This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Giachelli, C. M. Articles by Yang, H. Search for Related Content PubMed PubMed Citation Articles by Giachelli, C. M. Articles by Yang, H. Related Collections Mechanism of atherosclerosis/growth factors Other Vascular biology Smooth muscle proliferation and differentiation

(Circulation Research. 2005;96:717.)
© 2005 American Heart Association, Inc.

Review

Regulation of Vascular Calcification

Roles of Phosphate and Osteopontin

Cecilia M. Giachelli, Mei Y. Speer, Xianwu Li, Rupak M. Rajachar, Hsueh Yang

From the Bioengineering Department, University of Washington, Seattle, Wash.

Correspondence to Cecilia M. Giachelli, PhD, Professor, Bioengineering, Box 351720, University of Washington, Seattle, WA 98195. E-mail ceci{at}u.washington.edu

This Review is part of a thematic series on Mechanisms of Vascular Calcificiation, which includes the following articles:

The Pathophysiology of Vascular Calcification in Chronic Kidney Disease

Mesenchymal Stem Cells and the Artery Wall

Regulation of Vascular Calcification by Osteoclast Regulatory Factors RANKL and OPG

Regulation of Vascular Calcification: Roles of Phosphate and Osteopontin

Angiogenesis and Pericytes in Initiation of Ectopic Calcification

Role of Bone Morphogenetic Proteins in Vascular Calcification
Linda Demer Guest Editor

	Abstract

Top Abstract Introduction Role of Phosphate Role of Osteopontin Conclusions References

 
Vascular calcification is prevalent in aging as well as a number of pathological conditions, and it is now recognized as a strong predictor of cardiovascular events in the general population as well as diabetic and end-stage renal disease patients. Vascular calcification is a highly regulated process involving inductive and inhibitory mechanisms. This article focuses on two molecules, phosphate and osteopontin, that have been implicated in the induction or inhibition of vascular calcification, respectively. Elevated phosphate is of interest because hyperphosphatemia is recognized as a major nonconventional risk factor for cardiovascular disease mortality in end-stage renal disease patients. Studies to date suggest that elevated phosphate stimulates smooth muscle cell phenotypic transition and mineralization via the activity of a sodium-dependent phosphate cotransporter. Osteopontin, however, appears to block vascular calcification most likely by preventing calcium phosphate crystal growth and inducing cellular mineral resorption.

Key Words: cardiovascular disease • osteopontin • phosphate • pyrophosphate • smooth muscle cell • vascular calcification

	Introduction

Top Abstract Introduction Role of Phosphate Role of Osteopontin Conclusions References

 
Calcification of the cardiovascular system is associated with a number of diseases, including end-stage renal disease (ESRD) and cardiovascular disease. Calcium phosphate deposition is the hallmark of vascular calcification and can occur in the blood vessels, myocardium, and cardiac valves. Calcium phosphate deposits are found in distinct layers of the blood vessel and are associated with specific pathologies. Intimal calcification is observed in atherosclerotic lesions,^1,2 whereas medial calcification is common to the arteriosclerosis observed with age, diabetes, and ESRD.^3,4 Intimal and medial calcification may occur independently of each other. In ESRD patients, intimal and medial calcification have been observed in affected vessels,^5,6 although the etiological and clinical significance of this finding is not yet clear.

Vascular calcification can lead to life-threatening organ dysfunction depending on its extent and the organ affected. For example, calcification of cardiac valve leaflets is recognized as a major mode of failure of native and bioprosthetic valves.^7,8 Furthermore, vascular calcification is responsible for calcific uremic arteriolopathy, a necrotizing skin condition associated with extremely high mortality rates.⁹ Finally, idiopathic infantile arterial calcification, a genetic disease characterized by arterial calcification, fibrosis, and stenosis, leads to premature death in afflicted neonates.¹⁰

In contrast, age and vascular disease-related vascular calcifications were previously considered benign. However, recent clinical studies have challenged this dogma. Calcification has been positively correlated with coronary atherosclerotic plaque burden,^11,12 increased risk of myocardial infarction,^13–15 and plaque instability.^2,16 Furthermore, in the Rotterdam Coronary Calcification Study, a large population-based study, graded associations between coronary calcification score and stroke were identified.¹⁷ Similarly, medial arterial calcification is strongly correlated with coronary artery disease and future cardiovascular events in type I diabetic subjects,^18,19 and is a strong prognostic marker of cardiovascular disease mortality in ESRD patients.²⁰ These findings may be explained by growing evidence that vascular medial calcification in large arteries leads to increased stiffness and therefore decreased compliance of these vessels. These mechanical changes are associated with increased arterial pulse wave velocity and pulse pressure, and lead to impaired arterial distensibility, increased afterload favoring left ventricular hypertrophy, and compromised coronary perfusion.²¹ Thus, intimal and medial calcifications may contribute to the morbidity and mortality associated with cardiovascular disease.

It is becoming increasingly clear that vascular calcification is an actively regulated process that may be initiated by a number of different, nonmutually exclusive mechanisms. These mechanisms have been extensively reviewed elsewhere²² and include: (1) loss of mineral inhibiting factors; (2) induction of bone formation; (3) cell death; and (4) circulating nucleational complexes (ie, aggregates of calcium phosphate and proteins released from remodeling bone that may initiate ectopic mineralization). Abnormalities in mineral metabolism that enhance the calcium x phosphate product (Ca x P) may further exacerbate vascular calcification initiated by any of these mechanisms. This article focuses on recent evidence implicating elevated phosphate as a major inductive factor for vascular calcification and osteopontin as an inducible inhibitor of vascular calcification, and our current understanding of their mechanisms of action.

