Vascular calcification and bone disease: the calcification paradox

Vascular calcification and bonedisease: the calcification paradoxVeerle Persy and Patrick D’Haese

Laboratory of Pathophysiology, University of Antwerp, Antwerp, Belgium

Review

Glossary

Atherosclerosis: inflammatory process characterized by the formation of

vascular plaques in the tunica intima, for which hypercholesterolemia is an

important causal factor.

Bisphosphonate: non-hydrolyzable pyrophosphate analog therapeutically

used as a bone resorption inhibitor in adult-onset osteoporosis.

Bone turnover: bone remodeling process characterized by a balance between

bone formation by osteoblasts and bone resorption by osteoclasts.

Calcification paradox: association of ectopic mineralization in the vasculature

with decreased or impaired bone turnover found in adult-onset osteoporosis

and chronic renal disease.

CKD: chronic kidney disease.

FGF-23: fibroblast growth factor 23; phosphaturic hormone inhibiting renal

phosphate reabsorption and regulating renal 1a-hydroxylase activity.

Klotho: anti-aging protein; cofactor for binding of FGF-23 to its receptor.

Monckeberg’s sclerosis: generalized stiffening and calcification process in the

tunica media of the large arteries. Synonyms are media calcification,

arteriosclerosis and elastocalcinosis.

Osteopenia: condition of low bone mineral density, 1.0–2.5 standard deviations

below peak bone density.

Osteoporosis: condition with bone mineral density more than 2.5 standard

deviations below peak bone density; accompanied by increased fracture risk.

Osteoprotegerin (OPG): cytokine of the TNF-a family, decoy receptor for

RANKL. Inhibits bone resorption by preventing binding of RANKL to RANK.

RANK: receptor activator of nuclear factor kB. Expressed on osteoclasts and

promotes osteoclast differentiation and activation on binding of RANKL.

RANKL: receptor activator of nuclear factor kB. Secreted by active osteoblasts

and forms the coupling agent linking bone formation and bone resorption.

Renal osteodystrophy: term for the metabolic bone diseases accompanying

chronic kidney disease.

Statin: drug of the HMG-CoA reductase inhibitor family used to treat

hypercholesterolemia.

TGF-b: transforming growth factor b fibrogenic hormone.

TNAP: tissue-nonspecific alkaline phosphatase.

TRAP: tartrate-resistant acid phosphatase.

Vascular calcification or ectopic mineralization in bloodvessels is an active, cell-regulated process, increasinglyrecognized as a general cardiovascular risk factor.Remarkably, ectopic artery mineralization is frequentlyaccompanied by decreased bone mineral density or dis-turbed bone turnover. This contradictory association,observed mainly in osteoporosis and chronic kidneydisease, is called the ‘calcification paradox’. Here, wereview recent advances in our understanding of thecalcification paradox, including protein expression pat-terns governing both normal and ectopic mineralization,the conversion of vascular smooth muscle cells to bone-like cells, and the regulatory pathways involved in bothbone and vessel mineralization. Further elucidation ofthe mechanisms underlying the calcification paradox iscrucial in order to develop preventive and therapeuticstrategies to deal with vascular calcification and reducethe associated cardiovascular risk.

IntroductionVascular calcification is a process in which mineral isectopically deposited in blood vessels, mainly in the largeelastic and muscular arteries, such as the aorta, the cor-onary, carotid and iliofemoral arteries, or in the cardiacvalves. This review focuses on the mechanisms underlyingthe paradoxical association of vascular tunica media(middle layer of artery wall) calcification with decreasedbone density or disturbed bone turnover. This so-called‘calcification paradox’ has mainly been observed in osteo-porosis and chronic kidney disease (CKD).

Vascular calcification: clinical context andpathophysiological implicationsCoronary artery calcification is recognized as an indepen-dent predictor of cardiovascular disease [1] and mortality[2]. A recent American College of Cardiology Foundation/American Heart Association consensus documentindicates that screening for coronary calcification providesincremental risk prediction information in patients withintermediate risk of coronary heart disease [3]. It wasshown that calcification of the abdominal aorta predictssubsequent cardiovascular events in the general popu-lation [4] and in hemodialysis patients [5].

Ectopic vessel mineralization can be localized either inthe tunica intima or in the tunica media of the vessel (Box1). Intima calcification is associated with atherosclerosisand results in focal calcification of atherosclerotic plaques,whereas media calcification (arteriosclerosis or Moncke-

Corresponding author: D’Haese, P. ([email protected]).

1471-4914/$ – see front matter � 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.molmed.2

berg’s sclerosis) is more generalized and is found mainly inthe elderly and in patients with CKD, osteoporosis, hyper-tension or diabetes mellitus. Monckeberg’s sclerosis leadsto vessel stiffening, which is characterized by increases inpulse pressure and pulse wave velocity and is associatedwith increased cardiovascular risk [6,7].

The distinctive characteristics of intima and mediacalcification are described in Box 1. However, in general,intima and media calcification are not distinguished inepidemiological studies, a confounding factor thatmust betaken into accountwhen interpreting results. In a study byLondon et al. in which both forms of calcification weredistinguishedmorphologically onplainX-ray images, bothintima and media calcification were shown to entail anincreased mortality risk in hemodialysis patients [8].Although the role of inflammation in atheroscleroticintima calcification is well established, no studies havereported inflammation as an etiologic factor in mediacalcification.

VSMC: vascular smooth muscle cell.

