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This article is part of a thematic series on Pathobiology of Calcific Vasculopathy and Valvulopathy, which includes
the following articles:
Thematic Series on the Pathobiology of Vascular Calcification: An Introduction [Circ Res. 2011;108:1378 1380]
Molecular Imaging Insights Into Early Inflammatory Stages of Arterial and Aortic Valve Calcification [Circ Res.
2011;108:13811391]
Calcific Aortic Valve Stenosis: Methods, Models, and Mechanisms [Circ Res. 2011;108:13921412]
The Roles of Lipid Oxidation Products and Receptor Activator of Nuclear Factor-kappa B Signaling in Atherosclerotic
Calcification [Circ Res. 2011;108:14821493]
Fetuin-A Regulation of Calcified Matrix Metabolism [Circ Res. 2011;108:1494 1509]
CellMatrix Interactions in the Pathobiology of Calcific Aortic Valve Disease: Critical Roles for Matricellular,
Matricrine, and Matrix Mechanics Cues [Circ Res. 2011;108:1510 1524]
The Regulation of Valvular and Vascular Sclerosis by Osteogenic Morphogens [Circ Res. 2011;109:564 577]
Genetics in Arterial Calcification: Pieces of a Puzzle and Cogs in a Wheel [Circ Res. 2011;109:578 592]
Arterial Calcification in Chronic Kidney Disease: Key Roles for Calcium and Phosphate
Abstract: Vascular calcification contributes to the high risk of cardiovascular mortality in chronic kidney disease
(CKD) patients. Dysregulation of calcium (Ca) and phosphate (P) metabolism is common in CKD patients and
drives vascular calcification. In this article, we review the physiological regulatory mechanisms for Ca and P
homeostasis and the basis for their dysregulation in CKD. In addition, we highlight recent findings indicating
that elevated Ca and P have direct effects on vascular smooth muscle cells (VSMCs) that promote vascular
calcification, including stimulation of osteogenic/chondrogenic differentiation, vesicle release, apoptosis, loss of
inhibitors, and extracellular matrix degradation. These studies suggest a major role for elevated P in promoting
osteogenic/chondrogenic differentiation of VSMC, whereas elevated Ca has a predominant role in promoting
VSMC apoptosis and vesicle release. Furthermore, the effects of elevated Ca and P are synergistic, providing a
major stimulus for vascular calcification in CKD. Unraveling the complex regulatory pathways that mediate the
effects of both Ca and P on VSMCs will ultimately provide novel targets and therapies to limit the destructive
effects of vascular calcification in CKD patients. (Circ Res. 2011;109:697-711.)
Key Words: calcium chronic kidney disease phosphate vascular calcification
Original received April 19, 2011; revision received July 28, 2011; accepted July 29, 2011. In June 2011, the average time from submission to first
decision for all original research papers submitted to Circulation Research was 14.48 days.
From the British Heart Foundation Centre of Excellence (C.M.S., A.K.), Cardiovascular Division, Kings College London, London UK; Bioengineering
Department (M.H.C., C.M.G.), University of Washington, Seattle, WA.
Correspondence to Cecilia Giachelli, Department of Bioengineering, University of Washington, Box 355061, Seattle, WA 98195. E-mail
ceci@u.washington.edu
2011 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org
DOI: 10.1161/CIRCRESAHA.110.234914
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September 2, 2011
alkaline phosphatase
bone morphogenetic protein 2
calcium
calcium sensing receptor
calcium channel blocker
chronic kidney disease
extracellular matrix
fibroblast growth factor 23
matrix Gla protein
matrix metalloproteinase
sodium-dependent phosphate cotransporter
osteopontin
phosphate
inorganic phosphate
parathyroid hormone
reactive oxygen species
vascular smooth muscle cells
cation rapidly progresses in patients on dialysis.1 The development of calcification in CKD patients is strongly linked to
dysregulated mineral metabolism characterized by long-term
elevation of serum phosphate (P) levels as well as transient
Figure 1. Vascular calcification is mediated by vascular smooth muscle cells (VSMCs). Alterations in calcium (Ca) and phosphate
(P) levels or vascular insult lead to osteogenic/chondrogenic conversion of VSMCs in the vascular wall. This is associated with dramatic
loss of mineralization inhibitors, the production of calcifying matrix vesicles, and extracellular matrix (ECM) degradation. In addition, Ca
and P induce VSMC apoptosis and release of apoptotic bodies, which, in turn, form the initial nidus for vascular calcification. (Illustration Credit: Cosmocyte/Ben Smith).
