RESUMEN
Inflammatory cytokines and growth factors drive angiogenesis independently; however, their integrated role in pathologic and physiologic angiogenesis is not fully understood. Suppressor of cytokine signaling-3 (SOCS3) is an inducible negative feedback regulator of inflammation and growth factor signaling. In the present study, we show that SOCS3 curbs pathologic angiogenesis. Using a Cre/Lox system, we deleted SOCS3 in vessels and studied developmental and pathologic angiogenesis in murine models of oxygen-induced retinopathy and cancer. Conditional loss of SOCS3 leads to increased pathologic neovascularization, resulting in pronounced retinopathy and increased tumor size. In contrast, physiologic vascularization is not regulated by SOCS3. In vitro, SOCS3 knockdown increases proliferation and sprouting of endothelial cells costimulated with IGF-1 and TNFα via reduced feedback inhibition of the STAT3 and mTOR pathways. These results identify SOCS3 as a pivotal endogenous feedback inhibitor of pathologic angiogenesis and a potential therapeutic target acting at the converging crossroads of growth factor- and cytokine-induced vessel growth.
Asunto(s)
Carcinoma Pulmonar de Lewis/prevención & control , Hipoxia/patología , Melanoma Experimental/prevención & control , Neovascularización Patológica/prevención & control , Síndromes Paraneoplásicos Oculares/prevención & control , Proteínas Supresoras de la Señalización de Citocinas/fisiología , Animales , Western Blotting , Carcinoma Pulmonar de Lewis/irrigación sanguínea , Carcinoma Pulmonar de Lewis/patología , Proliferación Celular , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Factor I del Crecimiento Similar a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/metabolismo , Integrasas/metabolismo , Masculino , Melanoma Experimental/irrigación sanguínea , Melanoma Experimental/patología , Ratones , Ratones Endogámicos C57BL , Neovascularización Patológica/etiología , Síndromes Paraneoplásicos Oculares/patología , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Proteína 3 Supresora de la Señalización de Citocinas , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
OBJECTIVE: Angiogenesis is essential physiologically in growth and pathologically in tumor development, chronic inflammatory disorders, and proliferative retinopathies. Activation of protease-activated receptor 2 (PAR2) leads to a proangiogenic response, but its mechanisms have yet to be specifically described. Here, we investigated the mode of action of PAR2 in retinal angiogenesis. METHODS AND RESULTS: PAR2-activating peptide, SLIGRL, increased retinal angiogenesis associated with an induction of vascular endothelial growth factor and angiopoetin-2 and most notably tie2 in the retina in vivo as well as in cultured neuroretinal endothelial cells. SLIGRL also induced release of the proinflammatory and angiogenic mediator tumor necrosis factor-alpha (TNF-alpha) via the MEK/extracellular signal-regulated kinase (ERK) (MEK/ERK) pathway in these endothelial cells. TNF-alpha, in turn, elicited tie2 expression by activating the MEK/ERK pathway. PAR2-evoked tie2 expression, endothelium proliferation (in vitro), and retinal neovascularization (in vivo) were abrogated by selective TNF-alpha blockers (neutralizing antibody infliximab and soluble TNF-alpha receptor-Fc fusion protein etanercept) as well as the MEK inhibitor PD98059. CONCLUSIONS: The proangiogenic properties of PAR2 are intertwined with its proinflammatory effects, such that in retinal vasculature, they depend on TNF-alpha and subsequent induction of tie2 via the MEK/ERK pathway.
Asunto(s)
Neovascularización Fisiológica , Receptor PAR-2/fisiología , Angiopoyetina 2/metabolismo , Animales , Animales Recién Nacidos , Células Cultivadas , Endotelio Vascular/fisiología , Oligopéptidos/fisiología , Ratas , Ratas Sprague-Dawley , Receptor TIE-2/metabolismo , Retina/fisiología , Factor de Necrosis Tumoral alfa/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Semaphorins are known modulators of axonal sprouting and angiogenesis. In the retina, we identified a distinct and almost exclusive expression of Semaphorin 3F in the outer layers. Interestingly, these outer retinal layers are physiologically avascular. Using functional in vitro models, we report potent anti-angiogenic effects of Semaphorin 3F on both retinal and choroidal vessels. In addition, human retinal pigment epithelium isolates from patients with pathologic neovascularization of the outer retina displayed reduced Semaphorin 3F expression in 10 out of 15 patients. Combined, these results elucidate a functional role for Semaphorin 3F in the outer retina where it acts as a vasorepulsive cue to maintain physiologic avascularity.
