Thesis
Reference
Etude du rôle de PARP-1 dans la mort cellulaire induite par l'oxygène
MÉTRAILLER-RUCHONNET, Isabelle
Abstract
Les lésions aigues du poumon sont consécutives à différentes étiologies, dont l'exposition à des concentrations élevées en oxygène. Le stress oxydatif résultant de l'hyperoxie induit la mort des cellules endothéliales, formant la barrière alvéolo-capillaire, responsable des échanges gazeux pulmonaires. Dans ce travail nous avons étudié le rôle de la molécule Poly- (ADP-Ribose) Polymerase-1 (PARP-1) impliqué dans la réparation de l'ADN, ainsi que dans la mort cellulaire. Dans cette optique, nous avons étudié le rôle de PARP-1 dans la mort cellulaire induite par l'oxygène, ainsi que dans la répartition cellulaire "in vitro" et "in vivo". Nos résultats démontrent le rôle essentiel de PARP-1 dans le contrôle de la réparation cellulaire et le remodelage tissulaire, induit lors de lésions pulmonaires dues à l'oxygène. Cependant, le rôle de PARP-1 dans le mécanisme aigu de mort cellulaire semble complexe et dépendant du type cellulaire analysé.
MÉTRAILLER-RUCHONNET, Isabelle. Etude du rôle de PARP-1 dans la mort cellulaire induite par l'oxygène. Thèse de doctorat : Univ. Genève, 2008, no. Méd. 10539
URN : urn:nbn:ch:unige-5996
DOI : 10.13097/archive-ouverte/unige:599
Available at:
http://archive-ouverte.unige.ch/unige:599
Disclaimer: layout of this document may differ from the published version.
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Département de pédiatrie
Unité de pneumologie pédiatrique Thèse proposée sous la direction du Professeur Constance Barazzone-Argiroffo
Etude du Rôle de PARP-1 dans la Mort Cellulaire Induite par l’Oxygène
THÈSE
présentéeàlaFacultéde médecine de l’Universitéde Genève pourobtenirlegrade de Docteur èsmédecine
par
Isabelle Métrailler-Ruchonnet
de Lancy (GE)
Thèsen°10539
GENÈVE 2008
RéSUMé
Les lésions aigues du poumon mènent parfois à un syndrome respiratoire défini par des critères radiologiques, présence d’infiltrats et physiopathologiques caractérisés par une hypoxémie, une dyspnée et une détresse respiratoire. Les diverses étiologies peuvent être soit la conséquence de lésions directes (pneumonies, aspiration, hyperoxie), soit de lésions indirectes du poumon (sepsis, brûlure) menant au relargage de molécules pro-inflammatoires, responsables de lésions pulmonaires. L’expression physiopathologique de ces lésions aigues provient d’un trouble de la perméabilité capillaire affectant la capacité de diffusion des alvéoles. Dans les formes les plus sévères de ce syndrome, la destruction complète de la barrière alvéolo- capillaire est la conséquence de lésions cellulaires majeures, entraînant une altération structurale pulmonaire, parfois irréversible (Bernard et al., 1994).
La barrière alvéolo-capillaire est formée de trois différentes couches : l’endothélium, l’épithélium et le tissu conjonctif. L’endothélium est composé de cellules endothéliales qui sont en contact étroit avec l’épithélium, afin de permettre une diffusion gazeuse adéquate entre les alvéoles et le sang.
Ces cellules sont très sensibles à l’oxygène et leur destruction provoque une augmentation de la perméabilité vasculaire (Zimmerman et al., 1999).
L’épithélium participe au développement, ainsi qu’à la résolution des lésions aigües du poumon.
Il est composé de deux types cellulaires : les cellules épithéliales de type I, qui représentent 75% de la surface de l’épithélium, et les cellules épithéliales de type II, qui représentent les 25%
restants. Les pneumocytes de type I sont larges et aplatis avec peu d’organelles. Ces cellules sont primordiales dans l’échange O2/CO2. À l’opposé, les pneumocytes de type II ont une forme cuboidale et contiennent beaucoup d’organelles, ainsi que des corps lamellaires. Ceux-ci permettent la production des protéines du surfactant qui sont essentielles, avec les phospholipides au maintien de la tension de surface du poumon permettant son expansion, ainsi que des mouvements de fluides dans les voies aériennes. Finalement, le tissu conjonctif et les membranes basales des alvéoles sont formés par les fibroblastes (Figure 1, partie gauche).
Le développement des lésions pulmonaires se produit selon deux phases. La première, nommée
« phase exsudative » est caractérisée par le développement de lésions alvéolaires diffuses.
Ces lésions alvéolaires se manifestent elles- mêmes par la mort des pneumocytes et des cellules endothéliales, un influx de cellules inflammatoires extravasant du plasma dans l’interstice (œdème), ainsi que d’un dépôt de fibrino-protéique dans les interstices (les membranes hyalines). Ces lésions surviennent entre le premier et le troisième jour (Figure 1, partie droite). Entre le troisième et le septième jour survient la seconde phase dite
« fibroproliférative ». Durant cette phase, les pneumocytes s’hyperplasient et les fibroblastes prolifèrent. En outre, de nombreuses cytokines, facteurs de croissance et oxidants sont également produits. D’un point de vue pathologique, cette seconde phase est cruciale pour le développement d’une phase résolutive ou au contraire d’une phase chronique de réparation (fibrose). Le degré de lésions des cellules épithéliales et la capacité de produire une réparation correcte du poumon seront les facteurs capables d’influencer le développement d’une fibrose pulmonaires ou non.
Dans un cadre thérapeutique, l’oxygène est indispensable dans le traitement des patients souffrant de lésions aiguës du poumon. Cependant, l’exposition à un taux élevé d’oxygène induit la production de radicaux libres à plus ou moins long terme. Ces radicaux sont générés lors de réactions métaboliques conduisant à la formation de molécules dotées d’un ou de plusieurs électrons solitaires. Dans des conditions normales, 90% de l’oxygène consommé par les cellules est dégradé en H2O par la cytochrome C oxidase, tandis qu’une une infime partie (1-2%) de cet oxygène est converti en radicaux libres par la mitochondrie.
Lorsque ce système est dépassé par l’exposition à quantité importante d’oxygène, le système de détoxification cellulaire n’est plus capable de faire face à la production exagérée de radicaux libres. Ceux-ci peuvent être produits à différents niveaux dans la cellule : par la chaîne respiratoire mitochondriale, par les NADPH oxidases membranaires et par quelques composants cellulaires (thioles, catécholamines ou flavines).
Les radicaux libres endommagent les lipides membranaires, les protéines, ainsi que l’intégrité de l’ADN cellulaires. Ils peuvent également affecter la cellule jusqu’à provoquer sa mort.
Deux types de mort cellulaires sont décrits à ce jour : l’apoptose et la nécrose. L’apoptose est une mort cellulaire génétiquement déterminée se produisant dans le but de remplacer des cellules endommagées, vieilles ou superflues (Kroemer et al., 1995). Les
Résumé
Figure 2 Représentation schématique des voies intrinsèques et extrinsèques d’apoptose. Adapté de MacFarlane et Williams, 2004.
Figure 1 Alvéole normale (partie droite) et alvéole détruite (partie gauche) dans la phase aigüe de lésion du pou- mon. Dans la phase aigüe, nous observons une destruction des cellules bronchiques et épitheliales alvéolaires avec la formation de membranes hyalines riches en protéines. Les neutrophiles peuvent être observés comme adhérents à l’épithelium. Dans la partie en contact avec l’air, des macrophages sécrètent des cytokines pro-inflamatoires (IL-1, IL-6, TNFa) qui permettent de stimuler le chemotaxisme cellulaire et d’activer les neutrophiles. La production de matrice extra-cellulaire est induite par les macrophages menant à la production d’oedeèmes interstitiels. Adapté de Ware et Matthay, 2000.
cellules apoptotiques présentent une condensation et une fragmentation de la chromatine, alors que la membrane plasmatique reste intacte. A l’inverse, la nécrose est une mort accidentelle survenant après une lésion physicochimique extrême, une baisse subite du glucose ou une catastrophe énergétique cellulaire (Kerr et al., 1972; Wyllie et al., 1980).
