RESUMO
Treatment of vitamin D deficiency with vitamin D is a common procedure when taking care of elderly patients, calcium supplementation being added only when calcium dietary intake is insufficient. Here, we report the case of a 58-year-old female who was referred to our unit because of suspicion of Paget's disease of the skull, based on elevated serum alkaline phosphatase and high skull methylene diphosphonate-technetium uptake. She had been prescribed cholecalciferol (100,000 IU/month) and calcium salts for the past 7 months after discovery of severe vitamin D deficiency by her primary care physician. No specific skull bone lesions were observed on both X-ray and computerized tomography. Serum calcium, phosphate and 25(OH) vitamin D levels were normal, while serum C-terminal cross-linked telopeptide, bone alkaline phosphatase and calcitriol were high and daily urinary calcium excretion was low. We found that she had not been compliant with the calcium prescription while vitamin D had been thoroughly taken. We suspected osteomalacia due to calcium deficiency. Both skull uptake and biological abnormalities normalised in few months after adding calcium supplementation to the vitamin D treatment, and spine bone mineral density increased by 9.5 % after 14 months of full treatment. The present case illustrates the necessity for adequate calcium intake during vitamin D repletion to normalise bone mineralisation and turnover and maintain the skeletal integrity.
Assuntos
Remodelação Óssea/efeitos dos fármacos , Cálcio/deficiência , Colecalciferol/uso terapêutico , Osteomalacia/etiologia , Deficiência de Vitamina D/tratamento farmacológico , Fosfatase Alcalina/sangue , Densidade Óssea/efeitos dos fármacos , Densidade Óssea/fisiologia , Remodelação Óssea/fisiologia , Cálcio/sangue , Cálcio/uso terapêutico , Suplementos Nutricionais , Feminino , Humanos , Adesão à Medicação , Pessoa de Meia-Idade , Osteomalacia/diagnóstico , Osteomalacia/fisiopatologia , Deficiência de Vitamina D/fisiopatologiaRESUMO
Patients suffering from chronic kidney disease (CKD) often experience bone loss and arterial calcifications. It is unclear if hypogonadism contributes to the development of these complications and whether androgen therapy might prevent them. Male adult rats were randomized into four groups. The first group received standard chow (control), while three other groups were fed a 0.25% adenine/low vitamin K diet (CKD). Two CKD groups were treated with testosterone or dihydrotestosterone (DHT), whereas the control group and one CKD group received vehicle (VEH). CKD animals had 10-fold higher serum creatinine and more than 15-fold higher parathyroid hormone levels compared to controls. Serum testosterone levels were more than two-fold lower in the CKDVEH group compared to control + VEH and CKD + testosterone groups. Seminal vesicle weight was reduced by 50% in CKDVEH animals and restored by testosterone and DHT. CKD animals showed a low bone mass phenotype with decreased trabecular bone volume fraction and increased cortical porosity, which was not rescued by androgen treatment. Aortic calcification was much more prominent in CKD animals and not unequivocally prevented by androgens. Messenger RNA expression of the androgen receptor-responsive genes Acta1 and Col1a1 was reduced by CKD and stimulated by androgen treatment in levator ani muscle but not in the bone or aortic tissue. We conclude that adenine-induced CKD results in the development of hypogonadism in male rats. Androgen therapy is effective in restoring serum testosterone levels and androgen-sensitive organ weights but does not prevent bone loss or arterial calcifications, at least not in the presence of severe hyperparathyroidism.
Assuntos
Doenças Ósseas Metabólicas , Hipogonadismo , Insuficiência Renal Crônica , Ratos , Masculino , Animais , Androgênios/metabolismo , Testosterona , Di-Hidrotestosterona/farmacologia , Insuficiência Renal Crônica/complicações , Insuficiência Renal Crônica/tratamento farmacológico , Hipogonadismo/complicações , AdeninaRESUMO
Myeloid cells are known mediators of hypertension, but their role in initiating renin-induced hypertension has not been studied. Vitamin D deficiency causes pro-inflammatory macrophage infiltration in metabolic tissues and is linked to renin-mediated hypertension. We tested the hypothesis that impaired vitamin D signaling in macrophages causes hypertension using conditional knockout of the myeloid vitamin D receptor in mice (KODMAC). These mice develop renin-dependent hypertension due to macrophage infiltration of the vasculature and direct activation of renal juxtaglomerular (JG) cell renin production. Induction of endoplasmic reticulum stress in knockout macrophages increases miR-106b-5p secretion, which stimulates JG cell renin production via repression of transcription factors E2f1 and Pde3b. Moreover, in wild-type recipient mice of KODMAC/miR106b-/- bone marrow, knockout of miR-106b-5p prevents the hypertension and JG cell renin production induced by KODMAC macrophages, suggesting myeloid-specific, miR-106b-5p-dependent effects. These findings confirm macrophage miR-106b-5p secretion from impaired vitamin D receptor signaling causes inflammation-induced hypertension.
