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1.
Environ Sci Pollut Res Int ; 26(36): 36322-36332, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31713825

RESUMO

The widespread use of thiram has raised concerns for health and its toxic effects, but the underlying toxicity mechanism on platelets and bones is poorly defined. Here, we found a significant increase in the number of platelets in chickens with the thiram intake, due to the increased expression of thrombopoietin mRNA in the dysfunction liver. Furthermore, the decreased vascular distribution and cell death of chondrocytes in the tibial growth plates (TGPs) were observed, resulting in bone growth inhibition, which is associated with the abnormal activation of platelets leading to the extraordinary decrease of vascular endothelial growth factor A (VEGFA) and angiopoietin-1 protein were released and their corresponding receptors VEGFR2 and Tie-2 expressions were also reduced in the TGPs. Taken together, these findings revealed that thiram has an adverse effect on bones and platelets, which may have a high risk of thrombosis and osteoarthritis.


Assuntos
Galinhas/sangue , Galinhas/crescimento & desenvolvimento , Lâmina de Crescimento/efeitos dos fármacos , Praguicidas/toxicidade , Tiram/toxicidade , Proteínas Angiogênicas/metabolismo , Animais , Lâmina de Crescimento/irrigação sanguínea , Lâmina de Crescimento/metabolismo , Lâmina de Crescimento/patologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Contagem de Plaquetas , Trombopoetina/metabolismo , Tíbia/efeitos dos fármacos , Tíbia/crescimento & desenvolvimento
2.
Int J Mol Sci ; 20(23)2019 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-31757091

RESUMO

The longitudinal growth of long bone, regulated by an epiphyseal cartilaginous component known as the "growth plate", is generated by epiphyseal chondrocytes. The growth plate provides a continuous supply of chondrocytes for endochondral ossification, a sequential bone replacement of cartilaginous tissue, and any failure in this process causes a wide range of skeletal disorders. Therefore, the cellular and molecular characteristics of the growth plate are of interest to many researchers. Hedgehog (Hh), well known as a mitogen and morphogen during development, is one of the best known regulatory signals in the developmental regulation of the growth plate. Numerous animal studies have revealed that signaling through the Hh pathway plays multiple roles in regulating the proliferation, differentiation, and maintenance of growth plate chondrocytes throughout the skeletal growth period. Furthermore, over the past few years, a growing body of evidence has emerged demonstrating that a limited number of growth plate chondrocytes transdifferentiate directly into the full osteogenic and multiple mesenchymal lineages during postnatal bone development and reside in the bone marrow until late adulthood. Current studies with the genetic fate mapping approach have shown that the commitment of growth plate chondrocytes into the skeletal lineage occurs under the influence of epiphyseal chondrocyte-derived Hh signals during endochondral bone formation. Here, we discuss the valuable observations on the role of the Hh signaling pathway in the growth plate based on mouse genetic studies, with some emphasis on recent advances.


Assuntos
Desenvolvimento Ósseo , Ossos da Extremidade Inferior/metabolismo , Ossos da Extremidade Superior/metabolismo , Lâmina de Crescimento/metabolismo , Proteínas Hedgehog/metabolismo , Animais , Ossos da Extremidade Inferior/crescimento & desenvolvimento , Ossos da Extremidade Superior/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Lâmina de Crescimento/crescimento & desenvolvimento , Proteínas Hedgehog/genética , Humanos , Transdução de Sinais
3.
Int J Mol Sci ; 20(18)2019 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-31514268

RESUMO

In the development of the skeleton, the long bones are arising from the process of endochondral ossification (EO) in which cartilage is replaced by bone. This complex process is regulated by various factors including genetic, epigenetic, and environmental elements. It is recognized that DNA methylation, higher-order chromatin structure, and post-translational modifications of histones regulate the EO. With emerging understanding, non-coding RNAs (ncRNAs) have been identified as another mode of EO regulation, which is consist of microRNAs (miRNAs or miRs) and long non-coding RNAs (lncRNAs). There is expanding experimental evidence to unlock the role of ncRNAs in the differentiation of cartilage cells, as well as the pathogenesis of several skeletal disorders including osteoarthritis. Cutting-edge technologies such as epigenome-wide association studies have been employed to reveal disease-specific patterns regarding ncRNAs. This opens a new avenue of our understanding of skeletal cell biology, and may also identify potential epigenetic-based biomarkers. In this review, we provide an updated overview of recent advances in the role of ncRNAs especially focus on miRNA and lncRNA in the development of bone from cartilage, as well as their roles in skeletal pathophysiology.


