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Glycation endproducts in osteoporosis — Is there a pathophysiologic importance?
Clinica Chimica Acta 371 (2006) 32 – 36 www.elsevier.com/locate/clinchim
Invited critical review
Glycation endproducts in osteoporosis — Is there a pathophysiologic importance? Gert E. Hein Division of Rheumatology and Osteology, Department of Internal Medicine III, Friedrich-Schiller-University of Jena, Germany Received 22 December 2005; received in revised form 16 February 2006; accepted 6 March 2006 Available online 13 June 2006
Abstract Advanced glycation endproducts (AGEs) are chemical modifications of proteins by carbohydrates including those metabolic intermediates formed during the Maillard reaction. The generation of AGEs is an inevitable process in vivo. AGEs constitute a heterogeneous class of compounds characterized by brown color, fluorescence and a tendency to polymerize. These unique compounds are specifically recognized by AGE receptors (RAGE) present on different cell types. A remarkable feature of AGE-mediated cross-linked proteins is decreased solubility and resistance to proteolytic digestion. This effect results in altered biomechanical properties in affected tissues including increased stiffness and rigidity. The AGE–RAGE interaction additionally induces activation of nuclear factor kB (NF-kB) in RAGE bearing cells (e.g., cells participating in bone turnover). This interaction results e.g. in increased expression of cytokines, growth factors and adhesion molecules. Recent findings provide important evidence that bone proteins are also affected by AGE modification. Investigations conducted by other groups, as well as ours, support the hypothesis that bone protein glycation influences osteoclasts (bone resorption) and osteoblasts (bone formation). © 2006 Elsevier B.V. All rights reserved. Keywords: Osteoporosis; Bone remodelling; AGE-modification of proteins; Osteoblast; Osteoclast
Contents 1. Osteoporosis and bone remodeling 2. Advanced glycation endproducts . 3. AGEs and bone, what is known? . References . . . . . . . . . . . . . . .
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1. Osteoporosis and bone remodeling According to the 2001 NIH consensus conference, osteoporosis is defined as a systemic bone disease characterized by insufficient bone strength, which predisposes to a higher risk of bone fracture [1]. The socioeconomic importance of osteoporosis is already high and is estimated to significantly increase in the future due to the changing age pyramid. It should be noted, however, that osteoporosis influences not only quality of life, but also life expectancy [2].
E-mail address: [email protected]. 0009-8981/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2006.03.017
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Osteoporosis is divided into primary (without a definitive basic disease) and secondary (basic disease leading to osteoporosis) forms. Bone strength is not only determined by quantitative parameters of bone such as size, mass and mineral density, but also by the quality of the material and structure of the bone tissue. Such qualitative parameters of bone tissue include its micro-architecture and geometry, extent of trabecular cross-linking, size and orientation of hydroxyapatite crystals, and integrity of collagen network [3]. It is generally accepted that osteoporosis is caused by disturbed bone remodeling. By the second half of the 19th century, the Austrian physician Victor von Ebner recognized that adult bone does not remain unchanged, but is in fact continually renewed in a
G.E. Hein / Clinica Chimica Acta 371 (2006) 32–36
33
coupled process of formation and resorption [4]. It is now known that adult bone requires permanent renewal for maintaining quality and optimum strength. About 10% of bone mass is replaced by this process every year. In contrast to cortical bone, trabecular bone is modified and replaced more quickly due to its continual structural adaptation and reorganization in response to new loads [5]. Bone reorganization starts with the recruitment and activation of osteoclasts. These cells resorb a defined bone volume (approximately 0.025 mm3) in a “basic multicellular unit” (BMU) resulting in a “resorption cavity.” By means of a coupling mechanism, osteoblasts are later attracted to fill the resorption lacuna with osteoid, i.e., unmineralized matrix. Primary and secondary mineralization later complete remodeling. Despite the progress in our understanding of bone biology, the precise mechanism(s) that initiate the remodeling process remain unclear to date. For example, what factors induce the movement of the osteoclasts to a defined place and how are they activated? A plausible hypothesis is that chemical changes in the bone protein result in biophysical/ biomechanical alterations of the bone matrix and are thereby responsible for the initiation of the remodeling process. Modifications of osseous proteins could result in functional alterations of osteoclasts and osteoblasts and be of significant pathophysiologic importance for the development of osteoporosis, as well as other metabolic bone diseases. A possible mechanism by which bone protein may be altered is via glycation, or in the generation and accumulation of advanced glycation endproducts (AGEs).
