RESUMO
INTRODUCTION: Hernia repair with prosthetic meshes represents one of the most common surgical procedures in the field of surgery. This intervention is always associated with an ensuing inflammatory response, angiogenesis and fibrotic encapsulation forming a foreign body granuloma (FBG) around the mesh fibres. Several studies have described this inflammatory reaction by characterising inflammatory cell infiltrate around the FBG after mesh explantation. However, very little is known about the real-time progression of such an inflammatory response. The aim of this study was to investigate the feasibility of monitoring the ongoing inflammatory response to mesh implantation using bioluminescence in vivo. MATERIALS AND METHODS: Three luciferase transgenic mice strains (FVB/N-Tg(Vegfr2-luc)-Xen, BALB/C-Tg(NFκB-RE-luc)-Xen and Tg(INS/EpRE-Luc)T20Rbl) were used. Mice were anaesthetized with 2 % isoflurane, and two incisions were made on the left and right sides of the abdomen of the mice. A 1-cm(2) propylene mesh was implanted subcutaneously in the right incision wound of each mouse, and the left wound served as control. Two hundred microliters of D-luciferin was injected into the mice, and bioluminescence measurements were done prior to the surgical intervention and subsequently every 3 days. After mesh explantation, histological analysis was done. Statistical analysis was done using prism GraphPad software. RESULTS: Bioluminescence results revealed different time points of maximum signal for the different mice strains. VEGFR2 gene expression peaked on day 6, NFkB on day 12 and ARE on day 3 post mesh implantation. We also observed much higher bioluminescent signal around the FBG surrounding the mesh as compared to the control wound, with p < 0.05 for all the different mice strains. CONCLUSION: Our results prove the possibility of monitoring the inflammatory reaction after mesh implantation in vivo using bioluminescence signal release. This provides a novel method of accessing and accurately describing the ongoing inflammatory response over a given period of time.
Assuntos
Benzotiazóis , Reação a Corpo Estranho/patologia , Implantação de Prótese/efeitos adversos , Telas Cirúrgicas/efeitos adversos , Animais , Biópsia por Agulha , Modelos Animais de Doenças , Imuno-Histoquímica , Inflamação/patologia , Mediadores da Inflamação/sangue , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Implantação de Prótese/métodos , Distribuição Aleatória , Medição de Risco , Estatísticas não Paramétricas , Cicatrização/fisiologiaRESUMO
BACKGROUND: Fracture healing is a complex biological process with specific temporal expression patterns. During this process new bone tissue is formed, which is similar to the original bone in quality and structure. This occurs in four phases: inflammation, formation of a soft tissue callus, formation of a bony callus and remodelling of the bony callus. This needs the precise orchestration of each cell type involved. OBJECTIVES: This article presents details of the fracture healing phases and the relevant factors. During the aging process there is an increase of reactive oxygen species and a change in expression pattern of growth factors that have a negative effect on the fracture healing process. METHODS: A selective review of the literature was carried out in PubMed concerning the influence of aging on fracture healing. CONCLUSION: The healing process is regulated by systemic and local factors. An understanding of these processes and the changes during aging is necessary in order to improve the knowledge of delayed or lack of fracture healing during aging to decide when an intervention is needed.
Assuntos
Envelhecimento/metabolismo , Remodelação Óssea/fisiologia , Consolidação da Fratura/fisiologia , Fraturas Ósseas/fisiopatologia , Fraturas Ósseas/terapia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Feminino , Humanos , Modelos Biológicos , Estresse OxidativoRESUMO
BACKGROUND: Xenon has profound neuroprotective effects after neurological injury and is currently undergoing phase 2 clinical trials in cardiac arrest patients. However, xenon is very costly, which might preclude its widespread use. We hypothesized argon, which is more available, might also protect central nervous tissues and allow better functional recovery in a rodent model of global cerebral ischaemia. METHODS: Fourteen male Sprague-Dawley rats were subjected to 7 min of cardiac arrest and 3 min of cardiopulmonary resuscitation (CPR). One hour after successful CPR, animals were randomized to either ventilation with 70% argon in oxygen (n = 7) for 1 h or 70% nitrogen (controls, n=7). A neurological deficit score (NDS) was calculated daily for the following 7 days, then the animals were killed and the brains harvested for histopathological analyses. RESULTS: All animals survived. Control rats had severe neurological dysfunction, while argon-treated animals showed significant improvements in the NDS at all time points. This was paralleled by a significant reduction in the neuronal damage index in the neocortex and the hippocampal CA 3/4 region. CONCLUSIONS: Our study demonstrates that a single 1 h application of 70% argon significantly reduced histopathological damage of the neocortex and hippocampus, associated with a marked improvement in functional neurological recovery.
