Navegando por Palavras-chave "Proteoglycans"

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    Avaliação do colágeno no osso de ratas diabéticas tipo 1 tratadas com isoflavonas
    (Universidade Federal de São Paulo, 2022-11-29) Vieira, Magno César [UNIFESP]; Simões, Manuel de Jesus [UNIFESP]; Carbone, Adriana Aparecida Ferraz [UNIFESP]; http://lattes.cnpq.br/9174521191304809; http://lattes.cnpq.br/5987164343458678; http://lattes.cnpq.br/5998705224419140
    Neste trabalho, o modelo “ratas diabéticas ovariectomizadas” foi utilizado pensando nas mulheres em menopausa e diabéticas com um tratamento alternativo com isoflavonas da soja, baseamo-nos em dados que mostram um aumento no número de mulheres que procuram utilizar-se de terapias hormonais. Objetivo: Analisar a matriz extracelular no osso de ratas diabéticas tratadas com isoflavonas ou 17β- estradiol. Métodos: Foram utilizadas 60 ratas (Rattus norvegicus albinus), fêmeas, adultas, ±3 meses de idade. Os animais foram separados em seis (6) grupos, a saber: controle Sham (n=10) animais não ovariectomizados na fase de estro; Sham+DM (n=10) controle Sham ratas diabéticas não ovariectomizadas na fase de estro; OVX (n=10) controle, ratas ovariectomizadas que receberam veículo propilenoglicol; OVX+DM (n=10) ratas diabéticas ovariectomizadas que receberam veículo propilenoglicol (fase de estro); OVX+DM+ISO (n=10) animais diabéticos ovariectomizados tratados com isoflavonas da soja (150mg/Kg, por gavagem); OVX+DM+E2 (n=10) animais diabéticos ovariectomizados tratados com estrogênio (17β-estradiol, 10μg/Kg, por via subcutânea). Para a indução do diabetes Tipo 1, as ratas receberam uma única injeção intraperitoneal de 60 mg/Kg de estreptozotocina (STZ, Sigma-Aldrich). O diabetes foi confirmado três dias após a injeção da estreptozotocina. Todos os animais foram tratados durante 30 dias consecutivos, e ao final, os animais foram anestesiados e os fêmures removidos e processados para estudo em H.E. e Picro Sirius Red. Para a análise dos resultados foi utilizado Oneway ANOVA seguido do teste de Tukey. Foram considerados estatisticamente significativos experimentos cujo valor de p foi menor que 5% (p ≤ 0,05) para significância estatística. Os cálculos foram feitos com o programa SPSS versão13 (SPSS, Chicago, IL). Resultados: Os resultados obtidos foram os seguintes: Peso corporal: não foi diferente entre os grupos não diabéticos Sham e OVX e, os grupos de ratas diabéticas Sham+DM, OVX+DM, OVX+DM+ISO e OVX+DM+E2 (p>0,05). No entanto, houve uma diminuição significativa do peso corporal nos grupos de ratas diabéticas em comparação com os grupos não diabéticas Sham e OVX (p<0,001). Sensibilidade à Insulina: Os valores mais baixos de insulina tolerância foram observadas no OVX+DM (2,41±0,95). Os grupos Sham (4,64±0,95) e OVX (4,57±0,58) apresentaram os maiores valores de sensibilidade à insulina (p<0,001). x Os grupos OVX+DM+ISO e OVX+DM+E2 apresentaram valores semelhantes, inferiores aos Sham e OVX e superior ao grupo OVX+DM. Histomorfometria e Análise Bioquímica de Glicosaminoglicanos Sulfatados: O volume do osso trabecular foi maior nos grupos de ratas diabéticas ovariectomizadas OVX+DM+E2 (26,35±2,13) e OVX+DM+ISO (18,36±173), tratadas, respectivamente, com 17β estradiol e isoflavonas e menor no OVX+DM (8,32±2,52), grupo de ratas diabéticas ovariectomizadas que receberam veículo propilenoglicol em fase de estro. Resultados semelhantes foram encontrados em relação à espessura do osso cortical, que foi maior no OVX+DM+E2 (398,4±1,74) e OVX+DM+ISO (295,6±1,45) e menor no OVX+DM (142,6±2,74) (p<0,05). Com relação ao sulfato de condroitina foi encontrado nos ossos de todos os grupos de animais estudados. Picro Sirius Red: As fibras colágenas de substância óssea trabecular e cortical das epífises dos fêmures apresentaram maior intensidade de birrefringência nos grupos (OVX+DM+ISO 330,2±2 e OVX+DM+E2 414,8± 33,7) tratados, respectivamente, com isoflavonas e 17β estradiol
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    Avaliação histológica, ultraestrutural e molecular de córneas com ceratocone e dos efeitos do crosslinking rápido sobre os ceratócitos cultivados em modelo 3D
    (Universidade Federal de São Paulo (UNIFESP), 2021) Covre, Joyce Luciana [UNIFESP]; Gomes, Jose Alvaro Pereira [UNIFESP]; Universidade Federal de São Paulo
    Purpose: Characterize healthy and keratoconus human corneas and keratocytes using histochemical, ultrastructural and molecular analysis and to evaluate the effects of crosslinking treatment at different times on healthy and KC keratocytes cultured in the 3D model. Methods: Cornea samples from control and KC patients were collected and processed for histological, biochemical, immunohistochemical and ultrastructural analyses. Primary keratocytes from control and KC corneas were isolated by collagenase digestion and cultured in DMEM/Ham\'s F12 medium supplemented with 2 % fetal bovine serum. After these cells reach the confluence state (~30 days), they were processed for Western blot analysis to evaluate protein levels implicated in cell structure, adhesion and signaling and an analysis of the lipid profile was performed. Cultured keratocytes were also subjected to CXL treatment at different times. After 24 hours viable cells were quantified by MTT and LDH techniques and apoptotic cells were counted using the caspase 3 marker. The levels of pro-inflammatory cytokines IL-6 and IL-8 and MMP-3, MMP-9 from the supernatants were tested by an enzymelinked immunosorbent assay (ELISA). Results: Histological analysis showed that stroma from KC corneas presented disorganized collagen fibers compared to control samples. Picrosirius-polarization analysis demonstrated enhanced birefringence of collagen networks from control corneas compared to KC. Densitometry of yellow-red/ green birefringence of collagen fibers confirmed these findings and indicates a loss of collagen alignment in the KC. Ultrastructural analysis of KC cornea demonstrated loose fibrillar networks of stroma with small and thin collagen fibril diameters compared to the control samples. In addition, cultured KC cells showed reduced levels of vimentin, focal adhesion kinase (FAK), β1-integrin, keratocan and the activation of Src tyrosine. By contrast, lumican levels were increased in the KC cells and in the corneal stroma. In the analysis of the lipid profile, the potential biomarkers were shown to be completely different in cells with keratoconus compared to control cells (normal). After 24 hours of treatment with CXL, cell viability assessed by MTT and LDH assays indicated that there were no significant changes in the healthy and keratoconus groups. On the other hand, the immunofluorescence analysis showed increased levels of caspase 3 cleaved in healthy cells and keratoconus after 24 hours of treatment with CXL for 30 minutes. The quantification of the levels of pro-inflammatory cytokines IL- 8 and IL-6 and MMP-3 and -9 did not change. Conclusion: Our findings provide that KC is associated with a marked alteration of structural and molecular patterns of corneal stroma and keratocytes. This may be of significance for explanation of pathogenesis of corneal ectactic diseases.
