RESUMO
Cartilages are unique in the family of connective tissues in that they contain a high concentration of the glycosaminoglycans, chondroitin sulfate and keratan sulfate attached to the core protein of the proteoglycan, aggrecan. Multiple aggrecan molecules are organized in the extracellular matrix via a domain-specific molecular interaction with hyaluronan and a link protein, and these high molecular weight aggregates are immobilized within the collagen and glycoprotein network. The high negative charge density of glycosaminoglycans provides hydrophilicity, high osmotic swelling pressure and conformational flexibility, which together function to absorb fluctuations in biomechanical stresses on cartilage during movement of an articular joint. We have summarized information on the history and current knowledge obtained by biochemical and genetic approaches, on cell-mediated regulation of aggrecan metabolism and its role in skeletal development, growth as well as during the development of joint disease. In addition, we describe the pathways for hyaluronan metabolism, with particular focus on the role as a "metabolic rheostat" during chondrocyte responses in cartilage remodeling in growth and disease.Future advances in effective therapeutic targeting of cartilage loss during osteoarthritic diseases of the joint as an organ as well as in cartilage tissue engineering would benefit from 'big data' approaches and bioinformatics, to uncover novel feed-forward and feed-back mechanisms for regulating transcription and translation of genes and their integration into cell-specific pathways.
Assuntos
Cartilagem Articular , Ácido Hialurônico , Agrecanas/genética , Agrecanas/análise , Agrecanas/metabolismo , Ácido Hialurônico/metabolismo , Polieletrólitos/análise , Polieletrólitos/metabolismo , Polieletrólitos/farmacologia , Cartilagem Articular/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Glicosaminoglicanos , Lectinas Tipo C/metabolismoRESUMO
Purpose/Aim of the study: Healthy tendons are maintained in homeostasis through controlled usage of glucose for energy and redox equilibrium. Tendon cell stress imposed by overuse injury or vascular insufficiency is accompanied by activation of wound healing pathways which facilitate an adaptive response and the restoration of homeostasis. To understand this response at the gene expression level we have studied the in vivo effects of injected TGF-ß1 in a murine model of tendinopathy, as well as treatment of murine tendon explants with either TGF-ß1 or hypoxia in vitro. METHODS AND RESULTS: We provide evidence (from expression patterns and immunohistochemistry) that both in vivo and in vitro, the stress response in tendon cells may be metabolically controlled in part by glycolytic reprogramming. A major feature of the response to TGF-ß1 or hypoxia is activation of the Warburg pathway which generates lactate from glucose under normoxia and thereby inhibits mitochondrial energy production. CONCLUSIONS: We discuss the likely outcome of this major metabolic shift in terms of the potential benefits and damage to tendon and suggest how incorporation of this metabolic response into our understanding of initiation and progression of tendinopathies may offer new opportunities for diagnosis and the monitoring of therapies.
Assuntos
Glucose/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Ácido Láctico/biossíntese , Transdução de Sinais , Tendões/citologia , Tendões/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Proteína ADAMTS5/deficiência , Proteína ADAMTS5/metabolismo , Aerobiose/efeitos dos fármacos , Animais , Hipóxia Celular/efeitos dos fármacos , Hipóxia Celular/genética , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Glicólise/efeitos dos fármacos , Glicólise/genética , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Masculino , Camundongos Knockout , Neovascularização Fisiológica/efeitos dos fármacos , Neovascularização Fisiológica/genética , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
ADAMTS5 has been implicated in the degradation of cartilage aggrecan in human osteoarthritis. Here, we describe a novel role for the enzyme in the regulation of TGFß1 signaling in dermal fibroblasts both in vivo and in vitro. Adamts5(-/-) mice, generated by deletion of exon 2, exhibit impaired contraction and dermal collagen deposition in an excisional wound healing model. This was accompanied by accumulation in the dermal layer of cell aggregates and fibroblastic cells surrounded by a pericellular matrix enriched in full-length aggrecan. Adamts5(-/-) wounds exhibit low expression (relative to wild type) of collagen type I and type III but show a persistently elevated expression of tgfbRII and alk1. Aggrecan deposition and impaired dermal repair in Adamts5(-/-) mice are both dependent on CD44, and Cd44(-/-)/Adamts5(-/-) mice display robust activation of TGFß receptor II and collagen type III expression and the dermal regeneration seen in WT mice. TGFß1 treatment of newborn fibroblasts from wild type mice results in Smad2/3 phosphorylation, whereas cells from Adamts5(-/-) mice phosphorylate Smad1/5/8. The altered TGFß1 response in the Adamts5(-/-) cells is dependent on the presence of aggrecan and expression of CD44, because Cd44(-/-)/Adamts5(-/-) cells respond like WT cells. We propose that ADAMTS5 deficiency in fibrous tissues results in a poor repair response due to the accumulation of aggrecan in the pericellular matrix of fibroblast progenitor cells, which prevents their transition to mature fibroblasts. Thus, the capacity of ADAMTS5 to modulate critical tissue repair signaling events suggests a unique role for this enzyme, which sets it apart from other members of the ADAMTS family of proteases.
