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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:547-552.)
© 1997 American Heart Association, Inc.

Articles

Detection of Osteopontin in Calcified Human Aortic Valves

Emile R. Mohler, III; Leonard P. Adam; Pam McClelland; Lori Graham; ; David R. Hathaway

From the Department of Medicine, Indiana University Medical Center, Indianapolis, and the University of Pennsylvania, Philadelphia.

Correspondence to Emile R. Mohler III, MD, Presbyterian Hospital of the University of Pennsylvania Healthcare System, Wright-Saunders Building, 39th and Market Streets, Philadelphia, PA 19104. E-mail mohlere{at}mail.med.upenn.edu.

	Abstract

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Abstract Cardiac valve calcification often results in obstruction of blood flow, which eventually leads to valve replacement. The molecular mechanisms resulting in valve calcification are unknown. Collagen and specific bone matrix proteins are thought to provide the framework for ectopic tissue calcification. This investigation was performed to determine whether the bone matrix protein osteopontin was present in calcified human aortic valves. Proteins extracted from human aortic valve tissue were subjected to polyacrylamide gel electrophoresis followed by Western blotting, using polyclonal antibodies directed against osteopontin. Fresh frozen tissue sections were also screened for osteopontin and macrophages using immunohistochemical techniques. Osteopontin was present in both heavily and minimally calcified aortic valves and absent in noncalcified purely regurgitant or normal aortic valves by both radioimmunoassay (n=16) and immunohistochemical techniques (n=8). Osteopontin colocalized with valvular calcific deposits, and macrophages were identified in the vicinity of osteopontin. These results, in addition to showing that osteopontin is present in calcified human aortic valves, suggest that osteopontin is a regulatory protein in pathological calcification. Identification of the cells producing osteopontin in abnormal cardiac valves and of proximate stimuli for its secretion may lead to novel therapeutic strategies to prevent and/or reverse calcific valve disease.

Key Words: arotic stenosis • valve • calcification • osteopontin

	Introduction

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Calcific aortic valve stenosis is the most common valvular abnormality necessitating aortic valve replacement. The pathophysiological mechanisms underlying the development of valve calcification are multifactorial. One known determinant is age; the incidence of calcific valve stenosis increases with age.^{1 2} The advancing age of the United States population is likely to expand the magnitude of this already significant cardiovascular problem. A second factor is intrinsic valve architecture. Both congenitally malformed and previously normal valves may calcify. The congenital form (eg, bicuspid aortic valve) is thought to calcify secondary to "wear and tear" of the valve.¹ Inflammation (eg, rheumatic fever) or degeneration with aging (senile calcification) can lead to calcification of a normal trileaflet valve.¹ Despite detailed gross pathological description of calcified cardiac valves, the molecular mechanisms underlying valve calcification are unknown.

The calcification of any tissue is not random but results from an orchestrated complex of events ending in calcium deposition and accumulation.^{3 4} Tissue calcification develops at a nucleation site where Ca²⁺, PO₄^3-, other minerals, and specific molecules are assembled in the correct spatial orientation necessary for crystal formation. Bone matrix proteins provide the framework for the nucleation process. Recently, it was shown that selected matrix proteins involved in bone formation are expressed by proliferating fibroblasts, macrophages, platelets, and vascular cells.^{5 6 7 8 9} In particular, osteopontin is thought to be an important regulator of calcium deposition in bone and ectopic calcified tissue such as calcified coronary artery atherosclerotic plaques.^{6 10 11} Thus, bone matrix proteins are not just found in bone but are present in diseases with ectopic calcification.

A common factor in cardiac valvular calcification is thought to be injury to the cardiac valve (eg, inflammation or shear stress).^{12 13} We hypothesize that such injury leads to ectopic expression and/or adsorption of bone matrix proteins in calcified aortic valves. In addition, propagation of the calcium deposit may be facilitated by the presence of bone matrix proteins. To investigate this possibility, we surveyed aortic valves, both calcified and noncalcified, for the presence of the bone matrix protein osteopontin. Our principal findings are that osteopontin is present in both heavily and minimally calcified aortic valves and is primarily located in zones of calcification.

	Methods

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Peptide Synthesis and Antibody Production
A peptide (LVVDPKSKEEDKHLFRISHELDS) based on a particular sequence in osteopontin was synthesized with an automated peptide synthesizer (Applied Biosystems model 431 A peptide synthesizer), using the multiple antigenic peptide synthesis system, and purified by reverse-phase liquid chromatography (Applied Biosystems model 151).¹⁴ Rabbits were immunized with 300 µg of the multiple antigenic peptide synthesis peptide, and the immunoglobulin fraction was purified from serum by using a column of DEAE Affi-Gel blue (Bio-Rad Laboratories). The antibody-enriched fraction was then further purified by using a column of peptide coupled to cyanogen bromide–activated Sepharose 4B.¹⁵ An additional polyclonal antibody directed against human bone-derived osteopontin was kindly provided by Larry W. Fisher, PhD (National Institute of Dental Research).