AGEs are able to alter physiological processes in vivo by different mechanisms:
2. Advanced glycation endproducts
Collagen molecules in bone have an exceptionally long lifetime, making them susceptible to AGE modification. Thus it may be hypothesized that the generation and accumulation of AGEs in bone tissue could be a reason for deterioration of bone tissue quality (e.g. increased stiffness). This may also be a cause of disturbed cell proliferation and development of functional alterations of cells. On this way AGEs could especially contribute to disturbed bone remodeling via osteoclasts and osteoblasts activation of NF-kB, induced by the binding of AGEs or the known pro-inflammatory molecules on the more intensively expressed RAGE. In our studies we found significantly higher levels of the AGE pentosidine as well as of carboxymethyl lysine (CML) in the serum of patients with osteoporosis [28]. In contrast, we were only able to find a significantly positive correlation between AGE level and age in the group of healthy controls. Thus it seems that osteoporosis itself is connected with a more intensified generation of AGEs. Odetti et al. [29] additionally evaluated the concentration of pentosidine in cortical and trabecular bone. Interestingly, the concentration of cortical pentosidine showed not only a significant exponential increase with age but was also negatively correlated to bone density and mineralization (Singh score). Despite these findings, it was unclear if increased AGE generation was a causal phenomenon of osteoporosis or an epiphenomenon. In order to further investigate this issue, AGE levels were evaluated relative to histomorphometric findings in these patients. A significant positive correlation between serum pentosidine and specific cellular parameters of the bone turnover
Advanced glycation endproducts (AGEs) are chemical modifications of proteins by sugar derived aldehyde groups generated during the Maillard reaction. However, it has recently become evident that ascorbic acid [6], reactive carbonyl intermediates such as 3-deoxyglucosone (3-DG), as well as products of sugar auto-oxidation (arabinose, glyoxal) and lipid peroxidation (malondialdehyde) are also precursors that cause chemical modifications of proteins. The formation of reactive carbonyl compounds resulting from auto-oxidation or lipid peroxidation is closely related to oxidative processes [7–9]. The generation of AGEs is an inevitable process in vivo. These compounds constitute a heterogeneous class of molecules characterized by a brown color, fluorescence, and a tendency to polymerize. The unique structures are specifically recognized by AGE-binding proteins or receptors (RAGEs). Today there are several known AGE-binding proteins with different characteristics and qualities. These include AGE-R1, AGE-R2, AGE-R3, ScR-II, CD36 and RAGE. The latter three are multi-ligand receptors of the immunoglobulin superfamily [10]. These receptors have already been identified in a variety of cell types [11–14] (see also Figs. 1A, B). Oxidative stress accelerates the generation of AGEs, and in turn the intracellular accumulation of AGEs accelerates oxidative stress. It can be appreciated that proteins with long half-lives are particularly susceptible to chemical modification such as the formation of AGEs.
1. AGE-mediated cross-linked proteins have decreased solubility and high resistance to proteolytic digestion, with the consequence of altered physicochemical and mechanical properties of tissue components (e.g. increased stiffness of collagen) [15]. 2. The AGE-mediated additional cross-linking (in addition to the physiologically cross-linking by pyridinium compounds) may alter the secondary and tertiary structures of the proteins and thereby generate new immunogenic epitopes [16,17]. 3. The binding of AGEs on the RAGE induces activation of the nuclear factor kB (NFkB) of the RAGE bearing cells, resulting in increased expressions of, e.g. cytokines, growth factors and adhesion molecules [18–20]. 4. The accumulation of AGEs intensifies the expression of RAGE. RAGE is not only a receptor for AGEs but also for other pro-inflammatory molecules such as HMBG 1 and the S 100/calgranulin family, possibly increasing inflammatory reactions by this indirect way [21–23]. 5. AGEs may alter the gene expression, and function, of multipotential stem cells (MSC) [24]. AGEs are now generally accepted as playing a pivotal pathophysiological role in several chronic diseases [25–27]. 3. AGEs and bone, what is known?
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G.E. Hein / Clinica Chimica Acta 371 (2006) 32–36
general, a different staining pattern for both AGEs was observed in the bone specimens. Whereas imidazolon staining was much more intense in the bone tissue than in the marrow, CML showed a stronger immunoreactivity in the bone marrow compared to tissue (see Figs. 2A, B). There was no significant correlation between staining intensity of either AGE and cellular markers of bone resorption (i.e., ES/BS = eroded surface as percentage of trabecular surface or OcS/BS = osteoclast covered surface as percentage of the trabecular surface containing resorptive cavities). There was also no correlation to the mineral apposition rate (MAR). However, a significant negative correlation was found between the intensity of AGE immunoreactivity and the cellular bone formation marker ObS/BS (percentage of osteoblast covered bone surface). These results are in agreement with those of Katayama et al. [35] who reported that AGE modified collagen is able to inhibit phenotypic expression of osteoblasts. Thus it seems that the glycation of bone proteins, an inevitable process in vivo and occurring more intensely in osteoporotic bone, is able to influence both bone remodeling processes, osteoclastic bone resorption and osteoblastic bone formation. Whether this results in accelerated bone resorption (with development of a “high turnover osteoporosis”), or decreased bone formation (with development of a “low turnover osteoporosis”), may depend on the specificity of the generated glycation endproduct.
