Inhibition of Reactive Gliosis Prevents Neovascular Growth in the Mouse Model of Oxygen-Induced Retinopathy

Retinal neovascularization (NV) is a major cause of blindness in ischemic retinopathies. Previous investigations have indicated that ischemia upregulates GFAP and PDGF-B expression. GFAP overexpression is a hallmark of reactive gliosis (RG), which is the major pathophysiological feature of retinal damage. In addition, PDGF-B has been implicated in proliferative retinopathies. It was the aim of this study to gain insights on the possible pharmacological interventions to modulate PDGF-B and GFAP expression, and its influence on RG and NV. We used an array of assays to evaluate the effects of YC-1, a small molecule inhibitor of HIF-1 and a novel NO-independent activator of soluble guanylyl cyclase (sGC), on RG and NV, in vivo and in vitro. When compared to the DMSO-treated retinas, dual-intravitreal injections of YC-1, in vivo: (1) suppressed the development and elongation of neovascular sprouts in the retinas of the oxygen-induced retinopathy (OIR) mouse model; and (2) reduced ischemia-induced overexpression of GFAP and PDGF-B at the message (by 64.14±0.5% and 70.27±0.04%) and the protein levels (by 65.52±0.02% and 57.59±0.01%), respectively. In addition, at 100 µM, YC-1 treatment downregulated the hypoxia-induced overexpression of GFAP and PDGF-B at the message level in rMC-1 cells (by 71.42±0.02% and 75±0.03%), and R28 cells (by 58.62±0.02% and 50.00±0.02%), respectively; whereas, the protein levels of GFAP and PDGF-B were reduced (by 78.57±0.02% and 77.55±0.01%) in rMC-1cells, and (by 81.44±0.02% and 79.16±0.01%) in R28 cells, respectively. We demonstrate that YC-1 reversed RG during ischemic retinopathy via impairing the expression of GFAP and PDGF-B in glial cells. This is the first investigation that delves into the reversal of RG during ischemic retinal vasculopathies. In addition, the study reveals that YC-1 may exert promising therapeutic effects in the treatment of retinal and neuronal pathologies

The interplay of intracellular calcium and zinc ions in response to electric field stimulation in primary rat cortical neurons in vitro

Recent pharmacological studies demonstrate a role for zinc (Zn2+) in shaping intracellular calcium (Ca2+) dynamics and vice versa in excitable cells including neurons and cardiomyocytes. Herein, we sought to examine the dynamic of intracellular release of Ca2+ and Zn2+ upon modifying excitability of primary rat cortical neurons using electric field stimulation (EFS) in vitro. We show that exposure to EFS with an intensity of 7.69 V/cm induces transient membrane hyperpolarization together with transient elevations in the cytosolic levels of Ca2+ and Zn2+ ions. The EFS-induced hyperpolarization was inhibited by prior treatment of cells with the K+ channel opener diazoxide. Chemical hyperpolarization had no apparent effect on either Ca2+ or Zn2+. The source of EFS-induced rise in Ca2+ and Zn2+ seemed to be intracellular, and that the dynamic inferred of an interplay between Ca2+ and Zn2+ ions, whereby the removal of extracellular Ca2+ augmented the release of intracellular Ca2+ and Zn2+ and caused a stronger and more sustained hyperpolarization. We demonstrate that Zn2+ is released from intracellular vesicles located in the soma, with major co-localizations in the lysosomes and endoplasmic reticulum. These studies further support the use of EFS as a tool to interrogate the kinetics of intracellular ions in response to changing membrane potential in vitro

Gender Dimorphism in Aspartame-Induced Impairment of Spatial Cognition and Insulin Sensitivity

Previous studies have linked aspartame consumption to impaired retention of learned behavior in rodents. Prenatal exposure to aspartame has also been shown to impair odor-associative learning in guinea pigs; and recently, aspartame-fed hyperlipidemic zebrafish exhibited weight gain, hyperglycemia and acute swimming defects. We therefore investigated the effects of chronic lifetime exposure to aspartame, commencing in utero, on changes in blood glucose parameters, spatial learning and memory in C57BL/6J mice. Morris Water Maze (MWM) testing was used to assess learning and memory, and a random-fed insulin tolerance test was performed to assess glucose homeostasis. Pearson correlation analysis was used to investigate the associations between body characteristics and MWM performance outcome variables. At 17 weeks of age, male aspartame-fed mice exhibited weight gain, elevated fasting glucose levels and decreased insulin sensitivity compared to controls (P<0.05). Females were less affected, but had significantly raised fasting glucose levels. During spatial learning trials in the MWM (acquisition training), the escape latencies of male aspartame-fed mice were consistently higher than controls, indicative of learning impairment. Thigmotactic behavior and time spent floating directionless was increased in aspartame mice, who also spent less time searching in the target quadrant of the maze (P<0.05). Spatial learning of female aspartame-fed mice was not significantly different from controls. Reference memory during a probe test was affected in both genders, with the aspartame-fed mice spending significantly less time searching for the former location of the platform. Interestingly, the extent of visceral fat deposition correlated positively with non-spatial search strategies such as floating and thigmotaxis, and negatively with time spent in the target quadrant and swimming across the location of the escape platform. These data suggest that lifetime exposure to aspartame, commencing in utero, may affect spatial cognition and glucose homeostasis in C57BL/6J mice, particularly in males

