RESUMO
The article Second International Consensus Conference on lesions of uncertain malignant potential in the breast (B3 lesions), written by Christoph J Rageth, Elizabeth AM O'Flynn, Katja Pinker, Rahel A Kubik-Huch, Alexander Mundinger, Thomas Decker, Christoph Tausch, Florian Dammann, Pascal A. Baltzer, Eva Maria Fallenberg, Maria P Foschini, Sophie Dellas, Michael Knauer, Caroline Malhaire, Martin Sonnenschein, Andreas Boos, Elisabeth Morris, Zsuzsanna Varga, was originally published electronically on the publisher's internet portal (currently SpringerLink) on November 30, 2018 without open access.
RESUMO
PURPOSE: The second International Consensus Conference on B3 lesions was held in Zurich, Switzerland, in March 2018, organized by the International Breast Ultrasound School to re-evaluate the consensus recommendations. METHODS: This study (1) evaluated how management recommendations of the first Zurich Consensus Conference of 2016 on B3 lesions had influenced daily practice and (2) reviewed current literature towards recommendations to biopsy. RESULTS: In 2018, the consensus recommendations for management of B3 lesions remained almost unchanged: For flat epithelial atypia (FEA), classical lobular neoplasia (LN), papillary lesions (PL) and radial scars (RS) diagnosed on core-needle biopsy (CNB) or vacuum-assisted biopsy (VAB), excision by VAB in preference to open surgery, and for atypical ductal hyperplasia (ADH) and phyllodes tumors (PT) diagnosed at VAB or CNB, first-line open surgical excision (OE) with follow-up surveillance imaging for 5 years. Analyzing the Database of the Swiss Minimally Invasive Breast Biopsies (MIBB) with more than 30,000 procedures recorded, there was a significant increase in recommending more frequent surveillance of LN [65% in 2018 vs. 51% in 2016 (p = 0.004)], FEA (72% in 2018 vs. 62% in 2016 (p = 0.005)), and PL [(76% in 2018 vs. 70% in 2016 (p = 0.04)] diagnosed on VAB. A trend to more frequent surveillance was also noted also for RS [77% in 2018 vs. 67% in 2016 (p = 0.07)]. CONCLUSIONS: Minimally invasive management of B3 lesions (except ADH and PT) with VAB continues to be appropriate as an alternative to first-line OE in most cases, but with more frequent surveillance, especially for LN.
Assuntos
Biópsia com Agulha de Grande Calibre/métodos , Neoplasias da Mama/diagnóstico , Biópsia Guiada por Imagem/métodos , Neoplasias da Mama/patologia , Neoplasias da Mama/cirurgia , Carcinoma Intraductal não Infiltrante/patologia , Carcinoma Intraductal não Infiltrante/cirurgia , Carcinoma Lobular/patologia , Carcinoma Lobular/cirurgia , Bases de Dados Factuais , Feminino , Humanos , Procedimentos Cirúrgicos Minimamente Invasivos , Tumor Filoide/patologia , Tumor Filoide/cirurgia , Vigilância da População , Guias de Prática Clínica como AssuntoRESUMO
The purpose of this study is to obtain a consensus for the therapy of B3 lesions. The first International Consensus Conference on lesions of uncertain malignant potential in the breast (B3 lesions) including atypical ductal hyperplasia (ADH), flat epithelial atypia (FEA), classical lobular neoplasia (LN), papillary lesions (PL), benign phyllodes tumors (PT), and radial scars (RS) took place in January 2016 in Zurich, Switzerland organized by the International Breast Ultrasound School and the Swiss Minimally Invasive Breast Biopsy group-a subgroup of the Swiss Society of Senology. Consensus recommendations for the management and follow-up surveillance of these B3 lesions were developed and areas of research priorities were identified. The consensus recommendation for FEA, LN, PL, and RS diagnosed on core needle biopsy or vacuum-assisted biopsy (VAB) is to therapeutically excise the lesion seen on imaging by VAB and no longer by open surgery, with follow-up surveillance imaging for 5 years. The consensus recommendation for ADH and PT is, with some exceptions, therapeutic first-line open surgical excision. Minimally invasive management of selected B3 lesions with therapeutic VAB is acceptable as an alternative to first-line surgical excision.
