Prenatal Alcohol Exposure Impairs the Placenta-Cortex Transcriptomic Signature, Leading to Dysregulation of Angiogenic Pathways.

Although alcohol consumption during pregnancy is a major cause of behavioral and learning disabilities, most FASD infants are late- or even misdiagnosed due to clinician's difficulties achieving early detection of alcohol-induced neurodevelopmental impairments. Neuroplacentology has emerged as a new field of research focusing on the role of the placenta in fetal brain development. Several studies have reported that prenatal alcohol exposure (PAE) dysregulates a functional placenta-cortex axis, which is involved in the control of angiogenesis and leads to neurovascular-related defects. However, these studies were focused on PlGF, a pro-angiogenic factor. The aim of the present study is to provide the first transcriptomic "placenta-cortex" signature of the effects of PAE on fetal angiogenesis. Whole mouse genome microarrays of paired placentas and cortices were performed to establish the transcriptomic inter-organ "placenta-cortex" signature in control and PAE groups at gestational day 20. Genespring comparison of the control and PAE signatures revealed that 895 and 1501 genes were only detected in one of two placenta-cortex expression profiles, respectively. Gene ontology analysis indicated that 107 of these genes were associated with vascular development, and String protein-protein interaction analysis showed that they were associated with three functional clusters. PANTHER functional classification analysis indicated that "intercellular communication" was a significantly enriched biological process, and 27 genes were encoded for neuroactive ligand/receptors interactors. Protein validation experiments involving Western blot for one ligand-receptor couple (Agt/AGTR1/2) confirmed the transcriptomic data, and Pearson statistical analysis of paired placentas and fetal cortices revealed a negative correlation between placental Atg and cortical AGTR1, which was significantly impacted by PAE. In humans, a comparison of a 38WG control placenta with a 36WG alcohol-exposed placenta revealed low Agt immunolabeling in the syncytiotrophoblast layer of the alcohol case. In conclusion, this study establishes the first transcriptomic placenta-cortex signature of a developing mouse. The data show that PAE markedly unbalances this inter-organ signature; in particular, several ligands and/or receptors involved in the control of angiogenesis. These data support that PAE modifies the existing communication between the two organs and opens new research avenues regarding the impact of placental dysfunction on the neurovascular development of fetuses. Such a signature would present a clinical value for early diagnosis of brain defects in FASD.

Influence of Probiotics Administration Before Liver Resection in Patients with Liver Disease: A Randomized Controlled Trial.

BACKGROUND: By inhibiting the growth of pathogenic bacteria and modulating the local intestinal immune system, probiotics may reduce bacterial translocation and systemic endotoxaemia, factors partially responsible for post-operative complications following liver resection for hepatocellular carcinoma in patients with cirrhosis. METHODS: Patients with resectable hepatocellular carcinoma developed in the setting of chronic liver disease were prospectively divided into two equal-sized groups: one receiving probiotic treatment 14 days prior to surgery and the other receiving placebo. The primary endpoint was the level of circulating endotoxins after hepatectomy. Secondary endpoints were systemic inflammation (inflammatory cytokine levels), post-operative liver function and overall post-operative complication rate. RESULTS: From May 2013 to December 2018, 64 patients were randomized, and 54 patients were included in the analysis, 27 in each arm. No significant change in endotoxin levels was observed over time in either group (P = 0.299). No difference between the groups in terms of post-operative liver function and overall complication rates was observed. The only differences observed were significant increases in the levels of TNFalpha (P = 0.019) and interleukin 1-b (P = 0.028) in the probiotic group in the post-operative period. CONCLUSION: Contrary to the modest data reported in the literature, the administration of probiotics before minor liver resection for hepatocellular carcinoma developed in the setting of compensated chronic liver disease does not seem to have an impact on circulating endotoxin levels or post-operative complication rates. TRIAL REGISTRATION: Trial registration: NCT02021253.

In utero alcohol exposure exacerbates endothelial protease activity from pial microvessels and impairs GABA interneuron positioning.

