The soluble glycoprotein

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Osteoprotegerin (OPG) is a soluble glycoprotein and member of the TNF-receptor superfamily that is characterized by its ability to bind to receptor activator of NF-κB Ligand (RANKL) and (TNF)-related apoptosis inducing ligand (TRAIL) (Corallini, Rimondi, & Secchiero, 2008e), (Emery et al., 1998). It exists as either a 60-kd monomeric structure or as a disulfide linked 120-kd homodimer and is encoded on chromosome 8q (Yun et al., 1998a). In contrast to other members of the TNF receptor superfamily, OPG does not have specific transmembrane or cytoplasmic domains. It is instead secreted into the circulation as a soluble receptor (Yun et al., 1998b), (Corallini, Rimondi, & Secchiero, 2008d). OPG consists of 401 amino acids, however the cleaving of a 21-amino acid signal peptide leads to the formation of a mature 380 amino acid form (Simonet et al., 1997f). OPG also distinguishes itself from other members of TNF-receptor superfamily because of the maintenance of its biological activity in its soluble, circulating form. It was identified in 1997-1998 simultaneously by two separate groups (Simonet et al., 1997e), (Tsuda et al., 1997a) and has had a number of synonyms including osteoclastogenesis inhibitory factor (OCIF), TNF receptor like molecule 1 (TR1), and follicular dendritic cell-associated receptor 1 (FDCR-1). However, OPG is now the accepted term for this glycoprotein. At the time of discovery both groups demonstrated an important role for OPG in the regulation of bone turnover as a result of its direct inhibition of osteoclastogenesis (Simonet et al., 1997d), (Tsuda et al., 1997b), (Reid & Holen, 2009a). It consists of 4 amino-terminal cysteine rich domains that are structurally similar to the extracellular portions of other associates in the TNF receptor superfamily. The carboxy-terminal incorporates portions 5 and 6, that are death domain homologous regions (Baker & Reddy, 1998).

Evidence for the role of the OPG/RANK/RANKL axis in bone turnover from mouse studies

A physiological role for OPG in regulating bone formation and resorption was initially demonstrated when OPG deficient mice, produced by targeted disruption of the gene were viable and fertile but developed profound bone loss, marked destruction of growth plates and reduced trabecular femur bone mass (Bucay et al., 1998b). In this study the authors further noted that the elevated mortality of these adolescent mice was related to an increased occurrence of vertebral or endochondral fractures. Interestingly the offspring of surviving, female double knockout mice gave birth to histologicaly normal double knockout offspring, suggesting that OPG is not essential for normal foetal development. Besides the effect on bone quality and elevated alkaline phosphatase, mice who survived to 6 months appeared to have no untypical haematological or biochemical characteristics. In a similar study Mizuno et al (1998) also created an OPG homozygous mouse; they found no histopathological abnormalities in the femurs of these mice at 5 weeks. However there was a marked increase in osteoclast size, number and proliferation, coupled with a progressive loss of trabecular femoral bone found between 8 and 13 weeks. Suggesting that the early osteoporotic phenotype observed in these adolescent mice is likely due to an increase in osteoclastogenesis. (Mizuno et al., 1998b). A putative role for OPG in this process was first elucidated in a classic study by Simonet et al., (1997) who created OPG-overexpressing mice. At 10 weeks, other than an enlarged spleen (~38%), these mice were phenotypically no different from their normal littermates; however they showed signs of profound osteopetrosis characterized by significant radio-opacity of the long bones, vertebrae, and pelvis when compared to their ordinary littermates. Mice which appeared to highly express the OPG transgene displayed obvious signs of osteopetrosis by x-ray at birth, the severity of which increased significantly into adolescence and adulthood. Despite this increase in radio-density, there was no irregularity in tooth eruption, a symptom commonly observed in ostepetrotic mice (Yoshida et al., 1990), (Soriano, Montgomery, Geske, & Bradley, 1991). Additionally in order to investigate the effect of OPG on healthy mice, Simonet et al., (1997) administered recombinant OPG to 4 week old wild-type mice and found that after 7 days they had a 3 fold increase (31.1% versus 12.0%) in trabecular bone of the proximal tibial metaphysis when compared to controls (Simonet et al., 1997c). The authors further clarified a role for OPG in the regulation of bone formation demonstrating that the administration of recombinant OPG blocks differentiation of precursor cells into osteoclasts in a dose dependant manner in vitro. Additionally the authors underlined a possible clinical application of recombinant OPG by illustrating the potential for OPG therapy to ameliorate the bone loss that one would expect in ovariectomized rats, where bone volume in the proximal tibial metaphysis was increased in OPG treated rats relative to controls (Simonet et al., 1997b).

