RESUMEN
Alu RNA accumulation due to DICER1 deficiency in the retinal pigmented epithelium (RPE) is implicated in geographic atrophy (GA), an advanced form of age-related macular degeneration that causes blindness in millions of individuals. The mechanism of Alu RNA-induced cytotoxicity is unknown. Here we show that DICER1 deficit or Alu RNA exposure activates the NLRP3 inflammasome and triggers TLR-independent MyD88 signaling via IL18 in the RPE. Genetic or pharmacological inhibition of inflammasome components (NLRP3, Pycard, Caspase-1), MyD88, or IL18 prevents RPE degeneration induced by DICER1 loss or Alu RNA exposure. These findings, coupled with our observation that human GA RPE contains elevated amounts of NLRP3, PYCARD, and IL18 and evidence of increased Caspase-1 and MyD88 activation, provide a rationale for targeting this pathway in GA. Our findings also reveal a function of the inflammasome outside the immune system and an immunomodulatory action of mobile elements.
Asunto(s)
Elementos Alu , ARN Helicasas DEAD-box/metabolismo , Atrofia Geográfica/inmunología , Atrofia Geográfica/patología , Inflamasomas/inmunología , Factor 88 de Diferenciación Mieloide/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Ribonucleasa III/metabolismo , Animales , Proteínas Portadoras/metabolismo , Atrofia Geográfica/metabolismo , Humanos , Inflamasomas/metabolismo , Ratones , Proteína con Dominio Pirina 3 de la Familia NLR , Epitelio Pigmentado de la Retina/patología , Receptores Toll-Like/metabolismoRESUMEN
Prolyl 3-hydroxylase 2 (P3H2) catalyzes the post-translational formation of 3-hydroxyproline on collagens, mainly on type IV. Its activity has never been directly associated to angiogenesis. Here, we identified P3H2 gene through a deep-sequencing transcriptome analysis of human umbilical vein endothelial cells (HUVECs) stimulated with vascular endothelial growth factor A (VEGF-A). Differently from many previous studies we carried out the stimulation not on starved HUVECs, but on cells grown to maintain the best condition for their in vitro survival and propagation. We showed that P3H2 is induced by VEGF-A in two primary human endothelial cell lines and that its transcription is modulated by VEGF-A/VEGF receptor 2 (VEGFR-2) signaling pathway through p38 mitogen-activated protein kinase (MAPK). Then, we demonstrated that P3H2, through its activity on type IV Collagen, is essential for angiogenesis properties of endothelial cells in vitro by performing experiments of gain- and loss-of-function. Immunofluorescence studies showed that the overexpression of P3H2 induced a more condensed status of Collagen IV, accompanied by an alignment of the cells along the Collagen IV bundles, so towards an evident pro-angiogenic status. Finally, we found that P3H2 knockdown prevents pathological angiogenesis in vivo, in the model of laser-induced choroid neovascularization. Together these findings reveal that P3H2 is a new molecular player involved in new vessels formation and could be considered as a potential target for anti-angiogenesis therapy.
Asunto(s)
Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Neovascularización Fisiológica , Procolágeno-Prolina Dioxigenasa/genética , Procolágeno-Prolina Dioxigenasa/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Animales , Neovascularización Coroidal/diagnóstico por imagen , Neovascularización Coroidal/etiología , Neovascularización Coroidal/metabolismo , Neovascularización Coroidal/patología , Colágeno Tipo IV/genética , Colágeno Tipo IV/metabolismo , Modelos Animales de Enfermedad , Técnica del Anticuerpo Fluorescente , Regulación de la Expresión Génica , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Ratones , Unión Proteica , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Eye drop formulations allowing topical treatment of retinal pathologies have long been sought as alternatives to intravitreal administration. This study aimed to assess whether a novel nanostructured microemulsions system (NaMESys) could be usefully employed to deliver sorafenib to the retina following topical instillation. NaMESys carrying 0.3% sorafenib (NaMESys-SOR) proved to be cytocompatible in vitro on rabbit corneal cells, and well-tolerated following b.i.d. ocular administration to rabbits during a 3-month study. In rats subject to retinal ischemia-reperfusion, NaMESys-SOR significantly inhibited retinal expression of tumor necrosis factor-alpha (TNFα, 20.7%) and inducible nitric oxide synthase (iNos, 87.3%) mRNAs in comparison to controls. Similarly, in streptozotocin-induced diabetic rats, NaMESys-SOR inhibited retinal expression of nuclear factor kappa B (NFκB), TNFα, insulin like growth factor 1 (IGF1), IGF1 receptor (IGF1R), vascular endothelial growth factor receptor 1 (VEGFR1) and 2 (VEGFR2) mRNAs by three-fold on average compared to controls. Furthermore, a reduction in TNFα, VEGFR1 and VEGFR2 protein expression was observed by western blot. Moreover, in mice subject to laser-induced choroidal neovascularization, NaMESys-SOR significantly inhibited neovascular lesions by 54%. In conclusion, NaMESys-SOR was shown to be a well-tolerated ophthalmic formulation able to deliver effective amounts of sorafenib to the retina, reducing proinflammatory and pro-angiogenic mediators in reliable models of proliferative retinopathies. These findings warrant further investigations on the full therapeutic potential of NaMESys-SOR eye drops, aiming to address unmet needs in the pharmacotherapy of retinal neovascular diseases.
