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1.
Cell ; 142(4): 531-43, 2010 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-20723755

RESUMO

Muscle wasting and cachexia have long been postulated to be key determinants of cancer-related death, but there has been no direct experimental evidence to substantiate this hypothesis. Here, we show that in several cancer cachexia models, pharmacological blockade of ActRIIB pathway not only prevents further muscle wasting but also completely reverses prior loss of skeletal muscle and cancer-induced cardiac atrophy. This treatment dramatically prolongs survival, even of animals in which tumor growth is not inhibited and fat loss and production of proinflammatory cytokines are not reduced. ActRIIB pathway blockade abolished the activation of the ubiquitin-proteasome system and the induction of atrophy-specific ubiquitin ligases in muscles and also markedly stimulated muscle stem cell growth. These findings establish a crucial link between activation of the ActRIIB pathway and the development of cancer cachexia. Thus ActRIIB antagonism is a promising new approach for treating cancer cachexia, whose inhibition per se prolongs survival.


Assuntos
Receptores de Activinas Tipo II/antagonistas & inibidores , Caquexia/tratamento farmacológico , Atrofia Muscular/tratamento farmacológico , Miocárdio/patologia , Neoplasias/complicações , Receptores de Activinas Tipo II/genética , Ativinas/metabolismo , Animais , Anorexia/tratamento farmacológico , Anorexia/etiologia , Atrofia/tratamento farmacológico , Atrofia/etiologia , Caquexia/etiologia , Feminino , Humanos , Inibinas/genética , Inibinas/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Musculares/metabolismo , Músculo Esquelético/patologia , Atrofia Muscular/etiologia , Mioblastos/patologia , Transplante de Neoplasias , Neoplasias/mortalidade , Transdução de Sinais , Transplante Heterólogo , Fator de Necrose Tumoral alfa/metabolismo
2.
J Appl Physiol (1985) ; 106(2): 582-95, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19074574

RESUMO

Spaceflight results in a number of adaptations to skeletal muscle, including atrophy and shifts toward faster muscle fiber types. To identify changes in gene expression that may underlie these adaptations, we used both microarray expression analysis and real-time polymerase chain reaction to quantify shifts in mRNA levels in the gastrocnemius from mice flown on the 11-day, 19-h STS-108 shuttle flight and from normal gravity controls. Spaceflight data also were compared with the ground-based unloading model of hindlimb suspension, with one group of pure suspension and one of suspension followed by 3.5 h of reloading to mimic the time between landing and euthanization of the spaceflight mice. Analysis of microarray data revealed that 272 mRNAs were significantly altered by spaceflight, the majority of which displayed similar responses to hindlimb suspension, whereas reloading tended to counteract these responses. Several mRNAs altered by spaceflight were associated with muscle growth, including the phosphatidylinositol 3-kinase regulatory subunit p85alpha, insulin response substrate-1, the forkhead box O1 transcription factor, and MAFbx/atrogin1. Moreover, myostatin mRNA expression tended to increase, whereas mRNA levels of the myostatin inhibitor FSTL3 tended to decrease, in response to spaceflight. In addition, mRNA levels of the slow oxidative fiber-associated transcriptional coactivator peroxisome proliferator-associated receptor (PPAR)-gamma coactivator-1alpha and the transcription factor PPAR-alpha were significantly decreased in spaceflight gastrocnemius. Finally, spaceflight resulted in a significant decrease in levels of the microRNA miR-206. Together these data demonstrate that spaceflight induces significant changes in mRNA expression of genes associated with muscle growth and fiber type.


Assuntos
Regulação da Expressão Gênica , Músculo Esquelético/metabolismo , Atrofia Muscular/genética , Voo Espacial , Ausência de Peso , Adaptação Fisiológica/genética , Animais , Análise por Conglomerados , Feminino , Perfilação da Expressão Gênica/métodos , Elevação dos Membros Posteriores , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/fisiopatologia , Atrofia Muscular/fisiopatologia , Miostatina/genética , Análise de Sequência com Séries de Oligonucleotídeos , Fosfatidilinositol 3-Quinases/genética , Reação em Cadeia da Polimerase , Proteínas Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Serina-Treonina Quinases TOR , Fatores de Tempo
3.
J Clin Invest ; 111(8): 1221-30, 2003 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-12697741

RESUMO

Studies in rodents have implicated various cytokines as paracrine mediators of increased osteoclastogenesis during estrogen deficiency, but increases in RANKL, the final effector of osteoclastogenesis, have not been demonstrated. Thus, we isolated bone marrow mononuclear cells expressing RANKL on their surfaces by two-color flow cytometry using FITC-conjugated osteoprotegerin-Fc (OPG-Fc-FITC) as a probe. The cells were characterized as preosteoblastic marrow stromal cells (MSCs), T lymphocytes, or B lymphocytes by using Ab's against bone alkaline phosphatase (BAP), CD3, and CD20, respectively, in 12 premenopausal women (Group A), 12 early postmenopausal women (Group B), and 12 age-matched, estrogen-treated postmenopausal women (Group C). Fluorescence intensity of OPG-Fc-FITC, an index of the surface concentration of RANKL per cell, was increased in Group B over Groups A and C by two- to threefold for MSCs, T cells, B cells, and total RANKL-expressing cells. Moreover, in the merged groups, RANKL expression per cell correlated directly with the bone resorption markers, serum C-terminal telopeptide of type I collagen and urine N-telopeptide of type I collagen, in all three cell types and inversely with serum 17beta-estradiol for total RANKL-expressing cells. The data suggest that upregulation of RANKL on bone marrow cells is an important determinant of increased bone resorption induced by estrogen deficiency.


