Volume 45, Issue 6, Supplement, Pages A57-A63 (June 2007)

13 of 25

ABSTRACT

FULL TEXT

FULL-TEXT PDF (130 KB)

Clinical, cellular, and molecular aspects of arterial calcification

Received 19 January 2007; accepted 17 February 2007.

Arterial calcification is a complex and independently regulated process with risk factors similar to those for atherosclerotic occlusive disease. It may develop either within the atherosclerotic intima or in the media. When calcification is found in coronary or lower extremity arteries, it is an independent predictor of cardiovascular events and lower extremity amputation. Recent evidence suggests a role for several endogenous stimulators and inhibitors in the pathogenesis of arterial calcification. Inflammatory mediators and matrix-degrading enzymes are also thought to control the progression of calcification in humans. Current research involves efforts to define the complex interactions between cellular and molecular mediators of arterial calcification.

Article Outline

• Abstract

• Clinical aspects

• Population studies

• Histology

• Cellular aspects

• The media: smooth muscle cells and calcifying vascular cells

• The adventitia: pericytes, fibroblasts, and mesenchymal stem cells

• Angiogenesis and the role of endothelial cells

• Molecular aspects

• Smooth muscle cell calcification in vitro

• Arterial calcification in rats: calcium, phosphorous, and bone resorption

• Arterial calcification in mice: role of endogenous inhibitors

• Matrix degradation in arterial calcification

• Conclusions

• References

• Copyright

Arterial calcification was previously viewed as an inevitable, passive, and degenerative process that occurred at the end stages of atherosclerosis. Recent studies, however, have demonstrated that calcification of arteries is a complex and regulated process.1, 2 It may occur in conjunction with atherosclerosis or in an isolated form that is commonly associated with diabetes and renal failure.3, 4 Higher artery calcium scores are associated with increased cardiovascular events, and some aspects of arterial calcification are similar to the biology of forming bone.5, 6 Arterial calcification can thus be viewed as a distinct inflammatory arteriopathy, much like atherosclerosis and aneurysms, with its own contribution to cardiovascular morbidity and mortality.7

Current research involves efforts to define the complex interactions between cellular and molecular mediators of arterial calcification and, in particular, the role of endogenous calcification inhibitors. This review discusses the clinical relevance, cellular events, and suspected molecular pathways that control arterial calcification and highlight some potential avenues for future research.

Clinical aspects

Population studies

At least one third of Americans aged >45 years have arterial calcification when assessed by computed tomography (CT).8, 9 Risk factors for the development of early coronary artery calcification are similar to those for atherosclerotic occlusive disease and include hypertension, increased body mass index, and low high-density lipoprotein cholesterol.10 In asymptomatic volunteers aged >50 years, coronary artery calcification is associated with increasing age, triglycerides, cholesterol, and increased intra-abdominal fat.11

The presence of diabetes significantly raises the risk of coronary calcification, and end-stage renal disease is thought to result in increased arterial calcium accumulation due to abnormalities in calcium and phosphorus homeostasis.12, 13, 14 Common risk factors between atherosclerosis and calcification, and the high incidence of concurrent disease, have complicated our ability to separate the contribution of each to clinical events.

Recent data on lower extremity amputation, mortality, and coronary events confirm a strong predictive role of arterial calcium accumulation that persists even after correction for traditional risk factors. For example, when high coronary artery calcification is identified by electron beam or spiral CT, it predicts a fivefold to sevenfold increase in the risk of hard coronary events and this risk persists despite correction for age.15, 16 Similar predictive value is associated with calcification identified in the aortic arch.17 When arterial calcification is specifically identified in the mid-thigh femoral artery, it is associated with a nearly threefold increase in the risk of amputation.18, 19

Although the mechanisms whereby arterial calcification leads to increased vascular events are currently unknown, recent data suggest that coronary events and end-organ damage are related more to increased arterial stiffness rather than to the associated occlusive lesions.20, 21 Further attempts to characterize the effects of increased arterial stiffness on end organs are needed, as are methods to distinguish the effects of calcification from those of atherosclerotic lesions.

Histology

Vascular calcification has been divided into four forms that involve (1) the atherosclerotic intima, (2) the media of medium and large sized arteries, (3) cardiac valves, and (4) a widespread form known as calciphylaxis.22 The second form, known as medial artery calcification or Mönckeberg sclerosis, is of primary importance in the diabetic population and particularly in patients with diabetic tibial artery disease. Medial artery calcification may be found in conjunction with atherosclerotic lesions, or it may occur independently.2 It occurs in patients with normal serum calcium and phosphorus levels, and this distinguishes it from vascular calciphylaxis, which is related to high serum calcium and phosphate levels that result in passive mineral deposition onto elastic fibers.22 Of these forms, medial artery calcification has been most strongly associated with cardiovascular events.18

The expression of bone-related genes in atherosclerotic lesions was described more than a decade ago.23, 24 Arteries from patients with medial calcification have increased expression of osteoprotegrin, a member of the tumor necrosis factor (TNF) ligand and receptor-signaling family. This is associated with apoptosis that occurs next to areas of recent calcification.25 In clinical specimens, medial artery calcification occurred next to medial smooth muscle cells (SMCs) and in the absence of macrophages or lipids. Compared with control arteries, these vessels expressed less matrix Gla protein (MGP) and osteonectin and more alkaline phosphatase, bone sialoprotein, bone Gla protein, and collagen II, all indicators of osteogenesis or chondrogenesis.26

Cells residing in the media of human calcified arteries lose their expression of SMC markers and begin to express more osteogenic or chondrogenic markers, including the osteochondrogenic transcription factors Cbfa1, Msx2, and Sox9.27 These studies on human specimens show arterial changes that resemble the biology of developing bone. They have helped us form hypotheses on the basic mechanisms that initiate and regulate arterial calcification.

Although our understanding of the specific pathologic events that incite arterial calcification remains limited, the factors that predispose to its development have recently been defined, and attempts to place these findings in perspective have begun.2 It is thought that poor glucose control leads to a low-grade inflammatory response in the adventitia that is related to chronic metabolic, osmotic, and oxidative stresses.28 These oxidative and hyperlipidemic signals may then induce the secretion of inflammatory mediators such as TNF-α, interleukin (IL) 6, and adipokines from macrophages in the arterial wall.29, 30 Moreover, patients with elevated levels of soluble IL-2 receptor, a marker of T-cell activation, have increased rates of coronary calcium progression compared with controls.31 The complex interplay between risk factors, inflammatory mediators, and the development of occlusive vs calcific lesions will need further evaluation and scrutiny.

