Vascular calcification and osteoporosis—from clinical observation ...

Osteoporos Int (2007) 18:251–259 

DOI 10.1007/s00198-006-0282-z 

REVIEW 

Vascular calcification and osteoporosis—from clinical 

observation towards molecular understanding 

L. C. Hofbauer & C. C. Brueck & C. M. Shanahan & 

M. Schoppet & H. Dobnig 

Received: 7 August 2006 /Accepted: 26 October 2006 / Published online: 7 December 2006 

# International Osteoporosis Foundation and National Osteoporosis Foundation 2006 

Abstract Patients with osteoporosis frequently suffer 

from vascular calcification, which was shown to predict 

both cardiovascular morbidity/mortality and osteoporotic 

fractures. Various common risk factors and mechanisms 

have been suggested to cause both bone loss and 

vascular calcification, including aging, estrogen deficiency, 

vitamin D and K abnormalities, chronic inflammation 

and oxidative stress. Major breakthroughs in molecular 

and cellular biology of bone metabolism and the 

characterization of knockout animals with deletion of 

bone-related genes have led to the concept that common 

signaling pathways, transcription factors and extracellular 

matrix interactions may account for both skeletal and 

vascular abnormalities. For example, mice that lack the 

L. C. Hofbauer : C. C. Brueck : M. Schoppet 

Department of Internal Medicine (LCH, CCB, MS), 

Philipps-University, 

Marburg, Germany 

C. M. Shanahan : M. Schoppet 

Division of Cardiovascular Medicine (CMS, MS), 

Addenbrooke’s Hospital, 

Cambridge CB2 2QQ, UK 

C. C. Brueck : H. Dobnig 

Department of Internal Medicine (CCB, HD), 

Division of Endocrinology and Nuclear Medicine, 

University of Graz, 

Graz, Austria 

L. C. Hofbauer (*) 

Division of Gastroenterology and Endocrinology, 

Department of Internal Medicine, Philipps-University, 

Baldingerstrasse, 

35033 Marburg, Germany 

e-mail: hofbauer@post.med.uni-marburg.de 

cytokine decoy receptor osteoprotegerin or the hormone 

Klotho display a combined osteoporosis-arterial calcification 

phenotype. In this review, we summarize the current 

data and evaluate potential mechanisms of the osteoporosis-arterial 

calcification syndrome. We propose a unifying 

hypothesis of vascular calcification that combines 

both active and passive mechanisms of vascular mineralization 

with aspects of bone resorption and age-related 

changes. 

Keywords Fetuin-A . Klotho . Matrix Gla protein . 

Osteoporosis . Osteoprotegerin . Vascular calcification 

Introduction 

For more than a century, clinicians have observed that 

patients with osteoporosis frequently display vascular diseases, 

including atherosclerosis and arterial calcification 

(Fig. 1). In fact, daily clinical practice reveals that patients 

hospitalized for osteoporotic fractures frequently suffer from 

cardiovascular events, including acute myocardial infarction 

and stroke. With respect to vascular calcification, we need to 

distinguish between (neo)-intimal calcification, which is a 

passive deposition of calcium into necrotic and apoptotic 

lesions of late-stage atherosclerotic plaques, and medial 

calcification, such as in Mönckeberg’s medial sclerosis, 

which results from active processes with some similarities to 

osteogenesis. 

Longitudinal analysis of bone loss and vascular calcification 

over a 25-year period in the Framingham Heart 

Study demonstrated that cortical bone loss measured at the 

metacarpal was associated with the progression of atherosclerotic 

aortic calcification in women [1]. A crosssectional 

study in 2,348 postmenopausal women from

252 Osteoporos Int (2007) 18:251–259 

Fig. 1 The osteoporosis/arterial calcification syndrome. Computed 

tomography demonstrating severe aortic calcification (arrow) in a 71- 

year-old man with an osteoporotic hip fracture (T score by DEXA: 

