Mitochondrial Roles and Cytoprotection in Chronic Liver Injury

The liver is one of the richest organs in terms of number and density of mitochondria. Most chronic liver diseases are associated with the accumulation of damaged mitochondria. Hepatic mitochondria have unique features compared to other organs' mitochondria, since they are the hub that integrates hepatic metabolism of carbohydrates, lipids and proteins. Mitochondria are also essential in hepatocyte survival as mediator of apoptosis and necrosis. Hepatocytes have developed different mechanisms to keep mitochondrial integrity or to prevent the effects of mitochondrial lesions, in particular regulating organelle biogenesis and degradation. In this paper, we will focus on the role of mitochondria in liver physiology, such as hepatic metabolism, reactive oxygen species homeostasis and cell survival. We will also focus on chronic liver pathologies, especially those linked to alcohol, virus, drugs or metabolic syndrome and we will discuss how mitochondria could provide a promising therapeutic target in these contexts

Etude protéomique de deux types de myofibroblastes hépatiques chez le rat

La fibrose hépatique est la conséquence du dépôt de matrice extra-cellulaire anormale par des cellules absentes du foie normal, les myofibroblastes. Il existe plusieurs hypothèses concernant leur origine, la plus courante étant qu'ils proviendraient de la trans-différenciation des cellules étoilées du foie (CEF). Récemment, les fibroblastes de l'espace porte ont été impliqués dans les fibroses portales liées aux pathologies biliaires et virales. Ces cellules pourraient jouer un rôle plus important et sans doute différent des CEF au cours de la fibrose. Le but de ce travail était de comparer les profils protéomiques de myofibroblastes de CEF (mCEF) et de myofibroblastes portaux (MP) par électrophorèse bidimensionnelle (2-DE) pour identifier des protéines exprimées différemment entre ces deux types cellulaires. Ces marqueurs pourraient permettre de déterminer in vivo, la participation de chaque type de myofibroblastes à la fibrose en fonction de son étiologie et nous renseigner sur les fonctions spécifiques de chacun. Les cellules ont été isolées à partir de foies de rats normaux et cultivées jusqu'à P2 où elles présentent un phénotype myofibroblastique. Des gels 2D à gradient de pH 4-7 puis 5,5-6,7 ont été réalisés pour les deux types cellulaires. Les profils protéiques ont été comparés et les protéines exprimées de manière différentielle ont été prélevées pour être analysées par spectrométrie de masse. Dix protéines exprimées différemment ont été relevées dont 3 ont pu être identifiées ; la cytoglobine présente dans les mCEF pourrait être un marqueur spécifique de ce type de myofibroblastes. La guanase surexprimée dans les mCEF pourrait être un témoin de phénomènes d'apoptose importants dans ces cellules. Enfin, la SPI 6 ou PI-9 chez l'homme, surexprimée dans les MP, est un inhibiteur du granzyme B et pourrait protéger ces cellules contre l'apoptose induite par les cellules natural killers. Elle pourrait devenir une cible thérapeutique intéressantePARIS6-Bibl.Pitié-Salpêtrie (751132101) / SudocPARIS-BIUM (751062103) / SudocSudocFranceF

Rôle des myofibroblastes issus des cellules étoilées du foie et des myofibroblastes portaux dans la fibrose hépatique (étude comparative par analyse protéomique et transcriptomique)

Mon travail a consisté à comparer les phénotypes de deux types de myofibroblastes hépatiques, les myofibroblastes issus de cellules étoilées du foie (MF-CEFs) et les myofibroblastes portaux (MFPs). Pour cela, nous avons utilisé deux approches complémentaires, la protéomique et la transcriptomique, qui nous ont permis de déterminer des caractéristiques phénotypiques et fonctionnelles spécifiques de chaque population de myofibroblastes. Nous avons mis en évidence que, si les MF-CEFs semblent plus fortement impliqués dans la régénération hépatique, l angiogenèse et la régulation du tonus vasculaire, ils expriment également beaucoup de protéines associées au stress. Les MFPs présenteraient un phénotype contractile plus différencié et semblent très impliqués dans le recrutement des leucocytes. D autre part, l utilisation de marqueurs identifiés dans ce travail a confirmé, dans un modèle de cirrhose, que les MFPs sont localisés dans les espaces portes et les septa. Enfin, l étude par microarray a permis d identifier un transcrit dont la protéine, l ostéoprotégérine, est synthétisée par les MF-CEFs ; cette protéine peut être dosée dans le sérum et en particulier, elle est augmentée chez les patients fibrotiques. Incluse dans un nouveau score de fibrose, sa performance diagnostique pour l évaluation du stade fibrose chez les patients atteints d hépatite C chronique semble supérieure à celle des scores existants. Ce travail apporte donc des éléments nouveaux pour la compréhension des rôles respectifs des MF-CEFs et MFPs dans la fibrose hépatique, en suggèrant notamment une contribution importante des MFPs, et ouvre également des perspectives cliniques intéressantes.PARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Proteomics Analysis of Morphogenic Changes of Human Umbilical Vein Endothelial Cells Induced by a Phorbol-Ester Mimicking Angiogenesis

Phorbol 12-myristate 13-acetate (PMA) can induce proliferation and migration of endothelial cells, mimicking vessel formation. We analysed by two-dimensional electrophoresis and MALDI-TOF/TOF the effects of PMA on cultured Human Umbilical Vein Endothelial Cells (HUVECs) to further investigate the complex mechanisms related to protein kinase C activation in this angiogenesis model. At 1 μg/ml for 24 hours, PMA induced transition of HUVECs from quiescent type into the proliferative-migrating phenotype. After 2D gel analysis, 15 differences were detected between PMA-treated samples and controls, including 8 increased proteins and 7 decreased proteins. The three main proteins identified by mass spectrometry and increased after PMA are directly involved in cell stress (α-glucosidase, heat-shock protein 70, and 150 kDa oxygen-regulated protein). Four other proteins varied in function of time, two increasing after PMA (heat shock protein 90β, protein-disulfide isomerase A3), and two other decreasing after this treatment (glucose-related protein 75, cathepsin B). These four proteins are involved in protein folding, apoptosis or tumour dissemination. Our data show that phorbol esters modify a number of proteins involved in multiple and intricate pathways for promoting a phenotype ensuring cell survival and cell migration for new vessels formation

