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1.
Curr Osteoporos Rep ; 20(2): 141-152, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35156183

RESUMO

PURPOSE OF REVIEW: RNA-sequencing (RNA-seq) is a novel and highly sought-after tool in the field of musculoskeletal regenerative medicine. The technology is being used to better understand pathological processes, as well as elucidate mechanisms governing development and regeneration. It has allowed in-depth characterization of stem cell populations and discovery of molecular mechanisms that regulate stem cell development, maintenance, and differentiation in a way that was not possible with previous technology. This review introduces RNA-seq technology and how it has paved the way for advances in musculoskeletal regenerative medicine. RECENT FINDINGS: Recent studies in regenerative medicine have utilized RNA-seq to decipher mechanisms of pathophysiology and identify novel targets for regenerative medicine. The technology has also advanced stem cell biology through in-depth characterization of stem cells, identifying differentiation trajectories and optimizing cell culture conditions. It has also provided new knowledge that has led to improved growth factor use and scaffold design for musculoskeletal regenerative medicine. This article reviews recent studies utilizing RNA-seq in the field of musculoskeletal regenerative medicine. It demonstrates how transcriptomic analysis can be used to provide insights that can aid in formulating a regenerative strategy.


Assuntos
Sistema Musculoesquelético , Medicina Regenerativa , Técnicas de Cultura de Células , Humanos , Células-Tronco , Engenharia Tecidual , Transcriptoma
2.
Curr Rheumatol Rep ; 23(11): 78, 2021 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-34716494

RESUMO

PURPOSE OF REVIEW: Osteoarthritis (OA) is the most common forms of arthritis in the general population, accounting for more pain and functional disability than any other musculoskeletal disease. There are currently no approved disease modifying drugs for OA. In the absence of effective pharmacotherapy, many patients with OA turn to nutritional supplements and nutraceuticals, including collagen derivatives. Collagen hydrolyzates and ultrahydrolyzates are terms used to describe collagens that have been broken down into small peptides and amino acids in the presence of collagenases and high pressure. RECENT FINDINGS: This article reviews the relevant literature and serves as a White Paper on collagen hydrolyzates and ultrahydrolyzates as emerging supplements often advertised to support joint health in OA. Collagen hydrolyzates have demonstrated some evidence of efficacy in a handful of small scale clinical trials, but their ability to treat and reverse advanced joint disease remains highly speculative, as is the case for other nutritional supplements. The aim of this White Paper is to stimulate research and development of collagen-based supplements for patients with OA and other musculoskeletal diseases at academic and industrial levels. This White Paper does not make any treatment recommendations for OA patients in the clinical context, but simply aims to highlight opportunities for scientific innovation and interdisciplinary collaboration, which are crucial for the development of novel products and nutritional interventions based on the best available and published evidence.


Assuntos
Artropatias , Osteoartrite , Colágeno , Suplementos Nutricionais , Humanos , Osteoartrite/tratamento farmacológico , Dor
3.
Curr Opin Rheumatol ; 32(1): 92-101, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31724973

RESUMO

PURPOSE OF REVIEW: Osteoarthritis is a debilitating disease leading to joint degeneration, inflammation, pain, and disability. Despite efforts to develop a disease modifying treatment, the only accepted and available clinical approaches involve palliation. Although many factors contribute to the development of osteoarthritis, the gut microbiome has recently emerged as an important pathogenic factor in osteoarthritis initiation and progression. This review examines the literature to date regarding the link between the gut microbiome and osteoarthritis. RECENT FINDINGS: Studies showing correlations between serum levels of bacterial metabolites and joint degeneration were the first links connecting a dysbiosis of the gut microbiome with osteoarthritis. Further investigations have demonstrated that microbial community shifts induced by antibiotics, a germ-free environment or high-fat are important underlying factors in joint homeostasis and osteoarthritis. It follows that strategies to manipulate the microbiome have demonstrated efficacy in mitigating joint degeneration in osteoarthritis. Moreover, we have observed that dietary supplementation with nutraceuticals that are joint protective may exert their influence via shifts in the gut microbiome. SUMMARY: Although role of the microbiome in osteoarthritis is an area of intense study, no clear mechanism of action has been determined. Increased understanding of how the two factors interact may provide mechanistic insight into osteoarthritis and lead to disease modifying treatments.


