Oxymatrine alleviates periodontitis in rats by inhibiting inflammatory factor secretion and regulating MMPs/TIMP protein expression

Abstract Purpose: To investigate the effect of oxymatrine on periodontitis in rats and related mechanism. Methods: Ninety SD rats were divided into control, model, 10, 20 and 40 mg/kg oxymatrine and tinidazole groups. The periodontitis model was established in later 5 groups. The 10, 20 and 40 mg/kg oxymatrine groups were intragastrically administrated with 10, 20 and 40 mg/kg oxymatrine, respectively. The tinidazole group was intragastrically administrated with 100 mg/kg tinidazole. The treatment duration was 4 weeks. The tooth mobility, gingival and plaque indexes, serum inflammatory factor levels and gingival tissue matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinase (TIMP) protein levels were detected. Results: After treatment, compared with model group, in 40 mg/kg oxymatrine group the rat general conditions were obviously improved, the tooth mobility, gingival index and plaque index were significantly decreased (P<0.05), the serum tumor necrosis factor-α, interleukin-1β and prostaglandin E2 levels were significantly decreased (P<0.05), the MMP-2 and MMP-9 protein levels were significantly decreased (P<0.05), and the TIMP-2 protein level was significantly increased (P<0.05). Conclusions: Oxymatrine can alleviate the experimental periodontitis in rats. The mechanism may be related to its inhibiting inflammatory factor secretion and regulating MMPs/TIMP protein expression.

Etiologic role of root canal infection in apical periodontitis and its relationship with clinical symptomatology

Abstract: Evidence shows the polymicrobial etiology of endodontic infections, in which bacteria and their products are the main agents for the development, progression, and dissemination of apical periodontitis. Microbial factors in necrotic root canals (e.g., endotoxin) may spread into apical tissue, evoking and supporting a chronic inflammatory load. Thus, apical periodontitis is the result of the complex interplay between microbial factors and host defense against invasion of periradicular tissues. This review of the literature aims to discuss the complex network between endodontic infectious content and host immune response in apical periodontitis. A better understanding of the relationship of microbial factors with clinical symptomatology is important to establish appropriate therapeutic procedures for a more predictable outcome of endodontic treatment.

Expressão de metaloproteinases de matriz e PCNA em úlceras de córnea profundas, induzidas em coelhos, tratadas com plasma rico em plaquetas / Expression of matrix metalloproteinases and PCNA in deep corneal ulcers induced in rabbits, treated with platelet-rich plasma

O objetivo deste estudo foi avaliar a influência do plasma rico (PRP) e pobre (PPP) em plaquetas na proliferação celular e expressão de metaloproteinases de matriz (MMPs), durante a reparação de úlceras corneais profundas. Foram utilizadas 45 coelhas, distribuídas em 3 grupos (G) experimentais (n=15), designados como grupos PRP (GR), PPP (GP) e Controle (GC), de acordo com o tratamento. Todos os animais foram submetidos à indução cirúrgica unilateral de úlcera corneal. No GR e GP, o sangue autólogo foi centrifugado, utilizando-se protocolo padronizado, e foram confeccionados os colírios de PRP e PPP, e instilados cinco vezes ao dia. No GC, foi utilizado colírio lubrificante. Cada grupo foi subdividido (n=5), segundo o momento final de avaliação, sendo 4 (M4), 7 (M7) e 30 dias (M30). As córneas dos animais foram processadas para avaliação morfológica e imuno-histoquímica para PCNA, MMP1, MMP2, MMP9, MT1-MMP e TIMP1. No M4, os níveis de MMP2 foram maiores no GP e GR, sendo que, no M7, esse comportamento foi observado apenas no GP. No M30, no GR, verificou-se maior número de células epiteliais e marcação para MMP1 que o GP. No GR, a proliferação celular foi maior no M4 que nos demais momentos, e a marcação para MMP2 foi maior no M4 que no M30. O PRP estimula a proliferação celular na fase inicial (M4) do tratamento quando comparado aos demais momentos, diferentemente dos demais tratamentos. O uso de colírios de plasma rico e pobre em plaquetas influencia a expressão de metaloproteinases de matriz envolvidas no processo de reparação corneal.

