RESUMO
Pathogenic variants disrupting the binding between sclerostin (encoded by SOST) and its receptor LRP4 have previously been described to cause sclerosteosis, a rare high bone mass disorder. The sclerostin-LRP4 complex inhibits canonical WNT signaling, a key pathway regulating osteoblastic bone formation and a promising therapeutic target for common bone disorders, such as osteoporosis. In the current study, we crossed mice deficient for Sost (Sost-/-) with our p.Arg1170Gln Lrp4 knock-in (Lrp4KI/KI) mouse model to create double mutant Sost-/-;Lrp4KI/KI mice. We compared the phenotype of Sost-/- mice with that of Sost-/-;Lrp4KI/KI mice, to investigate a possible synergistic effect of the disease-causing p.Arg1170Trp variant in Lrp4 on Sost deficiency. Interestingly, presence of Lrp4KI alleles partially mitigated the Sost-/- phenotype. Cellular and dynamic histomorphometry did not reveal mechanistic insights into the observed phenotypic differences. We therefore determined the molecular effect of the Lrp4KI allele by performing bulk RNA sequencing on Lrp4KI/KI primary osteoblasts. Unexpectedly, mostly genes related to bone resorption or remodeling (Acp5, Rankl, Mmp9) were upregulated in Lrp4KI/KI primary osteoblasts. Verification of these markers in Lrp4KI/KI, Sost-/- and Sost-/-;Lrp4KI/KI mice revealed that sclerostin deficiency counteracts this Lrp4KI/KI effect in Sost-/-;Lrp4KI/KI mice. We therefore hypothesize that models with two inactivating Lrp4KI alleles rather activate bone remodeling, with a net gain in bone mass, whereas sclerostin deficiency has more robust anabolic effects on bone formation. Moreover, these effects of sclerostin and Lrp4 are stronger in female mice, contributing to a more severe phenotype than in males and more detectable phenotypic differences among different genotypes.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Remodelação Óssea , Hiperostose , Sindactilia , Masculino , Feminino , Animais , Camundongos , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Camundongos Knockout , Fenótipo , Mutação , Remodelação Óssea/genética , Proteínas Relacionadas a Receptor de LDL/genética , Proteínas Relacionadas a Receptor de LDL/metabolismoRESUMO
A new therapeutic option to treat osteoporosis is focused on Wnt signaling and its inhibitor sclerostin, a product of the Sost gene. In this work, we study the effect of sclerostin deficiency on trabecular bone formation and resorption in male and female mice and whether it affects mechano-responsiveness. Male and female 10- and 26-week-old Sost knockout (KO) and littermate controls (LCs) were subjected to in vivo mechanical loading of the left tibia for 2 weeks. The right tibia served as internal control. The mice were imaged using in vivo micro-computed tomography at days 0, 5, 10, and 15 and tibiae were collected for histomorphometric analyses after euthanasia. Histomorphometry and micro-CT-based 3D time-lapse morphometry revealed an anabolic and anti-catabolic effect of Sost deficiency although increased trabecular bone resorption accompanied by diminished trabecular bone formation occurred with age. Loading led to diminished resorption in adult female but not in male mice. A net gain in bone volume could be achieved with mechanical loading in Sost KO adult female mice, which occurred through a further reduction in resorbed bone volume. Our data show that sclerostin deficiency has a particularly positive effect in adult female mice. Sclerostin antibodies are approved to treat postmenopausal women with high risk of osteoporotic fractures. Further studies are required to clarify whether both sexes benefit equally from sclerostin inhibition.
Assuntos
Reabsorção Óssea/metabolismo , Osso e Ossos/metabolismo , Osso Esponjoso/metabolismo , Osteoporose/metabolismo , Tempo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Feminino , Glicoproteínas/metabolismo , Masculino , Camundongos , Osteogênese/efeitos dos fármacos , Osteogênese/fisiologia , Microtomografia por Raio-X/métodosRESUMO
Multiple myeloma (MM) is a plasma cell cancer that develops in the skeleton causing profound bone destruction and fractures. The bone disease is mediated by increased osteoclastic bone resorption and suppressed bone formation. Bisphosphonates used for treatment inhibit bone resorption and prevent bone loss but fail to influence bone formation and do not replace lost bone, so patients continue to fracture. Stimulating bone formation to increase bone mass and fracture resistance is a priority; however, targeting tumor-derived modulators of bone formation has had limited success. Sclerostin is an osteocyte-specific Wnt antagonist that inhibits bone formation. We hypothesized that inhibiting sclerostin would prevent development of bone disease and increase resistance to fracture in MM. Sclerostin was expressed in osteocytes from bones from naive and myeloma-bearing mice. In contrast, sclerostin was not expressed by plasma cells from 630 patients with myeloma or 54 myeloma cell lines. Mice injected with 5TGM1-eGFP, 5T2MM, or MM1.S myeloma cells demonstrated significant bone loss, which was associated with a decrease in fracture resistance in the vertebrae. Treatment with anti-sclerostin antibody increased osteoblast numbers and bone formation rate but did not inhibit bone resorption or reduce tumor burden. Treatment with anti-sclerostin antibody prevented myeloma-induced bone loss, reduced osteolytic bone lesions, and increased fracture resistance. Treatment with anti-sclerostin antibody and zoledronic acid combined increased bone mass and fracture resistance when compared with treatment with zoledronic acid alone. This study defines a therapeutic strategy superior to the current standard of care that will reduce fractures for patients with MM.