Cette mort n’est pas génétiquement déterminée et correspond morphologiquement à un gonflement des organelles découlant d’un changement dans la perméabilité membranaire. Jusqu’à récemment, ces types de mort étaient considérés comme étant distincts l’un de l’autre. Aujourd’hui, pourtant, un passage entre ces deux voies, donnant naissance à un nouveau type de mort cellulaire : l’aponécrose a été décrit (Formigli et al., 2000).
L’apoptose possède des voies d’activation clairement définies : la voie extrinsèque (dépendant de récepteurs cellulaires) et la voie intrinsèque (dépendant de la mitochondrie). Ces deux voies sont liées à l’activation de protéases, connues sous le nom de caspases. Les caspases sont une famille d’enzymes capables de cliver diffèrentes protéines une fois activées. Deux différents types de caspases sont décrites : les caspases initiatrices (caspase-2, -8, -9, -10) capables de s’auto-activer et d’activer d’autres caspases, et les caspases effectrices (caspase -3, -6, -7) dépendantes des premières pour être activées.
Une autre voie, ne dépendant pas de l’actavation des caspases (caspase-indépendante) peut être
activée, en aval de la mitochondrie, menant également à l’apoptose (Boise and Thompson, 1997, McCarthy et al., 1997). Dans des conditions normales, les molécules effectrices responsables de cette voie se trouvent attachées à la membrane interne de la mitochondrie. L’une de ces molécules se nomme AIF (apoptotic-inducing factor) (Daugas et al., 2000a) et contribue à la formation de fragments d’ADN de haut poids moléculaire, ainsi qu’à la condensation de la chromatine (Susin et al., 1999). Une autre enzyme appelée endonuclease G (EndoG), se trouvant également attachée à la membrane interne de la mitochondrie est responsable de la dégradation de l’ADN de manière caspase-indépendante.
Cette enzyme produit également des cassures de l’ADN et coopère avec la DNAse I pour faciliter la dégradation de l’ADN.
Les molécules provoquant la mort cellulaire de manière caspase-dépendante ou caspase- indépendante sont localisées au niveau de l’espace intermembranaire de la mitochondrie. Dans le cadre de l’activation de la voie intrinsèque caspase- dépendante, le relargage du cytochrome C est un élément essentiel. En effet, une fois relâché dans le cytosol, le cytochrome C est capable d’activer la caspase-3 en coordination avec d’autres molécules telles qu’Apaf-1, l’ATP et la pro-caspase-9. Le relargage du cytochrome C se produit par deux méchanismes différents : soit par l’ouverture du pore mitochondrial (MPTP), soit par la formation d’un canal après l’oligomérisation et l’insertion
Figure 3 L’intensité des stimulis menant aux dommages de l’ADN déterminent la survie, l’apoptose ou la nécrose cel- lulaire. Dépendant de l’intensité du stimulus, les agents génotoxiques peuvent mener à différentes voies. Lors de dom- mages légers de l’ADN, la poly(ADP-ribolysation) facilite la réparation de l’ADN et la survie cellulaie (voie 1). Des stimuli génotoxiques plus importants activent la voie de mort apoptotique caspase dépendante (voie 2). Des lésions sévères de l’ADN peuvent induire une activation excessive de PARP, diminuant le stock de NAD/ATP cellulaire. Cette diminution de stock bloque l’apoptose cellulaire résultant en nécrose (voie 3). Modifié de Virag et Szabo, 2002.
Résumé
de la molécule Bax ou du complexe Bax/Bak. Ces méchanismes sont activés à partir de différents stimuli, dont l’exposition à un stress oxidatif. En outre, l’ouverture du MPTP permet également le mouvement de solutés vers l’intérieur de la mitochondrie et inhibent la production d’ATP menant à un gonflement de la mitochondrie susceptible de conduire à la nécrose. Lors de l’ouverture du MPTP, AIF est également relâché.
L’effet de l’oxygène et ses conséquences sur la vie cellulaire a été largement étudié à ce jour. In vitro, on peut observer suivant le type cellulaire étudié de la nécrose ou de l’apoptose avec une exposition à l’oxygène à faible concentration (Budinger et al., 2002; Katoh et al., 1997b; Kazzaz et al., 1996). Les différentes voies d’activation,Les différentes voies d’activation, ainsi que leur conséquences sur le métabolisme, ont permis d’envisager des cibles moléculaires à protéger pour retarder ou éviter les mécanismes de mort cellulaire (Ahmad et al., 2002; Nicotera et al., 1999; O’Reilly, 2001; Wang et al., 2003).
In vivo, différentes stratégies ont égalmenet déjà été étudiées dans l’optique de diminuer les lésions aigues dues à l’oxygène, ainsi que d’empêcher la destruction de la barrière alvéolo-capillaire survenant lors de la mort cellulaire (Lu et al., 2001; (Lu et al., 2001;(Lu et al., 2001;
Pagano and Barazzone-Argiroffo, 2003; White et al., 1991).
Les Poly-(ADP-ribosyl) polymerases (PARP)(ADP-ribosyl) polymerases (PARP) font parties d’une famille d’enzymes nucléaires,
comptant 18 membres, impliquées dans la réparation de l’ADN, ainsi que dans la mort cellulaire. PARP-1 est la molécule la plus importante au point de vue de l’activité de cette famille, ainsi que la molécule la plus étudiée.
Son activité induit la poly(ADP-ribosyl)ation de protéines nucléaires en utilisant la catalyse de la nicotinamide adénine dinucléotide (NAD) et de l’ADP-ribose, permettant ainsi la réparation des cassures de l’ADN. Le rôle de PARP-1 dans la mort cellulaire dépend de l’importance des lésions nucléaires et celui-ci joue un rôle clé dans la balance entre l’apoptose et la nécrose (Figure 3).
Dans ce contexte, la présente étude est dédiée àa présente étude est dédiée à l’analyse du rôle de l’oxygène dans les dommages de l’ADN et de mieux comprendre les mécanismes permettant leur réduction. D’un point de vue méthodologique, ce travail est divisé en deux parties distinctes. La première est consacrée à l’étude de la molécule PARP-1 et de son action sur la mort cellulaire aigue induite pas l’oxygène in vivo et in vitro. Dans la seconde partie, nous avons étudié l’influence de PARP-1 dans le contrôle de la prolifération cellulaire et les mécanismes de réparation après destructions aigeus du poumon.
Nos résultats ont révèlé un rôle essentiel de PARP-os résultats ont révèlé un rôle essentiel de PARP- 1 dans le contrôle de la réparation cellulaire et le remodelage tissulaire, induit lors de lésions pulmonaires dues à l’oxygène. Cependant, le rôle de PARP-1 dans le mécanisme aigu de mort cellulaire semble complexe et dépendant du type cellulaire analysé.
CONTENTS
RéSUMé 1
CONTENTS 5
REMERCIEMENTS 7
ABBREVIATIONS 9
1-INTRODUCTION 11
1.1 Hyperoxia-induced lung damage: from clinical context to animal model 12
1.2 Oxygen toxicity: the role of ROS 13
1.3 Mechanism of cell death 15
1.3.1 Apoptosis versus necrosis 15
1.3.2 The pathways leading to apoptosis 16
1.4 Hyperoxia and mechanisms of cell death 17
1.5 The Poly (ADP-Ribose) Polymerase molecule 18
1.6 PARP-1 implication in cell death 19
1.7 The role of the PARP-1 inhibition in oxidative-induced cell death 20
1.8 Introduction to the thesis work 21
2-RESULTS 23
Poly (ADP-ribose) polymerase activation mediates lung epithelial cell death in vitreo but is not essential in hyperoxia-induced cell death
Poly (ADP-ribose) polymerase-1 (PARP-1) controls lung cell proliferation and repair after hyperoxia-induced lung damage
3-CONCLUSIONS AND PERSPECTIVES 47
REFERENCES CITED 49
REMERCIEMENTS
Un travail de thèse est rarement le résultat d’un seul individu. Il est le résultat de personnes dont la con- tribution tend vers un intérêt commun. A travers ces quelques lignes, je souhaite remercier les personnes qui ont participé à ce travail et sans qui ce dernier n’aurait été possible. Je souhaite leur exprimer toute ma recon- naissance et leur témoigner toute mon amitié.