009.07.001 Available online 3 September 2009 405

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http://dx.doi.org/10.1016/j.molmed.2009.07.001

Box 1. Intima versus media calcification

Intima calcification Media calcification

Atherosclerosis Arteriosclerosis or Monckeberg’s sclerosis

Calcification pattern Focal, in plaques Generalized

Risk factors Dyslipidemia, hypercholesterolemia Aging, diabetes, renal failure, osteoporosis, hypertension

Molecular mechanisms Lipid accumulation Transdifferentiation of VSMCs into bone-like cells

(osteoblast-chondrocyte and osteoclast-like cells)Foam cell formationInflammation Ca, P, vitamin D metabolismOxidative stress Loss of calcification inhibitors (pyrophosphate, MGP, fetuin)Apoptosis

Consequences Plaque formation: stenosis Arterial stiffening: increased pulse pressure, elevated

pulse wave velocityPlaque calcification: controversial

effect on plaque stability, possibly

relating to the localization of

calcification [117]Complications Ischemia, infarction Systolic hypertension, left ventricular hypertrophy

Review Trends in Molecular Medicine Vol.15 No.9

The calcification paradox: definition and epidemiologyRemarkably, ectopic calcification in blood vessels is oftenaccompanied by either decreased bone mineral density ordisturbed bone turnover. This contradictory associationhas been observed in the general population [9], in osteo-porosis (Table 1) and CKD patients [10,11] and in lessfrequent bone disorders such as Paget’s disease [12]. Theterm calcification paradox has been coined to refer to thiscounterintuitive observation.

In postmenopausal osteoporosis, a negative correlationbetween bone mineral density and vascular calcificationhas been detected in several cross-sectional and longitudi-nal studies using different techniques (Table 1). Moreover,some studies also indicated that vascular calcification wasassociated with the risk of fragility fractures [13,14]. In alarge prospective study of postmenopausal women, serialdual-energy X-ray absorptiometry (DEXA) scans revealedthat for every standard deviation of decrease in bonemineral density, the cardiovascular mortality riskincreased by 30% [15].

The inverse correlation between vascular calcificationand bone mineral density is not restricted to severe boneloss as in osteoporosis, because this association was alsoobserved in a cohort of healthy perimenopausal womenwith a low prevalence of osteoporosis [16] and in a mixedpopulation of healthy middle-aged men and women [9].

Vascular calcification is a prominent feature of theincreased cardiovascular morbidity and mortalityobserved in CKD patients [17]. In addition to increasedcalcification of atherosclerotic plaques, patients on dialysisalso show characteristic calcifications of the vasculartunica media, which contribute to the high cardiovascularmortality in this population [8]. The vascular calcificationprocess shows rapid progression in both dialysis [18] andCKD stage 4 patients [19]. Elevated serum phosphatelevels, increased levels of calcium and/or phosphate (cal-cium�phosphorus product) and high parathyroid hormone

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(PTH) levels have been identified as risk factors for vesselcalcification and mortality in the CKD population, as wellas treatment with calcium-containing phosphate bindersand vitamin D metabolites [20,21].

A decline in renal function often leads to the develop-ment of metabolic bone disease, traditionally groupedunder the name renal osteodystrophy and now morebroadly captured under the term chronic kidney dis-ease–mineral and bone disorder (CKD-MBD) [22] (Box2). This array of bone diseases is characterized by disturb-ances in the bone remodeling process (Box 3), which isresponsible for maintaining the dynamic equilibrium be-tween bone deposition and resorption. Renal osteodystro-phy comprises both high (secondary hyperparathyroidismand osteitis fibrosa) and low turnover forms (osteomalacia,adynamic bone disease).

The paradoxical association of vascular calcification andbone disease was also observed in CKD; studies revealedthat vascular calcification is increased in both high-turn-over and low-turnover [23] renal osteodystrophy. However,the effects of the underlying bone turnover rate and PTHlevels on the development of vascular calcification are stillunclear. Moreover, although the definition of CKD-MBDincorporates vascular calcification, the Kidney Disease:Improving Global Outcomes (KDIGO) document explicitlystates that ‘the link between mineral disturbances andvascular calcification in CKD is not yet fully established’[22].

London et al. [23] found the highest calcification scoresin dialysis patients with the lowest PTH values and histo-logical signs of adynamic bone disease. Moreover, hemo-dialysis patients treated with calcium carbonate as aphosphate binding agent showed a significantly greaterincrease in coronary calcium score and decrease in trabe-cular bone density in comparison with patients whoreceived the calcium-free phosphate binder sevelamer[24]. In LDL receptor knockout mice with chronic renal

Table 1. Epidemiological evidence of the calcification paradoxa

Study Study type Population Follow up Imaging modality Bone

parameter

Vascular

parameter

Association Associated

risk

Hak

et al. [109]

Cross-sectional+

longitudinal

Perimenopausal

(n=236),

postmenopausal

women (n=720)

9 years

(n=236)

Rx lumbar

spine(AC),

hand (BMD)

MCA AC Negative

Kado

et al. [15]

Prospective Postmenopausal

women (n=6046)

BMD

(heel, hip)

CV mortality

Aoyagi

et al. [110]

Cross sectional Women (n=524) NA DEXA (BMD),

Rx (AC)

BMD AC None

Kiel

et al. [111]

Longitudinal Framingham

subpopulation

(n=554)

25 years Rx lumbar spine

(AC), hand (BMD)

MCA AC Negative in

women,

none in men

Tanko

et al. [112]

Cross sectional Postmenopausal

women (n=963)

NA DEXA (BMD), Rx

lumbar spine (AC)

