP and HP CTR compared with control cells (P 0.05 and
P 0.05, respectively; Fig. 3C).
The relevance of Wnt/-catenin signaling activation in cal-
cification was tested by addition to culture medium (100 ng/ml)
of DKK-1, a commercially available specific endogenous ex-
tracellular antagonist of the Wnt signaling (1, 26). As shown in
Fig. 4, the addition of DKK-1 reduced the level of calcification
induced by high phosphate (P 0.05 vs. HP cells) and
prevented the increase in calcification induced by calcitriol
(P 0.05 vs. HP CTR cells); a trend toward a decreased
calcification, although not significant, was also observed after
addition of DKK-1 to HP PC cells. The effect of DKK-1 on
-catenin translocation was also evaluated by confocal immu-
nofluorescence (Fig. 3, B and C). The increase of nuclear
-catenin expression in HP and HP CTR cells was abolished
by coincubation with DKK-1 (P 0.05 vs. controls).
Additional direct Wnt/-catenin transcriptional targets were
examined, including cyclin D1 (an early marker of cells enter-
ing the cell cycle), VCAN/versican (a large chondroitin sulfate
proteoglycan; a member of the aggrecan/versican proteoglycan
family), and axin 2 (axis inhibition protein 2 or conductin; a
member of the Wnt/-catenin signaling pathway that regulates
the stability of -catenin). Effects of cotreatment with the
inhibitor DKK-1 on the expression of genes were also tested
(Fig. 5). The mRNA expression of cyclin D1 was similarly
elevated in cells in HP and HP CTR compared with control
cells (P 0.05). In cells treated with HP PC, the cyclin D1
mRNA level was greater than in control (P 0.05) but lower
than in HP and HP CTR cells (P 0.05). Addition of
DKK-1 dramatically reduced cyclin D1 mRNA to a level not
different from that of control. VCAN expression was also
increased in HP, HP CTR, and HP PC cells over control
(P 0.05). Cotreatment with DKK-1 significantly reduced
VCAN mRNA levels to control values in all the groups.
Finally, compared with control, axin 2 mRNA expression was
elevated in HP and HP CTR (P 0.05) but not in HP PC
group; this increase was abolished by DKK-1 (P 0.05).
DISCUSSION
In the present study, we have explored the differential effect
of two VDR agonists, calcitriol and paricalcitol, on VSMC
A
C
HP HP+CTR
HP+PC
Nuclear
beta-catenin
TFIIB
0
40
80
120
160
200
Nuclear beta-catenin
(integrated OD vs
. Control)
Control HP HP+CTR HP+PC
*
*
#
B
C
-catenin
3
4
5
Nuclear beta
(Mander’s coefficient vs. Control)
0
1
2
#
#
Fig. 3. Effect of calcitriol and paricalcitol on the canonical Wnt/-catenin signaling pathway during HASMC calcification. HASMCs are incubated for 24 h in
a high (3.3 mmol/l) phosphate (HP) medium (calcification medium) alone or supplemented with either calcitriol 10
8
M (HP CTR) or paricalcitol 3·10
8
M
(HP PC). Cells incubated in normal phosphate (0.9 mmol/l) medium are used as controls. A: levels of nuclear -catenin is assessed by western blotting of
nuclear extracts. Image represents 3 different experiments. Quantification is performed by measurement of the integrated optical density (OD) and normalized
to TFIIB levels. Data are means SE of the 3 independent experiments. *P 0.05 vs. control. #P 0.05 vs. HP. B: HASMCs are incubated for 24 h in a
high (3.3 mmol/l) phosphate (HP) medium (calcification medium) alone or supplemented with either calcitriol 10
8
M (HP CTR) or paricalcitol 3·10
8
M
(HP PC) alone or with the addition to culture medium (100 ng/ml) of commercially available Dickkopf-related protein 1 (DKK-1), a specific endogenous
extracellular antagonist of the Wnt signaling. Intracellular localization of -catenin is visualized by immunofluorescence using confocal microscopy. For each
treatment, -catenin staining (green immunofluorescence) is shown at left;atmiddle, the same sample is counterstained with DAPI (blue) for nuclear stain; the
merged image is shown at right. Images represent 3 different experiments. C: quantification of nuclear -catenin staining is performed by the Mander’s coefficient
(M2 plugin: DAPI vs. green). *P 0.05 vs. control. #P 0.05 vs. the same treatment without DKK-1.
F1139PARICALCITOL AND Wnt/-CATENIN
AJP-Renal Physiol doi:10.1152/ajprenal.00684.2011 www.ajprenal.org
by 10.220.32.246 on November 21, 2017http://ajprenal.physiology.org/Downloaded from

