RESUMEN
KEY POINTS: Combining nitric oxide (NO)-mediated increased blood flow with angiopoietin-1-Tie2 receptor signalling induces arteriolargenesis - the formation of arterioles from capillaries - in a model of physiological angiogenesis. This NO-Tie-mediated arteriolargenesis requires endogenous vascular endothelial growth factor (VEGF) signalling. Inhibition of VEGF signalling increases pericyte coverage in microvessels. Together these findings indicate that generation of functional neovasculature requires close titration of NO-Tie2 signalling and localized VEGF induction, suggesting that the use of exogenous VEGF expression as a therapeutic for neovascularization may not be successful. ABSTRACT: Signalling through vascular endothelial growth factor (VEGF) receptors and the tyrosine kinase with IgG and EGF domains-2 (Tie2) receptor by angiopoietins is required in combination with blood flow for the formation of a functional vascular network. We tested the hypothesis that VEGF and angiopoietin-1 (Ang1) contribute differentially to neovascularization induced by nitric oxide (NO)-mediated vasodilatation, by comparing the phenotype of new microvessels in the mesentery during induction of vascular remodelling by over-expression of endothelial nitric oxide synthase in the fat pad of the adult rat mesentery during inhibition of angiopoietin signalling with soluble Tie2 (sTie2) and VEGF signalling with soluble Fms-like tyrosine kinase receptor-1 (sFlt1). We found that NO-mediated angiogenesis was blocked by inhibition of VEGF with sFlt1 (from 881 ± 98% increase in functional vessel area to 279 ± 72%) and by inhibition of angiopoietin with sTie2 (to 337 ± 67%). Exogenous angiopoietin-1 was required to induce arteriolargenesis (8.6 ± 1.3% of vessels with recruitment of vascular smooth muscle cells; VSMCs) in the presence of enhanced flow. sTie2 and sFlt1 both inhibited VSMC recruitment (both 0%), and VEGF inhibition increased pericyte recruitment to newly formed vessels (from 27 ± 2 to 54 ± 3% pericyte ensheathment). We demonstrate that a fine balance of VEGF and angiopoietin signalling is required for the formation of a functional vascular network. Endogenous VEGF signalling prevents excess neovessel pericyte coverage, and is required for VSMC recruitment during increased nitric oxide-mediated vasodilatation and angiopoietin signalling (NO-Tie-mediated arteriogenesis). Therapeutic vascular remodelling paradigms may therefore require treatments that modulate blood flow to utilize endogenous VEGF, in combination with exogenous Ang1, for effective neovascularization.
Asunto(s)
Angiopoyetina 1/fisiología , Neovascularización Fisiológica/fisiología , Factor A de Crecimiento Endotelial Vascular/fisiología , Animales , Masculino , Mesenterio/irrigación sanguínea , Mesenterio/fisiología , Ratas Wistar , Receptor TIE-2/fisiología , Flujo Sanguíneo Regional , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Receptor 1 de Factores de Crecimiento Endotelial Vascular/fisiologíaRESUMEN
Complement C1q is part of the C1 macromolecular complex that mediates the classical complement activation pathway: a major arm of innate immune defense. C1q is composed of A, B, and C chains that require post-translational prolyl 4-hydroxylation of their N-terminal collagen-like domain to enable the formation of the functional triple helical multimers. The prolyl 4-hydroxylase(s) that hydroxylate C1q have not previously been identified. Recognized prolyl 4-hydroxylases include collagen prolyl-4-hydroxylases (CP4H) and the more recently described prolyl hydroxylase domain (PHD) enzymes that act as oxygen sensors regulating hypoxia-inducible factor (HIF). We show that several small-molecule prolyl hydroxylase inhibitors that activate HIF also potently suppress C1q secretion by human macrophages. However, reducing oxygenation to a level that activates HIF does not compromise C1q hydroxylation. In vitro studies showed that a C1q A chain peptide is not a substrate for PHD2 but is a substrate for CP4H1. Circulating levels of C1q did not differ between wild-type mice or mice with genetic deficits in PHD enzymes, but were reduced by prolyl hydroxylase inhibitors. Thus, C1q is hydroxylated by CP4H, but not the structurally related PHD hydroxylases. Hence, reduction of C1q levels may be an important off-target side effect of small molecule PHD inhibitors developed as treatments for renal anemia.
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Complemento C1q/metabolismo , Prolina Dioxigenasas del Factor Inducible por Hipoxia/metabolismo , Hipoxia/metabolismo , Macrófagos/metabolismo , Procolágeno-Prolina Dioxigenasa/metabolismo , Inhibidores de Prolil-Hidroxilasa/farmacología , Anemia/tratamiento farmacológico , Anemia/etiología , Animales , Línea Celular , Complemento C1q/análisis , Vía Clásica del Complemento , Femenino , Humanos , Hidroxilación , Enfermedades Renales/sangre , Enfermedades Renales/tratamiento farmacológico , Enfermedades Renales/patología , Macrófagos/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Procolágeno-Prolina Dioxigenasa/antagonistas & inhibidores , Inhibidores de Prolil-Hidroxilasa/uso terapéutico , Procesamiento Proteico-PostraduccionalRESUMEN
Factor-inhibiting hypoxia-inducible factor (HIF)-1 (FIH-1) is an asparaginyl ß-hydroxylase enzyme that was initially found to hydroxylate the HIF-α, preventing its transcriptional activity and leading to adaptive responses to hypoxia. More recently, other substrates, such as neurogenic locus notch homolog (Notch), have been found to be alternative FIH targets, but the biologic relevance of this regulation was never investigated. Given the key function of Notch in angiogenesis, we here investigate the role of FIH/Notch signaling in endothelial cells. We report that FIH-1 silencing in HUVECs results in reduced growth and increased apoptosis. The knockdown of FIH is associated with increased Notch2 activity, leading to enhanced expression of the Notch target hairy/enhancer-of-split related with YRPW motif protein 1 (Hey-1). Consistent with recent findings showing that Notch2 suppresses survivin (a key inhibitor of apoptosis), FIH targeting in HUVECs leads to selective repression of survivin in endothelial cells, thus promoting cell apoptosis and growth arrest. Our data support the concept that FIH-1 may interact with Notch2 and repress its activity, thereby playing a critical role in controlling the survival of vascular endothelial cells. These findings might pave the way toward novel, antiangiogenic strategies in disorders that are characterized by excessive vascular growth, such as cancer and rheumatoid arthritis.
