Iron-enriched diet contributes to early onset of osteoporotic phenotype in a mouse model of hereditary hemochromatosis

Osteoporosis is associated with chronic iron overload secondary to hereditary hemochromatosis (HH), but the causative mechanisms are incompletely understood. The main objective of this study was to investigate the role of dietary iron on osteoporosis, using as biological model the Hfe-KO mice, which have a systemic iron overload. We showed that these mice show an increased susceptibility for developing a bone loss phenotype compared to WT mice, which can be exacerbated by an iron rich diet. The dietary iron overload caused an increase in inflammation and iron incorporation within the trabecular bone in both WT and Hfe-KO mice. However, the osteoporotic phenotype was only evident in Hfe-KO mice fed the iron-enriched diet. This appeared to result from an imbalance between bone formation and bone resorption driven by iron toxicity associated to Hfe-KO and confirmed by a decrease in bone microarchitecture parameters (identified by micro-CT) and osteoblast number. These findings were supported by the observed downregulation of bone metabolism markers and upregulation of ferritin heavy polypeptide 1 (Fth1) and transferrin receptor-1 (Tfrc), which are associated with iron toxicity and bone loss phenotype. In WT mice the iron rich diet was not enough to promote a bone loss phenotype, essentially due to the concomitant depression of bone resorption observed in those animals. In conclusion the dietary challenge influences the development of osteoporosis in the HH mice model thus suggesting that the iron content in the diet may influence the osteoporotic phenotype in systemic iron overload conditions.National Funds through Foundation for Science and Technology (FCT) Norte-01-0145-FEDER-000012 Portuguese Foundation for Science and Technology (FCT) SFRH/BD/77056/2011 European Regional Development Fund (FEDER) Norte-01-0145-FEDER-000012info:eu-repo/semantics/publishedVersio

Oestrogen deficiency modulates particle-induced osteolysis

International audienceABSTRACT: INTRODUCTION: Postmenopausal osteoporosis may modulate bone response to wear debris. In this article, we evaluate the influence of oestrogen deficiency on experimental particle-induced osteolysis. METHODS: Polyethylene (PE) particles were implanted onto the calvaria of normal controls, sham-ovariectomized (OVX), OVX mice and OVX mice supplemented with oestrogen (OVX+E). After 14 days, seven skulls per group were analyzed using a high-resolution micro-computed tomography (micro-CT) and histomorphometry, and for tartrate-specific alkaline phosphatase. Five calvariae per group were cultured for the assay of IL-1β, IL-6, TNF-α and receptor activator of the nuclear factor κB (RANKL) secretion using quantitative ELISA. Serum IL-6 concentrations were obtained. The expression of RANKL and osteoprotegerin (OPG) mRNA were evaluated using real-time PCR. RESULTS: As assessed by μCT and by histomorphometry, PE particles induced extensive bone resorption and an intense inflammatory reaction in normal controls, sham-OVX and OVX+E mice, but not in the OVX mice group. In normal controls, sham-OVX and OVX+E mice, PE particles induced an increase in serum IL-6, in TNF-α and RANKL local concentrations, and resulted in a significant increase in RANKL/OPG messenger RNA (mRNA) ratio. Conversely, these parameters remained unchanged in OVX mice after PE implantation. CONCLUSIONS: Oestrogen privation in the osteolysis murine model ultimately attenuated osteolytic response to PE particles, suggesting a protective effect. This paradoxical phenomenon was associated with a down-regulation of pro-resorptive cytokines. It is hypothesized that excessive inflammatory response was controlled, illustrated by the absence of increase of serum IL-6 in OVX mice after PE implantation

Por que estrógeno e raloxifeno melhoram a densidade mineral óssea?: mecanismo de ação do estrógeno e de um modulador seletivo do receptor de estrógeno (SERM) no osso

