G Hedouin

Vaiva

MD Guillaume Vaiva
email: guillaume.vaiva@chru-lille.fr

MD Sébastien Dambry
email: sebastien.dambry@chru-lille.fr

First trends of change in mortality by suicide in Nord-Pas-de-Calais since VigilanS

Keywords: suicide, suicide attempt, mortality, prevention

diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

« Premières analyses de mortalité par suicide en Nord -Pas de Calais depuis l'implantation de VigilanS »

First trends of change of mortality by suicide in Nord -Pas de Calais since VigilanS

Stéphane DUHEM 1 , Sébastien DAMBRY 2 *, Elodie MARCHAND 3 , Anne CRETON 4 , Christophe DEBIEN 2 , Valéry HEDOUIN 3 (2012[START_REF] Qualitativement | Nous ne disposions que du nombre total de levées de corps et du sexe. Résultats Les données détaillées apparaissent dans le tableau 1. Nous observons une diminution de la moyenne des décès d'environ 10 % depuis[END_REF]( ) comparés aux 3 ans depuis VigilanS (2015( -2017)). Les décès des patients de la cohorte VigilanS ont été recensés en interpellant les mairies de naissance et de domicile de tous les patients intégrant le dispositif sur l'année 2016. La cause du décès a ensuite été caractérisée par contact du médecin traitant. Résultats. Nous observons une baisse moyenne de la mortalité par suicide de 9,9 % (-12 % chez les hommes ; +0,3 % chez les femmes). L'analyse exhaustive de la mortalité par suicide dans la cohorte VigilanS en 2016 retrouve 19 décès, dont 14 par suicide, soit 0.4 % des patients. Ce taux observé de mortalité dans la cohorte VigilanS en 2016 est significativement en dessous du seuil attendu dans la littérature (1 à 2 % dans l'année qui suit la tentative de suicide). Conclusion : Il semble possible de toucher l'objectif demandé par l'OMS à l'horizon 2020 d'une diminution de 10 % des conduites suicidaires dans les pays industrialisés. Le dispositif VigilanS pourrait donc se montrer protecteur en termes de mortalité par suicide.

MOTS-CLÉS : suicide, tentative de suicide, mortalité, prévention

Abstract

Introduction: In terms of suicide prevention, population-based prevention devices often struggle to demonstrate an effect in terms of reducing suicidal acts of suicide, often focusing on the evolution of the number of suicide attempts, on time too short to statistically interpret changes in mortality. The consolidated figures for mortality in France are provided by the CepiDC with a delay of approximately three years. We therefore had to try to work with an approximation of the phenomenon, and we tested the proxy value of the enumeration of corpses, body examinations and medico-legal autopsies for suicide by the forensic doctors of the Nord-Pas-de-Calais region in France. In parallel, we conducted a comprehensive mortality study for 2016 in the VigilanS cohort. Method: By measuring the evolution of the number of body lifts for suicide by the Department of Forensic Medicine of the region, for the 3 years before VigilanS (2012VigilanS ( -2014) ) compared to the 3 years since VigilanS (2015)(2016)(2017). The deaths of patients of the VigilanS cohort were identified by calling the city halls of birth and domicile of all patients integrating the device in 2016. The cause of death was then characterized by contact of the attending physician. Results. We observe an average decrease in suicide mortality of 9.9 % (-12 % for men, + 0.3 % for women). The exhaustive analysis of suicide mortality in the VigilanS cohort in 2016 found 19 deaths, of which 14 by suicide, or 0.4 % of patients. This observed mortality rate in the VigilanS cohort in 2016 is significantly below the expected threshold in the literature (1 to

Introduction

La notion d'un antécédent de tentative de suicide est un facteur de risque déterminant pour la survenue d'une autre tentative, mais aussi d'un décès par suicide [START_REF] Christiansen | Risk of repetition of suicide attempt, suicide or all deaths after an episode of attempted suicide: a register-based survival analysis[END_REF].

La mortalité par suicide peut apparaître comme un phénomène en partie différent sur le plan épidémiologique de la morbidité par suicide (tentatives de suicide, crises suicidaires, idéation suicidaire) : la mortalité par suicide touche plus les hommes que les femmes (2 femmes pour 1 homme pour les tentatives de suicide), est en lien avec l'occurrence d'un ou plusieurs troubles mentaux au moment du décès dans 95 % des cas (alors que le poids de la maladie mentale semble moins marqué pour les tentatives de suicide) (2) (3). En termes de prévention, une partie des acteurs de santé adoptent l'adage qui dit que les dispositifs de prévention populationnels ont pour cible la réduction de la souffrance psychique, des situations de crise et donc des tentatives de suicide, alors que la diminution des décès par suicide doit découler d'une meilleure prise en charge de la maladie mentale par les médecins généralistes et les systèmes de soin psychiatriques (4). D'ailleurs, les dispositifs de prévention du suicide peinent souvent à démontrer un effet populationnel en termes de réduction des passages à l'acte suicidaires (tentatives de suicide ou suicides). Souvent, le suivi de ces dispositifs porte sur un an, voire deux, et se concentre sur l'évolution du chiffre des tentatives de suicide ; de plus, les suivis de cohorte sur quelques centaines d'individus ne sont pas assez puissants statistiquement pour interpréter des variations de mortalité (phénomène rare) sur ces laps de temps. Ces temps sont trop courts pour observer une éventuelle variation en termes de mortalité. Quand le temps d'observation est assez long et les effectifs populationnels suffisants, la baisse des conduites suicidaires est parfois retrouvée en termes de diminution des tentatives de suicide et peu ou pas en termes de mortalité par suicide, comme dans le programme de l'European Alliance Again Depression [START_REF] Hegerl | Focus on mental health care reforms in Europe: the European alliance against depression: a multilevel approach to the prevention of suicidal behaviour[END_REF].

En reprenant l'une des données fortes de notre première étude d'impact en Nord -Pas de Calais (article précédent), nous constatons une baisse des passages aux urgences pour tentative de suicide (moins 13,5 % entre 2014 et 2017) et cette baisse apparaît aussi marquée chez les hommes que chez les femmes (moins 14 % chez les hommes et moins 10 % chez les femmes). Les décès par suicide connaissant un sex ratio de 3 hommes pour 1 femme, nous

Figure 1 .

 1 Figure 1. Evolution des levées de corps pour suicide dans 3 zones géographiques représentatives du NPC entre 2012 et 2017

Figure 2 .

