RESUMO
3M syndrome is characterized by severe pre- and postnatal growth retardation, typical facial features, and normal intelligence. Homozygous or compound heterozygous mutations in either CUL7, OBSL1, or CCDC8 have been identified in the etiology so far. Clinical and molecular features of 24 patients (23 patients and a fetus) from 19 unrelated families with a clinical diagnosis of 3M syndrome were evaluated and genotype-phenotype correlations were investigated with the use of DNA sequencing, chromosomal microarray, and whole exome sequencing accordingly. A genetic etiology could be established in 20 patients (n = 20/24, 83%). Eleven distinct CUL7 or OBSL1 mutations, among which eight was novel, were identified in 18 patients (n = 18/24, 75%). Ten patients had CUL7 (n = 10/18, 56%) while eight had OBSL1 (n = 8/18, 44%) mutations. Birth weight and height standard deviation scores at admission were significantly (p < 0.05) lower in patients with CUL7 mutation compared to that of patients with OBSL1 mutation. Two patients with a similar phenotype had a de novo 20p13p deletion involving BMP2. No genetic etiology could be established in four patients (n = 4/28, 17%). This study yet represents the largest cohort of 3M syndrome patients from a single center in Turkey. Microdeletions involving BMP2 may cause a phenotype similar to 3M syndrome with some distinctive features. Larger cohort of patients are required to establish genotype-phenotype correlations in 3M syndrome.
Assuntos
Proteína Morfogenética Óssea 2/genética , Proteínas Culina/genética , Proteínas do Citoesqueleto/genética , Nanismo/genética , Estudos de Associação Genética , Hipotonia Muscular/genética , Mutação , Coluna Vertebral/anormalidades , Adolescente , Sequência de Bases , Proteína Morfogenética Óssea 2/deficiência , Criança , Pré-Escolar , Cromossomos Humanos Par 20 , Estudos de Coortes , Proteínas Culina/metabolismo , Proteínas do Citoesqueleto/metabolismo , Nanismo/diagnóstico , Nanismo/metabolismo , Nanismo/patologia , Feminino , Feto , Expressão Gênica , Genótipo , Humanos , Lactente , Recém-Nascido , Masculino , Hipotonia Muscular/diagnóstico , Hipotonia Muscular/metabolismo , Hipotonia Muscular/patologia , Fenótipo , Coluna Vertebral/metabolismo , Coluna Vertebral/patologia , Sequenciamento do ExomaRESUMO
The bone morphogenetic protein (BMP)-SMAD signaling pathway is a key transcriptional regulator of hepcidin in response to tissue iron stores, serum iron, erythropoietic drive and inflammation to increase the iron supply when needed for erythropoiesis, but to prevent the toxicity of iron excess. Recently, BMP2 was reported to play a non-redundant role in hepcidin regulation in addition to BMP6. Here, we used a newly validated BMP2 ELISA assay and mice with a global or endothelial conditional knockout (CKO) of Bmp2 or Bmp6 to examine how BMP2 is regulated and functionally contributes to hepcidin regulation by its major stimuli. Erythropoietin (EPO) did not influence BMP2 expression in control mice, and still suppressed hepcidin in Bmp2 CKO mice. Lipopolysaccharide (LPS) reduced BMP2 expression in control mice, but still induced hepcidin in Bmp2 CKO mice. Chronic dietary iron loading that increased liver iron induced BMP2 expression, whereas acute oral iron gavage that increased serum iron without influencing liver iron did not impact BMP2. However, hepcidin was still induced by both iron loading methods in Bmp2 CKO mice, although the degree of hepcidin induction was blunted relative to control mice. Conversely, acute oral iron gavage failed to induce hepcidin in Bmp6 -/- or CKO mice. Thus, BMP2 has at least a partially redundant role in hepcidin regulation by serum iron, tissue iron, inflammation and erythropoietic drive. In contrast, BMP6 is absolutely required for hepcidin regulation by serum iron.
