Pathogenesis Of Bone Disease Research Articles

Assessing Energy Substrate Oxidation In Vitro with 14CO2 Trapping.

Mitochondria host the machinery for the tricarboxylic acid (TCA) cycle and electron transportchain (ETC), which generate adenosine triphosphate (ATP) to maintain energy homeostasis. Glucose, fatty acids, and amino acids are the major energy substrates fueling mitochondrial respiration in most somatic cells. Evidence shows that different cell types may have a distinct preference for certain substrates. However, substrate utilization by various cells in the skeleton has not been studied in detail. Moreover, as cellular metabolism is attuned to physiological and pathophysiological changes, direct assessments of substrate dependence in skeletal cells may provide important insights into the pathogenesis of bone diseases. The following protocol is based on the principle of carbon dioxide release from substrate molecules following oxidative phosphorylation. By using substrates containing radioactively labeled carbon atoms (14C), the method provides a sensitive and easy-to-use assay for the rate of substrate oxidation in cell culture. A case study with primary calvarial preosteoblasts versus bone marrow-derived macrophages (BMMs) demonstrates different utilization of the main substrates between the two cell types.

Assessment of the prevalence of osteoporosis among children with hemophilia and its relation to serum 25(oh) vitamin D and serum rankl

Osteoporosis is one of the comorbidities that complicating hemophilia. Recurrent hemarthrosis, chronic arthropathy and immobilization all are factors that make patients with hemophilia prone to this complication. Till recently the major emphasis of the osteoporosis diagnosis is the dual-energy X-ray absorptiometry (DEXA) scan, however, the definition of osteoporosis in pediatrics not only low bone mineral density measured by DEXA scan but also required the presence of clinically significant fracture history. Investigation target is evaluating the osteoporosis prevalence between cases with hemophilia, assessing the risk factors increasing its incidence including diet habits, the severity of hemophilia, duration between the first diagnosis of the disease and enrollment in the study and chronic hepatitis C infection. Also, considering the role of vitamin D deficiency, and another potential indirect mechanism mediated through Receptor activator of NF-Kappa-B, the Receptor activator of NF-Kappa-B ligand (RANK-RANKL) pathway has been suggested in the pathogenesis of bone disease and osteoclastic activity but remains controversial.Thirty-nine hemophilia pediatric patients were recruited from hematology clinic, Cairo university hospital, history taking, and examination were done focusing on the musculoskeletal system and dietetic data. Twenty normal age-matching children enrolled as a control group. DEXA scan results showed normal bone mineral density in 28 patients (71.8%) and osteoporosis in 11 patients (28.2%). The median Z score of patients was -1.40 (-2.2 – -0.6). There was a statistically significant decrease of bone mineral density in patients with hemophilia comparing with a control group with P-value <0.001, also we observed significantly higher serum RANKL in the group of patients with low bone mineral density ensuring the relation between RANKL and osteoclastic activity.

MiR-206 inhibits osteogenic differentiation of bone marrow mesenchymal stem cells by targetting glutaminase

Osteoblast-mediated bone formation is a complex process involving various pathways and regulatory factors, including cytokines, growth factors, and hormones. Investigating the regulatory mechanisms behind osteoblast differentiation is important for bone regeneration therapy. miRNAs are known as important regulators, not only in a variety of cellular processes, but also in the pathogenesis of bone diseases. In the present study, we investigated the potential roles of miR-206 during osteoblast differentiation. We report that miR-206 expression was significantly down-regulated in human bone marrow mesenchymal stem cells (BMSCs) at days 7 and 14 during osteogenic induction. Furthermore, miR-206 overexpressing BMSCs showed attenuated alkaline phosphatase (ALP) activity, Alizarin Red staining, and osteocalcin secretion. The mRNA levels of osteogenic markers, Runx2 and Osteopontin (OPN), were significantly down-regulated in miR-206 overexpressing BMSCs. We observed that significantly increased glutamine uptake at days 7 and 14 during the osteogenic induction and inhibition of glutamine metabolism by knocking down glutaminase (GLS)-suppressed osteogenic differentiation of BMSCs. Here, we discover that miR-206 could directly bind to the 3′-UTR region of GLS mRNA, resulting in suppressed GLS expression and glutamine metabolism. Finally, restoration of GLS in miR-206 overexpressing BMSCs led to recovery of glutamine metabolism and osteogenic differentiation. In summary, these results reveal a new insight into the mechanisms of the miR-206-mediated osteogenesis through regulating glutamine metabolism. Our study may contribute to the development of therapeutic agents against bone diseases.

