Osteopontin Inhibits Mineral Deposition and Promotes Regression of Ectopic Calcification
2002; Elsevier BV; Volume: 161; Issue: 6 Linguagem: Inglês
10.1016/s0002-9440(10)64482-3
ISSN1525-2191
AutoresSusan A. Steitz, Mei Y. Speer, Marc D. McKee, Lucy Liaw, Manuela Almeida, Hsueh Yang, Cecilia M. Giachelli,
Tópico(s)Dermatological and Skeletal Disorders
ResumoEctopic calcification, the abnormal calcification of soft tissues, can have severe clinical consequences especially when localized to vital organs such as heart valves, arteries, and kidneys. Recent observations suggest that ectopic calcification, like bone biomineralization, is an actively regulated process. These observations have led a search for molecular determinants of ectopic calcification. A candidate molecule is osteopontin (OPN), a secreted phosphoprotein invariantly associated with both normal and pathological mineral deposits. In the present study, OPN was found to be a natural inhibitor of ectopic calcification in vivo. Glutaraldehyde-fixed aortic valve leaflets showed accelerated and fourfold to fivefold greater calcification after subcutaneous implantation into OPN-null mice compared to wild-type mice. In vitro and in vivo studies suggest that OPN not only inhibits mineral deposition but also actively promotes its dissolution by physically blocking hydroxyapatite crystal growth and inducing expression of carbonic anhydrase II in monocytic cells and promoting acidification of the extracellular milieu. These findings suggest a novel mechanism of OPN action and potential therapeutic approach to the treatment of ectopic calcification. Ectopic calcification, the abnormal calcification of soft tissues, can have severe clinical consequences especially when localized to vital organs such as heart valves, arteries, and kidneys. Recent observations suggest that ectopic calcification, like bone biomineralization, is an actively regulated process. These observations have led a search for molecular determinants of ectopic calcification. A candidate molecule is osteopontin (OPN), a secreted phosphoprotein invariantly associated with both normal and pathological mineral deposits. In the present study, OPN was found to be a natural inhibitor of ectopic calcification in vivo. Glutaraldehyde-fixed aortic valve leaflets showed accelerated and fourfold to fivefold greater calcification after subcutaneous implantation into OPN-null mice compared to wild-type mice. In vitro and in vivo studies suggest that OPN not only inhibits mineral deposition but also actively promotes its dissolution by physically blocking hydroxyapatite crystal growth and inducing expression of carbonic anhydrase II in monocytic cells and promoting acidification of the extracellular milieu. These findings suggest a novel mechanism of OPN action and potential therapeutic approach to the treatment of ectopic calcification. Biomineralization is an exquisitely regulated physiological process normally restricted to bones and teeth. Under certain pathological conditions, however, calcium salts may deposit in soft tissues with detrimental effects. Although most soft tissues can undergo ectopic calcification as a result of injury, aging, disease or metabolic imbalance, blood vessels, heart valves, and the kidney seem particularly susceptible. Ectopic calcification of blood vessels is widespread in individuals with atherosclerosis, diabetes, and end-stage renal disease and is correlated with several poor outcomes. In arteries, calcification is positively correlated with atherosclerotic plaque burden and increased risk of myocardial infarction,1Beadenkopf WG Daoud AS Love BM Calcification in the coronary arteries and its relationship to arteriosclerosis and myocardial infarction.Am J Roentgenol. 1964; 92: 865-871Google Scholar, 2Locker TH Schwartz RS Cotta CW Hickman JR Fluoroscopic coronary artery calcification and associated coronary disease in asymptomatic young men.J Am Coll Cardiol. 1992; 19: 1167-1172Abstract Full Text PDF PubMed Scopus (119) Google Scholar, 3Puentes G Detrano R Tang W Wong N French W Narahara K Brundage B Baksheshi H Estimation of coronary calcium mass using electron beam computed tomography: a promising approach for predicting coronary events?.Circulation. 