C4b Binding Protein Acts as an Innate Immune Effector Against Influenza A Virus

C4b Binding Protein (C4BP) is a major fluid phase inhibitor of the classical and lectin pathways of the complement system. Complement inhibition is achieved by binding to and restricting the role of activated complement component C4b. C4BP functions as a co-factor for factor I in proteolytic inactivation of both soluble and cell surface-bound C4b, thus restricting the formation of the C3-convertase, C4b2a. C4BP also accelerates the natural decay/dissociation of the C3 convertase. This makes C4BP a prime target for exploitation by pathogens to escape complement attack, as seen in Streptococcus pyogenes or Flavivirus. Here, we examined whether C4BP can act on its own in a complement independent manner, against pathogens. C4BP bound H1N1 and H3N2 subtypes of Influenza A Virus (IAV) most likely via multiple sites in Complement Control Protein (CCP) 1-2, 4-5, and 7-8 domains of its α-chain. In addition, C4BP CCP1-2 bound H3N2 better than H1N1. C4BP bound three IAV envelope proteins: Haemagglutinin (~70 kDa), Neuraminidase (~55 kDa), and Matrix protein 1 (~25kDa). C4BP suppressed H1N1 subtype infection into the lung epithelial cell line, A549, while it promoted infection by H3N2 subtype. C4BP restricted viral entry for H1N1 but had the opposite effect on H3N2, as evident from experiments using pseudo-typed viral particles. C4BP downregulated mRNA levels of pro-inflammatory IFN-α, IL-12, and NFκB in the case of H1N1, while it promoted a pro-inflammatory immune response by upregulating IFN- α, TNF-α, RANTES, and IL-6 in the case of H3N2. We conclude that C4BP differentially modulates the efficacy of IAV entry, and hence, replication in a target cell in a strain-dependent manner, and acts as an entry inhibitor for H1N1. Thus, CCP containing complement proteins such as factor H and C4BP may have additional defense roles against IAV that do not rely on the regulation of complement activation

Clinical, genetic, and functional characterization of the glycine receptor β-subunit A455P variant in a family affected by hyperekplexia syndrome

Hyperekplexia is a rare neurological disorder characterized by exaggerated startle response affecting newborns with the hallmark characteristics of hypertonia, apnea, and noise or touch-induced non-epileptic seizures. The genetic causes of the disease can vary and several associated genes and mutations have been reported to affect glycine receptors (GlyRs); however, the mechanistic links between GlyRs and hyperekplexia are not yet understood. Here, we describe a patient with hyperekplexia from a consanguineous family. Extensive genetic screening using exome sequencing coupled with autozygome analysis and iterative filtering supplemented by in silico prediction identified that the patient carries the homozygous missense mutation A455P in GLRB, which encodes the GlyR β-subunit. To unravel the physiological and molecular effects of A455P on GlyRs, we used electrophysiology in a heterologous system as well as immunocytochemistry, confocal microscopy, and cellular biochemistry. We found a reduction in glycine-evoked currents in N2A cells expressing the mutation compared to wild type cells. Western blot analysis also revealed a reduced amount of GlyR β protein both in cell lysates and isolated membrane fractions. In line with the above observations, co-immunoprecipitation assays suggested that the GlyR α1-subunit retained co-assembly with βA455P to form membrane-bound heteromeric receptors. Finally, structural modelling showed that the A455P mutation affected the interaction between the GlyR β-subunit transmembrane domain 4 and the other helices of the subunit. Taken together, our study identifies and validates a novel loss-of-function mutation in GlyRs whose pathogenicity is likely to cause hyperekplexia in affected individuals

Sugar-sweetened carbonated beverage consumption correlates with BMI, waist circumference, and poor dietary choices in school children

<p>Abstract</p> <p>Background</p> <p>The prevalence of obesity and overweight is increasing globally. Frequently coexisting with under-nutrition in developing countries, obesity is a major contributor to chronic disease, and will become a serious healthcare burden especially in countries with a larger percentage of youthful population. 35% of the population of Saudi Arabia are under the age of 16, and adult dietary preferences are often established during early childhood years. Our objective was to examine the dietary habits in relation to body-mass-index (BMI) and waist circumference (W_C), together with exercise and sleep patterns in a cohort of male and female Saudi school children, in order to ascertain whether dietary patterns are associated with obesity phenotypes in this population.</p> <p>Methods</p> <p>5033 boys and 4400 girls aged 10 to 19 years old participated in a designed Food Frequency Questionnaire. BMI and W_C measurements were obtained and correlated with dietary intake.</p> <p>Results</p> <p>The overall prevalence of overweight and obesity was 12.2% and 27.0% respectively, with boys having higher obesity rates than girls (P ≤ 0.001). W_C and BMI was positively correlated with sugar-sweetened carbonated beverage (SSCB) intake in boys only. The association between male BMI and SSCB consumption was significant in a multivariate regression model (P < 0.0001). SSCB intake was positively associated with poor dietary choices in both males and females. Fast food meal intake, savory snacks, iced desserts and total sugar consumption correlated with SSCB intake in both boys (r = 0.39, 0.13, 0.10 and 0.52 respectively, P < 0.001) and girls (r = 0.45, 0.23, 0.16 and 0.55 respectively, P < 0.001). Older children reported eating significantly less fruit and vegetables than younger children; and less eggs, fish and cereals. Conversely, consumption of SSCB and sugar-sweetened hot beverages were higher in older versus younger children (P < 0.001). BMI and W_C were negatively correlated with hours of night-time sleep and exercise in boys, but only with night time sleep in girls, who also showed the lowest frequency of exercise.</p> <p>Conclusions</p> <p>A higher intake of SSCB is associated with poor dietary choices. Male SSCB intake correlates with a higher W_C and BMI. Limiting exposure to SSCB could therefore have a large public health impact.</p

