RESUMO
Although insulin-like growth factor 1 (IGF-1) has been associated with retinopathy, proof of a direct relationship has been lacking. Here we show that an IGF-1 receptor antagonist suppresses retinal neovascularization in vivo, and infer that interactions between IGF-1 and the IGF-1 receptor are necessary for induction of maximal neovascularization by vascular endothelial growth factor (VEGF). IGF-1 receptor regulation of VEGF action is mediated at least in part through control of VEGF activation of p44/42 mitogen-activated protein kinase, establishing a hierarchical relationship between IGF-1 and VEGF receptors. These findings establish an essential role for IGF-1 in angiogenesis and demonstrate a new target for control of retinopathy. They also explain why diabetic retinopathy initially increases with the onset of insulin treatment. IGF-1 levels, low in untreated diabetes, rise with insulin therapy, permitting VEGF-induced retinopathy.
Assuntos
Fatores de Crescimento Endotelial/fisiologia , Linfocinas/fisiologia , Neovascularização Patológica/fisiopatologia , Receptor IGF Tipo 1/fisiologia , Vasos Retinianos/fisiologia , Animais , Inibidores do Crescimento/farmacologia , Humanos , Insulina/farmacologia , Fator de Crescimento Insulin-Like I/análogos & derivados , Fator de Crescimento Insulin-Like I/farmacologia , Isquemia/etiologia , Isquemia/fisiopatologia , Isquemia/prevenção & controle , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Patológica/etiologia , Neovascularização Patológica/prevenção & controle , Proteínas Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/fisiologia , Receptor IGF Tipo 1/antagonistas & inibidores , Receptores de Fatores de Crescimento/fisiologia , Receptores de Fatores de Crescimento do Endotélio Vascular , Vasos Retinianos/efeitos dos fármacos , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Mangroves are effective blue carbon sinks and are the most carbon rich ecosystems on earth. However, their areal extent has declined by over one-third in recent decades. Degraded mangrove forests result in reduced carbon captured and lead to release of stored carbon into the atmosphere by CO2 emission. The aim of this study was to assess changes in carbon dynamics in a gradually degrading mangrove forest on Bonaire, Dutch Caribbean. Remote sensing techniques were applied to estimate the distribution of intact and degraded mangroves. Forest structure, sediment carbon storage, sediment CO2 effluxes and dissolved organic and inorganic carbon in pore and surface waters across intact and degraded parts were assessed. On average intact mangroves showed 31% sediment organic carbon in the upper 30 cm compared to 20% in degraded mangrove areas. A loss of 1.51 MgCO2 ha-1 yr-1 for degraded sites was calculated. Water samples showed a hypersaline environment in the degraded mangrove area averaging 93 which may have caused mangrove dieback. Sediment CO2 efflux within degraded sites was lower than values from other studies where degradation was caused by clearing or cutting, giving new insights into carbon dynamics in slowly degrading mangrove systems. Results of water samples agreed with previous studies where inorganic carbon outwelled from mangroves might enhance ecosystem connectivity by potentially buffering ocean acidification locally. Wetlands will be impacted by a variety of stressors resulting from a changing climate. Results from this study could inform scientists and stakeholders on how combined stresses, such as climate change with salinity intrusion may impact mangrove's blue carbon sink potential and highlight the need of future comparative studies of intact versus degraded mangrove stands.
RESUMO
Persistent microvascular hyperpermeability to plasma proteins even after the cessation of injury is a characteristic but poorly understood feature of normal wound healing. It results in extravasation of fibrinogen that clots to form fibrin, which serves as a provisional matrix and promotes angiogenesis and scar formation. We present evidence indicating that vascular permeability factor (VPF; also known as vascular endothelial growth factor) may be responsible for the hyperpermeable state, as well as the angiogenesis, that are characteristic of healing wounds. Hyperpermeable blood vessels were identified in healing split-thickness guinea pig and rat punch biopsy skin wounds by their capacity to extravasate circulating macromolecular tracers (colloidal carbon, fluoresceinated dextran). Vascular permeability was maximal at 2-3 d, but persisted as late as 7 d after wounding. Leaky vessels were found initially at the wound edges and later in the subepidermal granulation tissue as keratinocytes migrated to cover the denuded wound surface. Angiogenesis was also prominent within this 7-d interval. In situ hybridization revealed that greatly increased amounts of VPF mRNA were expressed by keratinocytes, initially those at the wound edge, and, at later intervals, keratinocytes that migrated to cover the wound surface; occasional mononuclear cells also expressed VPF mRNA. Secreted VPF was detected by immunofluoroassay of medium from cultured human keratinocytes. These data identify keratinocytes as an important source of VPF gene transcript and protein, correlate VPF expression with persistent vascular hyperpermeability and angiogenesis, and suggest that VPF is an important cytokine in wound healing.