	Role of Phosphate

Top Abstract Introduction Role of Phosphate Role of Osteopontin Conclusions References

 
Hyperphosphatemia is commonly observed in renal disease, especially in ESRD patients.²³ Elevated serum phosphorus (in the form of phosphate) is a major risk factor for vascular calcification and cardiovascular mortality in these patients.^23,24 Although elevations in the Ca x P may thermodynamically drive calcification, growing evidence indicates that direct effects of elevated phosphate on vessel wall cells may be more important in regulating the propensity of the vessel to calcify. We and others have found that heterogeneous, uncloned populations of vascular smooth muscle cells (VSMCs) do not spontaneously mineralize in culture, but can be induced to mineralize by elevating phosphate levels in the culture medium to those typically observed in hyperphosphatemic individuals (>2 mmol/L).^25–29 Under these conditions, the extracellular matrix surrounding the VSMCs undergoes calcification with features similar to that observed in bone and in pathological vascular calcification in vivo, including the presence of calcifying collagen fibers, matrix vesicles, and bioapatite.³⁰ In VSMC cultures, calcification does not appear to require apoptosis or to be associated with apoptotic cells³⁰ (Giachelli and Li unpublished observations), although induction of apoptosis may accelerate calcification.³¹

Concomitant with induction of VSMC mineralization, treatment with elevated phosphate induces cultured VSMCs to undergo a profound phenotypic transition. Under normal phosphate conditions, VSMCs express smooth muscle lineage markers representative of the contractile phenotype, including smooth muscle (SM) -actin and SM22. After treatment with elevated phosphate, there is a dramatic loss of these smooth muscle cell (SMC) lineage markers, and simultaneous gain of osteochondrogenic markers such as osteopontin, Cbfa-1/Runx2, alkaline phosphatase, and osteocalcin.^27,32 Importantly, almost identical changes in smooth muscle gene expression are observed in biopsy specimens from ESRD patients with calciphylaxis³² and with calcified inferior epigastric arteries.³³ Finally, in an experimental mouse model of vascular calcification, the matrix Gla protein null (MGP^–/–) mice, spontaneous vascular calcification was found in mice older than 2 weeks of age.³⁴ Before mineralization, the vessels appear normal and medial SMCs express abundant SM lineage genes. However, as mineralization of the elastic lamellae ensues, medial cells lose SM -actin and SM22 expression and gain expression of osteopontin, alkaline phosphatase, and Cbfa1/Runx2³⁰ (Speer and Giachelli, unpublished observation). Furthermore, analysis of older MGP^–/– mice with advanced vascular calcification showed clusters of cells with chondrocytic features including type II collagen expression.³⁴

Based on these findings, we have hypothesized that VSMCs have the capacity to undergo modulation from a contractile to an osteochondrogenic phenotypic state that is controlled by local environmental cues such as elevated phosphate levels (Figure). According to gene expression patterns, this phenotypic state appears to be distinct from the previously characterized synthetic/dedifferentiated state seen in arteries injured by chemicals, disease, or trauma.³⁵ The osteochondrogenic state may be exquisitely designed to repair and/or adapt to a mineralizing microenvironment, with enhanced expression of a number of mineral regulating molecules including the mineralization inhibitor, osteopontin, discussed later. Recent studies suggest that other molecules that promote or inhibit vascular calcification may also act, in part, by regulating VSMC phenotypic change, including elevated calcium²⁹ and BMP-7.³⁶

View larger version (15K): [in this window] [in a new window]   SMC phenotypic modulation associated with vascular calcification. It is hypothesized that VSMCs have the capacity to undergo modulation from a contractile to an osteochondrogenic phenotypic state that is controlled by local environmental cues such as elevated phosphate (P) or calcium (Ca) levels. The osteochondrogenic state is proposed to regulate matrix mineralization and to participate in the formation of bone and cartilage-like structures observed in calcified vascular lesions. SM indicates smooth muscle; alk phos, alkaline phosphatase; OPN, osteopontin; MHC, myosin heavy chain. The (+) indicates expression and (–) indicates lack of expression.

Mechanisms controlling this phenotypic transition in response to elevated phosphate in VSMCs are currently undergoing active investigation. We have determined that mineralization and VSMC phenotypic modulation in response to elevated phosphate are dependent on the activity of sodium-dependent phosphate cotransporters in the cells. Sodium-dependent phosphate cotransporters use the sodium gradient to actively transport phosphate into the cell. Three types of sodium dependent phosphate cotransporters have been identified based on structure, tissue expression, and regulation, and each family contains several members identified from various species. The type I and type II sodium-dependent phosphate cotransporters are predominantly expressed in intestine and kidney, and function to control phosphate reabsorption by these tissues.^37,38 The type III sodium-dependent phosphate cotransporters are represented by Pit-1 (also named Glvr-1 and SLC20A1) and Pit-2 (also named Ram-1 and SLC20A2).³⁹ These proteins are more ubiquitously expressed in tissues including kidney, heart, lung, brain, liver, and bone.⁴⁰ Although the physiological functions of type III sodium-dependent phosphate cotransporters have not yet been identified, they may serve a more generalized function to allow phosphate movement into cells in support of oxidative phosphorylation. Although type III sodium-dependent phosphate cotransporters appear to be constitutively expressed in many tissues, phosphate deficiency³⁹ and certain cytokines like insulin-like growth factor, transforming growth factor-ß, and platelet-derived growth factor^41–43 induce expression, whereas PTH reduces expression.⁴⁴ We have found that Pit-1 and Pit-2 are the only sodium-dependent phosphate cotransporters expressed in human VSMCs²⁹ (Li and Giachelli, unpublished data). Inhibition of sodium-dependent phosphate transport by phosphonoformic acid, a generic sodium-dependent phosphate cotransporter inhibitor, blocked elevated phosphate-induced SMC mineralization.^26,29,45 Furthermore, elevated phosphate-induced Cbfa-1 and osteocalcin expression were also inhibited by phosphonoformic acid.^26,45 Most recently, we have found that suppression of endogenous Pit-1 expression by small interfering RNAs inhibits SMC mineralization in response to elevated phosphate, and overexpression of either Pit-1 or Pit-2 is able to rescue the phosphate-induced mineralization in Pit-1–deficient cells.⁴⁶ In addition, Suzuki et al showed that vasopressin induces SMC mineralization through the enhancement of the phosphate transport activity of Pit-1.⁴⁷ These findings suggest that Pit-1 and phosphate transport play a crucial role in SMC mineralization and phenotypic modulation by elevated phosphate.