−3.1 at the spine and −2.6 at the proximal femur). His risk profile 

includes type 2 diabetes mellitus, arterial hypertension and a 60-packyear 

history of cigarette smoking 

California revealed that aortic calcifications represent a 

strong predictor for low bone density and fragility fractures 

[2]. A longitudinal subgroup analysis showed a graded 

association between atherosclerotic vascular calcification 

and vertebral bone loss [2]. Another prospective study on 

2,662 postmenopausal women from Denmark demonstrated 

that advanced aortic calcification, a surrogate marker for 

atherosclerosis, was significantly associated with lower 

BMD and rapid bone loss of the proximal femur [3]. Of 

note, a study on 963 Danish women aged 60–85 years 

revealed that BMD at the proximal femur, but not at the 

distal radius or the lumbar spine, was inversely correlated 

with the severity of aortic calcification [4]. This phenomenon 

could also indicate that the proximal femur, which is 

supplied by end-arteries, is more vulnerable to atherosclerosis 

than the lumbar spine with its redundant arterial 

supply [3, 4]. A small study in 36 patients also demonstrated 

a high occurrence of osteoporosis in patients with 

advanced atherosclerotic involvement of the carotid and/or 

femoral artery [5]. 

However, common mechanisms in the pathogenesis of 

osteoporosis and vascular diseases, especially arterial 

calcification, have been suggested [6–9], which include: 

– age-related mechanisms 

– chronic inflammation (e.g., in rheumatoid arthritis) 

– cigarette smoking 

– diabetes mellitus 

– estrogen deficiency 

– hypovitaminosis C, D and K 

– oxidized lipids and free radicals 

– renal failure 

During the last decade, various research tools have 

become available that allow clinicians and scientists to take 

a closer look at the molecular and cellular mechanisms 

underlying these two disorders. This has led to the 

characterization of osteoblastic and osteoclastic lineage cell 

differentiation from pluripotent stem cells and the discovery 

of essential factors and transcriptional regulators, including 

receptor activator of NF-κB ligand (RANKL) and corebinding 

factor a-1 (cbfa-1). In addition, signaling pathways 

such as c-jun N-terminal kinase, nuclear factor-κB and Wnt 

signaling have been identified and their role in animal 

models of human disease dissected. This has led to major 

advancements in the understanding of bone metabolism. 

Moreover, major similarities between vascular biology and 

the evolution of vascular calcification and the process of 

bone formation were subsequently described [10–12]. In 

addition, some knockout mouse models with targeted 

deletion of bone-related genes yielded a combined skeletal 

and vascular phenotype [13, 14]. 

In recent years, sophisticated imaging techniques that 

combine non-invasive evaluation with high resolution 

[electron-beam computed tomography (EBCT), multi-detector 

computed tomography (CT) and ultrafast spiral CT] 

now allow better and more detailed assessment of coronary 

artery disease and arterial calcification in vivo. The 

advantage of EBCT scanning of coronary calcifications 

include a shorter acquisition time and lower radiation 

exposure, whereas multislice CT scanners yield better 

reproducibility [15]. With technical improvements in CT 

imaging techniques such as rapid gantry rotation, multidetector 

arrays and electrocardiographic gating, these 

techniques are virtually equivalent to EBCT (if calcification 

is present) and are becoming more accessible [16, 17]. 

Another technical innovation is the use of dual-energy 

X-ray absorptiometry (DXA) commonly used to detect 

prevalent vertebral fractures on lateral spine imaging, but 

which can also be simultaneously employed to assess aortic 

arterial calcification [18]. 

In this review, we will discuss common molecular and 

cellular mechanisms of bone metabolism and vascular biology 

as they relate to the osteoporosis/arterial calcification syndrome, 

based on the phenotypes of knockout mice that are 

lacking matrix Gla protein (MGP), osteopontin, fetuin-A 

(α 2 -Heremans-Schmid glycoprotein), Smad-6, Klotho and 

osteoprotegerin (OPG) and present a unifying hypothesis. 