Assuntos
Proteínas ADAM/deficiência , Agrecanas/metabolismo , Derme/fisiopatologia , Receptores de Hialuronatos/metabolismo , Deleção de Sequência , Fator de Crescimento Transformador beta1/metabolismo , Cicatrização/genética , Proteínas ADAM/genética , Proteína ADAMTS5 , Receptores de Ativinas Tipo I/genética , Receptores de Ativinas Tipo I/metabolismo , Receptores de Activinas Tipo II , Agrecanas/genética , Animais , Animais Recém-Nascidos , Agregação Celular/efeitos dos fármacos , Derme/efeitos dos fármacos , Derme/metabolismo , Derme/patologia , Células Epitelioides/efeitos dos fármacos , Células Epitelioides/metabolismo , Células Epitelioides/patologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrose , Regulação da Expressão Gênica/efeitos dos fármacos , Genótipo , Humanos , Masculino , Camundongos , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Proteínas Smad Reguladas por Receptor/genética , Proteínas Smad Reguladas por Receptor/metabolismo , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Células-Tronco/patologia , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/farmacologia , Cicatrização/efeitos dos fármacosRESUMO
Previous work has shown that versican, decorin and a catabolic fragment of decorin, termed decorunt, are the most abundant proteoglycans in human skin. Further analysis of versican indicates that four major core protein species are present in human skin at all ages examined from fetal to adult. Two of these are identified as the V0 and V1 isoforms, with the latter predominating. The other two species are catabolic fragments of V0 and V1, which have the amino acid sequence DPEAAE as their carboxyl terminus. Although the core proteins of human skin versican show no major age-related differences, the glycosaminoglycans (GAGs) of adult skin versican are smaller in size and show differences in their sulfation pattern relative to those in fetal skin versican. In contrast to human skin versican, human skin decorin shows minimal age-related differences in its sulfation pattern, although, like versican, the GAGs of adult skin decorin are smaller than those of fetal skin decorin. Analysis of the catabolic fragments of decorin from adult skin reveals the presence of other fragments in addition to decorunt, although the core proteins of these additional decorin catabolic fragments have not been identified. Thus, versican and decorin of human skin show age-related differences, versican primarily in the size and the sulfation pattern of its GAGs and decorin in the size of its GAGs. The catabolic fragments of versican are detected at all ages examined, but appear to be in lower abundance in adult skin compared with fetal skin. In contrast, the catabolic fragments of decorin are present in adult skin, but are virtually absent from fetal skin. Taken together, these data suggest that there are age-related differences in the catabolism of proteoglycans in human skin. These age-related differences in proteoglycan patterns and catabolism may play a role in the age-related changes in the physical properties and injury response of human skin.
Assuntos
Envelhecimento , Decorina , Envelhecimento da Pele , Pele , Versicanas , Adulto , Envelhecimento/metabolismo , Sequência de Aminoácidos , Sítios de Ligação de Anticorpos/genética , Decorina/genética , Decorina/metabolismo , Combinação de Medicamentos , Eletroforese em Gel de Poliacrilamida , Feto/metabolismo , Humanos , Immunoblotting , Pessoa de Meia-Idade , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Pele/metabolismo , Sulfamonometoxina/metabolismo , Trimetoprima/metabolismo , Versicanas/genética , Versicanas/metabolismo , Adulto JovemRESUMO
BACKGROUND: The proteoglycan 4 (PRG4) gene encodes for a mucin-like O-linked glycosylated protein with several names, including lubricin and superficial zone protein. The objective of this study was to analyze PRG4 in normal bovine calf and steer synovial fluids for evidence of native multimers formed by intermolecular disulfide bonds. METHODS: A combination of mucin biochemical techniques, with antibodies to both terminal domains and the mucin-like domain of PRG4, were used for analyses. RESULTS: Multimers were present in both calf and steer fluids, and reduction and alkylation converts the multimeric complex (likely dimeric) into monomeric subunits. Tandem mass spectrometry analyses supported the Western blot data and identified PRG4 in the reduced approximately 345 kDa monomeric form. Interestingly, approximately 70 kDa fragments released upon reduction contained peptides from both the N and C terminal regions, which most likely represent fragments of a sparsely glycosylated PRG4 population that are disulfide-linked to extensively glycosylated, intact monomers. CONCLUSIONS: The analyses described here have demonstrated the presence of native disulfide-bonded multimers of PRG4 in normal bovine synovial fluids. GENERAL SIGNIFICANCE: These structures are similar to those described for multimerization of mucins in general. Such multimerization and proteolytic cleavage of PRG4 may have functional significance in joint health and disease.