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    Biologia vascular: síntese e remodelamento de moléculas da matriz extracelular e de superfície de células endoteliais em artéria carótida de camundongos ApoE-/-.
    (Universidade Federal de São Paulo (UNIFESP), 2021) Russo, Thatiane Amaral [UNIFESP]; Regatieri, Juliana Luporini Dreyfuss [UNIFESP]; Universidade Federal de São Paulo
    The endothelium is composed of a monolayer of cells and their basemant membrane, which line the inside of the blood vessels and are responsible for maintaining vascular homeostasis. Under physiological conditions, it maintains vascular tone, laminar blood flow, coagulation process, cell proliferation and migration and control of the inflammatory response. Endothelial cells (ECs) respond to mechanical forces such as cell stretching and shear stress, influencing cellular behavior such as: morphology, migration, adhesion and angiogenesis, where many extracellular matrix (ECM) and cell surface components play a fundamental role. Endothelial dysfunction can cause cardiovascular diseases, such as hypertension, thrombosis and atherosclerosis, and these diseases can be modulated by the mechanical forces applied to the artery wall. This study aimed to investigate the remodeling of ECM and molecules associated with this remodeling in ECs submitted in vitro to two mechanical stimuli, cell stretching and shear stress, both in physiological and pathological conditions. After the stimuli, tests of cell behavior (adhesion, cell migration and formation of capillary structures), analysis of the biosynthesis of glycosaminoglycans by incorporating 35S, immunofluorescence, qPCR and qPCR array were performed. Both mechanical forces induced changes the ECs morphology, cell adhesion, migration and formation of capillary structures in culture were modulated positively in physiological conditions and negatively in pathological situations. The pathological mechanical forces applied to ECs induced a higher gene expression of syndecan-4, perlecan, versican, decorin, fibronectin, collagen III α1, connexin-43, VEGFA, TGF-β1 and TGF-β3. In addition, in vivo assays with ApoE-/- deficient mice were used as an animal model to study ECM remodeling in atherosclerotic disease. For this purpose the carotids of ApoE-/- mice were analyzed by high resolution ultrasound, demonstrating that there was an increase in the internal diameter and internal thickness (IMT) of these vessels, characterizing an artery dysfunction. Then these carotids were removed and subjected to analysis of gene expression by qPCR and qPCR array, immunofluorescence and histopathology. It was possible to observe a higher gene expression and a higher fluorescence intensity of syndecan-4, perlecan, versican and connexin-43 in carotids of ApoE-/- mice when compared to wild type control mice. The results of the qPCR array demonstrated higher expression of adhesion molecules, laminins and integrins and genes related to ECM proteolysis in carotids of ApoE-/- mice when compared to wild type control mice. This study helps to understand the remodeling and associated molecules of the ECM in in vitro and in vivo models of cardiovascular diseases, and how the endothelium can be affected by the mechanical forces of cell stretching and shear stress.
