BASIC SCIENCE

Vascular Calcification in Uremia: What Is Newand Where Are We Going?Ziyad Al-Aly

Arterial calcification is very common in patients with chronic kidney disease (CKD) and other chronic

inflammatory disorders such as diabetes mellitus. Arterial calcification is associated with significant

morbidity and increased early mortality. Vascular calcification is a highly orchestrated process that en-

trains a repertoire of transcription factors and involves the activation of an osteogenic program that re-

capitulates the molecular fingerprints seen in bone formation. Recent studies have implicated the

inflammatory cytokine tumor necrosis factor a in the pathobiology of arterial calcification. Metabolic

acidosis, which is prevalent in patients with advanced kidney disease, has also been shown in some

recent studies to attenuate vascular calcification in animal models. In this review, we summarize the

recent advances in understanding the molecular mechanisms underpinning vascular mineralization

and discuss their implications in terms of translational opportunities, unmet needs, and future direc-

tions.

Published by Elsevier Inc.

Index Words: Vascular calcification; Uremia; Chronic kidney disease; Vascular disease; Tumor necrosis

factor a; Metabolic acidosis

Cardiovascular disease is the leading causeof death in patients with chronic kidney

disease (CKD).1 It is disconcerting to notethat the mortality of a 30-year-old patient ondialysis is equal to that of an 80-year old inthe general population.1 Traditional risk fac-tors such as hypertension, diabetes, olderage, left ventricular hypertrophy, elevatedlevels of low-density lipoprotein, or reducedlevels of high-density lipoprotein alone donot account for the increased early mortalityamong patients with CKD.2 A minimal re-duction in kidney function is independentlyassociated with an increased risk for cardio-vascular mortality. What is it about reducedcreatinine clearance that confers an increasedrisk of vascular disease and death? We andothers have suggested that nontraditionalrisk factors such as elevated levels of proin-flammatory cytokines and the presence ofvascular calcification, both of which are char-acteristic of the uremic state, could accountfor this dramatic increase in patient mortal-ity.1-3 Calcification of the cardiovascularsystem is a very common finding in patientswith CKD and other inflammatory condi-tions.1 Cardiovascular calcification is asso-ciated with significant morbidities andincreased early mortality. Cardiovascularcalcification may contribute to a variety ofdevastating complications such as gangrene,myocardial infarction, sudden death, andstroke.4

Advances in Chronic Kidney Disease, Vo

The pathobiology of vascular calcification iscomplex and involves a variety of factorsincluding derangements in calcium and phos-phorus homeostasis, increased proinflamma-tory cytokines, increased levels of procalcificmoieties, and decreased levels of calcificationinhibitors. Vascular calcification once thoughtto be caused by a passive degenerative pro-cess appears to be a highly orchestrated pro-cess that involves molecular reprogrammingand phenotypic changes in the vessel wall.1

The mechanisms underpinning vascular min-eralization resemble to a large extent mem-branous and endochondral bone formationand entrain a host of osteo/chondrogenic fac-tors leading to increased expression of osteo-genic and matrix protein and subsequentmineralization.5 In this review, we addressthe recent advances in understanding thepathobiology and the mechanisms of vascularcalcification and discuss the potential transla-tional opportunities, unmet needs, and futuredirections.

From the Section of Nephrology, St Louis Veterans Affairs

Medical Center, St Louis, MO.

Address correspondence to Ziyad Al-Aly, MD, Section of

Nephrology, St Louis Veterans Affairs Medical Center, 915North Grand BLVD, Saint Louis, MO 63106. E-mail: zalaly@

hotmail.com and zalaly@yahoo.com.

Published by Elsevier Inc.1548-5595/08/1504-0011$34.00/0

doi:10.1053/j.ackd.2008.07.011

l 15, No 4 (October), 2008: pp 413–419 413

Ziyad Al-Aly414

Molecular Mechanisms of VascularCalcification: An Overview

Extraskeletal calcification occurs in associa-tion with or as a result of multiple biologicprocesses. Calcification often accompaniestissue/cell necrosis, damaged proteins, ormechanical injury. Arterial calcification caninvolve the intimal or medial layer of the arte-rial wall. Medial calcification is often observedin patients with advanced CKD, diabetes, andother chronic inflammatory disorders. Medialcalcification, also known as Monckeberg’s cal-cification, leads to increased arterial stiffness,elevated blood pressure, left ventricular hy-pertrophy, and an increased risk of mortality.Intimal calcification is calcification of the ath-eromatous plaque in patients with atheroscle-rotic vascular disease where it may serve asa nidus for further plaque growth and possi-bly rupture. Intimal calcification is also associ-ated with increased mortality risk. Medial andintimal calcification can often coexist in thesame patient.