 2 Figure 2. Nombre de jours entre la sortie du système de soin et le décès par suicide

, Guillaume VAIVA 2 * 1

  ) Centre d'Investigation Clinique, CHU de Lille & Inserm, F-59000 Lille, France 2) Pôle de Psychiatrie, Médecine Légale et Médecine en milieu pénitentiaire, CNRS UMR 9193 -SCA-Lab -Sciences Cognitives et Sciences Affectives, CHU & Université de Lille, F-59000 Lille, France 3) Service de Médecine Légale, EA 7367 -UTML -Unité de Taphonomie Médico-Légale, CHU & Université de Lille, F-59000 Lille, France 4) Centre Hospitalier de Valenciennes, FranceRésuméIntroduction : En termes de prévention du suicide, les dispositifs de prévention populationnels peinent souvent à démontrer un effet en termes de réduction des passages à l'acte suicidaires, se concentrant souvent sur l'évolution du chiffre des tentatives de suicide, sur des temps trop courts pour interpréter statistiquement des variations de mortalité. Les chiffres consolidés de la mortalité en France sont fournis par le CepiDC avec un délai d'environ trois ans. Il nous fallait donc tenter de travailler avec une approximation du phénomène et nous avons testé la valeur de proxy du dénombrement des levées de corps, examens de corps et autopsies médico-légales pour suicide par les médecins légistes de la Région Nord-Pas-de-Calais. De façon parallèle, nous avons mené une étude exhaustive de mortalité sur l'année 2016 dans la cohorte VigilanS. Méthode : En mesurant l'évolution du nombre de levées de corps pour suicide par le département de Médecine Légale de la région, sur les 3 ans avant VigilanS

  Tableau 1. Evolution du nombre de levées de corps pour suicide dans le NPC entre 2012 et 2017.

						-2104 H	F	2015	2016	2017	Moyenne 2015-2017 H	F
	Boulogne	58	55	44	52	43,0 9,3	43	55	57	52	40,0	9,0
	Dunkerque	47	40	30	39	28,7 8,7	24	23	18	22	19,5	4,0
	Lille	209	208	191	203	156,3 46,3	209	196	171	192	141,0	51,5
	TOTAL	314	303	265	294	230,7 64,3	276	274	246	265	200,5	64,5
										variation	-9,9%	-12,1%	0,3%
	NPC CepiDC	815	719	728	754	583,3 170,7


Florian Alonso

Pirjo Spuul

Thomas Daubon

Ijsbrand Kramer

Elisabeth Génot
email: elisabeth.genot@inserm.fr

Variations on the theme of podosomes: A matter of context

Keywords: podosomes, microenvironment, extracellular matrix, basement membrane, invasion, mechanosensing

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

2 soluble, matrix and mechanical factors. For instance, cells probe their surroundings with filopodia, adhere to the extracellular matrix (ECM) through focal or fibrillar adhesion sites and deploy lamellipodia in order to migrate. Podosomes form another class of structures restricted to certain cells and/or certain contexts but endowed with multiple capabilities. Unlike the structures mentioned above, they are endowed with proteolytic activities that allow them to degrade matrix proteins. Podosomes are therefore intimately associated with the process of cellular invasion, where cells breach anatomical barriers and penetrate tissues. Recent advances in the field have established that these dynamic structures also exhibit mechanosensory attributes allowing the exploration of the pericellular environment. This property must now be integrated in understanding how they participate in physiological processes. This review does not deal with related structures described in tumor cells and named invadopodia, which are associated with cancer pathology.

One essential component of the microenvironment is the ECM, a collection of extracellular molecules secreted by cells that provides them with structural and biochemical support. The interstitial matrix is predominant, consisting in structural components such as collagen-I (fibrillar) and elastin, whose main role is to resist tension, and components that facilitate contacts with cells such as fibronectin and laminins. Other key components are proteoglycans such as aggrecan and perlecan that, through their long glycosaminoglycan chains, capture water and make the matrix resistant to compression.

Basement membranes (BMs) are specialized ECM that delimit all epithelia and endothelia but also envelop muscle, fat and nerve cells, and serve as structural barriers [START_REF] Rowe | Breaching the basement membrane: who, when and how?[END_REF]. They consist primarily of laminins (various isoforms depending on tissue type) and collagen-IV, crosslinked by non-covalent interactions with nidogens and perlecan [START_REF] Pozzi | The nature and biology of basement membranes[END_REF], thus forming a compact and resistant mesh that plays an essential role in maintaining tissue integrity while delivering homeostasis signals. Tissue maintenance, repair and expansion as well as tissue surveillance by immune-competent cells, are processes that require matrix remodeling [START_REF] Rowe | Breaching the basement membrane: who, when and how?[END_REF]. These are the physiological processes in which podosomes play a key role.

Structurally, podosomes are actin-based membrane structures in direct contact with the ECM. First characterized as autonomous entities, it is now recognized that they are building blocks of a network, interconnected by contractile actin filaments and linking the actin cytoskeleton to the plasma membrane [START_REF] Labernadie | Protrusion force microscopy reveals oscillatory force generation and mechanosensing activity of human macrophage podosomes[END_REF][START_REF] Luxenburg | The architecture of the adhesive apparatus of cultured osteoclasts: from podosome formation to sealing zone assembly[END_REF][START_REF] Van Den Dries | Interplay between myosin IIA-mediated contractility and actin network integrity orchestrates podosome composition and oscillations[END_REF]. As plasma membrane domains, podosomes are privileged sites of interactions with the pericellular environment that regulates their formation, lifespan, subcellular distribution and architecture. As cytoskeleton domains, podosomes derive their morphological and functional characteristics from the dynamics of actin polymerization and adopt a dynamic collective behavior [START_REF] Van Den Dries | Interplay between myosin IIA-mediated contractility and actin network integrity orchestrates podosome composition and oscillations[END_REF][START_REF] Proag | Working together: spatial synchrony in the force and actin dynamics of podosome first neighbors[END_REF].

Podosomes are particularly abundant in cells of the myelomonocytic lineage, such as macrophages [START_REF] Linder | Wiskott-Aldrich syndrome protein regulates podosomes in primary human macrophages[END_REF], immature dendritic cells [START_REF] Burns | Configuration of human dendritic cell cytoskeleton by Rho GTPases, the WAS protein, and differentiation[END_REF] and osteoclasts [START_REF] Destaing | Podosomes display actin turnover and dynamic self-organization in osteoclasts expressing actin-green fluorescent protein[END_REF]. They are also prominent in megakaryocytes [START_REF] Sabri | Deficiency in the Wiskott Aldrich protein induces premature proplatelet formation and platelet production in the bone marrow compartment[END_REF][START_REF] Schachtner | Megakaryocytes assemble podosomes that degrade matrix and protrude through basement membrane[END_REF]. In these cell types, they constitute a major part of the actin cytoskeleton and are formed constitutively upon cell adhesion. They are also found in non-hematopoietic cells. In these cell types, their formation involves ECM-derived signals but also requires specific extracellular induction. These can be mechanical cues and/or specific soluble factors that depend on the cell tissue origin.

