miR-199a-5p Is Upregulated during Fibrogenic Response to Tissue Injury and Mediates TGFbeta-Induced Lung Fibroblast Activation by Targeting Caveolin-1

As miRNAs are associated with normal cellular processes, deregulation of miRNAs is thought to play a causative role in many complex diseases. Nevertheless, the precise contribution of miRNAs in fibrotic lung diseases, especially the idiopathic form (IPF), remains poorly understood. Given the poor response rate of IPF patients to current therapy, new insights into the pathogenic mechanisms controlling lung fibroblasts activation, the key cell type driving the fibrogenic process, are essential to develop new therapeutic strategies for this devastating disease. To identify miRNAs with potential roles in lung fibrogenesis, we performed a genome-wide assessment of miRNA expression in lungs from two different mouse strains known for their distinct susceptibility to develop lung fibrosis after bleomycin exposure. This led to the identification of miR-199a-5p as the best miRNA candidate associated with bleomycin response. Importantly, miR-199a-5p pulmonary expression was also significantly increased in IPF patients (94 IPF versus 83 controls). In particular, levels of miR-199a-5p were selectively increased in myofibroblasts from injured mouse lungs and fibroblastic foci, a histologic feature associated with IPF. Therefore, miR-199a-5p profibrotic effects were further investigated in cultured lung fibroblasts: miR-199a-5p expression was induced upon TGFβ exposure, and ectopic expression of miR-199a-5p was sufficient to promote the pathogenic activation of pulmonary fibroblasts including proliferation, migration, invasion, and differentiation into myofibroblasts. In addition, we demonstrated that miR-199a-5p is a key effector of TGFβ signaling in lung fibroblasts by regulating CAV1, a critical mediator of pulmonary fibrosis. Remarkably, aberrant expression of miR-199a-5p was also found in unilateral ureteral obstruction mouse model of kidney fibrosis, as well as in both bile duct ligation and CCl4-induced mouse models of liver fibrosis, suggesting that dysregulation of miR-199a-5p represents a general mechanism contributing to the fibrotic process. MiR-199a-5p thus behaves as a major regulator of tissue fibrosis with therapeutic potency to treat fibroproliferative diseases. © 2013 Lino Cardenas et al

Identification et caractérisation du polymorphisme génétique des cytochromes P450 4A11 et 4A22 (CYP4A11 et CYP4A22) et de la glycine N-acyltransférase (GLYAT)