Assuntos
Cartilagem/crescimento & desenvolvimento , Cartilagem/metabolismo , RNA não Traduzido/genética , Animais , Condrócitos/citologia , Condrócitos/metabolismo , Epigênese Genética , Lâmina de Crescimento/metabolismo , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , RNA não Traduzido/metabolismo
4.
J Pediatr Orthop ; 39(9): 479-486, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31503237

RESUMO

BACKGROUND: Although physeal fractures and physeal bars can result in significant clinical consequences to growth and development of the injured physis, little orthopaedic research has focused upon this topic. Our objective was to extend a previously developed rat model to examine the immunohistochemical features following surgical application of techniques disrupting the physis. METHODS: Physes were surgically disrupted using fracture (control), epiphyseal scrape (ES), or epiphyseal drill (ED). After 1, 3, 6, 10, or 21 days, animals were euthanized, sites processed for histology and immunohistochemical localization of vascular endothelial growth factor (VEGF), Factor VIII, Sox-9, PTHrP (parathyroid hormone-related protein) and PTHrP-R (parathyroid hormone-related protein receptor) in resting, proliferative, and hypertrophic physeal zones. Incidence of physeal bars, vertical septa and islands within the metaphysis was quantified. Semiquantitative analysis of immunohistochemistry was performed. RESULTS: Physeal bars, vertical septa, and displaced cartilage islands were present each of the surgical treatments. Fisher's exact test showed a statistically significant increase in the presence of physeal bars (P=0.002) and vertical septa (P=0.012) in the ED group at 10 and 21 days. Analysis of VEGF showed significant differences among the surgical treatments involving the resting zone, and the proliferative zone for days 1, 6, and 21 (P≤0.02) with greater mean scores present in the fracture (control) group, followed by the ED group; the lowest scores were present in the ES group. PTHrP-R immunolocalization showed significant differences among treatments in the hypertrophic zone at days 6 and 21 (P=0.022 and 0.044, respectively). CONCLUSIONS: On the basis of the type of surgical treatment, results show significant differences in the presence of VEGF (reflecting the vascular bed) in the resting and proliferating zones at days 1, 6, and 21. VEGF localization was less abundant in the ED group (which had more physeal bars), suggesting that lack of vascular ingrowth plays a role in physeal bar formation. CLINICAL RELEVANCE: Basic science data presented here provide insight into the importance of the various regions of the physis and its repair and continued growth after physeal fracture. We suggest that a better understanding of the cellular basis of physeal arrest following physeal fracture may have future relevance for the development of treatments to prevent or correct arrest.


Assuntos
Lâmina de Crescimento/metabolismo , Fraturas Salter-Harris/metabolismo , Técnicas de Ablação , Animais , Epífises/lesões , Epífises/metabolismo , Fator VIII/metabolismo , Lâmina de Crescimento/cirurgia , Imuno-Histoquímica , Proteína Relacionada ao Hormônio Paratireóideo/metabolismo , Ratos , Receptor Tipo 1 de Hormônio Paratireóideo/metabolismo , Fatores de Transcrição SOX9/metabolismo , Fraturas Salter-Harris/cirurgia , Fator A de Crescimento do Endotélio Vascular/metabolismo
5.
J Immunol ; 203(5): 1356-1368, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31366715

RESUMO

Singleton-Merten syndrome (SMS) is a type I interferonopathy characterized by dental dysplasia, aortic calcification, skeletal abnormalities, glaucoma, and psoriasis. A missense mutation in IFIH1 encoding a cytoplasmic viral RNA sensor MDA5 has recently been identified in the SMS patients as well as in patients with a monogenic form of lupus. We previously reported that Ifih1gs/+ mice express a constitutively active MDA5 and spontaneously develop lupus-like nephritis. In this study, we demonstrate that the Ifih1gs/+ mice also exhibit SMS-like bone abnormalities, including decreased bone mineral density and thin cortical bone. Histological analysis revealed a low number of osteoclasts, low bone formation rate, and abnormal development of growth plate cartilages in Ifih1gs/+ mice. These abnormalities were not observed in Ifih1gs/+ ・Mavs-/- and Ifih1gs/+ ・Ifnar1-/- mice, indicating the critical role of type I IFNs induced by MDA5/MAVS-dependent signaling in the bone pathogenesis of Ifih1gs/+ mice, affecting bone turnover. Taken together, our findings suggest the inhibition of type I IFN signaling as a possible effective therapeutic strategy for bone disorders in SMS patients.


Assuntos
Doenças da Aorta/metabolismo , Doenças Ósseas/metabolismo , Osso e Ossos/anormalidades , Osso e Ossos/metabolismo , Hipoplasia do Esmalte Dentário/metabolismo , Helicase IFIH1 Induzida por Interferon/metabolismo , Metacarpo/anormalidades , Doenças Musculares/metabolismo , Odontodisplasia/metabolismo , Osteoporose/metabolismo , Calcificação Vascular/metabolismo , Animais , Doenças da Aorta/genética , Doenças Ósseas/genética , Cartilagem/metabolismo , Hipoplasia do Esmalte Dentário/genética , Lâmina de Crescimento/metabolismo , Masculino , Metacarpo/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Doenças Musculares/genética , Mutação de Sentido Incorreto/genética , Odontodisplasia/genética , Osteoporose/genética , Calcificação Vascular/genética
6.
J Cell Biol ; 218(9): 3134-3152, 2019 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-31371388

