RESUMO
UNLABELLED: In many diseases, expression and ligand-dependent activity of the p75(NTR) receptor can promote pericyte and vascular dysfunction, inflammation, glial activation, and neurodegeneration. Diabetic retinopathy (DR) is characterized by all of these pathological events. However, the mechanisms by which p75(NTR) may be implicated at each stage of DR pathology remain poorly understood. Using a streptozotocin mouse model of diabetic retinopathy, we report that p75(NTR) is upregulated very early in glia and in pericytes to mediate ligand-dependent induction of inflammatory cytokines, disruption of the neuro-glia-vascular unit, promotion of blood-retina barrier breakdown, edema, and neuronal death. In a mouse model of oxygen-induced retinopathy, mimicking proliferative DR, p75(NTR)-dependent inflammation leads to ischemia and pathological angiogenesis through Semaphorin 3A. The acute use of antagonists of p75(NTR) or antagonists of the ligand proNGF suppresses each distinct phase of pathology, ameliorate disease, and prevent disease progression. Thus, our study documents novel disease mechanisms and validates druggable targets for diabetic retinopathy. SIGNIFICANCE STATEMENT: Diabetic retinopathy (DR) affects an estimated 250 million people and has no effective treatment. Stages of progression comprise pericyte/vascular dysfunction, inflammation, glial activation, and neurodegeneration. The pathophysiology of each stage remains unclear. We postulated that the activity of p75NTR may be implicated. We show that p75NTR in glia and in pericytes mediate ligand-dependent induction of inflammatory cytokines, disruption of the neuro-glia-vascular unit, promotion of blood-retina barrier breakdown, edema, and neuronal death. p75NTR-promoted inflammation leads to ischemia and angiogenesis through Semaphorin 3A. Antagonists of p75NTR or antagonists of proNGF suppress each distinct phase of pathology, ameliorate disease, and prevent disease progression. Our study documents novel mechanisms in a pervasive disease and validates druggable targets for treatment.
Assuntos
Retinopatia Diabética/complicações , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Inflamação/etiologia , Fator de Crescimento Neural/metabolismo , Doenças Neurodegenerativas/etiologia , Precursores de Proteínas/metabolismo , Receptores de Fator de Crescimento Neural/metabolismo , Doenças Vasculares/etiologia , Animais , Animais Recém-Nascidos , Anticorpos/farmacologia , Astrócitos/química , Células Cultivadas , Meios de Cultivo Condicionados/farmacologia , Citocinas/genética , Citocinas/metabolismo , Retinopatia Diabética/induzido quimicamente , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/fisiologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Marcação In Situ das Extremidades Cortadas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fator de Crescimento Neural/imunologia , Precursores de Proteínas/imunologia , Ratos , Receptores de Fator de Crescimento Neural/imunologia , Retina/patologia , Estreptozocina/toxicidade , Tomografia de Coerência Óptica , Vias Visuais/patologiaRESUMO
Ischemic retinopathies are characterized by sequential vaso-obliteration followed by abnormal intravitreal neovascularization predisposing patients to retinal detachment and blindness. Ischemic retinopathies are associated with robust inflammation that leads to generation of IL-1ß, which causes vascular degeneration and impairs retinal revascularization in part through the liberation of repulsive guidance cue semaphorin 3A (Sema3A). However, retinal revascularization begins as inflammation culminates in ischemic retinopathies. Because inflammation leads to activation of proteases involved in the formation of vasculature, we hypothesized that proteinase-activated receptor (Par)-2 (official name F2rl1) may modulate deleterious effects of IL-1ß. Par2, detected mostly in retinal ganglion cells, was up-regulated in oxygen-induced retinopathy. Surprisingly, oxygen-induced retinopathy-induced vaso-obliteration and neovascularization were unaltered in Par2 knockout mice, suggesting compensatory mechanisms. We therefore conditionally knocked down retinal Par2 with shRNA-Par2-encoded lentivirus. Par2 knockdown interfered with normal revascularization, resulting in pronounced intravitreal neovascularization; conversely, the Par2 agonist peptide (SLIGRL) accelerated normal revascularization. In vitro and in vivo exploration of mechanisms revealed that IL-1ß induced Par2 expression, which in turn down-regulated sequentially IL-1 receptor type I and Sema3A expression through Erk/Jnk-dependent processes. Collectively, our findings unveil an important mechanism by which IL-1ß regulates its own endothelial cytotoxic actions by augmenting neuronal Par2 expression to repress sequentially IL-1 receptor type I and Sema3A expression. Timely activation of Par2 may be a promising therapeutic avenue in ischemic retinopathies.
