(Circulation. 1997;95:297-298.)
© 1997 American Heart Association, Inc.
Articles |
Lipid Hypothesis of Cardiovascular Calcification
the Division of Cardiology, Departments of Medicine and Physiology, UCLA School of Medicine.
Correspondence to Linda L. Demer, MD, PhD, Division of Cardiology, Box 951679, UCLA School of Medicine, Los Angeles, CA 90095-1679.
Key Words: Editorials • lipids • calcium • valves • atherosclerosis • calcification
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Introduction |
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 Top Introduction MechanismRole of Lipids in...Link of Lipids to...Similarity of Prosthetic Valve...Are Valvular and Bone...References |
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Prosthetic heart valve calcification can be a devastating event, especially in young children. In this issue of Circulation, Vyavahare et al1 make the important observation that bioprosthetic heart valve calcification is prevented by valvular pretreatment with ethanol. Not only do these findings have a direct impact on treatment of patients with valvular heart disease, they also provide clues about the fundamental mechanisms underlying calcification of bioprosthetic valves as well as native valve disease and atherosclerotic plaque in general. A particularly important question raised by this study is whether lipids and/or protein modification is an etiologic factor in cardiovascular calcification.
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Mechanism |
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TopIntroductionMechanismRole of Lipids in...Link of Lipids to...Similarity of Prosthetic Valve...Are Valvular and Bone...References |
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To explain the negative effects of ethanol on bioprosthetic calcification, Vyavahare and colleagues1 identified three possible mechanisms: elimination of phospholipids and cholesterol, changes in collagen fiber conformation, and possible direct effects of ethanol on osteocalcin and/or the calcification process. Although they provide evidence for the first two possibilities, the following analysis suggests that the first mechanism predominates.
Lipid Extraction
Although ethanol treatment extracted almost all the lipid elements, the authors did not consider this the sole mechanism because the alternative method of lipid extraction, chloroform-methanol treatment, had less effect on calcification than ethanol despite an even more effective extraction. However, technical factors may need to be considered further because other investigators2 3 4 using chloroform-methanol extraction in a similar model have reported 8- to 100-fold inhibition of calcification, nearly the same as that achieved with ethanol.
Direct Effects of Ethanol on Calcification
Because direct effects of ethanol on mineralization generally require prolonged exposure, they are less likely to explain the inhibitory effect on calcification, which presumably begins after implantation when the ethanol is no longer present. Thus, although ethanol-fed rats have lower osteocalcin levels, any ethanol remaining in the valve would not be expected to produce a circulating level capable of affecting osteocalcin. Moreover, because the osteocalcin gene null mouse5 has increased bone mineralization, lower osteocalcin levels are not likely to explain inhibition of valvular calcification.
Protein Alterations
The authors1 suggest that protein alterations are important in inhibition of calcification on the basis that 30% ethanol treatment, which did not result in protein conformational changes, also did not prevent calcification. However, this same treatment also failed to extract lipids (Table 1).1
These considerations suggest that lipids are a key factor in calcification of prosthetic valves and potentially of native valves. Lipids may be present in the form of lipoproteins, which were shown by ultrastructural studies to accumulate in cardiac valves of the rabbit.6 In native valves, calcification occurs along a zone of lipid accumulation, in which lipids appear ultrastructurally as membranous vesicles. In atherosclerotic plaque and valves, calcium deposition is invariably associated with these matrix vesicles.7
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Role of Lipids in Bone Calcification |
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TopIntroductionMechanismRole of Lipids in...Link of Lipids to...Similarity of Prosthetic Valve...Are Valvular and Bone...References |
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A role of cholesterol and phospholipids in prosthetic valve calcification is consistent with their known involvement in bone calcification, which shares many features with vascular and native valve calcification.8 9 10 The process involves lipids at the level of nidus formation, the initial step in crystal formation in biomineralization. Even bone nodules formed in vitro contain lipids within their extracellular matrix.11 Matrix vesicles released by osteoblasts into the extracellular matrix contain phospholipids, phosphoproteins, and enzymes such as alkaline phosphatase, which are believed to function in the initiation and regulation of hydroxyapatite mineral formation. It is within these matrix vesicles that the nidus for crystallization is believed to form.