The aim of this study was to evaluate the influence of platelet-rich (PRP) and poor (PPP) plasma in cell proliferation and matrix metalloproteinases (MMPs) expression during the repair of deep corneal ulcers. Forty-five female rabbits were distributed in 3 experimental groups (G) (n = 15), referred to as PRP (GR), PPP (GP) and Control (GC) groups, in accordance with the treatment. All animals underwent surgical induction of unilateral corneal ulcer. PRP and PPP eye drops were made by using centrifuged blood through standardized protocol, and instilled five times a day. In GC, lubricant eye drops were used. Each group was subdivided (n = 5) according to the final time point, 4 (M4), 7 (M7) and 30 days (M30). The animals' corneas were processed for morphological and immunohistochemical analysis for PCNA, MMP1, MMP2, MMP9, MT1-MMP and TIMP1. In M4, the levels of MMP2 were higher in GP and GR, and in M7, this behavior was only observed in the GP. In M30, more epithelial cells and MMP1 expression were found in GR than GP. In GR, cell proliferation was higher in M4 than at other time points and MMP2 expression was higher in M4 than M30. The PRP stimulates cell proliferation in the early phase (M4) of treatment when compared to other time points, different from other treatments. The use of eye drops of platelet-rich and poor plasma influences the expression of matrix metalloproteinases involved in the corneal repair process.

Loss of TIMP-1 immune expression and tumor recurrence in localized prostate cancer

Introduction and objective: Overexpression of MMPs has been related to biochemical recurrence after radical prostatectomy. TIMP1 and TIMP2 are controllers of MMPs and the aim of this study is to evaluate the expression levels of MMPs and their regulators using immunohistochemistry in tissue microarray of localized prostate cancer (PC). Materials and Methods: Immune-expression of MMP-9, MMP-2, TIMP1, TIMP-2, MMP-14 and IL8, were analyzed by immunohistochemistry in radical prostatectomy specimens of 40 patients with localized PC who underwent surgery between September 1997 and February 2000. Protein expression was considered as categorical variables, negative or positive. The results of the immune-expression were correlated to Gleason score (GS), pathological stage (TNM), pre-operatory PSA serum levels and biochemical recurrence in a mean follow up period of 92.5 months. Results: The loss of TIMP1 immune-expression was related to biochemical recurrence. When TIMP1 was negative, 56.3% patients recurred versus 22.2% of those whose TIMP1 was positive (p=0.042). MMP-9, MMP-2, IL8 and MMP-14 were positive in the majority of PC. TIMP-2 was negative in all cases. Conclusion: Negative immune-expression of TIMP1 is correlated with biochemical recurrence in patients with PC possibly by failing to control MMP-9, an important MMP related to cancer progression.

Biochemical evaluation of the supporting structure of pelvic organs in selected numbers of premenopausal and postmenopausal Malaysian women.

Context: Pelvic organ prolapse (POP) is associated with menopause and changes in the proteins of the pelvic supporting system, but there is scant data on the precise alterations in Malaysian women. Aim: The aim of this study is to determine the differences in the extracellular matrices (ECM) of uterosacral ligaments in premenopausal and postmenopausal Malaysian women with or without POP. Settings and Design: The observational study was conducted for 9 months in three general hospitals involving 30 women who underwent hysterectomies for various indications except for carcinoma of pelvic organs. Materials and Methods: Three groups were identified: Premenopausal women (Group 1), postmenopausal women without POP (Group 2), and postmenopausal women with POP (Group 3). Age, duration of menopause, body mass index (BMI), parity, and vaginal deliveries were documented. Only 21 samples of the uterosacral ligaments were stained immunohistochemically for collagen I and III, matrix metalloproteinases (MMPs) 1 and 2, elastin, and tenascin. Statistical Analysis Used: Image J software analysis was utilized for quantification, while non-parametric statistics (Kruskal-Wallis with post-hoc Dunns Multiple Comparison test) was used for result analysis. Results: The profile parameters were not significantly different except for mean age and duration of menopause in Group 3. Samples from Group 2 showed lower expression of almost all proteins except MMP1 and tenascin (higher) as compared to Group 1. The changes appeared to be exaggerated in Group 3, though statistically insignificant. Conclusion: A significant difference in the expression of ECM was apparent in postmenopausal subjects as compared to premenopausal ( P = 0.05), compromising the uterosacral ligament tensile strength. The findings are proven similar as those changes in women from other studies.