Circulating miR-103a-3p and miR-660-5p are associated with bone parameters in patients with controlled acromegaly.

BackgroundBiochemical control of GH/IGF-I excess in acromegaly (ACRO) is associated with persistent impairment of trabecular microstructure leading to increased risk of vertebral fractures. Circulating miRNAs modulate the activity of osteoblasts and osteoclasts, and may be potential biomarkers of osteoporosis.AimsIdentify differentially expressed miRNAs in the serum of patients with controlled ACRO vs controls and correlate miRNA levels with both biochemical and structural bone parameters.Patients and methodsTwenty-seven patients with controlled ACRO (11 males, 16 females; mean age, 48 ± 5 years; BMI, 28 ± 4 kg/m2) and 27 age-, gender- and BMI-matched controls were recruited. Areal BMD at lumbar spine and femur, and trabecular bone score were assessed; volumetric BMD was measured by quantitative computed tomography QCT-Pro (Mindways). Twenty miRNAs, chosen by their putative role in bone, were quantified in serum using real-time qPCR.ResultsIn ACRO patients, miR-103a-3p and miR-191-5p were found overexpressed, whereas miR-660-5p was underexpressed (P < 0.001). miR-103a-3p levels were negatively associated with both trabecular vBMD at trochanter and serum osteoprotegerin concentrations (P < 0.05) and positively with vitamin D concentrations (P < 0.01) and total cross-sectional area of the femoral neck (P < 0.05). miR-660-5p levels were correlated with both trabecular vBMD at trochanter and OPG concentrations (P < 0.05), but were negatively associated with vitamin D levels (P < 0.05). A negative correlation between miR-103-a-3p and miR-660-5p was found in both groups (P < 0.001).ConclusionsCirculating miR-103a-3p and miR-660-5p are differentially expressed in controlled ACRO patients and associated with bone structural parameters. miRNAs may be one of the mechanisms involved in the pathogenesis of bone disease and could be used as biomarkers in ACRO patients.

Disrupted radial and tibial microarchitecture in patients with monoclonal gammopathy of undetermined significance.

Fracture risk is elevated in patients with monoclonal gammopathy of undetermined significance (MGUS). However, the pathogenesis of bone disease in these patients is poorly understood. Prior work using high-resolution peripheral CT (HRpQCT) demonstrated abnormal microarchitecture at the radius, with predominantly cortical abnormalities. We hypothesized that patients with MGUS have abnormal microarchitecture at both radius and tibia compared to controls, reflecting global skeletal effects of the disease. This case-control study enrolled 36 subjects; patients with MGUS (n = 12) were matched 1:2 by age, sex, and race to controls (n = 24). Areal BMD (aBMD) was measured by DXA, vBMD, and microarchitecture by HRpQCT, and whole bone stiffness by finite element analysis. Serum was drawn for markers of bone metabolism and inflammation. By DXA, MGUS patients had lower aBMD at the lumbar spine, femoral neck, and 1/3 radius. Markers of bone metabolism and inflammation did not differ. By HRpQCT at the radius, MGUS patients had lower total, trabecular and cortical density, lower trabecular number, and greater trabecular separation and heterogeneity. At the tibia, MGUS patients had lower total and trabecular density, lower trabecular number, greater separation and heterogeneity, and lower whole bone stiffness. Patients with MGUS had lower vBMD, cortical, and trabecular abnormalities at the radius compared to matched controls. At the tibia, trabecular abnormalities predominated. These results suggest that in addition to previously described cortical deficits, deterioration of trabecular bone may contribute to a generalized skeletal fragility in patients with MGUS.

Osteoporosis in primary biliary cholangitis.