Assuntos
Argônio/uso terapêutico , Parada Cardíaca/complicações , Hipóxia-Isquemia Encefálica/prevenção & controle , Fármacos Neuroprotetores/uso terapêutico , Administração por Inalação , Animais , Reanimação Cardiopulmonar , Avaliação Pré-Clínica de Medicamentos/métodos , Parada Cardíaca/terapia , Hipocampo/patologia , Hipóxia-Isquemia Encefálica/etiologia , Hipóxia-Isquemia Encefálica/patologia , Masculino , Aprendizagem em Labirinto , Neocórtex/patologia , Ratos , Ratos Sprague-Dawley , Recuperação de Função Fisiológica/efeitos dos fármacosRESUMO
Pregnancy-related complications not only represent a risk for maternal and fetal morbidity and mortality, but are also a risk for several diseases later in life. Many epidemiological studies have shown clear associations between an adverse intrauterine environment and an increased risk of diabetes, hypertension, cardiovascular disease, depression, obesity, and other chronic diseases in the adult. Some of these syndromes could be prevented by avoiding adverse stimuli or insults including psychological stress during pregnancy, intake of drugs, insufficient diet and substandard working conditions. Hence, all of these stimuli have the potential to alter health later in life. The placenta plays a key role in regulating the nutrient supply to the fetus and producing hormones that control the fetal as well as the maternal metabolism. Thus, any factor or stimulus that alters the function of the hormone producing placental trophoblast will provoke critical alterations of placental function and hence could induce programming of the fetus. The factors that change placental development may interfere with nutrient and oxygen supply to the fetus. This may be achieved by a direct disturbance of the placental barrier or more indirectly by, e. g., disturbing trophoblast invasion. For both path-ways, the respective pathologies are known: while preeclampsia is caused by alterations of the villous trophoblast, intra-uterine growth restriction is caused by insufficient invasion of the extravillous trophoblast. In both cases the effect can be undernutrition and/or fetal hypoxia, both of which adversely affect organ development, especially of brain and heart. However, the mechanisms responsible for disturbances of trophoblast differentiation and function remain elusive.
Assuntos
Desenvolvimento Fetal , Doenças Fetais/fisiopatologia , Troca Materno-Fetal , Modelos Biológicos , Placenta/fisiopatologia , Adulto , Feminino , Humanos , Masculino , GravidezRESUMO
Additively manufactured (AM) degradable porous metallic biomaterials offer unique opportunities for satisfying the design requirements of an ideal bone substitute. Among the currently available biodegradable metals, iron has the highest elastic modulus, meaning that it would benefit the most from porous design. Given the successful preclinical applications of such biomaterials for the treatment of cardiovascular diseases, the moderate compatibility of AM porous iron with osteoblast-like cells, reported in earlier studies, has been surprising. This may be because, as opposed to static in vitro conditions, the biodegradation products of iron in vivo are transported away and excreted. To better mimic the in situ situations of biodegradable biomaterials after implantation, we compared the biodegradation behavior and cytocompatibility of AM porous iron under static conditions to the conditions with dynamic in situ-like fluid flow perfusion in a bioreactor. Furthermore, the compatibility of these scaffolds with four different cell types was evaluated to better understand the implications of these implants for the complex process of natural wound healing. These included endothelial cells, L929 fibroblasts, RAW264.7 macrophage-like cells, and osteoblastic MG-63 cells. The biodegradation rate of the scaffolds was significantly increased in the perfusion bioreactor as compared to static immersion. Under either condition, the compatibility with L929 cells was the best. Moreover, the compatibility with all the cell types was much enhanced under physiomimetic dynamic flow conditions as compared to static biodegradation. Our study highlights the importance of physiomimetic culture conditions and cell type selection when evaluating the cytocompatibility of degradable biomaterials in vitro. STATEMENT OF SIGNIFICANCE: Additively manufactured (AM) degradable porous metals offer unique opportunities for the treatment of large bony defects. Despite the successful preclinical applications of biodegradable iron in the cardiovascular field, the moderate compatibility of AM porous iron with osteoblast-like cells was reported. To better mimic the in vivo condition, we compared the biodegradation behavior and cytocompatibility of AM porous iron under static condition to dynamic perfusion. Furthermore, the compatibility of these scaffolds with various cell types was evaluated to better simulate the process of natural wound healing. Our study suggests that AM porous iron holds great promise for orthopedic applications, while also highlighting the importance of physio-mimetic culture conditions and cell type selection when evaluating the cytocompatibility of degradable biomaterials in vitro.
