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B Ligand in Mönckeberg’s Sclerosis and Atherosclerosis
Departments of Internal Medicine and Cardiology (M.S., B.M.), Gastroenterology, Endocrinology and Metabolism (L.C.H.), and Pathology (P.J.B.), Philipps University, D-35033 Marburg, Germany; and Departments of Clinical Chemistry (N.A.-F., N.K.), Pathology (F.E.F.), and Biochemistry (K.T.P.), Justus Liebig University, D-35390 Giessen, Germany
Address all correspondence and requests for reprints to: Dr. Michael Schoppet, Department of Internal Medicine and Cardiology, Philipps University, Baldingerstrasse, D-35033 Marburg, Germany. E-mail: schoppet{at}mailer.uni-marburg.de.
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Abstract |
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B ligand are essential modulators of bone homeostasis and may be involved in the process of vascular calcification. In this study we investigated arteries from patients with Mönckeberg’s sclerosis and atherosclerosis. Apoptosis, which precedes vascular calcification in vitro, was assessed by an in situ ligation assay and was localized to the medial layer of arteries (Mönckeberg’s sclerosis) and the neointima (atherosclerosis). Immunohistochemistry and in situ hybridization revealed OPG immunoreactivity and mRNA expression surrounding calcified areas in the medial layer (Mönckeberg’s sclerosis), whereas OPG was mainly expressed adjacent to calcified neointimal lesions (atherosclerosis). Receptor activator of nuclear factor-B ligand protein and mRNA were barely or not detectable. Of note, TNF-related apoptosis-inducing ligand, an inducer of apoptosis that is also blocked by OPG, displayed a similar spatial distribution as OPG. In summary, we demonstrate enhanced apoptosis adjacent to vascular calcification, and the concurrent expression of regulators of apoptosis and osteoclastic differentiation, TNF-related apoptosis-inducing ligand and OPG, suggesting their involvement in the pathogenesis of vascular calcification. |
Introduction |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Osteoprotegerin (OPG) and receptor activator of nuclear factor-B (RANK) ligand (RANKL) are novel members of the TNF ligand and receptor signaling family, respectively, that constitute the final effectors of osteoclast function and therefore represent essential regulators of bone mass and bone homeostasis (5). RANKL is expressed by osteoblastic lineage cells and T lymphocytes and acts by binding to its physiological receptor, RANK, which is expressed on osteoclasts (6). Ligation of the receptor stimulates all aspects of osteoclast function, such as differentiation, maturation, fusion, survival, and activity. By contrast, OPG is produced by a variety of tissues, including the cardiovascular system, and acts as a soluble decoy receptor by neutralizing RANKL. OPG also represents a receptor for the cytotoxic ligand TNF-related apoptosis-inducing ligand (TRAIL) (7), a potent activator of apoptosis after binding to death domain homolog-containing receptors (8).
OPG-deficient mice exhibit a phenotype of osteoporosis, and two thirds of the animals also display medial calcifications of the aorta and the renal arteries (9) that can be successfully treated by an OPG transgene delivered during midgestation (10). In addition, RANKL and RANK transcripts could be demonstrated in calcified arterial lesions of OPG-deficient mice, but not wild-type mice (10). We hypothesized that regulators of bone metabolism, such as OPG and RANKL, may be expressed in the vicinity of calcified regions in Mönckeberg’s sclerosis and atherosclerosis, and that apoptosis may be involved in the process of calcification.
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Patients and Methods |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Mönckeberg’s medial sclerosis was diagnosed according to defined histological criteria (11, 12), including widespread medial calcifications and partially ossification. Specimens were termed atherosclerotic if only neointimal lesions were found; these specimens showed advanced atherosclerotic lesion types and calcifications in the neointima (types IV–VIa) (13). All specimens were examined by two experienced investigators. Specimens of Mönckeberg’s sclerosis were obtained from six patients immediately after lower limb amputation or at autopsy. Four of these specimens had only medial calcifications and minor or no intimal thickening; two specimens had medial and neointimal calcifications. Atherosclerotic tissues were obtained from two carotid artery specimens collected during carotid eversion endarterectomy of patients with carotid stenosis and from four femoral and iliac artery specimens collected during lower limb bypass surgery from patients with femoral artery stenosis. Two control specimens were obtained from normal femoral arteries, two controls derived from normal internal mammary arteries. Control arteries showed no histopathological signs of neointima development or medial sclerosis. The study was approved by the institutional review board of Justus Liebig University Giessen. The tissue samples used were waste material that would have otherwise been discarded after the surgical procedure.
