RESUMO
OBJECTIVES: To evaluate the effect of isotretinoin on orthodontic tooth movement (OTM) and wound healing following exodontia. SETTING AND SAMPLE POPULATION: Sixteen 40-day-old male Wistar rats were divided into two groups: (a) OTM and (b) tooth extraction (TE) of the upper 1st molar and OTM. The experimental animals were treated with isotretinoin (7.5 mg/kg) and the control animals with oil solution for 37 days. MATERIALS AND METHODS: The OTM and bone volume were evaluated by the micro-CT and the periodontium healing was assessed by immunohistochemistry for VEGF-C, COX-2 and IL-1ß. RESULTS: The animals of both groups submitted to the TE showed a statistically significant decrease in the bone volume percentage and increase in OTM. No significant difference of OTM and bone volume was observed between the control and experimental group. However, the alveolar bone of the isotretinoin group revealed more medullary spaces with inflammatory, hematopoietic cells, blood vessels and intense immunolabeling for VEGF-C. This group also showed faster gingival regeneration. No significant difference was observed in the COX-2 and IL-1ß labelings following TE between both groups. CONCLUSION: The isotretinoin did not affect the OTM nor did it cause an alteration in maxillary bone volume. This exogenous acid may contribute to the acceleration of gingival healing.
Assuntos
Periodonto/efeitos dos fármacos , Técnicas de Movimentação Dentária , Tretinoína/farmacologia , Animais , Ciclo-Oxigenase 2/metabolismo , Imuno-Histoquímica , Interleucina-1beta/metabolismo , Masculino , Maxila , Ratos , Ratos Wistar , Extração Dentária , Fator C de Crescimento do Endotélio Vascular/metabolismoRESUMO
Fluorescent-based live/dead labelling combined with fluorescent microscopy is one of the widely used and reliable methods for assessment of cell viability. This method is, however, not quantitative. Many image-processing methods have been proposed for cell quantification in an image. Among all these methods, several of them are capable of quantifying the number of cells in high-resolution images with closely packed cells. However, no method has addressed the quantification of the number of cells in low-resolution images containing closely packed cells with variable sizes. This paper presents a novel method for automatic quantification of live/dead cells in 2D fluorescent low-resolution images containing closely packed cells with variable sizes using a mean shift-based gradient flow tracking. Accuracy and performance of the method was tested on growth plate confocal images. Experimental results show that our algorithm has a better performance in comparison to other methods used in similar detection conditions.
Assuntos
Sobrevivência Celular , Processamento de Imagem Assistida por Computador/métodos , Microscopia Confocal/métodos , Reconhecimento Automatizado de Padrão/métodos , Algoritmos , Animais , Contagem de Células , Corantes Fluorescentes , SuínosRESUMO
We used a microarray approach to evaluate gene expression profiles in human AIS osteoblasts, and to identify genes that are differentially expressed following estrogen exposure in non-AIS and AIS human osteoblasts. We found that more than one gene is likely responsible for AIS. Furthermore, some of these genes are estrogen-regulated, suggesting a possible role of estrogens in the etiology of scoliosis.
Assuntos
Predisposição Genética para Doença , Escoliose/genética , Adolescente , Perfilação da Expressão Gênica , Pesquisa em Genética , Humanos , Escoliose/etiologiaRESUMO
Spinal deformities, and particularly scoliosis, are the most frequent forms of orthopedic deformities in children and adolescents. About 1-6% of the population has scoliosis. This disorder leads to severe spinal deformities and predominantly affects adolescent girls.Although the multifactorial origin of adolescent idiopathic scoliosis (AIS) is broadly recognized, the genetic causes of AIS are still largely unknown. Our previous studies suggested a generalized dysfunction of melatonin transduction (the hormone that is primarily produced in the brain and epiphysis). In the meantime we have demonstrated that such a defect of signal transduction is caused by chemical alterations, which inactivate the function of the inhibitory G protein-coupled melatonin receptors. This discovery has led to the development of the first blood test to detect children without symptoms who are at risk of developing scoliosis. Since a single function (cellular reaction to melatonin) is determined, the unique advantage of this test is that it can be performed without knowledge of mutations in defective genes that could provoke the onset of AIS.
