RESUMO
BACKGROUND: Studies suggest that a single prophylactic dose of amoxicillin reduces early implant complications, but it is unclear whether other antibiotics are also effective. This study compared the local antimicrobial and anti-inflammatory effects resulting from a single dose of azithromycin or amoxicillin before surgical placement of one-stage dental implants. METHODS: Healthy adult patients requiring one-stage dental implant placement were allocated randomly to receive either 2 g amoxicillin (n = 7) or 500 mg azithromycin (n = 6) before surgery. Peri-implant crevicular fluid (PICF) samples from the new implant and gingival crevicular fluid (GCF) from adjacent teeth were sampled on postoperative days 6, 13, and 20. Inflammatory mediators in the samples were analyzed by immunoassay, and antibiotic levels were measured by bioassay. RESULTS: On day 6, azithromycin concentrations in GCF and PICF were 3.39 ± 0.73 and 2.77 ± 0.90 µg/mL, respectively, whereas amoxicillin was below the limit of detection. During early healing, patents in the azithromycin group exhibited a significantly greater decrease in GCF volume (P = 0.03, analysis of variance). At specific times during healing, the azithromycin group exhibited significantly lower levels of interleukin (IL)-6 and IL-8 in GCF than the amoxicillin group and exhibited significantly lower levels of granulocyte colony stimulating factor, IL-8, macrophage inflammatory protein-1ß, and interferon-gamma-inducible protein-10 in PICF. CONCLUSIONS: Azithromycin was available at the surgical site for a longer period of time than amoxicillin, and patients taking azithromycin exhibited lower levels of specific proinflammatory cytokines and chemokines in GCF and PICF. Thus, preoperative azithromycin may enhance resolution of postoperative inflammation to a greater extent than amoxicillin.
Assuntos
Amoxicilina/uso terapêutico , Antibacterianos/uso terapêutico , Azitromicina/uso terapêutico , Implantes Dentários , Líquido do Sulco Gengival , Inflamação/prevenção & controle , Adulto , Disponibilidade Biológica , HumanosRESUMO
A chemiluminescent method is proposed for quantitation of NO generation in cell cultures. The method is based on activation of soluble guanylyl cyclase by NO. The product of the guanylyl cyclase reaction, pyrophosphate, is converted to ATP by ATP sulfurylase and ATP is detected in a luciferin-luciferase system. The method has been applied to the measurement of NO generated by activated murine macrophages (RAW 264.7) and bovine aortic endothelial cells. For macrophages activated by lipopolysaccharide and γ-interferon, the rate of NO production is about 100 amol/(cell·min). The rate was confirmed by the measurements of nitrite, the product of NO oxidation. For endothelial cells, the basal rate of NO generation is 5 amol/(cell·min); the rate approximately doubles upon activation by bradykinin, Ca(2+) ionophore A23187 or mechanical stress. For both types of cells the measured rate of NO generation is strongly affected by inhibitors of NO synthase. The sensitivity of the method is about 50 pM/min, allowing the registration of NO generated by 10(2)-10(4) cells. The enzyme-linked chemiluminescent method is two orders of magnitude more sensitive than fluorescent detection using 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM).