Assuntos
Reabsorção Óssea/etiologia , Proteínas de Transporte/fisiologia , Glicoproteínas de Membrana/fisiologia , Osteoporose Pós-Menopausa/etiologia , Adulto , Proteínas Reguladoras de Apoptose , Feminino , Citometria de Fluxo , Glicoproteínas/metabolismo , Humanos , Glicoproteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Osteoprotegerina , Ligante RANK , Receptor Ativador de Fator Nuclear kappa-B , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores do Fator de Necrose Tumoral , Ligante Indutor de Apoptose Relacionado a TNF , Fator de Necrose Tumoral alfa/metabolismo
4.
Bone ; 39(4): 754-66, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16730481

RESUMO

Mutations affecting the activity of the Wnt co-receptors LRP5 and LRP6 that cause alterations in skeletal biology confirmed the involvement of Wnt signaling in bone formation. We evaluated the potential role of Dkk1, an inhibitor of LRP5/6 activity, in bone formation by examining the normal expression pattern of Dkk1 in normal young mice and by assessing the consequences of osteoblast overexpression of Dkk1 in transgenic mice. Endogenous Dkk1 expression was detected primarily in osteoblasts and osteocytes. Transgenic over-expression of Dkk1 using two different rat collagen 1A1 promoters resulted in distinct bone phenotypes. More widespread Dkk1 expression (driven by the Col1A1 3.6 kb promoter) yielded osteopenia with forelimb deformities and hairlessness, while expression restricted to osteoblasts (driven by the Col1A1 2.3 kb promoter) induced severe osteopenia without limb defects or alopecia. The decrease in bone mass in vivo resulted from a significant 49% reduction in osteoblast numbers and was reflected in a 45% reduction in serum osteocalcin concentration; an in vitro study revealed that Dkk1 caused a dose-dependent suppression of osteoblast matrix mineralization. These data indicate that Dkk1 may directly influence bone formation and suggest that osteopenia develops in mice over-expressing Dkk1 at least in part due to diminished bone formation resulting from reduced osteoblast numbers.


Assuntos
Doenças Ósseas Metabólicas/fisiopatologia , Osso e Ossos/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteínas Wnt/fisiologia , Células 3T3 , Animais , Densidade Óssea , Doenças Ósseas Metabólicas/genética , Doenças Ósseas Metabólicas/metabolismo , Osso e Ossos/patologia , Osso e Ossos/fisiopatologia , Células Cultivadas , Embrião de Mamíferos/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Hibridização In Situ , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Proteínas Relacionadas a Receptor de LDL/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteocalcina/sangue , Osteogênese/genética , Osteogênese/fisiologia , Gravidez , Ratos , Proteínas Recombinantes/metabolismo , Transdução de Sinais/fisiologia , Proteínas Wnt/metabolismo
5.
Cancer Res ; 64(22): 8193-8, 2004 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-15548684

RESUMO

The progressive depletion of skeletal muscle is a hallmark of many types of advanced cancer and frequently is associated with debility, morbidity, and mortality. Muscle wasting is primarily mediated by the activation of the ubiquitin-proteasome system, which is responsible for degrading the bulk of intracellular proteins. E3 ubiquitin ligases control polyubiquitination, a rate-limiting step in the ubiquitin-proteasome system, but their direct involvement in muscle protein catabolism in cancer remains obscure. Here, we report the full-length cloning of E3alpha-II, a novel "N-end rule" ubiquitin ligase, and its functional involvement in cancer cachexia. E3alpha-II is highly enriched in skeletal muscle, and its expression is regulated by proinflammatory cytokines. In two different animal models of cancer cachexia, E3alpha-II was significantly induced at the onset and during the progression of muscle wasting. The E3alpha-II activation in skeletal muscle was accompanied by a sharp increase in protein ubiquitination, which could be blocked by arginine methylester, an E3alpha-selective inhibitor. Treatment of myotubes with tumor necrosis factor alpha or interleukin 6 elicited marked increases in E3alpha-II but not E3alpha-I expression and ubiquitin conjugation activity in parallel. E3alpha-II transfection markedly accelerated ubiquitin conjugation to endogenous cellular proteins in muscle cultures. These findings show that E3alpha-II plays an important role in muscle protein catabolism during cancer cachexia and suggest that E3alpha-II is a potential therapeutic target for muscle wasting.


