Effect of epithelial debridement on human cornea proteoglycans

Corneal transparency is attributed to the regular spacing and diameter of collagen fibrils, and proteoglycans may play a role in fibrillogenesis and matrix assembly. Corneal scar tissue is opaque and this opacity is explained by decreased ultrastructural order that may be related to proteoglycan composition. Thus, the objectives of the present study were to characterize the proteoglycans synthesized by human corneal explants and to investigate the effect of mechanical epithelial debridement. Human corneas unsuitable for transplants were immersed in F-12 culture medium and maintained under tissue culture conditions. The proteoglycans synthesized in 24 h were labeled metabolically by the addition of 35S-sulfate to the medium. These compounds were extracted by 4 M GuHCl and identified by a combination of agarose gel electrophoresis, enzymatic degradation with protease and mucopolysaccharidases, and immunoblotting. Decorin was identified as the main dermatan sulfate proteoglycan and keratan sulfate proteoglycans were also prominent components. When the glycosaminoglycan side chains were analyzed, only keratan sulfate and dermatan sulfate were detected (~50% each). Nevertheless, when these compounds were 35S-labeled metabolically, the label in dermatan sulfate was greater than in keratan sulfate, suggesting a lower synthesis rate for keratan sulfate. 35S-Heparan sulfate also appeared. The removal of the epithelial layer caused a decrease in heparan sulfate labeling and induced the synthesis of dermatan sulfate by the stroma. The increased deposit of dermatan sulfate proteoglycans in the stroma suggests a functional relationship between epithelium and stroma that could be related to the corneal opacity that may appear after epithelial cell debridement.Universidade Federal de São Paulo (UNIFESP) Escola Paulista de Medicina Departamento de BioquímicaUniversidade Federal de São Paulo (UNIFESP) Escola Paulista de Medicina Departamento de OftalmologiaUNIFESP, EPM, Depto. de BioquímicaUNIFESP, EPM, Depto. de OftalmologiaSciEL

Caracterização e imunolocalização de proteoglicanos de condroitim sulfato e/ou dermatam sulfato de matriz extracelular

-Proteoglicanos são macromoléculas complexas formadas por um esqueleto protéico ao qual está covalentemente ligada pelo menos uma cadeia de glicosaminoglicano. Glicosaminoglicanos (GAGs) são heteropolissacarídeos lineares formados por unidades dissacarídicas repetitivas em que um dos açúcares é uma hexosamina e o outro um açúcar não nitrogenado. Os PGs são componentes essenciais da matriz extracelular (MEC) e possuem papéis altamente diversificados agindo como organizadores celulares e influenciando o crescimento da célula e a maturação de tecidos especializados. Atuam também no armazenamento e modulação da atividade de fatores de crescimento. Alterações significativas no conteúdo, estrutura e concentração de PGs foram descritas em diferentes condições fisiopatológicas, como em artrose, diversos tipos de tumorais, além de células transformadas e mantidas em cultura. Nos tumores, estas modificações podem facilitar o crescimento, a progressão e a invasão tumoral. Estudos para a determinação das causas dessas modificações têm sido intensificados, mas questões relevantes permanecem ainda sem respostas. Por exemplo, que alterações ocorrem nas doenças que possam explicar tais modificações e como elas afetam a estrutura da matriz extracelular? Qual é o papel dos proteoglicanos nesse contexto? Com base nessas questões, o objetivo do presente trabalho foi caracterizar e analisar a distribuição de PGs de condroitim e/ou dermatam sulfato de MEC em 15 diferentes tecidos de coelhos da linhagem New Zealand. Proteoglicanos obtidos por extrações sucessivas com GuaHCl 4 M em tampão acetato 0,05 M, pH 6,4, foram submetidos à cromatografia de troca-iônica em Q-Sepharose para eliminação de outras moléculas de matriz, tais como, colágenos e proteínas não fibrosas. PGs purificados foram submetidos às análises por eletroforese em gel de agarose e SDS-PAGE e degradação proteolítica. Foram observadas bandas com migração característica de PGs em todos os tecidos. Por imunoblotting foi detectado decorim em aorta, intestino grosso músculo, pâncreas, traquéia e estômago e PGs de queratam sulfato em coração, fígado. Em aorta, cérebro, intestino delgado, córnea, útero e bexiga foram detectados ambos PGs. Análise histológica por imunofluorescência em aorta, útero revelou distribuição diferencial de PGs de condrotim e de dermatam sulfato na matriz desses tecidos

