Flavio Rezende

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

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.

Retinal lipid and glucose metabolism dictates angiogenesis through the lipid sensor Ffar1

Tissues with high metabolic rates often use lipids, as well as glucose, for energy, conferring a survival advantage during feast and famine. Current dogma suggests that high-energy–consuming photoreceptors depend on glucose. Here we show that the retina also uses fatty acid β-oxidation for energy. Moreover, we identify a lipid sensor, free fatty acid receptor 1 (Ffar1), that curbs glucose uptake when fatty acids are available. Very-low-density lipoprotein receptor (Vldlr), which is present in photoreceptors and is expressed in other tissues with a high metabolic rate, facilitates the uptake of triglyceride-derived fatty acid. In the retinas of Vldlr−/− mice with low fatty acid uptake but high circulating lipid levels, we found that Ffar1 suppresses expression of the glucose transporter Glut1. Impaired glucose entry into photoreceptors results in a dual (lipid and glucose) fuel shortage and a reduction in the levels of the Krebs cycle intermediate α-ketoglutarate (α-KG). Low α-KG levels promotes stabilization of hypoxia-induced factor 1a (Hif1a) and secretion of vascular endothelial growth factor A (Vegfa) by starved Vldlr−/− photoreceptors, leading to neovascularization. The aberrant vessels in the Vldlr−/− retinas, which invade normally avascular photoreceptors, are reminiscent of the vascular defects in retinal angiomatous proliferation, a subset of neovascular age-related macular degeneration (AMD), which is associated with high vitreous VEGFA levels in humans. Dysregulated lipid and glucose photoreceptor energy metabolism may therefore be a driving force in macular telangiectasia, neovascular AMD and other retinal diseases.

Neuronal ER stress impedes myeloid-cell-induced vascular regeneration through IRE1α degradation of netrin-1.

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.

Neuropilin-1 mediates myeloid cell chemoattraction and influences retinal neuroimmune crosstalk

Immunological activity in the CNS is largely dependent on an innate immune response and is heightened in diseases, such as diabetic retinopathy, multiple sclerosis, amyotrophic lateral sclerosis, and Alzheimers disease. The molecular dynamics governing immune cell recruitment to sites of injury and disease in the CNS during sterile inflammation remain poorly defined. Here, we identified a subset of mononuclear phagocytes (MPs) that responds to local chemotactic cues that are conserved among central neurons, vessels, and immune cells. Patients suffering from late-stage proliferative diabetic retinopathy (PDR) had elevated vitreous semaphorin 3A (SEMA3A). Using a murine model, we found that SEMA3A acts as a potent attractant for neuropilin-1-positive (NRP-1-positive) MPs. These proangiogenic MPs were selectively recruited to sites of pathological neovascularization in response to locally produced SEMA3A as well as VEGF. NRP-1-positive MPs were essential for disease progression, as NRP-1-deficient MPs failed to enter the retina in a murine model of oxygen-induced retinopathy (OIR), a proxy for PDR. OIR mice with NRP-1-deficient MPs exhibited decreased vascular degeneration and diminished pathological preretinal neovascularization. Intravitreal administration of a NRP-1-derived trap effectively mimicked the therapeutic benefits observed in mice lacking NRP-1-expressing MPs. Our findings indicate that NRP-1 is an obligate receptor for MP chemotaxis, bridging neural ischemia to an innate immune response in neovascular retinal disease.

Senescence-associated secretory phenotype contributes to pathological angiogenesis in retinopathy