Through evolution, in order to adapt to its chemical environment, the human organism has developed enzymatic systems that can transform exogenous molecules or xenobiotic (drugs, toxins, carcinogens…), generally of hydrophobic nature, in metabolites more easily excretable via urinary or biliary tract. Some of these enzymes are also involved in catabolic processes or in the biosynthesis of endogenous compounds (fatty acids, retinoids, steroids, prostaglandins…). These enzymes thus play a major role in the protective response of the body toward chemicals and in essential physiological processes. The existence of anomalies in the sequence or structure of the genes encoding these enzymes can expose carriers of these anomalies to particular susceptibility toward xenobiotics or to impairment of essential biological reactions. In a first step, we investigated the nature and extent of the sequence variability of three genes coding for the enzymes CYP4A11, CYP4A22 and Glycine N-acyltransferase (GLYAT). In a second step, functional analyses of sequence variations were carried out, by in silico and in vitro experiments. The CYP4A11 and CYP4A22 genes are the only members of the human CYP4A subfamily. The activity of the recently identified CYP4A22 isoform is still unknown, but the CYP4A11 isoform is know as a ω-hydroxylase of the arachidonic acid, which converted into 20-hydroxyeicosatetraenoic acid (20-HETE). Several studies have shown that genetic anomalies of CYP4A are likely to contribute for susceptibility to hypertension in humans. We analyzed the sequence variations of the CYP4A11 and CYP4A22 genes in genomic DNA samples of healthy volunteers. A total of 26 polymorphisms were identified and 5 novel CYP4A* alleles were characterized for each CYP4A gene. The CYP4A 3D models were built and validated to analyse the potential impact of sequence variations identified. This work represents the first description and characterisation of genetic polymorphism of the human CYP4A genes in a French population. The glycine N-acyltranferase or GLYAT plays an important role in the detoxification of xenobiotics containing a carboxylic group via conjugation with a glycine residue. Seven sequence variations of the GLYAT gene were identified and four novel GLYAT* alleles were characterized. Localisation of missense mutations in predicted secondary structures suggest that these variants might have a potential role on the GLYAT protein activity. These results could be helpful in investigating the potential association of GLYAT variants with an incidence of reduced efficiency in xenobiotic carboxylic acids detoxification in humans, such as acetylsalicylic acid, pesticides, and solvents (Toluene).Afin de s'adapter à son environnement chimique, l'organisme a développé au cours de l'évolution des systèmes enzymatiques capables de transformer de nombreuses molécules étrangères ou xénobiotiques (médicaments, composés toxiques, carcinogènes...), le plus souvent de nature hydrophobe, en métabolites suffisamment hydrophiles pour être plus facilement excrétés par voie urinaire et/ou biliaire. Certaines de ces enzymes sont également impliquées dans des processus cataboliques ou de biosynthèses de composés endogènes (acides gras, rétinoïdes, stéroïdes, prostaglandines…). Ces enzymes jouent ainsi un rôle fondamental à la fois dans la défense de l'organisme face à son environnement chimique et dans des processus physiologiques essentiels. On comprend dès lors que s'il existe, chez certains individus, des anomalies de séquence ou de structure des gènes codant pour ces enzymes, une partie de la population présentera une susceptibilité particulière à certaines molécules de l'environnement, voire des dysfonctionnements de certaines réactions biologiques indispensables. Les travaux de cette thèse s'inscrivent dans cette démarche. Dans un premier temps, ils ont consisté à évaluer la nature et l'étendue de la variabilité de la séquence nucléotidique de trois gènes codants pour les enzymes CYP4A11, CYP4A22 et la Glycine N-acyltransférase (GLYAT). Dans un deuxième temps, les analyses fonctionnelles des variations de séquence identifiées ont été abordées par des approches in silico et in vitro. Les cytochromes P450 CYP4A11 et CYP4A22, participent à la biotransformation de composés endogènes et sont impliqués plus particulièrement dans la voie d’activation de l’acide arachidonique. Des travaux récents suggèrent que des anomalies génétiques de ces enzymes constituent des facteurs de susceptibilité à l’hypertension artérielle chez l’homme. Nous avons ainsi analysé les variations de séquence du gène CYP4A11 et CYP4A22 dans des échantillons d'ADN provenant de volontaires sains. Au total, 26 polymorphismes ont été identifiés et 5 nouveaux CYP4A* allèles ont été caractérisés pour chaque isoforme CYP4A. Les structures 3D des protéines CYP4A ont été construites et validées pour l’analyse de l’impact des mutations identifiées. Bien que des travaux supplémentaires soient nécessaires pour confirmer le lien entre le polymorphisme génétique du CYP4A11 et du CYP4A22 et l’hypertension artérielle, ce travail représente la première description et caractérisation du polymorphisme génétique des isoformes CYP4A dans une population Française. De plus, nous avons mise en évidence une variabilité interethnique de ce polymorphisme génétique dans différentes populations testées. La glycine N-acyltransférase ou GLYAT est une enzyme impliquée dans la détoxication de xénobiotiques contenant un groupement carboxylique par conjugaison d’un résidu de glycine. Sept variations de séquence de la GLYAT ont été identifiées et quatre nouveaux GLYAT* allèles ont été caractérisés. La localisation des certaines mutations dans des structures secondaires très conservées de la protéine suggère un impact sur l’activité catalytique de cette enzyme. Bien que les conséquences cliniques potentielles de ces variations restent encore à étudier, ces résultats seront utiles pour de futures études d’association de ce polymorphisme génétique de la GLYAT avec les altérations de détoxications de xénobiotiques contenant un groupement carboxylique comme l’aspirine, certains pesticides ou le toluène

Identification and characterisation of genetic polymorphism of the cytochrome P450s 4A11 and 4A22 (CYP4A11 and CYP4A22) and Glycine N-acyltransferase (GLYAT) genes