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    Biomolecular analysis of matrix proteoglycans as biomarkers in non small cell lung cancer
    (Springer, 2018) Rangel, Maristela P.; Sa, Vanessa K. de; Prieto, Tabatha; Martins, João Roberto Maciel [UNIFESP]; Olivieri, Eloisa R.; Carraro, Dirce; Takagaki, Teresa; Capelozzi, Vera Luiza
    Matrix proteoglycans (PGs) have shown promise as biomarker in malignancies. We employed agarose gel eletrophoresis, quantitative real- time reverse transcription-polymerase chain reaction and immunohistochemistry to evaluate the content of sulfated glicosaminoglycans (chondroitin sulfate and heparan sulfate) and expression of PG (biglycan, glypican, perlecan, syndecan e versican) in patient-matched normal and tumor tissues obtained from resected specimens of lung cancer. A significant increase of heparan sulfate (HS) and chondroitin sulfate (CS) concentrations was found in tumor tissue samples when compared to normal lung tissue samples. HS was also significantly increased in adenocarcinomas compared to squamous cell carcinomas. PG gene expression, with exception of syndecan, were significantly decreased in tumor tissue compared to normal lung, coinciding with significant decrease of PG protein levels in tumor cells and stroma compared to normal lung tissue (Kappa coefficient 0.41, 0.42 and 0,28, respectively). Women patients (p = 0.02), non smokers (p = 0.05), T stage (p = 0.009), N stage (p = 0.03) and adenocarcinoma (p = 0.05) were associated with improved overall survival (OS). Patients presenting tumors with low concentration of sulfated GAG and high PGs levels presented better OS compared to patients with high concentration of sulfated GAG and low expression of PGs. Cox regression model controlled by gender, tobacco history and histological type, showed that patients with high perlecan and versican expression in tumor presented respectively high probability of life (beta risk 11.64
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    Biophysical and pharmacological characterization of a full-length synthetic analog of the antitumor polypeptide crotamine
    (Springer, 2020-09-07) Porta, Lucas de Carvalho [UNIFESP]; Fadel, Valmir; Campeiro, Joana D'Arc [UNIFESP]; Godinho, Rosely Oliveira [UNIFESP]; Hayashi, Mirian Akemi Furuie [UNIFESP]; Oliveira, Eduardo Brandt; http://lattes.cnpq.br/5559309395232147
    Crotamine is a polypeptide isolated from the venom of a South American rattlesnake. Among the properties and biological activities of crotamine, the most extraordinary is its ability to enter cells with unique selective affinity and cytotoxic activity against actively proliferating cells, such as tumor cells. This peptide is also a cargo carrier, and anticipating commercial application of this native polypeptide as a potential theranostic compound against cancer, we performed here a side-by-side characterization of a chemically synthesized full-length crotamine compared with its native counterpart. The structural, biophysical, and pharmacological properties were evaluated. Comparative NMR studies showed structural conservation of synthetic crotamine. Moreover, similarly to native crotamine, the synthetic polypeptide was also capable of inhibiting tumor growth in vivo, increasing the survival of mice bearing subcutaneous tumor. We also confirmed the ability of synthetic crotamine to transfect and transport DNA into eukaryotic cells, in addition to the importance of proteoglycans on cell surface for its internalization. This work opens new opportunities for future evaluation of chimeric and/or point-mutated analogs of this snake polypeptide, aiming for improving crotamine properties and applications, as well as possibly diminishing its potential toxic effects.
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    Caracterização morfométrica e de organização das fibras colágenas e proteoglicanos na cabeça do fêmur de camundongos MDX submetidos ou não à exacerbação da doença por exercício físico
    (Universidade Federal de São Paulo, 2021-09-22) Marangoni, Kevin Pereira [UNIFESP]; De Oliveira, Flavia [UNIFESP]; http://lattes.cnpq.br/3387760393535776; http://lattes.cnpq.br/5620060448568039; Universidade Federal de São Paulo (UNIFESP)
    Introdução: A Distrofia Muscular de Duchenne (DMD) é uma doença genética causada por uma alteração do gene codificador de distrofina no cromossomo X de seres humanos e causa fraqueza muscular progressiva, causada pela atrofia do tecido muscular e intensa inflamação. A DMD acomete principalmente os músculos esqueléticos, contudo, alterações no tecido ósseo também são descritas. Dessa maneira, o objetivo do estudo é avaliar, na epífise proximal do fêmur de camundongos mdx submetidos ou não ao exercício físico, a morfometria, bem como a estrutura do colágeno e proteoglicanos da cartilagem articular, com o intuito de se verificar possíveis alterações morfológicas consequentes da ausência de distrofina. Materiais e métodos: Utilizando o exercício físico como meio para exacerbação da progressão da doença, foram utilizados camundongos C57BL/10 e C57BL10-DMD/mdx com 24 semanas de idade, os quais foram distribuídos em 3 grupos (n=10 cada): Controle (Ctrl), mdx sedentários (Mdx-S) e mdx treinados (Mdx-T), sendo que o grupo Mdx-T foi induzido à prática de exercícios físicos intensos diariamente. Após o período de 8 semanas os animais foram eutanasiados com 32 semanas de idade e o fêmur coletado para análise histológica (hematoxilina/eosina, picrosirius, Safranina-O) e morfométrica. Resultados: Os principais resultados mostraram que apenas o grupo Mdx-S apresentou área da cartilagem articular menor que o grupo controle. Ademais, verificaram-se alterações na organização das fibras colágenas e dos proteoglicanos na cartilagem articular dos animais mdx, independente do grupo (Mdx-S ou MDX-T). Conclusão: Os animais distróficos demonstraram meio extracelular deficiente, caracterizado pela diminuição da síntese de proteoglicanos e desorganização das fibras colágenas, o que pode significar que o processo natural da distrofia muscular pode ter efeito negativo sobre a homeostasia da cartilagem articular de camundongos distróficos, contudo, a exacerbação da doença por exercício físico não demonstrou ter envolvimento com estes achados.
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    Colorectal cancer desmoplastic reaction up-regulates collagen synthesis and restricts cancer cell invasion
    (Springer, 2011-11-01) Coulson-Thomas, Vivien Jane [UNIFESP]; Coulson-Thomas, Yvette May [UNIFESP]; Gesteira, Tarsis F. [UNIFESP]; Paula, Claudia A. A. de [UNIFESP]; Mader, Ana M.; Waisberg, Jaques; Pinhal, Maria A. [UNIFESP]; Friedl, Andreas; Toma, Leny [UNIFESP]; Nader, Helena B. [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Fac Med ABC; Univ Wisconsin
    During cancer cell growth many tumors exhibit various grades of desmoplasia, unorganized production of fibrous or connective tissue, composed mainly of collagen fibers and myofibroblasts. the accumulation of an extracellular matrix (ECM) surrounding tumors directly affects cancer cell proliferation, migration and spread; therefore the study of desmoplasia is of vital importance. Stromal fibroblasts surrounding tumors are activated to myofibroblasts and become the primary producers of ECM during desmoplasia. the composition, density and organization of this ECM accumulation play a major role on the influence desmoplasia has upon tumor cells. in this study, we analyzed desmoplasia in vivo in human colorectal carcinoma tissue, detecting an up-regulation of collagen I, collagen IV and collagen V in human colorectal cancer desmoplastic reaction. These components were then analyzed in vitro co-cultivating colorectal cancer cells (Caco-2 and HCT116) and fibroblasts utilizing various co-culture techniques. Our findings demonstrate that direct cell-cell contact between fibroblasts and colorectal cancer cells evokes an increase in ECM density, composed of unorganized collagens (I, III, IV and V) and proteoglycans (biglycan, fibromodulin, perlecan and versican). the desmoplastic collagen fibers were thick, with an altered orientation, as well as deposited as bundles. This increased ECM density inhibited the migration and invasion of the colorectal tumor cells in both 2D and 3D co-culture systems. Therefore this study sheds light on a possible restricting role desmoplasia could play in colorectal cancer invasion.