RESUMO

Regulated growth plate activity is essential for postnatal bone development and body stature, yet the systems regulating epiphyseal fusion are poorly understood. Here, we show that the tissue inhibitors of metalloprotease (TIMP) gene family is essential for normal bone growth after birth. Whole-body quadruple-knockout mice lacking all four TIMPs have growth plate closure in long bones, precipitating limb shortening, epiphyseal distortion, and widespread chondrodysplasia. We identify TIMP/FGF-2/IHH as a novel nexus underlying bone lengthening where TIMPs negatively regulate the release of FGF-2 from chondrocytes to allow IHH expression. Using a knock-in approach that combines MMP-resistant or ADAMTS-resistant aggrecans with TIMP deficiency, we uncouple growth plate activity in axial and appendicular bones. Thus, natural metalloprotease inhibitors are crucial regulators of chondrocyte maturation program, growth plate integrity, and skeletal proportionality. Furthermore, individual and combinatorial TIMP-deficient mice demonstrate the redundancy of metalloprotease inhibitor function in embryonic and postnatal development.


Assuntos
Desenvolvimento Ósseo , Osso e Ossos/metabolismo , Condrócitos/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Lâmina de Crescimento/metabolismo , Inibidores Teciduais de Metaloproteinases/metabolismo , Animais , Fator 2 de Crescimento de Fibroblastos/genética , Camundongos , Camundongos Knockout , Inibidores Teciduais de Metaloproteinases/genética
7.
Curr Med Sci ; 39(4): 604-608, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31346997

RESUMO

Children presenting with partial physeal arrest and significant remaining growth may benefit from physeal bar resection, although the operation is a technique demanding procedure. This study evaluates the treatment of post-traumatic pediatric ankle varus deformity using physeal bar resection and hemi-epiphysiodesis with the assistance of two operative methods. Forty-five patients presenting with a distal tibial medial physeal bridge as well as ankle varus deformity following traumatic ankle physeal injury between 2009 and 2017 were followed. These patients were treated with physeal bar resection and hemi-epiphysiodesis, with the assistance of either fluoroscopy (10 cases) or intraoperative three-dimensional navigation (35 cases). Of the 45 cases, the median age was 9.0 years (range: 3-14 years) with 28 male and 17 female patients. The median of pre-operation ankle varus angle was 20 degrees (IQR 15-25) and 5 degrees (IQR 0-20) at the time of final follow up, representing a statistically significant difference (P<0.05). No differences were observed with regards to age, gender, and surgical history between effective group and ineffective group (P>0.05). The median of pre-operative ankle varus angles of the navigation and fluoroscopy groups were both 20 degrees (P>0.05). The median correction angle of the navigation and fluoroscopy groups was 10 and 15 degrees, respectively (P>0.05). Our results indicate that physeal bar resection and hemiepiphysiodesis are effective treatments for correcting ankle varus deformity due to traumatic medial physeal arrest of the distal tibia. We observe no difference in outcome between fluoroscopy group and three-dimensional navigation group during the procedures.


Assuntos
Tornozelo/patologia , Lâmina de Crescimento/metabolismo , Metatarso Varo/terapia , Tíbia/metabolismo , Adolescente , Criança , Pré-Escolar , Feminino , Lâmina de Crescimento/patologia , Humanos , Masculino , Metatarso Varo/genética , Metatarso Varo/patologia , Período Pré-Operatório , Tíbia/patologia , Resultado do Tratamento
8.
PLoS Genet ; 15(6): e1008216, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31246957

RESUMO

ASAP1 is a multi-domain adaptor protein that regulates cytoskeletal dynamics, receptor recycling and intracellular vesicle trafficking. Its expression is associated with poor prognosis for a variety of cancers, and promotes cell migration, invasion and metastasis. Little is known about its physiological role. In this study, we used mice with a gene-trap inactivated ASAP1 locus to study the functional role of ASAP1 in vivo, and found defects in tissues derived from mesenchymal progenitor cells. Loss of ASAP1 led to growth retardation and delayed ossification typified by enlarged hypertrophic zones in growth plates and disorganized chondro-osseous junctions. Furthermore, loss of ASAP1 led to delayed adipocyte development and reduced fat depot formation. Consistently, deletion of ASAP1 resulted in accelerated chondrogenic differentiation of mesenchymal cells in vitro, but suppressed osteo- and adipogenic differentiation. Mechanistically, we found that FAK/Src and PI3K/AKT signaling is compromised in Asap1GT/GT MEFs, leading to impaired adipogenic differentiation. Dysregulated FAK/Src and PI3K/AKT signaling is also associated with attenuated osteogenic differentiation. Together these observations suggest that ASAP1 plays a decisive role during the differentiation of mesenchymal progenitor cells.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Adipogenia/genética , Condrogênese/genética , Osteogênese/genética , Animais , Diferenciação Celular/genética , Quinase 1 de Adesão Focal/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Lâmina de Crescimento/crescimento & desenvolvimento , Lâmina de Crescimento/metabolismo , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Proteína Oncogênica v-akt/genética , Fosfatidilinositol 3-Quinases/genética , Quinases da Família src/genética
9.
J Cell Biol ; 218(6): 1853-1870, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31085560