BMD AC AC negative

Schulz

et al. [13]

Cross-sectional+

longitudinal

Postmenopausal

women,

OP Prev. 70%

(n=2348)

9 months–8

years (n=228)

CT BMD AC Negative # hip,

# vertebra

Bagger

et al. [14]

Prospective Postmenopausal

women (n=2662)

7.5 years DEXA (BMD),

Rx (AC)

BMD AC Negative # hip,

#vertebra

NS

Farhat

et al. [16]

Cross sectional Perimenopausal

women, SWAN

subpopulation

(n=490)

NA EBCT vBMD AC, CAC AC negative,

CAC NS

Samelson

et al. [113]

Longitudinal Framingham

subpopulation

(n=2499)

35 years Rx (AC) AC None

Shen

et al. [114]

Cross sectional Amish population

(n=682)

BMD AC, CAC None

Naves

et al. [115]

Prospective Population >50

years (n=624)

4 years DEXA (BMD),

Rx thoracic

and lumbar

spine (AC)

BMD AC Negative All fractures,

# vertebra

Reddy

et al. [116]

Retrospective Women (n=228) DEXA (BMD),

mammography

(BAC)

BMD BAC Negative

aSummary of studies investigating the relationship between osteoporosis or bone mineral density and vascular calcification, including the risk factors associated with this

combination. Abbreviations: #, fracture; AC, aortic calcification; BMD, bone mineral density; CAC, coronary artery calcification; CT, computed tomography; CV, cardiovas-

cular; DEXA, dual energy X-ray absorptiometry; EBCT, electron beam computed tomography; MCA, metacarpal cortical area; NA, not applicable; NS, not significant; OP,

osteoporosis; Prev., prevalence; Rx, X-ray; SWAN, the Study of Women’s Health Across the Nation; vBMD, volumetric bone mineral density.


failure, administration of bone morphogenetic protein(BMP)-7 not only reduced the severity of adynamic bonedisease, but also decreased vascular calcification [25].However, high-turnover bone disease of secondary hyper-parathyroidism might also contribute to the developmentand progression of uremia-related vascular calcificationboth experimentally [26] and in dialysis patients [21].

Parallels between bone and vessel mineralizationMonckeberg’s sclerosis was first described in the 1850s, butuntil approximately a decade ago it was generallyconsidered a degenerative process accompanied by passivemineral precipitation. However, with accumulating exper-imental evidence indicating that media calcification is anactive, cell-mediated process [27] in which vascular smoothmuscle cells (VSMCs) actively deposit hydroxyapatite [28],this topic has begun to attract considerable scientific in-terest.

Bonemineralization and ectopic mineralization in bloodvessels exhibit many parallels, with conversion of vascularcells to bone-like cells that express a number of bone-related proteins, such as core binding factor a 1 (CBFA-1 or RUNX2), bone sialoprotein, osteocalcin and alkalinephosphatase. However, in vivo these parallel expression

patterns add up to an opposite net result. In other words,they lead to pathological calcification of blood vessels,whereas bone mineralization is impaired or disturbed.

Important insights into the molecular process of tissuemineralization in general have come from the work ofMurshed and colleagues [29], who used several knockoutand transgenic cell lines and mouse strains to determinewhy physiological mineralization is spatially restricted tobone and teeth and the parallels between physiologicalmineralization and pathological ectopic mineralization.Their results indicate that mineralization occurs in anytissue that lays down an extracellular matrix rich infibrillar (e.g. type 1) collagen, provided there is concurrentexpression of alkaline phosphatase (or other pyrophospha-tases) to cleave the ubiquitous mineralization inhibitorpyrophosphate and enough phosphate is present as asubstrate for hydroxyapatite formation [29]. Pyropho-sphate is generated by nucleotide pyrophosphatase phos-phodiesterase 1 (NPP1) and is secreted extracellularlythrough the ANK pyrophosphate transporter (Figure 1).Cellular pyrophosphate production in step with pyropho-sphate cleavage by alkaline phosphatase determines thephosphate/pyrophosphate (Pi/PPi) ratio, an important reg-ulator of mineralization.

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Box 2. Spectrum of metabolic bone disease accompanying

chronic kidney disease

According to the new KDIGO (Kidney Disease Improving Global

Outcomes) TMV classification system, metabolic bone disorders

accompanying chronic kidney disease are categorized according to

changes in 3 parameters: bone turnover (T, X-axis), mineralization

(M, Y-axis) and bone volume (V, Z-axis). The colored bars represent

the most common forms of renal osteodystrophy. Low turnover

forms of renal osteodystrophy are osteomalacia (red bar), with

abnormal mineralization and adynamic bone disease (green bar)

where mineralization is normal. High turnover diseases are osteitis

fibrosa (purple bar), an extreme form of hyperparathyroid bone

disease with still normal mineralization and mixed uremic osteody-

strophy (blue bar) which is accompanied by a mineralization defect.

Figure reproduced with permission from Ref. [22]. Abbreviations:

AD, adynamic bone disease; HPT, hyperparathyroid bone disease;

MUO, mixed uremic osteodystrophy; OF, osteitis fibrosa; OM,

osteomalacia.


Although both type I collagen and alkaline phosphataseare widely expressed, under normal physiological con-ditions their coexpression is restricted to mineralizingtissues such as bone and teeth. Transgenic expression ofboth alkaline phosphatase and type I collagen in liver cellsinduced mineralization around these cells [29].