calcification in vitro. We found that calcitriol increased calci-
fication, which appeared to be associated with the activation of
the Wnt/-catenin and BMP2 signaling pathways. By contrast,
paricalcitol decreased calcification, which was accompanied by
an inhibition of the Wnt/-catenin pathway and downregula-
tion of osteoblastic gene expression.
As previously demonstrated by us and other researchers (6,
36, 40, 44, 47, 49, 58), VSMCs cultured in high phosphate
undergo osteogenic transformation and calcification. BMPs
and Wnt ligands have been implicated in the regulation of both
osteoblastic transdifferentiation of aortic VSMCs in vitro and
VC (7, 17, 31, 49, 51, 57). In our study, we found that the
calcification process was associated with the upregulation of
osteogenic factors such as BMP2, the transcription factors
Runx2/Cbfa1 and Msx2, and the procalcificant protein OC.
This is in agreement with previous results (56) showing that the
expression of all these markers was significantly induced in
calcified vessels. BMP2 is a key factor for osteogenic differ-
entiation of mesenchymal cells that upregulates Runx2 and
Msx2 (30). OC is a downstream target gene of Runx2 and
Msx2 (16).
In the present study, we also show that calcification of the
HASMCs is also associated with a concomitant activation of
the canonical Wnt/-catenin signaling pathway. This was
quantitatively demonstrated both immunocytochemically by
the translocation of -catenin into the nucleus and by the
significant increase of the expression of nuclear -catenin by
Western blotting. Furthermore, the addition of DKK-1, a se-
creted, endogenous extracellular Wnt/-catenin inhibitory
gene product (1, 26) that is commercially available, inhibited
the calcification and the concomitant cellular changes induced
by high phosphate.
The activation of -catenin signaling modulates osteoblast
proliferation and differentiation (32, 41). Other authors (22)
observed VSMC proliferation in vessels developing calcifica-
tion. Notably, we demonstrated that the calcification observed
in the high phosphate-treated cells was accompanied by a
*
*
e
ls
*
*
e
ls
2
e
ls
#
Cyclin D mRNA lev
e
(vs. Control)
*
$
#
#
Cyclin D mRNA lev
e
(vs. Control)
0
0.5
1.5
1
Cyclin D mRNA lev
e
(vs. Control)
*
$
#
levels
ol)
*
#
*
#
1.5
2
levels
ol)
*
#
*
#
Control HP HP+CTR HP+PC HP
+
DKK1
HP+CTR
+
DKK1
HP+PC
+
DKK1
Control HP HP+CTR HP+PC HP
+
DKK1
HP+CTR
+
DKK1
HP+PC
+
DKK1
Control HP HP+CTR HP+PC HP
+
DKK1
HP+CTR
+
DKK1
HP+PC
