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'Wet' age-related macular degeneration (AMD) is characterized by pathologic choroidal neovascularization (CNV) that destroys central vision. Abundant evidence points to inflammation and immune cell dysfunction in the progression of CNV in AMD. Mast cells are resident immune cells that control the inflammatory response. Mast cells accumulate and degranulate in the choroid of patients with AMD, suggesting they play a role in CNV. Activated mast cells secrete various biologically active mediators, including inflammatory cytokines and proteolytic enzymes such as tryptase. We investigated the role of mast cells in AMD using a model of CNV. Conditioned media from activated mast cells exerts proangiogenic effects on choroidal endothelial cells and choroidal explants. Laser-induced CNV in vivo was markedly attenuated in mice genetically depleted of mast cells (Kit W-sh/W-sh ) and in wild-type mice treated with mast cell stabilizer, ketotifen fumarate. Tryptase was found to elicit pronounced choroidal endothelial cell sprouting, migration and tubulogenesis; while tryptase inhibition diminished CNV. Transcriptomic analysis of laser-treated RPE/choroid complex revealed collagen catabolism and extracellular matrix (ECM) reorganization as significant events correlated in clusters of mast cell activation. Consistent with these analyses, compared to wildtype mice choroids of laser-treated mast cell-deficient mice displayed less ECM remodelling evaluated using collagen hybridizing peptide tissue binding. Findings herein provide strong support for mast cells as key players in the progression of pathologic choroidal angiogenesis and as potential therapeutic targets to prevent pathological neovascularization in 'wet' AMD., (© 2024. The Author(s).)

The subretina, composed of the choroid and the retinal pigment epithelium (RPE), bears a critical role in proper vision. In addition to phagocytosis of photoreceptor debris, the RPE shuttles oxygen and nutrients to the neuroretina. For their own energy production, RPE cells mainly rely on lactate, a major by-product of glycolysis. Lactate, in turn, is believed to convey most of its biological effects via the hydroxycarboxylic acid receptor 1 (HCAR1). Here, the lactate-specific receptor, HCAR1, is found to be exclusively expressed in the RPE cells within the subretina, and Hcar1 -/- mice exhibit a substantially thinner choroidal vasculature during development. Notably, the angiogenic properties of lactate on the choroid are impacted by the absence of Hcar1. HCAR1-deficient mice exhibit elevated endoplasmic reticulum stress along with eukaryotic initiation factor 2α phosphorylation, a significant decrease in the global protein translation rate, and a lower proliferation rate of choroidal vasculature. Strikingly, inhibition of the integrated stress response using an inhibitor that reverses the effect of eukaryotic initiation factor 2α phosphorylation restores protein translation and rescues choroidal thinning. These results provide evidence that lactate signalling via HCAR1 is important for choroidal development/angiogenesis and highlight the importance of this receptor in establishing mature vision., Competing Interests: Disclosure Statement None declared., (Copyright © 2024 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Preterm birth is a major public health concern, requiring a deeper understanding of its underlying inflammatory mechanisms and to develop effective therapeutic strategies. This review explores the complex interaction between inflammation and preterm labor, highlighting the pivotal role of the dysregulation of inflammation in triggering premature delivery. The immunological environment of pregnancy, characterized by a fragile balance of immune tolerance and resistance, is disrupted in preterm labor, leading to a pathological inflammatory response. Feto-maternal infections, among other pro-inflammatory stimuli, trigger the activation of toll-like receptors and the production of pro-inflammatory mediators, promoting uterine contractility and cervical ripening. Emerging anti-inflammatory therapeutics offer promising approaches for the prevention of preterm birth by targeting key inflammatory pathways. From TLR-4 antagonists to chemokine and interleukin receptor antagonists, these interventions aim to modulate the inflammatory environment and prevent adverse pregnancy outcomes. In conclusion, a comprehensive understanding of the inflammatory mechanisms leading to preterm labor is crucial for the development of targeted interventions in hope of reducing the incidence of preterm birth and improving neonatal health outcomes., Competing Interests: Declaration of Competing Interest S.C. holds a patent on composition of matter for the use of 101.10/rytvela (IL-1 receptor antagonists, compositions, and methods of treatment, United States patent no. USPTO8618054, May 5, 2005). S.C. and D.M.O. hold a patent on methods for reducing perinatal morbidity and/or mortality (US20210322509, March 9, 2016). The remaining authors have no financial conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

MicroRNAs (miRs) regulate physiological and pathological processes, including ischemia-induced angiogenesis and neovascularization. They can be transferred between cells by extracellular vesicles (EVs). However, the specific miRs that are packaged in EVs released from skeletal muscles, and how this process is modulated by ischemia, remain to be determined. We used a mouse model of hindlimb ischemia and next generation sequencing (NGS) to perform a complete profiling of miR expression and determine the effect of ischemia in skeletal muscles, and in EVs of different sizes (microvesicles (MVs) and exosomes) released from these muscles. Ischemia significantly modulated miR expression in whole muscles and EVs, increasing the levels of several miRs that can have pro-angiogenic effects (angiomiRs). We found that specific angiomiRs are selectively enriched in MVs and/or exosomes in response to ischemia. In silico approaches indicate that these miRs modulate pathways that play key roles in angiogenesis and neovascularization, including HIF1/VEGF signaling, regulation of actin cytoskeleton and focal adhesion, NOTCH, PI3K/AKT, RAS/MAPK, JAK/STAT, TGFb/SMAD signaling and the NO/cGMP/PKG pathway. Thus, we show for the first time that angiomiRs are selectively enriched in MVs and exosomes released from ischemic muscles. These angiomiRs could be targeted in order to improve the angiogenic function of EVs for potential novel therapeutic applications in patients with severe ischemic vascular diseases.

This review examines the complexities of preterm birth (PTB), emphasizes the pivotal role of inflammation in the pathogenesis of preterm labor, and assesses current available interventions. Antibiotics, progesterone analogs, mechanical approaches, nonsteroidal anti-inflammatory drugs, and nutritional supplementation demonstrate a limited efficacy. Tocolytic agents, targeting uterine activity and contractility, inadequately prevent PTB by neglecting to act on uteroplacental inflammation. Emerging therapies targeting toll-like receptors, chemokines, and interleukin receptors exhibit promise in mitigating inflammation and preventing PTB., Competing Interests: Disclosure S. Chemtob and W.D. Lubell hold a patent on composition of matter for the use of 101.10/rytvela (IL-1 receptor antagonists, compositions, and methods of treatment, United States patent no. USPTO8618054, May 5, 2005). S Chemtob, WD Lubell, DM Olson, and S Girard hold a patent on methods for reducing perinatal morbidity and/or mortality (US20210322509, March 9, 2016)., (Copyright © 2024 Elsevier Inc. All rights reserved.)

G-coupled protein receptors (GPCRs) are the ultimate refuge of pharmacology and medicine as more than 40% of all marketed drugs are directly targeting these receptors. Through cell surface expression, they are at the forefront of cellular communication with the outside world. Metabolites among the conveyors of this communication are becoming more prominent with the recognition of them as ligands for GPCRs. HCAR1 is a GPCR conveyor of lactate. It is a class A GPCR coupled to G αi which reduces cellular cAMP along with the downstream G βγ signaling. It was first found to inhibit lipolysis, and lately has been implicated in diverse cellular processes, including neural activities, angiogenesis, inflammation, vision, cardiovascular function, stem cell proliferation, and involved in promoting pathogenesis for different conditions, such as cancer. Other than signaling from the plasma membrane, HCAR1 shows nuclear localization with different location-biased activities therein. Although different functions for HCAR1 are being discovered, its cell and molecular mechanisms are yet ill understood. Here, we provide a comprehensive review on HCAR1, which covers the literature on the subject, and discusses its importance and relevance in various biological phenomena.