Assuntos
Dissulfetos/metabolismo , Multimerização Proteica , Proteoglicanas/metabolismo , Líquido Sinovial/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos , Western Blotting , Bovinos , Epitopos , Espectrometria de Massas , Dados de Sequência Molecular , Peptídeos/química , Proteoglicanas/química , Proteoglicanas/isolamento & purificação , Coloração pela PrataRESUMO
Pro-inflammatory cytokines induce meniscal matrix degradation and inhibition of endogenous repair mechanisms, but the pathogenic mechanisms behind this are mostly unknown. Therefore, we investigated details of interleukin-1 (IL-1alpha)-induced aggrecan turnover in mature meniscal tissue explants. Fibro-cartilagenous disks (3 mm diameter x 1 mm thickness) were isolated from the central, weight-bearing region of menisci from 2-year-old cattle. After 3 or 6 days of IL-1alpha-treatment, GAG loss (DMMB assay), biosynthetic activity ([(35)SO(4)]-sulfate and [(3)H]-proline incorporation), gene expression (quantitative RT-PCR) and the abundance (zymography, Western blot) of matrix-degrading enzymes and specific aggrecan products were determined. Meniscal fibrocartilage had a 4-fold lower GAG content (per wet weight) than adjacent articular cartilage, and expressed MMPs-1, -2, -3 and ADAMTS4 constitutively, whereas ADAMTS5 m-RNA was essentially undetectable. Significant IL-1 effects were a decrease in biosynthetic activity, an increase in GAG release and in the expression/abundance of MMP-2, MMP-3 and ADAMTS4. Fresh tissue contained aggrecan core protein products similar to those previously described for bovine articular cartilage of this age. IL-1 induced the release of aggrecanase-generated CS-substituted products including both high (>250 kDa) and low molecular weight (about 75 kDa) species. TIMP-3 (but not TIMP-1 and -2 or a broad spectrum MMP inhibitor) inhibited IL-1-dependent GAG loss. In addition, IL-1 induced the release of preformed pools of three known G1-bearing products. We conclude that aggrecanases are responsible for IL-1-stimulated GAG release from meniscal explants, and that IL-1 also stimulates release of G1-bearing products, by a process possibly involving hyaluronan fragmentation.
Assuntos
Agrecanas/metabolismo , Artrite/imunologia , Glicosaminoglicanos/metabolismo , Mediadores da Inflamação/metabolismo , Interleucina-1alfa/metabolismo , Meniscos Tibiais/imunologia , Proteínas ADAM/efeitos dos fármacos , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Proteína ADAMTS4 , Agrecanas/efeitos dos fármacos , Animais , Artrite/metabolismo , Artrite/fisiopatologia , Calpaína/efeitos dos fármacos , Calpaína/genética , Calpaína/metabolismo , Bovinos , Endopeptidases/efeitos dos fármacos , Endopeptidases/genética , Endopeptidases/metabolismo , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Ácido Hialurônico/metabolismo , Mediadores da Inflamação/farmacologia , Interleucina-1alfa/farmacologia , Metaloproteinases da Matriz/efeitos dos fármacos , Metaloproteinases da Matriz/genética , Metaloproteinases da Matriz/metabolismo , Meniscos Tibiais/efeitos dos fármacos , Meniscos Tibiais/metabolismo , Modelos Biológicos , Pró-Colágeno N-Endopeptidase/efeitos dos fármacos , Pró-Colágeno N-Endopeptidase/genética , Pró-Colágeno N-Endopeptidase/metabolismo , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Inibidor Tecidual de Metaloproteinase-3/efeitos dos fármacos , Inibidor Tecidual de Metaloproteinase-3/genética , Inibidor Tecidual de Metaloproteinase-3/metabolismoRESUMO
The deposition of aggrecan/hyaluronan (HA)-rich matrix within the tendon body and surrounding peritenon impede tendon healing and result in compromised biomechanical properties. Hence, the development of novel strategies to achieve targeted removal of the aggrecan-HA pericellular matrix may be effective in treating tendinopathy. The current study examined the therapeutic potential of a recombinant human hyaluronidase, rHuPH20 (FDA approved for reducing HA accumulation in tumors) for treating murine Achilles tendinopathy. The 12-week-old C57Bl/6 male mice were injected with two doses of rHuTGF-ß1 into the retrocalcaneal bursa (RCB) to induce a combined bursitis and tendinopathy. Twenty-four hours following induction of injury, treatment groups were administered rHuPH20 Hyaluronidase (rHuPH20; Halozyme Therapeutics) into the RCB. At either 6 h (acute), 9 days, or 25 days following hyaluronidase treatment, Achilles tendons were analyzed for gene expression, histology and immunohistochemistry, fluorophore-assisted carbohydrate electrophoresis, and biomechanical properties. The rHuPH20 treatment was effective, particularly at the acute and 9-day time points, in (a) removing HA deposits from the Achilles tendon and surrounding tissues, (b) improving biomechanical properties of the healing tendon, and (c) eliciting targeted increases in expression of specific cell fate, extracellular matrix metabolism, and inflammatory genes. The potential of rHuPH20 to effectively clear the pro-inflammatory, HA-rich matrix within the RCB and tendon strongly supports the future refinement of injectable glycosidase preparations as potential treatments to protect or regenerate tendon tissue by reducing inflammation and scarring in the presence of bursitis or other inducers of damage such as mechanical overuse. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:59-69, 2020.
