Publications by authors named "Loïc Blanchon"

40 Publications

Halogenated Agent Delivery in Porcine Model of Acute Respiratory Distress Syndrome via an Intensive Care Unit Type Device.

J Vis Exp 2020 09 24(163). Epub 2020 Sep 24.

Department of Perioperative Medicine, CHU Clermont-Ferrand; GReD, CNRS, INSERM, Université Clermont Auvergne; Division of Allergy, Pulmonary, and Critical Care Medicine, Vanderbilt University Medical Center.

Acute respiratory distress syndrome (ARDS) is a common cause of hypoxemic respiratory failure and death in critically ill patients, and there is an urgent need to find effective therapies. Preclinical studies have shown that inhaled halogenated agents may have beneficial effects in animal models of ARDS. The development of new devices to administer halogenated agents using modern intensive care unit (ICU) ventilators has significantly simplified the dispensing of halogenated agents to ICU patients. Because previous experimental and clinical research suggested potential benefits of halogenated volatiles, such as sevoflurane or isoflurane, for lung alveolar epithelial injury and inflammation, two pathophysiologic landmarks of diffuse alveolar damage during ARDS, we designed an animal model to understand the mechanisms of the effects of halogenated agents on lung injury and repair. After general anesthesia, tracheal intubation, and the initiation of mechanical ventilation, ARDS was induced in piglets via the intratracheal instillation of hydrochloric acid. Then, the piglets were sedated with inhaled sevoflurane or isoflurane using an ICU-type device, and the animals were ventilated with lung-protective mechanical ventilation during a 4 h period. During the study period, blood and alveolar samples were collected to evaluate arterial oxygenation, the permeability of the alveolar-capillary membrane, alveolar fluid clearance, and lung inflammation. Mechanical ventilation parameters were also collected throughout the experiment. Although this model induced a marked decrease in arterial oxygenation with altered alveolar-capillary permeability, it is reproducible and is characterized by a rapid onset, good stability over time, and no fatal complications. We have developed a piglet model of acid aspiration that reproduces most of the physiological, biological, and pathological features of clinical ARDS, and it will be helpful to further our understanding of the potential lung-protective effects of halogenated agents delivered through devices used for inhaled ICU sedation.
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http://dx.doi.org/10.3791/61644DOI Listing
September 2020

Human Amnion Epithelial Cells (AECs) Respond to the FSL-1 Lipopeptide by Engaging the NLRP7 Inflammasome.

Front Immunol 2020 7;11:1645. Epub 2020 Aug 7.

Genetics, Reproduction and Development (GReD) Laboratory, Clermont Auvergne University, CNRS UMR 6293, INSERM U1103, Translational Approach to Epithelial Injury and Repair Team, Clermont-Ferrand, France.

Inflammation is the leading mechanism involved in both physiological and pathological rupture of fetal membranes. Our aim was to obtain a better characterization of the inflammasome-dependent inflammation processes in these tissues, with a particular focus on the nucleotide-binding oligomerization domain (NOD)-like receptor, pyrin domain containing protein 7 (NLRP7) inflammasome. The presence of NLRP7 inflammasome actors [NLRP7, apoptosis-associated speck-like protein containing a CARD domain (ASC), and caspase-1] was confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR) in human amnion and choriodecidua at the three trimesters and at term. The protein concentrations were then determined by enzyme-linked immunosorbent assay in term tissues, with or without labor. The presence of and in human fetal membranes was investigated using a PCR approach. Human amnion epithelial cells (AECs) were treated for 4 or 20 h with fibroblast-stimulating lipopeptide-1 (FSL-1), a -derived ligand. Transcripts and proteins quantity was then measured by RT-quantitative PCR and Western blotting, respectively. NLRP7 and ASC colocalization was confirmed by immunofluorescence. Western blots allowed analysis of pro-caspase-1 and gasdermin D cleavage. NLRP7, ASC, and caspase-1 transcripts were expressed in both sheets of human fetal membranes during all pregnancy stages, but only ASC protein expression was increased with labor. In addition, and were detected for the first time in human fetal membranes. NLRP7 and caspase-1 transcripts, as well as NLRP7, ASC, and pro-caspase-1 protein levels, were increased in FSL-1-treated AECs. The NLRP7 inflammasome assembled around the nucleus, and pro-caspase-1 and gasdermin D were cleaved into their mature forms after FSL-1 stimulation. Two new mycoplasmas, and , were identified in human fetal membranes, and a lipopeptide derived from was found to induce NLRP7 inflammasome formation in AECs.
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http://dx.doi.org/10.3389/fimmu.2020.01645DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7426397PMC
April 2021

Occurrence of a RAGE-Mediated Inflammatory Response in Human Fetal Membranes.

Front Physiol 2020 25;11:581. Epub 2020 Jun 25.

CNRS, INSERM, GReD, Université Clermont Auvergne, Clermont-Ferrand, France.

Context: Sterile inflammation has been shown to play a key role in the rupture of the fetal membranes (FMs). Moreover, an early and exacerbated runaway inflammation can evolve into a preterm premature rupture of membranes and lead to potential preterm birth. In this context, we investigated the receptor for advanced glycation end products (RAGE), an axis implied in physiological sterile inflammation, in conjunction with two major ligands: AGEs and High-Mobility Group Box 1 (HMGB1). Our first objective was to determine the spatiotemporal expression profiles of the different actors of the RAGE-signaling axis in human FMs, including its intracellular adaptors Diaphanous-1 and Myd88. Our second goal was to evaluate the functionality of RAGE signaling in terms of FMs inflammation.

Methods: The presence of the actors (RAGE, HMGB1, Myd88, and Diaphanous-1) at the mRNA level was investigated by reverse transcription quantitative polymerase chain reaction (RT-qPCR) in the human amnion and choriodecidua at the three trimesters and at term. Measurements were conducted at two distinct zones: the zone of intact morphology (ZIM) and the zone of altered morphology (ZAM). Then, proteins were quantified using Western blot analysis, and their localization was evaluated by immunofluorescence in term tissues. In addition, pro-inflammatory cytokine secretion was quantified using a Multiplex assay after the treatment of amnion and choriodecidua explants with two RAGE ligands (AGEs and HMGB1) in the absence or presence of a RAGE inhibitor (SAGEs).

Results: The FMs expressed the RAGE-signaling actors throughout pregnancy. At term, RNA and protein overexpression of the RAGE, HMGB1, and Diaphanous-1 were found in the amnion when compared to the choriodecidua, and the RAGE was overexpressed in the ZAM when compared to the ZIM. The two RAGE ligands (AGEs and HMGB1) induced differential cytokine production (IL1β and TNFα) in the amnion and choriodecidua.

Conclusion: Considered together, these results indicate that RAGE signaling is present and functional in human FMs. Our work opens the way to a better understanding of FMs weakening dependent on a RAGE-based sterile inflammation.
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http://dx.doi.org/10.3389/fphys.2020.00581DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7330021PMC
June 2020

Study of sRAGE, HMGB1, AGE, and S100A8/A9 Concentrations in Plasma and in Serum-Extracted Extracellular Vesicles of Pregnant Women With Preterm Premature Rupture of Membranes.