Assuntos
Densidade Óssea/efeitos dos fármacos , Proteínas Morfogenéticas Ósseas/antagonistas & inibidores , Fraturas Ósseas/prevenção & controle , Osteócitos/química , Osteogênese/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal , Animais , Anticorpos/farmacologia , Anticorpos/uso terapêutico , Proteínas Morfogenéticas Ósseas/imunologia , Linhagem Celular Tumoral , Difosfonatos/uso terapêutico , Marcadores Genéticos/imunologia , Humanos , Imidazóis/uso terapêutico , Camundongos , Mieloma Múltiplo/complicações , Células Tumorais Cultivadas , Ácido ZoledrônicoRESUMO
The bone marrow niche is a dynamic and complex microenvironment that can both regulate, and be regulated by the bone matrix. Within the bone marrow (BM), mesenchymal stromal cell (MSC) precursors reside in a multi-potent state and retain the capacity to differentiate down osteoblastic, adipogenic, or chondrogenic lineages in response to numerous biochemical cues. These signals can be altered in various pathological states including, but not limited to, osteoporotic-induced fracture, systemic adiposity, and the presence of bone-homing cancers. Herein we provide evidence that signals from the bone matrix (osteocytes) determine marrow adiposity by regulating adipogenesis in the bone marrow. Specifically, we found that physiologically relevant levels of Sclerostin (SOST), which is a Wnt-inhibitory molecule secreted from bone matrix-embedded osteocytes, can induce adipogenesis in 3T3-L1 cells, mouse ear- and BM-derived MSCs, and human BM-derived MSCs. We demonstrate that the mechanism of SOST induction of adipogenesis is through inhibition of Wnt signaling in pre-adipocytes. We also demonstrate that a decrease of sclerostin in vivo, via both genetic and pharmaceutical methods, significantly decreases bone marrow adipose tissue (BMAT) formation. Overall, this work demonstrates a direct role for SOST in regulating fate determination of BM-adipocyte progenitors. This provides a novel mechanism for which BMAT is governed by the local bone microenvironment, which may prove relevant in the pathogenesis of certain diseases involving marrow adipose. Importantly, with anti-sclerostin therapy at the forefront of osteoporosis treatment and a greater recognition of the role of BMAT in disease, these data are likely to have important clinical implications.
Assuntos
Adipócitos/metabolismo , Adipogenia , Tecido Adiposo/metabolismo , Células da Medula Óssea/metabolismo , Glicoproteínas/metabolismo , Células-Tronco Mesenquimais/metabolismo , Osteócitos/metabolismo , Células 3T3-L1 , Proteínas Adaptadoras de Transdução de Sinal , Tecido Adiposo/citologia , Adiposidade , Animais , Meios de Cultivo Condicionados/metabolismo , Glicoproteínas/deficiência , Glicoproteínas/genética , Peptídeos e Proteínas de Sinalização Intercelular , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Comunicação Parácrina , Fenótipo , Nicho de Células-Tronco , Via de Sinalização WntRESUMO
Activating FGFR3 mutations in human result in achondroplasia (ACH), the most frequent form of dwarfism, where cartilages are severely disturbed causing long bones, cranial base and vertebrae defects. Because mandibular development and growth rely on cartilages that guide or directly participate to the ossification process, we investigated the impact of FGFR3 mutations on mandibular shape, size and position. By using CT scan imaging of ACH children and by analyzing Fgfr3Y367C/+ mice, a model of ACH, we show that FGFR3 gain-of-function mutations lead to structural anomalies of primary (Meckel's) and secondary (condylar) cartilages of the mandible, resulting in mandibular hypoplasia and dysmorphogenesis. These defects are likely related to a defective chondrocyte proliferation and differentiation and pan-FGFR tyrosine kinase inhibitor NVP-BGJ398 corrects Meckel's and condylar cartilages defects ex vivo. Moreover, we show that low dose of NVP-BGJ398 improves in vivo condyle growth and corrects dysmorphologies in Fgfr3Y367C/+ mice, suggesting that postnatal treatment with NVP-BGJ398 mice might offer a new therapeutic strategy to improve mandible anomalies in ACH and others FGFR3-related disorders.