Assuntos
Fatores de Crescimento Endotelial/análise , Queratinócitos/metabolismo , Linfocinas/análise , Cicatrização , Animais , Sequência de Bases , Células Cultivadas , Fatores de Crescimento Endotelial/genética , Feminino , Cobaias , Humanos , Linfocinas/genética , Dados de Sequência Molecular , RNA Mensageiro/análise , Ratos , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Vascular permeability factor (VPF, also known as vascular endothelial growth factor or VEGF), is a potent microvascular permeability enhancing cytokine and a selective mitogen for endothelial cells. It has been implicated in tumor angiogenesis and ascites fluid accumulation. Since development of the destructive synovial pannus in rheumatoid arthritis (RA) is associated with changes in vascular permeability (synovial fluid accumulation), synovial cell hyperplasia, and angiogenesis, we examined synovial fluids (SFs) and joint tissue for the expression and local accumulation of VPF/VEGF. VPF/VEGF was detected in all of 21 synovial fluids examined and when measured by an immunofluorimetric assay, ranged from 6.9 to 180.5 pM. These levels are biologically significant, since < 1 pM VPF/VEGF can elicit responses from its target cells, endothelial cells. Levels of VPF/VEGF were highest in rheumatoid arthritis fluids (n = 10), with a mean value (+/- SEM) of 59.1 +/- 18.0 pM, vs. 21.4 +/- 2.3 pM for 11 SFs from patients with other forms of arthritis (p = 0.042). In situ hybridization studies that were performed on joint tissues from patients with active RA revealed that synovial lining macrophages strongly expressed VPF/VEGF mRNA, and that microvascular endothelial cells of nearby blood vessels strongly expressed mRNA for the VPF/VEGF receptors, flt-1 and KDR. Immunohistochemistry performed on inflamed rheumatoid synovial tissue revealed that the VPF/VEGF peptide was localized to macrophages within inflamed synovium, as well as to microvascular endothelium, its putative target in the tissue. Together, these findings indicate that VPF/VEGF may have an important role in the pathogenesis of RA.
Assuntos
Artrite Reumatoide/metabolismo , Fatores de Crescimento Endotelial/análise , Linfocinas/análise , Líquido Sinovial/química , Membrana Sinovial/química , Adulto , Idoso , Artrite Reumatoide/etiologia , Fatores de Crescimento Endotelial/genética , Fatores de Crescimento Endotelial/fisiologia , Feminino , Humanos , Linfocinas/genética , Linfocinas/fisiologia , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/análise , Receptores Proteína Tirosina Quinases/análise , Receptores Proteína Tirosina Quinases/genética , Receptores de Fatores de Crescimento/análise , Receptores de Fatores de Crescimento/genética , Receptores de Fatores de Crescimento do Endotélio Vascular , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Vascular permeability factor (VPF) is a highly conserved 34-42-kD protein secreted by many tumor cells. Among the most potent vascular permeability-enhancing factors known, VPF is also a selective vascular endothelial cell mitogen, and therefore has been called vascular endothelial cell growth factor (VEGF). Our goal was to define the cellular sites of VPF (VEGF) synthesis and accumulation in tumors in vivo. Immunohistochemical studies were performed on solid and ascites guinea pig line 1 and line 10 bile duct carcinomas using antibodies directed against peptides synthesized to represent the NH2-terminal and internal sequences of VPF. These antibodies stained tumor cells and, uniformly and most intensely, the endothelium of immediately adjacent blood vessels, both preexisting and those newly induced by tumor angiogenesis. A similar pattern of VPF staining was observed in autochthonous human lymphoma. In situ hybridization demonstrated VPF mRNA in nearly all line 10 tumor cells but not in tumor blood vessels, indicating that immunohistochemical labeling of tumor vessels with antibodies to VPF peptides reflects uptake of VPF, not endogenous synthesis. VPF protein staining was evident in adjacent preexisting venules and small veins as early as 5 h after tumor transplant and plateaued at maximally intense levels in newly induced tumor vessels by approximately 5 d. VPF-stained vessels were also hyperpermeable to macromolecules as judged by their capacity to accumulate circulating colloidal carbon. In contrast, vessels more than approximately 0.5 mm distant from tumors were not hyperpermeable and did not exhibit immunohistochemical staining for VPF. Vessel staining disappeared within 24-48 h of tumor rejection. These studies indicate that VPF is synthesized by tumor cells in vivo and accumulates in nearby blood vessels, its target of action. Because leaky tumor vessels initiate a cascade of events, which include plasma extravasation and which lead ultimately to angiogenesis and tumor stroma formation, VPF may have a pivotal role in promoting tumor growth. Also, VPF immunostaining provides a new marker for tumor blood vessels that may be exploitable for tumor imaging or therapy.
