RESUMEN
Interferon-γ (IFNG) is one of the key cytokines that regulates both innate and adaptive immune responses in the body. However, the role of IFNG in the regulation of vascularization, especially in the context of Vascular endothelial growth factor A (VEGFa)-induced angiogenesis is not clarified. Here, we report that IFNG shows potent anti-angiogenic potential against VEGFa-induced angiogenesis. IFNG significantly inhibited proliferation, migration, and tube formation of Human umbilical vein endothelial cells (HUVECs) both under basal and VEGFa-treated conditions. Intriguingly, Knockdown (KD) of STAT1 abolished the inhibitory effect of IFNG on VEGFa-induced angiogenic processes in HUVECs. Furthermore, IFNG exhibited potent anti-angiogenic efficacy in the mouse model of oxygen-induced retinopathy (OIR), an in vivo model for hypoxia-induced retinal neovascularization, without induction of functional side effects. Taken together, these results show that IFNG plays a crucial role in the regulation of VEGFa-dependent angiogenesis, suggesting its potential therapeutic applicability in neovascular diseases.
Asunto(s)
Interferón gamma/uso terapéutico , Isquemia/complicaciones , Neovascularización Retiniana/complicaciones , Neovascularización Retiniana/tratamiento farmacológico , Animales , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Regulación hacia Abajo/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Hipoxia/complicaciones , Interferón gamma/administración & dosificación , Interferón gamma/farmacología , Inyecciones Intravítreas , Ratones , Neovascularización Fisiológica/efectos de los fármacos , Retina/efectos de los fármacos , Retina/patología , Retina/fisiopatología , Neovascularización Retiniana/fisiopatología , Factor de Transcripción STAT1/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Purpose: Ciliary neurotrophic factor (CNTF) is a well-characterized neurotrophic factor currently in clinical trials for the treatment of macular telangiectasia type II. Our previous work showed that CNTF-induced STAT3 signaling is a potent inhibitor of pathologic preretinal neovascular tuft formation in the mouse model of oxygen-induced retinopathy. In this study, we investigated the effect of CNTF on outer retinal and choroidal angiogenesis and the mechanisms that underpin the observed decrease in outer retinal neovascularization following CNTF treatment. Methods: In the Vldlr-/- and laser-CNV mouse models, mice received a one-time injection (on postnatal day [P] 12 in the Vldlr-/- model and 1 day after laser in the Choroidal Neovascularization (CNV) model) of recombinant CNTF or CxCl10, and the extent of neovascular lesions was assessed 6 days posttreatment. STAT3 downstream targets affected by CNTF treatment were identified using quantitative PCR analysis. A proteome array was used to compare media conditioned by CNTF-treated and control-treated primary Müller cells to screen for CNTF-induced changes in secreted angiogenic factors. Results: Intravitreal treatment with recombinant CNTF led to significant reduction in neovascularization in the Vldlr-/- and laser-CNV mouse models. Treatment effect in the Vldlr-/- was long-lasting but time sensitive, requiring intravitreal treatment before P19. Mechanistic workup in vitro as well as in vivo confirmed significant activation of the STAT3-signaling pathway in Müller cells in response to CNTF treatment and upregulation of CxCl10. Intravitreal injections of recombinant CxCl10 significantly reduced outer retinal neovascularization in vivo in both the Vldlr-/- and laser-CNV mouse models. Conclusions: CNTF treatment indirectly affects outer retinal and choroidal neovascularization by inducing CxCl10 secretion from retinal Müller cells.
Asunto(s)
Quimiocina CXCL10/metabolismo , Factor Neurotrófico Ciliar/uso terapéutico , Neovascularización Retiniana/prevención & control , Animales , Western Blotting , Células Cultivadas , Neovascularización Coroidal/metabolismo , Neovascularización Coroidal/patología , Neovascularización Coroidal/prevención & control , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Células Ependimogliales , Inmunohistoquímica , Coagulación con Láser , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Neovascularización Retiniana/metabolismo , Neovascularización Retiniana/patología , Factor de Transcripción STAT3/metabolismo , Regulación hacia ArribaRESUMEN
Oxygen-induced retinopathy (OIR) is a widely used model to study ischemia-driven neovascularization (NV) in the retina and to serve in proof-of-concept studies in evaluating antiangiogenic drugs for ocular, as well as nonocular, diseases. The primary parameters that are analyzed in this mouse model include the percentage of retina with vaso-obliteration (VO) and NV areas. However, quantification of these two key variables comes with a great challenge due to the requirement of human experts to read the images. Human readers are costly, time-consuming, and subject to bias. Using recent advances in machine learning and computer vision, we trained deep learning neural networks using over a thousand segmentations to fully automate segmentation in OIR images. While determining the percentage area of VO, our algorithm achieved a similar range of correlation coefficients to that of expert inter-human correlation coefficients. In addition, our algorithm achieved a higher range of correlation coefficients compared with inter-expert correlation coefficients for quantification of the percentage area of neovascular tufts. In summary, we have created an open-source, fully automated pipeline for the quantification of key values of OIR images using deep learning neural networks.