Influência de materiais odontológicos na capacidade de resposta de fibroblastos cultivados de polpa dental humana / Influence of dental materials on the response capability of cultured fibroblasts from human dental pulp

O presente trabalho tem como objetivo investigar a influência de materiais utilizados na prática odontológica (Single Bond, HEMA, Vitrebond, Ketac Molar e Dycal) na resposta inflamatória de fibroblastos cultivados de polpa dental humana de dentes permanentes em relação à expressão e produção de mediadores da inflamação. As culturas primárias de fibroblastos foram estabelecidas a partir do tecido pulpar de terceiros molares hígidos. Após a quarta passagem, os fibroblastos foram estimulados pelos materiais e pelos materiais seguidos por LPS de E. coli pelos tempos de 6 e 24 horas. Os testes utilizados foram: MTT, Trypan Blue, Análise de Griess, PCR quantitativo e ELISA. Os dados foram analisados estatisticamente aplicando-se o teste ANOVA a 1 critério e pós-teste de Tukey e ANOVA a 2 critérios e teste de correção de Bonferroni (p<0,05). Os materiais SB10 (Single Bond 1:100) e DY (Dycal) afetaram a viabilidade celular com diminuição do metabolismo. Os materiais SB1 (Single Bond 1:1.000), SB10 (Single Bond 1:100) e VB (Vitrebond) seguidos de LPS de E. coli diminuíram o metabolismo celular de maneira estatisticamente significativa. Os níveis de óxido nítrico produzidos foram diminuídos quando os fibroblastos foram estimulados pelo KM (Ketac Molar). A expressão gênica para pró-colágeno tipo I foi diminuída quando os fibroblastos foram estimulados pelos materiais SB10 (Single Bond 1:100), SB (Single Bond polimerizado) e DY (Dycal). Para o SDF-1_/CXCL12 houve um aumento da expressão para o grupo estimulado apenas por LPS de E. coli, SB10 (Single Bond 1:100) e DY (Dycal). Para o IL-6 notou-se uma diminuição significativa para o grupo estimulado por H1000 (HEMA 1000 nM) e um aumento para o grupo SB10 (Single Bond 1:100). A expressão gênica de IL- 8/CXCL8 diminuiu para os fibroblastos estimulados pelas três concentrações de HEMA e de Single Bond, VB (Vitrebond) e DY (Dycal) no período de 6 horas e houve um aumento para os materiais SB10 (Single Bond 1:100) e VB...

The aim of the present study is to investigate the influence of dental materials (Single Bond, HEMA, Vitrebond, Ketac Molar e Dycal) in the inflammatory response of human dental pulp fibroblasts from permanent teeth in relation to inflammatory mediators expression. and production. Primary cultures were established from third molars pulp tissue. After the fourth passage, the fibroblasts were stimulated only by materials and also by the materials followed by LPS from E. coli for 6 and 24 hours. Data were statistically analyzed using Oneway ANOVA and Tukey post-test and Two-way ANOVA followed by Bonferroni post-test (p<0.05). SB10 (Single Bond 1: 100) and DY (Dycal) affected cell viability and consequently decreased cell metabolism. SB1 (Single Bond 1:1,000), SB10 (Single Bond 1:100) and VB (Vitrebond) followed by LPS E. coli decreased cell metabolism. Nitric oxide levels were reduced when fibroblasts were stimulated by KM (Ketac Molar). Pro-collagen type I expression was reduced when fibroblasts were stimulated by SB10 (Single Bond 1:100), SB (polymerized Single Bond) and DY (Dycal). SDF-1_/CXCL12 expression was increased for the group stimulated only by LPS from E. coli, SB10 (Single Bond 1:100) and DY (Dycal). IL-6 expression had a significant decrease in the group stimulated by H1000 (HEMA 1000 nM) and an increase for SB10 (Single Bond 1:100) group. The expression of IL-8/CXCL8 decreased when fibroblasts were stimulated by the three concentrations of HEMA and of Single Bond, VB (Vitrebond) and DY (Dycal) at 6 hours and increased for SB10 (Single Bond 1:100) and VB (Vitrebond) at 24 hours. There was decrease in SDF-1_/CXCL12 production for the three concentrations of HEMA and DY (Dycal) and a declining trend for the other materials tested. The production of IL-6 was increased by VB (Vitrebond) and KM (Ketac Molar). The production of IL-8/CXCL8 increased by SB1 (Single Bond 1:1,000), VB...