Assuntos
Bioensaio , Células Endoteliais/metabolismo , Luciferina de Vaga-Lumes/química , Luciferases/química , Medições Luminescentes/normas , Macrófagos/metabolismo , Óxido Nítrico/análise , Animais , Aorta/citologia , Aorta/efeitos dos fármacos , Aorta/metabolismo , Bradicinina/farmacologia , Bovinos , Linhagem Celular , GMP Cíclico/metabolismo , Difosfatos/química , Difosfatos/metabolismo , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Luciferina de Vaga-Lumes/metabolismo , Guanosina Trifosfato/metabolismo , Guanilato Ciclase/metabolismo , Lipopolissacarídeos/farmacologia , Luciferases/metabolismo , Luminescência , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Camundongos , Óxido Nítrico/biossíntese , Doadores de Óxido Nítrico/metabolismo , Doadores de Óxido Nítrico/farmacologia , Nitritos/química , Nitritos/metabolismo , Compostos Nitrosos/metabolismo , Compostos Nitrosos/farmacologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Sensibilidade e Especificidade , Guanilil Ciclase Solúvel , Sulfato Adenililtransferase/metabolismoRESUMO
BACKGROUND: Aggressive metastatic breast cancer cells seemingly evade surgical resection and current therapies, leading to colonization in distant organs and tissues and poor patient prognosis. Therefore, high-throughput in vitro tools allowing rapid, accurate, and novel anti-metastatic drug screening are grossly overdue. Conversely, aligned nanofiber constitutes a prominent component of the late-stage breast tumor margin extracellular matrix. This parallel suggests that the use of a synthetic ECM in the form of a nanoscale model could provide a convenient means of testing the migration potentials of cancer cells to achieve a long-term goal of providing clinicians an in vitro platform technology to test the efficacy of novel experimental anti-metastatic compounds. METHODS: Electrospinning produces highly aligned, cell-adhesive nanofiber matrices by applying a strong electric field to a polymer-containing solution. The resulting fibrous microstructure and morphology closely resembles in vivo tumor microenvironments suggesting their use in analysis of migratory potentials of metastatic cancer cells. Additionally, a novel interface with a gel-based delivery system creates CXCL12 chemotactic gradients to enhance CXCR4-expressing cell migration. RESULTS: Cellular dispersions of MCF-10A normal mammary epithelial cells or human breast cancer cells (MCF-7 and MDA-MB-231) seeded on randomly-oriented nanofiber exhibited no significant differences in total or net distance traveled as a result of the underlying topography. Cells traveled ~2-5 fold greater distances on aligned fiber. Highly-sensitive MDA-MB-231 cells displayed an 82% increase in net distance traversed in the presence of a CXCL12 gradient. In contrast, MCF-7 cells exhibited only 31% increase and MCF-10A cells showed no statistical difference versus control or vehicle conditions. MCF-10A cells displayed little sensitivity to CXCL12 gradients, while MCF-7 cells displayed early sensitivity when CXCL12 concentrations were higher. MDA-MB-231 cells displayed low relative expression levels of CXCR4, but high sensitivity resulting in 55-fold increase at late time points due to CXCL12 gradient dissipation. CONCLUSIONS: This model could create clinical impact as an in vitro diagnostic tool for rapid assessment of tumor needle biopsies to confirm metastatic tumors, their invasiveness, and allow high-throughput drug screening providing rapid development of personalized therapies.
Assuntos
Materiais Biomiméticos , Neoplasias da Mama/patologia , Movimento Celular , Nanofibras/ultraestrutura , Materiais Biomiméticos/síntese química , Neoplasias da Mama/química , Movimento Celular/efeitos dos fármacos , Quimiocina CXCL12/farmacologia , Fatores Quimiotáticos/farmacologia , Matriz Extracelular/ultraestrutura , Feminino , Ensaios de Triagem em Larga Escala , Humanos , Células MCF-7 , Invasividade Neoplásica , Metástase Neoplásica , RNA Mensageiro/análise , Receptores CXCR4/genética , Microambiente TumoralRESUMO
Reports demonstrate the role of M-CSF (CSF1) in tumor progression in mouse models as well as the prognostic value of macrophage numbers in breast cancer patients. Recently, a subset of CD14+ monocytes expressing the Tie2 receptor, once thought to be predominantly expressed on endothelial cells, has been characterized. We hypothesized that increased levels of CSF1 in breast tumors can regulate differentiation of Tie2- monocytes to a Tie2+ phenotype. We treated CD14+ human monocytes with CSF1 and found a significant increase in CD14+/Tie2+ positivity. To understand if CSF1-induced Tie2 expression on these cells improved their migratory ability, we pre-treated CD14+ monocytes with CSF1 and used Boyden chemotaxis chambers to observe enhanced response to angiopoietin-2 (ANG2), the chemotactic ligand for the Tie2 receptor. We found that CSF1 pre-treatment significantly augmented chemotaxis and that Tie2 receptor upregulation was responsible as siRNA targeting Tie2 receptor abrogated this effect. To understand any augmented angiogenic effect produced by treating these cells with CSF1, we cultured human umbilical vein endothelial cells (HUVECs) with conditioned supernatants from CSF1-pre-treated CD14+ monocytes for a tube formation assay. While supernatants from CSF1-pre-treated TEMs increased HUVEC branching, a neutralizing antibody against the CSF1R abrogated this activity, as did siRNA against the Tie2 receptor. To test our hypothesis in vivo, we treated PyMT tumor-bearing mice with CSF1 and observed an expansion in the TEM population relative to total F4/80+ cells, which resulted in increased angiogenesis. Investigation into the mechanism of Tie2 receptor upregulation on CD14+ monocytes by CSF1 revealed a synergistic contribution from the PI3 kinase and HIF pathways as the PI3 kinase inhibitor LY294002, as well as HIF-1α-deficient macrophages differentiated from the bone marrow of HIF-1αfl/fl/LysMcre mice, diminished CSF1-stimulated Tie2 receptor expression.