Many metabolites possess covalent and noncovalent signaling functions. However, ongoing research considers them mostly as ligands, neglecting their potential involvement in post-translational modifications. In this forum article, we discuss the dual signaling functions of metabolites, using lactate as a case study, and advocate for the use of multiple complementary techniques to disentangle their functions., Competing Interests: Declaration of interests No interests are declared., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The GPCR HCAR1 is known to be the sole receptor for lactate, which modulates its metabolic effects. Despite its significant role in many processes, mice deficient in HCAR1 exhibit no visible phenotype and are healthy and fertile. We performed transcriptomic analysis on HCAR1 deficient cells, in combination with lactate, to explore pathophysiologically altered processes. Processes such as immune regulation, various cancers, and neurodegenerative diseases were significantly enriched for HCAR1 transcriptomic signature. However, the most affected process of all was autism spectrum disorder. We performed behavioral tests on HCAR1 KO mice and observed that these mice manifest autistic-like behavior. Our data opens new avenues for research on HCAR1 and lactate effect at a pathological level. Video Abstract., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Background: Intraamniotic inflammation is associated with preterm birth, especially in cases occurring before 32 weeks' gestation, and is causally linked with an increased risk for neonatal mortality and morbidity. Targeted anti-inflammatory interventions may assist in improving the outcomes for pregnancies impacted by intrauterine inflammation. Interleukin-1 is a central upstream mediator of inflammation. Accordingly, interleukin-1 is a promising candidate target for intervention therapies and has been targeted previously using the interleukin-1 receptor antagonist, anakinra. Recent studies have shown that the novel, noncompetitive, allosteric interleukin-1 receptor inhibitor, rytvela, partially resolved inflammation associated with preterm birth and fetal injury. In this study, we used a preterm sheep model of chorioamnionitis to investigate the anti-inflammatory efficacy of rytvela and anakinra, administered in the amniotic fluid in the setting of intraamniotic Escherichia coli lipopolysaccharide exposure., Objective: We hypothesized that both rytvela and anakinra would reduce lipopolysaccharide-induced intrauterine inflammation and protect the fetal brain., Study Design: Ewes with a singleton fetus at 105 days of gestation (term is ∼150 days) were randomized to one of the following groups: (1) intraamniotic injections of 2 mL saline at time=0 and time=24 hours as a negative control group (saline group, n=12); (2) intraamniotic injection of 10 mg Escherichia coli lipopolysaccharide in 2 mL saline and intraamniotic injections of 2 mL saline at time=0 hours and time=24 hours as an inflammation positive control group (lipopolysaccharide group, n=11); (3) intraamniotic injection of Escherichia coli lipopolysaccharide in 2 mL saline and intraamniotic injections of 2.5 mg rytvela at time=0 hours and time=24 hours to test the anti-inflammatory efficacy of rytvela (lipopolysaccharide + rytvela group, n=10); or (4) intraamniotic injection of Escherichia coli lipopolysaccharide in 2 mL saline and intraamniotic injections of 100 mg anakinra at time=0 hours and time=24 hours to test the anti-inflammatory efficacy of anakinra (lipopolysaccharide + anakinra group, n=12). Amniotic fluid was sampled at time 0, 24, and 48 hours (ie, at each intervention and at delivery). Fetal umbilical cord blood was collected at delivery for differential blood counts and chemical studies. Inflammation was characterized by the analysis of fetal tissue cytokine and chemokine levels using quantitative polymerase chain reaction, enzyme-linked inmmunosorbent assay, and histology. The primary study outcome of interest was the assessment of anakinra and rytvela brain-protective effects in the setting of Escherichia coli lipopolysaccharide-induced intrauterine inflammation. Secondary outcomes of interest were to assess protection from fetal and intrauterine (ie, amniotic fluid, chorioamnion) inflammation., Results: Intraamniotic administration of lipopolysaccharide caused inflammation of the fetal lung, brain, and chorioamnionitis in preterm fetal sheep. Relative to treatment with saline only in the setting of lipopolysaccharide exposure, intraamniotic administration of both rytvela and anakinra both significantly prevented periventricular white matter injury, microglial activation, and histologic chorioamnionitis. Anakinra showed additional efficacy in inhibiting fetal lung myeloperoxidase activity, but its use was associated with metabolic acidaemia and reduced fetal plasma insulin-like growth factor-1 levels at delivery., Conclusion: Intraamniotic administration of rytvela or anakinra significantly inhibited fetal brain inflammation and chorioamnionitis in preterm fetal sheep exposed to intraamniotic lipopolysaccharide. In addition, anakinra treatment was associated with potential negative impacts on the developing fetus., (Copyright © 2023. Published by Elsevier Inc.)

Background: Perinatal infection/inflammation is associated with a high risk for neurological injury and neurodevelopmental impairment after birth. Despite a growing preclinical evidence base, anti-inflammatory interventions have not been established in clinical practice, partly because of the range of potential targets. We therefore systematically reviewed preclinical studies of immunomodulation to improve neurological outcomes in the perinatal brain and assessed their therapeutic potential., Methods: We reviewed relevant studies published from January 2012 to July 2023 using PubMed, Medline (OvidSP) and EMBASE databases. Studies were assessed for risk of bias using the SYRCLE risk of bias assessment tool (PROSPERO; registration number CRD42023395690)., Results: Forty preclinical publications using 12 models of perinatal neuroinflammation were identified and divided into 59 individual studies. Twenty-seven anti-inflammatory agents in 19 categories were investigated. Forty-five (76%) of 59 studies reported neuroprotection, from all 19 categories of therapeutics. Notably, 10/10 (100%) studies investigating anti-interleukin (IL)-1 therapies reported improved outcome, whereas half of the studies using corticosteroids (5/10; 50%) reported no improvement or worse outcomes with treatment. Most studies (49/59, 83%) did not control core body temperature (a known potential confounder), and 25 of 59 studies (42%) did not report the sex of subjects. Many studies did not clearly state whether they controlled for potential study bias., Conclusion: Anti-inflammatory therapies are promising candidates for treatment or even prevention of perinatal brain injury. Our analysis highlights key knowledge gaps and opportunities to improve preclinical study design that must be addressed to support clinical translation., (© 2023. BioMed Central Ltd., part of Springer Nature.)

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.