Purification and utilization of chondroitinases AC, B and C from Flavobacterium in the study of proteoglycans the extracellular matrix

BV UNIFESP: Teses e dissertaçõe

Reduced urinary excretion of sulfated polysaccharides in diabetic rats

The aim of the present study was to further understand the changes in renal filtration that occur in the early stages of diabetes mellitus. Diabetes was induced in male Wistar rats by a single injection of streptozotocin. Glycemia, body weight, 24-h urine volume and urinary excretion of creatinine, protein and glycosaminoglycans were measured 10 and 30 days after diabetes induction. All the diabetic animals used in the present study were hyperglycemic, did not gain weight, and presented proteinuria and creatinine hyperfiltration. in contrast, the glycosaminoglycan excretion decreased. Dextrain sulfates of different molecular weights (6.0 to 11.5 kDa) were administered to the diabetic rats, and to age-matched, sham-treated controls. Most of the dextran sulfate was excreted during the first 24 h, and the amounts excreted in the urine were inversely proportional to the dextran sulfate molecular weight for all groups. Nevertheless, diabetic rats excreted less and accumulated more dextran sulfate in kidney and liver, as compared to controls. These differences, which were observed only for the dextran sulfates of higher molecular weights (> 7 kDa), increased with the duration of diabetes. Our findings suggest differential renal processing mechanisms for proteins and sulfated polysaccharides, with the possible involvement of kidney cells. (c) 2004 Elsevier B.V. All rights reserved

Cecropia pachystachya Leaves Present Potential to Be Used as New Ingredient for Antiaging Dermocosmetics

Several biological activities have been reported for leaf extracts of Cecropia pachystachya species, including antioxidant and wound healing activities. This study aims to report, for the first time, the antiaging potential of the hydroethanolic (HE) and the ethanolic (EE) extracts obtained from the leaves of C. pachystachya using different in vitro assays. Both HE and EE presented relevant antioxidant capacity in different models, including phosphomolybdenum, 1,1-diphenyl-2-picryl-hydrazyl (DPPH), carotene/linoleic acid bleaching, and thiobarbituric acid reactive substances (TBARS) assays. Their ability to prevent the production of advanced glycation end products (AGEs) was also evaluated, and both extracts showed important activity, especially HE. The extracts also stimulated the fibroblasts proliferation in vitro, specialized cells that produce several mediators which maintain the skin integrity and youthfulness. Cytotoxicity of the extracts was not observed for this lineage or HEK-293, human embryonic kidney cells widely used to evaluate cytotoxicity of chemical compounds. HE also exhibited the ability to inhibit the collagenase (metalloproteinase MMP-2) and elastase activities. The total phenolic and flavonoids contents were also determined. HPLC analysis revealed the presence of the flavonoids orientin and iso-orientin, which were quantified to be used as chemical markers. The results suggested that the extracts of C. pachystachya leaves present the potential to be used in dermocosmetic formulations to prevent the skin aging process, which attracts the attention of pharmaceutical companies and researchers interested in the development of novel ingredients likely to be used as active principles in antiaging products

POSITIVE CORRELATION BETWEEN DISEASE ACTIVITY INDEX AND MATRIX METALLOPROTEINASES ACTIVITY IN A RAT MODEL OF COLITIS

ContextInflammatory bowel disease, including ulcerative colitis and Crohn’s disease, comprising a broad spectrum of diseases those have in common chronic inflammation of the gastrointestinal tract, histological alterations and an increased activity levels of certain enzymes, such as, metalloproteinases.ObjectivesEvaluate a possible correlation of disease activity index with the severity of colonic mucosal damage and increased activity of metalloproteinases in a model of ulcerative colitis induced by dextran sulfate sodium.MethodsColitis was induced by oral administration of 5% dextran sulfate sodium for seven days in this group (n=10), whereas control group (n=16) received water. Effects were analyzed daily by disease activity index. In the seventh day, animals were euthanized and hematological measurements, histological changes (hematoxylin and eosin and Alcian Blue staining), myeloperoxidase and metalloproteinase activities (MMP-2 and MMP-9) were determined.ResultsDextran sulfate sodium group showed elevated disease activity index and reduced hematological parameters. Induction of colitis caused tissue injury with loss of mucin and increased myeloperoxidase (P<0.001) and MMP-9 activities (45 fold) compared to the control group.ConclusionsIn this study, we observed a disease activity index correlation with the degree of histopathological changes after induction of colitis, and this result may be related mainly to the increased activity of MMP-9 and mieloperoxidase