Assuntos
Caquexia/metabolismo , Proteínas Musculares/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Sequência de Aminoácidos , Caquexia/enzimologia , Clonagem Molecular , DNA Complementar , Hidrólise , Interleucina-6/fisiologia , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Fator de Necrose Tumoral alfa/fisiologia , Ubiquitina-Proteína Ligases/biossíntese , Ubiquitina-Proteína Ligases/química
6.
Bone ; 81: 562-572, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26318907

RESUMO

Bone loss associated with microgravity exposure poses a significant barrier to long-duration spaceflight. Osteoprotegerin-Fc (OPG-Fc) is a receptor activator of nuclear factor kappa-B ligand (RANKL) inhibitor that causes sustained inhibition of bone resorption after a single subcutaneous injection. We tested the ability of OPG-Fc to preserve bone mass during 12 days of spaceflight (SF). 64-day-old female C57BL/6J mice (n=12/group) were injected subcutaneously with OPG-Fc (20mg/kg) or an inert vehicle (VEH), 24h prior to launch. Ground control (GC) mice (VEH or OPG-Fc) were maintained under environmental conditions that mimicked those in the space shuttle middeck. Age-matched baseline (BL) controls were sacrificed at launch. GC/VEH, but not SF/VEH mice, gained tibia BMD and trabecular volume fraction (BV/TV) during the mission (P<0.05 vs. BL). SF/VEH mice had lower BV/TV vs. GC/VEH mice, while SF/OPG-Fc mice had greater BV/TV than SF/VEH or GC/VEH. SF reduced femur elastic and maximum strength in VEH mice, with OPG-Fc increasing elastic strength in SF mice. Serum TRAP5b was elevated in SF/VEH mice vs. GC/VEH mice. Conversely, SF/OPG-Fc mice had lower TRAP5b levels, suggesting that OPG-Fc preserved bone during spaceflight via inhibition of osteoclast-mediated bone resorption. Decreased bone formation also contributed to the observed osteopenia, based on the reduced femur periosteal bone formation rate and serum osteocalcin level. Overall, these observations suggest that the beneficial effects of OPG-Fc during SF are primarily due to dramatic and sustained suppression of bone resorption. In growing mice, this effect appears to compensate for the SF-related inhibition of bone formation, while preventing any SF-related increase in bone resorption. We have demonstrated that the young mouse is an appropriate new model for SF-induced osteopenia, and that a single pre-flight treatment with OPG-Fc can effectively prevent the deleterious effects of SF on mouse bone.


Assuntos
Reabsorção Óssea/prevenção & controle , Fragmentos Fc das Imunoglobulinas/farmacologia , Osteoprotegerina/farmacologia , Proteínas Recombinantes de Fusão/farmacologia , Voo Espacial , Ausência de Peso/efeitos adversos , Fosfatase Alcalina/sangue , Animais , Biomarcadores/sangue , Fenômenos Biomecânicos , Densidade Óssea/efeitos dos fármacos , Conservadores da Densidade Óssea/farmacologia , Reabsorção Óssea/etiologia , Reabsorção Óssea/fisiopatologia , Modelos Animais de Doenças , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Osteocalcina/sangue , Ligante RANK/antagonistas & inibidores
7.
J Appl Physiol (1985) ; 94(5): 2085-94, 2003 May.
Artigo em Inglês | MEDLINE | ID: mdl-12514166

RESUMO

There are several aspects of the spaceflight environment that may lead to changes in immunity: mission-related psychological stress, radiation, and changes in gravity. On December 5, 2001, the space shuttle Endeavor launched for a 12-day mission to examine these effects on C57BL/6 mice for the first time. On their return, assays were performed on the spleen, blood, and bone marrow. In response to flight, there were no significant differences in the general circulating leukocyte proportions. In contrast, there was an increase in splenic lymphocyte percentages, with a corresponding decrease in granulocytes. There was an overall shift in splenic lymphocytes away from T cells toward B cells, and a decrease in the CD4-to-CD8 ratios due to a decrease in T helpers. In contrast, there were proportional increases in bone marrow T cells, with decreases in B cells. Although the blast percentage and count were decreased in flight mice, the CD34(+) population was increased. The data were more consistent with a shift in bone marrow populations rather than a response to changes in the periphery. Many of the results are similar to those using other models. Clearly, spaceflight can influence immune parameters ranging from hematopoiesis to mature leukocyte mechanisms.


