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  • Caractrisation des microparticules, des mitochondries libres et des mito-MPs produites par des cellules dadnocarcinome rnal et de leur effet sur la polarisation des macrophages

    Sous la supervision de:Luc Boudreau

    Alain SimardSandra Turcotte

    Guillaume Pelletier

  • 2

  • 1. INTRODUCTIONMicrovsicules de RCC et immunomodulation

    3

  • Objectifs gnraux

    1. Caractriser les microparticules produites par les cellules dadnocarcinome rnal (RCC)

    2. Caractriser limpact des diffrentes populations de microparticules de RCC sur la polarisation des macrophages

    4

  • Cancer du reinIntroduction

    5

  • RCC

    Renal Cell Carcinoma ou adnocarcinome rnal Cancer du rein le plus frquent (~90%) Caus par une mutation du gne VHL

    6

  • RCC

    25 30 % des patients diagnostiqus avec le RCC prsentent dj des mtastases.

    Le taux de survie 5 ans pour le mRCC est infrieur 10 %.

    7

  • n engl j med

    353;23

    www.nejm.org december

    8

    ,

    2005

    The

    new england journal

    of

    medicine

    2478

    the cases of sporadic clear-cell renal-cell carcino-ma,

    8

    which represents a major portion of all casesof renal-cell carcinoma.

    VHL protein, the product of the

    VHL

    gene, func-tions as a tumor suppressor, inhibiting growthwhen reintroduced into cultures of renal-cell carci-noma.

    9,10

    Hypoxia-inducible genes are normally in-hibited by VHL protein,

    11

    including several encod-ing proteins involved in angiogenesis (e.g., vascularendothelial growth factor [VEGF]), cell growth(e.g., transforming growth factor

    a

    [TGF-

    a

    ]), glu-cose uptake (e.g., the GLUT-1 glucose transporter),and acidbase balance (e.g., carbonic anhydrase IX[CA9]). When VHL protein is lost, these proteins areoverexpressed, creating a microenvironment favor-able for epithelial-cell proliferation (Fig. 4A). Thus,cells deficient in VHL protein behave as if they are hy-poxic, even in conditions of normoxia. VHL protein,with elongin proteins C and B, binds cul2 protein (amember of the cullin family of ubiquitin ligase pro-teins), indicating that some VHL protein serves asthe receptor subunit of a ubiquitin ligase complex

    that promotes the ubiquitination and destructionof proteins (Fig. 4B).

    12,13

    VHL protein binds thetranscriptional activators hypoxia-inducible factor1

    a

    (HIF-1

    a

    ) and 2

    a

    (HIF-2

    a

    ) directly and destabiliz-es them.

    14

    Furthermore, VHL protein promotes theubiquitination and destruction of HIF-

    a

    .

    15-17

    TheseVHL-regulated pathways are being studied as po-tential targets of therapies for clear-cell renal-cellcarcinoma.

    HIF is the key regulator of the hypoxic responsein multicellular organisms. Thus, VHL protein hasa central role in oxygen sensing. For HIF-

    a

    to bindVHL protein, a proline residue must undergo hy-droxylation, which is an unusual protein modifi-cation

    18,19

    (Fig. 4B). A family of proline hydroxy-lases operates on HIF-

    a

    in a graded fashion, so thatthe extent of hydroxylation depends on oxygen ten-sion.

    20,21

    Hydroxylation of an asparagine residueblocks the interaction of HIF-

    a

    with the transcrip-tional coactivator p300.

    22

    Thus, multiple hydroxyl-ation steps cooperate to inhibit HIF-

    a

    activity.To correlate the genotype with the disease phe-

    Figure 1. Staging Overview and Five-Year Survival Rates for Renal Cancer.

    Survival data

    3

    are based on the 1997 tumornodemetastasis (TNM) staging guidelines.

    4

    More recent renal-cancer staging is described elsewhere.

    5

    The New England Journal of Medicine Downloaded from nejm.org at UNIVERSITY OF OTTAWA on October 18, 2015. For personal use only. No other uses without permission.

    Copyright 2005 Massachusetts Medical Society. All rights reserved.

    8

  • n engl j med 353;23 www.nejm.org december 8, 2005

    medical progress

    2483

    The New England Journal of Medicine Downloaded from nejm.org at UNIVERSITY OF OTTAWA on October 18, 2015. For personal use only. No other uses without permission.

    Copyright 2005 Massachusetts Medical Society. All rights reserved.

    9

  • n engl j med 353;23 www.nejm.org december 8, 2005

    medical progress

    2483

    The New England Journal of Medicine Downloaded from nejm.org at UNIVERSITY OF OTTAWA on October 18, 2015. For personal use only. No other uses without permission.