Assuntos
Células Endoteliais , Ferro , Ferro/farmacologia , Porosidade , Materiais Biocompatíveis/farmacologia , MetaisRESUMO
Owing to limited self-healing capacity, tendon ruptures and healing remain major orthopedic challenges. Increasing evidence suggests that post-traumatic inflammatory responses, and hence, cytokines are involved in both cases, and also in tendon exercise and homeostasis. This review summarizes interrelations known between the cytokines interleukin (IL)-1ß, tumor necrosis factor (TNF)α, IL-6 and vascular endothelial growth factor (VEGF) in tendon to assess their role in tendon damage and healing. Exogenic cytokine sources are blood-derived leukocytes that immigrate in damaged tendon. Endogenous expression of IL-1ß, TNFα, IL-6, IL-10 and VEGF was demonstrated in tendon-derived cells. As tendon is a highly mechanosensitive tissue, cytokine homeostasis and cell survival underlie an intimate balance between adequate biomechanical stimuli and disturbance through load deprivation and overload. Multiple interrelations between cytokines and tendon extracellular matrix (ECM) synthesis, catabolic mediators e.g. matrix-degrading enzymes, inflammatory and angiogenic factors (COX-2, PGE2, VEGF, NO) and cytoskeleton assembly are evident. Pro-inflammatory cytokines affect ECM homeostasis, accelerate remodeling, amplify biomechanical adaptiveness and promote tenocyte apoptosis. This multifaceted interplay might both contribute to and interfere with healing. Much work must be undertaken to understand the particular interrelation of these inflammatory and regulatory mediators in ruptured tendon and healing, which has relevance for the development of novel immunoregulatory therapeutic strategies.
Assuntos
Citocinas/fisiologia , Traumatismos dos Tendões/imunologia , Cicatrização/imunologia , Humanos , Ruptura/imunologia , Traumatismos dos Tendões/fisiopatologiaRESUMO
Gram-positive bacterial bone infections are an important cause of morbidity particularly in immunocompromised patients. Antimicrobial peptides (AP) are effectors of the innate immune system and directly kill microorganisms in the first hours after microbial infection. The aim of the present investigation was to study the expression and regulation of gram-positive specialized human beta-defensin-3 (HBD-3) in bone. Samples of healthy and osteomyelitic human bone were assessed for the expression of HBD-3. Using primary and immortalized osteoblasts (SAOS-2 cells), release and regulation of HBD-3 was evaluated after exposure to Staphylococcus aureus supernatant and/or corticosteroids using PCR, immunohistochemistry, Western blot and ELISA. To determine the role of toll-like-receptors-2 and -4 (TLR-2/-4), shRNA was used to downregulate TLRs. An osteomyelitis mouse model was created performed to investigate the release of murine beta-defensins using immunohistochemistry and RT-PCR. Cultured osteoblasts and human bone produce HBD-3 under standard conditions. The release increases within hours of bacterial supernatant exposure in cultured osteoblasts. This observation was not made in chronically infected bone samples. The shRNA-technology revealed the necessity of TLR-2 and -4 in HBD-3 induction in osteoblasts. Blocking protein synthesis with cycloheximide showed that the rapid release of HBD-3 is not dependent on a translational de novo synthesis and is not affected by glucocorticoids. The murine osteomyelitis model confirmed the in vivo release uptake of mouse beta-defensins-4 (MBD-4) in bone. This report shows the bacterial induction of HBD-3 via TLR-2 and -4 in osteoblasts and suggests a central role of antimicrobial peptides in the prevention of bacterial bone infection. The rapid and effective induction of HBD-3 in osteoblasts incubated with conditioned media from bacteria is more likely a result of a rapid secretion of preformed HBD-3 by osteoblasts rather than a result of enhanced biosynthesis. The increased incidence of gram-positive bacterial bone infection in patients with regular intake of glucocorticoids does not seem to be caused by a deranged HBD-3 release in osteoblasts.
Assuntos
Osso e Ossos/química , Osteoblastos/metabolismo , Osteomielite/imunologia , Receptor 2 Toll-Like/fisiologia , Receptor 4 Toll-Like/fisiologia , beta-Defensinas/genética , Corticosteroides/farmacologia , Animais , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/microbiologia , Regulação da Expressão Gênica , Humanos , Cinética , Camundongos , Osteoblastos/química , Staphylococcus aureus/imunologia , beta-Defensinas/biossínteseRESUMO
Recent studies suggest that the formyl-peptide-receptor-like-1 (FPRL1) plays an essential role in the inflammatory responses of host defense mechanisms and neurodegenerative disorders such as Alzheimer's disease (AD). We therefore analyzed whether amyloid beta1-42 (Abeta1-42) increased the activity of phospholipase D (PLD) via FPRL1, which is an enzyme involved in the secretion, endocytosis and receptor signaling. PLD activity was determined using a transphosphatidylation assay. The internalization of Abeta1-42 via FPRL1 was visualized using fluorescence microscopy and quantified by ELISA (Enzyme Linked Immunosorbent Assay). Determining receptor activity by extracellular-signal regulated kinases 1/2 (ERK1/2) phosphorylation and cAMP level measurement verified the Abeta1-42-induced activation of FPRL1. We were able to show that Abeta1-42 is rapidly internalized via FPRL1 in astrocytes and microglia. PLD was additionally activated by Abeta1-42 and via FPRL1 in rat glial cells. Furthermore, the ERK1/2 phosphorylation by FPRL1 agonists was dependent on the PLD product phosphatidic acid (PA). Together, these data suggest that PLD plays an important role in the regulation of Abeta1-42-induced endocytosis and FPRL1 receptor signaling.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Endocitose/fisiologia , Neuroglia/metabolismo , Fragmentos de Peptídeos/metabolismo , Fosfolipase D/metabolismo , Receptores de Formil Peptídeo/metabolismo , Transdução de Sinais/fisiologia , Peptídeos beta-Amiloides/agonistas , Peptídeos beta-Amiloides/farmacologia , Análise de Variância , Animais , Animais Recém-Nascidos , Encéfalo/citologia , Células Cultivadas , AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Interações Medicamentosas , Endocitose/efeitos dos fármacos , Humanos , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Neuroglia/efeitos dos fármacos , Oligopeptídeos/farmacologia , Fragmentos de Peptídeos/agonistas , Fragmentos de Peptídeos/farmacologia , Ratos , Receptores de Formil Peptídeo/agonistas , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , terc-Butil Álcool/farmacologiaRESUMO