Primary biliary cholangitis (PBC) is an autoimmune cholestatic liver disease with multiple debilitating complications. Osteoporosis is a common complication of PBC resulting in frequent fractures and leading to significant morbidity in this population, yet evidence for effective therapy is lacking. We sought to summarize our current understanding of the pathophysiology of osteoporosis in PBC, as well as current and emerging therapies in order to guide future research directions. A complete search with a comprehensive literature review was performed with studies from PubMed, EMBASE, Web of Science, Cochrane database, and the Countway Library. Osteoporosis in PBC is driven primarily by decreased bone formation, which differs from the increased bone resorption seen in postmenopausal osteoporosis. Despite this fundamental difference, current treatment recommendations are based primarily on experience with postmenopausal osteoporosis. Trials specific to PBC-related osteoporosis are small and have not consistently demonstrated a benefit in this population. As it stands, prevention of osteoporosis in PBC relies on the mitigation of risk factors such as smoking and alcohol use, as well as encouraging a healthy diet and weight-bearing exercise. The primary medical intervention for the treatment of osteoporosis in PBC remains bisphosphonates though a benefit in terms of fracture reduction has never been shown. This review outlines what is known regarding the pathogenesis of bone disease in PBC and summarizes current and emerging therapies.

Ecophysiological determinants of the human skeletal system

This review assessed relationships between external factors and the level of joint pathology (hip, knee, ankle, elbow, shoulder, hands and feet) in humans with dysplastic arthritis, psoriatic arthritis, rheumatoid arthritis, Sjogren’s syndrome, systemic lupus erythematosus, systemic sclerosis, degenerative joint disease, ankylosing spondylitis, osteoporosis, osteomalacia and gout. The accumulation of physiologically different chemical elements in different types of bones in humans of different ages allows for a more accurate assessment of the causal quoted response from parallel biochemical systems. These allow a better understanding of the link between exposure arising from smoking, alcohol, drugs, diseases, heredity, effects of amalgam dental fillings, diet, food preferences, chemical elements, occupational and environmental exposure to toxicants, lipoperoxidation and pro-antioxidant reactions. Physiology of the osteoarticular system determines what is responsible for bone and long-term body accumulation of toxic metals, which may be involved in the pathogenesis of bone diseases. While relationships between stressors, antioxidant system and bone condition predict potential risks in certain abnormalities and change oxidative stress. Elemental instability in the environment combined with salinity, acidity, redox potential and local changes in hydrochemical balance, enhances adverse effects. Processes of remodeling and bone mineralization continue throughout life and therefore may be a determinant of long-term accumulation of toxicants. Environmental issues affecting bioaccumulation of chemical elements in the osteoarticular system in humans is poorly understood. Alloplastic procedures, including the need for prostheses, supplemented by image layouts oxidative enzyme activities, as well as lipoperoxidation and the level of stress proteins, give a complete picture of skeletal response to external stressors. Simultaneously, the analysis of the impact of stressors on bones allows a more accurate tracing of causal quoted responses from parallel reactions. They have a direct relationship with stressors and affect the nature and degree of responses and defense capabilities. Thus they have a role in the diagnosis of diseases of the skeletal system.

Development of PEGylated carboxylic acid-modified polyamidoamine dendrimers as bone-targeting carriers for the treatment of bone diseases

In this study, we aimed to develop a polyethylene glycol (PEG)-conjugated third generation polyamidoamine (PAMAM) dendrimer with multiple carboxylic acids as a bone-targeting carrier for the treatment of bone diseases. We conjugated PAMAM backbones to various carboxylic acids [aspartic acid (Asp), glutamic acid (Glu), succinic acid (Suc), or aconitic acid (Aco)] to obtain four different types of carboxylic acid-modified PAMAMs. PEG was covalently bound to carboxylic acid-modified PAMAMs to obtain PEGylated carboxylic acid-modified PAMAMs. In a tissue distribution study, the amount of 111In-labeled unmodified PAMAM taken up by the bone after intravenous injection in mice was 11.3%. In contrast, the dose of 111In-labeled PEG(5)-Asp-PAMAM, PEG(5)-Glu-PAMAM, PEG(5)-Suc-PAMAM, or PEG(5)-Aco-PAMAM that accumulated in the bone after injection was approximately 46.0, 15.6, 22.6, and 24.5%, respectively. The bone clearance rates of 111In-labeled PEGylated carboxylic acid-modified PAMAMs were proportional to their affinities to hydroxyapatite and Ca2+. An intra-bone distribution study showed that fluorescein isothiocyanate-labeled PEG(5)-Asp-PAMAM predominantly accumulated on eroded and quiescent surfaces, a pattern associated with the pathogenesis of bone diseases, such as rheumatoid arthritis and osteoporosis. Our findings indicate that PEG(5)-Asp-PAMAM is a promising drug carrier for efficient drug targeting to the bones.