Asunto(s)
Neovascularización Coroidal/tratamiento farmacológico , Diabetes Mellitus Experimental/complicaciones , Retinopatía Diabética/tratamiento farmacológico , Nanoestructuras/administración & dosificación , Enfermedades de la Retina/tratamiento farmacológico , Neovascularización Retiniana/tratamiento farmacológico , Sorafenib/farmacología , Administración Oftálmica , Animales , Retinopatía Diabética/etiología , Retinopatía Diabética/patología , Modelos Animales de Enfermedad , Emulsiones , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Nanoestructuras/química , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/farmacología , Conejos , Ratas , Ratas Sprague-Dawley , Enfermedades de la Retina/patología , Sorafenib/administración & dosificaciónRESUMEN
Age-related macular degeneration (AMD) is the primary cause of blindness in advanced countries. Repeated intravitreal delivery of anti-vascular endothelial growth factor (VEGF) agents has represented an important advancement for the therapy of wet AMD with significative results in terms of blindness prevention and partial vision restore. Nonetheless, some patients are not responsive or do not attain significant visual improvement, intravitreal injection may cause serious complications and important side effects have been reported for the prolonged block of VEGF-A. In order to evaluate new anti-angiogenic strategies, we focused our attention on VEGF receptor 1 (VEGFR1) developing a specific VEGFR-1 antagonist, a tetrameric tripeptide named inhibitor of VEGFR 1 (iVR1). We have evaluated its anti-angiogenic activity in the preclinical model of AMD, the laser-induced choroid neovascularization (CNV). iVR1 is able to potently inhibit CNV when delivered by intravitreal injection. Surprisingly, it is able to significantly reduce CNV also when delivered by gavage. Our data show that the specific block of VEGFR1 in vivo represents a valid alternative to the block of VEGF-A and that the inhibition of the pathological neovascularization at ocular level is also possible by systemic delivery of compounds not targeting VEGF-A.
Asunto(s)
Neovascularización Coroidal/tratamiento farmacológico , Neovascularización Coroidal/patología , Oligopéptidos/administración & dosificación , Oligopéptidos/uso terapéutico , Receptor 1 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Administración Oral , Animales , Inyecciones Intravítreas , Rayos Láser , Ratones Endogámicos C57BL , Oligopéptidos/química , Espectroscopía Infrarroja por Transformada de Fourier , Ácido Trifluoroacético/química , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Geographic atrophy (GA), an untreatable advanced form of age-related macular degeneration, results from retinal pigmented epithelium (RPE) cell degeneration. Here we show that the microRNA (miRNA)-processing enzyme DICER1 is reduced in the RPE of humans with GA, and that conditional ablation of Dicer1, but not seven other miRNA-processing enzymes, induces RPE degeneration in mice. DICER1 knockdown induces accumulation of Alu RNA in human RPE cells and Alu-like B1 and B2 RNAs in mouse RPE. Alu RNA is increased in the RPE of humans with GA, and this pathogenic RNA induces human RPE cytotoxicity and RPE degeneration in mice. Antisense oligonucleotides targeting Alu/B1/B2 RNAs prevent DICER1 depletion-induced RPE degeneration despite global miRNA downregulation. DICER1 degrades Alu RNA, and this digested Alu RNA cannot induce RPE degeneration in mice. These findings reveal a miRNA-independent cell survival function for DICER1 involving retrotransposon transcript degradation, show that Alu RNA can directly cause human pathology, and identify new targets for a major cause of blindness.