Osteomyelitis often causes functional impairment due to tissue destruction. This report demonstrates a novel previously unappreciated role of osteoblasts. Samples of osteomyelitic bone and bacterially challenged osteoblasts produce increased amounts of antimicrobial peptides in order to combat bacterial bone infection. An osteomyelitis mouse model confirmed the osseous induction of the murine homologue of human beta-defensin-2, suggesting a central role in the prevention of bacterial bone infection. Antimicrobial peptides are effectors of the innate defence system and play a key role in host protection at cellular surfaces. Some of them are produced constitutively, whereas others are induced during infection. Human beta-defensins represent a major subclass of antimicrobial peptides and act as a first line of defence through their broad spectrum of potent antimicrobial activity. The aim of the present in-vitro and in-vivo investigations was to study the expression and regulation of human beta-defensin-2 in the case of bacterial bone infection and to analyse the effects of immunosuppressive drugs on bone-derived antimicrobial peptide expression. Samples of healthy human bone, osteomyelitic bone and cultured osteoblasts (hFOB cells) were assessed for the expression of human beta-defensin-2. Regulation of human beta-defensin-2 was studied in hFOB cells after exposure to bacterial supernatants, proinflammatory cytokines and immunosuppressive drugs (glucocorticoids and methotrexate) and was assayed by enzyme-linked immunosorbent assay. An osteomyelitis mouse model was performed to demonstrate the regulation of the murine homologue of human beta-defensin-2, named murine beta-defensin-3, by real-time reverse transcription-polymerase chain reaction and immunohistochemistry. Healthy human bone and cultured osteoblasts are able to produce human beta-defensin-2 under standard conditions. Samples of infected bone produce higher levels of endogenous antibiotics, such as human beta-defensin-2, when compared with samples of healthy bone. A clear induction of human beta-defensin-2 was observed after exposure of cultured osteoblasts to gram-positive bacteria or proinflammatory cytokines. Additional treatment with glucocorticoids or methotrexate prevented bacteria-mediated antimicrobial peptide induction in cultured osteoblasts. The osteomyelitis mouse model demonstrated transcriptional upregulation of the murine homologue of human beta-defensin-2, namely murine beta-defensin-3, in bone after intraosseous contamination of the tibia. Human and murine bone have the ability to produce broad-spectrum endogenous antibiotics when challenged by micro-organisms in vitro and in vivo. Immunosuppressive drugs, such as glucocorticoids or methotrexate, may increase the susceptibility to bone infection by decreasing antimicrobial peptide expression levels in case of microbial challenge. The induction of human beta-defensin-2 following bacterial contact suggests a central role of antimicrobial peptides in the prevention of bacterial bone infection.
Assuntos
Anti-Infecciosos/metabolismo , Osso e Ossos/metabolismo , beta-Defensinas/metabolismo , Idoso , Animais , Estudos de Casos e Controles , Linhagem Celular , Dexametasona/uso terapêutico , Ensaio de Imunoadsorção Enzimática/métodos , Expressão Gênica/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Imunossupressores/uso terapêutico , Masculino , Metotrexato/uso terapêutico , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Modelos Animais , Osteoblastos/metabolismo , Osteomielite/tratamento farmacológico , Osteomielite/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Infecções Estafilocócicas/metabolismo , Staphylococcus aureus , beta-Defensinas/genéticaRESUMO
Oxidative stress is central to neuronal damage in neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease. In consequence, activation of the cerebral oxidative stress defence is considered as a promising strategy of therapeutic intervention. Here we demonstrate that the flavone luteolin confers neuroprotection against oxidative stress via activation of the nuclear factor erythroid-2-related factor 2 (Nrf2), a transcription factor central to the maintenance of the cellular redox homeostasis. Luteolin protects rat neural PC12 and glial C6 cells from N-methyl-4-phenyl-pyridinium (MPP+) induced toxicity in vitro and effectively activates Nrf2 as shown by ARE-reporter gene assays. This protection critically depends on the activation of Nrf2 since downregulation of Nrf2 by shRNA completely abrogates the protection of luteolin in vitro. Furthermore, the neuroprotective effect of luteolin is abolished by the inhibition of the luteolin-induced ERK1/2-activation. Our results highlight the relevance of Nrf2 for neural cell survival conferred by flavones. In particular, we identified luteolin as a promising lead for the search of orally available, blood brain barrier permeable compounds to support the therapy of neurodegenerative disorders.