Assuntos
Acondroplasia/genética , Cartilagem/anormalidades , Mandíbula/anormalidades , Côndilo Mandibular/anormalidades , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Acondroplasia/diagnóstico por imagem , Acondroplasia/tratamento farmacológico , Acondroplasia/fisiopatologia , Animais , Cartilagem/crescimento & desenvolvimento , Cartilagem/fisiopatologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Condrócitos/metabolismo , Condrócitos/patologia , Modelos Animais de Doenças , Humanos , Mandíbula/crescimento & desenvolvimento , Mandíbula/fisiopatologia , Côndilo Mandibular/crescimento & desenvolvimento , Côndilo Mandibular/fisiopatologia , Camundongos , Osteogênese/efeitos dos fármacos , Osteogênese/genética , Compostos de Fenilureia/administração & dosagem , Inibidores de Proteínas Quinases/administração & dosagem , Pirimidinas/administração & dosagemRESUMO
Dickkopf-1 (DKK1) and sclerostin are antagonists of the Wnt/ß-catenin pathway and decreased expression of either results in increased bone formation and mass. As both affect the same signaling pathway, we aimed to elucidate the redundancy and/or compensation of sclerostin and DKK1. Weekly sclerostin antibody (Scl-Ab) was used to treat 9-week-old female Dkk1 KO (Dkk1-/-:Wnt3+/-) mice and compared to Scl-Ab-treated wild-type mice as well as vehicle-treated Dkk1 KO and wild-type animals. While Wnt3 heterozygote (Wnt3+/-) mice show no bone phenotype, Scl-Ab and vehicle-treated control groups of this genotype were included. Specimens were harvested after 3 weeks for microCT, bone histomorphometry, anti-sclerostin immunohistochemistry, and biomechanical testing. Scl-Ab enhanced bone anabolism in all treatment groups, but with synergistic enhancement seen in the cancellous compartment of Dkk1 KO mice (bone volume + 55% Dkk1 KO p < 0.01; + 22% wild type p < 0.05). Scl-Ab treatment produced less marked increases in cortical bone of the tibiae, with anabolic effects similar across genotypes. Mechanical testing confirmed that Scl-Ab improved strength across all genotypes; however, no enhancement was seen within Dkk1 KO mice. Dynamic bone labeling showed that Scl-Ab treatment was associated with increased bone formation, regardless of genotype. Immunohistochemical staining for sclerostin protein indicated no differences in the Dkk1 KO mice, indicating that the increased Wnt signaling associated with DKK1 deficiency was not compensated by upregulation of sclerostin protein. These data suggest complex interactions between Wnt signaling factors in bone, but critically illustrate synergy between DKK1 deficiency and Scl-Ab treatment. These data support the application of dual-targeted therapeutics in the modulation of bone anabolism.
Assuntos
Osso e Ossos/efeitos dos fármacos , Osso e Ossos/fisiologia , Glicoproteínas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Osteogênese/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Anticorpos Monoclonais/farmacologia , Feminino , Camundongos , Camundongos Knockout , Osteogênese/efeitos dos fármacosRESUMO
We identified previously in vitro LRP4 (low-density lipoprotein receptor-related protein 4) as a facilitator of the WNT (Wingless-type) antagonist sclerostin and found mutations disrupting this function to be associated with high bone mass in humans similar to patients lacking sclerostin. To further delineate the role of LRP4 in bone in vivo, we generated mice lacking Lrp4 in osteoblasts/osteocytes or osteocytes only. Lrp4 deficiency promoted progressive cancellous and cortical bone gain in both mutants, although more pronouncedly in mice deficient in osteoblast/osteocyte Lrp4, consistent with our observation in human bone that LRP4 is most strongly expressed by osteoblasts and early osteocytes. Bone gain was related primarily to increased bone formation. Interestingly, Lrp4 deficiency in bone dramatically elevated serum sclerostin levels whereas bone expression of Sost encoding for sclerostin was unaltered, indicating that osteoblastic Lrp4 retains sclerostin within bone. Moreover, we generated anti-LRP4 antibodies selectively blocking sclerostin facilitator function while leaving unperturbed LRP4-agrin interaction, which is essential for neuromuscular junction function. These antibodies increased bone formation and thus cancellous and cortical bone mass in skeletally mature rodents. Together, we demonstrate a pivotal role of LRP4 in bone homeostasis by retaining and facilitating sclerostin action locally and provide a novel avenue to bone anabolic therapy by antagonizing LRP4 sclerostin facilitator function.