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Células Endoteliales/citología , Células Endoteliales/metabolismo , Oxigenasas de Función Mixta/metabolismo , Proteínas Represoras/metabolismo , Apoptosis , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Proteínas de Ciclo Celular/genética , Hipoxia de la Célula , Proliferación Celular , Supervivencia Celular , Técnicas de Silenciamiento del Gen , Células Endoteliales de la Vena Umbilical Humana , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Proteínas Inhibidoras de la Apoptosis/genética , Oxigenasas de Función Mixta/antagonistas & inhibidores , Oxigenasas de Función Mixta/genética , Interferencia de ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptor Notch2/metabolismo , Proteínas Represoras/antagonistas & inhibidores , Proteínas Represoras/genética , Transducción de Señal , SurvivinRESUMEN
BACKGROUND: Hypoxia may contribute to the pathogenesis of various diseases of the vascular wall. Hypoxia-inducible factors (HIFs) are nuclear transcriptional factors that regulate the transcription of genes that mediate cellular and tissue homeostatic responses to altered oxygenation. This article reviews the published literature on and discusses the role of the HIF pathway in diseases involving the vascular wall, including atherosclerosis, arterial aneurysms, pulmonary hypertension, vascular graft failure, chronic venous diseases, and vascular malformation. METHODS: PubMed was searched with the terms "hypoxia-inducible factor" or "HIF" and "atherosclerosis," "carotid stenosis," "aneurysm," "pulmonary artery hypertension," "varicose veins," "venous thrombosis," "graft thrombosis," and "vascular malformation." RESULTS: In atherosclerotic plaque, HIF-1α was localized in macrophages and smooth muscle cells bordering the necrotic core. Increased HIF-1α may contribute to atherosclerosis through alteration of smooth muscle cell proliferation and migration, angiogenesis, and lipid metabolism. The expression of HIF-1α is significantly elevated in aortic aneurysms compared with nonaneurysmal arteries. In pulmonary hypertension, HIF-1α contributes to the increase of intracellular K(+) and Ca(2+) leading to vasoconstriction of pulmonary smooth muscle cells. Alteration of the HIF pathway may contribute to vascular graft failure through the formation of intimal hyperplasia. In chronic venous disease, HIF pathway dysregulation contributes to formation of varicose veins and venous thromboembolism. However, whether the activation of the HIF pathway is protective or destructive to the venous wall is unclear. Increased activation of the HIF pathway causes aberrant expression of angiogenic factors contributing to the formation and maintenance of vascular malformations. CONCLUSIONS: Pathologic vascular wall remodelling of many common diseases of the blood vessels has been found to be associated with altered activity of the HIF pathway. Therefore, understanding the role of the HIF pathway in diseases of the vascular wall is important to identify novel therapeutic strategies in the management of these pathologies.
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Vasos Sanguíneos/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Enfermedades Vasculares/metabolismo , Animales , Vasos Sanguíneos/patología , Humanos , Oxígeno/metabolismo , Pronóstico , Transducción de Señal , Enfermedades Vasculares/patología , Enfermedades Vasculares/terapiaRESUMEN
BACKGROUND: Colorectal cancer (CRC) is characterised by hypoxia, which activates gene transcription through hypoxia-inducible factors (HIF), as well as by expression of epidermal growth factor (EGF) and EGF receptors, targeting of which has been demonstrated to provide therapeutic benefit in CRC. Although EGF has been demonstrated to induce expression of angiogenic mediators, potential interactions in CRC between EGF-mediated signalling and the hypoxia/HIF pathway remain uncharacterised. METHODS: PCR-based profiling was applied to identify angiogenic genes in Caco-2 CRC cells regulated by hypoxia, the hypoxia mimetic dimethyloxallylglycine (DMOG) and/or EGF. Western blotting was used to determine the role of HIF-1alpha, HIF-2alpha and MAPK cell signalling in mediating the angiogenic responses. RESULTS: We identified a total of 9 angiogenic genes, including angiopoietin-like (ANGPTL) 4, ephrin (EFNA) 3, transforming growth factor (TGF) ß1 and vascular endothelial growth factor (VEGF), to be upregulated in a HIF dependent manner in Caco-2 CRC cells in response to both hypoxia and the hypoxia mimetic dimethyloxallylglycine (DMOG). Stimulation with EGF resulted in EGFR tyrosine autophosphorylation, activation of p42/p44 MAP kinases and stabilisation of HIF-1α and HIF-2α proteins. However, expression of 84 angiogenic genes remained unchanged in response to EGF alone. Crucially, addition of DMOG in combination with EGF significantly increased expression of a further 11 genes (in addition to the 9 genes upregulated in response to either DMOG alone or hypoxia alone). These additional genes included chemokines (CCL-11/eotaxin-1 and interleukin-8), collagen type IV α3 chain, integrin ß3 chain, TGFα and VEGF receptor KDR. CONCLUSION: These findings suggest that although EGFR phosphorylation activates the MAP kinase signalling and promotes HIF stabilisation in CRC, this alone is not sufficient to induce angiogenic gene expression. In contrast, HIF activation downstream of hypoxia/DMOG drives expression of genes such as ANGPTL4, EFNA3, TGFß1 and VEGF. Finally, HIF activation synergises with EGF-mediated signalling to additionally induce a unique sub-group of candidate angiogenic genes. Our data highlight the complex interrelationship between tumour hypoxia, EGF and angiogenesis in the pathogenesis of CRC.