Podosomes are often arranged in clusters or in ring-shaped superstructures called "rosettes".

Podosomes have been extensively described for over three decades and in a wide variety of cell types: in vitro, in the bidimensional context of the culture dish, under hyperoxic atmosphere and often using a gelatin film to mimic ECM. These explorations have revealed their large repertoire of functions [START_REF] Veillat | Podosomes: Multipurpose organelles?[END_REF].

More recently, podosome presentation and functions have been investigated ex vivo or in conditions that better mimic physiological processes in vivo. These studies have highlighted the predominant role of the microenvironment in the acquisition of podosome characteristics and functioning. Thus, it turns out that although the different types of podosomes share many components, fine details of their composition, characteristics and subcellular arrangement are determined by the context. Below we describe the main features of podosomes updated with the latest advances in the field, including the characterization of the elements that have led to better understanding of their organization and dynamics and their functional employment in the context of the various functions associated with them. We then describe three physiological processes in which podosomes described in vitro or ex vivo could occur in vivo: macrophage migration across tissues, antigen presentation and uptake by immature dendritic cells and endothelial cell sprouting during angiogenesis. This review does not deal with intracellular signaling pathways, which are known to involve Src and RhoGTPases as pivotal actors of podosome assembly. They have been reviewed elsewhere [START_REF] Spuul | Importance of RhoGTPases in formation, characteristics, and functions of invadosomes[END_REF].

The podosome is an actin structure that connects the cytoskeleton with the plasma membrane

Typically, a podosome is a punctate (0.5-1.0 µm in diameter) structure located at the plasma membrane and is principally made of filamentous actin. It is a highly dynamic organelle undergoing constant disassembly and reformation on a minute time-scale, and characterized by a rapid turnover of actin within the structure [START_REF] Destaing | Podosomes display actin turnover and dynamic self-organization in osteoclasts expressing actin-green fluorescent protein[END_REF] (Figure 1A-D). Actin polymerization begins at the plasma membrane and is mediated by the Arp2/3 complex activated by Wiskott-Aldrich syndrome protein (WASp or N-WASp) downstream of the small G protein Cdc42. While the podosome enlarges through the generation of branched actin on a rigid matrix, the core structure grows perpendicular to the substratum over a height of about 0.6 μm (Figure 1C,D). Cortactin (CTTN) and the multi-domain scaffold protein Tks5 (SH3PXD2A) are often used as podosome markers. Cortactin consolidates the Arp2/3-generated actin filaments, whereas Tks5 plays an essential, but yet poorly understood, role in the subcellular orientation of the actin nucleation sites [START_REF] Oikawa | Tks5dependent formation of circumferential podosomes/invadopodia mediates cell-cell fusion[END_REF]. Integrins and CD44 are the dominant transmembrane adhesion molecules of podosomes and constitute the bridge that connects the inside with the outside. The transmembrane metalloprotease MT1-MMP (MMP-14) is selectively delivered in transport vesicles to the basal side of the podosome through a microtubule/motor-protein network [START_REF] Wiesner | KIF5B and KIF3A/KIF3B kinesins drive MT1-MMP surface exposure, CD44 shedding, and extracellular matrix degradation in primary macrophages[END_REF]. The enzyme remains spatially confined to the site of podosome formation through interactions with actin filaments [START_REF] El Azzouzi | Metalloproteinase MT1-MMP islets act as memory devices for podosome reemergence[END_REF]. Surrounding the columnar actin core, numerous structural proteins associated with integrins have been identified, including paxillin, vinculin and talin (Figure 1 C,D) [START_REF] Veillat | Podosomes: Multipurpose organelles?[END_REF][START_REF] Saltel | Invadosomes: intriguing structures with promise[END_REF]. The actin core and adhesion ring give the podosome the characteristic bipartite appearance in immunofluorescent microscopy (Figure 1A,B) which is also useful for recognizing the structures.