Assuntos
Hipertensão Renal/metabolismo , Hipertensão/metabolismo , Macrófagos/metabolismo , MicroRNAs/metabolismo , Nefrite/metabolismo , Renina/metabolismo , Animais , Medula Óssea , Transplante de Medula Óssea , Modelos Animais de Doenças , Fator de Transcrição E2F1/metabolismo , Estresse do Retículo Endoplasmático , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Mieloides , Receptores de Calcitriol , Vitamina DRESUMO
The plasminogen (Plg)/plasminogen activator (PA) system plays a key role in cancer progression, presumably via mediating extracellular matrix degradation and tumor cell migration. Consequently, urokinase-type PA (uPA)/plasmin antagonists are currently being developed for suppression of tumor growth and angiogenesis. Paradoxically, however, high levels of PA inhibitor 1 (PAI-1) are predictive of a poor prognosis for survival of patients with cancer. We demonstrated previously that PAI-1 promoted tumor angiogenesis, but by an unresolved mechanism. We anticipated that PAI-1 facilitated endothelial cell migration via its known interaction with vitronectin (VN) and integrins. However, using adenoviral gene transfer of PAI-1 mutants, we observed that PAI-1 promoted tumor angiogenesis, not by interacting with VN, but rather by inhibiting proteolytic activity, suggesting that excessive plasmin proteolysis prevents assembly of tumor vessels. Single deficiency of uPA, tissue-type PA (tPA), uPA receptor, or VN, as well as combined deficiencies of uPA and tPA did not impair tumor angiogenesis, whereas lack of Plg reduced it. Overall, these data indicate that plasmin proteolysis, even though essential, must be tightly controlled during tumor angiogenesis, probably to allow vessel stabilization and maturation. These data provide insights into the clinical paradox whereby PAI-1 promotes tumor progression and warrant against the uncontrolled use of uPA/plasmin antagonists as tumor angiogenesis inhibitors.
Assuntos
Inibidores da Angiogênese/farmacologia , Endopeptidases/metabolismo , Neoplasias Experimentais/tratamento farmacológico , Neovascularização Patológica/tratamento farmacológico , Inibidor 1 de Ativador de Plasminogênio/farmacologia , Vitronectina/metabolismo , Animais , Endotélio Vascular/efeitos dos fármacos , Fibrinolisina/metabolismo , Queratinócitos/patologia , Camundongos , Camundongos Mutantes , Neoplasias Musculares/irrigação sanguínea , Invasividade Neoplásica , Neoplasias Experimentais/irrigação sanguínea , Neovascularização Patológica/etiologia , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Ligação Proteica , Vitronectina/genéticaRESUMO
High resolution microfocus X-ray computed tomography (HR-microCT) was employed to characterize the structural alterations of the cortical and trabecular bone in a mouse model of obesity-driven type 2 diabetes (T2DM). C57Bl/6J mice were randomly assigned for 14 weeks to either a control diet-fed (CTRL) or a high fat diet (HFD)-fed group developing obesity, hyperglycaemia and insulin resistance. The HFD group showed an increased trabecular thickness and a decreased trabecular number compared to CTRL animals. Midshaft tibia intracortical porosity was assessed at two spatial image resolutions. At 2 µm scale, no change was observed in the intracortical structure. At 1 µm scale, a decrease in the cortical vascular porosity of the HFD bone was evidenced. The study of a group of 8 week old animals corresponding to animals at the start of the diet challenge revealed that the decreased vascular porosity was T2DM-dependant and not related to the ageing process. Our results offer an unprecedented ultra-characterization of the T2DM compromised skeletal micro-architecture and highlight an unrevealed T2DM-related decrease in the cortical vascular porosity, potentially affecting the bone health and fragility. Additionally, it provides some insights into the technical challenge facing the assessment of the rodent bone structure using HR-microCT imaging.
Assuntos
Diabetes Mellitus Tipo 2/diagnóstico , Tíbia/diagnóstico por imagem , Microtomografia por Raio-X/métodos , Animais , Densidade Óssea , Dieta Hiperlipídica , Modelos Animais de Doenças , Hiperglicemia , Resistência à Insulina , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade , Tíbia/patologiaRESUMO
The differentiation of embryonic mesenchymal cells into chondrocytes and the subsequent formation of a cartilaginous scaffold that enables the formation of long bones are hallmarks of endochondral ossification. During this process, chondrocytes undergo a remarkable sequence of events involving proliferation, differentiation, hypertrophy and eventually apoptosis. Forkhead Box O (FoxO) transcription factors (TFs) are well-known regulators of such cellular processes. Although FoxO3a was previously shown to be regulated by 1,25-dihydroxyvitamin D3 in osteoblasts, a possible role for this family of TFs in chondrocytes during endochondral ossification remains largely unstudied. By crossing Collagen2-Cre mice with FoxO1lox/lox;FoxO3alox/lox;FoxO4lox/lox mice, we generated mice in which the three main FoxO isoforms were deleted in growth plate chondrocytes (chondrocyte triple knock-out; CTKO). Intriguingly, CTKO neonates showed a distinct elongation of the hypertrophic zone of the growth plate. CTKO mice had increased overall body and tail length at eight weeks of age and suffered from severe skeletal deformities at older ages. CTKO chondrocytes displayed decreased expression of genes involved in redox homeostasis. These observations illustrate the importance of FoxO signaling in chondrocytes during endochondral ossification.