RESUMO

In childhood, skeletal growth is driven by transient expansion of cartilage in the growth plate. The common belief is that energy production in this hypoxic tissue mainly relies on anaerobic glycolysis and not on mitochondrial respiratory chain (RC) activity. However, children with mitochondrial diseases causing RC dysfunction often present with short stature, which indicates that RC activity may be essential for cartilage-mediated skeletal growth. To elucidate the role of the mitochondrial RC in cartilage growth and pathology, we generated mice with impaired RC function in cartilage. These mice develop normally until birth, but their later growth is retarded. A detailed molecular analysis revealed that metabolic signaling and extracellular matrix formation is disturbed and induces cell death at the cartilage-bone junction to cause a chondrodysplasia-like phenotype. Hence, the results demonstrate the overall importance of the metabolic switch from fetal glycolysis to postnatal RC activation in growth plate cartilage and explain why RC dysfunction can cause short stature in children with mitochondrial diseases.


Assuntos
Cartilagem/patologia , Condrócitos/patologia , Complexo de Proteínas da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Transtornos do Crescimento/complicações , Lâmina de Crescimento/patologia , Doenças Mitocondriais/etiologia , Animais , Cartilagem/metabolismo , Diferenciação Celular , Condrócitos/metabolismo , Colágeno Tipo II/fisiologia , DNA Helicases/fisiologia , Transporte de Elétrons , Metabolismo Energético , Transtornos do Crescimento/metabolismo , Transtornos do Crescimento/patologia , Lâmina de Crescimento/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Doenças Mitocondriais/metabolismo , Doenças Mitocondriais/patologia , Proteínas Mitocondriais/fisiologia , Transdução de Sinais
10.
PLoS One ; 14(4): e0213483, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30947269

RESUMO

Regenerated cartilage formed after Autologous Chondrocyte Implantation may be of suboptimal quality due to postulated hypertrophic changes. Parathyroid hormone-related peptide, containing the parathyroid hormone sequence (PTHrP 1-34), enhances cartilage growth during development and inhibits hypertrophic differentiation of mesenchymal stromal cells (MSCs) and growth plate chondrocytes. This study aims to determine the possible anabolic and/or hypertrophic effect of PTH on human articular chondrocytes. Healthy human articular cartilage-derived chondrocytes (n = 6 donors) were cultured on type II collagen-coated transwells with/without 0.1 or 1.0 µM PTH from day 0, 9, or 21 until the end of culture (day 28). Extracellular matrix production, (pre)hypertrophy and PTH signaling were assessed by RT-qPCR and/or immunohistochemistry for collagen type I, II, X, RUNX2, MMP13, PTHR1 and IHH and by determining glycosaminoglycan production and DNA content. The Bern score assessed cartilage quality by histology. Regardless of the concentration and initiation of supplementation, PTH treatment significantly decreased DNA and glycosaminoglycan content and reduced the Bern score compared with controls. Type I collagen deposition was increased, whereas PTHR1 expression and type II collagen deposition were decreased by PTH supplementation. Expression of the (pre)hypertrophic markers MMP13, RUNX2, IHH and type X collagen were not affected by PTH. In conclusion, PTH supplementation to healthy human articular chondrocytes did not affect hypertrophic differentiation, but negatively influenced cartilage quality, the tissues' extracellular matrix and cell content. Although PTH may be an effective inhibitor of hypertrophic differentiation in MSC-based cartilage repair, care may be warranted in applying accessory PTH treatment due to its effects on articular chondrocytes.


Assuntos
Cartilagem/metabolismo , Proteína Relacionada ao Hormônio Paratireóideo/farmacologia , Fragmentos de Peptídeos/farmacologia , Receptor Tipo 1 de Hormônio Paratireóideo/genética , Regeneração/genética , Autoenxertos/crescimento & desenvolvimento , Autoenxertos/metabolismo , Cartilagem/crescimento & desenvolvimento , Diferenciação Celular/genética , Condrócitos/metabolismo , Colágeno Tipo X/genética , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Lâmina de Crescimento/crescimento & desenvolvimento , Lâmina de Crescimento/metabolismo , Proteínas Hedgehog/genética , Humanos , Metaloproteinase 13 da Matriz/genética , Células-Tronco Mesenquimais/metabolismo , Proteína Relacionada ao Hormônio Paratireóideo/genética , Fragmentos de Peptídeos/genética , Transdução de Sinais/genética
11.
Life Sci Alliance ; 2(2)2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30971423