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Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Factor de Crecimiento Epidérmico/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Hipoxia/genética , Neovascularización Patológica/genética , Transcriptoma , Hipoxia de la Célula , Línea Celular Tumoral , Neoplasias Colorrectales/metabolismo , Receptores ErbB/metabolismo , Humanos , Factor 1 Inducible por Hipoxia/metabolismo , Neovascularización Patológica/metabolismoRESUMEN
OBJECTIVE: Rheumatoid arthritis (RA) is characterized by hypoxia and the expression of hypoxia-inducible transcription factors (HIFs), which coordinate cellular responses to hypoxia. The objective of this study was to analyze the expression and regulation of prolyl hydroxylase domain (PHD) enzymes and factor-inhibiting HIF-1α (FIH-1), which regulate cellular HIF levels, and to study the roles of these enzymes in RA fibroblast-like synoviocytes (RA FLS). METHODS: The expression of PHD and FIH and downstream target genes was assessed by quantitative polymerase chain reaction and Western blotting. A small interfering RNA (siRNA) approach and an in vitro endothelial cell angiogenesis assay were used to analyze the roles of HIF hydroxylases. RESULTS: In human RA FLS, knockdown of PHD-2, but not knockdown of PHD-1 or FIH-1, dramatically augmented HIF-1α expression, modestly increased HIF-2α protein expression under normoxic conditions, and up-regulated HIF-dependent gene expression. In contrast, silencing of PHD-3 up-regulated HIF-2α but reduced HIF-1α, thereby decreasing the expression of HIF-regulated genes. A similar effect of PHD-2 knockdown was observed in osteoarthritis FLS (OA FLS) but not in nondiseased primary human dermal fibroblasts. These findings correlated with the induction of in vitro angiogenesis by supernatants from RA FLS and OA FLS transfected with siPHD-2 but not by supernatants from nondiseased fibroblasts or from siPHD-3-transfected cells. CONCLUSION: Our data suggest that PHD-2 is the major hydroxylase regulating HIF levels and the expression of angiogenic genes in arthritic cells. PHD-2 appears to regulate responses relevant to arthritis via HIF-α, highlighting the major importance of this enzyme in hypoxia- and angiogenesis-dependent inflammatory diseases such as RA.
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Artritis Reumatoide/enzimología , Hipoxia de la Célula/fisiología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Neovascularización Patológica/enzimología , Procolágeno-Prolina Dioxigenasa/metabolismo , Membrana Sinovial/enzimología , Células Cultivadas , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Prolina Dioxigenasas del Factor Inducible por Hipoxia , Neovascularización Patológica/genética , Procolágeno-Prolina Dioxigenasa/genética , Membrana Sinovial/citologíaRESUMEN
The vascular endothelial lining of blood vessels plays a key 'target-effector' role in vivo, integrating the body's response to inflammatory cytokines, chemokines and growth factors (derived from both endothelial cells themselves and from other cells such as leukocytes and fibroblasts), to allow leukocyte activation, adhesion and extravasation from the flowing blood into underlying tissue. Endothelial proliferation, through the process of angiogenesis, results in an increased cell surface area for these events to occur, and further functions to deliver oxygen and nutrients, and to remove waste products. In addition to playing an important role in physiology, the endothelium is thus an active participant in inflammatory pathologies. One of the best understood diseases in which inflammation and angiogenesis play a part is rheumatoid arthritis (RA). Blockade of the inflammatory cascade in RA has significant consequences for the vasculature, highlighting the links between reducing endothelial activation and therapeutic benefit in chronic inflammatory disorders.
