RESUMO
Aging is an established risk factor for vascular diseases, but vascular aging itself may contribute to the progressive deterioration of organ function. Here, we show in aged mice that vascular endothelial growth factor (VEGF) signaling insufficiency, which is caused by increased production of decoy receptors, may drive physiological aging across multiple organ systems. Increasing VEGF signaling prevented age-associated capillary loss, improved organ perfusion and function, and extended life span. Healthier aging was evidenced by favorable metabolism and body composition and amelioration of aging-associated pathologies including hepatic steatosis, sarcopenia, osteoporosis, "inflammaging" (age-related multiorgan chronic inflammation), and increased tumor burden. These results indicate that VEGF signaling insufficiency affects organ aging in mice and suggest that modulating this pathway may result in increased mammalian life span and improved overall health.
Assuntos
Envelhecimento/fisiologia , Envelhecimento Saudável , Longevidade , Fator A de Crescimento do Endotélio Vascular/metabolismo , Tecido Adiposo , Animais , Vasos Sanguíneos/fisiologia , Composição Corporal , Distribuição da Gordura Corporal , Metabolismo dos Carboidratos , Carcinogênese , Endotélio Vascular/metabolismo , Fígado Gorduroso/patologia , Feminino , Inflamação/prevenção & controle , Fígado/patologia , Masculino , Camundongos , Densidade Microvascular , Microvasos/fisiologia , Osteoporose/prevenção & controle , Consumo de Oxigênio , Sarcopenia/prevenção & controle , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/sangueAssuntos
Fatores de Crescimento Endotelial/fisiologia , Neovascularização Fisiológica , Proteínas da Gravidez/fisiologia , Animais , Feminino , Humanos , Microcirculação/crescimento & desenvolvimento , Neoplasias/irrigação sanguínea , Neovascularização Patológica , Fator de Crescimento Placentário , Gravidez , Transdução de Sinais , Fator A de Crescimento do Endotélio VascularRESUMO
Expressional patterns of the endothelial and neuronal forms of nitric oxide synthase (NOS) in cerebral ischemia were studied utilizing a permanent middle cerebral artery occlusion (PMCAO) model. Motor performance and infarct volumes were determined in the rats. Immunohistochemical staining for eNOS, nNOS and neurofilament were performed at 1, 2, 3, 5, 7 and 14 days after PMCAO. Vascular endothelial growth factor (VEGF) expression was determined by in-situ hybridization. PMCAO caused a reproducible cortical infarct with motor deficits in the rats. Double immunohistochemical stainings indicated that eNOS and nNOS were induced in ischemic neurons. Most stained neurons were positive for both NOS forms but some reacted with only one NOS antibody. nNOS expression peaked at 24-48 h after PMCAO, stained mainly the cytoplasm of core neurons, and disappeared after the 3rd day. eNOS expression increased until the 7th day, stained mainly the cytoplasm and membrane of penumbral cells and disappeared by the 14th day after PMCAO. VEGF expression was significantly induced in the penumbral zone in a similar distribution to eNOS. The anatomical and temporal pattern of VEGF and eNOS induction in the brain after permanent ischemia suggest that these mediators may play a role in protecting penumbral tissue from additional ischemic damage.