Assuntos
Neoplasias da Mama/metabolismo , Fator Estimulador de Colônias de Macrófagos/farmacologia , Monócitos/citologia , Monócitos/efeitos dos fármacos , Receptor TIE-2/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Feminino , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Receptores de Lipopolissacarídeos/metabolismo , Camundongos , Monócitos/metabolismoRESUMO
Studies suggest that psychosocial factors can impact cancer progression. Parallel work in the fields of psychoneuroimmunology and developmental neuroscience have led to the implication of catecholamine hormones (norepinephrine and epinephrine) and their receptors (the ß-adrenergic receptors; ß-ARs) in regulating cancer progression. In this review we discuss studies that describe the effects of psychological stress as mediated by factors including the catecholamines norepinephrine and epinephrine on various aspects of tumor progression including proliferation, angiogenesis, and metastasis. We discuss the role of ß-ARs in facilitating these effects and the potential use of ß-blockers in adjuvant cancer therapy.
Assuntos
Antagonistas Adrenérgicos beta/uso terapêutico , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/metabolismo , Estresse Psicológico/metabolismo , Antagonistas Adrenérgicos beta/farmacologia , Animais , Progressão da Doença , Humanos , Transdução de Sinais , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/psicologia , Estresse Psicológico/patologiaRESUMO
Stressor exposure has been shown to enhance host susceptibility and the severity of a plethora of illnesses, including gastrointestinal disease. In mice, susceptibility to Citrobacter rodentium has been shown to be dependent on host genetics as well as the composition of the intestinal microbiota, but the effects of stressor exposure on this gastrointestinal pathogen have not been elucidated fully. Previously, our lab showed that exposure to the prolonged-restraint stressor prior to a challenge with C. rodentium alters the intestinal microbiota community structure, including a reduction of beneficial genera such as Lactobacillus, which may contribute to stressor-enhanced C. rodentium-induced infectious colitis. To test the effects of stressor exposure on C. rodentium infection, we exposed resistant mice to a prolonged-restraint stressor concurrent with pathogen challenge. Exposure to prolonged restraint significantly enhanced C. rodentium-induced infectious colitis in resistant mice, as measured by increases in colonic histopathology, colonic inflammatory mediator gene production, and pathogen translocation from the colon to the spleen. It was further tested if the beneficial bacterium Lactobacillus reuteri could reduce the stressor-enhanced susceptibility to C. rodentium-enhanced infectious colitis. While L. reuteri treatment did not reduce all aspects of stressor-enhanced infectious colitis, it did significantly reduce pathogen translocation from the colon to the spleen. Taken together, these data demonstrate the deleterious effects that prolonged stressor exposure can have at the onset of a gastrointestinal infection by its ability to render a resistant mouse highly susceptible to C. rodentium. Probiotic treatment ameliorated the systemic manifestations of stress on colonic infection.
