RESUMO
Hypercoagulability, a major complication of metastatic cancers, has usually been treated with heparins from natural sources, or with their synthetic derivatives, which are under intense investigation in clinical oncology. However, the use of heparin has been challenging for patients with risk of severe bleeding. While the systemic administration of heparins, in preclinical models, has shown primarily attenuating effects on metastasis, their direct effect on established solid tumors has generated contradictory outcomes. We investigated the direct antitumoral properties of two sulfated fucans isolated from marine echinoderms, FucSulf1 and FucSulf2, which exhibit anticoagulant activity with mild hemorrhagic potential. Unlike heparin, sulfated fucans significantly inhibited tumor cell proliferation (by ~30-50%), and inhibited tumor migration and invasion in vitro. We found that FucSulf1 and FucSulf2 interacted with fibronectin as efficiently as heparin, leading to loss of prostate cancer and melanoma cell spreading. The sulfated fucans increased the endocytosis of ß1 integrin and neuropilin-1 chains, two cell receptors implicated in fibronectin-dependent adhesion. The treatment of cancer cells with both sulfated fucans, but not with heparin, also triggered intracellular focal adhesion kinase (FAK) degradation, with a consequent overall decrease in activated focal adhesion kinase levels. Finally, only sulfated fucans inhibited the growth of B16-F10 melanoma cells implanted in the dermis of syngeneic C57/BL6 mice. FucSulf1 and FucSulf2 arise from this study as candidates for the design of possible alternatives to long-term treatments of cancer patients with heparins, with the advantage of also controlling local growth and invasion of malignant cells.
Assuntos
Integrina beta1 , Melanoma , Masculino , Animais , Humanos , Camundongos , Proteína-Tirosina Quinases de Adesão Focal , Integrina beta1/metabolismo , Fibronectinas/metabolismo , Neuropilina-1 , Heparina/farmacologia , EndocitoseRESUMO
The thrombospondins (TSPs) are a family of multimeric extracellular matrix proteins that dynamically regulate cellular behavior and response to stimuli. In so doing, the TSPs directly and indirectly affect biological processes such as embryonic development, wound healing, immune response, angiogenesis, and cancer progression. Many of the direct effects of Thrombospondin 1 (TSP-1) result from the engagement of a wide range of cell surface receptors including syndecans, low density lipoprotein receptor-related protein 1 (LRP1), CD36, integrins, and CD47. Different or even opposing outcomes of TSP-1 actions in certain pathologic contexts may occur, depending on the structural/functional domain involved. To expedite response to external stimuli, these receptors, along with vascular endothelial growth factor receptor 2 (VEGFR2) and Src family kinases, are present in specific membrane microdomains, such as lipid rafts or tetraspanin-enriched microdomains. The molecular organization of these membrane microdomains and their constituents is modulated by TSP-1. In this review, we will describe how the presence of TSP-1 at the plasma membrane affects endothelial cell signal transduction and angiogenesis.
RESUMO
: Nanodrugs have in recent years been a subject of great debate. In 2017 alone, almost 50 nanodrugs were approved for clinical use worldwide. Despite the advantages related to nanodrugs/nanomedicine, there is still a lack of information regarding the biological safety, as the real behavior of these nanodrugs in the body. In order to better understand these aspects, in this study, we evaluated the effect of polylactic acid (PLA) nanoparticles (NPs) and magnetic core mesoporous silica nanoparticles (MMSN), of 1000 nm and 50 nm, respectively, on human cells. In this direction we evaluated the cell cycle, cytochemistry, proliferation and tubulogenesis on tumor cells lines: from melanoma (MV3), breast cancer (MCF-7, MDA-MB-213), glioma (U373MG), prostate (PC3), gastric (AGS) and colon adenocarcinoma (HT-29) and non-tumor cell lines: from human melanocyte (NGM), fibroblast (FGH) and endothelial (HUVEC), respectively. The data showed that an acute exposure to both, polymeric nanoparticles or MMSN, did not show any relevant toxic effects on neither tumor cells nor non-tumor cells, suggesting that although nanodrugs may present unrevealed aspects, under acute exposition to human cells they are harmless.