The mechanisms underpinning the biologyof medial and intimal calcification are likely toshare some resemblance but are not identical.The mechanisms of vascular calcification in-volve multiple processes including pure phys-icochemical deposition of amorphous calciumphosphate and finely orchestrated cellularprocesses that entrain a repertoire of genesand transcription factors, some of which reca-pitulate to a large extent the process of bonemodeling. The mineral composition of extra-skeletal calcified tissue involves basic calciumphosphate in apatite form and hydroxyapa-tite, which is normally found in bone. Arterialwall vascular smooth muscle cells, adventitialcells, pericytes, and fibroblasts as well as cir-culating mesenchymal cells appear to playa role in orchestrating the calcification pro-cess. In response to procalcific cues, vascularsmooth muscle cells and mesenchymal stemcells upregulate the osteo/chondrogenic rep-ertoire of genes including core-binding factoralpha (1) (Cbfa-1) also called Runx2, osterix,bone morphogenetic protein 2 (BMP2),Msx2, Sox9, and alkaline phosphatase (ALP).Recent advances have shed some light oncalcification inhibitors, the effect of metabolicacidosis, the lack of calcification inhibitors,

and the presence of putative procalcificmoieties.1,6,7

Earlier studies provided a good under-standing of the role of phosphorus in thebiology of calcification. Elevated levels ofphosphorus, which are characteristic of theadvanced uremic state, have been implicatedin vascular calcification. Both in vitro and invivo models have shown that high phospho-rus is conducive of vascular calcification andthat this process involves osteoblastic differ-entiation markers osteocalcin, osteopontin,and Cbfa-1 and is mainly mediated via the so-dium-dependent phosphate transporter Pit-1.However, it has been noted both in animalmodels and in humans that vascular calcifica-tion sometimes occurs in the face of normal di-valent ion homeostasis, suggesting perhapsthat, independent of derangements in calciumand phosphorus, other factors in the uremicenvironment may fuel the process of calcifica-tion. This notion is further supported by evi-dence in which experimentally raising serumphosphorus concentrations alone in animalmodels does not result in vascular calcifica-tion. This failure to induce mineralization isattributed to the presence of inhibitors ofextracellular matrix mineralization.8 In an invitro model of calcification in which aorticrings were induced to calcify after physical in-jury, the calcification of injured aortas wassubstantially reduced when they were coincu-bated with uninjured aortas or incubated inconditioned medium from uninjured aortas.The inhibition of calcification by conditionedmedium was reversed by adding alkalinephosphatase. The elimination of pyrophos-phate by adding inorganic pyrophosphatase-induced calcification of normal aortas andthe addition of pyrophosphate prevented cal-cification in injured aortas. Thus, pyrophos-phate is an inhibitor of vascular calcificationin this model. These findings are furthersupported by the recent findings that idio-pathic infantile arterial calcification, a geneticdisorder that is characterized by calcificationof the internal elastic lamina of musculararteries, is associated with inactivatingmutations of the enzyme ectonucleotide pyro-phosphatase/phosphodiesterase 1. This cell-surface enzyme normally generates inorganicpyrophosphate, a solute that serves as an

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essential physiologic inhibitor of hydro-xyapatite crystal growth and calcification.9

Interestingly, recent studies suggest that pyro-phosphate is reduced in patients on hemodial-ysis and that the dialysis procedure itselfenhances the clearance of pyrophosphate.

Other inhibitors of calcification includefetuin A, an acute-phase reactant that is syn-thesized by the liver. Fetuin A knockout micedevelop extensive extraskeletal calcification.Epidemiologic evidence suggests that fetuinA is decreased in patients with advancedCKD on renal replacement therapy, and lowlevels of fetuin A are associated with vascularcalcification and increased cardiovascularmortality.