Assuntos
Citrobacter rodentium/metabolismo , Infecções por Enterobacteriaceae/microbiologia , Infecções por Enterobacteriaceae/patologia , Limosilactobacillus reuteri/metabolismo , Probióticos/metabolismo , Estresse Fisiológico/fisiologia , Animais , Ansiedade/complicações , Comportamento Animal , Colite/metabolismo , Colite/microbiologia , Colite/patologia , Colo/metabolismo , Colo/microbiologia , Colo/patologia , Suscetibilidade a Doenças/metabolismo , Suscetibilidade a Doenças/microbiologia , Interleucina-6/metabolismo , Masculino , Camundongos , Baço/metabolismo , Baço/microbiologia , Baço/patologiaRESUMO
AIM: Peri-implant gingival healing following one-stage implant placement was investigated and compared to periodontal healing. METHODS: Healing at surgical sites [implant (I) and adjacent teeth (T+)] was compared to non-operated tooth (T-) in non-smokers receiving one-stage implant. Periodontal Indices (PI, GI) were recorded at surgery and up to 12 weeks post-operatively. Peri-implant (PICF) and gingival crevicular fluids (GCF) were analysed for cytokines, collagenases and inhibitors. Data were analysed by linear mixed model regression analysis and repeated measures anova. RESULTS: Forty patients (22 females; 21-74 years old) completed the study. Surgical site GI, increased at week 1, decreased significantly during early healing (weeks 1-3; p = 0.0003) and continually decreased during late healing (weeks 6-12) for I (p < 0.01). PICF volume decreased threefold by week 12 (p = 0.0003). IL-6, IL-8, MIP-1ß and TIMP-1 levels significantly increased at surgical sites at week one, significantly decreasing thereafter (p < 0.016). Week one IL-6, IL-8 and MIP-1ß levels were ~threefold higher and TIMP-1 levels 63% higher, at I compared to T+ (p = 0.001). CONCLUSION: Peri-implant gingival healing, as determined by crevicular fluid molecular composition, differs from periodontal healing. The observed differences suggest that peri-implant tissues, compared to periodontal tissues, represent a higher pro-inflammatory state.
Assuntos
Implantes Dentários , Gengiva/patologia , Periodonto/patologia , Adulto , Idoso , Quimiocina CCL4/análise , Estudos de Coortes , Feminino , Seguimentos , Gengiva/cirurgia , Líquido do Sulco Gengival/química , Humanos , Interleucina-6/análise , Interleucina-8/análise , Masculino , Metaloproteinase 8 da Matriz , Metaloproteinase 9 da Matriz , Inibidores de Metaloproteinases de Matriz/análise , Pessoa de Meia-Idade , Índice Periodontal , Periodonto/cirurgia , Estudos Prospectivos , Retalhos Cirúrgicos/patologia , Inibidor Tecidual de Metaloproteinase-1/análise , Inibidor Tecidual de Metaloproteinase-2 , Fator A de Crescimento do Endotélio Vascular/análise , Cicatrização/fisiologia , Adulto JovemRESUMO
Pancreatic stellate cells (PSC) are a subset of pancreatic cancer-associated fibroblasts. These cells provide prosurvival signals to tumors; however, little is known regarding their interactions with immune cells within the tumor microenvironment. We hypothesized that factors produced by human PSC could enhance myeloid-derived suppressor cell (MDSC) differentiation and function, which promotes an immunosuppressive microenvironment. Primary PSC cell lines (n = 7) were generated from human specimens and phenotypically confirmed via expression of vimentin, α-smooth muscle actin (α-SMA), and glial fibrillary acidic protein (GFAP). Luminex analysis indicated that PSC but not human fetal primary pancreatic fibroblast cells (HPF; negative controls) produced MDSC-promoting cytokines [interleukin (IL-6), VEGF, macrophage colony-stimulating factor (M-CSF) ] and chemokines (SDF-1, MCP-1). Culture of peripheral blood mononuclear cells [peripheral blood mononuclear cell (PBMC), n = 3 donors] with PSC supernatants or IL-6/granulocyte macrophage colony-stimulating factor (GM-CSF; positive control) for 7 days promoted PBMC differentiation into an MDSC (CD11b+CD33+) phenotype and a subpopulation of polymorphonuclear CD11b+CD33+CD15+ cells. The resulting CD11b+CD33+ cells functionally suppressed autologous T-lymphocyte proliferation. In contrast, supernatants from HPF did not induce an MDSC phenotype in PBMCs. Culture of normal PBMCs with PSC supernatants led to STAT3 but not STAT1 or STAT5 phosphorylation. IL-6 was an important mediator as its neutralization inhibited PSC supernatant-mediated STAT3 phosphorylation and MDSC differentiation. Finally, the FLLL32 STAT3 inhibitor abrogated PSC supernatant-mediated MDSC differentiation, PSC viability, and reduced autocrine IL-6 production indicating these processes are STAT3 dependent. These results identify a novel role for PSC in driving immune escape in pancreatic cancer and extend the evidence that STAT3 acts as a driver of stromal immunosuppression to enhance its interest as a therapeutic target.