Assuntos
Nanopartículas/toxicidade , Ciclo Celular , Proliferação de Células , Óxido Ferroso-Férrico/química , Fibroblastos/metabolismo , Fibroblastos/fisiologia , Células HT29 , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , Células MCF-7 , Nanopartículas/química , Poliésteres/química , Dióxido de Silício/químicaRESUMO
The unique composition of tumor-produced extracellular matrix (ECM) can be a determining factor in changing the profile of endothelial cells in the tumor microenvironment. As the main receptor for ECM proteins, integrins can activate a series of signaling pathways related to cell adhesion, migration, and differentiation of endothelial cells that interact with ECM proteins. We studied the direct impact of the decellularized ECM produced by a highly metastatic human melanoma cell line (MV3) on the activation of endothelial cells and identified the intracellular signaling pathways associated with cell differentiation. Our data show that compared to the ECM derived from a human melanocyte cell line (NGM-ECM), ECM produced by a melanoma cell line (MV3-ECM) is considerably different in ultrastructural organization and composition and possesses a higher content of tenascin-C and laminin and a lower expression of fibronectin. When cultured directly on MV3-ECM, endothelial cells change morphology and show increased adhesion, migration, proliferation, and tubulogenesis. Interaction of endothelial cells with MV3-ECM induces the activation of integrin signaling, increasing FAK phosphorylation and its association with Src, which activates VEGFR2, potentiating the receptor response to VEGF. The blockage of αvß3 integrin inhibited the FAK-Src association and VEGFR activation, thus reducing tubulogenesis. Together, our data suggest that the interaction of endothelial cells with the melanoma-ECM triggers integrin-dependent signaling, leading to Src pathway activation that may potentiate VEGFR2 activation and up-regulate angiogenesis. J. Cell. Physiol. 231: 2464-2473, 2016. © 2016 Wiley Periodicals, Inc.
Assuntos
Células Endoteliais/metabolismo , Matriz Extracelular/metabolismo , Integrina alfaVbeta3/metabolismo , Melanoma/metabolismo , Transdução de Sinais , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Células Endoteliais/enzimologia , Ativação Enzimática , Matriz Extracelular/ultraestrutura , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Humanos , Melanócitos/metabolismo , Neovascularização Fisiológica , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
Intravital microscopy was used to assess the involvement of ExoU, a Pseudomonas aeruginosa cytotoxin with phospholipase A2 activity, in dysfunction of cerebral microcirculation during experimental pneumosepsis. Cortical vessels from mice intratracheally infected with low density of the ExoU-producing PA103 P. aeruginosa strain exhibited increased leukocyte rolling and adhesion to venule endothelium, decreased capillar density and impaired arteriolar response to vasoactive acetylcholine. These phenomena were mediated by the platelet activating factor receptor (PAFR) pathway because they were reversed in mice treated with a PAFR antagonist prior to infection. Brains from PA103-infected animals exhibited a perivascular inflammatory infiltration that was not detected in animals infected with an exoU deficient mutant or in mice treated with the PAFR antagonist and infected with the wild type bacteria. No effect on brain capillary density was detected in mice infected with the PAO1 P. aeruginosa strain, which do not produce ExoU. Finally, after PA103 infection, mice with a targeted deletion of the PAFR gene exhibited higher brain capillary density and lower leukocyte adhesion to venule endothelium, as well as lower increase of systemic inflammatory cytokines, when compared to wild-type mice. Altogether, our results establish a role for PAFR in mediating ExoU-induced cerebral microvascular failure in a murine model of sepsis.
Assuntos
Proteínas de Bactérias/metabolismo , Encéfalo/patologia , Microcirculação/fisiologia , Fator de Ativação de Plaquetas/metabolismo , Infecções por Pseudomonas/patologia , Pseudomonas aeruginosa/metabolismo , Sepse/patologia , Animais , Adesão Celular , Citocinas/análise , Feminino , Microscopia Intravital , Leucócitos/imunologia , Camundongos , Glicoproteínas da Membrana de Plaquetas/metabolismo , Pseudomonas aeruginosa/crescimento & desenvolvimento , Receptores Acoplados a Proteínas G/metabolismo , Transdução de SinaisRESUMO
OBJECTIVE: The body adiposity index (BAI) has been recently proposed as an alternative index to body mass index (BMI) and waist circumference (WC) to evaluate adiposity in adults, with special focus on its ability to discriminate gender specificities on adiposity. Endothelial dysfunction, circulating endothelial cells (CECs), endothelin-1 and adipocytokines are all related to atherosclerosis and nowadays considered as markers of emerging cardiovascular (CV) risk. This study aimed to determine in normal weight and obese adolescents which measures of body composition (BAI and z-BMI) or distribution (WC) correlate better with emerging CV risk markers. PATIENTS: Forty adolescents were selected according to BMI: normal weight (n = 20; 7 girls/13 boys, 14·7 ± 1·4 years, 53·4 ± 6·0 kg, z-BMI 0·6 ± 0·1) and obese ones (n = 20; 13 girls/7 boys, 14·1± 1·0 years, 86·7 ± 11·5 kg, z-BMI 2·7 ± 0·4). MEASUREMENTS: Body fat and fat mass were measured by dual-energy X-ray absorptiometry (DXA). Non-nutritive skin microvascular reactivity was evaluated by laser Doppler flowmetry with iontophoretic release of vasoactive drugs. Activated CECs were assessed by flow cytometric analysis. RESULTS: In adolescents, the measurement of % fat by DXA showed high correlation with BAI (ρ = 0·75, P < 0·0001), z-BMI (r = 0·84, P < 0·0001) and WC (r = 0·83, P < 0·0001). Endothelin-1 and activated CECs did not correlate with any anthropometric measures while adipocytokines expressed variable associations among them. Endothelium-dependent vasodilation showed higher correlation with BAI (r = -0·51, P < 0·0001) compared to z-BMI (r = -0·40, P < 0·001) or WC (r = -0·45, P < 0·001), specially on females. CONCLUSIONS: BAI was associated with emerging CV risk markers in adolescents but further research is needed to evaluate its potential in clinical and epidemiological sets.
