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1.
Clin Exp Immunol ; 161(3): 459-70, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20646000

RESUMO

Anti-endothelial cell antibodies (AECA) have been frequently detected in systemic vasculitis, which affects blood vessels of various sizes. To understand the pathogenic roles of AECA in systemic vasculitis, we attempted to identify target antigens for AECA comprehensively by a proteomic approach. Proteins extracted from human umbilical vein endothelial cells (HUVEC) were separated by two-dimensional electrophoresis, and Western blotting was subsequently conducted using sera from patients with systemic vasculitis. As a result, 53 autoantigenic protein spots for AECA were detected, nine of which were identified by mass spectrometry. One of the identified proteins was peroxiredoxin 2 (Prx2), an anti-oxidant enzyme. Frequency of anti-Prx2 autoantibodies, measured by enzyme-linked immunosorbent assay (ELISA), was significantly higher in systemic vasculitis (60%) compared to those in collagen diseases without clinical vasculitis (7%, P < 0·01) and healthy individuals (0%, P < 0·01). Further, the titres changed in parallel with the disease activity during time-courses. The presence of anti-Prx2 autoantibodies correlated significantly with elevation of serum d-dimers and thrombin-antithrombin complex (P < 0·05). Immunocytochemical analysis revealed that live endothelial cells expressed Prx2 on their surface. Interestingly, stimulation of HUVEC with rabbit anti-Prx2 antibodies increased secretion of interleukin (IL)-6, IL-1ß, IL-1ra, growth regulated oncogene (GRO)-α, granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), IL-8 and monocyte chemoattractant protein (MCP)-1 more than twofold compared to that of with rabbit immunoglobulin (Ig)G. Taken together, our data suggest that anti-Prx2 autoantibodies would be a useful marker for systemic vasculitis and would be involved in the inflammatory processes of systemic vasculitis.


Assuntos
Autoanticorpos/imunologia , Autoantígenos/imunologia , Peroxirredoxinas/imunologia , Vasculite/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Autoanticorpos/sangue , Autoanticorpos/farmacologia , Western Blotting , Linhagem Celular , Células Cultivadas , Quimiocinas/metabolismo , Citocinas/metabolismo , Eletroforese em Gel Bidimensional , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/imunologia , Células Endoteliais/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Células HeLa , Humanos , Masculino , Pessoa de Meia-Idade , Peroxirredoxinas/metabolismo , Proteínas/imunologia , Proteínas/metabolismo , Vasculite/sangue , Vasculite/metabolismo , Adulto Jovem
2.
Clin Exp Rheumatol ; 27(2): 347-53, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19473582

RESUMO

Osteoarthritis (OA) is considered to be linked to obesity and body fat mass. Recent investigations, however, are aimed at clarifying the roles of adipose tissue-derived proteins and a wide variety of lipid mediators, including fatty acids, sphingolipids, and eicosanoids, in cartilage degradation in OA, in addition to the effects body weight itself. Here, we review recent progress in studies of OA, focusing on the potential role of lipid mediators in articular cartilage and introducing the concept that "OA is a metabolic disease" in which lipids essentially contribute to the pathophysiology of cartilage degradation.


Assuntos
Condrócitos/metabolismo , Metabolismo dos Lipídeos , Osteoartrite/metabolismo , Adipocinas/fisiologia , Animais , Modelos Animais de Doenças , Humanos , Síndrome Metabólica/fisiopatologia , Camundongos , Obesidade/fisiopatologia , Osteoartrite/etiologia
3.
Clin Exp Rheumatol ; 26(4): 589-95, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18799089

RESUMO

OBJECTIVES: Pannus is invasive granulation tissue found on the articular cartilage having rheumatoid arthritis (RA). However, pannus-like tissue has also been found in osteoarthritis (OA). Our previous study showed that pannus-like tissue in OA (OA pannus) was frequently found in human OA samples. The purpose of the study is to investigate the development and the characteristics of OA pannus in a rat OA model. DESIGN: Ligaments of the knee joint were transected in Wister rats to induce OA. The knee joints were removed at weeks 1, 2, 4 and 6, and subjected to histological study. Samples were stained with hematoxylin and eosin (HE), Safranin-O and immuno-stained for vimentin, CD34, type II collagen and MMP-3. The whole knee joint of OA rats was implanted in SCID mice and kept for a further 3 weeks. Then the histological findings were evaluated in HE sections. RESULT: OA pannus appeared at week 2 and extend over the articular surface. OA pannus cells were positive for vimentin and/or CD34. At week 6, a part of articular surface was restored with matrix. OA pannus cells expressed MMP-3 as well as type II collagen. Histological study of rat OA knees implanted in SCID mice showed that OA pannus cells filled the joint space and invaded articular cartilage. CONCLUSIONS: The presence of OA pannus was found in a rat OA model and its features were similar to those in human OA. OA pannus had both catabolic and reparative features, and the latter feature were speculated to be dominant in the later phase of the disease under a certain environmental condition.