Assuntos
Osso e Ossos/metabolismo , Condrócitos/metabolismo , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O3/genética , Fatores de Transcrição Forkhead/genética , Osteogênese/genética , Animais , Osso e Ossos/citologia , Proteínas de Ciclo Celular , Diferenciação Celular , Proliferação de Células , Condrócitos/citologia , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Cruzamentos Genéticos , Feminino , Proteína Forkhead Box O1/deficiência , Proteína Forkhead Box O3/deficiência , Fatores de Transcrição Forkhead/deficiência , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Integrases/genética , Integrases/metabolismo , Masculino , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Transgênicos , Oxirredutases/genética , Oxirredutases/metabolismo , Cultura Primária de Células , Transdução de SinaisRESUMO
Spaceflight leads to osteopenia in both humans and animals, principally as a result of decreased bone formation, which might be the consequence of impaired osteoblast differentiation. The effect of microgravity on osteoblast differentiation in vitro was investigated using the human osteosarcoma cell line MG-63. Genes related to matrix formation and maturation were quantified both at the protein and mRNA level in untreated and hormone-treated (dihydroxyvitamin D3 [1,25(OH)2D3], 10(-7) M; transforming growth factor beta2 (TGF-beta2), 10 ng/ml) cells cultured for 9 days under microgravity conditions aboard the Foton 10 satellite and compared with ground and inflight unit-gravity cultures. The expression of alkaline phosphatase (ALP) activity following treatment at microgravity increased only by a factor of 1.8 compared with the 3.8-fold increase at unit-gravity (p < 0.01), whereas no alteration was detected in the production of collagen type I between unit- and microgravity. In addition, gene expression for collagen Ialpha1, ALP, and osteocalcin following treatment at microgravity was reduced to 51, 62, and 19%, respectively, of unit-gravity levels (p < 0.02). The lack of correlation between collagen type I gene and protein expression induced by microgravity is most likely related to the different kinetics of gene and protein expression observed at unit-gravity: following treatment with 1,25(OH)2D3 and TGF-beta2, collagen Ialpha1 mRNA increased gradually during 72 h, but collagen type I production was already maximal after treatment for 48 h. In conclusion, microgravity decreases the activity of osteoblasts in vitro; in particular the differentiation of osteoblasts in response to systemic hormones and growth factors is reduced by microgravity.
Assuntos
Matriz Óssea/efeitos dos fármacos , Calcitriol/farmacologia , Osteoblastos/efeitos dos fármacos , Biossíntese de Proteínas , Fator de Crescimento Transformador beta/farmacologia , Ausência de Peso , Fosfatase Alcalina/metabolismo , Matriz Óssea/citologia , Matriz Óssea/metabolismo , Diferenciação Celular/fisiologia , Colágeno/biossíntese , Expressão Gênica , Humanos , Osteoblastos/citologia , Osteocalcina/biossíntese , Células Tumorais CultivadasRESUMO
Bone turnover requires the interaction of several proteases during the resorption phase. Indirect evidence suggests that the plasminogen activator/plasmin pathway is involved in bone resorption and turnover, and recently we have shown that this cascade plays a role in the degradation of nonmineralized bone matrix in vitro. To elucidate the role of the plasminogen activator inhibitor 1 (PAI-1) in bone turnover in vivo, bone metabolism was analyzed in mice deficient in the expression of PAI-1 gene (PAI-1-/-) at baseline (8-week-old mice) and 4 weeks after ovariectomy (OVX) or sham operation (Sham) and compared with wild-type (WT) mice. PAI-1 inactivation was without any effect on bone metabolism at baseline or in Sham mice. However, significant differences were observed in the response of WT and PAI-1-/- mice to ovariectomy. The OVX WT mice showed, as expected, decreased trabecular bone volume (BV/TV) and increased osteoid surface (OS/BS) and bone formation rate (BFR), as assessed by histomorphometric analysis of the proximal tibial metaphysis. In contrast, no significant change in any of the histomorphometric variables studied was detected in PAI-1-/- mice after ovariectomy. As a result, the OVX PAI-1-/- had a significantly higher BV/TV, lower OS/BS, lower mineral apposition rate (MAR) and BFR when compared with the OVX WT mice. However, a comparable decrease in the cortical thickness was observed in OVX PAI-1-/- and WT mice. In addition, the cortical mineral content and density assessed in the distal femoral metaphysis by peripheral quantitative computed tomography (pQCT), decreased significantly after ovariectomy, without difference between PAI-1-/- mice and WT mice. In conclusion, basal bone turnover and bone mass are only minimally affected by PAI-1 inactivation. In conditions of estrogen deficiency, PAI-1 inactivation protects against trabecular bone loss but does not affect cortical bone loss, suggesting a site-specific role for PAI-1 in bone turnover.