Assuntos
Análise Química do Sangue/métodos , Programas de Rastreamento/métodos , Melatonina/sangue , Escoliose/sangue , Escoliose/diagnóstico , Biomarcadores/sangue , Predisposição Genética para Doença/genética , Humanos , Escoliose/fisiopatologiaRESUMO
Diabetes Mellitus leads to pain neuropathy and cardiovascular complications which remain resistant to current therapies involving the control of glycaemia. This study aims at defining the contribution of kinin B(1) receptor (B(1)R) and the oxidative stress on sensory abnormalities and arterial hypertension in a rat model of insulin resistance. Rats were fed with 10% d-glucose for a chronic period of 12-14 weeks and the impact of a diet supplemented with alpha-lipoic acid, a potent antioxidant, was determined on tactile and cold allodynia, arterial hypertension and the expression of kinin B(1)R (real-time PCR and autoradiography) in several tissues. Acute effects of brain penetrant (LF22-0542) and peripherally acting (R-715) B(1)R antagonists were also assessed. Glucose-fed rats exhibited tactile and cold allodynia along with increases in systolic blood pressure between 4 and 12 weeks; these alterations were alleviated by alpha-lipoic acid. The latter regimen also decreased significantly increased plasma levels of insulin and glucose and insulin resistance (HOMA index) at 14 weeks. B(1)R mRNA was virtually absent in liver, aorta, lung, kidney and spinal cord isolated from control rats, yet B(1)R mRNA was markedly increased in all tissues in glucose-fed rats. Up-regulated B(1)R mRNA and B(1)R binding sites (spinal cord) were significantly reduced by alpha-lipoic acid in glucose-fed rats. LF22-0542 reduced tactile and cold allodynia (3h) and reversed arterial hypertension (3-48h) in glucose-fed rats. R-715 abolished tactile and cold allodynia but had not effect on blood pressure. Data suggest that the oxidative stress contributes to the induction and up-regulation of B(1)R in the model of insulin resistance induced by glucose feeding. The over expressed B(1)R contributes centrally to arterial hypertension and in the periphery to sensory abnormalities.
Assuntos
Hipertensão/etiologia , Resistência à Insulina , Estresse Oxidativo/fisiologia , Receptor B1 da Bradicinina/fisiologia , Transtornos de Sensação/etiologia , Animais , Modelos Animais de Doenças , Glucose/administração & dosagem , Glucose/farmacologia , Hipertensão/metabolismo , Cininas , Ratos , Ratos Sprague-Dawley , Receptor B1 da Bradicinina/genética , Transtornos de Sensação/metabolismo , Regulação para Cima/genéticaRESUMO
RATIONALE: Repetitive transcranial magnetic stimulation (rTMS) is used alone or in combination with physiotherapy for rehabilitation of stroke patients. TMS mapping can also quantify the excitability of the motor area in both the ipsilesional (IL) and contralateral (CL) hemisphere. OBJECTIVE: This study is the first to measure the dynamics of cortical excitability by TMS mapping before and after treatment with low-frequency (LF) rTMS in the contralesional hemisphere at three different timepoints. Furthermore, the patients were clinically evaluated during the same visit as the mapping to establish both short and long-term outcomes after rTMS treatment. METHODS AND RESULTS: A total of 16 participants with acute ischemic stroke were assessed 10 days post-stroke by TMS mapping. The patients were randomized into two equal groups: a real rTMS group and a sham group. The rTMS group received LF-rTMS to the contralesional hemisphere for 10 days, starting on the first day after the first mapping. Each subject was also evaluated by mapping on days 45 and 90 after stroke onset. The primary clinical outcome measured was the Fugl-Meyer Assessment for Upper Extremity (FMA-UE) on days 10, 45 and 90 post-stroke. At 10 days after stroke onset, both groups presented low excitability in the lesion side and high excitability in the non-affected side. In the real rTMS group, at 45 days after stroke, a downward trend in the excitability of the contralesional hemisphere and an upward trend in the excitability of the lesioned side were observed. At 90 days after stroke, a tendency toward balanced excitability between both hemispheres was observed. In the sham group, at both 45 and 90 days, we observed increased excitability in the non-affected side compared to the side with the lesioned motor area. At 45 days, the real rTMS group demonstrated a better recovery of the upper limb motor function than the sham group, but at 90 days, there was no significant difference between the two groups. DISCUSSION: These results demonstrated that LF-rTMS treatment enhances rebalance of the excitability patterns in both hemispheres and led us to question the "one size fits all" approach widely used in rTMS interventions. ABBREVIATIONS: Amax = maximum amplitude, Amean = AM = averaged amplitude, APB = abductor pollicis brevis, CL = contralesional, DTI = diffusion tensor imaging, EEG = electroencephalography, EMG = electromyography, FMA-UE = Fugl-Meyer Assessment for Upper Extremity, HS = hot spot, IHC = interhemispheric functional connectivity, IL = ipsilesional, LF-rTMS = low-frequency repetitive transcranial magnetic stimulation, MCA = middle cerebral artery, MEP(s) = motor evoked potential(s), NIBS = non-invasive brain stimulation, rMT = resting motor threshold, RP = responsive points, rTMS = repetitive transcranial magnetic stimulation, TMS = transcranial magnetic stimulation.