Front Physiol 2020 23;11:609. Epub 2020 Jun 23.

Centre de Recherche du Centre Hospitalier Universitaire (CHU) de Québec-Université Laval, Québec City, QC, Canada.

Preterm premature rupture of membranes (PPROM), defined as rupture of fetal membranes prior to 37 weeks of gestation, complicates approximately 2-4% of pregnancies and is responsible for 40% of all spontaneous preterm births. PPROM arises from complex pathophysiological pathways with a key actor: inflammation. Sterile inflammation is a feature of senescence-associated fetal membrane maturity. During specific steps of sterile inflammation, cells also release highly inflammatory damage-associated molecular pattern markers (DAMPs), such as high-mobility group box 1 (HMGB1) or S100A8/A9, known to link and activate the receptor for advanced glycation end products (RAGE). The objective of this study was to measure longitudinally during pregnancy concentrations of the soluble form of RAGE (sRAGE) and its main ligands (AGE, HMGB1, S100A8/A9) in blood specimens. We studied 246 pregnant women (82 with PPROM and 164 matched control pregnant women without complications) from a cohort of 7,866 pregnant women recruited in the first trimester and followed during pregnancy until delivery. sRAGE, AGE, HMGB1, and S100A8/A9 concentrations were measured in plasma and in serum-extracted extracellular vesicles from first trimester (T1), second trimester (T2), and delivery (D). In plasma, we observed, in both PPROM and control groups, (i) a significant increase of HMGB1 concentrations between T1 vs. T2, T1 vs. D, but not between T2 vs. D; (ii) a significant decrease of sRAGE concentrations between T1 and T2 and a significant increase between T2 and D; (iii) a significant decrease of AGE from T1 to D; (iv) no significant variation of S100A8/A9 between trimesters. In intergroup comparisons (PPROM vs. control group), there were no significant differences in time variation taking into account the matching effects. There was a correlation between plasma and serum-extracted extracellular vesicle concentrations of sRAGE, AGE, HMGB1, and S100A8/A9. Our results suggest that the rupture of fetal membranes (physiological or premature) is accompanied by a variation in plasma concentrations of sRAGE, HMGB1, and AGE. The study of RAGE and its main ligands in extracellular vesicles did not give additional insight into the pathophysiological process conducting to PPROM.
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http://dx.doi.org/10.3389/fphys.2020.00609DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7324632PMC
June 2020

The receptor for advanced glycation end-products enhances lung epithelial wound repair: An in vitro study.

Exp Cell Res 2020 06 21;391(2):112030. Epub 2020 Apr 21.

Université Clermont Auvergne, CNRS, INSERM, GReD, Clermont-Ferrand, France; Department of Perioperative Medicine, CHU Clermont-Ferrand, Clermont-Ferrand, France; Division of Allergy, Pulmonary, and Critical Care Medicine, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, USA. Electronic address:

Re-epithelialization of the alveolar surface is a key process of lung alveolar epithelial barrier repair after acute lung injury. The receptor for advanced glycation end-products (RAGE) pathway plays key roles in lung homeostasis, and its involvement in wound repair has been already reported in human bronchial epithelial cells. However, its effects on lung alveolar epithelial repair after injury remain unknown. We investigated whether RAGE stimulation with its ligands high-mobility group box 1 protein (HMGB1) or advanced glycation end-products (AGEs), alone or associated with RAGE inhibition using RAGE antagonist peptide, affects in vitro wound healing in human alveolar epithelial A549 cells. We further asked whether these effects could be associated with changes in cell proliferation and migration. We found that treatment of A549 cells with HMGB1 or AGEs promotes RAGE-dependent wound healing after a scratch assay. In addition, both RAGE ligands increased cell proliferation in a RAGE-dependent manner. Treatment with HMGB1 increased migration of alveolar epithelial cells at 12 h, independently of RAGE, whereas AGEs stimulated migration as measured 48 h after injury in a RAGE-dependent manner. Taken together, these results suggest that RAGE pathway is involved in lung alveolar epithelial wound repair, possibly through enhanced cell migration and proliferation.
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http://dx.doi.org/10.1016/j.yexcr.2020.112030DOI Listing
June 2020

Advanced Glycation End Products and Receptor (RAGE) Promote Wound Healing of Human Corneal Epithelial Cells.

Invest Ophthalmol Vis Sci 2020 03;61(3):14

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Purpose: We used a human corneal epithelial cell (HCE) line to determine the involvement of the advanced glycation end products (AGEs) / receptor for AGEs (RAGE) couple in corneal epithelium wound healing.

Methods: After wounding, HCE cells were exposed to two major RAGE ligands (HMGB1 and AGEs), and wound healing was evaluated using the in vitro scratch assay. Following wound healing, the HCE cells were used to study the influence of the RAGE ligands on HCE proliferation, invasion, and migration. Activation of the nuclear factor (NF)-κB signaling pathway by the AGEs/RAGE couple was tested using a luciferase reporter assay. Functional transcriptional regulation by this pathway was confirmed by quantification of expression of the connexin 43 target gene. For each experiment, specific RAGE involvement was confirmed by small interfering RNA treatments.

Results: AGEs treatment at a dose of 100 µg/mL significantly improved the wound healing process in a RAGE-dependent manner by promoting cell migration, whereas HMGB1 had no effect. No significant influence of the AGEs/RAGE couple was observed on cell proliferation and invasion. However, this treatment induced an early activation of the NF-κB pathway and positively regulated the expression of the target gene, connexin 43, at both the mRNA and protein levels.

Conclusions: Our results demonstrate that the RAGE pathway is activated by AGEs treatment and is involved in the promotion of corneal epithelial wound healing. This positive action is observed only during the early stages of wound healing, as illustrated by the quick activation of the NF-κB pathway and induction of connexin 43 expression.
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http://dx.doi.org/10.1167/iovs.61.3.14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7401750PMC
March 2020

Risk Factors and Outcomes of Preterm Premature Rupture of Membranes in a Cohort of 6968 Pregnant Women Prospectively Recruited.

J Clin Med 2019 Nov 15;8(11). Epub 2019 Nov 15.

Department of Molecular Biology, Medical biochemistry and Pathology, Faculty of Medicine, Centre de recherche du CHU de Québec-Université Laval, Québec City, QC G1V 0A6, Canada.