Assuntos
Isquemia Encefálica/enzimologia , Córtex Cerebral/enzimologia , Fatores de Crescimento Endotelial/genética , Endotélio Vascular/enzimologia , Linfocinas/genética , Neurônios/enzimologia , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico/biossíntese , Animais , Isquemia Encefálica/patologia , Isquemia Encefálica/fisiopatologia , Córtex Cerebral/patologia , Córtex Cerebral/fisiopatologia , Modelos Animais de Doenças , Endotélio Vascular/patologia , Endotélio Vascular/fisiopatologia , Imuno-Histoquímica , Infarto da Artéria Cerebral Média/enzimologia , Infarto da Artéria Cerebral Média/patologia , Infarto da Artéria Cerebral Média/fisiopatologia , Masculino , Neurônios/patologia , RNA Mensageiro/metabolismo , Ratos , Ratos Endogâmicos SHR , Fatores de Tempo , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
AIMS: To test the feasibility of gene transfer into hyaloid blood vessels and into preretinal neovascularisation in a rat model of retinopathy of prematurity (ROP), using different viral vectors. METHODS: Newborn rats were exposed to alternating hypoxic and hyperoxic conditions in order to induce ocular neovascularisation (ROP rats). Adenovirus, herpes simplex, vaccinia, and retroviral (MuLV based) vectors, all carrying the beta galactosidase (beta-gal) gene, were injected intravitreally on postnatal day 18 (P18). Two sets of controls were also examined: P18 ROP rats injected with saline and P18 rats that were raised in room air before the viral vectors or saline were injected. Two days after injection, the rats were killed, eyes enucleated, and beta-gal expression was examined by X-gal staining in whole mounts and in histological sections. RESULTS: Intravitreal injection of the adenovirus and vaccinia vectors yielded marked beta-gal expression in hyaloid blood vessels in the rat ROP model. Retinal expression of beta-gal with these vectors was limited almost exclusively to the vicinity of the injection site. Injection of herpes simplex yielded a punctuate pattern of beta-gal expression in the retina but not in blood vessels. No significant beta-gal expression occurred in rat eyes injected with the retroviral vector. CONCLUSIONS: Adenovirus is an efficient vector for gene transfer into blood vessels in an animal model of ROP. This may be a first step towards utilising gene transfer as a tool for modulating ocular neovascularisation for experimental and therapeutic purposes.
Assuntos
Técnicas de Transferência de Genes , Terapia Genética/métodos , Vasos Retinianos , Retinopatia da Prematuridade/terapia , Animais , Animais Recém-Nascidos , Modelos Animais de Doenças , Estudos de Viabilidade , Expressão Gênica , Vetores Genéticos/administração & dosagem , Humanos , Recém-Nascido , Mastadenovirus/genética , Ratos , Ratos Endogâmicos , Retroviridae/genética , Simplexvirus/genética , Vaccinia virus/genética , Corpo Vítreo , beta-Galactosidase/genéticaRESUMO
Development of microvascular networks is set to meet the metabolic requirements of the tissue they perfuse. Accordingly, impairment of oxygen homeostasis, either due to increased oxygen consumption or as a result of blood vessel occlusion, triggers compensatory neovascularization. This feedback reaction is mediated by a hypoxia- and hypoglycemia-induced vascular endothelial growth factor (VEGF). VEGF accumulates under stress as a result of increased hypoxia-inducible factor-1alpha-mediated transcription, stabilization of the mRNA, and the function of a hypoxia-refractory internal ribosome entry site within its 5'-untranslated region. Matching of vascular density to the metabolic needs of the tissue may include a process of hyperoxia-induced vessel regression. Thus newly formed vascular networks may undergo a natural process of vascular pruning that takes place whenever VEGF, acting as a vascular survival factor, is downregulated below the level required to sustain immature vessels. Immature vessels are particularly vulnerable and are selectively obliterated upon withdrawal of VEGF. The plasticity window for vessel regression is determined by a delay in the recruitment of periendothelial cells to the preformed endothelial plexus. Thus fine-tuning of microvascular density takes place mostly in the newly formed plexus, but the mature system is refractory to episodic changes in tissue oxygenation. These regulatory links may malfunction in certain pathological settings.
Assuntos
Fatores de Crescimento Endotelial/fisiologia , Homeostase/fisiologia , Hipóxia/fisiopatologia , Linfocinas/fisiologia , Neovascularização Fisiológica/fisiologia , Oxigênio/fisiologia , Animais , Humanos , Hiperóxia/fisiopatologia , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Normal cardiovascular development is exquisitely dependent on the correct dosage of the angiogenic growth factor and vascular morphogen vascular endothelial growth factor (VEGF). However, cardiac expression of VEGF is also robustly augmented during hypoxic insults, potentially mediating the well-established teratogenic effects of hypoxia on heart development. We report that during normal heart morphogenesis VEGF is specifically upregulated in the atrioventricular (AV) field of the heart tube soon after the onset of endocardial cushion formation (i.e. the endocardium-derived structures that build the heart septa and valves). To model hypoxia-dependent induction of VEGF in vivo, we conditionally induced VEGF expression in the myocardium using a tetracycline-regulated transgenic system. Premature induction of myocardial VEGF in E9.5 embryos to levels comparable with those induced by hypoxia prevented formation of endocardial cushions. When added to explanted embryonic AV tissue, VEGF fully inhibited endocardial-to-mesenchymal transformation. Transformation was also abrogated in AV explants subjected to experimental hypoxia but fully restored in the presence of an inhibitory soluble VEGF receptor 1 chimeric protein. Together, these results suggest a novel developmental role for VEGF as a negative regulator of endocardial-to-mesenchymal transformation that underlies the formation of endocardial cushions. Moreover, ischemia-induced VEGF may be the molecular link between hypoxia and congenital defects in heart septation.