Assuntos
Reabsorção Óssea/prevenção & controle , Estrogênios/metabolismo , Inibidor 1 de Ativador de Plasminogênio/fisiologia , Animais , Densidade Óssea , Feminino , Fêmur/fisiopatologia , Úmero/fisiopatologia , Camundongos , Camundongos Knockout , Ovariectomia , Inibidor 1 de Ativador de Plasminogênio/genética , Tíbia/fisiopatologia , Tomografia Computadorizada por Raios X/métodosRESUMO
The plasminogen/plasmin proteolytic cascade plays an important role in extracellular matrix remodeling. The presence of the two plasminogen activators (PAs), tissue-type plasminogen activator (tPA), and urokinase-type plasminogen activator (uPA), and their inhibitor type 1 (PAI-1) in bone cells, suggests a role in one or more aspects of bone resorption such as osteoclast formation, mineral dissolution, and degradation of the organic matrix. These different processes were assayed in vitro using cells derived from mice with either tPA (tPA-/-), uPA (uPA-/-), PAI-1 (PAI-1-/-) inactivation or with a combined inactivation (tPA-/-:uPA-/-) and compared with wild-type mice (WT). First, osteoclast formation, assessed by investigating the number and characteristics of tartrate-resistant acid phosphatase-positive multinucleated cells formed in cocultures of primary osteoblasts and bone marrow cells treated with 1alpha,25-dihydroxyvitamin D3, was not different between the different cell types. Second, dentine resorption, an assay for osteoclast activity, was not affected by the combined deficiency of both tPA and uPA. Finally, the ability to degrade nonmineralized bone-like matrix was however, significantly reduced in tPA-/-:uPA-/- cells compared with WT cells (28.1 +/- 0.6%, n = 6 vs. 56.4 +/- 3.1%, n = 6, respectively, p < 0.0001). Surprisingly, collagen proteolysis by bone cells was not dependent on the presence of plasmin as suggested by degradation assays performed on type I 3H-collagen films. Taken together, these data suggest that the plasminogen activator/plasmin system is not required for osteoclast formation, nor for the resorption of the mineral phase, but is involved in the removal of noncollagenous proteins present in the nonmineralized bone matrix.
Assuntos
Reabsorção Óssea/fisiopatologia , Ativadores de Plasminogênio/fisiologia , Plasminogênio/fisiologia , Animais , Linhagem Celular , Colágeno/metabolismo , Técnicas In Vitro , Camundongos , Osteoclastos/fisiologia , Inibidor 1 de Ativador de Plasminogênio/fisiologia , Ativador de Plasminogênio Tecidual/fisiologia , Trítio , Ativador de Plasminogênio Tipo Uroquinase/fisiologiaRESUMO
UNLABELLED: Plasminogen activators tPA and uPA are involved in tissue remodeling, but their role in bone growth is undefined. Mice lacking tPA and uPA show increased bone formation and bone mass. The noncollagenous components of bone matrix are also increased, probably from defective degradation. This study underlines the importance of controlled bone matrix remodeling for normal endochondral ossification. INTRODUCTION: Proteolytic pathways are suggested to play a role in endochondral ossification. To elucidate the involvement of the plasminogen activators tPA and uPA in this process, we characterized the long bone phenotype in mice deficient in both tPA and uPA (tPA-/-:uPA-/-). MATERIALS AND METHODS: Bones of 2- to 7-day-old tPA-/-:uPA-/- and wild-type (WT) mice were studied using bone histomorphometry, electron microscopy analysis, and biochemical assessment of bone matrix components. Cell-mediated degradation of metabolically labeled bone matrix, osteoblast proliferation, and osteoblast differentiation, both at the gene and protein level, were studied in vitro using cells derived from both genotypes. RESULTS: Deficiency of the plasminogen activators led to elongation of the bones and to increased bone mass (25% more trabecular bone in the proximal tibial metaphysis), without altering the morphology of the growth plate. In addition, the composition of bone matrix was modified in plasminogen activator deficient mice, because an increased amount of proteoglycans (2x), osteocalcin (+45%), and fibronectin (+36%) was detected. Matrix degradation assays showed that plasminogen activators, by generating plasmin, participate in osteoblast-mediated degradation of the noncollagenous components of bone matrix. In addition, proliferation of primary osteoblasts derived from plasminogen activator-deficient mice was increased by 35%. Finally, osteoblast differentiation and formation of a mineralized bone matrix were enhanced in osteoblast cultures derived from tPA-/-:uPA-/- mice. CONCLUSIONS: The data presented indicate the importance of the plasminogen system in degradation of the noncollagenous components of bone matrix and suggest that the accumulation of these proteins in bone matrix--as occurs during plasminogen activator deficiency--may in turn stimulate osteoblast function, resulting in increased bone formation.