Assuntos
Fatores de Crescimento Endotelial/análise , Linfocinas/análise , Neoplasias Experimentais/química , Sequência de Aminoácidos , Animais , Vasos Sanguíneos/química , Fatores de Crescimento Endotelial/genética , Fatores de Crescimento Endotelial/metabolismo , Cobaias , Imuno-Histoquímica , Linfocinas/genética , Linfocinas/metabolismo , Dados de Sequência Molecular , Neoplasias Experimentais/irrigação sanguínea , Hibridização de Ácido Nucleico , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
According to the current theory of retrograde signaling, NGF binds to receptors on the axon terminals and is internalized by receptor-mediated endocytosis. Vesicles with NGF in their lumina, activating receptors in their membranes, travel to the cell bodies and initiate signaling cascades that reach the nucleus. This theory predicts that the retrograde appearance of activated signaling molecules in the cell bodies should coincide with the retrograde appearance of the NGF that initiated the signals. However, we observed that NGF applied locally to distal axons of rat sympathetic neurons in compartmented cultures produced increased tyrosine phosphorylation of trkA in cell bodies/ proximal axons within 1 min. Other proximal proteins, including several apparently localized in cell bodies, displayed increased tyrosine phosphorylation within 5-15 min. However, no detectable 125I-NGF appeared in the cell bodies/proximal axons within 30-60 min of its addition to distal axons. Even if a small, undetectable fraction of transported 125I-NGF was internalized and loaded onto the retrograde transport system immediately after NGF application, at least 3-6 min would be required for the NGF that binds to receptors on distal axons just outside the barrier to be transported to the proximal axons just inside the barrier. Moreover, it is unlikely that the tiny fraction of distal axon trk receptors located near the barrier alone could produce a measurable retrograde trk phosphorylation even if enough time was allowed for internalization and transport of these receptors. Thus, our results provide strong evidence that NGF-induced retrograde signals precede the arrival of endocytotic vesicles containing the NGF that induced them. We further suggest that at least some components of the retrograde signal are carried by a propagation mechanism.
Assuntos
Fatores de Crescimento Neural/metabolismo , Neurônios/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Fator de Crescimento Neural/metabolismo , Tirosina/metabolismo , Animais , Transporte Biológico , Compartimento Celular , Células Cultivadas , Fatores de Crescimento Neural/farmacologia , Neuritos , Fosforilação , Ratos , Ratos Sprague-Dawley , Receptor trkA , Transdução de Sinais/fisiologia , Gânglio Cervical Superior/citologiaRESUMO
Growing axons receive a substantial supply of tubulin and other proteins delivered from sites of synthesis in the cell body by slow axonal transport. To investigate the mechanism of tubulin transport most previous studies have used in vitro models in which the transport of microtubules can be visualized during brief periods of growth. To investigate total tubulin transport in neurons displaying substantial growth over longer periods, we used rat sympathetic neurons in compartmented cultures. Tubulin synthesized during pulses of [35S]methionine was separated from other proteins by immunoprecipitation with monoclonal antibodies to alpha and beta tubulin, further separated on SDS-PAGE, and quantified by phosphorimaging. Results showed that 90% of newly synthesized tubulin moved into the distal axons within 2 d. Furthermore, the leading edge of tubulin was transported at a velocity faster than 4 mm/d, more than four times the rate of axon elongation. This velocity did not diminish with distance from the cell body, suggesting that the transport system is capable of distributing newly synthesized tubulin to growth cones throughout the axonal tree. Neither diffusion nor the an mass transport of axonal microtubules can account for the velocity and magnitude of tubulin transport that was observed. Thus, it is likely that most of the newly synthesized tubulin was supplied to the growing axonal tree in subunit form such as a heterodimer or an oligomer considerably smaller than a microtubule.