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Artritis Reumatoide/fisiopatología , Endotelio Vascular/fisiopatología , Inflamación/fisiopatología , Artritis Reumatoide/tratamiento farmacológico , Enfermedades Cardiovasculares/tratamiento farmacológico , Enfermedades Cardiovasculares/fisiopatología , Quimiocinas/metabolismo , Enfermedad Crónica , Citocinas/metabolismo , Humanos , Inflamación/tratamiento farmacológico , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Leucocitos/metabolismoRESUMEN
BACKGROUND: Doxycycline and micronized purified flavonoid fraction (MPFF) modulate vein wall remodeling that may be associated with hypoxia in varicose veins (VVs), vein graft stenosis, and deep venous thrombosis. We recently reported that in vitro exposure of non-VV (NVVs) and VVs to hypoxic conditions activates the hypoxia-inducible factor (HIF) pathway. This study investigated the in vitro effects of doxycycline and MPFF on the HIF pathway in hypoxic NVVs and VVs. METHODS: Six NVVs and six VVs obtained from surgery were used to prepare vein organ cultures, which were exposed to hypoxia (1% O(2)), with and without MPFF (10(-5) mol/L) or doxycycline (5 µg/mL) for 16 hours. The veins were analyzed for HIF-1α, HIF-2α, and their target gene expression, with real-time polymerase chain reaction and Western blot. The differences between gene expressions were tested with one-way analysis of variance with repeated measures, followed by the Dunnett test for multiple comparisons. P < .05 was considered significant. RESULTS: Treatment of NVV organ cultures exposed to hypoxia with doxycycline or MPFF did not significantly alter the expression of HIF-1α and HIF-2α messenger (m)RNA and protein compared with untreated. Doxycycline also did not significantly affect the expression of HIF-1α and HIF-2α mRNA and protein in VVs exposed to hypoxia compared with untreated VVs. However, MPFF significantly reduced the expression of HIF-1α but not HIF-2α mRNA in VVs exposed to hypoxia compared with untreated VVs. Interestingly, the reduction of the expression of HIF-1α mRNA in VVs by MPFF was not reflected at the protein level. The mRNA expression of HIF target genes, namely glucose transporter-1, carbonic anhydrase-9, vascular endothelial growth factor, B-cell lymphoma 2/adenovirus E1B 19-kDa protein-interacting protein 3, prolyl hydroxylase domain-2, and prolyl hydroxylase domain-3, was not significantly altered in NVVs and VVs exposed to hypoxia and treated with doxycycline or MPFF compared with those untreated. CONCLUSIONS: Doxycycline and MPFF at a concentration corresponding to a therapeutic dose do not alter the activation of the HIF pathway in NVV and VV organ cultures exposed to hypoxia. Our findings suggest vein wall remodeling actions in NVVs and VVs are likely not HIF-dependent.
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Antibacterianos/farmacología , Diosmina/farmacología , Doxiciclina/farmacología , Hesperidina/farmacología , Vena Safena/efectos de los fármacos , Várices/metabolismo , Adulto , Anciano , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Hipoxia de la Célula/fisiología , Femenino , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Masculino , Persona de Mediana Edad , Vena Safena/metabolismo , Vena Safena/patología , Transducción de Señal/fisiología , Técnicas de Cultivo de Tejidos , Várices/patologíaRESUMEN
BACKGROUND: Venous hypoxia has been postulated to contribute to varicose vein (VV) formation. Direct measurements of vein wall oxygen tension have previously demonstrated that the average minimum oxygen tensions were significantly lower in VVs compared with non-varicose veins (NVVs). Hypoxia-inducible factors (HIFs) are nuclear transcriptional factors that regulate the expression of several genes of oxygen homeostasis. This study aimed to investigate if hypoxia was associated with VVs by assessing the expression of HIF-1α, HIF-2α, HIF target genes, and upstream HIF regulatory enzymes in VVs and NVVs, and their regulation by hypoxia. METHODS: VVs and NVVs were surgically retrieved and immediately snap-frozen or used for organ culture preparation. The relative expression of HIF-1α, HIF-2α, HIF target genes, and HIF regulatory enzymes in VVs and NVVs was analyzed with quantitative polymerase chain reaction (Q-PCR) and Western blot. VV and NVV organ ex vivo cultures were exposed to 16 hours of normoxia, hypoxia (oxygen tension 1%), or the hypoxia mimetic dimethyloxallyl glycine (DMOG) 1 mM in normoxia. The vein organ cultures were then analyzed for HIF-1α, HIF-2α, and their target gene expression with Q-PCR and Western blot. RESULTS: HIF-1α and HIF-2α mRNA were significantly upregulated in VVs compared with NVVs (89.8 ± 18.6 vs 10.4 ± 7.2 and 384.9 ± 209.4 vs 8.1 ± 4.2, respectively). HIF target gene mRNA expression was also significantly elevated in VVs compared with NVVs, namely glucose transporter-1 (GLUT-1; 8.7 ± 2.1 vs 1.0 ± 0.3), carbonic anhydrase-9 (CA9; 8.5 ± 2.1 vs 2.8 ± 1.2), vascular endothelial growth factor (VEGF; 7.5 ± 2.1 vs 0.9 ± 0.2), and BCL2/adenovirus E1B 19-kDa protein-interacting protein 3 (BNIP-3; 4.5 ± 0.7 vs 1.4 ± 0.3). The upregulation of HIF-1α, HIF-2α, and HIF target genes in VVs was also reflected at protein level. Of the HIF regulatory enzymes, the expression of prolyl-hydroxylase domain (PHD)-2 and PHD-3 was found to be elevated in VVs compared with NVVs. Exposure of VV and NVV organ cultures to hypoxia or DMOG was associated with increases in HIF-1α and HIF-2α protein and HIF target gene expression compared with normoxia only. CONCLUSIONS: The study concluded, we believe for the first time, an increased activation of the HIF pathway, with upregulation of the expression of HIF-1α and HIF-2α transcription factors, and HIF target genes, in VVs compared with NVVs. Exposure of VVs and NVVs to hypoxic conditions was associated with increased expression of HIF-1α and HIF-2α protein and HIF target genes. The data suggest that the HIF pathway may be associated with several pathophysiologic changes in the VV wall, and that hypoxia may be a feature contributing to VV pathogenesis.