Assuntos
Imunidade/fisiologia , Voo Espacial , Animais , Medula Óssea/imunologia , Medula Óssea/fisiologia , Feminino , Citometria de Fluxo , Granulócitos/imunologia , Imunidade Celular/imunologia , Imunidade Celular/fisiologia , Contagem de Leucócitos , Sistema Linfático/imunologia , Sistema Linfático/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Baço/citologia , Baço/imunologia , Células-Tronco/fisiologia
8.
J Appl Physiol (1985) ; 94(5): 2095-103, 2003 May.
Artigo em Inglês | MEDLINE | ID: mdl-12506046

RESUMO

This portion of the study quantified the effects of a 12-day space shuttle mission (Space Transport System-108/UF-1) on body and lymphoid organ masses, activation marker expression, cytokine secretion, and erythrocyte and thrombocyte characteristics in C57BL/6 mice. Animals in flight (Flt group) had 10-12% lower body mass compared with ground controls housed either in animal enclosure modules or under standard vivarium conditions (P < 0.001) and the smallest thymus and spleen masses. Percentages of CD25(+) lymphocytes, CD3(+)/CD25(+) T cells, and NK1.1(+)/CD25(+) natural killer cells from Flt mice were higher compared with both controls (P < 0.05). In contrast, CD71 expression was depressed in the Flt and animal enclosure module control mice compared with vivarium control animals (P < 0.001). Secretion of interferon-gamma, IL-2, and IL-4, but not tumor necrosis factor-alpha and IL-5, by splenocytes from Flt mice was decreased relative to either one or both ground controls (P < 0.05). Flt mice also had high red blood cell and thrombocyte counts compared with both sets of controls; low red blood cell volume and distribution width, percentage of reticulocytes, and platelet volume were also noted (P < 0.05) and were consistent with dehydration. These data indicate that relatively short exposure to the spaceflight environment can induce profound changes that may become significant during long-term space missions.


Assuntos
Plaquetas/fisiologia , Citocinas/biossíntese , Eritrócitos/fisiologia , Imunidade/fisiologia , Voo Espacial , Animais , Biomarcadores , Peso Corporal/fisiologia , Sobrevivência Celular/fisiologia , Ingestão de Líquidos/fisiologia , Ingestão de Alimentos/fisiologia , Contagem de Eritrócitos , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Tamanho do Órgão/fisiologia , Contagem de Plaquetas , Baço/citologia
9.
Nat Rev Drug Discov ; 11(5): 401-19, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22543469

RESUMO

Bone is a complex tissue that provides mechanical support for muscles and joints, protection for vital organs, a mineral reservoir that is essential for calcium homeostasis, and the environment and niches required for haematopoiesis. The regulation of bone mass in mammals is governed by a complex interplay between bone-forming cells termed osteoblasts and bone-resorbing cells termed osteoclasts, and is guided physiologically by a diverse set of hormones, cytokines and growth factors. The balance between these processes changes over time, causing an elevated risk of fractures with age. Osteoclasts may also be activated in the cancer setting, leading to bone pain, fracture, spinal cord compression and other significant morbidities. This Review chronicles the events that led to an increased understanding of bone resorption, the elucidation of the signalling pathway mediated by osteoprotegerin, receptor activator of NF-κB (RANK) and RANK ligand (RANKL) and its role in osteoclast biology, as well as the evolution of recombinant RANKL antagonists, which culminated in the development of the therapeutic RANKL-targeted antibody denosumab.


Assuntos
Anticorpos Monoclonais/farmacologia , Osteoprotegerina/metabolismo , Ligante RANK/antagonistas & inibidores , Receptor Ativador de Fator Nuclear kappa-B/metabolismo , Animais , Anticorpos Monoclonais Humanizados , Reabsorção Óssea/fisiopatologia , Osso e Ossos/metabolismo , Denosumab , Desenho de Fármacos , Humanos , Osteoclastos/fisiologia , Ligante RANK/metabolismo , Transdução de Sinais
10.
J Bone Miner Res ; 26(5): 1012-21, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21542004

RESUMO

Therapeutic enhancement of fracture healing would help to prevent the occurrence of orthopedic complications such as nonunion and revision surgery. Sclerostin is a negative regulator of bone formation, and treatment with a sclerostin monoclonal antibody (Scl-Ab) results in increased bone formation and bone mass in animal models. Our objective was to investigate the effects of systemic administration of Scl-Ab in two models of fracture healing. In both a closed femoral fracture model in rats and a fibular osteotomy model in cynomolgus monkeys, Scl-Ab significantly increased bone mass and bone strength at the site of fracture. After 10 weeks of healing in nonhuman primates, the fractures in the Scl-Ab group had less callus cartilage and smaller fracture gaps containing more bone and less fibrovascular tissue. These improvements at the fracture site corresponded with improvements in bone formation, bone mass, and bone strength at nonfractured cortical and trabecular sites in both studies. Thus the potent anabolic activity of Scl-Ab throughout the skeleton also was associated with an anabolic effect at the site of fracture. These results support the potential for systemic Scl-Ab administration to enhance fracture healing in patients.


