Insights into the mechanism of vascular calcification

Insights into the Mechanism of Vascular Calcification Kristina Bostro ¨ m,

MD, PhD

Vascular calcification is common and clinically significant in atherosclerosis and heart failure. It was long believed to be an end-stage process of “passive” mineral precipitation. However, there is now a growing awareness that vascular calcification is a biologically regulated phenomenon. It has many similarities to bone formation, and ectopic bone is a well-documented part of vascular calcification. This implies that alterations in vascular cell differentiation, extensive or localized, are

an integral part of vascular calcification. Matrix ␥-carboxylated glutamate (GLA) protein (MGP)-deficient mice develop extensive vascular calcification with replacement of the media by progressively calcifying cartilage. A potential mechanism that explains these findings is MGP interference with bone morphogenetic proteins— potent inducers of cartilage and bone. 䊚2001 by Excerpta Medica, Inc. Am J Cardiol 2001;88(suppl):20E–22E

ascular calcification, including coronary and aortic calcification, is very common and clinically V significant in atherosclerosis and heart failure. Coro-

other hypothesis is that inhibition, not formation, is the “active” part of vascular calcification.4,5 Specific proteins with a high affinity for calcium may act as calcium binders and prevent mineral precipitation from occurring in the extracellular matrix. However, in this model, calcification remains a passive process that occurs as soon as inhibitors are rendered nonfunctional or are removed from the matrix. This hypothesis does not readily explain the findings of ectopic-tissue formation in calcified vessels.

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nary calcification is present in most patients with coronary artery disease (CAD), in individual significant coronary artery lesions, and in ruptured lesions causing sudden death.1 It is also the single most important risk factor for dissection in angioplasty.1 Aortic calcification causes decreased aortic compliance and elastic recoil, which, in severe cases, results in cardiac ischemia due to significantly impaired reverse aortic flow and coronary perfusion.1 In combination with coronary stenosis, it results in even greater ischemia, hypoxemia, and ventricular dysfunction, and it is closely associated with increased mortality and morbidity through increased risk of myocardial infarction and mortality.1 Systolic hypertension and left ventricular hypertrophy are other consequences of aortic calcification.1 Vascular calcification was long believed to be the end-stage of a “passive” crystallization process in which mineral precipitates in extracellular fluids nearly saturated in calcium and phosphate. However, there is a growing awareness that vascular calcification is a biologically regulated phenomenon, and as such, may be subject to prevention and reversal. The common and well-documented findings of ectopic tissue and cartilage formation in vascular calcification1,2 suggest that cell differentiation plays a pivotal role by proceeding along osteogenic and chondrogenic lineages resulting in calcified tissues. Calcification in calcified tissue is known to occur as a well-regulated process—a controlled formation of hydroxyapatite rather than passive precipitation.3 AnFrom the Division of Cardiology, Department of Medicine, and the Department of Physiology, University of California, Los Angeles, School of Medicine, Los Angeles, California, USA. This work was funded in part by National Institutes of Health grant HL04270 and the Atorvastatin Research Award Program. Address for reprints: Kristina Bostro¨m, MD, PhD, Division of Cardiology, University of California, Los Angeles, School of Medicine, Box 951679, Room 47-123 CHS, Los Angeles, California 900951679. E-mail: [email protected].

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©2001 by Excerpta Medica, Inc. All rights reserved.

SIMILARITIES BETWEEN BONE AND VASCULAR CALCIFICATION Vascular calcification has many similarities to embryonic bone formation and bone repair. To form calcified bone, osteoblasts produce matrix vesicles and osteoid, an extracellular matrix containing collagen type I, osteocalcin, osteopontin, bone sialoprotein, and other bone matrix proteins.3 The osteoid provides a permissive milieu for mineralization in which hydroxyapatite mineral crystallizes in a controlled fashion, a process which occurs about 10 days after the production of the osteoid. Vessels, atherosclerotic lesions, and calcified heart valves contain both matrix vesicles and all the major components of bone osteoid that are instrumental in allowing this mineralization, as follows: (1) bone morphogenetic protein (BMP)-2, (2) osteopontin, (3) collagen I, (4) matrix ␥-carboxylated glutamate (GLA) protein (MGP), (5) osteonectin, (6) biglycan, and (7) osteocalcin.1 Thus, calcification of these structures may proceed in the same way as bone formation. Osteoid may also add bulk to lesions and increase vascular obstruction, even before it is calcified.