1995; 92: I313Google Scholar increased ischemic episodes in peripheral vascular disease4Niskanen LK Suhonen M Siitonen O Lehtinen JM Uusitupa MI Aortic and lower limb artery calcification in type II (non-insulin-dependent) diabetic patients and non-diabetic control subjects: a five year follow-up study.Atherosclerosis. 1990; 84: 61-71Abstract Full Text PDF PubMed Scopus (65) Google Scholar and increased risk of dissection after angioplasty.5Fitzgerald PJ Ports TA Yock PG Contribution of localized calcium deposits to dissection after angioplasty: an observational study using intravascular ultrasound.Circulation. 1992; 86: 64-70Crossref PubMed Scopus (460) Google Scholar In addition, medial arterial calcification is strongly correlated with coronary artery disease in type I diabetics6Olson 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: 1571-1578Crossref PubMed Scopus (142) Google Scholar and is a strong independent marker of future cardiovascular events in diabetic patients.7Lehto S Niskanen L Suhonen L 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: 978-983Crossref PubMed Scopus (478) Google Scholar In the heart, calcific aortic stenosis, characterized by mineralization of aortic valve leaflets resulting in their deterioration and subsequent mechanical failure, occurs in ∼1 to 2% of the elderly population.8O'Keefe JH Lavie CJ Nishimura RA Edwards WD Degenerative aortic stenosis. One effect of the graying of America.Postgrad Med. 1991; 89: 143-154PubMed Google Scholar Although a common therapy for aortic stenosis is bioprosthetic tissue valve implantation, these replacement valves also suffer from ectopic mineralization, which is the leading cause of implant failure.9Schoen FJ Levy RJ Piehler HR Pathological considerations in replacement cardiac valves.Cardiovasc Pathol. 1992; 1: 29-52Abstract Full Text PDF Scopus (120) Google Scholar In the kidney, renal stones affect millions yearly, and can lead to incapacitating pain as well as hydronephrosis and hydroureter.10Cotran RS Kumar V Robbins SL Cellular injury and cellular death.in: Robbins SL Pathological Basis of Disease. ed 6. WB Saunders, Philadelphia1999: 1-35Google Scholar Recent insights into the mechanisms regulating ectopic calcification have predominantly come from studies of cardiovascular calcification. Similarities to bone mineralization were suggested by the finding that bone morphogenetic protein-2 and bone matrix proteins including osteopontin (OPN), osteonectin, osteocalcin, and matrix GLA protein are found in calcified vascular tissues.11Giachelli CM Ectopic calcification: gathering hard facts about soft tissue mineralization.Am J Pathol. 1999; 154: 671-675Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar In addition, smooth muscle cells and other vascular media derived-cells demonstrate osteoblast-like properties and can mineralize their extracellular matrices under appropriate conditions in vitro.12Bostrom K Watson KE Horn S Wortham C Herman IM Demer LL Bone morphogenic protein expression in human atherosclerotic lesions.J Clin Invest. 1993; 91: 1800-1809Crossref PubMed Scopus (891) Google Scholar, 13Wada T McKee MD Stietz S Giachelli CM Calcification of vascular smooth muscle cell cultures: inhibition by osteopontin.Circ Res. 1999; 84: 1-6Crossref PubMed Scopus (396) Google Scholar, 14Shioi 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: 2003-2009Crossref PubMed Scopus (240) Google Scholar Moreover, ectopic bone formation has occasionally been found in calcified vascular lesions.15Virchow R Cellular Pathology: As Based upon Physiological and Pathological Histology. Dover, New York1863: 404-408Google Scholar Finally, matrix vesicles similar to those proposed to nucleate mineral in bone have been identified in calcified vascular tissues.16Kim KM Calcification of matrix vesicles in human aortic valve and aortic media.Fed Proc. 1976; 35: 156-162PubMed Google Scholar, 17Tanimura A McGregor DH Anderson HC Calcification in atherosclerosis. I Human studies.J Exp Path. 1986; 2: 261-297Google Scholar, 18Hsu HHT Camacho N Isolation of calcifiable vesicles from human atherosclerotic aortas.Atherosclerosis. 