+
DKK1
Control HP HP+CTR HP+PC
Axin 2 mRNA
(vs. Contro
HP
+
DKK1
HP+CTR
+
DKK1
HP+PC
+
DKK1
#
0
0.5
1
Control HP HP+CTR HP+PC
Axin 2 mRNA
(vs. Contro
HP
+
DKK1
HP+CTR
+
DKK1
HP+PC
+
DKK1
#
A
N
mRNA
levels
(vs. Control)
#
#
*
*
AN
mRNA
levels
(vs. Control)
1
2
1.5
AN
mRNA
levels
(vs. Control)
*
#
#
*
*
Control HP HP+CTR HP+PC
VCA
HP
+
DKK1
HP+CTR
+
DKK1
HP+PC
+
DKK1
Control HP HP+CTR HP+PC
VC
A
HP
+
DKK1
HP+CTR
+
DKK1
HP+PC
+
DKK1
0
0.5
Control HP HP+CTR HP+PC
VC
A
HP
+
DKK1
HP+CTR
+
DKK1
HP+PC
+
DKK1
Fig. 5. Effect of calcitriol and paricalcitol on the expression of -catenin direct
transcriptional target genes, cyclin D1, axin 2, and VCAN/versican, during
HASMC. HASMCs are incubated for 24 h in a high (3.3 mmol/l) phosphate
(HP) medium (calcification medium) alone or supplemented with either cal-
citriol 10
8
M (HP CTR) or paricalcitol 3·10
8
M (HP PC) alone or with
the addition to culture medium (100 ng/ml) of commercially available DKK-1,
a specific endogenous extracellular antagonist of the Wnt signaling. Cells
incubated in normal phosphate (0.9 mmol/l) medium are used as controls.
mRNA levels are analyzed by real-time RT-PCR technique. Expression of
target gene mRNA is normalized to the expression of GAPDH. Bars are
means SE (3 independent experiments; 6 repetitions in each experiment).
*P 0.05 vs. control. $P 0.05 vs. HP. #P 0.05 vs. the same treatment
without DKK-1.
5
* $
*
#
2
3
4
* $
#
$
Calcium deposition
g
/mg protein vs. Control)
0
1
Control HP HP+CTR HP+PC HP
+
DKK1
HP+CTR
+
DKK1
HP+PC
+
DKK1
(µ
g
Fig. 4. Effect of the inhibition of the canonical Wnt/-catenin signaling
pathway by DKK-1 on HASMC. HASMCs are incubated for 24 h in a high
(3.3 mmol/l) phosphate (HP) medium (calcification medium) alone or supple-
mented with either calcitriol 10
8
M (HP CTR) or paricalcitol 3·10
8
M
(HP PC) alone or with the addition to culture medium (100 ng/ml) of
commercially available DKK-1, a specific endogenous extracellular antagonist
of the Wnt signaling. Cells incubated in normal phosphate (0.9 mmol/l)
medium are used as controls. Calcium content is determined with the phenol-
sulphonephthalein dye. Bars are means SE (3 independent experiments; 6
repetitions in each experiment). *P 0.05 vs. control. $P 0.05 vs. HP.
#P 0.05 vs. the same treatment without DKK-1.
F1140 PARICALCITOL AND Wnt/-CATENIN
AJP-Renal Physiol doi:10.1152/ajprenal.00684.2011 www.ajprenal.org
by 10.220.32.246 on November 21, 2017http://ajprenal.physiology.org/Downloaded from