Assuntos
Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/farmacologia , Densidade Óssea/efeitos dos fármacos , Fraturas do Fêmur/fisiopatologia , Consolidação da Fratura/efeitos dos fármacos , Glicoproteínas/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal , Animais , Fenômenos Biomecânicos/efeitos dos fármacos , Diáfises/efeitos dos fármacos , Diáfises/patologia , Diáfises/fisiopatologia , Modelos Animais de Doenças , Fêmur/efeitos dos fármacos , Fêmur/patologia , Fêmur/fisiopatologia , Fíbula/efeitos dos fármacos , Fíbula/patologia , Fíbula/fisiopatologia , Glicoproteínas/imunologia , Peptídeos e Proteínas de Sinalização Intercelular , Macaca fascicularis , Masculino , Tamanho do Órgão/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Osteotomia , Ratos , Ratos Sprague-Dawley
11.
J Bone Miner Res ; 26(11): 2610-21, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21773994

RESUMO

The physiological role of Dickkopf-1 (Dkk1) during postnatal bone growth in rodents and in adult rodents was examined utilizing an antibody to Dkk1 (Dkk1-Ab) that blocked Dkk1 binding to both low density lipoprotein receptor-related protein 6 (LRP6) and Kremen2, thereby preventing the Wnt inhibitory activity of Dkk1. Treatment of growing mice and rats with Dkk1-Ab resulted in a significant increase in bone mineral density because of increased bone formation. In contrast, treatment of adult ovariectomized rats did not appreciably impact bone, an effect that was associated with decreased Dkk1 expression in the serum and bone of older rats. Finally, we showed that Dkk1 plays a prominent role in adult bone by mediating fracture healing in adult rodents. These data suggest that, whereas Dkk1 significantly regulates bone formation in younger animals, its role in older animals is limited to pathologies that lead to the induction of Dkk1 expression in bone and/or serum, such as traumatic injury.


Assuntos
Envelhecimento/metabolismo , Osso e Ossos/lesões , Osso e Ossos/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Osteogênese/fisiologia , Envelhecimento/efeitos dos fármacos , Animais , Anticorpos Bloqueadores/administração & dosagem , Anticorpos Bloqueadores/farmacologia , Densidade Óssea/efeitos dos fármacos , Doenças Ósseas Metabólicas/sangue , Doenças Ósseas Metabólicas/fisiopatologia , Osso e Ossos/diagnóstico por imagem , Osso e Ossos/patologia , Linhagem Celular , Estrogênios/deficiência , Feminino , Fêmur/diagnóstico por imagem , Fêmur/efeitos dos fármacos , Fêmur/patologia , Consolidação da Fratura/efeitos dos fármacos , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/sangue , Vértebras Lombares/efeitos dos fármacos , Vértebras Lombares/patologia , Masculino , Camundongos , Osteogênese/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Regulação para Cima/efeitos dos fármacos , Via de Sinalização Wnt/efeitos dos fármacos , Microtomografia por Raio-X
12.
J Bone Miner Res ; 25(5): 948-59, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20200929

RESUMO

The development of bone-rebuilding anabolic agents for treating bone-related conditions has been a long-standing goal. Genetic studies in humans and mice have shown that the secreted protein sclerostin is a key negative regulator of bone formation. More recently, administration of sclerostin-neutralizing monoclonal antibodies in rodent studies has shown that pharmacologic inhibition of sclerostin results in increased bone formation, bone mass, and bone strength. To explore the effects of sclerostin inhibition in primates, we administered a humanized sclerostin-neutralizing monoclonal antibody (Scl-AbIV) to gonad-intact female cynomolgus monkeys. Two once-monthly subcutaneous injections of Scl-AbIV were administered at three dose levels (3, 10, and 30 mg/kg), with study termination at 2 months. Scl-AbIV treatment had clear anabolic effects, with marked dose-dependent increases in bone formation on trabecular, periosteal, endocortical, and intracortical surfaces. Bone densitometry showed that the increases in bone formation with Scl-AbIV treatment resulted in significant increases in bone mineral content (BMC) and/or bone mineral density (BMD) at several skeletal sites (ie, femoral neck, radial metaphysis, and tibial metaphysis). These increases, expressed as percent changes from baseline were 11 to 29 percentage points higher than those found in the vehicle-treated group. Additionally, significant increases in trabecular thickness and bone strength were found at the lumbar vertebrae in the highest-dose group. Taken together, the marked bone-building effects achieved in this short-term monkey study suggest that sclerostin inhibition represents a promising new therapeutic approach for medical conditions where increases in bone formation might be desirable, such as in fracture healing and osteoporosis.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Densidade Óssea/efeitos dos fármacos , Proteínas Morfogenéticas Ósseas/imunologia , Osso e Ossos/metabolismo , Marcadores Genéticos/imunologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Osso e Ossos/patologia , Feminino , Macaca fascicularis , Osteogênese
13.
J Bone Miner Res ; 24(4): 578-88, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19049336

RESUMO

The development of bone-rebuilding anabolic agents for potential use in the treatment of bone loss conditions, such as osteoporosis, has been a long-standing goal. Genetic studies in humans and mice have shown that the secreted protein sclerostin is a key negative regulator of bone formation, although the magnitude and extent of sclerostin's role in the control of bone formation in the aging skeleton is still unclear. To study this unexplored area of sclerostin biology and to assess the pharmacologic effects of sclerostin inhibition, we used a cell culture model of bone formation to identify a sclerostin neutralizing monoclonal antibody (Scl-AbII) for testing in an aged ovariectomized rat model of postmenopausal osteoporosis. Six-month-old female rats were ovariectomized and left untreated for 1 yr to allow for significant estrogen deficiency-induced bone loss, at which point Scl-AbII was administered for 5 wk. Scl-AbII treatment in these animals had robust anabolic effects, with marked increases in bone formation on trabecular, periosteal, endocortical, and intracortical surfaces. This not only resulted in complete reversal, at several skeletal sites, of the 1 yr of estrogen deficiency-induced bone loss, but also further increased bone mass and bone strength to levels greater than those found in non-ovariectomized control rats. Taken together, these preclinical results establish sclerostin's role as a pivotal negative regulator of bone formation in the aging skeleton and, furthermore, suggest that antibody-mediated inhibition of sclerostin represents a promising new therapeutic approach for the anabolic treatment of bone-related disorders, such as postmenopausal osteoporosis.