We revisited risk factors and outcomes related to the preterm premature rupture of membranes (PPROM). A total of 7866 pregnant women were recruited during 5 years at their first prenatal visit to the perinatal clinic of the institution. We compared three groups (women without prematurity, women with spontaneous preterm labor with intact membranes (sPL with IM), women with PPROM) regarding 60 criteria about characteristics, lifestyle, medical, gynecological, obstetrical history of mothers, medication during pregnancy, events at delivery, and complications in neonates. Logistic regression analyses adjusting for potential confounding factors were used. Of the 6968 women selected, 189 (2.8%) presented a PPROM, and 225 (3.2%) an sPL with IM. The specific risk factors for PPROM were body mass index (BMI) <18.5 kg/m (adjusted odds ratio, aOR: 2.00 (1.09-3.67)), history of PPROM (aOR: 2.75 (1.19-6.36)), nulliparity (aOR: 2.52 (1.77-3.60)), gestational diabetes (aOR: 1.87 (1.16-2.99)), and low level of education (aOR: 2.39 (1.20-4.78)). The complications associated with PPROM were abruption placentae, cesarean, APGAR 5' <4, birth weight <2500 g, stillbirth, neonatal jaundice, and hospitalization of mother and neonates. All these complications were also associated with sPL with IM. Our study confirms some of the risk factors of PPROM and highlights a new one: gestational diabetes. Outcomes of PPROM are related to prematurity.
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http://dx.doi.org/10.3390/jcm8111987DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6912547PMC
November 2019

Inhibition of the Receptor for Advanced Glycation End-Products in Acute Respiratory Distress Syndrome: A Randomised Laboratory Trial in Piglets.

Sci Rep 2019 06 25;9(1):9227. Epub 2019 Jun 25.

Department of Perioperative Medicine, CHU Clermont-Ferrand, Clermont, Ferrand, France.

The receptor for advanced glycation end-products (RAGE) modulates the pathogenesis of acute respiratory distress syndrome (ARDS). RAGE inhibition attenuated lung injury and restored alveolar fluid clearance (AFC) in a mouse model of ARDS. However, clinical translation will require assessment of this strategy in larger animals. Forty-eight anaesthetised Landrace piglets were randomised into a control group and three treatment groups. Animals allocated to treatment groups underwent orotracheal instillation of hydrochloric acid (i) alone; (ii) in combination with intravenous administration of a RAGE antagonist peptide (RAP), or (iii) recombinant soluble (s)RAGE. The primary outcome was net AFC at 4 h. Arterial oxygenation was assessed hourly and alveolar-capillary permeability, alveolar inflammation and lung histology were assessed at 4 h. Treatment with either RAP or sRAGE improved net AFC (median [interquartile range], 21.2 [18.8-21.7] and 19.5 [17.1-21.5] %/h, respectively, versus 12.6 [3.2-18.8] %/h in injured, untreated controls), oxygenation and decreased alveolar inflammation and histological evidence of tissue injury after ARDS. These findings suggest that RAGE inhibition restored AFC and attenuated lung injury in a piglet model of acid-induced ARDS.
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http://dx.doi.org/10.1038/s41598-019-45798-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6592897PMC
June 2019

Pathological Implications of Receptor for Advanced Glycation End-Product () Gene Polymorphism.

Dis Markers 2019 4;2019:2067353. Epub 2019 Feb 4.

CHU Clermont-Ferrand, Department of Medical Biochemistry and Molecular Biology, 63000 Clermont-Ferrand, France.

The receptor for advanced glycation end-products (RAGE) is a cell surface transmembrane multiligand receptor, encoded by the gene. RAGE presents many transcripts, is expressed mainly in the lung, and involves multiple pathways (such as NFB, Akt, p38, and MAP kinases) that initiate and perpetuate an unfavorable proinflammatory state. Due to these numerous functional activities, RAGE is implicated in multiple diseases. is a highly polymorphic gene, with polymorphisms or SNP (single-nucleotide polymorphism) that could be responsible or co-responsible for disease development. This review was designed to shed light on the pathological implications of polymorphisms. Five polymorphisms are described: rs2070600, rs1800624, rs1800625, rs184003, and a 63 bp deletion. The rs2070600 SNP may be associated with the development of human autoimmune disease, diabetes complications, cancer, and lung diseases such as chronic obstructive pulmonary disease and acute respiratory distress syndrome. The rs1800624 SNP involves gene regulation and may be related to reduced risk of heart disease, cancer, Crohn's disease, and type 1 diabetes complications. The rs1800625 SNP may be associated with the development of diabetic retinopathy, cancer, and lupus but may be protective against cardiovascular risk. The rs184003 SNP seems related to coronary artery disease, breast cancer, and diabetes. The 63 bp deletion may be associated with reduced survival from heart diseases during diabetic nephropathy. Here, these potential associations between polymorphisms and the development of diseases are discussed, as there have been conflicting findings on the pathological impact of SNPs in the literature. These contradictory results might be explained by distinct SNP frequencies depending on ethnicity.
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http://dx.doi.org/10.1155/2019/2067353DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6378764PMC
July 2019

In Vitro Method to Control Concentrations of Halogenated Gases in Cultured Alveolar Epithelial Cells.

J Vis Exp 2018 10 23(140). Epub 2018 Oct 23.

Department of Perioperative Medicine, CHU Clermont-Ferrand; Centre National de la Recherche Scientifique Unité Mixte de Recherche (CNRS UMR) 6293, Institut National de la Santé et de la Recherche Médicale (INSERM) U1103, Laboratoire de Génétique, Reproduction et Développement (GReD), Université Clermont Auvergne.

Acute respiratory distress syndrome (ARDS) is a syndrome of diffuse alveolar injury with impaired alveolar fluid clearance and severe inflammation. The use of halogenated agents, such as sevoflurane or isoflurane, for the sedation of intensive care unit (ICU) patients can improve gas exchange, reduce alveolar edema, and attenuate inflammation during ARDS. However, data on the use of inhaled agents for continuous sedation in the ICU to treat or prevent lung damage is lacking. To study the effects of halogenated agents on alveolar epithelial cells under "physiologic" conditions, we describe an easy system to culture cells at the air-liquid interface and expose them to halogenated agents to provide precise controlled "air" fractions and "medium" concentrations for these agents. We developed a sealed air-tight chamber in which plates with human alveolar epithelial immortalized cells could be exposed to a precise, controlled fraction of sevoflurane or isoflurane using a continuous gas flow provided by an anesthetic machine circuit. Cells were exposed to 4% of sevoflurane and 1% of isoflurane for 24 hours. Gas mass spectrometry was performed to determine the concentration of halogenated agents dissolved in the medium. After the first hour, the concentrations of sevoflurane and isoflurane in the medium were 251 mg/L and 25 mg/L, respectively. The curves representing the concentrations of both sevoflurane and isoflurane dissolved in the medium showed similar courses over time, with a plateau reached at one hour after exposure. This protocol was specifically designed to reach precise and controlled concentrations of sevoflurane or isoflurane in vitro to improve our understanding of mechanisms involved in epithelial lung injury during ARDS and to test novel therapies for the syndrome.
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http://dx.doi.org/10.3791/58554DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6235595PMC
October 2018

Driving pressure and acute respiratory distress syndrome in critically ill patients.

Respirology 2019 02 5;24(2):137-145. Epub 2018 Sep 5.

Department of Perioperative Medicine, CHU Clermont-Ferrand, Clermont-Ferrand, France.