Assuntos
1-Metil-4-fenilpiridínio/toxicidade , Sobrevivência Celular/efeitos dos fármacos , Flavonoides/farmacologia , Genes Reporter/genética , Herbicidas/toxicidade , Luteolina/farmacologia , Fator 2 Relacionado a NF-E2/genética , Estresse Oxidativo/fisiologia , Proteínas/genética , Células Tumorais Cultivadas/efeitos dos fármacos , Animais , Antioxidantes , Encéfalo/metabolismo , Sobrevivência Celular/genética , Expressão Gênica/efeitos dos fármacos , Técnicas In Vitro , Peptídeos e Proteínas de Sinalização Intracelular , Proteína 1 Associada a ECH Semelhante a Kelch , Estresse Oxidativo/genética , Células PC12 , RNA Interferente Pequeno/genética , Ratos , Regulação para Cima/efeitos dos fármacosRESUMO
PURPOSE: To determine the expression and production of antimicrobial peptides by mucosal cells of the lacrimal passage in healthy and pathologic states. METHODS: Detection of bactericidal-permeability-increasing protein (BPI), heparin-binding protein (CAP37), human cationic antimicrobial protein (LL-37), human alpha-defensin 5 (HD5), human alpha-defensin 6 (HD6), human beta-defensin 1 (HBD-1), and human beta-defensin 2 (HBD-2) was performed by reverse transcription-polymerase chain reaction (RT-PCR). Intracellular deposition of lysozyme, lactoferrin, secretory phospholipase A(2), human neutrophil defensins (HNP-1, -2, and -3), human beta-defensin 1 (HBD-1), and human beta-defensin 2 (HBD-2) was analyzed immunohistochemically. Samples were obtained from 15 patients by surgery and from 10 cadavers. RESULTS: RT-PCR revealed BPI, CAP37, and HBD-1 mRNA in samples of healthy nasolacrimal duct epithelium. Additionally, HBD-2 mRNA was detected in epithelial samples from patients with dacryocystitis. Messenger RNAs for LL-37 and alpha-defensin 5 and 6 were absent in all samples investigated. Immunohistochemistry revealed lysozyme, lactoferrin, secretory phospholipase A(2), and HNP-1, -2, and -3 to be present in all samples, whereas HBD-1 was present only in some of the healthy and inflamed samples. Immunoreactive HBD-2 peptide was visible only in some of the inflamed samples. CONCLUSIONS: The data suggest that the human efferent tear ducts produce a broad spectrum of antimicrobial peptides. Under inflammatory conditions, changes in the expression pattern occurred, revealing induction of the human inducible defensin HBD-2 and in some cases downregulation of HBD-1 and CAP37. Antimicrobial peptides have a therapeutic potential in dacryocystitis, in that they have a broad spectrum of antimicrobial activity and accelerate epithelial healing. However, caution is appropriate, because defensins also promote fibrin formation and cell proliferation, which are key elements in scarring processes, such as dacryostenosis.
Assuntos
Anti-Infecciosos/metabolismo , Proteínas Sanguíneas/biossíntese , Proteínas de Transporte/biossíntese , Dacriocistite/metabolismo , Defensinas/biossíntese , Proteínas do Olho/biossíntese , Obstrução dos Ductos Lacrimais/metabolismo , Proteínas de Membrana , Ducto Nasolacrimal/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Peptídeos Catiônicos Antimicrobianos , Proteínas Sanguíneas/genética , Proteínas de Transporte/genética , Criança , Pré-Escolar , Dacriocistite/patologia , Defensinas/genética , Regulação para Baixo , Células Epiteliais/metabolismo , Proteínas do Olho/genética , Feminino , Humanos , Técnicas Imunoenzimáticas , Obstrução dos Ductos Lacrimais/patologia , Masculino , Pessoa de Meia-Idade , Ducto Nasolacrimal/patologia , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Lágrimas/metabolismoRESUMO
The somatostatin receptor subtype sst2A is highly expressed, non-mutated and functionally active in gliomas. After stimulation of cultivated human U343 glioma cells with somatostatin, octreotide (sst2-, sst3- and sst5-selective peptide agonist) or the sst2-selective non-peptide agonist L-054,522 multiple signal transduction pathways are induced: elevated cAMP levels are reduced, protein tyrosine phosphatases (especially SHP2) are activated and mitogen-activated protein kinases are inhibited. Stimulation of the phosphatases resulted in dephosphorylation of activated receptors for EGF and PDGF (epidermal and platelet-derived growth factor), and as a consequence the mitogen-activated protein kinases ERK 1 and 2 (p42/p44) were de-phosphorylated in co-stimulation experiments. Furthermore, somatostatin or sst2-selective agonists reduced EGF-stimulated expression of the AP-1 complex (c-jun/c-jun) on the transcriptional and translational level. These experiments show that the interaction of stimulatory and inhibitory receptors are important mechanisms for the regulation of signal cascades and gene expression.