Assuntos
Transporte Axonal/fisiologia , Axônios/metabolismo , Gânglio Cervical Superior/citologia , Tubulina (Proteína)/metabolismo , Animais , Animais Recém-Nascidos , Transporte Biológico , Técnicas de Cultura de Células , Células Cultivadas , Microtúbulos/metabolismo , Neurônios/citologia , Ratos , Ratos Sprague-Dawley , Tubulina (Proteína)/biossínteseRESUMO
Extravascular coagulation is a prominent feature of such important pathological processes as cellular immunity and neoplasia and has been thought to result from procoagulants associated with the inflammatory or tumor cells peculiar to these entities. It was found that increased microvascular permeability alone is sufficient to induce equivalent extravascular coagulation in several normal tissues. The results indicate that saturating levels of procoagulant are present even in normal tissues and that microvascular permeability is a rate-limiting step in extravascular coagulation.
Assuntos
Coagulação Sanguínea , Permeabilidade Capilar , Animais , Bradicinina/farmacologia , Permeabilidade Capilar/efeitos dos fármacos , Fibrina/fisiologia , Fibrinogênio/farmacologia , Cobaias , Histamina/farmacologia , Neoplasias/fisiopatologia , Pele/irrigação sanguínea , Pele/efeitos dos fármacos , Fenômenos Fisiológicos da PeleRESUMO
Tumor ascites fluids from guinea pigs, hamsters, and mice contain activity that rapidly increases microvascular permeability. Similar activity is also secreted by these tumor cells and a variety of other tumor cell lines in vitro. The permeability-increasing activity purified from either the culture medium or ascites fluid of one tumor, the guinea pig line 10 hepatocarcinoma, is a 34,000- to 42,000-dalton protein distinct from other known permeability factors.
Assuntos
Permeabilidade Capilar , Neoplasias Experimentais/fisiopatologia , Animais , Ascite/fisiopatologia , Líquido Ascítico/fisiologia , Cricetinae , Cobaias , CamundongosRESUMO
Osteopontin (OPN) is a secreted adhesive glycoprotein with a functional glycine-arginine-glycine-aspartate-serine (GRGDS) cell-binding domain. An interesting feature of OPN structure is the presence of a thrombin-cleavage site in close proximity to the GRGDS region. Cleavage of OPN by thrombin is likely to be of physiological importance, because cleavage of blood plasma OPN occurs naturally after activation of the blood coagulation pathway. To investigate functional consequences of OPN cleavage by thrombin, cell attachment and spreading assays were performed with uncleaved and cleaved forms of OPN. For all cell lines examined, thrombin-cleaved OPN promoted markedly greater cell attachment and spreading than uncleaved OPN. Cell attachment and spreading on thrombin-cleaved OPN was inhibited both by the soluble GRGDS peptides and an OPN-specific antibody raised to the GRGDS domain of OPN, thus implicating the GRGDS region in mediating the increased cell attachment and spreading observed on thrombin-cleaved OPN. Because the GRGDS sequence in OPN is only six residues from the thrombin-cleavage site, the data suggest that possibility that thrombin cleavage allows greater accessibility of the GRGDS domain to cell surface receptors. To investigate receptors that recognize uncleaved and thrombin-cleaved OPN, affinity chromatography was performed on placental extracts; the cell surface integrin alpha v beta 3 bound to columns constructed either with native or thrombin-cleaved OPN and was selectively eluted from each with soluble GRGDS peptide and EDTA. Moreover, adhesion assays performed in the presence of alpha v beta 3 blocking monoclonal antibody LM609 identified alpha v beta 3 as a major functional receptor for thrombin-cleaved OPN. Several lines of evidence suggest that cleavage of OPN by thrombin occurs in vivo, such as in tumors and at sites of tissue injury, and adhesion assay data presented here indicate that such cleavage is important in the regulation of OPN function.