Background And Objective: Elevated driving pressure (ΔP) may be associated with increased risk of acute respiratory distress syndrome (ARDS) in patients admitted via the emergency department and with post-operative pulmonary complications in surgical patients. This study investigated the association of higher ΔP with the onset of ARDS in a high-risk, intensive care unit (ICU) population.

Methods: This is a secondary analysis of a prospective multicentre observational study. Data for this ancillary study were obtained from intubated adult patients with at least one ARDS risk factor upon ICU admission enrolled in a previous multicentre observational study. Patients were followed up for the development of ARDS within 7 days (primary outcome). Univariate and multivariate analyses tested the association between ΔP (measured at ICU admission (baseline) or 24 h later (day 1)) and the development of ARDS.

Results: A total of 221 patients were included in this study, among whom 34 (15%) developed ARDS within 7 days. These patients had higher baseline ΔP than those who did not (mean ± SD: 12.5 ± 3.1 vs 9.8 ± 3.4 cm H O, respectively, P = 0.0001). The association between baseline ΔP and the risk of developing ARDS was robust to adjustment for baseline tidal volume, positive-end expiratory pressure, illness severity, serum lactate and sepsis, pneumonia, severe trauma and shock as primary ARDS risk factors (odds ratio: 1.20; 95% CI: 1.03-1.41; P = 0.02). The same results were found with day 1 ΔP.

Conclusion: Among at-risk ICU patients, higher ΔP may identify those who are more likely to develop ARDS.
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http://dx.doi.org/10.1111/resp.13394DOI Listing
February 2019

Clinical and Biological Predictors of Plasma Levels of Soluble RAGE in Critically Ill Patients: Secondary Analysis of a Prospective Multicenter Observational Study.

Dis Markers 2018 10;2018:7849675. Epub 2018 May 10.

Department of Perioperative Medicine, CHU Clermont-Ferrand, Clermont-Ferrand, France.

Rationale: Although soluble forms of the receptor for advanced glycation end products (RAGE) have been recently proposed as biomarkers in multiple acute or chronic diseases, few studies evaluated the influence of usual clinical and biological parameters, or of patient characteristics and comorbidities, on circulating levels of soluble RAGE in the intensive care unit (ICU) setting.

Objectives: To determine, among clinical and biological parameters that are usually recorded upon ICU admission, which variables, if any, could be associated with plasma levels of soluble RAGE.

Methods: Data for this ancillary study were prospectively obtained from adult patients with at least one ARDS risk factor upon ICU admission enrolled in a large multicenter observational study. At ICU admission, plasma levels of total soluble RAGE (sRAGE) and endogenous secretory (es)RAGE were measured by duplicate ELISA and baseline patient characteristics, comorbidities, and usual clinical and biological indices were recorded. After univariate analyses, significant variables were used in multivariate, multidimensional analyses.

Measurements And Main Results: 294 patients were included in this ancillary study, among whom 62% were admitted for medical reasons, including septic shock (11%), coma (11%), and pneumonia (6%). Although some variables were associated with plasma levels of RAGE soluble forms in univariate analysis, multidimensional analyses showed no significant association between admission parameters and baseline plasma sRAGE or esRAGE.

Conclusions: We found no obvious association between circulating levels of soluble RAGE and clinical and biological indices that are usually recorded upon ICU admission. This trial is registered with NCT02070536.
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http://dx.doi.org/10.1155/2018/7849675DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5971347PMC
October 2018

Retinoic Acid Pathway Regulation of Vascular Endothelial Growth Factor in Ovine Amnion.

Reprod Sci 2019 10 27;26(10):1351-1359. Epub 2018 Mar 27.

Department of Obstetrics and Gynecology, Oregon Health & Science University, Portland, OR, USA.

Vascular endothelial growth factor (VEGF) has been proposed as an important regulator of amniotic fluid absorption across the amnion into the fetal vasculature on the surface of the placenta. However, the activators of VEGF expression and action in the amnion have not been identified. Using the pregnant sheep model, we aimed to investigate the presence of the retinoic acid (RA) pathway in ovine amnion and to determine its effect on VEGF expression. Further, we explored relationships between RA receptors and VEGF and tested the hypothesis that RA modulates intramembranous absorption (IMA) through induction of amnion VEGF in sheep fetuses subjected to altered IMA rates. Our study showed that RA receptor isoforms were expressed in sheep amnion, and RA response elements (RAREs) were identified in ovine RARβ and VEGF gene promoters. In ovine amnion cells, RA treatment upregulated RARβ messenger RNA (mRNA) and increased VEGF transcript levels. In sheep fetuses, increases in IMA rate was associated with elevated VEGF mRNA levels in the amnion but not in the chorion. Further, RARβ mRNA was positively correlated with VEGF mRNA levels in the amnion and not chorion. We conclude that an RA pathway is present in ovine fetal membranes and that RA is capable of inducing VEGF. The finding of a positive relationship between amnion VEGF and RARβ during altered IMA rate suggests that the retinoid pathway may play a role through VEGF in regulating intramembranous transport across the amnion.
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http://dx.doi.org/10.1177/1933719118765979DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6949972PMC
October 2019

Retinoic acid and tracheal occlusion for diaphragmatic hernia treatment in rabbit fetuses.

Prenat Diagn 2018 06;38(7):482-492

"Translational approach to epithelial injury and repair" team, Université Clermont Auvergne, CNRS, Inserm, GReD, 63000, Clermont-Ferrand, France.

Introduction: Lung hypoplasia and pulmonary arterial hypertension in congenital diaphragmatic hernia lead to a high perinatal mortality. Although sustained fetoscopic tracheal occlusion (TO) improves lung development, a major side effect is abnormal pneumocyte differentiation. This study evaluated the potential ability of intratracheal retinoic acid (RA) administration to reduce adverse effects of sustained TO in a rabbit model of diaphragmatic hernia.

Methods: A left diaphragmatic defect was created on day 23 in time-dated pregnant rabbits. On day 28, the same rabbits underwent sham surgery or TO, with an injection of empty or RA-loaded liposomes. On day 30, the fetuses were harvested, and the lungs were processed for histology, immunohistochemistry, and gene expression quantification.

Results: A tracheal RA injection at the time of TO had no effect on the lung-to-body-weight ratio, radial alveolar count or lung connective tissue composition. Retinoic acid plus TO had synergic effects on vascular measurements, proportional medial thickness, and endothelin-1 receptor type-A gene expression. The most noticeable effect was recovery of normal pneumocyte differentiation.

Conclusion: Retinoic acid plus TO prevented abnormal pneumocyte differentiation and seemed to have a beneficial effect on pulmonary vascularization.
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http://dx.doi.org/10.1002/pd.5256DOI Listing
June 2018

Receptor for advanced glycation end-products and ARDS prediction: a multicentre observational study.

Sci Rep 2018 02 8;8(1):2603. Epub 2018 Feb 8.

CHU Clermont-Ferrand, Department of Perioperative Medicine, Clermont-Ferrand, France.