Figures (3)
Citations
More filters

Journal ArticleDOI
Ting Cai1, Danqin Sun1, Ying Duan1, Ping Wen1  +3 moreInstitutions (1)
TL;DR: It is suggested that high-phosphate may activate WNT/β-catenin signaling through different pathways, and the activated WNT-3A/ β-catanin signaling, through direct downstream target Runx2, could play an important role in promoting VOT and AMC.
Abstract: Arterial medial calcification (AMC) is prevalent in patients with chronic kidney disease (CKD) and contributes to elevated risk of cardiovascular events and mortality. Vascular smooth muscle cells (VSMCs) to osteogenic transdifferentiation (VOT) in a high-phosphate environment is involved in the pathogenesis of AMC in CKD. WNT/β-catenin signaling is indicated to play a crucial role in osteogenesis via promoting Runx2 expression in osteoprogenitor cells, however, its role in Runx2 regulation and VOT remains incompletely clarified. In this study, Runx2 was induced and β-catenin was activated by high-phosphate in VSMCs. Two forms of active β-catenin, dephosphorylated on Ser37/Thr41 and phosphorylated on Ser675 sites, were upregulated by high-phosphate. Activation of β-catenin, through ectopic expression of stabilized β-catenin, inhibition of GSK-3β, or WNT-3A protein, induced Runx2 expression, whereas blockade of WNT/β-catenin signaling with Porcupine (PORCN) inhibitor or Dickkopf-1 (DKK1) protein inhibited Runx2 induction by high-phosphate. WNT-3A promoted osteocalcin expression and calcium deposition in VSMCs, whereas DKK1 ameliorated calcification of VSMCs induced by high-phosphate. Two functional T cell factor (TCF)/lymphoid enhancer-binding factor binding sites were identified in the promoter region of Runx2 gene in VSMCs, which interacted with TCF upon β-catenin activation. Site-directed mutation of each of them attenuated Runx2 response to β-catenin, and deletion or destruction of both of them completely abolished this responsiveness. In the aortic tunica media of rats with chronic renal failure, followed by AMC, Runx2 and β-catenin was induced, and the Runx2 mRNA level was positively associated with the abundance of phosphorylated β-catenin (Ser675). Collectively, our study suggested that high-phosphate may activate WNT/β-catenin signaling through different pathways, and the activated WNT/β-catenin signaling, through direct downstream target Runx2, could play an important role in promoting VOT and AMC.

121 citations


Cites background from "In vascular smooth muscle cells par..."

  • ...As a master osteoblast transcription factor and the earliest osteoblastic marker, Runx2 has been generally recognized as an early feature of VOT [24,25,27,28]....

    [...]

  • ...Several studies indicate that high-phosphate can activate β-catenin signaling, which is evidenced by the upregulation of βcatenin expression [25,26], the decrease of phosphorylated β-catenin (Ser33/37/Thr41) [26], or the translocation of β-catenin into the nuclei [24,27] in VSMCs....

    [...]


Journal ArticleDOI
25 Feb 2014-PLOS ONE
TL;DR: In conclusion, magnesium transport through the cell membrane is important to inhibit VSMC calcification in vitro and inhibition of Wnt/β-catenin by magnesium is one potential intracellular mechanism by which this anti-calcifying effect is achieved.
Abstract: Magnesium reduces vascular smooth muscle cell (VSMC) calcification in vitro but the mechanism has not been revealed so far. This work used only slightly increased magnesium levels and aimed at determining: a) whether inhibition of magnesium transport into the cell influences VSMC calcification, b) whether Wnt/β-catenin signaling, a key mediator of osteogenic differentiation, is modified by magnesium and c) whether magnesium can influence already established vascular calcification. Human VSMC incubated with high phosphate (3.3 mM) and moderately elevated magnesium (1.4 mM) significantly reduced VSMC calcification and expression of the osteogenic transcription factors Cbfa-1 and osterix, and up-regulated expression of the natural calcification inhibitors matrix Gla protein (MGP) and osteoprotegerin (OPG). The protective effects of magnesium on calcification and expression of osteogenic markers were no longer observed in VSMC cultured with an inhibitor of cellular magnesium transport (2-aminoethoxy-diphenylborate [2-APB]). High phosphate induced activation of Wnt/β-catenin pathway as demonstrated by the translocation of β-catenin into the nucleus, increased expression of the frizzled-3 gene, and downregulation of Dkk-1 gene, a specific antagonist of the Wnt/β-catenin signaling pathway. The addition of magnesium however inhibited phosphate-induced activation of Wnt/β-catenin signaling pathway. Furthermore, TRPM7 silencing using siRNA resulted in activation of Wnt/β-catenin signaling pathway. Additional experiments were performed to test the ability of magnesium to halt the progression of already established VSMC calcification in vitro. The delayed addition of magnesium decreased calcium content, down-regulated Cbfa-1 and osterix and up-regulated MGP and OPG, when compared with a control group. This effect was not observed when 2-APB was added. In conclusion, magnesium transport through the cell membrane is important to inhibit VSMC calcification in vitro. Inhibition of Wnt/β-catenin by magnesium is one potential intracellular mechanism by which this anti-calcifying effect is achieved.