Assuntos
Endocárdio/embriologia , Fatores de Crescimento Endotelial/isolamento & purificação , Cardiopatias Congênitas/etiologia , Septos Cardíacos/embriologia , Valvas Cardíacas/embriologia , Linfocinas/isolamento & purificação , Animais , Endocárdio/citologia , Hipóxia/complicações , Técnicas In Vitro , Mesoderma/citologia , Camundongos , Camundongos Transgênicos , Morfogênese , Distribuição Tecidual , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularAssuntos
Fatores de Crescimento Endotelial/metabolismo , Linfocinas/metabolismo , Óxido Nítrico Sintase/fisiologia , Retinopatia da Prematuridade/metabolismo , Animais , Humanos , Recém-Nascido , Isquemia/tratamento farmacológico , Isquemia/metabolismo , Neovascularização Patológica , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II , Óxido Nítrico Sintase Tipo III , Doenças Retinianas/tratamento farmacológico , Doenças Retinianas/metabolismo , Retinopatia da Prematuridade/tratamento farmacológico , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Astrocytes play a key role in the development of retinal vessels by detecting hypoxia in developing retina and secreting the hypoxia-induced angiogenic factor VEGF to induce vessel formation. The astrocytes which play this role are themselves spreading over the retina, just ahead of the growing vessels. To understand the mechanisms which keep astrocytes in this strategic 'just ahead' position we have studied the effects of hyperoxia and hypoxia on astrocyte differentiation and movement in situ in neonatal rat retina and in primary culture. Hyperoxia in situ inhibited the stellation of astrocytes, so that they persisted in a relatively unbranched form, which accumulated at the edge of their spreading population; hyperoxia permitted but did not accelerate migration. Conversely, hypoxia induced unstellated astrocytes to stellate within 6 h. If the hypoxia was abnormally severe, it caused the astrocytes to hyperstellate and slowed their spread. Astrocytes in primary culture did not change morphology or motility when challenged by hypoxia. When treated with medium conditioned by retina however, astrocytes became mobile and, if the medium was conditioned by hypoxic retina, became stellate. These results suggest that the oxygen released by retinal vessels maintains the mobility of astrocytes, via a diffusible factor released by other retinal cells. Conversely, naturally generated hypoxia of developing retina plays a triple role, inducing astrocytes to stellate, to end their migration and to produce VEGF, thereby inducing vessel formation. The induction of stellation is mediated by a diffusible factor released by other retinal cells. Thus hypoxia of the retina generated by neural maturation induces key events in both the differentiation of astrocytes and the formation of blood vessels.
Assuntos
Envelhecimento/fisiologia , Astrócitos/citologia , Astrócitos/fisiologia , Oxigênio/metabolismo , Retina/fisiologia , Animais , Diferenciação Celular/fisiologia , Hipóxia Celular/fisiologia , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Células Cultivadas , Meios de Cultivo Condicionados/farmacologia , Mitose/fisiologia , Ratos , Ratos Sprague-Dawley , Retina/citologia , Retina/crescimento & desenvolvimentoAssuntos
Inibidores da Angiogênese/uso terapêutico , Antineoplásicos/uso terapêutico , Neoplasias/tratamento farmacológico , Neovascularização Patológica/prevenção & controle , Angiostatinas , Animais , Colágeno/farmacologia , Endostatinas , Fatores de Crescimento Endotelial/farmacologia , Endotélio Vascular/efeitos dos fármacos , Matriz Extracelular/efeitos dos fármacos , Humanos , Linfocinas/farmacologia , Neoplasias/irrigação sanguínea , Fragmentos de Peptídeos/farmacologia , Plasminogênio/farmacologia , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Cardiac rupture is a fatal complication of acute myocardial infarction lacking treatment. Here, acute myocardial infarction resulted in rupture in wild-type mice and in mice lacking tissue-type plasminogen activator, urokinase receptor, matrix metalloproteinase stromelysin-1 or metalloelastase. Instead, deficiency of urokinase-type plasminogen activator (u-PA-/-) completely protected against rupture, whereas lack of gelatinase-B partially protected against rupture. However, u-PA-/- mice showed impaired scar formation and infarct revascularization, even after treatment with vascular endothelial growth factor, and died of cardiac failure due to depressed contractility, arrhythmias and ischemia. Temporary administration of PA inhibitor-1 or the matrix metalloproteinase-inhibitor TIMP-1 completely protected wild-type mice against rupture but did not abort infarct healing, thus constituting a new approach to prevent cardiac rupture after acute myocardial infarction.