Acute respiratory distress syndrome (ARDS) prediction remains challenging despite available clinical scores. To assess soluble receptor for advanced glycation end-products (sRAGE), a marker of lung epithelial injury, as a predictor of ARDS in a high-risk population, adult patients with at least one ARDS risk factor upon admission to participating intensive care units (ICUs) were enrolled in a multicentre, prospective study between June 2014 and January 2015. Plasma sRAGE and endogenous secretory RAGE (esRAGE) were measured at baseline (ICU admission) and 24 hours later (day one). Four AGER candidate single nucleotide polymorphisms (SNPs) were also assayed because of previous reports of functionality (rs1800625, rs1800624, rs3134940, and rs2070600). The primary outcome was ARDS development within seven days. Of 500 patients enrolled, 464 patients were analysed, and 59 developed ARDS by day seven. Higher baseline and day one plasma sRAGE, but not esRAGE, were independently associated with increased ARDS risk. AGER SNP rs2070600 (Ser/Ser) was associated with increased ARDS risk and higher plasma sRAGE in this cohort, although confirmatory studies are needed to assess the role of AGER SNPs in ARDS prediction. These findings suggest that among at-risk ICU patients, higher plasma sRAGE may identify those who are more likely to develop ARDS.
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http://dx.doi.org/10.1038/s41598-018-20994-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5805783PMC
February 2018

Cigarette smoke condensate affects the retinoid pathway in human amnion.

Placenta 2017 Oct 1;58:98-104. Epub 2017 Sep 1.

Team "Translational Approach to Epithelial Injury and Repair", Université Clermont Auvergne, CNRS, Inserm, GReD, F-63000 Clermont-Ferrand, France.

Introduction: The preterm premature rupture of membranes (PPROM) is a frequent pathology responsible of more than 30% of preterm births. Tobacco smoking is one of the most frequently described risk factors identified and contributes to the pre term weakening of fetal membranes. As previously demonstrated, all-trans retinoic acid (atRA) regulates several genes involved in the extracellular matrix dynamics, an essential actor in fetal membrane ruptures. We hypothesized that cigarette smoke may affect this pathway in human amnion.

Methods: Amnion was obtained from full-term fetal membranes collected from non-smoking women after cesarean births and used either as explants or for the isolation of derived epithelial cells. The pro-healing and transcriptomic effects of atRA were studied by a scratch assay experiment and quantitative RT-PCR, respectively, after treatment with dimethyl sulfoxyde (DMSO), atRA, DMSO + cigarette smoke condensate (CSC), or atRA + CSC.

Results: Our results show a strong alteration of the retinoid pathway after CSC treatment on amnion-derived epithelial cells and explants. We first demonstrated that CSC inhibits the activity of the RARE reporter gene in amnion-derived epithelial cells. Then, atRA's effects on both the transcription of its target genes and wound healing were demonstrated to be inhibited or at least decreased by the CSC in human amnion epithelial cells.

Discussion: Here, we demonstrated that CSC altered the retinoid signal, already known to have roles in fetal membrane physiopathology. These results highlight a potential negative action of maternal smoking on the retinoid pathway in human amnion and more generally on pregnancy.
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http://dx.doi.org/10.1016/j.placenta.2017.08.076DOI Listing
October 2017

RAGE inhibition reduces acute lung injury in mice.

Sci Rep 2017 08 3;7(1):7208. Epub 2017 Aug 3.

Department of Perioperative Medicine, CHU Clermont-Ferrand, Clermont-Ferrand, France.

The receptor for advanced glycation end-products (RAGE) is involved in inflammatory response during acute respiratory distress syndrome (ARDS). Growing body of evidence support strategies of RAGE inhibition in experimental lung injury, but its modalities and effects remain underinvestigated. Anesthetised C57BL/6JRj mice were divided in four groups; three of them underwent orotracheal instillation of acid and were treated with anti-RAGE monoclonal antibody (mAb) or recombinant soluble RAGE (sRAGE), acting as a decoy receptor. The fourth group served as a control. Lung injury was assessed by the analysis of blood gases, alveolar permeability, histology, AFC, and cytokines. Lung expression and distribution epithelial channels ENaC, Na,K-ATPase, and aquaporin (AQP)-5 were assessed. Treatment with either anti-RAGE mAb or sRAGE improved lung injury, arterial oxygenation and decreased alveolar inflammation in acid-injured animals. Anti-RAGE therapies were associated with restored AFC and increased lung expression of AQP-5 in alveolar cell. Blocking RAGE had potential therapeutic effects in a translational mouse model of ARDS, possibly through a decrease in alveolar type 1 epithelial cell injury as shown by restored AFC and lung AQP-5 expression. Further mechanistic studies are warranted to describe intracellular pathways that may control such effects of RAGE on lung epithelial injury and repair.
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http://dx.doi.org/10.1038/s41598-017-07638-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5543147PMC
August 2017

Retinoic Acid Engineered Amniotic Membrane Used as Graft or Homogenate: Positive Effects on Corneal Alkali Burns.

Invest Ophthalmol Vis Sci 2017 07;58(9):3513-3518

Translational Approach to Epithelial Injury and Repair Team, Université Clermont Auvergne, Centre National de Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale, Génétique Reproduction et Développement, Clermont-Ferrand, France.

Purpose: Alkali burns are the most common, severe chemical ocular injuries, their functional prognosis depending on corneal wound healing efficiency. The purpose of our study was to compare the benefits of amniotic membrane (AM) grafts and homogenates for wound healing in the presence or absence of previous all-trans retinoic acid (atRA) treatment.

Methods: Fifty male CD1 mice with reproducible corneal chemical burn were divided into five groups, as follows: group 1 was treated with saline solution; groups 2 and 3 received untreated AM grafts or grafts treated with atRA, respectively; and groups 4 and 5 received untreated AM homogenates or homogenates treated with atRA, respectively. After 7 days of treatment, ulcer area and depth were measured, and vascular endothelial growth factor (VEGF) and matrix metalloproteinase 9 (MMP-9) were quantified.

Results: AM induction by atRA was confirmed via quantification of retinoic acid receptor β (RARβ), a well-established retinoic acid-induced gene. Significant improvements of corneal wound healing in terms of ulcer area and depth were obtained with both strategies. No major differences were found between the efficiency of AM homogenates and grafts. This positive action was increased when AM was pretreated with atRA. Furthermore, AM induced a decrease in VEGF and MMP-9 levels during the wound healing process. The atRA treatment led to an even greater decrease in the expression of both proteins.

Conclusions: Amnion homogenate is as effective as AM grafts in promoting corneal wound healing in a mouse model. A higher positive effect was obtained with atRA treatment.
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http://dx.doi.org/10.1167/iovs.17-21810DOI Listing
July 2017

Myocilin expression is regulated by retinoic acid in the trabecular meshwork-derived cellular environment.

Exp Eye Res 2017 02 30;155:91-98. Epub 2017 Jan 30.