Uremic Serum Impairs Osteogenic Differentiation of Human Bone Marrow Mesenchymal Stromal Cells.

Chronic Kidney Disease-Mineral and Bone Disorder (CKD-MBD) is characterized by an increased fracture risk. Bone marrow mesenchymal stromal cells (BMSCs) may be involved in the pathogenesis of bone disease and, in view of their promising potential applications in bone tissue engineering, the effect of uremia on BMSCs regenerative potential represents a central issue. The present study evaluated in vitro the effect of a serum pool from hemodialysis patients on BMSCs to observe its influence on osteogenic differentiation. Besides alterations in spatial organization and cytotoxicity along with hyperproliferation, gene expression analysis suggested an impairment in the osteogenic differentiation. More importantly, Receptor activator of nuclear factor kappa-B ligand (RANKL) was upregulated with a mild reduction in osteoprotegerin levels. In summary, uremic environment seems to impair BMSCs osteogenic differentiation. Moreover BMSCs themselves may enhance osteoclastogenesis, feasibly contributing to the altered bone remodeling in CKD-MBD patients. J. Cell. Physiol. 232: 2201-2209, 2017. © 2016 Wiley Periodicals, Inc.

The effect of nephrectomy on Klotho, FGF-23 and bone metabolism

Background Increased concentration of fibroblast growth factor 23 (FGF-23) and decreased levels of soluble Klotho (sKL) are linked to negative clinical outcomes among patients with chronic kidney disease and acute kidney injury. Therefore, it is reasonable to hypothesize that GFR reduction caused by nephrectomy might alter mineral metabolism and induces adverse consequences. Whether nephrectomy due to urological indications causes derangements in FGF-23 and sKL has not been studied. The aim of the study was to evaluate the effect of acute GFR decline due to unilateral nephrectomy on bone metabolism, FGF-23 and sKL levels.MethodsThis is a prospective, single-centre observational study of patients undergoing nephrectomy due to urological indications. Levels of C-terminal FGF-23 (c-FGF-23), sKL and bone turnover markers [β-crosslaps (CTX), bone-specific alkaline phosphatase (bALP) and tartrate-resistant acid phosphatase 5b (TRAP 5b)] were measured before and after surgery (5 ± 2 days).ResultsTwenty-nine patients were studied (14 females, age 63.0 ± 11.6, eGFR 87.3 ± 19.2 ml/min/1.73 m2). After surgery, eGFR significantly declined (p < 0.0001). Nephrectomy significantly decreased sKL level [709.8 (599.9–831.2) vs. 583.0 (411.7–752.6) pg/ml, p < 0.001] and did not change c-FGF-23 concentration [70.5 (49.8–103.3) vs. 77.1 (60.5–109.1) RU/ml, p = 0.9]. Simultaneously, alterations in bone turnover markers were observed. Serum concentration of CTX increased [0.49 (0.4–0.64) vs. 0.59 (0.46–0.85) ng/ml, p = 0.001], while bALP and TRAP 5b decreased [23.6 (18.8–31.4) vs. 17.9 (15.0–22.0) U/l, p < 0.0001 and 3.3 (3.0–3.7) vs. 2.8 (2.3–3.2) U/l, p < 0.001, respectively].ConclusionsNephrectomy among patients with preserved renal function before surgery does not increase c-FGF-23 but reduces sKL. Moreover, nephrectomy results in derangements in bone turnover markers in short-term follow-up. These changes may participate in pathogenesis of bone disease after nephrectomy.

A single nucleotide polymorphism in the TGF-β1 gene (rs1982073 C>T) may contribute to increased risks of bone fracture, osteoporosis, and osteoarthritis: a meta-analysis.

