ABSTRACT
Background Matrix metalloproteinases (MMPs) are key players in tumor progression, helping tumor cells to modify their microenvironment, which allows cell migration to secondary sites. The role of integrins, adhesion receptors that connect cells to the extracellular matrix, in MMP expression and activity has been previously suggested. However, the mechanisms by which integrins control MMP expression are not completely understood. Particularly, the role of 21 integrin, one of the major collagen I receptors, in MMP activity and expression has not been studied. Alternagin-C (ALT-C), a glutamate-cysteine-aspartate-disintegrin from Bothrops alternatus venom, has high affinity for an 21 integrin. Herein, we used ALT-C as a 21 integrin ligand to study the effect of ALT-C on MMP-9 and MMP-2 expression as well as on tumor cells, fibroblats and endothelial cell migration. Methods ALT-C was purified by two steps of gel filtration followed by anion exchange chromatography. The 21, integrin binding properties of ALT-C, its dissociation constant (Kd) relative to this integrin and to collagen I (Col I) were determined by surface plasmon resonance. The effects of ALT-C (10, 40, 100 and 1000 nM) in migration assays were studied using three human cell lines: human fibroblasts, breast tumor cell line MDA-MB-231, and microvascular endothelial cells HMEC-1, considering cells found in the tumor microenvironment. ALT-C effects on MMP-9 and MMP-2 expression and activity were analyzed by quantitative PCR and gelatin zymography, respectively. Focal adhesion kinase activation was determined by western blotting. Results Our data demonstrate that ALT-C, after binding to 21 integrin...
Subject(s)
Humans , /physiology , /physiology , Tumor Microenvironment/physiology , Breast Neoplasms/physiopathology , Proto-Oncogene Proteins c-myc/physiology , Cell Adhesion/physiology , Cell Movement/physiologyABSTRACT
Background: Matrix metalloproteinases (MMPs) are key players in tumor progression, helping tumor cells to modify their microenvironment, which allows cell migration to secondary sites. The role of integrins, adhesion receptors that connect cells to the extracellular matrix, in MMP expression and activity has been previously suggested. However, the mechanisms by which integrins control MMP expression are not completely understood. Particularly, the role of α2ß1 integrin, one of the major collagen I receptors, in MMP activity and expression has not been studied. Alternagin-C (ALT-C), a glutamate-cysteine-aspartate-disintegrin from Bothrops alternatus venom, has high affinity for an α2ß1 integrin. Herein, we used ALT-C as a α2ß1 integrin ligand to study the effect of ALT-C on MMP-9 and MMP-2 expression as well as on tumor cells, fibroblats and endothelial cell migration. Methods: ALT-C was purified by two steps of gel filtration followed by anion exchange chromatography. The α2ß1, integrin binding properties of ALT-C, its dissociation constant (Kd) relative to this integrin and to collagen I (Col I) were determined by surface plasmon resonance. The effects of ALT-C (10, 40, 100 and 1000 nM) in migration assays were studied using three human cell lines: human fibroblasts, breast tumor cell line MDA-MB-231, and microvascular endothelial cells HMEC-1, considering cells found in the tumor microenvironment. ALT-C effects on MMP-9 and MMP-2 expression and activity were analyzed by quantitative PCR and gelatin zymography, respectively. Focal adhesion kinase activation was determined by western blotting. Results: Our data demonstrate that ALT-C, after binding to α2ß1 integrin, acts by two distinct mechanisms against tumor progression, depending on the cell type: in tumor cells, ALT-C decreases MMP-9 and MMP-2 contents and activity, but increases focal adhesion kinase phosphorylation and transmigration; and in endothelial cells, ALT-C inhibits MMP-2, which is necessary for tumor angiogenesis. ALT-C also upregulates c-Myc mRNA level, which is related to tumor suppression. Conclusion: These results demonstrate that α2ß1 integrin controls MMP expression and reveal this integrin as a target for the development of antiangiogenic and antimetastatic therapies.(AU)
Subject(s)
Humans , Animals , Breast Neoplasms , Matrix Metalloproteinase 2/pharmacology , Matrix Metalloproteinase 9/pharmacology , Crotalid Venoms/pharmacology , Integrin alpha2beta1/metabolism , Endothelial Cells , Blotting, Western/methods , Polymerase Chain Reaction/methods , Bothrops , Receptors, Collagen , Tumor Microenvironment , Flow CytometryABSTRACT
OBJECTIVE: The purpose of this study was to investigate the effects of resistance training on angiogenesis markers of visceral adipose tissue in ovariectomized rats. METHOD: Adult Sprague-Dawley female rats were divided into four groups (n=6 per group): sham-sedentary, ovariectomized sedentary, sham-resistance training and ovariectomized resistance training. The rats were allowed to climb a 1.1-m vertical ladder with weights attached to their tails and the weights were progressively increased. Sessions were performed three times per week for 10 weeks. Visceral adipose tissue angiogenesis and morphology were analyzed by histology. VEGF-A mRNA and protein levels were analyzed by real-time PCR and ELISA, respectively. RESULTS: Ovariectomy resulted in higher body mass (p=0.0003), adipocyte hypertrophy (p=0.0003), decreased VEGF-A mRNA (p=0.0004) and protein levels (p=0.0009), and decreased micro-vascular density (p=0.0181) in the visceral adipose tissue of the rats. Resistance training for 10 weeks was not able to attenuate the reduced angiogenesis in the visceral adipose tissue of the ovariectomized rats. CONCLUSION: Our findings indicate that the resistance training program used in this study could not ameliorate low angiogenesis in the visceral adipose tissue of ovariectomized rats.