Single nucleotide polymorphisms in patients 

with osteoporosis and/or vascular disease 

Genetic studies in monozygotic twins have indicated that 

approximately 70% of the variability of human bone 

mineral density is genetically determined [19]. Candidate

Osteoporos Int (2007) 18:251–259 253 

genes that may in part explain this variability can be 

categorized into receptors, cytokines/growth factors and 

matrix proteins, and are summarized in Table 1. However, 

only a few of these single nucleotide polymorphisms have 

been implicated in both osteoporosis and vascular disease. 

Gene polymorphisms in the promoter region (G395A) and 

exon 4 (C1818T) of klotho, which encodes a hormone with 

anti-aging properties that acts by repressing intracellular 

signals of insulin and insulin-like growth factor-1 (IGF-1) 

were associated with bone density in aged Caucasian and 

Japanese postmenopausal women [20]. Of interest, the 

klotho promoter polymorphism G395A was also found to 

be a risk factor for coronary artery disease in Japanese 

patients [21]. Another gene for which single nucleotide 

polymorphisms have been implicated in both osteoporosis 

and vascular disease is OPG, a decoy receptor for the 

essential osteoclast cytokine RANKL. Single nucleotide 

polymorphisms within the promoter region (A163G and 

T245G) were more frequently detected in patients with 

vertebral fractures [22]. In addition, linkage of polymorphisms 

T950C (promoter) and G1181C (exon 1) within the 

OPG gene were associated with an increased risk of 

coronary artery disease in Caucasian men [23], while the 

promoter polymorphism T950C was also found to be 

related to vascular morphology and function [24]. 

Concurrent analysis of MGP T138C and osteopontin 

T443C gene polymorphisms revealed no statistical association 

with coronary artery calcification and bone density 

[25]. There are no studies on fetuin-A polymorphisms and 

the risk of osteoporosis and vascular disease. However, one 

study suggested that fetuin-A polymorphisms are associated 

with fetuin-A serum levels [26]. Patients with low fetuin-A 

serum levels were found to have high free phosphate levels, 

which may affect both bone metabolism and vascular 

calcification [26]. 

Table 1 Single nucleotide polymorphisms in patients with osteoporosis 

and/or vascular disease. IL, interleukin 

Candidate genes that take part in single nucleotide polymorphisms 

Receptors Estrogen receptors-α and -β 

Calcium-sensing receptor 

Vitamin D receptor 

Cytokines and cytokine antagonists Interleukin-1β 

IL-1 receptor antagonist 

IL-4 

IL-6 

Osteoprotegerin 

Transforming growth factor-β 

Tumor necrosis factor-α 

Bone-associated proteins 

Collagen Iα1 

Matrix Gla protein 

Others 

Apolipoprotein E 

Klotho 

Is vascular calcification an active or passive process? 

Two schools of thought 

Vascular calcification may arise as a result of two distinct 

disease entities: atherosclerosis (typically intimal location) 

and vascular ossification (typically medial location). Atherosclerosis 

is characterized by a sequence of events, 

including endothelial dysfunction, intimal edema, foam cell 

formation and migration of leukocytes and macrophages 

culminating in the rupture of a plaque with subsequent 

thrombus formation [27]. Atherosclerotic lesions are predominantly 

observed in curved arteries and adjacent to 

bifurcations, i.e., in the beginning of carotid arteries, 

lumbar aorta and coronary arteries, which may be explained 

by the hemodynamics of blood flow in these areas. 

Arterial calcification can occur without (medial calcification) 

or within (neo-intimal calcification) atherosclerotic 

lesions. Intimal calcification in late-stage atherosclerotic 

plaques predominantly occurs in the coronary and carotid 

arteries as well as the aorta, whereas medial calcification 

such as in Mönckeberg’s sclerosis is most frequent in the 

femoral arteries [10]. While clinical practice suggests a 

high incidence of atherosclerotic lesions in patients with the 

metabolic syndrome and pronounced arterial calcification in 

smokers and patients on hemodialysis, there is considerable 

overlap between atherosclerosis and arterial calcification, 

since both processes are not mutually exclusive. The 

coronary calcification score as determined by electron 

beam and multislice CT scanners has been demonstrated 

to be a good predictor of coronary heart disease events. Its 

use and its correlation with other cardiovascular risk scores, 

e.g., the Framingham risk score, were recently reviewed 

[15]. 