Matrix Gla protein (MGP) is usually elabo-rated by vascular smooth muscle cells andchondrocytes and serves as an inhibitor ofcalcification as evidenced by the pronounceddiffuse arterial calcification found in MGP-deficient mice. Similarly, OPG, Klotho, andFGF23 knockout mice exhibit extensive me-dial calcification. Molecular signals entrainedand proteins regulated by these genes appearto negatively regulate the process of calcifica-tion. Further studies are needed to understandthe relevance of these findings in patients withkidney disease.

The Role of ALP

ALP is a downstream genomic target of theosteogenic program. ALP is then localized tothe plasma membrane and oriented such thatits catalytic subunit is ectoplasmic. Withinthe extracellular environment, ALP cleavesa phosphate from beta-glycerol phosphate.Guided by the observations that under normalcircumstances extracellular mineralization isnormally spatially restricted only to bone,Murshed et al reported that the spatial restric-tion of extracellular matrix mineralization tobone is caused by the exclusive coexpressionin osteoblasts of type I collagen and tissuenonspecific alkaline phosphatase Tnap (an en-zyme that cleaves pyrophosphate).8 Medialvascular calcification occurs in collagen-richlamellae in the tunica media in which theexpression of ALP is induced under the influ-ence of a variety of stimuli. The understandingthat ALP, the expression of which is amenable

to modulation, is indeed a necessary step forvessel wall mineralization underscores the im-portance of this enzyme and offers a potentialtarget for intervention.

BMP2 as a Novel Uremic Toxin

Targeted deletion of the gene that encodes forSmad6, a crucial protein that is expressed inthe vasculature and negatively regulates intra-cellular BMP signals, results in cartilaginousmetaplasia and ossification of the medial layerof the vessel wall, and, thus, the inhibitory roleof Smad 6 limits the osteogenic responsivenessof the vasculature to BMP signals and distur-bances in BMP levels are likely to influencethis intricate system. Uremic serum is charac-terized by increased levels of BMP2 and in-duces the expression of Cbfa-1 and ALPactivity in bovine vascular smooth musclecells. Uremic serum induction of Cbfa-1 is ab-rogated by the BMP inhibitor noggin. Interest-ingly, the downstream response of ALP touremic serum is dependent on Cbfa-1 tran-scriptional activity. The addition of inorganicphosphorus does not augment Cbfa-1 expres-sion in vascular smooth muscle cells incu-bated in uremic serum. Phosphonoformicacid, which acts as a specific competitive in-hibitor of sodium phosphate cotransport,only partially blocks Cbfa-1 expression in vas-cular cells incubated with uremic serum andtotally abrogates the effect in vascular smoothmuscle cells incubated with normal serum.10

These observations suggest that uremic serumharbors elevated levels of BMP2, a noveluremic toxin, and may also contain someyet-unidentified factors that are inherentlyprocalcific and that their procalcific effect isnot abrogated by phosphonoformic acid.

Novel Osteogenic Program: BMP2-Msx2-Wnt

This calcification process recruits bone/vascu-lar morphogens and autocrine and paracrineWnt signals.5 The pro-osteogenic BMP-2 hasbeen shown to drive the osteogenic differenti-ation of calcifying vascular cells in vitro. Theinjection of recombinant BMP2 in mice resultsin increased aortic calcium accumulation.Aortic calcification in animal models is charac-terized by an increased expression of the

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osteoblast transcription factor Msx2, whichentrains canonical Wnt signals (Wnt1,Wnt3a, and Wnt7a), and augmenting aorticMsx2 activity with the CMV-Msx2 transgeneenhances vascular Wnt signaling in the media.The BMP2-Msx2-Wnt molecular signals ap-pear to be working as an orchestrated pro-gram of osteogenic nature that drives thecalcification in the vessel wall.

The Role of Tumor Necrosis Factor a

Tumor necrosis factor a (TNF-a) appears toplay an important role in vascular calcificationin vitro. Calcifying vascular cells incubatedwith increasing concentrations of TNF-a show a dose-dependent increase in alkalinephosphatase activity and calcium mineral de-position as assayed by the incorporation of45Ca in matrix.11 These calcifying vascularcells undergo a phenotypic transformation tocuboidal osteoblast-like cells in the presenceof TNF-a.11 TNF-a has also been shown tostimulate the expression of a component ofthe osteogenic BMP2 in mesenchymal cells.12