Assuntos
Densidade Óssea , Osso e Ossos/metabolismo , Glicoproteínas/sangue , Receptores de LDL/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Idoso , Agrina/metabolismo , Animais , Anticorpos Bloqueadores/farmacologia , Linhagem Celular , Feminino , Colo do Fêmur/microbiologia , Expressão Gênica , Glicoproteínas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Proteínas Relacionadas a Receptor de LDL , Masculino , Camundongos Knockout , Microscopia Confocal , Junção Neuromuscular/metabolismo , Osteoblastos/metabolismo , Osteócitos/metabolismo , Osteogênese/genética , Ligação Proteica , Ratos Wistar , Receptores de LDL/antagonistas & inibidores , Receptores de LDL/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Microtomografia por Raio-XRESUMO
Renal osteodystrophy affects the majority of patients with advanced chronic kidney disease (CKD) and is characterized by progressive bone loss. This study evaluated the effects of sclerostin knockout on bone in a murine model of severe, surgically induced CKD in both sclerostin knockout and wild-type mice. Mice of both genotypes with normal kidney function served as controls. Tibiae were analyzed using micro-computed tomography, and lumbar vertebrae were analyzed by histomorphometry. Results were tested for statistical significance by 2-way ANOVA to investigate whether bone of the knockout mice reacted differently to CKD compared with bone of wild-type mice. In the tibiae, there was no difference after creation of CKD between wild-type and knockout animals for cortical thickness or cross-sectional moment of inertia. Increases in cortical porosity induced by CKD differed significantly between genotypes in the tibial metaphysis but not in the diaphysis. In the trabecular compartment, no difference in reaction to CKD between genotypes was found for bone volume, trabecular number, trabecular thickness, and trabecular separation. In the lumbar vertebrae, significant differences in response to CKD between wild-type and knockout mice were seen for both bone volume and trabecular thickness. Osteoblast parameters did not differ significantly, whereas osteoclast numbers significantly increased in the wild-type but significantly decreased in knockout mice with CKD. No differences in response to CKD between genotypes were found for bone formation rate or mineral apposition rate. Thus, complete absence of sclerostin has only minor effects on CKD-induced bone loss in mice.
Assuntos
Densidade Óssea , Distúrbio Mineral e Ósseo na Doença Renal Crônica/etiologia , Glicoproteínas/genética , Osteogênese , Insuficiência Renal Crônica/complicações , Proteínas Adaptadoras de Transdução de Sinal , Animais , Osso e Ossos/citologia , Osso e Ossos/patologia , Distúrbio Mineral e Ósseo na Doença Renal Crônica/genética , Distúrbio Mineral e Ósseo na Doença Renal Crônica/metabolismo , Modelos Animais de Doenças , Feminino , Genótipo , Glicoproteínas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteoblastos/patologia , Insuficiência Renal Crônica/metabolismo , Tíbia , Microtomografia por Raio-XRESUMO
BACKGROUND: No treatment to date is available which specifically targets bone formation in ankylosing spondylitis (AS). Several recent studies have shown that sclerostin (SOST), a Wnt inhibitor specific to osteocytes and chondrocytes, is down-regulated in AS patients. This suggests Wnt signalling may be upregulated, and application of exogenous recombinant SOST (rSOST) may inhibit Wnt signalling and slow pathological bone formation. METHODS: The proteoglycan-induced spondylitis (PGISp) mouse model in which we have previously demonstrated downregulated SOST expression, was used for this study. Mice were injected with 2.5 ug rSOST/day for a period of 8 weeks following induction of disease. Axial skeleton disease development was assessed by histology and skeletal changes examined using DEXA. RESULTS: rSOST treatment had no effect on peripheral or axial disease development, bone density or disease severity. Injected rSOST was stable over 8 h and residual levels were evident 24 h after injection, resulting in a cumulative increase in SOST serum levels over the treatment time course. Immunohistochemical examination of SOST levels within the joints in non-rSOST treated PGISp mice showed a significant decrease in the percentage of positive osteocytes in the unaffected joints compared to the affected joints, while no difference was seen in rSOST treated mice. This suggests that rSOST treatment increases the number of SOST-positive osteocytes in unaffected joints but not affected joints, despite having no impact on the number of joints affected by disease. CONCLUSIONS: Although not disease-modifying, rSOST treatment did appear to regulate SOST levels in the joints suggesting biological activity. Further dose response studies are required and SOST may require modifications to improve its bone targeting ability in order to affect tissue formation to a meaningful level in this model.