Tissue embedding, processing, and conventional staining
All specimens were fixed for 12 h with 4% formaldehyde/PBS from 2–5 h after surgical excision or at autopsy 8–12 h postmortem and then paraffin-embedded. Specimens were cut in serial sections of 6 µm without prior decalcification to avoid denaturation of protein or RNA (14). Tissue integrity was demonstrated by hematoxylin and eosin staining of specimens. Sections were mounted on slides coated with aminopropyltriethoxysilane (Sigma-Aldrich Corp., Taufkirchen, Germany). After deparaffinization and rehydration, serial sections of all specimens were subjected to in situ ligation assay (ISL), immunohistochemistry, and in situ hybridization. Additional serial sections of all specimens were subjected to von Kossa staining and kernechtrot (4-amino-9,10-dihydro-1,3-dihydroxy-9,10-dioxo-2-anthracen sulfonate) counterstaining to demonstrate calcifications as well as to conventional hematoxylin and eosin staining for histopathological analysis. Five sites per specimen, 50 µm distance from each other, were investigated; all analyses were repeated four to six times. All specimens were analyzed by two experienced investigators by assessing semiquantitatively the average staining intensity and the typical staining pattern of the whole section circumference.
ISL
The ISL (or T4 DNA ligation assay) (15) was employed as described previously (16). It demonstrates the apoptosis-specific form of DNA fragmentation, i.e. 3' single base overhang, double-stranded DNA breaks. Briefly, a 3' single base overhang DNA probe was prepared by PCR using primers 5'-TCACTAAAGGGAACAAAAGC-3' and 5'-ACTATAGGGCGAATTGGAG-3', complementary to the T3 and T7 promoters in the multicloning site of plasmid Bluescript II KS+/– (Stratagene, La Jolla, CA). The PCR product was separated by electrophoresis on 2% agarose gels (Pharmacia Biotech, Erlangen, Germany) in Tris-acetate-EDTA buffer, stained with ethidium bromide, excised from the gel, and purified with the Qiaex II gel extraction kit (Qiagen, Hilden, Germany).
After deparaffinization and rehydration, slides were treated with proteinase K (20 µg/ml) for 10 min and postfixed in 4% methanol-free formaldehyde/PBS. For the positive control, a specimen was preincubated with Tris buffer (pH 7.9) containing 1 µg/ml deoxyribonuclease I (DNase I) for 10 min and processed separately thereafter. All slides were then incubated with a mixture of 50 mM Tris-HCl (pH 7.8), 10 mM MgCl2, 10 mM dithiothreitol, 1 mM ATP, 25 µg/ml BSA, 15% polyethylene glycol, 3 µg/ml digoxigenin (DIG)-labeled DNA fragment, and 32 U/ml DNA T4 ligase (Sigma-Aldrich Corp.) in a humidified chamber for 1 h at room temperature. For the negative control, either the DIG-labeled DNA fragment or DNA T4 ligase was omitted in the incubation mixture. The slides were then washed in 60 C deionized water. Slides were preblocked with normal sheep serum and incubated with sheep anti-DIG Fab antibody conjugated to alkaline phosphatase (Roche, Mannheim, Germany; 1:500) for 30 min. Color development was performed with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate [NBT/BCIP; 100 mM Tris (pH 9.5), 100 mM NaCl, 50 mM MgCl2, 0.38 mg/ml NBT, 0.18 mg/ml BCIP, and 1 mM levamisole (Sigma-Aldrich Corp.)] for 70 min at room temperature. The reaction was terminated by deionized water, ISL and conventionally stained serial sections were examined for the scoring of cells undergoing apoptosis by light microscopy at 400-fold magnification. The apoptotic index was determined by counting the number of ISL-positive nuclei on a total of 500 cell nuclei/slide and calculating the mean as a percentage of all slides.