Assuntos
Cartilagem Articular/patologia , Meniscos Tibiais/patologia , Osteoartrite do Joelho/patologia , Tíbia/patologia , Animais , Antígenos CD34 , Cartilagem Articular/metabolismo , Colágeno Tipo II/metabolismo , Modelos Animais de Doenças , Feminino , Metaloproteinase 3 da Matriz/metabolismo , Camundongos , Camundongos SCID , Osteoartrite do Joelho/metabolismo , Ratos , Ratos Wistar , Vimentina/metabolismo
4.
Osteoarthritis Cartilage ; 16(3): 279-86, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17945514

RESUMO

Although adult human cartilage is physiologically avascular tissue, angiogenesis can be observed during the process of endochondral bone development. Inflammation in articular joints can also lead to neovascularization in cartilage. In such conditions, the expression of angiogenic factors, such as vascular endothelial growth factor (VEGF), has been shown to play a key role, controlling not only angiogenesis but also chondrocyte metabolism. Here we review recent research findings concerning the potential role of VEGF in cartilage, focusing in particular on its possible involvement in the pathogenesis of osteoarthritis.


Assuntos
Cartilagem Articular/metabolismo , Condrócitos/citologia , Neovascularização Fisiológica/fisiologia , Osteoartrite/patologia , Fator A de Crescimento do Endotélio Vascular/fisiologia , Adulto , Apoptose/fisiologia , Desenvolvimento Ósseo/fisiologia , Cartilagem Articular/irrigação sanguínea , Sobrevivência Celular/fisiologia , Condrócitos/química , Condrócitos/metabolismo , Humanos , Hipóxia/patologia , Fator 1 Induzível por Hipóxia/fisiologia , Recém-Nascido , Osteoartrite/metabolismo , Osteófito/patologia , Estresse Mecânico
5.
Clin Exp Rheumatol ; 25(1): 11-6, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17417984

RESUMO

OBJECTIVE: The purpose of this study was to examine the effects of a selective cyclooxigenase-2 (COX-2) inhibitor (celecoxib) comparing diclofenac. METHODS: Using chondrocytes derived from cartilage of non-arthritic (NA) subjects or patients with osteoarthritis (OA) or rheumatoid arthritis (RA), we examined the effects of celecoxib on incorporation of 3H-thymidine and 35S-sulfate, apoptosis, and production of matrix metalloproteinase (MMP)-1, MMP-3, MMP-13, and regulated upon activation, normal T cell expressed and secreted (RANTES), macrophage inflammatory protein (MIP)-1a and nitric oxide (NO). RESULTS: Celecoxib and diclofenac tended to reduce 3H-tymidine incorporation of chondrocytes. Celecoxib induced apoptosis in a dose-dependent manner, but to a lesser degree than diclofenac. Celecoxib inhibited proteoglycan synthesis (indicated by 35S-sulfate incorporation) in NA chondrocytes, but not in OA and RA chondrocytes. Celecoxib increased interleukin-1 (IL-1)-induced production of RANTES and MIP-1alpha by chondrocytes and decreased IL-1-induced NO production by chondrocytes, whereas it did not affect MMP production. CONCLUSION: Celecoxib had both beneficial and adverse effects on chondrocytes. RA, OA and NA chondrocytes showed different responses. Interestingly, celecoxib enhanced the production of chemokines.


Assuntos
Artrite Reumatoide/tratamento farmacológico , Quimiocinas/metabolismo , Condrócitos/efeitos dos fármacos , Inibidores de Ciclo-Oxigenase 2/farmacologia , Osteoartrite/tratamento farmacológico , Pirazóis/farmacologia , Sulfonamidas/farmacologia , Idoso , Idoso de 80 Anos ou mais , Anti-Inflamatórios não Esteroides/farmacologia , Celecoxib , Células Cultivadas , Quimiocina CCL3 , Quimiocina CCL5/metabolismo , Quimiocinas CC/metabolismo , Diclofenaco/farmacologia , Humanos , Metaloproteinases da Matriz Secretadas/efeitos dos fármacos , Óxido Nítrico/metabolismo
6.
Osteoarthritis Cartilage ; 15(6): 673-81, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17336549

RESUMO

OBJECTIVES: To evaluate osteoarthritis (OA) of the knee using positron emission tomography (PET) with 2-(18)F-fluoro-2-deoxy-D-glucose ((18)F-FDG) as a tracer. MATERIALS AND METHODS: Fifteen patients with medial-type knee OA and three healthy subjects were enrolled in the study. After clinical examination and conventional radiography, (18)F-FDG PET and magnetic resonance imaging (MRI) were performed. (18)F-FDG uptake was quantified as a standardized uptake value (SUV) and the localization of (18)F-FDG uptake was identified using fusion images created with MRI scans. RESULTS: (18)F-FDG generally accumulated in periarticular lesions and was absent in the articular cartilage. SUVs of the whole knee were higher in OA than in controls, and those in the medial condyle were higher than in the lateral condyle in OA. Prominent (18)F-FDG uptake was found in the intercondylar notch in OA and extended along the posterior cruciate ligament (PCL) in some cases. Periosteophytic accumulation was found in one-half of cases with definite osteophytes. Accumulation was also found in subchondral lesions and bone marrow, which corresponded with bone edema diagnosed by MRI. No significant correlation was found between SUV and clinical manifestations. CONCLUSIONS: (18)F-FDG uptake was upregulated in OA and generally accumulated in periarticular lesions. Increased uptake was found in the intercondylar notch extending along the PCL, periosteophytic lesions, and bone marrow. These results provide in vivo pathognomonic insights into OA.