Assuntos
Adiposidade/fisiologia , Doenças Cardiovasculares/metabolismo , Circunferência da Cintura/fisiologia , Absorciometria de Fóton , Tecido Adiposo/metabolismo , Tecido Adiposo/fisiologia , Adolescente , Índice de Massa Corporal , Feminino , Humanos , Masculino , Fatores de RiscoRESUMO
An increased plasma concentration of von Willebrand factor (vWF) is detected in individuals with many infectious diseases and is accepted as a marker of endothelium activation and prothrombotic condition. To determine whether ExoU, a Pseudomonas aeruginosa cytotoxin with proinflammatory activity, enhances the release of vWF, microvascular endothelial cells were infected with the ExoU-producing PA103 P. aeruginosa strain or an exoU-deficient mutant. Significantly increased vWF concentrations were detected in conditioned medium and subendothelial extracellular matrix from cultures infected with the wild-type bacteria, as determined by enzyme-linked immunoassays. PA103-infected cells also released higher concentrations of procoagulant microparticles containing increased amounts of membrane-associated vWF, as determined by flow cytometric analyses of cell culture supernatants. Both flow cytometry and confocal microscopy showed that increased amounts of vWF were associated with cytoplasmic membranes from cells infected with the ExoU-producing bacteria. PA103-infected cultures exposed to platelet suspensions exhibited increased percentages of cells with platelet adhesion. Because no modulation of the vWF mRNA levels was detected by reverse transcription-polymerase chain reaction assays in PA103-infected cells, ExoU is likely to have induced the release of vWF from cytoplasmic stores rather than vWF gene transcription. Such release is likely to modify the thromboresistance of microvascular endothelial cells.
Assuntos
Proteínas de Bactérias/metabolismo , Células Endoteliais/microbiologia , Endotélio Vascular/microbiologia , Pseudomonas aeruginosa/metabolismo , Fator de von Willebrand/metabolismo , Células Cultivadas , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Humanos , Adesividade PlaquetáriaRESUMO
An increased plasma concentration of von Willebrand factor (vWF) is detected in individuals with many infectious diseases and is accepted as a marker of endothelium activation and prothrombotic condition. To determine whether ExoU, a Pseudomonas aeruginosa cytotoxin with proinflammatory activity, enhances the release of vWF, microvascular endothelial cells were infected with the ExoU-producing PA103 P. aeruginosa strain or an exoU-deficient mutant. Significantly increased vWF concentrations were detected in conditioned medium and subendothelial extracellular matrix from cultures infected with the wild-type bacteria, as determined by enzyme-linked immunoassays. PA103-infected cells also released higher concentrations of procoagulant microparticles containing increased amounts of membrane-associated vWF, as determined by flow cytometric analyses of cell culture supernatants. Both flow cytometry and confocal microscopy showed that increased amounts of vWF were associated with cytoplasmic membranes from cells infected with the ExoU-producing bacteria. PA103-infected cultures exposed to platelet suspensions exhibited increased percentages of cells with platelet adhesion. Because no modulation of the vWF mRNA levels was detected by reverse transcription-polymerase chain reaction assays in PA103-infected cells, ExoU is likely to have induced the release of vWF from cytoplasmic stores rather than vWF gene transcription. Such release is likely to modify the thromboresistance of microvascular endothelial cells.
