Strategies for revascularizing the ischemic retina

Les rétinopathies ischémiques (RI) sont la cause majeure de cécité chez les personnes âgées de moins de 65 ans. Il existe deux types de RIs soit la rétinopathie du prématuré (ROP) ainsi que la rétinopathie diabétique (RD). Les RIs sont décrites en deux phases soit la phase de vasooblitération, marquée par une perte importante de vaisseaux sanguins, et une phase de néovascularisation secondaire à lʼischémie menant à une croissance pathologique de vaisseaux. Cette seconde phase peut générer des complications cliniques telles quʼun oedème dans lʼhumeur vitré ainsi que le détachement de la rétine chez les patients déjà atteints dʼune RI. Les traitements approuvés pour les RIs visent à réduire la formation des vaisseaux pathologiques ou lʼoedème; mais ceux-ci malheureusement ne règlent pas les problèmes sous-jacents tels que la perte vasculaire et lʼischémie. La rétine est un tissu hautement vascularisé qui contribue à lʼirrigation et à lʼhoméostasie des neurones. Lʼinteraction neurovasculaire, comprenant de neurones, vaisseaux et cellules gliales, contribue au maintien de cette homéostasie. Durant le développement, les neurones et les cellules gliales jouent un rôle important dans la vascularisation de la rétine en sécrétant des facteurs qui stimulent l'angiogenèse. Cependant, nos connaissances sur lʼinteraction neurovasculaire dans les RIs sont limitées. En identifiant les interactions importantes entre les cellules composant cette unité neurovasculaire dans la rétine, nous pourrons viser des cibles qui engendreront une revascularisation seine afin de diminuer les signes pathologiques chez les patients atteints dʼune RI. Les travaux présentés dans cette thèse visent à mieux expliquer cette interaction neurovasculaire en soulignant des concepts importants propres aux RIs. En utilisant un modèle de rétinopathie induite par lʼoxygène chez la souris, qui reproduit les caractéristiques importantes de la ROP (et en certaines instances, la RD), nous identifions quelques molécules clés jouant un rôle significatif dans les RIs soit la sémaphorine 3A (sema3A), lʼIL-1β, ainsi que le récepteur PAR2. Nos résultats démontrent que Sema3A, sécrétée par les cellules ganglionnaires rétiniennes (CGRs) durant une ischémie, empêche la revascularisation normale et que cette expression est induite par lʼIL-1β provenant des microglies activées. En bloquant Sema3A directement ou via lʼinhibition de lʼIL- 1β, nous remarquons une revascularisation seine ainsi quʼune diminution importante des vaisseaux pathologiques. Cela nous indique que Sema3A est impliquée dans la guidance vasculaire et quʼelle contribue à la pathogenèse des RIs. Lʼactivation de façon exogène de PAR2, identifié aussi comme régulateur du récepteur de lʼIL-1β (IL- 1RI) sur les CGRs, se traduit par une diminution séquentielle de lʼIL-1RI et de Sema3A ce qui mène également à une revascularisation seine. En conclusion, ces travaux soulignent lʼimportance de lʼinteraction neurovasculaire ainsi que la guidance vasculaire dans les RIs. Ils renforcent lʼimportance de la communication entre neurone, vaisseau et microglie dans la pathogenèse des RIs. Finalement, nous identifions quelques molécules clés qui pourront servir comme cibles afin de lutter contre lʼischémie qui cause des problèmes vasculaires chez les patients atteints dʼune RI.Ischemic retinopathies (IRs), namely, retinopathy of prematurity (ROP) and diabetic retinopathy (DR), are the major cause of blindness in persons under the age of 65. IRs are biphasic disorders described by an initial vasoobliterative phase marked by a persistent microvascular degeneration, which leads to ischemia. Retinal ischemia, secondary to vessel loss, incites a second neovascularization phase represented by an aberrant, misdirected neovessel formation into the vitreous, which can cause adverse clinical complications including vitreous hemorrhaging and tractional retinal detachment. While current treatments aim at reducing vitreous/retinal hemorrhaging and/or pathological pre-retinal neovascularization, these regimens fail to address the underlying problem; that is, microvascular decay and retinal ischemia. The retina is a highly metabolic tissue that requires a significant amount of nutrients and oxygen. This is supplied by an intricate and highly regulated vascular network required to maintain homeostasis and proper function. The intricate cellular interactions in the neurovascular unit – the consortium of vessel, neurons and support glia – are required for regulating and maintaining homeostasis under normal conditions. However, the understanding of how this unit functions under ischemic stress, that which is seen in patients suffering from IRs, is not well defined. The present work underlines several important concepts of neurovascular coupling in IRs in efforts to identify potential therapeutic agents that may help curb retinal ischemia by stimulating normal revascularization. Using a mouse model of oxygen-induced retinopathy (OIR), which reproduces the salient features of ROP (and in some instances DR), we identified key players involved in generating the pathophysiological signatures associated with IRs; namely, semaphorin3A (Sema3A), interleukin-1β (IL-1β) and protease-activated receptor 2 (PAR2). Our results show that neuronal-derived Sema3A, secreted by ischemic retinal ganglion cells (RGCs), acts as a potent vaso-repulsive molecules that impedes normal revascularization. Activated microglia contribute to this process by secreting IL-1β, which induces paracrine release of Sema3A expression contributing to microvascular decay as well as pathological pre-retinal neovascularization. Inhibition of Sema3A or IL-1β translates to rapid revascularization and, as a result, a significant reduction in pathological neovessel formation. These results demonstrate that Sema3A is directly involved in vascular guidance and precipitates the pathophysiological features associated with IRs. PAR2, found on RGCs, was also identified as a key regulatory mechanism involved in dampening IL-1β induced Sema3A mediated vascular decay by reducing IL-1 receptor (IL-1RI). Exogenous activation of neuronal PAR2 translates to a sequential reduction of both IL-1RI and Sema3A resulting in accelerated revascularization and consequentially pre-retinal neovascularization. In conclusion, these studies highlight the importance of neurovascular coupling associated with IRs. Herein, we demonstrate the consorted interaction between neuron, vessel and glia and its impact on shaping the retinal vasculature during disease. Moreover, we underscore the significant impact of neuronal guidance cues in manifesting the salient vascular features of IRs. Finally, we identify key players that may serve as potential therapeutic avenues in curbing retinal ischemia in efforts to reduce vascular complications associated with IRs