Subsequently the mechanism through which this activity is mediated has been well described. Osteoblasts and their precursor cells, stromal cells express the homotrimeric, transmembrane protein; RANKL, particularly in regions where there is active bone remodeling or inflammatory osteolysis (Hofbauer & Schoppet, 2004a). RANKL consists of 316 amino acids and in addition to osteoblastic/ stromal cells it is abundantly expressed by T cells in lymph tissue. RANKL is found in the circulation in two discrete forms, it is either secreted by T cells or as a result of proteolytic cleavage from cell surfaces (Hofbauer & Heufelder, 2001), (Walsh & Choi, 2003), (Schoppet, Preissner, & Hofbauer, 2002). RANKL stimulates receptor activator of nuclear factor ?B (RANK). RANK is a transmembrane receptor, consisting of 616 amino acids which is found on the surface of cells of monocyte/macrophage lineage, such as dendritic cells and osteoclasts and their precursors (Dougall et al., 1999). RANKL binds to RANK on osteoclast precursors and more mature osteoclasts, upregulating intracellular pathways that lead to increased proliferation and survival of osteoclasts leading to activation of osteoclastogenic processes, which results in increased bone resorption and bone loss. Generally an increase in RANKL is associated with decrease in OPG, such that the ratio of RANKL to OPG changes in favor of osteoclastogenesis. Many reports have supported the assertion that the RANKL/OPG ratio is a major determinant of bone mass (Hofbauer & Schoppet, 2004b). Both stromal cells and osteoblasts secrete OPG as a homodimer, which acts as a decoy receptor, binding to RANKL, thus blocking the resultant inhibition of osteoclastogenesis and bone loss (Corallini, Rimondi, & Secchiero, 2008b). In vitro investigations have demonstrated the importance of OPG dimerisation for this process. Homodimeric OPG binds strongly (KD 10nM) with homotrimeric RANKL to form stable dimmer-trimer compounds.

Schneeweis et al., (2005) showed, using sedimentation velocity analysis that 1:2 OPG-RANKL complexes were not formed in mixtures containing a 2-fold molar excess of RANKL over OPG, implying that both of the OPG monomers in the homodimer cannot bind to a separate RANKL trimer simultaneously. However, 2:1 OPG-RANKL complexes did emerge when OPG was present at a 2-fold molar excess over RANKL. Moreover, the authors found that the second OPG dimer displayed a significant loss of affinity (KD - 3µM). The authors concluded that the most likely explanation based on these findings was that the high affinity OPG-RANKL binding is dependant on avidity. Two of the OPG monomers in each dimer bind to two out of three of the RANKL monomers in each trimereric structure. Only one monomer in the second OPG molecule is able to weakly interact with the third and only available RANKL monomer (Schneeweis, Willard, & Milla, 2005).

RANKL / RANK Molecular pathway inducing Osteoclastogenesis

The regulatory role of RANKL in bone resorption and formation has also been shown in vivo, Baud'huin et al., (2007) demonstrated that administration of RANKL to adult mice induces bone resorption, whilst mice deficient in functional RANKL develop osteoporosis (Baud'huin et al., 2007). A crucial mechanism in promoting the resorptive action of osteoclasts is the binding of RANK and RANKL. As an affiliate of the TNF receptor superfamily, RANK does not have any kinase activity, therefore it is necessary for RANK to enlist the help of associated factors to transduce the signals after binding to its ligand (Leibbrandt & Penninger, 2008c). Binding of RANK to its Ligand leads to the translocation of TNF receptor-associated factors (TRAFs) to the intracellular surface of RANK. RANK has been shown to associate with TRAFs 1 - 6 during in vitro experiments (Darnay, Haridas, Ni, Moore, & Aggarwal, 1998c), (Galibert, Tometsko, Anderson, Cosman, & Dougall, 1998a), (Wong et al., 1998c), (Leibbrandt & Penninger, 2008b). RANK encodes a cytoplasmic domain which encloses several TRAF binding sites that cluster in specific domains, the areas enclosed by the amino acids 235-358 and 359-531 bind the TRAF6 adaptor molecule, the 532-625 region contains several binding locations for TRAFs 2, 5, and 6. (Darnay, Haridas, Ni, Moore, & Aggarwal, 1998b), (Wong et al., 1998b), (Wong, Josien, & Choi, 1999). However, only TRAF6 interacts with the membrane-proximal region of the RANK cytoplasmic domain which is distinct from other TRAFs. The functional significance of these TRAF binding domains is to initiate RANK-induced NF-κB and c-Jun NH2-terminal kinase (JNK) activation. Deletion of the TRAF6 binding site of RANK almost completely blocked the RANK-dependent activation of NF-kB (Galibert, Tometsko, Anderson, Cosman, & Dougall, 1998b). Nevertheless, JNK activation can still progress, implying that interactions with TRAF6 are essential for activation of the NF-κB but not for the activation of the JNK pathway. (Darnay, Haridas, Ni, Moore, & Aggarwal, 1998a), (Wong et al., 1998a), (Galibert, Tometsko, Anderson, Cosman, & Dougall, 1998c), (Lee, Kwack, Kim, Lee, & Kim, 2000). Armstrong et al (2009) demonstrated using genetically modified gene constructs of RANK that are selectively incapable of binding different TRAF proteins that TRAF pathways with the exception of TRAF6, downstream of RANK, affecting osteoclast differentiation were functionally redundant. The interaction of RANK with TRAF6 however was extremely important for the formation of cytoskeletal structures and the resorptive activity of osteoclasts. (Armstrong et al., 2002). Lomaga et al., (1999) found that viable TRAF6 double knockout mice appeared phenotypically normal at birth but did not mature and died soon after birth. Those TRAF6-/- animals that lived longer than 14 days had a 20 - 30% reduction in body mass and length. In addition, they had modest enlargement of the heart and liver which was accompanied by significant splenomegaly, represented by an increase in organ size of 2 - 6 fold compared to wild type littermates. X-ray examination of these mice showed that their long bones and vertebral bodies were radio-opaque. The long bones, especially the femur, were reduced in length and exhibited a distinct broadening at the ends attributable to a failure in bone modelling, indicative of osteopetrosis. Molars and incisors of the double knockout animals had failed to erupt which is, again, common in osteopetrotic mice (Popoff & Marks, Jr., 1995), as bone resorption allows for the opening of a channel through the jawbone for teeth to grow. Peripheral quantitative computed tomography analysis of the proximal tibial bone metaphyisis showed a significant increase in bone mass in double knockout compared to the transgenic mice (Lomaga et al., 1999b). These findings were further strengthened by Naito et al., (1999) who found that in addition to premature mortality and runting of young knockout mice, they also had limited bone marrow cavities which consisted of mostly spongy bone. Further histological analysis highlighted abnormal bone formation and thickened epiphyseal growth plates. Like Lomaga et al (1998), the authors attributed this profound osteopetrosis to a failure of osteoclast precursors to differentiate into mature osteoclasts in response to RANKL. (Lomaga et al., 1999a), (Naito et al., 1999).

