NOTCH1 signaling induces pathological vascular permeability in diabetic retinopathy.

Diabetic macular edema is a major complication of diabetes resulting in loss of central vision. Although heightened vessel leakiness has been linked to glial and neuronal-derived factors, relatively little is known on the mechanisms by which mature endothelial cells exit from a quiescent state and compromise barrier function. Here we report that endothelial NOTCH1 signaling in mature diabetic retinas contributes to increased vascular permeability. By providing both human and mouse data, we show that NOTCH1 ligands JAGGED1 and DELTA LIKE-4 are up-regulated secondary to hyperglycemia and activate both canonical and rapid noncanonical NOTCH1 pathways that ultimately disrupt endothelial adherens junctions in diabetic retinas by causing dissociation of vascular endothelial-cadherin from ß-catenin. We further demonstrate that neutralization of NOTCH1 ligands prevents diabetes-induced retinal edema. Collectively, these results identify a fundamental process in diabetes-mediated vascular permeability and provide translational rational for targeting the NOTCH pathway (primarily JAGGED1) in conditions characterized by compromised vascular barrier function.

Hexapeptides from human milk prevent the induction of oxidative stress from parenteral nutrition in the newborn guinea pig.

INTRODUCTION: In preterm neonates, peroxides contaminating total parenteral nutrition (TPN) contribute to oxidative stress, which is suspected to be a strong inducer of hepatic complications related to prematurity. Recently, others reported that hexapeptides derived from human milk (HM) exerted free radical-scavenging activities in vitro. Therefore, the aim of this study was to assess the capacity of these hexapeptides to limit the generation of peroxides in TPN and to prevent TPN-induced hepatic oxidative stress. METHODS: At 3 d of life, guinea pigs were infused, through a catheter in jugular vein, with TPN containing or not peptide-A (YGYTGA) or peptide-B (ISELGW). Peroxide concentrations were measured in TPN solutions, whereas glutathione, glutathionyl-1,4-dihydroxynonenal (GS-HNE) and mRNA levels of interleukin-1 (IL-1) and tumor necrosis factor-α (TNFα) were determined in liver after 4 d of infusion. RESULTS: The addition of peptide-A to TPN allowed a reduction in peroxide contamination by half. In vivo, peptide-A or peptide-B corrected the hepatic oxidative status induced by TPN. Indeed, both peptides lowered the hepatic redox potential of glutathione and the level of GS-HNE, a marker of lipid peroxidation. As compared with animals infused with TPN without peptide, the hepatic mRNA levels of IL-1 and TNFα were lower in animals infused with TPN containing peptide-A or peptide-B. DISCUSSION: These results suggest that the addition of YGYTGA or ISELGW to TPN will reduce oxidative stress in newborns. The reduction in mRNA of two proinflammatory cytokines could be important for the incidence of hepatic complications related to TPN.

miR-106b suppresses pathological retinal angiogenesis.

MicroRNAs are small non-coding RNAs that post-transcriptionally regulate gene expression. We recently demonstrated that levels of miR-106b were significantly decreased in the vitreous and plasma of patients with neovascular age-related macular degeneration (AMD). Here we show that expression of the miR-106b-25 cluster is negatively regulated by the unfolded protein response pathway of protein kinase RNA-like ER kinase (PERK) in a mouse model of neovascular AMD. A reduction in levels of miR-106b triggers vascular growth both in vivo and in vitro by inducing production of pro-angiogenic factors. We demonstrate that therapeutic delivery of miR-106b to the retina with lentiviral vectors protects against aberrant retinal angiogenesis in two distinct mouse models of pathological retinal neovascularization. Results from this study suggest that miRNAs such as miR-106b have the potential to be used as multitarget therapeutics for conditions characterized by pathological retinal angiogenesis.

Isolation and characterization of four antibacterial peptides from bovine hemoglobin.