Assuntos
Tendão do Calcâneo/patologia , Bursite/tratamento farmacológico , Hialuronoglucosaminidase/uso terapêutico , Tendinopatia/tratamento farmacológico , Animais , Fenômenos Biomecânicos , Proteínas da Matriz Extracelular/metabolismo , Humanos , Hialuronoglucosaminidase/administração & dosagem , Injeções , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Recombinantes/uso terapêutico , Regeneração , Tendinopatia/metabolismo , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
The secreted metalloprotease ADAMTS5 is implicated in destruction of the cartilage proteoglycan aggrecan in arthritis, but its physiological functions are unknown. Its expression profile during embryogenesis and in adult tissues is therefore of considerable interest. beta-Galactosidase (beta-gal) histochemistry, enabled by a LacZ cassette inserted in the Adamts5 locus, and validated by in situ hybridization with an Adamts5 cRNA probe and ADAMTS5 immunohistochemistry, was used to profile Adamts5 expression during mouse embryogenesis and in adult mouse tissues. Embryonic expression was scarce prior to 11.5 days of gestation (E11.5) and noted only in the floor plate of the developing brain at E 9.5. After E11.5 there was continued expression in brain, especially in the choroid plexus, peripheral nerves, dorsal root ganglia, cranial nerve ganglia, spinal and cranial nerves, and neural plexuses of the gut. In addition to nerves, developing limbs have Adamts5 expression in skeletal muscle (from E13.5), tendons (from E16.5), and inter-digital mesenchyme of the developing autopod (E13.5-15.5). In adult tissues, there is constitutive Adamts5 expression in arterial smooth muscle cells, mesothelium lining the peritoneal, pericardial and pleural cavities, smooth muscle cells in bronchi and pancreatic ducts, glomerular mesangial cells in the kidney, dorsal root ganglia, and in Schwann cells of the peripheral and autonomic nervous system. Expression of Adamts5 during neuromuscular development and in smooth muscle cells coincides with the broadly distributed proteoglycan versican, an ADAMTS5 substrate. These observations suggest the major contexts in which developmental and physiological roles could be sought for this protease.
Assuntos
Proteínas ADAM/genética , Embrião de Mamíferos/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Proteínas ADAM/metabolismo , Proteína ADAMTS5 , Animais , Artérias/embriologia , Artérias/crescimento & desenvolvimento , Artérias/metabolismo , Linhagem da Célula/genética , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Feminino , Imunofluorescência , Imuno-Histoquímica , Hibridização In Situ , Mucosa Intestinal/metabolismo , Intestinos/embriologia , Intestinos/crescimento & desenvolvimento , Pulmão/embriologia , Pulmão/crescimento & desenvolvimento , Pulmão/metabolismo , Masculino , Camundongos , Camundongos Knockout , Músculo Esquelético/embriologia , Músculo Esquelético/crescimento & desenvolvimento , Músculo Esquelético/metabolismo , Sistema Nervoso/embriologia , Sistema Nervoso/crescimento & desenvolvimento , Sistema Nervoso/metabolismo , Fatores de Tempo , Versicanas/metabolismoRESUMO
High blood flow through baboon polytetrafluorethylene aorto-iliac grafts increases neointimal vascular smooth muscle cell (SMC) death, neointimal atrophy, and cleavage of versican to generate the DPEAAE neoepitope, a marker of ADAMTS-mediated proteolysis. In this study, we have determined the effect of high blood flow on transcript abundance in the neointima for ADAMTS1, -4, -5, -8, -9, -15, and -20. We found that after 24 hr of flow, the mRNA for ADAMTS4 was significantly increased, whereas that for the other family members was unchanged. Because vascular SMC death is markedly increased in the graft after 24 hr of high flow, we next examined the possibility that the ADAMTS4 induction and the cell death are causally related. The addition of Fas ligand to SMC cultures increased both ADAMTS4 mRNA and cell death approximately 5-fold, consistent with the idea that ADAMTS4-dependent cleavage of versican may be partly responsible for cell death and tissue atrophy under these conditions.