In utero alcohol exposure can induce severe neurodevelopmental disabilities leading to long-term behavioral deficits. Because alcohol induces brain defects, many studies have focused on nervous cells. However, recent reports have shown that alcohol markedly affects cortical angiogenesis in both animal models and infants with fetal alcohol spectrum disorder (FASD). In addition, the vascular system is known to contribute to controlling gamma-aminobutyric acid (GABA)ergic interneuron migration in the developing neocortex. Thus, alcohol-induced vascular dysfunction may contribute to the neurodevelopmental defects in FASD. The present study aimed at investigating the effects of alcohol on endothelial activity of pial microvessels. Ex vivo experiments on cortical slices from mouse neonates revealed that in endothelial cells from pial microvessels acute alcohol exposure inhibits both glutamate-induced calcium mobilization and activities of matrix metalloproteinase-9 (MMP-9) and tissue plasminogen activator (tPA). The inhibitory effect of alcohol on glutamate-induced MMP-9 activity was abrogated in tPA-knockout and Grin1flox/VeCadcre mice suggesting that alcohol interacts through the endothelial NMDAR/tPA/MMP-9 vascular pathway. Contrasting with the effects from acute alcohol exposure, in mouse neonates exposed to alcohol in utero during the last gestational week, glutamate exacerbated both calcium mobilization and endothelial protease activities from pial microvessels. This alcohol-induced vascular dysfunction was associated with strong overexpression of the N-methyl-d-aspartate receptor subunit GluN1 and mispositioning of the Gad67-GFP interneurons that normally populate the superficial cortical layers. By comparing several human control fetuses with a fetus chronically exposed to alcohol revealed that alcohol exposure led to mispositioning of the calretinin-positive interneurons, whose density was decreased in the superficial cortical layers II-III and increased in deepest layers. This study provides the first mechanistic and functional evidence that alcohol impairs glutamate-regulated activity of pial microvessels. Endothelial dysfunction is characterized by altered metalloproteinase activity and interneuron mispositioning, which was also observed in a fetus with fetal alcohol syndrome. These data suggest that alcohol-induced endothelial dysfunction may contribute in ectopic cortical GABAergic interneurons, that has previously been described in infants with FASD.

Clinical and Molecular Characterization of Patients with Mucopolysaccharidosis Type I in an Algerian Series.

Mucopolysaccharidoses (MPS's) represent a subgroup of lysosomal storage diseases related to a deficiency of enzymes that catalyze glycosaminoglycans degradation. Mucopolysaccharidosis type I (MPS I) is a rare autosomal recessive disorder caused by a deficiency of α-l-iduronidase encoded by the IDUA gene. Partially degraded heparan sulfate and dermatan sulfate accumulate progressively and lead to multiorgan dysfunction and damage. The aim of this study is to describe the clinical, biochemical, and molecular characteristics of 13 Algerian patients from 11 distinct families. MPS I diagnosis was confirmed by molecular study of the patients' IDUA gene. Clinical features at the diagnosis and during the follow-up are reported. Eighty-four percent of the studied patients presented with a mild clinical phenotype. Molecular study of the IDUA gene allowed the characterization of four pathological variations at the homozygous or compound heterozygote status: IDUA NM_00203.4:c.1598C>G-p.(Pro533Arg) in 21/26 alleles, IDUA NM_00203.4:c.532G>A-p.(Glu178Lys) in 2/26 alleles, IDUA NM_00203.4:c.501C>G-p.(Tyr167*) in 2/26 alleles, and IDUA NM_00203. 4: c.1743C>G-p.(Tyr581*) in 1/26 alleles. This molecular study unveils the predominance of p.(Pro533Arg) variation in our MPS I patients. In this series, the occurrence of some clinical features linked to the Scheie syndrome is consistent with the literature, such as systematic valvulopathies, corneal opacity, and umbilical hernia; however, storage signs, facial dysmorphic features, and hepatomegaly were more frequent in our series. Screening measures for these debilitating diseases in highly consanguineous at-risk populations must be considered a priority health problem.

High levels of soluble serum hemojuvelin in patients with congenital dyserythropoietic anemia type I.