Assuntos
Isquemia Encefálica/complicações , Isquemia Encefálica/fisiopatologia , Córtex Cerebral/fisiopatologia , Acidente Vascular Cerebral/complicações , Acidente Vascular Cerebral/fisiopatologia , Estimulação Magnética Transcraniana , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fenômenos Fisiológicos do Sistema Nervoso , Reabilitação do Acidente Vascular Cerebral , Fatores de Tempo , Resultado do TratamentoRESUMO
The plasmid pMK16 containing-SV40 replicated origin defective gene was efficiently introduced into early-passage human umbilical vein endothelial cells (HUVEC) using positively charged liposomes. The resulting cell line acquired an almost infinite lifespan, was morphologically unchanged, expressed SV40-antigen, and coexpressed von Willebrand factor (vWF), tissue plasminogen activator (t-PA), plasminogen activator inhibitor-1 (PAI-1), angiotensin conversion enzyme (ACE), and endothelin converting enzyme (ECE). In addition, these are the first immortalized human endothelial cells, to our knowledge, that biosynthesized and secreted interleukins (IL-1 beta and IL-6) in both a constitutive and regulated fashion and endothelin-1 (ET-1), the most potent vasoactive peptide, which has been suggested to be implicated in the pathogenesis of hypertension. Interestingly enough, both of the immortalized cells and the early-passage HUVEC from which the immortalized cells were obtained biosynthesized and secreted the same levels of ET-1 suggesting full maintenance of its biosynthetic pathway including the presence of active ECE, which cleaves big endothelin-1 (big-ET-1) to ET-1 and regulation factors. Moreover, the immortalized cells retained the ability to express the functional specific amino acid Na(+)-independent system Y+ transporter, which mediates L-arginine transport into endothelial cells from which endothelium-derived relaxing factor (EDRF, nitric oxide) is formed via the action of nitric oxide-synthase. Obtaining these immortalized human endothelial cells without alteration of the differentiated characteristics constitutes a useful model: (a) to study ET-1 secretion, gene regulation, and human ECE, which may be an important therapeutic target in disease conditions in which ET-1 is to be implicated; (b) to study L-arginine transport, which is a key step in the formation of EDRF; (c) to study IL-1 beta and IL-6 secretions, and gene regulations; (d) to substitute large quantities of HUVEC; and, finally, (e) to reproduce, starting with different primary endothelial cells both from human and animal origin.
Assuntos
Antígenos Transformantes de Poliomavirus/genética , Endotélio Vascular/crescimento & desenvolvimento , Transfecção , Veias Umbilicais/crescimento & desenvolvimento , Arginina/metabolismo , Transporte Biológico , Diferenciação Celular , Células Cultivadas , Endotélio Vascular/citologia , Humanos , Fenótipo , Sódio/metabolismo , Veias Umbilicais/citologiaRESUMO
OBJECTIVE: To compare in cell culture endothelin-1 (ET-1) production, receptor density, and effect on macromolecular synthesis by articular chondrocytes (AC). METHODS: AC were isolated from 1-month and 18-month old rats and cultured as monolayers. They were incubated with ET-1 without or with iNOS inhibitors, nitro-L-arginine methyl ester (L-NAME) or guanylate cyclase inhibitor, LY83583 and then [3H]thymidine, 35SO4 and [3H]proline incorporations were measured. The density and affinity for 125I-ET-1 of binding sites, and receptor isotypes were determined. The cells were also treated with interleukin-1beta (IL-1beta) or tumor necrosis factor-alpha (TNF-alpha), and then ET-1 productions measured. As well, the cells were challenged with NOC-5 (nitric oxide donor) or ET-1 and then ET-1 and NO respectively were measured. RESULTS: A concentration-dependent stimulation of DNA, PG, collagen and NO synthesis was obtained when cells were incubated with ET-1 for 24-h. Eighteen-month old chondrocytes incorporated per microg DNA more [3H]thymidine, 35SO4 and [3H]proline but less NO when challenged with ET-1 than the 1-month old cells. However, strong inhibition of this initial stimulation was seen after 48-h. L-NAME and LY83583 enhanced basal-, and ET-1-induced initial stimulation and completely suppressed late (at 48-h to 72-h) ET-1-induced inhibition, suggesting NO was responsible for this inhibitory effect. Eighteen-month old chondrocytes expressed per mug DNA more high affinity receptors of predominantly ET(A) subtype. They also produced more ET-1 but less NO under basal conditions and more ET-1 when challenged with IL-1beta and TNF-alpha. NOC-5 inhibited the production of ET-1. CONCLUSIONS: Eighteen-month old chondrocytes produce more ET-1, possess more ET-1-specific receptors, and increase more DNA, PG and collagen synthesis when challenged during 24-h with ET-1. NO, which suppresses ET-1 production and the production of which is increased by ET-1, seems to account for the late ET-1-induced inhibition of macromolecular synthesis. The possible implication of ET-1 in aging as related to osteoarthritis is discussed.