Assuntos
Neoplasias da Mama/diagnóstico , Carcinoma Intraductal não Infiltrante/patologia , Carcinoma Lobular/patologia , Mamografia/métodos , Tumor Filoide/patologia , Biópsia com Agulha de Grande Calibre , Mama/patologia , Gerenciamento Clínico , Feminino , Humanos , Biópsia Guiada por Imagem , Vigilância da População/métodos , Guias de Prática Clínica como AssuntoRESUMO
Osteoarthritis (OA), the most common arthritic condition in humans, is characterized by the progressive degeneration of articular cartilage accompanied by chronic joint pain. Inflammatory mediators, such as cytokines and prostaglandin E(2) (PGE(2)) that are elevated in OA joints, play important roles in the progression of cartilage degradation and pain-associated nociceptor sensitivity. We have found that the nuclear receptor family transcription factors Liver X Receptors (LXRalpha and -beta) are expressed in cartilage, with LXRbeta being the predominant isoform. Here we show that genetic disruption of Lxrbeta gene expression in mice results in significantly increased proteoglycan (aggrecan) degradation and PGE(2) production in articular cartilage treated with IL-1beta, indicating a protective role of LXRbeta in cartilage. Using human cartilage explants, we found that activation of LXRs by the synthetic ligand GW3965 significantly reduced cytokine-induced degradation and loss of aggrecan from the tissue. Furthermore, LXR activation dramatically inhibited cytokine-induced PGE(2) production by human osteoarthritic cartilage as well as by a synovial sarcoma cell line. These effects were achieved at least partly by repression of the expression of ADAMTS4, a physiological cartilage aggrecanase, and of cyclooxygenase-2 and microsomal prostaglandin E synthase-1, key enzymes in the PGE(2) synthesis pathway. Consistent with our in vitro observations, oral administration of GW3965 potently alleviated joint pain in a rat meniscal tear model of osteoarthritis.
Assuntos
Cartilagem Articular/metabolismo , Dinoprostona/antagonistas & inibidores , Receptores Nucleares Órfãos/agonistas , Osteoartrite/complicações , Dor/metabolismo , Proteínas ADAM/antagonistas & inibidores , Proteína ADAMTS4 , Animais , Benzoatos/farmacologia , Benzilaminas/farmacologia , Linhagem Celular Tumoral , Ciclo-Oxigenase 2/metabolismo , Dinoprostona/metabolismo , Humanos , Oxirredutases Intramoleculares/antagonistas & inibidores , Ligantes , Receptores X do Fígado , Camundongos , Camundongos Mutantes , Receptores Nucleares Órfãos/genética , Receptores Nucleares Órfãos/fisiologia , Osteoartrite/metabolismo , Dor/etiologia , Pró-Colágeno N-Endopeptidase/antagonistas & inibidores , Prostaglandina-E Sintases , RatosRESUMO
Design, synthesis and structure-activity relationship of a series of biphenylsulfonamido-3-methylbutanoic acid based aggrecanase-1 inhibitors are described. In addition to robust aggrecanase-1 inhibition, these compounds also exhibit potent MMP-13 activity. In cell-based cartilage explants assay compound 48 produced 87% inhibition of proteoglycan degradation at 10 µg/mL. Good pharmacokinetic properties were demonstrated by 46 with a half-life of 6h and bioavailability of 23%.
Assuntos
Proteínas ADAM/antagonistas & inibidores , Proteínas ADAM/metabolismo , Compostos de Bifenilo/farmacologia , Pró-Colágeno N-Endopeptidase/antagonistas & inibidores , Pró-Colágeno N-Endopeptidase/metabolismo , Inibidores de Proteases/farmacologia , Sulfonamidas/farmacologia , Proteína ADAMTS4 , Animais , Compostos de Bifenilo/química , Compostos de Bifenilo/farmacocinética , Desenho de Fármacos , Humanos , Masculino , Metaloproteinase 13 da Matriz/metabolismo , Modelos Moleculares , Osteoartrite/tratamento farmacológico , Inibidores de Proteases/química , Inibidores de Proteases/farmacocinética , Proteoglicanas/metabolismo , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Sulfonamidas/química , Sulfonamidas/farmacocinéticaRESUMO
Human osteoarthritis is a progressive disease of the joints characterized by degradation of articular cartilage. Although disease initiation may be multifactorial, the cartilage destruction appears to be a result of uncontrolled proteolytic extracellular matrix destruction. A major component of the cartilage extracellular matrix is aggrecan, a proteoglycan that imparts compressive resistance to the tissue. Aggrecan is cleaved at a specific 'aggrecanase' site in human osteoarthritic cartilage; this cleavage can be performed by several members of ADAMTS family of metalloproteases. The relative contribution of individual ADAMTS proteases to cartilage destruction during osteoarthritis has not been resolved. Here we describe experiments with a genetically modified mouse in which the catalytic domain of ADAMTS5 (aggrecanase-2) was deleted. After surgically induced joint instability, there was significant reduction in the severity of cartilage destruction in the ADAMTS5 knockout mice compared with wild-type mice. This is the first report of a single gene deletion capable of abrogating the course of cartilage destruction in an animal model of osteoarthritis. These results demonstrate that ADAMTS5 is the primary 'aggrecanase' responsible for aggrecan degradation in a murine model of osteoarthritis, and suggest rational strategies for therapeutic intervention in osteoarthritis.