OBJECTIVE: Congenital dyserythropoietic anemia (CDA) is a rare group of red blood cell disorders with ineffective erythropoiesis and secondary hemochromatosis. Inappropriate suppression of hepcidin and high levels of growth differentiation factor 15 (GDF15) have been described in CDA I and II patients, probably contributing to secondary hemochromatosis. Hemojuvelin (HJV) is an important regulator of serum hepcidin, while soluble form of HJV (s-HJV) competitively down-regulates hepcidin. METHODS: We determined the soluble hemojuvelin (s-HJV) levels in 17 patients with CDA I and in 17 healthy volunteers (HV) and looked for correlations with other parameters of iron overload and erythropoiesis. RESULTS: Significantly higher levels of s-HJV were found in patients (2.32 ± 1.40 mg/L) compared with healthy volunteers (0. 69 ± 0.44 mg/L) (P = 0.001). Western blot analysis confirmed the presence of high levels of s-HJV in CDA I patients. s-HJV positively correlated with serum ferritin, erythropoietin, soluble transferrin receptor, and GDF15 and negatively correlated with hepcidin to ferritin ratios. CONCLUSIONS: We for the first time documented high levels of serum s-HJV in CDA I patients, suggesting that it may contribute to iron loading pathology in CDA I and eventually in other anemias with ineffective erythropoiesis.

Serum hepcidin levels and muscle iron proteins in humans injected with low- or high-dose erythropoietin.

Inhibition of hepcidin expression by erythropoietic signals is of great physiological importance; however, the inhibitory pathways remain poorly understood. To investigate (i) the direct effect of erythropoietin (Epo) and (ii) the contribution of putative mediators on hepcidin repression, healthy volunteers were injected with a single dose of Epo, either low (63 IU/kg, n = 8) or high (400 IU/kg, n = 6). Low-dose Epo provoked hepcidin down-modulation within 24 h; the effect was not immediate as hepcidin circadian variations were still present following injection. High-dose Epo induced no additional effect on the hepcidin response, that is hepcidin diurnal fluctuations were not abolished in spite of extremely high Epo levels. We did not find significant changes in putative mediators of hepcidin repression, such as transferrin saturation, soluble transferrin receptor, or growth differentiation factor 15. Furthermore, the potential hepcidin inhibitor, soluble hemojuvelin, was found unaltered by Epo stimulation. This finding was consistent with the absence of signs of iron deficiency observed at the level of skeletal muscle tissue. Our data suggest that hepcidin repression by erythropoietic signals in humans may not be controlled directly by Epo, but mediated by a still undefined factor.

Obesity does not influence hepcidin and hemojuvelin levels in hemodialysis patients.

BACKGROUND/AIMS: Clinical studies have shown increased levels of hepcidin causing functional iron deficiency in obese individuals. This study examined whether obesity contributes to increased hepcidin and hemojuvelin levels in adult hemodialysis patients. METHODS: In a case-control design, 37 obese [body mass index (BMI) >30 kg/m(2)] stable hemodialysis patients and 37 patients with normal BMI (20-25 kg/m(2)), matched for age, gender and race, who fulfilled a strict set of inclusion and exclusion criteria were included in the study. Serum hepcidin and hemojuvelin, markers of iron status and inflammation, and routine hematological and biochemical variables were measured on samples obtained prior to the midweek hemodialysis session. RESULTS: Obese and nonobese patients (BMI 35.1 ± 3.4 vs. 22.8 ± 1.4 kg/m(2); p < 0.001) were similar with regard to basic comorbidities and use of erythropoietin and iron. Levels of hemoglobin, hypochromic red cells and reticulocytes were similar in the two groups. Serum iron and transferrin saturation levels were on the low side and not different between obese and lean individuals; total iron-binding capacity showed a trend towards higher levels in obese patients (48.4 ± 8.3 vs. 44.9 ± 7.4 µmol/l; p = 0.065). Levels of serum ferritin (651 ± 302 vs. 705 ± 327 µg/l; p = 0.46), hepcidin (118.3 ± 67.7 vs. 119.3 ± 78.0 ng/ml; p = 0.95) and hemojuvelin (1.90 ± 1.11 vs. 1.94 ± 1.24 mg/l; p = 0.90) were high but similar between the two groups. Serum hepcidin showed a significant correlation only with ferritin (r = 0.287, p = 0.013). CONCLUSIONS: Hepcidin and hemojuvelin levels are already considerably elevated in dialysis patients, but obesity does not have an additional impact. Further studies should examine whether increased weight contributes towards hepcidin elevation in predialysis individuals, in whom there is a lesser burden of systemic inflammation.