Growing evidence suggests that type III sodium-dependent phosphate cotransporters are likely to be important mediators of cell-mediated matrix mineralization in general. In osteoblasts, Pit-1 mRNA levels increase and correlate with differentiation and mineralization.⁴⁸ Furthermore, inhibition of sodium-dependent phosphate cotransporters by phosphonoformic acid also blocked phosphate-induced mineralization and osteopontin (OPN) expression in this cell type.^49,50 Finally, phosphate uptake as well as mineralization of chondrocyte-derived matrix vesicles have been shown to depend on the activity of phosphate transporters, including type III sodium-dependent phosphate cotransporters.^51,52 Matrix vesicles are thought to be key nucleating structures during endochondral ossification, and matrix vesicles have been described in calcified vascular lesions⁵³ and cultured SMCs.²⁸ Thus, these studies suggest that phosphate must be transported through mineral-forming cells, perhaps via matrix vesicles, to participate in matrix mineralization, and additionally that phosphate may have a unique signaling role(s) in these cells.

Although this review has focused on potential roles of VSMCs, it is important to stress that other cell types may also contribute to the osteochondrogenic processes observed in vascular calcification. In a series of elegant experiments, Demer et al identified and cloned a population of bovine arterial medial cells, termed calcifying vascular cells, that spontaneously form nodules that mineralize in vitro under long-term culture.^54,55 These cells lack characteristic VSMC markers and display pericyte-like properties early in culture, and develop osteoblastic features, including expression of alkaline phosphatase, osteocalcin, and mineralization, with time in culture. Nodulation and mineralization of calcifying vascular cells are modulated by a large number of proatherogenic factors, and have more recently been shown to undergo additional developmental fates, including leiomyogenesis, depending on the culture conditions.⁵⁵ Thus, these cells behave like pericytes that have long been postulated as a reservoir of multipotent stem cells in adults and can be induced to differentiate into multiple lineages, including osteoblasts.⁵⁶ Likewise, Towler et al have described vascular myofibroblasts expressing Msx2 associated with mineralization in diabetic low-density lipoprotein receptor-null mice.^57–59 These data support the presence of non-SM–derived, pluripotent stem cell-like populations within the artery wall capable of osteogenic differentiation that might be involved in vascular calcification under pathological conditions. Thus, lineage studies are critically needed to determine conditions and disease states under which pluripotent stem cells, phenotypic modulation of SMCs, or both contribute to vascular calcification.

	Role of Osteopontin

Top Abstract Introduction Role of Phosphate Role of Osteopontin Conclusions References

 
It has long been known that blood and body fluids are at or near saturation with respect to calcium and phosphate levels, suggesting that mechanisms must exist to prevent ectopic calcification. Human and mouse genetic findings have now determined that most tissues, including blood vessels, normally express inhibitors of mineralization, and lack of these molecules ("loss of inhibition") leads to calcification. In humans, a dramatic example of the importance of this mechanism is genetic deficiency of the small molecule, pyrophosphate, that leads to idiopathic infantile arterial calcification.^10,60 In this condition, calcification of the arteries leads to a severe fibroproliferative vascular disease that culminates in heart failure soon after birth. Thus, pyrophosphate has emerged as a major regulator of vascular calcification during human development. A model mimicking the human disease, the tip-toe walking mouse, has a naturally occurring mutation in nucleotide pyrophosphate/phosphodiesterase I, which leads to pyrophosphate deficiency. In addition to articular cartilage calcification, ankylosis, and increased cementum, these animals display vascular calcification.^61–65 A growing number of other putative calcification inhibitory molecules have been identified using mouse mutational analyses, including MGP, ß-glucosidase, carbonic anhydrase II, fetuin, osteoprotegerin, desmin, and Smad 6.^34,66–73 Mutant mice deficient in these molecules present with enhanced cardiovascular calcification as part of their phenotype and demonstrate that specific proteins and small molecules are normally important in suppressing ectopic calcification, including vascular calcification.

Another important molecule is OPN. OPN is an acidic phosphoprotein normally found in mineralized tissues such as bones and teeth, and it is involved in regulation of mineralization by acting as an inhibitor of apatite crystal growth, as well as promoting osteoclast function through the _vß₃ integrin.⁷⁴ Although OPN is not found in normal arteries, we^75–77 and others^78–82 have reported that OPN is abundant at sites of calcification in human atherosclerotic plaques and in calcified aortic valves. In addition, OPN levels are greatly elevated in the spontaneously mineralizing arteries of MGP^–/– mice.²⁷ These findings suggest that OPN may be an important regulator of arterial mineral deposition under conditions of injury and disease.

To examine the role of OPN in vascular calcification, we bred OPN-null mice (OPN^–/–) that have no overt vascular phenotype to MGP^–/– mice in which vascular calcification spontaneously develops. Mice deficient in both MGP and OPN (MGP^–/–OPN^–/–) showed accelerated and enhanced vascular calcification compared with mice deficient in MGP alone (MGP^–/–OPN^+/+).⁷¹ These studies indicate that OPN is an inducible inhibitor of vascular calcification in vivo and may play an important role in the adaptive response of the body to injury and disease. In light of our previous in vitro findings, part of the inhibition of arterial calcification in MGP^–/– mice may be accounted for by the potent apatite inhibitory activity of phosphorylated OPN.^30,83 Furthermore, our most recent studies point to a novel role for OPN in promoting ectopic mineral resorption as well.