Assuntos
Humanos , Proteínas de Bactérias/metabolismo , Células Endoteliais/microbiologia , Endotélio Vascular/microbiologia , Pseudomonas aeruginosa/metabolismo , Fator de von Willebrand/metabolismo , Células Cultivadas , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Adesividade PlaquetáriaRESUMO
Thrombospondin-1 (TSP-1) gives rise to fragments that have both pro- and anti-angiogenic effects in vitro and in vivo. The TSP-HepI peptide (2.3 kDa), located in the N-terminal domain of TSP-1, has proangiogenic effects on endothelial cells. We have previously shown that TSP-1 itself exhibits a dual effect on endothelial colony-forming cells (ECFC) by enhancing their adhesion through its TSP-HepI fragment while reducing their proliferation and differentiation into vascular tubes (tubulogenesis) in vitro. This effect is likely mediated through CD47 binding to the TSP-1 C-terminal domain. Here we investigated the effect of TSP-HepI peptide on the angiogenic properties of ECFC in vitro and in vivo. TSP-HepI peptide potentiated FGF-2-induced neovascularisation by enhancing ECFC chemotaxis and tubulogenesis in a Matrigel plug assay. ECFC exposure to 20 µg/mL of TSP-HepI peptide for 18 h enhanced cell migration (p < 0.001 versus VEGF exposure), upregulated alpha 6-integrin expression, and enhanced their cell adhesion to activated endothelium under physiological shear stress conditions at levels comparable to those of SDF-1α. The adhesion enhancement appeared to be mediated by the heparan sulfate proteoglycan (HSPG) syndecan-4, as ECFC adhesion was significantly reduced by a syndecan-4-neutralising antibody. ECFC migration and tubulogenesis were stimulated neither by a TSP-HepI peptide with a modified heparin-binding site (S/TSP-HepI) nor when the glycosaminoglycans (GAGs) moieties were removed from the ECFC surface by enzymatic treatment. Ex vivo TSP-HepI priming could potentially serve to enhance the effectiveness of therapeutic neovascularisation with ECFC.
Assuntos
Endotélio Vascular/citologia , Neovascularização Fisiológica/fisiologia , Trombospondina 1/fisiologia , Animais , Células Cultivadas , Feminino , Citometria de Fluxo , Humanos , Camundongos , Trombospondina 1/químicaRESUMO
Vascular endothelial growth factor (VEGF) and αvß3 integrin are key molecules that actively participate in tumor angiogenesis and metastasis. Some integrin-blocking molecules are currently under clinical trials for cancer and metastasis treatment. However, the mechanism of action of such inhibitors is not completely understood. We have previously demonstrated the anti-angiogenic and anti-metastatic properties of DisBa-01, a recombinant His-tag RGD-disintegrin from Bothrops alternatus snake venom in some experimental models. DisBa-01 blocks αvß3 integrin binding to vitronectin and inhibits integrin-mediated downstream signaling cascades and cell migration. Here we add some new information on the mechanism of action of DisBa-01 in the tumor microenvironment. DisBa-01 supports the adhesion of fibroblasts and MDA-MB-231 breast cancer cells but it inhibits the adhesion of these cells to type I collagen under flow in high shear conditions, as a simulation of the blood stream. DisBa-01 does not affect the release of VEGF by fibroblasts or breast cancer cells but it strongly decreases the expression of VEGF mRNA and of its receptors, vascular endothelial growth factor receptors 1 and 2 (VEGFR1 and VEGFR2) in endothelial cells. DisBa-01 at nanomolar concentrations also modulates metalloprotease 2 (MMP-2) and 9 (MMP-9) activity, the latter being decreased in fibroblasts and increased in MDA-MB-231 cells. In conclusion, these results demonstrate that αvß3 integrin inhibitors may induce distinct effects in the cells of the tumor microenvironment, resulting in blockade of angiogenesis by impairing of VEGF signaling and in inhibition of tumor cell motility.