Assuntos
Osteogênese , Ativador de Plasminogênio Tecidual/deficiência , Ativador de Plasminogênio Tipo Uroquinase/deficiência , Animais , Matriz Óssea/metabolismo , Deleção de Genes , Regulação da Expressão Gênica , Camundongos , Camundongos Knockout , Tamanho do Órgão , Osteoblastos/metabolismo , Plasminogênio/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ativador de Plasminogênio Tecidual/genética , Ativador de Plasminogênio Tipo Uroquinase/genéticaRESUMO
UNLABELLED: 1alpha,25(OH)2-vitamin D strongly regulates the expression of the epithelial calcium channel CaT1. CaT1 expression is reduced in ERKOalpha mice and induced by estrogen treatment, pregnancy, or lactation in VDR WT and KO mice. Estrogens and vitamin D are thus independent potent regulators of the expression of this calcium influx mechanism, which is involved in active intestinal calcium absorption. INTRODUCTION: Active duodenal calcium absorption consists of three major steps: calcium influx into, transfer through, and extrusion out of the enterocyte. These steps are carried out by the calcium transport protein 1 (CaT1), calbindin-D9K, and the plasma membrane calcium ATPase (PMCA1b), respectively. We investigated whether estrogens or hormonal changes during the female reproductive cycle influence the expression of these genes, and if so, whether these effects are vitamin D-vitamin D receptor (VDR) dependent. MATERIALS AND METHODS: We evaluated duodenal expression patterns in estrogen receptor (ER)alpha and -beta knockout (KO) mice, as well as in ovariectomized, estrogen-treated, pregnant, and lactating VDR wild-type (WT) and VDR KO mice. RESULTS: Expression of calcium transporter genes was not altered in ERKObeta mice. CaT1 mRNA expression was reduced by 55% in ERKOalpha mice, while the two other calcium transporter genes were not affected. Ovariectomy caused no change in duodenal expression pattern of VDR WT and KO mice, whereas treatment with a pharmacologic dose of estrogens induced CaT1 mRNA expression in VDR WT (4-fold) and KO (8-fold) mice. Pregnancy enhanced CaTI expression equally in VDR WT and KO mice (12-fold). Calbindin-D9K and PMCA1b expression increased to a lesser extent and solely in pregnant VDR WT animals. In lactating VDR WT and KO mice, CaT1 mRNA expression increased 13 times, which was associated with a smaller increase in calbindin-D9K protein content and PMCA1b mRNA expression. CONCLUSIONS: Estrogens or hormonal changes during pregnancy or lactation have distinct, vitamin D-independent effects at the genomic level on active duodenal calcium absorption mechanisms, mainly through a major upregulation of the calcium influx channel CaT1. The estrogen effects seem to be mediated solely by ERalpha.
Assuntos
Canais de Cálcio/fisiologia , Cálcio/metabolismo , Estrogênios/metabolismo , Receptores de Calcitriol/metabolismo , Regulação para Cima , Animais , Transporte Biológico , Enterócitos/metabolismo , Receptor alfa de Estrogênio , Receptor beta de Estrogênio , Camundongos , Camundongos Knockout , Modelos Genéticos , Mutagênese , RNA Mensageiro/metabolismo , Receptores de Estrogênio/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Canais de Cátion TRPV , Vitamina D/metabolismoRESUMO
Space flight results in loss of bone mass, especially in weight-bearing bones, a condition that is suggested to be similar to disuse osteoporosis. As models to elucidate the underlying mechanism, bed rest studies were performed and bone metabolism in the rat both during space flight and during hindlimb unloading was investigated. The general picture is that bone formation is decreased partly as a result of reduced osteoblast function, whereas bone resorption is unaltered or increased. This deficit in bone mass can be replaced, but the time span for restoration exceeds the period of unloading. Changes in blood flow, systemic hormones, and locally produced factors are contributing in a yet undefined way to the response of osteoblastic cells to loading. The pathway by which loading and/or gravity are transduced into biochemical signals is still unknown. In vitro studies with osteoblastic cells show that their differentiation and cell morphology are altered during space flight. Elucidation of the involved signaling pathways has only recently been started. It is hoped that as the mechanisms by which bone responds to mechanical (un)loading are further understood, this insight will influence the treatment of other etiologies of osteoporosis.