Assuntos
Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Proteínas Morfogenéticas Ósseas/imunologia , Osso e Ossos/efeitos dos fármacos , Marcadores Genéticos/imunologia , Osteogênese/efeitos dos fármacos , Osteoporose Pós-Menopausa/tratamento farmacológico , Animais , Bioensaio , Fenômenos Biomecânicos , Densidade Óssea/efeitos dos fármacos , Osso e Ossos/patologia , Linhagem da Célula/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Fêmur/efeitos dos fármacos , Fêmur/patologia , Humanos , Vértebras Lombares/efeitos dos fármacos , Vértebras Lombares/patologia , Camundongos , Testes de Neutralização , Tamanho do Órgão/efeitos dos fármacos , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteocalcina/sangue , Osteoporose Pós-Menopausa/sangue , Osteoporose Pós-Menopausa/patologia , Osteoporose Pós-Menopausa/fisiopatologia , Ovariectomia , Ratos , Ratos Sprague-Dawley , Tíbia/efeitos dos fármacos , Tíbia/patologia , Tomografia Computadorizada por Raios X
14.
J Bone Miner Res ; 23(6): 860-9, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18269310

RESUMO

INTRODUCTION: Sclerosteosis is a rare high bone mass genetic disorder in humans caused by inactivating mutations in SOST, the gene encoding sclerostin. Based on these data, sclerostin has emerged as a key negative regulator of bone mass. We generated SOST knockout (KO) mice to gain a more detailed understanding of the effects of sclerostin deficiency on bone. MATERIALS AND METHODS: Gene targeting was used to inactivate SOST and generate a line of SOST KO mice. Radiography, densitometry, microCT, histomorphometry, and mechanical testing were used to characterize the impact of sclerostin deficiency on bone in male and female mice. Comparisons were made between same sex KO and wildtype (WT) mice. RESULTS: The results for male and female SOST KO mice were similar, with differences only in the magnitude of some effects. SOST KO mice had increased radiodensity throughout the skeleton, with general skeletal morphology being normal in appearance. DXA analysis of lumbar vertebrae and whole leg showed that there was a significant increase in BMD (>50%) at both sites. microCT analysis of femur showed that bone volume was significantly increased in both the trabecular and cortical compartments. Histomorphometry of trabecular bone revealed a significant increase in osteoblast surface and no significant change in osteoclast surface in SOST KO mice. The bone formation rate in SOST KO mice was significantly increased for trabecular bone (>9-fold) at the distal femur, as well as for the endocortical and periosteal surfaces of the femur midshaft. Mechanical testing of lumbar vertebrae and femur showed that bone strength was significantly increased at both sites in SOST KO mice. CONCLUSIONS: SOST KO mice have a high bone mass phenotype characterized by marked increases in BMD, bone volume, bone formation, and bone strength. These results show that sclerostin is a key negative regulator of a powerful, evolutionarily conserved bone formation pathway that acts on both trabecular and cortical bone.


Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Osso e Ossos/metabolismo , Deleção de Genes , Osteogênese , Proteínas Adaptadoras de Transdução de Sinal , Animais , Biomarcadores/sangue , Densidade Óssea , Proteínas Morfogenéticas Ósseas/deficiência , Proteínas Morfogenéticas Ósseas/genética , Osso e Ossos/diagnóstico por imagem , Cálcio/sangue , Feminino , Marcadores Genéticos/genética , Glicoproteínas , Peptídeos e Proteínas de Sinalização Intercelular , Masculino , Camundongos , Camundongos Knockout , Fenótipo , Fosfatos/sangue , Estresse Mecânico , Tomografia Computadorizada por Raios X
15.
Nature ; 423(6937): 337-42, 2003 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-12748652

RESUMO

Osteoclasts are specialized cells derived from the monocyte/macrophage haematopoietic lineage that develop and adhere to bone matrix, then secrete acid and lytic enzymes that degrade it in a specialized, extracellular compartment. Discovery of the RANK signalling pathway in the osteoclast has provided insight into the mechanisms of osteoclastogenesis and activation of bone resorption, and how hormonal signals impact bone structure and mass. Further study of this pathway is providing the molecular basis for developing therapeutics to treat osteoporosis and other diseases of bone loss.


