Novel Function of Nogo-A as Negative Regulator of Endothelial Progenitor Cell Angiogenic Activity: Impact in Oxygen-Induced Retinopathy.

Endothelial Progenitor Cells (EPCs) can actively participate in revascularization in oxygen-induced retinopathy (OIR). Yet the mechanisms responsible for their dysfunction is unclear. Nogo-A, whose function is traditionally related to the inhibition of neurite function in the central nervous system, has recently been documented to display anti-angiogenic pro-repellent properties. Based on the significant impact of EPCs in retinal vascularization, we surmised that Nogo-A affects EPC function, and proceeded to investigate the role of Nogo-A on EPC function in OIR. The expression of Nogo-A and its specific receptor NgR1 was significantly increased in isolated EPCs exposed to hyperoxia, as well as in EPCs isolated from rats subjected to OIR compared with respective controls (EPCs exposed to normoxia). EPCs exposed to hyperoxia displayed reduced migratory and tubulogenic activity, associated with the suppressed expression of prominent EPC-recruitment factors SDF-1/CXCR4. The inhibition of Nogo-A (using a Nogo-66 neutralizing antagonist peptide) or siRNA-NGR1 in hyperoxia-exposed EPCs restored SDF-1/CXCR4 expression and, in turn, rescued the curtailed neovascular functions of EPCs in hyperoxia. The in vivo intraperitoneal injection of engineered EPCs (Nogo-A-inhibited or NgR1-suppressed) in OIR rats at P5 (prior to exposure to hyperoxia) prevented retinal and choroidal vaso-obliteration upon localization adjacent to vasculature; coherently, the inhibition of Nogo-A/NgR1 in EPCs enhanced the expression of key angiogenic factors VEGF, SDF-1, PDGF, and EPO in retina; CXCR4 knock-down abrogated suppressed NgR1 pro-angiogenic effects. The findings revealed that hyperoxia-induced EPC malfunction is mediated to a significant extent by Nogo-A/NgR1 signaling via CXCR4 suppression; the inhibition of Nogo-A in EPCs restores specific angiogenic growth factors in retina and the ensuing vascularization of the retina in an OIR model.

Tyrosine-Protein Phosphatase Non-receptor Type 9 (PTPN9) Negatively Regulates the Paracrine Vasoprotective Activity of Bone-Marrow Derived Pro-angiogenic Cells: Impact on Vascular Degeneration in Oxygen-Induced Retinopathy.

BACKGROUND AND AIM: Insufficient post-ischemic neovascularization is an initial key step in the pathogenesis of Oxygen-Induced Retinopathy (OIR). During neovascularization, pro-angiogenic cells (PACs) are mobilized from the bone marrow and integrate into ischemic tissues to promote angiogenesis. However, the modulation of PAC paracrine activity during OIR and the specific mechanisms involved remain to be explored. Because Tyrosine-protein phosphatase non-receptor type 9 (PTPN9) is reported to be a negative regulator of stem cell differentiation and angiogenesis signaling, we investigated its effect on PAC activity in the context of OIR. METHODS AND RESULTS: In a rat model of OIR, higher levels of PTPN9 in the retina and in bone marrow derived PACs are associated with retinal avascular areas, lower levels of the mobilization factor SDF-1 and decreased number of CD34+/CD117+/CD133+ PACs. PACs exposed ex vivo to hyperoxia display increased PTPN9 expression, which is associated with impaired ability of PAC secretome to promote angiogenesis ex vivo (choroidal vascular sprouting) and in vitro (endothelial cell tubule formation) compared to the secretome of PACs maintained in normoxia. Suppression of PTPN9 (using siRNA) increases VEGF and SDF-1 expression to normalize PAC secretome during hyperoxia, leading to restored angiogenic ability of PAC secretome. Moreover, endothelial cells exposed to the secretome of siPTPN9-treated PACs expressed increased levels of activated form of VEGF receptor 2 (VEGFR2). In the rat model of OIR, intravitreal injection of secretome from siPTPN9-treated PACs significantly reduced retinal vaso-obliteration; this was associated with higher retinal levels of VEGF/SDF-1, and increased recruitment of PACs (CD34+ cells) to the retinal and choroidal vessels. CONCLUSION: Our results suggest that hyperoxia alters the paracrine proangiogenic activity of BM-PACs by inducing PTPN9, which can contribute to impair post-ischemic revascularization in the context of OIR. Targeting PTPN9 restores PAC angiogenic properties, and provide a new target for vessel integrity in ischemic retinopathies.