Assuntos
Fluordesoxiglucose F18 , Articulação do Joelho/patologia , Imageamento por Ressonância Magnética/métodos , Osteoartrite do Joelho/diagnóstico , Tomografia por Emissão de Pósitrons/métodos , Humanos , Articulação do Joelho/diagnóstico por imagem , Osteoartrite do Joelho/patologia
7.
Clin Exp Rheumatol ; 25(6): 860-5, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-18173920

RESUMO

OBJECTIVE: A contribution of mast cells and its mediators in the pathogenesis of arthritis has been postulated. We aimed to clarify the role of mast cell-derived serine protease tryptase and proteinase activated receptor (PAR)-2-mediated signaling in chondrocytes. METHODS: Human articular cartilage specimens were obtained from patients with osteoarthritis (OA), rheumatoid arthritis (RA) and with traumatic fracture without arthritis (PT; as controls) who underwent joint surgery. Isolated chondrocytes were cultured in vitro by monolayer, and confluent cells were incubated with recombinant human lung Beta tryptase or with a PAR-2 agonist peptide. The secreted level of vascular endothelial growth factor (VEGF) in culture supernatant was measured using commercially available ELISA kits, and expression of VEGF mRNA was analyzed using real-time PCR. RESULTS: The tryptase-stimulated chondrocytes from OA or RA, but not from PT patients, produced significantly higher amount of VEGF in their supernatants. The response was blocked by a G-protein receptor inhibitor pertussis toxin, however, was not reproduced by incubation of cells with the PAR-2 agonist, suggesting a presence of non-PAR-2 dependent signals for the VEGF induction. In addition, actinomycin D and cycloheximide did not exert significant inhibition, indicating a regulation of VEGF release by tryptase. CONCLUSION: The inflammatory mediator, mast cell-derived protease tryptase may modulate chondrocyte metabolism through induction of VEGF release.


Assuntos
Condrócitos/efeitos dos fármacos , Osteoartrite/metabolismo , Triptases/farmacologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Artrite Reumatoide/metabolismo , Células Cultivadas , Condrócitos/metabolismo , Cicloeximida/farmacologia , Dactinomicina/farmacologia , Feminino , Fraturas Ósseas/metabolismo , Humanos , Masculino , Toxina Pertussis/farmacologia , Reação em Cadeia da Polimerase , Receptor PAR-2/agonistas
8.
Osteoarthritis Cartilage ; 14(11): 1163-73, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16757188

RESUMO

OBJECTIVE: To investigate the modulation of expression of proteinase-activated receptor-2 (PAR-2) in articular chondrocytes by inflammatory cytokines. DESIGN: Articular synovium and cartilage tissues were collected from eight patients with osteoarthritis (OA), and three patients without arthropathy ("normal"). Chondrocytes were stimulated with interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha or transforming growth factor (TGF)-beta1. The expression of PAR-2 was detected using reverse transcriptase-polymerase chain reaction (PCR), Western blotting and immunofluorescence. Quantitative PCR was performed to assess the expression levels of PAR-2 messenger RNA (mRNA). RESULTS: The expression of PAR-2 mRNA was demonstrated in both OA and normal chondrocytes as well as in synovial fibroblasts. However, the level of PAR-2 in OA chondrocytes was much higher than in normal chondrocytes. Long-term culture revealed that PAR-2 mRNA expression was maintained up to three passages in OA but not in normal chondrocytes. IL-1beta and TNF-alpha both upregulated PAR-2 expression in normal and OA chondrocytes. In contrast, TGF-beta1 significantly decreased expression of PAR-2 in OA chondrocytes but increased PAR-2 in normal chondrocytes. CONCLUSIONS: Overexpression of PAR-2 in OA chondrocytes is upregulated by proinflammatory cytokines IL-1beta and TNF-alpha, and down-regulated by regulatory cytokine TGF-beta1. PAR-2 may be involved in the pathogenesis of OA.


Assuntos
Cartilagem Articular/imunologia , Condrócitos/imunologia , Citocinas/imunologia , Osteoartrite/imunologia , Receptor PAR-2/análise , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular , Membrana Celular/imunologia , Células Cultivadas , Citoplasma/imunologia , Relação Dose-Resposta Imunológica , Feminino , Fibroblastos/imunologia , Humanos , Interleucina-1beta/imunologia , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/análise , Membrana Sinovial/imunologia , Fatores de Tempo , Fator de Crescimento Transformador beta/imunologia , Fator de Necrose Tumoral alfa/imunologia , Regulação para Cima/imunologia
9.
Osteoarthritis Cartilage ; 13(8): 679-87, 2005 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15961327

RESUMO

OBJECTIVE: The vascular invasion of bone marrow tissue into the subchondral plate is often observed in articular cartilage and we named it the subchondral bone absorption pit; however, its implication in the pathogenesis of osteoarthritis (OA) has been poorly understood. The purpose of this study was to evaluate its characteristics and roles in osteoarthritic conditions. METHODS: Articular cartilage specimens from 11 patients with medial type knee OA and 7 non-arthritic cadavers were analyzed with HE staining. OA sections were stained with safranin-O, TRAP (tartrate resistant acid phosphatase) and immunostained with anti-MMP-1, MMP-3, MMP-13, vitronectin receptor (VNR)-alpha chain, vimentin and bone morphogenic protein (BMP) 2/4 antibodies. RESULTS: Subchondral bone resorption pits were classified according to the extent of invasion: pits with bone marrow tissue were located within uncalcified cartilage below the tidemark in grade I and invaded beyond the tidemark in grade II, while no invasion was seen in grade 0. Grade II pits were dominant in OA compared to non-arthritic joints, especially medial condyles. Proteoglycan detected with safranin-O staining was lost around the tip of grade II pits and the density of pits was related to the modified Mankin Score. Cells in pits expressed vimentin, MMP-1, MMP-3 and MMP-13. Some polynuclear cells co-expressed VNR-alpha chain and MMP-13, whereas pits showed reparative features expressing BMP. CONCLUSION: These results suggest that subchondral bone resorption pits contribute to cartilage degradation by expressing matrix metalloproteinases in OA.