In bone, expression of the mineralization inhibitorpyrophosphate is countered by local expression of mem-brane-bound alkaline phosphatase, which eliminates thismineralization inhibitor, thereby producing free phos-phate that contributes to hydroxyapatite formation. Thearterial tunica media, which is composed of VSMCsembedded in a lamellarly organized extracellular matrixconsisting of collagen (including type I) and elastin fibers[30], also contains pyrophosphate, but VSMCs do notnormally express alkaline phosphatase. However, undercalcifying conditions, increased expression and activity ofalkaline phosphatase have been detected in VSMCs [31],whereas cultured VSMCs can produce matrix vesicles [32],as well as alkaline phosphatase.

Lack of pyrophosphate gives rise to a combination ofincreased bone mineralization and spontaneous ectopic cal-cification in mice deficient in both NPP1 and ANK [33],whereas lack of alkaline phosphatase results in hypopho-sphatasia, elevated pyrophosphate levels and decreased

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bone mineralization [34]. Correction of the Pi/PPi ratio inmice with double knockout of NPP1 and alkaline phospha-tase rescues the major phenotypic characteristics in theseanimals [35]. Cultured rat aortas secrete pyrophosphateand their calcification can be induced by addition of alkalinephosphatase to the culture medium and inhibited byaddition of pyrophosphate [36].

The studies described above highlight the importance ofthe Pi/PPi ratio in regulating mineralization processes.However, prominent differences between long bones andspongious bones such as vertebrae and calvaria reveal thatthe complexity of the interplay between these genes is stillincompletely understood, even for bone. In mice withdouble knockout of tissue-nonspecific alkaline phospha-tase (TNAP) and NNP1, mineralization defects invertebrae and calvaria were rescued in comparison withthe hypomineralization in TNAP knockouts, but mineral-ization impairment in long bones was not [37].

The most abundant protein in the extracellular matrixof the arterial tunica media is elastin. This is a fibrillarprotein and thus might contribute to vessel calcification byacting as a nucleation scaffold for hydroxyapatite depo-sition. The elastin content of arterial walls decreases withvessel size towards the periphery, concurrent withdecreased occurrence of media calcification. It has beendemonstrated that risk factors for media calcification suchas age and diabetes are accompanied by degenerativechanges in the arterial elastin network. Moreover, thecalcification process mostly starts around elastin sheetsin the vessel wall and elastin degradation products induceosteoblastic conversion in cultured smooth muscle cells[38] and dermal fibroblasts [39]. Subdermal elastinimplants calcified with increased expression of transform-ing growth factor (TGF)-b and matrix metalloproteinase(MMP)-2 and expression of CBFA-1, alkaline phosphataseand osteopontin, whereas control Dacron implants did not[40].

Adequate phosphate load is a prerequisite for bothnormal bone mineralization and ectopic vascular calcifica-tion. Elegant in vitro research has demonstrated the cau-sative role of phosphate in vascular calcification [41].Exposure of cultured VSMCs to elevated phosphate levelsinduces mineralization accompanied by downregulation ofvascular smooth muscle differentiation genes and de novoexpression of the central osteoblast transcription factorCBFA-1 and a number of its downstream genes [27]. Whenphosphate entry into cells was prevented by blocking ordownregulating the sodium-dependent phosphate cotran-sporter PIT-1 [42], both the switch to an osteoblast-likephenotype and concomitant mineralization were inhibited,whereas transgenic overexpression of mouse PIT-1 inVSMCs restored these effects [42]. Exposure to elevatedphosphate levels not only favors increased phosphatetransport into the cell, but also upregulates PIT-1 expres-sion, thereby initiating a positive feedback loop thatfurther enhances cellular phosphate uptake. UpregulatedPIT-1 expression was also demonstrated in calcified aortasof uremic rats [43].

This mechanism explains why increased serum phos-phate levels represent a risk factor for vascular calcifica-tion in CKD [20] and hemodialysis patients [21,44] and

Box 3. Bone remodeling and bone turnover

Bone is not a static tissue. To maintain mechanical integrity, adapt to changes in mechanical load and react to the need to incorporate or

release calcium or phosphate, bone is continuously remodeled. Bone remodeling is an active, regulated process consisting of an ordered

sequence of bone resorption and formation at discrete sites in the bone. The remodeling process per se does not affect net bone mass, but is

responsible for yearly renewal of approximately 30% of cancellous bone.

The bone remodeling cycle is initiated by bone lining cells that allow pre-osteoclasts access to the mineralized bone surface. Active, bone-

resorbing osteoclasts then excavate a resorption lacuna. During the reversal phase (leaving cement lines or reversal lines in the remodeled bone)

the osteoclasts are replaced by osteoblasts, which fill the resorption lacuna by active deposition of unmineralized bone matrix or osteoid. When

the osteoid is subsequently mineralized in a concerted process involving extracellular release of matrix vesicles, the remodeling site has been

replaced with new bone.


why efficient phosphate binding can slow down or preventthe calcification process in animal models [45] and CKDpatients [46].

Molecules or pathways influencing phosphate homeo-stasis such as fibroblast growth factor-23 (FGF-23), andKlotho, also affect both vascular calcification and bonemineralization. FGF-23 is a phosphaturic hormone thatinhibits renal phosphate reabsorption and negativelyregulates kidney 1a-hydroxylase activity. FGF-23 nullmice exhibit hyperphosphatemia, in combination withskeletal abnormalities (decreased bone mineral densityand osteoidosis) and vascular calcification [47]. A phos-phate-deficient diet both normalized hyperphosphatemiaand prevented vascular calcifications in these animals,whereas a diet deficient in vitamin D did not [48]. Ablationof vitamin D upregulation in FGF-23 knockout mice byadditional knockout of either 1a-hydroxylase or vitamin Dreceptor expression prevented vascular calcification, butbone mineral density was still reduced [49,50].