Assuntos
Adesão Celular/efeitos dos fármacos , Desintegrinas/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Integrina alfaVbeta3 , Venenos de Serpentes/farmacologia , Fator A de Crescimento do Endotélio Vascular , Animais , Bothrops , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Colágeno Tipo I/metabolismo , Desintegrinas/química , Desintegrinas/genética , Células Endoteliais/metabolismo , Feminino , Fibroblastos/efeitos dos fármacos , Humanos , Integrina alfaVbeta3/antagonistas & inibidores , Integrina alfaVbeta3/metabolismo , Neovascularização Fisiológica , Peptídeos/química , Peptídeos/genética , Ligação Proteica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Venenos de Serpentes/química , Microambiente Tumoral , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
BACKGROUND AND PURPOSE: Independent studies in experimental models of Trypanosoma cruzi appointed different roles for endothelin-1 (ET-1) and bradykinin (BK) in the immunopathogenesis of Chagas disease. Here, we addressed the hypothesis that pathogenic outcome is influenced by functional interplay between endothelin receptors (ET(A)R and ET(B)R) and bradykinin B(2) receptors (B(2)R). EXPERIMENTAL APPROACH: Intravital microscopy was used to determine whether ETR/B(2)R drives the accumulation of rhodamine-labelled leucocytes in the hamster cheek pouch (HCP). Inflammatory oedema was measured in the infected BALB/c paw of mice. Parasite invasion was assessed in CHO over-expressing ETRs, mouse cardiomyocytes, endothelium (human umbilical vein endothelial cells) or smooth muscle cells (HSMCs), in the presence/absence of antagonists of B(2)R (HOE-140), ET(A)R (BQ-123) and ET(B)R (BQ-788), specific IgG antibodies to each GPCRs; cholesterol or calcium-depleting drugs. RNA interference (ET(A)R or ET(B)R genes) in parasite infectivity was investigated in HSMCs. KEY RESULTS: BQ-123, BQ-788 and HOE-140 reduced leucocyte accumulation in HCP topically exposed to trypomastigotes and blocked inflammatory oedema in infected mice. Acting synergistically, ET(A)R and ET(B)R antagonists reduced parasite invasion of HSMCs to the same extent as HOE-140. Exogenous ET-1 potentiated T. cruzi uptake by HSMCs via ETRs/B(2)R, whereas RNA interference of ET(A)R and ET(B)R genes conversely reduced parasite internalization. ETRs/B(2)R-driven infection in HSMCs was reduced in HSMC pretreated with methyl-ß-cyclodextrin, a cholesterol-depleting drug, or in thapsigargin- or verapamil-treated target cells. CONCLUSIONS AND IMPLICATIONS: Our findings suggest that plasma leakage, a neutrophil-driven inflammatory response evoked by trypomastigotes via the kinin/endothelin pathways, may offer a window of opportunity for enhanced parasite invasion of cardiovascular cells.
Assuntos
Doença de Chagas/metabolismo , Doença de Chagas/parasitologia , Receptor B2 da Bradicinina/metabolismo , Receptor de Endotelina A/metabolismo , Receptor de Endotelina B/metabolismo , Trypanosoma cruzi/metabolismo , Animais , Antagonistas de Receptor B2 da Bradicinina , Células CHO , Cálcio/metabolismo , Células Cultivadas , Doença de Chagas/imunologia , Doença de Chagas/patologia , Cricetinae , Edema/metabolismo , Edema/patologia , Antagonistas do Receptor de Endotelina A , Antagonistas do Receptor de Endotelina B , Endotelina-1/metabolismo , Células Endoteliais da Veia Umbilical Humana/parasitologia , Humanos , Inflamação/metabolismo , Inflamação/patologia , Cininas/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Trypanosoma cruzi/imunologiaRESUMO
The extracellular matrix (ECM) contains important cues for tissue homeostasis and morphogenesis. The matricellular protein tenascin-C (TN-C) is overexpressed in remodeling tissues and cancer. In the present work, we studied the effect of different ECM-which exhibited a significant diversity in their TN-C content-in endothelial survival, proliferation and tubulogenic differentiation: autologous (endothelial) ECM devoid of TN-C, but bearing large amounts of FN; fibroblast ECM, bearing both high TN-C and FN contents; and finally, glioma-derived matrices, usually poor in FN, but very rich in TN-C. HUVECs initially adhered to the immobilized matrix produced by U373 MG glioma cells, but significantly detached and died by anoikis (50 to 80%) after 24h, as compared with cells incubated with endothelial and fibroblast matrices. Surviving endothelial cells (20 to 50%) became up to 6-fold more proliferative and formed 74-97% less tube-like structures in vitro than cells grown on non-tumoral matrices. An antibody against the EGF-like repeats of tenascin-C (TN-C) partially rescued cells from the tubulogenic defect, indicating that this molecule is responsible for the selection of highly proliferative and tubulogenic defective endothelial cells. Interestingly, by using defined substrata, in conditions that mimic glioma and normal cell ECM composition, we observed that fibronectin (FN) modulates the TN-C-induced selection of endothelial cells. Our data show that TN-C is able to modulate endothelial branching morphogenesis in vitro and, since it is prevalent in matrices of injured and tumor tissues, also suggest a role for this protein in vascular morphogenesis, in these physiological contexts.