Le Prof. Constance Barazzone, directrice de cette thèse et de mon P.h.D m’a accueillie durant plusieurs années dans son laboratoire. Durant toute cette période, j’ai bé- néficié de son soutien et de sa confiance tandis que sa supervision a représenté pour moi une source de moti- vation et d’encouragement.
Ce travail n’aurait pu être mené à bien sans l’aide de mes collègues de laboratoires. Tout particulièrement, le Dr. Alessandra Pagano (Université Aix-Marseille) m’a encadrée tout au long de mon travail. Sa persévérance, sa rigueure scientifique et sa capacité d’analyse ont été une véritable source d’inspiration pour mon travail et resterons un modèle pour l’avenir. Je dois à Yves Do- nati de m’avoir enseigné la base de multiples tech-
niques de laboratoire. Toujours disponible, il a effectué maintes fois des expériences sonvent infructueuses, en conservant intacte sa bonne humeur. Le Dr. Stéphanie Carnesecchi a été présente lors des dernières expéri- ences de ce travail. Ses remarques et son expérience ont contribué à l’aboutissement de celui-ci. A tous les trois, je souhaite leur témoigner ma plus profonde am- itié et ma plus grande reconnaissance.
De nombreuses autres personnes ont par leurs conseils et leur expérience permis de mener à bien mes projet.
Parmi celles-ci, je souhaite remercier particulièrement le Dr. Michel Aurrand-Lions (Inserm, Marseille) dont les suggestions et les critiques pertinentes ont été déter- minantes à la publication de certains resultats ainsi que les Professeurs Dominique Belin, Shozo Izui et Paolo Meda.
Ce travail a été financé par le Fond National de la Re- cherche Scientifique (FNRS), la Faculté de Médecine de l’Université de Genève, la bourse Wölfermann Nae- gele et la Société Suisse de Pneumologie que je remer- cie également.
ABBREVIATIONS
AIF Apoptosis Inducing Factor ALI/ARDS Acute Lung Injury
ATP Adenosine –Tri-Phosphate BPD Bronchopneumodysplasia CAD Caspase-activated DNAse EndoG Endonuclease G
HUVEC Human Umbilical Endothelial Cells
IL Interleukine
NAD Nicotinamide Adenine Dinucleotide NFkB Nuclear Factor Kappa B
OMM Outer Mitochondrial Membrane
PAR Pol y(ADP-ribose)
PARP Poly-ADP Ribose Polymearse PCNA Proliferation of Cell Nuclear Antigen ROS Radical Oxygen Species
SP-A/-B/-C Surfactant Protein -A/-B/-C
STAT Signal Transducer and Activator of Transcription
0
-INTRODUCTION
A large number of studies have demonstrated that increased oxidative burden occurs in lung and airway diseases, as shown by increased markers of oxidative stress either in animal models or in patients (Yu, 1994b). In most pulmonary diseases, such as asthma, tobacco exposure, cystic fibrosis and acute respiratory distress syndrome (ARDS), oxidant burden is enhanced resulting from the concomitant direct effects of oxidants in the extracellular space as well as the increased release of reactive oxygen species (ROS) from macrophages and neutrophils (MacNee, 2001).
Acute lung injury (ALI/ARDS) is a syndrome of respiratory failure defined by radiological (lung infiltrates) and physiological (hypoxemia, dyspnea, respiratory failure) criteria consequent to critical illness. ALI/ARDS can arise either from direct lung injuries (e.g., pneumonia, aspiration, toxic inhalation, lung trauma and hyperoxia), or through indirect mechanisms, such as sepsis or burns. The pathophysiological expression of ALI/ARDS consists in troubles of the capillary permeability and the alveolar diffusion capacity. The most severe forms of ALI/ARDS are characterized by the full destruction of the alveolo-capillary barrier, caused by an extensive cellular damage and a subsequent pulmonary edema (Bernard et al., 1994).
The alveolo-capillary barrier is composed of three different layers: the endothelium, the connective tissues and the epithelium (see Figure 1A, left side). Endothelial cells are a key constituent of the capillaries and are in close contact to the epithelium.
These cells are sensitive to cellular injuries and their destruction increases vascular permeability through the formation of intercellular gaps. Their injury induces also the up-regulation of surface adhesion molecules, which allows the recruitment of inflammatory cells and widens the cellular damage (Zimmerman et al., 1999). The epithelium is composed by two different types of epithelial cells: the type I and the type II pneumocytes.
Type I pneumocytes, the main components of the epithelium, are the largest and the thinnest, and have a low density of subcellular organelles. They operate as a semi-permeable barrier allowing O2 and CO2 to flow rapidly across. These cells are neither able to regenerate nor reproduce. On the other hand, type II pneumocytes represent 25% of the total epithelial cells. They are cuboidal-shaped with a high density of subcellular organelles, the lamellar bodies, which contains layers of pulmonary surfactant. The surfactant is secreted at the surface of the alveoli and operates as an ion
and fluid pump, which allows physiological fluids to move out of the air spaces. It is constituted of phospholipids, proteins (SP-A, SP-B and SP- C) and lysosomal enzymes. The surfactant also enables to decrease the alveolar surface tension impeding pulmonary collapse and has an anti- microbial effect. In consequence, an insufficiency of surfactant production usually affects correct gas exchange due to the development of lung atelectasis. The epithelial barrier is normally tighter than the endothelial barrier. ALI/ARDS has been described to occur in two distinct phases. The exudative phase (1-3 days after the initial insult) is characterized by diffuse alveolar damage with pneumocytes death, microvascular injury, influx of inflammatory cells and proteinaceous fluid deposit (hyaline membrane) in the interstitium (Figure 1, right side) (Bachofen et al., 1982; Tomashefski, 1990). This phase is principally destructive with an important level of cell death. From the 3rd to the 7th days, the fibroproliferative phase embraces a process of lung repair characterized by the hyperplasia of pneumocytes II and the proliferation of fibroblasts (Tomashefski, 1990). In the case of epithelial injury, type-II pneumocytes migrate to the denuded area and proliferate into new alveolar type II cells, or differentiate into alveolar type I cells (Adamson and Bowden, 1974; Mason et al., 2002; Nielsen et al., 1997). Type II cells are therefore considered as the progenitors of alveolar epithelial cells involved in the development and repair of ARDS. During this process, fibroblasts, epithelial type II and inflammatory cells replace the altered lung part by a new arrangement. Moreover, throughout this period, inflammatory cytokines, proteases, oxidants, growth factors and lipid mediators are secreted. Depending on the degree of alteration, this new architecture may exhibit abnormal features (Adamson et al., 1988a, Yee et al., 2006). Based on this discrepancy, the degree of epithelial injury can be deduced according to the outcome, from a complete resolution to an inadequate repair leading to fibrosis (Sznajder, 1999; Zapol et al., 1979).
ALI/ARDS affecting preterm babies (characterized by low birth weight and gestational age) is called idiopathic respiratory distress syndrome (IRDS).
It is caused by lung immaturity and insufficient surfactant production by type II cells, leading to alveolar collapse and poor oxygenation. As a therapy, surfactant and supplemental oxygen are provided with mechanical ventilatory support.
However, these treatments have also a deleterious effect favouring the development of IRDS
Chapter 1
(Jobe and Ikegami, 1998). In extreme cases, it evolves towards a more chronic form called bronchopulmonary dysplasia (BPD). Through time, this form can resolve partially, but can also persist as a chronic pulmonary illness during adulthood (Hulskamp et al., 2005).