In a subcutaneous implantation model, a 5- to 10-fold greater calcification was observed in glutaraldehyde-fixed porcine aortic valve leaflets explanted from OPN^–/– mice versus OPN wild-types (OPN^+/+), verifying again the inhibitory effect of OPN in calcification in vivo. More importantly, heterozygous mice showed early calcification of implants at 14 days, with subsequent regression at 30 days. The regression was found to correlate with the accumulation of OPN and carbonic anhydrase II expressing monocyte-derived cells, including macrophages and foreign body giant cells, and with subsequent acidification of the implants.⁷⁰ Rescue of the calcification phenotype in the OPN^–/– subcutaneous implantation model could be achieved by the administration of exogenous OPN. Significant inhibition of calcification in glutaraldehyde-fixed bovine pericardial implants was achieved compared with controls by delivering soluble phosphorylated rat recombinant OPN via injection at the implant site (72% inhibition) or adsorption to the implant surface (91% inhibition). Reduced phosphorylation and inactivation of the arginine-glycine-aspartate motif in adsorbed OPN resulted in significant loss of inhibition, indicating that the optimal anti-calcific effect required sufficient phosphorylation (10 to 14 phosphate groups/molecule of OPN) and a functional RGD motif. More importantly, quantitative immunostaining showed a strong positive correlation between carbonic anhydrase II expression localized to glutaraldehyde-fixed bovine pericardial implants and adsorbed OPN having sufficient phosphorylation and a functional RGD motif.⁸⁴ These studies suggest that OPN acts as an inducible inhibitor of calcification not only by inhibiting crystal growth but also by promoting active regression.

	Conclusions

Top Abstract Introduction Role of Phosphate Role of Osteopontin Conclusions References

 
Vascular calcification is highly correlated with cardiovascular disease mortality, especially in ESRD and diabetic patients. In addition to the devastating effects of inappropriate biomineralization seen in cardiac valvulopathies, calciphylaxis, and idiopathic infantile arterial calcification, vascular calcification is now recognized as a marker of atherosclerotic plaque burden as well as a major contributor to loss of arterial compliance and increased pulse pressure seen with age, diabetes, and renal insufficiency. The presence of inducers, such as phosphate, and inhibitors, such as pyrophosphate and osteopontin, are likely to control whether calcification occurs under pathological conditions. Furthermore, arterial wall cells appear to play a particularly important role in mediating vascular calcification. Understanding the origins of the cells participating in osteochondral tissue formation, and mechanisms controlling their differentiation may aid in the development of novel therapeutic strategies to prevent and potentially reverse vascular calcification, which is an urgent need in the ESRD population.

	Acknowledgments

 
This work was supported by National Institutes of Health grant AR 48798 and R01 HL62329, and National Science Foundation grant EEC-9529161 to C.M.G.

	Footnotes

 
Original received January 4, 2005; resubmission received March 1, 2005; accepted March 1, 2005.

	References

Top Abstract Introduction Role of Phosphate Role of Osteopontin Conclusions References

 