Background: Preterm birth is the leading cause of neonatal morbidity and mortality. Studies have shown that interleukin 1 plays a major role in the pathophysiology of preterm birth by inducing the production of proinflammatory mediators and uterine activation proteins leading to labor. More importantly, uteroplacental inflammation, associated with preterm birth parturition pathways, is detrimental to fetal tissues and leads to long-term sequelae. Our group has developed an allosteric antagonist of the interleukin 1 receptor, rytvela, found to be potent and safe in preventing preterm birth by suppressing inflammation via the inhibition of the mitogen-activated protein kinase pathway while preserving the Nuclear factor kappa B pathway (important in immune vigilance). Rytvela has been shown to inhibit inflammatory up-regulation and uterine activation while preserving fetal development., Objective: This study aimed to further the preclinical development of rytvela by evaluating its optimal dose and minimal duration of treatment to inhibit the inflammatory cascade, prolong gestation, and promote neonatal outcomes., Study Design: Pregnant CD-1 mice were administered with lipopolysaccharide (10 μg, intraperitoneal administration) or interleukin 1 (1 μg/kg, intrauterine administration) on gestational day 16 to induce preterm labor. Rytvela was administered at different doses (0.1, 0.5, 1.0, 2.0, 4.0 mg/kg/d subcutaneously) from gestational days 16 to 18.5. To evaluate the minimal duration of treatment, the mice were administered with rytvela (2 mg/kg/d subcutaneously) for 24, 36, or 48 hours. The rate of prematurity (gestational day <18.5) and neonate survival and weight were evaluated. Gestational tissues were collected at gestational day 17.5 to quantify cytokines, proinflammatory mediators, and uterine activating proteins by real-time quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. The neonatal lungs and intestines were collected from postnatal days 5 to 7 and analyzed by histology., Results: Rytvela exhibited a dose-response profile and achieved maximum efficacy at a dose of 2 mg/kg/d by reducing 70% of lipopolysaccharide-induced preterm births and 60% of interleukin 1β-induced preterm births. In addition, rytvela attained maximum efficacy at a dose of 1 mg/kg/d by increasing neonate survival by up to 65% in both models of preterm birth. Rytvela protected fetuses from inflammatory insult as of 24 hours, preserving lung and intestinal integrity, and prevented preterm birth and fetal mortality by 60% and 50%, respectively, as of 36 hours of treatment., Conclusion: The maximum efficacy of rytvela was achieved at 2 mg/kg/d with improved birth outcomes and prevented inflammatory up-regulation upon 36 hours (only) of treatment. Rytvela exhibited desirable properties for the safe prevention of preterm birth and fetal protection., (Copyright © 2022 Elsevier Inc. All rights reserved.)

In pursuit of more effective-labor delaying tocolytic agents, the prostaglandin F2α (PGF2α) receptor (FP) modulator PDC113.824 [(6S)-2] represents a potent lead for developing therapy to treat preterm birth. Derivatives of FP modulator (6S)-2 were synthesized, possessing respectively 5- and 7-hydroxyl groups on the indolizidin-2-one amino acid (I 2 aa) residue. The effects of the alcohol substituents were examined in a PGF2α-induced myometrial contraction assay. Based on knowledge of dihedral angle values of model I 2 aa peptides from X-ray analyses, the results of the study indicate respectively encouraging and limited potential for creating improved tocolytic agents by modifications at the 5- and 7-positions., (© 2022 European Peptide Society and John Wiley & Sons Ltd.)

Corneal wound healing involves communication between the different cell types that constitute the three cellular layers of the cornea (epithelium, stroma and endothelium), a process ensured in part by a category of extracellular vesicles called exosomes. In the present study, we isolated exosomes released by primary cultured human corneal epithelial cells (hCECs), corneal fibroblasts (hCFs) and corneal endothelial cells (hCEnCs) and determined whether they have wound healing characteristics of their own and to which point they modify the genetic and proteomic pattern of these cell types. Exosomes released by all three cell types significantly accelerated wound closure of scratch-wounded hCECs in vitro compared to controls (without exosomes). Profiling of activated kinases revealed that exosomes from human corneal cells caused the activation of signal transduction mediators that belong to the HSP27, STAT, β-catenin, GSK-3β and p38 pathways. Most of all, data from gene profiling analyses indicated that exosomes, irrespective of their cellular origin, alter a restricted subset of genes that are completely different between each targeted cell type (hCECs, hCFS, hCEnCs). Analysis of the genes specifically differentially regulated for a given cell-type in the microarray data using the Ingenuity Pathway Analysis (IPA) software revealed that the mean gene expression profile of hCECs cultured in the presence of exosomes would likely promote cell proliferation and migration whereas it would reduce differentiation when compared to control cells. Collectively, our findings represent a conceptual advance in understanding the mechanisms of corneal wound repair that may ultimately open new avenues for the development of novel therapeutic approaches to improve closure of corneal wounds.

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 PGE 2 EP 4 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.

Perinatal hypoxic/ischemic (HI) brain injury is a major clinical problem with devastating neurodevelopmental outcomes in neonates. During HI brain injury, dysregulated factor production contributes to microvascular impairment. Glycolysis-derived lactate accumulated during ischemia has been proposed to protect against ischemic injury, but its mechanism of action is poorly understood. Herein, we hypothesize that lactate via its G-protein coupled receptor (GPR81) controls postnatal brain angiogenesis and plays a protective role after HI injury. We show that GPR81 is predominantly expressed in neurons of the cerebral cortex and hippocampus. GPR81-null mice displayed a delay in cerebral microvascular development linked to reduced levels of various major angiogenic factors and augmented expression of anti-angiogenic Thrombospondin-1 (TSP-1) in comparison to their WT littermates. Coherently, lactate stimulation induced an increase in growth factors (VEGF, Ang1 and 2, PDGF) and reduced TSP-1 expression in neurons, which contributed to accelerating angiogenesis. HI injury in GPR81-null animals curtailed vascular density and consequently increased infarct size compared to changes seen in WT mice; conversely intracerebroventricular lactate injection increased vascular density and diminished infarct size in WT but not in GPR81-null mice. Collectively, we show that lactate acting via GPR81 participates in developmental brain angiogenesis, and attenuates HI injury by restoring compromised microvasculature.

Neutrophils and other leukocytes invade the mouse uterus at term birth, which is normal for activating the uterus for labor. To better understand the regulation of this migration at term and interleukin (IL)-1β-induced preterm birth, we developed a mouse leukocyte migration assay (mLMA) and used it with rytvela, an IL-1 receptor allosteric antagonist. The mLMA uses term peripheral blood leukocytes that migrate in a Boyden chamber in response to a chemoattractant. We tested several mouse uterine tissues after homogenization and sedimentation of debris for chemoattractant activity. The most active chemoattractant homogenate came from the mouse lower uterus on gestational day (GD) 18.5. Using flow cytometry, we demonstrated that 99% of the cells that migrate are neutrophils. IL-1β administered on GD 16 stimulated neutrophil migration and invasion into the uterus and the fetal brain along with preterm birth on GD 17. Preterm birth and the increased leukocyte invasion of the maternal uterus and fetal brain were all blocked by the co-administration of rytvela. To test where the site of IL-1β action might be, we examined the potency of lower uterine chemoattractant and the activation of leukocytes following IL-1β +/- rytvela administration. IL-1β did not increase lower uterus homogenate chemoattractant activity, but it significantly ( p < 0.05) increased leukocyte activation as defined by cytokine and chemokine expression. Rytvela blocked this activation of leukocytes by IL-1β. We conclude that IL-1β stimulates preterm birth in mice by increasing leukocyte activation leading to increased uterine and fetal brain leukocyte invasion., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lee, Patel, Onushko, Fang, Chemtob and Olson.)