Today severe BPD is still considered as a several illness associated with co-morbidity. However, it is difficult to find a good relationship between the severity of IRDS and the subsequent development of BPD. Indeed, BPD development is complex and secondary to multiple factors of prematurity involving not only surfactant insufficiency, but also lung inflammation, barotrauma, oxygen toxicity and probable genetic susceptibility. High levels of oxidative stress markers, such as 8-isoprostane or malondialdehyde, and low antioxidant levels have been reported in preterm ventilated babies and were found to be good predictors of BPD development (Ahola et al., 2004; Collard et al., 2004). Therefore, assessment of normal or abnormal repair after oxidative stress induced- acute lung injury seems a good track to follow in order to better understand the physiopathology of BPD. In humans, investigations of BPD depend on indirect sampling such as bronchoalveolar lavages (BAL) or on autopsies. In this context, animal models are useful complementary approaches to study the acute and the repair phases of diseases,
and to identify molecular changes that may provide targets to possible therapeutical interventions.
1.1 Hyperoxia-induced lung damage:
from clinical context to animal model
As stated previously, exogenous oxygen treatment is used to cure critical ill patient or preterm infants in order to restore adequate gas exchange.
However, oxygen remains the main source of reactive oxygen species (ROS) that are toxic for most cells when used at high concentration (Crapo, 1986b; Freeman and Crapo, 1982; Harris et al., 1991).
Exposure to high oxygen concentrations (100%) has been used as a valuable model of ALI/ARDS in different animal models according to the point of interest. Lung morphology and damages triggered by mechanical ventilatory have been studied in baboons and sheep (Albertine et al., 1999;
King, 1989, Maniscalco et al., 2005). Hyperoxia- induced inflammation has been analyzed in rabbits, whereas molecular aspects have been explored in rat and mouse models (Barazzone et al., 1998b; Crapo, 1986b; Crapo and Tierrney, 1974). Survival rate varies accordingto the studied species. Adult rats survive between 60 to 66 hours Figure 1 The normal alveolus (left side) and the injured alveolus (right side). In the acute phase, there is sloughing of both bronchidal and alveolar epithelial cells, with the formation of protein-rich hyaline membrane. Neutrophils are shown adhering to the injured epithelium. In the air-space, macrophages secrete pro-inflamatory cytokines (IL- 1, -6 and TNFa) acting locally to stimulate chemotaxis and to activate neutrophils. The production of extracellular matrix by macrophages is induced and interstitial edema occur. Adapted from Ware and Matthay, 2000.
and mice survive between 90-96 hours (Clark and Lambertsen, 1971; Crapo et al., 1980), and adult monkey survive much longer (13 days) (Kapanci et al., 1969). From a morphological point of view, the sequence of changes occurring in lungs in response to oxygen toxicity remains very similar from a species to another (Crapo, 1986a). Indeed, hyperoxia-induced acute lung damage has been recognized to occur in 3 steps. During the first hours of exposure, cells are affected by both extracellular ROS and by a synthesis of intracellular ROS. Indeed, mitochondria, microsomes and nuclear membranes are the main source of ROS and are responsible for the first cellular damages (Freeman and Crapo, 1982; Turrens et al., 1982;
Yusa et al., 1984). Analysis of rat lungs by electron microscopy (EM) showed fluid accumulation around the capillary, resulting from increased vascular permeability (Crapo, 1986b). According to the animal model, damage occurring during this stage is reversible and no lesions are observable on optical microscopy. The second stage is interpreted as the inflammatory phase and spans from 24 to 48 hours of exposure. It is usually associated with an accumulation of inflammatory cells namely neutrophils and the release of inflammatory mediators in the lung increasing the stress damage (Figure 2). The combination of these mechanisms leads to pulmonary cellular infiltrates and perivascular edema (recognizable on optical microscopy at 48 hours), which amplify cell injury (Barry and Crapo, 1985). Cell death is associated with complete destruction of the alveolo-capillary barrier (Barazzone et al., 1998b;
Crapo et al., 1980). The ending of this second stage is considered as the limit for the injury reversibility.
Beyond, the third stage is characterized by the alveolar cell destruction. In rodents, it usually takes place between 72 to 96 h of exposure and corresponds to the irreversible alteration of the pulmonary capillary endothelium and epithelium (Figure 2). All these damages bring to the disruption of the alveolo-capillary barrier and to the deposition of fibrin and cellular debris forming the hyaline membrane layer in the alveoli. In rats, approximately 50% of capillary endothelial cells
are destroyed in the hours preceding death. The total capillary surface decreases in direct proportion to the loss of endothelial cells (Crapo et al., 1980).
Following the oxidative damage, injured and dead cells are replaced through increased proliferation and differentiation. However, failure to modulate these processes leads to excessive proliferation of epithelial cells and fibroblasts, increased collagen deposition and chronic lung disease (CLD).
Models that combine immaturity, hyperoxia and barotrauma, more representative of BPD, exist only in lambs and primates and are therefore not easy to produce (Coalson et al., 2000).
Acute exposure to hyperoxia 100% O2 or chronic exposure to lower oxygen tension of premature rats results in lesions that are close to BPD (Tierney et al., 1977; Wagenaar et al., 2004). Recently, it has been reported that the selective induction of TGF-b in mouse pups epithelial cells was able to induce lesions resembling BPD (Lee et al., 2004).
In mice, the most studied models of pulmonary- induced fibrosis are bleomycin-induced fibrosis and radiation-induced fibrosis (Beinert et al., 1999; Piguet et al., 1989). None of these two models are really close to BPD, but they are rather useful to study the process of epithelial injury and myofibroblasts/fibroblast proliferation, two features common to varieties of CLD. Recovery from acute exposure to oxygen is considered more representative of BPD, although it has not been well studied in mice, since these animals often die rapidly from oxygen toxicity without completely repairing acute lesions (Adamson et al., 1988b).
In spite of this disadvantage, genetically modified mice are irreplaceable to dissect the molecular and cellular process of lung damage. Indeed, while the progression of pulmonary oxygen toxicity is chronologically ordered, the sequence of events and the different mechanisms involved at single cell level remain far from being understood.
1.2 oxygen toxicity: tHe role of roS Oxygen-derived reactive oxygen species (ROS) are produced by intermediate mechanisms of metabolic reactions characterized by the presence of one or more unpaired electrons. ROS play a
Figure 2 Representative electronic picture of air (A) and hyperoxia 84h (B) exposed mouse lung. Note the destruction of epitelial cells. The arrow points proteacinous deposits. Magnification 10000x. Barazzone, unpublished.
Chapter 1
regulatory role in multiple cellular metabolic processes (such as cell growth, cell death or inflammation) and modulate physiological cell signaling (such as cell migration) (Lee and Choi, 2003; Zaher et al., 2007). They participate also to the activation of various enzymatic cascades (e.g., MAPK pathway), and transcriptional and redox sensitive factors (e.g., NFkB, AP-1) (Janssen et al., 1993).
A high and abnormal ROS production disturbs physiological cellular functions due to its toxic effect. In normal conditions, 90% of oxygen consumed by cells is degraded into H2O by the cytochrome C oxidase system, avoiding significant production of ROS. In these conditions, the majority of ROS derives from mitochondrial respiration and corresponds to 1-2% of the molecular oxygen consumption (Richter et al., 1995). During hyperoxia, ROS are generated through the metabolism of oxygen and are responsible for tissue damage (Fridovich, 1978). ROS are composed of inorganic molecules including hydrogen, transitional metals and oxygen (O2) (Fleury et al. 2002). In oxidative conditions, O2 can accept a single electron transfer, allowing the formation of free radicals. Superoxide radical (O2-) is found in different aerobic cells, such as phagocytic cells, neutrophils, monocytes, and macrophages (Fridovich, 1978). O2- is extremely unstable and can be either converted into H2O2, spontaneously or catalytically by superoxide dismutase (Freeman and Crapo, 1981,Raha and Robinson, 2001). Only 1-2% of oxygen is converted to superoxide anion by the mitochondrial electron transport chain. The remaining oxygen is converted into other radical oxygen intermediates (ROI) in different cell compartments. Additional reactive species such as hydroxyl free radical (.OH) and perhydroxyl radical (HO2.) are also produced, but in a minor proportion. Altogether, these ROS have a stronger deleterious effect than the superoxide itself, and interact with transitional metals and also with almost every type of cellular molecules (e.g., organic lipids, sugars and amino
acids) (Halliwell and Gutteridge, 1984; Yu, 1994a).