The contributions of TRAF2 and TRAF5 to osteoclastogenesis seem to be relatively small. TRAF2-/- liver derived progenitor cells demonstrate only marginally reduced accumulation of multinuclear osteoclasts, and activation of NF-κB and JNK by RANKL was comparable to normal controls, Likewise, TRAF5 deficient cells have only a mild defect in osteoclastogenesis, and NF-κB and JNK activation is not affected upon RANK stimulation. (Kanazawa & Kudo, 2005), (Kanazawa, Azuma, Nakano, & Kudo, 2003). There are at least seven distinct pathways activated by RANK-induced protein kinase signalling; four of them directly induce osteoclastogenesis; inhibitor of NF-κB kinase/NF-κB, c-Jun amino-terminal kinase/activator protein-1, c-myc, calcineurin/nuclear factor of activated T cells (NFATc1). There are three others that directly mediate osteoclast activation, they include (src and MKK6/p38/ MITF) and survival (src and extracellular signal-regulated kinase). (Boyce & Xing, 2007a)

These studies indicate that TRAF6 is likely the most important adaptor molecule linking RANK signalling to the NF-κB osteoclastogenesis pathway but that other TRAFs may potentially circumvent and compensate for the consequences of a TRAF6-de?ciency (Leibbrandt & Penninger, 2008a). In addition to TRAFs, there are other adapter molecules that bind to RANK to induce signalling in this pathway. This in turn results in the activation of the transcription factor NF-κB. (Matsumoto, Sudo, Saito, Osada, & Tsujimoto, 2000), (Xing et al., 2002). Growth factor receptor-bound protein 2 (Grb-2) associated binder 2 (Gab2) is one of a family of adapter proteins, phosphorylated at tyrosine residues that leads to the recruitment of a variety of signalling molecules that have Sarcoma 2 (Src 2) homologous domains. Loss of Gab2 results in reduced RANKL/RANK-induced osteoclast differentiation, decreased bone resorption, and mild osteopetrosis (Boyce et al., 2007a), (Wada et al., 2005a), suggesting that it is an important player in RANKL-induced osteoclastogenesis (Wada et al., 2005b).

The vital role of NF-κB/activator protein-1/ NFATc1 signalling for osteoclast formation was revealed after the engineering of mice with genetic disruption of the p50 and p52 subunits of NF-κB and of the immediate early gene transcript, c-Fos (Karsenty & Wagner, 2002), and subsequently by a study in which adoptive transfer of NFATc1-/- stem cells to cFos-/- mice resulted in osteoclast formation (Takayanagi et al., 2002b). Over expression of a constitutively active form of NFATc1 induces osteoclast formation by M-CSF treated Fos-/- or NF-?b p50/p52-/- osteoclast precursors in the absence of RANKL (Yao, Matsuo, Nishimura, Xing, & Boyce, 2005) indicating that it is downstream from NF-κB and c-Fos (Figure 1). On the basis of all of these studies, NFATc1 has been described as the master regulator of osteoclastogenesis (Boyce & Xing, 2007b), (Takayanagi et al., 2002a).