Influência de materiais odontológicos na capacidade de resposta de fibroblastos cultivados de polpa dental humana / Influence of dental materials on the response capability of cultured fibroblasts from human dental pulp

O presente trabalho tem como objetivo investigar a influência de materiais utilizados na prática odontológica (Single Bond, HEMA, Vitrebond, Ketac Molar e Dycal) na resposta inflamatória de fibroblastos cultivados de polpa dental humana de dentes permanentes em relação à expressão e produção de mediadores da inflamação. As culturas primárias de fibroblastos foram estabelecidas a partir do tecido pulpar de terceiros molares hígidos. Após a quarta passagem, os fibroblastos foram estimulados pelos materiais e pelos materiais seguidos por LPS de E. coli pelos tempos de 6 e 24 horas. Os testes utilizados foram: MTT, Trypan Blue, Análise de Griess, PCR quantitativo e ELISA. Os dados foram analisados estatisticamente aplicando-se o teste ANOVA a 1 critério e pós-teste de Tukey e ANOVA a 2 critérios e teste de correção de Bonferroni (p<0,05). Os materiais SB10 (Single Bond 1:100) e DY (Dycal) afetaram a viabilidade celular com diminuição do metabolismo. Os materiais SB1 (Single Bond 1:1.000), SB10 (Single Bond 1:100) e VB (Vitrebond) seguidos de LPS de E. coli diminuíram o metabolismo celular de maneira estatisticamente significativa. Os níveis de óxido nítrico produzidos foram diminuídos quando os fibroblastos foram estimulados pelo KM (Ketac Molar). A expressão gênica para pró-colágeno tipo I foi diminuída quando os fibroblastos foram estimulados pelos materiais SB10 (Single Bond 1:100), SB (Single Bond polimerizado) e DY (Dycal). Para o SDF-1_/CXCL12 houve um aumento da expressão para o grupo estimulado apenas por LPS de E. coli, SB10 (Single Bond 1:100) e DY (Dycal). Para o IL-6 notou-se uma diminuição significativa para o grupo estimulado por H1000 (HEMA 1000 nM) e um aumento para o grupo SB10 (Single Bond 1:100). A expressão gênica de IL- 8/CXCL8 diminuiu para os fibroblastos estimulados pelas três concentrações de HEMA e de Single Bond, VB (Vitrebond) e DY (Dycal) no período de 6 horas e houve um aumento para os materiais SB10 (Single Bond 1:100) e VB...

The aim of the present study is to investigate the influence of dental materials (Single Bond, HEMA, Vitrebond, Ketac Molar e Dycal) in the inflammatory response of human dental pulp fibroblasts from permanent teeth in relation to inflammatory mediators expression. and production. Primary cultures were established from third molars pulp tissue. After the fourth passage, the fibroblasts were stimulated only by materials and also by the materials followed by LPS from E. coli for 6 and 24 hours. Data were statistically analyzed using Oneway ANOVA and Tukey post-test and Two-way ANOVA followed by Bonferroni post-test (p<0.05). SB10 (Single Bond 1: 100) and DY (Dycal) affected cell viability and consequently decreased cell metabolism. SB1 (Single Bond 1:1,000), SB10 (Single Bond 1:100) and VB (Vitrebond) followed by LPS E. coli decreased cell metabolism. Nitric oxide levels were reduced when fibroblasts were stimulated by KM (Ketac Molar). Pro-collagen type I expression was reduced when fibroblasts were stimulated by SB10 (Single Bond 1:100), SB (polymerized Single Bond) and DY (Dycal). SDF-1_/CXCL12 expression was increased for the group stimulated only by LPS from E. coli, SB10 (Single Bond 1:100) and DY (Dycal). IL-6 expression had a significant decrease in the group stimulated by H1000 (HEMA 1000 nM) and an increase for SB10 (Single Bond 1:100) group. The expression of IL-8/CXCL8 decreased when fibroblasts were stimulated by the three concentrations of HEMA and of Single Bond, VB (Vitrebond) and DY (Dycal) at 6 hours and increased for SB10 (Single Bond 1:100) and VB (Vitrebond) at 24 hours. There was decrease in SDF-1_/CXCL12 production for the three concentrations of HEMA and DY (Dycal) and a declining trend for the other materials tested. The production of IL-6 was increased by VB (Vitrebond) and KM (Ketac Molar). The production of IL-8/CXCL8 increased by SB1 (Single Bond 1:1,000), VB...