Nutraceutical Targeting of Inflammation-Modulating microRNAs in Severe Forms of COVID-19: A Novel Approach to Prevent the Cytokine Storm.

The coronavirus disease 2019 (COVID-19) pandemic has become the number one health problem worldwide. As of August 2020, it has affected more than 18 million humans and caused over 700,000 deaths worldwide. COVID-19 is an infectious disease that can lead to severe acute respiratory syndrome. Under certain circumstances, the viral infection leads to excessive and uncontrolled inflammatory response, which is associated with the massive release of inflammatory cytokines in pulmonary alveolar structures. This phenomenon has been referred to as the "cytokine storm," and it is closely linked to lung injury, acute respiratory syndrome and mortality. Unfortunately, there is currently no vaccine available to prevent the infection, and no effective treatment is available to reduce the mortality associated with the severe form of the disease. The cytokine storm associate with COVID-19 shows similarities with those observed in other pathologies such as sepsis, acute respiratory distress syndrome, acute lung injury and other viral infection including severe cases of influenza. However, the specific mechanisms that cause and modulate the cytokine storm in the different conditions remain to be determined. micro-RNAs are important regulators of gene expression, including key inflammatory cytokines involved in the massive recruitment of immune cells to the lungs such as IL1ß, IL6, and TNFα. In recent years, it has been shown that nutraceutical agents can modulate the expression of miRs involved in the regulation of cytokines in various inflammatory diseases. Here we review the potential role of inflammatory-regulating-miRs in the cytokine storm associated with COVID-19, and propose that nutraceutical agents may represent a supportive therapeutic approach to modulate dysregulated miRs in this condition, providing benefits in severe respiratory diseases.

Novel Anti-Interleukin-1ß Therapy Preserves Retinal Integrity: A Longitudinal Investigation Using OCT Imaging and Automated Retinal Segmentation in Small Rodents.

Retinopathy of prematurity (ROP) is the leading cause of blindness in neonates. Inflammation, in particular interleukin-1ß (IL-1ß), is increased in early stages of the disorder, and contributes to inner and outer retinal vasoobliteration in the oxygen-induced retinopathy (OIR) model of ROP. A small peptide antagonist of IL-1 receptor, composed of the amino acid sequence, rytvela, has been shown to exert beneficial anti-inflammatory effects without compromising immunovigilance-related NF-κB in reproductive tissues. We conducted a longitudinal study to determine the efficacy of "rytvela" in preserving the integrity of the retina in OIR model, using optical coherence tomography (OCT) which provides high-resolution cross-sectional imaging of ocular structures in vivo. Sprague-Dawley rats subjected to OIR and treated or not with "rytvela" were compared to IL-1 receptor antagonist (Kineret). OCT imaging and custom automated segmentation algorithm used to measure retinal thickness (RT) were obtained at P14 and P30; gold-standard immunohistochemistry (IHC) was used to confirm retinal anatomical changes. OCT revealed significant retinal thinning in untreated animals by P30, confirmed by IHC; these changes were coherently associated with increased apoptosis. Both rytvela and Kineret subsided apoptosis and preserved RT. As anticipated, Kineret diminished both SAPK/JNK and NF-κB axes, whereas rytvela selectively abated the former which resulted in preserved monocyte phagocytic function. Altogether, OCT imaging with automated segmentation is a reliable non-invasive approach to study longitudinally retinal pathology in small animal models of retinopathy.

MicroRNA-96 Promotes Vascular Repair in Oxygen-Induced Retinopathy-A Novel Uncovered Vasoprotective Function.