Asunto(s)
Elementos Alu/genética , ARN Helicasas DEAD-box/deficiencia , Degeneración Macular/genética , Degeneración Macular/patología , ARN/genética , ARN/metabolismo , Ribonucleasa III/deficiencia , Animales , Muerte Celular , Supervivencia Celular , Células Cultivadas , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Ratones , MicroARNs/metabolismo , Datos de Secuencia Molecular , Oligonucleótidos Antisentido , Fenotipo , Epitelio Pigmentado de la Retina/enzimología , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/patología , Ribonucleasa III/genética , Ribonucleasa III/metabolismoRESUMEN
Geographic atrophy, an advanced form of age-related macular degeneration (AMD) characterized by death of the retinal pigmented epithelium (RPE), causes untreatable blindness in millions worldwide. The RPE of human eyes with geographic atrophy accumulates toxic Alu RNA in response to a deficit in the enzyme DICER1, which in turn leads to activation of the NLRP3 inflammasome and elaboration of IL-18. Despite these recent insights, it is still unclear how RPE cells die during the course of the disease. In this study, we implicate the involvement of Caspase-8 as a critical mediator of RPE degeneration. Here we show that DICER1 deficiency, Alu RNA accumulation, and IL-18 up-regulation lead to RPE cell death via activation of Caspase-8 through a Fas ligand-dependent mechanism. Coupled with our observation of increased Caspase-8 expression in the RPE of human eyes with geographic atrophy, our findings provide a rationale for targeting this apoptotic pathway in this disease.
Asunto(s)
Elementos Alu , Apoptosis , Caspasa 8/metabolismo , ARN Helicasas DEAD-box/metabolismo , Proteínas del Ojo/metabolismo , Degeneración Macular/metabolismo , ARN/metabolismo , Ribonucleasa III/metabolismo , Animales , Caspasa 8/genética , ARN Helicasas DEAD-box/genética , Proteínas del Ojo/genética , Humanos , Interleucina-18/genética , Interleucina-18/metabolismo , Degeneración Macular/patología , Ratones , Ratones Noqueados , ARN/genética , Ribonucleasa III/genética , Regulación hacia Arriba/genéticaRESUMEN
Deficient expression of the RNase III DICER1, which leads to the accumulation of cytotoxic Alu RNA, has been implicated in degeneration of the retinal pigmented epithelium (RPE) in geographic atrophy (GA), a late stage of age-related macular degeneration that causes blindness in millions of people worldwide. Here we show increased extracellular-signal-regulated kinase (ERK) 1/2 phosphorylation in the RPE of human eyes with GA and that RPE degeneration in mouse eyes and in human cell culture induced by DICER1 depletion or Alu RNA exposure is mediated via ERK1/2 signaling. Alu RNA overexpression or DICER1 knockdown increases ERK1/2 phosphorylation in the RPE in mice and in human cell culture. Alu RNA-induced RPE degeneration in mice is rescued by intravitreous administration of PD98059, an inhibitor of the ERK1/2-activating kinase MEK1, but not by inhibitors of other MAP kinases such as p38 or JNK. These findings reveal a previously unrecognized function of ERK1/2 in the pathogenesis of GA and provide a mechanistic basis for evaluation of ERK1/2 inhibition in treatment of this disease.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Degeneración Macular/enzimología , Degeneración Macular/terapia , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Animales , ARN Helicasas DEAD-box/metabolismo , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Flavonoides/farmacología , Humanos , Ratones , Fosforilación , Epitelio Pigmentado de la Retina/metabolismo , Ribonucleasa III/metabolismo , Transducción de SeñalRESUMEN