High-resolution microscopy has now shown that the adhesion ring of integrin-associated proteins is not really continuous but instead forms a polygon of discrete complexes [START_REF] Van Den Dries | Interplay between myosin IIA-mediated contractility and actin network integrity orchestrates podosome composition and oscillations[END_REF][START_REF] Cox | Bayesian localization microscopy reveals nanoscale podosome dynamics[END_REF][START_REF] Joosten | Super-Resolution Correlative Light and Electron Microscopy (SR-CLEM) Reveals Novel Ultrastructural Insights Into Dendritic Cell Podosomes[END_REF][START_REF] Walde | Vinculin binding angle in podosomes revealed by high resolution microscopy[END_REF]. Characterization of podosomes in the orthogonal plane (perpendicular to the basal membrane) has revealed the existence of a third element, the podosome cap. It covers the column of actin filaments like an umbrella and contains proteins such as formin-like protein, FMNL1 [START_REF] Mersich | The formin FRL1 (FMNL1) is an essential component of macrophage podosomes[END_REF], inverted formin, INF2 [START_REF] Panzer | The formins FHOD1 and INF2 regulate inter-and intra-structural contractility of podosomes[END_REF], supervillin, SVIL [START_REF] Bhuwania | Supervillin couples myosin-dependent contractility to podosomes and enables their turnover[END_REF], and the sphingolipid long-chain base-responsive protein LSP1 [START_REF] Cervero | Lymphocyte-specific protein 1 regulates mechanosensory oscillation of podosomes and actin isoform-based actomyosin symmetry breaking[END_REF]. This structure may serve as a hub for incoming transport vesicles, regulate podosome growth or channel the myosin contractile forces associated with podosomes [START_REF] Bhuwania | Supervillin couples myosin-dependent contractility to podosomes and enables their turnover[END_REF][START_REF] Cervero | Lymphocyte-specific protein 1 regulates mechanosensory oscillation of podosomes and actin isoform-based actomyosin symmetry breaking[END_REF] (Figure 1C,D). Detailed analysis of the actin network has allowed a better understanding of the architecture of the organelle and brought to light the mechanics of their sensory properties. At the base of the columnar structure, the detection of Arp2/3 serves as a signature for the formation of a branched actin network [START_REF] Linder | The polarization defect of Wiskott-Aldrich syndrome macrophages is linked to dislocalization of the Arp2/3 complex[END_REF]. This branched actin core is surrounded by unbranched antiparallel contractile filaments that radiate from the top of the podosome near the cap to the ring of adhesion sites [START_REF] Luxenburg | The architecture of the adhesive apparatus of cultured osteoclasts: from podosome formation to sealing zone assembly[END_REF][START_REF] Destaing | Podosomes display actin turnover and dynamic self-organization in osteoclasts expressing actin-green fluorescent protein[END_REF][START_REF] Akisaka | Adhesion structures and their cytoskeleton-membrane interactions at podosomes of osteoclasts in culture[END_REF] (Figure 1D). Pioneering work using atomic force microscopy has shown that podosomes exert forces on the matrix in an oscillating movement by using the myosin IIA motor of these contractile filaments [START_REF] Labernadie | Dynamics of podosome stiffness revealed by atomic force microscopy[END_REF]. The core of actin filaments grows upwards from the basal side. In turn, the acto-myosin contractile cables tethered onto the matrix at the level of the ring stretch, inducing the recruitment of mechano-sensitive protein vinculin at these sites. The stored forces are restored in protruding forces at the core and are in turn relayed onto the ECM [START_REF] Labernadie | Protrusion force microscopy reveals oscillatory force generation and mechanosensing activity of human macrophage podosomes[END_REF][START_REF] Van Den Dries | Interplay between myosin IIA-mediated contractility and actin network integrity orchestrates podosome composition and oscillations[END_REF][START_REF] Labernadie | Dynamics of podosome stiffness revealed by atomic force microscopy[END_REF]. Talin, vinculin and paxillin are required for actin polymerization and protrusion force generation [START_REF] Bouissou | Podosome Force Generation Machinery: A Local Balance between Protrusion at the Core and Traction at the Ring[END_REF]. Myosin IIA contracts the actin network filaments and applies tension to the bound vinculin molecules, counterbalancing core growth and eventually reducing podosome size and protrusion. Depending on the rigidity of the matrix, the magnitude of the forces leads to distinct levels of recruitment of the mechano-sensitive protein vinculin at sites where the actin filaments connect to the cell membrane [START_REF] Van Den Dries | Interplay between myosin IIA-mediated contractility and actin network integrity orchestrates podosome composition and oscillations[END_REF]. In this way, the exerted forces translate the physical property of the substrate into biochemical signals to the podosomes, which in turn are transmitted to the cell and may lead to differential podosome composition. The formin INF2 was recently shown to regulate contracting events at individual podosomes from the cap structure [START_REF] Panzer | The formins FHOD1 and INF2 regulate inter-and intra-structural contractility of podosomes[END_REF]. The adhesive ring also undergoes pressure oscillations resulting from the movements of the core [START_REF] Van Den Dries | Interplay between myosin IIA-mediated contractility and actin network integrity orchestrates podosome composition and oscillations[END_REF][START_REF] Proag | Working together: spatial synchrony in the force and actin dynamics of podosome first neighbors[END_REF]. Thus, adhesion proteins sustain tension and feedback on actin-driven protrusion and the integrity and dynamics of protrusive cores and adhesive rings are coordinated by the actomyosin apparatus [START_REF] Bouissou | Podosome Force Generation Machinery: A Local Balance between Protrusion at the Core and Traction at the Ring[END_REF].

Another set of contractile actin/myosin-filaments has been revealed by the use of super-resolution microscopy and these connect podosomes with each other [START_REF] Labernadie | Protrusion force microscopy reveals oscillatory force generation and mechanosensing activity of human macrophage podosomes[END_REF][START_REF] Van Den Dries | Interplay between myosin IIA-mediated contractility and actin network integrity orchestrates podosome composition and oscillations[END_REF][START_REF] Bhuwania | Supervillin couples myosin-dependent contractility to podosomes and enables their turnover[END_REF]. These connections ensure their regular distribution [START_REF] Luxenburg | The architecture of the adhesive apparatus of cultured osteoclasts: from podosome formation to sealing zone assembly[END_REF][START_REF] Bhuwania | Supervillin couples myosin-dependent contractility to podosomes and enables their turnover[END_REF][START_REF] Burgstaller | Podosome-mediated matrix resorption and cell motility in vascular smooth muscle cells[END_REF] and enable their collective and synchronized functioning [START_REF] Proag | Working together: spatial synchrony in the force and actin dynamics of podosome first neighbors[END_REF][START_REF] Bhuwania | Supervillin couples myosin-dependent contractility to podosomes and enables their turnover[END_REF] (Figure 1B,D). In fact, connectivity extends well beyond the nearest neighbors of podosomes, as shown by the continuous redistribution of podosome components throughout podosome clusters [START_REF] Meddens | Actomyosin-dependent dynamic spatial patterns of cytoskeletal components drive mesoscale podosome organization[END_REF]. The formin FHOD1 was recently shown to regulate the actomyosin-based contractility of these connecting filaments within podosome clusters in primary macrophages [START_REF] Panzer | The formins FHOD1 and INF2 regulate inter-and intra-structural contractility of podosomes[END_REF]. These interconnecting filaments appear to be associated with the radiating filaments described above. Super-resolution correlative light and electron microscopy (SR-CLEM) has allowed the detection of another network of filaments that has no apparent connection with podosome cores. These short filaments are restricted to the ventral side of the plasma membrane and form a thick layer of cortical actin at the bottom of the entire podosome cluster [START_REF] Joosten | Super-Resolution Correlative Light and Electron Microscopy (SR-CLEM) Reveals Novel Ultrastructural Insights Into Dendritic Cell Podosomes[END_REF].

The dynamic behavior of podosomes comprises de novo formation, dissolution and reformation.

Podosomes also undergo continuous fission and fusion events, where neighboring structures are in close spatial vicinity. In between these podosomes, SR-CLEM detects a thick bundle of unbranched filaments that may originate from the actin core. The filaments close to and on top of the core are decorated with zyxin but not with vinculin, suggesting that they do not associate with the plasma membrane. In contrast to the radiating fibers and interconnecting cables, these interpodosomal connections are insensitive to inhibition of actin polymerization, suggesting that these pools of actin are not dynamically coupled [START_REF] Joosten | Super-Resolution Correlative Light and Electron Microscopy (SR-CLEM) Reveals Novel Ultrastructural Insights Into Dendritic Cell Podosomes[END_REF].

Podosomes are thus endowed with mechano-sensory qualities and podosome clusters function as a coordinated mechanosensory area [START_REF] Meddens | Podosomes revealed by advanced bioimaging: what did we learn?[END_REF]. These mechano-sensory properties may either combine or, more likely, alternate with their matrix-degrading activity (Figure 1C,D).