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    Concentration of glycosaminoglycan in ovariectomized mice uterus after treatment with ovarian steroids
    (Hindawi Publishing Corp, 2016) Maioral, Gabriela C. C. C. [UNIFESP]; Gomes, Regina Celia T. [UNIFESP]; Verna, Carina [UNIFESP]; Simoes, Manuel de J. [UNIFESP]; Nader, Helena B. [UNIFESP]; Simoes, Ricardo S.; Baracat, Edmund C.; Soares-, Jose Maria, Jr.
    The aim of this study was to evaluate the amount of non-and sulfated glycosaminoglycans in the ovariectomized mice uterus, after treatment with ovarian steroids. For this purpose, 50 adult female mice were divided into five groups with 10 animals/each: control group: CG (ovary intact), and ovariectomized groups: OG (vehicle), EG (estradiol), PG (progesterone) and EPG (estradiol combined to progesterone). The treatments started 30 days after ovariectomy. All the animals were treated for 50 consecutive days. These hormones were administered in a sterile oily solution via gavage. Twenty-four hours after the last treatment, all animals were euthanized, removing the uterine horn for biochemical analyses. To quantify, the hyaluronic acid (HA) used ELISA-like fluorometric assay, and the sulfated glycosaminoglycans (GAGs) used agarose gel electrophoresis. The amount of HA was significantly higher in the group treated with progesterone (PG) compared to the others groups (p < 0.05), and in the group treated with estradiol (EG), the amount of chondroitin/dermatan sulfate was significantly higher compared to the others groups (p < 0.05), and in the group treated with progesterone (PG), the amount of heparan sulfate was significantly lower compared to the others groups, except to control group (p < 0.05). Our results showed that the estroprogestative therapy after long time (50 days) profoundly affected the amount of glycosaminoglycans in uterine. These changes may be indicative of uterine pathology such as the development of tumor.
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    The critical interaction of the metallopeptidase PHEX with heparan sulfate proteoglycans
    (Elsevier B.V., 2008-01-01) Barros, Nilana M. T. [UNIFESP]; Nascimento, Fabio D. [UNIFESP]; Oliveira, Vitor [UNIFESP]; Juliano, Maria Aparecida [UNIFESP]; Juliano, Luiz [UNIFESP]; Loisel, Thomas; Nader, Helena B. [UNIFESP]; Boileau, Guy; Tersariol, Ivarne L. S.; Carmona, Adriana K. [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Enobia Pharma Inc; Univ Montreal; Univ Mogi das Cruzes
    The PHEX gene (phosphate-regulating gene with homologies to endopeptidase on the X chromosome) identified as a mutated gene in patients with X-linked hypophosphatemia (XLH), encodes a protein (PHEX) that shows striking homologies to members of the M13 family of zinc metallopeptidases. in the present work the interaction of glycosaminoglycans with PHEX has been investigated by affinity chromatography, circular dichroism, protein intrinsic fluorescence analysis, hydrolysis of FRET substrates flow cytometry and confocal microscopy. PHEX was eluted from a heparin-Sepharose chromatography column at 0.8 M NaCl showing a strong interaction with heparin. Circular dichroism spectra and intrinsic fluorescence analysis showed that PHEX is protected by glycosaminoglycans against thermal denaturation. Heparin, heparan sulfate and chondroitin sulfate inhibited PHEX catalytic activity, however among them; heparin presented the highest inhibitory activity (K-i = 2.5 +/- 0.2 nM). Flow cytometry analysis showed that PHEX conjugated to Alexa Fluor 488 binds to the cell surface of CHO-K1, but did not bind to glycosaminoglycans defective cells CHO-745. Endogenous PHEX was detected at the cell surface of CHO-K1 colocalized with heparan sulfate proteoglycans, but was not found at the cell surface of glycosaminoglycans defective cells CHO-745. in permeabilized cells, PHEX was detected in endoplasmic reticulum of both cells. in addition, we observed that PHEX colocalizes with heparan sulfate at the cell surface of osteoblasts. This is the first report that the metallopeptidase PHEX is a heparin binding protein and that the interaction with GAGs modulates its enzymatic activity, protein stability and cellular trafficking. (c) 2008 Elsevier B.V. All rights reserved.