Immunohistochemistry
Slides were preincubated with 5% normal goat or rabbit serum, then incubated with antibodies against OPG (two antihuman OPG mouse monoclonal antibodies, clones 69146.11 and 69127.11; R&D Systems, Minneapolis, MN; 12.5 µg/ml), RANKL (antihuman RANKL mouse monoclonal antibody, clone 70525.11; R&D Systems; 25 µg/ml), TRAIL (antihuman TRAIL goat polyclonal antibody, K-18; Santa Cruz Biotechnology, Santa Cruz, CA; 10 µg/ml; and antihuman TRAIL mouse monoclonal antibody, clone 75411.11, R&D Systems; 50 µg/ml), human smooth muscle 
-actin (mouse monoclonal antibody, clone 1A4; Dakocytomation, Hamburg, Germany; 1.5 µg/ml), human macrophage antigen CD68 (mouse monoclonal antibody, clone KP1; Dakocytomation; 4.3 µg/ml), human T lymphocyte antigen CD3 (mouse monoclonal antibody, clone T3-4B5; Dakocytomation; 2.3 µg/ml), and human B lymphocyte antigen CD21 (mouse monoclonal antibody, clone 1F8; Dakocytomation; 3.5 µg/ml). Negative controls were conducted by substituting the specific primary antibody with an irrelevant antibody. Subsequently, specimens were incubated with biotinylated goat antimouse antibody (Sigma-Aldrich Corp.) or biotinylated rabbit antigoat antibody (Dakocytomation), streptavidin-alkaline phosphatase-conjugate, Fast Red stain (all reagents from Sigma-Aldrich Corp.), and Mayer’s hemalum counterstaining. Slides were covered with glycerol-gelatin and coverslips and finally examined by light microscopy.
RNA preparation, RT, and cDNA synthesis
Total cellular RNA was extracted using a modified single step isolation protocol based on established methods (17). For the preparation of RNA to be subjected to cDNA transcription, we employed a vascular smooth muscle cell line for OPG and TRAIL cRNA probes and an osteosarcoma cell line, MG-63, for RANKL cRNA probe (purchased from American Type Culture Collection, Manassas, VA). Cultures of human vascular smooth muscle cells were established from saphenous veins, characterized, and grown exactly as described previously (16). Cells were frozen in liquid nitrogen, homogenized, and lysed in 500 µl TRIzol (Sigma-Aldrich Corp.). RNA concentrations were measured by spectrophotometry and calculated on the basis of OD260 values.
Genomic DNA contamination was eliminated by a protocol adapted from previously described methods (18, 19). The solution containing 1 µg/µl RNA was incubated for 30 min at 37 C with 0.09 U/µl ribonuclease (RNase)-free deoxyribonuclease (Promega Corp., Mannheim, Germany), 40 U RNase inhibitors (RNasin, Promega Corp.), and 0.66 mM MnCl2 in 10 mM Tris-HCl (pH 7.8). Before heat inactivation of DNase at 75 C for 5 min, 3 mM EDTA and 0.15 mM dithiothreitol were added to avoid subsequent DNase reactivation (18). Afterward, the reaction mixture was cooled to 42 C. RT was performed at 42 C for 60 min after the addition of 8 U avian myeloblastosis virus reverse transcriptase, 10.55 mM dithiothreitol, 11.34 mM MgCl2, 0.57 µg oligo(deoxythymidine) primer, 0.5 mM deoxy-NTP, and 32 U RNase inhibitors (RT system from Promega Corp.) in a total volume of 21 µl. The reaction was terminated by heat inactivation of reverse transcriptase at 90 C for 5 min. RT products were diluted to 10 ng cDNA/µl in sterile distilled water for subsequent PCR.
The primer pairs (MWG Biotech, Ebersberg, Germany) used for PCR amplification of the cloned sequence that was finally employed for cRNA probe preparation are shown in Table 1
. PCR was performed in a final volume of 20 µl containing 20 ng cDNA of a defined specimen, 0.2 mM deoxy-NTP-Mix (Pharmacia Biotech), 0.5 U Taq DNA polymerase (Eppendorf, Hamburg, Germany), 10 pmol of each primer, 50 mM KCl in 10 mM Tris-HCl (pH 8.3), and 1.5 mM MgCl2. Individual PCR protocols were employed for every PCR target. PCR products were electrophoresed in 2% agarose gels (Pharmacia Biotech). Specific products were identified after ethidium bromide staining under UV light, excised from the gel, and extracted with Qiaex II agarose gel extraction kit (Qiagen).