The various signals regulating podosome formation 1.3.1. Matrix signals transmitted at the level of the plasma membrane

Under conventional cell culture conditions, podosomes appear in their characteristic form in cells seeded on a smooth and rigid plastic dish, as shown in Figure 1. For myelomonocytic cells, integrinmediated adhesion to the ECM-coated substratum triggers the assembly of podosomes, which form the predominant adhesive structures for these cells. Different matrices engage different integrins and thus trigger distinct signaling cascades: integrin β2 bound to fibrinogen drives podosome formation in macrophages [START_REF] Labernadie | Dynamics of podosome stiffness revealed by atomic force microscopy[END_REF], integrin β3 bound to osteopontin is involved in osteoclasts [START_REF] Horton | Interaction of osteopontin with osteoclast integrins[END_REF], and integrin β1 bound to collagen-I is a potent triggering signal for megakaryocytes [START_REF] Sabri | Deficiency in the Wiskott Aldrich protein induces premature proplatelet formation and platelet production in the bone marrow compartment[END_REF][START_REF] Schachtner | Megakaryocytes assemble podosomes that degrade matrix and protrude through basement membrane[END_REF]. The matrix also regulates the lifespan of podosomes: when megakaryocytes are seeded on collagen-I, podosomes triple their longevity as compared to cells seeded on fibrinogen [START_REF] Schachtner | Megakaryocytes assemble podosomes that degrade matrix and protrude through basement membrane[END_REF]. The geometry of the substrate dictates podosome subcellular localization, arrangement and morphology, while also impacting their lifetime.

On a surface lined with reliefs, dendritic cells accumulate podosomes along ridges and these podosomes are more stable than those formed in non-rugged areas [START_REF] Van Den Dries | Dual-color superresolution microscopy reveals nanoscale organization of mechanosensory podosomes[END_REF]. These properties are well illustrated by cells seeded on a collagen-I matrix: podosomes tend to line up on the ridges and follow the meanders of the deposited collagen-I fibers [START_REF] Schachtner | Megakaryocytes assemble podosomes that degrade matrix and protrude through basement membrane[END_REF] and may even change their architecture and composition [START_REF] Juin | Physiological type I collagen organization induces the formation of a novel class of linear invadosomes[END_REF]. Here, podosome accumulation and presentation dictated by collagen-I topography could be mediated by the newly discovered interpodosomal network [START_REF] Joosten | Super-Resolution Correlative Light and Electron Microscopy (SR-CLEM) Reveals Novel Ultrastructural Insights Into Dendritic Cell Podosomes[END_REF]. As another example to illustrate the prominent role of the ECM, podosomes barely form when endothelial cells derived from liver sinusoids are seeded on a soft matrix. In contrast, on a stiff substratum corresponding to a matrix associated with liver fibrosis, they form abundantly and with a highly amplified proteolytic power [START_REF] Juin | Extracellular matrix rigidity controls podosome induction in microvascular endothelial cells[END_REF]. Matrix signals also play a decisive role in podosome formation induced by cytokines. Thus, collagen-IV is essential for podosome induction by the vascular endothelial growth factor VEGF in microvascular endothelial cells [START_REF] Daubon | VEGF-A stimulates podosomemediated collagen-IV proteolysis in microvascular endothelial cells[END_REF], while fibronectin is the preferred ECM for TGF-β-induced podosome formation in endothelial cells derived from large arterial vessels [START_REF] Varon | Transforming growth factor beta induces rosettes of podosomes in primary aortic endothelial cells[END_REF].

Podosomes may also be induced by suppressing the tractive forces exerted by cells on a matrix.

Fibroblasts do not normally present podosomes on a rigid matrix but classical focal adhesion contacts.

When the cells are spread on a fluid matrix composed of lipid-attached Arg-Gly-Asp (RGD) peptides, they assemble podosome-like structures that convert into focal adhesion contacts when a force is applied to the fluid lipid-RGD bilayer [START_REF] Yu | Integrin-matrix clusters form podosome-like adhesions in the absence of traction forces[END_REF]. These findings highlight the role of mechano-signals in the regulation of podosome formation. A constricted environment also promotes podosome formation.

Arterial endothelial cells confined into a micro-channel scaffold device spontaneously assemble podosomes [START_REF] Spuul | Microfluidic devices for the study of actin cytoskeleton in constricted environments: Evidence for podosome formation in endothelial cells exposed to a confined environment[END_REF]. Within the channel, the podosomes are mainly arranged in clusters, while they mainly form rosettes when entering or exiting the confined space. Therefore, physical constraints influence podosome organization and disturbance of cell polarity may be involved in their formation.

Although adhesion is a pre-requisite for podosome formation and podosomes gather information about matrix composition by the engagement of specific integrins, kinetic studies performed in osteoclasts suggest that this interaction is not the prime event that leads to their assembly as cortactin and paxillin are detected first at the site of podosome formation and precede integrin recruitment [START_REF] Luxenburg | Involvement of actin polymerization in podosome dynamics[END_REF]. Podosome formation is stimulated by a local loss of contractility and the recruitment of negative regulators of RhoA such as p190RhoGAP [START_REF] Yu | Integrin-matrix clusters form podosome-like adhesions in the absence of traction forces[END_REF][START_REF] Burgstaller | Actin cytoskeleton remodelling via local inhibition of contractility at discrete microdomains[END_REF][START_REF] Lener | Matrix-degrading podosomes in smooth muscle cells[END_REF]. As F-actin depolymerization promotes cortactin phosphorylation [START_REF] Fan | Actin depolymerization-induced tyrosine phosphorylation of cortactin: the role of Fer kinase[END_REF], the nucleation of the filaments could be triggered by Arp2/3 and/or a cortactinenriched primer complex, on which ECM signals converge [START_REF] Gunst | Actions by actin: reciprocal regulation of cortactin activity by tyrosine kinases and F-actin[END_REF][START_REF] Webb | Cortactin regulates podosome formation: roles of the protein interaction domains[END_REF]. As mentioned above, the dynamic behavior of podosomes includes podosome dissolution and reformation. Beside its proteolytic activity, MT1-MMP also serves to imprint spatial memory for podosome reformation after dissolution, and therefore contributes to the construction of the structure [START_REF] El Azzouzi | Metalloproteinase MT1-MMP islets act as memory devices for podosome reemergence[END_REF]. More recently, cell fate specification pathways were also shown to regulate podosome formation.

Alteration of gene expression and other signals

Blocking Notch signalling frees the way for the formation of podosomes in endothelial tip cells, one of the first steps in the process of angiogenesis. As Notch mainly interferes with gene transcription, podosome induction is very likely the immediate consequence of altered gene expression [START_REF] Spuul | VEGF-A/Notch-Induced Podosomes Proteolyse Basement Membrane Collagen-IV during Retinal Sprouting Angiogenesis[END_REF].