Sex-dimorphism in Cardiac Nutrigenomics: effect of Trans fat and/or Monosodium Glutamate consumption

<p>Abstract</p> <p>Background</p> <p>A paucity of information on biological sex-specific differences in cardiac gene expression in response to diet has prompted this present nutrigenomics investigation.</p> <p>Sexual dimorphism exists in the physiological and transcriptional response to diet, particularly in response to high-fat feeding. Consumption of <it>Trans</it>-fatty acids (TFA) has been linked to substantially increased risk of heart disease, in which sexual dimorphism is apparent, with males suffering a higher disease rate. Impairment of the cardiovascular system has been noted in animals exposed to Monosodium Glutamate (MSG) during the neonatal period, and sexual dimorphism in the growth axis of MSG-treated animals has previously been noted. Processed foods may contain both TFA and MSG.</p> <p>Methods</p> <p>We examined physiological differences and changes in gene expression in response to TFA and/or MSG consumption compared to a control diet, in male and female C57BL/6J mice.</p> <p>Results</p> <p>Heart and % body weight increases were greater in TFA-fed mice, who also exhibited dyslipidemia (P < 0.05). Hearts from MSG-fed females weighed less than males (P < 0.05). 2-factor ANOVA indicated that the TFA diet induced over twice as many cardiac differentially expressed genes (DEGs) in males compared to females (P < 0.001); and 4 times as many male DEGs were downregulated including <it>Gata4</it>, <it>Mef2d </it>and <it>Srebf2</it>. Enrichment of functional Gene Ontology (GO) categories were related to transcription, phosphorylation and anatomic structure (P < 0.01). A number of genes were upregulated in males and downregulated in females, including pro-apoptotic histone deacetylase-2 (HDAC2). Sexual dimorphism was also observed in cardiac transcription from MSG-fed animals, with both sexes upregulating approximately 100 DEGs exhibiting sex-specific differences in GO categories. A comparison of cardiac gene expression between all diet combinations together identified a subset of 111 DEGs significant only in males, 64 DEGs significant in females only, and 74 transcripts identified as differentially expressed in response to dietary manipulation in both sexes.</p> <p>Conclusion</p> <p>Our model identified major changes in the cardiac transcriptional profile of TFA and/or MSG-fed mice compared to controls, which was reflected by significant differences in the physiological profile within the 4 diet groups. Identification of sexual dimorphism in cardiac transcription may provide the basis for sex-specific medicine in the future.</p

The nexus between VEGF and NFκB orchestrates a hypoxia-independent neovasculogenesis.

Nuclear Factor-Kappa B [NFκB] activation triggers the elevation of various pro-angiogenic factors that contribute to the development and progression of diabetic vasculopathies. It has been demonstrated that vascular endothelial growth factor [VEGF] activates NFκB signaling pathway. Under the ischemic microenvironments, hypoxia-inducible factor-1 [HIF-1] upregulates the expression of several proangiogenic mediators, which play crucial roles in ocular pathologies. Whereas YC-1, a soluble guanylyl cyclase [sGC] agonist, inhibits HIF-1 and NFκB signaling pathways in various cell and animal models. Throughout this investigation, we examined the molecular link between VEGF and NFκB under a hypoxia-independent microenvironment in human retinal microvascular endothelial cells [hRMVECs]. Our data indicate that VEGF promoted retinal neovasculogenesis via NFκB activation, enhancement of its DNA-binding activity, and upregulating NFκB/p65, SDF-1, CXCR4, FAK, αVβ3, α5β1, EPO, ET-1, and MMP-9 expression. Conversely, YC-1 impaired the activation of NFκB and its downstream signaling pathways, via attenuating IκB kinase phosphorylation, degradation and activation, and thus suppressing p65 phosphorylation, nuclear translocation, and inhibiting NFκB-DNA binding activity. We report for the first time that the nexus between VEGF and NFκB is implicated in coordinating a scheme that upregulates several pro-angiogenic molecules, which promotes retinal neovasculogenesis. Our data may suggest the potential use of YC-1 to attenuate the deleterious effects that are associated with hypoxia/ischemia-independent retinal vasculopathies