Assuntos
Células da Medula Óssea/enzimologia , Reabsorção Óssea/fisiopatologia , Metaloproteinases da Matriz/análise , Osteoartrite do Joelho/fisiopatologia , Idoso , Proteínas Morfogenéticas Ósseas/análise , Remodelação Óssea/fisiologia , Reabsorção Óssea/metabolismo , Cadáver , Cartilagem Articular/irrigação sanguínea , Cartilagem Articular/metabolismo , Cartilagem Articular/fisiopatologia , Colagenases/análise , Humanos , Imuno-Histoquímica/métodos , Metaloproteinase 1 da Matriz/análise , Metaloproteinase 13 da Matriz , Metaloproteinase 3 da Matriz/análise , Neovascularização Patológica/fisiopatologia , Osteoartrite do Joelho/metabolismo , Proteoglicanas/metabolismo , Vimentina/análise , Vitronectina/análise
10.
Ann Rheum Dis ; 64(5): 735-42, 2005 May.
Artigo em Inglês | MEDLINE | ID: mdl-15834055

RESUMO

OBJECTIVE: To clarify the effect of interleukin (IL) 18 on cartilage degeneration by studying the profile of IL18 receptor (IL18R) on chondrocytes and the direct effect of IL18 on production of matrix metalloproteinases (MMPs), aggrecanases, and tissue inhibitors of metalloproteinases (TIMPs) in articular chondrocytes. METHODS: Monolayer cultured human articular chondrocytes were isolated from non-arthritic subjects and patients with rheumatoid arthritis or osteoarthritis. Gene expression of IL18, IL18Ralpha, IL18Rbeta, MMPs, and aggrecanases was detected by RT-PCR. Protein levels of IL18Ralpha were analysed by flow cytometry. Protein levels of IL18, MMPs, and TIMPs were measured by ELISA. Aggrecanase-2 mRNA expression was quantitatively analysed by real time RT-PCR. Protein levels of signalling molecules were assayed by western blotting. RESULTS: IL18 mRNA was constitutively expressed in chondrocytes, and was enhanced by IL1beta stimulation. Flow cytometric analysis showed that IL1beta, tumour necrosis factor alpha, and IL18 up regulated IL18Ralpha expression levels. The level of IL18Rbeta mRNA was much lower than that of IL18Ralpha, and was slightly up regulated by IL1beta. In chondrocytes responding to IL18, IL18 (1-100 ng/ml) slightly increased the production of MMP-1, MMP-3, and MMP-13, which was blocked by NF-kappaB inhibitor and p38 mitogen activated protein kinase inhibitor. IL18 up regulated mRNA expression of aggrecanase-2, but not aggrecanase-1. IL18 also slightly stimulated TIMP-1 production?through extracellular signal regulated kinase activation. CONCLUSION: IL18 induces production of MMPs from chondrocytes in inflammatory arthritis. Although the direct effect of IL18 on chondrocytes may not be pivotal for the induction of cartilage degeneration, IL18 seems to play some part in the degradation of articular cartilage in arthritis.


Assuntos
Artrite Reumatoide/enzimologia , Cartilagem Articular/enzimologia , Condrócitos/enzimologia , Interleucina-18/farmacologia , Metaloproteinases da Matriz/biossíntese , Osteoartrite/enzimologia , Proteínas ADAM , Proteína ADAMTS5 , Adulto , Idoso , Idoso de 80 Anos ou mais , Artrite/enzimologia , Artrite/metabolismo , Artrite/patologia , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Cartilagem Articular/patologia , Células Cultivadas , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Ensaio de Imunoadsorção Enzimática/métodos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-18/biossíntese , Interleucina-18/genética , Metaloendopeptidases/biossíntese , Metaloendopeptidases/genética , Pessoa de Meia-Idade , Osteoartrite/metabolismo , Osteoartrite/patologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Transdução de Sinais , Inibidor Tecidual de Metaloproteinase-1/biossíntese
11.
Ann Rheum Dis ; 63(11): 1379-86, 2004 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-15479886