Podosomes are also involved in embryonic development where cell migration is instrumental for generating tissues in the appropriate position, as shown in the zebrafish model [START_REF] Murphy | A Src-Tks5 pathway is required for neural crest cell migration during embryonic development[END_REF]. In the nematode C elegans, podosomes are used by the anchor cell, a specialized somatic gonadal cell, to migrate across the basement membrane in order to connect the developing uterine with vulval tissues [START_REF] Hagedorn | The netrin receptor DCC focuses invadopodia-driven basement membrane transmigration in vivo[END_REF]. .

Another means to control podosome induction is oxygen or nutrient deficiency [START_REF] Vanwinkle | Hypoxia-induced alterations in cytoskeleton coincide with collagenase expression in cultured neonatal rat cardiomyocytes[END_REF], or the production of reactive oxygen species (ROS) [START_REF] Mu | Circulating Exosomes Isolated from Septic Mice Induce Cardiovascular Hyperpermeability Through Promoting Podosome Cluster Formation[END_REF]. However, the how and why of these observations have not yet been explored. There are thus several ways in which podosome formation may be induced or modulated. Table 1 summarizes the various signals that have been shown to regulate podosome assembly in endothelial cells. These regulations are dynamic as podosomes in turn act on the microenvironment.

Macrophage podosomes and trans-tissue migration

Macrophages originate predominantly from blood-borne monocytes that migrate into tissues. They differentiate in a tissue-dependent manner, giving rise to tissue-specific cells (such as Kupffer cells in the liver or glial cells in the brain). Besides tissue-specific characteristics, two broad distinctions are made in the macrophage population, namely M1 and M2. The two subpopulations distinguish themselves by expression of specific cell surface markers and the secretion of specific sets of cytokines which lead to two opposite functional characteristics. The M1 signature is associated with elimination of (damaged) tissue and of foreign organisms (initiation phase of inflammation) whereas the M2 signature is associated with tissue repair (resolution phase of inflammation) [START_REF] Mills | Anatomy of a discovery: m1 and m2 macrophages[END_REF]. In fact, M1

and M2 are the two extreme of a continuum of phenotypes in vivo and subpopulations exist that do not fit these two categories [START_REF] Chavez-Galan | Much More than M1 and M2 Macrophages, There are also CD169(+) and TCR(+) Macrophages[END_REF]. Macrophages are confronted by matrices whose rigidity and topography must be decoded for an "all-terrain" migration. In porous collagen-I based matrices, cellular deformability is sufficient for their progression and they move in an amoeboid fashion. In more dense matrices, macrophages form podosomes and use their proteolytic properties to forge ahead through the stroma in a mesenchymal migration mode [START_REF] Van Goethem | Matrix architecture dictates three-dimensional migration modes of human macrophages: differential involvement of proteases and podosome-like structures[END_REF]. Both M1 and M2 populations produce podosomes but those associated with the M2 signature tend to be more dynamic and were shown to contribute to mesenchymal mobility [START_REF] Cougoule | Blood leukocytes and macrophages of various phenotypes have distinct abilities to form podosomes and to migrate in 3D environments[END_REF].

On a two-dimensional surface under in vitro conditions, podosomes are evenly distributed over the whole ventral (attached side) of macrophages. As mentioned above, podosomes predominantly serve as adhesion sites because they lack other matrix contacts. The even distribution is lost when macrophages are set to migrate in response to chemokines such as CSF-1. A new population of large podosomes with a rapid turnover (short half-life) accumulates at the leading edge. The enhanced dynamics of M2 macrophage podosomes is essential for the migration process, in particular when cells migrate across the endothelium (process of diapedesis) [START_REF] Burns | Configuration of human dendritic cell cytoskeleton by Rho GTPases, the WAS protein, and differentiation[END_REF]. When analyzed in more detail, the chemokine-exposed macrophages may be seen to possess two distinct populations of podosomes: i) large dynamic "precursors" located at the periphery and at the leading edge; and ii) smaller and more stable "successors", located more centrally and often derived from splitting precursors [START_REF] Bhuwania | Supervillin couples myosin-dependent contractility to podosomes and enables their turnover[END_REF]. The differences between the two podosome populations may be due to the differential regulation of myosin-II by the cap components, LSP1 dominating in precursors and SVIL in successors [START_REF] Cervero | Lymphocyte-specific protein 1 regulates mechanosensory oscillation of podosomes and actin isoform-based actomyosin symmetry breaking[END_REF].

Interestingly, LSP1 regulates podosome oscillations, protrusive force generation and podosome lifespan [START_REF] Cervero | Lymphocyte-specific protein 1 regulates mechanosensory oscillation of podosomes and actin isoform-based actomyosin symmetry breaking[END_REF].

An example of an in vitro two-dimensional environment is where cells are crawling on an endothelium or certain epithelia attached to their basement membrane such as in lung, bladder, intestine or certain glands. However, most environments are three-dimensional. In a collagen-I gel mimicking the environment of macrophages in connective tissues in vivo, podosomes take on a totally different appearance and are consequently named "3D podosomes" [START_REF] Van Goethem | Macrophage podosomes go 3D[END_REF]. Instead of having the typical core/ring/cap moieties, they present as globular structures at the end of long protrusions with less distinct boundaries between the substructures. Nevertheless, they remain recognizable as podosomes thanks to their specific markers, their proteolytic capacity and their dynamics. They are larger, less numerous, and their composition slightly differs from their 2D counterparts.

In vitro, macrophages carrying 3D podosomes and exposing MT1-MMP at the plasma membrane dig tunnels through which they migrate (Figure 2A) [START_REF] Cabec | Phagocyte migration: an overview[END_REF], a phenomenon that reflects trans-tissular migration. Indeed, the podosomes in HIV-1 infected macrophages are more stable than those of uninfected cells and their size and degradation potential are increased as a result of Hck (hematopoietic cell kinase, the predominant member of the Src-family of tyrosine kinases in these cells) stimulation by the viral protein Nef. Such changes are reflected by the accelerated migration of these cells towards inflammatory sites [START_REF] Lener | Matrix-degrading podosomes in smooth muscle cells[END_REF]. Conversely, diminishing podosome size and degradation potential through Hck inhibition reduces cell migration [START_REF] Cougoule | Threedimensional migration of macrophages requires Hck for podosome organization and extracellular matrix proteolysis[END_REF][START_REF] Verollet | HIV-1 Infection of T Lymphocytes and Macrophages Affects Their Migration via Nef[END_REF][START_REF] Verollet | HIV-1 reprograms the migration of macrophages[END_REF]. It is likely that 3D podosomes also possess the adhesion properties of "2D" podosomes because integrins and CD44 localize at these structures [START_REF] Van Goethem | Macrophage podosomes go 3D[END_REF]. However, the function of mechanosensory and matrix signal transduction remains difficult to explore in three-dimensional contexts.