Assuntos
Proteína Morfogenética Óssea 2/fisiologia , Proteína Morfogenética Óssea 6/fisiologia , Hepcidinas/metabolismo , Animais , Proteína Morfogenética Óssea 2/deficiência , Proteína Morfogenética Óssea 6/deficiência , Eritropoetina/farmacologia , Hepcidinas/efeitos dos fármacos , Inflamação , Ferro/sangue , Ferro/farmacologia , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos KnockoutRESUMO
Pulmonary arterial hypertension (PAH) is a devastating cardiopulmonary disorder characterized by pulmonary arterial remodeling mainly due to excess cellular proliferation and apoptosis resistance of pulmonary arterial smooth muscle cells (PASMCs). Reduced bone morphogenetic protein receptor 2 (BMPR2) expression in patients with PAH impairs pulmonary arterial endothelial cells (PAECs) function. This can adversely affect PAEC survival and promote PASMCs proliferation. We hypothesized that interventions to normalize the expression of genes that are targets of the BMPR2 signaling could restore PAECs function and prevent or reverse PAH. Here we characterized for the first time, in human PAECs, chemokine (C-C motif) ligand 5 (CCL5/RANTES) deficiency restore BMP-mediated PAECs function. In the cell culture experiments, we found that CCL5 deficiency increased apoptosis and tube formation of PAECs, but suppressed proliferation and migration of PASMCs. Silencing CCL5 expression in PAH PAECs restored bone morphogenetic protein (BMP) signaling responses and promoted phosphorylation of SMADs and transcription of ID genes. Moreover, CCL5 deficiency inhibited angiogenesis by increasing pSMAD-dependent and-independent BMPR2 signaling. This was linked mechanistically to enhanced interaction of BMPR2 with caveolin-1 via CCL5 deficiency-mediated stabilization of endothelial surface caveolin-1. Consistent with these functions, deletion of CCL5 significantly attenuated development of Sugen5416/hypoxia-induced PAH by restoring BMPR2 signaling in mice. Taken together, our findings suggest that CCL5 deficiency could reverse obliterative changes in pulmonary arteries via caveolin-1-dependent amplification of BMPR2 signaling. Our results shed light on better understanding of the disease pathobiology and provide a possible novel target for the treatment of PAH.
Assuntos
Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Caveolina 1/metabolismo , Quimiocina CCL5/deficiência , Hipertensão Pulmonar/metabolismo , Hipertensão Pulmonar/fisiopatologia , Artéria Pulmonar/metabolismo , Artéria Pulmonar/fisiopatologia , Animais , Proteína Morfogenética Óssea 2/deficiência , Proteína Morfogenética Óssea 2/metabolismo , Movimento Celular , Proliferação de Células , Quimiocina CCL5/metabolismo , Doença Crônica , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Endotélio Vascular/fisiopatologia , Feminino , Técnicas de Silenciamento de Genes , Hemodinâmica , Humanos , Hipertensão Pulmonar/etiologia , Hipertensão Pulmonar/patologia , Hipóxia/complicações , Ligantes , Pulmão/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Artéria Pulmonar/patologia , Receptores de Quimiocinas/metabolismo , Transdução de SinaisRESUMO
BACKGROUND AND AIMS: Obesity and diabetes potentiate vascular calcification by increasing vascular smooth muscle cells osteoblastic differentiation mediated by the transcription factor Msx2 and bone morphogenetic protein-2 signaling. However, Bmp-2/Msx2 crosstalk to induce VSMC osteogenic phenotype transition and calcification is poorly understood in diabetes. We aimed to investigate mechanisms underlying Bmp-2-driven VSMC osteogenic differentiation and calcification in leptin-deficient ob/ob mice. METHODS: We incubated VSMC from ob/ob mice and wild type C57BL/6 littermates with or without Bmp-2. We used loss-of-function experiments to investigate the role of Msx2 in Bmp-2-induced ob/ob VSMC osteochondrogenic differentiation and calcification by transfecting Msx2 siRNA into VSMC. RESULTS: Baseline ob/ob VSMC and aorta showed increased Msx2, Runx2, alkaline phosphatase mRNA and protein expression, which further increased in Bmp-2-incubated ob/ob VSMC, therefore augmenting ob/ob VSMC calcification in comparison to wild type VSMC. Accordingly, signaling pathways to induce VSMC osteogenic differentiation, such as Smad1/5 phosphorylation increased in ob/ob versus wild type aorta. In comparison to wild type VSMC, Msx2 siRNA transfected VSMC decreased Bmp-2-dependent osteochondrogenic differentiation response by abrogating Msx2, Runx2, Alpl expression in ob/ob but not in wild type VSMC. Nonetheless, Msx2 inhibition did not decrease calcification in Bmp-2 stimulated ob/ob VSMC in vitro. CONCLUSIONS: Our data support a crucial role of Msx2 for ob/ob VSMC osteochondrogenic differentiation, however, Msx2 signaling alone is not sufficient for ob/ob VSMC calcification after Bmp-2 stimulation in vitro. These findings can be translated into novel perspectives for the understanding of the mechanisms and to provide therapeutic targets underlying vascular calcification in type 2 diabetes.