Assuntos
Diferenciação Celular , Osteoclastos/citologia , Osteoclastos/metabolismo , Animais , Reabsorção Óssea , Proteínas de Transporte/metabolismo , Glicoproteínas/metabolismo , Humanos , Glicoproteínas de Membrana/metabolismo , Osteoporose/tratamento farmacológico , Osteoporose/metabolismo , Osteoprotegerina , Ligante RANK , Receptor Ativador de Fator Nuclear kappa-B , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores do Fator de Necrose Tumoral , Transdução de Sinais
16.
Am J Physiol Lung Cell Mol Physiol ; 285(3): L602-10, 2003 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-12740217

RESUMO

We reported an association between the ability of recombinant human keratinocyte growth factor (rHuKGF) to upregulate the expression of surfactant protein A (SP-A) and to downregulate pulmonary inflammation that occurs after allogeneic bone marrow transplantation (BMT). To establish a causal relationship, rHuKGF (5 mg/kg) was administered subcutaneously for three consecutive days before irradiation to SP-A-sufficient and -deficient [SP-A(+/+) and SP-A(-/-), respectively] mice given inflammation-inducing allogeneic spleen T cells at the time of BMT. In contrast with SP-A(+/+) mice, rHuKGF failed to suppress the high levels of TNF-alpha, IFN-gamma, and nitric oxide contained in bronchoalveolar lavage fluids collected on day 7 after BMT from SP-A(-/-) mice. Early post-BMT weight loss was attenuated by rHuKGF in both SP-A(+/+) and SP-A(-/-) recipients. In the absence of supportive respiratory care, however, SP-A deficiency eventually abolished the ability of rHuKGF to prevent weight loss and to improve survival monitored for 1 mo after allogeneic BMT. In further experiments, the addition of cyclophosphamide (which is known to cause severe injury to the alveolar epithelium in donor T cell-recipient mice) to the conditioning regimen prevented rHuKGF-induced upregulation of SP-A and suppression of lung inflammation in both SP-A(+/+) and SP-A(-/-) mice. We conclude that endogenous baseline SP-A levels and optimal upregulation of SP-A are required for the anti-inflammatory protective effects of KGF after allogeneic transplantation.


Assuntos
Transplante de Medula Óssea/imunologia , Fatores de Crescimento de Fibroblastos/farmacologia , Doença Enxerto-Hospedeiro/tratamento farmacológico , Doença Enxerto-Hospedeiro/metabolismo , Pulmão/metabolismo , Proteína A Associada a Surfactante Pulmonar/genética , Animais , Transplante de Medula Óssea/mortalidade , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Fator 7 de Crescimento de Fibroblastos , Doença Enxerto-Hospedeiro/imunologia , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Complacência Pulmonar/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Proteína A Associada a Surfactante Pulmonar/metabolismo , Proteínas Recombinantes/farmacologia , Linfócitos T/imunologia , Condicionamento Pré-Transplante , Transplante Homólogo , Regulação para Cima/imunologia , Redução de Peso
17.
Blood ; 99(12): 4592-600, 2002 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-12036893

RESUMO

Decreased thymopoietic capacity contributes to the severe and clinically significant immune deficiency seen after bone marrow transplantation (BMT). One mechanism for thymopoietic failure is damage to the interleukin 7 (IL-7)-producing thymic epithelial cells (TECs) by irradiation and chemotherapy, which can be partially treated by IL-7 administration. Pretreatment of BMT recipients with keratinocyte growth factor (KGF, or Fgf7), an epithelial cell-specific growth factor, protects mucosal, cutaneous, and pulmonary epithelial cells from cytotoxic therapy-induced damage in experimental murine models. Like other epithelial cells, TECs specifically express KGF receptors. Because KGF specifically protects KGF receptor-bearing epithelial cells and post-BMT immune deficiency is caused by loss of TECs, we hypothesized that KGF pretreatment would improve post-BMT thymic function. To test the hypothesis, BMT recipient mice were given KGF or placebo prior to congenic or allogeneic BMT. Administration of KGF before murine BMT significantly increased the capacity of the thymus to generate donor-derived thymocytes. KGF pretreatment also normalized the proportion of thymic subpopulations, increased the number of naive T cells in the periphery, and improved the response to neoantigen immunization. KGF treatment caused increased production of intrathymic IL-7, and the thymopoietic effects of KGF required an intact IL-7 signaling pathway. These results demonstrate that KGF may have immunomodulatory effects by a unique mechanism of protection of TECs. Furthermore, thymic injury and prolonged posttransplantation immune deficiency in BMT recipients can be prevented by KGF administration.