1999; 143: 353-362Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar Although these studies suggest cell-mediated regulation of ectopic mineralization, the most compelling data have come from studies of mutant mice. The matrix GLA protein-null mouse displays extensive calcification of the arterial tree, and animals die within 2 months apparently from aortic rupture.19Luo GDP 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: 78-81Crossref PubMed Scopus (1714) Google Scholar In addition, several other mutant mouse strains show enhanced susceptibility to ectopic calcification, including mice deficient in osteoprotegerin;20Bucay 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: 1260-1268Crossref PubMed Scopus (2091) Google Scholar β-glucosidase; carbonic anhydrase II;21Spicer 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: 947-954PubMed Google Scholar desmin;22Thornell LE Carlsson L Li Z Mericskay M Paulin D Null mutation in the desmin gene gives rise to cardiomyopathy.J Mol Cell Cardiol. 1997; 29: 2107-2124Abstract Full Text PDF PubMed Scopus (166) Google Scholar fetuin;23Jahnen-Dechent W Schinke T Trindl A Muller-Esterl W Sablitzky F Kaiser S Blessing M Cloning and targeted deletion of the mouse fetuin gene.J Biol Chem. 1997; 272: 31496-31503Crossref PubMed Scopus (229) Google Scholar the progressive ankylosis gene, ANK;24Ho AM Johnson MD Kingsley DM Role of the mouse ank gene in control of tissue calcification and arthritis.Science. 2000; 289: 265-270Crossref PubMed Scopus (534) Google Scholar Npps, a nucleotide triphosphate pyrophosphohydrolase;25Okawa 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.Nat Genet. 1998; 19: 271-273Crossref PubMed Scopus (342) Google Scholar and an intracellular mediator of transforming growth factor-β signaling, Smad6.26Galvin KM Donovan MJ Lynch CA Meyer RI Paul RJ Lorenz JN Fairchild-Huntress V Dixon KL Dunmore JH Gimbrone Jr, MA Falb D Huszar D A role for Smad6 in development and homeostasis of the cardiovascular system.Nat Genet. 2000; 24: 171-174Crossref PubMed Scopus (394) Google Scholar These findings highlight the fundamental importance and potential diversity of mechanisms that are genetically programmed in animals to prevent ectopic calcification. One molecule that consistently co-localizes with ectopic calcification is OPN. OPN is an acidic phosphoprotein normally found in bone, teeth, kidney, and epithelial lining tissues. OPN′s expression is increased under conditions of injury and disease in many tissues, and it is closely associated with the calcified deposits found in numerous pathologies including atherosclerotic lesions, aortic stenosis, kidney stones, and tumors.27Giachelli CM Schwartz SM Liaw L Molecular and cellular biology of osteopontin.Trends Cardiovasc Med. 1995; 5: 88-95Abstract Full Text PDF PubMed Scopus (116) Google Scholar OPN is a multifunctional protein containing several structural domains including an integrin-binding (RGD) adhesive domain and aspartic acid-rich calcium-binding regions. In addition, OPN can be highly phosphorylated on serine and threonine residues. The combination of electronegative glutamic and aspartic acid residues, serine/threonine kinase substrate sites, and the putative calcium-binding motifs endow OPN with the ability to bind significant amounts of Ca2+ (50 mol calcium to 1 mol osteopontin).28Chen Y Bal BS Gorski JP Calcium and collagen binding properties of osteopontin, bone sialoprotein, and bone acidic glycoprotein-75 from bone.J Biol Chem. 1992; 276: 24871-24878Google Scholar These properties likely contribute to OPN′s ability to bind and regulate apatite crystal growth, the predominant calcium-phosphate mineral phase found in bones and teeth as well as at sites of ectopic calcification. Indeed, in vitro studies support a role for OPN as an inhibitor of apatite growth in both cell-free and cell-dependent in vitro models.13Wada T McKee MD Stietz S Giachelli CM Calcification of vascular smooth muscle cell cultures: inhibition by osteopontin.Circ Res. 1999; 84: 1-6Crossref PubMed Scopus (396) Google Scholar, 29Hunter GK Kyle CL Goldberg HA Modulation of crystal formation by bone phosphoproteins: structural specificity of the osteopontin-mediated inhibition of hydroxyapatite formation.Biochem J. 1994; 300: 723-728Crossref PubMed Scopus (363) Google Scholar, 30Boskey AL Maresca M Ullrich W Doty SB Butler WT Prince CW Osteopontin-hydroxyapatite interactions in vitro: inhibition of hydroxyapatite formation and growth in a gelatin-gel.Bone Miner. 