Control Tissue
Purified osteopontin from human milk was chosen for osteopontin control. Human milk was obtained from the milk bank at Riley Children's Hospital, Indianapolis, Ind. Osteopontin was purified according to a previously published method.¹⁶ Briefly, milk was adsorbed to barium citrate by adding 0.1 vol sodium citrate solution (3.8 g/100 mL H₂O) and 0.1 vol barium chloride solution (15 g/100 mL H₂O). The phosphoprotein was eluted from the barium citrate precipitate with 0.2 mol/L barium chloride (pH 6.8).

Collection and Processing of Aortic Valves
Diseased human aortic valves were obtained from patients undergoing surgical cardiac valve replacement. Normal cardiac valves were removed from the explanted heart of patients undergoing cardiac transplant (without a history of valve disease) and from cadaveric hearts at autopsy. The collection of these valves was approved by the Indiana University investigational review board. Valves from subjects removed at valve replacement or cardiac transplant were immediately washed with normal physiological saline solution and frozen at -80°C. Cadaveric valves were removed, washed with normal physiological saline solution, and frozen no longer than 8 hours after death. The tissues were then frozen with liquid nitrogen, ground to a powder with mortar and pestle, and stored at -80°C until used. Additionally, some valves were lyophilized shortly after surgical removal followed by EDTA and guanidine hydrochloride (GdHCl) extraction as described below.

Aortic Valve Classification
Cardiac valves obtained at the time of aortic valve replacement were initially separated into those with and those without calcification on gross examination. The noncalcified valves were purely regurgitant by clinical and echocardiographic criteria and were thin, pliable, and without gross evidence of calcific nodules. In addition, a select group of purely regurgitant aortic valves were analyzed radiographically to assess for microscopic calcification. Radiographs were performed using magnification mammography. The valves were placed on a magnification stand and a manual technique of 25 kV (peak) and 10 mA was utilized.

The calcified valves were either (1) congenitally bicuspid, (2) degenerative, or (3) rheumatic in etiology. The congenital bicuspid etiology was identified by observing two valve leaflets, one leaflet with a false raphe that was somewhat larger than the other leaflet. The degenerative etiology was identified by observing three valve leaflets with calcification not involving leaflet tip fusion in individuals greater than age 65 years. The rheumatic etiology was identified by clinical history and calcification of the mitral valve leaflets as well as aortic valve leaflets. The clinical history of autopsy and cardiac transplant patients was reviewed for cardiac valve disease. Also, the autopsy and transplant valves were inspected for any gross evidence of calcification.

Immunohistochemical Staining
Freshly obtained aortic valves were frozen in Tris-buffered saline (Triangle Biomedical Sciences), 8-µm cryosections were prepared, and slides were postfixed in 100% methanol. Endogenous peroxidase activity was then quenched with 1% H₂O₂. The presence of osteopontin was assessed by incubating specimens for 30 minutes with a blocking solution containing 10% nonimmune goat serum. Polyclonal antibodies directed against an osteopontin peptide (described above) were used at a concentration of 1:200 and applied to sections for 1 hour. A goat anti-rabbit biotinylated antibody was incubated with sections for 30 minutes followed by incubation with avidin-biotinylated peroxidase (Vector Laboratories) for 60 minutes. The reaction was developed by using a diaminobenzamidine reagent set (Kirkegard & Perry Laboratories). Macrophage staining was done with CD68 and HAM56 anti-human mouse monoclonal antibodies (DAKO Corp), using the same methodology as described above for osteopontin, except a goat anti-mouse biotinylated antibody was used.

For calcium staining, sequential frozen tissue sections were air dried and then submerged in distilled water. The sections were then removed from the water and covered with alizarin red S stain for 60 seconds. Excess stain was blotted away and slides were quickly washed in distilled water, dehydrated, cleared, and coverslipped.

Protein Purification
Valvular proteins were extracted into a solution containing 3% SDS and separated by SDS-polyacrylamide gel electrophoresis. Alternatively, osteopontin was purified from the mixture of proteins extracted by demineralization of valve tissue according to a previously published method.¹⁷ Briefly, valve tissue was initially washed in normal physiological saline solution and lyophilized. The tissue pieces were ground to powder with mortar and pestle and extracted successively with 4 mol/L GdHCl in 50 mmol/L Tris-HCl buffer, pH 7.2 (5 mL/g, dry weight), for 24 hours followed by two 48-hour extractions with 4 mol/L GdHCl containing 0.5 mol/L EDTA and 50 mmol/L Tris-HCL buffer, pH 7.2 (10 mL/g, dry weight). Each extraction was at 4°C, and the solutions contained the following protease inhibitors and alkaline phosphatase inhibitor: 5 mmol/L benzamidine hydrochloride, 1 mmol/L sodium iodoacetate, 5 mg/L pepstatin, 10 mmol/L PMSF, 1 mg/L L-1-chloro-3-(4-tosylamido)-7-amino-2-heptanone hydrochloride, and 5 mmol/L levamisole.