Estrogen deficiency is responsible for increased bone turnover in the postmenopausal period, and it can be prevented by estrogen replacement therapy. The way by which estrogen acts on bone cells is not fully understood and there are still many unsolved questions: (1) What is the target-cell of estrogen in bone? Estrogen receptor has been described in monocyte, osteoclast, bone marrow stromal cell and osteoblast, but it is still not clear what cell mediates the effect of estrogen in bone. (2) What are the mediators of estrogen action on bone? There is some controversy about the role of interleukin-6; most of the positive results were in animals, not in human; other cytokines are also involved, as tumoral necrosis factor and interleukin-1. (3) Is the anti-osteoclast effect of estrogen related to the apoptosis of osteoclast precursors? Some authors had already reported that estrogen increase apoptosis of osteoclasts precursors in animals, but it is not clear whether this effect is also present in humans. (4) What is the role of the bone marrow in osteoclastogenesis and in its inhibition by estrogen? Recently, it has been shown that stromal cells produce a membrane-associated factor (RANK-ligand) that stimulates osteoclast precursors, showing the importance of stromal cells in osteoclastogenesis and probably in the effect of estrogen in bone. (5) What is the transcription mechanism of estrogen action into the cell? NF-kappaB proteins may have an important role in post-menopausal osteoporosis, by regulating the secretion of cytokines involved on osteoclastogenesis. These questions on the mechanism of action of estrogens and also SERMs will be discussed in this review based on studies of literature and on recent studies of our group.A deficiência de estrógeno é responsável pelo aumento na remodelação óssea após a menopausa, cuja prevenção é feita pela terapia de reposição hormonal com estrógeno; porém, ainda não está esclarecido o mecanismo da ação anti-reabsortiva do estrógeno no osso e permanecem várias questões: (1) Qual a célula-alvo de ação do estrógeno no osso? O receptor de estrógeno já foi descrito em monócito, osteo-clasto, células do estroma da medula óssea e osteoblasto, mas é desconhecido o papel dessas células no efeito do estrógeno. (2) Quais os mediadores do efeito do estrógeno no osso? Os resultados da literatura são controversos quanto ao papel da interleucina-6, sendo a maioria dos resultados positivos em animais e não no homem. Outras citoquinas como interleucina-1 e fator de necrose tumoral parecem estar envolvidos. (3) O efeito anti-osteoclástico do estrógeno está relacionado à apoptose de precursores dos osteoclastos? Já foi relatado, em animais, que o estrógeno aumenta apoptose dos precursores dos osteoclastos, porém não é conhecido esse efeito no homem. (4) Qual o papel do estroma da medula óssea na osteoclastogênese e na sua inibição pelo estrógeno? Recentemente foi descrito um fator, produzido pelas células do estroma (RANK ligante) que induz a formação de osteoclasto, sugerindo a importância dessas células na osteoclastogênese e no efeito do estrógeno no osso. (5) Qual a via de transcrição intracelular do efeito do estrógeno? Proteínas NF-kapaB podem ter um papel significante na osteoporose pós-menopausa, podendo corresponder à via pelo qual o estrógeno regula a produção de citoquinas envolvidas na osteoclastogênese, porém ainda não está esclarecido este efeito. Estas questões, sobre o mecanismo de ação do estrógeno, como também dos SERMs serão discutidas nessa revisão.Universidade Federal de São Paulo (UNIFESP) Departamento de MedicinaHospital Lariboisière INSERM U349UNIFESP, Depto. de MedicinaSciEL

Effect of C282Y genotype on self-reported musculoskeletal complications in hereditary hemochromatosis

Objective Arthropathy that mimics osteoarthritis (OA) and osteoporosis (OP) is considered a complication of hereditary hemochromatosis (HH). We have limited data comparing OA and OP prevalence among HH patients with different hemochromatosis type 1 (HFE) genotypes. We investigated the prevalence of OA and OP in patients with HH by C282Y homozygosity and compound heterozygosity (C282Y/H63D) genotype. Methods A total of 306 patients with HH completed a questionnaire. Clinical and demographic characteristics and presence of OA, OP and related complications were compared by genotype, adjusting for age, sex, body mass index (BMI), current smoking and menopausal status. Results In total, 266 of the 306 patients (87%) were homozygous for C282Y, and 40 (13%) were compound heterozygous. The 2 groups did not differ by median age [60 (interquartile range [IQR] 53 to 68) vs. 61 (55 to 67) years, P=0.8], sex (female: 48.8% vs. 37.5%, P=0.18) or current smoking habits (12.4% vs. 10%, P=0.3). As compared with compound heterozygous patients, C282Y homozygous patients had higher median serum ferritin concentration at diagnosis [1090 (IQR 610 to 2210) vs. 603 (362 to 950) mu g/L, P<0.001], higher median transferrin saturation [80% (IQR 66 to 91%) vs. 63% (55 to 72%), P<0.001]) and lower median BMI [24.8 (22.1 to 26.9) vs. 26.2 (23.5 to 30.3) kg/m2, P<0.003]. The overall prevalence of self-reported OA was significantly higher with C282Y homozygosity than compound heterozygosity (53.4% vs. 32.5%; adjusted odds ratio [aOR] 2.4 [95% confidence interval 1.2-5.0]), as was self-reported OP (25.6% vs. 7.5%; aOR 3.5 [1.1-12.1]). Conclusion Patients with C282Y homozygosity may be at increased risk of musculoskeletal complications of HH.Association Rhumatisme et Travail (Centre Viggo Petersen, Hospital Lariboisiere, Paris