Impact of country of birth on genetic testing of metastatic lung adenocarcinomas in France : African women exhibit a mutational spectrum more similar to Asians than to Caucasians

Background: Limited data are available on the prevalence of oncogenic driver mutations in Caucasian populations, and especially in Europeans. Aim: To evaluate the targetable mutational spectra in unselected patients with lung adenocarcinoma in routine clinical practice from several French hospitals, using the same molecular platform. Patients and Methods: Samples from 2,219 consecutive patients with histologically-proven advanced lung adenocarcinoma were centrally analysed at a referenced and certified diagnostic platform in order to test for activating and resistance mutations in EGFR, KRAS, BRAF, ERBB2 and PI3KCA. Demographic and clinical features were retrieved from the medical charts. Multivariate binary logistic regression was used to determine the independent predictive factors for the occurrence of specific mutations, in the whole study population or in selected subgroups. Findings: The overall respective incidence of EGFR, KRAS, BRAF, ERBB2 and PI3KCA mutations was 10.5%, 0.9%, 25%, 1.5%, 2.1% and 1.4%, in our study sample including 87.4% white Caucasians, 10.8% Africans and 1.8% Asians; 60.6% men, 30.7% never smoker (median age: 68.3 years). Ethnicity was an independent predictor for EGFR, KRAS and ERBB2 gene abnormalities. In all cases, a significantly higher prevalence of targetable EGFR and ERBB2, and a lower prevalence of resistance KRAS mutations were observed in African women as compared to African men or Caucasians. Conclusions: In real life conditions of routine genetic testing, we have identified subsets of patients with specific targetable activating somatic mutations according to ethnicity, who could preferentially benefit from anti-EGFR and anti-ERBB2 targeted therapies

Chronological occurrence of PI3KCA mutations in breast cancer liver metastases after repeat partial liver resection

International audienceBACKGROUND:Liver metastases of breast cancer are frequent and can recur even after "complete/R0" resection in combination with systemic and hormonal treatments. The aim of this study was to analyze throughout repeat hepatectomies for liver metastases the evolution of PI3KCA gene mutational status.METHODS:All liver metastases nodules (n = 70) from 19 women who underwent at least 2 liver resections were reexamined. DNA extraction from archived tumoral tissue was performed and the major 'hot spot' mutations in the helical and catalytic domains of PI3KCA have been analyzed using Massarray platform (Agena Bioscience) based on allelic discrimination PCR amplification followed by sensitive mass spectrometry detection.RESULTS:The two major somatic hot spot PI3KCA mutations were found in 27 (38.6%) nodules corresponding to 8 of the 19 patients (42%). The frequency of women whose breast cancer liver metastases (BCLM) carries PI3KCA mutations increased from the first to the third hepatectomy. Tumors carrying PI3KCA mutations are significantly larger and more frequently observed when resections were R0 compared to patients with no PI3KCA mutation.CONCLUSION:PI3KCA mutations are frequently observed in BCLM and persist along with the recurrence. Their identification in circulating tumor cells should become a useful biomarker in the routine practice of breast cancer management to prevent tumor recurrence and overcome the problems of intra- and inter-tumoral heterogeneity of the current biomarkers

Including osteoprotegerin and collagen IV in a score-based blood test for liver fibrosis increases diagnostic accuracy

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Proteomic analysis of NME1/NDPK A null mouse liver: evidence for a post-translational regulation of annexin IV and EF-1Bα

International audienceNME/NDPK family proteins are involved in the control of intracellular nucleotide homeostasis as well as in both physiological and pathological cellular processes, such as proliferation, differentiation, development, apoptosis, and metastasis dissemination, through mechanisms still largely unknown. One family member, NME1/NDPK-A, is a metastasis suppressor, yet the primary physiological functions of this protein are still missing. The purpose of this study was to identify new NME1/NDPK-A-dependent biological functions and pathways regulated by this gene in the liver. We analyzed the proteomes of wild-type and transgenic NME1-null mouse livers by combining two-dimensional gel electrophoresis and mass spectrometry (matrix-assisted laser desorption/ionization time of flight and liquid chromatography-tandem mass spectrometry). We found that the levels of three proteins, namely, phenylalanine hydroxylase, annexin IV, and elongation factor 1 Bα (EF-1Bα), were strongly reduced in the cytosolic fraction of NME1(-/-) mouse livers when compared to the wild type. This was confirmed by immunoblotting analysis. No concomitant reduction in the corresponding messenger RNAs or of total protein level was observed, however, suggesting that NME1 controls annexin IV and EF-1Bα amounts by post-translational mechanisms. NME1 deletion induced a change in the subcellular location of annexin IV in hepatocytes resulting in enrichment of this protein at the plasma membrane. We also observed a redistribution of EF-1Bα in NME1(-/-) hepatocytes to an intracytoplasmic compartment that colocalized with a marker of the reticulum endoplasmic. Finally, we found reduced expression of annexin IV coincident with decreased NME1 expression in a panel of different carcinoma cell lines. Taken together, our data suggest for the first time that NME1 might regulate the subcellular trafficking of annexin IV and EF-1Bα. The potential role of these proteins in metastatic dissemination is discussed