Assuntos
Disbiose/complicações , Microbioma Gastrointestinal/fisiologia , Inflamação/microbiologia , Osteoartrite/microbiologia , Disbiose/metabolismo , Disbiose/microbiologia , Humanos , Inflamação/metabolismo , Osteoartrite/metabolismo
4.
J Immunol ; 201(2): 560-572, 2018 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-29858265

RESUMO

Obese patients with type 2 diabetes (T2D) are at an increased risk of foot infection, with impaired immune function believed to be a critical factor in the infectious process. In this study, we test the hypothesis that humoral immune defects contribute to exacerbated foot infection in a murine model of obesity/T2D. C57BL/6J mice were rendered obese and T2D by a high-fat diet for 3 mo and were compared with controls receiving a low-fat diet. Following injection of Staphylococcus aureus into the footpad, obese/T2D mice had greater foot swelling and reduced S. aureus clearance than controls. Obese/T2D mice also had impaired humoral immune responses as indicated by lower total IgG levels and lower anti-S. aureus Ab production. Within the draining popliteal lymph nodes of obese/T2D mice, germinal center formation was reduced, and the percentage of germinal center T and B cells was decreased by 40-50%. Activation of both T and B lymphocytes was similarly suppressed in obese/T2D mice. Impaired humoral immunity in obesity/T2D was independent of active S. aureus infection, as a similarly impaired humoral immune response was demonstrated when mice were administered an S. aureus digest. Isolated splenic B cells from obese/T2D mice activated normally but had markedly suppressed expression of Aicda, with diminished IgG and IgE responses. These results demonstrate impaired humoral immune responses in obesity/T2D, including B cell-specific defects in Ab production and class-switch recombination. Together, the defects in humoral immunity may contribute to the increased risk of foot infection in obese/T2D patients.


Assuntos
Linfócitos B/fisiologia , Diabetes Mellitus Tipo 2/imunologia , Pé/microbiologia , Centro Germinativo/imunologia , Obesidade/imunologia , Infecções Estafilocócicas/imunologia , Staphylococcus aureus/imunologia , Animais , Diferenciação Celular , Células Cultivadas , Citidina Desaminase/metabolismo , Diabetes Mellitus Tipo 2/microbiologia , Dieta Hiperlipídica , Modelos Animais de Doenças , Progressão da Doença , Pé/patologia , Humanos , Imunidade Humoral , Switching de Imunoglobulina , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/microbiologia , Infecções Estafilocócicas/microbiologia
5.
Infect Immun ; 85(6)2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28320836

RESUMO

Obesity and associated type 2 diabetes (T2D) are important risk factors for infection following orthopedic implant surgery. Staphylococcus aureus, the most common pathogen in bone infections, adapts to multiple environments to survive and evade host immune responses. Whether adaptation of S. aureus to the unique environment of the obese/T2D host accounts for its increased virulence and persistence in this population is unknown. Thus, we assessed implant-associated osteomyelitis in normal versus high-fat-diet obese/T2D mice and found that S. aureus infection was more severe, including increases in bone abscesses relative to nondiabetic controls. S. aureus isolated from bone of obese/T2D mice displayed marked upregulation of four adhesion genes (clfA, clfB, bbp, and sdrC), all with binding affinity for fibrin(ogen). Immunostaining of infected bone revealed increased fibrin deposition surrounding bacterial abscesses in obese/T2D mice. In vitro coagulation assays demonstrated a hypercoagulable state in obese/T2D mice that was comparable to that of diabetic patients. S. aureus with an inactivating mutation in clumping factor A (clfA) showed a reduction in bone infection severity that eliminated the effect of obesity/T2D, while infections in control mice were unchanged. In infected mice that overexpress plasminogen activator inhibitor-1 (PAI-1), S. aureusclfA expression and fibrin-encapsulated abscess communities in bone were also increased, further linking fibrin deposition to S. aureus expression of clfA and infection severity. Together, these results demonstrate an adaptation by S. aureus to obesity/T2D with increased expression of clfA that is associated with the hypercoagulable state of the host and increased virulence of S. aureus.


Assuntos
Coagulase/metabolismo , Diabetes Mellitus Tipo 2/complicações , Obesidade/complicações , Osteomielite/patologia , Infecções Estafilocócicas/microbiologia , Abscesso/patologia , Animais , Anticorpos Antibacterianos/genética , Anticorpos Antibacterianos/metabolismo , Coagulase/genética , Diabetes Mellitus Tipo 2/microbiologia , Modelos Animais de Doenças , Fibrinogênio/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/microbiologia , Osteomielite/microbiologia , Análise de Sequência de RNA , Ativação Transcricional , Regulação para Cima , Virulência
6.
Anal Biochem ; 518: 134-138, 2017 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-27913164

RESUMO

Extracting high-quality RNA from articular cartilage is challenging due to low cellularity and high proteoglycan content. This problem hinders efficient application of RNA sequencing (RNA-seq) analysis in studying cartilage homeostasis. Here we developed a method that purifies high-quality RNA directly from cartilage. Our method optimized the collection and homogenization steps so as to minimize RNA degradation, and modified the conventional TRIzol protocol to enhance RNA purity. Cartilage RNA purified using our method has appropriate quality for RNA-seq experiments including an RNA integrity number of ∼8. Our method also proved efficient in extracting high-quality RNA from subchondral bone.