Purpose: Recruitment and activation of inflammatory cells, such as retinal microglia/macrophages, in the subretinal space contribute significantly to the pathogenesis of age-related macular degeneration (AMD). This study aims to explore the functional role of vascular endothelial growth factor (VEGF-A), placental growth factor (PlGF) and VEGF-A/PlGF heterodimer in immune homeostasis and activation during pathological laser-induced choroidal neovascularization (CNV). Methods: To investigate these roles, we utilized the PlGF-DE knockin (KI) mouse model, which is the full functional knockout (KO) of PlGF. In this model, mice express a variant of PlGF, named PlGF-DE, that is unable to bind and activate VEGFR-1 but can still form heterodimer with VEGF-A. Results: Our findings demonstrate that, although there is no difference in healthy conditions, PlGF-DE-KI mice exhibit decreased microglia reactivity and reduced recruitment of both microglia and monocyte-macrophages, compared to wild-type mice during laser-induced CNV. This impairment is associated with a reduction in VEGF receptor 1 (VEGFR-1) phosphorylation in the retinae of PlGF-DE-KI mice compared to C57Bl6/J mice. Corroborating these data, intravitreal delivery of PlGF or VEGF-A/PlGF heterodimer in PlGF-DE-KI mice rescued the immune cell response at the early phase of CNV compared to VEGF-A delivery. Conclusions: In summary, our study suggests that targeting PlGF and the VEGF-A/PlGF heterodimer, thereby preventing VEGFR-1 activation, could represent a potential therapeutic approach for the management of inflammatory processes in diseases such as AMD.
Asunto(s)
Neovascularización Coroidal , Microglía , Factor de Crecimiento Placentario , Factor A de Crecimiento Endotelial Vascular , Animales , Ratones , Neovascularización Coroidal/metabolismo , Modelos Animales de Enfermedad , Macrófagos/metabolismo , Macrófagos/inmunología , Ratones Endogámicos C57BL , Ratones Noqueados , Microglía/metabolismo , Factor de Crecimiento Placentario/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Inflammation plays a crucial role in cancer progression, but the relevance of the inflammasome remains unclear. Alu RNA was the first endogenous nucleic acid shown to activate the NLRP3 (nucleotide-binding domain leucine-rich repeat containing 3) inflammasome. Here, we showed that Alu RNA can induce epithelial-to-mesenchymal transition (EMT) through NLRP3 inflammasome activation and IL-1ß release in colorectal cancer (CRC) cells. Alu RNA is stored, transported and transferred to CRC cells by exosomes. Exosomal Alu RNA promotes tumorigenesis by inducing invasion, metastasis and EMT via NLRP3 inflammasome activation. Consistent with these data, we found that significantly increased Alu RNA expression correlates with the induction of NLRP3 priming in human CRC patients. Furthermore, the level of Alu RNA in circulating exosomes correlates with CRC progression in a preclinical model. These findings reveal the direct involvement of Alu RNA in cancer pathogenesis, and its presence in CRC cell-derived exosomes could be used as a noninvasive diagnostic biomarker.
Asunto(s)
Neoplasias Colorrectales , Exosomas , Humanos , ARN/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Inflamasomas/metabolismo , Carcinogénesis/metabolismo , Neoplasias Colorrectales/metabolismo , Exosomas/metabolismoRESUMEN
The discovery of sequence-specific gene silencing by endogenous double-stranded RNAs (dsRNA) has propelled synthetic short-interfering RNAs (siRNAs) to the forefront of targeted pharmaceutical engineering. The first clinical trials utilized 21-nucleotide (nt) siRNAs for the treatment of neovascular age-related macular degeneration (AMD). Surprisingly, these compounds were not formulated for cell permeation, which is required for bona fide RNA interference (RNAi). We showed that these "naked" siRNAs suppress neovascularization in mice not via RNAi but via sequence-independent activation of cell surface Toll-like receptor-3 (TLR3). Here, we demonstrate that noninternalized siRNAs induce retinal degeneration in mice by activating surface TLR3 on retinal pigmented epithelial cells. Cholesterol conjugated siRNAs capable of cell permeation and triggering RNAi also induce the same phenotype. Retinal degeneration was not observed after treatment with siRNAs shorter than 21-nts. Other cytosolic dsRNA sensors are not critical to this response. TLR3 activation triggers caspase-3-mediated apoptotic death of the retinal pigment epithelium (RPE) via nuclear translocation of interferon regulatory factor-3. While this unexpected adverse effect of siRNAs has implications for future clinical trials, these findings also introduce a new preclinical model of geographic atrophy (GA), a late stage of dry AMD that causes blindness in millions worldwide.