The essential signaling pathway for normal osteoclastogenesis. Under physiologic conditions, RANKL produced by osteoblasts binds to RANK on the surface of osteoclast precursors and recruits the adaptor protein TRAF6, leading to NF-κB activation and translocation to the nucleus. NF-κB increases c-Fos expression and c-Fos interacts with NFATc1 to trigger the transcription of osteoclastogenic genes. OPG inhibits the initiation of the process by binding to RANKL. NFAT, nuclear factor of activated T cells; NF-κB, nuclear factor-?B; OPG, osteoprotegerin; RANKL, receptor activator of nuclear factor-?B ligand; TRAF, tumor necrosis factor receptor associated factor. (adapted from (Boyce et al., 2007a) and (Hofbauer & Schoppet, 2004c) Need to explain yellow in the text

Disequilibrium in the RANKL/OPG fraction or signaling is a major instigator in the pathology of many disorders of the skeleton where increased bone resorption/formation, or inappropriate bone remodeling are a factor (Hofbauer & Schoppet, 2004d). This is supported by Whyte el al., (2002) who demonstrated a loss in osteoprotective function for homozygous deletions of 100 kb of OPG in patients with the autosomal-recessive disorder; juvenile Paget's disease a condition in which increased resorption, severe osteopaenia, and persistent fractures are primary symptoms (Whyte et al., 2002). It is further supported by the identification of an inactivating deletion in exon 3 of OPG in with idiopathic hyperphosphatasia, which is also an autosomal-recessive disease typified by increased bone resorption, deformities of long bones, kyphosis, and acetabular protrusion (Cundy et al., 2002), (Daroszewska et al., 2004), (Boyce et al., 2007b). The central role of defective OPG signaling and secretion in Juvenile Paget's Disease was verified by Cundy et al., (2005) where the administration of recombinant OPG to sufferers of Juvenile Paget's Disease led to a decrease in the speed of bone resorption and improved radio density upon examination by x-ray (Cundy et al., 2005). In addition, in vivo models, such as in a T-cell-dependent model of rat adjuvant arthritis (Kong et al., 1999b) and collagen induced arthritis (Schett et al., 2003) which are both characterized by severe joint inflammation, bone and cartilage destruction and crippling, blocking of RANKL through osteoprotegerin treatment at the onset of disease prevents bone and cartilage destruction but not inflammation (Kong et al., 1999a), and has been show to prevent bone and tooth loss in an animal model of periodontal disease, without having any significant effect on the immune process (Teng et al., 2000). More recent animal models have used combination therapy with blockade of RANKL with the administration of OPG in conjunction with the blockade of various inflammatory agents, including; IL-1 and TNF-α and found that with the use of these two treatments as a tandem therapy, bone loss and systemic inflammation could be substantially reduced (Zwerina et al., 2004).

There have been several studies in postmenopausal women that have attempted to investigate the relationship between circulating OPG concentrations and Bone Mineral Density. However the findings from some of these studies at this point have been somewhat at odds. Several studies have demonstrated that OPG appears to be increased in osteoporosis, (Rogers, Saleh, Hannon, Greenfield, & Eastell, 2002), Whilst in some instances OPG serum levels appear to be decreased in osteoporosis, other studies suggest OPG serum levels to be negatively correlated to BMD. In a study by Mezquita et al., (2005) conducted in a cohort of 206 postmenopausal women the authors revealed that lower concentrations of circulating OPG were positively related to low BMD as well as prevalence of vertebral fracture (Mezquita-Raya et al., 2005). However a study by Yano et al., (1999) comparing OPG serum levels in Japanese men and women spanning a large age range of ages demonstrated that serum OPG was significantly increased in those postmenopausal women who were osteoporotic (Yano et al., 1999). A possible reason for the differences between these studies could be the difference in experimental design and different populations utilized. (Reid & Holen, 2009b)

In addition to the severe osteoporosis observed in OPG deficient mice (Mizuno et al., 1998a), OPG knockout mice appear to exhibit significant renal and aortic calcification. Furthermore, administration of recombinant OPG to rats appears to prevent the onset of arterial calcifications induced by warfarin treatment or high doses of vitamin D (Price, June, Buckley, & Williamson, 2001). Arterial calcification usually complicates chronic atherosclerosis and it appears to be accelerated in these mice, suggesting that OPG may play an important role in protecting large blood vessels from medial calcification and other complications of atherosclerosis (Bucay et al., 1998a). The relationship between osteoporosis and vascular calcification in these animal models of OPG deficiency is somewhat reminiscent of the clinical setting where these conditions often occur congruently (Hofbaeur 2007) Longitudinal analysis of bone loss and vascular calcification over a 25-year period in the Framingham Heart Study showed women with the greatest magnitude of bone loss also had the most severe progression of abdominal aortic calcification (Kiel et al., 2001). Furthermore A cross sectional study in 2,348 postmenopausal women revealed that aortic calcification strongly predicts low bone mineral density and occurrence of fractures. A subgroup of 228 women within this cohort who were longitudinally observed showed that the percentage yearly increase in aortic calcification accounted for almost half of the variance in the percentage rate of bone loss. Additionally a strong graded association was observed between the progression of vascular calcification and bone loss for each quartile. Women in the highest aortic calcification-quartile had four times greater yearly bone loss than women in the lowest quartile (Schulz, Arfai, Liu, Sayre, & Gilsanz, 2004).