BACKGROUND AND AIMS: Vascular degeneration is a hallmark in the pathogenesis of oxygen-induced retinopathy (OIR). Dysregulation of microRNAs (miRNAs), key regulators of genes expressions, has been implicated in the regulation of ocular angiogenesis. However, miRNAs specific functions in impaired vascular development during OIR are poorly understood. Herein, we identified miR-96 as one of the most highly expressed miRNAs in the retina and choroid during vascular development and investigated the potential role of miR-96 on microvascular degeneration in a rat OIR model. METHODS AND RESULTS: Next generation sequencing (NGS) and qRT-PCR analysis showed that miR-96 maintain high levels of expression during ocular vascular development. Nevertheless, miR-96 was significantly downregulated in the retina and choroid of OIR rats (80% O2 from P5 to P10) during the phase of microvascular degeneration. Similarly, human retinal microvascular endothelial cells (HRMEC) subjected to hyperoxia (80% O2) showed a significant downregulation of miR-96 evaluated by qPCR. Interestingly, HRMEC supplemented with miR-96 regulated positively the expression of several key angiogenic factors including VEGF and ANG-2. To explore the angiogenic activity of miR-96 on HRMEC, we performed a gain/loss of function study. In a similar way to hyperoxia exposure, we observed a robust angiogenic impairment (tubulogenesis and migration) on HRMEC transfected with an antagomiR-96. Conversely, overexpression of miR-96 stimulated the angiogenic activity of HRMEC and protected against hyperoxia-induced endothelial dysfunction. Finally, we evaluated the potential vasoprotective function of miR-96 in OIR animals. Rat pups intravitreally supplemented with miR-96 mimic (1 mg/kg) displayed a significant preservation of retinal/choroidal microvessels at P10 compared to controls. This result was consistent with the maintenance of physiologic levels of VEGF and ANG-2 in the OIR retina. CONCLUSION: This study demonstrates that miR-96 regulates the expression of angiogenic factors (VEGF/ANG-2) associated to the maintenance of retinal and choroidal microvasculature during physiological and pathological conditions. Intravitreal supplementation of miR-96 mimic could constitute a novel therapeutic strategy to improve vascular repair in OIR and other ischemic retinopathies.

The Inability of the Choroid to Revascularize in Oxygen-Induced Retinopathy Results from Increased p53/miR-Let-7b Activity.

Retinopathy of prematurity (ROP) is characterized by an initial retinal avascularization, followed by pathologic neovascularization. Recently, choroidal thinning has also been detected in children formerly diagnosed with ROP; a similar sustained choroidal thinning is observed in ROP models. But the mechanism underlying the lack of choroidal revascularization remains unclear and was investigated in an oxygen-induced retinopathy (OIR) model. In OIR, evidence of senescence was detected, preceded by oxidative stress in the choroid and the retinal pigment epithelium. This was associated with a global reduction of proangiogenic factors, including insulin-like growth factor 1 receptor (Igf1R). Coincidentally, tumor suppressor p53 was highly expressed in the OIR retinae. Curtailing p53 activity resulted in reversal of senescence, normalization of Igf1r expression, and preservation of choroidal integrity. OIR-induced down-regulation of Igf1r was mediated at least partly by miR-let-7b as i) let-7b expression was augmented throughout and beyond the period of oxygen exposure, ii) let-7b directly targeted Igf1r mRNA, and iii) p53 knock-down blunted let-7b expression, restored Igf1r expression, and elicited choroidal revascularization. Finally, restoration of Igf1r expression rescued choroid thickness. Altogether, this study uncovers a significant mechanism for defective choroidal revascularization in OIR, revealing a new role for p53/let-7b/IGF-1R axis in the retina. Future investigations on this (and connected) pathway could further our understanding of other degenerative choroidopathies, such as geographic atrophy.

Visual Maturation at Term Equivalent Age in Very Premature Infants According to Factors Influencing Its Development.

Introduction: Visual impairment is a concern in premature infants as perinatal factors may alter maturation during visual development. This observational study aimed at evaluating visual maturation at term equivalent age and factors associated with impaired visual maturation. Methods: Infants born before 32 weeks' gestation were evaluated with routine brain MRI, visual acuity, refraction, fundus, and clinical eye examination. Environmental factors were collected from infant's files. Results: Fifty-four infants (29.5 ± 1.7 weeks' gestation, birth weight 1194 ± 288 g) were studied at term equivalent age. Visual acuity was higher in premature infants at term equivalent age than in a reference publication with the same method in term newborns at birth (1.54 ± 0.67 vs. 0.99 ± 0.40 cycles/degree, p = 0.008). In multivariate analysis, abnormal brain MRI was the only factor associated with visual acuity (r 2= 0.203; p = 0.026). Incomplete retinal vascularization was observed in 29/53 of infants at term equivalent age and associated with MRI abnormalities of the posterior fossa (p = 0.027) and larger refractive sphere difference between both eyes (1.2 ± 0.8 vs. 0.6 ± 0.4 diopters; p = 0.0005). Retinopathy of prematurity was associated with indices of smaller cerebral volume (p = 0.035). Conclusion: Higher visual acuity in premature infants at term equivalent age than in term newborns at birth may be related to longer visual experience from birth. Lower visual acuity was correlated with abnormal MRI in preterm infants at term equivalent age.