EA7281 - Retinoids, Reproduction Developmental Diseases, School of Medicine, Clermont Université, Université d'Auvergne, F-63000 Clermont-Ferrand, France. Electronic address:

Glaucoma is the leading cause of irreversible blindness and is usually classified as angle closure and open angle glaucoma (OAG). Primary open angle glaucoma represents the most frequent clinical presentation leading to ganglion cell death and optic nerve degeneration as a main consequence of an intraocular pressure' (IOP) increase. The mechanisms of this IOP increase in such pathology remain unclear but one protein called Myocilin could be a part of the puzzle in the trabecular meshwork (TM). Previously described to be transcriptionally regulated by glucocorticoids, the comprehension of the trabecular regulation of Myocilin' expression has only weakly progressed since 15 years. Due to the essential molecular and cellular implications of retinoids' pathway in eye development and physiology, we investigate the potential role of the retinoic acid in such regulation and expression. This study demonstrates that the global retinoids signaling machinery is present in immortalized TM cells and that Myocilin (MYOC) expression is upregulated by retinoic acid alone or combined with a glucocorticoid co-treatment. This regulation by retinoic acid acts through the MYOC promoter which contains a critical cluster of four retinoic acid responsive elements (RAREs), with the RARE-DR2 presenting the strongest effect and binding the RARα/RXRα heterodimer. All together, these results open up new perspectives for the molecular understanding glaucoma pathophysiology and provide further actionable clues on Myocilin gene regulation.
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http://dx.doi.org/10.1016/j.exer.2017.01.006DOI Listing
February 2017

Effects of tracheal occlusion with retinoic acid administration on normal lung development.

Prenat Diagn 2017 May 16;37(5):427-434. Epub 2017 Apr 16.

EA7281 - Retinoids, Reproduction Developmental Diseases, Auvergne University, Clermont-Ferrand, France.

Introduction: Tracheal occlusion (TO) is an investigational therapy for severe congenital diaphragmatic hernia that decreases pulmonary hypoplasia, but sustained TO also induces deficient surfactant synthesis. Intramuscular maternal administration of retinoic acid (RA) in a surgical rabbit model of congenital diaphragmatic hernia showed a beneficial effect on lung maturation. We evaluated the potential of RA delivery into the trachea and studied the combined effects of TO and RA on normal lung development.

Methods: Experiments were performed on normal rabbit fetuses. Liposomes and capric triglyceride (Miglyol ), alone and with RA, were administered in the trachea just before TO (d26). Lung morphology and surfactant production were studied at term (d30).

Results: Tracheal occlusion increased lung weight and enhanced alveolar development but increased apoptotic activity and decreased surfactant expression. Tracheal injection of RA improved surfactant production to levels of normal controls.

Conclusion: We established the potential of liposome and Miglyol as RA vehicle for delivering this bioactive molecule in the fetal airways. Tracheal RA injection seems to oppose the effects of TO in fetuses with normal lungs. © 2017 John Wiley & Sons, Ltd.
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http://dx.doi.org/10.1002/pd.5012DOI Listing
May 2017

All-trans retinoic acid promotes wound healing of primary amniocytes through the induction of LOXL4, a member of the lysyl oxidase family.

Int J Biochem Cell Biol 2016 12 18;81(Pt A):10-19. Epub 2016 Oct 18.

Clermont Université, Auvergne University, EA7281- Retinoids, Reproduction, Developmental Diseases, Medicine School, 63000 Clermont-Ferrand, France.

Thirty percent of preterm births directly result from preterm premature rupture of fetal membranes (PPROM). Clinical management currently proposes using a collagen plug to mechanically stop loss of amniotic fluid. Vitamin A and its active metabolite (retinoic acid) have well-known pro-healing properties and could thus make good candidates as a proposable adjuvant to this mechanical approach. Here we investigate the molecular mechanisms involved in the pro-healing properties of all-trans retinoic acid (atRA) in fetal membranes via an approach using an in vitro primary amniocyte wound model and transcriptomics. The results demonstrate that atRA promotes migration in primary amniocytes, improving wound healing in vitro by up to 90%. This effect is mediated by the induction of LOXL4, which plays a crucial role in the dynamics of the extracellular matrix by regulating collagen reticulation. This new insight into how atRA exerts its pro-healing properties prompts us to propose using atRA as a candidate strategy to help prevent future PPROM.
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http://dx.doi.org/10.1016/j.biocel.2016.10.007DOI Listing
December 2016

Lysyl oxidase-like 4 involvement in retinoic acid epithelial wound healing.

Sci Rep 2016 09 6;6:32688. Epub 2016 Sep 6.

Clermont Université, Université d'Auvergne, EA7281 - Retinoids, Reproduction Developmental Diseases, School of Medicine, F-63000 Clermont-Ferrand, France.

Vitamin A and its active forms (retinoic acids/RAs) are known to have pro-healing properties, but their mechanisms of action are still poorly understood. This work aimed to identify the cellular and molecular processes by which atRA (all-trans RA) improves wound healing, using an in vivo model of mouse corneal alkali burns and an in vitro cellular human corneal epithelial injury model. Regulation by atRA has been studied on most of the cellular events that occur in wound healing. We investigated the direct influence of atRA on a specific target gene known to be involved in the extracellular matrix (ECM) dynamics, one of the pathways contributing to epithelial repair. Our results demonstrate that atRA promotes corneal epithelial wound healing by acting preferentially on migration. The induction of lysyl oxidase-like 4 (LOXL4) expression by atRA in the corneal epithelium environment was established as essential in the mechanism of atRA-dependent wound healing. Our study describes for the first time a direct link between a retinoic-induced gene and protein, LOXL4, and its general clinical pro-healing properties in ECM dynamics.
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http://dx.doi.org/10.1038/srep32688DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5011693PMC
September 2016

Nuclear retinoid receptors and pregnancy: placental transfer, functions, and pharmacological aspects.

Cell Mol Life Sci 2016 10 9;73(20):3823-37. Epub 2016 Aug 9.

EA7281, Retinoids, Reproduction Developmental Diseases, School of Medicine, Clermont Université, Université d'Auvergne, 63000, Clermont-Ferrand, France.

Animal models of vitamin A (retinol) deficiency have highlighted its crucial role in reproduction and placentation, whereas an excess of retinoids (structurally or functionally related entities) can cause toxic and teratogenic effects in the embryo and foetus, especially in the first trimester of human pregnancy. Knock-out experimental strategies-targeting retinoid nuclear receptors RARs and RXRs have confirmed that the effects of vitamin A are mediated by retinoic acid (especially all-trans retinoic acid) and that this vitamin is essential for the developmental process. All these data show that the vitamin A pathway and metabolism are as important for the well-being of the foetus, as they are for that of the adult. Accordingly, during this last decade, extensive research on retinoid metabolism has yielded detailed knowledge on all the actors in this pathway, spurring the development of antagonists and agonists for therapeutic and research applications. Natural and synthetic retinoids are currently used in clinical practice, most often on the skin for the treatment of acne, and as anti-oncogenic agents in acute promyelocytic leukaemia. However, because of the toxicity and teratogenicity of retinoids during pregnancy, their pharmacological use needs a sound knowledge of their metabolism, molecular aspects, placental transfer, and action.
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http://dx.doi.org/10.1007/s00018-016-2332-9DOI Listing
October 2016

Net alveolar fluid clearance is associated with lung morphology phenotypes in acute respiratory distress syndrome.