Shanahan et al
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Shanahan et al
701
Figure 3. Role of phosphate (P) in vascular smooth muscle cell (VSMC) calcification. Elevated extracellular P affects multiple signaling pathways that increase the susceptibility of VSMC to calcification, including decreased calcification inhibitors, increased extracellular matrix (ECM) degradation, osteogenic/chondrogenic differentiation, apoptosis, and vesicle release. Some of the effects of P are
mediated through sodium-dependent phosphate cotransporters, Pit-1 and Pit-2, potentially via P transport-dependent and P transportindependent activities. Whether other receptors exist that mediate specific downstream signaling pathways in response to P is not yet
known but cannot be excluded.
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epigastric arteries of renal transplantation patients.76,77 Finally, increased Runx2, osterix, and ALP expressions were
observed in arteries from pediatric predialysis and dialysis
patients.78
Shanahan et al
vivo, elastin degradation alone in the absence of a P load was
insufficient to induce vascular calcification in uremic mice.83
In vivo evidence supporting a role for matrix remodeling in
elevated P-induced vascular calcification include studies in
uremic high-P-fed mice that observed elastin remodeling and
elevated elastase levels including MMP-2, MMP-9, and
cathepsin S in calcified arterial medias.83 Furthermore, cathepsin S was required for arterial calcification in apolipoprotein E/ mice with chronic renal insufficiency.91 Finally,
a strong correlation between MMP-2 upregulation and elastic
fiber disorganization, stiffness, calcification, and vasomotor
dysfunction was observed in the arterial vasculature in
dialysis patients,92 and upregulation of arterial MMP-2 and
MMP-9 were correlated with arterial stiffening in diabetic
CKD patients.92
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Channel-Mediated Ca Uptake
Plasma membrane voltage-dependent L-type Ca channels
have long been recognized as important for Ca homeostasis in
VSMCs. These channels as well as T-type Ca channels can be
targeted by drugs known as Ca channel blockers (CCBs)
widely used in the treatment of hypertension.116,117 In a
prospective clinical trial, CCBs were shown to slow the
progression of calcification in hypertensive patients with and
without renal failure,118 121 whereas association studies have
shown they can reduce mortality in renal patients.122
Pioneering studies by Fleckenstein119 showed that in response to vitamin D overload in rats vascular calcification
could be prevented by treatment with the CCBs verapamil or
nifedipine. Although it was assumed that the mechanism of
action was via prevention of Ca uptake in the hypercalcemic
environment induced in the rats in response to vitamin D,
other direct VSMC effects have not been excluded. For
example, CCBs are effective in blocking calcification induced by either gluteraldehyde or warfarin treatments, and
these are not associated with hypercalcemia.123,124 CCBs can
also partially block atherogenesis in animal models and this
may be attributable to their known effects on VSMC differentiation, proliferation, and matrix synthesis.125,126 Despite
the importance of Ca uptake in VSMCs, few studies have
investigated CCBs in the context of calcification in vitro. In
a recent study, Chen et al127 using bovine VSMCs showed
that verapamil and not nifedipine could block VSMC calcification, potentially via downregulation of ALP activity.