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Várices/metabolismo , Venas/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Aminoácidos Dicarboxílicos/farmacología , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Western Blotting , Anhidrasa Carbónica IX , Anhidrasas Carbónicas/genética , Anhidrasas Carbónicas/metabolismo , Estudios de Casos y Controles , Hipoxia de la Célula , Dioxigenasas/genética , Dioxigenasas/metabolismo , Femenino , Transportador de Glucosa de Tipo 1/genética , Transportador de Glucosa de Tipo 1/metabolismo , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Prolina Dioxigenasas del Factor Inducible por Hipoxia , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Persona de Mediana Edad , Técnicas de Cultivo de Órganos , Procolágeno-Prolina Dioxigenasa/genética , Procolágeno-Prolina Dioxigenasa/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Factores de Tiempo , Regulación hacia Arriba , Várices/genética , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Venas/efectos de los fármacosRESUMEN
OBJECTIVE: To test whether ETS-related gene (Erg) inhibits tumor necrosis factor (TNF)-α-dependent endothelial activation and inflammation. METHODS AND RESULTS: Endothelial activation underlies many vascular diseases, including atherosclerosis. Endothelial activation by proinflammatory cytokines decreases expression of the ETS transcription factor Erg. By using human umbilical vein endothelial cells (HUVECs), we showed that Erg overexpression by adenovirus (AdErg) repressed basal and TNF-α-induced expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule (VCAM), and interleukin 8 (IL-8). Erg inhibited TNF-α-dependent activation of the ICAM-1 promoter, nuclear factor (NF)-κB activity, and NF-κB p65 phosphorylation. Basal NF-κB activity was also inhibited by Erg overexpression. Chromatin immunoprecipitation showed that Erg binds to the ICAM-1 proximal promoter region, which contains 7 putative ETS binding sites. To test the anti-inflammatory role of Erg in vivo, we used a murine model of TNF-α-dependent acute inflammation. The injection of AdErg into the paw decreased TNF-α-induced inflammation compared with control. Finally, staining of human coronary plaques showed loss of Erg expression from the endothelium overlaying active plaque shoulders. CONCLUSIONS: We have identified a novel physiological anti-inflammatory pathway under the control of the transcription factor Erg; this pathway inhibits NF-κB-dependent transcription and TNF-α-induced inflammation in vivo. These results suggest a novel approach to anti-inflammatory therapies.
Asunto(s)
Células Endoteliales/inmunología , Mediadores de Inflamación/metabolismo , Inflamación/prevención & control , FN-kappa B/metabolismo , Transactivadores/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Células Cultivadas , Enfermedad de la Arteria Coronaria/metabolismo , Modelos Animales de Enfermedad , Regulación hacia Abajo , Humanos , Inflamación/genética , Inflamación/inmunología , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Fosforilación , Regiones Promotoras Genéticas , Interferencia de ARN , Factores de Tiempo , Transactivadores/genética , Factor de Transcripción ReIA/metabolismo , Regulador Transcripcional ERG , Transfección , Factor de Necrosis Tumoral alfa/metabolismo , Molécula 1 de Adhesión Celular Vascular/genética , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
OBJECTIVE: In vivo optical imaging can delineate at the macroscopic level processes that are occurring at the cellular and molecular levels. E-selectin, a leukocyte adhesion molecule expressed on endothelium, is induced by tumor necrosis factor α (TNFα) and other cytokines involved in the pathogenesis of rheumatoid arthritis (RA). Collagen-induced arthritis (CIA) in mice is widely used to study the disease mechanisms and identify new treatments for RA. The purpose of this study was to demonstrate E-selectin-targeted fluorescence imaging in vivo in a mouse model of paw edema generated by local injection of TNFα as well as in mice with CIA. METHODS: Animals with either CIA or TNFα-induced paw edema were injected with anti-E-selectin or control antibodies labeled with a DyLight 750-nm near-infrared (NIR) probe. In vivo imaging studies were undertaken using an NIR optical imaging system, and images were coregistered with plain radiographic images. RESULTS: The mean fluorescence intensity measured over the time-course of TNFα-induced edema demonstrated a 1.97-fold increase (P<0.001) in signal in inflamed paws at 8 hours following injection of anti-E-selectin antibody, as compared to that in the isotype control. In the CIA model, a 2.34-fold increase in E-selectin-targeted signal was demonstrated (P<0.01). Furthermore, significant E-selectin-targeted signal was observed in the paws of animals immunized with collagen that did not display overt signs of arthritis. CONCLUSION: E-selectin-targeted fluorescence in vivo imaging is a quantifiable method of detecting endothelial activation in arthritis and can potentially be applied to the quantification of disease and the investigation of the effects of new therapies. Importantly, this approach may also be useful for the detection of subclinical disease in RA.