Asunto(s)
Factor 3 Regulador del Interferón/metabolismo , ARN Interferente Pequeño/toxicidad , Degeneración Retiniana/inducido químicamente , Receptor Toll-Like 3/metabolismo , Animales , Caspasa 3/metabolismo , Muerte Celular/genética , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Unión Proteica , ARN Interferente Pequeño/metabolismo , Degeneración Retiniana/metabolismo , Degeneración Retiniana/patología , Epitelio Pigmentado de la Retina/metabolismo , Transducción de SeñalRESUMEN
The proangiogenic members of VEGF family and related receptors play a central role in the modulation of pathological angiogenesis. Recent insights indicate that, due to the strict biochemical and functional relationship between VEGFs and related receptors, the development of a new generation of agents able to target contemporarily more than one member of VEGFs might amplify the antiangiogenic response representing an advantage in term of therapeutic outcome. To identify molecules that are able to prevent the interaction of VEGFs with related receptors, we have screened small molecule collections consisting of >100 plant extracts. Here, we report the isolation and identification from an extract of the Malian plant Chrozophora senegalensis of the biflavonoid amentoflavone as an antiangiogenic bioactive molecule. Amentoflavone can to bind VEGFs preventing the interaction and phosphorylation of VEGF receptor 1 and 2 (VEGFR-1,VEGFR-2) and to inhibit endothelial cell migration and capillary-like tube formation induced by VEGF-A or placental growth factor 1 (PlGF-1) at low µm concentration. In vivo, amentoflavone is able to inhibit VEGF-A-induced chorioallantoic membrane neovascularization as well as tumor growth and associated neovascularization, as assessed in orthotropic melanoma and xenograft colon carcinoma models. In addition structural studies performed on the amentoflavone·PlGF-1 complex have provided evidence that this biflavonoid effectively interacts with the growth factor area crucial for VEGFR-1 receptor recognition. In conclusion, our results demonstrate that amentoflavone represents an interesting new antiangiogenic molecule that is able to prevent the activity of proangiogenic VEGF family members and that the biflavonoid structure is a new chemical scaffold to develop powerful new antiangiogenic molecules.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Antineoplásicos Fitogénicos/farmacología , Biflavonoides/farmacología , Neoplasias del Colon/tratamiento farmacológico , Neovascularización Patológica/tratamiento farmacológico , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Animales , Antineoplásicos Fitogénicos/química , Biflavonoides/química , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Células HEK293 , Humanos , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Fosforilación , Trasplante Heterólogo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Neovascularization in response to tissue injury consists of the dual invasion of blood (hemangiogenesis) and lymphatic (lymphangiogenesis) vessels. We reported recently that 21-nt or longer small interfering RNAs (siRNAs) can suppress hemangiogenesis in mouse models of choroidal neovascularization and dermal wound healing independently of RNA interference by directly activating Toll-like receptor 3 (TLR3), a double-stranded RNA immune receptor, on the cell surface of blood endothelial cells. Here, we show that a 21-nt nontargeted siRNA suppresses both hemangiogenesis and lymphangiogenesis in mouse models of neovascularization induced by corneal sutures or hindlimb ischemia as efficiently as a 21-nt siRNA targeting vascular endothelial growth factor-A. In contrast, a 7-nt nontargeted siRNA, which is too short to activate TLR3, does not block hemangiogenesis or lymphangiogenesis in these models. Exposure to 21-nt siRNA, which we demonstrate is not internalized unless cell-permeating moieties are used, triggers phosphorylation of cell surface TLR3 on lymphatic endothelial cells and induces apoptosis. These findings introduce TLR3 activation as a method of jointly suppressing blood and lymphatic neovascularization and simultaneously raise new concerns about the undesirable effects of siRNAs on both circulatory systems.