Assuntos
Adesão Celular/fisiologia , Sialoglicoproteínas/genética , Sialoglicoproteínas/metabolismo , Trombina/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Linhagem Celular , Humanos , Integrinas/metabolismo , Dados de Sequência Molecular , Estrutura Molecular , Peso Molecular , Osteopontina , Ratos , Receptores de Citoadesina/metabolismo , Receptores de Vitronectina , Sialoglicoproteínas/químicaRESUMO
Vascular permeability factor (VPF), also known as vascular endothelial growth factor (VEGF), increases microvascular permeability and is a specific mitogen for endothelial cells. Expression of VPF/VEGF previously was demonstrated in a variety of tumor cells, in cultures of pituitary-derived cells, and in corpus luteum. Here we present evidence, by Northern analysis and in situ hybridization, that the VPF/VEGF gene is expressed in many adult organs, including lung, kidney, adrenal gland, heart, liver, and stomach mucosa, as well as in elicited peritoneal macrophages. The highest levels of VPF/VEGF transcripts were found in epithelial cells of lung alveoli, renal glomeruli and adrenal cortex, and in cardiac myocytes. The prominence of VPF/VEGF mRNA in these tissues suggests a possible role for VPF/VEGF in regulating baseline microvascular permeability, which is essential for tissue nutrition and waste removal. We also demonstrate particularly high VPF/VEGF mRNA levels in several human tumors, where it may be involved in promoting tumor angiogenesis and stroma generation, both as an endothelial cell mitogen and indirectly by its permeability enhancing effect that leads to the deposition of a provisional fibrin gel matrix.
Assuntos
Fatores de Crescimento Endotelial/biossíntese , Linfocinas/biossíntese , Macrófagos/metabolismo , Neoplasias/metabolismo , Animais , Sequência de Bases , Células Cultivadas , Fatores de Crescimento Endotelial/genética , Fatores de Crescimento Endotelial/metabolismo , Expressão Gênica/fisiologia , Cobaias , Humanos , Linfocinas/genética , Linfocinas/metabolismo , Dados de Sequência Molecular , Especificidade de Órgãos , RNA Mensageiro/análise , Células Tumorais Cultivadas , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Osteopontin, a glycoprotein with a glycine-arginine-glycine-aspartate-serine (GRGDS) cell-binding domain, has been described in bone and is also known to be expressed in other organs, particularly kidney. The goal of the present work was to define the distribution of osteopontin synthesis and deposition in a wide variety of normal adult human tissues using a multifaceted approach that included immunohistochemistry, in situ hybridization, and Northern analysis. Immunohistochemical studies have revealed the unexpected finding that osteopontin is deposited as a prominent layer at the luminal surfaces of specific populations of epithelial cells of the gastrointestinal tract, gall bladder, pancreas, urinary and reproductive tracts, lung, breast, salivary glands, and sweat glands. Northern analyses identified gallbladder as a major site of osteopontin gene transcription comparable in magnitude with that of kidney, and immunoblotting identified osteopontin in bile. In situ hybridization localized osteopontin gene transcripts predominantly to the epithelium of a variety of organs as well as to ganglion cells of bowel wall. Osteopontin of epithelial cell origin, like bone-derived osteopontin, promoted GRGDS-dependent cell spreading in attachment assays. We postulate that osteopontin secreted by epithelium binds integrins on luminal surfaces. Collectively, these findings suggest an important role for osteopontin on many luminal epithelial surfaces communicating with the external environment.
Assuntos
Sialoglicoproteínas/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Adesão Celular/fisiologia , Sistema Digestório/metabolismo , Epitélio/metabolismo , Feminino , Genitália/metabolismo , Humanos , Masculino , Dados de Sequência Molecular , Osteopontina , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sialoglicoproteínas/genética , Distribuição Tecidual , Sistema Urinário/metabolismoRESUMO
An average of 50-80% of patients treated in the intensive care unit is affected by disturbances of neuromuscular functions due to damage to the nerves and muscles, which has led to the terms critical illness polyneuropathy and myopathy. Both components occur in 30-50% of patients, while the others predominantly show a pure myopathy, while pure neuropathy is rare. Meanwhile, the descriptive term of the concept as intensive care unit-acquired weakness (ICUAW) is preferred. The most significant risk factors for the development of ICUAW are sepsis, multiorgan dysfunction and acute respiratory distress syndrome (ARDS). In at least one third of patients, persistent impairment by paralysis, sensory disturbances and balance problems persist when they leave the ICU. At approximately 10%, these leg-accentuated and highly everyday relevant disorders persist over the first year after ICU therapy. Pure myopathy rarely leads to residual disturbances, while the neuropathic component is responsible for long-term impairments.