A sample of the partially purified extract was further purified using column separation to determine whether more than one protein was present at the 66-kD level. This was accomplished by passing the extract over a Sephacryl S-200 column according to a previously published method,¹⁸ which yielded a high- and low-molecular-weight fraction. The high-molecular-weight fraction was then placed on a DEAE-Sephacel column and eluted with a 0 to 500 mmol/L NaCl gradient in a buffer of 20 mmol/L MOPS, pH 7.0, and 1 mmol/L each EDTA, EGTA, and DTT.

Gel Electrophoresis and Immunoassay
Valve proteins were separated with 7.5% SDS–polyacrylamide gel electrophoresis, using the buffer system of Porzio and Pearson¹⁹ and transferred to nitrocellulose.²⁰ Radioimmunoassays, using the polyclonal antibodies, were then performed to screen for osteopontin.²⁰ Immunoblot analysis was performed using an appropriate dilution of antiserum with detection by autoradiographs after the addition of ¹²⁵I-protein A (Dupont New England Nuclear).

	Results

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Demographic Data
The demographic data for patients, according to valve etiology, are listed in the Table. As expected, the patients with a calcified aortic valve of degenerative etiology comprised the oldest age group. The underlying pathology of patients with a purely regurgitant etiology included congenital bicuspid aortic valve, annuloaortic ectasia, and Marfan's syndrome. The transplant aortic valve was removed from a patient with a history of idiopathic dilated cardiomyopathy. The autopsy aortic valves were removed from a patient who died secondary to acute lymphoblastic leukemia and a patient who died of a myocardial infarction.

View this table: [in this window] [in a new window]   Table 1. Demographics of Patients According to Valve Etiology

Protein Identification
Calcific deposits obtained from aortic valves removed at surgery and frozen with liquid nitrogen contained an array of proteins that could be extracted with 3% SDS and separated by SDS–polyacrylamide gel electrophoresis (Fig 1). Osteopontin is reported to migrate from 50 to 70 kD, depending on gel conditions, and positive antibody labeling of osteopontin was found in this molecular-weight range, as described below.

View larger version (56K): [in this window] [in a new window]   Figure 1. Coomassie blue–stained SDS-polyacrylamide gel of aortic valvular proteins. CA indicates calcified aortic valve; REG, noncalcified purely regurgitant aortic valve; TP, noncalcified transplant valve; and AUT, normal autopsy valve.

Immunoblot analysis of SDS protein extracts with the antibodies directed against an osteopontin peptide identified a band at 66 kD in tissue from all calcified aortic valves and human milk (data not shown). The heavily calcified aortic valves tested included both the congenital bicuspid (n=6) and degenerative (n=2) valve types.

The noncalcified valves, including the purely regurgitant, transplant, and autopsy valves, had no antibody labeling when SDS protein extracts from the same amount or much higher amounts of tissue were loaded on the gel for immunoblot analysis. The polyclonal antibodies directed against human bone-derived osteopontin also showed the same results as above (data not shown). A fainter, lower-molecular-weight band (60 kD), in addition to the prominent 66-kD band, was consistently present with human milk. Soluble osteopontin is known to exist in a variety of size isoforms which are thought due to posttranslational modification (personal communication from Mary Farach-Carson, PhD). Thus, this lower-molecular-weight band in human milk is most likely an isoform species.

Because the immunoblot signal from SDS extracts was not prominent, we sought to demineralize the valve tissue with EDTA and extract protein with GdHCl to determine whether osteopontin could be observed more overtly. Using these techniques, we easily detected a prominent band at the 66-kD level in the extracts of lyophilized calcified aortic valves (Fig 2). This finding indicates that more osteopontin is extracted with a 3-day EDTA/GdHCl method than from tissue incubated in SDS for 1 hour. Also, there was no antibody labeling of noncalcified valve tissue after EDTA/GdHCl extraction, even when higher amounts of noncalcified than calcified tissue extracts were loaded on the gel for immunoblot analysis (Fig 2). Osteopontin was also identified in a purely regurgitant valve with a small (2 mm) calcific nodule (data not shown).

View larger version (67K): [in this window] [in a new window]   Figure 2. Immunoblot analysis of aortic valves and control tissue using polyclonal osteopontin antibody directed against the synthetic peptide. The arrow indicates the 66-kD protein thought to be osteopontin; control, human milk; calcified, calcified aortic valve; regurgitant, noncalcified purely regurgitant aortic valve; transplant, noncalcified aortic valve explant removed from a patient who underwent cardiac transplantation; and autopsy, normal aortic valve collected at autopsy.