Asunto(s)
Vasos Linfáticos/metabolismo , Neovascularización Fisiológica , ARN Interferente Pequeño/genética , Receptor Toll-Like 3/metabolismo , Animales , Apoptosis , Proliferación Celular , Células Endoteliales/citología , Miembro Posterior/irrigación sanguínea , Miembro Posterior/metabolismo , Ratones , Fosforilación , Receptor Toll-Like 3/genéticaRESUMEN
The presence of α-synuclein aggregates in the retina of Parkinson's disease patients has been associated with vision impairment. In this study we sought to determine the effects of α-synuclein overexpression on the survival and function of dopaminergic amacrine cells (DACs) in the retina. Adult mice were intravitreally injected with an adeno-associated viral (AAV) vector to overexpress human wild-type α-synuclein in the inner retina. Before and after systemic injections of levodopa (L-DOPA), retinal responses and visual acuity-driven behavior were measured by electroretinography (ERG) and a water maze task, respectively. Amacrine cells and ganglion cells were counted at different time points after the injection. α-synuclein overexpression led to an early loss of DACs associated with a decrease of light-adapted ERG responses and visual acuity that could be rescued by systemic injections of L-DOPA. The data show that α-synuclein overexpression affects dopamine neurons in the retina. The approach provides a novel accessible method to model the underlying mechanisms implicated in the pathogenesis of synucleinopathies and for testing novel treatments.
Asunto(s)
Células Amacrinas/metabolismo , Neuronas Dopaminérgicas/metabolismo , Retina/metabolismo , Degeneración Retiniana/metabolismo , Trastornos de la Visión/metabolismo , alfa-Sinucleína/metabolismo , Células Amacrinas/patología , Animales , Neuronas Dopaminérgicas/patología , Femenino , Técnica del Anticuerpo Fluorescente , Levodopa/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Retina/efectos de los fármacos , Retina/patología , Degeneración Retiniana/patología , Trastornos de la Visión/patología , Agudeza VisualRESUMEN
PURPOSE: The resistance to selective EGFR inhibitors involves the activation of alternative signaling pathways, and Akt activation and VEGF induction have been described in EGFR inhibitor-resistant tumors. Combined inhibition of EGFR and other signaling proteins has become a successful therapeutic approach, stimulating the search for further determinants of resistance as basis for novel therapeutic strategies. EXPERIMENTAL DESIGN: We established human cancer cell lines with various degrees of EGFR expression and sensitivity to EGFR inhibitors and analyzed signal transducers under the control of EGFR-dependent and EGFR-independent pathways. RESULTS: Multitargeted inhibitor vandetanib (ZD6474) inhibited the growth and the phosphorylation of Akt and its effector p70S6 kinase in both wild-type and EGFR inhibitor-resistant human colon, prostate, and breast cancer cells. We found that the resistant cell lines exhibit, as common feature, VEGFR-1/Flt-1 overexpression, increased secretion of VEGF and placental growth factor, and augmented migration capabilities and that vandetanib is able to antagonize them. Accordingly, a new kinase assay revealed that in addition to VEGF receptor (VEGFR)-2, RET, and EGFR, vandetanib efficiently inhibits also VEGFR-1. The contribution of VEGFR-1 to the resistant phenotype was further supported by the demonstration that VEGFR-1 silencing in resistant cells restored sensitivity to anti-EGFR drugs and impaired migration capabilities, whereas exogenous VEGFR-1 overexpression in wild-type cells conferred resistance to these agents. CONCLUSIONS: This study shows that VEGFR-1 contributes to anti-EGFR drug resistance in different human cancer cells. Moreover, vandetanib inhibits VEGFR-1 activation, cell proliferation, and migration, suggesting its potential utility in patients resistant to EGFR inhibitors.
