Impact de la signalisation calcique dans les maladies de la chaine respiratoire mitochondriale (étude du déficit du complexe II associé au syndrôme de Leigh)

Nonobstant les avancées des connaissances sur les bases génétiques de la phosphorylation oxydative (OXPHOS) liées aux maladies les facteurs déterminants à la moléculaire et / ou cellulaire, pour dysfonctionnement de tissus spécifiques ne sont pas complètement compris. Nous avons étudié les événements cellulaires associés, déficit du complexe II de respiratoire mitochondriale dans deux modèles: fïbroblastes humains issus d'un patient avec mutation SDHA (succinate dehydrogenase subunit A) et les fïbroblastes et les cellules neuronales dérivées des cellules chroniquement traitées avec des inhibiteurs du complexe II. La mutation ou l'inhibition du complexe II ont été présentés afin de déterminer une augmentation importante signaux Ca2+ basal et provoqué par des agonistes dans le cytosol et les mitochondries, en parallèle avec le dysfonctionnement mitochondrial (la perte du potentiel de membrane mitochondrial ( mit), réduction de la production de l'ATP mitochondrial ([ATP]mt) et des espèces réactives d'oxygène (ROS)). Le surcharge Ca2+ Cytosolique et mitochondrial a été associé à: i / une baisse l'expression de SERCA2b et PMCA; ii / augmentation de la fuite du Ca2+ RE; iii / diminution de la motilité mitochondriale; iv / augmentation des sites contacts RE-mitochondrie et v / augmentation de la capacité d'absorption du calcium mitochondrial. Fait intéressant, nous avons montré que les cellules déficientes en complexe II ont développé une production d'ATP glycolytique Ca2+ dépendant. En outre, l'augmentation de la charge Ça2 *mitochondriale a lieu avant la perte Aymit et a été montré pour être impliquée dans le développement de la pathologie mitochondriale. Ces résultats ont révélés l'importance de la signalisation Ca2+ dans le contrôle des bioénergétiques cellulaires liés au déficit complexe II de la chaîne respiratoire. Nos résultats peuvent contribuer à la compréhension de la pathologie liée à la carence en complexe II et des maladies de la chaîne respiratoire mitochondriale en général.Despite advanced knowledge on the genetic basis of oxidative phosphorylation (OXPHOS)-related diseases, the molecular and/or cellular detenninants for tissue specific dysfunction are not completely understood. We studied the cellular events associated with initochondrial respiratory complex II deficiency in two models: human fibroblasts derived from a patient harbouring SDHA (succinate dehydrogenase subunit A) mutation and fibroblasts and neuronal derived cells treated chronically with complex II inhibitors. Mutation or inhibition of complex II were shown to determine a large increase of basal and agonist-evoked Ca2+ signals in the cytosol and the mitochondria, in parallel with initochondrial dysfunction (membrane potential ( mit) loss, [ATP] reduction and Reactive Oxygen Species (ROS) production). Cytosolic and initochondrial Ca2+ overload was shown to be associated with: i/ down- expression of SERCA2b and PMCA; ii/ increased ER Ca2+ leakage; iii/ decreased initochondrial motility; iv/ increased ER-mitochondria contact sites; and v/ increased initochondrial calcium uptake capacity. Interestingly, we showed that complex II deficient cells developed a Ca2+ dependent glycolytic ATP production. Moreover, increased mitochondria! Ca2+ load occurred prior to Av|/nl), loss and was shown to be implicated in the development of initochondrial pathology. These results revealed the importance of Ca2+ signalling in the control of the cellular bioenergetics outcomes linked to respiratory chain complex II deficiency. Our findings may help in the understanding of the pathology linked to complex II deficiency and to mitochondrial respiratory chain diseases in generalPARIS5-BU Méd.Cochin (751142101) / SudocSudocFranceF

Serum microRNAs are altered in various stages of chronic kidney disease: a preliminary study

International audienceBackground: MicroRNAs (miRNAs) are innovative and informative blood-based biomarkers involved in numerous pathophysiological processes. In this study and based on our previous experimental data, we investigated miR-126, miR-143, miR-145, miR-155 and miR-223 as potential circulating biomarkers for the diagnosis and prognosis of patients with chronic kidney disease (CKD). The primary objective of this study was to assess the levels of miRNA expression at various stages of CKD.Methods: RNA was extracted from serum, and RT-qPCR was performed for the five miRNAs and cel-miR-39 (internal control).Results: Serum levels of miR-143, -145 and -223 were elevated in patients with CKD compared with healthy controls. They were further increased in chronic haemodialysis patients, but were below control levels in renal transplant recipients. In contrast, circulating levels of miR-126 and miR-155 levels, which were also elevated in CKD patients, were lower in the haemodialysis group and even lower in the transplant group. Four of the five miRNA species were correlated with estimated glomerular filtration rate, and three were correlated with circulating uraemic toxins.Conclusions: This exploratory study suggests that specific miRNAs could be biomarkers for complications of CKD, justifying further studies to link changes of miRNA levels with outcomes in CKD patients

A multi-omics analysis of the regulatory changes induced by miR-223 in a monocyte/macrophage cell line

International audienc

Over-expression of miR-223 enhances VSMC migration.

<p>To upregulate and knock-down the expression of miR-223, VSMCs were transfected for 48 h with pre-miR-223 and anti-miR-223, respectively. Scrambled, unrelated miRNA was used as a control. The migration rate was studied by (A) wound healing assay and (B) Boyden chambers (n = 3, two independent experiments).</p

Effect of miR-223 up- and down regulation and Pi on miR-223 specific targets Mef2c and RhoB.