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Proliferação de Células , Células Endoteliais/metabolismo , Endotélio Vascular/citologia , Matriz Extracelular/metabolismo , Tenascina/metabolismo , Animais , Adesão Celular , Glioma/metabolismo , Humanos , Ratos , Ratos WistarRESUMO
The data presented in this article suggest that intact TSP-1 may act, in normal vessel homeostasis, as an angiostatic factor favoring vessel quiescence, or even vessel regression, but this activity would be largely impaired in the presence of an excess of angiogenic stimuli and proteases. The fact that the N-terminal domain of TSP-1 (heparin-binding domain or HBD) is recognized by a plethora of cell receptors, all of them engaged in proangiogenic responses, strongly suggests that the proteolytic cleavage of HBD may be relevant in certain pathophysiological conditions.
Assuntos
Neoplasias/irrigação sanguínea , Neoplasias/metabolismo , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Trombospondina 1/metabolismo , Animais , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias/genética , Neoplasias/patologia , Neovascularização Patológica/genética , Trombospondina 1/genéticaRESUMO
Group B Streptococcus (GBS) is a major etiologic agent of neonatal bacterial infections and is the most common cause of sepsis and pneumonia in newborns. Surface and secreted molecules of GBS are often essential virulence factors which are involved in the adherence of the bacteria to host cells or are required to suppress the defense mechanisms of hosts. We analyzed the peptidase profiles of GBS by detection of proteolytic activities on SDS-PAGE containing copolymerized gelatin as substrate. Based on the inhibition by o-phenathroline and EGTA, three distinct peptidases of 220, 200 and 180 kDa were identified in the culture medium, besides one major cell-associated proteolytic activity, a 200-kDa metallopeptidase, suggesting that all were zinc-metallopeptidases. GBS culture supernatants, rich in metallotype peptidases, also cleaved fibronectin, laminin, type IV collagen, fibrinogen and albumin. Cleavage of the host extracellular matrix by GBS may be a relevant factor in the process of bacterial dissemination and/or invasion. Notably, metallopeptidase inhibitors strongly blocked GBS growth as well as its interaction with human cell lineages. Understanding the contribution of peptidases to the pathogenesis of GBS disease may broaden our perception of how this significant pathogen causes severe infections in newborn infants.
Assuntos
Linhagem da Célula/efeitos dos fármacos , Metaloproteases/metabolismo , Inibidores de Proteases/farmacologia , Streptococcus agalactiae/citologia , Streptococcus agalactiae/enzimologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Humanos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Streptococcus agalactiae/efeitos dos fármacosRESUMO
Thrombospondin-1 (TSP-1) is an extracellular matrix protein that modulates focal adhesion in mammalian cells and exhibits dual roles in angiogenesis. In a previous work, we showed that a recombinant 18 kDa protein encompassing the N-terminal residues 1-174 of human TSP-1 (TSP18) induced tubulogenesis of human umbilical vein endothelial cells and protected them from apoptosis. Our results indicated that these effects were possibly mediated by syndecan-4 proteoglycan, since binding of TSP18 to endothelial extracts was inhibited by anti-syndecan-4 antibody. Syndecan-4 is a heparan-sulfate proteoglycan that regulates cell-matrix interactions and is the only member of its family present in focal adhesions. In this report, we demonstrate that a monoclonal antibody against syndecan-4 blocks TSP18-induced tubulogenesis. Furthermore, through 2D adhesion and 3D angiogenic assays, we demonstrate that two sequences, TSP Hep I and II, retain the major pro-angiogenic activity of TSP18. These TSP-1 motifs also compete with the fibronectin Hep II domain for binding to syndecan-4 on endothelial cell surface, indicating that they may exert their effects by interfering with the recognition of fibronectin by syndecan-4. Additionally, TSP18 and its derived peptides activate the PKC-dependent Akt-PKB signaling pathway. Blockage of PKC activation prevented HUVEC spreading when seeded on TSP18 fragment, and on TSP Hep I and TSP Hep II peptides, but not on gelatin-coated substrates. Our results identify syndecan-4 as a novel receptor for the N-terminus of TSP-1 and suggest that TSP-1 N-terminal pro-angiogenic activity is linked to its capacity of interfering with syndecan-4 functions in the course of cell adhesion.