Cellular aspects

The media: smooth muscle cells and calcifying vascular cells

The ability of medial SMCs to undergo phenotypic transformation was recognized >25 years ago by Campbell et al.32 Phenotypic SMC changes that occur during calcification have only recently been described, however.33 SMCs cultured from the aortic wall have multilineage potential with the ability to express chondrogenic, leiomyogenic, and stromogenic markers, but not adipogenic markers.34

Studies by Demer et al35 have demonstrated that certain populations of medial SMCs, when grown in culture, are capable of undergoing calcification. These cells, known as calcifying vascular cells (CVCs), can differentiate into osteoblast-like cells while losing their ability to express smooth muscle-specific markers.36 This suggests that the changes in cellular phenotype that occur during calcification may occur through the transformation of a subpopulation of medial SMCs into more osteogenic cells, and these changes may occur in the setting of diminished inhibitors or increased stimulators of calcification. The in vivo origin of these osteoblastic cells, however, has not been determined.

The adventitia: pericytes, fibroblasts, and mesenchymal stem cells

A program of osteogenic expression has been described in the arterial wall of LDLR–/– mice fed a high fat, diabetogenic diet.28 This work, done by Towler et al,37 demonstrated up-regulation of osteoblastic transcription factors Msx2 and Msx1 in perivascular adventitial cells. Msx2, which is thought to act in response to stimulation by bone morphogenetic proteins (BMPs), stimulates osteogenesis in adventitial myofibroblasts and in cells from a mesenchymal stem cell line while inhibiting adipogenic differentiation. In addition, mice carrying the CMV-Msx2 transgene have increased expression of Msx2 only in the adventitia, whereas alkaline phosphatase expression is increased only in the media.38

They further evaluated conditioned media from Msx2-stimulated mesenchymal cells and found that it induced expression of Wnt signaling molecules, TCF/Lef transcription, and nuclear β-catenin localization with concomitant alkaline phosphatase induction.38 This strongly suggests that a subset of adventitial cells expressing a bone morphogenic protein 2 (BMP-2)-Msx2 signaling program can influence the phenotype of medial cells and may be responsible for inducing calcification.

In cell culture experiments on microvascular pericytes, dexamethasone, a steroid with recognized stimulatory effects on vascular calcification, down-regulates molecules thought to act as calcification inhibitors including MGP, osteopontin, and vascular calcification-associated factor (VCAF).39 Advanced glycation end products increase calcification and osteoblastic differentiation of microvascular pericytes.40 Therefore, cells expressing osteochondrogenic markers during calcification may represent medial cells responding to adventitial signals or precursor cells that are recruited into the media from surrounding tissues or from the blood stream.

Angiogenesis and the role of endothelial cells

A potential association between angiogenesis and the progression of arterial calcification has recently been proposed.41 According to this hypothesis, osteogenic signals or osteogenic precursor cells may be conveyed into the arterial wall by collateral vessels that develop in response to inflammatory signals. This theory is based on the known relationship between angiogenesis and the progression of atherosclerotic plaques.6 Inhibitors of angiogenesis prevent the progression of atherosclerotic plaques,42 whereas stimulators of angiogenesis enhance plaque progression.43

Inflammatory collaterals have been shown to originate from the adventitia.44 Once established, it is proposed that these collateral vessels may allow for the migration of multipotent adventitial myofibroblasts or pericytes into the atherosclerotic intima or media where they can differentiate into osteoblast-like cells through the effects of bone-signaling proteins such as the BMPs and osteoprotegrin. Alternatively, the collaterals can be used to convey signals secreted by adventitial cells to the media, where they can affect the phenotype of calcifying vascular cells.

Of note is that arteries from rats that underwent removal of the adventitia had reduced amounts of medial calcification on a calcification-inducing diet.45 In addition, endothelial cells exposed to atherogenic stimuli in culture demonstrated increased expression of MGP and BMP2.46 Endothelial cells grown in culture with CVCs can either inhibit or stimulate calcification, depending on the specific culture conditions, and this is thought to be related to their expression of BMP2.47 However, direct in vivo confirmation of a relationship between angiogenesis and arterial calcification has not been demonstrated.

Molecular aspects

Smooth muscle cell calcification in vitro

Numerous factors have been shown to affect the ability of media-derived cells to undergo calcification in vitro (Table).48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77 Shortly after their identification of CVCs from bovine aorta, Demer et al51 demonstrated that in vitro calcification was enhanced by the addition of two atherosclerosis-promoting proteins—transforming growth factor β1 (TGF-β1) and 25-hydroxycholesterol—and they suggested a link between calcification and atherosclerosis.

Table.

Factors involved in arterial calcification


	Stimulators	Inhibitors
	Inorganic phosphate48, 49, 50	Pyrophosphate61
	TGF-β151	Statins62
	25-hydroxycholesterol51	N-3 fatty acids63
	Estrogen35	Tropoelastin64, 65
	cAMP36	BMP-766
	MAP kinase52	Bisphosphonates67, 68, 69
	Acetylated LDL53, 54	⁎Matrix Gla Protein70
	Homocysteine37	Osteopontin71, 72
	Glucose 55	⁎Osteoprotegrin73
	Endothelin-156	⁎NPP-1 via Pip73
	Elastin degradation products43	⁎Ahsg (Fetuin-A)74
	Pit-157	⁎Smad675
	Leptin58	⁎Klotho76
	BMP2-Msx2-Wnt38	⁎Carbonic anhydrase-277
	MMPs59, 60

TGF, Transforming growth factor; cAMP, cyclic adenosine monophosphate; BMP, bone morphogenic protein; MAP, mitogen-activated protein kinase; LDL, low-density lipoprotein; NPP, nucleotide pyrophosphatase phosphodiesterase; PPi, inorganic pyrophosphate.

Factors shown to affect calcification in vivo are in bold.

⁎

Denotes data from mice with spontaneous or targeted gene alteration.