Genetic factors have been shown to be of great importance for the pathogenesis of bone diseases, such as fracture, osteoporosis (OP), and osteoarthritis (OA). However, published studies on the correlations of transforming growth factor-β1 (TGF-β1) gene polymorphisms with bone diseases have been hampered by small sample sizes or inconclusive findings. We hence aimed at examining the relationships between a single nucleotide polymorphism in the TGF-β1 gene (rs1982073 C>T) with bone fracture, OP, and OA risks in this meta-analysis. A systematic electronic search of literature was conducted to identify all published studies in English or Chinese on the association between the TGF-β1 gene and fracture, OP, or OA risks. Data were abstracted independently by two reviewers. To investigate the strength of this relationship, crude odds ratios with 95% confidence intervals were used. An updated meta-analysis based on nine independent case-control studies were chosen (patients with fracture, OP, or OA = 1569; healthy controls = 1638). Results identified a higher frequency of rs1982073 C>T in patients with fracture, OP, or OA than in healthy controls. Ethnicity and genotyping method-stratified analysis under both models implied that the rs1982073 C>T polymorphism was positively correlated with the risk of fracture, OP, and OA among Asians under detection via the non-PCR-RFLP method. Disease-stratified results yielded that rs1982073 C>T may increase the risk of fracture, OP, and OA under the allele model, but was only significantly related to OP under the dominant model. According to the sample size-stratified analysis, subjects with the rs1982073 C>T polymorphism in the allele model were more likely to develop the three bone diseases in both the small and large sample size groups, and only in the large sample size under the dominant model. Our findings show that TGF-β1 rs1982073 C>T has a modest effect in increasing susceptibility to bone fracture, OP, and OA.

In vitro and in vivo angiogenic activity of osteoarthritic and osteoporotic osteoblasts is modulated by VEGF and vitamin D3 treatment

Vascular Endothelial Growth Factor (VEGF) is a potent angiogenic factor, which also regulates bone remodeling. Osteoblasts not only respond to VEGF stimulation, but also express and synthesize this factor. The present study was aimed to evaluate in vitro differences in VEGF production and expression of cultured human osteoblastic cells derived from healthy donors and from subjects affected by osteoarthritis and osteoporosis, under basal conditions than after vitamin D3, and to investigate the angiogenic activity of culture media obtained by these cells in chick embryo chorioallantoic membrane (CAM) assay. The results showed that normal and pathological osteoblasts produce and express VEGF and 1,25 dihydroxy-vitamin D3 treatment increases protein and m-RNA VEGF levels. In addition culture media of pathological osteoblasts induce a strong angiogenic response, greater than observed with culture medium of normal cells, suggesting the involvement of osteoblast-derived VEGF in the pathogenesis of bone diseases.

TNF-alpha modulates alveolar bone repair under homeostatic conditions in mice (44.1)

Abstract Cytokines have been implicated in the pathogenesis of bone diseases, but its role in bone repair process remains unknown. Our objective was to characterize the role of TNF-alpha in alveolar bone healing after tooth extraction in mice. Following the extraction of the right upper incisor of C57Bl/6(WT) and TNFp55KO strains, the maxilla containing the remaining alveolus was collected for histomorphometric and molecular (RealTimePCR) analysis. In WT mice the initial formation of clot was followed by the transient inflammatory infiltrate and a granulation tissue, gradually substituted by bone. In WT mice TNF-a mRNA was detected with an initial peak at 7 days, followed by a decreasing expression over time and a second peak at 28 days. Bone markers expression presented an initial high expression of COL-I, followed by CBFA-1 and ALP and subsequently by OCN and PHEX. TNFp55KO strain presented a delayed angiogenesis and osteogenesis processes, increased inflammatory reaction, despite of a decrease in CXCL1, CCL-2 and CCL3 levels; and increased COL-I mRNA levels and decreased CBFA-1, ALP, OCN and PHEX expression. The results presented here demonstrate that the absence of TNF-alpha interferes in alveolar bone repair through mechanisms that involve the modulation of inflammatory cell migration and osteogenic markers expression during the course of alveolar bone repair.