Assuntos
Proteínas Morfogenéticas Ósseas/administração & dosagem , Modelos Animais de Doenças , Progressão da Doença , Espondilite Anquilosante/tratamento farmacológico , Espondilite Anquilosante/patologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Feminino , Marcadores Genéticos , Células HEK293 , Humanos , Injeções Subcutâneas , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Resultado do TratamentoRESUMO
Intermittent parathyroid hormone (iPTH) treatment stimulates T-cell production of the osteogenic Wnt ligand Wnt10b, a factor required for iPTH to activate Wnt signaling in osteoblasts and stimulate bone formation. However, it is unknown whether iPTH induces Wnt10b production and bone anabolism through direct activation of the parathyroid hormone (PTH)/PTH-related protein receptor (PPR) in T cells. Here, we show that conditional silencing of PPR in T cells blunts the capacity of iPTH to induce T-cell production of Wnt10b; activate Wnt signaling in osteoblasts; expand the osteoblastic pool; and increase bone turnover, bone mineral density, and trabecular bone volume. These findings demonstrate that direct PPR signaling in T cells plays an important role in PTH-induced bone anabolism by promoting T-cell production of Wnt10b and suggest that T cells may provide pharmacological targets for bone anabolism.
Assuntos
Osso e Ossos/metabolismo , Hormônio Paratireóideo/metabolismo , Receptor Tipo 1 de Hormônio Paratireóideo/genética , Receptor Tipo 1 de Hormônio Paratireóideo/metabolismo , Animais , Densidade Óssea , Feminino , Inativação Gênica , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Biológicos , Osteoblastos/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Proteínas Wnt/metabolismo , Microtomografia por Raio-X/métodosRESUMO
Proper regulation of Wnt signaling is critical for normal bone development and homeostasis. Mutations in several Wnt signaling components, which increase the activity of the pathway in the skeleton, cause high bone mass in human subjects and mouse models. Increased bone mass is often accompanied by severe headaches from increased intracranial pressure, which can lead to fatality and loss of vision or hearing due to the entrapment of cranial nerves. In addition, progressive forehead bossing and mandibular overgrowth occur in almost all subjects. Treatments that would provide symptomatic relief in these subjects are limited. Porcupine-mediated palmitoylation is necessary for Wnt secretion and binding to the frizzled receptor. Chemical inhibition of porcupine is a highly selective method of Wnt signaling inhibition. We treated three different mouse models of high bone mass caused by aberrant Wnt signaling, including homozygosity for loss-of-function in Sost, which models sclerosteosis, and two strains of mice carrying different point mutations in Lrp5 (equivalent to human G171V and A214V), at 3 months of age with porcupine inhibitors for 5-6 weeks. Treatment significantly reduced both trabecular and cortical bone mass in all three models. This demonstrates that porcupine inhibition is potentially therapeutic for symptomatic relief in subjects who suffer from these disorders and further establishes that the continued production of Wnts is necessary for sustaining high bone mass in these models.
Assuntos
Mutação com Ganho de Função , Hiperostose , Animais , Humanos , Camundongos , Proteínas Adaptadoras de Transdução de Sinal , Secreções Corporais , Modelos Animais de Doenças , Hiperostose/genética , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , MutaçãoRESUMO
Humans lacking sclerostin display progressive bone overgrowth due to increased bone formation. Although it is well established that sclerostin is an osteocyte-secreted bone formation inhibitor, the underlying molecular mechanisms are not fully elucidated. We identified in tandem affinity purification proteomics screens LRP4 (low density lipoprotein-related protein 4) as a sclerostin interaction partner. Biochemical assays with recombinant proteins confirmed that sclerostin LRP4 interaction is direct. Interestingly, in vitro overexpression and RNAi-mediated knockdown experiments revealed that LRP4 specifically facilitates the previously described inhibitory action of sclerostin on Wnt1/ß-catenin signaling. We found the extracellular ß-propeller structured domain of LRP4 to be required for this sclerostin facilitator activity. Immunohistochemistry demonstrated that LRP4 protein is present in human and rodent osteoblasts and osteocytes, both presumed target cells of sclerostin action. Silencing of LRP4 by lentivirus-mediated shRNA delivery blocked sclerostin inhibitory action on in vitro bone mineralization. Notably, we identified two mutations in LRP4 (R1170W and W1186S) in patients suffering from bone overgrowth. We found that these mutations impair LRP4 interaction with sclerostin and its concomitant sclerostin facilitator effect. Together these data indicate that the interaction of sclerostin with LRP4 is required to mediate the inhibitory function of sclerostin on bone formation, thus identifying a novel role for LRP4 in bone.