Assuntos
Proteínas ADAM/biossíntese , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Versicanas/metabolismo , Proteínas ADAM/genética , Animais , Aorta Abdominal/metabolismo , Aorta Abdominal/fisiopatologia , Aorta Torácica/metabolismo , Atrofia , Prótese Vascular , Morte Celular , Células Cultivadas , Modelos Animais de Doenças , Proteína Ligante Fas/farmacologia , Humanos , Artéria Ilíaca/fisiopatologia , Masculino , Músculo Liso Vascular/patologia , Músculo Liso Vascular/fisiopatologia , Miócitos de Músculo Liso/patologia , Papio cynocephalus , Politetrafluoretileno , RNA Mensageiro/biossíntese , Fluxo Sanguíneo Regional , Túnica Íntima/metabolismo , Túnica Íntima/patologiaRESUMO
The TTR (transforming growth factor ß1 (TGFß1) injection with treadmill running) model of murine joint injury was used to examine effects of intra-articular Hyaluronan (IA HA) on the metabolism of subchondral bone. HA was injected 24 h after TGFß1 injection and its effects on the mRNA of 80 genes in the Nfkb pathway, and bone remodeling genes, Acp5, Nos2 and Arg1, in femoral and tibial epiphyses/metaphyses of injected and contralateral legs was assessed. Structural bone parameters at those sites were determined by Micro-computed tomography (micro CT) and bone remodeling cells identified with histochemistry for tartrate-resistant acid phosphatase and immunohistochemistry for Nitric oxide synthase 2 (NOS2) and Arginase 1. Gene expression responses in femoral compartments were generally inhibitory and notably biphasic whereas the tibia was relatively non-responsive. Gene expression was also altered in the contralateral femoral compartment but were predominantly activated. IA TGFb did not alter bone structure in the injected leg, but resulted in a statistically significant reduction (25-40%) in trabecular bone of the contralateral limb. IA HA did not affect such changes. This bone loss was associated with an acute decrease in transcript abundance for Acp5, Nos2, Arg1 and this decrease persisted for Nos2 and Arg1. In conclusion, the data illustrate that in this model, IA TGFß1 injection results in marked biphasic changes in NfKb-regulated apoptosis, IL1 and IL12 pathways, which were transiently altered after IA HA therapy. The finding that all modulations are essentially restricted to the femoral compartment is consistent with the predominant localization and clearance of injected HA from this site.
RESUMO
Fibrillar collagens are highly prevalent in the extracellular matrix of all connective tissues and therefore commonly used as a biomaterial in tissue engineering applications. In the native environment, collagen fibers are arranged in a complex hierarchical structure that is often difficult to recreate in a tissue engineered construct. Small leucine rich proteoglycans as well as hyaluronan binding proteoglycans, aggrecan and versican, have been implicated in regulating fiber formation. In this study, we modified proteoglycan production in vitro by altering culture medium glucose concentrations (4500, 1000, 500, 250, and 125â¯mg/L), and evaluated its effect on the formation of collagen fibers inside tissue engineered meniscal constructs. Reduction of extracellular glucose resulted in a dose dependent decrease in total sulfated glycosaminoglycan (GAG) production, but minimal decreases of decorin and biglycan. However, fibromodulin doubled in production between 125 and 4500â¯mg/L glucose concentration. A peak in fiber formation was observed at 500â¯mg/L glucose concentration and corresponded with reductions in total GAG production. Fiber formation reduction at 125 and 250â¯mg/L glucose concentrations are likely due to changes in metabolic activity associated with a limited supply of glucose. These results point to proteoglycan production as a means to manipulate fiber architecture in tissue engineered constructs. STATEMENT OF SIGNIFICANCE: Fibrillar collagens are highly prevalent in the extracellular matrix of all connective tissues; however achieving appropriate assembly and organization of collagen fibers in engineered connective tissues is a persistent challenge. Proteoglycans have been implicated in regulating collagen fiber organization both in vivo and in vitro, however little is known about methods to control proteoglycan production and the subsequent fiber organization in tissue engineered menisci. Here, we show that media glucose content can be optimized to control proteoglycan production and collagen fiber assembly, with optimal collagen fiber assembly occurring at sub-physiologic levels of glucose.
Assuntos
Colágenos Fibrilares/metabolismo , Glucose/farmacologia , Menisco/fisiologia , Proteoglicanas/biossíntese , Engenharia Tecidual/métodos , Animais , Bovinos , Decorina/metabolismo , Fibromodulina/metabolismo , Menisco/efeitos dos fármacos , Alicerces Teciduais/químicaRESUMO
BACKGROUND: The attempted healing of tendon after acute injury (overloading, partial tear or complete rupture) proceeds via the normal wound healing cascade involving hemostasis, inflammation, matrix synthesis and matrix remodeling. Depending on the degree of trauma and the nature of the post-injury milieu, a variable degree of healing and recovery of function occurs. Post-injury analgesia is often achieved with NSAIDs such as Ibuprofen, however there is increasing evidence that NSAID usage may interfere with the healing process. This study aimed to investigate the cellular mechanism by which IBU therapy might lead to a worsening of tendon pathology. METHODS: We have examined the effect of oral Ibuprofen, on Achilles tendon healing in a TGFb1-induced murine tendinopathy model. Dosing was started 3 days after initial injury (acute cellular response phase) and continued for 22 days or started at 9 days after injury (transition to matrix regeneration phase) and given for 16 days. Cellular