Other factors thought to be involved in atherosclerosis progression were also found to affect the ability of CVCs to calcify. For example, CVCs are responsive to the effects of estrogen35 and to the effects of the intracellular-signaling molecules cyclic adenosine monophosphate36 and mitogen-activated protein kinase (MAPK).52 N-3 fatty acids inhibit calcification of CVCs though p38-MAPK and peroxisome proliferator-activated receptor-γ pathways,63 and leptin, the circulating protein product of the ob gene, promotes osteoblastic differentiation and calcification of CVCs by increasing alkaline phosphatase activity.58 In addition, macrophages are thought to exert their procalcification effects on CVCs through secretion of TNF-α.29

Cultured SMCs are also capable of undergoing calcification and expressing osteogenic phenotypes when they are exposed to high levels of inorganic phosphates.78, 79 This process is stimulated by high glucose, which increases the expression of Cbfa1 and BMP-2,55 and it is inhibited by osteopontin and inorganic pyrophosphate.48, 49, 50 Acetylated low-density lipoprotein and homocysteine stimulate vascular SMC calcification,53, 54 whereas statins prevent calcification through effects on the Gas6-Axl survival pathway.62 Endothelin-1 blockade prevents calcification of cultured rat SMCs and it also has effects in vivo.56 Matrix proteins, including elastin precursors, elastin degradation products, and decorin, a proteoglycan found in human calcified plaques, are also known to enhance SMC calcification in vitro.65, 65, 80

Of interest is that BMP-7 may counter the calcification-inducing effects of BMP-2 by promoting the SMC phenotype.66 SMC calcification and phenotypic transformation into chondrocyte-like cells is also affected by altering levels of the sodium-dependent phosphate cotransporter Pit-1, suggesting that phosphate uptake by Pit-1 is essential for phosphate-induced SMC calcification and phenotype changes.67

Arterial calcification in rats: calcium, phosphorous, and bone resorption

Much of the early experimental data on medial calcification came from studies in rats overdosed with vitamin D.81 Arterial calcification in this model is accompanied by calcification in the lungs and other tissues. It is most similar to human calciphylaxis.2, 82 High serum levels of calcium and phosphate are similar to levels seen in dialysis patients and those with parathyroid axis dysfunction.22 Vitamin D3 also increases vascular SMC calcification in vitro, and this effect is partially mediated by inhibition of parathyroid hormone–related peptide expression.83

The earliest finding in calcification induced by vitamin D3 is that of matrix vesicles developing along elastin fibers, and this is similar to what has been seen in human specimens.84, 85 This is followed by deposition of calcium hydroxyapatite crystals along the elastic lamellae and degeneration of medial elastin.84, 86 Once the calcification process is initiated, it is thought to spread along the elastin fibers, ultimately leading to destruction of the elastin fibers themselves and then spreading to the surrounding medial smooth muscle cells.59 Calcification in this model is generally thought to occur in the absence of an inflammatory cell infiltrate and is linked to bone resorption.87

Characterized by decreased levels of circulating fetuin-A, it can be delayed by inhibitors of bone resorption, including the cytokine osteoprotegrin and the amino bisphosphonates.67, 68, 69 Calcification can also be reduced by V-H+-adenosine triphosphatase inhibitor SB 24278488 and sevelamer, a phosphate binder that does not contain calcium.89 These studies by Price et al suggest that arterial calcification may be related to serum factors emanating from resorbing bone.

In humans, there is a known association between osteoporosis and arterial calcification.90, 91 Although calcification in these models is not thought to involve an inflammatory intermediate, it is likely that certain mechanisms overlap with those of other forms of calcification. For example, localized changes in extracellular calcium or phosphorus concentrations may be involved in the initial stages of medial and intimal calcification or in their progression.33 In addition, we have recently shown that systemic administration of the antibiotic doxycycline, which has properties that inhibit matrix metalloproteinase activity, can reduce arterial calcification in rats treated with vitamin D.60

Another model of arterial calcification in rats involves the use of warfarin and vitamin K that are thought to work by reducing levels of MGP. Arterial calcification in warfarin-treated rats is accelerated by growth and vitamin D.92 It can be reversed by treatment with an endothelin receptor antagonist and this appears to be dependent on activation of membrane bound carbonic anhydrase IV.93 Finally, arterial calcification can also be induced in rats by feeding with an adenine-containing diet that causes uremia.94 It has recently been demonstrated that calcification in this model is increased by diets low in protein and reduced by the bone resorption inhibitor ibandronate.95

Studies using rats injected with vitamin D have yielded significant information during the last 40 years; however, calcification in this model appears to be most similar to human vascular calciphylaxis because rats do not develop intimal lesions or similar inflammatory responses. Therefore, recent work in genetically altered mouse strains that spontaneously develop arterial calcification gives new opportunities to study this complex process.

Arterial calcification in mice: role of endogenous inhibitors

Evidence for endogenous calcification inhibitors comes from targeted or spontaneous gene alterations in mice. Mice deficient in the vitamin K–dependent protein MGP, a mineral-binding extracellular matrix protein, have chondrocytes within the arterial wall. They develop arterial calcification shortly after birth, and this results in death from arterial rupture in <3 months.70 The mechanisms by which MGP deficiency leads to increased calcification remain unknown but may be related to interactions between MGP and BMP2.96, 97 Another potential interaction occurs between MGP and osteopontin, which also accumulates adjacent to calcifying areas. Mice deficient in both MGP and osteopontin had twice as much arterial calcification and died significantly earlier than control MGP-deficient mice.70, 98 Thyroid hormone is also thought to increase vascular SMC calcification through upregulation of MGP.99

Mice with a genetic deficiency leading to decreased inorganic pyrophosphate production develop spontaneous calcification through an endochondral process.61 The decrease in inorganic pyrophosphate production is related to a mutation that inhibits the catalytic activity of nucleotide pyrophosphatase phosphodiesterase 1 (NPP-1). This mutation was initially identified as a cause of most of the cases of human idiopathic infantile arterial calcification.100 Cultured aortic vascular SMCs isolated from mice deficient in NPP-1 had increased expression of the chondrogenic marker type II collagen and increased expression in aortic specimens of the mature chondrocyte marker type IX/XI collagen. In an organ culture model of aortic calcification, high phosphate and calcium levels are unable to induce calcification because of inhibition by pyrophosphate.50