Fig. 1. (A) Myeloid cells ( ) are stained with Anti-RAGE antibodies (redbrown color), 2 Megakaryocytes ( ) are RAGE negative. (B) 2 Osteoclasts (large cells with multiple nuclei) ( ) RAGE positive.
was found [28]. These correlations provide evidence that, initially, pentosidine contributes to the increased osteoclast formation and/or activity and subsequent intensified osteoblast recruitment. Interestingly, over ten years ago Miyata et al. [30] showed that AGEs were able to enhance osteoclast-induced bone resorption in cultured mouse bone cells and in rats implanted subcutaneously with devitalized bone particles. Although the underlying mechanisms were unclear, addition of osteoclast inhibitors, such as calcitonin, into the culture medium neutralized the effects of AGEs. The process of bone formation may be influenced and altered by AGE modification of bone proteins. McCarthy et al. demonstrated that AGE modified collagen was able to regulate proliferation and differentiation of osteoblastic cells depending on developmental stage [31,32]. The addition of AGE–BSA to cultures of human osteoblast-like cells resulted in a significantly reduced synthesis of osteocalcin and collagen I. Receptors of AGE (RAGE) have been identified in these cells [33]. In our own recent investigation [34], we looked for the intensity of AGE immunoreactivity of osseous proteins in bone specimens taken from osteoporotic patients and for correlation to parameters of bone remodelling based on histomorphometric findings. In all the investigated bone particles (from the iliac crest), AGEs were present as imidazolon and CML. In
Fig. 2. (A) The AGE Imidazolone is enriched more in bone tissue than in bone marrow. Staining of trabecular bone tissue with Anti-Imidazolone (brown color) in a case of low turnover osteoporosis. Imidazolon 1:10,000–40×. (B) The AGE Carboxymethyl lysin (CML) especially is found in bone marrow. CML 1:100,000–40×.
G.E. Hein / Clinica Chimica Acta 371 (2006) 32–36
It should be noted, however, that the quality of the bone matrix may also be altered by nonenzymatic glycation (NEG) of bone protein. Recently, Wang et al. [36] reported on age-related changes in noncalcified collagen molecules in osteoid and its likely effects on the mechanical integrity of human cortical bone. The results of this study indicated that the denaturation of noncalcified collagenous matrix in bone increased with age. Such collagen denaturation in osteoid was correlated to nonenzymatic collagen cross-links as well as with strength and toughness of bone. New investigations by Hermandez et al. [37] have shown that structural ductility (defined as the ultimate strain of the whole trabecula) is significantly related to the content of pentosidine (which explained 9% of the variance in ultimate strain). However pentosidine represents only a small portion of the intermolecular NEG–collagen cross links in bone. Finally there may be an additional mechanism of AGEmediated influence on bone remodeling processes via alteration of multipotential stem cells (MSC) by AGEs. MSCs in the bone marrow are able to differentiate into different mesenchymal phenotypic cells, including osteoblasts, chondroblasts, muscle cells, and fibroblasts [38,39]. Because the number of MSCs in the bone marrow declines with aging, it is possible that an age-related accumulation (in osteoporotic bone and bone marrow intensified) of AGE modified proteins could influence the process of MSC generation and their differentiation. AGEs are able to alter biological responses of microvascular cells in vitro, particularly by mediation through RAGE and other AGE receptors [40,41]. These effects may further suppress repair processes in the microvasculature [42]. Thus, it can be appreciated that the cell proliferation and differentiation could be affected by disturbance of the microvasculature as well as by RAGE-mediated alterations of MSCs. AGE accumulation and increased cross-linking of bone proteins could also impair the MSC interaction with extracellular matrix proteins and soluble chemokines, growth factors, etc. Finally intracellular AGE formation could profoundly alter MSC gene expression and cell signalling. Kume et al. [24] recently investigated the influence of AGEs on proliferation, apoptosis, and reactive oxygen species generation in MSCs. They found that AGEs inhibited proliferation of MSCs, induced apoptosis, and prevented cognate differentiation into adipose tissue, cartilage, and bone, suggesting a deleterious effect of AGEs in the pathogenesis of musculoskeletal disorders. In summary, there is a wide spectrum of possibilities as to how AGEs may be able to affect the quality of bone via impaired remodelling thereby contributing to the development of osteoporosis. Further clarification of this problem and the development of therapeutic strategies against the generation, accumulation, and other influences of AGEs are topics of ongoing research. References [1] NIH Consensus Development Panel on Osteoporosis. JAMA 2000:785–95. [2] Cauley JA, Thompson DE, Ensrud KC, Scott JC, Black D. Risk of mortality following clinical fractures. Osteoporos Int 2000;11(7):556–61.