In Utero Alcohol Exposure Impairs Retinal Angiogenesis and the Microvessel-Associated Positioning of Calretinin Interneurons.

In addition to brain disorders, which constitute a devastating consequence of prenatal alcohol exposure (PAE), eye development is also significantly affected. Given that the retina is a readily accessible part of the central nervous system, a better understanding of the impact of ethanol on retinal development might provide ophthalmological landmarks helpful for early diagnosis of fetal alcohol syndrome. This study aimed to provide a fine morphometric and cellular characterization of the development of retinal microvasculature and neurovascular interactions in a mouse model of fetal alcohol spectrum disorder (FASD). The data revealed that PAE impaired superficial vascular plexus development. In particular, progression of the vascular migration front was significantly decreased in PAE retinas, supporting a delay in plexus progression. Moreover, a significant decrease in the vessel density and number of perforating vessels was quantified in PAE mice, supporting less angiogenesis. The present study provides also the first evidence of a close interaction between migrating calretinin-positive interneurons and perforating microvessels in the inner nuclear layer of the developing retina. This neurovascular association was significantly impaired by PAE. Moreover, projections of amacrine cells were abnormally distributed and densified in stratum S1 and S2. In humans, comparison of a five-month-old control infant with a three-month-old alcohol-exposed case revealed a similar mispositioning of calretinin-positive interneurons. This opens new research avenues regarding a neurovascular contribution in the deleterious effects of alcohol in the developing retina and support that ophthalmological examination could become a promising approach for early detection of alcohol-exposed infants presenting with neurovascular brain defects.

Expression of placental CD146 is dysregulated by prenatal alcohol exposure and contributes in cortical vasculature development and positioning of vessel-associated oligodendrocytes.

Recent data showed that prenatal alcohol exposure (PAE) impairs the "placenta-brain" axis controlling fetal brain angiogenesis in human and preclinical models. Placental growth factor (PlGF) has been identified as a proangiogenic messenger between these two organs. CD146, a partner of the VEGFR-1/2 signalosome, is involved in placental angiogenesis and exists as a soluble circulating form. The aim of the present study was to investigate whether placental CD146 may contribute to brain vascular defects described in fetal alcohol spectrum disorder. At a physiological level, quantitative reverse transcription polymerase chain reaction experiments performed in human placenta showed that CD146 is expressed in developing villi and that membrane and soluble forms of CD146 are differentially expressed from the first trimester to term. In the mouse placenta, a similar expression pattern of CD146 was found. CD146 immunoreactivity was detected in the labyrinth zone and colocalized with CD31-positive endothelial cells. Significant amounts of soluble CD146 were quantified by ELISA in fetal blood, and the levels decreased after birth. In the fetal brain, the membrane form of CD146 was the majority and colocalized with microvessels. At a pathophysiological level, PAE induced marked dysregulation of CD146 expression. The soluble form of CD146 decreased in both placenta and fetal blood, whereas it increased in the fetal brain. Similarly, the expression of several members of the CD146 signalosome, such as VEGFR2 and PSEN, was differentially impaired between the two organs by PAE. At a functional level, targeted repression of placental CD146 by in utero electroporation (IUE) of CRISPR/Cas9 lentiviral plasmids resulted in (i) a decrease in cortical vessel density, (ii) a loss of radial vascular organization, and (iii) a reduced density of oligodendrocytes. Statistical analysis showed that the more the vasculature was impaired, the more the cortical oligodendrocyte density was reduced. Altogether, these data support that placental CD146 contributes to the proangiogenic "placenta-brain" axis and that placental CD146 dysfunction contributes to the cortical oligo-vascular development. Soluble CD146 would represent a promising placental biomarker candidate representative of alcohol-induced neurovascular defects in neonates, as recently suggested by PlGF (patents WO2016207253 and WO2018100143).