A recent study in rats showed that FGF-23 acts directlyon the parathyroid through the mitogen-activated proteinkinase pathway to decrease serum PTH [51]. This findingis rather surprising in light of the fact that both FGF-23and serum PTH are consistently increased CKD patients

and might point to resistance of the parathyroid gland toincreased levels of FGF-23.

The anti-aging protein Klotho acts as a cofactor forbinding of FGF-23 to the FGF receptor. Klotho-deficientmice show an accelerated aging syndrome, with osteopeniaand vascular calcification, in combination with both hyper-phosphatemia and hypercalcemia [52].

Vascular calcification can mimic intramembranous andendochondral bone formationThe body has two distinct processes in which bone isformed. In endochondral bone formation, a cartilage inter-mediate is laid down to allow length growth and is sub-sequently invaded by osteoblasts and replaced bymineralized bone. In intramembranous bone formation,mesenchymal cells are directly converted to bone-formingosteoblasts.

VSMCs in the tunica media of calcifying vessels fromdialysis patients were found to express a number of osteo-blast-related proteins such as osteopontin, type I collagen,bone sialoprotein and alkaline phosphatase [53], as well asthe central osteoblast transcription factor CBFA-1 [54].Acquirement of an osteoblast-like phenotype is accom-panied by downregulation of the typical smooth muscle

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Figure 1. Pathways and mediators involved in vascular calcification and the calcification paradox. Vascular calcification is an active cell-regulated process in which

mineralization is the net result of a transdifferentiation process of vascular smooth muscle cells to chondrocyte-like or osteoblast-like cells. This transdifferentiation process

is initiated by a number of stimuli causing damage to the arterial wall, including fibrosis, elastin degradation and hyperphosphatemia. This transdifferentiation process

creates the conditions necessary for mineralization, including the presence of alkaline phosphatase to cleave the mineralization inhibitor pyrophosphate and of a fibrillar

matrix rich in collagen type I. Finally, deposited vessel calcifications can potentially be resorbed or remodeled by osteoclast-like cells in the vessel wall.

Abbreviations: ALP, alkaline phosphatase; BMP-2, bone morphogenetic protein type 2; CA IV, carbonic anhydrase type IV; Cbfa-1, core binding factor 1; Ch, chondrocyte-like

cell; Coll I (II), collagen type I (II); MGP, matrix Gla protein; MMP-2, matrix metalloproteinase type 2; MV, matrix vesicles; NPP1, nucleotide pyrophosphatase

phosphodiesterase 1; OB, osteoblast-like cell; OC, osteoclast-like cell; OPG, osteoprotegerin; Pi, phosphate; PPi, pyrophosphate; SM22, smooth muscle cell 22; VSMC,

vascular smooth muscle cell.


differentiation markers SM22a and smooth muscle a-actin[27] and the secretion of matrix vesicles [32].

This transdifferentiation process of VSMCs to an osteo-genic phenotype can be explained by the commonmesench-ymal origin of VSMC and bone cells. Induction of thisprocess by a variety of stimuli has been reported, includingelevated phosphate and calcium levels, alkaline phospha-tase, BMP-2, oxidized low-density lipoprotein (LDL), inter-leukin (IL)-4, TGF-b, mechanical stimulation and serumfrom uremic patients.

In addition to transdifferentiation to osteoblast-likecells, VSMCs can acquire a chondrocyte-like phenotype.Expression of the chondrocyte-specific transcription factorSOX9 and collagen type II, a chondrocyte matrix protein,was observed in calcified aortas of chronic renal failurerats, as well as in human aortic tissue [55], indicating thatvascular calcification resembles, at least in part, endochon-dral bone formation. Arterial cartilaginous metaplasia andconcurrent vascular calcification, in combination with cal-cification of articular cartilage, are shared characteristicsin the phenotype of genetic ablation mouse models forNPP1 [33] and matrix Gla protein (MGP) [56]. In the latter

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model, genetic fate mapping convincingly proved thatchondrocytes in calcifying vessels arise from VSMCsbecause 99% of the cells expressing chondrocyte-specifictype II collagen in arteries of MGP knockout mice were ofsmooth muscle cell origin [57].

In bone, the Pi/PPi ratio is an important regulator ofchondrogenesis. PPi deficiency in NPP1 knockout micecontributes to an endochondral-type vascular calcificationbecause bone marrow stromal cells from these animalsshowed increased spontaneous chondrogenesis, inhibitedby correction of the Pi/PPi ratio by PPi supplementation[33]. Inmice lacking the PPi transporterANK, extracellularPPi deficiency is coupled to increased chondrogenesis.Johnson et al. [58] recently revealed another piece ofthe puzzle by providing a mechanistic insight linkingchondrogenesis and vascular calcification. Increasedchondrocyte differentiation of ANK-deficient bone marrowstromal cells could be corrected by knocking out vanin-1pantetheinase, an enzyme that inhibits synthesis ofthe chondrogenesis regulator glutathione. The same inter-vention corrected chondrogenic transdifferentiation andcalcification of cultured ANK-deficient VSMCs, whereas


increased osteoblast mineralization could not be correctedby eliminating vanin-1 pantetheinase [58]. SOX9 is alsoimplicated in fibrotic processes: ectopic SOX9 expressionis capable of inducing aberrant extracellular matrixdeposition in liver cells. In conjunction with TGF-b sig-naling, a well-known profibrotic stimulus, SOX9 expressionleads to induction of collagen type I expression [59], whichmight be one of the mechanisms by which enhanced SOX9expression in blood vessels [55] contributes to vascularcalcification. It has been shown that TGF-b itself enhancesexpression of osteogenic proteins in VSMCs [38,39] andcauses cartilaginousmetaplasia in rat arterial endothelium[60].