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas Relacionadas a Receptor de LDL/metabolismo , Osteócitos/metabolismo , Osteogênese , Proteínas Adaptadoras de Transdução de Sinal , Substituição de Aminoácidos , Animais , Proteínas Morfogenéticas Ósseas/genética , Marcadores Genéticos/genética , Células HEK293 , Humanos , Proteínas Relacionadas a Receptor de LDL/genética , Camundongos , Mutação de Sentido Incorreto , Transdução de Sinais/genética , Proteína Wnt1/genética , Proteína Wnt1/metabolismo , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Osteoarthritis (OA) is a common, debilitating, chronic disease with no disease-modifying drug approved to date. We discovered LNA043-a derivative of angiopoietin-like 3 (ANGPTL3)-as a potent chondrogenesis inducer using a phenotypic screen with human mesenchymal stem cells. We show that LNA043 promotes chondrogenesis and cartilage matrix synthesis in vitro and regenerates hyaline articular cartilage in preclinical OA and cartilage injury models in vivo. LNA043 exerts at least part of these effects through binding to the fibronectin receptor, integrin α5ß1 on mesenchymal stem cells and chondrocytes. In a first-in-human (phase 1), randomized, double-blinded, placebo-controlled, single ascending dose, single-center trial ( NCT02491281 ; sponsored by Novartis Pharmaceuticals), 28 patients with knee OA were injected intra-articularly with LNA043 or placebo (3:1 ratio) either 2 h, 7 d or 21 d before total knee replacement. LNA043 met its primary safety endpoint and showed short serum pharmacokinetics, cartilage penetration and a lack of immunogenicity (secondary endpoints). Post-hoc transcriptomics profiling of cartilage revealed that a single LNA043 injection reverses the OA transcriptome signature over at least 21 d, inducing the expression of hyaline cartilage matrix components and anabolic signaling pathways, while suppressing mediators of OA progression. LNA043 is a novel disease-modifying OA drug candidate that is currently in a phase 2b trial ( NCT04864392 ) in patients with knee OA.
Assuntos
Cartilagem Articular , Osteoartrite do Joelho , Humanos , Osteoartrite do Joelho/tratamento farmacológico , Condrócitos , Transdução de Sinais , Angiopoietinas/metabolismo , Angiopoietinas/farmacologia , Angiopoietinas/uso terapêutico , Proteína 3 Semelhante a AngiopoietinaRESUMO
A series of novel benzimidazole derivatives has been designed via a scaffold morphing approach based on known calcilytics chemotypes. Subsequent lead optimisation led to the discovery of penta-substituted benzimidazoles that exhibit attractive in vitro and in vivo calcium-sensing receptor (CaSR) inhibitory profiles. In addition, synthesis and structure-activity relationship data are provided.
Assuntos
Benzimidazóis/farmacologia , Receptores de Detecção de Cálcio/antagonistas & inibidores , Benzimidazóis/química , Benzimidazóis/farmacocinética , Modelos Moleculares , Relação Estrutura-AtividadeRESUMO
Loss-of-function mutations in the Sost gene lead to high bone mass phenotypes. Pharmacological inhibition of Sost/sclerostin provides a new drug strategy for treating osteoporosis. Questions remain as to how physical activity may affect bone mass under sclerostin inhibition and if that effect differs between males and females. We previously observed in female Sost knockout (KO) mice an enhanced cortical bone formation response to a moderate level of applied loading (900 µÎµ at the tibial midshaft). The purpose of the present study was to examine cortical bone adaptation to the same strain level applied to male Sost KO mice. Strain-matched in vivo compressive loading was applied to the tibiae of 10-, 26- and 52-week-old male Sost KO and littermate control (LC) mice. The effect of tibial loading on bone (re)modeling was measured by microCT, 3D time-lapse in vivo morphometry, 2D histomorphometry and gene expression analyses. As expected, Sost deficiency led to high cortical bone mass in 10- and 26-week-old male mice as a result of increased bone formation. However, the enhanced bone formation associated with Sost deficiency did not appear to diminish with skeletal maturation. An increase in bone resorption was observed with skeletal maturation in male LC and