Assuntos
Diferenciação Celular , Células Mieloides/imunologia , Neoplasias Pancreáticas/imunologia , Células Estreladas do Pâncreas/fisiologia , Fator de Transcrição STAT3/fisiologia , Linhagem Celular Tumoral , Humanos , Interleucina-6/fisiologia , Ativação Linfocitária , Células Mieloides/citologia , Neoplasias Pancreáticas/patologia , Células Estreladas do Pâncreas/patologia , Transdução de Sinais , Linfócitos T/imunologia , Evasão TumoralRESUMO
Microvesicles are small membrane-bound particles comprised of exosomes and various-sized extracellular vesicles. These are released by several cell types. Microvesicles have a variety of cellular functions from communication to mediating growth and differentiation. Microvesicles contain proteins and nucleic acids. Previously, we showed that plasma microvesicles contain microRNAs (miRNAs). Based on our previous report, the majority of peripheral blood microvesicles are derived from platelets, while mononuclear phagocytes, including macrophages, are the second most abundant population. Here, we characterized macrophage-derived microvesicles and explored their role in the differentiation of naive monocytes. We also identified the miRNA content of the macrophage-derived microvesicles. We found that RNA molecules contained in the macrophage-derived microvesicles were transported to target cells, including mono cytes, endothelial cells, epithelial cells, and fibroblasts. Furthermore, we found that miR-223 was transported to target cells and was functionally active. Based on our observations, we hypothesize that microvesicles bind to and activate target cells. Furthermore, we find that microvesicles induce the differentiation of macrophages. Thus, defining key components of this response may identify novel targets to regulate host defense and inflammation.
Assuntos
Diferenciação Celular , Micropartículas Derivadas de Células/metabolismo , Exossomos/metabolismo , Macrófagos/metabolismo , MicroRNAs/metabolismo , Comunicação Celular , Linhagem Celular , Linhagem Celular Tumoral , Células Cultivadas , Perfilação da Expressão Gênica , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Macrófagos/citologia , Macrófagos/ultraestrutura , MicroRNAs/genética , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Monócitos/citologia , Monócitos/metabolismo , Monócitos/ultraestrutura , Análise de Sequência com Séries de Oligonucleotídeos , Transporte de RNA/efeitos dos fármacosRESUMO
M-CSF is overexpressed in breast cancer and is known to stimulate macrophages to produce VEGF resulting in angiogenesis. It has recently been shown that the growth factor GM-CSF injected into murine breast tumors slowed tumor growth by secreting soluble VEGF receptor-1 (sVEGFR-1) that binds and inactivates VEGF. This study presents a mathematical model that includes all the components above, as well as MCP-1, tumor cells, and oxygen. The model simulations are representative of the in vivo data through predictions of tumor growth using different protocol strategies for GM-CSF for the purpose of predicting higher degrees of treatment success. For example, our model predicts that once a week dosing of GM-CSF would be less effective than daily, twice a week, or three times a week treatment because of the presence of essential factors required for the anti-tumor effect of GM-CSF.