1. Hunt JL, Fairman R, Mitchell ME, Carpenter JP, Golden M, Khalapyan T, Wolfe M, Neschis D, Milner R, Scoll B, Cusack A, Mohler ER III. Bone formation in carotid plaques: a clinicopathological study. Stroke. 2002; 33: 1214–1219.[Abstract/Free Full Text]
2. Burke AP, Taylor A, Farb A, Malcom GT, Virmani R. Coronary calcification: insights from sudden coronary death victims. Z Kardiol. 2000; 89: 49–53.[Medline] [Order article via Infotrieve]
3. Monckeberg JG. Uber die reine mediaverkalkung der extremitatenarterien und ihr verhalten zur arteriosklerose. Virchows Arch Pathol Anat. 1902; 171: 141–167.
4. Edmonds ME, Morrison N, Laws JW, Watkins PJ. Medial arterial calcification and diabetic neuropathy. BMJ (Clin Res Ed). 1982; 284: 6928–6930.
5. Schwarz U, Buzello M, Ritz E, Stein G, Raabe G, Wiest G, Mall G, Amann K. Morphology of coronary atherosclerotic lesions in patients with end-stage renal failure. Nephrol Dial Transplant. 2000; 15: 218–223.[Abstract/Free Full Text]
6. Ibels LS, Alfrey AC, Huffer WE, Craswell PW, Anderson JT, Weil R III. Arterial calcification and pathology in uremic patients undergoing dialysis. Am J Med. 1979; 66: 790–796.[CrossRef][Medline] [Order article via Infotrieve]
7. Schoen FJ, Levy RJ. Tissue heart valves: current challenges and future research perspectives. J Biomed Mater Res. 1999; 47: 439–465.[CrossRef][Medline] [Order article via Infotrieve]
8. O’Keefe JH Jr, Lavie CJ, Nishimura RA, Edwards WD. Degenerative aortic stenosis. One effect of the graying of America. Postgrad Med. 1991; 89: 143–144.
9. Coates T, Kirkland GS, Dymock RB, Murphy BF, Brealey JK, Mathew TH, Disney AP. Cutaneous necrosis from calcific uremic arteriolopathy. Am J Kidney Dis. 1998; 32: 384–391.[Medline] [Order article via Infotrieve]
10. Rutsch F, Ruf N, Vaingankar S, Toliat MR, Suk A, Hohne W, Schauer G, Lehmann M, Roscioli T, Schnabel D, Epplen JT, Knisely A, Superti-Furga A, McGill J, Filippone M, Sinaiko AR, Vallance H, Hinrichs B, Smith W, Ferre M, Terkeltaub R, Nurnberg P. Mutations in ENPP1 are associated with ‘idiopathic’ infantile arterial calcification. Nat Genet. 2003; 34: 379–381.[CrossRef][Medline] [Order article via Infotrieve]
11. Rumberger JA, Simons DB, Fitzpatrick LA, Sheedy PF, Schwartz RS. Coronary artery calcium area by electron-beam computed tomography and coronary atherosclerotic plaque area. A histopathologic correlative study. Circulation. 1995; 92: 2157–2162.[Abstract/Free Full Text]
12. Sangiorgi G, Rumberger JA, Severson A, Edwards WD, Gregoire J, Fitzpatrick LA, Schwartz RS. Arterial calcification and not lumen stenosis is highly correlated with atherosclerotic plaque burden in humans: a histologic study of 723 coronary artery segments using nondecalcifying methodology. J Am Coll Cardiol. 1998; 31: 126–133.[Abstract/Free Full Text]
13. Beadenkopf WG, Daoud AS, Love BM. Calcification in the coronary arteries and its relationship to arterioscler and myocardial infarction. AJR Am J Roentgenel. 1964; 92: 865–871.
14. Locker TH, Schwartz RS, Cotta CW, Hickman JR. Fluoroscopic coronary artery calcificcation and associated coronary disease in asymptomatic young men. J Am Coll Cardiol. 1992; 19: 1167–1192.[Abstract]
15. Puentes G, Detrano R, Tang W, Wong N, French W, Narahara K, Burndage B, Baksheshi H. Estimation of coronary calcium mass using electron beam computed tomography: a promising approach for predicting coronary events? Circulation. 1995; 92: I313.
16. Fitzgerald PJ, Ports TA, Yock PG. Contribution of localized calcium deposits to dissection after angioplasty. An observational study using intravascular ultrasound. Circulation. 1992; 86: 64–70.[Abstract/Free Full Text]
17. Vliegenthart R, Hollander M, Breteler MM, van der Kuip DA, Hofman A, Oudkerk M, Witteman JC. Stroke is associated with coronary calcification as detected by electron-beam CT: the Rotterdam Coronary Calcification Study. Stroke. 2002; 33: 462–465.[Abstract/Free Full Text]
18. Olson JC, Edmundowicz D, Becker DJ, Kuller LH, Orchard TJ. Coronary calcium in adults with type 1 diabetes: a stronger correlate of clinical coronary artery disease in men than in women. Diabetes. 2000; 49: 1571–1578.[Abstract]
19. Lehto S, Niskanen L, Suhonen M, Ronnemaa T, Laakso M. Medial artery calcification. A neglected harbinger of cardiovascular complications in non–insulin-dependent diabetes mellitus. Arterioscler Thromb Vasc Biol. 1996; 16: 978–983.[Abstract/Free Full Text]
20. London GM, Guerin AP, Marchais SJ, Metivier F, Pannier B, Adda H. Arterial media calcification in end-stage renal disease: impact on all-cause and cardiovascular mortality. Nephrol Dial Transplant. 2003; 18: 1731–1740.[Abstract/Free Full Text]
21. Guerin AP, London GM, Marchais SJ, Metivier F. Arterial stiffening and vascular calcifications in end-stage renal disease. Nephrol Dial Transplant. 2000; 15: 1014–1021.[Abstract/Free Full Text]
22. Speer MY, Giachelli CM. Regulation of cardiovascular calcification. Cardiovasc Pathol. 2004; 13: 63–70.[Medline] [Order article via Infotrieve]
23. Block GA, Hulbert-Shearon TE, Levin NW, Port FK. Association of serum phosphorus and calcium x phosphate product with mortality risk in chronic hemodialysis patients: a national study. Am J Kidney Dis. 1998; 31: 607–617.[Medline] [Order article via Infotrieve]
24. Raggi P. Detection and quantification of cardiovascular calcifications with electron beam tomography to estimate risk in hemodialysis patients. Clin Nephrol. 2000; 54: 325–333.[Medline] [Order article via Infotrieve]
25. Shioi A, Nishizawa Y, Jono S, Koyama H, Hosoi M, Morii H. Beta-glycerophosphate accelerates calcification in cultured bovine vascular smooth muscle cells. Arterioscler Thromb Vasc Biol. 1995; 15: 2003–2009.[Abstract/Free Full Text]
26. Jono S, McKee MD, Murry CE, Shioi A, Nishizawa Y, Mori K, Morii H, Giachelli CM. Phosphate regulation of vascular smooth muscle cell calcification. Circ Res. 2000; 87: e10–e17.[Medline] [Order article via Infotrieve]
27. Steitz SA, Speer MY, Curinga G, Yang HY, Haynes P, Aebersold R, Schinke T, Karsenty G, Giachelli CM. Smooth muscle cell phenotypic transition associated with calcification - Upregulation of Cbfa1 and downregulation of smooth muscle lineage markers. Circ Res. 2001; 89: 1147–1154.[Abstract/Free Full Text]