The question whether arterial calcification is an active or 

passive process is still under debate [10, 29]. The active 

hypothesis is supported by histological findings of bona 

fide bone tissue with bone marrow, the presence of 

osteoblast- and osteoclast-like cells and the secretion of 

various bone-related proteins in lesions affected by arterial 

calcification [10]. In analogy to bone formation, it is 

hypothesized that pluripotent mesenchymal stem cells 

actively differentiate towards osteoblast-like cells capable 

of forming extracellular matrix that is subsequently mineralized 

[10]. This process involves activation of osteoblastic 

transcription factors, including Cbfa-1 [28]. Whether the 

putative stem cells are smooth muscle cells that transdifferentiate 

or a phenotypically distinct cell type known as 

calcifying vascular cells (CVC) [10, 29, 30] is still under 

discussion. The active process is physiologically inhibited 

by factors that suppress osteogenic differentiation. Some of 

the histological features of ectopically formed bone and the 

presence of bone-like cells and their secretion of bonerelated 

proteins are arguments in favor of the active process.


The passive hypothesis is based on the concept that—in 

an appropriate microenvironment—calcium and phosphate 

physiochemically precipitate in areas of advanced tissue 

degeneration or necrosis within the vascular wall when the 

physiological calcium phosphate solubility threshold is 

exceeded [11]. This largely acellular extracellular process 

that occurs in association with membrane debris is further 

enhanced by a deficient macrophage-mediated clearance 

mechanism [31]. There are several potential calcification 

inhibitors that may act either locally or systemically [11]. 

MGP is a vitamin K-dependent inhibitor of calcification 

that acts locally, while fetuin-A is a circulating factor that 

prevents mineral precipitation and serves as an opsonin, 

thus enhancing phagocytosis of mineral precipitates [11]. In 

addition, pyrophosphate is a potent inhibitor of vascular 

calcification, as exemplified by a syndrome termed generalized 

arterial calcification of infancy that is caused by lossof-function 

mutations of ecto-nucleotide pyrophosphatase/ 

phosphodiesterase-1 (ENPP1), the enzyme that generates 

pyrophosphate [32]. Several lines of evidence indicate that 

active and passive mechanisms occur in parallel and are not 

mutually exclusive [10]. 

Table 2 Knockout mice with a combined skeletal and vascular 

phenotype or vascular calcification 

Gene References Phenotype 

Matrix Gla 

protein 

[33] Enchondral ossification defects, 

arterial calcification 

Osteopontin [39] Protection against ovariectomyinduced 

osteoporosis 

[44] Aggravation of arterial calcification 

in MGP-deficient mice 

Fetuin-A [47] Secondary hyperparathyroidism due 

to chronic renal failure, arterial 

calcification, calciphylaxis and 

extensive ectopic calcification a 

Smad6 [50] Heart valve hyperplasia, arterial 

ossification b 

Klotho [13] Osteoporosis, atherosclerosis, 

arterial calcification, other agingrelated 

disorders c 

Osteoprotegerin [14] Osteoporosis, arterial calcification 

a Ectopic calcification precedes renal failure in calcification-prone 

DBA/2 mice; b no skeletal abnormalities; c including gonadal and 

skin atrophy, growth hormone deficiency, physical inactivity and 

pulmonary emphysema 

Lessons from the skeletal and vascular phenotype 

of knockout mice 

The generation and characterization of knockout mice with 

targeted deletions of bone-related genes have provided 

important mechanistic insights in bone metabolism, and 

surprisingly, some of these animal models display a 

combined skeletal and vascular phenotype (Table 2). The 

physiological function of these genes and the skeletal and 

vascular phenotypes of six distinct knockout mice will be 

briefly discussed. 