Anaesth Crit Care Pain Med 2016 Apr 27;35(2):81-6. Epub 2016 Feb 27.

Department of Perioperative Medicine, CHU de Clermont-Ferrand, Intensive Care Unit, University Hospital of Clermont-Ferrand, 63003 Clermont-Ferrand, France; EA 7281, R2D2, Clermont Université, Université d'Auvergne, 63003 Clermont-Ferrand, France.

Background: The acute respiratory distress syndrome (ARDS) is a heterogeneous syndrome that encompasses multiple phenotypes, e.g. with regards to lung morphology as assessed by computed tomography (CT). Focal or non-focal lung morphology may influence the response to positive end-expiratory pressure (PEEP), recruitment manoeuvres and prone position. Lung morphology has been hypothesized to be associated with alveolar fluid clearance (AFC), thus explaining various responses to such therapeutic interventions; however, this hypothesis has not been specifically studied in humans.

Methods: We measured net AFC rates in 30 patients with ARDS as a secondary data analysis of a prospective single-centre study. Net AFC rates were compared between patients with focal ARDS and those with non-focal ARDS, as assessed by lung CT-scans.

Results: Net AFC rates were significantly lower in patients with non-focal ARDS (n=23; median [interquartile range], 1.5 [0-5.5] %/h) as compared to those with focal ARDS (n=7; 10.3 [4.5-15] %/h) (P=0.01). The area under the receiver-operating characteristic curve when net AFC rates were used to differentiate the presence from absence of non-focal ARDS was 0.93 (95% confidence interval, 0.81-1). Tidal volumes and PEEP levels differed between focal and non-focal ARDS patients, but there was no difference in arterial oxygenation or in alveolar-capillary permeability.

Conclusions: Non-focal lung morphology may be characterized by a functional endotype consistent with marked AFC impairment. Despite study limitations and the need for validating studies in larger cohorts, such novel findings may reinforce our understanding of the association between ARDS phenotypes and therapeutic responses.
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http://dx.doi.org/10.1016/j.accpm.2015.11.006DOI Listing
April 2016

Soluble Forms and Ligands of the Receptor for Advanced Glycation End-Products in Patients with Acute Respiratory Distress Syndrome: An Observational Prospective Study.

PLoS One 2015 14;10(8):e0135857. Epub 2015 Aug 14.

CHU Clermont-Ferrand, Intensive Care Unit, Department of Anesthesiology, Critical Care and Perioperative Medicine, Estaing Hospital, Clermont-Ferrand, France; Clermont Université, Université d'Auvergne, Clermont-Ferrand, France.

Background: The main soluble form of the receptor for advanced glycation end-products (sRAGE) is elevated during acute respiratory distress syndrome (ARDS). However other RAGE isoforms and multiple ligands have been poorly reported in the clinical setting, and their respective contribution to RAGE activation during ARDS remains unclear. Our goal was therefore to describe main RAGE isoforms and ligands levels during ARDS.

Methods: 30 ARDS patients and 30 mechanically ventilated controls were prospectively included in this monocenter observational study. Arterial, superior vena cava and alveolar fluid levels of sRAGE, endogenous-secretory RAGE (esRAGE), high mobility group box-1 protein (HMGB1), S100A12 and advanced glycation end-products (AGEs) were measured in duplicate ELISA on day 0, day 3 and day 6. In patients with ARDS, baseline lung morphology was assessed with computed tomography.

Results: ARDS patients had higher arterial, central venous and alveolar levels of sRAGE, HMGB1 and S100A12, but lower levels of esRAGE and AGEs, than controls. Baseline arterial sRAGE, HMGB1 and S100A12 were correlated with nonfocal ARDS (AUC 0.79, 0.65 and 0.63, respectively). Baseline arterial sRAGE, esRAGE, S100A12 and AGEs were associated with severity as assessed by PaO2/FiO2.

Conclusions: This is the first kinetics study of levels of RAGE main isoforms and ligands during ARDS. Elevated sRAGE, HMGB1 and S100A12, with decreased esRAGE and AGEs, were found to distinguish patients with ARDS from those without. Our findings should prompt future studies aimed at elucidating RAGE/HMGB1/S100A12 axis involvement in ARDS.

Trial Registration: clinicaltrials.gov Identifier: NCT01270295.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0135857PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4537285PMC
May 2016

Aquaporins and Fetal Membranes From Diabetic Parturient Women: Expression Abnormalities and Regulation by Insulin.

J Clin Endocrinol Metab 2015 Oct 24;100(10):E1270-9. Epub 2015 Jul 24.

Retinoids, Reproduction Developmental Diseases (D.B., M.R., G.M., C.P., D.G., L.B., V.S.), School of Medicine, Clermont Université, Université d'Auvergne, F-63000 Clermont-Ferrand, France; Biochemistry and Molecular Biology Department (D.B., G.M., R.L. V.S.), CHU Clermont-Ferrand, F-63000 Clermont-Ferrand, France; Biostatistics Unit Department (B.P.), CHU Clermont-Ferrand, F-63000 Clermont-Ferrand, France; School of Medicine Henri-Warembourg (P.D., I.F.), Université Lille 2, PRES Lille Nord de France, F-59000 Lille, France; and Integrative Genomics and Modelization of Metabolic Diseases (A.V.), EGID, School of Medicine Henri-Warembourg, Université Lille 2, PRES Lille Nord de France, F-59000 Lille, France.

Context: During pregnancy, aquaporins (AQPs) expressed in fetal membranes are essential for controlling the homeostasis of the amniotic volume, but their regulation by insulin was never explored in diabetic women.

Objective: The aim of our study was to investigate the involvement of AQPs 1, 3, 8, and 9 expressed in fetal membranes in diabetic parturient women and the control of their expression by insulin.

Design And Participants: From 129 fetal membranes in four populations (controls, type 1, type 2 [T2D], and gestational diabetes [GD]), we established an expression AQP profile. In a second step, the amnion was used to study the control of the expression and functions of AQPs 3 and 9 by insulin.

Main Outcomes And Measures: The expression of transcripts and proteins of AQPs was studied by quantitative RT-PCR and ELISA. We analyzed the regulation by insulin of the expression of AQPs 3 and 9 in the amnion. A tritiated glycerol test enabled us to measure the impact of insulin on the functional characteristics. Using an inhibitor of phosphatidylinositol 3-kinase, we analyzed the insulin intracellular signaling pathway.