Assuntos
Baixo Débito Cardíaco/etiologia , Ruptura Cardíaca/etiologia , Metaloendopeptidases/antagonistas & inibidores , Infarto do Miocárdio/complicações , Infarto do Miocárdio/tratamento farmacológico , Inativadores de Plasminogênio/uso terapêutico , Inibidores de Proteases/uso terapêutico , Animais , Arritmias Cardíacas , Transplante de Medula Óssea , Movimento Celular , Colagenases/metabolismo , Técnicas de Transferência de Genes , Leucócitos/citologia , Leucócitos/metabolismo , Metaloproteinase 3 da Matriz/genética , Metaloproteinase 9 da Matriz , Camundongos , Camundongos Mutantes , Neovascularização Fisiológica/efeitos dos fármacos , Inibidor 1 de Ativador de Plasminogênio/genética , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Ativadores de Plasminogênio/genética , Inibidor Tecidual de Metaloproteinase-1/genética , Inibidor Tecidual de Metaloproteinase-1/metabolismoRESUMO
Features that distinguish tumor vasculatures from normal blood vessels are sought to enable the destruction of preformed tumor vessels. We show that blood vessels in both a xenografted tumor and primary human tumors contain a sizable fraction of immature blood vessels that have not yet recruited periendothelial cells. These immature vessels are selectively obliterated as a consequence of vascular endothelial growth factor (VEGF) withdrawal. In a xenografted glioma, the selective vulnerability of immature vessels to VEGF loss was demonstrated by downregulating VEGF transgene expression using a tetracycline-regulated expression system. In human prostate cancer, the constitutive production of VEGF by the glandular epithelium was suppressed as a consequence of androgen-ablation therapy. VEGF loss led, in turn, to selective apoptosis of endothelial cells in vessels devoid of periendothelial cells. These results suggest that the unique dependence on VEGF of blood vessels lacking periendothelial cells can be exploited to reduce an existing tumor vasculature.
Assuntos
Vasos Sanguíneos/metabolismo , Fatores de Crescimento Endotelial/metabolismo , Glioma/irrigação sanguínea , Linfocinas/metabolismo , Neoplasias Experimentais/irrigação sanguínea , Androgênios/metabolismo , Animais , Apoptose/fisiologia , Regulação para Baixo/genética , Fatores de Crescimento Endotelial/genética , Regulação Neoplásica da Expressão Gênica/genética , Glioma/patologia , Humanos , Marcação In Situ das Extremidades Cortadas , Linfocinas/genética , Masculino , Camundongos , Camundongos Nus , Neoplasias Experimentais/patologia , Neoplasias da Próstata/irrigação sanguínea , Neoplasias da Próstata/metabolismo , RNA Mensageiro/genética , Tetraciclina/farmacologia , Transplante Heterólogo , Células Tumorais Cultivadas , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
The sequence of events that leads to tumor vessel regression and the functional characteristics of these vessels during hormone-ablation therapy are not known. This is because of the lack of an appropriate animal model and monitoring technology. By using in vivo microscopy and in situ molecular analysis of the androgen-dependent Shionogi carcinoma grown in severe combined immunodeficient mice, we show that castration of these mice leads to tumor regression and a concomitant decrease in vascular endothelial growth factor (VEGF) expression. Androgen withdrawal is known to induce apoptosis in Shionogi tumor cells. Surprisingly, tumor endothelial cells begin to undergo apoptosis before neoplastic cells, and rarefaction of tumor vessels precedes the decrease in tumor size. The regressing vessels begin to exhibit normal phenotype, i.e., lower diameter, tortuosity, vascular permeability, and leukocyte adhesion. Two weeks after castration, a second wave of angiogenesis and tumor growth begins with a concomitant increase in VEGF expression. Because human tumors often relapse following hormone-ablation therapy, our data suggest that these patients may benefit from combined anti-VEGF therapy.