Introduction: Inflammation-induced white matter injury (WMI) in preterm infants is characterized by microglia activation, astrogliosis, oxidative stress and neurodevelopmental impairments. Microglia and astrocytes activation can be described under a broad spectrum of activation profile with extremes described as pro-inflammatory/neurotoxic (M1 microglia or A1 astrocyte) or anti-inflammatory/neuroprotective (M2 microglia or A2 astrocyte) in response to stimuli including lipopolysaccharide (LPS) and interleukin 1 (IL-1). As IL-1 signalling pathway has been posited as a major driver of inflammation-induced perinatal WMI, our aim was to evaluate the contribution of IL-1 modulation in LPS-induced microglia and astrocyte activation., Methods: Primary neonatal cell co-cultures of astrocytes and microglia were treated with LPS (100 ng/ml) for 8 h or 24 h. Two distinct IL-1 receptor antagonists, Rytvela or Kineret (1 μg/ml), were added simultaneously with LPS to respectively modulate or block IL-1 receptor. Medium was collected to measure levels of IL-1β. Expression of markers related to pro- and anti-inflammatory microglia and astrocyte activation profiles and antioxidant genes were assessed., Results: At 8 h, LPS exposure induced pro- (M1/A1) and anti-inflammatory (M2/A2) marker expression and inhibited antioxidant gene expression in microglia and astrocytes. By 24 h, continuous LPS exposure increased pro-inflammatory and neurotoxic microglial and astrocytic markers (M1/A1), as well as antioxidant genes. Administration of IL-1 antagonists Rytvela and Kineret with continuous LPS exposure had no significant effect on modulation of specific microglia and astrocyte activation pathways., Discussion/conclusion: We show that LPS effects on in vitro neonatal microglia and astrocytes co-cultures are time dependent eliciting a number of pro- and anti-inflammatory responses during the acute phase of inflammation (8 h), which shift towards pro-inflammatory and neurotoxic factors by 24 h. Although LPS-induced inflammation led to abundant IL-1 expression, IL-1 inhibition had no significant impact on in vitro modulation of microglia and astrocyte activation pathways towards M2 and A2 activation profile., (© 2022 International Society for Developmental Neuroscience.)

Dyslipidemia and autophagy have been implicated in the pathogenesis of blinding neovascular age-related macular degeneration (NV-AMD). VLDL receptor (VLDLR), expressed in photoreceptors with a high metabolic rate, facilitates the uptake of triglyceride-derived fatty acids. Since fatty acid uptake is reduced in Vldlr-/- tissues, more remain in circulation, and the retina is fuel deficient, driving the formation in mice of neovascular lesions reminiscent of retinal angiomatous proliferation (RAP), a subtype of NV-AMD. Nutrient scarcity and energy failure are classically mitigated by increasing autophagy. We found that excess circulating lipids restrained retinal autophagy, which contributed to pathological angiogenesis in the Vldlr-/- RAP model. Triglyceride-derived fatty acid sensed by free fatty acid receptor 1 (FFAR1) restricted autophagy and oxidative metabolism in photoreceptors. FFAR1 suppressed transcription factor EB (TFEB), a master regulator of autophagy and lipid metabolism. Reduced TFEB, in turn, decreased sirtuin-3 expression and mitochondrial respiration. Metabolomic signatures of mouse RAP-like retinas were consistent with a role in promoting angiogenesis. This signature was also found in human NV-AMD vitreous. Restoring photoreceptor autophagy in Vldlr-/- retinas, either pharmacologically or by deleting Ffar1, enhanced metabolic efficiency and suppressed pathological angiogenesis. Dysregulated autophagy by circulating lipids might therefore contribute to the energy failure of photoreceptors driving neovascular eye diseases, and FFAR1 may be a target for intervention.

Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Purpose: The purpose of this study was to develop an in vivo optical coherence tomography (OCT) system capable of imaging the developing mouse retina and its associated morphometric and microstructural changes., Methods: Thirty-four wild-type mice (129S1/SvlmJ) were anesthetized and imaged between postnatal (P) day 7 and P21. OCT instrumentation was developed to optimize signal intensity and image quality. Semi-automatic segmentation tools were developed to quantify the retinal thickness of the nerve fiber layer (NFL), inner plexiform layer (IPL), inner nuclear layer (INL), and the outer retinal layers (ORL), in addition to the total retina. The retinal maturation was characterized by comparing layer thicknesses between consecutive time points., Results: From P7 to P10, the IPL increased significantly, consistent with retinal synaptogenesis. From P10 to P12, the IPL and ORL also increased, which is coherent with synaptic connectivity and photoreceptor maturation. In contrast, during these periods, the INL decreased significantly, consistent with cellular densification and selective apoptotic "pruning" of the tissue during nuclear migration. Thereafter from P12 to P21, the INL continued to thin (significantly from P17 to P21) whereas the other layers remained unchanged. No time-dependent changes were observed in the NFL. Overall, changes in the total retina were attributed to those in the IPL, INL, and ORL. Regions of the retina adjacent to the optic nerve head were thinner than distal regions during maturation., Conclusions: Changes in retinal layer thickness are consistent with retinal developmental mechanisms. Accordingly, this report opens new horizons in using our system in the mouse to characterize longitudinally developmental digressions in models of human diseases.

Background: Intraamniotic inflammation is associated with up to 40% of preterm births, most notably in deliveries occurring prior to 32 weeks' gestation. Despite this, there are few treatment options allowing the prevention of preterm birth and associated fetal injury. Recent studies have shown that the small, non-competitive allosteric interleukin (IL)-1 receptor inhibitor, rytvela, may be of use in resolving inflammation associated with preterm birth (PTB) and fetal injury. We aimed to use an extremely preterm sheep model of chorioamnionitis to investigate the anti-inflammatory efficacy of rytvela in response to established intra-amniotic (IA) lipopolysaccharide (LPS) exposure. We hypothesized that rytvela would reduce LPS-induced IA inflammation in amniotic fluid (AF) and fetal tissues., Methods: Sheep with a single fetus at 95 days gestation (estimated fetal weight 1.0 kg) had surgery to place fetal jugular and IA catheters. Animals were recovered for 48 hours before being randomized to either: i) IA administration of 2 ml saline 24 hours before 2 ml IA and 2 ml fetal intravenous (IV) administration of saline (Saline Group, n = 7); ii) IA administration of 10 mg LPS in 2 ml saline 24 hours before 2 ml IA and 2 ml fetal IV saline (LPS Group, n = 10); 3) IA administration of 10 mg LPS in 2 ml saline 24 hours before 0.3 mg/fetal kg IA and 1 mg/fetal kg fetal IV rytvela in 2 ml saline, respectively (LPS + rytvela Group, n = 7). Serial AF samples were collected for 120 h. Inflammatory responses were characterized by quantitative polymerase chain reaction (qPCR), histology, fluorescent immunohistochemistry, enzyme-linked inmmunosorbent assay (ELISA), fluorescent western blotting and blood chemistry analysis., Results: LPS-treated animals had endotoxin and AF monocyte chemoattractant protein (MCP)-1 concentrations that were significantly higher at 24 hours (immediately prior to rytvela administration) relative to values from Saline Group animals. Following rytvela administration, the average MCP-1 concentrations in the AF were significantly lower in the LPS + rytvela Group relative to in the LPS Group. In delivery samples, the expression of IL-1β in fetal skin was significantly lower in the LPS + rytvela Group compared to the LPS Group., Conclusion: A single dose of rytvela was associated with partial, modest inhibition in the expression of a panel of cytokines/chemokines in fetal tissues undergoing an active inflammatory response., Competing Interests: DO and SC are founders and directors of Maternica Therapeutics, which has a commercial interest in the development of rytvela.