We further fractionated the EDTA/GdHCl extracts by column chromatography to determine whether osteopontin was a prominent component of the mixture of proteins. Chromatography on columns of Sephacyl S-200 and DEAE separated osteopontin from other, more abundant, proteins that migrated with an apparent molecular weight in the range of 66 kD. A prominent band at the 66-kD level was detected on nitrocellulose paper with naphthol blue-black staining in 100 to 250 mmol/L NaCl fractions collected. This band was not detected in the 300 to 500 mmol/L NaCl fractions (data not shown). However, the reverse was found with osteopontin antibody labeling of the Western blots generated from the various DEAE fractions. Osteopontin was prominent in the 250 to 300 mmol/L NaCl fractions but not with the fractions of lower dilution (Fig 3). These results indicate that although osteopontin migrates at the 66-kD level, another more abundant protein comigrates at the same level.

View larger version (49K): [in this window] [in a new window]   Figure 3. Immunoblot analysis of a calcified aortic valve, using polyclonal antibodies after separation with a DEAE column and NaCl gradient. A prominent 66-kD band, indicating osteopontin, is seen with the 200 to 400 mmol/L NaCl fractions. A prominent band at the 66-kD level was detected on the Western blot in the wash and 100 to 200 mmol/L NaCl fractions.

Immunohistochemistry
Colocalization of Osteopontin and Calcification
Osteopontin antibody colocalized with focal areas of calcification in all stained stenotic valves, which included two degenerative and two congenital bicuspid aortic valves. Fig 4 is a representative section from a calcified congenital bicuspid aortic valve. The aortic surface is at the top of the leaflet, with a nodular lesion below. Staining with alizarin red S (red staining) demonstrated regions of extracellular calcification centered predominately around the lesion. Osteopontin (brown immunoreaction product labeling the anti-osteopontin peptide antibody) is also present predominantly around the lesion in a similar pattern as alizarin red S staining. Osteopontin also colocalized with a small calcific nodule present in a purely regurgitant congenital bicuspid aortic valve (Fig 5). There was no positive staining with alizarin red S or osteopontin of a normal-appearing aortic valve obtained at autopsy from a patient who died of a myocardial infarction (data not shown). The amount of osteopontin labeling varied proportionally with the amount of alizarin red S staining.

View larger version (129K): [in this window] [in a new window]   Figure 4. Colocalization of osteopontin with calcium deposits. A and B, Section of calcified aortic valve stained with alizarin red S, indicating the distribution of calcium. C and D, Section of calcified aortic valve with anti-osteopontin peptide antibody.

View larger version (139K): [in this window] [in a new window]   Figure 5. Association of osteopontin and calcification with minimal valvular calcification. A, Radiograph, using mammography, of a congenital bicuspid aortic valve replaced due to severe regurgitation. Calcific deposits are seen as bright white regions at the midportion of the tip and base of the left leaflet. B, Photomicrograph of a section of the calcified tip of the left leaflet that was stained with alizarin red S. C and D, Low- and high-power photomicrographs, respectively, of a section of the calcified tip of the left leaflet, using anti-osteopontin antibody to identify osteopontin. These findings indicate osteopontin is present in aortic valves that have minimal calcification.

The specificity of the anti-osteopontin peptide antibody was verified using the avidin-biotin protocol to stain a sequential section but omitting the primary antibody. There was no staining without the primary antibody (data not shown). Also, incubation of the primary antibody with osteopontin peptide (0.1 mg/mL) for 1 hour before staining produced no tissue staining, indicating a specific interaction of antibody and protein. Thus, these results demonstrate colocalization of osteopontin and extracellular calcium.

Macrophage Staining
To determine whether macrophages are associated with osteopontin and calcified tissue, adjacent frozen sections from five calcified aortic valves were stained with two different monoclonal antibodies directed against human macrophages (CD68 and HAM56). Macrophages were associated with osteopontin and calcific deposits (Fig 6) with both CD68 and HAM56 monoclonal antibodies in all valves examined.

View larger version (90K): [in this window] [in a new window]   Figure 6. Photomicrographs showing the association of osteopontin and macrophages in a calcified aortic valve. A, Calcium staining with alizarin red S. B, Osteopontin identified with anti-osteopontin peptide antibody. C, CD68-positive macrophages (brown) with hematoxylin and eosin counterstaining. Macrophages were associated with osteopontin and calcific deposits.

	Discussion

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Calcific aortic valve disease was described as early as 1904 by Monckeburg,²¹ yet the mechanism of leaflet calcification is still poorly understood. Ashworth,²² in 1946, first described the similarities between aortovalvular disease and atherosclerotic coronary artery disease. Both diseases have calcific deposition, stainable neutral lipid, and inflammatory cells (ie, macrophages). The primary changes of calcific aortic valves have been described as an alteration of valvular collagen with extracellular droplets of neutral fat occurring in collagen bundles.^{23 24} This pathological alteration was presumably followed by calcification.