Assuntos
Neoplasias Encefálicas , Glioma , Receptores de Somatostatina/genética , Receptores de Somatostatina/metabolismo , Benzimidazóis/farmacologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Receptores ErbB/metabolismo , Regulação Neoplásica da Expressão Gênica , Hormônios/farmacologia , Humanos , Indóis/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular , Sistema de Sinalização das MAP Quinases/fisiologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Octreotida/farmacologia , Fosforilação , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Proto-Oncogênicas c-jun/genética , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Somatostatina/farmacologia , Fator de Transcrição AP-1/metabolismo , Células Tumorais CultivadasRESUMO
The Achilles tendon is one of the most common sites of injury and rupture. Evidence suggests that local vascularisation is involved in this aetiology. We investigated the expression of one important angiogenic factor, the vascular endothelial growth factor (VEGF), in normal and pathologic human Achilles tendons using immunohistochemical, biochemical, molecular and cell biology methods. VEGF could be immunostained in tenocytes of ruptured and foetal Achilles tendons, but not in normal adult ones. In microvessels, the VEGF receptor VEGFR-1 (flt-1) could be visualised as well. High VEGF levels were measured in homogenates from ruptured adult, lower ones in foetal and negligible concentrations in normal adult Achilles tendons using enzyme-linked immunoassay (ELISA) and Western blot experiments. Reverse transcriptase-polymerase chain reaction (RT-PCR) showed that the splice variants VEGF121 and VEGF165 are exclusively expressed. In tenocytes cultivated from newborn rat Achilles tendons, hypoxia or epidermal growth factor (EGF) raised VEGF production moderately whereas their combination resulted in a strong, synergistic induction. These results prove the presence of an angiogenic peptide and vascularisation in ruptured and foetal tendons and support the view that microtrauma or degeneration in the Achilles tendon precedes its rupture.
Assuntos
Tendão do Calcâneo , Fatores de Crescimento Endotelial/metabolismo , Linfocinas/metabolismo , Traumatismos dos Tendões/metabolismo , Tendão do Calcâneo/efeitos dos fármacos , Tendão do Calcâneo/lesões , Tendão do Calcâneo/metabolismo , Tendão do Calcâneo/patologia , Adulto , Processamento Alternativo , Animais , Animais Recém-Nascidos , Western Blotting , Hipóxia Celular/fisiologia , Células Cultivadas , Relação Dose-Resposta a Droga , Fatores de Crescimento Endotelial/análise , Fatores de Crescimento Endotelial/genética , Ensaio de Imunoadsorção Enzimática , Fator de Crescimento Epidérmico/farmacologia , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Técnicas Imunoenzimáticas , Linfocinas/análise , Linfocinas/genética , Proteínas Proto-Oncogênicas/metabolismo , RNA Mensageiro/genética , Ratos , Receptores Proteína Tirosina Quinases/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Traumatismos dos Tendões/patologia , Fator A de Crescimento do Endotélio Vascular , Receptor 1 de Fatores de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Our aim was to investigate vascular endothelial growth factor (VEGF) expression after lacerations of a meniscus in a rabbit model. Specimens of meniscus were examined using immunohistochemistry, enzyme-linked immunoassay and the reverse transcription polymerase chain reaction after one, two, five or ten weeks. In the periphery of the meniscus 90% of the lacerations had healed after five and ten weeks, but no healing was observed in the avascular area. Expression of VEGF protein and VEGF mRNA was found in the meniscus of both the operated and the contralateral sites but both were absent in control rabbits which had not undergone operation. The highest expression of VEGF was found in the avascular area after one week (p < 0.001). It then lessened at both the vascular and avascular areas, but still remained greater in comparison with the control meniscus (p < 0.05). Despite greater expression of VEGF, angiogenesis failed at the inner portion. These findings demonstrated the poor healing response in the avascular area which may not be caused by an intrinsic cellular insufficiency to stimulate angiogenesis.