Aim: To explore the current literature on prenatal inflammation-associated risk factors for retinopathy of prematurity (ROP)., Methods: Subjective summary of selected experimental and epidemiological publications that support the authors' central hypothesis that the aetiology of ROP begins before birth., Results: Based on current evidence we suggest that, contrary to current aetiological models, the process of ROP development begins with a prephase in utero. This beginning is likely initiated by inflammatory responses that are associated with intrauterine infection., Conclusion: We propose a novel aetio-pathogenetic model of ROP and suggest that the effects of postnatal exposure to inflammatory stressors (resulting from infection or hyperoxia or both) as well as those of other pre- and postnatal contributors to the complex pathogenesis of ROP might be modified by the prenatal phase of the disease., (© 2021 The Authors. Acta Paediatrica published by John Wiley & Sons Ltd on behalf of Foundation Acta Paediatrica.)

In preparation for delivery, the uterus transitions from actively maintaining quiescence during pregnancy to an active parturient state. This transition occurs as a result of the accumulation of pro-inflammatory signals which are amplified by positive feedback interactions involving paracrine and autocrine signaling at the level of each intrauterine cell and tissue. The amplification events occur in parallel until they reach a certain threshold, 'tipping the scale' and contributing to processes of uterine activation and functional progesterone withdrawal. The described signaling interactions all occur upstream from the presentation of clinical labor symptoms. In this review, we will: 1) describe the different physiological processes involved in uterine transition for each intrauterine tissue; 2) compare and contrast the current models of labor initiation; 3) introduce innovative models for measuring paracrine inflammatory interactions; and 4) discuss the therapeutic value in identifying and targeting key players in this crucial event for preterm birth., Competing Interests: DO and SC are founders of Maternica Therapeutics, Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Leimert, Xu, Princ, Chemtob and Olson.)

Cyclic peptide diversity has been broadened by elaborating the A 3 -macrocyclization to include various di-amino carboxylate components with different N ε -amine substituents. Triple-bond reduction provided new cyclic peptide macrocycles with Z -olefin and completely saturated structures. Moreover, cyclic azasulfurylpeptides were prepared by exchanging the propargylglycine (Pra) component for an amino sulfamide surrogate. Examination of such diversity-oriented methods on potent cyclic azapeptide modulators of the cluster of differentiation 36 receptor (CD36) identified the importance of the triple bond as well as the N ε -allyl lysine and azaPra residues for high CD36 binding affinity. Cyclic azapeptides which engaged CD36 effectively reduced pro-inflammatory nitric oxide and downstream cytokine and chemokine production in macrophages stimulated with a Toll-like receptor-2 agonist. Studying the triple bond and amine components in the multiple-component A 3 -macrocyclization has given a diverse array of macrocycles and pertinent information to guide the development of ideal CD36 modulators with biomedical potential for curbing macrophage-driven inflammation.

6-Hydroxymethyl indolizidin-2-one amino acids were synthesized in 10 steps from l-serine by intramolecular ring opening of a symmetrical epoxide and lactam formation. X-ray analyses indicated the bicycles replicated ideal peptide type II' β-turn central dihedral angle geometry. Inside a prostaglandin-F 2α receptor modulator, the 6-hydroxymethyl analogue retained inhibitory activity on myometrial contractility.

During the development of the retina and the nervous system, high levels of energy are required by the axons of retinal ganglion cells (RGCs) to grow towards their brain targets. This energy demand leads to an increase of glycolysis and L-lactate concentrations in the retina. L-lactate is known to be the endogenous ligand of the GPR81 receptor. However, the role of L-lactate and its receptor in the development of the nervous system has not been studied in depth. In the present study, we used immunohistochemistry to show that GPR81 is localized in different retinal layers during development, but is predominantly expressed in the RGC of the adult rodent. Treatment of retinal explants with L-lactate or the exogenous GPR81 agonist 3,5-DHBA altered RGC growth cone (GC) morphology (increasing in size and number of filopodia) and promoted RGC axon growth. These GPR81-mediated modifications of GC morphology and axon growth were mediated by protein kinases A and C, but were absent in explants from gpr81 -/- transgenic mice. Living gpr81 -/- mice showed a decrease in ipsilateral projections of RGCs to the dorsal lateral geniculate nucleus (dLGN). In conclusion, present results suggest that L-lactate and its receptor GPR81 play an important role in the development of the visual nervous system.

Preterm birth (PTB) remains the leading cause of infant morbidity and mortality. Despite 50 years of research, therapeutic options are limited and many lack clear efficacy. Tocolytic agents are drugs that briefly delay PTB, typically to allow antenatal corticosteroid administration for accelerating fetal lung maturity or to transfer patients to high-level care facilities. Globally, there is an unmet need for better tocolytic agents, particularly in low- and middle-income countries. Although most tocolytics, such as betamimetics and indomethacin, suppress downstream mediators of the parturition pathway, newer therapeutics are being designed to selectively target inflammatory checkpoints with the goal of providing broader and more effective tocolysis. However, the relatively small market for new PTB therapeutics and formidable regulatory hurdles have led to minimal pharmaceutical interest and a stagnant drug pipeline. In this review, we present the current landscape of PTB therapeutics, assessing the history of drug development, mechanisms of action, adverse effects, and the updated literature on drug efficacy. We also review the regulatory hurdles and other obstacles impairing novel tocolytic development. Ultimately, we present possible steps to expedite drug development and meet the growing need for effective preterm birth therapeutics.

Preterm birth (PTB) and its consequences are a major public health concern as preterm delivery is the main cause of mortality and morbidity at birth. There are many causes of PTB, but inflammation is undeniably associated with the process of premature childbirth and fetal injury. At present, treatments clinically available mostly involve attempt to arrest contractions (tocolytics) but do not directly address upstream maternal inflammation on development of the fetus. One of the possible solutions may lie in the modulation of inflammatory mediators. Of the many pro-inflammatory cytokines involved in the induction of PTB, IL-6 stands out for its pleiotropic effects and its involvement in both acute and chronic inflammation. Here, we provide a detailed review of the effects of IL-6 on the timing of childbirth, its occurrence during PTB and its indissociable roles with associated fetal tissue damage., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Desjarlais, Ruknudin, Wirth, Lahaie, Dabouz, Rivera, Habelrih, Omri, Hardy, Rivard and Chemtob.)

Traumatic brain injury is one of the leading causes of morbidity and mortality throughout the world. Its increasing incidence, in addition to its fundamental role in the development of neurodegenerative disease, proves especially concerning. Despite extensive preclinical and clinical studies, researchers have yet to identify a safe and effective neuroprotective strategy. Following brain trauma, secondary injury from molecular, metabolic, and cellular changes causes progressive cerebral tissue damage. Chronic neuroinflammation following traumatic brain injuries is a key player in the development of secondary injury. Targeting this phenomenon for development of effective neuroprotective therapies holds promise. This strategy warrants a concrete understanding of complex neuroinflammatory mechanisms. In this review, we discuss pathophysiological mechanisms such as the innate immune response, glial activation, blood-brain barrier disruption, activation of immune mediators, as well as biological markers of traumatic brain injury. We then review existing and emerging pharmacological therapies that target neuroinflammation to improve functional outcome.