Matrix Gla protein (MGP) 

MGP is a member of a family of mineral-binding proteins 

that includes several coagulation factors (factors VII and 

IX), anti-coagulation factors (proteins C and S) and 

osteocalcin, a constituent of bone matrix that inhibits bone 

formation [12]. Like other family members, MGP contains 

γ-carboxy-glutamic acid (Gla) residues, which account for 

its high affinity for hydroxyapatite that forms via γ- 

carboxylation, a process that is inhibited by warfarin [11]. 

The crucial role of MGP for bone and cartilage 

metabolism is underlined by the phenotype of MGPdeficient 

mice. These mice exhibit tachycardia, short stature 

and die prematurely at 2 months [33]. The direct cause of 

death in these animals is severe hemorrhage due to a 

ruptured thoracic or abdominal aorta. Detailed analysis 

using alizarin red staining for detection of mineralized 

tissues revealed extensive vascular calcification in mice as 

early as 2 to 3 weeks postnatally [33]. The elastic lamellae 

of the aorta and the internal elastic lamina of the coronary 

arteries were calcified as detected by von Kossa staining. In 

addition, the aorta of MGP-deficient mice displayed 

features of cartilaginous metaplasia, with the presence of 

chondrocytes and a metachromic cartilaginous matrix, 

including type II collagen and proteoglycans [33]. These 

changes reduced vascular compliance with stiffening of the 

vascular wall and a higher propensity for rupture and 

thrombus formation. 

The skeletal phenotype of MGP-deficient mice is characterized 

by calcifications of cartilage in the proliferating 

chondrocyte zone, which is not present in wild-type mice 

[33]. Moreover, proliferating chondrocytes of MGP-deficient 

mice are not organized in columns, and hypertrophic 

chondrocytes are absent, indicating a severe enchondral 

ossification defect at the growth plate that resulted in short 

stature and osteopenia [33]. 

Osteopontin 

Osteopontin is an abundant acidic non-collagenous bone 

matrix glycoprotein that binds to integrins, including α v β 3 - 

integrin, which is the major integrin type on the osteoclastic 

cell surface [34, 35]. Integrins are crucial for osteoclast 

migration to resorption sites, attachment to bone and 

formation of the sealing zone [34, 35]. Osteopontin bound 

to substrate enhances attachment of osteoclasts [34, 35]. By


contrast, soluble osteopontin modulates intracellular calcium 

levels in osteoclasts, serves as a chemoattractant and 

inhibits endothelial cell apoptosis [36–38]. 

Targeted deletion of osteopontin in knockout mice 

revealed that the absence of osteopontin is associated with 

impaired osteoclast function, and thus confers protection 

against bone loss in animal models characterized by 

enhanced osteoclastic activity. These disorders include 

ovariectomy-induced bone loss [39], bone loss following 

prolonged immobilization [40], bone loss and erosions in 

collagen-induced arthritis [41] and development of skeletal 

metastases [42]. 

Osteopontin has also been linked to arterial calcification in 

patients with advanced atherosclerosis [43]. While osteopontin 

deficiency is protective against bone loss, it aggravates 

arterial calcification of MGP-deficient mice [44]. Aortic 

calcification of MGP-deficient mice at 4 months was more 

extensive in MGP/osteopontin double knockouts, and 

histological findings included elastic laminae fragmentation, 

vessel rupture and aneurysm formation [44]. Parallel to 

progressive calcification, smooth muscle cells (SMC) from 

calcified areas of MGP/osteopontin double knockouts 

appeared to lose their SMC lineage marker SM α-actin 

[44]. These data indicate that SMC may have some cellular 

plasticity, and—in the absence of MGP and osteopontin— 

may actively differentiate towards an osteoblast-like cell type. 

Fetuin-A 

Fetuin-A is identical to α 2 -Heremans-Schmid glycoprotein, 

an abundant serum protein with a high affinity for 

mineral. Of note, fetuin-A circulates in the serum and 

serves as an inhibitor of the formation and precipitation of 

apatite precursor mineral by forming a soluble “calciprotein” 

peptide [45]. In addition, fetuin-A promotes endocytosis 

and serves as an opsonin to promote phagocytosis, 

thus favoring the removal of insoluble calcium remnants 

[45]. Clinically, low fetuin-A serum concentrations may 

predispose to vascular and ectopic calcification in patients 

with chronic renal failure and were found to be associated 

with increased cardiovascular mortality in patients on 

hemodialysis [46]. 