28. Reynolds JL, Joannides AJ, Skepper JN, McNair R, Schurgers LJ, Proudfoot D, Jahnen-Dechent W, Weissberg PL, Shanahan CM. Human vascular smooth muscle cells undergo vesicle-mediated calcification in response to changes in extracellular calcium and phosphate concentrations: a potential mechanism for accelerated vascular calcification in ESRD. J Am Soc Nephrol. 2004; 15: 2857–2867.[Abstract/Free Full Text]
29. Yang H, Curinga G, Giachelli CM. Elevated extracellular calcium levels induce smooth muscle cell matrix mineralization in vitro. Kidney Int. 2004; 66: 2293–2299.[CrossRef][Medline] [Order article via Infotrieve]
30. Wada T, McKee MD, Steitz S, Giachelli CM. Calcification of vascular smooth muscle cell cultures: inhibition by osteopontin. Circ Res. 1999; 84: 166–178.[Abstract/Free Full Text]
31. Proudfoot D, Skepper JN, Hegyi L, Bennett MR, Shanahan CM, Weissberg PL. Apoptosis regulates human vascular calcification in vitro: evidence for initiation of vascular calcification by apoptotic bodies. Circ Res. 2000; 87: 1055–1062.[Abstract/Free Full Text]
32. Moe SM, Duan D, Doehle BP, O’Neill KD, Chen NX. Uremia induces the osteoblast differentiation factor Cbfa1 in human blood vessels. Kidney Int. 2003; 63: 1003–1011.[CrossRef][Medline] [Order article via Infotrieve]
33. Moe SM, O’Neill KD, Duan D, Ahmed S, Chen NX, Leapman SB, Fineberg N, Kopecky K. Medial artery calcification in ESRD patients is associated with deposition of bone matrix proteins. Kidney Int. 2002; 61: 638–647.[CrossRef][Medline] [Order article via Infotrieve]
34. Luo G, Ducy P, McKee MD, Pinero GJ, Loyer E, Behringer RR, Karsenty G. Spontaneous calcification of arteries and cartilage in mice lacking matrix GLA protein. Nature. 1997; 386: 78–81.[CrossRef][Medline] [Order article via Infotrieve]
35. Owens GK, Kumar MS, Wamhoff BR. Molecular regulation of vascular smooth muscle cell differentiation in development and disease. Physiol Rev. 2004; 84: 767–801.[Abstract/Free Full Text]
36. Davies 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.[Abstract/Free Full Text]
37. Murer H, Forster I, Biber J. The sodium phosphate cotransporter family SLC34. Pflugers Arch. 2004; 447: 763–767.[CrossRef][Medline] [Order article via Infotrieve]
38. Reimer RJ, Edwards RH. Organic anion transport is the primary function of the SLC17/type I phosphate transporter family. Pflugers Arch. 2004; 447: 629–635.[CrossRef][Medline] [Order article via Infotrieve]
39. Collins JF, Bai L, Ghishan FK. The SLC20 family of proteins: dual functions as sodium-phosphate cotransporters and viral receptors. Pflugers Arch. 2004; 447: 647–652.[CrossRef][Medline] [Order article via Infotrieve]
40. Kavanaugh MP, Kabat D. Identification and characterization of a widely expressed phosphate transporter/retrovirus receptor family. Kidney Int. 1996; 49: 959–963.[Medline] [Order article via Infotrieve]
41. Palmer G, Bonjour JP, Caverzasio J. Expression of a newly identified phosphate transporter/retrovirus receptor in human SaOS-2 osteoblast-like cells and its regulation by insulin-like growth factor I. Endocrinology. 1997; 138: 5202–5209.[Abstract/Free Full Text]
42. Palmer G, Guicheux J, Bonjour JP, Caverzasio J. Transforming growth factor-beta stimulates inorganic phosphate transport and expression of the type III phosphate transporter Glvr-1 in chondrogenic ATDC5 cells. Endocrinology. 2000; 141: 2236–2243.[Abstract/Free Full Text]
43. Giachelli CM, Jono S, Shioi A, Nishizawa Y, Mori K, Morii H. Vascular calcification and inorganic phosphate. Am J Kidney Dis. 2001; 38: S34–S37.[Medline] [Order article via Infotrieve]
44. Tatsumi S, Segawa H, Morita K, Haga H, Kouda T, Yamamoto H, Inoue Y, Nii T, Katai K, Taketani Y, Miyamoto KI, Takeda E. Molecular cloning and hormonal regulation of PiT-1, a sodium-dependent phosphate cotransporter from rat parathyroid glands. Endocrinology. 1998; 139: 1692–1699.[Abstract/Free Full Text]
45. Chen NX, O’Neill KD, Duan D, Moe SM. Phosphorus and uremic serum up-regulate osteopontin expression in vascular smooth muscle cells. Kidney Int. 2002; 62: 1724–1731.[CrossRef][Medline] [Order article via Infotrieve]
46. Li X, Giachelli CM. The role of type III sodium-dependent phosphate cotransporter Pit-1in smooth muscle cell calcification. Cardiovasc Pathol. 2004; 13 (Suppl 1): 185.[Medline] [Order article via Infotrieve]
47. Suzuki A, Nishiwaki-Yasuda K, Ono Y, Kakita A, Ishiwata Y, Matsumoto T, Imamura S, Kato T, Hayakawa N, Oda N, Oiso Y. Vasopressin enhanced Na-dependent Pi transport activity and the mineralization in vascular smooth muscle cells. J Bone Miner Res. 2004; 19 (Suppl 1): S114.
48. Nielsen LB, Pedersen FS, Pedersen L. Expression of type III sodium-dependent phosphate transporters/retroviral receptors mRNAs during osteoblast differentiation. Bone. 2001; 28: 160–166.[Medline] [Order article via Infotrieve]
49. Beck GR Jr, Moran E, Knecht N. Inorganic phosphate regulates multiple genes during osteoblast differentiation, including Nrf2. Exp Cell Res. 2003; 288: 288–300.[CrossRef][Medline] [Order article via Infotrieve]
50. Beck GR Jr, Zerler B, Moran E. Phosphate is a specific signal for induction of osteopontin gene expression. Proc Natl Acad Sci U S A. 2000; 97: 8352–8357.[Abstract/Free Full Text]
51. Guicheux J, Palmer G, Shukunami C, Hiraki Y, Bonjour JP, Caverzasio J. A novel in vitro culture system for analysis of functional role of phosphate transport in endochondral ossification. Bone. 2000; 27: 69–74.[Medline] [Order article via Infotrieve]
52. Wu LN, Guo Y, Genge BR, Ishikawa Y, Wuthier RE. Transport of inorganic phosphate in primary cultures of chondrocytes isolated from the tibial growth plate of normal adolescent chickens. J Cell Biochem. 2002; 86: 475–489.[CrossRef][Medline] [Order article via Infotrieve]
53. Kim KM. Calcification of matrix vesicles in human aortic valve and aortic media. Fed Proc. 1976; 35: 156–162.[Medline] [Order article via Infotrieve]
54. Bostrom 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] [Order article via Infotrieve]
55. Abedin M, Tintut Y, Demer LL. Mesenchymal stem cells and the artery wall. Circ Res. 2004; 95: 671–676.[Abstract/Free Full Text]
56. Brighton CT, Lorich DG, Kupcha R, Reilly TM, Jones AR, Woodbury RA. The pericyte as a possible osteoblast progenitor cell. Clin Orthop. 1992; 287–299.
57. Cheng SL, Shao JS, 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.[Abstract/Free Full Text]
58. Towler 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.[Abstract/Free Full Text]