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Artritis Experimental/metabolismo , Selectina E/metabolismo , Endotelio Vascular/metabolismo , Análisis de Varianza , Animales , Artritis Experimental/inmunología , Línea Celular , Células Cultivadas , Relación Dosis-Respuesta a Droga , Selectina E/inmunología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/inmunología , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente , Inmunohistoquímica , Masculino , Ratones , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
The paradigm of a therapy aimed at inhibiting the formation of blood vessels, which would consequentially deprive cells and tissues of oxygen and nutrients, was born from the concept pioneered by the late Judah Folkman that blood vessel formation is central to the progression and maintenance of diseases which involve cellular metabolism and tissue expansion, and cancer in particular. The prototype targeted angiogenesis inhibitor anti-vascular endothelial growth factor (VEGF) antibody bevacizumab was approved in 2004 for colorectal cancer, and has since been approved for other cancers. Rheumatoid arthritis (RA) is a chronic inflammatory disease, during which inflamed tissue invades and destroys cartilage and bone. The tissue expansion, invasion, expression of cytokines and growth factors and areas of hypoxia which are a feature of RA have resulted in the hypothesis that angiogenesis inhibition may also be beneficial in RA, drawing on the success of bevacizumab. This review focuses on our current understanding of the importance of angiogenesis in RA, and on the lessons which may be learnt from the clinical experiences of angiogenesis blockade, particularly in colorectal cancer.
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Inhibidores de la Angiogénesis/uso terapéutico , Anticuerpos Monoclonales Humanizados/uso terapéutico , Artritis Reumatoide/tratamiento farmacológico , Neoplasias Colorrectales/tratamiento farmacológico , Neovascularización Patológica/tratamiento farmacológico , Animales , Artritis Reumatoide/metabolismo , Artritis Reumatoide/patología , Bevacizumab , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Humanos , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patologíaRESUMEN
BACKGROUND: Decreased venous tone and vein wall dilation may contribute to varicose vein formation. We have shown that prolonged vein wall stretch is associated with upregulation of matrix metalloproteases (MMPs) and decreased contraction. Because hypoxia-inducible factors (HIFs) expression also increases with mechanical stretch, this study tested whether upregulation of HIFs is an intermediary mechanism linking prolonged vein wall stretch to the changes in MMP expression and venous contraction. METHODS: Segments of rat inferior vena cava (IVC) were suspended in tissue bath under 0.5-g basal tension for 1 hour, and a control contraction to phenylephrine (PHE, 10(-5)M) and KCl (96 mM) was elicited. The veins were then exposed to prolonged 18 hours of tension at 0.5 g, 2 g, 2 g plus HIF inhibitor U0126 (10(-5)M), 17-[2-(dimethylamino)ethyl] amino-17-desmethoxygeldanamycin (17-DMAG, 10(-5)M), or echinomycin (10(-6)M), or 2 g plus dimethyloxallyl glycine (DMOG; 10(-4)M), a prolyl-hydroxylase inhibitor that stabilizes HIF. The fold-change in PHE and KCl contraction was compared with the control contraction at 0.5-g tension for 1 hour. Vein tissue homogenates were analyzed for HIF-1α, HIF-2α, MMP-2, and MMP-9 messenger RNA (mRNA) and protein amount using real-time reverse transcription polymerase chain reaction and Western blots. RESULTS: Compared with control IVC contraction at 0.5-g tension for 1 hour, the PHE and KCl contraction after prolonged 0.5-g tension was 2.0 ± 0.35 and 1.1 ± 0.06, respectively. Vein contraction to PHE and KCl after prolonged 2-g tension was significantly reduced (0.87 ± 0.13 and 0.72 ± 0.05, respectively). PHE-induced contraction was restored in IVC exposed to prolonged 2-g tension plus the HIF inhibitor U0126 (1.38 ± 0.15) or echinomycin (1.99 ± 0.40). U0126 and echinomycin also restored KCl-induced contraction in IVC exposed to prolonged 2-g tension (1.14 ± 0.05 and 1.11 ± 0.15, respectively). Treatment with DMOG further reduced PHE- and KCl-induced contraction in veins subjected to prolonged 2-g tension (0.47 ± 0.06 and 0.57 ± 0.01, respectively). HIF-1α and HIF-2α mRNA were overexpressed in IVC exposed to prolonged 2-g tension, and the overexpression was reversed by U0126. The overexpression of HIF-1α and HIF-2α in stretched IVC was associated with increased MMP-2 and MMP-9 mRNA. The protein amount of HIF-1α, HIF-2α, MMP-2, and MMP-9 was also increased in IVC exposed to prolonged 2-g wall tension. CONCLUSIONS: Prolonged increases in vein wall tension are associated with overexpression of HIF-1α and HIF-2α, increased MMP-2 and MMP-9 expression, and reduced venous contraction in rat IVC. Together with our report that MMP-2 and MMP-9 inhibit IVC contraction, the data suggest that increased vein wall tension induces HIF overexpression and causes an increase in MMP expression and reduction of venous contraction, leading to progressive venous dilation and varicose vein formation.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Mecanotransducción Celular , Presorreceptores/metabolismo , Vasoconstricción , Vena Cava Inferior/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/antagonistas & inhibidores , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Benzoquinonas/farmacología , Western Blotting , Butadienos/farmacología , Equinomicina/farmacología , Subunidad alfa del Factor 1 Inducible por Hipoxia/antagonistas & inhibidores , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Técnicas In Vitro , Lactamas Macrocíclicas/farmacología , Masculino , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Mecanotransducción Celular/efectos de los fármacos , Nitrilos/farmacología , Fenilefrina/farmacología , Cloruro de Potasio/farmacología , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Regulación hacia Arriba , Vasoconstricción/efectos de los fármacos , Vasoconstrictores/farmacología , Vena Cava Inferior/efectos de los fármacosRESUMEN
Kynurenine is a small molecule derived from tryptophan when this amino acid is metabolised via the kynurenine pathway. The biological activity of kynurenine and its metabolites (kynurenines) is well recognised. Therefore, understanding the regulation of the subsequent biochemical reactions is essential for the design of therapeutic strategies which aim to interfere with the kynurenine pathway. However, kynurenine concentration in the body may not only be determined by the efficiency of kynurenine synthesis but also by the rate of kynurenine clearance. In this review, current knowledge about the mechanisms of kynurenine production and routes of its clearance is presented. In addition, the involvement of kynurenine and its metabolites in the biology of different T cell subsets (including Th17 cells and regulatory T cells) and neuronal cells is discussed.