Assuntos
Proteínas Angiogênicas/metabolismo , Endotélio/irrigação sanguínea , Neovascularização Fisiológica , Sindecana-4/metabolismo , Trombospondina 1/química , Trombospondina 1/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Anticorpos/farmacologia , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Endotélio/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Fibronectinas/metabolismo , Humanos , Dados de Sequência Molecular , Neovascularização Fisiológica/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Proteína Quinase C/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Relação Estrutura-Atividade , SuínosRESUMO
Sporotrichosis, a mycosis caused by Sporothrix schenckii, is characterized by lymphocutaneous lesions. In immunocompromised hosts, this fungus may invade the bloodstream and disseminate to other tissues, such as lung and bone. Our group previously showed that S. schenckii yeasts adhere to endothelial monolayers and that this interaction is modulated by cytokines. Using 3.0 mum-pore culture inserts, the present work shows that transforming growth factor (TGF)-beta1 led to a 80+/-26 % increase in fungal migration across endothelial monolayers and inhibited fungus internalization by 55+/-23.5 %, when compared to untreated cells. The major surface endothelial molecules recognized by S. schenckii were not modulated by TGF-beta1. These data suggested that a paracellular route is preferentially used by S. schenckii during the transmigration of cultured endothelial cells. It was further observed that TGF-beta1 increased the subendothelial matrix exposure and that anti-fibronectin (anti-FN) and anti-laminin (anti-LM) antibodies abolished the increase in S. schenckii association with endothelial monolayers induced by TGF-beta1. These antibodies also inhibited (38.2+/-4.29 % and 50.8+/-17.3 %, respectively) the adhesion of S. schenckii to freshly prepared native endothelial matrices. Furthermore, transendothelial migration of S. schenckii was blocked by anti-FN and anti-LM antibodies. These data indicate that TGF-beta1-induced S. schenckii adhesion to endothelial monolayers results from the increased exposure of the subendothelial extracellular matrix and that this event may contribute to the enhancement of transendothelial migration.
Assuntos
Adesão Celular , Movimento Celular , Células Endoteliais/microbiologia , Proteínas da Matriz Extracelular/fisiologia , Sporothrix/fisiologia , Sporothrix/patogenicidade , Fator de Crescimento Transformador beta1/farmacologia , Células Cultivadas , Endotélio Vascular/citologia , Endotélio Vascular/microbiologia , Proteínas da Matriz Extracelular/metabolismo , Humanos , Transdução de Sinais , Fator de Crescimento Transformador beta1/metabolismoRESUMO
We have previously demonstrated that alternagin-C (ALT-C), a disintegrin-like protein from the venom of the Brazilian snake Bothrops alternatus, induces human vascular endothelial cell (HUVEC) proliferation by up-regulating the expression of vascular endothelial growth factor (VEGF). Here, we show that ALT-C is also able to induce in vivo angiogenesis using the model of matrigel plug in nude mice. Fibroblast growth factor (FGF) alone or supplemented with ALT-C was mixed with melted matrigel and subcutaneously injected in nude mice. After two weeks, the matrigel plugs were removed and analyzed to verify endothelial cell migration and new vessel formation. ALT-C (1 and 10 ng) strongly induced endothelial cell migration as well as the formation of new vessels. However, in higher concentrations, ALT-C strongly inhibited angiogenesis. In low concentrations (1 and 10nM), ALT-C also up-regulates the expression of VEGF receptor 2 (VEGFR2, KDR) mostly after 48 h, but it did not affect VEGFR1 (Ftl-1) in HUVEC cells as demonstrated by real-time PCR analysis. However, in higher concentrations (100 nM) the expression of both receptors is down-regulated. A peptide derived from ALT-C primary structure also affects HUVEC proliferation in vitro and angiogenesis in vivo. In conclusion, the present study shows for the first time the in vivo angiogenesis induced by a disintegrin-like molecule and the modulation of VEGFRs as well.
Assuntos
Bothrops/fisiologia , Venenos de Crotalídeos/farmacologia , Desintegrinas/fisiologia , Neovascularização Fisiológica/fisiologia , Fragmentos de Peptídeos/fisiologia , Sequência de Aminoácidos , Animais , Células Cultivadas , Humanos , Camundongos , Camundongos NusRESUMO
The harmonious development of the central nervous system depends on the interactions of the neuronal and glial cells. Extracellular matrix elements play important roles in these interactions, especially laminin produced by astrocytes, which has been shown to be a good substrate for neuron growth and axonal guidance. Glioblastomas are the most common subtypes of primary brain tumors and may be astrocytes in origin. As normal laminin-producing glial cells are the preferential substrate for neurons, and glial tumors have been shown to produce laminin, we questioned whether glioblastoma retained the same normal glial-neuron interactive properties with respect to neuronal growth and differentiation. Then, rat neurons were co-cultured onto rat normal astrocytes or onto three human glioblastoma cell lines obtained from neurosurgery. The co-culture confirmed that human glioblastoma cells as well as astrocytes maintained the ability to support neuritogenesis, but non-neural normal or tumoral cells failed to do so. However, glioblastoma cells did not distinguish embryonic from post-natal neurons in relation to neurite pattern in the co-cultures, as normal astrocytes did. Further, the laminin organization on both normal and tumoral glial cells was altered from a filamentous arrangement to a mixed punctuate/filamentous pattern when in co-culture with neurons. Together, these results suggest that glioblastoma cells could identify neuronal cells as partners, to support their growth and induce complex neurites, but they lost the normal glia property to distinguish neuronal age. In addition, our results show for the first time that neurons modulate the organization of astrocytes and glioblastoma laminin on the extracellular matrix.