Afin de s'adapter à son environnement chimique, l'organisme a développé au cours de l'évolution des systèmes enzymatiques capables de transformer de nombreuses molécules étrangères ou xénobiotiques (médicaments, composés toxiques, carcinogènes...), le plus souvent de nature hydrophobe, en métabolites suffisamment hydrophiles pour être plus facilement excrétés par voie urinaire et/ou biliaire. Certaines de ces enzymes sont également impliquées dans des processus cataboliques ou de biosynthèses de composés endogènes (acides gras, rétinoïdes, stéroïdes, prostaglandines…). Ces enzymes jouent ainsi un rôle fondamental à la fois dans la défense de l'organisme face à son environnement chimique et dans des processus physiologiques essentiels. On comprend dès lors que s'il existe, chez certains individus, des anomalies de séquence ou de structure des gènes codant pour ces enzymes, une partie de la population présentera une susceptibilité particulière à certaines molécules de l'environnement, voire des dysfonctionnements de certaines réactions biologiques indispensables. Les travaux de cette thèse s'inscrivent dans cette démarche. Dans un premier temps, ils ont consisté à évaluer la nature et l'étendue de la variabilité de la séquence nucléotidique de trois gènes codants pour les enzymes CYP4A11, CYP4A22 et la Glycine N-acyltransférase (GLYAT). Dans un deuxième temps, les analyses fonctionnelles des variations de séquence identifiées ont été abordées par des approches in silico et in vitro. Les cytochromes P450 CYP4A11 et CYP4A22, participent à la biotransformation de composés endogènes et sont impliqués plus particulièrement dans la voie d’activation de l’acide arachidonique. Des travaux récents suggèrent que des anomalies génétiques de ces enzymes constituent des facteurs de susceptibilité à l’hypertension artérielle chez l’homme. Nous avons ainsi analysé les variations de séquence du gène CYP4A11 et CYP4A22 dans des échantillons d'ADN provenant de volontaires sains. Au total, 26 polymorphismes ont été identifiés et 5 nouveaux CYP4A* allèles ont été caractérisés pour chaque isoforme CYP4A. Les structures 3D des protéines CYP4A ont été construites et validées pour l’analyse de l’impact des mutations identifiées. Bien que des travaux supplémentaires soient nécessaires pour confirmer le lien entre le polymorphisme génétique du CYP4A11 et du CYP4A22 et l’hypertension artérielle, ce travail représente la première description et caractérisation du polymorphisme génétique des isoformes CYP4A dans une population Française. De plus, nous avons mise en évidence une variabilité interethnique de ce polymorphisme génétique dans différentes populations testées. La glycine N-acyltransférase ou GLYAT est une enzyme impliquée dans la détoxication de xénobiotiques contenant un groupement carboxylique par conjugaison d’un résidu de glycine. Sept variations de séquence de la GLYAT ont été identifiées et quatre nouveaux GLYAT* allèles ont été caractérisés. La localisation des certaines mutations dans des structures secondaires très conservées de la protéine suggère un impact sur l’activité catalytique de cette enzyme. Bien que les conséquences cliniques potentielles de ces variations restent encore à étudier, ces résultats seront utiles pour de futures études d’association de ce polymorphisme génétique de la GLYAT avec les altérations de détoxications de xénobiotiques contenant un groupement carboxylique comme l’aspirine, certains pesticides ou le toluène.Through evolution, in order to adapt to its chemical environment, the human organism has developed enzymatic systems that can transform exogenous molecules or xenobiotic (drugs, toxins, carcinogens…), generally of hydrophobic nature, in metabolites more easily excretable via urinary or biliary tract. Some of these enzymes are also involved in catabolic processes or in the biosynthesis of endogenous compounds (fatty acids, retinoids, steroids, prostaglandins…). These enzymes thus play a major role in the protective response of the body toward chemicals and in essential physiological processes. The existence of anomalies in the sequence or structure of the genes encoding these enzymes can expose carriers of these anomalies to particular susceptibility toward xenobiotics or to impairment of essential biological reactions. In a first step, we investigated the nature and extent of the sequence variability of three genes coding for the enzymes CYP4A11, CYP4A22 and Glycine N-acyltransferase (GLYAT). In a second step, functional analyses of sequence variations were carried out, by in silico and in vitro experiments. The CYP4A11 and CYP4A22 genes are the only members of the human CYP4A subfamily. The activity of the recently identified CYP4A22 isoform is still unknown, but the CYP4A11 isoform is know as a ω-hydroxylase of the arachidonic acid, which converted into 20-hydroxyeicosatetraenoic acid (20-HETE). Several studies have shown that genetic anomalies of CYP4A are likely to contribute for susceptibility to hypertension in humans. We analyzed the sequence variations of the CYP4A11 and CYP4A22 genes in genomic DNA samples of healthy volunteers. A total of 26 polymorphisms were identified and 5 novel CYP4A* alleles were characterized for each CYP4A gene. The CYP4A 3D models were built and validated to analyse the potential impact of sequence variations identified. This work represents the first description and characterisation of genetic polymorphism of the human CYP4A genes in a French population. The glycine N-acyltranferase or GLYAT plays an important role in the detoxification of xenobiotics containing a carboxylic group via conjugation with a glycine residue. Seven sequence variations of the GLYAT gene were identified and four novel GLYAT* alleles were characterized. Localisation of missense mutations in predicted secondary structures suggest that these variants might have a potential role on the GLYAT protein activity. These results could be helpful in investigating the potential association of GLYAT variants with an incidence of reduced efficiency in xenobiotic carboxylic acids detoxification in humans, such as acetylsalicylic acid, pesticides, and solvents (Toluene)