RESUMO

OBJECTIVE: To determine whether IL18 has any indirect effects on osteoclastogenesis mediated by T cells in RA synovium, and compare its effects with those of IL1 beta and TNF alpha. METHODS: Resting T cells were isolated from peripheral blood of healthy donors, and stimulated with 2 microg/ml phytohaemagglutinin (PHA) and 0.5 ng/ml IL2 for 24 hours. Synovial T cells were isolated from RA synovial tissue. The levels of soluble receptor activator of the NF-kappa B ligand (RANKL), osteoprotegerin (OPG), IFN gamma, M-CSF, and GM-CSF were determined by ELISA. Membrane bound RANKL expression was analysed by flow cytometry. Commercially available human osteoclast precursors were cocultured with T cells to induce osteoclast formation, which was determined with tartrate resistant acid phosphatase staining and pit formation assay. RESULTS: In PHA prestimulated T cells or RA synovial T cells, IL18, IL1 beta, or TNFalpha increased soluble RANKL production and membrane bound RANKL expression in a dose dependent manner. IL18, IL1 beta, and TNF alpha did not induce M-CSF, GM-CSF, IFN gamma, or OPG production in PHA prestimulated T cells or RA synovial T cells. IL18 increased the number of osteoclasts and bone resorption area on dentine slices in the coculture of human osteoclast precursors with PHA prestimulated T cells or RA synovial T cells; its ability was equivalent to that of IL1 beta, but less potent than that of TNF alpha. In the coculture system, OPG completely blocked osteoclast induction by IL18 or IL1 beta, and greatly inhibited induction by TNF alpha. CONCLUSION: IL18, IL1 beta, or TNF alpha can indirectly stimulate osteoclast formation through up regulation of RANKL production from T cells in RA synovitis; IL18 is as effective as IL1 beta, but less potent than TNF alpha.


Assuntos
Artrite Reumatoide/imunologia , Interleucina-18/farmacologia , Osteoclastos/patologia , Membrana Sinovial/imunologia , Linfócitos T/imunologia , Análise de Variância , Artrite Reumatoide/patologia , Reabsorção Óssea/patologia , Proteínas de Transporte/análise , Células Cultivadas , Citometria de Fluxo , Glicoproteínas/análise , Fator Estimulador de Colônias de Granulócitos e Macrófagos/análise , Humanos , Interferon gama/análise , Interleucina-1/farmacologia , Ativação Linfocitária , Fator Estimulador de Colônias de Macrófagos/análise , Glicoproteínas de Membrana/análise , Mitógenos/farmacologia , Osteoclastos/efeitos dos fármacos , Osteoprotegerina , Fito-Hemaglutininas/farmacologia , Ligante RANK , Receptor Ativador de Fator Nuclear kappa-B , Receptores Citoplasmáticos e Nucleares/análise , Receptores do Fator de Necrose Tumoral , Linfócitos T/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia
12.
Gene Ther ; 11(11): 909-15, 2004 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15057262

RESUMO

Previously, we showed that human osteoblasts expressing the human telomerase reverse transcriptase (hTERT) gene exhibited specific survival advantages--the result of breaching the replicative senescence barrier and maintaining the phenotypic and functional properties of primary osteoblasts in vitro over the total replicative capacity of primary osteoblasts. We postulated that rejuvenated osteoblasts may have a potential to correct bone loss or osteopenia in age-related osteoporotic diseases. In the present study, we studied whether telomerized presenescent osteoblasts prevent bone mass loss in vivo. After obtaining the informed consent from a patient with osteoarthritis who underwent the arthroplastic knee surgery, osteoblastic cells were isolated from donor bone sample. We transfected the gene encoding hTERT into human osteoblastic cells. Human bone fragments from a donor were incubated with human hTERT-transfected presenescent (in vitro aged) osteoblasts or mock-transfected presenescent osteoblasts in culture medium containing Matrigel. We subcutaneously implanted human bone fragments with telomerized presenescent osteoblasts or primary presenescent osteoblasts as three-dimensional Matrigel xenografts in severe combined immunodeficiency (SCID) mice (each group: six mice) and analyzed the grafts at 6 weeks after implantation. We also determined whether telomerized osteoblasts affect the bone-forming capacity in vivo, using a well-established mouse transplantation model in which ceramic hydroxyapatite/tricalcium phosphate particles are used as carrier vehicle. Telomerized presenescent osteoblasts were rejuvenated, and maintained the functional properties of young osteoblasts in vitro. Bone mineral content (BMC) and bone mineral density (BMD) were measured by ash weight and dual-energy X-ray absorptiometry, respectively. Whereas BMC and BMD of human bone fragments, which were inoculated with aged osteoblasts in SCID mice, decreased with time, telomerized presenescent osteoblasts maintained the BMC and BMD of human bone fragments, indicating that telomerized and rejuvenated osteoblasts may be functional to prevent bone mass loss in vivo. In xenogenic transplants, telomerized osteoblasts generated more bone tissue with lamellar bone structure and cellular components, than did control osteoblasts. These findings suggest that telomerized/rejuvenated presenescent osteoblasts may be used in the development of tissue engineering or cell-based therapy for bone regeneration and repair.


Assuntos
Regeneração Óssea/genética , Terapia Genética/métodos , Osteoblastos/ultraestrutura , Osteoporose/terapia , Telômero/transplante , Animais , Densidade Óssea , Sobrevivência Celular , Senescência Celular , Proteínas de Ligação a DNA , Humanos , Camundongos , Camundongos SCID , Osteoblastos/transplante , Osteoporose/patologia , Osteoporose/fisiopatologia , Telomerase/análise , Telomerase/genética , Telômero/ultraestrutura , Transplante Heterólogo
13.
Rheumatology (Oxford) ; 41(3): 329-37, 2002 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11934972