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Sistema Inmunológico/metabolismo , Quinurenina/biosíntesis , Animales , Humanos , Inmunidad , Triptófano/metabolismoRESUMEN
PURPOSE: The cause of ulnar drift in patients with rheumatoid arthritis (RA) is unknown. It may occur because of external forces applied to the fingers during normal use. Alternatively, it may arise after changes in the internal forces on the anatomy of the digits owing to alterations in the supporting structures of the joints or their control mechanisms, or both. Intrinsic muscle tightness, which is commonly seen in RA hands, may be the result of adaptive shortening or a direct consequence of RA. Previous studies carried out by our group have shown that joints, tendons, and associated synovium in RA hands are consistently hypoxic. Therefore, we formed the hypothesis that there is a difference in hand/forearm muscle oxygen tension in RA versus non-RA. METHODS: We measured tissue oxygen levels in the intrinsic muscles of the hands and forearm muscles of 29 patients with a diagnosis of RA, who were undergoing elective surgery. We measured oxygen levels using a microelectrode technique. A total of 31 patients without RA undergoing elective surgery served as matched controls. RESULTS: Our results show that the intrinsic muscles of RA patients are significantly more hypoxic than in non-RA controls. Moreover, there is a trend in the RA group for increasing hypoxia in a radial-to-ulnar direction when comparing the different intrinsic muscle groups. We also demonstrate that forearm and thenar and hypothenar muscles are significantly more hypoxic in RA versus non-RA patients. CONCLUSIONS: The intrinsic muscle weakness, intrinsic tightness, and muscle wasting observed in RA may not be due to disuse atrophy resulting from joint disease. From our data, we speculate that these changes may be the result of direct muscular involvement in RA leading to muscle hypoxia.
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Artritis Reumatoide/complicaciones , Deformidades Adquiridas de la Mano/diagnóstico , Hipoxia/fisiopatología , Músculo Esquelético/fisiopatología , Oxígeno/metabolismo , Adulto , Anciano , Análisis de Varianza , Antirreumáticos , Artritis Reumatoide/diagnóstico , Artritis Reumatoide/tratamiento farmacológico , Estudios de Casos y Controles , Procedimientos Quirúrgicos Electivos/métodos , Femenino , Deformidades Adquiridas de la Mano/etiología , Deformidades Adquiridas de la Mano/cirugía , Humanos , Hipoxia/etiología , Masculino , Persona de Mediana Edad , Músculo Esquelético/irrigación sanguínea , Consumo de Oxígeno/fisiología , Radiografía , Valores de Referencia , Índice de Severidad de la Enfermedad , Estadísticas no Paramétricas , Cúbito/diagnóstico por imagen , Cúbito/fisiopatologíaRESUMEN
In vivo molecular optical imaging has significant potential to delineate and measure, at the macroscopic level, in vivo biological processes that are occurring at the cellular and molecular level. Optical imaging has already been developed for in vitro and ex vivo applications in molecular and cellular biology (e.g. fluorescence confocal microscopy), but is still at an early stage of development as a whole-animal in vivo imaging technique. Both sensitivity and spatial resolution remain incompletely defined. Rapid advances in hardware technology and highly innovative reporter probes and dyes will be expected to deliver significant insight into perturbations of molecular pathways that occur in disease, ultimately with the potential of translating into future molecular imaging techniques for patients with arthritis. This review will focus on currently available technologies for live in vivo animal optical imaging, including fluorescence reflectance imaging, potential novel tomographic techniques, bioluminescence reporter technology and potential novel labelling techniques, highlighting in particular the potential application of in vivo fluorescence imaging in arthritis.