Assuntos
Lesões do Menisco Tibial , Fator A de Crescimento do Endotélio Vascular/metabolismo , Cicatrização/fisiologia , Animais , Capilares/patologia , Modelos Animais de Doenças , Expressão Gênica , Técnicas Imunoenzimáticas , Meniscos Tibiais/irrigação sanguínea , Meniscos Tibiais/patologia , RNA Mensageiro/genética , Coelhos , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
INTRODUCTION: Vascular endothelial growth factor (VEGF) is detectable in later stages of human osteoarthritis (OA), but not in the healthy articular cartilage. Due to its capacity to increase matrix metalloproteinases and to decrease their inhibitors (tissue inhibitors of metalloproteinases or TIMPs) VEGF seems to play an important role in the development of osteoarthrosis. In late stages of osteoarthritis, invasion of blood vessels from the subchondral growth plate, synovitis with angiogenesis and osteophyte growth is observable. Several studies have revealed a central role for VEGF in all these phenomena. In order to investigate whether VEGF participates in early changes of OA or may even possess characteristics of a marker of OA, we developed an experimental posttraumatic OA New Zealand White rabbit animal model. MATERIALS AND METHODS: In four skeletally mature New Zealand White rabbits, OA was induced by joint instability after transsection of the anterior cruciate ligament in both knees. After eight weeks the animals were killed. OA was verified histologically using the Mankin scale. Expression of VEGF was detected by immunohistochemistry and RT-PCR. Proteoglycans were evaluated by using HE and safranin-O staining. Four non-surgically treated animals acted as a control. RESULTS: The mean Mankin score was 5.11 (±2.14), corresponding to a moderate OA. VEGF and VEGF transcripts were detectable in the cartilage of early experimental posttraumatic OA rabbits. Control samples remained negative for VEGF mRNA and protein. DISCUSSION: The results of this study are promising concerning the role of VEGF as a diagnostic marker. VEGF could further be participated in early changes of OA. A therapeutic approach by modulation of VEGF production could be a possibility for the future.
Assuntos
Osteoartrite/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Cartilagem Articular/patologia , Condrócitos/metabolismo , Modelos Animais de Doenças , Imuno-Histoquímica , Masculino , Neovascularização Patológica/patologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Coelhos , Reação em Cadeia da Polimerase em Tempo Real , Fator A de Crescimento do Endotélio Vascular/análiseRESUMO
INTRODUCTION: Impaired trophoblast invasion into the uteroplacental arteries is accompanied with an evidence of oxidative stress in the extravillous trophoblast in preeclampsia complicated with IUGR. OBJECTIVES: Preeclampsia is characterised by increased lipid oxidation and diminished antioxidant capacity; recently, we have shown that PE is associated with an increased expression of the nuclear factor erythroid 2-related factor 2 (Nrf2) in villous cytotrophoblast. A possible relationship between the vascular endothelial growth factor (VEGF) and Nrf2 was established in vitro and the activation of Nrf2 pathway could lead to upregulation of VEGF synthesis through the induction of Nrf2-dependent Heme oxygenase-1 (HO-1). In this study the expression of Nrf2 and VEGF was determined in the interstitial and intramural extravillous trophoblast in normal pregnancies and those complicated by preeclampsia and intra-uterine growth restriction (IUGR). METHODS: Full-thickness uterine tissues were obtained from caesarean hysterectomies performed in 5 healthy normotensive women delivering term infants and from 5 women with severe early-onset preeclampsia and IUGR (29-34 week's gestation). The interstitial and intramural trophoblasts were studied by immunohistochemical analysis of paraffin sections stained with anti VEGF and anti Nrf2. RESULTS: Cases suffering from preeclampsia with IUGR were characterised by reduced invasion of extravillous trophoblast into uteroplacental arteries in the endometrial and myometrial segments. In addition, these cells showed an increased expression of Nrf2 in the pathological sections. The overexpression of Nrf2 in cases with preeclampsia was associated with restricted expression of VEGF in these cells compared to controls. CONCLUSION: Our data suggest that besides villous cytotrophoblast, also the extravillous trophoblast is a source of Nrf2-dependent genes. VEGF deficiency may cause higher oxidative stress in extravillous trophoblast in cases with preeclampsia with IUGR. The resulting reduced basal defence against oxidative stress and the higher vulnerability to oxidative damage may play a role in the limited trophoblast invasion into uteroplacental arteries in cases suffering from early onset preeclampsia and IUGR.
RESUMO
INTRODUCTION: Preeclampsia is a multi-organ syndrome characterized by maternal endothelial damage, is an independent long-term risk factor for hypertension and cardiovascular disease. OBJECTIVES: In animal models the administration of the Vascular Endothelial Growth Factor (VEGF) could reverse the hypertensive signs accompanying this disease. In addition VEGF is implicated in placental oxidative stress during preeclampsia. One of the major cellular defence mechanisms against oxidative stress is the activation of the nuclear factor erythroid 2-related factor 2 (Nrf2). Therefore, the activation of Nrf2 up regulates the HO-1/CO system. The principal aim of this work is to investigate whether the activation of Nrf2 raises VEGF levels by up regulation of CO release. METHODS: This study took place in vitro, the choriocarcinoma cell line BeWo cells and the primary human umbilical vein endothelial cells (HUVECs) were used to study the relationship between VEGF and an Nrf2 inducer Sulforaphane, a naturally occurring compound derived from broccoli. ELISA, Western blot assay and the Dual Luciferase Assay were both mainly applied for protein and VEGF activity analysis. RESULTS: It was found that activation of HO-1 expression via Nrf2/ARE pathway augmented the production of CO, which in turn up-regulated the gene expression of VEGF, and down regulated the production of the antiangiogenic protein, the VEGF antagonist sFlt-1. CONCLUSION: Nrf2 driven HO-1 expression elevates the levels of VEGF via CO production. In particular, activating of Nrf2 via sulforaphane, may have therapeutic potential in preeclampsia.