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    Efeitos do estiramento mecânico na síntese de matriz extracelular de células musculares lisas vasculares
    (Universidade Federal de São Paulo (UNIFESP), 2017-03-31) Ferreira, Mariana Machado Leiva [UNIFESP]; Dreyfuss, Juliana Luporini [UNIFESP]; http://lattes.cnpq.br/2390066977030420; http://lattes.cnpq.br/4589516020973403; Universidade Federal de São Paulo (UNIFESP)
    O estresse mecânico é capaz de alterar o fenótipo e a morfologia celulares, através de um processo chamado mecanotransdução, que é a promoção de respostas celulares decorrentes de estímulos físicos. O músculo liso vascular está sujeito a deformações mecânicas decorrentes da passagem do sangue pelos vasos sanguíneos, modulando processos como proliferação, migração e apoptose celulares. Deformações mecânicas ânormais/patológicas estão envolvidas em doenças cardiovasculares, uma vez que modulam negativamente o fenótipo de células musculares lisas vasculares. No presente trabalho foi realizada uma investigação da expressão de glicosaminoglicanos sulfatados e outras moléculas da matriz extracelular e de superfície celular em células musculares lisas vasculares em cultura submetidas ao estiramento celular, avaliando seus efeitos em condições fisiológicas e patológicas. Foi utilizado o equipamento Flexcell FX- 5000™ que é capaz de promover deformação biaxial controlada resultando em um estiramento mecânico das células musculares, avaliando seus efeitos em 5% de estiramento (condição fisiológica) e 15% de estiramento (condição patológica) por 4 ou 24 horas. Após estes estímulos mecânicos, as células foram submetidas a ensaios como imunofluorescência, PCR quantitativo, análise dos glicosaminoglicanos sulfatados e ensaios de comportamento ..'- celular. A exposição de células musculares lisas vasculares às forças mecânicas influencia no remodelamento da matriz extracelular e nas interações célula-matriz e célula-célula. As células musculares alteram sua morfologia quando submetidas a estiramento, adquirindo um direcionamento e se tornando alongadas, e têm sua viabilidade aumentada após estimulação mecânica, neste trabalho não se verificaram diferenças na migração celular após estiramentos mecânicos. A síntese de glicosaminoglicanos sulfatados também é influenciada pelas forças mecânicas, células musculares lisas submetidas a estiramentos apresentaram maior quantidade de heparam e condroitin sulfato nas frações celular e no meio de cultura. A quantidade de ácido hialurônico na fração celular e no meio de cultura das células estiradas também foi modificada pelo estiramento.As forças mecânicas modulam a expressão de de Sindecam-4, Colágeno tipo 3, Perlecam, Biglicam, Glipicam-1, TGF~1, Conexina 43, Fibronectina e FAK. O estiramento mecânico também levou à redistribuição de Biglicam, Versican e Perlecam na matriz extracelular e de Sindecam-4 na superfície celular. Tais resultados ajudam a compreender melhor como a hemostasia vascular e as moléculas de matriz extracelular e proteoglicanos são afetados pelas forças mecânicas, e como estas moléculas se comportam em doenças cardiovasculares.
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    Expressão dos glicosaminoglicanos e proteoglicanos na matriz extracelular da cérvice uterina da rata albina prenhe após injeção local de hialuronidase
    (Universidade Federal de São Paulo (UNIFESP), 2012) Souza, Guilherme Negrão de [UNIFESP]; Camano, Luiz [UNIFESP]
    Objetivo: Avaliar a expressao dos glicosaminoglicanos (GAGs) e proteoglicanos (PGs) na matriz extracelular (MEC) da cervice uterina da rata albina prenhe, apos injecao local de hialuronidase. Metodologia: Dez ratas prenhes foram distribuidas aleatoriamente em dois grupos, numericamente iguais. O grupo controle (Gc) foi constituido pelas ratas que receberam 1 mL de agua destilada, dose unica, no 18º dia da prenhez, sob anestesia, ministrado na cervice uterina. No grupo experimental (Gexp), os animais receberam, sob as mesmas condicoes do Gc, 0,02 mL de hialuronidase, diluido em 0,98 ml de agua destilada (total de 1ml). No 20º dia de prenhez, seis horas apos marcacao in vivo de [35SO4] de sulfato radioativo, as ratas foram sacrificadas por inalacao de monoxido de carbono e submetidas a disseccao, preparando-se a cervice uterina para caracterizacao e quantificacao dos GAGs recemsintetizados, condroitim sulfato (CS), dermatam sulfato (DS) e heparam sulfato (HS), extraidos apos proteolise e determinados e caracterizados, apos eletroforese em gel de agarose, por densitometria optica, antes e apos degradacao enzimatica com enzimas especificas. Foram avaliadas tambem a expressao qualitativa do acido hialuronico (AH) por histoquimica e sua quantificacao por ensaio tipo sanduiche em placa de ELISA. A analise estatistica de todas as informacoes coletadas nesta pesquisa foi inicialmente feita de forma descritiva. Para as variaveis de natureza quantitativa (numerica) foram calculadas mediana, valor minimo, valor maximo e confeccionados graficos do tipo diagrama de dispersao unidimensional. A analise inferencial, empregada com o intuito de comparar os valores da quantidade dos GAGs entre os grupos, foi o Teste t-Student para amostras independentes, com o nivel de significancia α igual a 5%. Tambem, nesse estudo, diferentes PGs foram avaliados por imunofluorescencia com anticorpos. Resultados: O comportamento da sintese destas moleculas ocorreu de forma similar e a degradacao enzimatica confirmou a presenca de CS/DS e HS na composicao das amostras estudadas; houve diminuicao significante na quantidade total dos GAGs sulfatados e do AH no Gexp se comparado ao Gc. Na histoquimica, ha dispersao na distribuicao do AH no Gexp. Paralelamente, observou-se no Gexp menor expressao (imunofluorescencia) de diversos PGs como o decorim, biglicam, sindecam e CD44 Conclusoes: O tratamento com hialuronidase (Gexp) promove queda significativa da concentracao dos GAGs sulfatados na cervice uterina de ratas albinas prenhes. Tanto a expressao qualitativa (por histoquimica) quanto a quantitativa (dosagem fluorometrica) do AH tambem se mostraram reduzidas no Gexp. O conjunto dos resultados permite inferir que as alteracoes desses compostos na cervice uterina no Gexp, comparado ao Gc, promovem modificacoes locais semelhantes ao processo de maturacao cervical fisiologica, fatores primordiais ao desencadeamento da parturicao