Asunto(s)
Resistencia a Antineoplásicos/fisiología , Receptores ErbB/antagonistas & inhibidores , Neoplasias/metabolismo , Transducción de Señal/fisiología , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismo , Antineoplásicos/farmacología , Western Blotting , Adhesión Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Receptores ErbB/efectos de los fármacos , Humanos , Inmunoprecipitación , Neoplasias/tratamiento farmacológico , Piperidinas/farmacología , Quinazolinas/farmacología , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección , Receptor 1 de Factores de Crecimiento Endotelial Vascular/efectos de los fármacosRESUMEN
Diabetic retinopathy (DR) is a secondary complication of diabetes. DR can cause irreversible blindness, and its pathogenesis is considered multifactorial. DR can progress from non-proliferative DR to proliferative DR, characterized by retinal neovascularization. The main cause of vision loss in diabetic patients is diabetic macular edema, caused by vessel leakage and blood retinal barrier breakdown. Currently, aflibercept is an anti-VEGF approved for diabetic macular edema. Aflibercept can bind several members of vascular permeability factors, namely VEGF-A, B, and PlGF. We analyzed the aflibercept-PlGF complex at molecular level, through an in silico approach. In order to explore the role of PlGF in DR, we treated primary human retinal endothelial cells (HRECs) and mouse retinal epithelial cells (RPEs) with aflibercept and an anti-PlGF antibody. We explored the hypothesis that aflibercept has anti-inflammatory action through blocking of PlGF signaling and the ERK axis in an in vitro and in vivo model of DR. Both aflibercept and the anti-PlGF antibody exerted protective effects on retinal cells, by inhibition of the ERK pathway. Moreover, aflibercept significantly decreased (pâ¯<â¯0.05) the expression of TNF-α in an in vitro and in vivo model of DR. Therefore, our data suggest that inhibition of PlGF signaling, or a selective blocking, may be useful in the management of early phases of DR when the inflammatory process is largely involved.
Asunto(s)
Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Glucosa/toxicidad , Inflamación/tratamiento farmacológico , Proteínas de la Membrana/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Enfermedades de la Retina/tratamiento farmacológico , Inhibidores de la Angiogénesis/farmacología , Animales , Células Cultivadas , Simulación por Computador , Células Endoteliales/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/genética , Humanos , Ratones , Modelos Biológicos , Modelos Moleculares , Unión Proteica , Conformación Proteica , Dominios Proteicos , Ratas , Receptores de Factores de Crecimiento Endotelial Vascular , Retina/citologíaRESUMEN
Placental growth factor (PlGF) is a proangiogenic member of the vascular endothelial growth factor (VEGF) family playing a central role in pathological angiogenesis. PlGF-DE is a PlGF variant unable to bind vascular endothelial growth factor receptor 1 (VEGFR-1) but still able to generate heterodimer with VEGF-A. We have generated PlGF-DE knockin mice that are vital and fertile and show unaltered expression of Plgf, Vegf-a, Vegfr-1, and Vegfr-2 compared with wild-type mice. Interestingly, these mutants showed additional and remarkable angiogenesis impairment in tumor growth, hindlimb ischemia, and choroidal neovascularization compared with both PlGF knockout and wild-type mice. These findings provided insights on VEGF-A/PlGF heterodimer function, which was able to rescue neovascularization and vascular leakage in PlGF-DE knockin mice. Collectively, these data show that PlGF-DE knockin mouse could be considered the full functional knockout of PlGF, suggesting a reassessment of the phenotypes of knockout mice for the genes whose products are able to generate heterodimeric proteins.
Asunto(s)
Técnicas de Sustitución del Gen , Factor de Crecimiento Placentario/metabolismo , Multimerización de Proteína , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Proliferación Celular , Neovascularización Coroidal/metabolismo , Neovascularización Coroidal/patología , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Óxido Nítrico Sintasa de Tipo III/metabolismo , Fosforilación , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Placental growth factor (PlGF), a proangiogenic member of vascular endothelial growth family, is active during pathological conditions like cancer, metastasis formation and hind limb ischemia and in wound healing. Endothelial cells express PlGF and hypoxia positively modulates in vitro its expression. To verify whether hypoxia modulates PlGF expression in different cellular contexts and in vivo, we first analyzed five human and five mouse cancer cell lines showing that in eight of them hypoxia positively modulates PlGF. Next, we analyzed xenograft colorectal cancer tumors showing that human cancer cells were able to express PlGF in hypoxic area of the tumor. Surprisingly, we did not visualize mouse PlGF in CD31 positive tumor vessels, but in low CD31 positive vessels, a characteristic of lymphatic vessels. We found that hypoxia effectively activates PlGF expression in lymphatic endothelial cells as well as in LYVE1 positive tumor vessels. We also investigated two additional mouse angiogenic models, hind limb ischemia and wound healing, and we confirmed that lymphatic vessels of both ischemic muscles and skin express PlGF. These results show for the first time that hypoxia activates PlGF expression in lymphatic endothelial cells, which have to be considered an additional source for PlGF production in pathological contexts.