59. Shao JS, Cheng SL, Charlton-Kachigian N, Loewy AP, Towler DA. Teriparatide (human parathyroid hormone (1–34)) inhibits osteogenic vascular calcification in diabetic low density lipoprotein receptor-deficient mice. J Biol Chem. 2003; 278: 50195–50202.[Abstract/Free Full Text]
60. Rutsch F, Vaingankar S, Johnson K, Goldfine I, Maddux B, Schauerte P, Kalhoff H, Sano K, Boisvert WA, Superti-Furga A, Terkeltaub R. PC-1 nucleoside triphosphate pyrophosphohydrolase deficiency in idiopathic infantile arterial calcification. Am J Pathol. 2001; 158: 543–554.[Abstract/Free Full Text]
61. Okawa A, Nakamura I, Goto S, Moriya H, Nakamura Y, Ikegawa S. Mutation in Npps in a mouse model of ossification of the posterior longitudinal ligament of the spine. Nat Genet. 1998; 19: 271–273.[CrossRef][Medline] [Order article via Infotrieve]
62. Sali A, Favaloro JM, Terkeltaub R, Goding JW. Germline deletion of the nucleoside triphosphosphate (NTPPPH) plasma cell membrane glycoprotein (PC-1) produces abnormal calcification of periarticular tissues. In: Vanduffel L, Lemmens R, eds. EctoATPase and related ectonucleotidases. Maastricht, The Netherlands: Shaker Publishing BV; 1999.
63. Harmey D, Hessle L, Narisawa S, Johnson KA, Terkeltaub R, Millan JL. Concerted regulation of inorganic pyrophosphate and osteopontin by akp2, enpp1, and ank: an integrated model of the pathogenesis of mineralization disorders. Am J Pathol. 2004; 164: 1199–1209.[Abstract/Free Full Text]
64. Johnson K, Goding J, Van Etten D, Sali A, Hu SI, Farley D, Krug H, Hessle L, Millan JL, Terkeltaub R. Linked deficiencies in extracellular PP(i) and osteopontin mediate pathologic calcification associated with defective PC-1 and ANK expression. J Bone Miner Res. 2003; 18: 994–1004.[CrossRef][Medline] [Order article via Infotrieve]
65. Nociti FH, Jr Berry JE, Foster BL, Gurley KA, Kingsley DM, Takata T, Miyauchi M, Somerman MJ. Cementum: a phosphate-sensitive tissue. J Dent Res. 2002; 81: 817–821.[Abstract/Free Full Text]
66. Spicer 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.[Abstract]
67. Schafer C, Heiss A, Schwarz A, Westenfeld R, Ketteler M, Floege J, Muller-Esterl W, Schinke T, Jahnen-Dechent W. 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.[CrossRef][Medline] [Order article via Infotrieve]
68. Pereira L, Andrikopoulos K, Tian J, Lee SY, Keene DR, Ono R, Reinhardt DP, Sakai LY, Biery NJ, Bunton T, Dietz HC, Ramirez F. Targetting of the gene encoding fibrillin-1 recapitulates the vascular aspect of Marfan syndrome. Nat Genet. 1997; 17: 218–222.[CrossRef][Medline] [Order article via Infotrieve]
69. Kuroo M, Matsumura Y, Aizawa H, Kawaguchi H, Suga T, Utsugi T, Ohyama Y, Kurabayashi M, Kaname T, Kume E, Iwasaki H, Iida A, ShirakiIida T, Nishikawa S, Nagai R, Nabeshima Y. Mutation of the mouse klotho gene leads to a syndrome resembling aging. Nature. 1997; 390: 45–51.[CrossRef][Medline] [Order article via Infotrieve]
70. Steitz SA, Speer MY, McKee MD, Liaw L, Almeida M, Yang H, Giachelli CM. Osteopontin inhibits mineral deposition and promotes regression of ectopic calcification. Am J Pathol. 2002; 161: 2035–2046.[Abstract/Free Full Text]
71. Speer MY, McKee MD, Guldberg RE, Liaw L, Yang HY, Tung E, Karsenty G, Giachelli CM. 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.[Abstract/Free Full Text]
72. Bucay N, Sarosi I, Dunstan CR, Morony S, Tarpley J, Capparelli C, Scully S, Tan HL, Xu W, Lacey DL, Boyle WJ, Simonet WS. osteoprotegerin-deficient mice develop early onset osteoporosis and arterial calcification. Genes Dev. 1998; 12: 1260–1268.[Abstract/Free Full Text]
73. Galvin KM, Donovan MJ, Lynch CA, Meyer RI, Paul RJ, Lorenz JN, Fairchild-Huntress V, Dixon KL, Dunmore JH, Gimbrone MA Jr, Falb D, Huszar D. A role for smad6 in development and homeostasis of the cardiovascular system. Nat Genet. 2000; 24: 171–174.[CrossRef][Medline] [Order article via Infotrieve]
74. Giachelli CM, Steitz S. Osteopontin: a versatile regulator of inflammation and biomineralization. Matrix Biology. 2000; 19: 622.
75. Giachelli CM, Bae N, Almeida M, Denhardt DT, Alpers CE, Schwartz SM. Osteopontin is elevated during neointima formation in rat arteries and is a novel component of human atherosclerotic plaques. J Clin Invest. 1993; 92: 1686–1696.[Medline] [Order article via Infotrieve]
76. O’Brien ER, Garvin MR, Stewart DK, Hinohara T, Simpson JB, Schwartz SM, Giachelli CM. Osteopontin is synthesized by macrophage, smooth muscle, and endothelial cells in primary and restenotic human coronary atherosclerotic plaques. Arterioscler Thromb. 1994; 14: 1648–1656.[Abstract/Free Full Text]
77. O’Brien KD, Kuusisto J, Reichenbach DD, Ferguson M, Giachelli C, Alpers CE, Otto CM. Osteopontin is expressed in human aortic valvular lesions. Circulation. 1995; 92: 2163–2168.[Abstract/Free Full Text]
78. Ikeda T, Shirasawa T, Esaki Y, Yoshiki S, Hirokawa K. Osteopontin mRNA is expressed by smooth muscle-derived foam cells in human atherosclerotic lesions of the aorta. J Clin Invest. 1993; 92: 2814–2820.[Medline] [Order article via Infotrieve]
79. Hirota S, Imakita M, Kohri K, Ito A, Morii E, Adachi S, Kim HM, Kitamura Y, Yutani C, Nomura S. Expression of osteopontin messenger RNA by macrophages in atherosclerotic plaques. A possible association with calcification. Am J Pathol. 1993; 143: 1003–1008.[Abstract]
80. Fitzpatrick LA, Severson A, Edwards WD, Ingram RT. Diffuse calcification in human coronary arteries. Association of osteopontin with atherosclerosis. J Clin Invest. 1994; 94: 1597–1604.[Medline] [Order article via Infotrieve]
81. Shen M, Marie P, Farge D, Carpentier S, De Pollak C, Hott M, Chen L, Martinet B, Carpentier A. Osteopontin is associated with bioprosthetic heart valve calcification in humans. C R Acad Sci III. 1997; 320: 49–57.[Medline] [Order article via Infotrieve]
82. Srivatsa SS, Harrity PJ, Maercklein PB, Kleppe L, Veinot J, Edwards WD, Johnson CM, Fitzpatrick LA. Increased cellular expression of matrix proteins that regulate mineralization is associated with calcification of native human and porcine xenograft bioprosthetic heart valves. J Clin Invest. 1997; 99: 996–1009.[Medline] [Order article via Infotrieve]
83. Jono 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.[Abstract/Free Full Text]
84. Ohri R, Tung E, Rajachar RM, Giachelli CM. Mitigation of ectopic calcification in osteopontin-null mice by exogenous osteopontin. Calcif Tissue Int. In press.