Assuntos
Doenças da Aorta/metabolismo , Proteínas de Homeodomínio/metabolismo , Resistência à Insulina , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Obesidade/metabolismo , Osteoblastos/metabolismo , Osteogênese , Calcificação Vascular/metabolismo , Animais , Aorta/metabolismo , Aorta/patologia , Doenças da Aorta/genética , Doenças da Aorta/patologia , Proteína Morfogenética Óssea 2/deficiência , Proteína Morfogenética Óssea 2/genética , Células Cultivadas , Modelos Animais de Doenças , Predisposição Genética para Doença , Proteínas de Homeodomínio/genética , Resistência à Insulina/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Obesos , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Obesidade/genética , Obesidade/patologia , Osteoblastos/patologia , Fenótipo , Interferência de RNA , Transdução de Sinais , Transfecção , Calcificação Vascular/genética , Calcificação Vascular/patologiaRESUMO
Microvascular endothelial cells (ECs) display a high degree of phenotypic and functional heterogeneity among different organs. Organ-specific ECs control their tissue microenvironment by angiocrine factors in health and disease. Liver sinusoidal endothelial cells (LSECs) are uniquely differentiated to fulfill important organ-specific functions in development, under homeostatic conditions, and in regeneration and liver pathology. Recently, Bmp2 has been identified by us as an organ-specific angiokine derived from LSECs. To study angiocrine Bmp2 signaling in the liver, we conditionally deleted Bmp2 in LSECs using EC subtype-specific Stab2-Cre mice. Genetic inactivation of hepatic angiocrine Bmp2 signaling in Stab2-Cre;Bmp2fl/fl (Bmp2LSECKO) mice caused massive iron overload in the liver and increased serum iron levels and iron deposition in several organs similar to classic hereditary hemochromatosis. Iron overload was mediated by decreased hepatic expression of hepcidin, a key regulator of iron homeostasis. Thus, angiocrine Bmp2 signaling within the hepatic vascular niche represents a constitutive pathway indispensable for iron homeostasis in vivo that is nonredundant with Bmp6. Notably, we demonstrate that organ-specific angiocrine signaling is essential not only for the homeostasis of the respective organ but also for the homeostasis of the whole organism.
Assuntos
Proteína Morfogenética Óssea 2/genética , Células Endoteliais/metabolismo , Hemocromatose/genética , Hepcidinas/genética , Homeostase/genética , Ferro/metabolismo , Fígado/metabolismo , Animais , Proteína Morfogenética Óssea 2/deficiência , Proteína Morfogenética Óssea 6/genética , Proteína Morfogenética Óssea 6/metabolismo , Capilares/metabolismo , Capilares/patologia , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Células Endoteliais/patologia , Feminino , Regulação da Expressão Gênica , Hemocromatose/metabolismo , Hemocromatose/patologia , Hepatócitos/metabolismo , Hepatócitos/patologia , Hepcidinas/metabolismo , Integrases/genética , Integrases/metabolismo , Fígado/irrigação sanguínea , Fígado/patologia , Masculino , Camundongos , Camundongos Transgênicos , Comunicação Parácrina , Transdução de Sinais , Transcrição GênicaRESUMO
Bone morphogenetic proteins 2 and 4 (BMP2/4) are essential for osteoblast differentiation and osteogenesis. Generation of a BMP2/4 dual knock-out ((ko/ko)) osteoblastic cell line is a valuable asset for studying effects of BMP2/4 on skeletal development. In this study, our goal was to create immortalized mouse deleted BMP2/4 osteoblasts by infecting adenoviruses with Cre recombinase and green fluorescent protein genes into immortalized murine floxed BMP2/4 osteoblasts. Transduced BMP2/4(ko/ko) cells were verified by green immunofluorescence and PCR. BMP2/4(ko/ko) osteoblasts exhibited small size, slow cell proliferation rate and cell growth was arrested in G1 and G2 phases. Expression of bone-relate genes was reduced in the BMP2/4(ko/ko) cells, resulting in delay of cell differentiation and mineralization. Importantly, extracellular matrix remodeling was impaired in the BMP2/4(ko/ko) osteoblasts as reflected by decreased Mmp-2 and Mmp-9 expressions. Cell differentiation and mineralization were rescued by exogenous BMP2 and/or BMP4. Therefore, we for the first time described establishment of an immortalized deleted BMP2/4 osteoblast line useful for study of mechanisms in regulating osteoblast lineages.