Assuntos
Cartilagem Articular/metabolismo , Modelos Animais de Doenças , Deleção de Genes , Metaloendopeptidases/deficiência , Metaloendopeptidases/genética , Osteoartrite/metabolismo , Proteínas ADAM , Proteína ADAMTS5 , Animais , Domínio Catalítico , Endopeptidases/química , Endopeptidases/deficiência , Endopeptidases/genética , Endopeptidases/metabolismo , Éxons/genética , Cabeça do Fêmur , Lâmina de Crescimento/metabolismo , Articulações/patologia , Articulações/fisiopatologia , Metaloendopeptidases/química , Metaloendopeptidases/metabolismo , Camundongos , Camundongos Knockout , Osteoartrite/genética , Osteoartrite/patologia , Osteoartrite/fisiopatologia , Proteoglicanas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
BACKGROUND: Tenascin-C (TN-C) is an extracellular matrix glycoprotein that is involved in tissue injury and repair processes. We analyzed TN-C expression in normal and osteoarthritic (OA) human cartilage, and evaluated its capacity to induce inflammatory and catabolic mediators in chondrocytes in vitro. The effect of TN-C on proteoglycan loss from articular cartilage in culture was also assessed. METHODS: TN-C in culture media, cartilage extracts, and synovial fluid of human and animal joints was quantified using a sandwich ELISA and/or analyzed by Western immunoblotting. mRNA expression of TN-C and aggrecanases were analyzed by Taqman assays. Human and bovine primary chondrocytes and/or explant culture systems were utilized to study TN-C induced inflammatory or catabolic mediators and proteoglycan loss. Total proteoglycan and aggrecanase -generated ARG-aggrecan fragments were quantified in human and rat synovial fluids by ELISA. RESULTS: TN-C protein and mRNA expression were significantly upregulated in OA cartilage with a concomitant elevation of TN-C levels in the synovial fluid of OA patients. IL-1 enhanced TN-C expression in articular cartilage. Addition of TN-C induced IL-6, PGE2, and nitrate release and upregulated ADAMTS4 mRNA in cultured primary human and bovine chondrocytes. TN-C treatment resulted in an increased loss of proteoglycan from cartilage explants in culture. A correlation was observed between TN-C and aggrecanase generated ARG-aggrecan fragment levels in the synovial fluid of human OA joints and in the lavage of rat joints that underwent surgical induction of OA. CONCLUSIONS: TN-C expression in the knee cartilage and TN-C levels measured in the synovial fluid are significantly enhanced in OA patients. Our findings suggest that the elevated levels of TN-C could induce inflammatory mediators and promote matrix degradation in OA joints.
Assuntos
Cartilagem Articular/patologia , Condrócitos/patologia , Matriz Extracelular/metabolismo , Mediadores da Inflamação/metabolismo , Osteoartrite do Joelho/patologia , Tenascina/fisiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Cartilagem Articular/metabolismo , Bovinos , Linhagem Celular Tumoral , Células Cultivadas , Condrócitos/metabolismo , Matriz Extracelular/patologia , Feminino , Humanos , Mediadores da Inflamação/fisiologia , Masculino , Pessoa de Meia-Idade , Osteoartrite do Joelho/etiologia , Osteoartrite do Joelho/metabolismo , Ratos , Ratos Endogâmicos Lew , Líquido Sinovial/metabolismo , Tenascina/biossíntese , Tenascina/genética , Regulação para Cima/fisiologiaRESUMO
Mass spectrometry-based proteomic analyses performed on cartilage tissue extracts identified the serine protease HtrA1/PRSS11 as a major protein component of human articular cartilage, with elevated levels occurring in association with osteoarthritis. Overexpression of a catalytically active form of HtrA1, but not an active site mutant (S328A), caused a marked reduction in proteoglycan content in chondrocyte-seeded alginate cultures. Aggrecan degradation fragments were detected in conditioned media from the alginate cultures overexpressing active HtrA1. Incubation of native or recombinant aggrecan with wild type HtrA1 resulted in distinct cleavage of these substrates. Cleavage of aggrecan by HtrA1 was strongly enhanced by HtrA1 agonists such as CPII, a C-terminal hexapeptide derived from the C-propeptide of procollagen IIalpha1 (i.e. chondrocalcin). A novel HtrA1-susceptible cleavage site within the interglobular domain (IGD) of aggrecan was identified, and an antibody that specifically recognizes the neoepitope sequence (VQTV(356)) generated at the HtrA1 cleavage site was developed. Western blot analysis demonstrated that HtrA1-generated aggrecan fragments containing the VQTV(356) neoepitope were significantly more abundant in osteoarthritic cartilage compared with cartilage from healthy joints, implicating HtrA1 as a critical protease involved in proteoglycan turnover and cartilage degradation during degenerative joint disease.