Assuntos
Remodelação Óssea/fisiologia , Homeostase , Voo Espacial , Animais , Repouso em Cama/efeitos adversos , Biomarcadores , Densidade Óssea , Doenças Ósseas Metabólicas/etiologia , Doenças Ósseas Metabólicas/prevenção & controle , Reabsorção Óssea/etiologia , Reabsorção Óssea/prevenção & controle , Osso e Ossos/embriologia , Divisão Celular , Dinoprostona/biossíntese , Matriz Extracelular/fisiologia , Regulação da Expressão Gênica , Glucocorticoides/metabolismo , Gravitação , Decúbito Inclinado com Rebaixamento da Cabeça , Humanos , Sistema de Sinalização das MAP Quinases , Técnicas de Cultura de Órgãos , Osteoblastos/fisiologia , Osteoclastos/fisiologia , Osteogênese , Ratos , Estresse Mecânico , Suporte de Carga , Ausência de Peso/efeitos adversosRESUMO
Tissue-type plasminogen activator (t-PA) is a positive modulator of the plasminogen-plasmin system, which is involved in bone remodeling. In the present study, 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] was found to stimulate t-PA gene expression in ROS17/2.8 osteosarcoma cells. Transient transfection analysis and in vitro DNA binding studies identified two vitamin D-responsive elements (VDRE) in the human t-PA enhancer. The first VDRE (bp -7175 to -7146) comprised an inverted palindrome separated by 9 bp (IP9) overlapping a palindrome separated by 3 bp. The second VDRE (bp -7315 to -7302) is an IP2 element overlapping the previously identified retinoic acid-responsive element. 1,25(OH)(2)D(3) treatment of primary osteoblasts derived from t-PAlacZ transgenic mice containing 9 kb of 5' sequence of the human t-PA gene increased the number of lacZ-positive cells, fitting with the probability model of enhancer function.
Assuntos
Calcitriol/farmacologia , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Elementos de Resposta , Ativador de Plasminogênio Tecidual/genética , Animais , Sequência de Bases , Sítios de Ligação , Calcitriol/genética , Células Cultivadas , Relação Dose-Resposta a Droga , Humanos , Camundongos , Camundongos Transgênicos , Modelos Genéticos , Dados de Sequência Molecular , Osteoblastos , Osteossarcoma/metabolismo , Regiões Promotoras Genéticas , Ratos , Crânio/metabolismo , Fatores de Tempo , Transfecção , Células Tumorais CultivadasRESUMO
A blind study was set up to examine the in vitro growth characteristics of skin fibroblasts from 2 individuals with and 9 at risk for familial Alzheimer disease, 4 individuals with sporadic Alzheimer disease, 18 with Down syndrome as well as 5 younger and 6 older controls. Several variables (biopsy size, number of explants, medium, passage procedure) were standardized. Two growth characteristics were examined quantitatively: (i) the actual in vitro replicating life span was determined by counting the number of cells plated the previous week at 500,000 cells/flask (cumulative population doublings); and (ii) the growth potential was examined by a colony size distribution assay. A difference from the age-matched controls in the growth characteristics of skin fibroblasts was only observed for two patients with and one older individual at risk for familial Alzheimer disease. The growth properties of skin fibroblast cultures from patients with sporadic Alzheimer disease or Down syndrome were not at variance with their age-matched controls. The decrease in the growth potential observed in the familial Alzheimer disease fibroblasts is however modest and needs confirmation. It is clear that the growth properties of skin fibroblasts, as examined in this study, do not provide a good marker for any form of Alzheimer disease, nor do they provide an appropriate in vitro system to study factors which may contribute to the etiopathogenesis of Alzheimer disease or Down syndrome.
Assuntos
Doença de Alzheimer/patologia , Síndrome de Down/patologia , Fibroblastos/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/genética , Biomarcadores , Divisão Celular , Sobrevivência Celular , Feminino , Humanos , Técnicas In Vitro , Masculino , Pessoa de Meia-Idade , Progéria/patologia , Pele/patologiaRESUMO
Bone loss is observed after exposure to weightlessness in both astronauts and inflight animals. Histological and biochemical studies on rats have shown a decrease in bone formation, probably as a result of altered osteoblast function. To investigate whether microgravity alters osteoblast differentiation in vitro, the human osteosarcoma cell line MG-63 was used as a model. MG-63 cells can be induced to differentiate by treating the cells with 1,25(OH)2D3 (10(-7) mol/L) and transforming growth factor-beta 2 (TGFbeta2) (10 ng/mL). The message level of differentiation-related genes was quantitated via competitive reverse transcription-polymerase chain reaction (RT-PCR), both in untreated and hormone-treated cells cultured under microgravity for 9 days aboard the unmanned Foton 10 spaceflight, and compared to ground and inflight unit-gravity cultures. At microgravity, gene expression for collagen Ialpha1 following treatment was reduced to 51% of unit-gravity levels (p < 0.05). The amount of alkaline phosphatase messenger ribonucleic acid (mRNA) following treatment at microgravity increased by only a factor of 5 compared to the tenfold increase at unit gravity (p < 0.02). The osteocalcin message level in treated cells cultured at microgravity was only 19% of the level found in cells grown at unit gravity (p < 0.02). In conclusion, microgravity reduces the differentiation of osteoblastic MG-63 cells in response to systemic hormones and growth factors.