105 citations


Journal ArticleDOI
TL;DR: Caution is however warranted as sclerostin not only opposes mineralization in the bone but possibly also in the vasculature, as additional studies are required to define determinants of Wnt inhibitors in CKD and to evaluate the efficacy and safety of recently introduced pharmaceuticals targeting these inhibitors.
Abstract: For more than a decade, the Wnt–β-catenin pathway has been the focus of intense basic and clinical research in the bone field because of its importance in skeletal development and maintenance of bone mass. Wnt activation increases bone formation and decreases bone resorption. The Wnt–β-catenin signaling pathway is tightly regulated by several inhibitors, among which Dickkopf-related protein 1 (DKK1) and sclerostin have been most comprehensively studied. Mounting evidence indicates that a disturbed Wnt–β-catenin signaling is also implicated in the pathogenesis of the chronic kidney disease–associated bone and mineral disorder (CKD-MBD) and affects its various components. DKK1 and sclerostin, more specifically, may be involved in the intense cross-talk between the kidneys, vasculature, and bone. Studies exploring clinical correlates of circulating sclerostin and DKK1 levels so far yielded conflicting results. Biological variability and analytical issues account at least partly for this inconsistency. Antibodies neutralizing Wnt inhibitors may be an appealing strategy to prevent or treat CKD-MBD. Caution is however warranted as sclerostin not only opposes mineralization in the bone but possibly also in the vasculature. Additional studies are required to define determinants of Wnt inhibitors in CKD and to evaluate the efficacy and safety of recently introduced pharmaceuticals targeting these inhibitors.

92 citations


Journal ArticleDOI
21 Oct 2013-Cancers
TL;DR: The molecular basis for the various mechanisms by which 1,25(OH)2D3 antagonizes Wnt/β-catenin signaling, preferentially in human colon carcinoma cells, and the consequences of this inhibition for the phenotype and proliferation rate are reviewed.
Abstract: The Wnt/b-catenin signaling pathway is abnormally activated in most colorectal cancers and in a proportion of other neoplasias. This activation initiates or contributes to carcinogenesis by regulating the expression of a large number of genes in tumor cells. The active vitamin D metabolite 1a,25-dihydroxyvitamin D3 (1,25(OH)2D3) inhibits Wnt/b-catenin signaling by several mechanisms at different points along the pathway. Additionally, paracrine actions of 1,25(OH)2D3 on stromal cells may also repress this pathway in neighbouring tumor cells. Here we review the molecular basis for the various mechanisms by which 1,25(OH)2D3 antagonizes Wnt/b-catenin signaling, preferentially in human colon carcinoma cells, and the consequences of this inhibition for the phenotype and proliferation rate. The effect of the vitamin D system on Wnt/b-catenin signaling and tumor growth in animal models will also be commented in detail. Finally, we revise existing data on the relation between vitamin D receptor expression and vitamin D status and the expression of Wnt/b-catenin pathway genes and targets in cancer patients.