Inmunodetección de metaloproteinasas de matriz extracelular (MMPs)-2, -9, -13 y -14 en lesiones apicales asociadas con periodontitis apical asintomática / Immunodetection of matrix metalloproteinases (MMPs)-2, -9, -13 and -14 in periapical lesions associated with asymptomatic apical periodontitis

La periodontitis apical asintomática (PAa) es una patología infecciosa caracterizada por destrucción ósea perirradicular asociada a un proceso inflamatorio crónico y producción de mediadores inflamatorios, entre los cuales se encuentran las metaloproteinasas de matriz extracelular (MMPs). Entre éstas, las MMPs-13, -14, -2 y -9, son producidas por el tejido óseo y degradan sinérgicamente el colágeno tipo I, principal componente de los tejidos periodontales, y gelatina, producto de la degradación y desnaturación del colágeno. El objetivo de este estudio fue determinar el patrón de expresión de las MMPs-2, -9, -13 y -14 en granulomas periapicales (GPAs), quistes radiculares inflamatorios (QRIs) y ligamento periodontal sano (LS). Materiales y Métodos: Se seleccionaron 12 pacientes con diagnóstico clínico de PAa e indicación de exodoncia a partir de los cuales se obtuvieron biopsias de lesiones periapicales (LPAs). Como controles, se seleccionaron 7 individuos con indicación de exodoncia de premolares por ortodoncia, obteniéndose biopsias de LS. Se efectuó el diagnóstico anátomo-patológico de los especímenes y se caracterizó la expresión de las MMPs en estudio mediante inmunohistoquímica. Resultados: Las MMPs en estudio sólo se detectaron en GPAs y QRIs, y se inmunolocalizaron principalmente en el infiltrado inflamatorio de éstos. Adicionalmente, la MMP-2 se identificó en fibroblastos del tejido conectivo. Conclusiones: MMPs-2, -9, -13 y -14 se expresan predominantemente en el infiltrado inflamatorio de las LPAs y no en LS, y por tanto se sugiere la participación de estos mediadores en la patogénesis de la PAa.

Asymptomatic apical periodontitis (aAP) is an infectious disease characterized by perirradicular bone destruction associated with chronic inflammation and release of inflammatory mediators, such as matrix metalloproteinases (MMPs). MMPs-13, -14 and -2, -9 are bone-expressed enzymes that can synergistically degrade collagen I, the main component of periodontal extracellular matrix, and gelatin, the product of degradation and denaturation of collagen. The aim of this study was to characterize the expression pattern of MMPs-2, -9, -13, and -14 in periapical granulomas (PGs), radicular cysts (RCs) and healthy periodontal ligament (PDL). Materials and Methods: Individuals with clinical diagnosis of aAP and indication of extraction were selected (N=12), and biopsies of periapical lesions (PLs) were obtained. For controls, 7 subjects with indication of premolar extraction for orthodontic reasons were selected, and PDL biopsies were obtained. Samples were diagnosed by anatomopathological examination and immunohistochemical staining was carried out to characterize MMPs expression. Results: MMPs-2, -9, -13 and -14 detection was limited to PLs and were localized mainly to inflammatory infiltrate on both, PGs and RCs. Additionally, MMP-2 was immunolocalized to fibroblasts from the connective tissue. Conclusions: Whereas MMPs-2, -9, -13 and -14 were not detected in healthy periodontal ligament, they were highly expressed on inflammatory infiltrate from PGs and RCs, suggesting a role of these mediators in aAP pathogenesis.