OPG expression and function in the vascular system

Evidently the RANKL/RANK/OPG triad is an important player involved in the homeostatic control of the immune and skeletal systems. Research in recent years has also begun to shed light on an equally intriguing roll for this axis in the homeostasis of the vascular environment. Many of the same signals that modulate RANKL and OPG, both immunomodulatory and osteogenic in origin may also regulate their expression in the vascular endothelium. As well as the typical activity of OPG in boney tissues, OPG expression and secretion is also found at high concentrations in the arterial wall, where the content in aortic extracts is reported to be 500 - 1000 times greater than those found in the circulation (Olesen, Ledet, & Rasmussen, 2005g), (Knudsen et al., 2003a) a similar concentration to that found in bone. It has also been demonstrated that both micro/macro vascular endothelial cells (VECs) and vascular smooth muscle cells (VSMCs) secrete OPG (Collin-Osdoby et al., 2001c), (Secchiero et al., 2006d), (Zhang et al., 2002d). A number of potential growth factors and inflammatory cytokines which are thought to be key players in the pathogenesis of atherosclerosis and coronary artery disease have also been implicated in the regulation of OPG in the vascular wall. VEC-expression of OPG can be induced by the addition of the inflammatory cytokines; TNF-α, IL-1a, IL-1β, activated integrin avβ3 and additionally porphyromonas gingivalis, an initiating activator of periodontal disease. (Kobayashi-Sakamoto, Hirose, Isogai, & Chiba, 2004b), (Secchiero et al., 2006c), (Ben-Tal et al., 2007b). Collin-Osdoby et al., (2001) demonstrated that human microvascular ECs express mRNA transcripts for both RANKL and OPG. In addition they showed that RANKL and OPG mRNA are significantly and dose-dependently upregulated in response to TNF-α and IL-1 as measured by semi-quantitative real time PCR. Upon further analysis of the time course of upregulation of OPG and RANKL mRNA expression, it was apparent that the rise in RANKL expression was first observed at 10 h after the addition of TNF-α and by 24 h had risen to a peak of 3-6-fold in comparison to untreated VEC. These levels of expression continued for between 48 and 72 h when continuously co-cultured with TNF-α. Removal of the cytokine after 24 h led to a sustained decline in RANKL expression, however levels were still elevated by as much as 2-fold after 48 h. OPG mRNA levels in VEC rose more swiftly in response to TNF-α. Elevated OPG mRNA levels were apparent within 1 h, reached their highest level by 10 h, but declined to approximately half their maximum values at 24 h, and thereafter fell more slowly up to 72 h. Despite this, OPG mRNA levels were ten times higher than the unconditioned VEC. OPG mRNA levels quickly returned to concentrations similar to that of untreated VEC after withdrawal of TNF-α treated media. (Collin-Osdoby et al., 2001b).

Expression of OPG in Vascular Endothelial Cells

Zannettino et al., 2005 have identified the site of OPG endothelial intracellular localisation to compartments known as Weibel-Palade Bodies (WPBs), they further observed that OPG is physically associated with von Willebrand Factor both in WPBs and in serum (Zannettino et al., 2005). Upon thrombogenic and in?ammatory insult with cytokines such as TNF-α, and IL-1β, the contents of WPBs quickly translocate to the plasma membrane and extracellular space, where they promote migration of leukocytes and platelets to inflammatory sites and areas of thrombus formation (Arnaout, 1993), (Wagner, 1993), strongly suggesting a vasoactive role for OPG in maintaining haemostasis and possibly in the prevention of vascular injury and inflammation. In VECs, activation of integrin αvβ3 and porphyromonas gingivalis appear to augment OPG expression levels via initiation of the NF-kB transcription pathway (Kobayashi-Sakamoto, Hirose, Isogai, & Chiba, 2004a); (Malyankar et al., 2000c); TNF-α and IL-1a also activate signalling pathways that result in NF-κB activation suggesting that activation of this transcription pathway may be an important step in modulating production of endothelial cell OPG (Baud & Karin, 2001), (Wesche et al., 1997).

Expression of OPG in Vascular Smooth Muscle Cells

Within the general vasculature however, OPG is more highly expressed in VSMCs compared to ECs, with VSMCs secreting up to 20-30 times that of endothelial cells. (Zhang et al., 2002c). Interestingly the specific area of OPG activity in the arterial architecture seems to be important as higher concentrations have been found in the tunica media of diabetics relative to normoglycaemic controls, however no difference in OPG concentration was observed in the same cohort when intimal tissue was compared (Golledge, McCann, Mangan, Lam, & Karan, 2004b). (Some quantitative data from this study). In vascular smooth muscle cells, a number of cytokines have been shown to augment OPG expression, including TNFa, IL-1β, insulin, basic ?broblast growth factor (bFGF), platelet-derived growth factor (PDGF), and angiotensin II (Collin-Osdoby et al., 2001a), (Olesen, Ledet, & Rasmussen, 2005f), (Ben-Tal et al., 2007a), (Zhang et al., 2002b)