Another genetic alteration resulting in spontaneous arterial calcification is a mutation in the gene encoding for osteoprotegrin, a member of the TNF superfamily of proteins, which is also expressed in human atherosclerotic lesions. It is a secreted osteoclast inhibitor, and mice deficient in osteoprotegrin demonstrate decreased bone density and increased aortic and renal artery medial calcification.73 This appears similar to the known association between osteoporosis and arterial calcification in humans.91 Apolipoprotein E–/– mice with inactivated osteoprotegrin demonstrate accelerated atherosclerotic lesion progression and calcification with reduced cellularity of lesions.101 Osteoprotegrin also inhibits calcification in rats given warfarin or high doses of vitamin D.82

More recently, the serum protein α2-Heremans-Schmid glycoprotein (Ahsg, also known as fetuin-A) has been identified as an important inhibitor of ectopic calcification. Mice deficient in Ahsg are phenotypically normal but develop calcification when placed on a diet containing increased calcium, phosphate, and vitamin D.74 Arterial calcification occurred in the absence of elevated serum calcium or phosphorus levels and resulted in increased blood pressure and renal failure. In vitro experiments showed that lack of Ahsg was associated with a lack of inhibitory activity of serum proteins on bone calcium phosphate precipitation, suggesting that the effects of Ahsg were systemic rather than specifically on the arterial wall. This study supports the notion that many factors in addition to serum calcium and phosphorus levels are important in the development of arterial calcification, even in patients with renal failure.

Three additional mouse strains are known to develop calcification, but mechanistic studies have not been reported. Mice without the gene encoding for the Smad6 homologue Madh6, an intracellular inhibitor of TGF-β signaling, develop arterial calcification and cartilaginous metaplasia possibly through release of the BMP-2 signaling cascade.75 Mutations in the mouse klotho gene cause a phenotype that resembles human aging and medial calcification develops that may be related to increased serum phosphate levels.76 Mice with a defect in the gene encoding for carbonic anhydrase 2 show calcification in small arteries.77 Attempts to define the mechanisms by which these factors, and others, lead to arterial calcification are ongoing.

Matrix degradation in arterial calcification

Matrix metalloproteinases (MMPs) are known to play diverse roles in arterial pathologic processes.102 They have been demonstrated to exert multiple effects during atherosclerosis, restenosis, and aneurysmal degeneration.102 A role of MMPs in arterial calcification was previously demonstrated by Basalyga et al,59 who focused on the importance of elastin degradation. Their work suggests that elastolytic MMPs are secreted by inflammatory cells in the adventitia or media, with consequent degradation of medial elastin. This leads to the release of elastin degradation products that, in turn, act as chemokines to recruit other inflammatory cells. A cycle of MMP-mediated elastin degradation, recruitment of inflammatory cells, and more MMP secretion ensues.

Inhibiting MMP activity would serve to stop this cycle and prevent the influx of new inflammatory cells. We have recently demonstrated efficacy of an MMP inhibitor in limiting calcium chloride–induced aortic calcification. We observed reduced aortic calcification in rats that were treated with GM6001, a nonselective, synthetic MMP inhibitor.92

MMPs are also known to have nonmatrix effects during osteogenesis,103 and they are involved in several aspects of mesenchymal stem cell renewal and differentiation.104 Recent work has demonstrated that osteopontin promotes pro-MMP9 activation in aortic mesenchymal cells.105 This suggests that the role of matrix-degrading enzymes during arterial calcification may go beyond that of degrading medial elastin. Current work in our laboratory is focused on addressing these issues.

Conclusions

Artery calcification is a complex and regulated process that may occur in conjunction with atherosclerosis or in a more isolated form. It is now known to be an independent predictor of cardiovascular events, including lower extremity amputation. Multiple stimulators and inhibitors control the progression of vascular calcification, and it is likely to involve inflammatory mediators emanating from cells located in the arterial wall or the surrounding adventitia. Matrix effects also control the progression of calcification. Future work will involve unraveling of the molecular events leading to calcification and the development of novel molecular-based therapies to prevent its progression.

References

1. 1Abedin M, Tintut Y, Demer LL. Vascular calcification: mechanisms and clinical ramifications. Arterioscler Thromb Vasc Biol. 2004;24:1161–1170. CrossRef

2. 2Shao J-S, Cai J, Towler DA. Molecular mechanisms of vascular calcification: lessons learned from the aorta. Arterioscler Thromb Vasc Biol. 2006;26:1423–1430. CrossRef

3. 3Budoff MJ, Yu D, Nasir K, Mehrotra R, Chen L, Takasu J, et al Diabetes and progression of coronary calcium under the influence of statin therapy. Am Heart J. 2005;149:695–700. Abstract | Full Text | Full-Text PDF (185 KB) | CrossRef

4. 4Block GA, Spiegel DM, Ehrlich J, Mehta R, Lindbergh J, Dreisbach A, et al. Effects of sevelamer and calcium on coronary artery calcification in patients new to hemodialysis. Kidney Int. 2005;68:1815–1824. MEDLINE | CrossRef

5. 5Raggi P, Callister TQ, Cooil B, He ZX, Lippolis NJ, Russo DJ, et al. Identification of patients at increased risk of first unheralded acute myocardial infarction by electron-beam computed tomography. Circulation. 2000;101:850–855.

6. 6Jeziorska M, McCollum C, Wooley DE. Observations on bone formation and remodelling in advanced atherosclerotic lesions of human carotid arteries. Virchows Arch. 1998;433:559–565. MEDLINE | CrossRef

7. 7Detrano RC, Wong ND, Doherty TM, Shavelle R. Prognostic significance of coronary calcific deposits in asymptomaic high-risk subjects. Am J Med. 1997;102:344–349. Abstract | Full Text | Full-Text PDF (539 KB) | CrossRef

8. 8Jain T, Peshock R, McGuire DK, Willett D, Yu Z, Vega GL, et al. African Americans and Caucasians have a similar prevalence of coronary calcium in the Dallas Heart Study. Journal of the American College of Cardiology. 2004;44:1011–1017. Abstract | Full Text | Full-Text PDF (143 KB) | CrossRef

9. 9Bild DE, Detrano R, Peterson D, Guerci A, Liu K, Shahar E, et al. Ethnic Differences in Coronary Calcification: The Multi-Ethnic Study of Atherosclerosis (MESA). Circulation. 2005;111:1313–1320. CrossRef

10. 10Mahoney LT, Burns TL, Stanford W, Thompson BH, Witt JD, Rost CA, et al. Coronary risk factors measured in childhood and young adult life are associated with coronary artery calcification in young adults: The muscatine study. J Am Coll Cardiol. 1996;27:277–284. Abstract | Full-Text PDF (959 KB) | CrossRef