Interestingly, verapamil also blocked mineralization of
The CaR
Elevated extracellular Ca also has a direct signaling role in
VSMC calcification without any need for uptake.128 The CaR
is a G-protein-coupled receptor capable of sensing changes in
extracellular Ca concentrations in the mmol/L range. The
CaR was first identified in the parathyroid, where it regulates
Ca homeostasis by suppressing PTH secretion and renal Ca
reabsorption. However, it is also widely expressed on tissues
that are not involved in the regulation of Ca homeostasis,
including VSMC and endothelial cells. In contractile VSMCs
within the vessel wall the CaR has been shown to play a
physiological role in regulating vascular myogenic tone.129 In
cultured VSMCs it regulates proliferation129,130 and survival
with stimulation of the CaR leading to ERK1/2 activation,
suggesting the CaR may be important in both contractile and
synthetic VSMC phenotypic contexts.131
In calcified arteries from CKD patients, expression of the
CaR is downregulated131 and studies in vitro have shown that
when VSMCs are induced to calcify in response to elevated
extracellular Ca, expression of the CaR is also downregulated.132 Moreover, ablation of CaR function increased
VSMC calcification in response to both Ca and P, whereas
calcimimetics, drugs that increase the sensitivity of the CaR
to Ca, can ameliorate calcification in the same model.132
Although these studies suggest that the CaR is deregulated in
CKD, they were unable to identify a mechanism to account
for reduced calcification in the presence of a functional CaR.
However, a role for the CaR in regulating expression of the
key calcification inhibitor MGP previously has been
shown.133 Increased extracellular Ca can increase MGP transcription, and this transcriptional activation can be mimicked
by treatment with CaR agonists. Thus, Ca sensing may be
critical for the feedback response of VSMCs to increased
extracellular Ca leading to the production of inhibitory
proteins. This notion is supported by in vivo studies showing
treatment of animals with or without renal failure with
calcimimetics increases aortic MGP production.134 If calcimimetics have direct effects on VSMC calcification, then these
drugs, which are in clinical use for hyperparathyroidism, also
may be useful in blocking progression of calcification.
Shanahan et al
SM contraction, whereas the mitochondria and lysosomes are
involved in longer-term events that also may be crucial for
VSMC calcification. Two recent studies have demonstrated
that the release of intracellular Ca from lysosomes may act to
promote calcification. In the first study, human VSMCs were
exposed to CaP nanocrystals of varying size. Phagocytotic
uptake of the smallest crystals by VSMCs was shown to
induce an intracellular Ca burst that resulted in necrotic/apoptotic cell death. This process could be inhibited by treatment
with the lysosomal proton pump inhibitor bafilomycin A1,
suggesting lysosomal processing of the mineral caused toxic
intracellular Ca release.110 Importantly, the vascular wall in
CKD patients has a very high Ca load and this Ca is deposited
in the extracellular matrix surrounding VSMCs as nanocrystals.78,135 Thus, at the early stages of mineral nidus formation
in the vessel wall, uptake of previously deposited mineral by
VSMCs would be predicted to promote cell death. This
notion is supported by evidence from an ex vivo model using
human vessels treated with Ca/P, where it was shown that
VSMCs undergo a wave of rapid apoptosis concomitantly
with the formation of the first crystalline nidus of calcification observable by electron microscopy.136
In a second study, Ca/P nanocrystals were found to
promote VSMC osteogenic differentiation. This phenotypic
change was restricted to upregulation of BMP-2 expression
within 24 hours and was not associated with elevated
Runx2.59 The mechanism that induced osteogenic gene expression remains unknown but may also involve Ca release
from intracellular stores. More speculatively, Ca overload has
been associated with aging of the vasculature and BMP-2 was
recently shown to be increased in senescent VSMCs.137 It is
plausible that uptake of nanocrystals promotes Ca-induced or
P-induced VSMC senescence, which drives further osteogenic differentiation via the BMP-2 pathway.138
ER stress can be induced by deregulated Ca homeostasis,
and emerging evidence indicates there may be a role for ER
stress in vascular calcification. First, ER stress recently has
been implicated as essential for bone development and
mineralization because it regulates osteoblast differentiation
via Runx2 and collagen I secretion.139 141 The ER stress
markers Grp78, Grp94, and CHOP were found in homogenates from rat aortas, where medial vascular calcification was
induced by vitamin D treatment, which is known to induce
intracellular Ca overload in VSMCs. These ER stress markers
were associated with increased VSMC apoptosis and signaling via JNK, as well as activation of caspases 3 and 12.142 So
far, expression of these markers has not been explored in
vessels from CKD patients.