Although the occurrence of processes parallel to bothintramembranous and endochondral bone formation hasbeen clearly demonstrated inmedia calcification, the inter-relationship of both ossification forms in calcifying vessels,as well as their regulation and impact on cardiovasculardisease parameters, remains to be investigated.

Vascular osteoclasts and calcification remodeling?A key issue in the clinical implications of vascular calcifi-cation is the question of whether vascular calcification isreversible or amenable to therapy; in other words, canestablished vascular calcifications diminish or disappearand, if so, what are the factors that could contribute to thisprocess? Although these questions remain largely unan-swered, some investigators believe that osteoclasts orosteoclast-like cells might be responsible for remodelingof vascular calcifications.

It is still unclear whether spontaneous regression ofvascular calcification occurs. Regression of establishedvascular calcifications is also likely to involve an activecell-regulated process because themineral phase depositedis crystalline hydroxyapatite [61], which is highly insolubleunder physiological conditions.

The vascular wall contains or can attract osteoclastprecursors in the form ofmonocytes; moreover, largemulti-nucleate osteoclast-like cells have been detected in calci-fied atherosclerotic lesions, as well as in media-typecalcified lesions of osteoprotegerin (OPG) knockout mice,in which the morphological evidence was further strength-ened by the expression of the osteoclast-specific proteinscathepsin K and tartrate-resistant acid phosphatase(TRAP) [62].

Differentiation and activity of bone osteoclasts isregulated by stimulating cytokines produced by osteo-blasts such as monocyte colony-stimulating factor(M-CSF) and receptor activator of NF-kB ligand (RANKL)and inhibitory cytokines such as OPG, IL-18 and IL-12.These molecules have also been found in the arterial wallunder certain circumstances [63]. M-CSF is constitutivelyexpressed by both endothelial and VSMCs. RANKL isgenerally not expressed in normal arteries, but has beendetected in atherosclerotic lesions [64,65] and media calci-fications [65]. Moreover, stimulated endothelial cells pro-duce RANKL [66].

In vitro evidence indicates that osteoclasts can resorbthe mineral phase associated with elastin fibers withoutcompromising elastin integrity and subdermal implan-tation of osteoclasts in the vicinity of calcifying elastin

reduced elastin mineralization, indicating that recruit-ment or production of osteoclast-like cells in the calcifyingmediamight be a potential pathway to induce regression ofthe vascular calcification process [67].

Moreover, VSMCs in vitro can decrease active osteoclastformation: in monocyte cultures stimulated with M-CSFand RANKL, osteoclast formation is induced. By contrast,co-cultures of monocytes and VSMCs decreased resorptionand no resorption pits were observed in co-culture withcalcifying vascular cells [68]. These findings indicate that,in contrast to bone, where active osteoblasts start the boneremodeling cycle by promoting recruitment and activationof osteoclasts, mineralizing vascular cells, albeit with anosteoblastic phenotype, actively inhibit resorption of vas-cular calcifications. This could possibly explain the pro-gressive character of vascular calcification in the presenceof increased osteoclast activation, as occurs during osteo-porotic bone loss.

The inhibiting effect of endothelin antagonists on vas-cular calcification [69,70] is, at least partially, mediated byupregulation of carbonic anhydrase type IV in the arterialmedia [71]. This enzyme is responsible for generatingprotons from carbonic acid, thereby creating an acidenvironment favorable for mineral resorption.

Role of the OPG–RANK–RANKL pathwayOPG, RANK and RANKL are a triad of key proteins for theregulation of bone mass and bone turnover. They also playa role in the immune system.

Osteoprotegerin is a member of the tumor necrosisfactor-related family and helps to regulate bone mass byinhibiting osteoclast differentiation and activation. Thissoluble protein acts as a decoy receptor, preventing thebinding of RANKL on bone marrow stromal cells andosteoblasts to RANK expressed on osteoclast precursors,which is a signal for osteoclast differentiation. Osteopro-tegerin is produced in a wide range of tissues, including thearterial wall [72,73], whereas RANK and RANKL are notexpressed in normal arteries [62].

Osteoprotegerin probably plays a central role in thecalcification paradox because OPG knockout mice showthe telltale phenotype combination of osteoporosis andvascular calcification: two-thirds of OPG knockout animalsexhibit severe media calcification in the aorta and renalarteries [74]. The osteoporotic phenotype can be rescued byboth intravenous administration and transgenic expres-sion of OPG, whereas vascular calcification in OPG nullmice can only be rescued by transgenic OPG expression[62]. Moreover, treatment with OPG can prevent thedevelopment of vascular calcification in rats with warfarin-and vitamin D-induced vascular calcification [75]. Thesefindings also suggest a direct role for OPG as an inhibitor ofthe calcification process, but not in regression or resorptionof calcifications that have already been formed. It ispossible that OPG inhibits the calcification processthrough downregulation of alkaline phosphatase activity[76].