Sost KO mice. Two weeks of in vivo loading (900 µÎµ at the tibial midshaft) induced only a mild anabolic response in 10- and 26-week-old male mice, independent of Sost deficiency. A decrease in the Wnt inhibitor Dkk1 expression was observed 3 h after loading in 52-week-old Sost KO and LC mice, and an increase in Lef1 expression was observed 8 h after loading in 10-week-old Sost KO mice. The current results suggest that long-term inhibition of sclerostin in male mice does not influence the adaptive response of cortical bone to moderate levels of loading. In contrast with our previous strain-matched study in females showing enhanced bone responses with Sost ablation, these results in males indicate that the influence of Sost deficiency on the cortical bone formation response to a moderate level of loading differs between males and females. Clinical studies examining antibodies to inhibit sclerostin may need to consider that the efficacy of additional physical activity regimens may be sex dependent.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Hiperostose/genética , Osteogênese/genética , Estresse Mecânico , Sindactilia/genética , Animais , Reabsorção Óssea/genética , Reabsorção Óssea/fisiopatologia , Osso e Ossos/fisiopatologia , Osso Cortical/fisiologia , Feminino , Glicoproteínas/genética , Hiperostose/fisiopatologia , Masculino , Camundongos , Camundongos Knockout , Osteogênese/fisiologia , Sindactilia/fisiopatologiaRESUMO
Microgravity exposure is associated with loss of muscle mass and strength. The E3 ubiquitin ligase MuRF1 plays an integral role in degrading the contractile apparatus of skeletal muscle; MuRF1 null (KO) mice have shown protection in ground-based models of muscle atrophy. In contrast, MuRF1 KO mice subjected to 21 days of microgravity on the International Space Station (ISS) were not protected from muscle atrophy. In a time course experiment microgravity-induced muscle loss on the ISS showed MuRF1 gene expression was not upregulated. A comparison of the soleus transcriptome profiles between spaceflight and a publicly available data set for hindlimb suspension, a claimed surrogate model of microgravity, showed only marginal commonalities between the models. These findings demonstrate spaceflight induced atrophy is unique, and that understanding of effects of space requires study situated beyond the Earth's mesosphere.
Assuntos
Hipogravidade , Proteínas Musculares/deficiência , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Atrofia Muscular/etiologia , Atrofia Muscular/metabolismo , Voo Espacial , Proteínas com Motivo Tripartido/deficiência , Ubiquitina-Proteína Ligases/deficiência , Animais , Suscetibilidade a Doenças , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Elevação dos Membros Posteriores , Camundongos , Camundongos Knockout , Atrofia Muscular/patologia , Tamanho do ÓrgãoRESUMO
Decreased activity or expression of sclerostin, an endogenous inhibitor of Wnt/ß-catenin signaling, results in increased bone formation and mass. Antibodies targeting and neutralizing sclerostin (Scl-Ab) have been shown to increase bone mass and reduce fracture risk. Sclerostin is also important in modulating the response of bone to changes in its biomechanical environment. However, the effects of Scl-Ab on mechanotransduction are unclear, and it was speculated that the loading response may be altered for individuals receiving Scl-Ab therapy. To address this, we carried out a 2-week study of tibial cyclic compressive loading on C57Bl/6 mice treated with vehicle or 100 mg/kg/wk Scl-Ab. Increases in bone volume, density, and dynamic bone formation were found with loading, and the anabolic response was further increased by the combination of load and Scl-Ab. To investigate the underlying mechanism, gene profiling by RNA sequencing (RNAseq) was performed on tibias isolated from mice from all four experimental groups. Major alterations in Wnt/ß-catenin gene expression were found with tibial loading, however not with Scl-Ab treatment alone. Notably, the combination of load and Scl-Ab elicited a synergistic response from a number of specific Wnt-related and mechanotransduction factors. An unexpected finding was significant upregulation of factors in the Rho GTPase signaling pathway with combination treatment. In summary, combination therapy had a more profound anabolic response than either Scl-Ab or loading treatment alone. The Wnt/ß-catenin and Rho GTPase pathways were implicated within bone mechanotransduction and support the concept that bone mechanotransduction is likely to encompass a number of interconnected signaling pathways. © 2017 American Society for Bone and Mineral Research.