Assuntos
Proteínas do Olho/metabolismo , Isquemia/metabolismo , Receptores de Trombina/metabolismo , Doenças Retinianas/metabolismo , Neurônios Retinianos/metabolismo , Animais , Proteínas do Olho/agonistas , Proteínas do Olho/genética , Inflamação/tratamento farmacológico , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Isquemia/tratamento farmacológico , Isquemia/genética , Isquemia/patologia , Camundongos , Camundongos Knockout , Oligopeptídeos/farmacologia , Receptores de Trombina/agonistas , Receptores de Trombina/genética , Doenças Retinianas/tratamento farmacológico , Doenças Retinianas/genética , Doenças Retinianas/patologia , Neurônios Retinianos/patologia , Semaforina-3A/genética , Semaforina-3A/metabolismoRESUMO
OBJECTIVE: Proinflammatory cytokines contribute to the development of retinal vasculopathies. However, the role of these factors and the mechanisms by which they elicit their effects in retina are not known. We investigated whether activated microglia during early stages of ischemic retinopathy produces excessive interleukin-1ß (IL-1ß), which elicits retinal microvascular degeneration not directly but rather by triggering the release of the proapoptotic/repulsive factor semaphorin-3A (Sema3A) from neurons. APPROACH AND RESULTS: Sprague Dawley rats subjected to retinopathy induced by hyperoxia (80% O2; O2-induced retinopathy) exhibited retinal vaso-obliteration associated with microglial activation, NLRP3 upregulation, and IL-1ß and Sema3A release; IL-1ß was mostly generated by microglia. Intraperitoneal administration of IL-1 receptor antagonists (Kineret, or rytvela [101.10]) decreased these effects and enhanced retinal revascularization; knockdown of Sema3A resulted in microvessel preservation and, conversely, administration of IL-1ß caused vaso-obliteration. In vitro, IL-1ß derived from activated primary microglial cells, cultured under hyperoxia, stimulated the release of Sema3A in retinal ganglion cells-5, which in turn induced apoptosis of microvascular endothelium; antagonism of IL-1 receptor decreased microglial activation and on retinal ganglion cells-5 abolished the release of Sema3A inhibiting ensuing endothelial cell apoptosis. IL-1ß was not directly cytotoxic to endothelial cells. CONCLUSIONS: Our findings suggest that in the early stages of O2-induced retinopathy, retinal microglia are activated to produce IL-1ß, which sustains the activation of microglia and induces microvascular injury through the release of Sema3A from adjacent neurons. Interference with IL-1 receptor or Sema3A actions preserves the microvascular bed in ischemic retinopathies and, consequently, decreases ensued pathological preretinal neovascularization.
Assuntos
Interleucina-1beta/metabolismo , Isquemia/patologia , Microglia/patologia , Doenças Retinianas/patologia , Retinite/patologia , Semaforina-3A/metabolismo , Animais , Antirreumáticos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Proteínas de Transporte , Células Endoteliais/imunologia , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Técnicas de Silenciamento de Genes , Hiperóxia/imunologia , Hiperóxia/metabolismo , Hiperóxia/patologia , Proteína Antagonista do Receptor de Interleucina 1/farmacologia , Interleucina-1beta/antagonistas & inibidores , Interleucina-1beta/imunologia , Isquemia/tratamento farmacológico , Isquemia/imunologia , Microcirculação/fisiologia , Microglia/imunologia , Microglia/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/imunologia , Peptídeos/farmacologia , Cultura Primária de Células , Ratos , Ratos Sprague-Dawley , Receptores Citoplasmáticos e Nucleares/imunologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Doenças Retinianas/tratamento farmacológico , Doenças Retinianas/imunologia , Células Ganglionares da Retina/imunologia , Células Ganglionares da Retina/metabolismo , Células Ganglionares da Retina/patologia , Retinite/tratamento farmacológico , Retinite/imunologia , Semaforina-3A/genética , Semaforina-3A/imunologiaRESUMO
The failure of blood vessels to revascularize ischemic neural tissue represents a significant challenge for vascular biology. Examples include proliferative retinopathies (PRs) such as retinopathy of prematurity and proliferative diabetic retinopathy, which are the leading causes of blindness in children and working-age adults. PRs are characterized by initial microvascular degeneration, followed by a compensatory albeit pathologic hypervascularization mounted by the hypoxic retina attempting to reinstate metabolic equilibrium. Paradoxically, this secondary revascularization fails to grow into the most ischemic regions of the retina. Instead, the new vessels are misdirected toward the vitreous, suggesting that vasorepulsive forces operate in the avascular hypoxic retina. In the present study, we demonstrate that the neuronal guidance cue semaphorin 3A (Sema3A) is secreted by hypoxic neurons in the avascular retina in response to the proinflammatory cytokine IL-1ß. Sema3A contributes to vascular decay and later forms a chemical barrier that repels neo-vessels toward the vitreous. Conversely, silencing Sema3A expression enhances normal vascular regeneration within the ischemic retina, thereby diminishing aberrant neovascularization and preserving neuroretinal function. Overcoming the chemical barrier (Sema3A) released by ischemic neurons accelerates the vascular regeneration of neural tissues, which restores metabolic supply and improves retinal function. Our findings may be applicable to other neurovascular ischemic conditions such as stroke.