Assuntos
Fosfatase Alcalina/metabolismo , Colágeno/metabolismo , Expressão Gênica , Osteoblastos/metabolismo , Osteocalcina/metabolismo , Ausência de Peso , Fosfatase Alcalina/genética , Calcitriol/farmacologia , Diferenciação Celular , Células Cultivadas , Colágeno/genética , Combinação de Medicamentos , Humanos , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteocalcina/genética , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , Fator de Crescimento Transformador beta/farmacologiaRESUMO
One of the proteolytic systems produced by bone cells is the plasminogen activator/plasmin pathway, which involves the two plasminogen activators and the type 1 plasminogen activator inhibitor (PAI-1) and results in plasmin generation. We have recently demonstrated that this pathway plays a specific role in the degradation of the nonmineralized matrix of bone in vitro. To evaluate whether PAI-1 is required during bone resorption in vivo, we studied the effects of PAI-1 inactivation on bone metabolism using systemic administration of 1alpha,25 dihydroxyvitamin D(3) [1, 25(OH)(2)D(3)] as model. PAI-1-deficient (PAI-1-/-) and wild-type (WT) mice were injected intraperitoneally with 1,25(OH)(2)D(3) (2 microg/kg) or vehicle every other day during 4 weeks and analyzed using biochemical parameters of bone turnover, histomorphometric analysis of the proximal tibial metaphysis, and pQCT analysis of the distal femoral metaphysis. PAI-1 inactivation did not affect bone metabolism in vehicle-treated mice. Treatment with 1,25(OH)(2)D(3) induced bone resorption similarly in PAI-1-/- and WT mice, as assessed by the increase in the urinary excretion of calcium (2. 2-fold and 2.3-fold, respectively) and of pyridinoline crosslinks (by 24% and 22%, respectively). In addition, a comparable reduction in bone mass was observed in PAI-1-/- and WT mice after treatment with 1,25(OH)(2)D(3), as evidenced by the decrease in the femoral calcium content (by 25% and 32%, respectively), in the trabecular bone volume (by 50% and 40%, respectively), in the trabecular mineral content (by 17% and 15%, respectively), and in the cortical mineral content (by 45% and 52%, respectively). The parameters of bone turnover also increased after 1,25(OH)(2)D(3) treatment. Serum osteocalcin was, respectively, 25% and 28% higher in PAI-1-/- and WT mice treated with 1,25(OH)(2)D(3) compared with the mice injected with vehicle. Similarly, the osteoid surface increased in 1, 25(OH)(2)D(3)-treated PAI-1-/- and WT mice by 40% and 51%, respectively, the mineral apposition rate increased by 15% and 8%, respectively, and the bone formation rate by 54% and 48%, respectively. These data indicate that PAI-1 is not critical during bone resorption induced by 1,25(OH)(2)D(3) in vivo.
Assuntos
Reabsorção Óssea , Calcitriol/farmacologia , Agonistas dos Canais de Cálcio/farmacologia , Inibidor 1 de Ativador de Plasminogênio/fisiologia , Animais , Remodelação Óssea/efeitos dos fármacos , Remodelação Óssea/fisiologia , Deleção de Genes , Masculino , Camundongos , Camundongos KnockoutRESUMO
The localization of alpha 4 integrin subunit and of fibronectin in Alzheimer's disease (AD), Down's syndrome, Lewy body dementia (DLB) and normal brains was immunohistochemically investigated. Antibodies against the alpha 4 subunit and a fibronectin specific antibody stained 'neuritic' plaques in AD and Down's syndrome, while 'preamyloid' and 'burned-out' plaques remained negative. No alpha 4 integrin subunit or fibronectin immunoreactivity was detected in the plaques of DLB consistent with the absence of neuritic plaques. In addition, hippocampal pyramidal neurons and some neocortical neurons showed immunoreactivity with alpha 4 subunit and fibronectin antibodies in all aged individuals, but not in younger controls. These results suggest an age-related and localized expression of alpha 4 integrin subunit and fibronectin in the brain.