Assuntos
Proteína Morfogenética Óssea 2/deficiência , Proteína Morfogenética Óssea 4/deficiência , Diferenciação Celular , Proliferação de Células , Técnicas de Silenciamento de Genes , Osteogênese , Animais , Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 4/genética , Linhagem Celular , Tamanho Celular , Matriz Extracelular/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular , Pontos de Checagem da Fase G2 do Ciclo Celular , Regulação da Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Integrases/genética , Integrases/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Osteoblastos , Fenótipo , Fatores de Tempo , Transdução GenéticaRESUMO
Post-natal osteogenesis after mechanical trauma or stimulus occurs through either endochondral healing, intramembranous healing or lamellar bone formation. Bone morphogenetic protein 2 (BMP2) is up-regulated in each of these osteogenic processes and is expressed by a variety of cells including osteoblasts and vascular cells. It is known that genetic knockout of Bmp2 in all cells or in osteo-chondroprogenitor cells completely abrogates endochondral healing after full fracture. However, the importance of BMP2 from differentiated osteoblasts and endothelial cells is not known. Moreover, the importance of BMP2 in non-endochondral bone formation such as intramembranous healing or lamellar bone formation is not known. Using inducible and tissue-specific Cre-lox mediated targeting of Bmp2 in adult (10-24 week old) mice, we assessed the role of BMP2 expression globally, by osteoblasts, and by vascular endothelial cells in endochondral healing, intramembranous healing and lamellar bone formation. These three osteogenic processes were modeled using full femur fracture, ulnar stress fracture, and ulnar non-damaging cyclic loading, respectively. Our results confirmed the requirement of BMP2 for endochondral fracture healing, as mice in which Bmp2 was knocked out in all cells prior to fracture failed to form a callus. Targeted deletion of Bmp2 in osteoblasts (osterix-expressing) or vascular endothelial cells (vascular endothelial cadherin-expressing) did not impact fracture healing in any way. Regarding non-endochondral bone formation, we found that BMP2 is largely dispensable for intramembranous bone formation after stress fracture and also not required for lamellar bone formation induced by mechanical loading. Taken together our results indicate that osteoblasts and endothelial cells are not a critical source of BMP2 in endochondral fracture healing, and that non-endochondral bone formation in the adult mouse is not as critically dependent on BMP2.
Assuntos
Proteína Morfogenética Óssea 2/deficiência , Consolidação da Fratura/fisiologia , Osteogênese/fisiologia , Animais , Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 2/fisiologia , Células Endoteliais/fisiologia , Feminino , Consolidação da Fratura/genética , Fraturas de Estresse/genética , Fraturas de Estresse/patologia , Fraturas de Estresse/fisiopatologia , Expressão Gênica , Masculino , Camundongos , Camundongos Knockout , Osteoblastos/fisiologia , Osteogênese/genética , Estresse MecânicoRESUMO
Depletion of Wnt signaling is a major requirement for the induction of the anterior prosencephalon. However, the molecular events driving the differential regionalization of this area into eye-field and telencephalon fates are still unknown. Here we show that the BMP pathway is active in the anterior neural ectoderm during late blastula to early gastrula stage in zebrafish. Bmp2b mutants and mosaic loss-of-function experiments reveal that BMP acts as a repressor of eye-field fate through inhibition of its key transcription factor Rx3, thereby protecting the future telencephalon from acquiring eye identity. This BMP-driven mechanism initiates the establishment of the telencephalon prior to the involvement of Wnt antagonists from the anterior neural border. Furthermore, we demonstrate that Rx3 and BMP are respectively required to maintain and restrict the chemokine receptor cxcr4a, which in turn contributes to the morphogenetic separation of eye-field and telencephalic cells during early neurulation.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Olho/embriologia , Morfogênese , Receptores CXCR4/metabolismo , Transdução de Sinais , Telencéfalo/embriologia , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Proteína Morfogenética Óssea 2/deficiência , Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 2/metabolismo , Proteínas Morfogenéticas Ósseas/genética , Olho/citologia , Olho/metabolismo , Proteínas de Homeodomínio/metabolismo , Mutação , Transdução de Sinais/genética , Telencéfalo/citologia , Telencéfalo/metabolismo , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/genéticaRESUMO
In amniotes, ventral folding morphogenesis achieves gut internalization, linear heart tube formation, ventral body wall closure, and encasement of the fetus in extraembryonic membranes. Impairment of ventral morphogenesis results in human birth defects involving body wall, gut, and heart malformations and in mouse misplacement of head and heart. Absence of knowledge about genetic pathways and cell populations directing ventral folding in mammals has precluded systematic study of cellular mechanisms driving this vital morphogenetic process. We report tissue-specific mouse mutant analyses identifying the bone morphogenetic protein (BMP) pathway as a key regulator of ventral morphogenesis. BMP2 expressed in anterior visceral endoderm (AVE) signals to epiblast derivatives during gastrulation to orchestrate initial stages of ventral morphogenesis, including foregut development and positioning of head and heart. These findings identify unanticipated functions for the AVE in organizing the gastrulating embryo and indicate that visceral endoderm-expressed BMP2 coordinates morphogenetic cell behaviors in multiple epiblast lineages.