Assuntos
Senescência Celular/fisiologia , Condrócitos/citologia , Condrócitos/metabolismo , Endotelina-1/metabolismo , Animais , Artérias/metabolismo , Células Cultivadas , Citocinas/metabolismo , Óxido Nítrico/metabolismo , Ligação Proteica , RatosAssuntos
Proteínas da Matriz Extracelular/metabolismo , Glicoproteínas/metabolismo , Escoliose/diagnóstico , Escoliose/metabolismo , Adolescente , Biomarcadores/metabolismo , Proteína de Matriz Oligomérica de Cartilagem , Feminino , Regulação da Expressão Gênica , Humanos , Proteínas Matrilinas , Modelos Biológicos , Modelos Teóricos , Mutação , Osteoblastos/metabolismo , Fenótipo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
This study showed that endothelins (ETs) stimulate DNA and proteoglycan synthesis in monolayer culture of rat articular chondrocytes (AC) by interacting with specific cell surface receptors. The high affinity receptors bound [125I]ET-1 with a Kd of 0.54 nM and Bmax of 81.4 pM/microgram DNA (approximately 40 000 binding sites per cell) was demonstrated. [125I]ET-1 binding was completely inhibited by unlabelled ET-1 or ET-2, and by BQ123 (ETA receptor antagonist), whereas ET-3 and IRL1038 (ETB receptor antagonist) did so only weakly. SDS-PAGE of cell extracts containing [125I]ET-1 cross-linked to the receptors, followed by autoradiography of the gels revealed a single 50-kDa band. These findings indicate that most of the receptors are subtype ETA. Although mRNA transcripts specific for both ETA and ETB receptors were found by RT-PCR, the ETA mRNA was more abundant. ET-1 increased the production of cAMP, cGMP and prostaglandin E2 (PGE2) and protein kinase C (PKC) activity in a concentration- and time-dependent manner. ET-1, and to a lesser degree ET-2, stimulated DNA synthesis, whereas ET-3 was inactive. Stimulation of DNA synthesis by ET-1 was strongly inhibited in a concentration-dependent manner by BQ123 and, to a much lesser degree, by IRL1038, which is consistent with an ETA receptor. ET-1 also stimulated proteoglycan synthesis and increased the amount of mRNA specific for the aggrecan gene. These findings strongly suggest that ET-1 is involved in regulating chondrocyte proliferation and metabolism in health, and presumably in disease.
Assuntos
Cartilagem Articular/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Endotelina-1/farmacologia , Proteoglicanas/biossíntese , Receptores de Endotelina/fisiologia , Transdução de Sinais/fisiologia , Animais , Cartilagem Articular/metabolismo , Divisão Celular/efeitos dos fármacos , Células Cultivadas , AMP Cíclico/biossíntese , GMP Cíclico/biossíntese , Replicação do DNA/fisiologia , Dinoprostona/biossíntese , Endotelina-1/fisiologia , Endotelina-2/farmacologia , Endotelina-3/farmacologia , Endotelinas/farmacologia , Fragmentos de Peptídeos/farmacologia , Peptídeos Cíclicos/farmacologia , Proteína Quinase C/metabolismo , RNA Mensageiro/análise , Ratos , Ratos Wistar , Receptor de Endotelina A , Receptor de Endotelina B , Receptores de Endotelina/efeitos dos fármacos , Receptores de Endotelina/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
The aim of this study was to determine the effects of endothelin-1 (ET-1) on proteoglycan (PG) and collagen synthesis by rat articular chondrocytes (RAC). PG and collagen synthesis was measured by [(35)S]-sulphate and [(3)H]-glycine incorporation, respectively into monolayers of confluent RAC exposed to ET-1 (10(-11) M-10(-7) M). ET-1 stimulated PG and collagen synthesis in these cells in a concentration-dependent manner during the first 24 h of incubation. Prolonged contact of the cells with ET-1 resulted in a gradual decrease, and finally, inhibition of ET-1 effects. This inhibition is mediated by nitric oxide (NO) released in response to ET-1 since: (1) nitric oxide synthase inhibitor, nitro-L-arginine-methyl ester (L-NAME), enhanced both basal and ET-1-induced [(35)S]-sulphate and [(3)H]-glycine incorporations; (2) sodium nitroprusside (SNP), which spontaneously releases NO, inhibited both basal and ET-1-induced incorporations, and was also able to suppress the effects of L-NAME; (3) NO levels in the culture media were also correlated with the inhibition of [(35)S]-sulphate and [(3)H]-glycine incorporation; and (4) SNP also inhibited aggrecan and collagen II transcriptions, probably via cGMP. This effect was mimicked with 8-bromo-cGMP. Interestingly, the LY83583, which blocks the NO-dependent production and release of cGMP, inhibited PG-collagen synthesis but had no effect on their mRNA expressions. Thus, normal levels of cGMP appeared to be necessary for PG-collagen synthesis, whereas decreased levels are detrimental. In conclusion, NO, produced by rat AC in response to ET-1, counteracts the stimulation and finally induces inhibition of PG-collagen synthesis by ET-1 in these cells but NO-induced cGMP is only partially responsible for this inhibition.