RESUMO

Longitudinal bone growth ceases with growth plate senescence during puberty. However, the molecular mechanisms of this phenomenon are largely unexplored. Here, we examined Wnt-responsive genes before and after growth plate senescence and found that CXXC finger protein 5 (CXXC5), a negative regulator of the Wnt/ß-catenin pathway, was gradually elevated with reduction of Wnt/ß-catenin signaling during senescent changes of rodent growth plate. Cxxc5 -/- mice demonstrated delayed growth plate senescence and tibial elongation. As CXXC5 functions by interacting with dishevelled (DVL), we sought to identify small molecules capable of disrupting this interaction. In vitro screening assay monitoring CXXC5-DVL interaction revealed that several indirubin analogs were effective antagonists of this interaction. A functionally improved indirubin derivative, KY19382, elongated tibial length through delayed senescence and further activation of the growth plate in adolescent mice. Collectively, our findings reveal an important role for CXXC5 as a suppressor of longitudinal bone growth involving growth plate activity.


Assuntos
Desenvolvimento Ósseo/fisiologia , Proteínas de Ligação a DNA/metabolismo , Lâmina de Crescimento/metabolismo , Fatores de Transcrição/metabolismo , Animais , Proteínas de Ligação a DNA/genética , Proteínas Desgrenhadas/metabolismo , Células HEK293 , Humanos , Indóis/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/genética , Transfecção , Via de Sinalização Wnt , beta Catenina/metabolismo
12.
Int J Mol Sci ; 20(5)2019 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-30871109

RESUMO

Eleutherococcus extract mixture (EEM) is an herbal mixture of dried stem of Eleutherococcus sessiliflorus and germinated barley, which has been highly effective, in previous screening and among the traditional medicines to tonify innate qi and acquired qi, respectively. In this study, we investigate the effects of EEM on endochondral bone formation. Female adolescent rats were given EEM, growth hormone or vehicle for 10 days. Tetracycline was intraperitoneally injected to light the fluorescent band 72 h before sacrifice to determine endochondral bone formation. In order to evaluate endocrine or paracrine/autocrine mechanisms, expressions of insulin-like growth factor 1 (IGF1), insulin-like growth factor binding protein 3 (IGFBP3), or bone morphogenetic protein 2 (BMP2) were evaluated after EEM administration in liver or growth plate (GP). EEM oral administration significantly increased endochondral bone formation and proliferative and hypertrophic zonal heights of tibial GP. EEM also upregulated hepatic IGF1 and IGFBP3 mRNA expressions, and IGF1 and BMP2 expressions in GP. Taken together, EEM increases endochondral bone formation through stimulating proliferation and hypertrophy with upregulation of hepatic IGF1 and IGFBP3 expressions. Considering immunohistochemical studies, the effect of EEM may be due to increased local IGF1 and BMP2 expression in GP, which may be considered growth hormone (GH)-dependent endocrine and autocrine/paracrine pathways.


Assuntos
Desenvolvimento Ósseo/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Eleutherococcus/química , Osteogênese/efeitos dos fármacos , Extratos Vegetais/farmacologia , Tíbia/efeitos dos fármacos , Animais , Proteína Morfogenética Óssea 2/metabolismo , Proliferação de Células/efeitos dos fármacos , Condrócitos/metabolismo , Feminino , Hormônio do Crescimento/metabolismo , Lâmina de Crescimento/efeitos dos fármacos , Lâmina de Crescimento/metabolismo , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Tíbia/metabolismo
13.
Ecotoxicol Environ Saf ; 175: 83-89, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-30889403

RESUMO

Tetramethyl thiuram disulfide (thiram) is a dithiocarbamate, which is widely used on seeds and storing food grains. The incorporation of thiram into the food chain could be a risk for both human beings and animals. Thiram-contaminated feed has been considered a common cause of tibial dyschondrolplasia (TD) in many avian species. The molecular mechanism of action of thiram on TD involving microRNA (miRNA) is not fully understood. For this purpose, the morbidity and pathologic changes were evaluated to understand the TD, and high-throughput RNA sequencing (RNA-Seq) was performed to explore the differentially expressed miRNAs (DEGs). RT-qPCR was used to confirm the validity as compared with sequencing data. The results showed that the marked alterations in the growth plate of the TD chickens were noticeable, with shrinking cells and irregular chondrocyte columns as compared with control group. In this study, we identified total 375 (p < 0.1), 340 (p < 0.05) and 266 (p < 0.01) significant DEGs between the TD and control groups. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of DEGs showed that the target miRNAs were significantly enriched in different treatment groups, such as apoptosis, mRNA surveillance pathway, mitophagy-animal, etc. This study provides theoretical basis for in-depth understanding the pathogenesis of thiram-induced TD and explore the new insights towards the proposed molecular mechanism of specific miRNA as biomarkers for effective gene diagnosis and treatment of TD in broilers.