Assuntos
Proteína Morfogenética Óssea 2/metabolismo , Endoderma/metabolismo , Cabeça/embriologia , Coração/embriologia , Morfogênese , Animais , Proteína Morfogenética Óssea 2/deficiência , Proteína Morfogenética Óssea 2/genética , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Endoderma/citologia , Hibridização In Situ , Camundongos , Camundongos KnockoutRESUMO
Bone morphogenetic proteins (BMPs) are critical molecules during gut morphogenesis. However, little is known about their participation in the homeostasis of adult gut and their possible role in diseases. Gastrointestinal complications occur during diabetes with loss of enteric neurons. In this study, we investigated the possible involvement of BMPs signaling pathway in diabetic enteric neuropathy in an experimental model of diabetes in rats. The expression of BMPs, BMPs receptors and intracellular Smad effectors were assessed in control and diabetic smooth muscle layer of jejunum by immunofluorescence, Western blot and RT-PCR methods. Myenteric neurons and glial cells were measured by immunofluorescence using specific markers. In addition, cell apoptosis was evaluated by means of direct and indirect techniques. We demonstrated that diabetic ganglia displayed a significant decrease in ganglion size due to enhanced apoptosis and loss of peripherin. A decrease in glial fibrillary acidic protein (GFAP protein) was also observed in enteric glial cells. BMP-2 was down-regulated in the myenteric plexus of diabetic rats at 3 and 9weeks. A loss of enteric neurons by apoptosis was correlated with an ectopic BMP-4, increased BMPR-Ia and nuclear p-Smad1 expression in the myenteric plexus. Insulin-treatment prevented the intestinal alterations observed. These findings suggest that diabetes is associated with an abnormal BMP/Smad signaling expression in the myenteric ganglia that affects the homeostasis of the enteric plexus.
Assuntos
Proteína Morfogenética Óssea 2/deficiência , Neuropatias Diabéticas/patologia , Neuropatias Diabéticas/fisiopatologia , Plexo Mientérico/patologia , Plexo Mientérico/fisiopatologia , Proteína Smad1/deficiência , Animais , Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 2/fisiologia , Neuropatias Diabéticas/metabolismo , Modelos Animais de Doenças , Homeostase/genética , Masculino , Plexo Mientérico/metabolismo , Ratos , Ratos Wistar , Proteína Smad1/genética , Proteína Smad1/fisiologiaRESUMO
Tooth development is regulated by epithelial-mesenchymal interactions and their reciprocal molecular signaling. Bone morphogenetic protein 2 (Bmp2) is essential for tooth formation. However, the role of Bmp2 during enamel formation remains unknown in vivo. In this study, the role of Bmp2 in the regulation of postnatal enamel formation was investigated via the conditional ablation of Bmp2 in enamel using the (Osx-Cre) mouse. Bmp2 gene ablation was confirmed by PCR analysis in Osx-Cre, Bmp2(flox/flox) mice. Bmp2-null mice displayed a severe and profound tooth phenotype with asymmetric and open forked incisors. Microradiographs revealed broken incisor tips and dental pulp chamber exposure. The enamel layer of incisors and molars was thin with hypomineralization. Scanning electron microscopy analysis showed that the enamel surface was rough with chipping and the enamel lacked a typical prismatic architecture. These results demonstrate that Bmp2 is essential for enamel formation.