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OPG, RANKL, and TRAIL PCR products were subcloned in pGEM-T (Promega Corp.). The plasmids were transformed in the XL1-Blue Escherichia coli strain (Stratagene, La Jolla, CA) and extracted by column purification according to the manufacturer’s instructions (Qiagen). The concentration and purity of the extracted plasmids were assessed on the basis of OD260 values. For cRNA probe synthesis, the vectors containing the insert were digested with NcoI or NotI (New England Biolabs, Beverly, MA) for the production of sense cRNA (NcoI) or antisense cRNA (NotI). In vitro transcription of DIG-labeled cRNA was performed using the DIG RNA labeling mix and RNA polymerases T7 and SP6 (all reagents from Roche). After precipitation with 8.0 mM LiCl and 100% ethanol extraction, the dried pellet was reconstituted in 100 µl RNase-free water. The concentrations of the DIG cRNA probes were estimated by semiquantitative dot blots.
In situ hybridization
After digestion with 20 µg/ml PBS proteinase K (Promega Corp.) for 25 min at 37 C, sections were incubated in 0.2% glycine and then in 20% acetic acid. The sections were postfixed in 4% paraformaldehyde for 10 min and prehybridized in 20% glycerol for 30 min. Sections were then incubated with the DIG-labeled antisense cRNA probes. All cRNA probes were used at a dilution of 1:100 in hybridization buffer containing 50% deionized formamide, 10% dextran sulfate, 0.3 mM sodium chloride/30 mM sodium citrate (2x SSC buffer), 0.2 mg/ml BSA/0.2 mg/ml Ficoll 400/0.2 mg/ml polyvinylpyrrolidone (Denhardt’s solution), 10 µg/ml salmon sperm DNA (Sigma-Aldrich Corp.), and 10 µg/ml yeast tRNA (Sigma-Aldrich Corp.). Hybridization was performed overnight at 37 C in a humidified chamber containing 50% formamide/2x SSC buffer.
Posthybridization washes were preceded by multiple washes in 4x SSC for 15 min at room temperature, slides were then incubated in 4x SSC containing 300 µg/ml RNase A and 10 U/ml RNase T1 (RNase A+T1 mix; BD Pharmingen, San Diego, CA) for 30 min at 37 C. The following washes were carried out: 4x SSC four times for 5 min each time at 37 C, 2x SSC for 15 min at 60 C, 0.2x SSC for 15 min at 42 C, 0.1x SSC for 5 min at room temperature, and 2x SSC for 5 min at room temperature. Slides were preincubated with 5% normal sheep serum and 3% BSA, then incubated overnight at 4 C with a sheep anti-DIG Fab antibody conjugated to alkaline phosphatase (Roche; 1:500). Staining was performed with NBT/BCIP and 1 mM levamisole (Sigma-Aldrich Corp.). Finally, sections were covered with glycerol-gelatin and coverslips. For every hybridization procedure, control incubations of various sections were performed using DIG-labeled cRNA sense probes. In addition, control incubations were performed by substituting the antisense cRNA probes with DIG to rule out unspecific binding.
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Results |
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Specimens of normal vessels (n = 4) were compared with Mönckeberg’s sclerosis (n = 6) and with atherosclerotic tissue samples (n = 6) by conventional hematoxylin and eosin and von Kossa staining. Calcification was detected in the medial layer in Mönckeberg’s sclerosis and in the neointimal layer in atherosclerosis, but not in normal control vessels (Fig. 1). Analysis of vascular lesions by characteristic markers, such as smooth muscle -actin, macrophage antigen CD68, T lymphocyte antigen CD3, and B lymphocyte antigen CD21, revealed that the medial tissue surrounding calcified lesions in Mönckeberg’s sclerosis contained only smooth muscle cells, whereas macrophages and lymphocytes were absent. By contrast, in atherosclerosis, the neointima exhibited foam cell accumulations composed of smooth muscle cells and macrophages, whereas lymphocytes were not detected (data not shown). Analysis of patient data for different variables, such as age, sex, the vascular region investigated, and atherosclerosis risk factors, revealed the well known higher incidence of diabetes and chronic renal failure in patients with Mönckeberg’s sclerosis (Table 2A) (4, 12). Comparison of clinical and histological characteristics of atherosclerosis and Mönckeberg’s sclerosis patients revealed no association of a single clinical parameter or the anatomical location (Table 2A) with histological findings (Table 2B). The results of the different histomorphological methods employed (von Kossa staining, ISL, immunohistochemistry, and in situ hybridization) were consistent within the same group (Table 2B). OPG, RANKL, and TRAIL protein and mRNA expression patterns were similar for atherosclerosis and Mönckeberg’s sclerosis patients, in that OPG and TRAIL were predominantly adjacent to calcifications and areas of apoptosis throughout the whole circumference of all specimens. Control experiments demonstrated that no unspecific binding of DIG to hydroxylapatite occurred (data not shown).