Assuntos
Agrecanas/química , Agrecanas/metabolismo , Serina Endopeptidases/metabolismo , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Agrecanas/análise , Agrecanas/imunologia , Alginatos , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cartilagem/metabolismo , Estudos de Casos e Controles , Células Cultivadas , Condrócitos/metabolismo , Condrócitos/patologia , Progressão da Doença , Epitopos/química , Epitopos/imunologia , Feminino , Regulação da Expressão Gênica , Ácido Glucurônico , Ácidos Hexurônicos , Serina Peptidase 1 de Requerimento de Alta Temperatura A , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Mutação , Osteoartrite/metabolismo , Osteoartrite/patologia , Serina Endopeptidases/genéticaRESUMO
Potent 3,4-disubstituted benzofuran P1' MMP-13 inhibitors have been prepared. Selectivity over MMP-2 was achieved through a substituent at the C4 position of the benzofuran P1' moiety of the molecule. By replacing a backbone benzene with a pyridine and valine with threonine, compounds (e.g., 44) with greatly reduced plasma protein binding were also obtained.
Assuntos
Benzofuranos/química , Inibidores de Metaloproteinases de Matriz , Inibidores de Proteases/química , Animais , Benzofuranos/síntese química , Benzofuranos/farmacologia , Metaloproteinase 13 da Matriz/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Inibidores de Proteases/síntese química , Inibidores de Proteases/farmacologia , Ligação Proteica , Coelhos , Albumina Sérica/química , Relação Estrutura-AtividadeRESUMO
The prevention of aggrecan (a key component of cartilage) cleavage via the inhibition of aggrecanase-1 may provide a unique opportunity to stop the progression of cartilage degradation in osteoarthritis. The evaluation of a series of biphenylsulfonamides resulted in the identification of the ((4-keto)-phenoxy)methyl biphenyl-4-sulfonamides analogs (19-21 and 24) with improved Agg-1 inhibition and MMP-2, MMP-13 activity.
Assuntos
Proteínas ADAM/antagonistas & inibidores , Proteínas ADAM/metabolismo , Química Farmacêutica/métodos , Osteoartrite/tratamento farmacológico , Pró-Colágeno N-Endopeptidase/antagonistas & inibidores , Pró-Colágeno N-Endopeptidase/metabolismo , Sulfonamidas/síntese química , Proteína ADAMTS4 , Cartilagem/efeitos dos fármacos , Cartilagem/metabolismo , Desenho de Fármacos , Humanos , Concentração Inibidora 50 , Metaloproteinase 13 da Matriz/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Modelos Químicos , Conformação Molecular , Proteoglicanas/química , Sulfonamidas/farmacologiaRESUMO
Articular cartilage chondrocytes help in the maintenance of tissue homeostasis and function of the articular joint. Study of primary chondrocytes in culture provides information closely related to in vivo functions of these cells. Limitations in the primary culture of chondrocytes have lead to the development of cells lines that serve as good surrogate models for the study of chondrocyte biology. In this study, we report the establishment and characterization of chondrocyte cell lines, MM-Sv/HP and MM-Sv/HP-2 from mouse articular cartilage. Cells were isolated from mouse femoral head articular cartilage, immortalized and maintained in culture through numerous passages. The morphology of the cells was from fibroblastic to polygonal in nature. Gene expression studies using quantitative PCR (Q-PCR) were performed on cells in monolayer culture and cells embedded in a three-dimensional alginate matrix. Stimulation of cells in monolayer culture with anabolic factor, BMP-2, resulted in increased gene expression of the extracellular matrix molecules, aggrecan and type II collagen and their regulator transcription factor, Sox9. Treatment by pro-inflammatory IL-1 resulted in increased gene expression of catabolic effectors including Aggrecanases (ADAMTS4, ADAMTS5), MMP-13 and nitric oxide synthase (Nos2). Cells in alginate treated with BMP-2 resulted in increased synthesis of proteoglycan which was released into the conditioned media on IL-1 stimulation. Western analysis of conditioned media showed the presence of Aggrecanase-cleaved aggrecan fragments. In summary, MM-Sv/HP and MM-Sv/HP-2 show preservation of important characteristics of articular chondrocytes as examined under multiple culture conditions and would provide a useful reagent in the study of chondrocyte biology.