In Silico Analysis of Metabolites from Peruvian Native Plants as Potential Therapeutics against Alzheimer’s Disease

Background: Despite research on the molecular bases of Alzheimer’s disease (AD), effective therapies against its progression are still needed. Recent studies have shown direct links between AD progression and neurovascular dysfunction, highlighting it as a potential target for new therapeutics development. In this work, we screened and evaluated the inhibitory effect of natural compounds from native Peruvian plants against tau protein, amyloid beta, and angiotensin II type 1 receptor (AT1R) pathologic AD markers. Methods: We applied in silico analysis, such as virtual screening, molecular docking, molecular dynamics simulation (MD), and MM/GBSA estimation, to identify metabolites from Peruvian plants with inhibitory properties, and compared them to nicotinamide, telmisartan, and grapeseed extract drugs in clinical trials. Results: Our results demonstrated the increased bioactivity of three plants’ metabolites against tau protein, amyloid beta, and AT1R. The MD simulations indicated the stability of the AT1R:floribundic acid, amyloid beta:rutin, and tau:brassicasterol systems. A polypharmaceutical potential was observed for rutin due to its high affinity to AT1R, amyloid beta, and tau. The metabolite floribundic acid showed bioactivity against the AT1R and tau, and the metabolite brassicasterol showed bioactivity against the amyloid beta and tau. Conclusions: This study has identified molecules from native Peruvian plants that have the potential to bind three pathologic markers of AD

miR-199a-5p and its target CAV1 are dysregulated in IPF.

<p>(A,B) Box plots showing the normalized expression of log₂-transformed CAV1 and miR-199a-5p in both IPF (n = 94) and control (n = 83) lungs. The box represents the 25–75% quartiles, the line in the box represents the median and whiskers represent the range. (**p<0.001). (C) Immunohistochemical analysis of CAV1 expression in lung tissue sections from IPF patients (i, ii and iii) and normal lung (iv). Three representative sections are shown (IPF n = 8 and control n = 8). (D) <i>In situ</i> hybridization (ISH) was performed to determine the localization of miR-199a-5p in lung tissue of IPF patients (i, ii and iii). ISH with scrambled probes (iv). Three representative sections are shown (n = 6).</p

Altered expression of miR-199a-5p and CAV1 in the unilateral ureteral obstruction (UUO) mouse model of kidney fibrosis.

<p>(A) miR-199a-5p expression in kidney from C57BL/6 mice after UUO at the indicated time points. n = 5 to 7 mice in each group. Data are expressed as mean ± SEM. * p<0.05 and ** p<0.01. (B) Paraffin sections were prepared from kidneys of C57BL/6 mice harvested 28 days after UUO. <i>In situ</i> hybridization assay was performed to determine the localization of miR-199a-5p in the kidney. Results represent one out of three independent experiments. (C) Immunohistochemical analysis of CAV1 expression in kidney tissue sections from C57BL/6 mice 28 days after UUO. One representative section out of two is shown.</p

Altered expression of miR-199a-5p in CCl₄ induced liver fibrosis mouse model.

<p>(A) Expression of miR-199a-5p in livers from 6 weeks CCl₄-treated or oil-treated BALB/C mice was analyzed by qPCR; n = 5 per group. Data are expressed as mean ± SEM. ** p<0.01. (B) Expression of miR-199a-5p in livers from 6 weeks CCl₄-treated or oil-treated C57BL/6 mice was analyzed by qPCR; n = 5 mice per group. Data are expressed as mean ± SEM. * p<0.05. (C) Expression of miR-199a-5p in livers from C57BL/6 mice 21 days following bile duct ligation or sham operation was analyzed by qPCR; n = 4 mice per group. Data are expressed as mean ± SEM. * p<0.05. (D) Expression of miR-199a-5p in liver of C57BL/6 mice during fibrosis regression. Liver fibrosis was induced by injection of CCl₄ and expression of miR-199a-5p was assessed 6 weeks after CCl₄ treatment as well as 2 and 4 weeks after the last injection. Data are expressed as mean ± SEM. * p<0.05; Expression of miR-199a-5p (E) and CAV1 (F) in primary stellate cells isolated from C57BL/6 mice and stimulated with 20 ng/ml TGFß for 48 h.</p

miR-199a-5p expression during bleomycin induced lung fibrosis.