Assuntos
Cartilagem Articular/química , Articulação do Joelho/química , RNA/química , RNA/isolamento & purificação , Humanos , Masculino
7.
Muscle Nerve ; 56(1): 143-151, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28168703

RESUMO

INTRODUCTION: Erythropoietin (EPO) has been identified as a neuroregenerative agent. We hypothesize that it may accelerate recovery after crush injury and may vary with crush severity. METHODS: Mice were randomized to mild, moderate, or severe crush of the sciatic nerve and were treated with EPO or vehicle control after injury. The sciatic function index (SFI) was monitored over the first week. Microstructural changes were analyzed by immunofluorescence for neurofilament (NF) and myelin (P0 ), and electron microscopy was used to assess ultrastructural changes. RESULTS: In moderate crush injuries, EPO significantly improved SFI at 7 days post-injury, an effect not observed with other severity levels. Increases in the ratio of P0 to NF were observed after EPO treatment in moderate crush injuries. Electron microscopy demonstrated endothelial cell hypertrophy in the EPO group. CONCLUSIONS: EPO accelerates recovery in moderately crushed nerves, which may be through effects on myelination and vascularization. Injury severity may influence the efficacy of EPO. Muscle Nerve 56: 143-151, 2017.


Assuntos
Eritropoetina/uso terapêutico , Recuperação de Função Fisiológica/efeitos dos fármacos , Neuropatia Ciática/tratamento farmacológico , Neuropatia Ciática/fisiopatologia , Análise de Variância , Animais , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Filamentos Intermediários/metabolismo , Filamentos Intermediários/patologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica , Proteína P0 da Mielina/metabolismo , Nervo Isquiático/efeitos dos fármacos , Nervo Isquiático/patologia , Nervo Isquiático/ultraestrutura , Índice de Gravidade de Doença
8.
Infect Immun ; 83(6): 2264-74, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25802056

RESUMO

Obesity and diabetes are among the greatest risk factors for infection following total joint arthroplasty. However, the underlying mechanism of susceptibility is unclear. We compared orthopedic implant-associated Staphylococcus aureus infections in type 1 (T1D) versus type 2 (T2D) diabetic mouse models and in patients with S. aureus infections, focusing on the adaptive immune response. Mice were fed a high-fat diet to initiate obesity and T2D. T1D was initiated with streptozotocin. Mice were then given a trans-tibial implant that was precoated with bioluminescent Xen36 S. aureus. Although both mouse models of diabetes demonstrated worse infection severity than controls, infection in T2D mice was more severe, as indicated by increases in bioluminescence, S. aureus CFU in tissue, and death within the first 7 days. Furthermore, T2D mice had an impaired humoral immune response at day 14 with reduced total IgG, decreased S. aureus-specific IgG, and increased IgM. These changes were not present in T1D mice. Similarly, T2D patients and obese nondiabetics with active S. aureus infections had a blunted IgG response to S. aureus. In conclusion, we report the first evidence of a humoral immune deficit, possibly due to an immunoglobulin class switch defect, in obesity and T2D during exacerbated S. aureus infection which may contribute to the increased infection risk following arthroplasty in patients with T2D and obesity.


Assuntos
Diabetes Mellitus Experimental/imunologia , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 2/imunologia , Imunidade Humoral , Obesidade/imunologia , Infecções Estafilocócicas/microbiologia , Imunidade Adaptativa , Animais , Intolerância à Glucose , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/induzido quimicamente , Osteomielite/microbiologia , Staphylococcus aureus
9.
J Cell Sci ; 126(Pt 24): 5704-13, 2013 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-24144697