Ischemic retinopathies (IRs), such as retinopathy of prematurity and diabetic retinopathy, are characterized by an initial phase of microvascular degeneration that results in retinal ischemia, followed by exaggerated pathologic neovascularization (NV). Mesenchymal stromal cells (MSCs) have potent pro-angiogenic and anti-inflammatory properties associated with tissue repair and regeneration, and in this regard exert protection to neurons in ischemic and degenerative conditions; however, the exact mechanisms underlying these functions remain largely unknown. Class III Semaphorins (A-G) are particularly implicated in regulating neural blood supply (as well as neurogenesis) by suppressing angiogenesis and affecting myeloid cell function; this is the case for distinct neuropillin-activating Sema3A as well as PlexinD1-activating Sema3E; but during IR the former Sema3A increases while Sema3E decreases. We investigated whether retinal vascular repair actions of MSCs are exerted by normalizing Semaphorin and downstream cytokines in IR. Intravitreal administration of MSCs or their secretome (MSCs-conditioned media [MSCs-CM]) significantly curtailed vasoobliteration as well as aberrant preretinal NV in a model of oxygen-induced retinopathy (OIR). The vascular repair effects of MSCs-CM in the ischemic retina were associated with restored levels of Sema3E. Vascular benefits of MSCs-CM were reversed by anti-Sema3E; while intravitreal injection of anti-angiogenic recombinant Sema3E (rSema3E) in OIR-subjected mice reproduced effects of MSCs-CM by inhibiting as expected preretinal NV but also by decreasing vasoobliteration. To explain these opposing vascular effects of Sema3E we found in OIR high retinal levels, respectively, of the pro- and anti-angiogenic IL-17A and Sema3A-regulating IL-1β; IL-17A positively affected expression of IL-1β. rSema3E decreased concentrations of these myeloid cell-derived pro-inflammatory cytokines in vitro and in vivo . Importantly, IL-17A suppression by MSCs-CM was abrogated by anti-Sema3E neutralizing antibody. Collectively, our findings provide novel evidence by which MSCs inhibit aberrant NV and diminish vasoobliteration (promoting revascularization) in retinopathy by restoring (at least in part) neuronal Sema3E levels that reduce pathological levels of IL-17A (and in turn other proinflammatory factors) in myeloid cells. The ability of MSCs to generate a microenvironment permissive for vascular regeneration by controlling the production of neuronal factors involved in immunomodulatory activities is a promising opportunity for stem cell therapy in ocular degenerative diseases., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Noueihed, Rivera, Dabouz, Abram, Omri, Lahaie and Chemtob.)

As a key cytokine mediator of inflammation, interleukin-1β (IL-1β) binds to the IL-1 receptor (IL-1R) and activates various downstream signaling mediators, including NF-κB, which is required for immune vigilance and cellular protection. Toward the development of IL-1-targeting therapeutics which exhibit functional selectivity, the all-D-amino acid peptide 1 (101.10, H-D-Arg-D-Tyr-D-Thr-D-Val-D-Glu-D-Leu-D-Ala-NH 2 ) was conceived as an allosteric IL-1R modulator that conserves NF-κB signaling while inhibiting other IL-1-activated pathways. Employing β-hydroxy-α-amino-γ-lactam (Hgl) stereoisomers to study the conformation about the Thr 3 residue in 1 , [(3 R ,4 S )-Hgl 3 ]- 1 ( 2b ), among all possible diastereomers, was found to exhibit identical in vitro and in vivo activity as the parent peptide and superior activity to the α-amino-γ-lactam (Agl) counterpart. Noting the relevance of the β-hydroxyl substituent and configuration for the activity of (3 R ,4 S )- 2b , fifteen different β-substituted-Agl 3 analogs of 1 (e.g., 2c-q ) have now been synthesized by a combination of solution- and solid-phase methods employing N -Fmoc-β-substituted-Agl 3 -Val-OH dipeptide building blocks. Introduction of a β-azido-Agl 3 residue into the resin bound peptide and subsequent reduction and CuAAC chemistry gave access to a series of amine and triazole derivatives (e.g., 2h-q ). β-Substituted-[Agl 3 ]- 1 analogs 2c-q exhibited generally similar circular dichroism (CD) spectra as that of Hgl analog 2b in water, presenting curve shapes indicative of β-turn structures. The relevance of the β-substituent was indicated in rodent models of preterm labor and retinopathy of prematurity (ROP), in which certain analogs inhibited preterm birth and vaso-obliteration, respectively, with activity similar to 1 and 2b . The β-substituted-[Agl 3 ]- 1 analogs exhibited functional selectivity on IL-1-induced signaling pathways. The described solid-phase method has provided discerning probes for exploring peptide structure-activity relationships and valuable leads for developing prototypes to treat inflammatory events leading to prematurity and retinopathy of prematurity, which are leading causes of infant morbidity and blindness respectively., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Geranurimi, Cheng, Quiniou, Côté, Hou, Lahaie, Boudreault, Chemtob and Lubell.)

Background: Hyperglycemia in preterm infants may be associated with severe retinopathy of prematurity (ROP) and other morbidities. However, it is uncertain which concentration of blood glucose is associated with increased risk of tissue damage, with little consensus on the cutoff level to treat hyperglycemia. The objective of our study was to examine the association between hyperglycemia and severe ROP in premature infants., Methods and Findings: In 2 independent, monocentric cohorts of preterm infants born at <30 weeks' gestation (Nantes University Hospital, 2006-2016, primary, and Lyon-HFME University Hospital, 2009-2017, validation), we first analyzed the association between severe (stage 3 or higher) ROP and 2 markers of glucose exposure between birth and day 21-maximum value of glycemia (MaxGly1-21) and mean of daily maximum values of glycemia (MeanMaxGly1-21)-using logistic regression models. In both the primary (n = 863 infants, mean gestational age 27.5 ± 1.4 weeks, boys 52.5%; 38 with severe ROP; 54,083 glucose measurements) and the validation cohort (n = 316 infants, mean gestational age 27.4 ± 1.4 weeks, boys 51.3%), MaxGly1-21 and MeanMaxGly1-21 were significantly associated with an increased risk of severe ROP: odds ratio (OR) 1.21 (95% CI 1.14-1.27, p < 0.001) and OR 1.70 (95% CI 1.48-1.94, p < 0.001), respectively, in the primary cohort and OR 1.17 (95% CI 1.05-1.32, p = 0.008) and OR 1.53 (95% CI 1.20-1.95, p < 0.001), respectively, in the validation cohort. These associations remained significant after adjustment for confounders in both cohorts. Second, we identified optimal cutoff values of duration of exposure above each concentration of glycemia between 7 and 13 mmol/l using receiver operating characteristic curve analyses in the primary cohort. Optimal cutoff values for predicting stage 3 or higher ROP were 9, 6, 5, 3, 2, 2, and 1 days above a glycemic threshold of 7, 8, 9, 10, 11, 12, and 13 mmol/l, respectively. Severe exposure was defined as at least 1 exposure above 1 of the optimal cutoffs. Severe ROP was significantly more common in infants with severe exposure in both the primary (10.9% versus 0.6%, p < 0.001) and validation (5.2% versus 0.9%, p = 0.030) cohorts. Finally, we analyzed the association between insulin therapy and severe ROP in a national population-based prospectively recruited cohort (EPIPAGE-2, 2011, n = 1,441, mean gestational age 27.3 ± 1.4, boys 52.5%) using propensity score weighting. Insulin use was significantly associated with severe ROP in overall cohort crude analyses (OR 2.51 [95% CI 1.13-5.58], p = 0.024). Adjustment for inverse propensity score (gestational age, sex, birth weight percentile, multiple birth, spontaneous preterm birth, main pregnancy complications, surfactant therapy, duration of oxygen exposure between birth and day 28, digestive state at day 7, caloric intake at day 7, and highest glycemia during the first week) and duration of oxygen therapy had a large but not significant effect on the association between insulin treatment and severe ROP (OR 0.40 [95% CI 0.13-1.24], p = 0.106). Limitations of this study include its observational nature and, despite the large number of patients included compared to earlier similar studies, the lack of power to analyze the association between insulin use and retinopathy., Conclusions: In this study, we observed that exposure to high glucose concentration is an independent risk factor for severe ROP, and we identified cutoff levels that are significantly associated with increased risk. The clinical impact of avoiding exceeding these thresholds to prevent ROP deserves further evaluation., Competing Interests: The authors have declared that no competing interests exist.