changes in tendon and surrounding peritenon were assessed using Hematoxylin/Eosin, chondroid accumulation with Safranin O and anti-aggrecan immunohistochemistry, and neo-vessel formation with GSI Lectin histochemistry. Markers of inflammation included histochemical localization of hyaluronan, immunohistochemistry of heavy chain 1 and TNFα-stimulated glycoprotein-6 (TSG6). Cell responses were further examined by RT-qPCR of 84 NFκB target genes and 84 wound healing genes. Biomechanical properties of tendons were evaluated by tensile testing. RESULTS: At a clinically-relevant dosage, Ibuprofen prevented the process of remodeling/removal of the inflammatory matrix components, hyaluronan, HC1 and TSG6. Furthermore, the aberrant matrix remodeling was accompanied by activation at day 28 of genes (Col1a2, Col5a3, Plat, Ccl12, Itga4, Stat3, Vegfa, Mif, Col4a1, Rhoa, Relb, F8, Cxcl9, Lta, Ltb, Ccl12, Cdkn1a, Ccl22, Sele, Cd80), which were not activated at any time without the drug, and so appear most likely to be involved in the pathology. Of these, Vegfa, Col4a1, F8, Cxcl9 and Sele, have been shown to play a role in vascular remodeling, consistent with the appearance at 25 days of vasculogenic cell groups in the peritenon and fat pad stroma surrounding the Achilles of the drug-dosed mice. Tensile stiffness (p = 0.004) and elastic modulus (p = 0.012) were both decreased (relative to age-matched uninjured and non-dosed mice) in mice dosed with Ibuprofen from day 3 to day 25, whether injured or not. CONCLUSION: We conclude that the use of Ibuprofen for pain relief during inflammatory phases of tendinopathy, might interfere with the normal processes of extracellular matrix remodeling and cellular control of expression of inflammatory and wound healing genes. It is proposed that the known COX2-mediated anti-inflammatory effect of ibuprofen has detrimental effects on the turnover of a pro-inflammatory HA matrix produced in response to soft-tissue injury, thus preventing the switch to cellular responses associated with functional matrix remodeling and eventual healing.
RESUMO
Hyaluronan (HA), a high molecular weight non-sulfated glycosaminoglycan, is an integral component of the extracellular matrix of developing and mature connective tissues including tendon. There are few published reports quantifying HA content during tendon growth and maturation, or detailing its effects on the mechanical properties of the tendon extracellular matrix. Therefore, the goal of the current study was to examine the role of HA synthesis during post-natal skeletal growth and maturation, and its influence on tendon structure and biomechanical function. For this purpose, the morphological, biochemical, and mechanical properties of Achilles tendons from wild type (WT) and hyaluronan synthase 1 and 3 deficient mouse strains (Has1-/- (Has1KO), Has3-/- (Has3KO), and Has1-/- 3-/- (Has1/3KO)) were determined at 4, 8, and 12 weeks of age. Overall, HAS-deficient mice did not show any marked differences from WT mice in Achilles tendon morphology or in the HA and chondroitin/dermatan sulfate (CS/DS) contents. However, HAS1-deficiency (in the single or Has1/3 double KO) impeded post-natal formation of the retrocalcaneal bursa, implicating HAS1 in regulating HA metabolism by cells lining the bursal cavity. Together, these data suggest that HA metabolism via HAS1 and HAS3 does not markedly influence the extracellular matrix structure or function of the tendon body, but plays a role in the formation/maintenance of peritendinous bursa. Additional studies are warranted to elucidate the relationship of HA and CS/DS metabolism to tendon healing and repair in vivo. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2622-2632, 2018.
Assuntos
Tendão do Calcâneo/crescimento & desenvolvimento , Bolsa Sinovial/crescimento & desenvolvimento , Calcâneo/crescimento & desenvolvimento , Hialuronan Sintases/fisiologia , Tendão do Calcâneo/anatomia & histologia , Tendão do Calcâneo/enzimologia , Animais , Bolsa Sinovial/enzimologia , Calcâneo/enzimologia , Sulfatos de Condroitina/metabolismo , Colágeno/metabolismo , Dermatan Sulfato/metabolismo , Ácido Hialurônico/metabolismo , Masculino , Camundongos Knockout , Distribuição Aleatória , Proteoglicanos Pequenos Ricos em Leucina/metabolismoRESUMO
The expression and potential functional role of aggrecan in adipogenesis and adipose tissue development was investigated in murine models of obesity. Aggrecan, as well as the two aggrecanases ADAMTS-4 and ADAMTS-5 (A Disintegrin And Metalloproteinase with Thrombospondin motif) mRNAs, are expressed in subcutaneous (SC) and gonadal (GON) adipose tissues of mice. Their presence was confirmed by western blotting using adipose tissue extracts. In mice with nutritionally induced obesity (high fat diet) as well as in lean controls, aggrecan mRNA expression was downregulated whereas ADAMTS-4 and ADAMTS-5 were upregulated with time. In mice with genetically determined obesity (ob/ob), ADAMTS-5 mRNA was upregulated in both SC and GON adipose tissues, as compared to wild-type (WT) mice (p<0.001). Enhanced aggrecanase expression levels in these tissues were associated with significantly elevated levels of G1-NITEGE, a degradation product of aggrecan. Thus, aggrecan levels were high at the early stages of adipose tissue development in mice, whereas its production decreased and its degradation increased during development of obesity. A functional role of aggrecan in promoting early stages of adipogenesis is supported by the findings that it stimulated the in vitro differentiation of 3T3-F442A preadipocytes and the de novo in vivo accumulation of fat in Matrigel plaques injected into WT mice. Proteoglycans in the extracellular matrix of adipose tissue, such as aggrecan, may contribute to the regulation of lipid uptake and obesity in mice.