Assuntos
Condrócitos/metabolismo , Colágeno/biossíntese , Endotelina-1/metabolismo , Proteoglicanas/antagonistas & inibidores , Aminoquinolinas/farmacologia , Animais , Células Cultivadas , Colágeno/metabolismo , Colágeno Tipo II/metabolismo , DNA/biossíntese , Relação Dose-Resposta a Droga , Masculino , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/metabolismo , Nitritos/metabolismo , Nitroprussiato/farmacologia , Prostaglandinas/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de TempoRESUMO
The purpose of the study was to describe endothelin (ET) production and to characterize the effect of hypoxia on preproendothelin-1 (preproET-1) messenger ribonucleic acid (mRNA) expression and ET secretion by rat type II pneumocytes in vitro. Rat type II pneumocytes were incubated in a sealed chamber containing a normoxic (21% O2) or hypoxic (1% O2) atmosphere for increasing durations. Immunoreactive ET (irET) was measured in cell supernatants using a radioimmunoassay. Rat preproET-1 mRNA was detected by Northern blot. Rat type II pneumocytes expressed preproET-1 mRNA, contained irET and secreted irET in a time-dependent manner. ET secretion was dependent on de novo ribonucleic acid (RNA) and protein synthesis. Hypoxia decreased irET secretion by 27% and reduced the steady-state level of preproET-1 mRNA by 60% whereas intracellular irET concentration was unchanged. Inhibition was partially reversible with the return to a normoxic atmosphere. Inhibition of nitric oxide synthesis did not prevent the inhibitory effect of hypoxia. In conclusion, rat type II pneumocytes in primary culture secreted immunoreactive endothelin and expressed preproendothelin-1 messenger ribonucleic acid. Hypoxia reversibly reduced endothelin-1 production through a reduction of the steady-state preproendothelin-1 messenger ribonucleic acid level. Nitric oxide synthesis did not mediate the inhibitory effect of hypoxia.
Assuntos
Endotelinas/metabolismo , Hipóxia/metabolismo , Alvéolos Pulmonares/metabolismo , Animais , Sobrevivência Celular/fisiologia , Células Cultivadas , Endotelina-1 , Endotelinas/genética , Hipóxia/patologia , Hipóxia/fisiopatologia , Masculino , Óxido Nítrico/farmacologia , Precursores de Proteínas/genética , Alvéolos Pulmonares/patologia , RNA Mensageiro/metabolismo , Radioimunoensaio , Ratos , Ratos Sprague-Dawley , Valores de Referência , Fatores de TempoRESUMO
OBJECTIVE: To examine, by immunohistochemistry, the localization and distribution of human collagenase-3 in normal, osteoarthritis (OA), and rheumatoid arthritis (RA) cartilage, and to investigate the effects of interleukin-1beta (IL-1beta) and transforming growth factor beta (TGFbeta) on the synthesis and distribution of collagenase-3. METHODS: Human cartilage specimens were obtained from tibial plateaus. In the first series of experiments, the OA specimens were excised from fibrillated and nonfibrillated areas of cartilage, and RA specimens were excised from lesional areas, including the cartilage-pannus junction when present. In the second series, full strips of cartilage were processed for culture in the presence or absence of IL-1beta (100 units/ml) or TGFbeta (150 ng/ml). Each specimen was processed for immunohistochemical analysis using a collagenase-3 monoclonal antibody. RESULTS: The number of cells that stained for collagenase-3 in normal cartilage was very low (approximately 3%). In OA cartilage, the percentage increased dramatically, and no difference was found between fibrillated and nonfibrillated areas. A statistically significant increase in the percentage of cells staining for collagenase-3 was found in the deep layer compared with the superficial layer. This finding was noted in both the fibrillated areas (mean +/- SEM 58.4 +/- 1.6% and 40.1 +/- 3.9%, respectively; P < 0.007) and the nonfibrillated areas (55.4 +/- 3.2% and 43.2 +/- 2.7%; P < 0.01). Similarly, RA cartilage showed a statistically significant (P < 0.001) increase in the level of chondrocytes staining positive for collagenase-3 in the deep layers (46.4 +/- 4.1%) compared with the superficial layers (26.2 +/- 3.4%). In these RA specimens, the numbers of positively staining chondrocytes were similar both close to and at a distance from the pannus junction. Both IL-1beta and TGFbeta increased the number of chondrocytes producing collagenase-3. Interestingly, in normal specimens, TGFbeta had a predominant effect in the deep layers, while IL-1beta had a greater effect on the superficial layers. CONCLUSION: This study demonstrates that, in situ, the increase in the level of chondrocytes synthesizing collagenase-3 in arthritic cartilage is predominant in the deep layers. The results further indicate that TGFbeta can up-regulate the level of this enzyme and, in normal cartilage in vitro, can cause a mimicking of the in situ distribution observed in arthritic cartilage.