Assuntos
Galinhas , Poluentes Ambientais/toxicidade , MicroRNAs/genética , Osteocondrodisplasias/induzido quimicamente , Doenças das Aves Domésticas/induzido quimicamente , Tiram/toxicidade , Tíbia/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Galinhas/genética , Lâmina de Crescimento/efeitos dos fármacos , Lâmina de Crescimento/metabolismo , Lâmina de Crescimento/patologia , MicroRNAs/metabolismo , Osteocondrodisplasias/veterinária , Doenças das Aves Domésticas/metabolismo , Tíbia/metabolismo , Tíbia/patologia , Transcriptoma/efeitos dos fármacos
14.
Nature ; 567(7747): 234-238, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30814736

RESUMO

Longitudinal bone growth in children is sustained by growth plates, narrow discs of cartilage that provide a continuous supply of chondrocytes for endochondral ossification1. However, it remains unknown how this supply is maintained throughout childhood growth. Chondroprogenitors in the resting zone are thought to be gradually consumed as they supply cells for longitudinal growth1,2, but this model has never been proved. Here, using clonal genetic tracing with multicolour reporters and functional perturbations, we demonstrate that longitudinal growth during the fetal and neonatal periods involves depletion of chondroprogenitors, whereas later in life, coinciding with the formation of the secondary ossification centre, chondroprogenitors acquire the capacity for self-renewal, resulting in the formation of large, stable monoclonal columns of chondrocytes. Simultaneously, chondroprogenitors begin to express stem cell markers and undergo symmetric cell division. Regulation of the pool of self-renewing progenitors involves the hedgehog and mammalian target of rapamycin complex 1 (mTORC1) signalling pathways. Our findings indicate that a stem cell niche develops postnatally in the epiphyseal growth plate, which provides a continuous supply of chondrocytes over a prolonged period.


Assuntos
Condrócitos/citologia , Células Clonais/citologia , Lâmina de Crescimento/citologia , Nicho de Células-Tronco/fisiologia , Envelhecimento , Animais , Cartilagem/citologia , Autorrenovação Celular , Células Clonais/metabolismo , Feminino , Lâmina de Crescimento/metabolismo , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos
15.
J Ethnopharmacol ; 236: 108-113, 2019 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-30849503

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: According to traditional Korean medicine theory in which children's growth retardation is attributed to blood deficiency, Siwu decoction (SWD), a representative treatment for blood deficiency, was chosen as a sample. AIM OF THE STUDY: To evaluate the effects of SWD on chondrocyte proliferation of growth plate in adolescent female rats. MATERIALS AND METHODS: Female adolescent rats were allocated to one of the following four groups; SWD 100 and 300 mg/kg, recombinant human growth hormone, and vehicle for 4 days. Tetracycline was intraperitoneally injected at 48 h before sacrifice to obtain a band exhibiting fluorescence by binding newly formed bone. Bromodeoxyuridine was injected at day 2-4 to mark proliferating chondrocytes. To evaluate possible mechanisms of SWD, expressions of insulin-like growth factor-1 (IGF-1) and bone morphogenetic protein-2 (BMP-2) in the growth plate were examined by immunohistochemistry. RESULTS: Treatment with SWD significantly increased the number of BrdU-positive chondrocytes and the new bone formation in the proximal growth plate of tibia compared to the vehicle treated control group. SWD also increased the expression of IGF-1 and BMP-2 in the proliferative and hypertrophic zones of the growth plate. CONCLUSIONS: SWD 300 mg/kg stimulates chondrocyte proliferation and new bone formation in the growth plate. Immunohistochemical studies indicate that the effects of SWD may be due to upregulation of local IGF-1 and BMP-2 expression in the growth plate, which may be considered as a GH-dependent paracrine-autocrine pathway.


Assuntos
Desenvolvimento Ósseo/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Lâmina de Crescimento/efeitos dos fármacos , Extratos Vegetais/farmacologia , Animais , Proteína Morfogenética Óssea 2/biossíntese , Condrócitos/metabolismo , Relação Dose-Resposta a Droga , Feminino , Lâmina de Crescimento/metabolismo , Fator de Crescimento Insulin-Like I/biossíntese , Medicina Tradicional Coreana , Ratos , Ratos Sprague-Dawley , Tíbia/efeitos dos fármacos , Tíbia/metabolismo
16.
Mol Ther ; 27(3): 673-680, 2019 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-30765323

RESUMO

Recombinant human growth hormone (GH) is commonly used to treat short stature in children. However, GH treatment has limited efficacy, particularly in severe, non-GH-deficient conditions such as chondrodysplasias, and potential off-target effects. Because short stature results from decreased growth plate chondrogenesis, we developed a cartilage-targeting single-chain human antibody fragment (CaAb) aiming to deliver therapeutic molecules to the growth plate, thereby increasing treatment efficacy while minimizing adverse effects on other tissues. To this end, we created fusion proteins of these CaAbs conjugated with insulin-like growth factor 1 (IGF-1), an endocrine and/or paracrine factor that positively regulates chondrogenesis. These CaAb-IGF-1 fusion proteins retained both cartilage binding and IGF-1 biological activity, and they were able to stimulate bone growth in an organ culture system. Using a GH-deficient (lit) mouse model, we found that subcutaneous injections of these CaAb-IGF-1 fusion proteins increased overall growth plate height without increasing proliferation in kidney cortical cells, suggesting on-target efficacy at the growth plate and less off-target effect on the kidney than IGF-1 alone. Alternate-day injections of these fusion proteins, unlike IGF-1 alone, were sufficient to produce a therapeutic effect. Our findings provide proof of principle that targeting therapeutics to growth plate cartilage can potentially improve treatment for childhood growth disorders.