Assuntos
Androgênios/fisiologia , Castração , Morte Celular , Fatores de Crescimento Endotelial/fisiologia , Endotélio Vascular/patologia , Linfocinas/fisiologia , Neoplasias Hormônio-Dependentes/patologia , Animais , Northern Blotting , Fatores de Crescimento Endotelial/genética , Endotélio Vascular/fisiopatologia , Humanos , Linfocinas/genética , Masculino , Camundongos , Camundongos SCID , Neoplasias Hormônio-Dependentes/irrigação sanguínea , Neovascularização Patológica , RNA Mensageiro/genética , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
As a result of deprivation of oxygen (hypoxia) and nutrients, the growth and viability of cells is reduced. Hypoxia-inducible factor (HIF)-1alpha helps to restore oxygen homeostasis by inducing glycolysis, erythropoiesis and angiogenesis. Here we show that hypoxia and hypoglycaemia reduce proliferation and increase apoptosis in wild-type (HIF-1alpha+/+) embryonic stem (ES) cells, but not in ES cells with inactivated HIF-1alpha genes (HIF-1alpha-/-); however, a deficiency of HIF-1alpha does not affect apoptosis induced by cytokines. We find that hypoxia/hypoglycaemia-regulated genes involved in controlling the cell cycle are either HIF-1alpha-dependent (those encoding the proteins p53, p21, Bcl-2) or HIF-1alpha-independent (p27, GADD153), suggesting that there are at least two different adaptive responses to being deprived of oxygen and nutrients. Loss of HIF-1alpha reduces hypoxia-induced expression of vascular endothelial growth factor, prevents formation of large vessels in ES-derived tumours, and impairs vascular function, resulting in hypoxic microenvironments within the tumour mass. However, growth of HIF-1alpha tumours was not retarded but was accelerated, owing to decreased hypoxia-induced apoptosis and increased stress-induced proliferation. As hypoxic stress contributes to many (patho)biological disorders, this new role for HIF-1alpha in hypoxic control of cell growth and death may be of general pathophysiological importance.
Assuntos
Apoptose/fisiologia , Divisão Celular/fisiologia , Proteínas de Ligação a DNA/fisiologia , Neoplasias Experimentais/irrigação sanguínea , Neovascularização Patológica/etiologia , Proteínas Nucleares/fisiologia , Oxigênio/fisiologia , Fatores de Transcrição , Animais , Células CHO , Hipóxia Celular , Linhagem Celular , Cricetinae , Proteínas de Ligação a DNA/genética , Fatores de Crescimento Endotelial/fisiologia , Marcação de Genes , Glucose/fisiologia , Fator 1 Induzível por Hipóxia , Subunidade alfa do Fator 1 Induzível por Hipóxia , Linfocinas/fisiologia , Camundongos , Camundongos Nus , Proteínas Nucleares/genética , Fluxo Sanguíneo Regional , Células-Tronco , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Plasminogen-activator inhibitor type I (PAI-1), the primary inhibitor of urinary-type plasminogen activator, is thought to play an important role in the control of stroma invasion by both endothelial and tumor cells. Using an in vitro angiogenesis model of capillary extension through a preformed monolayer, in conjunction with in situ hybridization analysis, we showed that PAI-1 mRNA is specifically induced in cells juxtaposed next to elongating sprouts. To further establish that PAI-1 expression is induced as a consequence of a direct contact with endothelial cells, coculture experiments were performed. PAI-1 mRNA was induced exclusively in fibroblasts (L-cells) contacting endothelial cell (LE-II) colonies. Reporter gene constructs driven by a PAI-1 promoter and stably transfected into L-cells were used to establish that both mouse and rat PAI-1 promoters mediate apposition-dependent regulation. This mode of PAI-1 regulation is not mediated by plasmin, as an identical spatial pattern of expression was detected in cocultures treated with plasmin inhibitors. Because endothelial cells may establish direct contacts with fibroblasts only during angiogenesis, we propose that focal induction of PAI-1 at the site of heterotypic cell contacts provides a mechanism to negate excessive pericellular proteolysis associated with endothelial cell invasion.