Caracterización molecular del síndrome respiratorio y reproductivo porcino (PRRS) del gen de la nucleocápside ORF 7 en granjas porcinas de Lima, Perú

The Betaarterivirus suid 1 and Betaarterivirus suid 2 species of the Porcine Reproductive and Respiratory Syndrome (PRRS) virus cause severe outbreaks due to genetic recombination with high epidemiological risk, which requires rapid identification for efficient molecular epidemiological surveillance. The objectives of the study were the analysis of the gene that synthesizes the nucleocapsid proteins (ORF 7), as well as to determine the phylogenetic relationships with specific primers. Molecular characterization was performed and 24 positive isolates of the Betaarterivirus suid 2 species were identified. Ten isolates were genotyped, relating 8 of them to lineage 1: sublineage 1.5 with the representative variant NADC34 and 2 to lineage 5: sublineage 5.1 with the representative variant RespPRRS_MLV. In addition, the molecular homology test was performed with the DNAMAN program on the 10 isolates, obtaining 99% (8) and 87% (2) of similarity with the NADC34 variant, confirming the aforementioned. The phylogenetic relationship with the reported isolates and the results obtained indicate that at least 10 belonged to the Betaarterivirus suid 2 species through the nucleocapsid gene (ORF 7) with the research's own primers and the parental relationship of 8 isolates with lineage 1: sublineage 1.5 and 2 isolated with lineage 5: sublineage 5.1 of the species Betaarterivirus suid 2.Las especies Betaarterivirus suid 1 y Betaarterivirus suid 2 2 del virus del Síndrome Reproductivo y Respiratorio Porcino (PRRS) ocasionan brotes severos debido a recombinaciones genéticas con alto riesgo epidemiológico, lo cual requiere de una rápida identificación para una eficiente vigilancia epidemiológica molecular. Los objetivos del estudio fueron el análisis del gen que sintetiza las proteínas de la nucleocápside (ORF 7), así como determinar las relaciones filogenéticas con cebadores específicos. Se realizó la caracterización molecular y se identificaron 24 aislados positivos de la especie Betaarterivirus suid 2. Se genotipificaron 10 aislados relacionando 8 de ellos al linaje 1: sublinaje 1.5 con la variante representativa NADC34 y 2 al linaje 5: sublinaje 5.1 con la variante representativa RespPRRS_MLV. Además, se realizó el test de homología molecular con el programa DNAMAN a los 10 aislados, obteniéndose 99% (8) y 87% (2) de similitud con la variante NADC34 confirmando lo antes mencionado. La relación filogenética con los aislados reportados y los resultados obtenidos indican que al menos 10 pertenecieron a la especie Betaarterivirus suid 2 mediante el gen de la nucleocápside (ORF 7) con los cebadores propios de la investigación y se confirmó la relación parental de 8 aislados con el linaje 1: sublinaje 1.5 y 2 aislados con el linaje 5: sublinaje 5.1 de la especie Betaarterivirus suid 2

Neuron-Derived Semaphorin 3A Is an Early Inducer of Vascular Permeability in Diabetic Retinopathy via Neuropilin-1

SummaryThe 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 TgCre-Esr1/Nrp1flox/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

Semaphorin 3F forms an anti-angiogenic barrier in outer retina

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

Understanding Retinopathy of Prematurity: Update on Pathogenesis

International audienceRetinopathy 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

p75NTR and Its Ligand ProNGF Activate Paracrine Mechanisms Etiological to the Vascular, Inflammatory, and Neurodegenerative Pathologies of Diabetic Retinopathy

In many diseases, expression and ligand-dependent activity of the p75NTR 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 p75NTR may be implicated at each stage of DR pathology remain poorly understood. Using a streptozotocin mouse model of diabetic retinopathy, we report that p75NTR 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, p75NTR-dependent inflammation leads to ischemia and pathological angiogenesis through Semaphorin 3A. The acute use of antagonists of p75NTR 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.This work was supported by the Canadian Institutes of Health Research and the Foundation to Fight Blindness to H.U.S. and the Canadian Diabetes Association to P.S

Choroidal Involution Is a Key Component of Oxygen-Induced Retinopathy

International audiencePurpose: 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

The Succinate Receptor SUCNR1 Resides at the Endoplasmic Reticulum and Relocates to the Plasma Membrane in Hypoxic Conditions

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

Subcellular localization of coagulation factor II receptor-like 1 in neurons governs angiogenesis

International audienceNeurons 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