Assuntos
Fator de Crescimento Insulin-Like I/farmacologia , Animais , Cartilagem/efeitos dos fármacos , Cartilagem/metabolismo , Condrogênese/efeitos dos fármacos , Lâmina de Crescimento/efeitos dos fármacos , Lâmina de Crescimento/metabolismo , Humanos , Células MCF-7 , Camundongos , Camundongos Endogâmicos C57BL , Mutação/genética
17.
PLoS One ; 14(2): e0212680, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30794654

RESUMO

C-type natriuretic peptide (CNP) and its receptor natriuretic peptide receptor B (NPR-B) are physiological potent positive regulators of endochondral bone growth; therefore, the CNP/NPR-B signaling pathway is one of the most promising therapeutic targets for treating growth failure and dwarfism. In this article, we summarized the pharmacological properties of a novel CNP analog peptide ASB20123 as a therapeutic agent for short stature. ASB20123, one of the CNP/ghrelin chimeric peptides, is composed of CNP(1-22) and human ghrelin(12-28, E17D). Compared to CNP(1-22), ASB20123 showed similar agonist activity for NPR-B and improved biokinetics with a longer plasma half-life in rats. In addition, the distribution of ASB20123 to the cartilage was higher than that of CNP(1-22) after single subcutaneous (sc) injection to mice. These results suggested that the C-terminal part of ghrelin, which has clusters of basic amino acid residues and a BX7B motif, might contribute to the retention of ASB20123 in the extracellular matrix of the growth plate. Multiple sc doses of ASB20123 potently stimulated skeletal growth in rats in a dose-dependent manner, and sc infusion was more effective than bolus injection at the same dose. Our data indicated that high plasma levels of ASB20123 would not necessarily be required for bone growth acceleration. Thus, pharmaceutical formulation approaches for sustained-release dosage forms to allow chronic exposure to ASB20123 might be suitable to ensure drug effectiveness and safety.


Assuntos
Desenvolvimento Ósseo/efeitos dos fármacos , Cartilagem , Nanismo , Lâmina de Crescimento , Peptídeo Natriurético Tipo C/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Cartilagem/crescimento & desenvolvimento , Cartilagem/patologia , Nanismo/tratamento farmacológico , Nanismo/metabolismo , Nanismo/patologia , Lâmina de Crescimento/metabolismo , Lâmina de Crescimento/patologia , Camundongos , Camundongos Endogâmicos ICR , Peptídeo Natriurético Tipo C/química , Ratos , Ratos Sprague-Dawley
18.
Int J Med Sci ; 16(2): 221-230, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30745802

RESUMO

Background: In a previous report, we demonstrated the presence of cells with a neural/glial phenotype on the concave side of the vertebral body growth plate in Idiopathic Scoliosis (IS) and proposed this phenotype alteration as the main etiological factor of IS. In the present study, we utilized the same specimens of vertebral body growth plates removed during surgery for Grade III-IV IS to analyse gene expression. We suggested that phenotype changes observed on the concave side of the vertebral body growth plate can be associated with altered expression of particular genes, which in turn compromise mechanical properties of the concave side. Methods: We used a Real-Time SYBR Green PCR assay to investigate gene expression in vertebral body growth plates removed during surgery for Grade III-IV IS; cartilage tissues from human fetal spine were used as a surrogate control. Special attention was given to genes responsible for growth regulation, chondrocyte differentiation, matrix synthesis, sulfation and transmembrane transport of sulfates. We performed morphological, histochemical, biochemical, and ultrastructural analysis of vertebral body growth plates. Results: Expression of genes that control chondroitin sulfate sulfation and corresponding protein synthesis was significantly lower in scoliotic specimens compared to controls. Biochemical analysis showed 1) a decrease in diffused proteoglycans in the total pool of proteoglycans; 2) a reduced level of their sulfation; 3) a reduction in the amount of chondroitin sulfate coinciding with raising the amount of keratan sulfate; and 4) reduced levels of sulfation on the concave side of the scoliotic deformity. Conclusion: The results suggested that altered expression of genes that control chondroitin sulfate sulfation and corresponding changes in protein synthesis on the concave side of vertebral body growth plates could be causal agents of the scoliotic deformity.