Assuntos
Transplante de Medula Óssea/métodos , Células Epiteliais/efeitos dos fármacos , Fatores de Crescimento de Fibroblastos/farmacologia , Substâncias Protetoras/farmacologia , Timo/efeitos dos fármacos , Animais , Células Sanguíneas/efeitos dos fármacos , Transplante de Medula Óssea/normas , Células Epiteliais/citologia , Fator 7 de Crescimento de Fibroblastos , Fatores de Crescimento de Fibroblastos/administração & dosagem , Sobrevivência de Enxerto/efeitos dos fármacos , Hematopoese/efeitos dos fármacos , Sistema Imunitário/efeitos dos fármacos , Sistema Imunitário/crescimento & desenvolvimento , Camundongos , Camundongos Endogâmicos BALB C , Substâncias Protetoras/administração & dosagem , Linfócitos T/efeitos dos fármacos , Timo/citologia
18.
Blood ; 100(2): 682-91, 2002 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-12091365

RESUMO

Thymus-dependent reconstitution of the peripheral T-cell compartment is critical for the successful outcome of bone marrow transplantation. However, graft-versus-host disease (GVHD) affects thymic stromal function and thus prevents normal T-cell maturation and selection. To determine whether cytoprotection of thymic epithelial cells (TECs) by keratinocyte growth factor (KGF) averts GVHD-related injury to the thymus, a nonirradiated murine parent-->F(1) transplantation model was investigated. Administration of KGF between days -3 and +3 of GVHD induction preserved normal thymic size, cellularity, and thymocyte phenotype when measured 2 weeks after transplantation and compared with saline-treated parent-->F(1) mice that received allogeneic transplants. Moreover, the characteristic GVHD-induced impairment in cell cycle progression of pro- and pre-T cells was prevented by KGF. However, the normal phenotypic and functional status of the thymus did not correlate with the higher number of GVHD-inducing mature donor T cells in thymi of KGF-treated mice. Importantly, extensive analysis of the different TEC populations within the thymic cortex and medulla revealed an almost normal stromal architecture and composition in GVHD mice treated with KGF. These observations are likely to reflect an indirect effect of KGF on thymopoiesis as KGF-receptor expression was demonstrated to be restricted to TECs. Thus, pharmacologic doses of KGF appear to exert a potent effect on TEC function, which in turn allows for normal T lymphopoiesis to occur during acute GVHD.


Assuntos
Células Epiteliais/efeitos dos fármacos , Fatores de Crescimento de Fibroblastos/farmacologia , Doença Enxerto-Hospedeiro/prevenção & controle , Timo/citologia , Animais , Antígeno B7-1/metabolismo , Ciclo Celular/efeitos dos fármacos , Transplante de Células/efeitos adversos , Modelos Animais de Doenças , Células Epiteliais/citologia , Fator 7 de Crescimento de Fibroblastos , Fatores de Crescimento de Fibroblastos/administração & dosagem , Doença Enxerto-Hospedeiro/tratamento farmacológico , Doença Enxerto-Hospedeiro/patologia , Haplótipos , Camundongos , Camundongos Endogâmicos C57BL , Substâncias Protetoras/administração & dosagem , Substâncias Protetoras/farmacologia , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Baço/citologia , Baço/efeitos dos fármacos , Timo/efeitos dos fármacos , Timo/imunologia
19.
Am J Physiol Gastrointest Liver Physiol ; 282(4): G690-701, 2002 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-11897629

RESUMO

There is an acute need for effective therapy for inflammatory bowel disease (IBD), particularly at the level of repair of the damaged epithelium. We evaluated the efficacy of recombinant human keratinocyte growth factor (rHuKGF) in both the dextran sodium sulfate (DSS) and the CD4(+)CD45RB(Hi) T cell transfer models of IBD. Disease was induced either by the ad libitum administration to normal mice of 4% DSS in the drinking water or by the injection of 4 x 10(5) CD4(+)CD45RB(Hi) T cells into immunodeficient scid/scid mice. rHuKGF was administered by subcutaneous injection at doses of 1.0 or 3.0 mg/kg in both preventative and therapeutic regimens during both studies. rHuKGF significantly improved survival and body weight loss in the DSS model in both preventative and therapeutic dosing regimens. It also improved diarrhea, hematochezia, and hematological parameters, as well as large intestine histopathology. In the T cell transfer model, rHuKGF improved body weight loss, diarrhea, and levels of serum amyloid A, as well as large intestine histopathology. In both models of IBD, the colonic levels of intestinal trefoil factor (ITF) were elevated by the disease state and further elevated by treatment with rHuKGF. These data suggest that rHuKGF may prove useful in the clinical management of IBD and its effects are likely mediated by its ability to locally increase the levels of ITF.


Assuntos
Linfócitos T CD4-Positivos/transplante , Sulfato de Dextrana , Fatores de Crescimento de Fibroblastos/uso terapêutico , Doenças Inflamatórias Intestinais/tratamento farmacológico , Antígenos Comuns de Leucócito/análise , Animais , Linfócitos T CD4-Positivos/imunologia , Diarreia , Modelos Animais de Doenças , Feminino , Fator 7 de Crescimento de Fibroblastos , Humanos , Doenças Inflamatórias Intestinais/patologia , Doenças Inflamatórias Intestinais/fisiopatologia , Mucosa Intestinal/patologia , Contagem de Leucócitos , Masculino , Camundongos , Camundongos SCID , Neutrófilos , Proteínas Recombinantes/uso terapêutico , Proteína Amiloide A Sérica/análise , Redução de Peso
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