Assuntos
Antineoplásicos/farmacologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Neoplasias Mamárias Experimentais/tratamento farmacológico , Modelos Biológicos , Animais , Antineoplásicos/administração & dosagem , Proliferação de Células/efeitos dos fármacos , Citocinas/biossíntese , Relação Dose-Resposta a Droga , Esquema de Medicação , Células Endoteliais/efeitos dos fármacos , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/administração & dosagem , Macrófagos/efeitos dos fármacos , Neoplasias Mamárias Experimentais/irrigação sanguínea , Neoplasias Mamárias Experimentais/patologia , Camundongos , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/patologia , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
Macrophages contribute to tumor growth through the secretion of the proangiogenic molecule vascular endothelial growth factor (VEGF). We previously observed that monocytes treated with the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) produce a soluble form of the VEGF receptor-1 (sVEGFR-1), which neutralizes VEGF biologic activity. The VEGF and VEGFR-1 promoters both contain a hypoxia regulatory element, which binds the hypoxia-inducible factor (HIF) transcription factors under hypoxic conditions. Based on this observation, we examined VEGF and sVEGFR-1 production from monocytes cultured at various O(2) concentrations. The amount of sVEGFR-1 production observed from GM-CSF-treated monocytes increased with decreasing levels of O(2). This sVEGFR-1 was biologically active and sequestered VEGF. To evaluate the role of the HIFs in sVEGFR-1 production, we used macrophages with a genetic deletion of HIF-1α. HIF-1α(-/-) macrophages cultured with GM-CSF at hypoxia secreted diminished amounts of VEGF compared with HIF-1α(+/+) macrophages, whereas sVEGFR-1 secretion was unaffected. In contrast, siRNA-mediated knockdown of HIF-2α inhibited the production of sVEGFR-1 in response to GM-CSF and low O(2), whereas VEGF production was unaffected. These studies suggest that hypoxia, generally thought to promote angiogenesis, can induce antiangiogenic behavior from macrophages within a GM-CSF-rich environment. Furthermore, these results suggest specific and independent roles for HIF-1α and HIF-2α in hypoxic macrophages.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Endotélio Vascular/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Hipóxia/metabolismo , Sistema Fagocitário Mononuclear/metabolismo , Neovascularização Fisiológica/fisiologia , Animais , Western Blotting , Citometria de Fluxo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/metabolismo , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT5 , Veias Umbilicais/citologia , 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/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Tumor-educated macrophages facilitate tumor metastasis and angiogenesis. We discovered that granulocyte macrophage colony-stimulating factor (GM-CSF) blocked macrophages vascular endothelial growth factor (VEGF) activity by producing soluble VEGF receptor-1 (sVEGFR-1) and determined the effect on tumor-associated macrophage behavior and tumor growth. We show GM-CSF treatment of murine mammary tumors slowed tumor growth and slowed metastasis. These tumors had more macrophages, fewer blood vessels, and lower oxygen concentrations. This effect was sVEGFR-1 dependent. In situ hybridization and flow cytometry identified macrophages as the primary source of sVEGFR-1. These data suggest that GM-CSF can re-educate macrophages to reduce angiogenesis and metastases in murine breast cancer.