Senescent cells encourage abnormal blood vessels and retinal disease. Old but deadly Diabetics often lose their sight, a result of poor blood supply to the retina. Oubaha and colleagues investigated how this oxygen deprivation leads to disease. They found that retinal cells do not simply die when oxygen-starved, rather they become senescent—a state in which the cells secrete a cocktail of undesirable molecules, in mice and humans. These cytokines drive more senescence, encourage abnormal blood vessel formation, and block healthy regeneration. Treatment of mice with a diabetes drug or a senescence inhibitor reduced retinal disease, suggesting that interrupting this process could be therapeutically beneficial. Pathological angiogenesis is the hallmark of diseases such as cancer and retinopathies. Although tissue hypoxia and inflammation are recognized as central drivers of vessel growth, relatively little is known about the process that bridges the two. In a mouse model of ischemic retinopathy, we found that hypoxic regions of the retina showed only modest rates of apoptosis despite severely compromised metabolic supply. Using transcriptomic analysis and inducible loss-of-function genetics, we demonstrated that ischemic retinal cells instead engage the endoplasmic reticulum stress inositol-requiring enzyme 1α (IRE1α) pathway that, through its endoribonuclease activity, induces a state of senescence in which cells adopt a senescence-associated secretory phenotype (SASP). We also detected SASP-associated cytokines (plasminogen activator inhibitor 1, interleukin-6, interleukin-8, and vascular endothelial growth factor) in the vitreous humor of patients suffering from proliferative diabetic retinopathy. Therapeutic inhibition of the SASP through intravitreal delivery of metformin or interference with effectors of senescence (semaphorin 3A or IRE1α) in mice reduced destructive retinal neovascularization in vivo. We conclude that the SASP contributes to pathological vessel growth, with ischemic retinal cells becoming prematurely senescent and secreting inflammatory cytokines that drive paracrine senescence, exacerbate destructive angiogenesis, and hinder reparative vascular regeneration. Reversal of this process may be therapeutically beneficial.

MicroRNA signatures in vitreous humour and plasma of patients with exudative AMD.

Age-related macular degeneration (AMD) is a leading cause of blindness worldwide affecting individuals over the age of 50. The neovascular form (NV AMD) is characterized by choroidal neovascularization (CNV) and responsible for the majority of central vision impairment. Using non-biased microRNA arrays and individual TaqMan qPCRs, we profiled miRNAs in the vitreous humour and plasma of patients with NV AMD. We identified a disease-associated increase in miR-146a and a decrease in miR-106b and miR-152 in the vitreous humour which was reproducible in plasma. Moreover, miR-146a/miR-106b ratios discriminated patients with NV AMD with an area under the Receiver Operating Characteristic curve (ROC AUC) of 0,977 in vitreous humour and 0,915 in plasma suggesting potential for a blood-based diagnostic. Furthermore, using the AMD Gene Consortium (AGC) we mapped a NV AMD-associated SNP (rs1063320) in a binding site for miR-152-3p in the HLA-G gene. The relationship between our detected miRNAs and NV AMD related genes was also investigated using gene sets derived from the Ingenuity Pathway Analysis (IPA). To our knowledge, our study is the first to correlate vitreal and plasma miRNA signatures with NV AMD, highlighting potential future worth as biomarkers and providing insight on NV AMD pathogenesis.

Omega-3 supplementation combined with anti-vascular endothelial growth factor lowers vitreal levels of vascular endothelial growth factor in wet age-related macular degeneration.

PURPOSE To determine the influence of omega-3 supplementation on vitreous vascular endothelial growth factor A (VEGF-A) levels in patients with exudative age-related macular degeneration (wet AMD) receiving intravitreal anti-VEGF treatment. DESIGN Prospective, randomized, open-label, single-center, clinical trial, consecutive interventional case series. METHODS The study included 3 cohorts with wet AMD and a control group with epiretinal membrane or macular hole. Twenty wet AMD patients being treated with anti-VEGF were randomized to daily supplementation of antioxidants, zinc, and carotenoids with omega-3 fatty acids (docosahexaenoic acid and eicosapentaenoic acid; group 1, n = 10) or without omega-3 fatty acids (group 2, n = 10). They were compared with an anti-VEGF treatment-naïve wet AMD group (group 3, n = 10) and an epiretinal membrane or macular hole group (group 4, n = 10). Primary outcome was vitreal VEGF-A levels (at the time of anti-VEGF injection). Secondary outcomes were plasma VEGF-A and central foveal thickness. Patients with new submacular hemorrhage or any other treatment within 3 months were excluded. Final analyses included 9, 6, 7, and 8 patients in groups 1 through 4, respectively. RESULTS Patients receiving omega-3s (group 1) had significantly lower levels of vitreal VEGF-A (141.11 ± 61.89 pg/mL) when compared with group 2 (626.09 ± 279.27 pg/mL; P = .036) and group 3 (735.48 ± 216.43 pg/mL; P = .013), but similar levels to group 4 (235.81 ± 33.99 pg/mL; P=.215). All groups showed similar values for plasma VEGF-A and central foveal thickness measurements. CONCLUSIONS This study demonstrated that omega-3 supplementation combined with anti-VEGF treatment is associated with decreased vitreal VEGF-A levels in wet AMD patients.