Assuntos
Proteína Morfogenética Óssea 2/deficiência , Esmalte Dentário/anormalidades , Animais , Densidade Óssea , Proteína Morfogenética Óssea 2/metabolismo , Esmalte Dentário/diagnóstico por imagem , Esmalte Dentário/patologia , Esmalte Dentário/ultraestrutura , Camundongos , Camundongos Knockout , Dente/diagnóstico por imagem , Dente/patologia , Dente/ultraestrutura , Microtomografia por Raio-XRESUMO
Analysis of animal models is indispensable to elucidate the molecular mechanism in vascular calcification (VC) as well as to develop new therapies for VC. Various gene-modified mice that show VC have been reported, and considerable progress has been made through the analyses of these animals. Mice of which bone-calcification regulatory factors were modified are the representative animal models for VC, indicating that these factors certainly regulate VC as well as bone-calcification. Inducible VC in wild-type animals is also an important research tool for developing preventive and therapeutic approach for VC.
Assuntos
Animais Geneticamente Modificados , Calcinose , Modelos Animais de Doenças , Camundongos/genética , Doenças Vasculares , Animais , Proteína Morfogenética Óssea 2/deficiência , Calcinose/genética , Proteínas de Ligação ao Cálcio/deficiência , Proteínas da Matriz Extracelular/deficiência , Proteínas de Membrana/deficiência , Osteoprotegerina/deficiência , Proteínas de Transporte de Fosfato , Diester Fosfórico Hidrolases/deficiência , Pirofosfatases/deficiência , Doenças Vasculares/genética , Proteína de Matriz GlaRESUMO
Although it is predicted that vitamin A and its active form, retinoic acid, regulate osteoblast lineage, this has not been elucidated in growing mammalians. To clarify the direct effect of retinoic acid on bone, we observed the process of filling up newly generating bone into a drill-hole of the bone, which is understood as membranous ossification, in vitamin A-deficient mice. Mice were assigned to three groups: a vitamin A-deficient group (VAD), which was fed a diet without vitamin A from the 10th day of gestation to the end of the experiments; a vitamin A-deficient-sufficient group (VADS), which was fed a diet without vitamin A from the 10th day of gestation to 4 weeks of age; and a vitamin A-sufficient group (VAS), which was fed a standard diet to the end of the experiment. In mice at 10 weeks of age (day 0), a drill-hole injury was made with a diameter of 1mm at the anterior portion of the diaphysis of the bilateral femurs. In VAD, retardation in repairing the drill-hole was demonstrated by in vivo micro-CT and histomorphometry from day 7 and after surgery. During repair of the bone defect, increases of bmp2, dlx5, msx2, col1a1, and osteocalcin mRNA expression were suppressed, and runx2-p2 mRNA expression was accelerated in VAD. Implantation of BMP2 in the bone defect of VAD normalized the delayed bone healing and mRNA expressions. We concluded that vitamin A regulates bmp2 mRNA expression and plays a crucial role in osteoblastogenesis and bone formation.
Assuntos
Proteína Morfogenética Óssea 2/deficiência , Fêmur/lesões , Fêmur/patologia , Deficiência de Vitamina A/fisiopatologia , Cicatrização , Animais , Fenômenos Biomecânicos , Peso Corporal , Densidade Óssea , Proteína Morfogenética Óssea 2/biossíntese , Regeneração Óssea , Feminino , Fêmur/diagnóstico por imagem , Regulação da Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tíbia/fisiopatologia , Vitamina A/sangue , Deficiência de Vitamina A/sangue , Deficiência de Vitamina A/patologia , Microtomografia por Raio-XRESUMO
Previous reports suggest the application of exogenous BMPs can accelerate bone formation during distraction osteogenesis (DO). However, there are drawbacks associated with the use of exogenous BMPs. A possible alternative to the use of exogenous BMPs is to upregulate the expression of endogenous BMPs. Since DO results in spontaneously generated de novo bone formation in a uniform radiographic, histological, and biomechanical temporal sequence, a genetically engineered model lacking endogenous BMP2 should have measurable deficits in bone formation at different time points. We performed DO on BMP2(fl/+) and BMP2(fl/+ cre) mice using a miniature Ilizarov fixator. Distracted samples were collected at various time points and analyzed using real time-quantitative PCR, lCT, radiology, immunohistochemistry, histology, and biomechanical testing. Immunohistochemical studies of 34-day heterozygous samples showed reduced expression of BMP2, BMP7, BMPR1a, ACTR1, and ACTR2b. lCT analysis of 51-day heterozygous samples revealed a decrease in trabecular number and increase in trabecular separation. Biomechanical testing of 51-day heterozygous samples revealed decreased stiffness and increased ultimate displacement. Radiological analysis showed the heterozygotes contained a decreased bone fill score at 17, 34, and 51 days. These data suggest endogenous BMPs are important for bone healing and manipulating endogenous BMPs may help accelerate bone consolidation during DO.