Papel de TGFß-1 na regulação da expressão de MMPs seus inibidores (TIMPs e Reck) em modelo de carcinoma mamário humano: análise funcional de RECK e sua correlação com dados clínico-patológicos / Role of TGFß-1 as a common regulator of MMPs and their inhibitors (TIMPs e RECK) in human breast cancer cell model: functional analysis of RECK and its correlation with clinical-pathological

A causa de morte da maioria das pacientes com câncer de mama se deve à doença metastática desenvolvida a partir do tumor primário. A degradação dos componentes da matriz extracelular (MEC), um dos principais eventos do processo metastático, é regulada pelo balanço entre as atividades das metaloproteinases de matriz (MMPs) e dos seus inibidores, tanto os inibidores teciduais (TIMPs) como o inibidor associado à membrana (RECK). Contudo, ainda existe pouca informação sobre os mecanismos moleculares responsáveis pela manutenção deste balanço. No presente trabalho, foi investigado o envolvimento de TGF-ß1 (Transforming Growth Factor-ß1), uma citocina multifuncional é capaz tanto de inibir o crescimento celular, quanto de promover invasão e metástase, dependendo do estadiamento e do tipo de tumor, na regulação da expressão de MMPs, TIMPs e RECK, em modelo de câncer de mama. Primeiramente, examinou-se os níveis de expressão de mRNA das isoformas e receptores de TGF-ß, em um painel de cinco linhagens de carcinoma mamário humano, com diferentes potenciais invasivos e metastáticos, por qRT-PCR. Os resultados obtidos demonstraram uma correlação positiva entre a expressão dessas moléculas, e a progressão do caráter invasivo e metastático celular. Em seguida, a linhagem altamente invasiva, MDA-MB-231, foi tratada com diferentes concentrações de TGF-ß1 recombinante. Esta citocina foi capaz de modular a expressão gênica de MMPs (MMP-2 e MMP-9) e de seus inibidores (TIMP- 2 e RECK). Tanto ERK½, quanto p38MAPK mostraram-se envolvidas neste mecanismo. Foi demonstrado que a inibição da atividade de ERK½ alterou a expressão das proteínas MMP-9, TIMP-2 e RECK, enquanto o bloqueio de p38 MAPK afetou os níveis protéicos de MMP-2 e TIMP-2. O aumento do potencial migratório e invasivo da linhagem MDA-MB-231, induzido por TGF-ß1, mostrou-se também dependente da atividade de MMPs, ERK½ e p38MAPK. Dada a ausência de informações sobre o papel de RECK em modelo mamário, a função deste inibidor de MMPs também foi investigada. Primeiramente, analisou-se a expressão de RECK ao longo do desenvolvimento da mama e, posteriormente, em 1040 amostras tumorais de mama humana, através da metodologia de Tissue Microarray, tendo sido possível demonstrar que a alta expressão de RECK associa-se a menor tempo de sobrevida global e livre de doença em 10 anos. Os resultados obtidos indicaram que a expressão da proteína RECK, em oposição ao verificado em outros tipos de tumores, está relacionada ao fenótipo mais agressivo de tumores de mama. Entretanto, a análise funcional de RECK, realizada por meio da utilização de vetores shRNA específicos para a inibição desta proteína, demonstrou que RECK também atua como um inibidor de invasão celular e da expressão de MMP-9, na linhagem MDA-MB-231. Em conjunto, os resultados obtidos neste trabalho contribuíram para a elucidação dos mecanismos moleculares de regulação de RECK, por clássicas moléculas associadas ao processo de tumorigênese (TGF-ß1 e MAPKs), bem como para o esclarecimento de suas funções em modelo mamário, sugerindo-o como mais um promissor candidato a marcador prognóstico e alvo molecular para a terapia do câncer de mama