Recently, through a series of gain of func-tion and loss of function experiments, wecharacterized a novel TNF-a–driven osteo-genic program in LDLr2/2 mice, a diabetesmellitus type 2 model of vascular calcification.LDLR2/2 mice, when having a high-fat diet(HFD), exhibit pronounced vascular minerali-zation in aortic and coronary arteries that ismost pronounced in the tunica media and ischaracterized by increased expression ofBMP2, Msx2, and the canonical Wnts Wnt3aand Wnt7a (Fig 1). The administration of theTNF-a–neutralizing antibody infliximab abro-gates these signals and arterial calcificationsignificantly.5 SM22 TNF-a transgenic micethat overexpress TNF-a predominantly in theaorta exhibit increased expression of the oste-ogenic BMP2, Msx2, Wnt3a, and Wnt7a andincreased aortic calcium accumulation. SM22TNF-a Tg:TOPGAL mice exhibited prominentLacZ histochemical staining of mural cells,whereas no LacZ staining was observed inTOPGAL siblings lacking the SM22 TNF-a transgene. These observations support andextend the current knowledge on the pro-oste-ogenic properties of TNF-a. Further studieshave shown that the use of DKK1 (an antago-

nist of Wnt signaling) abrogated the TNF-a in-duction of ALP (the genomic target of theosteogenic program) and, therefore, the effectof TNF-a on canonical Wnts is indeed function-ally relevant. The constellation of these findingsindicate that the vascular osteogenic programsdriven by TNF-a represent a disease state char-acterized by increased mural canonical Wnt sig-nals and, thus, it is a vascular Wnt-opathy.

The identification that aortic calcification intype 2 diabetes mellitus is in part a TNF-a–driven Wnt-opathy provides useful insightsinto the pathobiology of diabetic vascular dis-ease and paves the way to further test theseobservations in uremic animal models of calci-fication. If verified, these findings representa variety of translational targets that mayhave potential therapeutic applications.

The Role of Metabolic Acidosis

Metabolic acidosis, which is highly prevalentin patients with advanced renal disease, hasbeen shown to induce a net calcium effluxand decrease bone mineral content. In additionto its effect on bone, metabolic acidosis

Figure 1. Vascular calcification is a TNF-a–mediated vascular Wnt-opathy. The novel TNF-a–regulated BMP2-Msx2-Wnt osteogenic pro-gram. LDLR2/2 mice fed HFD upregulate theexpression of BMP2-Msx2-canonical Wnts(Wnt3a and Wnt7a) and exhibit pronounced vas-cular mineralization in aortic and coronaryarteries. The administration of the TNF-a–neutralizing antibody infliximab significantlyabrogates these signals and arterial calcification.Transgenic expression of TNF-a in the vesselwall results in increased expression of osteo-genic BMP2, Msx2, Wnt3a, and Wnt7a andincreased aortic calcium accumulation. Abbre-viations: LDL, low-density lipoprotein, TNF-a,tumor necrosis factor-alpha; BMP, bone mor-phogenetic protein.

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Figure 2. The effect of metabolic acidosis on cal-cium homeostasis, bone, and vascular mineralcontent. (A) In the setting of normal kidney func-tion, metabolic acidosis induces a net calcium ef-flux from bone; it stimulates urinary calcium andphosphorus excretion and reduces proximal tu-bular synthesis of 1,25 hydroxyvitamin D3, thuslimiting calcium absorption and, consequently,resulting in a negative calcium balance. (B) In thesetting of kidney failure and assuming an inverserelationship between bone and vascular minerali-zation (hypothetical model 1), the calcium effluxfrom bone is deposited in extraskeletal sites

stimulates urinary calcium and phosphorusexcretion and reduces proximal tubular syn-thesis of 1,25 hydroxyvitamin D3, thus limitingcalcium absorption and consequently result-ing in a negative calcium balance (Fig 2A).