Assuntos
Condrócitos/fisiologia , Lâmina de Crescimento/metabolismo , Escoliose/metabolismo , Coluna Vertebral/metabolismo , Adolescente , Diferenciação Celular , Criança , Condrócitos/ultraestrutura , Sulfatos de Condroitina/metabolismo , Lâmina de Crescimento/patologia , Humanos , Biossíntese de Proteínas , Escoliose/genética , Escoliose/patologia
19.
Nature ; 565(7740): 511-515, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30651640

RESUMO

Endochondral ossification, an important process in vertebrate bone formation, is highly dependent on correct functioning of growth plate chondrocytes1. Proliferation of these cells determines longitudinal bone growth and the matrix deposited provides a scaffold for future bone formation. However, these two energy-dependent anabolic processes occur in an avascular environment1,2. In addition, the centre of the expanding growth plate becomes hypoxic, and local activation of the hypoxia-inducible transcription factor HIF-1α is necessary for chondrocyte survival by unidentified cell-intrinsic mechanisms3-6. It is unknown whether there is a requirement for restriction of HIF-1α signalling in the other regions of the growth plate and whether chondrocyte metabolism controls cell function. Here we show that prolonged HIF-1α signalling in chondrocytes leads to skeletal dysplasia by interfering with cellular bioenergetics and biosynthesis. Decreased glucose oxidation results in an energy deficit, which limits proliferation, activates the unfolded protein response and reduces collagen synthesis. However, enhanced glutamine flux increases α-ketoglutarate levels, which in turn increases proline and lysine hydroxylation on collagen. This metabolically regulated collagen modification renders the cartilaginous matrix more resistant to protease-mediated degradation and thereby increases bone mass. Thus, inappropriate HIF-1α signalling results in skeletal dysplasia caused by collagen overmodification, an effect that may also contribute to other diseases involving the extracellular matrix such as cancer and fibrosis.


Assuntos
Doenças Ósseas/metabolismo , Doenças Ósseas/patologia , Condrócitos/metabolismo , Colágeno/biossíntese , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Animais , Cartilagem/metabolismo , Matriz Extracelular/metabolismo , Glucose/metabolismo , Glutamina/metabolismo , Lâmina de Crescimento/metabolismo , Hidroxilação , Prolina Dioxigenases do Fator Induzível por Hipóxia/deficiência , Prolina Dioxigenases do Fator Induzível por Hipóxia/genética , Ácidos Cetoglutáricos/metabolismo , Lisina/metabolismo , Masculino , Camundongos , Osteogênese , Oxirredução , Prolina/metabolismo
20.
Stem Cells ; 37(5): 677-689, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30681752

RESUMO

Long bone development involves the embryonic formation of a primary ossification center (POC) in the incipient diaphysis followed by postnatal development of a secondary ossification center (SOC) at each epiphysis. Studies have elucidated major basic mechanisms of POC development, but relatively little is known about SOC development. To gain insights into SOC formation, we used Col2-Cre Rosa-tdTomato (Col2/Tomato) reporter mice and found that their periarticular region contained numerous Tomato-positive lineage cells expressing much higher Tomato fluorescence (termed TomatoH ) than underlying epiphyseal chondrocytes (termed TomatoL ). With time, the TomatoH cells became evident at the SOC invagination site and cartilage canal, increased in number in the expanding SOC, and were present as mesenchymal lineage cells in the subchondral bone. These data were verified in two mouse lineage tracing models, Col2-CreER Rosa-tdTomato and Gli1-CreER Rosa-tdTomato. In vitro tests showed that the periarticular TomatoH cells from Col2/Tomato mice contained mesenchymal progenitors with multidifferentiation abilities. During canal initiation, the cells expressed vascular endothelial growth factor (VEGF) and migrated into epiphyseal cartilage ahead of individual or clusters of endothelial cells, suggesting a unique role in promoting vasculogenesis. Later during SOC expansion, chondrocytes in epiphyseal cartilage expressed VEGF, and angiogenic blood vessels preceded TomatoH cells. Gene expression analyses of microdissected samples revealed upregulation of MMPs in periarticular cells at the invagination site and suggested potential roles for novel kinase and growth factor signaling pathways in regulating SOC canal initiation. In summary, our data indicate that the periarticular region surrounding epiphyseal cartilage contains mesenchymal progenitors that initiate SOC development and form subchondral bone. Stem Cells 2019;37:677-689.


Assuntos
Desenvolvimento Ósseo/genética , Diferenciação Celular/genética , Células-Tronco Mesenquimais , Osteogênese/genética , Animais , Cartilagem/crescimento & desenvolvimento , Condrócitos/citologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Lâmina de Crescimento/crescimento & desenvolvimento , Lâmina de Crescimento/metabolismo , Camundongos , Transdução de Sinais/genética , Crânio/crescimento & desenvolvimento , Crânio/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Proteína GLI1 em Dedos de Zinco/genética
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