Contrary to the NF-kB pathway by which OPG seems to be upregulated in VECs Zhang et al., (2002) demonstrated that PDGF-induced OPG gene expression in VSMCs could be blocked by inhibition of the PI3-kinase/AKT and p38/MAPK signalling pathways but found that blockade of NF-kB activation did not attenuate PDGF-mediated OPG increases in VSMCs (Zhang et al., 2002a). Suggesting that in VSMCs, additional pathways to that of NF-kB activation may also be important in the upregulation of OPG production. Olesen et al., (2005) also found that TNF-α increased the amount of OPG produced from the VSMCs (Olesen, Ledet, & Rasmussen, 2005e), but interestingly OPG secretion was attenuated by the addition of insulin to the media. OPG production by VSMCs has also been shown to be reduced by peroxisome proliferator-activated receptor gamma (PPAR?) antagonists (Fu et al., 2002b). The authors found that OPG expression was inhibited by PPAR? ligands in human VSMCs and that this effect was completely abolished by a PPAR? antagonist. Moreover overexpression of PPAR? in these cells by transfection of an adenovirus considerably decreased OPG expression (Fu et al., 2002a).

There is now accumulating evidence demonstrating a role for OPG in the regulation of VEC survival (Malyankar et al., 2000b), (Cross et al., 2006a), (Pritzker, Scatena, & Giachelli, 2004c). However the specific means by which, OPG reduces VEC apoptosis has not yet been fully elucidated, however it is unlikely that it involves protection from TRAIL-induced apoptosis, as a number of studies have revealed that ECs are resistant to apoptosis induction by TRAIL, and are only sensitized to TRAIL-induced apoptosis under harsh conditions such as serum deprivation (Secchiero et al., 2003b), (Scatena & Giachelli, 2002c). Only one group have implicated the inhibition of TRAIL in the OPG-mediated reduction in EC apoptosis (Pritzker, Scatena, & Giachelli, 2004b), others groups have not found TRAIL to be present in EC cultures (Cross et al., 2006b) (Zauli et al., 2007a). Malyankar et al., (2000) reported that ECs plated on osteopontin had increasing OPG mRNA and protein secretion and a resultant reduction in EC apoptosis (Malyankar et al., 2000a). In addition Cross et al., (2006) demonstrated that OPG enhances EC growth and differentiation, in addition to promoting the growth of cord-like arrangements on a matrigel base. (Cross et al., 2006c)

Several studies have demonstrated that in chronic pathological conditions where there is continuous long-term exposure to inflammatory cytokines such as in rheumatoid arthritis, multiple myeloma, diabetes, or hyperlipidemia, OPG synthesis and storage in ECs has been shown to be low, or indeed, absent altogether (Browner, Lui, & Cummings, 2001b), (Giuliani, Bataille, Mancini, Lazzaretti, & Barille, 2001), (Wallin, Wajih, Greenwood, & Sane, 2001). One possibility is that this may be as a consequence of the continued chronic secretion of OPG leading to a significant depletion of vascular endothelial intacellular content after an extended time.

In the vascular system, RANKL and RANK are expressed by endothelial cells. RANKL /RANK interactions regulate endothelial survival and apoptosis. RANKL may be blocked by OPG, which is secreted by endothelial and smooth muscle cells. The physiological role of the OPG/RANKL /RANK system in the vascular wall and interactions with other ligands are currently under investigation. Adapted from (Hofbauer & Schoppet, 2004e).

Interestingly the specific area of OPG activity in the arterial architecture seems to be important, as higher concentrations have been found in the tunica media of diabetics relative to normoglycaemic controls, however no difference in OPG concentration was observed in the same cohort when intimal tissue was compared (Golledge, McCann, Mangan, Lam, & Karan, 2004a). (Some quantitative data from this study). This phenomenon of medial compared to intimal calcification was further studied by Schoppet et al., (2004) who found increased expression of OPG (but not RANKL) around areas of intimal and medial calcification in samples from patients with Monckeberg's sclerosis characterized medial calcification and atherosclerosis where intimal calcification is more common (Schoppet et al., 2004). These findings were similar to those of Dhore et al (Dhore et al., 2001), and again suggested that OPG may be involved in the process of vascular calcification.

Subsequent to their earlier work (Olesen, Ledet, & Rasmussen, 2005d), Olesen et al., (2007) showed that the addition of β-glycerophosphate to VSMC cultures led to significant calcification and as assessed by the measurement of total cellular calcium content was increased still further by the addition of insulin at a concentration of 1000 µU/ml. Interestingly the authors showed that their was a concomitant reduction in OPG expression, suggesting that this down-regulation of OPG may play some role in the increased calcification (Olesen, Nguyen, Wogensen, Ledet, & Rasmussen, 2007). Unlike their previous study (Olesen, Ledet, & Rasmussen, 2005c), lower levels of insulin (200 µU/ml) did not effect OPG secretion and the authors proposed this may have been due to the fact that the latter study was performed in the presence of serum or that the effects of insulin on OPG may be different depending on the degree of hyperinsulinaemia.