Assuntos
Isquemia/patologia , Neovascularização Patológica , Neurônios/patologia , Oxigênio/toxicidade , Regeneração , Doenças Retinianas/patologia , Semaforina-3A/fisiologia , Animais , Aorta/citologia , Aorta/efeitos dos fármacos , Aorta/metabolismo , Western Blotting , Adesão Celular , Movimento Celular , Proliferação de Células , Células Cultivadas , Modelos Animais de Doenças , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Técnicas Imunoenzimáticas , Interleucina-1beta/farmacologia , Isquemia/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , RNA Mensageiro/genética , Ratos , Doenças Retinianas/etiologia , Doenças Retinianas/metabolismo , Células Ganglionares da Retina/efeitos dos fármacos , Células Ganglionares da Retina/metabolismo , Neovascularização Retiniana , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
UNLABELLED: Retinopathy of prematurity (ROP) is a major cause of severe visual deficits in children. This review focuses on the role of newly identified factors from retinal neurons, which through their opposing actions on vascular development contribute to ROP. These hypoxia-generated mediators include the Krebs cycle intermediate, succinate acting via GPR91, and the neuronal guidance molecule Semaphorin 3A. CONCLUSION: Neuron-derived factors guide retinal vascularization and are major contributors to the pathogenesis of ROP.
Assuntos
Receptores Acoplados a Proteínas G/metabolismo , Neovascularização Retiniana/metabolismo , Retinopatia da Prematuridade/metabolismo , Semaforina-3A/metabolismo , Biomarcadores/metabolismo , Humanos , Recém-Nascido , Recém-Nascido Prematuro , Neovascularização Retiniana/etiologia , Neurônios Retinianos/metabolismo , Retinopatia da Prematuridade/patologiaRESUMO
The GPCR SUCNR1/GPR91 exerts proangiogenesis upon stimulation with the Krebs cycle metabolite succinate. GPCR signaling depends on the surrounding environment and intracellular localization through location bias. Here, we show by microscopy and by cell fractionation that in neurons, SUCNR1 resides at the endoplasmic reticulum (ER), while being fully functional, as shown by calcium release and the induction of the expression of the proangiogenic gene for VEGFA. ER localization was found to depend upon N-glycosylation, particularly at position N8; the nonglycosylated mutant receptor localizes at the plasma membrane shuttled by RAB11. This SUCNR1 glycosylation is physiologically regulated, so that during hypoxic conditions, SUCNR1 is deglycosylated and relocates to the plasma membrane. Downstream signal transduction of SUCNR1 was found to activate the prostaglandin synthesis pathway through direct interaction with COX-2 at the ER; pharmacologic antagonism of the PGE2 EP4 receptor (localized at the nucleus) was found to prevent VEGFA expression. Concordantly, restoring the expression of SUCNR1 in the retina of SUCNR1-null mice renormalized vascularization; this effect is markedly diminished after transfection of the plasma membrane-localized SUCNR1 N8A mutant, emphasizing that ER localization of the succinate receptor is necessary for proper vascularization. These findings uncover an unprecedented physiologic process where GPCR resides at the ER for signaling function.