CELL DIFFERENTIATION IN VASCULAR CALCIFICATION Alterations in cell differentiation are an integral part of vascular calcification, based on the observation of ectopic bone and cartilage in animal models of vascular calcification as well as in atherosclerotic lesions. Some animal models such as mice deficient in MGP6 or Smad6,7 show extensive ectopy with ossifi0002-9149/01/$ – see front matter PII S0002-9149(01)01718-0

cation involving entire layers of the vascular wall. Other models, such as mice deficient in apolipoprotein E,8 have localized etopic tissue associated with development of atherosclerotic lesions similar to human atherosclerosis. The difference may relate to the timing of the disturbance of normal cell differentiation (whether this occurs when all the cells in the vascular wall are susceptible to a specific trigger, or when just a small portion of cells in the adult vessel is involved). It is possible that replacement of entire vascular layers in genetically altered mice is triggered in early development when precursor cells are being recruited, whereas calcification in atherosclerotic plaques occurs when less differentiated cells appear as a result of the disease process or recruitment of circulating stem cells.1 Multipotent mesenchymal cells, sensitive to various growth factors, are present in both early vessel formation and in atherosclerotic lesions.9,10 The developing aorta is basically a simple tube of endothelial cells, which recruits immature precursors to smooth muscle cells from the surrounding mesenchyme to form the vascular media.9,11 In some cases, migrating cell populations, such as neural crest cells, also contribute to vessel formation.9 Alterations in smoothmuscle cell differentiation are well described in the atherosclerotic process,10 with less differentiated and more proliferative cell types giving rise to the neointima. There are reports showing significant differences among subpopulations of vascular cells, in both their response to growth factors and to their state of differentiation.9 Cells with osteoblastic characteristics, producing all the major components of osteoid described above, have been isolated from the vascular media.2,12 These cells have been termed “calcifying vascular cells” and have been used as an in vitro model for vascular calcification.

MATRIX ␥-CARBOXYLATED GLUTAMATE PROTEIN–DEFICIENT MICE MGP-deficient mice6 develop extensive vascular calcification resulting in severe hemodynamic changes and early death from congestive heart failure and/or aortic rupture. Lack of MGP causes the entire vascular media to be replaced by chondrocyte-like cells, producing a typical cartilage matrix that starts to progressively calcify at birth. MGP is a small matrix protein, initially isolated from bone and characterized by Price et al.5 It contains several GLA residues, most of which are clustered centrally in the protein, providing a negatively charged area for potential binding of metal ions.4,5 Although there are different theories about how MGP exerts its anticalcification effect, the molecular mechanism(s) of MGP remains unknown. Several investigators propose that MGP prevents mineral precipitation by binding calcium ions with high affinity through its GLA residues.4,5 Thus, it would constitute 1 of the active inhibitors (see above) that, once removed, would allow passive calcification. This may fit with the reported increase in MGP expression in vas-

cular calcification without ectopic tissue formation, such as Mo¨nckeberg’s media sclerosis13 and calcification induced by high doses of vitamin D and warfarin—an inhibitor of ␥-carboxylation.5 However, calcium binding may or may not have anything to do with mineral precipitation. It may simply be necessary to allow MGP to maintain its correct conformation and function.14 Where ectopic tissue is present, MGP expression is more likely to be involved in the initiation and development of the ectopic tissue, which is suggested by the histologic findings in MGP-deficient mice. The effect of MGP on cell differentiation in other locations is supported by the studies of Yagami et al,15 who found the MGP effect on chondrocyte mineralization was dependent on cell stage; it affected mineralization in hypertrophic chondrocytes, but not in proliferative chondrocytes. Overexpression of MGP in developing limb buds delayed chondrocyte maturation and blocked endochondral ossification. It is likely that the profound changes found in the vessel wall of MGPdeficient mice were initiated well before birth at a critical branching point in the determination of smooth-muscle cell versus chondrocyte differentiation.