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 IL1β, 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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Desjarlais, Wirth, Lahaie, Ruknudin, Hardy, Rivard and Chemtob.)

Background: Inflammation and particularly interleukin-1β (IL-1β), a pro-inflammatory cytokine highly secreted by activated immune cells during early AMD pathological events, contribute significantly to retinal neurodegeneration. Here, we identify specific cell types that generate IL-1β and harbor the IL-1 receptor (IL-1R) and pharmacologically validate IL-1β's contribution to neuro-retinal degeneration using the IL-1R allosteric modulator composed of the amino acid sequence rytvela (as well as the orthosteric antagonist, Kineret) in a model of blue light-induced retinal degeneration., Methods: Mice were exposed to blue light for 6 h and sacrificed 3 days later. Mice were intraperitoneally injected with rytvela, Kineret, or vehicle twice daily for 3 days. The inflammatory markers F4/80, NLRP3, caspase-1, and IL-1β were assessed in the retinas. Single-cell RNA sequencing was used to determine the cell-specific expression patterns of retinal Il1b and Il1r1. Macrophage-induced photoreceptor death was assessed ex vivo using retinal explants co-cultured with LPS-activated bone marrow-derived macrophages. Photoreceptor cell death was evaluated by the TUNEL assay. Retinal function was assessed by flash electroretinography., Results: Blue light markedly increased the mononuclear phagocyte recruitment and levels of inflammatory markers associated with photoreceptor death. Co-localization of NLRP3, caspase-1, and IL-1β with F4/80 + mononuclear phagocytes was clearly detected in the subretinal space, suggesting that these inflammatory cells are the main source of IL-1β. Single-cell RNA sequencing confirmed the immune-specific expression of Il1b and notably perivascular macrophages in light-challenged mice, while Il1r1 expression was found primarily in astrocytes, bipolar, and vascular cells. Retinal explants co-cultured with LPS/ATP-activated bone marrow-derived macrophages displayed a high number of TUNEL-positive photoreceptors, which was abrogated by rytvela treatment. IL-1R antagonism significantly mitigated the inflammatory response triggered in vivo by blue light exposure, and rytvela was superior to Kineret in preserving photoreceptor density and retinal function., Conclusion: These findings substantiate the importance of IL-1β in neuro-retinal degeneration and revealed specific sources of Il1b from perivascular MPs, with its receptor Ilr1 being separately expressed on surrounding neuro-vascular and astroglial cells. They also validate the efficacy of rytvela-induced IL-1R modulation in suppressing detrimental inflammatory responses and preserving photoreceptor density and function in these conditions, reinforcing the rationale for clinical translation.

GPCRs are the largest receptor family that are involved in virtually all biological processes. Pharmacologically, they are highly druggable targets, as they cover more than 40% of all drugs in the market. Our knowledge of biased signaling provided insight into pharmacology vastly improving drug design to avoid unwanted effects and achieve higher efficacy and selectivity. However, yet another feature of GPCR biology is left largely unexplored, location bias. Recent developments in this field show promising avenues for evolution of new class of pharmaceuticals with greater potential for higher level of precision medicine. Further consideration and understanding of this phenomenon with deep biochemical and molecular insights would pave the road to success. In this review, we critically analyze this perspective and discuss new avenues of investigation., (© 2020 The Author(s).)

The blood-brain barrier (BBB) is a major obstacle to the development of effective diagnostics and therapeutics for brain cancers and other central nervous system diseases. Peptide agonist analogs of kinin B1 and B2 receptors, acting as BBB permeabilizers, have been utilized to overcome this barrier. The purpose of the study was to provide new insights for the potential utility of kinin analogs as brain drug delivery adjuvants. In vivo imaging studies were conducted in various animal models (primary/secondary brain cancers, late radiation-induced brain injury) to quantify BBB permeability in response to kinin agonist administrations. Results showed that kinin B1 (B1R) and B2 receptors (B2R) agonists increase the BBB penetration of chemotherapeutic doxorubicin to glioma sites, with additive effects when applied in combination. B2R agonist also enabled extravasation of high-molecular-weight fluorescent dextrans (155 kDa and 2 MDa) in brains of normal mice. Moreover, a systemic single dose of B2R agonist did not increase the incidence of metastatic brain tumors originating from circulating breast cancer cells. Lastly, B2R agonist promoted the selective delivery of co-injected diagnostic MRI agent Magnevist in irradiated brain areas, depicting increased vascular B2R expression. Altogether, our findings suggest additional evidence for using kinin analogs to facilitate specific access of drugs to the brain.

Background: Classical cardiovascular risk factors (CRFs) are associated with impaired angiogenic activities of bone marrow-derived proangiogenic cells (PACs) related to peripheral artery diseases (PADs) and ischemia-induced neovascularization. MicroRNAs (miRs) are key regulators of gene expression, and they are involved in the modulation of PAC function and PAC paracrine activity. However, the effects of CRFs on the modulation of miR expression in PACs are unknown., Aims and Methods: We used a model of hindlimb ischemia and next-generation sequencing to perform a complete profiling of miRs in PACs isolated from the bone marrow of mice subjected to three models of CRFs: aging, smoking (SMK) and hypercholesterolemia (HC)., Results: Approximately 570 miRs were detected in PACs in the different CRF models. When excluding miRs with a very low expression level (<100 RPM), 40 to 61 miRs were found to be significantly modulated by aging, SMK, or HC. In each CRF condition, we identified downregulated proangiogenic miRs and upregulated antiangiogenic miRs that could contribute to explain PAC dysfunction. Interestingly, several miRs were similarly downregulated (e.g., miR-542-3p, miR-29) or upregulated (e.g., miR-501, miR-92a) in all CRF conditions. In silico approaches including Kyoto Encyclopedia of Genes and Genomes and cluster dendogram analyses identified predictive effects of these miRs on pathways having key roles in the modulation of angiogenesis and PAC function, including vascular endothelial growth factor signaling, extracellular matrix remodeling, PI3K/AKT/MAPK signaling, transforming growth factor beta (TGFb) pathway, p53, and cell cycle progression., Conclusion: This study describes for the first time the effects of CRFs on the modulation of miR profile in PACs related to PAD and ischemia-induced neovascularization. We found that several angiogenesis-modulating miRs are similarly altered in different CRF conditions. Our findings constitute a solid framework for the identification of miRs that could be targeted in PACs in order to improve their angiogenic function and for the future development of novel therapies to improve neovascularization and reduce tissue damage in patients with severe PAD., (Copyright © 2020 Desjarlais, Dussault, Rivera, Chemtob and Rivard.)