Assuntos
Endotélio Vascular/citologia , Regulação da Expressão Gênica , Neovascularização Fisiológica/fisiologia , Inibidor 1 de Ativador de Plasminogênio/biossíntese , Animais , Aorta/citologia , Capilares/citologia , Comunicação Celular , Técnicas de Cocultura , Fibrinolisina/antagonistas & inibidores , Fibrinolisina/farmacologia , Genes Reporter , Hibridização In Situ , Células L/metabolismo , Camundongos , Inibidor 1 de Ativador de Plasminogênio/genética , Regiões Promotoras Genéticas/genética , RNA Mensageiro/biossíntese , Ratos , Proteínas Recombinantes de Fusão/biossíntese , Especificidade da Espécie , TransfecçãoRESUMO
Little is known about how the initial endothelial plexus is remodelled into a mature and functioning vascular network. Studying postnatal remodelling of the retina vasculature, we show that a critical step in vascular maturation, namely pericyte recruitment, proceeds by outmigration of cells positive for (alpha)-smooth muscle actin from arterioles and that coverage of primary and smaller branches lags many days behind formation of the endothelial plexus. The transient existence of a pericyte-free endothelial plexus coincides temporally and spatially with the process of hyperoxia-induced vascular pruning, which is a mechanism for fine tuning of vascular density according to available oxygen. Acquisition of a pericyte coating marks the end of this plasticity window. To substantiate that association with pericytes stabilizes the vasculature, endothelial-pericyte associations were disrupted by intraocular injection of PDGF-BB. Ectopic PDGF-BB caused the detachment of PDGF-beta receptor-positive pericytes from newly coated vessels, presumably through interference with endogenous cues, but had no effect on mature vessels. Disruption of endothelial-pericyte associations resulted in excessive regression of vascular loops and abnormal remodelling. Conversely, intraocular injection of VEGF accelerated pericyte coverage of the preformed endothelial plexus, thereby revealing a novel function of this pleiotropic angiogenic growth factor. These findings also provide a cellular basis for clinical observations that vascular regression in premature neonates subjected to oxygen therapy [i.e. in retinopathy of prematurity] drops precipitously upon maturation of retina vessels and a mechanistic explanation to our previous findings that VEGF can rescue immature vessels from hyperoxia-induced regression.
Assuntos
Fatores de Crescimento Endotelial/farmacologia , Endotélio Vascular/crescimento & desenvolvimento , Linfocinas/farmacologia , Neovascularização Fisiológica/fisiologia , Lectinas de Plantas , Fator de Crescimento Derivado de Plaquetas/farmacologia , Vasos Retinianos/crescimento & desenvolvimento , Actinas/análise , Animais , Animais Recém-Nascidos , Arteríolas/citologia , Becaplermina , Movimento Celular , Endotélio Vascular/citologia , Humanos , Hiperóxia , Recém-Nascido , Lectinas/análise , Proteínas Proto-Oncogênicas c-sis , Ratos , Receptores do Fator de Crescimento Derivado de Plaquetas/análise , Retinopatia da Prematuridade/fisiopatologia , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Vascular endothelial growth factor (VEGF) is a hypoxia-inducible angiogenic growth factor that promotes compensatory angiogenesis in circumstances of oxygen shortage. The requirement for translational regulation of VEGF is imposed by the cumbersome structure of the 5' untranslated region (5'UTR), which is incompatible with efficient translation by ribosomal scanning, and by the physiologic requirement for maximal VEGF production under conditions of hypoxia, where overall protein synthesis is compromised. Using bicistronic reporter gene constructs, we show that the 1,014-bp 5'UTR of VEGF contains a functional internal ribosome entry site (IRES). Efficient cap-independent translation is maintained under hypoxia, thereby securing efficient production of VEGF even under unfavorable stress conditions. To identify sequences within the 5'UTR required for maximal IRES activity, deletion mutants were analyzed. Elimination of the majority (851 nucleotides) of internal 5'UTR sequences not only maintained full IRES activity but also generated a significantly more potent IRES. Activity of the 163-bp long "improved" IRES element was abrogated, however, following substitution of a few bases near the 5' terminus as well as substitutions close to the translation start codon. Both the full-length 5'UTR and its truncated version function as translational enhancers in the context of a monocistronic mRNA.