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[3] Mosekilde L, Ebbesen EN, Tornvig L, Thomsen JS. Trabecular bone structure and strength — remodeling and repair. J Musculoskelet Neuronal Interact Sep 2000;1(1):25–30. [4] Bauer J. Bemerkungen zur Frage der Osteoporose. Wien Klin Wochenschr 1963;39:691–2. [5] Parfitt AM. Quantum concept of bone remodeling and turnover Implications for the pathogenesis of osteoporosis. Calcif Tissue Int 1997;28:1–5. [6] Nagaraj RH, Monnier VM. Protein modification by the degradation products of ascorbate: formation of a novel pyrrole from the Maillard reaction of Lthreose with proteins. Biochim Biophys Acta 1995;15:75–84. [7] Niwa T, Katsuzuki T, Momoi T, et al. Modification of β2-m with advanced glycation end products as observed in dialysis-related amyloidosis by 3-DG accumulating in uremic serum. Kidney lnt 1996;49:861–7. [8] Wells-Knecht KJ, Zyzyk DV, Litchfield JE, et al. Mechanism of autoxidative glycosylation: identification of glyoxal and arabinose as intermediates in the autoxidative modification of proteins by glucose. Biochemistry 1995;34:3702–9. [9] Requena RJ, Fu MX, Ahmed MU, et al. Lipid peroxidation products as biomarkers of oxidative damage to proteins during lipid peroxidation reactions. Nephrol Dial Transplant 1996;11:48–53. [10] Vlassara H, Palace MR. Diabetes and advanced glycation endproducts. J Intern Med 2002;25:87–101. [11] Thornalley PJ. Cell activation by glycated proteins AGE receptors, receptor recognition factors and functional classification of AGEs. Cell Mol Biol Noisy le grand, vol. 44 (7); 1998. p. 1013–23. [12] Schmidt AM, Yan SD, Yan SF, Stern DM. The multiligand receptor RAGE as a progression factor amplifying immune and inflammatory responses. J Clin Invest 2001;108(7):949–55. [13] Lu C, He JC, Cai W, Liu H, Zhu L, Vlassara H. Advanced glycation endproduct (AGE) receptor 1 is a negative regulator of the inflammatory response to AGE in mesangial cells. Proc Natl Acad Sci U S A Aug 10 2004;101(32):11767–72. [14] Dumitriu IE, Baruah P, Valentinis B, et al. Release of high mobility group box 1 by dendritic cells controls T cell activation via the receptor for advanced glycation end products. J Immunol Jun 15 2005;174 (12):7506–15. [15] Wang X, Shen X, Li X, Agrawal CM. Age-related changes in the collagen network and toughness of bone. Bone 2002;31:107. [16] Corthay A, Backlund J, Broddefalk J, et al. Epitope glycosylation plays a critical role for T cell recognition of type II collagen in collagen-induced arthritis. Eur J Immunol 1998;28:2580–90. [17] Corthay A, Backlund J, Holmdahl R. Role of glycopeptide-specific T cells in collagen-induced arthritis: an example how post-translational modification of proteins may be involved in autoimmune disease. Ann Med 2001;33:456–65. [18] Hofmann MA, Schiekofer S, Isermann B, et al. Peripheral blood mononuclear cells isolated from patients with diabetic nephropathy demonstrated increased activation of the oxidative-stress sensitive transcription NF-kB. Diabetologia 1999;42:222–32. [19] Bowie A, O'Neill LA. Oxidative stress and nuclear factor-kappaB activation: a reassessment of the evidence in the light of recent discoveries. Biochem Pharmacol 2000;59:13–23. [20] Yuan Z, Feldman RJ, Sun M, et al. Inhibition of JNK by cellular stressand tumor necrosis factor a-induced AKT2 through activation of the NFkB pathway in human epithelial cells. J Biol Chem 2002;277:29973–82. [21] Gu L, Hagiwara S, Fan Q, et al. Role of receptor for advanced glycation end-products and signalling events in advanced glycation end-productinduced monocyte chemoattractant protein-1 expression in differentiated mouse podocytes. Nephrol Dial Transplant 2006;21:299–313. [22] Charoonpatrapong K, Shah R, Robling AG, et al. HMGB1 expression and release by bone cells. J Cell Physiol 2006;17 [Electronic publication ahead of print]. [23] Hermani A, De Servi B, Medunjanin S, Tessier PA, Mayer D. S100A8 and S100A9 activate MAP kinase and NF-kappaB signalling pathways and trigger translocation of RAGE in human prostate cancer cells. Exp Cell Res 2006;312:184–97.