The innovative development of azapeptide analogues of growth hormone releasing peptide-6 (GHRP-6) has produced selective modulators of the cluster of differentiation 36 receptor (CD36). The azapeptide CD36 modulators curb macrophage-driven inflammation and mitigate atherosclerotic and angiogenic pathology. In macrophages activated with Toll-like receptor-2 heterodimer agonist, they reduced nitric oxide production and proinflammatory cytokine release. In a mouse choroidal explant microvascular sprouting model, they inhibited neovascularization. In murine models of cardiovascular injury, CD36-selective azapeptide modulators exhibited cardioprotective and anti-atherosclerotic effects. In subretinal inflammation models, they altered activated mononuclear phagocyte metabolism and decreased immune responses to alleviate subsequent inflammation-dependent neuronal injury associated with retinitis pigmentosa, diabetic retinopathy and age-related macular degeneration. The translation of GHRP-6 to potent and selective linear and cyclic azapeptide modulators of CD36 is outlined in this review which highlights the relevance of turn geometry for activity and the biomedical potential of prototypes for the beneficial treatment of a wide range of cardiovascular, metabolic and immunological disorders.

Retinopathy of prematurity (ROP) is the leading cause of blindness in neonates. Inflammation, in particular interleukin-1β (IL-1β), 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., (Copyright © 2020 Sayah, Zhou, Omri, Mazzaferri, Quiniou, Wirth, Côté, Dabouz, Desjarlais, Costantino and Chemtob.)

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% O 2 from P5 to P10) during the phase of microvascular degeneration. Similarly, human retinal microvascular endothelial cells (HRMEC) subjected to hyperoxia (80% O 2 ) 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., (Copyright © 2020 Desjarlais, Wirth, Rivera, Lahaie, Dabouz, Omri, Ruknudin, Borras and Chemtob.)

Background: CD36, a member of the class B scavenger receptor family, participates in Toll-like receptor signaling on mononuclear phagocytes (MP) and can promote sterile pathogenic inflammation. We here analyzed the effect of CD36 deficiency on retinal inflammation and photoreceptor degeneration, the hallmarks of age-related macular degeneration (AMD), that characterize Cx3cr1 -/- mice. Methods: We analyzed subretinal MP accumulation, and cone- and rod-degeneration in light-challenged and aged, CD36 competent or deficient, hyper-inflammatory Cx3cr1 -/- mice, using histology and immune-stained retinal flatmounts. Monocytes (Mo) were subretinally adoptively transferred to evaluate their elimination rate from the subretinal space and Interleukin 6 (IL-6) secretion from cultured Mo-derived cells (MdCs) of the different mouse strains were analyzed. Results: CD36 deficient Cx3cr1 -/- mice were protected against age- and light-induced subretinal inflammation and associated cone and rod degeneration. CD36 deficiency in Cx3cr1 -/- MPs inhibited their prolonged survival in the immune-suppressive subretinal space and reduced the exaggerated IL-6 secretion observed in Cx3cr1 -/- MPs that we previously showed leads to increased subretinal MP survival. Conclusion: Cd36 deficiency significantly protected hyperinflammatory Cx3cr1 -/- mice against subretinal MP accumulation and associated photoreceptor degeneration. The observed CD36-dependent induction of pro-inflammatory IL-6 might be at least partially responsible for the prolonged MP survival in the immune-suppressive environment and its pathological consequences on photoreceptor homeostasis., (Copyright © 2020 Lavalette, Conart, Touhami, Roubeix, Houssier, Augustin, Raoul, Combadière, Febbraio, Ong, Chemtob, Sahel, Delarasse, Guillonneau and Sennlaub.)

Dynamic chirality influences numerous processes in nature from protein folding to catalysis. Azapeptides are peptidomimetics possessing semicarbazide residues that can interconvert between sp 2 and sp 3 hybridization, resulting in stereodynamic interconversions of pseudo- R and - S -configurations by means of a planar intermediate. Cyclic azapeptides have shown unprecedented binding affinity to the cluster of differentiation 36 receptor (CD36) and ability to mitigate macrophage-driven inflammation by modulation of the toll-like receptor 2/6 pathway. A novel approach to synthesize cyclic peptides via A 3 -macrocyclization has been used to make R - and S -configuration controls to study the relevance of semicarbazide hybridization for modulator activity. Nuclear magnetic resonance spectroscopy analysis of potent cyclic azapeptide CD36 modulators (e.g., 1 and 2 ) and related cyclic peptides demonstrated that binding affinity correlated with conformational rigidity, and a hybridization preference for sp 2 > S - > R -sp 3 semicarbazide nitrogen configuration was evaluated. Evidence of the active conformation and the relevance for dynamic chirality serve as insights for creating cyclic (aza)peptide CD36 modulators to curb inflammation.

N -Alkylation and N -acylation of the prostaglandin-F 2α allosteric modulator l-PDC31 were performed to install various alkyl, PEG and isoprenoid groups onto the l-enantiomer of the peptide. Among the different bio-conjugates studied, the N -dodecyl analog reduced prostaglandin-F 2α -induced mouse myometrium contractions ex vivo. Furthermore, N -dodecyl-l-PDC31 exhibited improved stability in a mouse serum assay, likely due to protection from protease degradation by the lipid chain.

Purpose: Targeting β-adrenergic receptor signaling with propranolol has emerged as a potential candidate to counteract choroidal neovascularization (CNV). Little is known of its effect on macrophages, which play a critical role in CNV. We investigated the effect of propranolol on angiogenic response of mononuclear phagocytes (MPs)., Methods: The angiogenic effect of propranolol was evaluated in laser-induced CNV model. Mice received intraperitoneal injections of propranolol (6 mg/kg/d) or vehicle. CNV area and inflammatory cells were determined respectively by using lectin staining and an anti-IBA-1 antibody on RPE/choroid flat mounts. Inflammatory gene expression was evaluated by quantitative (q) PCR analysis. Mechanisms of propranolol was studied in MP cell lines J774 and RAW264.7 and in primary peritoneal macrophages. Expression of pro- and antiangiogenic mediators was studied. In addition, effects of propranolol treatment of MPs was assessed on choroidal explant., Results: CNV was attenuated by propranolol and concomitantly associated with decreased inflammatory mediators IL-6 and TNFα, albeit with accumulation of (β-adrenoceptor harboring) MPs in the CNV area. Conditioned media from MPs preincubated with propranolol exerted antiangiogenic effects. Treatment of J774 confirmed the attenuation of inflammatory response to propranolol and increased cleaved caspase-3 on choroidal explant. We found that propranolol increased pigment epithelium-derived factor (PEDF) expression in MPs. Trapping of PEDF with an antibody abrogated antiangiogenic effects of propranolol. PEDF was also detected in CNV-associated MPs., Conclusions: We hereby show that propranolol confers on MPs antiangiogenic properties by increasing PEDF expression, which complements its effects on vascular tissue resulting in inhibition of choroidal vasoproliferation in inflammatory conditions. The study supports possible use of propranolol as a therapeutic modality for CNV.