RESUMO

OBJECTIVE: In order to elucidate which cytokine preferentially stimulates the synovium in patients with rheumatoid arthritis (RA), we investigated the roles of tumour necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6) using SCID mice engrafted with human RA tissue (SCID-HuRAg). METHODS: The SCID-HuRAg mice were prepared according to our previously described method. First, SCID-HuRAg mice were treated with chimeric anti-TNF-alpha monoclonal antibody (mAb, 100 microg/mouse) and histological changes were examined 4 weeks after the initial treatment. Secondly, a total of 100 microg of recombinant TNF-alpha or IL-6 (0.6 microg/h) was administered daily to mice using an osmium pump. The histological changes and serum cytokine levels were examined 4 weeks after the initial administration. Human immunoglobulin G (IgG) was administered to mice as a control. RESULTS: Synovial inflammatory cells were significantly decreased after the anti-TNF-alpha mAb treatment; conversely, the degree of synovial inflammation was significantly exacerbated by TNF-alpha administration. The levels of both IL-6 and TNF-alpha in sera were significantly increased by recombinant TNF-alpha administration, while TNF-alpha levels were unchanged by IL-6 administration. This suggests that TNF-alpha controls IL-6 production. Despite the profound changes in inflammation, we found no effects on bone and no articular cartilage damage was produced by TNF-alpha. CONCLUSION: This study provides strong evidence that TNF-alpha is a key molecule in the control of the inflammatory changes that occur in the RA synovium. In addition, TNF-alpha regulates IL-6 production. However, other inflammatory pathways independent of TNF-alpha may contribute to the bone and cartilage damage seen in RA.


Assuntos
Artrite Reumatoide/metabolismo , Membrana Sinovial/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Artrite Reumatoide/patologia , Células Cultivadas , Quimera , Meios de Cultivo Condicionados/metabolismo , Modelos Animais de Doenças , Interleucina-6/metabolismo , Interleucina-6/farmacologia , Masculino , Camundongos , Camundongos SCID , Proteínas Recombinantes/uso terapêutico , Organismos Livres de Patógenos Específicos , Membrana Sinovial/efeitos dos fármacos , Membrana Sinovial/transplante , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/farmacologia
14.
Clin Exp Rheumatol ; 20(6): 773-81, 2002.
Artigo em Inglês | MEDLINE | ID: mdl-12508768

RESUMO

OBJECT: In order to examine the mechanisms involved in steroid-induced arthropathy after intra-articular corticosteroid injection, a histological examination was performed in vivo using severe combined immunodeficiency (SCID) mice that were implanted with human articular cartilage into the back (SCID/hu model). In addition, the effect of corticosteroids on chondrocyte apoptosis was evaluated in vitro using cultured human chondrocytes. METHOD: Human articular cartilage was obtained during knee surgery and implanted subcutaneously into the backs of SCID mice. One month later, weekly injections of corticosteroid (hydrocortisone acatate: 1 mg/0.2 ml, triamcinolone acetonide: 0.2 mg/0.2 ml, dexamethasone acetate: 0.1 mg/0.2 ml) in the subcutaneous cavity around the grafted cartilage in SCID mice were initiated. After six weeks of treatment, the grafted cartilage pieces were removed from the SCID mice and examined histologically. Chondrocyte apoptosis after corticosteroid treatment was also investigated using cultured human chondrocytes. RESULT: In the corticosteroid treated, grafted articular cartilage, apoptotic chondrocytes were apparent in the superficial and middle layers of cartilage. But a reduced intensity of Safranin O staining was not remarkable. In the cultured chondrocytes, apoptotic changes were also observed after corticosteroid treatment. CONCLUSION: Corticosteroid treatment induces chondrocyte apoptosis and it may be important to understand the steroid-induced arthropathy.


Assuntos
Apoptose/efeitos dos fármacos , Cartilagem Articular/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Glucocorticoides/farmacologia , Animais , Cartilagem Articular/patologia , Cartilagem Articular/transplante , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/ultraestrutura , Células Cultivadas , Condrócitos/patologia , Condrócitos/transplante , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Citometria de Fluxo , Glucocorticoides/efeitos adversos , Humanos , Marcação In Situ das Extremidades Cortadas , Masculino , Camundongos , Camundongos SCID , Organelas/efeitos dos fármacos , Organelas/ultraestrutura , Osteoartrite do Joelho/complicações , Osteoartrite do Joelho/tratamento farmacológico , Osteoartrite do Joelho/patologia
15.
J Rheumatol ; 28(8): 1800-8, 2001 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-11508582

RESUMO

OBJECTIVE: To clarify the pharmacological action of methotrexate (MTX) on the synovium of patients with rheumatoid arthritis (RA) using severe combined immunodeficient (SCID) mice in which human RA synovial tissue had been grafted (SCID-HuRAg). METHODS: One month after engraftment of human RA tissue into SCID mice, MTX (0.3 mg/kg) was administered orally, then the appearance of apoptosis in the grafted tissue was examined by TdT mediated dUTP nick end labeling (TUNEL) staining and electron microscopy at various time points after MTX administration. In cultured synovial cells, synovial apoptotic changes after MTX treatment were studied by agarose gel electrophoresis and flow cytometric analysis. To compare the histological changes induced by MTX with those induced by other disease modifying antirheumatic drugs (DMARD) and a nonsteroidal antiinflammatory drug, histological examination of the grafted synovial tissues from SCID-HuRAg mice was conducted after 4 weeks of oral administration of MTX (0.3 mg/kg/week), salazosulfapyridine (30 mg/kg/day), auranofin (0.2 mg/kg/day), bucillamine (10 mg/kg/day), or indomethacin (2 mg/kg/day). RESULTS: A significant decrease in the number of inflammatory cells was observed in the grafted synovial tissue of MTX treated SCID-HuRAg. A similar antiinflammatory effect was not observed with the other DMARD. Induction of apoptosis was noted with MTX treatment but not with the others. The pro-apoptotic effect of MTX was also observed in synovial cell cultures. CONCLUSION: MTX induces apoptosis in RA synovium that, in turn, may contribute to its antiinflammatory effect on RA synovitis.