Recently, Mendoza et al13 examined theeffect of metabolic acidosis on extraskeletalcalcification in 5 of 6 nephrectomized ratstreated with calcitriol to induce calcification.The investigators showed that experimentalmetabolic acidosis induced by the additionof NH4Cl to drinking water prevented calci-triol-induced aortic calcium and phosphorusaccumulation in 5 of 6 nephrectomized ratsvia a mechanism that involves the abrogationof the sodium-dependent phosphate cotrans-porter Pit-1 gene expression. Metabolic acido-sis produces a net calcium efflux from bone. Ithas been postulated that there is an inverse re-lationship between bone and vascular miner-alization and that a calcium efflux from boneis sequestered in extraskeletal sites especiallyin patients with poor renal function. This con-tention is supported by multiple studiesshowing an inverse relationship betweenbone mineral density and the presence and se-verity of vascular calcification. The findingsthat ibandronate, which inhibits bone resorp-tion, also prevents medial artery calcificationin uremic animals further lends support tothe notion of an inverse relationship betweenbone and vascular mineralization. Viewedthrough this prism, metabolic acidosis, whichcontributes to a decreased mineral bone con-tent, would be expected to lead to worseningvascular calcification (Fig 2B). However, thefindings of Mendoza et al are built on the as-sumption that the molecular signature of vas-cular calcification lesions share a strikingresemblance to that of bone tissue and thatmany aspects of vascular calcification recapit-ulate the process of osteogenic bone

including the vasculature. The net calcium bal-ance isunknown. (C) In the settingofkidney failureand assuming that the response of vasculature tometabolic acidosis recapitulates that of bone(hypothetical model 2), metabolic acidosis pre-vents vascular calcification. The fate of calciumleached out of bone and the net calcium balanceare unknown.

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formation; the response of vascular calcifica-tion to metabolic acidosis may be similar tothat of bone and because metabolic acidosisimpairs bone mineralization it is likely to at-tenuate the calcification process in extraskele-tal sites (Fig 2C). The main problem in thishypothetic construct is that it does not accountfor the fate of calcium efflux from bone in met-abolic acidosis and highlights the need forcomprehensive and integrated studies to pro-vide a cohesive understanding of the effect ofmetabolic acidosis on the biology of bone andarterial mineralization and the interplay be-tween bone and vasculature.14

Dietary Protein Restriction and the Roleof Bisphosphonates

Recent observations by Price et al15 have sug-gested that dietary protein restriction inuremic rats leads to dramatically increasedfrequency and extent of arterial calcification.Interestingly, low dietary protein has beenshown to increase bone resorption and accel-erate bone loss, further cementing the notionthat there is perhaps an inverse relationshipbetween bone and vascular mineralization.Furthermore, treatment with the bisphospho-nate ibandronate significantly attenuates calci-fication in uremic animals fed a low-proteindiet.

Translational Opportunities, UnmetNeeds, and Future Directions

There is an unmet need to further elucidate therole of TNF-a in other models of vascularcalcification, particularly in CKD-related vas-cular calcification models. A better under-standing of the role of TNF-a in the biologyof calcification could lead to the developmentof new opportunities for intervention in pa-tients with vascular calcification and poten-tially reduce the increased early mortality inthis patient population.

The identification that osteogenic programsregulated by TNF-a entrain the canonical beta-catenin pathway and thus represent a Wnt-opathy is important and offers yet anotherpossible target for intervention.16 The Wnt/beta-catenin–signaling pathway is implicatedin numerous disease pathways. Nonsteroidal

anti-inflammatory agents, some vitamins,and imatinib mesylate have been shown tononspecifically inhibit Wnt signals.17 How-ever, our armamentarium of medical thera-peutics lacks agents that could specificallymodulate this pathway. Targeted tissue deliv-ery of therapeutic interventions tailored tospecifically stimulate or abrogate certain Wntsignals may be a promising opportunity inthe development of new drugs.

The molecular mechanisms underpinningthe induction of calcification in uremic ani-mals on a low-protein diet are important tounderstand and are highly relevant to patientcare. There is a definite need to confirm theseresults in multiple animal models and toexamine this question in human studies. Ifconfirmed as a risk factor for vascular calcifi-cation, there will be a need to discern the rela-tive importance and the quantitative relevanceof dietary protein restrictions vis-a-vis phos-phorous (which is often the result of increasedprotein intake) as risk factors for vascular cal-cification. Questions like what is the ideal pro-tein intake in patients on renal replacementtherapy become highly relevant.

This review also discussed the interestingfindings by Price et al15 that the bisphospho-nate ibandronate and the finding by Mendozaet al13 that experimentally induced metabolicacidosis both attenuate vascular calcificationin animals. Both of these findings inherentlyimply a relationship between bone and vascu-lar mineralization. Therefore, an integratedexperimental approach that simultaneouslyexamines bone and vascular response to an ex-perimental intervention is important in orderto fully elucidate the link between bone andvascular mineralization and its response tometabolic derangements.

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