Osteopetrosis and related osteoclast disorders in adults:A review and knowledge gapsOn behalf of the European calcified tissue society and ERN BOND

Osteopetrosis refers to a group of related rare bone diseases characterized by a high bone mass due to impaired bone resorption by osteoclasts. Despite the high bone mass, skeletal strength is compromised and the risk of fracture is high, particularly in the long bones. Osteopetrosis was classically categorized by inheritance pattern into autosomal recessive forms (ARO), which are severe and diagnosed within the first years of life, an intermediate form and an autosomal dominant (ADO) form; the latter with variable clinical severity and typically diagnosed during adolescence or in young adulthood. Subsequently, the AD form was shown to be a result of mutations in the gene CLCN7 encoding for the ClC-7 chloride channel). Traditionally, the diagnosis of osteopetrosis was made on radiograph appearance alone, but recent molecular and genetic advances have enabled a greater fidelity in classification of osteopetrosis subtypes. In the more severe ARO forms (e.g., malignant infantile osteopetrosis MIOP) typical clinical features have severe consequences and often result in death in early childhood. Major complications of ADO are atypical fractures with delay or failure of repair and challenge in orthopedic management. Bone marrow failure, dental abscess, deafness and visual loss are often underestimated and neglected in relation with lack of awareness and expertise. Accordingly, the care of adult patients with osteopetrosis requires a multidisciplinary approach ideally in specialized centers. Apart from hematopoietic stem cell transplantation in certain infantile forms, the treatment of patients with osteopetrosis, has not been standardized and remains supportive. Further clinical studies are needed to improve our knowledge of the natural history, optimum management and impact of osteopetrosis on the lives of patients living with the disorder

Bone Fragility Fractures in CKD Patients

Chronic kidney diseases (CKD) are associated with mineral and bone diseases (MBD), including pain, bone loss, and fractures. Bone fragility related to CKD includes the risk factors observed in osteoporosis in addition to those related to CKD, resulting in a higher risk of mortality related to fractures. Unawareness of such complications led to a poor management of fractures and a lack of preventive approaches. The current guidelines of the Kidney Disease Improving Global Outcomes (KDIGO) recommend the assessment of bone mineral density if results will impact treatment decision. In addition to bone density, circulating biomarkers of mineral, serum bone turnover markers, and imaging techniques are currently available to evaluate the fracture risk. The purpose of this review is to provide an overview of the epidemiology and pathogenesis of CKD-associated bone loss. The contribution of the current tools and other techniques in development are discussed. We here propose a current view of how to better predict bone fragility and the therapeutic options in CKD

Evaluation of bone density and microarchitecture in adult patients with X-linked hypophosphatemic rickets:A pilot longitudinal study

X-linked Hypophosphatemia (XLH) is the most common type of inherited rickets. Although the clinical features are well characterized, bone structure, mineralization, and biomechanical properties are poorly known. Our aim was to analyze bone properties in the appendicular and axial skeleton of adults with XLH. In this observational case-control study, each affected patient (N = 14; 9 females; age 50 ± 15 years) was matched by sex, age and body mass index to a minimum of two healthy controls (N = 34). Dual-energy X-ray Absorptiometry (DXA) analyses revealed that areal bone mineral density (aBMD) was higher in XLH patients at the lumbar spine (Z score mean difference = +2.47 SD, P value = 1.4 × 10 -3). Trabecular Bone Score was also higher at the lumbar spine (P value = 1.0 × 10 -4). High Resolution peripheral Quantitative Computed Tomography (HRpQCT) demonstrated that bone cross-sectional area was larger at the distal radius (P value = 6 × 10 -3). Total and trabecular volumetric BMD were lower at both sites. Trabecular bone volume fraction was also lower with fewer trabecular numbers at both sites. However, bone strength evaluated by micro-finite element analyzes revealed unaffected bone stiffness and maximum failure load. Evaluation of bone mineralization with aBMD by DXA at the distal radius correlated with vBMD by HRpQCT measurements at both sites. PTH levels were inversely correlated with trabecular vBMD and BV/TV at the tibia. We then followed a subset of nine patients (median follow-up of 4 years) and reassessed HRpQCT. At the tibia, we observed a greater decrease than expected from an age and sex standardized normal population in total and cortical vBMD as well as a trabecularization of the cortical compartment. In conclusion, in adult patients with XLH, bone mineral density is high at the axial skeleton but low at the appendicular skeleton. With time, microarchitectural alterations worsen. We propose that noninvasive evaluation methods of bone mineralization such as DXA including the radius should be part of the management of XLH patients. Larger studies are needed to evaluate the clinical significance of BMD changes in XLH patients under conventional or targeted therapies. </p