90 citations


Journal ArticleDOI
Demin Liu1, Wei Cui, Bin Liu, Haijuan Hu  +6 moreInstitutions (1)
TL;DR: It is demonstrated that atorvastatin protect VSMC from TGF-β1-stimulated calcification by inducing autophagy through suppression of the β-catenin pathway, which might be a therapeutic strategy for use in vascular calcification.
Abstract: Background: Arterial calcification is a major event in the progression of atherosclerosis. It is reported that statins exhibit various protective effects against vascular smooth muscle cell (VSMC) inflammation and proliferation in cardiovascular remodeling. Although statins counteract atherosclerosis, the molecular mechanisms of statins on the calcium release from VSMCs have not been clearly elucidated. Methods: Calcium content of VSMCs was measured using enzyme-linked immunosorbent assay (ELISA). The expression of proteins involved in cellular transdifferentiation was analyzed by western blot. Cell autophagy was measured by fluorescence microscopic analysis for acridine orange staining and transmission electron microscopy analysis. The autophagic inhibitors (3-MA, chloroquine, NH4Cl and bafilomycin A1) and β-catenin inhibitor JW74 were used to assess the effects of atorvastatin on autophagy and the involvement of β-catenin on cell calcification respectively. Furthermore, cell transfection was performed to overexpress β-catenin. Results: In VSMCs, atorvastatin significantly suppressed transforming growth factor-β1 (TGF-β1)-stimulated calcification, accompanied by the induction of autophagy. Downregulation of autophagy with autophagic inhibitors significantly suppressed the inhibitory effect of atorvastatin on cell calcification. Moreover, the beneficial effect of atorvastatin on calcification and autophagy was reversed by β-catenin overexpression. Conversely, JW74 supplement enhanced this effect. Conclusion: These data demonstrated that atorvastatin protect VSMC from TGF-β1-stimulated calcification by inducing autophagy through suppression of the β-catenin pathway, identifying autophagy induction might be a therapeutic strategy for use in vascular calcification.

87 citations


References
More filters

Journal ArticleDOI
TL;DR: This study concludes that a large percentage of hemodialysis patients who have a serum phosphorus level above 6.5 mg/dL and that this places them at increased risk of death, and supports the need for vigorous control of hyperphosphatemia to improve patient survival.
Abstract: Elevated serum phosphorus is a predictable accompaniment of end-stage renal disease (ESRD) in the absence of dietary phosphate restriction or supplemental phosphate binders. The consequences of hyperphosphatemia include the development and progression of secondary hyperparathyroidism and a predisposition to metastatic calcification when the product of serum calcium and phosphorus (Ca x PO4) is elevated. Both of these conditions may contribute to the substantial morbidity and mortality seen in patients with ESRD. We have analyzed the distribution of serum phosphorus in two large national, random, cross-sectional samples of hemodialysis patients who have been receiving dialysis for at least 1 year. Data were obtained from two special studies of the United States Renal Data System, the Case Mix Adequacy Study (1990) and the Dialysis Morbidity and Mortality Study Wave 1 (1993). The relative risk of death by serum phosphorus quintiles is described after adjusting for age at onset of ESRD, race, sex, smoking status, and the presence of diabetes, the acquired immunodeficiency syndrome, and/or neoplasm. Logistic regression analysis is then used to describe the demographic, comorbid, and laboratory parameters associated with high serum phosphorus. Serum phosphorus was similar in these two study populations and averaged 6.2 mg/dL. Ten percent of patients had levels greater than 9 mg/dL and at least 30% of each group had serum phosphorus levels greater than 7 mg/dL. The adjusted relative risk of death by serum phosphorus level was not uniform across all quintiles, being constant below a level of 6.5 mg/dL and increasing significantly above this level. The relative risk of death for those with a serum phosphorus greater than 6.5 mg/dL was 1.27 relative to those with a serum phosphorus of 2.4 to 6.5 mg/dL. This increased risk was not diminished by statistical adjustment for coexisting medical conditions, delivered dose of dialysis, nutritional parameters, or markers of noncompliance. Evaluation of predictors of serum phosphorus greater than 6.5 mg/dL revealed in multivariate analysis that younger age at onset of ESRD, female sex, white race, diabetes, active smoking, and higher serum creatinine levels were all significant predictors. Analysis of serum calcium revealed no correlation with relative risk of death. The Ca x PO4 product, however, showed a mortality risk trend similar to that seen with serum phosphorus alone. Those in the highest quintile of the Ca x PO4 product (>72 mg2/dL2) had a relative mortality risk of 1.34 relative to those with products of 42 to 52 mg2/dL2. The relative mortality risk by log parathyroid hormone (PTH) level was elevated for patients with higher levels, but the mortality risk associated with hyperphosphatemia was independent of PTH. For hemodialysis patients who have been receiving dialysis for at least 1 year, we conclude that a large percentage have a serum phosphorus level above 6.5 mg/dL and that this places them at increased risk of death. This increased risk is independent of PTH. The mechanism(s) responsible for death is unknown, but may be related to an abnormally high Ca x PO4 product. Although mechanisms are not clearly established, this study supports the need for vigorous control of hyperphosphatemia to improve patient survival.