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[24] Kume S, Kato S, Yamagishi S, et al. Advanced glycation end-products attenuate human mesenchymal stem cells and prevent cognate differentiation into adipose tissue, cartilage, and bone. J Bone Miner Res 2000:1647–58. [25] Singh R, Barden A, Mori T, Beilin L. Advanced glycation end-products: a review. Diabetologia 2001;44:129–46. [26] Raj DS, Choudhury D, Welbourne TC, Levi M. Advanced glycation endproducts: a nephrologist's perspective. Am J Kidney Dis 2000;35:365–80. [27] Takahashi M, Suzuki M, Kushida K, Miyamoto S, Inoue T. Relationship between pentosidine levels in serum and urine and activity in rheumatoid arthritis. Br J Rheumatol 1997;36:637–42. [28] Hein G, Wiegand R, Lehmann G, Stein G, Franke S. Advanced glycation end-products pentosidine and N-carboxymethyllysine are elevated in serum of patients with osteoporosis. Rheumatology 2003;42:1242–6. [29] Odetti P, Rossi S, Monacelli F, et al. Advanced glycation end-products and bone loss during aging. Ann N Y Acad Sci 2005;1043:710–7. [30] Miyata T, Notoya K, Yoshida K, et al. Advanced glycation end-products enhance osteoclast-induced bone resorption in cultured mouse unfractionated bone cells and in rats implanted subcutaneously with devitalized bone particles. J Am Soc Nephrol 1997;8:260–70. [31] McCarthy AD, Etcheverry SB, Bruzzone L, Lettieri G, Barrio DA, Cortizo AM. Nonenzymatic glycosylation of a type I collagen matrix: effects on osteoblastic development and oxidative stress. BMC Cell Biol 2001;2:16. [32] McCarthy AD, Etecheverry SB, Cortizo AM. Advanced glycation endproduct-specific receptors in rat and mouse osteoblast-like cells: regulation with stages of differentiation. Acta Diabetol 1999;36:45–52. [33] Cortizo AM, Lettieri MG, Barrio DA, Mercer N, Etcheverry SB, McCarthy AD. Advanced glycation end-products (AGEs) induce concerted changes in the osteoblastic expression of their receptor RAGE and in the activation of extracellular signal-regulated kinases (ERK). Mol Cell Biochem 2003;250:1–10.
[34] Hein G, Weiβ Ch, Lehmann G, Niwa T, Stein G, Franke S. Advanced glycation end product modification of bone proteins and bone remodelling: hypothesis and preliminary immunohistochemical findings. Ann Rheum Dis 2006;65:101–4. [35] Katayama Y, Akatsy T, Yamamoto M, Kugai N, Nagata N. Role of nonenzymatic glycosylation of type I collagen in diabetic osteopenia. J Bone Miner Res 1996;11:931–7. [36] Wang X, Li X, Shen C-X, Agrawal M. Age-related changes of noncalcified collagen in human cortical bone. Ann Biomed Eng 2003;31:1365–71. [37] Hernandez Ch-J, Tang S-Y, Baumbach B-M, et al. Trabecular microfracture and the influence of pyridinium and non-enzymatic glycationmediated collagen cross-links. Bone 2005;37:825–32. [38] Haynesworth SE, Goshima J, Goldberg VM, Caplan AI. Characterization of cells with osteogenic potential from human marrow. Bone 1992;13 (1):81–8. [39] Lazarus HM, Hayneswoth SE, Gerson SL, Rosenthal NS, Caplan AL. Ex vivo expansion and subsequent infusion of human bone marrow-derived stromal progenitor cells (mesenchymal progenitor cells): implications for therapeutic use. Bone Marrow Transplant 1995;16(4):557–64. [40] Rellier N, Ruggiero-Lopez D, Lecomte M, Lagarde M, Wiernsperger N. In vitro and in vivo acceleration of enzymatic glycosylation in diabetes. Life Sci 1999;64(17):1571–83. [41] MCKenna DJ, Nelson J, Stitt AW. Advanced glycation alters expression of the 67 kDa laminin receptor in retinal microvascular endothelial cells. Life Sci 2001;4, 68(24):2695–703. [42] Stitt AW, Hughes SJ, Canning P, et al. Substrates modified by advanced glycation end-products cause dysfunction and death in retinal pericytes by reducing survival signals mediated by platelet-derived growth factor. Diabetologia 2004;47(10):1735–46.