The metastatic disease is the main mortality cause of breast cancer patients. The metastatic process involves a complex cascade of events, including the organized breakdown of the extracellular matrix (ECM) compounds. The degradation of ECM is tightly regulated by the balance between the activities of matrix metalloproteinases (MMPs) and their inhibitors, the tissue inhibitors (TIMPs) and the membrane-associated inhibitor (RECK). Among the several molecules released and activated by ECM remodeling, TGF-ß1 (Transforming Growth Factor-ß1) is a multifunctional cytokine able to regulate both cell growth inhibition and invasion and metastasis promotion, depending on the tumor stage and type. Since the molecular mechanisms involved in the ECM remodeling control are still not completed understood, in this study, we investigated the involvement of TGF-ß1 in regulating of MMPs, TIMPs and RECK expression, in the breast cancer model. By qRT-PCR, we first examined the gene expression levels of TGF-ß isoforms and receptors, in a panel of five human breast cancer cell lines displaying different degrees of invasiveness and metastatic potential. Our results suggest a positive correlation between the mRNA expression of these molecules and the breast cancer progression. Moreover, the highly invasive breast cancer cell line MDA-MB-231 was treated with different concentrations of recombinant TGF-ß1. We described that this cytokine was able to modulate the gene expression of MMPs (MMP-2 and MMP-9) and MMPs inhibitors (TIMP-2 and RECK) at both the mRNA and protein levels, with ERK½ and p38 MAPK being involved in this molecular mechanism. However, while ERK½ activity inhibition altered MMP-9, TIMP-2 and RECK expression, the p38 MAPK blockage affected the protein levels of MMP-2 and TIMP-2. Finally, we reposted that the TGF-ß1-enhanced migration and invasion capacities of MDA-MB- 231 cells were blocked by MMPs, ERK½ and p38 MAPK inhibitors. Analysis of the RECK function in the breast model was also an objective of this study. We analyzed RECK expression during mammary gland development. We evaluated the RECK protein profile in 1040 breast tumor tissue samples using Tissue Microarray assays. We demonstrated that high expression levels of RECK were associated with shorter overall and disease-free survival in 10 years. Moreover, we verified that RECK is a biomarker of poor prognosis mainly for patients diagnosed with less aggressive breast tumor. Therefore, in contrast to other tumor types, our results indicate that high protein expression levels of RECK are related to a more aggressive phenotype. In fact, the RECK functional analysis, performed by using of shRNA vectors, showed that RECK function remains as an inhibitor of cellular invasion and MMP-9 expression, in MDA-MB-231 cells. Taken together, our results contribute to better understanding of the molecular mechanisms associated to RECK regulation by TGF-ß1 and MAPK as well as to clarify its role in breast model. Thus, we suggests RECK as a new and promising prognostic marker and molecular target candidate for breast cancer therapy

Avaliação das metaloproteinases de matriz -2 e -9 em gatos com desmineralização óssea secundária à tirotoxicose induzida / Evaluation of matrix metalloproteinases -2 and -9 in cats under bone demineralization secondary to induced thyrotoxicosis

Observou-se significativo aumento de atividade das formas ativas das metaloproteinases -2 e -9 em gatos com tirotoxicose induzida e desmineralização óssea. As formas pró e intermediária da metaloproteinase -2 elevaram-se com 14 dias de administração hormonal, porém, posteriormente, houve uma tendência de queda. Observou-se correlação negativa entre a forma ativa das metaloproteinases de matriz -2 e -9 e a densidade mineral óssea da extremidade distal do rádio. Os resultados sugerem aumento da degradação da matriz colágena secundária com a elevação dos hormônios tiroidianos.

Significant increase of activity of active forms of matrix metalloproteinases -2 and -9 in cats under induced thyrotoxicosis and bone demineralization was observed. Pro and intermediated forms of matrix metalloproteinases -2 and -9 increased at 14 days of hormonal treatment, followed by decrease tendency. A negative correlation between active forms of matrix metalloproteinases -2 and -9 and bone mineral density of radius distal extremity was also observed. The results suggest an increase of collagen matrix degradation secondary to high levels of thyroid hormones.