HRpn13, a newly identified component of the 19S particle, regulates proliferation, differentiation, and function in the human osteoblast-like cell line MG63

The 26S proteasome is a key component of the ubiquitin-proteasome system, a process responsible for the majority of cellular protein degradation. The function of the proteasomal ubiquitin receptor hRpn13, a component of the 26S proteasome, is not completely understood. To investigate the role of hRpn13 in the ubiquitin-proteasome system in osteoblasts, the effects of suppressing and overexpressing the hRpn13 gene on proliferation, differentiation, and function of human osteoblast-like MG63 cells were examined. After knockdown of hRpn13 by small interfering RNA, changes in osteoblast proliferation were evaluated by methyl-thiazolyl-tetrazolium assay. There was an increase in markers for osteoblast proliferation, specifically alkaline phosphatase activity, and elevated protein levels of osteocalcin, proliferating cell nuclear antigen (PCNA), and ubiquitin. Furthermore, hRpn13 knockdown also resulted in a decrease in the ratio between the gene expressions of RANKL and OPG, key players in the pathogenesis of bone diseases that influence the normal balance between bone formation and resorption. In contrast, overexpression of hRpn13 inhibited the proliferation of MG63 cells, and decreased alkaline phosphatase activity as well as protein levels of osteocalcin, PCNA, and ubiquitin while the ratio of RANKL to OPG expression increased. To confirm the function of hRpn13 in the ubiquitin-proteasome pathway, osteoblast proliferation enhancement and ubiquitin accumulation after hRpn2 knockdown was assessed. The results suggest that overexpression of hRpn13 negatively influences proliferation and osteogenic differentiation in MG63 cells. The evidence implies that hRpn13 modulates the influence of osteoblasts on osteoclasts by controlling the stability of regulatory proteins in osteoblasts. In summary, overexpression of hRpn13 promoted the activity of the ubiquitin-proteasome system.

Bone Mineral Density Changes In Patients with Paraproteinemia After Treatment with Bortezomib

AbstractAbstract 4989 Background:Bone disease is present at diagnosis in almost all patients with multiple myeloma (MM) and can impact substantially on patient morbidity and quality of life. Decreased bone mineral density is also observed not only in MM but also in patients with monoclonal gammopathy of undetermined significance (MGUS). The pathogenesis of bone disease in MM is complex. The activity of proteasome inhibitor bortezomib has been linked to increased bone formation and osteoblastic activation. Evidence from the available clinical data indicates that bortezomib has a positive impact on bone health in MM and demonstrates a bone anabolic effect. Methods:We analyzed retrospectively 53 patients with MM and 16 with MGUS who have completed bone density at least at diagnosis. 21 patients have completed two bone density (3 MGUS and 18 MM). The bone density was obtained in all patients at baseline and in 16 patients repeated after bortezomib treatement with a median time of bortezomib exposure of 6 months. We analyzed T-score values at lumbar spine and at femoral neck. Results:With a median age of 66 years, 41 male and 28 female were analyzed. At baseline the mean lumbar spine T-score of all subjects and of 16 MM treated with bortezomib was -0.50 and -0.76 respectively. At baseline the mean femoral neck T-score for all subjects and for 16 MM treated with Bortezomib was -1.56 and -1.31 respectively. The baseline mean lumbar spine T-score for MGUS and MM was -0.71 and -0.43 respectively. The baseline mean femoral neck T-score of MGUS and MM was -1.61 and -1.54 respectively. In the group of 16 patients treated with Bortezomib we observed from baseline a change in lumbar bone mineral density T-score of 0.36 and at femoral neck bone density T-score of 0.25. Conclusion:These data show that patients treated with proteasome inhibitor showed moderate increment in bone mineral density at lumbar spine and at femoral neck. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