RESUMO

TAK1 is a MAP3K that mediates non-canonical TGF-ß and BMP signaling. During the embryonic period, TAK1 is essential for cartilage and joint development as deletion of Tak1 in chondro-osteo progenitor cells leads to severe chondrodysplasia with defects in both chondrocyte proliferation and maturation. We have investigated the role of TAK1 in committed chondrocytes during early postnatal development. Using the Col2a1-CreER(T2); Tak1(f/f) mouse model, we induced deletion of Tak1 at postnatal day 7 and characterized the skeletal phenotypes of these mice at 1 and 3 months of age. Mice with chondrocyte-specific Tak1 deletion exhibited severe growth retardation and reduced proteoglycan and type II collagen content in the extracellular matrix of the articular cartilage. We found reduced Col2a1 and Acan expression, but increased Mmp13 and Adamts5 expression, in Tak1-deficient chondrocytes along with reduced expression of the SOX trio of transcription factors, SOX9, SOX5 and SOX6. In vitro, BMP2 stimulated Sox9 gene expression and Sox9 promoter activity. These effects were reduced; however, following Tak1 deletion or treatment with a TAK1 kinase inhibitor. TAK1 affects both canonical and non-canonical BMP signal transduction and we found that both of these pathways contribute to BMP2-mediated Sox9 promoter activation. Additionally, we found that ATF2 directly binds the Sox9 promoter in response to BMP signaling and that this effect is dependent upon TAK1 kinase activity. These novel findings establish that TAK1 contributes to BMP2-mediated Sox9 gene expression and is essential for the postnatal development of normal growth plate and articular cartilages.


Assuntos
Cartilagem Articular/metabolismo , Condrócitos/metabolismo , Lâmina de Crescimento/metabolismo , MAP Quinase Quinase Quinases/fisiologia , Fatores de Transcrição SOX9/metabolismo , Fator 2 Ativador da Transcrição/metabolismo , Animais , Proteína Morfogenética Óssea 2/fisiologia , Cartilagem Articular/citologia , Cartilagem Articular/crescimento & desenvolvimento , Proliferação de Células , Células Cultivadas , Matriz Extracelular/metabolismo , Deleção de Genes , Regulação da Expressão Gênica , Lâmina de Crescimento/crescimento & desenvolvimento , Camundongos , Camundongos Transgênicos , Regiões Promotoras Genéticas , Ligação Proteica , Proteoglicanas/metabolismo , Fatores de Transcrição SOX9/genética
10.
Development ; 139(6): 1198-212, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22354840

RESUMO

The Notch signaling pathway has emerged as an important regulator of endochondral bone formation. Although recent studies have examined the role of Notch in mesenchymal and chondro-osteo progenitor cell populations, there has yet to be a true examination of Notch signaling specifically within developing and committed chondrocytes, or a determination of whether cartilage and bone formation are regulated via RBPjκ-dependent or -independent Notch signaling mechanisms. To develop a complete understanding of Notch signaling during cartilage and bone development we generated and compared general Notch gain-of-function (Rosa-NICD(f/+)), RBPjκ-deficient (Rbpjκ(f/f)), and RBPjκ-deficient Notch gain-of-function (Rosa-NICD(f/+);Rbpjκ(f/f)) conditional mutant mice, where activation or deletion of floxed alleles were specifically targeted to mesenchymal progenitors (Prx1Cre) or committed chondrocytes (inducible Col2Cre(ERT2)). These data demonstrate, for the first time, that Notch regulation of chondrocyte maturation is solely mediated via the RBPjκ-dependent pathway, and that the perichodrium or osteogenic lineage probably influences chondrocyte terminal maturation and turnover of the cartilage matrix. Our study further identifies the cartilage-specific RBPjκ-independent pathway as crucial for the proper regulation of chondrocyte proliferation, survival and columnar chondrocyte organization. Unexpectedly, the RBPjκ-independent Notch pathway was also identified as an important long-range cell non-autonomous regulator of perichondral bone formation and an important cartilage-derived signal required for coordinating chondrocyte and osteoblast differentiation during endochondral bone development. Finally, cartilage-specific RBPjκ-independent Notch signaling likely regulates Ihh responsiveness during cartilage and bone development.


Assuntos
Cartilagem/embriologia , Condrogênese , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/genética , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Osteogênese , Receptores Notch/metabolismo , Animais , Osso e Ossos/embriologia , Cartilagem/metabolismo , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Condrócitos/citologia , Condrócitos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/deficiência , Células-Tronco Mesenquimais , Camundongos , Camundongos Transgênicos , Osteogênese/genética , Receptores Notch/genética , Transdução de Sinais
11.
Calcif Tissue Int ; 96(4): 313-23, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25673503

RESUMO

Obesity is a severe health problem in children, afflicting several organ systems including bone. However, the role of obesity on bone homeostasis and bone cell function in children has not been studied in detail. Here we used young mice fed a high-fat diet (HFD) to model childhood obesity and investigate the effect of HFD on the phenotype of cells within the bone marrow environment. Five-week-old male mice were fed a HFD for 3, 6, and 12 weeks. Decreased bone volume was detected after 3 weeks of HFD treatment. After 6 and 12 weeks, HFD-exposed mice had less bone mass and increased osteoclast numbers. Bone marrow cells, but not spleen cells, from HFD-fed mice had increased osteoclast precursor frequency, elevated osteoclast formation, and bone resorption activity, as well as increased expression of osteoclastogenic regulators including RANKL, TNF, and PPAR-gamma. Bone formation rate and osteoblast and adipocyte numbers were also increased in HFD-fed mice. Isolated bone marrow cells also had a corresponding elevation in the expression of positive regulators of osteoblast and adipocyte differentiation. Our findings indicate that in juvenile mice, HFD-induced bone loss is mainly due to increased osteoclast bone resorption by affecting the bone marrow microenvironment. Thus, targeting osteoclast formation may present a new therapeutic approach for bone complications in obese children.