    Copyright 2005 Massachusetts Medical Society. All rights reserved.

    10

  • Ligne cellulaire 786-O

    Cellules de RCC immortalises Mutes au gne du VHL Vendues par ATCC

    11

    786-O, 10 dcembre 2015

  • Cancer et systme immunitaireIntroduction

    12

  • Rle des macrophages

    doi:1

    0.10

    38/n

    ri307

    3

    13

  • Polarisation des macrophages

    14

  • Rle des macrophages dans linflammation

    15doi:10.1038/ni.1937

  • Vsicules extracellulairesIntroduction

    16

  • NATURE REVIEWS | GASTROENTEROLOGY & HEPATOLOGY VOLUME 11 | JUNE 2014 | 351

    Key points

    Microvesicles (MVs) are 0.11.0m vesicles containing lipids, proteins, RNAs and microRNAs; they are formed by budding from the cellular plasma membrane

    Circulating levels of several subpopulations of MVs are increased in patients with liver diseases, probably due to enhanced MV production and decreased MV clearance, related to the individual liver disorder

    MVs are now implicated at many stages of liver disease progression, including liver fibrogenesis, portal hypertension and activation of coagulation

    Several results suggest that MVs have a role in hepatocellular carcinoma by conveying information between tumour cells and between tumour and neighbouring cells

    High levels of circulating procoagulant MVs have been found in patients with acute liver failure and might contribute to normal or hypercoagulable global haemostasis in this setting

    MVs have promise as diagnostic and prognostic biomarkers in patients with liver diseases

    account for these changes. The potential roles of MVs in key processes of liver diseases, such as fibrosis, portal hypertension, complications of cirrhosis, thrombosis and hepatocellular carcinoma, are also presented. If specific studies in the context of liver diseases are lacking, we postulate on the potential effects of MVs on the basis ofavailable data from other organs. Finally, we interpret available results and propose clinical situations in which MVs could be useful as biomarkers.

    Increased MV levels in liver diseasesMVs have been detected in numerous human body fluids, including saliva,8 urine,9 bile,10 synovial fluid,11 vitreous fluid12 and semen,13 as well as in muscles,14 atherosclerotic plaques7 and liver tissue15 (Figure2a). Never theless, most studies have focused on plasma MVs, because they are easily accessible. Circulating levels of MVs are increased in patients with cardiovascular disor-ders, thrombosis and cancer.1618 Over the past ~10years, several groups have measured the levels of circulating MVs in patients with liver diseases (Table1).1930 The increased levels of several subpopulations of circu-lating MVs reported in these studies are a result of increased formation and/or decreased clearance of MVs (Figure2b;Box2).

    Enhanced MV formationSeveral causes of liver disease trigger MV production, in particular alcohol consumption,31 viral infection32 and features of the metabolic syndrome including dia-betes,33 obesity,34 dyslipidaemia35 and physical inactiv-ity.36 Liver disease itself might also induce MV release, as the main processes of MV formation (namely, apop-tosis and cell activation) are common in this context.37,38 Furthermore, many stimuli that promote MV release, including oxidative stress,39 shear stress,40,41 systemic inflammation and bacterial translocation, are present in liver diseases(Table2).10,39,4249

    Decrease in MV clearanceThe mechanisms of MV clearance have been reviewed elsewhere.5 Under healthy conditions, spleen and liver macrophages are the primary contributors to MV clear-ance from the circulation.5,50,51 Interestingly, several lines of evidence show that cirrhosis is associated with a defect in macrophage function. Indeed, the function of the macrophage Fc gamma receptor is impaired in patients with alcoholic cirrhosis and is correlated with the degree of liver insufficiency.52 Similarly, macrophage dysfunction has been observed in animal models of cir-rhosis53 and the clearance of certain molecules (radio-labelled colloid or microaggregated human serum albumin) has been shown to be decreased in patients with cirrhosis.54,55 As such, we speculate that clear-ance of MVs might also be decreased in these patients. During pathological conditions that are associated with elevated levels of circulating MVs, such as endotoxaemia, another pathway for MV clearance is turned on in the liver and lung endothelium.56 As cirrhosis is associated

    Cells

    Extracellularvesicles

    Examplesof similarsize

    MVB

    PS

    1 2

    Microvesicle

    0.11 m

    Bacteria

    Exosome

    40100 nm

    Virus

    Apoptotic body

    14 m

    Platelet

    Figure 1 | Definitions of microvesicles. Left: Exosomes. Cells release exosomes via two mechanisms. In the classic pathway (1), intracellular vesicles appear from inward membrane budding and form MVBs before fusing with the PM and being released into the extracellular space. Thedirect pathway (2) involves the release ofvesicles, in