11. 11Arad Y, Newstein D, Cadet F, Roth M, Guerci AD. Association of multiple risk factors and insulin resistance with increased prevalence of asymptomatic coronary artery disease by an electron-beam computed tomographic study. Arterioscler Thromb Vasc Biol. 2001;21:2051–2058. CrossRef

12. 12Schurgin S, Rich S, Mazzone T. Increased prevalence of significant coronary artery calcification in patients with diabetes. Diabetes Care. 2001;24:335–338. MEDLINE | CrossRef

13. 13Goodman WG, Goldin J, Kuizon BD, Yoon C, Gales B, Sider D, et al. Coronary-artery calcification in young adults with end-stage renal disease who are undergoing dialysis. N Engl J Med. 2000;342:1478–1483. MEDLINE | CrossRef

14. 14Allison MA, Wright CM. Age and gender are the strongest clinical correlates of prevalent coronary calcification (R1). Int J Cardiol. 2005;98:325–330. Abstract | Full Text | Full-Text PDF (130 KB) | CrossRef

15. 15Wayhs R, Zelinger A, Raggi P. High coronary artery calcium scores pose an extremely elevated risk for hard events. J Am Coll Cardiol. 2002;39:225–230. Abstract | Full Text | Full-Text PDF (87 KB) | CrossRef

16. 16Vliegenthart R, Oudkerk M, Song B, van der Kuip DAM, Hofman A, Witteman JCM. Coronary calcification detected by electron-beam computed tomography and myocardial infarction (The Rotterdam Coronary Calcification Study). Eur Heart J. 2002;23:1596–1603. CrossRef

17. 17Iribarren C, Sidney S, Sternfeld B, Browner WS. Calcification of the aortic arch: risk factors and association with coronary heart disease, stroke, and peripheral vascular disease. JAMA. 2000;283:2810–2815. MEDLINE | CrossRef

18. 18Lehto S, Niskanen L, Suhonen M, Ronnemaa T, Laakso M. Medial artery calcification (A neglected harbinger of cardiovascular complications in non-insulin-dependent diabetes mellitus). Arterioscl Thromb Vasc Biol. 1996;16:978–983. MEDLINE

19. 19Lehto S, Ronnemaa T, Pyorala K, Laakso M. Risk factors predicting lower extremity amputations in patients with NIDDM. Diabetes Care. 1996;19:607–612. MEDLINE

20. 20Leoncini G, Ratto E, Viazzi F, Vaccaro V, Parodi A, Falqui V, et al. Increased ambulatory arterial stiffness index is associated with target organ damage in primary hypertension. Hypertension. 2006;48:397–403. CrossRef

21. 21Wang L, Jerosch-Herold M, Jacobs DR, Shahar E, Detrano R, Folsom AR. Coronary artery calcification and myocardial perfusion in asymptomatic adults: the MESA (Multi-Ethnic Study of Atherosclerosis). J Am Coll Cardiol. 2006;48:1018–1026. Abstract | Full Text | Full-Text PDF (194 KB) | CrossRef

22. 22Vattikuti R, Towler DA. Osteogenic regulation of vascular calcification: an early perspective. Am J Physiol Endocrinol Metab. 2004;286:E686–E696. MEDLINE | CrossRef

23. 23Bostrom K, Watson KE, Horn S, Wortham C, Herman IM, Demer LL. Bone morphogenetic protein expression in human atherosclerotic lesions. J Clin Invest. 1993;91:1800–1809. MEDLINE | CrossRef

24. 24Shanahan CM, Cary NR, Metcalfe JC, Weissberg PL. High expression of genes for calcification-regulating proteins in human atherosclerotic plaques. J Clin Invest. 1994;93:2393–2402. MEDLINE | CrossRef

25. 25Schoppet M, Al-Fakhri N, Franke FE, Katz N, Barth PJ, Maisch B, et al. Localization of osteoprotegerin, tumor necrosis factor-related apoptosis-inducing ligand, and receptor activator of nuclear factor-kappaB ligand in Monckeberg’s sclerosis and atherosclerosis. J Clin Endocrinol Metab. 2004;89:4104–4112. CrossRef

26. 26Shanahan CM, Cary NRB, Salisbury JR, Proudfoot D, Weissberg PL, Edmonds ME. Medial localization of mineralization-regulating proteins in association with Monckeberg’s sclerosis: evidence for smooth muscle cell-mediated vascular calcification. Circulation. 1999;100:2168–2176.

27. 27Tyson KL, Reynolds JL, McNair R, Zhang Q, Weissberg PL, Shanahan CM. Osteo/chondrocytic transcription factors and their target genes exhibit distinct patterns of expression in human arterial calcification. Arterioscler Thromb Vasc Biol. 2003;23:489–494. CrossRef

28. 28Towler DA, Bidder M, Latifi T, Coleman T, Semenkovich CF. Diet-induced diabetes activates an osteogenic gene regulatory program in the aortas of low density lipoprotein receptor-deficient mice. J Biol Chem. 1998;273:30427–30434. MEDLINE | CrossRef

29. 29Tintut Y, Patel J, Territo M, Saini T, Parhami F, Demer LL. Monocyte/macrophage regulation of vascular calcification in vitro. Circulation. 2002;105:650–655. CrossRef

30. 30Zhang L, Zalewski A, Liu Y, Mazurek T, Cowan S, Martin JL, et al. Diabetes-induced oxidative stress and low-grade inflammation in porcine coronary arteries. Circulation. 2003;108:472–478. CrossRef

31. 31Wadwa RP, Kinney GL, Ogden L, Snell-Bergeon JK, Maahs DM, Cornell E, et al. Soluble interleukin-2 receptor as a marker for progression of coronary artery calcification in type 1 diabetes. Int J Biochem Cell Biol. 2006;38:996–1003. MEDLINE | CrossRef

32. 32Chamley-Campbell JH, Campbell GR, Ross R. Phenotype-dependent response of cultured aortic smooth muscle to serum mitogens. J Cell Biol. 1981;89:379–383. MEDLINE | CrossRef

33. 33Iyemere VP, Proudfoot D, Weissberg PL, Shanahan CM. Vascular smooth muscle cell phenotypic plasticity and the regulation of vascular calcification. J Intern Med. 2006;260:192–210. MEDLINE | CrossRef