Assuntos
Astrócitos/química , Neoplasias Encefálicas/fisiopatologia , Encéfalo/citologia , Glioblastoma/fisiopatologia , Laminina/análise , Neuritos/ultraestrutura , Neurônios/fisiologia , Animais , Astrócitos/citologia , Astrócitos/fisiologia , Neoplasias Encefálicas/química , Neoplasias Encefálicas/patologia , Diferenciação Celular , Células Cultivadas , Glioblastoma/química , Glioblastoma/patologia , Humanos , Neuritos/metabolismo , Neurônios/metabolismo , Ratos , Ratos WistarRESUMO
Sporothrix schenckii is the etiological agent of sporotrichosis, a subcutaneous mycosis that can evolve to systemic complications in immunocompromised patients. Interactions with endothelium are thought to be essential for systemic infections. In the present work, we studied the interaction between S. schenckii and human umbilical vein endothelial cells (HUVECs). S. schenckii interacts with HUVECs in a time-dependent manner. Morphological analysis showed that yeasts locate to interendothelial junctions. Ultrastructural studies showed that internalized yeasts were found inside endocytic vacuoles as early as 2 h, without causing any detectable damage to HUVECs after 24 h of infection. The viability of infected HUVECs was confirmed by the MTT assay. When HUVECs were treated with different concentrations of Interleukin-1beta or transforming growth factor-beta, a significant dose-dependent increase in cell-associated yeasts was observed. The preliminary analysis of the endothelial surface ligands for S. schenckii cells revealed two major molecules, with Mr of approximately 90 and 135 kDa. The interaction of endothelial cell surface molecules with S. schenckii yeast cells was modulated by divalent cations. This is the first demonstration that S. schenckii is able to adhere and invade endothelial cells without significantly affect cellular integrity. Our results suggest the contribution of cytokine-modulated calcium-dependent molecules to this process.
Assuntos
Citocinas/fisiologia , Células Endoteliais/química , Células Endoteliais/microbiologia , Sporothrix/patogenicidade , Cálcio/metabolismo , Adesão Celular , Moléculas de Adesão Celular/isolamento & purificação , Moléculas de Adesão Celular/metabolismo , Sobrevivência Celular , Células Cultivadas , Citocinas/farmacologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/ultraestrutura , Formazans/metabolismo , Humanos , Junções Intercelulares/microbiologia , Interleucina-1/farmacologia , Interleucina-1/fisiologia , Proteínas de Membrana/isolamento & purificação , Proteínas de Membrana/metabolismo , Ligação Proteica , Sporothrix/fisiologia , Sais de Tetrazólio/metabolismo , Fatores de Tempo , Fator de Crescimento Transformador beta/farmacologia , Fator de Crescimento Transformador beta/fisiologia , Vesículas Transportadoras/microbiologiaRESUMO
Alternagin-C (ALT-C), a disintegrin-like protein purified from the venom of the Brazilian snake Bothrops alternatus, interacts with the major collagen I receptor, the alpha(2)beta(1) integrin, inhibiting collagen binding. Here we show that ALT-C also inhibits the adhesion of a mouse fibroblast cell line (NIH-3T3) to collagen I (IC(50) 2.2 microm). In addition, when immobilized on plate wells, ALT-C supports the adhesion of this cell line as well as of human vein endothelial cell (HUVEC). ALT-C (3 microm) does not detach cells that were previously bound to collagen I. ALT-C (5 nm) induces HUVEC proliferation in vitro, and it inhibits the positive effect of vascular endothelial growth factor (VEGF) or FGF-2 on the proliferation of these cells, thus suggesting a common mechanism for these proteins. Gene expression analysis of human fibroblasts growing on ALT-C- or collagen-coated plates showed that ALT-C and collagen I induce a very similar pattern of gene expression. When compared with cells growing on plastic only, ALT-C up-regulates the expression of 45 genes including the VEGF gene and down-regulates the expression of 30 genes. Fibroblast VEGF expression was confirmed by RT-PCR and ELISA assay. Up-regulation of the VEGF gene and other growth factors could explain the positive effect on HUVEC proliferation. ALT-C also strongly activates Akt/PKB phosphorylation, a signaling event involved in endothelial survival and angiogenesis. In conclusion, ALT-C acts as a survival factor, promoting adhesion and endothelial cell proliferation.