Assuntos
Inibidores da Angiogênese/farmacologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Macrófagos/efeitos dos fármacos , Neoplasias Mamárias Experimentais/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Polaridade Celular/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/uso terapêutico , Neoplasias Pulmonares/prevenção & controle , Neoplasias Pulmonares/secundário , Macrófagos/fisiologia , Neoplasias Mamárias Experimentais/irrigação sanguínea , Neoplasias Mamárias Experimentais/patologia , Camundongos , Neutrófilos/efeitos dos fármacos , Oxigênio/análise , Fator A de Crescimento do Endotélio Vascular/análise , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
BACKGROUND: M-CSF recruits mononuclear phagocytes which regulate processes such as angiogenesis and metastases in tumors. VEGF is a potent activator of angiogenesis as it promotes endothelial cell proliferation and new blood vessel formation. Previously, we reported that in vitro M-CSF induces the expression of biologically-active VEGF from human monocytes. METHODOLOGY AND RESULTS: In this study, we demonstrate the molecular mechanism of M-CSF-induced VEGF production. Using a construct containing the VEGF promoter linked to a luciferase reporter, we found that a mutation reducing HIF binding to the VEGF promoter had no significant effect on luciferase production induced by M-CSF stimulation. Further analysis revealed that M-CSF induced VEGF through the MAPK/ERK signaling pathway via the transcription factor, Sp1. Thus, inhibition of either ERK or Sp1 suppressed M-CSF-induced VEGF at the mRNA and protein level. M-CSF also induced the nuclear localization of Sp1, which was blocked by ERK inhibition. Finally, mutating the Sp1 binding sites within the VEGF promoter or inhibiting ERK decreased VEGF promoter activity in M-CSF-treated human monocytes. To evaluate the biological significance of M-CSF induced VEGF production, we used an in vivo angiogenesis model to illustrate the ability of M-CSF to recruit mononuclear phagocytes, increase VEGF levels, and enhance angiogenesis. Importantly, the addition of a neutralizing VEGF antibody abolished M-CSF-induced blood vessel formation. CONCLUSION: These data delineate an ERK- and Sp1-dependent mechanism of M-CSF induced VEGF production and demonstrate for the first time the ability of M-CSF to induce angiogenesis via VEGF in vivo.
Assuntos
Sistema de Sinalização das MAP Quinases , Fator Estimulador de Colônias de Macrófagos/fisiologia , Neovascularização Fisiológica , Fator de Transcrição Sp1/fisiologia , Fator A de Crescimento do Endotélio Vascular/biossíntese , Transporte Ativo do Núcleo Celular , Animais , Células Cultivadas , Humanos , Camundongos , Monócitos , Fator de Transcrição Sp1/metabolismo , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
M-CSF induces PI 3-kinase activation, resulting in reactive oxygen species (ROS) production. Previously, we reported that ROS mediate macrophage colony-stimulating factor (M-CSF)-induced extracellular regulated kinase (Erk) activation and monocyte survival. In this work, we hypothesized that M-CSF-stimulated ROS products modulated Akt1 and p38 activation. Furthermore, we sought to clarify the source of these ROS and the role of ROS and Akt in monocyte/macrophage survival. Macrophages from p47(phox-/-) mice, lacking a key component of the NADPH oxidase complex required for ROS generation, had reduced cell survival and Akt1 and p38 mitogen-activated protein kinase (MAPK) phosphorylation compared with wild-type macrophages in response to M-CSF stimulation, but had no difference in M-CSF-stimulated Erk. To understand how ROS affected monocyte survival and signaling, we observed that NAC and DPI decreased cell survival and Akt1 and p38 MAPK phosphorylation. Using bone marrow-derived macrophages from mice expressing constitutively activated Akt1 (Myr-Akt1) or transfecting Myr-Akt1 constructs into human peripheral monocytes, we concluded that Akt is a positive regulator of monocyte survival. Moreover, the p38 MAPK inhibitor, SB203580, inhibited p38 activity and M-CSF-induced monocyte survival. These findings demonstrate that ROS generated from the NADPH oxidase complex contribute to monocyte/macrophage survival induced by M-CSF via regulation of Akt and p38 MAPK.
Assuntos
Monócitos/fisiologia , NADPH Oxidases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Células da Medula Óssea/fisiologia , Sobrevivência Celular , Células Cultivadas , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Imidazóis/farmacologia , Fator Estimulador de Colônias de Macrófagos/fisiologia , Macrófagos/fisiologia , Camundongos , Fosforilação , Piridinas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidoresRESUMO
Recent studies using ovarian cancer cells have shown that the catecholamine hormones norepinephrine (norepi) and epinephrine (epi) may influence cancer progression by modulating the expression of matrix metalloproteinases (MMP) and vascular endothelial growth factor (VEGF). The purpose of this study is to determine if the stress hormone norepi can influence the expression of MMP-2, MMP-9, and VEGF in nasopharyngeal carcinoma (NPC) tumors by using three NPC tumor cell lines. The NPC cell lines HONE-1, HNE-1, and CNE-1 were treated with norepi. The effects of norepi on MMP-2, MMP-9, and VEGF synthesis were measured by ELISA; functional MMP activity was measured by the invasive potential of the cells using a membrane invasion culture system whereas functional activity of VEGF was analyzed using a human umbilical vein endothelial cell tube formation assay. Norepi treatment increased MMP-2, MMP-9, and VEGF levels in culture supernatants of HONE-1 cells, which could be inhibited by the beta-blocker propranolol. Norepi induced the invasiveness of all NPC cell lines in a dose-dependent manner, which was blocked by CMT-3, an MMP inhibitor, and propranolol. Norepi stimulated the release of functional angiogenic VEGF by HONE-1 cells as well. Finally, HONE-1 cells were shown to express beta-adrenergic receptors as did seven of seven NPC biopsies examined. The data suggest that catecholamine hormones produced by the sympathetic-adrenal medullary axis may affect NPC tumor progression, in part, through modulation of key angiogenic cytokines.