34. 34Tintut Y, Alfonso Z, Saini T, Radcliff K, Watson K, Bostrom K, et al. Multilineage potential of cells from the artery wall. Circulation. 2003;108:2505–2510. CrossRef

35. 35Balica M, Bostrom K, Shin V, Tillisch K, Demer LL. Calcifying subpopulation of bovine aortic smooth muscle cells is responsive to 17 beta-estradiol. Circulation. 1997;95:1954–1960. MEDLINE

36. 36Tintut Y, Parhami F, Bostrom K, Jackson SM, Demer LL. cAmp stimulates osteoblast-like differentiation of calcifying vascular cells (Potential signaling pathway for vascular calcification). J Biol Chem. 1998;273:7547–7553. MEDLINE | CrossRef

37. 37Cheng S-L, Shao J-S, Charlton-Kachigian N, Loewy AP, Towler DA. Msx2 promotes osteogenesis and suppresses adipogenic differentiation of multipotent mesenchymal progenitors. J Biol Chem. 2003;278:45969–45977. MEDLINE | CrossRef

38. 38Shao JS, Cheng SL, Pingsterhaus JM, Charlton-Kachigian N, Loewy AP, Towler DA. Msx2 promotes cardiovascular calcification by activating paracrine Wnt signals. J Clin Invest. 2005;15:1210–1220.

39. 39Kirton JP, Wilkinson FL, Canfield AE, Alexander MY. Dexamethasone downregulates calcification-inhibitor molecules and accelerates osteogenic differentiation of vascular pericytes: implications for vascular calcification. Circ Res. 2006;98:1264–1272. CrossRef

40. 40Yamagishi S, Fujimori H, Yonekura H, Tanaka N, Yamamoto H. Advanced glycation endproducts accelerate calcification in microvascular pericytes. Biochem Biophys Res Commun. 1999;258:353–357. CrossRef

41. 41Collett GDM, Canfield AE. Angiogenesis and pericytes in the initiation of ectopic calcification. Circ Res. 2005;96:930–938. CrossRef

42. 42Moulton KS, Vakili K, Zurakowski D, Soliman M, Butterfield C, Sylvin E, et al. Inhibition of plaque neovascularization reduces macrophage accumulation and progression of advanced atherosclerosis. Proc Natl Acad Sci U S A. 2003;100:4736–4741. MEDLINE | CrossRef

43. 43Celletti FL, Waugh JM, Amabile PG, Brendolan A, Hilfiker PR, Dake MD. Vascular endothelial growth factor enhances atherosclerotic plaque progression. Nat Med. 2001;7:425–429. MEDLINE | CrossRef

44. 44Zhang Y, Cliff WJ, Schoefl GI, Higgins G. Immunohistochemical study of intimal microvessels in coronary atherosclerosis. Am J Pathol. 1993;143:164–172. MEDLINE

45. 45Bujan J, Bellon JM, Sabater C, Jurado F, Garcia-Honduvilla N, Dominguez B, et al. Modifications induced by atherogenic diet in the capacity of the arterial wall in rats to respond to surgical insult. Atherosclerosis. 1996;122:141–152. Abstract | Full-Text PDF (5068 KB) | CrossRef

46. 46Cola C, Almeida M, Li D, Romeo F, Mehta JL. Regulatory role of endothelium in the expression of genes affecting arterial calcification. Biochem Biophys Res Comm. 2004;320:424–427. CrossRef

47. 47Shin V, Zebboudj AF, Bostrom K. Endothelial cells modulate osteogenesis in calcifying vascular cells. J Vasc Res. 2004;41:193–201. MEDLINE | CrossRef

48. 48Wada T, McKee MD, Steitz S, Giachelli CM. Calcification of vascular smooth muscle cell cultures: inhibition by osteopontin. Circ Res. 1999;84:166–178. MEDLINE

49. 49Giachelli CM, Speer MY, Li X, Rajachar RM, Yang H. Regulation of vascular calcification: roles of phosphate and osteopontin. Circ Res. 2005;96:717–722. CrossRef

50. 50Lomashvili KA, Cobbs S, Hennigar RA, Hardcastle KI, O’Neill WC. Phosphate-induced vascular calcification: role of pyrophosphate and osteopontin. J Am Soc Nephrol. 2004;15:1392–1401. MEDLINE | CrossRef

51. 51Watson KE, Bostrom K, Ravindranath R, Lam T, Norton B, Demer LL. TGF-beta1 and 25-hydroxycholesterol stimulate osteoblast-like vascular cells to calcify. J Clin Invest. 1994;93:2106–2113. MEDLINE | CrossRef

52. 52Simmons CA, Nikolovski J, Thornton AJ, Matlis S, Mooney DJ. Mechanical stimulation and mitogen-activated protein kinase signaling independently regulate osteogenic differentiation and mineralization by calcifying vascular cells. J Biomech. 2004;37:1531–1541. Abstract | Full Text | Full-Text PDF (523 KB) | CrossRef

53. 53Proudfoot D, Davies JD, Skepper JN, Weissberg PL, Shanahan CM. Acetylated low-density lipoprotein stimulates human vascular smooth muscle cell calcification by promoting osteoblastic differentiation and inhibiting phagocytosis. Circulation. 2002;106:3044–3050. CrossRef

54. 54Li J, Chai S, Tang C, Du J. Homocysteine potentiates calcification of cultured rat aortic smooth muscle cells. Life Sci. 2003;74:451–461. MEDLINE | CrossRef

55. 55Chen NX, Duan D, O’Neill KD, Moe SM. High glucose increases the expression of Cbfa1 and BMP-2 and enhances the calcification of vascular smooth muscle cells. Nephrol Dial Transplant. 2006;21:3435–3442. MEDLINE | CrossRef

56. 56Wu SY, Zhang BH, Pan CS, Jiang HF, Pang YZ, Tang CS, et al. Endothelin-1 is a potent regulator in vivo in vascular calcification and in vitro in calcification of vascular smooth muscle cells. Peptides. 2003;24:1149–1156. MEDLINE | CrossRef

57. 57Li X, Yang H-Y, Giachelli CM. Role of the sodium-dependent phosphate cotransporter, Pit-1, in vascular smooth muscle cell calcification. Circ Res. 2006;14:905–912.