Assuntos
Artrite Reumatoide/metabolismo , Cartilagem Articular/enzimologia , Colagenases/metabolismo , Osteoartrite/metabolismo , Idoso , Cartilagem Articular/citologia , Cartilagem Articular/efeitos dos fármacos , Contagem de Células , Células Cultivadas , Humanos , Imuno-Histoquímica , Interleucina-1/farmacologia , Metaloproteinase 13 da Matriz , Pessoa de Meia-Idade , Fator de Crescimento Transformador beta/farmacologia , Regulação para CimaRESUMO
Endothelin-1 (ET-1) mRNA expression and protein production were examined in primary rat articular chondrocyte (AC) cultures by RT-PCR and radioimmunoassay, respectively. We found that serum-starved rat AC express ET-1 mRNA and produce the peptide constitutively. Treatment of cells with 10% FCS resulted in a marked increase in ET-1 levels with a peak at 48 h (5.6-fold). A similar concentration-dependent effect was also obtained in the presence of interleukin 1beta (3.1-fold), tumour necrosis factor alpha (3. 5-fold), lipopolysaccharide (2.7-fold), transforming growth factor beta1 (3.5-fold), epidermal growth factor (5.0-fold) and insulin-like growth factor-I (4.4-fold). In addition, ET-1 was found to induce, over a period of 24 h, a potent concentration-dependent stimulation of DNA synthesis in rat AC. These findings demonstrate for the first time the constitutive expression and production of ET-1 by rat AC which could be modulated by several cytokines and growth factors, suggesting a possible role for ET-1 in autocrine regulation of chondrocyte function.
Assuntos
Cartilagem Articular/metabolismo , Endotelina-1/biossíntese , Animais , Cartilagem Articular/citologia , Divisão Celular , Células Cultivadas , Endotelina-1/genética , Técnica Indireta de Fluorescência para Anticorpo , Expressão Gênica , Fenótipo , Reação em Cadeia da Polimerase , RNA Mensageiro , Ratos , Ratos Wistar , Coloração e Rotulagem , Transcrição GênicaRESUMO
OBJECTIVE: IL-1beta plays a fundamental role in osteoarthritis (OA) pathophysiology and cartilage destruction. Targeting the activation mechanism of this cytokine appears to be important as a therapeutic approach. As the interleukin-1 converting enzyme (ICE) is the physiologic modulator of the production of active IL-1beta, we investigated the effect of diacerhein and its active metabolite rhein used in the treatment of OA patients, on the enzyme expression and synthesis on human OA cartilage. Further, we looked at the effect of both drugs on the production of the active form of IL-1beta and IL-18. METHODS: The expression and synthesis of ICE were investigated on human OA cartilage explants using in-situ hybridization and immunohistochemical methods, respectively. The effect of the drugs on ICE OA chondrocytes was also determined by Northern blotting and a specific ELISA assay. Furthermore, the effect of both drugs on the level of active IL-1beta and IL-18 was examined by immunohistochemistry. RESULTS: Data showed that diacerhein and rhein have no true effect on reducing total ICE mRNA by both Northern blotting analysis and in-situ hybridization. A marked and statistically significant decrease was, however, found for protein production. ELISA showed a reduction of 31% (P< 0.04) for diacerhein and 50% (P< 0.02) for rhein. The drugs' immunohistological cell score reduction was similar to data from the ELISA, and a statistical significant reduction of ICE production was found at both superficial and deep zones of the cartilage. IL-1beta and IL-18 were both preferentially produced in chondrocytes of the superficial zone. For each of these cytokines, both drugs demonstrated a statistically significant decrease in this zone. A marked decrease was also noted in the deep zone, but statistical significance was reached only for rhein. CONCLUSION: These results provide a novel regulatory mechanism by which diacerhein and rhein could exert a down-regulation on IL-1's effect on OA cartilage.
Assuntos
Antraquinonas/farmacologia , Caspase 1/farmacologia , Interleucina-18/fisiologia , Interleucina-1/fisiologia , Osteoartrite/metabolismo , Anti-Inflamatórios não Esteroides/farmacologia , Northern Blotting , Cartilagem Articular/citologia , Cartilagem Articular/efeitos dos fármacos , Caspase 1/efeitos dos fármacos , Caspase 1/genética , Condrócitos/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Hibridização In Situ , Masculino , Osteoartrite/tratamento farmacológico , Osteoartrite/genética , RNA Mensageiro/efeitos dos fármacosRESUMO
Specific endothelin-1 (ET1) binding sites have been demonstrated in membranes derived form normal (NP) and benign hyperplasic (BPH) human prostate using an 125I-ET1 binding assay. 125I saturation experiments and Scatchard analysis demonstrated the existence of a homogeneous population of binding sites with high affinity (Kd(app)) and density (B(max)), respectively, 106 +/- 15 pM and 1086 +/- 399 fmol/mg protein for NP (n = 5) and 168 +/- 26 pM and 964 +/- 445 fmol/mg protein for BPH (n = 5). We demonstrated the presence of two subtypes of ET1 receptors, ETA and ETB, by means of the following ET1 competitors: ET2, ET3, and BQ123 (which is selective for the ETA receptor), and IRL1620 and sarafotoxine c (S6c) (which are selective for the ETB receptor). The displacement curves allowed us to conclude that the large majority (85%) of the ET1 receptors in normal and hyperplasic human prostate are of the A subtype.