1993; 22: 147-159Abstract Full Text PDF PubMed Scopus (378) Google Scholar, 31Jono S Peinado C Giachelli CM Phosphorylation of osteopontin is required for inhibition of vascular smooth muscle cell calcification.J Biol Chem. 2000; 275: 20197-20203Crossref PubMed Scopus (282) Google Scholar On the other hand, the co-localization of OPN with biomineralization in hard tissues, and its ability to bind and potentially orient calcium suggest that OPN might function to promote calcification in vivo.32Gorski JP Acidic phosphoproteins from bone matrix: a structural rationalization of their role in biomineralization.Calcif Tissue Int. 1992; 50: 391-396Crossref PubMed Scopus (205) Google Scholar To determine the role of OPN in ectopic calcification, we developed a model of ectopic calcification in OPN-replete and -deficient mice. Our studies indicate that OPN is a potent inhibitor of ectopic calcification, and suggest a novel function for OPN in controlling mineral-dissolving inflammatory cell function at sites of ectopic calcification. OPN mutant mice were generated in a 129/SvJ X Black Swiss background and genotyped as previously described by Liaw and colleagues.33Liaw L Birk DE Ballas CB Whitsitt JS Davidson JM Hogan BL Altered wound healing in mice lacking a functional osteopontin gene (spp1).J Clin Invest. 1998; 101: 1468-1478Crossref PubMed Google Scholar Animals were maintained in a specific pathogen-free environment, and fed standard chow and water ad libitum. Hybrid mutant mice were backcrossed onto the Black Swiss background for >7 generations. OPN homozygous wild-type (+/+), heterozygote (+/−), and homozygous null mice on the fixed Black Swiss background were used in these studies. Porcine aortic valve leaflets (kindly provided by St. Jude Medical, Inc., Minneapolis, MN) were aseptically dissected from porcine aortic valves obtained from the local abattoir, fixed in 0.6% glutaraldehyde in phosphate-buffered saline (PBS), pH 7.4, and stored in 0.6% glutaraldehyde in PBS, pH 7.4 until use. Four-mm2 biopsy punches of glutaraldehyde-fixed aortic valve leaflets (GFAVs) were prepared, washed extensively in sterile PBS, and subcutaneously implanted into the dorsal side of anesthetized 5- to 6-week-old, female OPN +/+, +/−, or −/− mice (two GFAVs per mouse). At the indicated times, mice were euthanized, and implants removed for either histological analysis or calcium quantitation. All protocols were approved by the animal use committee, University of Washington, Seattle, WA. Explants were fixed with methyl Carnoy's solution (3:1, methanol:acetic acid) and embedded in paraffin. Immunostaining was performed in 5-μm sections with goat anti-rat OPN antibody (OP199)34Liaw 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: 214-224Crossref PubMed Scopus (372) Google Scholar at 10 μg/ml, macrophage-specific rat anti-mouse BM-8 (Accurate Chemical & Scientific Corp., Westbury, NY) at 6 μg/ml, or sheep anti-human carbonic anhydrase II (CAII) (Biodesign International, Kennebunk, ME) at 5 μg/ml, and counterstained with methyl green as previously described.35Murry CE Giachelli CM Schwartz SM Vracko R Macrophages express osteopontin during repair of myocardial necrosis.Am J Pathol. 1994; 145: 1450-1462PubMed Google Scholar Calcium phosphate deposition was visualized by Alizarin Red S staining and von Kossa staining.36Meloan SN Puchtler H Valentine LS Alkaline and acid alizarin red S stains for alkali-soluble and alkali-insoluble calcium deposits.Arch Pathol. 