Assuntos
Receptores Acoplados a Proteínas G , Ácido Succínico , Animais , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Hipóxia , Camundongos , Receptores Acoplados a Proteínas G/metabolismo , Succinatos , Ácido Succínico/metabolismoRESUMO
Neurons have an important role in retinal vascular development. Here we show that the G protein-coupled receptor (GPCR) coagulation factor II receptor-like 1 (F2rl1, previously known as Par2) is abundant in retinal ganglion cells and is associated with new blood vessel formation during retinal development and in ischemic retinopathy. After stimulation, F2rl1 in retinal ganglion cells translocates from the plasma membrane to the cell nucleus using a microtubule-dependent shuttle that requires sorting nexin 11 (Snx11). At the nucleus, F2rl1 facilitates recruitment of the transcription factor Sp1 to trigger Vegfa expression and, in turn, neovascularization. In contrast, classical plasma membrane activation of F2rl1 leads to the expression of distinct genes, including Ang1, that are involved in vessel maturation. Mutant versions of F2rl1 that prevent nuclear relocalization but not plasma membrane activation interfere with Vegfa but not Ang1 expression. Complementary angiogenic factors are therefore regulated by the subcellular localization of a receptor (F2rl1) that governs angiogenesis. These findings may have implications for the selectivity of drug actions based on the subcellular distribution of their targets.
Assuntos
Neovascularização Fisiológica , Neurônios/metabolismo , Receptor PAR-2/metabolismo , Transporte Ativo do Núcleo Celular , Angiopoietina-1/genética , Angiopoietina-1/metabolismo , Animais , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microtúbulos/metabolismo , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Neovascularização Patológica , Neovascularização Fisiológica/genética , Regiões Promotoras Genéticas , Receptor PAR-2/deficiência , Receptor PAR-2/genética , Células Ganglionares da Retina/metabolismo , Vasos Retinianos/crescimento & desenvolvimento , Vasos Retinianos/metabolismo , Nexinas de Classificação/metabolismo , Fator de Transcrição Sp1/metabolismo , Frações Subcelulares/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
The deterioration of the inner blood-retinal barrier and consequent macular edema is a cardinal manifestation of diabetic retinopathy (DR) and the clinical feature most closely associated with loss of sight. We provide evidence from both human and animal studies for the critical role of the classical neuronal guidance cue, semaphorin 3A, in instigating pathological vascular permeability in diabetic retinas via its cognate receptor neuropilin-1. We reveal that semaphorin 3A is induced in early hyperglycemic phases of diabetes within the neuronal retina and precipitates initial breakdown of endothelial barrier function. We demonstrate, by a series of orthogonal approaches, that neutralization of semaphorin 3A efficiently prevents diabetes-induced retinal vascular leakage in a stage of the disease when vascular endothelial growth factor neutralization is inefficient. These observations were corroborated in Tg(Cre-Esr1)/Nrp1(flox/flox) conditional knockout mice. Our findings identify a therapeutic target for macular edema and provide further evidence for neurovascular crosstalk in the pathogenesis of DR.
Assuntos
Retinopatia Diabética/metabolismo , Neurônios/metabolismo , Semaforina-3A/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Células Cultivadas , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Retinopatia Diabética/patologia , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Obesos , Pessoa de Meia-Idade , Neuropilina-1/deficiência , Neuropilina-1/genética , Neuropilina-1/metabolismo , Permeabilidade/efeitos dos fármacos , RNA Mensageiro/metabolismo , Retina/metabolismo , Retina/fisiopatologia , Semaforina-3A/genética , Semaforina-3A/farmacologia , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
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.
Assuntos
Proteínas Angiostáticas/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Segmento Externo das Células Fotorreceptoras da Retina/metabolismo , Animais , Células Cultivadas , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Epitélio Pigmentado da Retina/metabolismo , Vasos Retinianos/metabolismo , Esferoides Celulares , Fator A de Crescimento do Endotélio Vascular/fisiologiaRESUMO
In stroke and proliferative retinopathy, despite hypoxia driven angiogenesis, delayed revascularization of ischemic tissue aggravates the loss of neuronal function. What hinders vascular regrowth in the ischemic central nervous system remains largely unknown. Using the ischemic retina as a model of neurovascular interaction in the CNS, we provide evidence that the failure of reparative angiogenesis is temporally and spatially associated with endoplasmic reticulum (ER) stress. The canonical ER stress pathways of protein kinase RNA-like ER kinase (PERK) and inositol-requiring enzyme-1α (IRE1α) are activated within hypoxic/ischemic retinal ganglion neurons, initiating a cascade that results in angiostatic signals. Our findings demonstrate that the endoribonuclease IRE1α degrades the classical guidance cue netrin-1. This neuron-derived cue triggers a critical reparative-angiogenic switch in neural macrophage/microglial cells. Degradation of netrin-1, by persistent neuronal ER stress, thereby hinders vascular regeneration. These data identify a neuronal-immune mechanism that directly regulates reparative angiogenesis.