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    Fibroblast and prostate tumor cell cross-talk: Fibroblast differentiation, TGF-beta, and extracellular matrix down-regulation
    (Elsevier B.V., 2010-11-15) Coulson-Thomas, Vivien Jane [UNIFESP]; Gesteira, Tarsis F.; Coulson-Thomas, Yvette May [UNIFESP]; Vicente, Carolina M.; Tersariol, Ivarne L. S.; Nader, Helena B.; Toma, Leny; Universidade Federal de São Paulo (UNIFESP)
    Growth and survival of tumors at a site of metastasis involve interactions with stromal cells in the surrounding environment. Stromal cells aid tumor cell growth by producing cytokines as well as by modifying the environment surrounding the tumor through modulation of the extracellular matrix (ECM). Small leucine-rich proteoglycans (SLRPs) are biologically active components of the ECM which can be altered in the stroma surrounding tumors. the influence tumor cells have on stromal cells has been well elucidated. However, little is understood about the effect metastatic cancer cells have on the cell biology and behavior of the local stromal cells. Our data reveal a significant downregulation in the expression of ECM components such as collagens I, II, III, and IV, and the SLRPs, decorin, biglycan, lumican, and fibromodulin in stromal cells when grown in the presence of two metastatic prostate cancer cell lines PO and DU145. Interestingly, TGF-beta down-regulation was observed in stromal cells, as well as actin depolymerization and increased vimentin and alpha 5 beta 1 integrin expression. MT1-MMP expression was upregulated and localized in stromal cell protrusions which extended into the ECM. Moreover, enhanced stromal cell migration was observed after crosstalk with metastatic prostate tumor cells. Xenografting metastatic prostate cancer cells together with activated stromal cells led to increased tumorigenicity of the prostate cancer cells. Our findings suggest that metastatic prostate cancer cells create a metastatic niche by altering the phenotype of local stromal cells, leading to changes in the ECM. (C) 2010 Elsevier Inc. All rights reserved.
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    GLYCOSAMINOGLYCANS AND PROTEOGLYCANS IN PALMAR FASCIA OF PATIENTS WITH DUPUYTREN
    (Atha Comunicacao & Editora, 2016) Hirai Nascimento, Priscilla Carneiro [UNIFESP]; Kobayashi, Elsa Yoko [UNIFESP]; de Saboya Lenzi, Luiz Guilherme [UNIFESP]; Gomes dos Santos, Joao Baptista [UNIFESP]; Nader, Helena Bonciani [UNIFESP]; Faloppa, Flavio [UNIFESP]
    Objective: To evaluate and compare the behavior of glycosaminoglycans (GAGs) in Dupuytren disease (DD). Methods: This is an experimental study with 23 patients diagnosed with DD. Tissue collected through fasciectomy with incision type Brunner or McCash were evaluated by electrophoresis for identification of GAGs. The quantification was carried out by immunofluorescence and dosage of proteins for different types of glycosaminoglycans. The results were expressed in percentage and statistically evaluated. Results: A significant increase was observed through eletrophoresis in GAGs, as compared to the control (p<0.05). Immunofluorescence of hyaluronic acid was reduced (23 times) when compared to the control (p<0.0001). Conclusion: An increase of sulfated GAGs in Dupuytren's disease, mainly dermatan sulfate, was evident from our results, as well as a pronounced decrease of hyaluronic acid in the palmar aponeurosis from the same patients.
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    Heparan sulfate mediates trastuzumab effect in breast cancer cells
    (Biomed Central Ltd, 2013-10-01) Suarez, Eloah Rabello [UNIFESP]; Paredes-Gamero, Edgar Julian [UNIFESP]; Del Giglio, Auro; Tersariol, Ivarne Luis dos Santos [UNIFESP]; Nader, Helena Bonciani [UNIFESP]; Pinhal, Maria Aparecida Silva [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Fac Med ABC
    Background: Trastuzumab is an antibody widely used in the treatment of breast cancer cases that test positive for the human epidermal growth factor receptor 2 (HER2). Many patients, however, become resistant to this antibody, whose resistance has become a major focus in breast cancer research. But despite this interest, there are still no reliable markers that can be used to identify resistant patients. A possible role of several extracellular matrix (ECM) components-heparan sulfate (HS), Syn-1(Syndecan-1) and heparanase (HPSE1)-in light of the influence of ECM alterations on the action of several compounds on the cells and cancer development, was therefore investigated in breast cancer cell resistance to trastuzumab.Methods: the cDNA of the enzyme responsible for cleaving HS chains from proteoglycans, HPSE1, was cloned in the pEGFP-N1 plasmid and transfected into a breast cancer cell lineage. We evaluated cell viability after trastuzumab treatment using different breast cancer cell lines. Trastuzumab and HS interaction was investigated by confocal microscopy and Fluorescence Resonance Energy Transfer (FRET). the profile of sulfated glycosaminoglycans was also investigated by [S-35]-sulfate incorporation. Quantitative RT-PCR and immunofluorescence were used to evaluate HPSE1, HER2 and Syn-1 mRNA expression. HPSE1 enzymatic activity was performed using biotinylated heparan sulfate.Results: Breast cancer cell lines responsive to trastuzumab present higher amounts of HER2, Syn-1 and HS on the cell surface, but lower levels of secreted HS. Trastuzumab and HS interaction was proven by FRET analysis. the addition of anti-HS to the cells or heparin to the culture medium induced resistance to trastuzumab in breast cancer cells previously sensitive to this monoclonal antibody. Breast cancer cells transfected with HPSE1 became resistant to trastuzumab, showing lower levels of HER2, Syn-1 and HS on the cell surface. in addition, HS shedding was increased significantly in these resistant cells.Conclusion: Trastuzumab action is dependent on the availability of heparan sulfate on the surface of breast cancer cells. Furthermore, our data suggest that high levels of heparan sulfate shed to the medium are able to capture trastuzumab, blocking the antibody action mediated by HER2. in addition to HER2 levels, heparan sulfate synthesis and shedding determine breast cancer cell susceptibility to trastuzumab.