In a series of elegant studies, Schäfer et al. evaluated the 

vascular and skeletal phenotype of fetuin-A knockout mice 

[47]. Fetuin-A-deficient mice when fed a vitamin D/ 

calcium/phosphate-rich diet developed severe ectopic calcification 

in small vessels, renal tubules and pulmonary 

alveoli [47]. In a second study, DBA/2 mice were used 

which represent a calcification-prone mouse strain that 

develops microsurgery-induced calcification of the heart 

and the tongue under regular diet [47]. The phenotype of 

these fetuin-A-deficient DBA/2 mice displayed severe 

ectopic calcification and calciphylaxis with extensive 

mineral deposition in the kidneys, the heart, the lungs and 

the skin. Further electron microscope analysis revealed the 

presence of calcium phosphate deposits in foam cell-like 

phagocytes [47]. The extensive ectopic calcifications in 

fetuin-A-deficient DBA/2 mice lead to arterial hypertension, 

renal failure, severe albuminuria, secondary hyperparathyroidism 

and secondary skeletal changes [47]. The 

bone of these mice is osteopenic with an increased number 

of osteoclasts [47]. Taken together, these findings are 

reminiscent of bone metabolism in patients with chronic 

renal failure on hemodialysis. Of note, ectopic calcification 

in these fetuin-A-deficient mice precedes renal failure [47]. 

Smad6 

Members of the bone morphogenetic protein (BMP)/transforming 

growth factor (TGF)-β family have pleiotropic 

effects on various tissues and organs [48]. Particularly 

BMPs play a crucial role in pattern formation during 

embryogenesis, control osteoblast differentiation and promote 

osteogenesis [48]. The spatial and temporal finetuning 

of local BMP effects is mediated by intracellular 

regulators that include stimulatory and inhibitory Smad 

proteins, of which Smad6 is an inhibitor of BMP signaling 

[49]. 

Targeted mutation of Madh6, the gene that encodes 

Smad6, in mice revealed an essential role of Smad6 in the 

development and integrity of the cardiovascular system 

[50]. Smad6-deficient mice had cardiac valve and outflow 

tract defects and suffered from considerable perinatal 

mortality [50]. Heart valves were hyperplastic, and the 

septation appeared misplaced. The aorta demonstrated 

cartilaginous metaplasia of the medial layer with ossification 

and the presence of bone marrow. As a result, vascular 

relaxation was impaired and the mean arterial blood 

pressure was increased [50]. These findings indicate that 

Smad6 may limit the osteogenic responsiveness of the 

cardiovascular system to TGF/BMP signals [50]. 

While Smad6 was also expressed in the gastrointestinal 

tract and developing bones, there were no obvious defects in 

these organs in Smad6-deficient mice, particularly no decreased 

bone mineral density or osteoporotic fractures [50]. 

Klotho 

Klotho is a circulating peptide hormone that is named after 

one of the Fates, a Greek goddess that spins the thread of 

life, thus determining the life span of humans. The overall 

biological effects of Klotho suppress aging, and there is an 

increased life span in mice that overexpress Klotho [51]. 

While Klotho has some homology (20–40%) to β- 

glucosidase enzyme, it lacks β-glucosidase activity. The 

mechanisms of Klotho include binding to a cell-surface


receptor and interference with insulin and IGF-1 signaling 

through inhibition of tyrosine phosphorylation, resulting in 

increased resistance to insulin and IGF-1 signaling. 

Mice with targeted deletion of Klotho display a short life 

span and show many features of premature aging, including 

infertility, gonadal and thymus atrophy, skin atrophy, 

decreased number of Purkinje cells, physical inactivity 

and pulmonary emphysema [13]. Of note, they also 

displayed severe osteoporosis and progressive atherosclerosis 

with associated medial calcification. Both the vascular 

and skeletal abnormalities in Klotho-deficient mice were 

prevented by transgenic overexpression of Klotho [13]. 