Invited critical review
Glycation endproducts in osteoporosis — Is there a pathophysiologic importance? Gert E. Hein Division of Rheumatology and Osteology, Department of Internal Medicine III, Friedrich-Schiller-University of Jena, Germany Received 22 December 2005; received in revised form 16 February 2006; accepted 6 March 2006 Available online 13 June 2006
Abstract Advanced glycation endproducts (AGEs) are chemical modifications of proteins by carbohydrates including those metabolic intermediates formed during the Maillard reaction. The generation of AGEs is an inevitable process in vivo. AGEs constitute a heterogeneous class of compounds characterized by brown color, fluorescence and a tendency to polymerize. These unique compounds are specifically recognized by AGE receptors (RAGE) present on different cell types. A remarkable feature of AGE-mediated cross-linked proteins is decreased solubility and resistance to proteolytic digestion. This effect results in altered biomechanical properties in affected tissues including increased stiffness and rigidity. The AGE–RAGE interaction additionally induces activation of nuclear factor kB (NF-kB) in RAGE bearing cells (e.g., cells participating in bone turnover). This interaction results e.g. in increased expression of cytokines, growth factors and adhesion molecules. Recent findings provide important evidence that bone proteins are also affected by AGE modification. Investigations conducted by other groups, as well as ours, support the hypothesis that bone protein glycation influences osteoclasts (bone resorption) and osteoblasts (bone formation). © 2006 Elsevier B.V. All rights reserved. Keywords: Osteoporosis; Bone remodelling; AGE-modification of proteins; Osteoblast; Osteoclast
Contents 1. Osteoporosis and bone remodeling 2. Advanced glycation endproducts . 3. AGEs and bone, what is known? . References . . . . . . . . . . . . . . .
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1. Osteoporosis and bone remodeling According to the 2001 NIH consensus conference, osteoporosis is defined as a systemic bone disease characterized by insufficient bone strength, which predisposes to a higher risk of bone fracture [1]. The socioeconomic importance of osteoporosis is already high and is estimated to significantly increase in the future due to the changing age pyramid. It should be noted, however, that osteoporosis influences not only quality of life, but also life expectancy [2].
E-mail address: [email protected]. 0009-8981/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2006.03.017
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32 33 33 35
Osteoporosis is divided into primary (without a definitive basic disease) and secondary (basic disease leading to osteoporosis) forms. Bone strength is not only determined by quantitative parameters of bone such as size, mass and mineral density, but also by the quality of the material and structure of the bone tissue. Such qualitative parameters of bone tissue include its micro-architecture and geometry, extent of trabecular cross-linking, size and orientation of hydroxyapatite crystals, and integrity of collagen network [3]. It is generally accepted that osteoporosis is caused by disturbed bone remodeling. By the second half of the 19th century, the Austrian physician Victor von Ebner recognized that adult bone does not remain unchanged, but is in fact continually renewed in a
G.E. Hein / Clinica Chimica Acta 371 (2006) 32–36
33
coupled process of formation and resorption [4]. It is now known that adult bone requires permanent renewal for maintaining quality and optimum strength. About 10% of bone mass is replaced by this process every year. In contrast to cortical bone, trabecular bone is modified and replaced more quickly due to its continual structural adaptation and reorganization in response to new loads [5]. Bone reorganization starts with the recruitment and activation of osteoclasts. These cells resorb a defined bone volume (approximately 0.025 mm3) in a “basic multicellular unit” (BMU) resulting in a “resorption cavity.” By means of a coupling mechanism, osteoblasts are later attracted to fill the resorption lacuna with osteoid, i.e., unmineralized matrix. Primary and secondary mineralization later complete remodeling. Despite the progress in our understanding of bone biology, the precise mechanism(s) that initiate the remodeling process remain unclear to date. For example, what factors induce the movement of the osteoclasts to a defined place and how are they activated? A plausible hypothesis is that chemical changes in the bone protein result in biophysical/ biomechanical alterations of the bone matrix and are thereby responsible for the initiation of the remodeling process. Modifications of osseous proteins could result in functional alterations of osteoclasts and osteoblasts and be of significant pathophysiologic importance for the development of osteoporosis, as well as other metabolic bone diseases. A possible mechanism by which bone protein may be altered is via glycation, or in the generation and accumulation of advanced glycation endproducts (AGEs).