Mitochondrial Ca overload is also a prominent feature of
many calcified tissues and, interestingly, this was observed in
normal arteries after long-term exposure to Ca and P ex
vivo.136,143,144 This contrasted to the predominant calcification of the extracellular matrix with no sign of mitochondrial
damage and Ca overload, which was observed in CKD
arteries exposed to the same conditions. One explanation for
this is possibly the different mechanisms of adaptation to
elevated Ca by contractile and phenotypically modified
VSMCs. Massive release of Ca-loaded vesicles by phenotypically modified VSMCs may mitigate intracellular Ca over-
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Figure 4. Role of calcium (Ca) in vascular smooth muscle cell (VSMC) calcification. Ca elevation induces vesicle production and
can lead to cell apoptosis and necrosis. High local levels of Ca trigger mineral nucleation in the matrix vesicles and apoptotic bodies.
Accumulation of mineralized matrix vesicles and apoptotic bodies, and their uptake accelerate cell death and facilitate further crystal
growth. Degradation of extracellular matrix (ECM) and nucleation of Ca apatite induces ECM protein mineralization.
Shanahan et al
MGP production in a number of studies, potentially as an
adaptive response aimed at inhibiting calcification.133,157159
However, constant stimulation may lead to ER stress and act
to deregulate carboxylation pathways localized in the ER,
leading to the production of unprocessed MGP. In addition to
this mechanism of MGP inactivation, a recent study has
demonstrated that exposure of VSMCs to elevated Ca eventually depletes MGP from matrix vesicles, further enhancing
their calcification capacity. This study also showed that
matrix vesicles are loaded with MMP-2 and Ca acts to
increase activation of this metalloproteinase, which would
further accelerate elastin degradation and calcification.148
Studies in vivo and ex vivo have led further support to
these in vitro findings. Using vessels obtained from children
with CKD or on dialysis, it was shown that Ca load in the
vessel wall correlated with time averaged CaP.78 Moreover,
in dialysis, vessels calcification correlated with increased
apoptosis and increased vesicle release shown by increased
annexin A6 deposition in the vessel wall.78 Extending these
studies by using vessels from these same children in organ
culture experiments, it was shown that control vessels were
entirely resistant to the induction of calcification by Ca or P.
In contrast, vessels obtained from children on dialysis extensively calcified in response to Ca/P. Again, the predominant
mechanism appeared to be increased apoptosis and increased
vesicle deposition, leading to extracellular matrix calcification.136 In addition, increased depositions of both fetuin-A
and uncarboxylated MGP were observed in vessels from
dialysis patients, and this deposition was dramatically increased in response to Ca/P treatment ex vivo. EM analysis of
the vessels showed large numbers of calcified and noncalcified matrix vesicles deposited in the extracellular matrix,
consistent with the induction of vesicular calcification once
inhibitors had been exhausted. Further studies are now
required to determine the cell biological mechanisms driving
vesicle calcification and inhibitor dysfunction in VSMCs in
response to Ca.
Conclusions
It is clear that dysregulated Ca and P homeostasis plays a
major role in driving VSMC calcification in CKD. In addition
to increasing the CaP, elevated Ca and P can act directly on
VSMC to drive distinct, as well as overlapping, pathways that
predispose to calcification (Figure 5). The body of data
presented in this review suggests that the maintenance of Ca
and P levels in the normal range is the most important aim in
CKD patients to minimize the vascular damage induced by
dysregulated mineral metabolism. However, there are many
other factors associated with the CKD milieu that may also
act to drive VSMC calcification. Some of these include
advanced glycation end products,160 ROS,70,161 and potentially vitamin D and PTH,162 although the calcific properties
of these factors may be dose-dependent and contextdependent. In addition to these factors, there are emerging
areas such as the FGF-23/klotho signaling axis that may
prove to be significant players in driving calcification. Although these factors are key to regulating Ca and P homeostasis, long-term exposure of VSMCs to elevated levels of
707
Figure 5. Overview of distinct and overlapping pathways initiated by elevated calcium (Ca) and phosphate (P) in vascular smooth muscle cells (VSMCs). Black background indicates
pathways specific to elevated serum inorganic phosphate (Pi),
white background indicates pathways specific to elevated
serum Ca, and gray area shows common pathways.
Sources of Funding
This work was supported by grants from the British Heart Foundation to C.M.S., NIH grants HL081785 and HL62329 to C.M.G., and
M.H.C. is funded by NIH training grant T32 HL07828.
Disclosures
None.
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