The ROI produced under these former conditions are directly scavenged or can react with cellular macromolecules. As a consequence, ROI are maintained at low concentrations in normal cellular steady-state. In a neutral aqueous medium, a wide variety of soluble cell components such as thiols, hydroquinones, catecholamines, or flavins undergo oxido-reduction reactions, and contribute to the intracellular free radical production. In addition, enzymes such as xanthine oxidase generate also free radicals during their normal catalytic cycling (Freeman et al., 1982; Yu, 1994a).
In hyperoxic conditions, levels of intracellular ROS are increased, modifying the cellular antioxidant capacity (Panus et al., 1988). In these conditions, ROS are generated by the mitochondrial electron transport chain and the plasma membrane NAD(P)H oxidase system added to the extracellular ROS production (Figure 3) (Chandel et al., 1998;
Freeman and Crapo, 1981).
Mitochondria consume about 90% of the inhaled oxygen and represent a rich source of ROS.
However, when the ROS production exceeds the cellular capacity of detoxification, an increased accumulation of cofactors allows the production of intracellular .O2- (Richter et al., 1995;
Thannickal and Fanburg, 2000). In addition to ROS production, heavy metal ions enclosed in mitochondria can also generate hydroxyl radical, leading to cytosolic damage. Free radicals are either produced in the intra-mitochondrial space or in the endoplasmic reticulum, and both alter the nuclear membrane (Freeman and Crapo, 1981).
In this case, extra-cellular-generated free radicals can cross the plasma membrane, react with cellular components (mainly lipids), and initiate toxic reactions at the membrane itself. These latter directly affect the unsaturated fatty acids and the transmembrane proteins including oxidable amino acids. Cell surfaces are thus both targets of reactive free radicals and gates for permeable radical species.
Figure 3 ROS generation in hyperoxic conditions: mitochondrial respiratory chain and NADPH oxidase system are the major source of ROS. Modified from Chandel and Budinger, 2007.
Lipid peroxidation arising from the reaction of free radicals with lipids is considered as a hallmark of the cellular injury. In the membrane, lipid or free fatty acid peroxidation is provoked by the attack of free radical species hosting enough reactivity to break the hydrogen-carbon bond. Through time, lipid peroxidation changes the lipid molecular structure, disrupting the cohesive lipid bilayer arrangement and the structural organization of biological membranes (Freeman and Crapo, 1982). Proteins can also be modified through oxidative reactions, such as protein fragmentation or aggregation, which render them susceptible to proteolytic digestion. This susceptibility of proteins towards free radical damage depends on their amino acid composition and on the importance and location of specific amino acids that mediate protein conformation and activity (Freeman and Crapo, 1982; Yu, 1994a). Finally, free radicals can damage DNA and in particular provoke strand breaks. The consequence is chromosomal aberrations and sister chromatin exchange either arising from nucleic acid base modifications or DNA strand scission (Dreher and Junod, 1996; Freeman and Crapo, 1982; Gille and Joenje, 1989). Mitochondrial DNA is particularly exposed to ROS damage, since mitochondria are a source of ROS poorly endowed with DNA repair processes (Backer and Weinstein, 1980). Many of these DNA modifications are mutagenic and favor cancer onset, aging or neurodegenerative diseases. When overproduced, the ROS can affect the different cellular compartment, avoiding normal signalling pathways and damaging the cells irreversibly.
In cultured cells exposed to high oxygen concentration, oxidation increases lactate production, perturbs mitochondrial respiration and depletes ATP content (Balin et al., 1976; Gille(Balin et al., 1976; Gille et al., 1989; Joenje et al., 1985; Schoonen et al., 1990a). Moreover, cells exhibit limited growth,Moreover, cells exhibit limited growth, reduced proliferation, decreased DNA and protein synthesis, and reduced plasma membrane integrity (Clement et al., 1985, Jornot et al., 1987; Junod et al., 1989; Junod et al., 1987). The decreasedThe decreased rate of proliferation is associated with a post- transcriptional control of two late cell-cycle- related genes: the histone and the thymidine kinase, leading to G1/S cell cycle arrest (Clement et al., 1992). The molecule p21 has been also described to block cell cycle in G1/S phase the G1 cyclins D and E and the S-phase cyclin A (Harper et al., 1993; Xiong et al., 1993). It can also inhibit the replication of DNA and the proliferation cell nuclear antigen (PCNA), which operates as an auxiliary factor for DNA replication and repair (Helt et al., 2004; O’Reilly et al., 1998). The increased expression of p21 has been described to be dependent of the accumulation of p53 in cells exhibiting DNA damage (O’Reilly et al., 1998;
Shenberger and Dixon, 1999). Like p53, several growth arrest and DNA damage (GADD) genes are induced in cells exposed to oxygen (O’Reilly et al., 2000). Among the existing molecules, GADD45 inhibits the cellular proliferation and stimulates the repair of DNA excision through a p53 dependent mechanism. Cell cycle stop at the G2 checkpoint has been studied in hyperoxia and has been linked with the up-regulation of the
ATM/ATR molecule (O’Reilly et al., 2003). The cell type seems to influence the checkpoint where cell stops upon oxygen exposure. Indeed, A549 or Mv1Lu or HCT116 have been described to interrupt in G1/S phase, whereas MLE15 stopped in G2 phase (Helt et al., 2001; Rancourt et al.,(Helt et al., 2001; Rancourt et al., 2001; Rancourt et al., 1999).
1.3 mecHaniSm of cell deatH
Cell death is a complex and regulated mechanism, which represents an essential step in normal (embryology development, cell turnover, cellular defence against external injuries) and pathological situations (ischemia-reperfusion, sepsis). These take place through two principal pathways: the apoptosis and the necrosis and these are briefly described hereafter.
1.3.1 apoptosis versus necrosis
Apoptosis can be defined as a genetically determined death responsible for the replacement of superfluous, aged or damaged cells (Kroemer et al., 1995). Apoptotic cells show a condensation (pyknosis) or a fragmentation (karyorrhexis) of their chromatin associated with a cellular shrinkage. The organelles and plasma membrane remain impermeable and exhibit a change in the orientation of the phosphatildyleserine residues at the plasma membrane (membrane flip-flop) (Kroemer et al., 1998).
On the other hand, necrosis is the outcome of a severe and acute injury marked by an abrupt anoxia, by a sudden decrease of glucose or extreme physicochemical injury (heat, detergents or strong bases) (Kerr et al., 1972, Wyllie et al., 1980).
This mechanism is not genetically controlled and corresponds to a morphological increase of cell volume caused by the permeabilization at the cell membrane. This increase of volume causes the rupture of the plasma membrane, dismantles the swollen mitochondria, and leads to ATP depletion and cell energy failure. As opposed to apoptosis, necrosis is not associated with specific markers of the DNA condensation, and can be detected rather by electronic microscopy. Local inflammation is frequently associated.
Although apoptosis and necrosis have long been viewed as opposed mechanisms, it is now generally agreed that they represent two end- members of a continuum. The evolution of injured tissues towards necrosis or apoptosis mainly depends on the abundance of energy stores within the affected cells. Apoptosis generally requires energy while necrosis occurs in ATP depleted cells (Nicotera et al., 1998). Consequently, preserved and maintained ATP levels support apoptosis over necrosis (Eguchi et al., 1997; Leist et al., 1997a), whereas an insufficient amount of ATP transforms apoptosis into necrosis (Hirsch et al., 1997; Xiang et al., 1996). As a consequence, a new definition of cell death embracing necrosis and apoptosis has been proposed: aponecrosis (Formigli et al., 2000). This mechanism results from an incomplete execution of internal apoptotic pathway followed by necrotic degeneration.