In 1863,²⁵ Virchow noted that coronary arteries calcify and the mineral component resembles that of bone. Interestingly, the mineral component of both calcified arteries and valves, hydroxyapatite, is similar biochemically and ultrastructurally to bone tissue.³ The similar mineral, lipid, and inflammatory components of atherosclerotic coronary arteries and calcified cardiac valves indicate a common calcific process may exist. Osteopontin and other bone matrix proteins, such as bone morphogenic protein-2, osteonectin, and osteocalcin, are involved in bone formation but also expressed by proliferating fibroblasts and endothelial vascular cells.^{5 8 9} This family of bone matrix proteins may participate in dystrophic tissue calcification, as seen with atherosclerotic artery calcification. Recently, osteopontin was identified in calcified human atherosclerotic plaques.^{11 26} We thus hypothesized that osteopontin may be present in calcified aortic valves.

Osteopontin (also known as secreted phosphoprotein-1 and bone sialoprotein-1, among others) is a noncollagenous, glycosylated phosphoprotein that is a prominent matrix component of mineralized bone. Although first identified in rat bone, osteopontin has subsequently been found in many tissues, including vascular smooth muscle cells and activated macrophages.^{6 27} Several structural characteristics lend support to the potential roles that osteopontin may play in the above tissues, including cellular adhesion via an Arg-Gly-Asp (RGD) motif,²⁸ hydroxyapatite binding via a sequence of nine consecutive aspartic acids, and a potential "E-F hand" calcium binding site.⁸ Also, osteopontin has been identified as a substrate for transglutaminase and factor XIII, which may serve to covalently anchor the protein to other extracellular matrix components.¹⁷

Our results indicate that osteopontin is present in both heavily and minimally calcified aortic valves. Furthermore, osteopontin colocalized with areas of calcium deposition. Our findings are consistent with the observations of O'Brien et al,²⁹ who found an association of calcium with osteopontin expression by using immunohistochemistry in human aortic valves. The novel aspect of this investigation is the isolation and identification of osteopontin protein in human calcified aortic valves. Also, osteopontin is present in all types of calcified aortic valves, regardless of etiology, and osteopontin varied in proportion to calcium present.

Ectopic tissue calcification is thought to begin at a nucleation site that places the essential components necessary for calcification in the correct spatial orientation required for crystal formation. The matrix protein that acts as the framing structure is the collagen fibril. Noncollagenous matrix proteins such as osteopontin, as well as various enzymes and growth factors, control the calcific process. Hormones also influence the calcific process, as seen with states of altered calcium metabolism, such as hyperparathyroidism, Padget's disease, and renal failure, in which ectopic calcification is not uncommon.^{30 31} This study lends support to the hypothesis that cardiac valve calcification involves a noncollagenous "bone" matrix protein that orchestrates tissue mineralization.

The cellular origin of osteopontin production may be the macrophage, as this inflammatory cell has been shown to secrete osteopontin.³² This possibility is supported by O'Brien et al,²⁹ who found a statistical association with the degree of osteopontin expression and the degrees of both calcification and macrophage accumulation in calcified aortic valves. Our investigation confirmed the results of O'Brien et al that macrophages are present in the vicinity of osteopontin and calcific deposits. Alternatively, other cellular constituents of the calcified valve, such as smooth muscle cells, may secrete osteopontin.³³ Watson et al³⁴ reported cloning of a calcifying vascular cell derived from the artery wall with characteristic features of osteoblasts. This osteoblastic-like cell may also be involved in the calcification of cardiac valves.

In conclusion, osteopontin was found in both heavily and minimally calcified valves and not in noncalcified purely regurgitant or normal-appearing valves. Also, osteopontin colocalized with valvular calcific deposits. These results suggest that osteopontin is a regulatory protein in pathological cardiac valve calcification. Identification of the cells producing osteopontin in abnormal cardiac valves and of proximate stimuli for its expression and secretion may lead to novel therapeutic strategies to prevent and/or reverse calcific valve disease.

	Acknowledgments

 
The polyclonal antibody directed against human bone-derived osteopontin was kindly provided by Larry W. Fisher, PhD (National Institute of Dental Research). We thank Mike Franklin for his technical assistance in antibody production and purification of osteopontin polyclonal antibodies. We also thank John W. Brown, MD; Mark W. Turrentine, MD; Yousuf Mahomed, MD; Ken A. Kesler, MD; and Margo Regas, RN, for their assistance in obtaining cardiac valves, and Handel E. Reynolds, MD, for his assistance with radiography. Dr Adam is now with Boston Biomedical Research Institute, Mass, and Dr Hathaway with Bristol-Myers Squibb, Princeton, NJ.