Assuntos
Antirreumáticos/farmacologia , Apoptose/efeitos dos fármacos , Artrite Reumatoide/tratamento farmacológico , Metotrexato/farmacologia , Sinovite/tratamento farmacológico , Animais , Apoptose/imunologia , Artrite Reumatoide/patologia , Citometria de Fluxo , Humanos , Marcação In Situ das Extremidades Cortadas , Masculino , Camundongos , Camundongos SCID , Microscopia Eletrônica , Membrana Sinovial/patologia , Membrana Sinovial/transplante , Membrana Sinovial/ultraestrutura , Sinovite/patologia
16.
J Bone Miner Res ; 16(8): 1453-64, 2001 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-11499868

RESUMO

The rate of bone formation is largely determined by the number of osteoblasts, which in turn is determined by the rate of replication of progenitors and the life span of mature cells, reflecting the timing of death by apoptosis. However, the exact age-dependent changes of the cellular activity, replicative potential, and life span of osteoblasts have not been investigated to date. Here, we present evidence that the cellular activity, telomere lengths, and replicative life span of osteoblastic cells obtained from juxta-articular bone marrow gradually decrease with the advance of donor age. Recently, telomerase reverse transcriptase (hTERT) has been identified as a human telomerase catalytic subunit. We transfected the gene encoding hTERT into telomerase-negative human osteoblastic cells from donors and osteoblastic cell strain NHOst 54881 cells and showed that expression of hTERT induces telomerase activity in these osteoblastic cells. In contrast to telomerase-negative control cells, which exhibited telomere shortening and senescence after 10-15 population doublings, telomerase-expressing osteoblastic cells had elongated telomere lengths and showed continued alkaline phosphatase activity and procollagen I C-terminal propeptide (PICP) secretion for more than 30 population doublings. These results indicate that osteoblasts with forced expression of hTERT may be used in cell-based therapies such as ex vivo gene therapy, tissue engineering, and transplantation of osteoblasts to correct bone loss or osteopenia in age-related osteoporotic diseases.


Assuntos
Envelhecimento/metabolismo , Osteoblastos/enzimologia , Telomerase/metabolismo , Telômero/fisiologia , Idoso , Envelhecimento/genética , Envelhecimento/fisiologia , Fosfatase Alcalina/metabolismo , Proteínas de Transporte/genética , Domínio Catalítico , Divisão Celular , Células Cultivadas , Proteínas de Ligação a DNA , Feminino , Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteocalcina/metabolismo , Fragmentos de Peptídeos/metabolismo , Pró-Colágeno/metabolismo , Proteínas de Ligação a RNA , Telomerase/genética , Doadores de Tecidos , Transfecção
17.
Br J Cancer ; 84(12): 1610-5, 2001 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-11401313

RESUMO

Previous studies have shown that the prognosis of patients who have tumours with high microvessel density (MVD) is worse than that of patients who have a lower density in a variety of cancers. In this study, we investigated the clinical relevance of neovascularity assessed by MVD and the concentration of vascular endothelial growth factor (VEGF) in the tumour tissue of patients with soft tissue sarcoma in comparison with major clinicohistologic parameters by univariate and multivariate analysis. In 115 patients with soft tissue sarcoma, MVD was measured by counting vessels stained with factor VIII antibody. The concentration of VEGF in the tumour tissue was determined by enzyme-linked immunosorbent assay. These parameters were then compared with disease outcome. The concentration of VEGF in the tumour tissue, but not MVD, was found to be correlated with disease outcome in patients with soft tissue, sarcoma. VEGF concentration in the tumour tissue showed a relationship with the clinical stage and histologic grade of the tumour. There was no significant difference in the levels of tissue VEGF concentration and MVD among soft tissue sarcomas classified according to histologic type. The level of tissue VEGF concentration in patients who had subsequent local recurrence and metastasis were significantly higher than the respective values in patients who did not have such disease outcome. No significant correlation existed between MVD and the concentration of VEGF in the tumour tissue. Univariate analysis showed that a high tissue VEGF concentration was associated with poor overall survival of the patient and a greater probability that local recurrence and metastasis had occurred. Multivariate analysis revealed that the tissue concentration of VEGF is an independent prognostic factor for the disease outcome of patients with soft tissue sarcoma. VEGF concentration in the tumour tissue, but not MVD, is an additional prognostic parameter for disease outcome in patients with soft tissue sarcoma, regardless of histologic type.