RESUMO
Critical sized bone defects have to be filled with material to allow bone healing. The golden standard for this treatment is autogenous bone grafting. Because of donor size morbidity, equivalent synthetic bone scaffolds should be developed. Different materials, especially ceramics and polymers are in the focus of research. Calcium phosphate ceramics show similar properties to bone and are degradable. Different modifications can improve the bioactive features. This article gives an overview about the current materials and their evidence of clinical use.
Assuntos
Materiais Biocompatíveis/uso terapêutico , Transplante Ósseo/métodos , Fixação Interna de Fraturas/instrumentação , Consolidação da Fratura/fisiologia , Osteogênese/fisiologia , Alicerces Teciduais , Transplante Ósseo/instrumentação , Fosfatos de Cálcio/uso terapêutico , Cerâmica/uso terapêutico , Medicina Baseada em Evidências , Humanos , Engenharia Tecidual/instrumentaçãoRESUMO
AIMS: Trophoblast fusion in the placenta is prerequisite to successful pregnancy and the pathological conditions related to it. The presence of syncytin-1, is not sufficient to explain the complete event and ADAM12 is a major co-player candidate. Via differential splicing, the ADAM12 gene produces a short and a long form, being the ADAM12-S and the ADAM12-L respectively. METHODS AND RESULTS: We investigated the localisation of both variants in the human placenta using whole mount in situ hybridisation, immunohistochemistry and Northern blotting in 1st (n=8) and 3rd (n=8) trimester placentae and in the case of NB in several cell lines. In Northern blotting, 1st and 3rd trimester placentae were positive for the ADAM12-S and Bewo, 293HEK, JAR, leucocytes, macrophages, 1st and 3rd trimester placentae were positive for ADAM12-L. In whole mount in situ hybridisation, the 1st and 3rd trimester placental syncytium was positive for both variants. In immunohistochemistry, ADAM12-L localised in the cytotrophoblast of both 1st and 3rd trimester placentae, while ADAM12-S localised in the complete syncytium, often including the cytotrophoblast. CONCLUSION: The different localisation of ADAM12-S and ADAM12-L indicates a possible different role making ADAM12-L a candidate for the fusion event, while the syncytial localisation of the ADAM12-S makes it a candidate for cell-cell and cell-matrix interactions between the placental syncytium and the maternal interface.
Assuntos
Proteínas ADAM/genética , Proteínas de Membrana/genética , Placenta/fisiologia , RNA Mensageiro/genética , Proteína ADAM12 , Feminino , Humanos , Gravidez , Isoformas de Proteínas/genética , Distribuição TecidualRESUMO
Femoral head necrosis is an ischaemic bone necrosis of traumatic or nontraumatic pathogenesis which can lead to hip joint destruction in young age. It is today the indication for 10 % of all the total hip joint replacements. Known aetiologies of nontraumatic femoral head necrosis are alcoholism, steroids, sickle cell anaemia, caisson, and Gaucher's disease. Further risk factors are chemotherapy, chronic inflammatory bowel disease, systemic lupus erythematosus, and multiple sclerosis, in which also steroids are involved. Gravidity is another risk factor, but still idiopathic pathogenesis is found. In diagnosis, the ARCO-classification of the Association for the Research of Osseous Circulation is essential. While stage 0 can only be found histologically, the reversible early stage 1 shows MR signal changes. In the irreversible early stage 2, first native x-ray changes are seen as lower radiolucency reflects new bone apposition on dead trabeculae. In stage 3, subchondral fracture follows, and in stage 4 secondary arthritis of the hip. Established therapy in stage 1 is core decompression, physiotherapy, and more and more also bisphosphonates. Sufficient data to support extracorporeal shock wave therapy are still lacking. Stem cell therapy seems to be a promising new therapy method in stage 2. In stage 2 and 3 mainly proximal femoral osteotomies and (non)vascularised bone transplantation are performed. In stage 4, depending on size and location of the necrotic zone and pathology of the adjacent bone, resurfacing or short stem hip arthroplasty can be performed. However, conventional THA is still golden standard. The problem and challenge, however, is the often young patient age in femoral head necrosis. Especially chemotherapy-associated osteonecrosis in leukaemia is found in patients in their second decade of life. Therefore, the hip should be preserved as long as possible.