58. 58Parhami F, Tintut Y, Ballard A, Fogelman AM, Demer LL. Leptin enhances the calcification of vascular cells: artery wall as a target of leptin. Circ Res. 2001;88:954–960. CrossRef

59. 59Basalyga DM, Simionescu DT, Xiong W, Baxter BT, Starcher BC, Vyavahare NR. Elastin degradation and calcification in an abdominal aorta injury model: role of matrix metalloproteinases. Circulation. 2004;110:3480–3487. CrossRef

60. 60Qin X, Corriere MA, Matrisian LM, Guzman RJ. Matrix metalloproteinase inhibition attenuates aortic calcification. Arterioscler Thromb Vasc Biol. 2006;26:1510–1516. CrossRef

61. 61Johnson K, Polewski M, van Etten D, Terkeltaub R. Chondrogenesis mediated by PPi depletion promotes spontaneous aortic calcification in NPP1-/- Mice. Arterioscler Thromb Vasc Biol. 2005;25:686–691. CrossRef

62. 62Son BK, Kozaki K, Iijima K, Eto M, Kojima T, Ota H, et al. Statins protect human aortic smooth muscle cells from inorganic phosphate-induced calcification by restoring Gas6-Axl survival pathway. Circ Res. 2006;98:1024–1031. CrossRef

63. 63Abedin M, Lim J, Tang TB, Park D, Demer LL, Tintut Y. N-3 fatty acids inhibit vascular calcification via the p38-mitogen-activated protein kinase and peroxisome proliferator-activated receptor-gamma pathways. Circ Res. 2006;98:727–729. CrossRef

64. 64Wachi H, Sugitani H, Murata H, Nakazawa J, Mecham RP, Seyama Y. Tropoelastin inhibits vascular calcification via 67-kDa elastin binding protein in cultured bovine aortic smooth muscle cells. J Atheroscler Thromb. 2004;11:159–166. MEDLINE

65. 65Simionescu A, Philips K, Vyavahare N. Elastin-derived peptides and TGF-beta1 induce osteogenic responses in smooth muscle cells. Biochem Biophys Res Commun. 2005;334:524–532. CrossRef

66. 66Davies MR, Lund RJ, Hruska KA. BMP-7 is an efficacious treatment of vascular calcification in a murine model of atherosclerosis and chronic renal failure. J Am Soc Nephrol. 2003;14:1559–1567. MEDLINE | CrossRef

67. 67Price PA, Faus SA, Williamson MK. Bisphosphonates alendronate and ibandronate inhibit artery calcification at doses comparable to those that inhibit bone resorption. Arterioscler Thromb Vasc Biol. 2001;21:817–824.

68. 68Price PA, Buckley JR, Williamson MK. The amino bisphosphonate ibandronate prevents vitamin D toxicity and inhibits vitamin D-induced calcification of arteries, cartilage, lungs and kidneys in rats. J Nutr. 2001;131:2910–2915. MEDLINE

69. 69Price PA, Omid N, Than TN, Williamson MK. The amino bisphosphonate ibandronate prevents calciphylaxis in the rat at doses that inhibit bone resorption. Calcif Tissue Int. 2002;71:356–363. CrossRef

70. 70Luo G, Ducy P, McKee MD, Pinero GJ, Loyer E, Behringer RR, et al. Spontaneous calcification of arteries and cartilage in mice lacking matrix GLA protein. Nature. 1997;386:78–81. MEDLINE | CrossRef

71. 71Speer MY, McKee MD, Guldberg RE, Liaw L, Yang H-Y, Tung E, et al. Inactivation of the osteopontin gene enhances vascular calcification of matrix Gla protein-deficient mice: evidence for osteopontin as an inducible inhibitor of vascular calcification in vivo. J Exp Med. 2002;196:1047–1055. MEDLINE | CrossRef

72. 72Speer MY, Chien YC, Quan M, Yang HY, Vali H, McKee MD, et al. Smooth muscle cells deficient in osteopontin have enhanced susceptibility to calcification in vitro. Cardiovasc Res. 2005;66:324–333. MEDLINE | CrossRef

73. 73Bucay N, Sarosi I, Dunstan CR, Morony S, Tarpley J, Capparelli C, et al. Osteoprotegerin-deficient mice develop early onset osteoporosis and arterial calcification. Genes Dev. 1998;12:1260–1268. MEDLINE | CrossRef

74. 74Schafer C, Heiss A, Schwarz A, Westenfeld R, Ketteler M, Floege J, et al. The serum protein {alpha}2-Heremans-Schmid glycoprotein/fetuin-A is a systemically acting inhibitor of ectopic calcification. J Clin Invest. 2003;112:357–366. MEDLINE | CrossRef

75. 75Galvin KM, Donovan MJ, Lynch CA, Meyer RI, Paul RJ, Lorenz JN, et al. A role for Smad6 in delvelopment and homeostasis of the cardiovascular system. Nat Genet. 2000;24:171–174. MEDLINE | CrossRef

76. 76Kuro-o M, Matsumura Y, Aizawa H, Kawaguchi H, Suga T, Utsugi T, et al. Mutation of the mouse klotho gene leads to a syndrome resembling ageing. Nature. 1997;390:45–51. MEDLINE | CrossRef

77. 77Spicer SS, Lewis SE, Tashian RE, Schulte BA. Mice carrying a CAR-2 null allele lack carbonic anhydrase II immunohistochemically and show vascular calcification. Am J Pathol. 1989;134:947–954. MEDLINE

78. 78Shioi A, Nishizawa Y, Jono S, Koyama H, Hosoi M, Morii H. Beta-glycerophosphate accelerates calcification in cultured bovine vascular smooth muscle cells. Arterioscl Thromb Vasc Biol. 1995;15:2003–2009. MEDLINE

79. 79Jono S, McKee MD, Murry CE, Shioi A, Nishizawa Y, Mori K, et al. Phosphate regulation of vascular smooth muscle cell calcification. Circ Res. 2000;87:E10–E17.

80. 80Fischer JW, Steitz SA, Johnson PY, Burke A, Kolodgie F, Virmani R, et al. Decorin promotes aortic smooth muscle cell calcification and colocalizes to calcified regions in human atherosclerotic lesions. Arterioscler Thromb Vasc Biol. 2004;24:2391–2396. CrossRef

81. 81Bonucci E, Sadun R. Dihydrotachysterol-induced aortic calcification (A histochemical and ultrastructural investigation). Clin Orthop Relat Res. 1975;283–294.