Asunto(s)
Aprendizaje Profundo , Neovascularización Retiniana/diagnóstico , Algoritmos , Animales , Modelos Animales de Enfermedad , Femenino , Procesamiento de Imagen Asistido por Computador/métodos , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Confocal/métodos , Variaciones Dependientes del Observador , Oxígeno , Neovascularización Retiniana/etiología , Neovascularización Retiniana/patologíaRESUMEN
BACKGROUND: Anti-angiogenic biologicals represent an important concept for the treatment of vasoproliferative diseases. However, the need for continued treatment, the presence of nonresponders, and the risk of long-term side effects limit the success of existing therapeutic agents. Although Tspan12 has been shown to regulate retinal vascular development, nothing is known about its involvement in neovascular disease and its potential as a novel therapeutic target for the treatment of vasoproliferative diseases. METHODS: Rodent models of retinal neovascular disease, including the mouse model of oxygen-induced retinopathy and the very low density lipoprotein receptor knockout mouse model were analyzed for Tspan/ß-catenin regulation. Screening of a phage display of a human combinatorial antibody (Ab) library was used for the development of a high-affinity Ab against Tspan12. Therapeutic effects of the newly developed Ab on vascular endothelial cells were tested in vitro and in vivo in the oxygen-induced retinopathy and very low density lipoprotein receptor knockout mouse model. RESULTS: The newly developed anti-Tspan12 Ab exhibited potent inhibitory effects on endothelial cell migration and tube formation. Mechanistic studies confirmed that the Ab inhibited the interaction between Tspan12 and Frizzled-4 and effectively modulates ß-catenin levels and target genes in vascular endothelial cells. Tspan12/ß-catenin signaling was activated in response to acute and chronic stress in the oxygen-induced retinopathy and very low density lipoprotein receptor mouse model of proliferative retinopathy. Intravitreal application of the Ab showed significant therapeutic effects in both models without inducing negative side effects on retina function. Moreover, combined intravitreal injection of the Ab with a known vascular endothelial growth factor inhibitor, Aflibercept, resulted in significant enhancement of the therapeutic efficacy of each monotherapy. Combination therapy with the Tspan12 blocking antibody can be used to reduce anti-vascular endothelial growth factor doses, thus decreasing the risk of long-term off-target effects. CONCLUSIONS: Tspan12/ß-catenin signaling is critical for the progression of vasoproliferative disease. The newly developed anti-Tspan12 antibody has therapeutic effects in vasoproliferative retinopathy and can enhance the potency of existing anti- vascular endothelial growth factor agents.
Asunto(s)
Neovascularización Retiniana/metabolismo , Transducción de Señal/fisiología , Tetraspaninas/antagonistas & inhibidores , Tetraspaninas/metabolismo , beta Catenina/antagonistas & inhibidores , beta Catenina/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos/genética , Anticuerpos/farmacología , Anticuerpos/uso terapéutico , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana , Humanos , Inyecciones Intravítreas , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Neovascularización Retiniana/tratamiento farmacológico , Transducción de Señal/efectos de los fármacosRESUMEN
PURPOSE: Retinal vascular disease represents a major cause for vision loss in the Western world. Recent research has shown that neuronal and vascular damage are closely related in retinal disease. Ciliary neurotrophic factor (CNTF) is a well-studied neurotrophic factor that is currently being tested in clinical trials for the treatment of retinal degenerative diseases and macular telangiectasia. However, little is known about its effect on retinal vasculature. In this study, we investigate the effects of CNTF in retinal neovascular disease using the mouse model of oxygen-induced retinopathy (OIR). METHODS: Newborn pups were exposed to 75% oxygen from postnatal day (P)7 to P12 and subsequently returned to room air. Ciliary neurotrophic factor was injected intravitreally at OIR P12 and the vaso-obliterated and neovascular areas were quantified at OIR P17. Immunohistochemistry, RNA, and protein analysis were used to identify CNTF-responsive cells. In vitro experiments were performed to analyze the effect of CNTF on endothelial and astroglial cells. RESULTS: In the OIR model, CNTF facilitated capillary regrowth and attenuated preretinal neovascularization in a dose-dependent manner. The protective effect of CNTF was mediated via activation of the JAK/STAT3/SOCS3 signaling pathway. Immunohistochemical studies identified endothelial cells among others as CNTF-responsive cells in the retina. In vitro studies confirmed the anti-angiogenic effect of CNTF on endothelial cell sprouting. CONCLUSIONS: This study provides evidence for a therapeutic potential of CNTF beyond degenerative retinal disease. Vasoproliferative retinopathies may benefit from a CNTF-dependent and SOCS3-mediated angiomodulatory effect.