Inhibition of insulin and IGF-1 signaling by knockout of 

insulin receptor substrate (IRS)-1 is able to rescue Klotho 

knockout mice and to restore the normal life span [51]. 

Further studies have indicated that Klotho confers antiapoptotic 

activity for endothelial cells [52] and protects 

against oxidative stress [53]. Further vascular studies of 

Klotho indicated that it is required for proper angiogenesis 

and vasculogenesis, since Klotho-deficient mice displayed 

reduced tissue capillary density, impaired angiogenesis and 

decreased endothelium-derived nitric oxide release after 

ischemic challenge [54]. Of note, histomorphometric analysis 

revealed low-turnover osteopenia in Klotho-deficient mice 

with a reduced number of osteoblast progenitors and a 

reduced osteogenic capacity as determined by matrix nodule 

formation [55]. Moreover, Klotho-deficient mice showed 

decreased osteoclastogenesis and upregulated expression of 

osteoprotegerin (OPG), an osteoclastogenesis inhibitor [55]. 

Thus, the Klotho-deficient mice are a promising murine 

model to assess age-related mechanisms in human diseases 

[13]. It also shows the essential role of aging and the 

protein Klotho in the common pathogenesis of osteoporosis 

and vascular disease. 

Osteoprotegerin (OPG) 

OPG is a glycoprotein that is abundantly expressed by 

various tissues, including the skeleton and the vascular 

wall. It circulates in serum and serves as a decoy receptor 

for the tumor necrosis factor (TNF) ligand superfamily 

members RANKL [56] and TNF-related apoptosis-inducing 

ligand (TRAIL) [57]. RANKL is an essential cytokine for 

osteoclast differentiation and activation, and thus, a 

stimulator of bone resorption, while OPG neutralizes 

RANKL and prevents bone resorption and bone loss. 

OPG knockout mice show severe bone loss and suffer 

from multiple osteoporotic fractures at the age of 1 month. 

These fractures include the longitudinal bones and the 

vertebral bodies and causes progressive crippling [14]. This 

phenotype is characterized by an increased number and 

activity of osteoclasts and can be completely rescued by an 

OPG transgene [58]. By contrast, mice carrying an OPG 

transgene or mice treated with an OPG fusion protein [59] 

or RANKL knockout mice [60] have no or fewer 

osteoclasts and develop osteopetrosis with hepatosplenomegaly 

due to extramedullary hematopoiesis. 

Surprisingly, the majority of the OPG-deficient mice 

developed severe medial calcifications of the renal arteries 

and the aorta that led to aneurysm formation and lethal 

vessel rupture and hemorrhage [14]. The vascular abnormalities 

were completely abolished using an OPG transgene 

approach, but not following postnatal administration of 

OPG protein, suggesting local production is important in 

the inhibition of vascular calcification [58]. The protective 

role of OPG in vascular calcification is also underscored by 

a rat model of vascular calcification, in which treatment 

with warfarin, an inhibitor of vitamin K-dependent γ- 

carboxylation, or supraphysiologic doses of vitamin D are 

used to induce diffuse vascular calcification [61]. In these 

two models, simultaneous administration of OPG fusion 

protein with mineralization-inducing agents prevented 

arterial calcification [61]. 

Unifying hypothesis and conclusions 

Here we propose a unifying hypothesis of vascular 

calcification, that combines both active and passive 

mechanisms, aspects of bone metabolism and age-related 

changes (Fig. 2). Under appropriate conditions, cells either 

residing in the vascular wall (smooth muscle cells) or 

precursor cells with mesenchymal differentiation potential 

(e.g., calcifying vascular cells) acquire osteogenic properties, 

which may involve BMP and cbfa-1 signaling pathways. 

This process is physiologically inhibited by factors 

such as Smad6 and others. These osteoblast-like cells 

deposit bone matrix proteins that subsequently become 

mineralized (1). In addition, matrix vesicles and apoptotic 

bodies from calcifying SMC form the nidus for passive 

calcification, unless physiological inhibitors are present. 