Chapter 1
1.3.2 the pathways leading to apoptosis
In human cells, the pathway leading to the apoptotic death is divided in three successive phases called initiation, integration/decision and execution/
degradation (Kroemer, 1997). The initiation phase varies according on the nature of the death-inducing signal, which can be either extrinsic (causing by the ligation of a death receptor) or intrinsic (affecting different organelles). The following integration/
decision phase involves the near-to-stimulation activation of caspases and the mitochondrial death effectors through a complex molecular interplay.
During this phase, the decision to die is taken and the “no return point” is trespassed. Finally, the execution/degradation phase, which is common to all types of cell death, corresponds to a post mortem process. It characterizes morphological and biochemical alterations associated with the latest stages of apoptosis independently from the initializing stimulus. Apoptosis is a complex mechanism ending with DNA fragmentation, which achieved through a caspase-dependent or a caspase-independent pathway.
The caspase-dependent pathway
This pathway is characterized by the activation of apoptotic specific cystein proteases (Boatright and Salvesen, 2003). Caspases are usually located in the cytosol and their activation is modulated by activators, such as Apaf-1 (Golstein and Kroemer,(Golstein and Kroemer, 2005; Kroemer and Jaattela, 2005; Papucci et al.,
2004; Vercammen et al., 1998). These proteases are known to cleave the aspartic acid residue of several proteins and the four amino-terminal residues of the caspase cleavage site, which determine their substrate specificity. Prior to evolve towards mature enzymes, caspases are synthesized as enzymatically inert zymogens weighting between 30 and 50 kDa. Morphologically, these zymogens are constituted of three distinct domains: an N- terminal prodomain, a large domain named p20, and a small domain named p10. Prodomains range from 23 to 219 a.a. depending on the caspase morphology. Caspases with a long prodomain are known as initiators (e.g., caspase-2, -8, - 9, and -10) and are able to self-activate. The mechanism responsible for this self-activation is either attributed to a cleavage of the N-terminal cell death effector domain (DED) (for pro-caspase -8 and -10), or to a complex formation with the caspase recruitment domain (CARD) (for pro- caspase-2 and -9) of the prodomain (Grutter, 2000).
Caspases with short prodomains, called effector caspases, are activated by initiator proteases: the caspase-3, -6, and -7. Their conversion into mature enzymes requires two cleavages, one separating the prodomain from the large subunit, and the other separating the large from the small subunits (Fuentes-Prior and Salvesen, 2004). Furthermore, several caspases have the capacity to activate other caspases creating a cascade (Fuentes-Prior and Salvesen, 2004). The involved caspases are those composed of a short prodomain (Boatright and Salvesen, 2003). Caspase activation occurs
Figure 4 Schematic representations of extrinsic apoptotic pathways. Adapted from Mac Farlane and Williams, 2004.
through two different mechanisms, called extrinsic and intrinsic pathways.
The extrinsic pathway is activated to eliminate discarded cells during the development or the immune response. It depends on apoptotis inducers (e.g., TNF, Fas-L, CD40), which bind to their cell surface specific receptor (Figure 5) (Boatright and Salvesen, 2003, Martin et al., 2005). Upon binding to their ligands, death receptors aggregate and form membrane-bound signaling complexes (Boatright and Salvesen, 2003) named Fas-associated death domain (FADD) or TNFR-associated death domain (TRADD). These complexes interact with the death domain (DED) located in the prodomain of pro-caspase-8. As a result, a new complex, called death-signaling complex (DISC) is formed on the cytosolic side of the plasma membrane. Through adaptator proteins, DISCS recruits additional molecules of pro-caspase-8, leading to an elevated concentration of zymogens.
On the other hand, the intrinsic pathway, also called mitochondrial pathway, occurs in cells injured by ionizing radiation, chemotherapeutic drugs or direct mitochondrial damage (e.g. oxidative stress).
In cellular stress conditions, the mitochondria become permeabilized due to the opening of MPTP (see paragraph 1.5.3). Depending on the cell type, caspase-8 can also directly activate procapase- 3 or a mitochondrion-mediated pathway by truncating the pro-apoptotic molecule Bid (tBid) molecule, which activates Bax to translocate to the mitochondrial membrane, forming a channel (Figure 5) (Desagher et al., 1999).This allows the subsequent release of the cytochrome C molecule located in the inner mitochondrial membrane space. Once released, the cytochrome C forms a complex called apoptosome with other molecules.
Among the selected molecules, Apaf-1 recruits itself the pro-caspase-9 through its N-terminal caspase-activation recruitment domain (CARD) (Figure 4). As a consequence, the apoptosome can activate the pro-caspase-3 and the pro-caspase-7 through the cytosolic dATP/ATP. The activated caspase-3 can activate other pro-capase-9 inducing a positive feedback pathway.
The late phases of the apoptotic death
Caspases are also described as selective enzymes damaging different cellular polypeptides. They can cleave key structural components of cytoskeleton such as cytoplasmic proteins (actinβ-catenin, or α-gesolin) or substrate in nucleus (including laminin B receptor, nuclear mitotic apparatus protein, U1 small ribonucleoprotein and p-53 associated with Mdm2). Additionally, these enzymes also degrade proteins involved in DNA metabolism, in the regulation of chromatin stability and DNA repair (such as poly-ADP-ribose polymerase, PARP).
Caspases also interfere with cell cycle regulating proteins (such as p21Cip1/Waf1) and signal transduction proteins or transcription factors (such as the pro-interleukin 1β , the pro-interleukin-16, the cytosolic phospholipase A2, NFκB, IκB or STAT1) (Earnshaw, 1999).
The hallmark of apoptosis is DNA fragmentation and condensation. The best-characterized enzyme
responsible for DNA fragmentation is the caspase-activated DNAse (CAD). It is located in the cytosol under an inactive heterodimeric form associated with its inhibitor named ICAD.
In apoptotic conditions, ICAD is proteolized by caspase-3 causing the dissociation of ICAD/
CAD heterodimer. At this point, CAD is able to translocate from the cytosol towards the nucleus, and to cleave double strands DNA (Yuste et al., 2005). This role has been demonstrated through in vitro experiments showing that cells derived from ICAD-null mice or expressing a caspase-resistant mutant ICAD have reduced DNA fragmentation (Sakahira et al., 1998; Zhang et al., 2000).
Caspase independent pathway
Apoptosis has also been described to occur in response to pro-apoptotic stimuli (e.g., etoposide, ceramide, staurosporin) despite the presence of caspases inhibitors, such as the over-expression of IAPs or ZVAD treatment (Daugas et al., 2000b;(Daugas et al., 2000b;
McCarthy et al., 1997; Xiang et al., 1996). ThisThis has been attributed to the release of specific caspase-independent death effectors located in the mitochondrial inter-membrane space such as the apoptotic-inducing factors (AIF) (Daugas et al., 2000b). AIF is a flavoprotein of 57 kDa inserted into the inner mitochondrial membrane, which is able to promote direct DNA fragmentation into high molecular weight (HMW) fragments and nuclear shrinkage, as observed after the use of recombinant AIF in vitro (Cregan et al., 2004;
Susin et al., 1999). This flavoprotein has been suggested to be caspase-independent and ATP- independent, allowing an alternative pathway to trigger nuclear apoptosis (Daugas et al., 2000b).
The endonuclease G molecule (EndoG) has also been recognized to be responsible for caspase- independent cell death (Li et al., 2001). In normal conditions, EndoG is tightly bound to the inter- membrane mitochondrial space, where it is involved in mitochondrial DNA repair (Lorenzo and Susin, 2004). Once released from mitochondria after the mitochondrial peremabilization or destruction, EndoG translocates to the nuclei and operates as a DNAse (Lorenzo and Susin, 2004; van Loo et al., 2001). Various studies have suggested that through such process, this molecule catalyzes both high molecular weight DNA cleavage and oligonucleosomal DNA breakdown (Widlak et al., 2001). Furthermore, Endo G cooperates with exonuclease and DNase I to facilitate DNA degradation (Li et al., 2001).