1972; 93: 190-197PubMed Google Scholar Transmission electron microscopy was performed as described by Wada and colleagues.13Wada T McKee MD Stietz S Giachelli CM Calcification of vascular smooth muscle cell cultures: inhibition by osteopontin.Circ Res. 1999; 84: 1-6Crossref PubMed Scopus (396) Google Scholar Four to 8 OPN+/+, +/− and −/− mice were subcutaneously implanted with two GFAVs as described above. At each time point, implants and adherent host tissues were retrieved, fixed, and embedded as described above. For each recovered specimen (two per mouse), two histological sections were prepared at least 50 μm apart, for a total of four sections per animal. OPN, BM-8, and CAII staining in sections was performed as described above and quantitated using the ProImage Analysis Program. The area containing the GFAV implant and the associated foreign body capsule was circumscribed and the percentage of this area stained with each antibody determined and averaged to generate the percent area stained per implant per mouse. The percent area stained per implant per mouse for four to eight mice were averaged to obtain the average percent area stained for each genotype ± SE. Explants were freeze-dried to constant weight and decalcified with 0.6 N HCl overnight at room temperature. Calcium quantitation was performed by the o-cresolphthalein complexone as directed in the Sigma Diagnostic kit (Sigma, St. Louis, MO) and normalized to dry weight, as previously described.13Wada T McKee MD Stietz S Giachelli CM Calcification of vascular smooth muscle cell cultures: inhibition by osteopontin.Circ Res. 1999; 84: 1-6Crossref PubMed Scopus (396) Google Scholar Accuracy of the kit was confirmed by atomic absorption spectroscopy (kindly performed by Baxter Health Care Corporation, Irvine, CA). GFAV explants were freeze-dried then immersed in Universal Indicator Solution (Fisher Scientific, Pittsburgh, PA). The pH of the GFAV solution was determined by three different, blinded readers by comparing the color of this solution to the manufacturer's provided graded color scale. Accuracy of the color indicator solution was determined using an MI-408 needle pH microelectrode (Microelectrodes, Inc., Bedford, NH). Control experiments indicated that tissue lyophilization did not significantly alter pH of GFAV using either the dye or microelectrode measurement method (data not shown). HL-60 cells (American Type Culture Collection, Rockville, MD) were maintained in RPMI media (Life Technologies, Inc., Grand Island, NY) containing 5% fetal bovine serum, 100 U/ml penicillin (Life Technologies, Inc.), and 100 mg/ml streptomycin (Life Technologies, Inc.). Primary human monocytes were differentially isolated from human blood,37Beezhold H Personius C Fibronectin fragments stimulate tumor necrosis factor secretion by human monocytes.J Leukoc Biol. 1992; 51: 59-64Crossref PubMed Scopus (67) Google Scholar, 38Defife KM Yun JK Azeez A Stack S Ishihara K Nakabayashi N Colton E Anderson JM Adhesion and cytokine production by monocytes on poly(2-methacryloyloxyethyl phosphorylcholine-co-alkyl methacrylate)-coated polymers.J Biomed Mater Res. 1995; 29: 431-439Crossref PubMed Scopus (77) Google Scholar and maintained in RPMI containing 5% autologous human serum. Preparation of protein extracts, gel electrophoresis, and Western blotting were performed as previously described39Malyankar UM Almeida M Johnson RJ Pichler RH Giachelli CM Osteopontin regulation in cultured rat renal epithelial cells.Kidney Int. 1997; 51: 1766-1773Crossref PubMed Scopus (64) Google Scholar using 5 μg/ml of sheep anti-human CA II (Biodesign International, Kennebunk, ME). Data were analyzed for statistical significance using analysis of variance statistics with Fischer's protected least significance difference test. Calculations were performed using the computer program StatView version 4.11 (Abacus Concepts, Berkeley, CA). Four-mm2 pieces of GFAV were subcutaneously implanted into mice carrying either the OPN homozygous wild-type (OPN+/+), heterozygous (OPN+/−), or homozygous null (OPN−/−) alleles.33Liaw L Birk DE Ballas CB Whitsitt JS Davidson JM Hogan BL Altered wound healing in mice lacking a functional osteopontin gene (spp1).J Clin Invest. 