<p>To up-regulate and knock-down the expression of miR-223, VSMCs were transfected for 48 h with pre-miR223 and anti-miR-223, respectively. Scrambled, unrelated miRNA was used as a control. For Pi effect, VSMCs were cultured for 2 days in DMEM supplemented with 1% FBS, in the presence or absence of 3.5 mM Pi. (A) After RNA extraction, expression of Mef2c and RhoB messengers was measured by RT-qPCR. Data are represented as the mean of 3 independent experiments +/− SD). (B) Immunofluorescence images showing the effect of miR-223 up- and down regulation and Pi on Mef2c and RhoB protein expression in VSMCs (magnification 20X). (C) Fluorescence intensity measurements from the immunofluorescence images displayed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047807#pone-0047807-g004" target="_blank">Figure 4B</a>, showing the effect of miR-223 up- and down regulation and Pi on Mef2c and RhoB protein expression (n = 3 for each marker, data are represented as the mean of 3 independent experiments +/− SD).</p

Physiological and molecular effects of Pi.

<p>VSMCs were cultured for 10 days in DMEM supplemented with 1% FBS in the presence or absence of 3.5 mM Pi. (<b>A</b>) A representative photograph of a calcification assay using alizarin staining, indicating greater calcification in Pi treated cells and the corresponding quantification. Alongside a control with cells incubated at 1.1 mM Pi and to check for non-specific calcium deposition, we also ran a cell-free control with the same Pi concentrations. (<b>B</b>) The VSMCs' metabolic activity was studied in a WST-1 colorimetric assay. Cells treated with 3.5 mM Pi showed low metabolic activity. (<b>C</b>) VSMC proliferation was measured in a BrdU ELISA. (<b>D</b>) The migration rate of VSMCs was determined using Boyden chambers and was found to be twice as high in Pi-treated VSMCs than in control cells. (<b>E</b>) Expression of miR-143, miR-145 and miR-223 was measured by qPCR. Data are represented as the mean of two or three independent experiments in triplicate.</p

Inorganic Phosphate Accelerates the Migration of Vascular Smooth Muscle Cells: Evidence for the Involvement of miR-223

<div><h3>Backgound</h3><p>An elevated serum inorganic phosphate (Pi) level is a major risk factor for kidney disease and downstream vascular complications. We focused on the effect of Pi levels on human aortic vascular smooth muscle cells (VSMCs), with an emphasis on the role of microRNAs (miRNAs).</p> <h3>Methodology/Principal Findings</h3><p>Exposure of human primary VSMCs <em>in vitro</em> to pathological levels of Pi increased calcification, migration rate and concomitantly reduced cell proliferation and the amount of the actin cytoskeleton. These changes were evidenced by significant downregulation of miRNA-143 (miR-143) and miR-145 and concomitant upregulation of their targets and key markers in synthetic VSMCs, such as Krüppel-like factors−4 and −5 and versican. Interestingly, we also found that miR-223 (a marker of muscle damage and a key factor in osteoclast differentiation) is expressed in VSMCs and is significantly upregulated in Pi-treated cells. Over-expressing miR-223 in VSMCs increased proliferation and markedly enhanced VSMC migration. Additionally, we found that the expression of two of the known miR-223 targets, Mef2c and RhoB, was highly reduced in Pi treated as well as miR-223 over-expressing VSMCs. To complement these <em>in vitro</em> findings, we also observed significant downregulation of miR-143 and miR-145 and upregulation of miR-223 in aorta samples collected from ApoE knock-out mice, which display vascular calcification.</p> <h3>Conclusions/Significance</h3><p>Our results suggest that (i) high levels of Pi increase VSMC migration and calcification, (ii) altered expression levels of miR-223 could play a part in this process and (iii) miR-223 is a potential new biomarker of VSMC damage.</p> </div

Schematic representation of the hypothetical molecular and cellular consequences of elevated phosphate exposure in VSMCs.

<p>Elevated extracellular Pi resulted in the downregulation of VSMC master regulators, MYO and SMαA. The expression of miR-143 and miR-145 was also downregulated in Pi-treated cells. These events affected downstream processes by reducing the size of the actin cytoskeleton, disturbing cell morphology, upregulating miR-143 and miR-145 targets and, ultimately, leading to increased calcification and a greater VSMC migration rate. Up-regulation of miR-223 further enhances VSMC migration and reduces the amount of actin cytoskeleton. ‘-’ indicates downregulation, whereas ‘+’ indicates upregulation of the respective miRNAs or genes. See Fig. 2 for abbreviations.</p

Elevated Pi affects VSMC phenotypic markers.

<p>VSMCs were cultured for 10 days in DMEM supplemented with 1% FBS, in the presence or absence of 3.5 mM Pi. (A) After RNA extraction, we performed RT-qPCR using specific primers for VSMC phenotypic marker genes. Contractile phenotype markers (such as myocardin (MYO) and Smooth muscle α–actin (SMαA) were highly downregulated), whereas synthetic phenotype markers like Krüppel-like factor 4 (KLF4), KLF5, versican (VSCN) and platelet-derived growth factor receptor-α (PDGFRα) were significantly upregulated (n = 3). (B) Immunofluorescence of microscopic images showing the actin cytoskeleton after Pi treatment of VSMCs (magnification 40X), as also assessed (C) by fluorescence intensity measurements in corresponding histogram. (D) Representative immunoblot indicating the downregulation of NFIA in Pi-treated VSMCs. β-actin was used as endogenous control. One representative experiment shown out of two or three independent experiments.</p