This article has been cited by other articles:

				G. Marhaug, V. Shah, R. Shroff, H. Varsani, L. R. Wedderburn, C. A. Pilkington, and P. A. Brogan Age-dependent inhibition of ectopic calcification: a possible role for fetuin-A and osteopontin in patients with juvenile dermatomyositis with calcinosis Rheumatology, July 1, 2008; 47(7): 1031 - 1037. [Abstract] [Full Text] [PDF]

				K. Kristiansson, E. Ilveskoski, T. Lehtimaki, L. Peltonen, M. Perola, and P. J. Karhunen Association Analysis of Allelic Variants of USF1 in Coronary Atherosclerosis Arterioscler. Thromb. Vasc. Biol., May 1, 2008; 28(5): 983 - 989. [Abstract] [Full Text] [PDF]

				E. VanBavel and E. N.T.P. Bakker A Vascular Bone Collector: Arterial Calcification Requires Tissue-Type Transglutaminase Circ. Res., March 14, 2008; 102(5): 507 - 509. [Full Text] [PDF]

				O. Phan, O. Ivanovski, I. G. Nikolov, N. Joki, J. Maizel, L. Louvet, M. Chasseraud, T. Nguyen-Khoa, B. Lacour, T. B. Drueke, et al. Effect of oral calcium carbonate on aortic calcification in apolipoprotein E-deficient (apoE-/-) mice with chronic renal failure Nephrol. Dial. Transplant., January 1, 2008; 23(1): 82 - 90. [Abstract] [Full Text] [PDF]

				P. Eller, K. Hochegger, G. M. Feuchtner, E. Zitt, I. Tancevski, A. Ritsch, F. Kronenberg, A. R. Rosenkranz, J. R. Patsch, and G. Mayer Impact of ENPP1 genotype on arterial calcification in patients with end-stage renal failure Nephrol. Dial. Transplant., January 1, 2008; 23(1): 321 - 327. [Abstract] [Full Text] [PDF]

				J. Cunningham, J. Floege, G. London, M. Rodriguez, and C. M. Shanahan Clinical Outcomes in Secondary Hyperparathyroidism and the Potential Role of Calcimimetics NDT Plus, January 1, 2008; 1(suppl_1): i29 - i35. [Abstract] [Full Text] [PDF]

				Y. Orita, H. Yamamoto, N. Kohno, M. Sugihara, H. Honda, S. Kawamata, S. Mito, N. N. Soe, and M. Yoshizumi Role of Osteoprotegerin in Arterial Calcification: Development of New Animal Model Arterioscler. Thromb. Vasc. Biol., September 1, 2007; 27(9): 2058 - 2064. [Abstract] [Full Text] [PDF]

				D. Fliser, B. Kollerits, U. Neyer, D. P. Ankerst, K. Lhotta, A. Lingenhel, E. Ritz, F. Kronenberg, and for the MMKD Study Group Fibroblast Growth Factor 23 (FGF23) Predicts Progression of Chronic Kidney Disease: The Mild to Moderate Kidney Disease (MMKD) Study J. Am. Soc. Nephrol., September 1, 2007; 18(9): 2600 - 2608. [Abstract] [Full Text] [PDF]

				K. Bostrom Osteopontin, a missing link in PDGF-induced smooth muscle cell migration Cardiovasc Res, September 1, 2007; 75(4): 634 - 635. [Full Text] [PDF]

				A. P. Burke, F. D. Kolodgie, and R. Virmani Fetuin-A, Valve Calcification, and Diabetes: What Do We Understand? Circulation, May 15, 2007; 115(19): 2464 - 2467. [Full Text] [PDF]

				R. Dhingra, L. M. Sullivan, C. S. Fox, T. J. Wang, R. B. D'Agostino Sr, J. M. Gaziano, and R. S. Vasan Relations of Serum Phosphorus and Calcium Levels to the Incidence of Cardiovascular Disease in the Community Arch Intern Med, May 14, 2007; 167(9): 879 - 885. [Abstract] [Full Text] [PDF]

A. C. Brisset, H. Hao, E. Camenzind, M. Bacchetta, A. Geinoz, J.-C. Sanchez, C. Chaponnier, G. Gabbiani, and M.-L. Bochaton-Piallat Intimal Smooth Muscle Cells of Porcine and Human Coronary Artery Express S100A4, a Marker of the Rhomboid Phenotype In Vitro Circ. Res., April 13, 2007; 1