An association between elevated serum OPG levels andcardiovascular disease, including vascular calcification,has been found in numerous patient studies [77] to theextent that some authors have suggested that serum OPG

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can be used as a biomarker of vascular disease. Osteoblastsproduce OPG in response to estradiol. In postmenopausalwomen, OPG serum levels in postmenopausal women aredecreased and increase with estrogen replacement therapyor oral contraceptives [72].

In bone, RANKL is expressed by osteoblasts and stro-mal cells, especially in trabecular bone undergoing activebone remodeling. Binding of RANKL to RANK on mono-cyte or macrophage lineage cells is, together with permiss-ive levels of M-CSF, necessary for the differentiation ofmultinucleate bone-resorbing osteoclasts. RANKL furtherpromotes the activity and survival of mature osteoclasts byinhibiting apoptosis. Because of the opposing effects ofOPG and RANKL on bone resorption, the OPG/RANKLratio is a major determinant of bone mass and bone turn-over. In calcified human arteries, OPG expression wasdownregulated [73], whereas elevated levels of RANKand RANKL have been reported in OPG knockout mice[62].

Opposite regulation of the OPG–RANK–RANKL systemin bone and vasculature by TFG-b might be a clue to thecalcification paradox. TGF-b increases RANKL expressionand suppresses OPG expression in endothelial cells,whereas it suppresses RANKL in osteoblastic lineage cells[78]. Thus, TGF-b increases the OPG/RANKL ratio inbone, inhibiting osteoclastic bone resorption, anddecreases the OPG/RANKL ratio in blood vessels, decreas-ing the calcification inhibition potential associated withOPG. This fits well with the decrease in OPG expression incalcifying VSMCs [73]. In contrast to these findings, ovari-ectomized rabbits with atherosclerosis showed enhancedplaque calcification concurrent with an increase in OPG/RANKL ratio [79], indicating that the net result might bedependent on the precise array of mediators present in thedisease process studied.

Role of bone resorption and turnoverInhibition of bone resorption can inhibit the developmentor progression of vascular calcification. A reduction invascular calcification was found across different animalmodels and different interventions used to inhibit boneresorption [75,80,81], indicating that inhibition of boneresorption itself was the factor responsible for this effect.

Inhibition of bone resorption temporarily reduces theavailability of calcium and phosphate as substrates fordeposition of hydroxyapatite crystals in the arterial wallby decreasing the efflux of calcium and phosphate frombone. A question that remains to be answered is whetherthe effect of bone resorption inhibition on vascular calcifi-cation is dependent on the rate of bone turnover [82].Under high turnover conditions, an abrupt decrease inbone resorption temporarily causes capture of extracalcium and phosphate in bone, when bone formation isstill high, until a new equilibriumwith lower bone turnoverhas been reached. However, when bone turnover is low, forexample in adynamic bone disease, inhibition of boneresorption means that excess calcium and phosphate arenot captured in bone. Moreover, further inhibition of thealready dramatically impaired bone resorption and/or boneformation in low turnover osteodystrophy might not bebeneficial because epidemiological studies revealed the

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highest calcification scores in patients with the lowestPTH levels, in other words, those with adynamic bonedisease [8]. Experimental support for this hypothesiswas the finding that administration of BMP-7 to uremicLDL receptor knockout mice – an experimental model ofadynamic bone disease – prevents vascular calcification byincreasing bone turnover [25]. Recently, a small prospec-tive study showed that improvement in bone turnover wasassociated with slower progression of coronary artery cal-cification [118].

Implications for therapyThe interrelationship between bone and the vasculatureimplies that treatment of bone conditions potentially hasan influence on cardiovascular calcification and risk,whereas, conversely, the potential influence on boneshould be taken into account when investigating treat-ments that can inhibit or reverse vascular calcification.

Bisphosphonate treatment provides examples illustrat-ing this point. These drugs are used to treat osteoporosisand have an inhibiting effect on vascular calcification.Bisphosphonates are non-hydrolyzable pyrophosphateanalogs that are part of the standard therapeutic arsenalfor the treatment of osteoporosis and other conditions withhigh levels of bone resorption, such as Paget’s disease,osteolytic lesions and hypercalcemia associated withmultiple myeloma. In high doses, bisphosphonates directlyinhibit mineralization by physicochemical interferencewith the formation and aggregation of calcium phosphatecrystals, thus acting more or less as pyrophosphate byblocking the transformation of amorphous calcium phos-phate to hydroxyapatite. However, their therapeutic usedepends on their inhibitory effect on osteoclast-mediatedbone resorption, which occurs at low doses that do notaffect physicochemical mineralization processes [83].

Bisphosphonates inhibit osteoclastic bone resorptionthrough several mechanisms: inhibition of terminal differ-entiation of osteoclasts, induction of osteoclast apoptosisand a decrease in osteoclastic activity by inhibition ofprotein prenylation [83]. However, bisphosphonates alsohave an effect on cells of the osteoblastic lineage [84] andthe effect of different bisphosphonate compounds on osteo-blastic cells correlates well with their different potencies invivo [83]. In a rat model of uremia-related vascular calci-fication, the inhibitory effect of bisphosphonates on vesselmineralization was more closely correlated with bone for-mation than with bone resorption parameters, which werenot decreased in this study [85]. Moreover, the doses ofetidronate and pamidronate necessary for inhibition ofvascular calcification in this study also decreased boneformation andmineralization, raising a question regardingthe practical therapeutic use of bisphosphonates as anti-calcification agents [45]. These findings are in agreementwith clinical observations of oversuppressed bone turn-over, decreased bone quality and increased microfracturerisk in patients on long-term bisphosphonate treatment[86,87].