Assuntos
Estado Terminal , Doenças Musculares , Polineuropatias , Sepse , Humanos , Unidades de Terapia Intensiva , Doenças Musculares/terapia , Polineuropatias/terapiaRESUMO
The p75 neurotrophin receptor (p75NTR, a.k.a. CD271), a transmembrane glycoprotein and a member of the tumor necrosis family (TNF) of receptors, was originally identified as a nerve growth factor receptor in the mid-1980s. While p75NTR is recognized to have important roles during neural development, its presence in both neural and nonneural tissues clearly supports the potential to mediate a broad range of functions depending on cellular context. Using an unbiased in vivo selection paradigm for genes underlying the invasive behavior of glioma, a critical characteristic that contributes to poor clinical outcome for glioma patients, we identified p75NTR as a central regulator of glioma invasion. Herein we review the expanding role that p75NTR plays in glioma progression with an emphasis on how p75NTR may contribute to the treatment refractory nature of glioma. Based on the observation that p75NTR is expressed and functional in two critical glioma disease reservoirs, namely, the highly infiltrative cells that evade surgical resection, and the radiation- and chemotherapy-resistant brain tumor-initiating cells (also referred to as brain tumor stem cells), we propose that p75NTR and its myriad of downstream signaling effectors represent rationale therapeutic targets for this devastating disease. Lastly, we provide the provocative hypothesis that, in addition to the well-documented cell autonomous signaling functions, the neurotrophins, and their respective receptors, contribute in a cell nonautonomous manner to drive the complex cellular and molecular composition of the brain tumor microenvironment, an environment that fuels tumorigenesis.
Assuntos
Carcinogênese/metabolismo , Glioma/metabolismo , Modelos Neurológicos , Fatores de Crescimento Neural/metabolismo , Proteínas do Tecido Nervoso/agonistas , Receptores de Fator de Crescimento Neural/agonistas , Transdução de Sinais , Animais , Antineoplásicos/uso terapêutico , Carcinogênese/patologia , Resistencia a Medicamentos Antineoplásicos , Glioma/tratamento farmacológico , Glioma/imunologia , Glioma/patologia , Humanos , Macrófagos/citologia , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/patologia , Microglia/citologia , Microglia/imunologia , Microglia/metabolismo , Microglia/patologia , Invasividade Neoplásica , Proteínas de Neoplasias/agonistas , Proteínas de Neoplasias/metabolismo , Células-Tronco Neoplásicas/imunologia , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/imunologia , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/patologia , Receptores de Fator de Crescimento Neural/metabolismo , Transdução de Sinais/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacosRESUMO
BACKGROUND: Reovirus is a naturally occurring oncolytic virus that usurps activated Ras-signaling pathways of tumor cells for its replication. Ras pathways are activated in most malignant gliomas via upstream signaling by receptor tyrosine kinases. The purpose of this study was to determine the effectiveness of reovirus as an experimental treatment for malignant gliomas. METHODS: We investigated whether reovirus would infect and lyse human glioma cell lines in vitro. We also tested the effect of injecting live reovirus in vivo on human gliomas grown subcutaneously or orthotopically (i.e., intracerebrally) in mice. Finally, reovirus was tested ex vivo against low-passage cell lines derived from human glioma specimens. All P values were two-sided. RESULTS: Reovirus killed 20 (83%) of 24 established malignant glioma cell lines tested. It caused a dramatic and often complete tumor regression in vivo in two subcutaneous (P =.0002 for both U251N and U87) and in two intracerebral (P =.0004 for U251N and P =.0009 for U87) human malignant glioma mouse models. As expected, serious toxic effects were found in these severely immunocompromised hosts. In a less immunocompromised mouse model, a single intratumoral inoculation of live reovirus led to a dramatic prolongation of survival (compared with control mice treated with dead virus; log-rank test, P<.0001 for both U251N and U87 cell lines). The animals treated with live virus also appeared to be healthier and gained body weight (P =.0001). We then tested the ability of reovirus to infect and kill primary cultures of brain tumors removed from patients and found that it killed nine (100%) of nine glioma specimens but none of the cultured meningiomas. CONCLUSIONS: Reovirus has potent activity against human malignant gliomas in vitro, in vivo, and ex vivo. Oncolysis with reovirus may be a potentially useful treatment for a broad range of human cancers.