Assuntos
Proteínas Morfogenéticas Ósseas/farmacologia , Cartilagem Articular/citologia , Cartilagem Articular/efeitos dos fármacos , Condrócitos/citologia , Condrócitos/efeitos dos fármacos , Mediadores da Inflamação/farmacologia , Interleucina-1/farmacologia , Fator de Crescimento Transformador beta/farmacologia , Agrecanas/metabolismo , Alginatos/farmacologia , Animais , Biomarcadores/metabolismo , Proteína Morfogenética Óssea 2 , Cartilagem Articular/enzimologia , Linhagem Celular Transformada , Condrócitos/enzimologia , Endopeptidases/metabolismo , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Fêmur/citologia , Fêmur/efeitos dos fármacos , Humanos , Camundongos , FenótipoRESUMO
N-((8-Hydroxy-5-substituted-quinolin-7-yl)(phenyl)methyl)-2-phenyloxy/amino-acetamide inhibitors of ADAMTS-5 (Aggrecanase-2) have been prepared. Selected compounds 10, 14, 25, and 53 show sub-microM ADAMTS-5 potency and good selectivity over the related metalloproteases ADAMTS-4 (Aggrecanase-1), MMP-13, and MMP-12. Compound 53 shows a good balance of potent ADAMTS-5 inhibition, moderate CYP3A4 inhibition and good rat liver microsome stability. This series of compounds represents progress towards selective ADAMTS-5 inhibitors as disease modifying osteoarthritis agents.
Assuntos
Proteínas ADAM/antagonistas & inibidores , Proteínas ADAM/química , Acetamidas/síntese química , Acetamidas/farmacologia , Proteínas ADAM/metabolismo , Proteína ADAMTS4 , Proteína ADAMTS5 , Animais , Química Farmacêutica/métodos , Citocromo P-450 CYP3A , Inibidores do Citocromo P-450 CYP3A , Inibidores das Enzimas do Citocromo P-450 , Desenho de Fármacos , Humanos , Concentração Inibidora 50 , Microssomos Hepáticos/efeitos dos fármacos , Modelos Químicos , Osteoartrite/tratamento farmacológico , Pró-Colágeno N-Endopeptidase/metabolismo , RatosRESUMO
Aggrecanases are ADAMTS (a disintegrin and metalloproteinase with thrombospondin type I motifs) proteases capable of primary (patho)physiological cleavage at specific Glu-Xaa bonds within the core protein of the hyaluronan-binding proteoglycan aggrecan. Accumulating evidence suggests that regulation of the activity of one such aggrecanase, ADAMTS-4 (or Aggrecanase-1), involves post-translational C-terminal processing (truncation) which modulates both glycosaminoglycan (GAG)-binding affinity and enzymatic activity. In the present study, we compared the effects of C-terminal truncation on the GAG-binding properties and aggrecanase activity of ADAMTS-5 (Aggrecanase-2) relative to three other ADAMTS family members, ADAMTS-9, ADAMTS-16 and ADAMTS-18. Full-length recombinant human ADAMTS-5 (M(r) approximately 85 kDa; ADAMTS-5p85) underwent autolytic cleavage during expression by CHO/A2 cells, and co-purified with C-terminally truncated (tr) isoforms of M(r) approximately 60 kDa (ADAMTS-5p60 and M(r) approximately 45 kDa (ADAMTS-5p45). All three ADAMTS-5 isoforms bound to sulfated GAGs (heparin and chondroitin sulfate (CS)). An ADAMTS-5p45 structural mimetic, terminating at Phe628 and comprising the catalytic domain, disintegrin-like domain and thrombospondin type I repeat (TSR)-1 domain (designated trADAMTS-5F628), also bound to heparin, and exhibited potent aggrecanase activity toward cleavage sites both in the aggrecan CS-2-attachment region (at Glu1771-Ala1772) and in the interglobular domain (at Glu373-Ala374). Further truncation (deletion of the TSR-1 domain) of ADAMTS-5 significantly reduced aggrecanase activity, although appreciable GAG (heparin)-binding affinity was maintained. Other TSR-1 domain-bearing truncated ADAMTS constructs demonstrating either positive GAG-binding ability (trADAMTS-9F649) or negligible GAG-affinity (trADAMTS-16F647 and trADAMTS-18F650) displayed comparably low aggrecanase activities. Thus, the presence of TSR-1 on truncated ADAMTSs appears to be necessary, but not sufficient, for effective aggrecanase-mediated catalysis of target Glu-Xaa bonds. Similarly, GAG-binding ability, irrespective of the presence of a TSR-1 domain, does not necessarily empower truncated ADAMTSs with proficient aggrecanase activity.