<p>(A) Heat map representing the statistically significant (adjusted p-value<0.05) differentially expressed microRNAs in lungs from BALB/C and C57BL/6 mice in response to bleomycin at the indicated time points. Up-regulated microRNAs are shown in progressively brighter shades of red, depending on the fold difference, and down-regulated microRNAs are shown in progressively brighter shades of green. miR-199a-5p is marked in red. n = 3 mice in each group. (B) miR-199a-5p expression in lungs from BALB/C and C57BL/6 mice in response to bleomycin at the indicated time points. n = 3 mice in each group. Data from microarrays experiments are expressed as mean of normalized fluorescence intensity ± SEM. **p<0.01 (C) Paraffin sections were prepared from lungs of C57BL/6 mice 14 days following bleomycin intra-tracheal instillation. <i>In situ</i> hybridization and immunohistochemistry assays were performed to determine the colocalization of miR-199a-5p and α-SMA. Results represent one out of three independently performed experiments.</p

Identification of miR-199a-5p candidate targets using a transcriptomic approach.

<p>Normal human pulmonary fibroblasts hFL1 were transfected with pre-miR-Neg, pre-miR-199a-5p or pre-miR-21 (n = 2). RNA samples were harvested at 48 h post-transfection and expression profiles were determined with pan genomic arrays. (A) Heatmap comparing the normalized log₂ of the ratios between the signal in the different conditions and the pre-miR-Neg signal. (B) Overrepresentation of miRNA predicted targets in the set of down-regulated transcripts following miR-199a-5p and miR-21 transfection using the webtool miRonTop. Graphs show the significance of the enrichment, represented as −log₁₀ (adjusted p-value), according to the fold enrichment using 2 different prediction tools for all known miRNAs: miR-199a-5p and miR-21 are represented as an open diamond and triangle, respectively. Threshold values used to define the set of up- and down-regulated genes: AveExp = 7.0; log FC = 0.7; adjusted p-value = 0.05. (C) Venn diagram comparing the number of miR-199a-5p targets among the set of highly-downregulated genes following pre-miR-199a-5p transfection according to 3 distinct target prediction tools. Cut-offs for selection are equal to 7.0 for the log₂ (signal), to −1.5 for the log₂ (ratio), and to 0.01 for the adjusted p-value.</p

Functional impact of miR-199a-5p on lung fibroblasts.

<p>Increased expression of miR-199a-5p in lung fibroblasts results in an enhanced ability of fibroblasts to migrate, invade matrigel, proliferate and differentiate into myofibroblasts. Lung fibroblasts were transfected with 10 nM of miR-199a-5p mimic or control. (A,B) <i>In vitro</i> scratch assay was performed to assess the rate of migration of A significant increase (p<0.01) in the migration rate was observed in miR-199a-5p transfected hFL1 lung fibroblasts compared with control. Data are representative of two independent experiments. (C) Invasion assay on matrigel showing that overexpression of miR-199a-5p increases MRC5 lung fibroblast invasiveness.. Data are representative of two independent experiments. (D) Proliferation assay was performed by bivariate flow cytometric analysis of EdU/DNA-stained MRC5 cells. The x-axis represents the linear intensity obtained from propidium iodide (total DNA content), and the y-axis represents the logarithmic intensity obtained from the Alexa Fluor 647. Cells are separated into G0/G1 phase and G2/M phase according to their DNA content, and into labeled undivided and labeled divided subgroups according to the DNA content of EdU-labeled cells. The % of cells in S phase is indicated on the upper right panel. One representative experiment out of three is shown. (E) Confocal microscopy of MRC5 cells overexpressing miR-199a-5p revealed that increasing miR-199a-5p levels in lung fibroblasts promotes their differentiation into myofibroblasts. Cells were stained with an antibody against α-SMA (green) and phalloidin (red). Experiments were performed twice. (F) Relative expression of COL1A1 was assessed by Taqman PCR in MRC5 cells overexpressing miR-199a-5p and exposed or not to TGFβ. Data are expressed as mean ± SEM and derived from 2 independent experiments. *p<0.05.</p