1.4 Hyperoxia and mecHaniSmS of cell deatH
Various cell culture models have been developed to understand the molecular mechanisms involved in hyperoxia-induced cell death. In general, experiments indicate that cells are usually killed when exposed to concentration higher than 40%
O2 (Schoonen et al., 1990b). Whereas, cells exposed to 95% of O2 usually exhibited clear and irreversible damage after 72 to 96 h depending on the cell type (Kazzaz et al., 1996; Metrailler- Ruchonnet et al., 2007). In terms of cell-death pathway, necrosis and apoptosis occur depending on the cell type: murine macrophages, endothelial
Chapter 1
and fibroblastic cell line are described to die by apoptosis (characterized by nuclear condensation and DNA fragmentation) (Budinger et al., 2002;
Katoh et al., 1997a; Metrailler-Ruchonnet et al., 2007; Petrache et al., 1999), while epithelial cells die by necrosis (MLE12) (corresponding to cytoplamsic, nuclear and mitochondrial swelling) (Kazzaz et al., 1996; Romashko et al., 2003), or apoptosis (A549 cells) (Kazzaz et al., 1996).(Kazzaz et al., 1996).
However, other studies conducted with A549 cells reveal aponecrosis features (O’Reilly et al., 2001;
Wang et al., 2003). Regardless of how cell die in hyperoxia, the identification and inhibition of cellular pathway that mediate hyperoxic cell death reduce cellular dysfunction occurring after oxygen exposition.
Several studies have been dedicated to the understanding of the cell death receptor-signaling pathway to attest its role in hyperoxia. To such conditions, MLE12 cells have an increased protein expression level of Fas/FasL advocating a possible role of the extrinsic pathway (De Paepe et al., 2005).
The extrinsic pathway activation has also been tied with the caspase-8/tBid mechanism. In A549 cells, the activation of the caspase-8/tBid mechanism leading to Bax translocation was observed (Wang et al., 2003). Moreover, the cleavage of Bid (tBid) emphasizes the Bax conformational change involved in oxygen-induced cellular damage, in primary rat alveolar epithelial cells (Buccellato et al., 2004). In A549 cells, the blockade of caspase- 8 by the FLIP molecule has been described with a decreased cell death, supporting the role of caspase after oxygen exposition. These results demonstrated a predominant role of the caspase- 8/tBid pathway and a definitive influence on the Bax activaion in hyperoxic conditions.
The importance of the caspase cascade has also been studied through various approaches.
Rat1a cells have been found to follow an apoptotic pathway characterized by the release of cytochrome C and the caspase-9 activation in oxidative conditions (Budinger et al., 2002). Zhang et al. (2003) documented the role of the caspase- 3 and -9 activation on MLE12 cells in hyperoxic conditions (Zhang et al., 2003b). The pro-survival Akt molecule, acting on the PI-3 kinase pathway, influence also the release of cytochrome C and the pro-apoptotic effect of Bad in hyperoxic A549 and in HLMVEC cells (Ahmad et al., 2006; Truong et al., 2004). The NADH/NADPH oxidase system is a major source of ROS generation whose activation depends on kinase pathways such as the extracellular signal-regulated kinase ½ (ERK) or the mitogen-activated protein kinase (MAPK). In MLE12 and endothelial cells, this system has been described to participate to hyperoxic cell death (Parinandi et al., 2003; Zhang et al., 2003a) and the ERK pathway has been found to modulate the pro- and anti-apoptotic members of the Bcl-2 family, affecting the cell death imbalance (Nyunoya et al., 2005; O’Reilly et al., 2000; Zaher et al., 2007).
Furthermore, hyperoxic conditions have also been recognized to influence transcription factors. As a redox-sensitive factor, NFkB is activated by the intracellular oxidant/antioxidant equilibrium, which acts as a cell pro-survival molecule (Piette et al., 1997). However, this protective property still needs to be considered with caution, since
it is influenced by the type of cells and cell death pathway (apoptosis or necrosis) (Franek et al., 2001; Li et al., 1997). NFκB regulates cytoprotective enzymes (such as MnSOD or GSH) in A549 and 16HBE cells (Rahman et al., 2001) and has also been tied with an increase of the Bcl-2 cellular level (Choi et al., 2006). The modulation of this increase is a crucial step in the generation of hyperoxia-induced cell damage (Barazzone-Argiroffo et al., 2003; Yang et al., 2004). Finally, hyperoxia modulates also various antioxidant enzymes (e.g. superoxide dismutase or catalase) on hyperoxia-exposed endothelial cells (HLMVEC) (Housset and Junod, 1982). The protective role of mitochondria towards oxidative stress derives from several factors and among which its influence on the antioxidant level. The amount of ROS produced within the cells modifies also the action of pro-apoptotic molecule such as Bax, making mitochondria sensitive to oxido- reduction status in terms of cell death (Buccellato et al., 2004). Consequently, mitochondria act as antioxidants and modulators in the release of apoptotic factors which regulate cell death in conditioning cell death (through channel formation by the Bax/Bak molecules).
Furthermore, it has also been demonstrated that hyperoxic conditions influence a signalling pathway tightly related to cell death: the cell cycle. Hyperoxia regulates cell cycle check points (e.g., p53 and p21) and the cyclins (O’Reilly, 2001; Shenberger and Dixon, 1999). In A549 hyperoxia-exposed cells, growth arrest has been linked with a decrease in the cyclin B1 protein and an increase in the p21 protein level. However, experiments conducted with p21-/- HCT116 cells demonstrated a similar cell cycle distribution than p21+/+ cells, suggesting rather the role of the p53 protein (McGrath-Morrow and Stahl, 2001). This protein has been associated with apoptosis due to its role in the Bax activation (Gotz and Montenarh, 1996; Miyashita and Reed, 1995).
1.5 tHe poly (adp-riboSe) polymeraSe molecule
The Poly(ADP-ribose) polymerase (PARP) is a family of eukaryotic nuclear enzyme known to be implicated in DNA repair, genomic integrity and cell death. The primary structure of enzyme is highly conserved in eukaryotes with a catalytic domain showing a high degree of homology between species (Boulares and Zoltoski 2003).
PARPs constitute a large family of 18 proteins which have been extensively studied (Ame et al., 2004). Among them, PARP-1 (75% of the PARP enzyme activity) and PARP-2 (25% of the PARP activity) are enzymes of the PARP family whose catalytic activity is stimulated by DNA strand breaks. PARP is therefore of crucial significance in maintaining genomic integrity and in triggering the cell survival pathway. However, its over- activation leads to cellular energy depletion and death (Berger, 1985).
PARP-1 functions as a DNA damage sensor and signalling molecule binding to both single- and double-stranded DNA break. Upon binding to
damaged DNA mainly through a zinc-finger domain, PARP-1 forms heterodimers and catalyzes the cleavage of nicotinamide adenine dinucleotide (NAD+) and ADP-ribose. This allows the synthesis of a linear or multi-branched polymer on various nuclear protein acceptors such as histones, thereby facilitating the work of DNA repair enzymes (Smith et al. 2001; de Murcia et al. 1994). The size of the branched polymer varies from a few to 200 ADP-ribose units. PARP is able to generate auto- modification and hetero-modification of acceptors which contribute positively to the survival of injured proliferating cells (D’Amours et al., 1999;
de Murcia and Menissier de Murcia, 1994). The auto-poly (ADP-ribosylation) represents a major regulatory mechanism for PARP-1 resulting in the down-regulation of the enzyme activity. PARP- 1 has been described to be able to poly-ADP- ribosylate several transcription factors, DNA replication and signalling molecules among which NF-κB, AP-2 or p53 (Kumari et al. 1998; Olivier et al. 1999; Kannan et al. 1999). PARP-1 regulates also cellular replication and differentiation.