Induction of diabetes by Streptozotocin led to an increase in detectable OPG levels and a fall in free RANKL concentration in apo-E null mice, and the addition of TNF-α (but not glucose or insulin) stimulated OPG release from human umbilical vein endothelial cells (Secchiero et al., 2006b). Using samples from human atherosclerotic plaques obtained at site of rupture during an acute myocardial infarction and plaques from apoE knockout mice, Sandberg et al showed increased activity of the OPG/RANKL/RANK system (Sandberg et al., 2006). In addition they showed that RANKL increased the release of chemoattractant peptide-1 in mononuclear cells of patients with unstable angina and also stimulated matrix metalloproteinase activity in VSMCs. Other factors influencing the secretion and expression of OPG and RANKL include the bone morphogenetic proteins BMP-2 and BMP-7 in addition to transforming growth factor β1 (TGFβ1). All reduce OPG secretion and mRNA expression; BMP-2 and BMP-7 increased RANKL mRNA but TGFβ1 reduced RANKL. To address the question of whether OPG is elevated in states of atherosclerosis and vascular calcification as a compensatory mechanism or if it is playing a negative role in the pathogenesis of these conditions, Zauli et al examined the effect of OPG on adhesion of pro-inflamatory cytokines to endothelial cells (Zauli et al., 2007b). They found that OPG promotes the adhesion of primary polymorphonuclear neutrophills and leukaemic HL60 cells to endothelial cells in vitro, and they confirmed these findings in vivo in rat mesentery. The authors concluded that OPG may play a deleterious role in endothelial pathophysiology by instigating the leucocyte adhesion to endothelium which is thought be an early step in the causation of endothelial dysfunction. On the other hand, Bennett et al found that OPG-deficient ApoE-/- mice developed larger atherosclerotic lesions in addition to more vascular calcification than their OPG+/+ littermates and that it acted as a survival factor for serum-deprived smooth muscle cells (Bennett et al., 2006). The exact role of OPG in the process of atherosclerosis was further examined by Moroney et al (Morony et al., 2008). They fed atherogenic LDL receptor knockout mice a high-fat diet and treated them with recombinant OPG or vehicle. The vehicle-treated mice developed atherosclerosis with associated calcification and their OPG levels rose in parallel. The degree of calcification, but not atherosclerosis was significantly reduced in the mice given recombinant OPG. The authors concluded that these results supported the theory that OPG inhibits vascular calcification, and may act as a marker (rather than a mediator) of atherosclerosis progression. It also appears that, in addition to slowing vascular calcification and possibly mediating atherosclerosis, OPG may be a pro-angiogenic factor (McGonigle, Giachelli, & Scatena, 2009). When added in vitro to a rat aortic ring model of angiogenesis, OPG led to an increase in neo-angiogenesis, an effect that was abrogated by pre-incubation with RANKL or TRAIL (see below). Additionally RANKL induced apoptosis on the endothelial cells. Circulating OPG has been shown to be significantly higher in patients with type 2 diabetes (Yaturu, Rains, & Jain, 2008) (Secchiero et al., 2006a) (Olesen, Ledet, & Rasmussen, 2005b) (Rasmussen, Tarnow, Hansen, Parving, & Flyvbjerg, 2006a), and is higher in the tunica media of type 2 diabetics than matched normal controls (Olesen, Ledet, & Rasmussen, 2005a). In addition, OPG is higher in individuals with severe Peripheral Artery Disease (PAD) than in those classified as having a mild to moderate PAD (Ziegler, Kudlacek, Luger, & Minar, 2005). Indeed it has also been shown that OPG can independently predict silent coronary artery disease (Griffin et al., 1999) in type 2 diabetic patients (Avignon et al., 2005).

Many of the same signals that modulate RANKL and OPG in bone or immune cells may also regulate their expression in vascular cells. From an indirect perspective, it is likely that the RANKL/ RANK/OPG axis exerts important effects on the vascular system through both immunomodulatory and osteogenesis- related mechanisms. Despite the seemingly therapeutic effect, the exact mechanism by which OPG acts to protect the vascular wall remains elusive, however there is growing data to suggest that OPG may play a part in the regulation of EC survival/apoptosis in cell models (Pritzker, Scatena, & Giachelli, 2004a), (Scatena & Giachelli, 2002b). It has been suggested that the pro-survival action of OPG on ECs may in part be due to inhibition of TRAIL-mediated apoptosis (Corallini, Rimondi, & Secchiero, 2008a), although several studies have suggested that ECs are resistant to TRAIL-induced apoptosis under normal physiological conditions (Scatena & Giachelli, 2002a), (Secchiero et al., 2003a).