AGEs are able to alter physiological processes in vivo by different mechanisms:
2. Advanced glycation endproducts
Collagen molecules in bone have an exceptionally long lifetime, making them susceptible to AGE modification. Thus it may be hypothesized that the generation and accumulation of AGEs in bone tissue could be a reason for deterioration of bone tissue quality (e.g. increased stiffness). This may also be a cause of disturbed cell proliferation and development of functional alterations of cells. On this way AGEs could especially contribute to disturbed bone remodeling via osteoclasts and osteoblasts activation of NF-kB, induced by the binding of AGEs or the known pro-inflammatory molecules on the more intensively expressed RAGE. In our studies we found significantly higher levels of the AGE pentosidine as well as of carboxymethyl lysine (CML) in the serum of patients with osteoporosis [28]. In contrast, we were only able to find a significantly positive correlation between AGE level and age in the group of healthy controls. Thus it seems that osteoporosis itself is connected with a more intensified generation of AGEs. Odetti et al. [29] additionally evaluated the concentration of pentosidine in cortical and trabecular bone. Interestingly, the concentration of cortical pentosidine showed not only a significant exponential increase with age but was also negatively correlated to bone density and mineralization (Singh score). Despite these findings, it was unclear if increased AGE generation was a causal phenomenon of osteoporosis or an epiphenomenon. In order to further investigate this issue, AGE levels were evaluated relative to histomorphometric findings in these patients. A significant positive correlation between serum pentosidine and specific cellular parameters of the bone turnover
Advanced glycation endproducts (AGEs) are chemical modifications of proteins by sugar derived aldehyde groups generated during the Maillard reaction. However, it has recently become evident that ascorbic acid [6], reactive carbonyl intermediates such as 3-deoxyglucosone (3-DG), as well as products of sugar auto-oxidation (arabinose, glyoxal) and lipid peroxidation (malondialdehyde) are also precursors that cause chemical modifications of proteins. The formation of reactive carbonyl compounds resulting from auto-oxidation or lipid peroxidation is closely related to oxidative processes [7–9]. The generation of AGEs is an inevitable process in vivo. These compounds constitute a heterogeneous class of molecules characterized by a brown color, fluorescence, and a tendency to polymerize. The unique structures are specifically recognized by AGE-binding proteins or receptors (RAGEs). Today there are several known AGE-binding proteins with different characteristics and qualities. These include AGE-R1, AGE-R2, AGE-R3, ScR-II, CD36 and RAGE. The latter three are multi-ligand receptors of the immunoglobulin superfamily [10]. These receptors have already been identified in a variety of cell types [11–14] (see also Figs. 1A, B). Oxidative stress accelerates the generation of AGEs, and in turn the intracellular accumulation of AGEs accelerates oxidative stress. It can be appreciated that proteins with long half-lives are particularly susceptible to chemical modification such as the formation of AGEs.
1. AGE-mediated cross-linked proteins have decreased solubility and high resistance to proteolytic digestion, with the consequence of altered physicochemical and mechanical properties of tissue components (e.g. increased stiffness of collagen) [15]. 2. The AGE-mediated additional cross-linking (in addition to the physiologically cross-linking by pyridinium compounds) may alter the secondary and tertiary structures of the proteins and thereby generate new immunogenic epitopes [16,17]. 3. The binding of AGEs on the RAGE induces activation of the nuclear factor kB (NFkB) of the RAGE bearing cells, resulting in increased expressions of, e.g. cytokines, growth factors and adhesion molecules [18–20]. 4. The accumulation of AGEs intensifies the expression of RAGE. RAGE is not only a receptor for AGEs but also for other pro-inflammatory molecules such as HMBG 1 and the S 100/calgranulin family, possibly increasing inflammatory reactions by this indirect way [21–23]. 5. AGEs may alter the gene expression, and function, of multipotential stem cells (MSC) [24]. AGEs are now generally accepted as playing a pivotal pathophysiological role in several chronic diseases [25–27]. 3. AGEs and bone, what is known?
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general, a different staining pattern for both AGEs was observed in the bone specimens. Whereas imidazolon staining was much more intense in the bone tissue than in the marrow, CML showed a stronger immunoreactivity in the bone marrow compared to tissue (see Figs. 2A, B). There was no significant correlation between staining intensity of either AGE and cellular markers of bone resorption (i.e., ES/BS = eroded surface as percentage of trabecular surface or OcS/BS = osteoclast covered surface as percentage of the trabecular surface containing resorptive cavities). There was also no correlation to the mineral apposition rate (MAR). However, a significant negative correlation was found between the intensity of AGE immunoreactivity and the cellular bone formation marker ObS/BS (percentage of osteoblast covered bone surface). These results are in agreement with those of Katayama et al. [35] who reported that AGE modified collagen is able to inhibit phenotypic expression of osteoblasts. Thus it seems that the glycation of bone proteins, an inevitable process in vivo and occurring more intensely in osteoporotic bone, is able to influence both bone remodeling processes, osteoclastic bone resorption and osteoblastic bone formation. Whether this results in accelerated bone resorption (with development of a “high turnover osteoporosis”), or decreased bone formation (with development of a “low turnover osteoporosis”), may depend on the specificity of the generated glycation endproduct.