RANKL inhibition as a targeted approach in the management of osteoporosis

Cortical and trabecular bone undergo a continuous and balanced remodeling process, consisting of an osteoclast-mediated boneresorption phase and an osteoblast-mediated bone-formation phase. An imbalance in this process, which favours bone resorption, results in bone loss and in damage to the skeletal microarchitecture, as seen in clinical conditions such as osteoporosis, rheumatoid arthritis and bone metastases. Currently, treatments are available for preventing or reversing excessive bone resorption through osteoclast inhibition. Among anti-resorptive treatments, nitrogen-containing bisphosphonates bind to hydroxyapatite with high affinity, are internalized by osteoclasts during the resorptionphase and inhibit osteoclast function, suppressing the mevalonate pathway. A more targeted anti-resorptive approach might be achieved through inhibition of RANK ligand (RANKL), which is one of the primary mediators of osteoclast activity, essential for osteoclast differentiation, function and survival. RANKL exerts direct catabolic effects on cortical and trabecular bone, leading to decreases in bone volume, mineral density and strength. Understanding the role of RANKL in the pathogenesis of bone disease has led to investigation of RANKL inhibition in conditions characterized by excessive bone resorption. In many preclinical models of postmenopausal osteoporosis, RANKL inhibition has prevented bone loss and microarchitecture damage, and has been associated with increased vertebral and femoral bone strength. A variety of innovative therapeutic approaches targeting RANKL inhibition are currently under investigation in humans. In randomized, controlled clinical trials, 6monthly subcutaneous administration of an experimental, fully human, monoclonal antibody against RANKL to womenwith osteopenia or postmenopausal osteoporosis significantly increased bone mineral density (BMD) at all skeletal sites (including the lumbar spine, femoral neck and distal radius) and decreased markers of bone turnover in a rapid, pronounced and sustained manner. Moreover, in two clinical studies, this therapeutic approach produced significantly greater increases in BMD than alendronate at all measured skeletal sites after 12 months of treatment. RANKL inhibition with this monoclonal antibody has recently been demonstrated to significantly reduce the risk of vertebral, non-vertebral and hip fractures inwomen with postmenopausal osteoporosis after 3 years of treatment. The frequency and pattern of reported adverse events have been similar to placebo or bisphosphonate treatment, suggesting a good tolerability profile. These data suggest that RANKL inhibition offers an additional and innovative therapeutic approach for the treatment of postmenopausal osteoporosis and, on the basis of comparative BMD results, may provide an incremental clinical benefit compared with currently available antiresorptive treatments.

RAGE deficiency induces a proinflammatory phenotype in bones and osteoblasts through PPAR-a suppression

Aims: Diabetes mellitus is associated with bone loss, increased fracture rate and reduced bone healing. Accumulation of advanced glycation end products (AGE) and associated overexpression of the receptor for AGE (RAGE) have been implicated in the pathogenesis of bone disease in diabetes. Although the AGE-RAGE interaction is well known for the sustained induction of inflammatory process, the physiologic role of RAGE in bone tissue is not clear. Remarkably, RAGE deficiency induces a proinflammatory phenotype in bones and osteoblasts. Therefore, we aimed to study the mechanism of underlying inflammatory processes in RAGE deficient osteoblasts as well as to reveal the intricate signaling events that link RAGE deficiency and inflammation in bones from RAGE knock-out (RAGE-/-) mice. Methods: Osteoblasts from wild type (WT, C57BL/6) and RAGE-/- mice were transiently transfected with different plasmids and siRNA. Reporter gene assay, western blotting, PCR, immunoprecipitation and chromatin immunoprecipitation techniques were employed to study the expression and interactions of transcription factors and coactivators involved in the signaling pathways. Results: The proinflammatory phenotype in the bones and osteoblasts from RAGE-/- mice was associated with reduced protein and gene expression and intranuclear translocation of the anti-inflammatory nuclear receptor PPAR-a. Moreover, PPAR-a activation by Wy14643 reversed inflammation in osteoblasts from RAGE-/- mice, suggesting that the lack of PPAR-a in RAGE deficiency is responsible for the proinflammatory phenotype. We therefore analyzed the PPAR-a promoter, which contains a series of Sp1 binding sites, to understand the regulation of PPAR-a gene expression. PPAR-a promoter driven reporter gene expression was much lower in RAGE-/- osteoblasts compared to WT osteoblasts. In addition, Sp1 overexpression significantly increased PPAR-a promoter activity in WT osteoblasts, but had only marginal effects in RAGE-/- osteoblasts. Sequential promoter deletions confirmed the strong Sp1 dependency in WT osteoblasts, which was largely blunted in RAGE-/- osteoblasts. However, suppression of Sp1 by siRNA transfection decreased PPAR-a promoter expression by around 50% in both WT and RAGE-/- osteoblasts. Remarkably, endogenous Sp1 gene and protein expression was similar in osteoblasts from WT and RAGE-/- mice, suggesting that a cofactor for PPAR-a gene expression is missing in RAGE-/- osteoblasts. Noteworthy, preliminary data show that the PPAR-g coactivator-1a (PGC-1a) is markedly reduced in RAGE-/- osteoblasts compared to WT osteoblasts. Conclusions: RAGE deficiency induces a proinflammatory phenotype in bones and osteoblasts through PPAR-a insufficiency. Transcription factor Sp1 is a strong regulator of PPAR-a expression however this regulation is inadequate in RAGE deficiency most likely due to the absence of an essential cofactor such as PGC-1a.