A POSSIBLE MECHANISM FOR MGP Clues to a potential role for MGP in cell differentiation comes from early observations made during purification of bone morphogenetic protein (BMP) from bone tissue16 and from recent binding studies.14 BMPs are potent bone- and cartilage-inducing growth factors, important in bone formation, but also expressed by endothelial cells in the developing aorta11 and in calcified atherosclerotic lesions.12 MGP was found to be closely associated with BMP during purification, requiring strong denaturants for dissociation.16 In addition, ligand blotting demonstrated MGP to be a binding protein for BMP-2.14 If this association has physiologic significance, MGP may be a modulator or inhibitor of BMP, capable of limiting bone formation to appropriate locations, while supporting smooth-muscle cell differentiation in early vessel formation. To study the effect of MGP on osteogenic and chondrogenic differentiation induced by BMP-2, we have used several multipotent cell lines sensitive to BMP-2. The cells were transfected with MGP, or MGP was exogenously added to change the MGP levels. The cells were then treated with BMP-2, and specific cell differentiation markers were monitored. We found that MGP does inhibit BMP-2–induced osteogenic and chondrogenic differentiation in a dosedependent fashion, as detected by multiple osteogenic and chondrogenic differentiation markers.17 This would correlate with a binding between MGP and BMP-2 in the culture media. Thus, the phenotype of MGP-deficient mice may be explained by absence of MGP, allowing BMP-2, secreted from endothelial cells in the developing vessel, to induce cartilage formation in cells intended for smooth-muscle cell differentiation. A SYMPOSIUM: FIRST INTERNATIONAL SAI MEETING

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FUTURE DIRECTIONS Overall, molecular regulation of vascular calcification is a growing research field with rapidly unfolding discoveries of new mechanisms and connections to other research fields. Regulation of vascular cell differentiation is a central part in many processes, including angiogenesis, normal bone vascularization, and pathologic neovascularization of atherosclerotic plaques. Interactions between MGP and BMP connect vascular calcification to the extracellular matrix field and constitute an example of matrix interfering in growth factor signaling. It is also likely that further research will increasingly find common mechanisms between embryonic development and adult pathology. 1. Bostro¨m K, Demer LL. Regulatory mechanisms in vascular calcification. Crit

Rev Eukaryot Gene Expr 2000;12:151–158. 2. Bostro¨m KI. Cell differentiation in vascular calcification. Z Kardiol 2000;

89(suppl 2):II-69 –II-74. 3. Stein GS, Lian JB. Molecular mechanisms mediating developmental and

hormone-regulated expression of genes in osteoblasts: an integrated relationship of cell growth and differentiation. In: Noda M, ed. Cellular and Molecular Biology of Bone. San Diego: Academic Press, 1993:48 –95. 4. Schinke T, McKee MD, Karsenty G. Extracellular matrix calcification: where is the action? Nat Genet 1999;21:150 –151. 5. Price PA, Faus SA, Williamson MK. Warfarin-induced artery calcification is accelerated by growth and vitamin D. Arterioscler Thromb Vasc Biol 2000;20: 317–327. 6. Luo G, Ducy P, McKee MD, Pinero GJ, Loyer E, Behringer RR, Karsenty G.

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Spontaneous calcification of arteries and cartilage in mice lacking matrix GLA protein. Nature 1997;385:78 – 81. 7. 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. 8. Qiao JH, Xie PZ, Fishbein MC, Kreuzer J, Drake TA, Demer LL, Lusis AJ. Pathology of atheromatous lesions in inbred and genetically engineered mice. Arterioscler Thromb 1994;14:1480 –1497. 9. Hungerford JE, Little CD. Developmental biology of the vascular smooth muscle cell: building a multilayered vessel wall. J Vasc Res 1999;36,2–27. 10. Owens G. Regulation of differentiation of vascular smooth muscle cells. Phys Rev 1995;75:487–517. 11. Shah NM, Groves AK, Anderson AJ. Alternative neural crest cell fates are instructively promoted by TGF␤ superfamily members. Cell 1996;85:331–343. 12. Bostro¨m 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. 13. Shanahan CM, Cary NRB, Salisbury JR, Proudfoot D, Weissberg PL, Edmonds ME. Medial localization of mineralization-regulating proteins in association with Mo¨nckeberg’s sclerosis: evidence for smooth muscle cell-mediated vascular calcification. Circulation 1999;100:2168 –2176. 14. Wallin R, Cain D, Hutson SM, Sane DC, Loeser R. Modulation of the binding of matrix Gla protein (MGP) to bone morphogenetic protein-2 (BMP-2). Thromb Haemost 2000;84:1039 –1044. 15. Yagami K, Suh J-Y, Motomi E-I, Koyama E, Abrams WR, Shapiro IM, Pacifici M, Iwamoto M. Matrix GLA protein is a developmental regulator of chondrocyte mineralization and, when constitutively expressed, blocks endochondral and intramembraneous ossification in the limb. J Cell Biol 1999;147: 1097–1108. 16. Urist MR, Huo YK, Brownell AG, Hohl WM, Buyske J, Lietze A, Tempst P, Hunkapiller M, DeLange RJ. Purification of bovine bone morphogenetic protein by hydroxyapatite chromatography. Proc Natl Acad Sci USA 1985;81:371–375. 17. Bostro¨m 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.

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