Fig. 1. (A) Myeloid cells ( ) are stained with Anti-RAGE antibodies (redbrown color), 2 Megakaryocytes ( ) are RAGE negative. (B) 2 Osteoclasts (large cells with multiple nuclei) ( ) RAGE positive.
was found [28]. These correlations provide evidence that, initially, pentosidine contributes to the increased osteoclast formation and/or activity and subsequent intensified osteoblast recruitment. Interestingly, over ten years ago Miyata et al. [30] showed that AGEs were able to enhance osteoclast-induced bone resorption in cultured mouse bone cells and in rats implanted subcutaneously with devitalized bone particles. Although the underlying mechanisms were unclear, addition of osteoclast inhibitors, such as calcitonin, into the culture medium neutralized the effects of AGEs. The process of bone formation may be influenced and altered by AGE modification of bone proteins. McCarthy et al. demonstrated that AGE modified collagen was able to regulate proliferation and differentiation of osteoblastic cells depending on developmental stage [31,32]. The addition of AGE–BSA to cultures of human osteoblast-like cells resulted in a significantly reduced synthesis of osteocalcin and collagen I. Receptors of AGE (RAGE) have been identified in these cells [33]. In our own recent investigation [34], we looked for the intensity of AGE immunoreactivity of osseous proteins in bone specimens taken from osteoporotic patients and for correlation to parameters of bone remodelling based on histomorphometric findings. In all the investigated bone particles (from the iliac crest), AGEs were present as imidazolon and CML. In
Fig. 2. (A) The AGE Imidazolone is enriched more in bone tissue than in bone marrow. Staining of trabecular bone tissue with Anti-Imidazolone (brown color) in a case of low turnover osteoporosis. Imidazolon 1:10,000–40×. (B) The AGE Carboxymethyl lysin (CML) especially is found in bone marrow. CML 1:100,000–40×.
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It should be noted, however, that the quality of the bone matrix may also be altered by nonenzymatic glycation (NEG) of bone protein. Recently, Wang et al. [36] reported on age-related changes in noncalcified collagen molecules in osteoid and its likely effects on the mechanical integrity of human cortical bone. The results of this study indicated that the denaturation of noncalcified collagenous matrix in bone increased with age. Such collagen denaturation in osteoid was correlated to nonenzymatic collagen cross-links as well as with strength and toughness of bone. New investigations by Hermandez et al. [37] have shown that structural ductility (defined as the ultimate strain of the whole trabecula) is significantly related to the content of pentosidine (which explained 9% of the variance in ultimate strain). However pentosidine represents only a small portion of the intermolecular NEG–collagen cross links in bone. Finally there may be an additional mechanism of AGEmediated influence on bone remodeling processes via alteration of multipotential stem cells (MSC) by AGEs. MSCs in the bone marrow are able to differentiate into different mesenchymal phenotypic cells, including osteoblasts, chondroblasts, muscle cells, and fibroblasts [38,39]. Because the number of MSCs in the bone marrow declines with aging, it is possible that an age-related accumulation (in osteoporotic bone and bone marrow intensified) of AGE modified proteins could influence the process of MSC generation and their differentiation. AGEs are able to alter biological responses of microvascular cells in vitro, particularly by mediation through RAGE and other AGE receptors [40,41]. These effects may further suppress repair processes in the microvasculature [42]. Thus, it can be appreciated that the cell proliferation and differentiation could be affected by disturbance of the microvasculature as well as by RAGE-mediated alterations of MSCs. AGE accumulation and increased cross-linking of bone proteins could also impair the MSC interaction with extracellular matrix proteins and soluble chemokines, growth factors, etc. Finally intracellular AGE formation could profoundly alter MSC gene expression and cell signalling. Kume et al. [24] recently investigated the influence of AGEs on proliferation, apoptosis, and reactive oxygen species generation in MSCs. They found that AGEs inhibited proliferation of MSCs, induced apoptosis, and prevented cognate differentiation into adipose tissue, cartilage, and bone, suggesting a deleterious effect of AGEs in the pathogenesis of musculoskeletal disorders. In summary, there is a wide spectrum of possibilities as to how AGEs may be able to affect the quality of bone via impaired remodelling thereby contributing to the development of osteoporosis. Further clarification of this problem and the development of therapeutic strategies against the generation, accumulation, and other influences of AGEs are topics of ongoing research. References [1] NIH Consensus Development Panel on Osteoporosis. JAMA 2000:785–95. [2] Cauley JA, Thompson DE, Ensrud KC, Scott JC, Black D. Risk of mortality following clinical fractures. Osteoporos Int 2000;11(7):556–61.
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