2,246 citations


"In vascular smooth muscle cells par..." refers background in this paper

  • ...Abnormal mineral metabolism, and particularly the accumulation of phosphate, plays a central role in the generation of VC (3, 25)....

    [...]


Journal ArticleDOI
TL;DR: Increased beta-catenin levels may promote neoplastic conversion by triggering cyclin D1 gene expression and, consequently, uncontrolled progression into the cell cycle through a LEF-1 binding site in the cyclinD1 promoter.
Abstract: β-Catenin plays a dual role in the cell: one in linking the cytoplasmic side of cadherin-mediated cell–cell contacts to the actin cytoskeleton and an additional role in signaling that involves transactivation in complex with transcription factors of the lymphoid enhancing factor (LEF-1) family. Elevated β-catenin levels in colorectal cancer caused by mutations in β-catenin or by the adenomatous polyposis coli molecule, which regulates β-catenin degradation, result in the binding of β-catenin to LEF-1 and increased transcriptional activation of mostly unknown target genes. Here, we show that the cyclin D1 gene is a direct target for transactivation by the β-catenin/LEF-1 pathway through a LEF-1 binding site in the cyclin D1 promoter. Inhibitors of β-catenin activation, wild-type adenomatous polyposis coli, axin, and the cytoplasmic tail of cadherin suppressed cyclin D1 promoter activity in colon cancer cells. Cyclin D1 protein levels were induced by β-catenin overexpression and reduced in cells overexpressing the cadherin cytoplasmic domain. Increased β-catenin levels may thus promote neoplastic conversion by triggering cyclin D1 gene expression and, consequently, uncontrolled progression into the cell cycle.

2,185 citations


Journal ArticleDOI
TL;DR: It is shown that mice with a targeted disruption of Lrp5 develop a low bone mass phenotype, and it is demonstrated that this phenotype becomes evident postnatally, and that it is secondary to decreased osteoblast proliferation and function in a Cbfa1-independent manner.
Abstract: The low-density lipoprotein receptor-related protein (Lrp)-5 functions as a Wnt coreceptor. Here we show that mice with a targeted disruption of Lrp5 develop a low bone mass phenotype. In vivo and in vitro analyses indicate that this phenotype becomes evident postnatally, and demonstrate that it is secondary to decreased osteoblast proliferation and function in a Cbfa1-independent manner. Lrp5 is expressed in osteoblasts and is required for optimal Wnt signaling in osteoblasts. In addition, Lrp5-deficient mice display persistent embryonic eye vascularization due to a failure of macrophage-induced endothelial cell apoptosis. These results implicate Wnt proteins in the postnatal control of vascular regression and bone formation, two functions affected in many diseases. Moreover, these features recapitulate human osteoporosis-pseudoglioma syndrome, caused by LRP5 inactivation.

1,068 citations


"In vascular smooth muscle cells par..." refers background in this paper

  • ...Interestingly, VDR activation by 1,25-(OH)2D3 caused upregulation of low-density lipoprotein receptor-related protein 5, a Wnt coreceptor that plays an important role in bone formation (10, 21), through its binding to a VDR element (10)....

    [...]