Results: The expression of AQP3 protein was significantly weaker in groups T2D and GD. In nondiabetic fetal membranes, we showed for the amnion (but not for the chorion) a significant repression by insulin of the transcriptional expression of AQPs 3 and 9, which was blocked by a phosphatidylinositol 3-kinase inhibitor.

Conclusion: In fetal membranes, the repression of AQP3 protein expression and functions observed in vivo is allowed by the hyperinsulinism described in pregnant women with T2D or GD.
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http://dx.doi.org/10.1210/jc.2015-2057DOI Listing
October 2015

Metabolic disturbances of the vitamin A pathway in human diaphragmatic hernia.

Am J Physiol Lung Cell Mol Physiol 2015 Jan;308(2):L147-57

Congenital diaphragmatic hernia (CDH) is a common life-threatening congenital anomaly resulting in high rates of perinatal death and neonatal respiratory distress. Some of the nonisolated forms are related to single-gene mutations or genomic rearrangements, but the genetics of the isolated forms (60% of cases) still remains a challenging issue. Retinoid signaling (RA) is critical for both diaphragm and lung development, and it has been hypothesized that subtle disruptions of this pathway could contribute to isolated CDH etiology. Here we used time series of normal and CDH lungs in humans, in nitrofen-exposed rats, and in surgically induced hernia in rabbits to perform a systematic transcriptional analysis of the RA pathway key components. The results point to CRPBP2, CY26B1, and ALDH1A2 as deregulated RA signaling genes in human CDH. Furthermore, the expression profile comparisons suggest that ALDH1A2 overexpression is not a primary event, but rather a consequence of the CDH-induced lung injury. Taken together, these data show that RA signaling disruption is part of CDH pathogenesis, and also that dysregulation of this pathway should be considered organ specifically.
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http://dx.doi.org/10.1152/ajplung.00108.2014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4338943PMC
January 2015

Regulation of DNA methylation patterns by CK2-mediated phosphorylation of Dnmt3a.

Cell Rep 2014 Aug 24;8(3):743-53. Epub 2014 Jul 24.

Laboratory of Cancer Epigenetics, Faculty of Medicine, Université Libre de Bruxelles, 808 route de Lennik, 1070 Brussels, Belgium. Electronic address:

DNA methylation is a central epigenetic modification that is established by de novo DNA methyltransferases. The mechanisms underlying the generation of genomic methylation patterns are still poorly understood. Using mass spectrometry and a phosphospecific Dnmt3a antibody, we demonstrate that CK2 phosphorylates endogenous Dnmt3a at two key residues located near its PWWP domain, thereby downregulating the ability of Dnmt3a to methylate DNA. Genome-wide DNA methylation analysis shows that CK2 primarily modulates CpG methylation of several repeats, most notably of Alu SINEs. This modulation can be directly attributed to CK2-mediated phosphorylation of Dnmt3a. We also find that CK2-mediated phosphorylation is required for localization of Dnmt3a to heterochromatin. By revealing phosphorylation as a mode of regulation of de novo DNA methyltransferase function and by uncovering a mechanism for the regulation of methylation at repetitive elements, our results shed light on the origin of DNA methylation patterns.
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http://dx.doi.org/10.1016/j.celrep.2014.06.048DOI Listing
August 2014

DNA methyl transferases are differentially expressed in the human anterior eye segment.

Acta Ophthalmol 2014 Aug 16;92(5):e366-71. Epub 2014 Feb 16.

EA 7281 R2D2, Biochemistry Laboratory, Medicine School, Auvergne University, F-63000 Clermont-Ferrand, France; Internal Medicine-Ophthalmology-ENT Department, Ophthalmology, Clermont-Ferrand University Hospital, Clermont-Ferrand, France.

Purpose: DNA methylation is an epigenetic mark involved in the control of genes expression. Abnormal epigenetic events have been reported in human pathologies but weakly documented in eye diseases. The purpose of this study was to establish DNMT mRNA and protein expression levels in the anterior eye segment tissues and their related (primary or immortalized) cell cultures as a first step towards future in vivo and in vitro methylomic studies.

Methods: Total mRNA was extracted from human cornea, conjunctiva, anterior lens capsule, trabeculum and related cell cultures (cornea epithelial, trabecular meshwork, keratocytes for primary cells; and HCE, Chang, B-3 for immortalized cells). cDNA was quantified by real-time PCR using specific primers for DNMT1, 2, 3A, 3B and 3L. Immunolocalization assays were carried out on human cornea using specific primary antibodies for DNMT1, 2 and 3A, 3B and 3L.

Results: All DNMT transcripts were detected in human cornea, conjunctiva, anterior lens capsule, trabeculum and related cells but showed statistically different expression patterns between tissues and cells. DNMT2 protein presented a specific and singular expression pattern in corneal endothelium.

Conclusions: This study produced the first inventory of the expression patterns of DNMTs in human adult anterior eye segment. Our research highlights that DNA methylation cannot be ruled out as a way to bring new insights into well-known ocular diseases. In addition, future DNA methylation studies using various cells as experimental models need to be conducted with attention to approach the results analysis from a global tissue perspective.
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http://dx.doi.org/10.1111/aos.12365DOI Listing
August 2014

A role for mesenchyme dynamics in mouse lung branching morphogenesis.

PLoS One 2012 23;7(7):e41643. Epub 2012 Jul 23.

Retinoids, Development and Developmental Diseases-EA 7281, Medicine School, Auvergne University, Clermont-Ferrand, France.

Mammalian airways are highly ramified tree-like structures that develop by the repetitive branching of the lung epithelium into the surrounding mesenchyme through reciprocal interactions. Based on a morphometric analysis of the epithelial tree, it has been recently proposed that the complete branching scheme is specified early in each lineage by a programme using elementary patterning routines at specific sites and times in the developing lung. However, the coupled dynamics of both the epithelium and mesenchyme have been overlooked in this process. Using a qualitative and quantitative in vivo morphometric analysis of the E11.25 to E13.5 mouse whole right cranial lobe structure, we show that beyond the first generations, the branching stereotypy relaxes and both spatial and temporal variations are common. The branching pattern and branching rate are sensitive to the dynamic changes of the mesoderm shape that is in turn mainly dependent upon the volume and shape of the surrounding intrathoracic organs. Spatial and temporal variations of the tree architecture are related to local and subtle modifications of the mesoderm growth. Remarkably, buds never meet after suffering branching variations and continue to homogenously fill the opening spaces in the mesenchyme. Moreover despite inter-specimen variations, the growth of the epithelial tree and the mesenchyme remains highly correlated over time at the whole lobe level, implying a long-range regulation of the lung lobe morphogenesis. Together, these findings indicate that the lung epithelial tree is likely to adapt in real time to fill the available space in the mesenchyme, rather than being rigidly specified and predefined by a global programme. Our results strongly support the idea that a comprehensive understanding of lung branching mechanisms cannot be inferred from the branching pattern or behavior alone. Rather it needs to be elaborated upon with the reconsideration of mesenchyme-epithelium coupled growth and lung tissues mechanics.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0041643PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3402475PMC
November 2012