Subject(s)
Animals , Female , Physical Conditioning, Animal/physiology , Ovariectomy/methods , Neovascularization, Physiologic/physiology , Intra-Abdominal Fat/blood supply , Estrogens/deficiency , Resistance Training/methods , Ribosomal Proteins/analysis , Time Factors , Enzyme-Linked Immunosorbent Assay , Immunohistochemistry , Biomarkers/analysis , Random Allocation , Reproducibility of Results , Rats, Sprague-Dawley , Adipocytes/physiology , Vascular Endothelial Growth Factor Receptor-2/analysis , Vascular Endothelial Growth Factor A/analysis , Intra-Abdominal Fat/metabolism , Real-Time Polymerase Chain ReactionABSTRACT
PURPOSE: To investigate the changes induced by DisBa-01 on repair of wound healing after induced incisional hernia (IH) in rats. METHODS: Thirty two male albino rats were submitted to IH and divided into four experimental groups: G1, placebo control; G2, DisBa-01-treated; G3, anti-αvβ3 antibodies-treated and G4, anti-α2 antibodies-treated. Histological, biochemical and extracellular matrix remodeling analysis of abdominal wall were evaluated. RESULTS: After 14 days, 100% of the G2 did not present hernia, and the hernia ring was closed by a thin membrane. In contrast, all groups maintained incisional hernia. DisBa-01 also increased the number macrophages and fibroblasts and induced the formation of new vessels. Additionally, MMP-2 was strongly activated only in G2 (p<0.05). Anti- αvβ3-integrin antibodies produced similar results than DisBa-01 but not anti-α2 integrin blocking antibodies. CONCLUSION: DisBa-01 has an important role in the control of wound healing and the blocking of this integrin may be an interesting therapeutically strategy in incisional hernia. .
Subject(s)
Animals , Male , Disintegrins/pharmacology , Hernia, Ventral/pathology , /antagonists & inhibitors , Platelet Aggregation Inhibitors/pharmacology , Wound Healing/drug effects , Abdominal Wall/pathology , Collagen/analysis , Collagen/drug effects , Disease Models, Animal , Fibroblasts/drug effects , Hernia, Ventral/drug therapy , Hernia, Ventral/surgery , /analysis , /physiology , Random Allocation , Rats, Wistar , Reproducibility of Results , Time Factors , Treatment OutcomeABSTRACT
PURPOSE: Incisional hernia (IH) is characterized by defective wound healing process. Disba-01, a αvb3 integrin blocker has shown to control the rate of wound repair and therefore it could be a target for new wound healing therapies.The objective of the study was to determine the changes induced by Disba-01 on repair of wound healing after induced IH in rats. METHODS: Thirty two male albino rats were submitted to IH and divided into 4 experimental groups: G1, placebo control; G2, DisBa-01-treated; G3, anti-αvβ3 antibodies-treated and G4, anti-α2 antibodies-treated. Histological. biochemical and extracellular matrix remodeling analysis of abdominal wall were evaluated. RESULTS: After 14 days, 100% of the G2 did not present hernia, and the hernia ring was closed by a thin membrane. In contrast, all groups maintained incisional hernia. DisBa-01 also increased the number macrophages and fibroblasts and induced the formation of new vessels. Additionally, MMP-2 was strongly activated only in G2 (P<0.05). Anti- αvβ3-integrin antibodies produced similar results than Disba-01 but not anti-α2 integrin blocking antibodies. CONCLUSION: These results strongly indicate that Disba-01 has an important role in the control of wound healing and the blocking of this integrin may be an interesting therapeutical strategy in IH. .