Asunto(s)
Endotelio Linfático/metabolismo , Neoplasias/metabolismo , Factor de Crecimiento Placentario/metabolismo , Células A549 , Animales , Hipoxia de la Célula , Línea Celular Tumoral , Células Endoteliales/citología , Células Endoteliales/metabolismo , Endotelio Linfático/citología , Células Endoteliales de la Vena Umbilical Humana , Humanos , Células MCF-7 , Ratones , Trasplante de Neoplasias , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Regulación hacia ArribaRESUMEN
Human intravenous immune globulin (IVIg), a purified IgG fraction composed of ~ 60% IgG1 and obtained from the pooled plasma of thousands of donors, is clinically used for a wide range of diseases. The biological actions of IVIg are incompletely understood and have been attributed both to the polyclonal antibodies therein and also to their IgG (IgG) Fc regions. Recently, we demonstrated that multiple therapeutic human IgG1 antibodies suppress angiogenesis in a target-independent manner via FcγRI, a high-affinity receptor for IgG1. Here we show that IVIg possesses similar anti-angiogenic activity and inhibited blood vessel growth in five different mouse models of prevalent human diseases, namely, neovascular age-related macular degeneration, corneal neovascularization, colorectal cancer, fibrosarcoma and peripheral arterial ischemic disease. Angioinhibition was mediated by the Fc region of IVIg, required FcγRI and had similar potency in transgenic mice expressing human FcγRs. Finally, IVIg therapy administered to humans for the treatment of inflammatory or autoimmune diseases reduced kidney and muscle blood vessel densities. These data place IVIg, an agent approved by the US Food and Drug Administration, as a novel angioinhibitory drug in doses that are currently administered in the clinical setting. In addition, they raise the possibility of an unintended effect of IVIg on blood vessels.
RESUMEN
Aberrant angiogenesis is implicated in diseases affecting nearly 10% of the world's population. The most widely used anti-angiogenic drug is bevacizumab, a humanized IgG1 monoclonal antibody that targets human VEGFA. Although bevacizumab does not recognize mouse Vegfa, it inhibits angiogenesis in mice. Here we show bevacizumab suppressed angiogenesis in three mouse models not via Vegfa blockade but rather Fc-mediated signaling through FcγRI (CD64) and c-Cbl, impairing macrophage migration. Other approved humanized or human IgG1 antibodies without mouse targets (adalimumab, alemtuzumab, ofatumumab, omalizumab, palivizumab and tocilizumab), mouse IgG2a, and overexpression of human IgG1-Fc or mouse IgG2a-Fc, also inhibited angiogenesis in wild-type and FcγR humanized mice. This anti-angiogenic effect was abolished by Fcgr1 ablation or knockdown, Fc cleavage, IgG-Fc inhibition, disruption of Fc-FcγR interaction, or elimination of FcRγ-initated signaling. Furthermore, bevacizumab's Fc region potentiated its anti-angiogenic activity in humanized VEGFA mice. Finally, mice deficient in FcγRI exhibited increased developmental and pathological angiogenesis. These findings reveal an unexpected anti-angiogenic function for FcγRI and a potentially concerning off-target effect of hIgG1 therapies.
RESUMEN
Angiogenesis is a complex biological phenomenon essential for a correct embryonic development and for post-natal growth. In adult life, it is a tightly regulated process but in several pathological conditions, angiogenesis results abnormal with either excessive or insufficient proliferation of blood vessels. The pro-angiogenic members of VEGF family, VEGF-A, VEGF-B and placental growth factor (PlGF), and the related receptors, VEGFR-1 and VEGFR-2, have a central and decisive role in pathological angiogenesis. Indeed, they are the targets for anti-angiogenic drugs currently approved: bevacizumab and ranibizumab, that specifically inhibit VEGF-A; aflibercept, that is able to prevent the activity of VEGF-A, VEGF-B and PlGF; several multirtarget tyrosine kinase inhibitors that are able to prevent VEGFR-1 and/or VEGFR-2 signaling. The anti-angiogenesis therapy has represented one of the most active fields of drug discovery of last decade and promises to be further expanded due the wide number of diseases for which it may by applied.