Consistent with the findings in animal models, it hasbeen shown that bisphosphonate treatment reduces vas-cular calcification in hemodialysis patients [88–90]. Inves-tigations into the effect of bisphosphonate treatment on


cardiovascular calcification in osteoporosis are scarce. Arelatively small study in osteoporotic women, however,found no effect of ibandronate treatment on the pro-gression rate of aortic calcification over a 3-year follow-up period [91].

Bisphosphonates have a very high affinity for hydro-xyapatite and are quickly cleared from the systemic circu-lation via absorption bymineralized bone, where they havea very long half-life. Remodeling mobilizes bisphospho-nates from bone, releasing them into the circulation andleading to secondary renal clearance or reabsorption bybone [10]. Both hydroxyapatite crystals and bone-like cellsare present in the calcified arterial wall, so a direct effect ofbisphosphonates on the vascular calcification process can-not be excluded. Bisphosphonates could absorb hydroxya-patitemineral in the arterial wall and subsequently exert alocal cellular effect when vascular calcifications areresorbed by macrophages or osteoclast-like cells.

Statins are examples of drugs aimed at vascular targetsthat also have an influence on bone. Statins are 3-hydroxy3-methylglutaryl coenzyme A (HMG-CoA) reductaseinhibitors that inhibit cholesterol synthesis by interferingwith the mevalonate pathway and as such are mainly usedto lower cholesterol levels. However, these compoundshave pleiotropic effects via interference with protein pre-nylation. Their pleiotropic actions include beneficial effectson cerebral vascular events, neurological disorders, cardiacarrythmias, heart failure, rheumatoid arthritis, auto-immune diseases and sepsis [92]. However, statins alsohave an anabolic effect on bone, as evidenced by increasedbone mass under statin therapy in a number of differentsettings [93], including acute coronary disease [94] anddiabetes mellitus type 2 [95]. The effect of statin therapy inosteoporosis is still somewhat controversial [93,96],although a recent meta-analysis reported modest but stat-istically significant beneficial effects of statins on hip bonemineral density [97].

The effect of statins on vascular calcification remainscontroversial. In apolipoprotein E knockout mice withchronic renal failure, low-dose simvastatin treatmentinhibited atherosclerotic plaque calcification without asignificant effect on atheroma formation [98]. In culturedVSMCs, both increased and decreased calcium depositionin response to statins were reported [99–101].

In patients with carotid artery stenosis, atorvastatindecreased serum osteopontin and OPG levels and hadbeneficial effects on plaques stability score [102], whereaspravastatin increased OPG levels in patients with type 2diabetes mellitus [103]. Daily simvastatin treatment for 12months did not reduce the progression of coronary artery orabdominal aorta calcification when compared with placebo[104]. Further research is needed to determine whetherstatins are useful for treating the paradoxical combinationof vascular calcification and bone disease.

Denosumab (AMG 162) is a fully human monoclonalantibody that binds to RANKL with high affinity andspecificity and blocks the interaction of RANKL withRANK, mimicking the endogenous effects of OPG. Whenadministered as a single subcutaneous injection, denosu-mab treatment resulted in a dose-dependent decrease inbone resorption, as measured by changes in serum and

urinary N-telopeptide, markers of osteoclastic bone resorp-tion [105]. No data are currently available on whether thisnew therapeutic compound can also regress vascular cal-cifications, an issue that is worth investigating in view ofthe potential role of the OPG–RANK–RANKL triad in thedevelopment of vascular calcification.

Vitamin K is an essential cofactor in the g-carboxylationof glutamate residues in a small group of proteins, in-cluding MGP, a potent inhibitor of arterial calcificationand osteocalcin that is thought to play a role in mineral-ization and calcium homeostasis. In view of this, vitamin Koffers interesting therapeutic perspectives and supple-mentation regressed warfarin-induced media calcificationin rats [106]; other evidence has shown that the compoundcan reduce fracture incidence in postmenopausal women[107].

Fetuin-A, a potent systemic inhibitor of soft tissuecalcification via its ability to form and stabilize protein–

mineral colloids (so-called calciproteins), was also indepen-dently associatedwith higher BMDwhen present at higherlevels in women [108]. The extent to which fetuin-A and/orother acidic bulk plasma proteins (e.g. albumin) might bevaluable in future therapeutic strategies deserves furtherinvestigation.

Concluding remarksAlthough vascular calcification and the calcification para-dox – the counterintuitive association of increased calciumdeposition in blood vessels and impaired bone mineraliz-ation – have been the focus of extensive research, theprecise mechanisms underlying the complex interplay ofbone and vasculature remain to be elucidated. Themediators and pathways involved in the calcification para-dox summarized in Figure 1 are centered around thetransdifferentiation of VSMCs to bone-like cells, includingcells with an osteoblastic and chondrocytic phenotype. It ispossible that osteoclast-like cells are also involved in limit-ing the calcification process. Differences in OPG/RANKLand Pi/PPI ratios, along with differences in the response toTGF-b in both tissues, have been implicated in tuning themany parallels between bone mineralization and ectopicvessel calcification into an opposite net result. The exist-ence of the calcification paradox implies that treatmentsaimed at bone disorders might have serious implicationsfor cardiovascular health and vice versa.

AcknowledgementsVeerle Persy was a Postdoctoral Fellow of the Fund for ScientificResearch – Flanders (Belgium). This research was supported by theFund for Scientific Research – Flanders, grant number G.0250.08. DirkDe Weerdt is warmly acknowledged for designing the graphics.

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Vascular calcification and bone disease: the calcification paradox

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