Journal ArticleDOI
TL;DR: It is proposed that WNT/TCF1 signaling, like bone morphogenetic protein/transforming growth factor-β signaling, activates Runx2 gene expression in mesenchymal cells for the control of osteoblast differentiation and skeletal development.
Abstract: Both activating and null mutations of proteins required for canonical WNT signaling have revealed the importance of this pathway for normal skeletal development. However, tissue-specific transcriptional mechanisms through which WNT signaling promotes the differentiation of bone-forming cells have yet to be identified. Here, we address the hypothesis that canonical WNT signaling and the bone-related transcription factor RUNX2/CBFA1/AML3 are functionally linked components of a pathway required for the onset of osteoblast differentiation. Our findings show that, in bone of the SFRP1 (secreted frizzled-related protein-1)-null mouse, which exhibits activated WNT signaling and a high bone mass phenotype, there is a significant increase in expression of T-cell factor (TCF)-1, Runx2, and the RUNX2 target gene osteocalcin. We demonstrate by mutational analysis that a functional TCF regulatory element responsive to canonical WNT signaling resides in the promoter of the Runx2 gene (-97 to -93). By chromatin immunoprecipitation, recruitment of beta-catenin and TCF1 to the endogenous Runx2 gene is shown. Coexpression of TCF1 with canonical WNT proteins resulted in a 2-5-fold activation of Runx2 promoter activity and a 7-8-fold induction of endogenous mRNA in mouse pluripotent mesenchymal and osteoprogenitor cells. This enhancement was abrogated by SFRP1. Taken together, our results provide evidence for direct regulation of Runx2 by canonical WNT signaling and suggest that Runx2 is a target of beta-catenin/TCF1 for the stimulation of bone formation. We propose that WNT/TCF1 signaling, like bone morphogenetic protein/transforming growth factor-beta signaling, activates Runx2 gene expression in mesenchymal cells for the control of osteoblast differentiation and skeletal development.

966 citations


"In vascular smooth muscle cells par..." refers background in this paper

  • ...Both contribute to the regulation of bone mass by promoting osteogenesis through the direct stimulation of Runx2 gene expression (12)....

    [...]


Journal ArticleDOI
TL;DR: In the context of raised Ca and P, vascular calcification is a modifiable, cell-mediated process regulated by vesicle release, and perturbation of the production or function of these inhibitors would lead to accelerated vascular calcifying.
Abstract: Patients with ESRD have a high circulating calcium (Ca) phosphate (P) product and develop extensive vascular calcification that may contribute to their high cardiovascular morbidity. However, the cellular mechanisms underlying vas- cular calcification in this context are poorly understood. In an in vitro model, elevated Ca or P induced human vascular smooth muscle cell (VSMC) calcification independently and synergistically, a process that was potently inhibited by serum. Calcification was initiated by release from living VSMC of membrane-bound matrix vesicles (MV) and also by apoptotic bodies from dying cells. Vesicles released by VSMC after prolonged exposure to Ca and P contained preformed basic calcium phosphate and calcified extensively. However, vesi- cles released in the presence of serum did not contain basic calcium phosphate, co-purified with the mineralization inhib- itor fetuin-A and calcified minimally. Importantly, MV re- leased under normal physiologic conditions did not calcify, and VSMC were also able to inhibit the spontaneous precipitation of Ca and P in solution. The potent mineralization inhibitor matrix Gla protein was found to be present in MV, and pre- treatment of VSMC with warfarin markedly enhanced vesicle calcification. These data suggest that in the context of raised Ca and P, vascular calcification is a modifiable, cell-mediated process regulated by vesicle release. These vesicles contain mineralization inhibitors derived from VSMC and serum, and perturbation of the production or function of these inhibitors would lead to accelerated vascular calcification.

829 citations


"In vascular smooth muscle cells par..." refers result in this paper

  • ...As previously demonstrated by us and other researchers (6, 36, 40, 44, 47, 49, 58), VSMCs cultured in high phosphate undergo osteogenic transformation and calcification....

    [...]


Performance
Metrics
No. of citations received by the Paper in previous years
YearCitations
20221
20211
20207
20199
201810
20178