Assuntos
Isquemia Encefálica/fisiopatologia , Estresse do Retículo Endoplasmático/fisiologia , Endorribonucleases/metabolismo , Neovascularização Fisiológica/fisiologia , Fatores de Crescimento Neural/metabolismo , Neurônios/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Retina/fisiopatologia , Proteínas Supressoras de Tumor/metabolismo , Animais , Western Blotting , Ácido Clodrônico , Eletroforese em Gel de Poliacrilamida , Citometria de Fluxo , Vetores Genéticos , Imuno-Histoquímica , Indóis , Lentivirus , Camundongos , Camundongos Endogâmicos C57BL , Microdissecção , Netrina-1 , Reação em Cadeia da Polimerase em Tempo Real , eIF-2 Quinase/metabolismoRESUMO
Retinopathy of prematurity (ROP), an ocular disease characterized by the onset of vascular abnormalities in the developing retina, is the major cause of visual impairment and blindness in premature neonates. ROP is a complex condition in which various factors participate at different stages of the disease leading to microvascular degeneration followed by neovascularization, which in turn predisposes to retinal detachment. Current ablative therapies (cryotherapy and laser photocoagulation) used in the clinic for the treatment of ROP have limitations and patients can still have long-term effects even after successful treatment. New treatment modalities are still emerging. The most promising are the therapies directed against VEGF; more recently the use of preventive dietary supplementation with ω-3 polyunsaturated fatty acid may also be promising. Other than pharmacologic and nutritional approaches, cell-based strategies for vascular repair are likely to arise from advances in regenerative medicine using stem cells. In addition to all of these, a greater understanding of other factors involved in regulating pathologic retinal angiogenesis continues to emerge, suggesting potential targets for therapeutic approaches. This review summarizes an update on the current state of knowledge on ROP from our and other laboratories, with particular focus on the role of nitro-oxidative stress and notably trans-arachidonic acids in microvascular degeneration, semaphorin 3 operating as vasorepulsive molecules in the avascular hypoxic retina and in turn impairing revascularization, succinate and its receptor GPR91 in neuron-mediated retinal neovascularization, and ω-3 lipids as modulators of preretinal neovascularization.
Assuntos
Recém-Nascido Prematuro , Retinopatia da Prematuridade/etiologia , Idade Gestacional , Humanos , Recém-Nascido , Peroxidação de Lipídeos , Neovascularização Patológica , Estresse Oxidativo , Oxigênio/fisiologia , Oxigênio/uso terapêutico , Receptores Acoplados a Proteínas G , Retina/embriologia , Vasos Retinianos/embriologia , Retinopatia da Prematuridade/prevenção & controle , Retinopatia da Prematuridade/terapia , Fatores de Risco , Semaforinas , Ácido Succínico , Fator A de Crescimento do Endotélio VascularRESUMO
PURPOSE: Retinopathy of prematurity (ROP) is a major cause of visual handicap in the pediatric population. To date, this disorder is thought to stem from deficient retinal vascularization. Intriguingly, functional electrophysiological studies in patients with mild or moderate ROP and in the oxygen-induced retinopathy (OIR) model in rats reveal central photoreceptor disruption that overlies modest retinal vessel loss; a paucity of retinal vasculature occurs predominantly at the periphery. Given that choroidal circulation is the major source of oxygen and nutrients to the photoreceptors, the authors set out to investigate whether the choroidal vasculature system may be affected in OIR. METHODS: Rat models of OIR treating newborn animals with 80% or 50/10% alternated oxygen level for the first two postnatal weeks were used to mimic ROP in humans. Immunohistology staining and vascular corrosion casts were used to investigate the vessel layout of the eye. To investigate the effect of 15-deoxy-Δ12,14-PGJ(2) (15d-PGJ(2); a nonenzymatic product of prostaglandin D(2)) on endothelial cells, in vitro cell culture and ex vivo choroid explants were employed and intravitreal injections were performed in animals. RESULTS: The authors herein demonstrate that deficient vascularity occurs not only in the retinal plexus but also in the choroid. This sustained, marked choroidal degeneration is specifically confined to central regions of the retina that present persistent photoreceptor loss and corresponding functional deficits. Moreover, the authors show that 15d-PGJ(2) is a prominent contributor to this choroidal decay. CONCLUSIONS: The authors demonstrate for the first time pronounced, sustained choroidal vascular involution during the development of ROP. Findings also suggest that effective therapeutic strategies to counter ROP should consider choroidal preservation.