1998; 101: 1468-1478Crossref PubMed Google Scholar At 7, 14, 30, and 90 days, implants were removed and assayed for mineral deposition, protein accumulation, and cell recruitment. In all animals, a robust foreign body response was induced by GFAV implantation. This host response was characterized by accumulation of macrophages, formation of giant cells, and fibrous encapsulation by days 14 and 30 (Figure 1; A to C). There were no obvious differences in quantity of the foreign body response between the different genotypes as determined by hematoxylin and eosin staining (Figure 1; A to C and data not shown), and this response was similar to the foreign body response typically observed after GFAV implantation in rats40Levy RJ Schoen FJ Levy JT Nelson AC Howard SL Oshry LJ Biologic determinants of dystrophic calcification and osteocalcin deposition in glutaraldehyde-preserved porcine aortic valve leaflets implanted subcutaneously in rats.Am J Pathol. 1983; 133: 143-155Google Scholar and rabbits.41Fishbein MC Levy RJ Ferrans VJ Dearden LC Nashef A Goodman AP Carpentier A Calcification of cardiac valve bioprostheses. Biochemical, histological and ultrastructural observations in a subcutaneous implantation model system.J Thorac Cardiovasc Surg. 1982; 83: 602-609PubMed Google Scholar However, differences in the quality of the foreign body response, especially in terms of macrophage number, was noted between genotypes as described below. As shown in Figure 1; D to I, OPN was highly expressed in host cells surrounding the GFAV implant in OPN+/+ mice at days 14 and 30. In contrast, OPN+/− mice showed little OPN staining at day 14 (Figure 1G), even when examined at high magnification (data not shown), but showed marked accumulation at day 30 (Figure 1H). Staining of adjacent sections with macrophage-specific marker, BM-8 (not shown), indicated that cells expressing OPN were predominantly of macrophage origin, and included a subset of mononuclear macrophages as well as multinucleated giant cells (Figure 1F). In addition, punctate OPN staining was also observed within the GFAV implant itself, especially in the 30-day OPN+/+ mice (Figure 1, E and F). This staining was co-localized with GFAV mineral deposits as determined by alizarin red staining of an adjacent section (data not shown, as well as the double-staining experiment shown in Figure 3, G and H, described in detail below). Levels of OPN associated with GFAV increased in OPN+/+ and OPN+/− mice with increasing time of implantation (Figure 1, Figure 2). As expected, no OPN was detected in OPN−/− mice at any time point examined (Figure 1, Figure 2).Figure 2Quantitation of OPN and mineral deposition associated with GFAV implants. A: OPN accumulation was quantitated using the Pro Image Analysis Program as described in Materials and Methods. Data are represented as percent area. B: Calcium deposition was quantitated from acid-hydrolyzed GFAV explants using Sigma's Calcium Diagnostic Kit and confirmed by atomic absorption spectroscopy. Numbers represent the average ± SE (n = 4 to 8). *, P < 0.005.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To determine whether OPN expression influenced the propensity of GFAVs to calcify in mice, implants were analyzed for calcium mineral deposition using histochemical and biochemical methods. The OPN null mutation affected both the extent and temporal appearance of calcification as measured by total calcium content (Figure 2B), alizarin red S staining (Figure 3), and von Kossa staining (not shown). None of the mice showed significant calcification at day 7. This did not appear to be because of a sensitivity problem, as we were unable to detect calcification using the sensitive fluorescent stain, calcein, in these samples (data not shown). Low levels of calcification were observed in GFAV implants at day 14 and day 30 in OPN+/+ mice (Figure 2B and Figure 3, A and B), and these levels were not statistically different. In contrast, significant calcification was observed in both OPN+/− and OPN−/− mice at day 14, at levels fourfold to fivefold greater than that observed at day 14 in OPN+/+ mice (Figure 2B and Figure 3, C and E). Finally, it did not appear that calcification was restricted to the OPN-deficient mice, as GFAVs implanted for 90 