Inflammatory Potential of Four Different Phases of Calcium Pyrophosphate Relies on NF-κB Activation and MAPK Pathways

Background: Calcium pyrophosphate (CPP) microcrystal deposition is associated with wide clinical phenotypes, including acute and chronic arthritis, that are interleukin 1β (IL-1β)-driven. Two CPP microcrystals, namely monoclinic and triclinic CPP dihydrates (m- and t-CPPD), have been identified in human tissues in different proportions according to clinical features. m-CPP tetrahydrate beta (m-CPPTβ) and amorphous CPP (a-CPP) phases are considered as m- and t-CPPD crystal precursors in vitro.Objectives: We aimed to decipher the inflammatory properties of the three crystalline phases and one amorphous CPP phase and the intracellular pathways involved.Methods: The four synthesized CPP phases and monosodium urate crystals (MSU, as a control) were used in vitro to stimulate the human monocytic leukemia THP-1 cell line or bone marrow-derived macrophages (BMDM) isolated from WT or NLRP3 KO mice. The gene expression of pro- and anti-inflammatory cytokines was evaluated by quantitative PCR; IL-1β, IL-6 and IL-8 production by ELISA; and mitogen-activated protein kinase (MAPK) activation by immunoblot analysis. NF-κB activation was determined in THP-1 cells containing a reporter plasmid. In vivo, the inflammatory potential of CPP phases was assessed with the murine air pouch model via cell analysis and production of IL-1β and CXCL1 in the exudate. The role of NF-κB was determined by a pharmacological approach, both in vivo and in vitro.Results:In vitro, IL-1β production induced by m- and t-CPPD and m-CPPTβ crystals was NLRP3 inflammasome dependent. m-CPPD crystals were the most inflammatory by inducing a faster and higher production and gene expression of IL-1β, IL-6, and IL-8 than t-CPPD, m-CPPTβ and MSU crystals. The a-CPP phase did not show an inflammatory property. Accordingly, m-CPPD crystals led to stronger activation of NF-κB, p38, extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK) MAPKs. Inhibition of NF-κB completely abrogated IL-1β and IL-8 synthesis and secretion induced by all CPP crystals. Also, inhibition of JNK and ERK1/2 MAPKs decreased both IL-1β secretion and NF-κB activation induced by CPP crystals. In vivo, IL-1β and CXCL1 production and neutrophil infiltration induced by m-CPPD crystals were greatly decreased by NF-κB inhibitor treatment.Conclusion: Our results suggest that the inflammatory potential of different CPP crystals relies on their ability to activate the MAPK-dependent NF-κB pathway. Studies are ongoing to investigate the underlying mechanisms

N-acetylcysteine treatment ameliorates the skeletal phenotype of a mouse model of diastrophic dysplasia

Diastrophic dysplasia (DTD) is a recessive chondrodysplasia caused by mutations in SLC26A2, a cell membrane sulfate-chloride antiporter. Sulfate uptake impairment results in low cytosolic sulfate, leading to cartilage proteoglycan (PG) undersulfation. In this work, we used the dtd mouse model to study the role of N-acetyl-l-cysteine (NAC), a well-known drug with antioxidant properties, as an intracellular sulfate source for macromolecular sulfation. Because of the important pre-natal phase of skeletal development and growth, we administered 30 g/l NAC in the drinking water to pregnant mice to explore a possible transplacental effect on the fetuses. When cartilage PG sulfation was evaluated by high-performance liquid chromatography disaccharide analysis in dtd newborn mice, a marked increase in PG sulfation was observed in newborns from NAC-treated pregnancies when compared with the placebo group. Morphometric studies of the femur, tibia and ilium after skeletal staining with alcian blue and alizarin red indicated a partial rescue of abnormal bone morphology in dtd newborns from treated females, compared with pups from untreated females. The beneficial effect of increased macromolecular sulfation was confirmed by chondrocyte proliferation studies in cryosections of the tibial epiphysis by proliferating cell nuclear antigen immunohistochemistry: the percentage of proliferating cells, significantly reduced in the placebo group, reached normal values in dtd newborns from NAC-treated females. In conclusion, NAC is a useful source of sulfate for macromolecular sulfation in vivo when extracellular sulfate supply is reduced, confirming the potential of therapeutic approaches with thiol compounds to improve skeletal deformity and short stature in human DTD and related disorder