Assuntos
Neoplasias Encefálicas/terapia , Glioma/terapia , Orthoreovirus Mamífero 3/fisiologia , Animais , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/virologia , Feminino , Glioma/patologia , Glioma/virologia , Humanos , Masculino , Orthoreovirus Mamífero 3/isolamento & purificação , Camundongos , Camundongos Endogâmicos NOD , Camundongos Nus , Camundongos SCID , Taxa de Sobrevida , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
A wide variety of rodent tumor cells of both fibroblastic and epithelial origins secrete a major transformation-related phosphoprotein with a molecular weight of approximately 62,000. Tumorigenic cells, regardless of the transforming agent, secrete 10-fold or more of this 32P-labeled protein as compared with their nontumorigenic counterparts. In this study we have extended these previous findings to tumorigenic human cells of diverse origins (both sarcomas and carcinomas). Metabolic labeling of cells with [32P]orthophosphate and immunoprecipitation with antibody specifically directed against the rat transformation-dependent secreted phosphoprotein have been used to identify antigenically related human phosphoproteins (Mr 66,000-69,000). Of the 14 human cell lines examined, all 8 of the lethal tumorigenic cell populations secreted these phosphoproteins either in continuous culture or as fresh explants from nude mice while the six nonmalignant cell lines did not (tumorigenicity in all cases was assayed in nude mice). Included in our study were three tumorigenic human cell lines (two sarcomas, one carcinoma), each with a matched, nontumorigenic control. The very close correlation between secretion of these phosphoproteins and the tumor cell phenotype of both rodents and humans raises the possibility that they may be important for tumor growth in vivo.
Assuntos
Transformação Celular Neoplásica/metabolismo , Fosfoproteínas/análise , Animais , Linhagem Celular , Epitélio/análise , Fibroblastos/análise , Humanos , Metionina/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Peso Molecular , Fosfoproteínas/biossíntese , Fosfoproteínas/metabolismoRESUMO
We have previously reported that rodent tumor cell lines secrete a potent vascular permeability factor with a molecular weight of 34,000-42,000 (Senger et al. Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid. Science (Wash. DC), 219: 983-985, 1983). This tumor-secreted vascular permeability factor (VPF) causes a rapid and completely reversible increase in microvascular permeability in the species (guinea pig or rat) from which the tumors were derived without causing mast cell degranulation or endothelial cell damage or exciting an inflammatory cell infiltrate. This VPF may be responsible, at least in part, for the increased permeability which is commonly displayed by solid and ascites tumor vessels. We have now examined 7 human tumor cell lines and have determined that 5 of them also secrete this same VPF. Antibody raised to guinea pig line 10 VPF neutralized more than 90% of the vascular permeability-increasing activity secreted by these 5 human tumor lines. Furthermore, VPFs from both guinea pig and human tumor sources bound to and were eluted similarly from immobilized heparin and comigrated identically on sodium dodecyl sulfate-polyacrylamide gels. Finally, 2 tumorigenic (in nude mice) human cell lines were found to secrete at least 14-fold more VPF than their directly matched, nontumorigenic counterparts, suggesting that elevated expression of this permeability factor may correlate with neoplastic transformation. These data suggest that a broad spectrum of tumor cells from several species, including humans, secretes a highly conserved molecule that enhances local vascular permeability and that this function may be important for tumor growth.
Assuntos
Permeabilidade Capilar , Linfocinas/metabolismo , Animais , Reações Antígeno-Anticorpo , Linhagem Celular , Meios de Cultura , Humanos , Linfocinas/imunologia , Peso Molecular , Neoplasias Experimentais/metabolismo , Ratos , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Rodent and human tumor cell lines secrete a potent vascular permeability factor (VPF) which causes a rapid and substantial increase in microvascular permeability to plasma proteins without causing mast cell degranulation, or endothelial cell damage or without exciting an inflammatory cell infiltrate [D. R. Senger, S. J. Galli, A. M. Dvorak, C. A. Perruzzi, V. S. Harvey, and H. F. Dvorak. Science (Wash. DC), 219: 983-985, 1983; D. R. Senger, C. A. Perruzzi, J. Feder, and H.F. Dvorak. Cancer Res., 46: 5629-5632, 1986]. VPF now has been purified to homogeneity from guinea pig tumor cell culture medium; it is a Mr 34,000-43,000 protein, and a NH2-terminal amino acid sequence has been derived. A synthetic peptide corresponding to amino acid residues 1-24 of the native protein was used to raise rabbit antibodies which bind all of the vessel permeability-increasing activity secreted by guinea pig tumor cells and which stain purified VPF on immunoblots. These findings establish that this NH2-terminal amino acid sequence was derived from the permeability factor. Homology searches found no identity or close similarity between VPF NH2-terminal sequence and database sequences, indicating that VPF is distinct from other proteins for which sequence data are available. In particular, no sequence similarity was found between tumor-secreted VPF and other mediators of increased vessel permeability including plasma and glandular kallikreins.