Assuntos
Medula Óssea/patologia , Reabsorção Óssea/fisiopatologia , Dieta Hiperlipídica/efeitos adversos , Osteoclastos/citologia , Adipócitos/citologia , Animais , Biomarcadores/sangue , Glicemia/análise , Densidade Óssea , Medula Óssea/metabolismo , Osso e Ossos/patologia , Diferenciação Celular , Separação Celular , Citometria de Fluxo , Antígenos Comuns de Leucócito/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/metabolismo , Osteoblastos/citologia , Osteoclastos/metabolismo , PPAR gama/metabolismo , Ligante RANK/metabolismo , Baço/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Microtomografia por Raio-X
12.
Mol Cell Biochem ; 397(1-2): 67-74, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25138700

RESUMO

Endochondral ossification, an important stage of fracture healing, is regulated by a variety of signaling pathways. Transforming growth factor ß (TGFß) superfamily plays important roles and comprises TGFßs, bone morphogenetic proteins (BMPs), and growth differentiation factors. TGFßs primarily regulate cartilage formation and endochondral ossification. BMP2 shows diverse efficacy, from the formation of skeleton and extraskeletal organs to the osteogenesis and remodeling of bone. G-protein-coupled receptor kinase 2-interacting protein-1 (GIT1), a shuttle protein in osteoblasts, facilitates fracture healing by promoting bone formation and increasing the secretion of vascular endothelial growth factor. Our study examined whether GIT1 regulates fracture healing through the BMP2 signaling pathway and/or through the TGFß signaling pathway. GIT1 knockout (KO) mice exhibited delayed fracture healing, chondrocyte accumulation in the fracture area, and reduced staining intensity of phosphorylated Smad1/5/8 (pSmad1/5/8) and Runx2. Endochondral mineralization diminished while the staining intensity of phosphorylated Smad2/3 (pSmad2/3) showed no significant change. Bone marrow mesenchymal stem cells extracted from GIT1 KO mice showed a decline of pSmad1/5/8 levels and of pSmad1/5/8 translocated into the cell nucleus after BMP2 stimulus. We detected no significant change in the pSmad2/3 level after TGFß1 stimulus. Data obtained from reporter gene analysis of C3H10T1/2 cells cultured in vitro confirmed these findings. GIT1-siRNA inhibited transcription in the cell nucleus via pSmad1/5/8 after BMP2 stimulus but had no significant effect on transcription via pSmad2/3 after TGFß1 stimulus. Our results indicate that GIT1 regulates Smad1/5/8 phosphorylation and mediates BMP2 regulation of Runx2 expression, thus affecting endochondral ossification at the fracture site.


Assuntos
Proteína Morfogenética Óssea 2/metabolismo , Regeneração Óssea/fisiologia , Proteínas de Ciclo Celular/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Transdução de Sinais/fisiologia , Animais , Proteína Morfogenética Óssea 2/genética , Proteínas de Ciclo Celular/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Proteínas Ativadoras de GTPase/genética , Camundongos , Camundongos Knockout , Proteínas Smad/genética , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo
13.
Arthritis Rheum ; 65(10): 2623-33, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23839930

RESUMO

OBJECTIVE: Osteoarthritis (OA) is a degenerative disease resulting in severe joint cartilage destruction and disability. While the mechanisms underlying the development and progression of OA are poorly understood, gene mutations have been identified within cartilage-related signaling molecules, implicating impaired cell signaling in OA and joint disease. The Notch pathway has recently been identified as a crucial regulator of growth plate cartilage development, and components are expressed in joint tissue. This study was undertaken to investigate a novel role for Notch signaling in joint cartilage development, maintenance, and the pathogenesis of joint disease in a mouse model. METHODS: We performed the first mouse gene study in which the core Notch signaling component, RBP-Jκ, was tissue specifically deleted within joints. The Prx1Cre transgene removed Rbpjk loxP-flanked alleles in mesenchymal joint precursor cells, while the Col2Cre(ERT2) transgene specifically deleted Rbpjk in postnatal chondrocytes. Murine articular chondrocyte cultures were also used to examine Notch regulation of gene expression. RESULTS: Loss of Notch signaling in mesenchymal joint precursor cells did not affect embryonic joint development in mice, but rather, resulted in an early, progressive OA-like pathology. Additionally, partial loss of Notch signaling in murine postnatal cartilage resulted in progressive joint cartilage degeneration and an age-related OA-like pathology. Inhibition of Notch signaling altered the expression of the extracellular matrix (ECM)-related factors type II collagen (COL2A1), proteoglycan 4, COL10A1, matrix metalloproteinase 13, and ADAMTS. CONCLUSION: Our findings indicate that the RBP-Jκ-dependent Notch pathway is a novel pathway involved in joint maintenance and articular cartilage homeostasis, a critical regulator of articular cartilage ECM-related molecules, and a potentially important therapeutic target for OA-like joint disease.