Dendritic cell podosomes, antigen presentation and uptake

Dendritic cells patrol the blood and tissues with the purpose of capturing and presenting foreign elements to naïve T cells to allow them to mount a defensive response. Dendritic cells are genuine professional antigen-presenting cells and they exist in several sub-populations: interstitial, dermal, plasmocytoid, inflammatory and Langerhans cells. In the absence of foreign bodies, they are said to be immature (iDC). They constantly probe their environment with pattern recognition receptors (PRR) that recognize molecules (mainly sugars, lipids, proteins and nucleic acids) shed by infectious agents such as bacteria, fungi and viruses [START_REF] Lipscomb | Dendritic cells: immune regulators in health and disease[END_REF]. The signaling events that ensue "pattern" binding to their receptors lead to the internalization of the antigen by endocytosis or phagocytosis, to the redistribution of the proteins involved in antigen processing, to enhanced expression of the major histocompatibility complex and to the release of cytokines (interferonβ, interleukins, TNFα and RANTES) that facilitate T-cell differentiation. In short, a set of reactions occur that lead to priming of the adaptive immune system [START_REF] Kramer | Signal Transduction[END_REF].

In vitro, iDCs podosomes are arranged in clusters (Figure 1A). In dense matrices of gelled collagen-I or Matrigel, iDCs form 3D podosomes at the tip of cell protrusions and adopt the mesenchymal migration mode similar to macrophages [START_REF] Cougoule | Podosomes, But Not the Maturation Status, Determine the Protease-Dependent 3D Migration in Human Dendritic Cells[END_REF]. As a consequence of antigen recognition, iDC turn into mature dendritic cells (mDC). Concomitantly, podosomes disappear and cells form long dendrites instead (hence the name of the cells). By reducing adhesion to the ECM, this transformation also marks a transition to a faster migration mode, like DCs migrating towards lymph nodes to present antigens to naïve T cells in vivo [START_REF] Van Helden | A critical role for prostaglandin E2 in podosome dissolution and induction of high-speed migration during dendritic cell maturation[END_REF].

Podosomes participate specifically in the antigen seeking, capture and uptake phases of T cell immune response triggering. First, the topographical cues seem to act as a triggering signal for podosome construction. When iDCs are seeded on a porous membrane, podosomes form precisely on the top of the pores [START_REF] Baranov | Podosomes of dendritic cells facilitate antigen sampling[END_REF][START_REF] Gawden-Bone | Dendritic cell podosomes are protrusive and invade the extracellular matrix using metalloproteinase MMP-14[END_REF]. How extracellular topographical cues are signaled into the cells is not clear. It may occur through the recruitment of curvature-sensing proteins, such as the BAR proteins CIP4, FBP17, ASAP1 or IRSp53 into the plasma membrane [START_REF] Suetsugu | Dynamic shaping of cellular membranes by phospholipids and membrane-deforming proteins[END_REF]. The topography also influences podosome morphology: the ring of adhesive proteins containing vinculin, paxillin, talin and other components remains positioned at the entry of the membrane pore, whereas the actin core descends into the pore at the same time as the protrusion (Figure 2B). The extension of the protruding actin core is dependent on myosin-II activity and therefore involves contractility. The protruding membrane exposes MT1-MMP and PRRs and becomes a privileged site for matrix proteolysis, antigen capture and endocytosis [START_REF] Baranov | Podosomes of dendritic cells facilitate antigen sampling[END_REF][START_REF] Gawden-Bone | Dendritic cell podosomes are protrusive and invade the extracellular matrix using metalloproteinase MMP-14[END_REF]. This podosome architecture seems ideally suited for iDCs to seek "pathological patterns" across epithelia, which may occur in the intestine, lung or nasal cavities [START_REF] Baranov | Podosomes of dendritic cells facilitate antigen sampling[END_REF].

Podosomes of endothelial cells in sprouting angiogenesis

Endothelial cells line the inner surface of all blood vessels. Besides their multiple functions at the blood/tissue interface, they also play a key role in the process of vascular bed formation during angiogenesis, i.e., the process whereby new vessels arise from existing ones and lead to the extension of the vascular network. This mainly concerns the micro-vasculature that buds to establish vascularization during development or restores irrigation of injured or ischemic tissues. The developing retina of a newborn mouse, in which vascularization is initiated in the first week after birth, has been a great source for studying the process of angiogenesis. The main angiogenic factor involved is VEGF and endothelial cells respond to it in different ways. Cells with the highest cell surface receptor (VEGFR2) density turn into "tip cells", whereas the adjacent cells become "stalk cells". Tip cells develop filopodia equipped with VEGFR2 that capture surrounding VEGF, and with integrins allowing them to dock into newly explored territories. They are the leading migratory cells.

The adjacent cells proliferate and extend a stalk that pushes tip cells further into the surrounding tissue. In this way, an initial vascular sprout gradually turns into a growing capillary. The fate of the stalk cells depends on a process referred to as lateral inhibition [START_REF] Kramer | Signal Transduction[END_REF]. VEGF enhances the membrane expression of Delta-1ike 4 (Dll4) in the tip cell and, by signaling to Notch receptors, imposes the "stalk phenotype" on adjacent endothelial cells (a juxtacrine signaling mode). The process described above suggests that tip cells must somehow suppress even low levels of Notch signaling. This may occur through a mechanism of cis-inhibition, where Notch and Notch-ligands are expressed on the same membrane and create non-productive signaling complexes that prevent trans-activation [START_REF] Del Alamo | Mechanism and significance of cisinhibition in Notch signalling[END_REF].

Thus, stalk cells have high Notch activity whereas tip cells have low Notch activity.

In vitro, microvascular endothelial cells exposed to VEGF form podosomes [START_REF] Daubon | VEGF-A stimulates podosomemediated collagen-IV proteolysis in microvascular endothelial cells[END_REF][START_REF] Spuul | VEGF-A/Notch-Induced Podosomes Proteolyse Basement Membrane Collagen-IV during Retinal Sprouting Angiogenesis[END_REF][START_REF] Fey | Impaired endothelial shear stress induces podosome assembly via VEGF up-regulation[END_REF][START_REF] Osiak | Subconfluent endothelial cells form podosomes downstream of cytokine and RhoGTPase signaling[END_REF][START_REF] Wang | GIT1 mediates VEGF-induced podosome formation in endothelial cells: critical role for PLCgamma[END_REF]. These podosomes are interconnected by contractile actin-myosin filaments in an annular superstructure called a rosette. They have a strong proteolytic activity especially against collagen-IV [START_REF] Daubon | VEGF-A stimulates podosomemediated collagen-IV proteolysis in microvascular endothelial cells[END_REF].