Assuntos
Linfocinas/isolamento & purificação , Neoplasias Experimentais/metabolismo , Sequência de Aminoácidos , Animais , Cromatografia , Cromatografia de Afinidade , Cromatografia Líquida de Alta Pressão , Durapatita , Cobaias , Hidroxiapatitas , Linfocinas/metabolismo , Dados de Sequência Molecular , Peso Molecular , Homologia de Sequência do Ácido Nucleico , Células Tumorais Cultivadas/metabolismo , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
We have previously demonstrated that a wide variety of rodent and human tumor cells secrete markedly elevated levels of a transformation-related phosphoprotein with a molecular weight (depending on animal species) of 58,000 to 69,000. With antibody raised to the tumor-secreted phosphoprotein (rat), we have now identified an antigenically related protein in normal rat and human plasma. The rat plasma protein and the rat tumor-secreted phosphoproteins comigrated on polyacrylamide gels and were identically cleaved during blood coagulation as well as by purified thrombin. Mouse macrophages expressed a similar or identical phosphoprotein suggesting that monocytic cells may be a source of the normal plasma protein. Consistent with elevated secretion of this protein by tumor cells, plasma and sera from cancer patients contained elevated levels of this protein, raising the possibility that this circulating marker may prove useful for monitoring tumor burden. Amino acid sequence derived from the amino terminus of the rat tumor phosphoprotein indicates that it is distinct from previously sequenced proteins, but that it may be related to protein-tyrosine kinases encoded by viral and proto-onc genes.
Assuntos
Coagulação Sanguínea , Proteínas Sanguíneas/metabolismo , Transformação Celular Neoplásica/metabolismo , Fosfoproteínas/metabolismo , Sequência de Aminoácidos , Animais , Western Blotting , Carcinoma/sangue , Linhagem Celular , Transformação Celular Viral , Humanos , Macrófagos/metabolismo , Camundongos , Peso Molecular , Ratos , Homologia de Sequência do Ácido Nucleico , Trombina/metabolismo , Células Tumorais Cultivadas/metabolismoRESUMO
Vascular permeability factor (VPF) is one of the most potent known inducers of microvascular hyperpermeability; in addition, it is a selective endothelial cell growth factor, hence its alternate name, vascular endothelial growth factor. VPF exerts its actions on the microvasculature by interacting with specific endothelial cell receptors. VPF is expressed by many transplantable animal tumors, by tumor cell lines in culture, and by certain normal cells in situ. The purpose of the present investigation was to determine whether and with what consistency VPF and its endothelial cell receptors are expressed in primary autochthonous human tumor of gastrointestinal tract origin, as determined by in situ hybridization and immunohistochemistry. Twenty-one primary adenocarcinomas (17 colon, 2 stomach, 1 small bowel, and 1 pancreas) were studied. The malignant epithelial cells expressed VPF mRNA strongly, in contrast to normal epithelium, hyperplastic polyps, and adenomas, which expressed little or no VPF mRNA. VPF expression was further increased in tumor cells immediately adjacent to zones of tumor necrosis; in such areas, occasional stromal cells also expressed VPF mRNA. In the ten colon carcinomas studied, tumor cells stained for VPF protein by immunohistochemistry. The endothelial cells of nearby stromal blood vessels also stained for VPF by immunohistochemistry and in addition expressed mRNAs encoding the VPF receptors flt-1 and kdr as determined by in situ hybridization. Endothelial cells away from the tumor did not stain for VPF and no definite mRNA expression for flt-1 or kdr was detected by in situ hybridization. The ganglion cells of the myenteric plexus of normal bowel expressed VPF mRNA and protein. These data indicate that primary autochthonous human tumors of gastrointestinal origin regularly express both VPF mRNA and VPF protein and that adjacent stromal vessels express mRNAs for both known VPF receptors. VPF is likely to contribute to tumor growth by promoting angiogenesis and stroma formation, both directly, through its action as an endothelial cell growth factor, and indirectly, by increasing vascular permeability, thereby leading to plasma protein extravasation, fibrin deposition, and the eventual replacement of the resulting matrix with vascularized stroma.