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Artritis Experimental/diagnóstico , Diagnóstico por Imagen/métodos , Imagenología Tridimensional/métodos , Tomografía Óptica/métodos , Animales , Modelos AnimalesRESUMEN
OBJECTIVES: To determine the function of the angiopoietin (Ang)-Tie ligand-receptor system, and to assess the effect of Tie1-751, a naturally occurring extracellular domain of the Tie1 receptor derived by alternative splicing, in an in vivo model of RA. METHODS: In the murine CIA model, expression of endogenous Ang1, Ang2, Tie1 and Tie2 in whole paws was analysed by quantitative RT-PCR. To assess the effect of inhibition of the Ang-Tie axis, Tie1-751 was expressed and fused to the Fc fragment of human IgG1. The effect of Tie1-751-Fc on human umbilical vein endothelial cell (HUVEC) cytoprotection and migration in response to Ang1, either alone or in combination with VEGF, was investigated. Furthermore, an in vitro angiogenesis assay was used to determine the effect of Tie1-751-Fc on Ang1-mediated angiogenesis. Finally, Tie1-751-Fc was administered in CIA, and the effects on clinical disease and joint architecture of hind foot specimens were determined. RESULTS: Gene expression levels of Ang1, Ang2, and receptors Tie1 and Tie2 in whole paws were significantly increased during the progression of arthritis. Tie1-751-Fc significantly inhibited HUVEC cytoprotection and migration in response to Ang1 alone, or Ang1 in combination with VEGF. Tie1-751-Fc also significantly inhibited angiogenesis induced by a combination of Ang1 plus VEGF. Finally, Tie1-751-Fc, when administered intra-peritoneally to arthritic mice, reduced clinical signs of arthritis, damage to joint architecture and infiltration of blood vessels into the synovium. CONCLUSIONS: Our data demonstrate that the Ang-Tie ligand-receptor system is dysregulated in CIA. Tie1-751, a novel splice variant of the Tie1 receptor, inhibits Ang1/VEGF signalling, suggesting that Ang inhibition may be of therapeutic benefit in inflammatory arthritis.
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Angiopoyetinas/genética , Artritis Experimental/metabolismo , Receptor TIE-1/genética , Análisis de Varianza , Angiopoyetinas/metabolismo , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Expresión Génica , Humanos , Masculino , Ratones , Reacción en Cadena de la Polimerasa , Receptor TIE-1/metabolismo , Transducción de SeñalRESUMEN
The expansion of the synovial lining of joints in rheumatoid arthritis (RA) necessitates an increase in the vascular supply to the synovium, to cope with the increased requirement for oxygen and nutrients. New blood vessel formation -'angiogenesis'- is recognized as a key event in the formation and maintenance of the pannus in RA, suggesting that targeting blood vessels in RA may be an effective future therapeutic strategy. Although many pro-angiogenic factors have been demonstrated to be expressed in RA synovium, vascular endothelial growth factor (VEGF) has been demonstrated to a have a central involvement in the angiogenic process in RA. Nevertheless, it is unclear whether angiogenesis - whether driven by VEGF and/or other factors - should be considered as a 'cause' or 'consequence' of disease. This ongoing 'chicken vs. egg' debate is difficult, as even the success of angiogenesis inhibition in models of RA does not provide a direct answer to the question. This review will focus on the role of the vasculature in RA, and the contribution of different angiogenic factors in promoting disease. Although no data regarding the effectiveness of anti-angiogenic therapy in RA have been reported to date, the blockade of angiogenesis nevertheless looks to be a promising therapeutic avenue.
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Artritis Reumatoide/etiología , Neovascularización Patológica/complicaciones , Membrana Sinovial/irrigación sanguínea , Inhibidores de la Angiogénesis/uso terapéutico , Antirreumáticos/uso terapéutico , Artritis Reumatoide/tratamiento farmacológico , Artritis Reumatoide/patología , Humanos , Neovascularización Patológica/tratamiento farmacológicoRESUMEN
The clinical success of TNFalpha blocking biologics in a growing number of immune-mediated pathologies, including rheumatoid arthritis, Crohn's disease, ankylosing spondylitis and psoriasis, confirms the importance of TNFalpha in driving chronic inflammation and represents an important step forward in the treatment of these conditions. TNFalpha blockade, however, is a treatment, rather than a cure, and is not effective in all patients or in all autoimmune diseases and further research is needed to get closer to a cure. Recently, the identification of a novel, IL-17 producing, T helper cell subset, that plays a dominant pathogenic role in animal models of autoimmunity, is a major advance on existing knowledge, although the role of these cells in human disease remains to be established. Cytokines driving angiogenesis are also important in disease chronicity and thus might be valid therapeutic targets.
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Artritis Reumatoide/tratamiento farmacológico , Enfermedades Autoinmunes/tratamiento farmacológico , Citocinas/antagonistas & inhibidores , Animales , Artritis Reumatoide/inmunología , Enfermedades Autoinmunes/inmunología , Citocinas/inmunología , Sistemas de Liberación de Medicamentos , Humanos , Interleucina-17/inmunología , Interleucina-17/metabolismo , Interleucinas/antagonistas & inhibidores , Interleucinas/inmunología , Neovascularización Patológica/metabolismo , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Rheumatoid arthritis (RA) is a chronic disabling autoimmune inflammatory disease of unknown aetiology with a prevalence of about 1% in most parts of the world. As a result of the debilitating nature of the disease, sufferers struggle with the simple activities of daily living and frequently fail to remain in full time employment. Furthermore, the mortality associated with the disease is equivalent to that seen in triple vessel coronary artery disease. Over the 10-15 years, advances in understanding the mechanisms of RA pathogenesis based on studies of human cells and animal models of arthritis have led to the identification of new targets for therapeutic intervention. Despite these advances, a significant proportion of patients continue to exhibit disease which is refractory to such therapy. As an alternative to anti-cytokine therapy, formation of new blood vessels ('angiogenesis') represents a potentially attractive target for therapy in RA. Angiogenesis has been a putative target in cancer since it was first linked to tumour growth and metastases in the 1970s. A number of significant advances have been made in the development of anti-cancer therapy using such an approach. This review focuses on the potential for targeting angiogenesis in RA, building upon the experience of angiogenesis inhibition in the oncological setting. Through this we hope to emphasise the potential value of anti-angiogenic therapy in RA and identify future directions for optimising treatment of this disabling disease.