Assuntos
Doença de Alzheimer/metabolismo , Química Encefálica/fisiologia , Fibronectinas/imunologia , Corpos de Lewy/metabolismo , Receptores de Fibronectina/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/imunologia , Doença de Alzheimer/patologia , Anticorpos Monoclonais/imunologia , Encéfalo/patologia , Síndrome de Down/imunologia , Síndrome de Down/patologia , Hipocampo/patologia , Humanos , Imuno-Histoquímica , Corpos de Lewy/imunologia , Corpos de Lewy/patologia , Proteínas tau/imunologiaRESUMO
In Alzheimer's disease, the typical clinical symptoms and the pathological findings are restricted to the nervous system. Nevertheless, like in some other neurologic-metabolic disorders, several alterations are found in peripheral tissues. The aim of this study was to examine whether cellular properties which can be studied in vitro on skin fibroblast cultures obtained from Alzheimer's disease patients differ from those of age-matched controls. Down syndrome patients were also included, since the same neuropathological findings are present in nearly 100% of Down syndrome patients. Since Alzheimer's disease is an age-related disorder, we examined the growth characteristics of skin fibroblast cultures. The in vitro senescence of cultured fibroblasts is widely accepted as a model for in vivo ageing. Normal growth properties were found. We can conclude that there is no premature ageing in Alzheimer's disease nor in Down syndrome and that the abnormalities found in peripheral tissues are related to the disease itself. The beta amyloid precursor protein (beta APP) has been shown to have adhesive interactions. We therefore investigated several parameters of adhesion in the skin fibroblast cultures: adhesion to a fibronectin coat, adhesion to extracellular matrix of Alzheimer's disease cultures and semi-quantification of adhesion-related molecules (beta 1-integrin, cell surface proteoglycans, extracellular matrix proteoglycans, extracellular matrix fibronectin). No significant difference was found in the parameters examined.
Assuntos
Doença de Alzheimer/patologia , Senescência Celular , Síndrome de Down/patologia , Envelhecimento/fisiologia , Doença de Alzheimer/genética , Precursor de Proteína beta-Amiloide/metabolismo , Adesão Celular , Células Cultivadas , Síndrome de Down/genética , Fibroblastos/citologia , HumanosRESUMO
Monoclonal antibodies were raised against platelet integrins purified by affinity chromatography. Two monoclonal antibodies (5C5 and 3H8) reacted with the purified alpha 2 subunit of VLA-2 in Western blotting. The monoclonal antibody 3H8 also reacted with fibrinogen in Western blots and in ELISA tests. CNBr fragmentation of the alpha chain of fibrinogen generated two peptides which were still recognized by this monoclonal antibody in Western blotting. N-terminus sequencing of these two fragments showed that they were non-overlapping fragments of the fibrinogen alpha-chain with as only common epitope an RGD(F)/RGD(S) sequence. Dot blots and ELISA tests showed that the antibody 3H8 also recognized, however with lower affinity, fibronectin and collagen IV, which are RGDS containing extracellular matrix proteins. The assumption that Mab 3H8 recognizes an RGD sequence, was further supported by the findings that the binding of Mab 3H8 to fibrinogen was partially inhibited by RGDF containing peptides and that the antibody was able to inhibit platelet aggregation.
Assuntos
Anticorpos Monoclonais/imunologia , Antígenos de Plaquetas Humanas/imunologia , Epitopos/imunologia , Fibrinogênio/imunologia , Integrinas/imunologia , Oligopeptídeos/imunologia , Receptores de Antígeno muito Tardio/imunologia , Sequência de Aminoácidos , Especificidade de Anticorpos , Reações Antígeno-Anticorpo , Plaquetas/imunologia , Western Blotting , Reações Cruzadas , Citometria de Fluxo , Integrina alfa2 , Dados de Sequência Molecular , Fragmentos de Peptídeos/imunologia , Agregação Plaquetária/efeitos dos fármacosRESUMO
Dynamic loading can affect the bone surrounding implants. For ultrastructural exploration of the peri-implant tissue response to dynamic loading, titanium implants were installed in rat tibiae, in which one implant was loaded while the contralateral served as the unloaded control. The loaded implants received stimulation either within 24 hrs after implantation (immediate loading) or after a 28-day healing period (delayed loading) for 4, 7, 14, 21, or 28 days. The samples were processed for histology and gene expression quantification. Compared with the unloaded control, bone-to-implant contact increased significantly by immediate loading for 28 days (p < .05), but not in case of delayed loading. No effect of loading was observed on the bone formation in the implant thread areas, on the blood vessel area, and on endosteal callus formation. Loading during healing (immediate) for 7 days induced, relative to the unloaded control, a 2.3-fold increase of Runx2 in peri-implant cortical bone (p < .01) without a change in the RANKL/Opg ratio. Loading after healing (delayed) for 7 days up-regulated Runx2 (4.3-fold, p < .01) as well as Opg (22.3-fold, p < .05) compared with the unloaded control, resulting in a significantly decreased RANKL/Opg ratio. These results indicate a stimulating effect of dynamic loading on implant osseointegration when applied during the healing phase. In addition, gene expression analyses revealed molecular adaptations favoring bone formation and, at the same time, affecting bone remodeling.