Assuntos
Fatores de Crescimento Endotelial/genética , Linfocinas/genética , Oxigênio/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Células 3T3 , Animais , Hipóxia Celular , Células Cultivadas , Fatores de Crescimento Endotelial/biossíntese , Humanos , Linfocinas/biossíntese , Camundongos , Neovascularização Patológica/genética , Ratos , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Heparin-binding epidermal growth factor-like growth factor (HB-EGF), a potent mitogen and migration factor for vascular smooth muscle cells (SMC), promoted neovascularization in vivo in the rabbit cornea. MRI demonstrated quantitatively the angiogenic effect of HB-EGF when introduced subcutaneously into nude mice. HB-EGF is not directly mitogenic to endothelial cells but it induced the migration of bovine endothelial cells and release of endothelial cell mitogenic activity from bovine vascular SMC. This mitogenic activity was specifically blocked by neutralizing anti-vascular endothelial growth factor (VEGF) antibodies. In contrast, EGF or transforming growth factor-alpha (TGF-alpha) had almost no effect on release of endothelial mitogenicity from SMC. In addition, RT-PCR analysis demonstrated that VEGF165 mRNA levels were increased in vascular SMC 4-10-fold by 0.35-2 nM of HB-EGF, respectively. Our data suggest that HB-EGF, as a mediator of intercellular communication, may play a new important role in supporting wound healing, tumor progression and atherosclerosis by stimulating angiogenesis.
Assuntos
Comunicação Celular/fisiologia , Córnea/irrigação sanguínea , Fatores de Crescimento Endotelial/metabolismo , Endotélio Vascular/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Linfocinas/metabolismo , Músculo Liso Vascular/metabolismo , Animais , Anticorpos/imunologia , Anticorpos/farmacologia , Bovinos , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Neovascularização da Córnea/sangue , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Peptídeos e Proteínas de Sinalização Intercelular , Imageamento por Ressonância Magnética , Camundongos , Camundongos Nus , Neovascularização Fisiológica/efeitos dos fármacos , RNA Mensageiro/metabolismo , Coelhos , Pele/irrigação sanguínea , Fator de Crescimento Transformador alfa/farmacologia , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
BACKGROUND AND OBJECTIVE: Vascular endothelial growth factor (VEGF), a key mediator of intraocular neovascularization, is triggered by hypoxia and has been shown in the eyes of animal models of central retinal vein occlusion (CRVO). However, there is little information on CRVO in humans, in particular, the identity of VEGF-producing cells. STUDY DESIGN: The study design was molecular localization of the site of VEGF production in the eyes of patients with CRVO. PARTICIPANTS: Ten formaldehyde solution-fixed and paraffin-embedded eyes removed surgically from patients with CRVO and neovascular glaucoma were studied. Five eyes with uveal melanoma and no neovascularization served as control specimens. METHODS: Thin whole-eye sections were hybridized in situ with a VEGF-specific probe to identify cells producing VEGF messenger RNA (mRNA). RESULTS: All ten eyes with CRVO showed evidence of intraretinal expression of VEGF mRNA. In all eyes, the inner nuclear layer showed VEGF-upregulated expression. Upregulation of VEGF mRNA was identified in four eyes in the ganglion cell layer and in two eyes with retinal detachment in the outer nuclear layer as well. CONCLUSIONS: The population of VEGF-producing retinal cells in each eye is likely to represent cells residing in ischemic regions of the retina. Hypoxia-induced VEGF is, most likely, the linking factor between retinal ischemia and iris and retinal neovascularization in CRVO.