Assuntos
Tecido Adiposo/crescimento & desenvolvimento , Agrecanas/metabolismo , Diferenciação Celular , Endopeptidases/metabolismo , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Proteína ADAMTS4 , Proteína ADAMTS5 , Tecido Adiposo/citologia , Agrecanas/genética , Animais , Sequência de Bases , Primers do DNA , Endopeptidases/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pró-Colágeno N-Endopeptidase/genética , Pró-Colágeno N-Endopeptidase/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Articular cartilage undergoes matrix degradation and loss of mechanical properties when stimulated with proinflammatory cytokines such as interleukin-1 (IL-1). Aggrecanases and matrix metalloproteinases (MMPs) are thought to be principal downstream effectors of cytokine-induced matrix catabolism, and aggrecanase- or MMP-selective inhibitors reduce or block matrix destruction in several model systems. The objective of this study was to use metalloproteinase inhibitors to perturb IL-1-induced matrix catabolism in bovine cartilage explants and examine their effects on changes in tissue compression and shear properties. Explanted tissue was stimulated with IL-1 for up to 24 days in the absence or presence of inhibitors that were aggrecanase-selective, MMP-selective, or non-selective. Analysis of conditioned media and explant digests revealed that aggrecanase-mediated aggrecanolysis was delayed to varying extents with all inhibitor treatments, but that aggrecan release persisted. Collagen degradation was abrogated by MMP- and non-selective inhibitors and reduced by the aggrecanase inhibitor. The inhibitors delayed but did not reduce loss of the equilibrium compression modulus, whereas the losses of dynamic compression and shear moduli were delayed and reduced. The data suggest that non-metalloproteinase mechanisms participate in IL-1-induced matrix degradation and loss of tissue material properties.
Assuntos
Cartilagem/metabolismo , Endopeptidases/metabolismo , Inibidores Enzimáticos/farmacologia , Interleucina-1/metabolismo , Metaloproteases/antagonistas & inibidores , Agrecanas/química , Agrecanas/metabolismo , Animais , Cartilagem/patologia , Cartilagem Articular/citologia , Bovinos , Relação Dose-Resposta a Droga , Concentração Inibidora 50 , Metaloproteases/metabolismo , Modelos Biológicos , Estresse Mecânico , Fatores de TempoRESUMO
Many studies in arthritis research require an evaluation of the cellular responses within the joint and the ensuing matrix degradation in articular cartilage. The early histochemical/histological scale of Mankin have opened new approaches to evaluating cartilage structure. Histological methods now include in situ hybridization for cell-specific gene expression and immunohistochemistry for the spatial organization of cartilage proteins and their processed forms. This chapter details of a method for immunohistochemical analysis of aggrecan degradation in articular cartilage samples which have been prepared by standard methods of formalin fixation and paraffin embedding. The procedure focuses on the application of antibodies (e.g., anti-ADAMTS4, anti-MT4MMP) which detect some of the proteinases most likely involved, and anti-NITEGE which detects the terminal product of the aggrecanase-mediated cleavage of aggrecan at Glu392-Ala393 (bovine, human, dog, rat, pig, sheep, horse, mouse) or Glu393-Ala394 (chick).
Assuntos
Agrecanas/metabolismo , Cartilagem Articular/metabolismo , Processamento de Imagem Assistida por Computador/métodos , Proteínas ADAM/metabolismo , Proteína ADAMTS4 , Agrecanas/química , Animais , Artrite/metabolismo , Artrite/patologia , Cartilagem Articular/patologia , Bovinos , Endopeptidases/metabolismo , Formaldeído , Humanos , Imuno-Histoquímica/métodos , Metaloproteinases da Matriz Associadas à Membrana/metabolismo , Camundongos , Inclusão em Parafina , Pró-Colágeno N-Endopeptidase/metabolismo , Fixação de TecidosRESUMO
We have used a murine Achilles tendinopathy model to investigate whether tissue changes (such as collagen disorganization, chondroid metaplasia, and loss of tensile properties) which are broadly characteristic of human tendinopathies, are accompanied by changes in the expression of chromatin-modifying enzymes and the methylation status of promoter regions of tendon cell DNA. Tendinopathy was induced by two intra-tendinous TGF-ß1 injections followed by cage activity or treadmill running for up to 28 days. Activation of DNA methyltransferases occurred at 3 days after the TGF-ß1 injections and also at 14 days, but only with treadmill activity. Genome-wide Methyl Mini-Seq™ analysis identified 19 genes with differentially methylated promoters, five of which perform functions with an apparent direct relevance to tendinopathy (Leprel2, Foxf1, Mmp25, Igfbp6, and Peg12). The functions of the genes identified included collagen fiber assembly and pericellular interactions, therefore their perturbation could play a role in the characteristic disorganization of fibers in affected tendons. We postulate that a study of the functional genomics of these genes in animal and human tendon could further delineate the pathogenesis of this multi-factorial complex disease. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:947-955, 2017.