Assuntos
Cartilagem Articular/fisiologia , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/fisiologia , Articulações/fisiologia , Receptores Notch/fisiologia , Transdução de Sinais/fisiologia , Animais , Células Cultivadas , Colágeno Tipo I/genética , Colágeno Tipo I/fisiologia , Cadeia alfa 1 do Colágeno Tipo I , Colágeno Tipo II/genética , Colágeno Tipo II/fisiologia , Homeostase/fisiologia , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/genética , Camundongos , Camundongos Endogâmicos , Camundongos Transgênicos , Modelos Animais , Osteoartrite/fisiopatologia
14.
Immune Netw ; 24(2): e17, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38725672

RESUMO

We have reported that anterior cruciate ligament (ACL) injury leads to the differential dysregulation of the complement system in the synovium as compared to meniscus tear (MT) and proposed this as a mechanism for a greater post-injury prevalence of post traumatic osteoarthritis (PTOA). To explore additional roles of complement proteins and regulators, we determined the presence of decay-accelerating factor (DAF), C5b, and membrane attack complexes (MACs, C5b-9) in discarded surgical synovial tissue (DSST) collected during arthroscopic ACL reconstructive surgery, MT-related meniscectomy, osteoarthritis (OA)-related knee replacement surgery and normal controls. Multiplexed immunohistochemistry was used to detect and quantify complement proteins. To explore the involvement of body mass index (BMI), after these 2 injuries, we examined correlations among DAF, C5b, MAC and BMI. Using these approaches, we found that synovial cells after ACL injury expressed a significantly lower level of DAF as compared to MT (p<0.049). In contrast, C5b staining synovial cells were significantly higher after ACL injury (p<0.0009) and in OA DSST (p<0.039) compared to MT. Interestingly, there were significantly positive correlations between DAF & C5b (r=0.75, p<0.018) and DAF & C5b (r=0.64 p<0.022) after ACL injury and MT, respectively. The data support that DAF, which should normally dampen C5b deposition due to its regulatory activities on C3/C5 convertases, does not appear to exhibit that function in inflamed synovia following either ACL injury or MT. Ineffective DAF regulation may be an additional mechanism by which relatively uncontrolled complement activation damages tissue in these injury states.

15.
bioRxiv ; 2024 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-38562830

RESUMO

Over 1,100 independent signals have been identified with genome-wide association studies (GWAS) for bone mineral density (BMD), a key risk factor for mortality-increasing fragility fractures; however, the effector gene(s) for most remain unknown. Informed by a variant-to-gene mapping strategy implicating 89 non-coding elements predicted to regulate osteoblast gene expression at BMD GWAS loci, we executed a single-cell CRISPRi screen in human fetal osteoblast 1.19 cells (hFOBs). The BMD relevance of hFOBs was supported by heritability enrichment from cross-cell type stratified LD-score regression involving 98 cell types grouped into 15 tissues. 24 genes showed perturbation in the screen, with four (ARID5B, CC2D1B, EIF4G2, and NCOA3) exhibiting consistent effects upon siRNA knockdown on three measures of osteoblast maturation and mineralization. Lastly, additional heritability enrichments, genetic correlations, and multi-trait fine-mapping revealed that many BMD GWAS signals are pleiotropic and likely mediate their effects via non-bone tissues that warrant attention in future screens.