Subject(s)
Animals , Male , Abdominal Wall , Disintegrins/pharmacology , Hernia, Ventral/drug therapy , /antagonists & inhibitors , /pharmacology , Wound Healing/drug effects , Collagen/drug effects , Fibroblasts/drug effects , Hernia, Ventral/metabolism , /chemistry , /metabolism , Macrophages/drug effects , /metabolism , Postoperative Complications/prevention & control , Random Allocation , Rats, Wistar , Wound Healing/physiologyABSTRACT
OBJECTIVE: The objective of this study was to assess the effects of resistance training on oxidative stress markers in the livers of ovariectomized rats. METHOD: Adult Sprague-Dawley rats were divided into the following four groups (n = 8 per group): sham-operated sedentary, ovariectomized sedentary, sham-operated resistance training, and ovariectomized resistance training. During the resistance training period, the animals climbed a 1.1-m vertical ladder with weights attached to their tails; the sessions were conducted 3 times per week, with 4-9 climbs and 8-12 dynamic movements per climb. The oxidative stress was assessed by measuring the levels of reduced glutathione and oxidized glutathione, the enzymatic activity of catalase and superoxide dismutase, lipid peroxidation, vitamin E concentrations, and the gene expression of glutathione peroxidase. RESULTS: The results showed significant reductions in the reduced glutathione/oxidized glutathione ratio (4.11±0.65 nmol/g tec), vitamin E concentration (55.36±11.11 nmol/g), and gene expression of glutathione peroxidase (0.49±0.16 arbitrary units) in the livers of ovariectomized rats compared with the livers of unovariectomized animals (5.71±0.71 nmol/g tec, 100.14±10.99 nmol/g, and 1.09±0.54 arbitrary units, respectively). Moreover, resistance training for 10 weeks was not able to reduce the oxidative stress in the livers of ovariectomized rats and induced negative changes in the hepatic anti-oxidative/oxidative balance. CONCLUSION: Our findings indicate that the resistance training program used in this study was not able to attenuate the hepatic oxidative damage caused by ovariectomy and increased the hepatic oxidative stress. .
Subject(s)
Animals , Female , Rats , Liver/metabolism , Ovariectomy , Oxidative Stress , Resistance Training , Biomarkers/metabolism , Catalase/analysis , Glutathione/analysis , Lipid Peroxidation , Physical Conditioning, Animal/methods , Random Allocation , Rats, Sprague-Dawley , Real-Time Polymerase Chain Reaction , Reproducibility of Results , Time Factors , Treatment Outcome , Thiobarbituric Acid Reactive Substances/analysis , Vitamin E/analysisABSTRACT
Introdução: O músculo esquelético é um tecido dinâmico com a habilidade intrínseca de se adaptar aos estímulos ambientais como resultado de mudanças qualitativas e quantitativas na expressão gênica, sendo esta capacidade definida como plasticidade. Este tecido é composto por populações de fibras rápidas e lentas que diferem fenotipicamente por expressarem diferentes isoformas de cadeias pesadas de miosina (CPM). Miosinas constituem uma família de proteínas chamadas "motores moleculares" com atividade ATOásica conhecidas por seu importante papel no processo de contração muscular. Objetivo: Realizar um levantamento, por meio de dados expostos na literatura, da relação existente entre a expressão de diferentes CPM no processo de remodelamento por meio de levantamento de dados descritos na literatura, senso consultados os bancos de dados internacionais "Pubmed" e "Highwire Press" e os bancos de dados nacionais Scielo e Lilacs, no período de janeiro a abril de 2008. Resultados: A fibra muscular pode alterar suas características contráteis por meio de mudanças nas quantidades das CPM. A velocidade de encurtamento do músculo esquelético varia de acordo com a isoforma de CPM que possui, conferindo assim ao tecido muscular a capacidade de adaptação frente a estímulos fisiológicos e patológicos. Conclusão: A fibra muscular pode alterar suas propriedades contráteis por meio de mudanças nas quantidades de isoformas de CPM que a constitui.
Subject(s)
Muscle Fibers, Skeletal , Muscle, Skeletal , Myosins , Neuronal Plasticity , Protein IsoformsABSTRACT
Agkistrodon contortrix laticinctus myotoxin (ACLMT) is a myotoxic Lys49 phospholipase A2 isolated from the venom of the broad-banded copperhead, A. c. laticinctus. We have previously shown that ACLMT affects water transport in toad bladders, but little in known about the mechanisms involved in the action of this toxin on membrane permeability. In this study, we examined the morphological alterations caused by ACLMT in toad bladder epithelium. The bladders were exposed to the toxin (20 nM) for 30 min at 23o C using Bentleys technique. Longitudinal and cross sections were obtained from paraffin-embedded bladders and stained with hematoxylin-eosin prior to analysis by light microscopy. Exposure to the toxin resulted in disorganization of the epithelial cell layer and damage to the smooth muscle bundles. The smooth muscle cells were swollen, with hypercontracted myofi laments and clear areas among the fibers. These findings suggest that ACLMT affects the structural integrity of the epithelium, and that the pathological changes induced by this toxin in smooth muscle cells may be caused by an increase in the cytosolic calcium concentration. These results contribute to our understanding of the mechanisms involved in the action of snake venom Lys49 PLA2 myotoxins in biological tissues.