Peptic digestion of bovine hemoglobin at low degree of hydrolysis yields several intermediate peptide fractions after separation by reversed phase HPLC exhibiting antibacterial activity against Micrococcus luteus A270, Listeria innocua, Escherichia coli, and Salmonella enteritidis. From these fractions, four new antibacterial peptides were isolated and analyzed by ESI-MS/MS. Three of these peptides correspond to fragments of the alpha-chain of bovine hemoglobin: alpha107-141, alpha137-141, and alpha133-141, and one peptide to the beta-chain: beta126-145. The minimum inhibitory concentrations (MIC) of these peptides towards the four strains and their hemolytic activity towards bovine erythrocytes were determined.

Gut microbiota influences pathological angiogenesis in obesity-driven choroidal neovascularization.

Age-related macular degeneration in its neovascular form (NV AMD) is the leading cause of vision loss among adults above the age of 60. Epidemiological data suggest that in men, overall abdominal obesity is the second most important environmental risk factor after smoking for progression to late-stage NV AMD To date, the mechanisms that underscore this observation remain ill-defined. Given the impact of high-fat diets on gut microbiota, we investigated whether commensal microbes influence the evolution of AMD Using mouse models of NV AMD, microbiotal transplants, and other paradigms that modify the gut microbiome, we uncoupled weight gain from confounding factors and demonstrate that high-fat diets exacerbate choroidal neovascularization (CNV) by altering gut microbiota. Gut dysbiosis leads to heightened intestinal permeability and chronic low-grade inflammation characteristic of inflammaging with elevated production of IL-6, IL-1ß, TNF-α, and VEGF-A that ultimately aggravate pathological angiogenesis.

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.

Neuropilin-1-Expressing Microglia Are Associated With Nascent Retinal Vasculature Yet Dispensable for Developmental Angiogenesis.

PURPOSE: Neuropilin-1 (NRP-1) is a transmembrane receptor that is critical for vascular development within the central nervous system (CNS). It binds and influences signaling of several key angiogenic factors, such as VEGF-165, semaphorin 3A, platelet derived growth factor, and more. Neuropilin-1 is expressed by neurons and endothelial cells as well as a subpopulation of proangiogenic macrophages/microglia that are thought to interact with endothelial tip cells to promote vascular anastomosis during brain vascularization. We previously demonstrated a significant role for NRP-1 in macrophage chemotaxis and showed that NRP-1-expressing microglia are major contributors to pathologic retinal angiogenesis. Given this influence on CNS angiogenesis, we now investigated the involvement of microglia-resident NRP-1 in developmental retinal vascularization. METHODS: We followed NRP-1 expressing microglia during retinal development. We used LysM-cre myeloid lineage-driver cre mice to reduce expression of NRP-1 in retinal myeloid-derived cells and performed a comprehensive morphometric analysis of retinal vasculature during development. RESULTS: We provide evidence that NRP-1+ microglia are present throughout the retina during vascular development with a preference for the non-vascularized retina. Using LysM-Cre/Nrp1(fl/fl) mice, we reduced NRP-1 expression by ~65% in retinal microglia and demonstrate that deficiency in NRP-1 in these microglia does not impair retinal angiogenesis. CONCLUSIONS: Our data draw a dichotomous role for NRP-1 in cells of myeloid lineage where it is dispensable for adequate retinal developmental vascularization yet obligate for pathologic retinal angiogenesis.

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.

Senescence-associated secretory phenotype contributes to pathological angiogenesis in retinopathy.

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.

Assessment of vascular regeneration in the CNS using the mouse retina.

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). It is increasingly being recognized that several neurodegenerative diseases such as Alzheimer's, multiple sclerosis, and amyotrophic lateral sclerosis present elements of vascular compromise. In addition, the most prominent causes of blindness in pediatric and working age populations (retinopathy of prematurity and diabetic retinopathy, respectively) are characterized by vascular degeneration and failure of physiological vascular regrowth. The aim of this technical paper is to provide a detailed protocol to study CNS vascular regeneration in the retina. The method can be employed to elucidate molecular mechanisms that lead to failure of vascular growth after ischemic injury. In addition, potential therapeutic modalities to accelerate and restore healthy vascular plexuses can be explored. Findings obtained using the described approach may provide therapeutic avenues for ischemic retinopathies such as that of diabetes or prematurity and possibly benefit other vascular disorders of the CNS.