Indeed, PARP-1 metabolism has been described to accelerate in the nuclei of proliferating cells (Kanai et al., 1981, Leduc et al., 1988). Poly (ADP-ribose) may also serve as a signal for protein degradation in oxidatively injured cells (Ciftci et al., 2001;
Ullrich and Grune, 2001).
The PARP-1 protein is composed by three structural domains, each of them associated with a particular function: 1) an N-terminal DNA-binding domain (DBD), containing two zinc finger structures responsible for PARP-1 interaction with DNA breaks 2) an auto-modification domain, containing a BRCT (BRCA1 C-Terminus) motif constituting the major protein interface with various nuclear partners and 3) a catalytic domain with the NAD+- binding site. The nuclear localization signal (NLS), responsible for the nuclear homing of PARP-1, is located between the DBD ant the auto-modification domain and contains the DEVD cleavage site recognized by caspase-3 and -7 (Figure 5).
Poly(ADP-ribosyl)ation by PARP at the auto- modification site is initiated by the binding of the zinc fingers to DNA breaks (Althaus and Richter, 1987; Lindahl et al., 1995). As a consequence of this auto-modification, the binding affinity of PARP for DNA is reduced, resulting in dissociation of PARP from DNA breaks (Zahradka and Ebisuzaki, 1982) and thereby allowing the DNA repair machinery to access the sites of DNA damage (Satoh and Lindahl, 1992). Moreover, poly (ADP-ribosyl)ation of
histones decreases their interaction with DNA and thus prevents chromatin condensation (Boulikas and Poirier, 1992; Poirier et al., 1982). During the caspase-dependent cell death pathway activation, PARP-1 molecule is cleaved by caspases (caspase- 3 and caspase-7) into two fragments at the DEVD site located in the NLS of PARP-1. These two resultant fragments are composed by the 24 kDa and 89 kDa parts (p24 and p89) (Figure 5) (Germain et al., 1999; Thornberry et al., 1997).. The p89 molecule contains the catalytic site and the auto-modification domain, while the p24 contains the DNA binding domain with the zinc fingers (Duriez and Shah, 1997; Kaufmann et al., 1993). In addition to be considered as a hallmark of apoptosis, cleaved fragments contribute to the suppression of PARP activity. Indeed the p89 and p24 are able to inhibit the homo-association and the DNA binding of intact PARP-1 respectively (D’Amours et al., 2001).
1.6 parp-1 implication in cell deatH Because of its immediate activation by DNA strand breaks, the degree of PARP-1 activation could be an important factor in the cell ability to repair the damage, survive or die. PARP-1 activation following limited DNA injury could constitute a signal to activate the repair and cell cycle control machineries. In contrast, if DNA damage is very extensive, the ensuing massive poly (ADP-ribosyl)ation can induce the activation of the death process. Indeed, it has been proposed Indeed, it has been proposed that excessive DNA damage, occurring during oxidative stress, induces massive PARP activation leading to NAD+ and ATP depletion, energy failure and necrotic cell death (Virag et al., 2002).
Conversely, PARP cleavage is a hallmark of apoptosis since PARP-1 is a substrate of caspase-3 (Eguchi et al., 1997; Leist et al., 1997b). Because. BecauseBecause the intracellular ATP level is directly affected by the catalytic activity of PARP-1, apoptosis which is an energy-requiring process is influenced by PARP activity. In contrast, excessive DNA damage induces massive PARP-1 activation leading to ATP store depletion and finally necrotic cell death induction. Both ATP and NAD+ are important determinants of the mode of cell death, especially in oxidative injured cells (Coppola et al., 1995;
Lelli et al., 1998; Ran et al., 1999; Crowley et al., 2000). From these observations, it was plausible to hypothesize that PARP as a NAD+ catabolizing enzyme may serve as a switch between apoptosis and necrosis. The decision between apoptosis and necrosis is thus modulated by the degree of
Figure 5 Structure of PARP-1. PARP-1 can be divided in 3 main domains: a DNA-binding domain (DBD) with 2 zinc fingers (Zn I and Zn II), an automodification domain with a BRCT motif, and a catalytic domain with the NAD+- binding site. The nuclear localisation signal (NLS) is located between the DBD and the auto modification domain. It comprises the DEVD cleeavage site recognized by caspase-3 and -7. Modified from Boulares and Zoltoski, 2003).
Chapter 1 0
activation of PARP-1 since this decision seems to be determined by the cellular ATP levels. Finally, recent data indicate that PARP-1 plays also a role in a caspase-independent apoptosis pathway mediated by apoptosis-inducing factor (AIF) (Yu et al., 2002).
A model can be proposed to unify the two seemingly opposite effects of PARP-1, the cell death promoting and the cytoprotective effects.
According to this concept (figure 6, modified from Virag, and Szabo 2002), cells exposed to DNA-damaging agents can enter three pathways determined by the intensity of stimulus: 1) PARP- 1 activated by mild genotoxic stimuli facilitates DNA repair by signalling cell-cycle arrest and by interacting with DNA-repair enzymes. 2) More severe DNA damage induces apoptotic cell death during which caspase-3 inactivates PARP-1 by cleaving it into two fragments (p89 and p24).
The caspase-dependent inactivation of PARP- 1 preserves cellular ATP and keeps the energy required for apoptosis. 3) The third pathway is induced by extensive DNA breakage which can be triggered by a massive degree of oxidative stress.
PARP-1 over-activation depletes the cellular stores of its substrate NAD+ and consequently ATP, preventing apoptotic cell death to proceed and leading to necrotic cell death.
1.7 tHe role of tHe parp-1 inHibition in oxidative-induced cell deatH
The initial studies on the role of PARP and cell death were performed using PARP- pharmacological
inhibitors allowing to maintain the cellular ATP and NAD+ pools in oxidative-stressed cells (Du et al., 2003; Halmosi et al., 2001). The inhibition of PARP-1 has also been described to inhibit replication, cell differentiation and proliferation (D’Amours et al., 1999). There is a wide variety of PARP-1 pharmacological inhibitors, with more or less specific inhibitory action. Endogenous inhibitor of PARP, such as nicotinamide or the compound 3-aminobenzamide (3-AB), a structural analogue of nicotinamide, are classic competitive PARP-1 inhibitors (Kuo et al., 1996).
It is generally assumed that benzamides inhibit PARP by interfering with the binding of NAD to the enzyme active site. An additional action of benzamides may be related to their binding to DNA and thereby preventing the recognition of DNA breaks by PARP and thus preventing its activation (McLick et al., 1987).
In vitro, both 3-AB and nicotinamide were effective in inhibiting the death of various cell types exposed to many forms of oxidative stress (Bowes et al., 1998; Chatterjee and Thiemermann, 1998).
Cultured rat cardiac myoblasts were protected from hydrogen peroxide and peroxynitrite-mediated necrosis by PARP inhibitors (Gilad et al., 1997).
Improved cellular survival was obtained in WRL- 68 cells treated with hydrogen peroxide during PARP inhibition due to pharmacological inhibitors or with siRNA techniques (Tapodi et al., 2005).
The PJ-34 a PARP inhibitor was also described to inhibit the oxidative damage following hypoxic- reoxygenation damage in cardio-myoblastic cells (Fiorillo et al., 2006). Finally, another approach
Figure 6 The intensity of DNA-damaging stimuli determines the fate of cells: survival, apoptosis or necrosis. Depend- ing on the intensity of the stimulus, genotoxic agents can trigger different pahways. In the case of mild DNA damage, poly (ADP-ribosylation) facilitates DNA repar and thus cell survival (pathway 1). More severe genotoxic stimuli ac- tivate the caspase apoptotic pathway (pathway 2). More severe genotoxic stimuli activate the caspase apoptotic path- way (pathway 2). Severe DNA damage may cause excessive PARP activation, depleting cellular NAD+/ATP stores.
NAD+/ATP depletion block apoptosis and result in necrosis (pathway 3). Modified fom Virag and Szabo, 2002.