Assuntos
Anticorpos/farmacologia , Glicoproteínas/imunologia , Osteogênese/efeitos dos fármacos , Tíbia/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Densidade Óssea/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Camundongos Endogâmicos C57BL , Modelos Animais , Tamanho do Órgão/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Tíbia/anatomia & histologia , Tíbia/diagnóstico por imagem , Tíbia/efeitos dos fármacos , Suporte de Carga/fisiologia , Microtomografia por Raio-XRESUMO
Sclerostin, a product of the Sost gene, is a Wnt-inhibitor and thus negatively regulates bone accrual. Canonical Wnt/ß-catenin signalling is also known to be activated in mechanotransduction. Sclerostin neutralizing antibodies are being tested in ongoing clinical trials to target osteoporosis and osteogenesis imperfecta but their interaction with mechanical stimuli on bone formation remains unclear. Sost knockout (KO) mice were examined to gain insight into how long-term Sost deficiency alters the local mechanical environment within the bone. This knowledge is crucial as the strain environment regulates bone adaptation. We characterized the bone geometry at the tibial midshaft of young and adult Sost KO and age-matched littermate control (LC) mice using microcomputed tomography imaging. The cortical area and the minimal and maximal moment of inertia were higher in Sost KO than in LC mice, whereas no difference was detected in either the anterior-posterior or medio-lateral bone curvature. Differences observed between age-matched genotypes were greater in adult mice. We analysed the local mechanical environment in the bone using finite-element models (FEMs), which showed that strains in the tibiae of Sost KO mice are lower than in age-matched LC mice at the diaphyseal midshaft, a region commonly used to assess cortical bone formation and resorption. Our FEMs also suggested that tissue mineral density is only a minor contributor to the strain distribution in tibial cortical bone from Sost KO mice compared to bone geometry. Furthermore, they indicated that although strain gauging experiments matched strains at the gauge site, strains along the tibial length were not comparable between age-matched Sost KO and LC mice or between young and adult animals within the same genotype.
Assuntos
Glicoproteínas/deficiência , Tíbia/crescimento & desenvolvimento , Proteínas Adaptadoras de Transdução de Sinal , Animais , Densidade Óssea , Desenvolvimento Ósseo/genética , Análise de Elementos Finitos , Glicoproteínas/genética , Peptídeos e Proteínas de Sinalização Intercelular , Camundongos , Camundongos Knockout , Estresse MecânicoRESUMO
Sclerostin is a soluble antagonist of canonical Wnt signaling and a strong inhibitor of bone formation. We present experimental data on the role of sclerostin in chronic kidney disease - bone mineral disorder (CKD-MBD). METHODS: We performed 5/6 nephrectomies in 36-week-old sclerostin-deficient (SOST-/-) B6-mice and in C57BL/6J wildtype (WT) mice. Animals received a high phosphate diet for 11weeks. The bones were analyzed by high-resolution micro-computed tomography (µCT) and quantitative bone histomorphometry. Aortic tissue was analyzed regarding the extent of vascular calcification. RESULTS: All nephrectomized mice had severe renal failure, and parathyroid hormone was highly increased compared to corresponding sham animals. All SOST-/- animals revealed the expected high bone mass phenotype. Overall, the bone compartment in WT and SOST-/- mice responded similarly to nephrectomy. In uremic WT animals, µCT data at both the distal femur and lumbar spine revealed significantly increased trabecular volume compared to non-uremic WTs. In SOST-/- mice, the differences between trabecular bone volume were less pronounced when comparing uremic with sham animals. Cortical thickness and cortical bone density at the distal femur decreased significantly and comparably in both genotypes after 5/6 nephrectomy compared to sham animals (cortical bone density -18% and cortical thickness -32%). Overall, 5/6 nephrectomy and concomitant hyperparathyroidism led to a genotype-independent loss of cortical bone volume and density. Overt vascular calcification was not detectable in either of the genotypes. CONCLUSION: Renal osteodystrophy changes were more pronounced in WT mice than in SOST-/- mice. The high bone mass phenotype of sclerostin deficiency was detectable also in the setting of chronic renal failure with severe secondary hyperparathyroidism.
Assuntos
Distúrbio Mineral e Ósseo na Doença Renal Crônica/metabolismo , Distúrbio Mineral e Ósseo na Doença Renal Crônica/patologia , Glicoproteínas/deficiência , Proteínas Adaptadoras de Transdução de Sinal , Animais , Feminino , Peptídeos e Proteínas de Sinalização Intercelular , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Hydrogels are under active development for controlled drug delivery, but their clinical translation is limited by low drug loading capacity, deficiencies in mechanical toughness and storage stability, and poor control over the drug release that often results in burst release and short release duration. This work reports a design of composite clay hydrogels, which simultaneously achieve a spectrum of mechanical, storage, and drug loading/releasing properties to address the critical needs from translational perspectives. The clay nanoparticles provide large surface areas to adsorb biological drugs, and assemble into microparticles that are physically trapped within and toughen hydrogel networks. The composite hydrogels demonstrate feasibility of storage, and extended release of large quantities of an insulin-like growth factor-1 mimetic protein (8 mg mL-1 ) over four weeks. The release rate is primarily governed by ionic exchange and can be upregulated by low pH, which is typical for injured tissues. A rodent model of Achilles tendon injury is used to demonstrate that the composite hydrogels allow for highly extended and localized release of biological drugs in vivo, while demonstrating biodegradation and biocompatibility. These attributes make the composite hydrogel a promising system for drug delivery and regenerative medicine.