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    Heparanase-2 Expression in Normal Ovarian Epithelium and in Benign and Malignant Ovarian Tumors
    (Lippincott Williams & Wilkins, 2009-12-01) Moura, Joel Pereira de [UNIFESP]; Nicolau, Sergio Mancini [UNIFESP]; Stavale, Joao Norberto [UNIFESP]; Silva Pinhal, Maria Aparecida da [UNIFESP]; Matos, Leandro Luongo de; Baracat, Edmund Chada [UNIFESP]; Lima, Geraldo Rodrigues de [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); ABC Fdn
    Introduction: Studies have highlighted the changes that take place in the environment between the cell and the extracellular matrix during the process of neoplastic expansion. Several papers have associated the expression of heparanase 1 with various malignant tumors. Heparanase 2 is probably related to loss of cell adhesion.Objective: the aim of this study was to evaluate the expression of heparanase 2 in epithelial neoplasia of the ovaries and in samples of normal ovarian tissue.Methods: Seventy-five ovary specimens were analyzed and divided into 3 groups: 23 malignant and 35 benign epithelial ovarian neoplasia and 17 without ovarian disease. We used 2 methodological techniques for evaluating the immunoexpression of heparanase 2. the first followed the qualitative criterion of positive or negative in relation to enzymatic expression, and the second involved computerized quantification of this expression, performed on the same slides.Results: in the quantitative analysis, we found positivity indices for heparanase 2 expression of 72.2% and 87.3% in the samples of benign and malignant neoplasias, respectively. in these,the intensity of expression and the expression index were 147.2 and 121.2. respectively, for the benign neoplasia and 134.1 and 118.0 for the malignant neoplasia. Qualitatively, its expression was strong or moderate in 44.2% of the benign and 78.2% of the malignant tumors; its expression in all of the nonneoplastic samples was negative, with the exception of one that was weakly positive.Conclusions: Heparanase 2 is involved in neoplastic proliferation, but it was not exclusively associated with the malignant process. Furthermore, there was no difference in its expression between benign and malignant ovarian epithelial neoplasia.
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    Heparina: interação com a matriz células endoteliais e atividade antitrombótica
    (Universidade Federal de São Paulo (UNIFESP), 2000) Trindade, Edvaldo da Silva [UNIFESP]; Nader, Helena Bonciani [UNIFESP]
    Heparina e drogas antitromboticas em geral estimulam, de forma especifica, a sintese do proteoglicano de heparam sulfato (PGHS) em celulas endoteliais. O efeito e composto e celula especificos. Ensaios cineticos com heparina mo que o estimulo e tempo e dose dependentes, e que esta se liga na superficie celular, sugerindo a existencia de possiveis receptores. Pelo presente trabalho, desenvolveu-se uma heparina marcada com biotina com o intuito de estudar a interacao com celulas endoteliais. A reacao de biotinilacao resultou num composto contendo uma biotina para cada residuo de acido urom'co. Essa heparina apresentou menor atividade anticoagulante (38U.I./mg), desprezivel atividade hemorragica, maior peso molecular (l8,6kDa), quando comparados com a heparina padrao, que possui 166U.I./mg, potente acao hemorragica e peso molecular de l3kDa. Essa heparina ainda, deixou de ser substrato para a heparinase e heparitinase H, porem manteve a capacidade de interacao com a Antitrombina III e a Fibronectina Os dados mostraram tambem que esta heparina e capaz de estimular a sintese de PGHS, como a heparina padrao. O(s) sitio(s) de ligacao para a heparina, nas celulas endoteliais, foram investigados utilizando-se como modelo, a heparina biotinilada em tecnicas de deteccao citoquimica e analise em microscopia confocal. Alem desta heparina, foram utilizados lectina WGA, que interage com a superficie celular e anticorpos especificos tanto para a superficie celular (anti-sindecam 4), como para a matriz extracelular (anti-fibronectina). Esses compostos foram ensaiados empregando-se tanto celulas recem-plaqueadas, sub-confluentes e confluentes, como em suspensao. Ainda a matriz extracelular livre de celulas foi investigada. Todos estes experimentos, revelaram que a ligacao da heparina biotinilada ocorreu somente nos componentes da matriz extracelular. Experimentos mantendo-se as celulas em presenca de heparina biotinilada por 22 horas, mostraram que ocorre o processo de internalizacao da mesma. Este conjunto de resultados sugere que o estimulo da sintese de PGHS, causado pela heparina possa ser independente da sua interacao com a superficie celular
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    Interação entre cininogênios humano e proteoglicanos na superfície celular: efeito nos processos de sinalização e migração
    (Universidade Federal de São Paulo (UNIFESP), 2017-02-22) Goncalves, Simone Pereira [UNIFESP]; Motta, Guacyara Da [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
    Objective: The General Objective Of The Present Study Is To Study The Relationship Between The Plasma Kallikrein-Kinin System And Tumor Progression, Associating The Presence Of Proteoglycans To The Process. Methods: To Identify The Enzymatic Activity Of Cho-745 Cells On Extracellular Hka (On The Surface Of Cultured Cells By Polyacrylamide Gel Electrophoresis In The Presence Of Sodium Dodecyl Sulfate And Immunodetection, To Study The Process Of Cell Death, Apoptosis And Necrosis Caused By H-Cinnogen And Hka On Cho-745 Cells In Culture By Assays Using Trypan Blue Dye And Annexin V Reagents, 7aad Respectively; (Wild-Type, Pgs) And Cho-745 (Mutant For Pg Production) In The Treatment With H-Kininogen, Hka And In The Presence Of The Peptides Hkh20 And Bk In The Absence Or Presence Of Kinase Inhibitors Captopril And Thiorphan; Characterize The Possible Signaling Pathways Involved In The Interaction Of H-Cininogen And Hka In Both Lineages Through The Flow Cytometry Technique. Results: The Hydrolysis Of Hka Was Studie
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    O papel dos proteoglicanos na adesão celular durante a tumorigênese: um modelo in vitro para o estudo da interação câncer-estroma