Redução da densidade mineral óssea em mulheres na menacme com prolactinoma

Tumoral hyperprolactinemia and consequent hypogonadism have been associated with osteoporosis. Bone mineral density (BMD) was measured by dual-energy RX absorptiometry in 24 patients with prolactinoma (15 macro and 9 micro adenomas; age range = 18 to 49 years). Student unpaired t or Mann-Whitney tests were used to compare groups, and Spearman test studied correlations. Lumbar spine (LS) was the most affected, as LS Z-score was < -2 SD in 20.83% of the patients. No difference was found in densitometric parameters for the comparison between macro and microprolactinoma, or those with normal prolactin versus hyperprolactinemia. LS BMD and LS Z-score were higher in the patients with > 8 menstrual cycles in the preceding year then in those with oligoamenorrhea (p = 0.030). The number of cycles was correlated to LS BMD (r = 0.515, p = 0.017) and body mass index to femoral neck BMD (r = 0.563, p = 0.006) and total femur BMD (r = 0.529, p = 0.011). Decreased bone mineral density was detected in 20.83% of our young patients with prolactinoma. The great involvement of trabecular bone skeletal regions, such as vertebrae, suggests the participation of hypogonadism in the pathogenesis of bone disease. Irrespective of prolactin levels, return to normal menses seems the best index of good control.

Congenital Bone Fractures in Spinal Muscular Atrophy: Functional Role for SMN Protein in Bone Remodeling

Spinal muscular atrophy is the second most common fatal childhood disorder. Core clinical features include muscle weakness caused by degenerating lower motor neurons and a high incidence of bone fractures and hypercalcemia. Fractures further compromise quality of life by progression of joint contractures or additional loss of motor function. Recent observations suggest that bone disease in spinal muscular atrophy may not be attributed entirely to lower motor neuron degeneration. The presence of the spinal muscular atrophy disease-determining survival motor neuron gene (SMN), SMN expression, and differential splicing in bone-resorbing osteoclasts was recently discovered. Its ubiquitous expression and the differential expression of splice variants suggest that SMN has specific roles in bone cell function. SMN protein also interacts with osteoclast stimulatory factor. Mouse models of human spinal muscular atrophy disease suggest a potential role of SMN protein in skeletal development. Dual energy x-ray absorptiometry analysis demonstrated a substantial decrease in total bone area and poorly developed caudal vertebra in the mouse model. These mice also had pelvic bone fractures. Studies delineating SMN signaling mechanisms and gene transcription in a cell-specific manner will provide important molecular insights into the pathogenesis of bone disease in children with spinal muscular atrophy. Moreover, understanding bone remodeling in spinal muscular atrophy may lead to novel therapeutic approaches to enhance skeletal health and quality of life. This article reviews the skeletal complications associated with spinal muscular atrophy and describes a functional role for SMN protein in osteoclast development and bone resorption activity.

Inhibition of RANKL as a treatment for osteoporosis: Preclinical and early clinical studies

Osteoporosis and several other bone disorders occur when there is an imbalance between the resorption and formation components of bone remodeling activity. Therapies available for some of these conditions modulate the activity of osteoclasts and/or osteoblasts. The recent discoveries of receptor activator of NF-kappaB ligand (RANKL), an endogenous activator of osteoclastogenenesis and osteoclast activity and its inhibitor, osteoprotegerin (OPG) as pivotal regulatory factors in the pathogenesis of bone diseases like osteoporosis provide unique targets for therapeutic agents. In laboratory animals and now in humans, administering forms of OPG markedly inhibits osteoclast activity and improves bone strength, documenting that the strategy of inhibiting RANKL activity has therapeutic promise. A highly specific, fully human antibody against RANKL has been produced (denosumab) that in early studies in humans reduces bone turnover and improves bone density. Attributes of denosumab in these clinical studies include a very rapid onset of action, sustained effects for several months after a single injection, and good tolerability. These results provide the basis for studies evaluating the effectiveness of denosumab in several clinical conditions characterized by increased osteoclastic activity.