Truncated netrin-1 contributes to pathological vascular permeability in diabetic retinopathy

Diabetic retinopathy (DR) is a major complication of diabetes and a leading cause of blindness in the working-age population. Impaired blood-retinal barrier function leads to macular edema that is closely associated with the deterioration of central vision. We previously demonstrated that the neuronal guidance cue netrin-1 activates a program of reparative angiogenesis in microglia within the ischemic retina. Here, we provide evidence in both vitreous humor of diabetic patients and in retina of a murine model of diabetes that netrin-1 is metabolized into a bioactive fragment corresponding to domains VI and V of the full-length molecule. In contrast to the protective effects of full-length netrin-1 on retinal microvasculature, the VI-V fragment promoted vascular permeability through the uncoordinated 5B (UNC5B) receptor. The collagenase matrix metalloprotease 9 (MMP-9), which is increased in patients with diabetic macular edema, was capable of cleaving netrin-1 into the VI-V fragment. Thus, MMP-9 may release netrin-1 fragments from the extracellular matrix and facilitate diffusion. Nonspecific inhibition of collagenases or selective inhibition of MMP-9 decreased pathological vascular permeability in a murine model of diabetic retinal edema. This study reveals that netrin-1 degradation products are capable of modulating vascular permeability, suggesting that these fragments are of potential therapeutic interest for the treatment of DR.

Corrigendum: Retinal lipid and glucose metabolism dictates angiogenesis through the lipid sensor Ffar1

Jean-Sébastien Joyal, Ye Sun, Marin L Gantner, Zhuo Shao, Lucy P Evans, Nicholas Saba, Thomas Fredrick, Samuel Burnim, Jin Sung Kim, Gauri Patel, Aimee M Juan, Christian G Hurst, Colman J Hatton, Zhenghao Cui, Kerry A Pierce, Patrick Bherer, Edith Aguilar, Michael B Powner, Kristis Vevis, Michel Boisvert, Zhongjie Fu, Emile Levy, Marcus Fruttiger, Alan Packard, Flavio A Rezende, Bruno Maranda, Przemyslaw Sapieha, Jing Chen, Martin Friedlander, Clary B Clish & Lois E H Smith Nat. Med.; doi:10.1038/nm.4059; corrected 24 March 2016

In Vivo Comparison of 23- and 25-Gauge Sutureless Vitrectomy Incision Architecture Using Spectral Domain Optical Coherence Tomography

Purpose. To investigate the in vivo incision architecture using spectral domain optical coherence tomography (SD-OCT) in 23-gauge and 25-gauge transconjunctival sutureless pars plana vitrectomy (TSPPV). Methods. A prospective observational study of 22 eyes of 22 patients that underwent three-port 25-gauge (10 eyes) or 23-gauge (12 eyes) TSPPV was performed. The three sclerotomies sites in each eye were analyzed by Corneal Adapter Model (CAM) RTVue SD-OCT (Optovue Inc., Fremont, CA, USA) with wound cross-section images (longitudinal and transversal) on days 1, 7, and 30 postoperatively. Transversal and longitudinal length, location, angle between the conjunctival surface tangent and the incision plane, and architecture deformations were evaluated. Results. All patients (22 eyes) completed the study and surgeries lasted less than 60 minutes. All wounds were obliquely performed, 23-gauge mean angle was 23 ± 5°, and 25-gauge angule was 21 ± 4°. Twenty-three-gauge sclerotomy transversal mean length was 1122 ± 242 μm and 25-gauge transversal sclerotomy mean length was 977 ± 174 μm; 23-gauge longitudinal mean length was 363 ± 42 μm and 25-gauge longitudinal sclerotomy mean length was 234 ±19 μm; 23-gauge open wound thickness mean was 61 ± 28 μm and 25-gauge open wound thickness mean was 22 ± 6 μm. All results were statistically significant (P < 0.05). No vitreous incarceration or silicone oil residue was observed in incision sites with both gauges. Conclusions. The 23-gauge and 25-gauge architectural wound constructions were well visualized using CAM SD-OCT. Statistical differences between the two gauges were observed throughout the study period.