Assuntos
Fatores de Crescimento Endotelial/análise , Linfocinas/análise , Neovascularização Patológica , Sarcoma/patologia , Neoplasias de Tecidos Moles/patologia , Adulto , Idoso , Feminino , Humanos , Masculino , Microcirculação , Pessoa de Meia-Idade , Metástase Neoplásica , Recidiva Local de Neoplasia , Prognóstico , Estudos Retrospectivos , Sarcoma/irrigação sanguínea , Neoplasias de Tecidos Moles/irrigação sanguínea , Análise de Sobrevida , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio Vascular
18.
Tumour Biol ; 22(4): 239-46, 2001.
Artigo em Inglês | MEDLINE | ID: mdl-11399949

RESUMO

The relationship between vascular endothelial growth factor (VEGF) and induction of angiogenesis in high-metastatic RCT(+) or low-metastatic RCT(-) clones of the poorly differentiated murine RCT sarcoma was investigated. The association with E-selectin in VEGF-induced angiogenesis was also evaluated. RCT(+) cells produced significantly larger amounts of VEGF than RCT(-) cells. In a tube formation assay with murine lung microvascular endothelial (MLE) cells, conditioned medium from RCT(+) cells showed a significantly greater effect on tube formation than that from RCT(-) cells. Induction of tube formation was suppressed by anti-mouse VEGF monoclonal antibody. Furthermore, anti-mouse E-selectin monoclonal antibody suppressed the tube formation induced by recombinant mouse VEGF. In a flow-cytometric analysis, the expression of E-selectin on MLE cells was upregulated after pretreatment with conditioned medium from RCT(+) and RCT(-) cells. Conditioned medium from RCT(+) cells induced a higher expression of E-selectin compared to medium from RCT(-) cells. Anti-VEGF monoclonal antibody prevented the upregulation of E-selectin by the RCT cell-conditioned medium. These findings suggest that E-selectin plays an important role in the angiogenesis induced by VEGF. VEGF derived from tumor cells may enhance angiogenesis by upregulating the expression of E-selectin on vascular endothelial cells.


Assuntos
Selectina E/fisiologia , Fatores de Crescimento Endotelial/fisiologia , Endotélio Vascular/fisiopatologia , Linfocinas/fisiologia , Neovascularização Patológica , Sarcoma/irrigação sanguínea , Animais , Anticorpos/imunologia , Meios de Cultivo Condicionados , Selectina E/imunologia , Fatores de Crescimento Endotelial/genética , Fatores de Crescimento Endotelial/imunologia , Linfocinas/genética , Linfocinas/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C3H , Metástase Neoplásica , Neovascularização Patológica/imunologia , Neovascularização Patológica/fisiopatologia , RNA Mensageiro/biossíntese , Sarcoma/secundário , Células Tumorais Cultivadas , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio Vascular
19.
J Rheumatol ; 28(1): 22-8, 2001 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-11196534

RESUMO

OBJECTIVE: To investigate the relationship between matrix metalloproteinases (MMP) and the soluble form of Fas ligand (sFasL) in the synovial fluid (SF) of patients with rheumatoid arthritis (RA), and to determine which MMP have a major role in cleaving FasL. METHODS: The concentrations of sFas and sFasL in SF from 48 patients with RA and 43 patients with osteoarthritis (OA) were measured using specific ELISA. The levels of different MMP (MMP-1, 2, 3, 7, 9) in SF were also measured by ELISA. The active forms of gelatinases were detected by gelatin zymogram. Human FasL-expressing transfected cells (hFasL/L5178Y) were used to investigate whether FasL is cleaved from membrane bound FasL. RESULTS: Significantly higher levels of MMP-1, 3, and 9 were found in SF from RA patients compared to OA patients, but MMP-7 was not detectable in either group. The concentrations of sFas and sFasL in SF were also higher in RA than in OA patients. However, there was no relationship between the concentration of sFas and sFasL. Among MMP, MMP-3 concentrations in SF were closely correlated with the level of sFasL and with disease activity of RA. Enzymatic cleavage assay indicated that MMP-3 has potential to cleave the FasL expressed on hFasL/L5178Y cells and to produce sFasL. CONCLUSION: There was significant correlation between the concentration of sFasL and MMP-3 in SF of patients with RA. In addition, our data indicate that the shedding of FasL may be regulated by MMP-3 in the joint of patients with RA.


Assuntos
Apoptose , Artrite Reumatoide/enzimologia , Metaloproteinase 3 da Matriz/metabolismo , Glicoproteínas de Membrana/metabolismo , Líquido Sinovial/enzimologia , Artrite Reumatoide/patologia , Ensaio de Imunoadsorção Enzimática , Proteína Ligante Fas , Feminino , Humanos , Elastase de Leucócito/metabolismo , Masculino , Pessoa de Meia-Idade
20.
Clin Calcium ; 11(5): 612-8, 2001 May.
Artigo em Japonês | MEDLINE | ID: mdl-15775563

RESUMO

The rate of bone formation is largely determined by the number of osteoblasts, which in turn is determined by the rate of replication of progenitors and the life-span of mature cells, reflecting the timing of death by apoptosis. However, the exact age-dependent changes of the cellular activity, replicative potential and life-span of osteoblasts have not so far been investigated to date. Here we present evidence that the cellular activity, telomere lengths and replicative life-span of osteoblastic cells obtained from juxta-articular bone marrow gradually decrease with the advance of donor age in patients with rheumatoid arthritis or osteoarthritis. Recently, human telomerase reverse transcriptase (hTERT) has been identified as a human teromerase catalytic subunit. We postulate that an expansion of the life-span of osteoblasts and their maintenance as differentiated bone matrix-producing cells may allow for autologus or allogenic cell and gene therapy in bone and joint diseases including osteoporosis. We therefore transfected human osteoblasts with a vector expressing hTERT cDNA, and investigated whether the replicative life-span can be expanded by the introduction of telomerase in human osteoblasts.

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