Received November 27, 1995; accepted May 24, 1996.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Selzer A. Changing aspects of the natural history of valvular aortic stenosis. N Engl J Med. 1987;317:91-98. [Medline] [Order article via Infotrieve]
2. Lindroos M, Kupari M, Heikkila J, Tilvis R. Prevalence of aortic valve abnormalities in the elderly: an echocardiographic study of a random population sample. J Am Coll Cardiol. 1993;21:1220-1225. [Abstract]
3. Anderson HC. Calcific diseases: a concept. Arch Pathol Lab Med. 1983;107:341-348. [Medline] [Order article via Infotrieve]
4. Christofferson J, Landis WJ. A contribution with the review to the description of mineralization of bone and other calcified tissues in vivo. Anat Rec. 1991;230:435-450. [Medline] [Order article via Infotrieve]
5. Bostrom K, Watson KE, Horn S, Worthham C, Herman IM, Demer LL. Bone morphogenic protein expression in human atherosclerotic lesions. J Clin Invest. 1994;91:1800-1809.
6. Hirota S, Imakita M, Kohri K, Ito A, Morii E, Adachi S, Kim H. Expression of osteopontin messenger RNA by macrophages in atherosclerotic plaques. Am J Pathol. 1993;143:1003-1008. [Abstract]
7. Thiede MA, Smock SL, Petersen DN, Grasser WA, Thompson DD, Nishimoto SK. Presence of messenger ribonucleic acid encoding osteocalcin, a marker of bone turnover, in bone marrow megakaryocytes and peripheral blood platelets. Endocrinology. 1994;135:929-937. [Abstract]
8. Denhardt DT, Guo X. Osteopontin: a protein with diverse functions. FASEB J. 1993;7:1475-1482. [Abstract]
9. Kelm RJ, Hair GA, Mann KG, Grant BW. Characterization of human osteoblast and megakaryocyte-derived osteonectin (SPARC). Blood. 1992;80:3112-3119. [Abstract/Free Full Text]
10. Roach HI. Why does bone matrix contain non-collagenous proteins? the possible roles of osteocalcin, osteonectin, osteopontin and bone sialoprotein in bone mineralisation and resorption. Cell Biol Int. 1994;18:617-628. [Medline] [Order article via Infotrieve]
11. 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.
12. Stein PD, Sabbah HN, Pitha JV. Continuing disease process of calcific aortic stenosis: role of microthrombi and turbulent flow. Am J Cardiol. 1977;39:159-163. [Medline] [Order article via Infotrieve]
13. Otto CM, Kuusisto J, Reichenbach DD, Gown AM, O'Brien KD. Characterization of the early lesion of "degenerative' valvular aortic stenosis: histological and immunohistochemical studies. Circulation. 1994;90:844-853. [Abstract/Free Full Text]
14. Tam JP. Synthetic peptide vaccine design: synthesis and properties of a high-density multiple antigenic peptide system. Proc Natl Acad Sci U S A. 1988;85:5409-5413. [Abstract/Free Full Text]
15. Hathaway DR, Adelstein RS. Human platelet myosin light chain kinase requires the calcium-binding protein calmodulin for activity. Proc Natl Acad Sci U S A. 1979;76:1653-1657. [Abstract/Free Full Text]
16. Senger DR, Perruzzi CA, Papadopoulos A, Tenen DG. Purification of a human milk protein closely similar to tumor-secreted phosphoproteins and osteopontin. Biochim Biophys Acta. 1989;996:43-48. [Medline] [Order article via Infotrieve]
17. Prince CW, Dickie D, Krumdieck CL. Osteopontin, a substrate for transglutaminase and factor XIII activity. Biochem Biophys Res Commun. 1991;177:1205-1210. [Medline] [Order article via Infotrieve]
18. Ritter NM, Farach-Carson MC, Butler WT. Evidence for the formation of a complex between osteopontin and osteocalcin. J Bone Miner Res. 1992;7:877-885. [Medline] [Order article via Infotrieve]
19. Porzio MA, Pearson AM. Improved resolution of myofibrillar proteins with sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Biochim Biophys Acta. 1977;490:27-34. [Medline] [Order article via Infotrieve]
20. Hathaway DR, Haeberle JR. A radioimmunoblotting method for measuring myosin light chain phosphorylation levels in smooth muscle. Am J Physiol. 1985;249:C345-C351. [Abstract/Free Full Text]
21. Monckeberg JG. Der normale histologische Bau und die Sklerose der Aortenklappen. Virchows Arch Pathol Anat Physiol. 1904;176:472-514.
22. Ashworth CT. Atherosclerotic valvular disease of the heart. Arch Pathol. 1946;42:285-298.
23. Walton KW, Williamson N, Johnson AG. The pathogenesis of atherosclerosis of the mitral and aortic valves. J Pathol. 1970;101:205-220. [Medline] [Order article via Infotrieve]
24. Edwards JE. On the etiology of calcific aortic stenosis. Circulation. 1962;26:17-18.
25. Virchow R. Cellular Pathology: As Based Upon Physiological and Pathological Histology. Mineola, NY: Dover Publications; 1863:404-408.
26. 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.
27. Giachelli CM, Bae N, Lombardi D, Majesky M, Schwartz S. Molecular cloning and characterization of 2B7, a rat mRNA which distinguishes smooth muscle cell phenotypes in vitro and is identical to osteopontin (secreted phosphoprotein I, 2aR). Biochem Biophys Res Commun. 1991;177:867-873. [Medline] [Order article via Infotrieve]
28. Liaw L, Almeida M, Hart CE, Schwartz SM, Giachelli CM. Osteopontin promotes vascular cell adhesion and spreading and is chemotactic for smooth muscle cells in vitro. Circ Res. 1994;74:214-224. [Abstract/Free Full Text]
29. O'Brien KD, Kuusisto J, Reichenbach DD, Ferguson M, Giachelli CM, Alpers CE, Otto CM. Osteopontin is expressed in human aortic valvular lesions. Circulation. 1995;92:2163-2168. [Abstract/Free Full Text]
30. Strickberger SA, Schulman SP, Hutchins GM. Association of Padget's disease of bone with calcific aortic valve disease. Am J Med. 1987;82:953-956. [Medline] [Order article via Infotrieve]
31. Maher ER, Pazianas M, Curtis JR. Calcific aortic stenosis: a complication of chronic uraemia. Nephron. 1987;47:119-122. [Medline] [Order article via Infotrieve]
32. Murry CE, Giachelli CM, Schwartz SM, Vracko R. Macrophages express osteopontin during repair of myocardial necrosis. Am J Pathol. 1994;145:1450-1462. [Abstract]
33. Giachelli C, Bae N, Lombardi D, Majesky M, Schwartz S. Molecular cloning and characterization of 2B7, a rat mRNA which distinguishes smooth muscle cell phenotypes in vitro and is identical to osteopontin (secreted phosphoprotein I, 2aR). Biochem Biophys Res Commun. 1991;177:867-873.
34. Watson KE, Bostrom K, Ravindranath R, Lam T, Norton B, Demer LL. TGF-ß1 and 25-hydroxycholesterol stimulate osteoblast-like vascular cells to calcify. J Clin Invest. 1994;93:2106-2113.