16.
Development ; 137(9): 1461-71, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20335360

RESUMO

The Notch pathway has recently been implicated in mesenchymal progenitor cell (MPC) differentiation from bone marrow-derived progenitors. However, whether Notch regulates MPC differentiation in an RBPjkappa-dependent manner, specifies a particular MPC cell fate, regulates MPC proliferation and differentiation during early skeletal development or controls specific Notch target genes to regulate these processes remains unclear. To determine the exact role and mode of action for the Notch pathway in MPCs during skeletal development, we analyzed tissue-specific loss-of-function (Prx1Cre; Rbpjk(f/f)), gain-of-function (Prx1Cre; Rosa-NICD(f/+)) and RBPjkappa-independent Notch gain-of-function (Prx1Cre; Rosa-NICD(f/+); Rbpjk(f/f)) mice for defects in MPC proliferation and differentiation. These data demonstrate for the first time that the RBPjkappa-dependent Notch signaling pathway is a crucial regulator of MPC proliferation and differentiation during skeletal development. Our study also implicates the Notch pathway as a general suppressor of MPC differentiation that does not bias lineage allocation. Finally, Hes1 was identified as an RBPjkappa-dependent Notch target gene important for MPC maintenance and the suppression of in vitro chondrogenesis.


Assuntos
Osso e Ossos/citologia , Diferenciação Celular/fisiologia , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais/fisiologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Western Blotting , Osso e Ossos/embriologia , Diferenciação Celular/genética , Proliferação de Células , Células Cultivadas , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Proteínas de Homeodomínio/fisiologia , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/genética , Botões de Extremidades/citologia , Botões de Extremidades/embriologia , Botões de Extremidades/metabolismo , Camundongos , Modelos Biológicos , Receptores Notch/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , Fatores de Transcrição HES-1
17.
Biotechnol Bioeng ; 110(5): 1476-86, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23239161

RESUMO

A major challenge in cartilage tissue engineering is the need to recreate the native tissue's anisotropic extracellular matrix structure. This anisotropy has important mechanical and biological consequences and could be crucial for integrative repair. Here, we report that hydrodynamic conditions that mimic the motion-induced flow fields in between the articular surfaces in the synovial joint induce the formation of a distinct superficial layer in tissue engineered cartilage hydrogels, with enhanced production of cartilage matrix proteoglycan and Type II collagen. Moreover, the flow stimulation at the surface induces the production of the surface zone protein Proteoglycan 4 (aka PRG4 or lubricin). Analysis of second harmonic generation signature of collagen in this superficial layer reveals a highly aligned fibrillar matrix that resembles the alignment pattern in native tissue's surface zone, suggesting that mimicking synovial fluid flow at the cartilage surface in hydrodynamic bioreactors could be key to creating engineered cartilage with superficial zone features.


Assuntos
Engenharia Tecidual/métodos , Animais , Reatores Biológicos , Cartilagem/citologia , Cartilagem/metabolismo , Condrócitos/metabolismo , Hidrodinâmica , Hidrogéis/química , Proteoglicanas/análise , Proteoglicanas/metabolismo , Suínos , Engenharia Tecidual/instrumentação
18.
Arthritis Rheum ; 64(8): 2611-23, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22422036

RESUMO

OBJECTIVE: The incidence of low back pain is extremely high and is often linked to intervertebral disc (IVD) degeneration. The mechanism of this disease is currently unknown. This study was undertaken to investigate the role of ß-catenin signaling in IVD tissue function. METHODS: ß-catenin protein levels were measured by immunohistochemical analysis of disc samples obtained from patients with disc degeneration and from normal subjects. To generate ß-catenin conditional activation (cAct) mice, Col2a1-CreER(T2) -transgenic mice were bred with ß-catenin(fx(Ex3)/fx(Ex3)) mice. Changes in disc tissue morphology and function were examined by micro-computed tomography, histologic analysis, and real-time polymerase chain reaction assays. RESULTS: ß-catenin protein was up-regulated in disc tissue samples from patients with disc degeneration. To assess the effects of increased ß-catenin levels on disc tissue, we generated ß-catenin cAct mice. Overexpression of ß-catenin in disc cells led to extensive osteophyte formation in 3- and 6-month-old ß-catenin cAct mice, which were associated with significant changes in the cells and extracellular matrix of disc tissue and growth plate. Gene expression analysis demonstrated that activation of ß-catenin enhanced runt-related transcription factor 2-dependent Mmp13 and Adamts5 expression. Moreover, genetic ablation of Mmp13 or Adamts5 on the ß-catenin cAct background, or treatment of ß-catenin cAct mice with a specific matrix metalloproteinase 13 inhibitor, ameliorated the mutant phenotype. CONCLUSION: Our findings indicate that the ß-catenin signaling pathway plays a critical role in disc tissue function.