OPG and Insulin resistance

Serum OPG and insulin sensitivity/resistance: (from Eoin: rewrite)

There have been a number of studies which have attempted to elucidate the relationship between serum OPG and insulin sensitivity/resistance. In a study of 286 women with a mean age of 52 years, Oh et al., (2005) found that LDL, total cholesterol, follicle stimulating hormone as well as age and waist to hip ratio were positively correlated with OPG, but there was no relationship between OPG and fasting glucose, fasting insulin, or insulin sensitivity (Oh et al., 2005). Ugur-Altun et al., (2004) also investigated the relationship between OPG and insulin resistance using the HOMA-IR model in 50 obese and 24 lean individuals who were not taking any medications. The authors found that OPG was lowest in the most insulin resistant obese group, and that OPG correlated negatively with insulin resistance, as measured by HOMA-IR (Ugur-Altun, Altun, Tatli, Arikan, & Tugrul, 2004d). Gannage-Yared et al., (2006) had similar findings in a study of 151 older men where they found a weak positive correlation between OPG and insulin sensitivity using the Quantitative Insulin Sensitivity Index (QUICKI), in addition the authors found a albeit weak correlations with (positive) adiponectin and (negative) triglycerides (Gannage-Yared, Fares, Semaan, Khalife, & Jambart, 2006a). The same group subsequently investigated a relationship between OPG and insulin resistance in an obese cohort of patients undergoing bariatric surgery (Gannage-Yared et al., 2008c). Unlike the matched non-obese group, OPG showed a correlation with HOMA-IR even with adjustment for age and presence of diabetes. Multiple linear regression revealed that the acute phase reactant and marker of vascular in inflammation, CRP in addition to HOMA-IR were independent predictors of OPG concentration - a relationships that had not been shown in previous studies, a relationship which had not been observed in previous studies (Ugur-Altun, Altun, Tatli, Arikan, & Tugrul, 2004c), (Browner, Lui, & Cummings, 2001a). This contrasted with the negative correlation seen between OPG and HOMA-IR in an obese population in an earlier study (Ugur-Altun, Altun, Tatli, Arikan, & Tugrul, 2004b), and the positive correlation between OPG and QUICKI in the same group's previous work (Gannage-Yared, Fares, Semaan, Khalife, & Jambart, 2006b). The authors speculated the small numbers in the HOMA paper (n=12 of obese with high HOMA) (Ugur-Altun, Altun, Tatli, Arikan, & Tugrul, 2004a), and the different population studied in their previous paper (ie elderly males) (Gannage-Yared, Fares, Semaan, Khalife, & Jambart, 2006c) might account for the differences (Gannage-Yared et al., 2008b). Considering these somewhat contradictory results some caution should be exercised when comparing findings different studies. Several studies have used commercially available assays that measure unbound and uncomplexed forms of both sRANKL or OPG (Xiang, Xu, Zhao, Yue, & Hou, 2006), (Knudsen et al., 2003b), (Rasmussen, Tarnow, Hansen, Parving, & Flyvbjerg, 2006b), (Jorgensen, Vind, Nybo, Rasmussen, & Hojlund, 2009). The data which is the subject of this body of work refers to free soluble RANKL and total OPG. This OPG assay measures both monomeric and dimeric isoforms of OPG, including OPG bound to RANKL and TRAIL and has been used to measure total OPG in many cohorts (Gannage-Yared, Fares, Semaan, Khalife, & Jambart, 2006d), (Gannage-Yared et al., 2008a), (Anand, Lahiri, Lim, Hopkins, & Corder, 2006),(Schoppet et al., 2003). In addition because of the non standard units of measurements used in other commercial ELISA assays and the difficulty in ascribing an exact molecular weight to the OPG-isoforms which they measure i.e. bound or unbound, monomeric or dimeric, the process of converting these values to SI units is somewhat complicated. Therefore previous studies that have exclusively measured uncomplexed OPG may unintentionally have excluded a large portion of the biologically active total circulating OPG which has either bound to TRAIL or RANKL or indeed has undergone some other unspecific binding.

From Shin et al 2008

Merge these two paragraphs

Simonet et al., (1997) first confirmed the role of OPG in the regulation of bone formation demonstrating that the administration of recombinant OPG blocks differentiation of precursor cells into osteoclasts in a dose dependant manner in vitro. Additionally the authors underlined a possible clinical application of recombinant OPG by illustrating the potential for OPG therapy to ameliorate the bone loss that one would expect in ovariectomized rats, where bone volume in the proximal tibial metaphysis was increased in OPG treated rats relative to controls (Simonet et al., 1997a). Since then there has been much interest in the use of OPG and or manipulation of the RANKL/RANK/OPG axis for the treatment of many bone related disorders. This interest has led to the development of a fully human monoclonal IgG2 antibody to RANKL, AMG162. (Denosumab). Denosumab selectively binds to RANKL but does not cross react with TNF-α, TNFβ, CD40 ligand, or TRAIL (Dougall & Chaisson, 2006). Upon binding to RANKL, denosumab blocksthe interaction between RANKL and RANK, a mechanism similar to that of endogenous OPG. Denosumab is currently in Phase II clinical trials for postmenopausal women with osteoporosis (Bone et al., 2008), breast cancer-related bone metastases (Lipton et al., 2007), structural damage in patients with rheumatoid arthritis (Cohen et al., 2008). I

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