82. 82Price PA, June HH, Buckley JR, Williamson MK. Osteoprotegerin inhibits artery calcification induced by warfarin and by vitamin D. Arterioscler Thromb Vasc Biol. 2001;21:1610–1616. CrossRef

83. 83Jono S, Nishizawa Y, Shioi A, Morii H. 1,25-Dihydroxyvitamin D3 increases in vitro vascular calcification by modulating secretion of endogenous parathyroid hormone-related peptide. Circulation. 1998;98:1302–1306. MEDLINE

84. 84Tanimura A, McGregor DH, Anderson HC. Matrix vesicles in atherosclerotic calcification. Proc Soc Exp Biol Med. 1983;172:173–177. MEDLINE

85. 85Kim KM. Calcification of matrix vesicles in human aortic valve and aortic media. Fed Proc. 1976;35:156–162. MEDLINE

86. 86Bobryshev YV. Calcification of elastic fibers in human atherosclerotic plaque. Atherosclerosis. 2005;180:293–303. Abstract | Full Text | Full-Text PDF (921 KB) | CrossRef

87. 87Price PA, Caputo JM, Williamson MK. Bone origin of the serum complex of calcium, phosphate, fetuin, and matrix Gla protein: biochemical evidence for the cancellous bone-remodeling compartment. J Bone Miner Res. 2002;17:1171–1179. MEDLINE | CrossRef

88. 88Price PA, June HH, Buckley JR, Williamson MK. SB 242784, a selective inhibitor of the osteoclastic V-H+ATPase, inhibits arterial calcification in the rat. Circ Res. 2002;91:547–552. CrossRef

89. 89Moe SM, Chen NX. Pathophysiology of vascular calcification in chronic kidney disease. Circ Res. 2004;95:560–567. CrossRef

90. 90Parhami F, Garfinkel A, Demer LL. Role of lipids in osteoporosis. Arterioscler Thromb Vasc Biol. 2000;20:2346–2348.

91. 91Parhami F, Demer LL. Arterial calcification in face of osteoporosis in ageing: can we blame oxidized lipids?. Current Opinion in Lipidology. 1997;8:312–314. MEDLINE | CrossRef

92. 92Price PA, Faus SA, Williamson MK. Warfarin-induced artery calcification is accelerated by growth and vitamin D. Arterioscler Thromb Vasc Biol. 2000;20:317–327. MEDLINE

93. 93Essalihi R, Dao HH, Gilbert LA, Bouvet C, Semerjian Y, McKee MD, et al. Regression of medial elastocalcinosis in rat aorta: a new vascular function for carbonic anhydrase. Circulation. 2005;112:1628–1635. CrossRef

94. 94Katsumata K, Kusano K, Hirata M, Tsunemi K, Nagano N, Burke SK, et al. Sevelamer hydrochloride prevents ectopic calcification and renal osteodystrophy in chronic renal failure rats. Kidney Int. 2003;64:441–450. MEDLINE | CrossRef

95. 95Price PA, Roublick AM, Williamson MK. Artery calcification in uremic rats is increased by a low protein diet and prevented by treatment with ibandronate. Kidney Internat. 2006;70:1577–1583.

96. 96Bostrom K, Tsao D, Shen S, Wang Y, Demer LL. Matrix GLA protein modulates differentiation induced by bone morphogenetic protein-2 in C3H10T1/2 Cells. J Biol Chem. 2001;276:14044–14052. MEDLINE

97. 97Zebboudj AF, Shin V, Bostrom K. Matrix GLA protein and BMP-2 regulate osteoinduction in calcifying vascular cells. J Cell Biochem. 2003;90:756–765. MEDLINE | CrossRef

98. 98Jono S, Peinado C, Giachelli CM. Phosphorylation of osteopontin is required for inhibition of vascular smooth muscle cell calcification. J Biol Chem. 2000;275:20197–20203. MEDLINE | CrossRef

99. 99Sato Y, Nakamura R, Satoh M, Fujishita K, Mori S, Ishida S, et al. Thyroid hormone targets matrix Gla protein gene associated with vascular smooth muscle calcification. Circ Res. 2005;97:550–557. CrossRef

100. 100Rutsch F, Ruf N, Vaingankar S, Toliat MR, Suk A, Hohne W, et al. Mutations in ENPP1 are associated with ‘idiopathic’ infantile arterial calcification. Nat Genet. 2003;34:379–381. MEDLINE | CrossRef

101. 101Bennett BJ, Scatena M, Kirk EA, Rattazzi M, Varon RM, Averill M, et al. Osteoprotegerin inactivation accelerates advanced atherosclerotic lesion progression and calcification in older ApoE-/- mice. Arterioscler Thromb Vasc Biol. 2006;26:2117–2124. CrossRef

102. 102Galis ZS, Khatri JJ. Matrix metalloproteinases in vascular remodeling and atherogenesis: the good, the bad, and the ugly. Circ Res. 2002;90:251–262.

103. 103Ortega N, Behonick D, Stickens D, Werb Z. How Proteases regulate bone morphogenesis. Ann N Y Acad Sci. 2003;995:109–116. MEDLINE | CrossRef

104. 104Mannello F, Tonti GAM, Bagnara GP, Papa S. Role and function of matrix metalloproteinases in the differentiation and biological characterization of mesenchymal stem cells. Stem Cells. 2006;24:475–481. MEDLINE | CrossRef

105. 105Lai C-F, Seshadri V, Huang K, Shao J-S, Cai J, Vattikuti R, et al. An osteopontin-NADPH oxidase signaling cascade promotes pro-matrix metalloproteinase 9 activation in aortic mesenchymal cells. Circ Res. 2006;98:1479–1489. CrossRef

Department of Surgery, Division of Vascular Surgery, Vanderbilt University Medical Center, Nashville, Tenn.

Reprint requests: Raul J. Guzman, MD, Department of Surgery, Division of Vascular Surgery, Vanderbilt University Medical Center, D-5237 MCN Bldg, 1161 21st Ave So, Nashville, TN 37235.

This research was supported by a Mentored Clinical Scientist Development Award from the NIH HL069926, a grant from the NIDDK DK067368, and grants from the Lifeline Foundation, and the William J. von Liebig Foundation.

Competition of interest: none.

PII: S0741-5214(07)00338-2

doi:10.1016/j.jvs.2007.02.049

13 of 25