NO-dependent protective effect of VEGF against excitotoxicity on layer VI of the developing cerebral cortex.

In industrialized countries, cerebral palsy affects 2.5‰ of preterm and term infants. At a neurochemical level, the massive release of glutamate constitutes a major process leading to excitotoxicity and neonatal brain lesions. Previous studies, conducted in the laboratory, revealed that, in (δ/δ)VEGF(A) transgenic mice, glutamate-induced brain lesions are exacerbated suggesting that VEGF(A) could play a protective action against excitotoxicity. Using a model of cultured cortical brain slices, the aim of the study was to characterize the central effects of VEGF against glutamate-induced excitotoxicity in neonates. Exposure of brain slices to glutamate induced a strong increase of necrotic cell death in the deep cortical layer VI and a decrease of apoptotic death in superficial layers II-IV. When administered alone, a 6-h treatment with VEGF(A) had no effect on both apoptotic and necrotic deaths. In contrast, VEGF(A) abolished the glutamate-induced necrosis observed in layer VI. While MEK and PI3-K inhibitors had no effect on the protective action of VEGF(A), L-NAME, a pan inhibitor of NOS, abrogated the effect of VEGF(A) and exacerbated the excitotoxic action of glutamate. Calcimetry experiments performed on brain slices revealed that VEGF(A) reduced the massive calcium influx induced by glutamate in layer VI and this effect was blocked by L-NAME. Neuroprotective effect of VEGF(A) was also blocked by LNIO and NPLA, two inhibitors of constitutive NOS, while AGH, an iNOS inhibitor, had no effect. Nitrite measurements, electron paramagnetic resonance spectroscopy and immunohistochemistry indicated that glutamate was a potent inducer of NO production via activation of nNOS in the cortical layer VI. In vivo administration of nNOS siRNA promoted excitotoxicity and mimicked the effects of L-NAME, LNIO and NPLA. A short-term glutamate treatment increased nNOS Ser1412 phosphorylation, while a long-term exposure inhibited nNOS/NR2B protein-protein interactions. Altogether, these findings indicate that, in deep cortical layers of mice neonates, glutamate stimulates nNOS activity. Contrasting with mature brain, NO production induced by high concentrations of glutamate is neuroprotective and is required for the anti-necrotic effect of VEGF(A).

Intestinal DMT1 cotransporter is down-regulated by hepcidin via proteasome internalization and degradation.

BACKGROUNDS & AIMS: The mechanism by which hepcidin regulates iron export from macrophages has been well established and is believed to involve degradation of ferroportin. However, in the small intestine, hepcidin's mechanisms of action are not known. We studied human polarized intestinal (Caco-2/TC7) cells and mouse duodenal segments, ex vivo, to investigate the molecular mechanisms by which hepcidin down-regulates intestinal transepithelial iron transport. METHODS: Iron transport was analyzed using 55FeNTA. Expression of Divalent Metal Transporter 1 (DMT1) and ferroportin was evaluated by reverse-transcription quantitative polymerase chain reaction and immunoblotting. Videomicroscopy analysis was performed on live cells that expressed either DMT1 or ferroportin fused to green fluorescent protein. RESULTS: In Caco-2/TC7 cells, physiologic doses of hepcidin (50-1000 nmol/L) inhibited transport of 55Fe in a dose-dependent manner; a half-maximum effect was observed at 75-100 nmol/L. However, 200 nmol/L hepcidin induced a significant decrease in DMT1 protein expression but no change in ferroportin protein levels, unlike macrophages. This result was confirmed ex vivo in isolated duodenal segments: 200 nmol/L hepcidin induced a significant reduction in iron transport and DMT1 protein levels but no change in ferroportin levels. In Caco-2/TC7 cells, the effect of hepcidin on the DMT1 protein level was completely abolished in the presence of a proteasome inhibitor (MG-132); DMT1 ubiquitination was induced by the addition of hepcidin. CONCLUSIONS: An acute increase in hepcidin concentration reduces intestinal iron absorption through ubiquitin-dependent proteasome degradation of DMT1.