Assuntos
Fatores de Crescimento Endotelial/metabolismo , Linfocinas/metabolismo , Retina/metabolismo , Oclusão da Veia Retiniana/metabolismo , Regulação para Cima , Idoso , Idoso de 80 Anos ou mais , Fatores de Crescimento Endotelial/genética , Enucleação Ocular , Feminino , Glaucoma Neovascular/metabolismo , Glaucoma Neovascular/patologia , Humanos , Hibridização In Situ , Linfocinas/genética , Masculino , RNA Mensageiro/metabolismo , Oclusão da Veia Retiniana/patologia , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
We show here that elevated levels of gonadotropins (luteinizing hormone and follicle stimulating hormone), as found in menopause or after ovariectomy, promote growth of human ovarian carcinoma by induction of tumor angiogenesis. Human epithelial ovarian cancer tumors progressed faster in ovariectomized mice. This induced growth could be attributed to the elevated levels of gonadotropins associated with loss of ovarian function because direct administration of gonadotropins also was effective in promoting tumor progression in vivo. On the other hand, gonadotropins had no direct effect on the proliferation of human ovarian cancer cells in vitro. Using MRI, we demonstrated that ovariectomy significantly (P < 0.02) induces neovascularization of human ovarian carcinoma spheroids implanted in nude mice. Moreover, conditioned medium of gonadotropin-treated human ovarian carcinoma cells showed increased mitogenic activity to bovine endothelial cells, and this activity could be blocked by neutralizing antibodies against luteinizing hormone and against vascular endothelial growth factor. Accordingly, gonadotropin stimulation resulted in a dose-dependent-induced expression of vascular endothelial growth factor in monolayer culture as well as in the outer proliferating cells of human ovarian cancer spheroids. These results demonstrate the significance of the elevated levels of gonadotropins, as found in menopause and in all ovarian cancer patients, on the progression of ovarian cancer and could explain the protective effect of estrogen replacement therapy. Based on these results, we suggest that hormonal therapy aimed at lowering the circulating levels of gonadotropins may possibly prolong remission in ovarian cancer by extending tumor dormancy.
Assuntos
Neovascularização Patológica/fisiopatologia , Neoplasias Ovarianas/irrigação sanguínea , Ovário/fisiopatologia , Animais , Fatores de Crescimento Endotelial/genética , Feminino , Hormônio Foliculoestimulante/metabolismo , Humanos , Hibridização In Situ , Hormônio Luteinizante/metabolismo , Linfocinas/genética , Imageamento por Ressonância Magnética , Menopausa , Camundongos , Camundongos Nus , Neoplasias Ovarianas/fisiopatologia , Ovariectomia , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Células Tumorais Cultivadas , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
PURPOSE: Iris neovascularization (rubeosis iridis) is a common finding in eyes harboring retinoblastoma. The purpose of the current study is to evaluate the histologic factors that may affect the development of rubeosis iridis in eyes with retinoblastoma and to examine whether vascular endothelial growth factor (VEGF), a hypoxia-induced angiogenic factor, is produced by hypoxic retinoblastoma and retinal cells in these eyes. MATERIALS AND METHODS: One hundred eighty-one enucleated eyes containing retinoblastoma were the source for the current study. Histologic slides were evaluated for the presence and degree of rubeosis iridis as well as for other histologic factors. Univariate and multivariate statistical analyses were performed to find a correlation between rubeosis iridis and the other histologic factors. Eight of the eyes underwent in situ hybridization with a specific VEGF mRNA probe to locate tumor and retinal cells that may produce this hypoxia-induced angiogenic factor. RESULTS: The amount of tumor necrosis as well as choroidal and optic nerve invasion was found to be one of the most important factors that correlated with the presence and degree of rubeosis iridis in the examined eyes. All eight eyes that underwent in situ hybridization analysis showed strong signals of VEGF mRNA in retinoblastoma cells around necrotic regions and in the outer nuclear layers in areas of detached retina. CONCLUSIONS: There exists an association between rubeosis iridis and histologic factors found in advanced stages of retinoblastoma, especially the amount of tumor necrosis. Vascular endothelial growth factor may well be an angiogenic factor that is secreted by the hypoxic retinoblastoma and retinal cells and, reaching the iris, causes (presumably in collaboration with other factors) rubeosis iridis.