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				N. M. Rajamannan Update on the pathophysiology of aortic stenosis Eur. Heart J. Suppl., July 1, 2008; 10(suppl_E): E4 - E10. [Abstract] [Full Text] [PDF]

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				J. N. Clark-Greuel, J. M. Connolly, E. Sorichillo, N. R. Narula, H. S. Rapoport, E. R. Mohler III, J. H. Gorman III, R. C. Gorman, and R. J. Levy Transforming Growth Factor-{beta}1 Mechanisms in Aortic Valve Calcification: Increased Alkaline Phosphatase and Related Events Ann. Thorac. Surg., March 1, 2007; 83(3): 946 - 953. [Abstract] [Full Text] [PDF]

				G. Van Nooten, P. Somers, M. Cornelissen, S. Bouchez, F. Gasthuys, E. Cox, L. Sparks, and K. Narine Acellular porcine and kangaroo aortic valve scaffolds show more intense immune-mediated calcification than cross-linked Toronto SPV(R) valves in the sheep model Interactive CardioVascular and Thoracic Surgery, October 1, 2006; 5(5): 544 - 549. [Abstract] [Full Text] [PDF]

				K. D. O'Brien Pathogenesis of Calcific Aortic Valve Disease: A Disease Process Comes of Age (and a Good Deal More) Arterioscler. Thromb. Vasc. Biol., August 1, 2006; 26(8): 1721 - 1728. [Abstract] [Full Text] [PDF]

				D M Shavelle Are angiotensin converting enzyme inhibitors beneficial in patients with aortic stenosis? Heart, October 1, 2005; 91(10): 1257 - 1259. [Abstract] [Full Text] [PDF]

				R. G. Seipelt, C. L. Backer, C. Mavroudis, V. Stellmach, M. Cornwell, I. M. Seipelt, F. A. Schoendube, and S. E. Crawford Local delivery of osteopontin attenuates vascular remodeling by altering matrix metalloproteinase-2 in a rabbit model of aortic injury J. Thorac. Cardiovasc. Surg., August 1, 2005; 130(2): 355 - 362. [Abstract] [Full Text] [PDF]

				R. V. Freeman and C. M. Otto Spectrum of Calcific Aortic Valve Disease: Pathogenesis, Disease Progression, and Treatment Strategies Circulation, June 21, 2005; 111(24): 3316 - 3326. [Full Text] [PDF]