Hepcidin and Iron Deficiency in Women One Year after Sleeve Gastrectomy: A Prospective Cohort Study.

Iron deficiency with or without anemia, needing continuous iron supplementation, is very common in obese patients, particularly those requiring bariatric surgery. The aim of this study was to address the impact of weight loss on the rescue of iron balance in patients who underwent sleeve gastrectomy (SG), a procedure that preserves the duodenum, the main site of iron absorption. The cohort included 88 obese women; sampling of blood and duodenal biopsies of 35 patients were performed before and one year after SG. An analysis of the 35 patients consisted in evaluating iron homeostasis including hepcidin, markers of erythroid iron deficiency (soluble transferrin receptor (sTfR) and erythrocyte protoporphyrin (PPIX)), expression of duodenal iron transporters (DMT1 and ferroportin) and inflammatory markers. After surgery, sTfR and PPIX were decreased. Serum hepcidin levels were increased despite the significant reduction in inflammation. DMT1 abundance was negatively correlated with higher level of serum hepcidin. Ferroportin abundance was not modified. This study shed a new light in effective iron recovery pathways after SG involving suppression of inflammation, improvement of iron absorption, iron supply and efficiency of erythropoiesis, and finally beneficial control of iron homeostasis by hepcidin. Thus, recommendations for iron supplementation of patients after SG should take into account these new parameters of iron status assessment.

Immunoassay for human serum hemojuvelin.

BACKGROUND: Hemojuvelin, a critical regulator of iron homeostasis, is involved in the regulation of hepcidin expression and iron homeostasis. It is expressed both as a membrane-bound form and as a soluble one. Serum hemojuvelin can be produced by secretion following furin cleavage or by proteolytic cleavage of the membrane-bound form by matriptase 2 (TMPRSS6). These forms contribute to down-regulation of hepcidin expression upon iron deficiency or hypoxia. This study describes the development and validation of the first enzyme-linked immunosorbent assay for hemojuvelin in human serum. DESIGN AND METHODS: This assay is based on the use of a recombinant human repulsive guidance molecule-c peptide and a polyclonal antibody against hemojuvelin able to recognize the recombinant peptide and the native soluble hemojuvelin by immunoprecipitation. RESULTS: The enzyme-linked immunosorbent assay was validated and appeared to be a robust method with intra- and inter-coefficients of variance ranging from 2.6% to 15%. The assay was able to quantify hemojuvelin levels in a control population within a range from 0.88 to 1.14 mg/L. Patients with iron-refractory iron-deficiency anemia with a mutation in the TMPRSS6 gene were found to have lower levels of circulating hemojuvelin than those in healthy patients. The enzyme-linked immunosorbent assay also showed that soluble hemojuvelin levels were significantly higher in patients with anemia of chronic disease than in control individuals. CONCLUSIONS: This enzyme-linked immunosorbent assay has a good specificity and sensitivity for the quantification of soluble hemojuvelin in human serum and could be a valuable aid to understanding the physiological role of this protein.

[Fetal alcohol exposure: when placenta would help to the early diagnosis of child brain impairments]. / Alcoolisation fœtale - Le placenta au secours du diagnostic précoce des troubles du développement cérébral de l'enfant.

Alcohol consumption during pregnancy constitutes a major cause of neurodevelopmental and behavioral disabilities. Whereas it is possible for clinicians to establish a perinatal diagnosis of fetal alcohol syndrome, the more severe expression of fetal alcohol spectrum disorder (FASD), most FASD children are late or mis-diagnosed due to a lack of clear morphological and neurodevelopmental abnormalities. Several precious years of care are consequently lost. Recent data revealed a functional placenta-brain axis involved in the control of the fetal brain angiogenesis which is impaired by in utero alcohol exposure. Because in the developing fetal brain a correct angiogenesis is required for a correct neurodevelopment, these preclinical and clinical advances pave the way for a new generation of placental biomarkers for early diagnosis of FASD.