Assuntos
Proteínas ADAM/metabolismo , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Endopeptidases/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Glicosaminoglicanos/metabolismo , Lectinas Tipo C/metabolismo , Proteínas ADAM/química , Proteínas ADAM/genética , Proteínas ADAMTS , Proteína ADAMTS5 , Proteína ADAMTS9 , Agrecanas , Animais , Células CHO , Bovinos , Cricetinae , Cricetulus , Humanos , Isoformas de Proteínas/metabolismoRESUMO
Lubricin is a secreted, cytoprotective glycoprotein that contributes to the essential boundary lubrication mechanisms necessary for maintaining low friction levels at articular cartilage surfaces. Diminishment of lubricin function is thereby implicated as an adverse contributing factor in degenerative joint diseases such as osteoarthritis. Lubricin occurs as a soluble component of synovial fluid, and is synthesized and localized in the superficial layer of articular cartilage (and thus has also been described as "superficial zone protein", or SZP); however, defined interactions responsible for lubricin retention at this site are not well characterized. In the current studies, we identified molecular determinants that enable lubricin to effectively bind to articular cartilage surfaces. Efficient and specific binding to the superficial zone was observed for synovial lubricin, as well as for recombinant full-length lubricin and a protein construct comprising the lubricin C-terminal (hemopexin-like) domain (LUB-C, encoded by exons 7-12). A construct representing the N-terminal region of lubricin (LUB-N, encoded by exons 2-5) exhibited no appreciable cartilage-binding ability, but displayed the capacity to dimerize, and thus potentially influence lubricin aggregation. Disulfide bond disruption significantly attenuated recombinant lubricin and LUB-C binding to cartilage surfaces, demonstrating a requirement for protein secondary structure in facilitating the appropriate localization of lubricin at relevant tissue interfaces. These findings help identify additional key attributes contributing to lubricin functionality, which would be expected to be instrumental in maintaining joint homeostasis.
Assuntos
Cartilagem Articular/metabolismo , Glicoproteínas/metabolismo , Animais , Bovinos , Glicoproteínas/química , Glicoproteínas/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
Insulin-like growth factor-I (IGF-I) is thought to play an important role in skeletal growth and development through its mitogenic and anabolic effects on epiphyseal growth plate chondrocytes. The bone morphogenetic proteins (BMPs) have been shown to promote endochondral osteogenesis, and some members of the BMP family, including BMP-2 and BMP-9, have anabolic effects on chondrocyte metabolism. We tested the hypothesis that BMP-2 and BMP-9 interact with IGF-I to modulate growth plate chondrocyte mitotic activity. IGF-I, but neither BMP-2 nor BMP-9, stimulated chondrocyte DNA synthesis. However, both BMP-2 and BMP-9 augmented the mitogenic action of IGF-I. BMP-2, but not BMP-9 increased IGF-I binding to growth plate chondrocytes in kinetic studies. In affinity labeling studies, 125I-IGF-I predominantly labeled an Mr approximately 135-kDa moiety, consistent with the alpha subunit of the type 1 IGF receptor and an Mr approximately 250-kDa moiety consistent with the type 2 IGF receptor. 125I-IGF-I labeling also appeared at Mr approximately 43 kDa, consistent with 125I-IGF-I binding to insulin-like growth binding protein-3. Treatment of chondrocytes with BMP-2, but not with BMP-9, increased the intensity of the Mr approximately 135-kDa band and decreased the intensity of the Mr approximately 43-kDa band. Taken together, these data suggest that the BMPs may modulate the action of IGF-I via the type 1 IGF receptor and/or IGF binding proteins.