    (Universidade Federal de São Paulo (UNIFESP), 2009-05-27) Vicente, Carolina Meloni [UNIFESP]; Toma, Leny [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
    During metastasis cells lose their original tissue contacts, move through the extracellular matrix (ECM), enter the lymphatic and/or blood system, extravasate and subsequently form new tumors. Therefore, these tumor cells must experience changes in cell-ECM adhesion. Adhesion receptors play crucial roles in the neoplastic transformation of normal cells through the induction of cancer-specific cellular behavior and morphology. This implies that cancer cells likely express and utilize a distinct set of adhesion receptors during carcinogenesis. Syndecan-2 is a transmembrane heparan sulfate (HS) proteoglycan which has been implicated in the formation of specialized membrane domains and functions as a direct link between the extracellular environment and the organization of the cortical cytoplasm. In several colon-rectal cancer cell lines, syndecan-2 is highly expressed compared to normal cell lines. This increase appears to be critical for cancerous cell behavior since it regulates adhesion and proliferation and therefore the tumorigenic activity. The results of this study showed that in a highly metastatic colon-rectal cancer cell line, HCT-116, both expression and synthesis of syndecan-2 are enhanced when grown on ECM produced by fibroblasts. The expression of the others syndecans decreased, as did HS in the ECM produced by the cancer cells. Among the stromal components of ECM, the fibronectin was shown to be important for the increase of syndecan-2. Co-localization between syndecan-2, integrin ƒÑ5ƒÒ1 and fibronectin, suggests the involvement of these molecules in the adhesion mechanism of HCT-116 cells. Furthermore, blocking syndecan-2 with an antibody resulted in the absence of stress fibers during cell adhesion, indicating its important role in the regulation of actin filaments. Thus, the stromal ECM has a fundamental role in regulating the expression of cell surface proteins and probably signaling to the interior of cancer cells by altering their proliferation and adhesion, and its format.
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    Quantitative evaluation of experimental choroidal neovascularization by confocal scanning laser ophthalmoscopy: fluorescein angiogram parallels heparan sulfate proteoglycan expression
    (Associação Brasileira de Divulgação Científica, 2010-07-01) Regatieri, Caio Vinicius Saito [UNIFESP]; Dreyfuss, Juliana Luporini [UNIFESP]; Melo, G.b. [UNIFESP]; Lavinsky, Daniel [UNIFESP]; Hossaka, S.k. [UNIFESP]; Rodrigues, Eduardo Buchele [UNIFESP]; Farah, Michel Eid [UNIFESP]; Nader, Helena Bonciani [UNIFESP]; Maia, Maurício [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
    The objective of the present study was to develop a quantitative method to evaluate laser-induced choroidal neovascularization (CNV) in a rat model using Heidelberg Retina Angiograph 2 (HRA2) imaging. The expression of two heparan sulfate proteoglycans (HSPG) related to inflammation and angiogenesis was also investigated. CNV lesions were induced with argon laser in 21 heterozygous Zucker rats and after three weeks a fluorescein angiogram and autofluorescence exams were performed using HRA2. The area and greatest linear dimension were measured by two observers not aware of the protocol. Bland-Altman plots showed agreement between the observers, suggesting that the technique was reproducible. After fluorescein angiogram, HSPG (perlecan and syndecan-4) were analyzed by real-time RT-PCR and immunohistochemistry. There was a significant increase in the expression of perlecan and syndecan-4 (P < 0.0001) in retinas bearing CNV lesions compared to control retinas. The expression of these two HSPG increased with increasing CNV area. Immunohistochemistry demonstrated that the rat retina damaged with laser shots presented increased expression of perlecan and syndecan-4. Moreover, we observed that the overexpression occurred in the outer layer of the retina, which is related to choroidal damage. It was possible to develop a standardized quantitative method to evaluate CNV in a rat model using HRA2. In addition, we presented data indicating that the expression of HSPG parallels the area of CNV lesion. The understanding of these events offers opportunities for studies of new therapeutic interventions targeting these HSPG.
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    Reação estromal e proteoglicanos de baixo peso molecular ricos em leucina
    (Universidade Federal de São Paulo (UNIFESP), 2010-01-27) Coulson-Thomas, Vivien Jane [UNIFESP]; Toma, Leny [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
    During cancer cell growth many tumors exhibit various grades of desmoplasia, unorganized production of fibrous or connective tissue, composed mainly of collagen fibers and myofibroblasts. The accumulation of extracellular matrix (ECM) surrounding tumors directly affects cancer cell proliferation, migration and spread, therefore the study of desmoplasia is of vital importance. Myofibroblasts synthesize an amalgam of products including collagens and other ECM proteins, such as proteoglycans and are activated during a desmoplastic reaction. Small leucine rich proteoglycans have been characterized surrounding breast and pancreatic tumors and have the ability to suppress cell proliferation. In this study we have analyzed desmoplasia co-cultivating colorectal cancer cells (Caco-2 and HCT116) and myofibroblasts using various co-culture systems. Our findings demonstrate that direct cell-cell contact between myofibroblasts and colorectal cancer cells evokes an upregulation of the expression of ECM components (collagen I, collagen III, collagen IV, collagen V, biglycan and fibromodulin) by myofibroblasts. The ECM accumulation produced when myofibroblasts are co-cultivated with colorectal cancer cells appears unorganized and in bundles. This ECM accumulation slowed the migration and invasion of the colorectal tumor cells in both monolayer and 3-D co-culture systems. The participation of the ECM components analyzed in this study in desmoplasia is also demonstrated in vivo in human colorectal carcinoma tissue, validating our in vitro system.