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Apoptosis was assessed using an ISL to demonstrate DNA fragmentation, a feature of programmed cell death. In Mönckeberg’ sclerosis, apoptotic cells (ISL-positive cells) were detected adjacent to calcified plaques and minor calcified lesions in the medial layer, which was composed of smooth muscle cells (Fig. 2F). The number of apoptotic cells in these areas ranged from 2–8.5% (mean, 4%).
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), whereas RANKL protein was scarcely scattered in these areas (data not shown). The results of OPG immunoreactivity were observed independently by two different monoclonal antibodies directed against OPG (data for clone 69146.11 are not shown).
TRAIL, an inducer of apoptosis in susceptible cells, exhibited a similar spatial staining pattern as OPG in the vicinity of apoptotic areas, suggesting the involvement of TRAIL in the apoptotic processes associated with vascular calcification (Fig. 2B). Of interest, a polyclonal antibody directed against the carboxyl terminus of the TRAIL molecule showed strong staining, whereas a monoclonal antibody directed against biologically active TRAIL showed only weak staining, probably due to TRAIL complexed to antigen (data not shown). Protein detection of OPG and TRAIL was paralleled by mRNA expression of the respective cytokines in Mönckeberg’s sclerosis (Fig. 2, D and E), whereas RANKL mRNA could not be detected (data not shown). Thus, OPG and TRAIL immunoreactivity and mRNA expression showed a similar localization, suggesting that OPG and TRAIL were produced by vessel wall-residing cells.
Atherosclerotic specimens
In contrast to Mönckeberg’s sclerosis samples, atherosclerotic specimens exhibited apoptosis mainly in the neointima, with apoptotic cells surrounding foam cell accumulations and calcified lesions (Fig. 3F), and the number of apoptotic cells ranged from 1.5–7% (mean, 3%). As in Mönckeberg’s sclerosis, OPG and TRAIL immunoreactivities were distributed predominantly around calcifications in the neointima in atherosclerosis and therefore in the same areas where apoptosis could be detected (Fig. 3, A and B), whereas only faint RANKL staining was seen (data not shown). As before, OPG and TRAIL mRNA were concomitantly detected in the neointima (Fig. 3, D and E), but RANKL mRNA was not found (data not shown).
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Tissue samples of normal control vessels were ISL negative and did not show apoptosis (Fig. 4F). No immunostaining for TRAIL (Fig. 4B) or RANKL (data not shown) was seen, whereas faint OPG protein expression by endothelial cells was detected (Fig. 4A). However, the low OPG protein expression was not paralleled by OPG mRNA expression (Fig. 4D), and neither TRAIL (Fig. 4E) nor RANKL (data not shown) mRNA was detected. Moreover, there were no differences between normal femoral or internal mammary arteries.
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Discussion |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Targeted deletion of OPG has been shown to result in both osteoporosis and calcifications of the medial layers of the aorta and renal arteries, affecting two thirds of OPG–/– mice (9). A phenotype of medial calcification is also seen in a number of mouse gene knockout studies, such as deficiencies of matrix GLA protein, a vitamin K-dependent circulating and bone-associated protein that can bind calcium in soft tissues (20); SMAD6, an intracellular mediator of bone morphogenetic protein signaling (21); and Klotho, a gene associated with aging (22). Furthermore, mice challenged with supraphysiological doses of vitamin D or warfarin treatment exhibit medial calcifications, a phenotype that can be prevented by inhibitors of osteoclasts, including OPG, bisphosphonates, and a selective inhibitor of the osteoclastic V-H+-adenosine triphosphatase, (23, 24, 25), providing further evidence for a mechanistic link between vascular calcification and bone metabolism.