Assuntos
Proteínas Morfogenéticas Ósseas/fisiologia , Condrócitos/metabolismo , Lâmina de Crescimento/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Fator de Crescimento Transformador beta/fisiologia , Animais , Proteína Morfogenética Óssea 2 , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas Morfogenéticas Ósseas/farmacologia , Células CHO , Cartilagem Articular/citologia , Bovinos , Células Cultivadas , Cricetinae , Cricetulus , Relação Dose-Resposta a Droga , Fator 2 de Diferenciação de Crescimento , Lâmina de Crescimento/citologia , Radioisótopos do Iodo , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta/farmacologiaRESUMO
Members of the bone morphogenetic protein (BMP) group of the TGF-beta superfamily have been shown to enhance matrix synthesis and maintain cartilage phenotype in long-term culture. These proteins have also been shown to augment cartilage repair in vivo, and may be of potential therapeutic benefit in the treatment of damaged articular cartilage. The present study was undertaken to examine the effects of BMP-9 on the metabolism of juvenile and adult bovine cartilage in vitro, and to compare the effects to those produced by two previously characterized BMPs: BMP-2 and 13 (CDMP-2). BMP-9 lead to a 7-8-fold stimulation of proteoglycan synthesis at the highest concentration tested, and a 6.4-fold stimulation of collagen synthesis at a concentration of 50 ng/mL in juvenile cartilage. BMP-2 also lead to a 7-8-fold increase in proteoglycan synthesis at the highest concentration tested, and was able to induce collagen synthesis 6.4-fold, but at a concentration of 1000 ng/mL. Proteoglycans isolated from BMP-9 treated cartilage exhibited an increased hydrodynamic size possibly due to increased glycosaminoglycan substitution or decreased C-terminal proteolysis. Consistent with the idea of limited C-terminal proteolysis, BMP-9 treatment lead to a significant reduction in the turnover rate of proteoglycans in juvenile explants. Interestingly, all three BMPs were unable to induce a measurable anabolic response in adult cartilage explants.
Assuntos
Proteínas Morfogenéticas Ósseas/farmacologia , Cartilagem/efeitos dos fármacos , Fatores Etários , Animais , Proteína Morfogenética Óssea 2 , Receptores de Proteínas Morfogenéticas Ósseas , Cartilagem/metabolismo , Bovinos , Fator 2 de Diferenciação de Crescimento , Proteoglicanas/biossíntese , Receptores de Fatores de Crescimento/análise , Fator de Crescimento Transformador beta/farmacologiaRESUMO
Members of the ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) family share common structural features including a disintegrin domain, a zinc metalloprotease domain, and at least one thrombospondin motif. Aberrant expression of several of these proteins has led to an understanding of their role in human disease; however, a link to function for many has not yet been made. One such uncharacterized family member, ADAMTS-8, shares significant protein sequence homology with a subgroup of ADAMTSs that includes ADAMTS-1, ADAMTS-4, ADAMTS-5, and ADAMTS-15. Each of these proteases has been shown to cleave 'aggrecanase-susceptible' site(s) within the extracellular matrix (ECM) proteoglycan aggrecan, and ADAMTS-4 and ADAMTS-5 have been postulated to play a role in the depletion of articular cartilage in osteoarthritic disease. Based on sequence relationships, in the present study we examined the ability of ADAMTS-8 to exhibit 'aggrecanase' activity. A neoepitope monoclonal antibody (MAb; AGG-C1; anti-NITEGE373) was developed and used to demonstrate the ability of ADAMTS-8 to cleave aggrecan at the aggrecanase-susceptible Glu373-Ala374 peptide bond. In addition, expression analyses demonstrated the presence of ADAMTS-8 mRNA transcripts in normal and osteoarthritic human cartilage.
Assuntos
Cartilagem Articular/enzimologia , Endopeptidases/metabolismo , Metaloendopeptidases/metabolismo , Proteínas ADAM , Proteína ADAMTS9 , Agrecanas , Animais , Anticorpos Monoclonais/imunologia , Western Blotting , Células CHO , Cricetinae , Cricetulus , Ensaio de Imunoadsorção Enzimática/métodos , Proteínas da Matriz Extracelular/metabolismo , Humanos , Lectinas Tipo C , Metaloendopeptidases/genética , Metaloendopeptidases/imunologia , Metaloendopeptidases/isolamento & purificação , Osteoartrite/metabolismo , Reação em Cadeia da Polimerase , Proteoglicanas/metabolismo , RNA Mensageiro/metabolismo , Homologia de Sequência de Aminoácidos , Distribuição TecidualRESUMO
Apoptosis may limit the utility of cell-based therapies for articular cartilage disorders. We tested the hypothesis that chondrocyte apoptosis can be reduced by optimizing the conditions employed to expand chondrocyte numbers in culture. Chondrocyte apoptosis was examined in monolayer and suspension culture, in the presence of fetal bovine serum (FBS) or autologous serum, and for culture periods of 2 or 4 days. The effect of these variables was assessed by measuring cell viability, Annexin V labeling and mitochondrial membrane potential. After 2 days of culture, the greatest increase in viable cell number (3.7-fold) occurred in monolayer cultures with autologous serum. After 4 days of culture, the greatest increase in cell number (9.0-fold) occurred in monolayer cultures supplemented with FBS. By Annexin V staining, the proportion of cells undergoing apoptosis after 2 days was not affected by the type of serum used or by culture in monolayer versus suspension. After 4 days of culture, the proportion of apoptotic cells was significantly reduced (35% to 13%, p<0.02) in suspension cultures with autologous serum. Apoptosis assessed by loss of mitochondrial membrane potential was decreased in the presence of autologous serum. These data suggest that suspension culture with autologous serum is useful in simultaneously maintaining cell proliferation and minimizing apoptosis in cultured human articular chondrocytes.