Mobility may impact attention abilities in healthy term or prematurely born children at 7-years of age: protocol for an intervention controlled trial.

BACKGROUND: Seven years of age is a milestone for learning basic knowledge that is strongly related to attention abilities such as Alerting, Orienting, and Inhibition function, allowing for appropriate adaptation to primary school. These attention abilities are also influenced by gestational age at birth in a complex manner, indicating an area of weakness in prematurely born children. Furthermore, recent studies suggest that allowing children to have freedom of movement during learning may improve their attention level and school performance. The purpose of the present study is to determine the influence of mobility on the attentional components that may impact learning abilities in children aged 7-years who were born at term and prematurely. METHODS: This prospective, randomized, controlled trial will focus on psychometric testing of attentional abilities assessed with the Attention Network Test for Child (Child ANT) and involves a mixed measurement design. Forty-eight children aged 7-years, half of whom were premature at birth and in their expected grade without learning difficulties will be included after parental consent. They will be equipped with a head-mounted display in which the Child ANT will be presented. The association of different flankers and pre-cues will allow the measurement of the development level of Alerting, Orienting, and Inhibition function. The task will be composed of one experimental block of trials randomly performed per posture: seated, standing, or free. DISCUSSION: This study will assess the contribution of mobility in specific attentional contexts that are usually present during fundamental learning in children. New pedagogical formats of teaching could consider these findings, and new pedagogical tools enabling free spontaneous child mobility might be designed. Moreover, a small percentage of children integrating into the educational system are born prematurely. These children, often considered immature and hyperactive, could benefit from educational innovations that enhance their attention abilities, thereby improving their adaptation to primary school. TRIAL REGISTRATION: This trial is registered at ClinicalTrials.gov ( NCT03125447 ).

Neonatal diabetes mellitus: improved screening and early management of an underestimated disease.

Neonatal diabetes mellitus is underdiagnosed in the neonatal period because of the metabolic adaptation capacities of the newborns. However, it is associated with increased risk of short- and long-term morbidity; when transient it may recurs in adulthood. It is important to improve screening and early management with appropriate guidelines.

Determinants of Indices of Cerebral Volume in Former Very Premature Infants at Term Equivalent Age.

Conventional magnetic resonance imaging (MRI) at term equivalent age (TEA) is suggested to be a reliable tool to predict the outcome of very premature infants. The objective of this study was to determine simple reproducible MRI indices, in premature infants and to analyze their neonatal determinants at TEA. A cohort of infants born before 32 weeks gestational age (GA) underwent a MRI at TEA in our center. Two axial images (T2 weighted), were chosen to realize nine measures. We defined 4 linear indices (MAfhlv: thickness of lateral ventricle; CSI: cortex-skull index; VCI: ventricular-cortex index; BOI: bi occipital index) and 1 surface index (VS.A: volume slice area). Perinatal data were recorded. Sixty-nine infants had a GA (median (interquartile range)) of 30.0 weeks GA (27.0; 30.0) and a birth weight of 1240 grams (986; 1477). MRI was done at 41.0 (40.0; 42.0) weeks post menstrual age (PMA). The inter-investigator reproducibility was good. Twenty one MRI (30.5%) were quoted abnormal. We observed an association with retinopathy of prematurity (OR [95CI] = 4.205 [1.231-14.368]; p = 0.017), surgery for patent ductus arteriosus (OR = 4.688 [1.01-21.89]; p = 0.036), early onset infection (OR = 4.688 [1.004-21.889]; p = 0.036) and neonatal treatment by cefotaxime (OR = 3.222 [1.093-9.497]; p = 0.03). There was a difference for VCI between normal and abnormal MRI (0.412 (0.388; 0.429) vs. 0.432 (0.418; 0.449); p = 0,019); BOI was higher when fossa posterior lesions were observed; VS.A seems to be the best surrogate for cerebral volume, 80% of VS.As' variance being explained by a multiple linear regression model including 7 variables (head circumference at birth and at TEA, PMA, dopamine, ibuprofen treatment, blood and platelets transfusions). These indices, easily and rapidly achievable, seem to be useful but need to be validated in a large population to allow generalization for diagnosis and follow-up of former premature infants.