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Link of Lipids to Calcification in Atherosclerosis |
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TopIntroductionMechanismRole of Lipids in...Link of Lipids to...Similarity of Prosthetic Valve...Are Valvular and Bone...References |
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There are several connections between lipids and tissue calcification.12 The calcium mineral of atherosclerotic plaques is intimately intertwined with cholesterol at the ultrastructural level. Sarig and colleagues13 showed cholesterol within the center of calcified granules of atherosclerotic plaque using confocal microscopy, suggesting that lipids may act to nucleate the calcium mineral crystals. In addition, a product of cholesterol oxidation found in atherosclerotic lesions, 25-hydroxycholesterol, accelerates in vitro vascular calcification,9 and high tissue lipid content facilitates mineralization in cholesterol-fed rabbits.14
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Similarity of Prosthetic Valve and Native Valve Calcification |
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TopIntroductionMechanismRole of Lipids in...Link of Lipids to...Similarity of Prosthetic Valve...Are Valvular and Bone...References |
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Maranto and Schoen15 and Schoen and colleagues16 previously noted that prosthetic valve calcification is an accelerated version of native valve calcification and that alkaline phosphatase–containing matrix vesicles are present in both fresh and fixed bioprosthetic tissue at sites at which mineralization occurs.
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Are Valvular and Bone Calcification Passive or Regulated? |
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TopIntroductionMechanismRole of Lipids in...Link of Lipids to...Similarity of Prosthetic Valve...Are Valvular and Bone...References |
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It may be argued that valvular calcification is just as passive and just as regulated as bone calcification. The fact that fixed, nonliving, valvular tissue can undergo calcification when implanted in the heart or subcutaneous space has fueled the impression that cardiovascular calcification in general is a "passive" process. However, the concept that "passive" crystal formation implies lack of cellular regulation is misleading.
First, in normal, regulated physiological mineralization such as bone growth, living cells are not normally present at the precise site of calcium-phosphate crystal formation but instead are at some distance. Rather than directly secreting calcium phosphate crystals, osteoblasts secrete a highly specialized extracellular matrix that with maturation has the architectural and physicobiochemical features needed to draw calcium and phosphate into the crystal phase. Thus, cellular regulation occurs at the level of matrix synthesis.
In skeletal bone, the matrix is called osteoid, and it may carry regulatory information directing architecture and consistency through one or more of its components: glycoproteins, phosphoproteins, proteoglycans, glycosaminoglycans, matrix vesicles, ectoenzymes, proteolipids, or phospholipids. In cartilage, chondrocytes actually undergo apoptosis before their matrix calcifies. Indeed, cell death, which is frequently associated with ectopic calcification, appears to be an integral, regulatory part of normal tissue mineralization, providing substrate such as matrix vesicles and other phospholipid elements that initiate crystal formation. Ultrastructural examination of bioprosthetic valve tissue has revealed cellular debris and membrane fragments serving as the initial nuclei of calcification.17 One may even question the earlier assumption that prosthetic valve tissue is acellular, since endogenous circulating cells could invade the prosthetic valve matrix. For instance, endogenous myofibroblast-like cells invade and produce islands of collagenous tissue in the linings of cardiac assist devices 30 days after implantation.18
If the matrix produced by the cells of the porcine valve contains lipids that are permissive for calcification, why doesn't the valve calcify while still in the pig? Two possible explanations are that the lipids may undergo oxidative modification during processing and that alterations in the proteins caused by glutaraldehyde fixation render it permissive for calcification. Both the lipids and the protein cross-linking may be necessary but not sufficient for calcification.
Thus, prosthetic valve calcification should be considered no more "passive" or unregulated than the physiological process of endochondral ossification of the axial skeleton. In both, mineralization occurs in an acellular tissue, with regulation most likely at the level of matrix organization and maturation.
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Footnotes |
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The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
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References |
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TopIntroductionMechanismRole of Lipids in...Link of Lipids to...Similarity of Prosthetic Valve...Are Valvular and Bone...References |
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1. Vyavahare N, Hirsch D, Lerner E, Baskin JZ, Schoen F, Bianco R, Kruth HS, Zand R, Levy RJ. Prevention of bioprosthetic heart valve calcification by ethanol preincubation: efficacy and mechanisms. Circulation.. 1997;95:479-488.