Asunto(s)
Proteínas Angiostáticas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Segmento Externo de las Células Fotorreceptoras Retinianas/metabolismo , Animales , Células Cultivadas , Humanos , Ratones , Ratones Endogámicos C57BL , Epitelio Pigmentado de la Retina/metabolismo , Vasos Retinianos/metabolismo , Esferoides Celulares , Factor A de Crecimiento Endotelial Vascular/fisiologíaRESUMEN
PURPOSE: Retinopathy of prematurity (ROP) is a leading cause of blindness in children and is, in its most severe form, characterized by uncontrolled growth of vision-threatening pathologic vessels. Propranolol, a nonselective ß-adrenergic receptor blocker, was reported to protect against pathologic retinal neovascularization in a mouse model of oxygen-induced retinopathy (OIR). Based on this single animal study using nonstandard evaluation of retinopathy, clinical trials are currently ongoing to evaluate propranolol treatment in stage 2 ROP patients who tend to experience spontaneous disease regression and are at low risk of blindness. Because these ROP patients are vulnerable premature infants who are still in a fragile state of incomplete development, the efficacy of propranolol treatment in retinopathy needs to be evaluated thoroughly in preclinical animal models of retinopathy and potential benefits weighed against potential adverse effects. METHODS: Retinopathy was induced by exposing neonatal mice to 75% oxygen from postnatal day (P) 7 to P12. Three routes of propranolol treatment were assessed from P12 to P16: oral gavage, intraperitoneal injection, or subcutaneous injection, with doses varying between 2 and 60 mg/kg/day. At P17, retinal flatmounts were stained with isolectin and quantified with a standard protocol to measure vasoobliteration and pathologic neovascularization. Retinal gene expression was analyzed with qRT-PCR using RNA isolated from retinas of control and propranolol-treated pups. RESULTS: None of the treatment approaches at any dose of propranolol (up to 60 mg/kg/day) were effective in preventing the development of retinopathy in a mouse model of OIR, evaluated using standard techniques. Propranolol treatment also did not change retinal expression of angiogenic factors including vascular endothelial growth factor. CONCLUSIONS: Propranolol treatment via three routes and up to 30 times the standard human dose failed to suppress retinopathy development in mice. These data bring into question whether propranolol through inhibition of ß-adrenergic receptors is an appropriate therapeutic approach for treating ROP.
Asunto(s)
Antagonistas Adrenérgicos beta/administración & dosificación , Propranolol/administración & dosificación , Neovascularización Retiniana/prevención & control , Retinopatía de la Prematuridad/prevención & control , Administración Oral , Angiopoyetina 1/genética , Angiopoyetina 1/metabolismo , Angiopoyetina 2/genética , Angiopoyetina 2/metabolismo , Animales , Animales Recién Nacidos , Proliferación Celular , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Células Endoteliales/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Eritropoyetina/genética , Eritropoyetina/metabolismo , Perfilación de la Expresión Génica , Humanos , Técnicas In Vitro , Recién Nacido , Inyecciones Intraperitoneales , Inyecciones Subcutáneas , Ratones , Oxígeno/toxicidad , ARN/metabolismo , Receptores Adrenérgicos beta/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular/genética , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismo , Retina/citología , Neovascularización Retiniana/metabolismo , Retinopatía de la Prematuridad/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Regulación hacia Arriba , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Mutations in low-density lipoprotein receptor-related protein 5 (Lrp5) impair retinal angiogenesis in patients with familial exudative vitreoretinopathy (FEVR), a rare type of blinding vascular eye disease. The defective retinal vasculature phenotype in human FEVR patients is recapitulated in Lrp5 knockout (Lrp5(-/-)) mouse with delayed and incomplete development of retinal vessels. In this study we examined gene expression changes in the developing Lrp5(-/-) mouse retina to gain insight into the molecular mechanisms that underlie the pathology of FEVR in humans. Gene expression levels were assessed with an Illumina microarray on total RNA from Lrp5(-/-) and WT retinas isolated on postnatal day (P) 8. Regulated genes were confirmed using RT-qPCR analysis. Consistent with a role in vascular development, we identified expression changes in genes involved in cell-cell adhesion, blood vessel morphogenesis and membrane transport in Lrp5(-/-) retina compared to WT retina. In particular, tight junction protein claudin5 and amino acid transporter slc38a5 are both highly down-regulated in Lrp5(-/-) retina. Similarly, several Wnt ligands including Wnt7b show decreased expression levels. Plasmalemma vesicle associated protein (plvap), an endothelial permeability marker, in contrast, is up-regulated consistent with increased permeability in Lrp5(-/-) retinas. Together these data suggest that Lrp5 regulates multiple groups of genes that influence retinal angiogenesis and may contribute to the pathogenesis of FEVR.