TITLE: Alcoolisation fœtale - Le placenta au secours du diagnostic précoce des troubles du développement cérébral de l'enfant. ABSTRACT: La consommation d'alcool au cours de la grossesse constitue une cause majeure de troubles du comportement et de handicap. Alors qu'il est possible pour un clinicien d'établir un diagnostic néonatal du syndrome d'alcoolisation fœtale, l'atteinte la plus sévère des troubles causés par l'alcoolisation fœtale (TCAF), une grande majorité des enfants échappe à un diagnostic précoce en raison de l'absence d'anomalies morphologiques évidentes. Plusieurs années de prise en charge sont alors perdues. Des avancées récentes ont permis d'établir l'existence d'un axe fonctionnel placenta-cerveau impliqué dans le contrôle de l'angiogenèse cérébrale, qui se trouve dérégulé chez les enfants exposés in utero à l'alcool. Une angiogenèse cérébrale normale étant un prérequis à l'établissement d'un neurodéveloppement correct, ces avancées ouvrent la voie à l'identification d'une nouvelle génération de biomarqueurs placentaires d'atteinte cérébrale pour le diagnostic précoce des enfants TCAF.

Glutamine and interleukin-1beta interact at the level of Sp1 and nuclear factor-kappaB to regulate argininosuccinate synthetase gene expression.

We previously demonstrated that the expression of the argininosuccinate synthetase (ASS) gene, a key step in nitric oxide production, is stimulated either by interleukin-1beta[Brasse-Lagnel et al. (2005) Biochimie 87, 403-9] or by glutamine in Caco-2 cells [Brasse-Lagnel et al. (2003) J. Biol. Chem. 278, 52504-10], through the activation of transcription factors nuclear factor-kappaB and Sp1, respectively. In these cells, the fact that glutamine stimulated the expression of a gene induced by pro-inflammatory factors appeared paradoxical as the amino acid is known to exert anti-inflammatory properties in intestinal cells. We therefore investigated the effect of simultaneous addition of both glutamine and interleukin-1beta on ASS gene expression in Caco-2 cells. In the presence of both compounds for 4 h, the increases in ASS activity, protein amount and mRNA level were almost totally inhibited, implying a reciprocal inhibition between the amino acid and the cytokine. The inhibition was exerted at the level of the transcription factors Sp1 and nuclear-kappaB: (a) interleukin-1beta inhibited the glutamine-stimulated DNA-binding of Sp1, which might be related to a decrease of its glutamine-induced O-glycosylation, and (b) glutamine induced per se a decrease in the amount of nuclear p65 protein without affecting the stimulating effect of interleukin-1beta on nuclear factor-kappaB, which might be related to the metabolism of glutamine into glutamate. The present results constitute the first demonstration of a reciprocal inhibition between the effects of an amino acid and a cytokine on gene expression, and provide a molecular basis for the protective role of glutamine against inflammation in the intestine.

Enhancing the diagnosis of fabry disease in cardiology with a targeted information: a before-after control-impact study.

BACKGROUND: Cardiac complications in Fabry disease are frequent and dominated by a high frequency of left ventricular hypertrophy; therefore, cardiologists may have an essential role in screening for this disease. Providing cardiologists with targeted information on Fabry disease would be valuable and could reduce both diagnostic and therapeutic delays. The aim of this study was to evaluate the efficiency of such strategy for Fabry screening. METHODS: We conducted a before-after control-impact study by comparing observations made before and after targeted information on Fabry disease among cardiologists. The information on Fabry disease consisted of (1) an educational booklet, (2) oral information and (3) screening kits. The programme was evaluated at the end of a 12-month study period. RESULTS: Forty-two cardiologists participated to this study. None of them had conducted screening test and new diagnostic for Fabry disease in the 3 years prior the information. After the information, screening with dried blood spots was performed in 55 patients (ranged 18-77 years, men: 39) with cardiac monitoring for supposed sarcomeric hypertrophic cardiomyopathy (n=41) or unexplained left ventricular hypertrophy (n=14) from January 2015 to January 2016. Two new cases of Fabry disease were diagnosed (3.4%) in two men (ages 58 and 51 years). The information was deemed relevant in both content and structure and was deemed useful for everyday practice. CONCLUSION: Cardiologists valued the targeted information on Fabry disease. This information had a direct clinical impact by allowing the diagnosis of two new families with Fabry disease.