				E. Rieder, G. Seebacher, M.-T. Kasimir, E. Eichmair, B. Winter, B. Dekan, E. Wolner, P. Simon, and G. Weigel Tissue Engineering of Heart Valves: Decellularized Porcine and Human Valve Scaffolds Differ Importantly in Residual Potential to Attract Monocytic Cells Circulation, May 31, 2005; 111(21): 2792 - 2797. [Abstract] [Full Text] [PDF]

				K. D. O'Brien, J. L. Probstfield, M. T. Caulfield, K. Nasir, J. Takasu, D. M. Shavelle, A. H. Wu, X.-Q. Zhao, and M. J. Budoff Angiotensin-Converting Enzyme Inhibitors and Change in Aortic Valve Calcium Arch Intern Med, April 25, 2005; 165(8): 858 - 862. [Abstract] [Full Text] [PDF]

				J. S. Borer Aortic Stenosis and Statins: More Evidence of "Pleotropy"? Arterioscler. Thromb. Vasc. Biol., March 1, 2005; 25(3): 476 - 477. [Full Text] [PDF]

				Z. Aherrahrou, S. B. Axtner, P. M. Kaczmarek, A. Jurat, S. Korff, L. C. Doehring, D. Weichenhan, H. A. Katus, and B. T. Ivandic A Locus on Chromosome 7 Determines Dramatic Up-Regulation of Osteopontin in Dystrophic Cardiac Calcification in Mice Am. J. Pathol., April 1, 2004; 164(4): 1379 - 1387. [Abstract] [Full Text] [PDF]

				Y. Matsui, S. R. Rittling, H. Okamoto, M. Inobe, N. Jia, T. Shimizu, M. Akino, T. Sugawara, J. Morimoto, C. Kimura, et al. Osteopontin Deficiency Attenuates Atherosclerosis in Female Apolipoprotein E-Deficient Mice Arterioscler. Thromb. Vasc. Biol., June 1, 2003; 23(6): 1029 - 1034. [Abstract] [Full Text] [PDF]

				T. Mihaljevic, S. Paul, L. H. Cohn, and A. Wechsler Pathophysiology of Aortic Valve Disease Card. Surg. Adult, January 1, 2003; 2(2003): 791 - 810. [Full Text]

				M. F. Bellamy, P. A. Pellikka, K. W. Klarich, A. J. Tajik, and M. Enriquez-Sarano Association of cholesterol levels, hydroxymethylglutaryl coenzyme-a reductase inhibitor treatment, and progression of aortic stenosis in the community J. Am. Coll. Cardiol., November 20, 2002; 40(10): 1723 - 1730. [Abstract] [Full Text] [PDF]

				K. D. O'Brien, D. M. Shavelle, M. T. Caulfield, T. O. McDonald, K. Olin-Lewis, C. M. Otto, and J. L. Probstfield Association of Angiotensin-Converting Enzyme With Low-Density Lipoprotein in Aortic Valvular Lesions and in Human Plasma Circulation, October 22, 2002; 106(17): 2224 - 2230. [Abstract] [Full Text] [PDF]

				E. Mohler III Vascular calcification: good, bad or ugly? Vascular Medicine, August 1, 2002; 7(3): 161 - 162. [PDF]

				N. M. Rajamannan, M. Subramaniam, M. Springett, T. C. Sebo, M. Niekrasz, J. P. McConnell, R. J. Singh, N. J. Stone, R. O. Bonow, and T. C. Spelsberg Atorvastatin Inhibits Hypercholesterolemia-Induced Cellular Proliferation and Bone Matrix Production in the Rabbit Aortic Valve Circulation, June 4, 2002; 105(22): 2660 - 2665. [Abstract] [Full Text] [PDF]

				B. Jian, P. L. Jones, Q. Li, E. R. Mohler III, F. J. Schoen, and R. J. Levy Matrix Metalloproteinase-2 Is Associated with Tenascin-C in Calcific Aortic Stenosis Am. J. Pathol., July 1, 2001; 159(1): 321 - 327. [Abstract] [Full Text] [PDF]

				C. M OTTO and K. D O'BRIEN Why is there discordance between calcific aortic stenosis and coronary artery disease? Heart, June 1, 2001; 85(6): 601 - 602. [Full Text]

				E. R. Mohler III, F. Gannon, C. Reynolds, R. Zimmerman, M. G. Keane, and F. S. Kaplan Bone Formation and Inflammation in Cardiac Valves Circulation, March 20, 2001; 103(11): 1522 - 1528. [Abstract] [Full Text] [PDF]

				W. Wojakowski, J. Gminski, K. Siemianowicz, M. Goss, and M. Machalski The influence of angiotensin-converting enzyme inhibitors on the aorta elastin metabolism in diet-induced hypercholesterolaemia in rabbits Journal of Renin-Angiotensin-Aldosterone System, March 1, 2001; 2(1): 37 - 42. [Abstract] [PDF]

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