days in OPN wild-type mice showed considerable calcification, although still less than OPN null mice (OPN+/+ = 3000 ± 600 mmol Ca/L/g protein versus OPN+/− = 1900 ± 500 mmol Ca/L/g protein versus OPN−/− = 6900 ± 250 mmol Ca/L/g protein). Hence, these studies indicate that OPN plays a role in modulating GFAV calcification in vivo. As shown in Figure 3, calcium deposits visualized by alizarin red staining were deposited along the periphery as well as throughout the interior of the implant. Calcification occurred predominantly in foci within the GFAV leaflet. OPN, in addition to being localized to cells of the foreign body reaction, was found associated with these punctate-mineralized deposits within the GFAV implant as shown by double staining with antibody to OPN and alizarin red S for mineral (Figure 3, G and H). By transmission electron microscopy, the foci of mineralization within the GFAV implant were found to be associated predominantly with cell remnants including membranous debris resembling matrix vesicles (Figure 4). This localization is consistent with reports describing experimental and patient GFAV mineralization on remnant cell membranes and organelles.42Schoen FJ Tsao JW Levy RJ Calcification of bovine pericardium used in cardiac valve bioprostheses. Implications for the mechanisms of bioprosthetic tissue mineralization.Am J Pathol. 1986; 123: 134-145PubMed Google Scholar, 43Ferrans VJ Boyce SW Billingham ME Jones M Ishihara T Roberts WC Calcific deposits in porcine bioprostheses: structure and pathogenesis.Am J Cardiol. 1980; 46: 721-734Abstract Full Text PDF PubMed Scopus (191) Google Scholar, 44Valente M Bortolotti U Thiene G Ultrastructural substrates of dystrophic calcification in porcine bioprosthetic failure.Am J Pathol. 1985; 119: 12-21PubMed Google Scholar Taken together, these studies implicate OPN, most likely derived from infiltrating macrophages and giant cells, as an inhibitor of ectopic calcification in this model system. Co-localization of OPN protein with mineralization within the GFAV suggest that OPN binding to nascent sites of mineralization may be one mechanism by which OPN exerts its anti-calcific effect. Comparison of calcification with OPN levels in GFAV at day 14 after implantation indicated that OPN was an inhibitor of ectopic calcification. These findings are consistent with previous in vitro studies showing that OPN can bind to and block hydroxyapatite crystal growth.29Hunter GK Kyle CL Goldberg HA Modulation of crystal formation by bone phosphoproteins: structural specificity of the osteopontin-mediated inhibition of hydroxyapatite formation.Biochem J. 1994; 300: 723-728Crossref PubMed Scopus (363) Google Scholar However, examination of the OPN mutants at day 30 after implantation suggested that simple physical inhibition alone could not explain the inhibitory effect of OPN on ectopic calcification in this model system. By 30 days, significant levels of OPN accumulated in OPN+/− mice throughout the GFAV implant and was similar to levels observed in OPN+/+ mice (Figure 2A, and immunostaining data not shown). Strikingly, OPN accumulation in OPN+/− mice at day 30 was concurrent with a significant reduction in GFAV mineralization from the levels observed at day 14 (Figure 2B and Figure 3, C and D). If OPN acted simply as a crystal poison, one would expect little difference in the day 14 and day 30 calcification values after implant in the OPN+/− mice. Thus, these findings suggested that OPN additionally acted to promote regression of ectopic calcification. Because OPN binding alone cannot mediate the dissolution of calcium phosphate, we hypothesized that the observed mineral regression was the result of an OPN-regulated cell-mediated dissolution of mineral. The only known mechanisms capable of removing calcium phosphate crystals are phagocytosis and acidification.45Fallon MD Teitelbaum SL Kahn AJ Multinucleation enhances macrophage-mediated bone resorption.Lab Invest. 1983; 49: 159-164PubMed Google Scholar To address the former possibility, we examined macrophage accumulation in our model. In all genotyp
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