Assuntos
Apoptose/fisiologia , Condrócitos/fisiologia , Anexina A5/metabolismo , Contagem de Células , Técnicas de Cultura de Células/métodos , Sobrevivência Celular , Células Cultivadas , Condrócitos/metabolismo , Citometria de Fluxo , Fluoresceína-5-Isotiocianato/metabolismo , Humanos , Potenciais da Membrana , Mitocôndrias/metabolismo , Mitocôndrias/fisiologia , Fatores de TempoRESUMO
OBJECTIVE: Lubricin, also referred to as superficial zone protein and PRG4, is a synovial glycoprotein that supplies a friction-resistant, antiadhesive coating to the surfaces of articular cartilage, thereby protecting against arthritis-associated tissue wear and degradation. This study was undertaken to generate and characterize a novel recombinant lubricin protein construct, LUB:1, and to evaluate its therapeutic efficacy following intraarticular delivery in a rat model of osteoarthritis (OA). METHODS: Binding and localization of LUB:1 to cartilage surfaces was assessed by immunohistochemistry. The cartilage-lubricating properties of LUB:1 were determined using a custom friction testing apparatus. A cell-binding assay was performed to quantify the ability of LUB:1 to prevent cell adhesion. Efficacy studies were conducted in a rat meniscal tear model of OA. One week after the surgical induction of OA, LUB:1 or phosphate buffered saline vehicle was administered by intraarticular injection for 4 weeks, with dosing intervals of either once per week or 3 times per week. OA pathology scores were determined by histologic analysis. RESULTS: LUB:1 was shown to bind effectively to cartilage surfaces, and facilitated both cartilage boundary lubrication and inhibition of synovial cell adhesion. Treatment of rat knee joints with LUB:1 resulted in significant disease-modifying, chondroprotective effects during the progression of OA, by markedly reducing cartilage degeneration and structural damage. CONCLUSION: Our findings demonstrate the potential use of recombinant lubricin molecules in novel biotherapeutic approaches to the treatment of OA and associated cartilage abnormalities.
Assuntos
Cartilagem Articular/patologia , Glicoproteínas/uso terapêutico , Osteoartrite/patologia , Osteoartrite/prevenção & controle , Proteínas Recombinantes/uso terapêutico , Animais , Cartilagem Articular/efeitos dos fármacos , Cartilagem Articular/lesões , Adesão Celular/efeitos dos fármacos , Modelos Animais de Doenças , Progressão da Doença , Glicoproteínas/administração & dosagem , Glicoproteínas/farmacologia , Injeções Intra-Articulares , Masculino , Distribuição Aleatória , Ratos , Ratos Endogâmicos Lew , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/farmacologia , Membrana Sinovial/efeitos dos fármacos , Membrana Sinovial/patologia , Resultado do TratamentoRESUMO
PURPOSE: In the treatment of osteoarthritis (OA), some of the therapeutic approaches require delivery of drug(s) to the diseased cartilage. Presence of adequate drug levels in the cartilage is one of the important criteria in selection and ranking of lead compounds. The purpose of this study was to investigate the correlation in cartilage compound levels between in vitro experiments and in vivo animal studies. MATERIALS AND METHODS: Bovine cartilage samples were incubated with test compounds of various concentrations in a culture medium, in the absence or presence of 25 mg/ml of serum albumin which served as an artificial synovial fluid (SF). The test compounds were also dosed to rabbits, the animal model used for efficacy studies, over a six-week treatment period. Test article concentrations in plasma, SF, and cartilage were determined by LC/MS/MS analysis. RESULTS AND CONCLUSIONS: A correlation in cartilage drug concentration was observed between in vitro and in vivo studies. Plasma protein binding and the test article's affinity to cartilage were the major determining factors for drug delivery to cartilage in vivo.