Assuntos
Próstata/química , Hiperplasia Prostática/metabolismo , Receptores de Endotelina/análise , Ligação Competitiva , Membrana Celular/química , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Cromatografia Líquida de Alta Pressão , Humanos , Radioisótopos do Iodo , Cinética , Ligantes , Masculino , Próstata/citologia , Próstata/metabolismo , Hiperplasia Prostática/patologia , Receptores de Endotelina/metabolismoRESUMO
OBJECTIVE: Treatment of normal cartilage with transforming growth factor beta (TGFbeta) can increase the synthesis of collagenase 3 by chondrocytes and mimic the in situ distribution of this enzyme in osteoarthritic (OA) cartilage, which occurs predominantly in the deep zone. In this study, we examined the elements of the TGFbeta system that are potentially relevant to this effect. METHODS: TGFbeta1 and TGFbeta2 levels in cultured cartilage explants were determined by enzyme-linked immunosorbent assay (ELISA). OA cartilage explants were treated with small latent TGFbeta1 complex in the presence of various inhibitors, and collagenase 3 levels were determined by ELISA. The inhibitors were against serine proteases, plasmin, cathepsins, furin, and a neutralizing antibody against the mannose-6 phosphate/ insulin-like growth factor 2 receptor (M6P/IGF-2R). Small latent TGFbeta1, TGFbeta receptor types I, II, and III (TGFbetaRI, RII, and RIII), M6P/IGF-2R, and furin were immunolocalized in cartilage. RESULTS: Our data showed that latent TGFbeta1 is the major isoform that is synthesized; levels of 17.2 +/-1.7 pg/mg and 1.1 +/- 0.3 pg/mg tissue wet weight (mean +/- SEM) were found for total TGFbeta1 and TGFbeta2, respectively, in OA cartilage. A general serine protease inhibitor abrogated activation of both endogenous and exogenous small latent TGFbeta1. Plasmin and furin inhibitors and anti-M6P/IGF-2R reduced the levels of exogenous small latent TGFbeta1 complex-induced collagenase 3 by 33%, 95%, and 76%, respectively, but the cathepsin inhibitor had no effect. Immunolocalization of the small latent TGFbeta1 complex as well as of TGFbetaRI and RII revealed a statistically significant increase in the chondrocyte score in only the deep zone of OA cartilage. The M6P/IGF-2R level was significantly higher in OA cartilage in both the superficial and deep zones. Furin was found in normal cartilage exclusively in the superficial zone, whereas in OA cartilage, a level similar to that in normal cartilage was found in the superficial zone, but a significantly higher cell score (mean +/- SEM 23.6 +/- 4.7%) was registered in the deep zone. CONCLUSION: The mechanisms of TGFbeta activation/ activity with regard to collagenase 3 modulation in cartilage appear to be controlled by furin convertase with or without M6P/IGF-2R. These factors and the small latent TGFbeta complex are increased in the deep zone of OA cartilage, corresponding to the preferential site of collagenase 3 production.
Assuntos
Cartilagem Articular/química , Colagenases/efeitos dos fármacos , Osteoartrite do Joelho/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Idoso , Colagenases/metabolismo , Feminino , Furina , Humanos , Masculino , Metaloproteinase 13 da Matriz , Subtilisinas/fisiologiaRESUMO
OBJECTIVE: To study the expression and production of interleukin-1beta-converting enzyme (ICE) in human normal and osteoarthritic (OA) cartilage and synovium, quantitate the level of ICE in OA chondrocytes, and examine the relationship between the topographic distribution of ICE, interleukin-1beta (IL-1beta), and IL-18, as well as apoptosis of chondrocytes. METHODS: The expression and synthesis of ICE were investigated in human normal and OA cartilage and synovial membrane using in situ hybridization and immunohistochemical methods. The intracellular level of ICE in OA chondrocytes was also measured by enzyme-linked immunosorbent assay (ELISA). Furthermore, the topographic relationship between the presence of ICE and mature IL-1beta and IL-18 was examined by immunohistochemistry, and apoptotic chondrocytes by the TUNEL technique. RESULTS: ICE was expressed and synthesized in both human synovial membrane and cartilage, with a significantly greater number of cells staining positive in OA tissue than in normal tissue. ICE production was preferentially located in the superficial and upper intermediate layers of articular cartilage. With a specific ELISA, a level of 230.2+/-22.5 pg/5 x 10(5) cells (mean +/- SEM) of ICE was found in OA chondrocytes. In cartilage, IL-1beta and IL-18 stained positive at a topographic location similar to that of ICE. The production of mature IL-1beta in OA cartilage explants and chondrocytes was completely blocked by treatment with a specific ICE inhibitor, which also markedly diminished the number of IL-18-positive cells. The data show that there was no close relationship between the presence of ICE and the presence of apoptotic chondrocytes in OA cartilage. CONCLUSION: This study shows, for the first time, the presence of active ICE in human articular cartilage, with a markedly increased cellular level in OA tissue. The relationship between active IL-1beta and ICE suggests that ICE may promote OA progression by activating this proinflammatory cytokine. The role of IL-18 in pathologic cartilage is discussed.