Prenatal alcohol exposure impairs autophagy in neonatal brain cortical microvessels.

Brain developmental lesions are a devastating consequence of prenatal alcohol exposure (PAE). We recently showed that PAE affects cortical vascular development with major effects on angiogenesis and endothelial cell survival. The underlying molecular mechanisms of these effects remain poorly understood. This study aimed at characterizing the ethanol exposure impact on the autophagic process in brain microvessels in human fetuses with fetal alcohol syndrome (FAS) and in a PAE mouse model. Our results indicate that PAE induces an increase of autophagic vacuole number in human fetal and neonatal mouse brain cortical microvessels. Subsequently, ex vivo studies using green fluorescent protein (GFP)-LC3 mouse microvessel preparations revealed that ethanol treatment alters autophagy in endothelial cells. Primary cultures of mouse brain microvascular endothelial cells were used to characterize the underlying molecular mechanisms. LC3 and p62 protein levels were significantly increased in endothelial cells treated with 50 mM ethanol. The increase of autophagic vacuole number may be due to excessive autophagosome formation associated with the partial inhibition of the mammalian target of rapamycin pathway upon ethanol exposure. In addition, the progression from autophagosomes to autolysosomes, which was monitored using autophagic flux inhibitors and mRFP-EGFP vector, showed a decrease in the autolysosome number. Besides, a decrease in the Rab7 protein level was observed that may underlie the impairment of autophagosome-lysosome fusion. In addition, our results showed that ethanol-induced cell death is likely to be mediated by decreased mitochondrial integrity and release of apoptosis-inducing factor. Interestingly, incubation of cultured cells with rapamycin prevented ethanol effects on autophagic flux, ethanol-induced cell death and vascular plasticity. Taken together, these results are consistent with autophagy dysregulation in cortical microvessels upon ethanol exposure, which could contribute to the defects in angiogenesis observed in patients with FAS. Moreover, our results suggest that rapamycin represents a potential therapeutic strategy to reduce PAE-related brain developmental disorders.

Biphasic effect of IL-1beta on the activity of argininosuccinate synthetase in Caco-2 cells. Involvement of nitric oxide production.

The expression of the argininosuccinate synthetase gene (ASS), the limiting enzyme of arginine synthesis, was previously shown to be rapidly induced by a short-term (4 h) exposure to IL-1beta in Caco-2 cells [Biochimie, 2005, 403-409]. The present report shows that, by contrast, a long-term (24 h) exposure to IL-1beta inhibited the ASS activity despite an increase in both specific mRNA level and protein amount, demonstrating a post-translational effect. Concerning the mechanism involved, we demonstrate that the inhibiting effect is linked to the production of nitric oxide (NO) induced by IL-1beta. Indeed, the inhibiting effect of IL-1beta was totally blocked in the presence of l-NMMA, an inhibitor of the inducible nitric oxide synthase, or by culturing the cells in an arginine-deprived medium. Moreover, a decrease in the ASS activity was induced by culturing the cells in the presence of SNAP, a NO donor. Conversely, blocking the action of NO by antioxidant agents, the stimulatory effect of IL-1beta on ASS activity was restored, as measured at 24 h. Finally, such an inhibiting effect of NO on ASS activity may be related, at least in part, to S-nitrosylation of the protein. The physiological relevance of the antagonistic effects of IL-1beta and NO on ASS is discussed.

IL-1beta stimulates argininosuccinate synthetase gene expression through NF-kappaB in Caco-2 cells.

Argininosuccinate synthetase (ASS) is limiting the arginine synthesis and can be stimulated by immunostimulants. We previously identified a putative NF-kappaB element in the human ASS gene promoter but its functionality was unknown (Husson et al., Eur. J. Biochem. 270 (2003) 1887). In the present study, using Caco-2 cells, a human enterocyte line, we demonstrate that IL-1beta rapidly induces the expression of the ASS gene at a transcriptional level through NF-kappaB activation. Using gel shift assay and double-strand oligonucleotide sequence of the identified putative NF-kappaB binding site of the ASS promoter, we provide evidence that NF-kappaB may functionally interact with this element.