Apoptosis has been described to precede calcification in vitro, and formation of apoptotic bodies may act as a nucleation site for calcium crystal formation in the vessel wall (1). The detection of TRAIL, a cytokine known to cause apoptosis in a variety of cell lines by ligating one of its several death receptors (8, 26), points to its role in initiating the apoptotic process, yet its role in vivo has not been fully elucidated. TRAIL is expressed by vascular smooth muscle cells and may promote cell death in an autocrine or paracrine fashion when induced in conditions such as diabetes, hyperlipidemia, chronic renal failure, or atherosclerosis (27). Of interest, OPG has been described to bind TRAIL and thereby to inhibit its cytotoxic capacity in an in vitro model (7). OPG can be detected in the vascular wall (5), and endothelial cells and vascular smooth muscle cells have been reported as its cellular source in the vessel wall (28, 29). The concomitant detection of OPG and TRAIL in apoptotic areas may indicate that increased OPG production at these sites represents a counterregulatory mechanism to limit apoptosis and thereby the initiation of calcification.
OPG and RANKL immunoreactivities in humans have been detected in the normal vascular wall and in early atherosclerotic lesions, whereas in advanced calcified lesions, OPG expression was found in the borders of calcified structures, and RANKL expression was found adjacent to calcium deposits (30). Of note, increased OPG serum levels have been found to be associated with the presence and severity of coronary artery disease (31, 32), which may indicate an insufficient counterregulatory mechanism in light of the protective role of OPG in the vascular system. However, a recent study suggests that OPG and matrix GLA protein may be down-regulated in calcified arteries (33), whereas the expression of transcription factors that regulate mineralization and osseous differentiation programs (34) and the up-regulation of osteogenic genes, such as alkaline phosphatase, bone sialoprotein, or bone GLA protein, in vascular smooth muscle cells in the course of medial calcification has been demonstrated (14). Of interest, dexamethasone enhances differentiation of vascular smooth muscle cells toward an osteoblastic phenotype in vitro (35) and concurrently inhibits OPG expression by osteoblasts (36).
In summary, we have found apoptosis around calcified regions of Mönckeberg’s sclerosis and atherosclerotic arteries, with the concurrent detection of OPG and TRAIL in these areas. Apoptotic cells may serve as a matrix for calcification, with TRAIL-induced apoptosis being an initiator of this process (Fig. 5). The OPG/RANKL system has been shown to regulate mineralization processes, and OPG may act as a potential counterregulatory and antiapoptotic factor. Further work is necessary to elucidate the exact mechanisms that lead to vascular calcification to develop preventive strategies.
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Acknowledgments |
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Footnotes |
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M.S. and N.A.-F. contributed equally to this work.
Abbreviations: DIG, Digoxigenin; DNase, deoxyribonuclease; EC, endothelial cells; ISL, in situ ligation assay; NBT/BCIP, nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate; OPG, osteoprotegerin; RANK, receptor activator of nuclear factor-B; RANKL, RANK ligand; RNase, ribonuclease; SSC, sodium citrate buffer; TRAIL, TNF-related apoptosis-inducing ligand; VSMC, vascular smooth muscle cells.
Received August 18, 2003.
Accepted May 14, 2004.
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B and osteoprotegerin expression by human microvascular endothelial cells, regulation by inflammatory cytokines, and role in human osteoclastogenesis. J Biol Chem 276:20659–20672This article has been cited by other articles:
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B. A. Mosheimer, N. C. Kaneider, C. Feistritzer, A. M. Djanani, D. H. Sturn, J. R. Patsch, and C. J. Wiedermann Syndecan-1 Is Involved in Osteoprotegerin-Induced Chemotaxis in Human Peripheral Blood Monocytes J. Clin. Endocrinol. Metab., May 1, 2005; 90(5): 2964 - 2971. [Abstract] [Full Text] [PDF]
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P. Collin-Osdoby Regulation of Vascular Calcification by Osteoclast Regulatory Factors RANKL and Osteoprotegerin Circ. Res., November 26, 2004; 95(11): 1046 - 1057. [Abstract] [Full Text] [PDF]
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