Assuntos
Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 9 da Matriz/genética , Neoplasias Nasofaríngeas/metabolismo , Norepinefrina/farmacologia , Fator A de Crescimento do Endotélio Vascular/genética , Biópsia , Linhagem Celular Tumoral , Humanos , Metaloproteinase 14 da Matriz/genética , Neoplasias Nasofaríngeas/patologia , Invasividade Neoplásica , Receptores Adrenérgicos beta/fisiologia , Regulação para CimaRESUMO
GM-CSF promotes homeostasis of myeloid cells. We report that GM-CSF upregulates mRNA and protein production of the soluble form of membrane bound VEGF receptor-1 (sVEGFR-1) in human monocytes. This sVEGFR-1 was biologically active, as cell-free supernatants from GM-CSF-stimulated monocytes blocked detection of endogenously expressed VEGF and inhibited endothelial cell migration and tube formation, even in the presence of exogenous rhVEGF. VEGF activity was recovered by neutralizing sVEGFR-1. To determine whether these events were important in vivo, Matrigel plugs were incubated with rhVEGF, rhGM-CSF, or rhGM-CSF/rhVEGF and injected into mice. Plugs containing GM-CSF or GM-CSF/VEGF had less endothelial cell invasion than plugs containing rhVEGF and were similar to plugs incubated with PBS alone. Neutralizing antibodies specific for sVEGFR-1 injected in these plugs reversed the effects of GM-CSF or GM-CSF/VEGF, while an isogenic antibody did not. Thus, GM-CSF and monocytes play a vital role in angiogenesis through the regulation of VEGF and sVEGFR-1.
Assuntos
Inibidores da Angiogênese/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Membrana Celular/metabolismo , Células Cultivadas , Colágeno/metabolismo , Meios de Cultivo Condicionados/química , Combinação de Medicamentos , Células Endoteliais/citologia , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Janus Quinase 2 , Laminina/metabolismo , Camundongos , Testes de Neutralização , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteoglicanas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , RNA Mensageiro/genética , Transdução de Sinais , Solubilidade , Fator A de Crescimento do Endotélio Vascular/análise , Fator A de Crescimento do Endotélio Vascular/farmacologia , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
The impact of the immune response in malignancy is poorly understood. While immune cells can destroy transformed cells, the targeting and accumulation of monocytes and macrophages at tumor sites may promote tumor metastases. The growth factor M-CSF is important in promoting monocyte survival. Since M-CSF(-/-) mice are protected against tumor metastases, we hypothesized that M-CSF induced monocytes to produce angiogenic factors that facilitate metastases. In this study we demonstrate that recombinant human M-CSF induces freshly isolated normal human monocytes to produce and release the growth factor vascular endothelial growth factor (VEGF) in a dose-dependent manner, which peaked at 5 days in culture. VEGF released by these monocytes is biologically active, as cell-free supernatants from these M-CSF-stimulated monocytes induced tube formation in HUVEC. Network formation by these HUVECs after treatment with supernatants from monocytes stimulated with M-CSF were inhibited by anti-VEGF, but not by the isogenic control, Abs. Collectively, these data support an important role for M-CSF and monocytes in VEGF production and angiogenesis.