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 Alzheimer's 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.

Systemic inflammation perturbs developmental retinal angiogenesis and neuroretinal function.

PURPOSE: Perinatal inflammatory stress in preterm babies is associated with increased rates of severe retinopathy of prematurity (ROP) and adverse neurological dysfunction. In this study, we set out to determine the consequences of severe systemic inflammatory stress on developmental retinal vascularization and evaluate the subsequent outcome on retinal function in later life. METHODS: Systemic inflammatory stress was induced in C57BL/6J mouse pups by an intraperitoneal injection of lipopolysaccharide (LPS; 1 mg/kg) at postnatal day 4. In response to LPS, retinal inflammation was confirmed by quantitative RT-PCR analysis of diverse inflammatory markers. A detailed and systematic analysis of retinal microglial infiltration, retinal vascular morphology, density, and growth rate was performed at key time points throughout retinal vascularization. Retinal function in adult life was assessed by using electroretinography at 6 weeks postinjection. RESULTS: As early as 48 hours after intraperitoneal administration of LPS, a significant increase in retinal vascular density was noted throughout the retina. A pronounced increase in the number of activated microglial cell was observed in the retinal ganglion cell layer and in the outer plexiform layer just prior to their vascularization; direct physical contact between activated microglia and sprouting vessels suggested that microglia partake in promoting the aberrant retinal vascularization. With maturity, animals subjected to perinatal inflammatory stress displayed depleted retinal vascular beds and had significantly decreased retinal function as determined by electroretinography. CONCLUSIONS: Our data reveal that early severe postnatal inflammatory stress leads to abnormal retinal vascular development and increased vessel anastomosis and, ultimately, permanently compromises retinal function. The aberrant and initially exaggerated retinal vascularization observed is associated with microglial activation, providing a cellular mechanism by which perinatal sepsis predisposes to ROP.

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.

The mode of administration of total parenteral nutrition and nature of lipid content influence the generation of peroxides and aldehydes.

BACKGROUND & AIMS: The absence of light protection of neonatal total parenteral nutrition (PN) contributes to the generation of 4-hydroxynonenal and peroxides. 4-Hydroxynonenal is suspected to be involved in PN-related liver complications. AIMS: To find a practical modality to reduce 4-hydroxynonenal in PN and assess in vivo the impact of PN containing low 4-hydroxynonenal concentration. METHODS: Six modalities of delivering PN were compared for the in vitro generation of peroxides and 4-hydroxynonenal: 1) MV-AA-L: light-protected (-L) solution containing multivitamin (MV) mixed with amino acids + dextrose (AA); 2) MV-AA+L: MV-AA without photo-protection (+L); 3) MV-LIP+L: MV mixed with lipid emulsion (LIP). LIP was a) Intralipid20%(®) or b) Omegaven(®). Hepatic markers of oxidative stress (glutathione, F(2α)-isoprostanes, GS-HNE) and inflammation (mRNA of TNF-α and IL-1) were measured in newborn guinea pigs infused during 4-days with MV-AA+L compounded with Intralipid20%(®) or Omegaven(®). RESULTS: Hydroperoxides and 4-hydroxynonenal were the lowest in MV-AA-L and the highest in MV-LIP+L. MV-AA+L with Omegaven(®) was associated with the lowest levels of markers of oxidative stress and inflammation. CONCLUSION: Compared to Intralipid20%(®), Omegaven(®) reduces oxidative stress associated with PN and prevents liver inflammation. These findings offer an alternative strategy to light protection of PN, which in the clinical setting is a cumbersome modality.