Assuntos
Degeneração do Disco Intervertebral/fisiopatologia , Deslocamento do Disco Intervertebral/fisiopatologia , Transdução de Sinais/fisiologia , Regulação para Cima/fisiologia , beta Catenina/metabolismo , Proteínas ADAM/metabolismo , Proteína ADAMTS5 , Animais , Colágeno Tipo II/genética , Modelos Animais de Doenças , Metaloproteinase 13 da Matriz/metabolismo , Camundongos , Camundongos Transgênicos , Modelos Animais
19.
Front Immunol ; 14: 1146563, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37207197

RESUMO

Anterior cruciate ligament (ACL) injury and meniscal tear (MT) are major causal factors for developing post-traumatic osteoarthritis (PTOA), but the biological mechanism(s) are uncertain. After these structural damages, the synovium could be affected by complement activation that normally occurs in response to tissue injury. We explored the presence of complement proteins, activation products, and immune cells, in discarded surgical synovial tissue (DSST) collected during arthroscopic ACL reconstructive surgery, MT-related meniscectomy and from patients with OA. Multiplexed immunohistochemistry (MIHC) was used to determine the presence of complement proteins, receptors and immune cells from ACL, MT, OA synovial tissue vs. uninjured controls. Examination of synovium from uninjured control tissues did not reveal the presence of complement or immune cells. However, DSST from patients undergoing ACL and MT repair demonstrated increases in both features. In ACL DSST, a significantly higher percentage of C4d+, CFH+, CFHR4+ and C5b-9+ synovial cells were present compared with MT DSST, but no major differences were seen between ACL and OA DSST. Increased cells expressing C3aR1 and C5aR1, and a significant increase in mast cells and macrophages, were found in ACL as compared to MT synovium. Conversely, the percentage of monocytes was increased in the MT synovium. Our data demonstrate that complement is activated in the synovium and is associated with immune cell infiltration, with a more pronounced effect following ACL as compared to MT injury. Complement activation, associated with an increase in mast cells and macrophages after ACL injury and/or MT, may contribute to the development of PTOA.


Assuntos
Lesões do Ligamento Cruzado Anterior , Artroplastia do Joelho , Menisco , Osteoartrite do Joelho , Humanos , Lesões do Ligamento Cruzado Anterior/complicações , Lesões do Ligamento Cruzado Anterior/cirurgia , Osteoartrite do Joelho/etiologia , Artroplastia do Joelho/efeitos adversos , Ativação do Complemento , Menisco/cirurgia
20.
J Cell Physiol ; 227(3): 1062-70, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21567390

RESUMO

The clinical literature strongly suggests that bone healing in cigarette smokers is impaired. Since cigarette smoke (CS) contains numerous polycyclic aromatic hydrocarbons (PAHs), and since dioxins impair bone formation in vivo via the Aryl Hydrocarbon Receptor (AHR), we investigated the impact of PAH/AHR signaling on chondrogenesis and on healing in a mouse tibial fracture model. We established that CS activates AHR signaling in fractures by up-regulating the AHR target gene cytochrome p4501A1 (Cyp1A1). For in vitro studies, we employed the mouse limb bud micromass chondrogenesis model. After confirming that chondrocytes express AHR during differentiation, we treated cells with a prototypical PAH found in CS, benzo(a)pyrene (BaP), or cigarette smoke extract (CSE). Both BaP and CSE strongly inhibited chondrogenesis in mesenchymal cells generated from E11 limb buds, with BaP also accelerating chondrocyte hypertrophy in cultures generated from E12 limb buds. Detection of DNA adducts in the BaP-treated cultures suggests that the distinct phenotypic effects of BaP may be due to the formation of reactive metabolites. Blockade of AHR signaling with the AHR antagonist MNF reverses the effects of BaP, but not CSE, suggesting that CSE inhibition of chondrogenesis is AHR-independent. Correlating with these results, tibial fracture calluses from BaP-treated mice were smaller and contained less mineralized tissue than vehicle controls. Overall, BaP is identified as a potent inhibitor of chondrogenesis in vitro with correlated effects on fracture healing similar to those of CS itself, suggesting a basis for PAHs as key compounds in the influence of CS on fracture repair.


Assuntos
Benzo(a)pireno/toxicidade , Condrogênese/efeitos dos fármacos , Consolidação da Fratura/efeitos dos fármacos , Receptores de Hidrocarboneto Arílico/fisiologia , Poluição por Fumaça de Tabaco/efeitos adversos , Animais , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Condrócitos/patologia , Condrócitos/fisiologia , Condrogênese/fisiologia , Modelos Animais de Doenças , Fraturas do Fêmur/metabolismo , Fraturas do Fêmur/patologia , Fraturas do Fêmur/fisiopatologia , Consolidação da Fratura/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Hidrocarboneto Arílico/genética , Fraturas da Tíbia/metabolismo , Fraturas da Tíbia/patologia , Fraturas da Tíbia/fisiopatologia
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