Assuntos
Metilação de DNA , Tendinopatia/metabolismo , Tendão do Calcâneo/patologia , Animais , Proteínas de Transporte/genética , Modelos Animais de Doenças , Fatores de Transcrição Forkhead/genética , Proteínas Ligadas por GPI/genética , Expressão Gênica , Estudo de Associação Genômica Ampla , Masculino , Metaloproteinases da Matriz Associadas à Membrana/genética , Camundongos Endogâmicos C57BL , Proteínas de Neoplasias/genética , Pró-Colágeno-Prolina Dioxigenase/genética , Regiões Promotoras Genéticas , Tendinopatia/patologiaRESUMO
Hutchinson-Gilford Progeria syndrome (HGPS) is a rare genetic disorder that displays features of segmental aging. It is manifested predominantly in connective tissue, with most prominent histological changes occurring in the skin, cartilage, bone and cardiovascular tissues. Detailed quantitative real time reverse-transcription polymerase chain reaction studies confirmed the previous observation that platelet-derived growth factor A-chain transcripts are consistently elevated 11+/-2- to 13+/-2-fold in two HGPS dermal fibroblast lines compared with age-matched controls. Furthermore, we identified two additional genes with substantially altered transcript levels. Nucleotide pyrophosphatase transcription was virtually shut down with decreased expression of 13+/-3- to 59+/-3-fold in HGPS, whereas aggrecan mRNA was elevated to 24+/-5 times to 41+/-4 times that of chronologically age-matched controls. Aggrecan, normally a component of cartilage and not always detectable in normal fibroblasts cultures, was secreted by HGPS fibroblast lines and was produced as a proteoglycan. This demonstrates that elevated aggrecan expression and its secretion are aberrant features of HGPS. We conclude that HGPS cells can display massively altered transcript levels leading to the secretion of inappropriate protein species.
Assuntos
Proteoglicanas de Sulfatos de Condroitina/genética , Proteínas da Matriz Extracelular/genética , Fibroblastos/fisiologia , Lectinas Tipo C/genética , Progéria/fisiopatologia , Pele/citologia , Actinas/genética , Agrecanas , Linhagem Celular , Criança , Pré-Escolar , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Humanos , Análise por Pareamento , Fator de Crescimento Derivado de Plaquetas/genética , Progéria/genética , Pirofosfatases/genética , RNA Mensageiro/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para CimaRESUMO
Equine degenerative suspensory ligament desmitis (DSLD) in Peruvian Paso horses typically presents at 7-15 years and is characterized by lameness, focal disorganization of collagen fibrils, and chondroid deposition in the body of the ligament. With the aim of developing a test for disease risk (that can be used to screen horses before breeding) we have quantified the expression of 76 TGFß-signaling target genes in adipose-derived stromal fibroblasts (ADSCs) from six DSLD-affected and five unaffected Paso horses. Remarkably, 35 of the genes showed lower expression (p<0.05) in cells from DSLD-affected animals and this differential was largely eliminated by addition of exogenous TGFß1. Moreover, TGFß1-mediated effects on expression were prevented by the TGFßR1/2 inhibitor LY2109761, showing that the signaling was via a TGFßR1/2 complex. The genes affected by the pathology indicate that it is associated with a generalized metabolic disturbance, since some of those most markedly altered in DSLD cells (ATF3, MAPK14, ACVRL1 (ALK1), SMAD6, FOS, CREBBP, NFKBIA, and TGFBR2) represent master-regulators in a wide range of cellular metabolic responses.
Assuntos
Tecido Adiposo/patologia , Fibroblastos/patologia , Regulação da Expressão Gênica , Doenças dos Cavalos/patologia , Ligamentos , Células Estromais/patologia , Fator de Crescimento Transformador beta/metabolismo , Animais , Cromatina/metabolismo , Fibroblastos/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Cavalos , Masculino , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Pirazóis/farmacologia , Pirróis/farmacologia , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
The composition of extracellular matrix during growth and regression of the neointima was analyzed during healing in a baboon aorto-iliac polytetrafluoroethylene graft. Graft neointimal thickening can be modulated by altering blood flow by construction of downstream arteriovenous fistulas. Normal flow with normal shear stress induces neointimal thickening, whereas high flow with high shear stress upstream of a fistula induces regression of established neointima. The neointima formed under normal shear stress is enriched in hyaluronan and proteoglycans, particularly versican. On the other hand, the neointima near the graft material is enriched in collagen and biglycan. Neointimal regression in response to high shear stress is associated with a loss of proteoglycans as detected by histochemical staining. Immunostaining with an antibody against an ADAMTS cleavage neoepitope of versican increases after switching to high flow, although immunostaining for versican core protein is not appreciably changed by high flow. The present data demonstrate that the graft neointima is enriched with proteoglycans, particularly versican and hyaluronan, as well as collagen, and there is a differential distribution of each. Neointimal atrophy occurs with an apparent loss of proteoglycans and evidence of versican degradation.