These include fetuin-A, MGP and osteopontin (2). In 

addition, fetuin-A forms soluble “calciproteins” and serves 

as an opsonin, thus facilitating phagocytic removal of 

mineral precipitates (3). The major protective effect of OPG 

on the vascular system seems to be due to its potent 

inhibition of RANKL, thus suppressing osteoclastic release 

of calcium and other minerals from bone (4). Whether OPG 

has additional beneficial direct effects on vascular wallresident 

cells (by inhibition of TRAIL or RANKL) has not 

been shown so far. The link between enhanced bone 

resorption and vascular calcification is further supported 

by the findings that other inhibitors of bone resorption 

(bisphosphonates, selective osteoclastic inhibitor V-H+ 

ATPase) have similar effects in a vitamin D-induced vascular 

calcification model [62, 63]. Aging and age-related changes


Fig. 2 Unifying hypothesis of arterial calcification. Smooth muscle 

cells (SMC) or calcifying vascular cells (CVC) actively differentiate 

towards osteoblast-like cells, a process that is stimulated by BMP and 

cbfa-1 and inhibited by Smad6 (1). Matrix vesicles and apoptotic 

bodies from altered SMC form the nidus for passive calcification, 

which is inhibited by fetuin-A, MGP, and osteopontin (2). Fetuin-A 

also facilitates phagocytic removal of mineral precipitates (3). The 

major protective effect of OPG on the vascular system may be due to 

its potent inhibition of osteoclastic release of calcium and other 

minerals from the bone (4). Aging and age-related changes adversely 

alter bone and vascular integrity, which is prevented by factors such as 

Klotho (5). BMP bone morphogenetic protein; CVC calcifying 

vascular cell; MGP matrix Gla protein; OPG osteoprotegerin; OPN 

osteopontin. Smad6 is an inhibitor of BMP signaling; cbfa-1 is an 

essential osteoblastic transcription factor 

may adversely affect both bone metabolism and vascular 

integrity, and inhibitors of age-related changes, e.g., the 

hormone Klotho (5) or antioxidants may have beneficial 

effects on the skeleton and the vascular system. Further 

research on the mechanisms of Klotho may define clinically 

relevant pathways and cross-talks with insulin-signaling and 

phosphate handling. 

How will these findings from mouse genomics benefit 

patients suffering from osteoporosis and arterial calcification? 

Clearly, genetic rodent models have some immanent 

peculiarities, including differences in bone growth pattern 

and a relative resistance against atherosclerosis and arterial 

calcification, thus limiting a mice-to-men extrapolation. 

However, progress in imaging techniques will allow noninvasive 

preclinical evaluation with high resolution, short 

acquisition time and reasonable radiation exposure [15]. 

These techniques are becoming more accessible and 

affordable, and should be employed in clinical studies to 

assess simultaneously arterial calcification and osteoporosis. 

Finally, intervention studies addressing the question 

whether treatment of osteoporosis benefits arterial calcification 

or vice versa [64] would be desirable, based on the 

molecular and cellular concepts of arterial calcification. 

In summary, the combined skeletal and vascular phenotypes 

of knockout mice have revealed important insights 

into the common pathogenesis of these two frequent agerelated 

disorders. For the reasons noted above, the findings 

in rodents cannot be directly extrapolated to the human 

osteoporosis/arterial calcification syndrome. However, 

these data highlight the need of clinicians to employ an 

open-minded approach with integrative thinking that may 

benefit patients with osteoporosis and arterial calcification. 

Acknowledgements Research is supported by grants from Deutsche 

Forschungsgemeinschaft Ho 1875/3-1 and Ho 1875/3-2 to Dr. 

Hofbauer, Ho 1875/5-2 to Dr. Hofbauer and Dr. Schoppet, and 

fellowship grants from ESCEO-Amgen to Dr. Brück and Professor A. 

Schmidtmann-Foundation to Dr. Schoppet. 

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