Assuntos
Osteoartrite/enzimologia , Adulto , Idoso , Apoptose/fisiologia , Northern Blotting , Cartilagem Articular/química , Cartilagem Articular/citologia , Cartilagem Articular/metabolismo , Caspase 1/genética , Caspase 1/isolamento & purificação , Caspase 1/fisiologia , Humanos , Imuno-Histoquímica , Hibridização In Situ , Interleucina-1/biossíntese , Interleucina-18/biossíntese , Pessoa de Meia-Idade , RNA Mensageiro/análise , Membrana Sinovial/química , Membrana Sinovial/metabolismoRESUMO
OBJECTIVE: The coexistence of different collagenases in cartilage suggests the possibility of specific roles for these enzymes in the degradation of the collagen network in osteoarthritis (OA). We investigated the in situ synthesis and distribution of collagenase- and collegenase-3 in normal and early experimental OA cartilage. METHODS: The OA model was created on 12 mongrel dogs by sectioning the anterior cruciate ligament of the right stifle joint with a stab wound. Dogs were divided into 3 groups of 4 animals each, and sacrificed at 4, 8, and 12 weeks, respectively. A 4th group (n = 4) of unoperated dogs was used as control. Articular cartilage from femoral condyles and tibial plateaus was examined histologically to grade severity of lesions, and immunohistochemical and morphometric analyses were performed to detect the presence of chondrocytes producing collagenase-1 and -3. RESULTS: In OA dogs, the histologic severity of lesions increased with time, being most severe at 8 and 12 weeks after surgery. In cartilage from OA compared to unoperated dogs, the immunoreactivity was 5-9 times higher (p < 0.0002) for collagenase-1, and 3-6 times higher (p < 0.0002) for collagenase-3, in both femoral condyles and tibial plateaus. Although the cell score increased throughout the cartilage, comparison of the superficial and upper intermediate layers (superficial) with the lower intermediate and deep layers (deep) revealed a significantly higher level for collagenase-1 (p < 0.007) in the superficial layers, contrary to the collagenase-3 data, which indicated a higher level (p < 0.007) in the deep layers. For collagenase- , the cell score increased steadily up to the 12th week, and for collagenase-3, the elevation peaked at 8 weeks. Correlation between the histologic severity and cell score in cartilage specimens from unoperated and OA dogs revealed the highest coefficient for collagenase- at the superficial layers (r = 0.69, p < 0.0001), while for collagenase-3, this was noted at the deep layers (r = 0.65, p < 0.0004). CONCLUSION: The number of chondrocytes involved in the synthesis of collagenase-1 and collegenase-3 increases dramatically in the early phase of OA. However, the difference in the topographic distribution of these enzymes, as well as the variation in their correlation pattern, may reflect a different function allocated for each collagenase in the OA cartilage degradation process.
Assuntos
Cartilagem/enzimologia , Colagenases/biossíntese , Osteoartrite/enzimologia , Animais , Cartilagem/patologia , Colagenases/análise , Cães , Imuno-Histoquímica/métodos , Metaloproteinase 1 da Matriz , Metaloproteinase 13 da Matriz , Osteoartrite/patologiaRESUMO
The goal of this study was to determine the efficacy of local IL-1Ra gene therapy by intra-articular plasmid injections on structural changes in the meniscectomy rabbit model of osteoarthritis. A partial meniscectomy of the right knee was performed on the rabbits through a medial parapatellar incision. The rabbits were then divided into four experimental groups. Group 1 received no treatment. Group 2 received three consecutive intra-articular injections at 24-hour intervals of 0.9% saline containing a lipid, gammaAP-DLRIE/DOPE, and a DNA plasmid, VR1012. Group 3 received three consecutive injections of saline containing 1000 microg of canine IL-1Ra plasmid and lipid. The injections were given starting 4 weeks post-surgery. Rabbits from Group 1 were killed 4 weeks post-surgery, and all other rabbits 8 weeks post-surgery. The severity of macroscopic and microscopic changes on cartilage on the medial and femoral condyles and tibial plateaus and synovium were graded separately. Specimens were also processed for immunohistochemical staining using a rabbit polyclonal antibody against canine IL-1Ra. The level of canine IL-1Ra in synovial fluid was determined using enzyme-linked immunosorbent assay. The presence of the DNA plasmid in the synovium was tested by polymerase chain reaction. A significant reduction in the width of osteophytes and size of macroscopic lesions (P < 0.04) was observed, and was dependent on the amount of IL-1Ra plasmid injected. A significant reduction was also noted in the severity of histologic cartilage lesions (P < 0.01) in the group that received the highest dosage (1000 microg) of IL-1Ra plasmid. IL-1Ra was detected in synovial fluid by enzyme-linked immunosorbent assay and by immunohistochemical staining in the synovium and cartilage of rabbits that received injections containing the IL-1Ra plasmid. Polymerase chain reaction analysis of synovial DNA revealed the presence of the cloned cDNA dog IL-1Ra up to 4 weeks after the first intra-articular injection. This study demonstrates that direct in vivo transfer of the IL-1Ra gene into osteoarthritis knee cells using intra-articular injections of a plasmid vector and lipids can significantly reduce the progression of experimental osteoarthritis. This avenue may therefore represent a promising future treatment for osteoarthritis.