2. Jorge-Herrero E, Fernandez P, Gutierrez M, Castillo-Olivares JL. Study of the calcification of bovine pericardium: analysis of the implication of lipids and proteoglycans. Biomaterials.. 1991;12:683-689.[Medline] [Order article via Infotrieve]
3. Jorge-Herrero E, Fernandez P, de la Torre N, Escudero C, Garcia-Paez JM, Bujan J, Castillo-Olivares JL. Inhibition of the calcification of porcine valve tissue by selective lipid removal. Biomaterials.. 1994;15:815-820.[Medline] [Order article via Infotrieve]
4. Rossi MA, Braile DM, Teixeira MDR, Souze DRS, Peres LC. Lipids extraction attenuates the calcific degeneration of bovine pericardium used in cardiac valve bioprostheses. J Exp Pathol.. 1990;71:187-196.
5. Ducy P, Desbois C, Boyce B, Pinero G, Story B, Dunstan C, Smith E, Bonadio J, Goldstein S, Gundberg C, Bradley A, Karsenty G. Increased bone formation in osteocalcin-deficient mice. Nature.. 1996;382:448-452.[Medline] [Order article via Infotrieve]
6.
Nievelstein-Post P, Mottino G, Fogelman A, Frank J. An ultrastructural study of lipoprotein accumulation in cardiac valves of the rabbit. Arterioscler Thromb.. 1994;14:1151-1161.
7. Kim KM, Huang SN. Ultrastructural study of calcification of human aortic valve. Lab Invest.. 1971;25:357-366.[Medline] [Order article via Infotrieve]
8. Bostrom K, Watson K, Horn S, Wortham C, Herman IM, Demer L. Bone morphogenetic protein expression in human atherosclerotic lesions. J Clin Invest.. 1993;91:1800-1809.
9. Watson E, Bostrom K, Ravindranath R, Lam T, Norton B, Demer L. TGF-ß and 25-hydroxycholesterol stimulate osteoblast-like vascular cells to calcify. J Clin Invest.. 1994;93:2106-2113.
10. Feldman T, Glagov S, Carroll JD. Restenosis following successful balloon valvuloplasty: bone formation in aortic valve leaflets. Cathet Cardiovasc Diagn.. 1993;29:1-7.[Medline] [Order article via Infotrieve]
11. Nefussi JR, Septier D, Sautier J-M, Forest N, Goldberg M. Localization of malachite green positive lipids in the matrix of bone nodule formed in vitro. Calcif Tissue Intl.. 1992;50:273-282.[Medline] [Order article via Infotrieve]
12. Watson KE, Demer LL. The atherosclerosis-calcification link? Curr Opin Lipidol.. 1996;7:101-104.[Medline] [Order article via Infotrieve]
13. Sarig S, Weiss TA, Katz I, Kahana F, Axouryt R, Okon E, Kruth H. Detection of cholesterol associated with calcium mineral using confocal fluorescence microscopy. Lab Invest.. 1994;71:782-787.[Medline] [Order article via Infotrieve]
14. Rokita E, Cichoki T, Heck D, Jarczyk L, Strzalkowski A. Calcification of aortic wall in cholesterol-fed rabbits. Atherosclerosis.. 1991;87:183-193.[Medline] [Order article via Infotrieve]
15.
Maranto AR, Schoen FJ. Alkaline phosphatase activity of glutaraldehyde-treated bovine pericardium used in bioprosthetic heart valves. Circ Res.. 1988;63:844-848.
16. Schoen FJ, Levy RJ, Piehler HR. Pathological considerations in replacement cardiac valves. J Soc Cardiovasc Pathol.. 1992;1:29-52.
17. Valente M, Bortolotti U, Thiene G. Ultrastructural substrates of dystrophic calcification in porcine bioprosthetic valve failure. Am J Pathol.. 1985;119:12-21.[Abstract]
18. Menconi MJ, Pockwinse S, Owen TA, Dasse KA, Stein GS, Lian JB. Properties of blood-contacting surfaces of clinically implanted cardiac assist devices: gene expression matrix composition and ultrastructural characterization of cellular linings. J Cell Biochem.. 1995;47:557-573.
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