Expressão de metaloproteinases de matriz e de seus inibidores teciduais em carcinomas basocelulares / Expression of matrix metalloproteinasis and their tissue inhibitors in basal cell carcinoma

INTRODUÇÃO: Aproximadamente 80 por cento das neoplasias malignas de pele não-melanomas são carcinomas basocelulares (CBC). Apesar das raras metástases, esses tumores são localmente agressivos. As metaloproteinases de matriz (MMPs), especialmente as MMP-2 e 9, são importantes no processo de invasão. Em contrapartida, os inibidores teciduais das MMPs (TIMPs) têm como principal função a inibição dessas enzimas. OBJETIVO: Investigar a associação de variáveis clinicopatológicas de pacientes portadores de CBC com a expressão de MMP-2, MMP-9, TIMP-1 e TIMP-2. MATERIAL E MÉTODOS: Foram selecionados 31 casos de CBC, sendo então obtidos, retrospectivamente, os dados referentes a idade, sexo e tamanho da lesão. Cortes histológicos das lesões foram submetidos a reação imuno-histoquímica pela técnica estreptavidina-biotina-peroxidase para detecção dos antígenos de interesse. Índices de imunomarcação foram construídos e comparados com os dados previamente obtidos. RESULTADOS: Observou-se correlação significativa entre idade e tamanho da lesão (R = 0,532; p = 0,008). Não foram observadas correlações significativas entre as outras variáveis e a expressão imuno-histoquímica dos antígenos de interesse. CONCLUSÃO: A expressão das metaloproteinases e de seus inibidores teciduais não parece ser influenciada pelos parâmetros investigados. Estudos adicionais são necessários para melhor compreensão de sua associação com o comportamento biológico do CBC.

INTRODUCTION: Approximately 80 percent of non-melanoma skin neoplasias are basal cell carcinomas (BCC). Although metastasis is rare, BBC carcinomas are locally aggressive tumors. Matrix metalloproteinases (MMPs), mainly MMP-2 and MMP-9, play an important role on the invasion process. On the other hand, tissue inhibitors of MMPs (TIMPs) have the main function of inhibiting these enzymes. OBJECTIVE: To investigate the association of clinical-pathological variables of BCC patients with the expression of MMP-2 and MMP-9, TIMP-1 and TIMP-2. Methods: Thirty-one BCC cases were selected. Gender, age of the patients and size of the lesions were obtained retrospectively. Histological cuts of the lesions were exposed to immunohistochemistry reaction by use of the streptavidine-biotin peroxidase technique in order to detect antigens. Immunomarking parameters were established and compared with previous data. RESULTS: A significant correlation between age and size of the lesion was observed (R = 0.532; p = 0.008). No significant correlations between other variables and immunohistochemical expression of antigens were observed. CONCLUSION: The expression of MMPs and TIMPs does not seem to be influenced by the parameters investigated in this work. Additional studies should be made to better understand its association with the biological behavior of basal cell carcinomas.

Tissue mRNA expression in rat of newly described matrix metalloproteinases

Matrix metalloproteinases (MMPs) comprise a large group of endoproteinases that degrade all protein components of the extracellular matrix. Functionally, MMPs contribute to several different physiological as well as pathological conditions. The number of newly described MMPs has increased in recent years, although current knowledge about their expression pattern in various tissues remains incomplete. Here we analyzed the relative mRNA expression of the most recently described MMPs _ MT5-MMP (MMP-24), MT6-MMP (MMP-25), MMP-27 and epilysin (MMP-28) _ in a broad selection of rat tissues using real time-PCR. MMP-24 mRNA was found to be widely expressed with predominance in the central nervous system. MMP-25 mRNA, in contrast, exhibited peak expression levels in testis, kidney and skeletal muscle, differing from previously described distribution patterns in humans. mRNAs for MMP-27 and MMP-28 were generally expressed at a lower level. All four MMPs studied were detected at higher mRNA levels in bone and kidney, suggesting a possible role of these MMPs in physiological processes within these two organs. The present study highlights the differential distribution pattern of newly described MMPs among different tissues and underlines differences in the mRNA expression between different species.