Pharmacological inhibition of Notch signaling produces the same effects [START_REF] Spuul | VEGF-A/Notch-Induced Podosomes Proteolyse Basement Membrane Collagen-IV during Retinal Sprouting Angiogenesis[END_REF]. These observations led us to investigate podosome formation in tip cells displaying low Notch activity and exposed to VEGF in situ using the model of the mouse retina.

Immunofluorescence staining of podosome markers detected podosomes in tip cells of the developing retinal vasculature. In their native microenvironment, podosomes are globular in shape, interact with the BM, and their occurrence coincides with reduced collagen-IV vessel coverage [START_REF] Spuul | VEGF-A/Notch-Induced Podosomes Proteolyse Basement Membrane Collagen-IV during Retinal Sprouting Angiogenesis[END_REF] (Figure 2C).

The cables connecting the podosomes are clearly visible without recourse to super-resolution microscopy. Based on in vitro data, we postulate that podosomes intervene in tip cells in vivo at two levels: initially in the process of sprouting to breach the BM and invade the ECM; and at a later stage in the process of anastomosis, where tip cells must first remove the basement membrane sheet of an existing capillary in order to adhere to the endothelial cells to allow the sprouts to interconnect.

Conclusions and perspectives

Our knowledge of podosomes has increased tremendously in recent years, thanks in particular to technological developments in microscopy [START_REF] Meddens | Podosomes revealed by advanced bioimaging: what did we learn?[END_REF]. Accumulating evidence of the role of podosomes in the degradation of collagen-IV and in the breaching of anatomical barriers during angiogenesis makes them potential therapeutic targets in aberrant neo-vascularization processes. The intervention of podosomes in macrophages and iDCs requires further exploration in vivo but their predicted role in transtissular invasion offers new avenues for intervention in poorly controlled innate and adaptive immune responses. Processes involving podosomes are not limited to the examples that we have presented in this review. For instance, they take center stage in the formation of the sealing zone in osteoclasts and engage them in the process of bone resorption. The maturation of the postsynaptic plate in the neuromuscular junction [START_REF] Proszynski | Podosomes are present in a postsynaptic apparatus and participate in its maturation[END_REF] and the fusion of myoblasts [START_REF] Chen | Invasive podosomes and myoblast fusion[END_REF] are also physiological phenomena in which podosomes are thought to play a decisive role. The investigation of podosomes in in vitro conditions has revealed their multiple potentialities but it is likely that the different functions of podosomes do not intervene simultaneously. Like iDCs and macrophages, osteoclasts and endothelial cells can also present antigens [START_REF] Li | Cross talk between the bone and immune systems: osteoclasts function as antigen-presenting cells and activate CD4+ and CD8+ T cells[END_REF][START_REF] Pober | Antigen Presentation by Vascular Cells[END_REF], and the latter can form elongated podosomes when cells are seeded on top of a porous membrane [START_REF] Varon | Transforming growth factor beta induces rosettes of podosomes in primary aortic endothelial cells[END_REF]. Thus, the presentation and properties of podosomes concern the microenvironment rather than cells. It is the microenvironment that determines the precise role that podosomes play in vivo by regulating their assembly, arrangement, architecture, Endothelial cells acquire invasive properties to allow the emergence of blood vessel branches. ( 1)

Developing vasculature at day 6 in the mouse retina stained with the isolectin B4 endothelial marker.

(2) Immunofluorescent staining of the boxed area in [START_REF] Rowe | Breaching the basement membrane: who, when and how?[END_REF] showing the angiogenic front at higher magnification F-actin (red), cortactin (green) and BM collagen-IV (white).

  MicroRNAs are small noncoding RNAs that play a critical role in regulating several cellular functions through transcriptional silencing. Either a loss or a gain of micro-RNA expression can regulate podosome formation. In vascular smooth muscle cells (VSMCs), miR-143 and -145 regulate a switch between differentiated (contractile/non-proliferative) and dedifferentiated (synthetic/migratory/proliferative) phenotypes. These micro-RNAs prevent the formation of podosomes in VSMCs by repressing the transcription of PKCα (PRKCA) and PDGFRα(PDGFRA, and of fascin (FSCN1), respectively[START_REF] Quintavalle | MicroRNA control of podosome formation in vascular smooth muscle cells in vivo and in vitro[END_REF]. Expression of miR-143 and -145 is repressed by the proto-oncogene Src, an event that may promote podosome formation. In contrast, miR-155 induces podosome assembly in arterial endothelial cells[START_REF] Curado | ALK5 and ALK1 play antagonistic roles in transforming growth factor beta-induced podosome formation in aortic endothelial cells[END_REF]. MiR-155 represses the transcription of the BMP-regulated transcription factor Smad5. As Smad5 signals to cells in the basal state, it is likely that miR-1 alters cell homeostasis by suppressing a regulatory signal. Consistent with this, miR-155 is known to influence endothelial to mesenchymal transition by counteracting RhoA function[START_REF] Bijkerk | MicroRNA-155 functions as a negative regulator of RhoA signaling in TGF-beta-induced endothelial to mesenchymal transition[END_REF].

Figure 1 .

 1 Figure 1. Structure and organization of podosomes. (A) In an immature dendritic cell, the immunofluorescent staining of actin (red) and vinculin (green), visualized in the basal plane, reveals the core and ring of podosomes and their group arrangement. (B) Schematic representation of (A). (C) Diagram of the cell with its podosomes and surrounding F-actin networks in sagittal view (along the dotted line shown in (B)), showing the actomyosin cables that interconnect the podosomes and the podosome cap. (D) Detail of podosomes: the core, seat of actin polymerization, the ring, consisting in plaque proteins allowing matrix adhesion, the cap, where the radiating actin filament bundles terminate, the cables of actomyosin that assemble the individual podosomes into a network and the recently discovered actin bundles associating neighbor podosomes in close vicinity. For the sake of clarity, the set of short filaments forming a thick layer of cortical actin at the bottom of the podosome cluster [19] is not shown. Podosomes are endowed with a mechanosensory activity orchestrated by forces generated at several points (arrows), which combines or alternates with their proteolytic activity depending on the matrix characteristics (not described in the figure). N.B., The schematic representation uses information gathered from data obtained in different cell types.

Figure 2 .

 2 Figure 2. Podosome presentation in different in vitro or ex vivo contexts. (A) Podosomes allowmacrophages to tunnel into the matrix to infiltrate the tissues. In a three-dimensional, compact collagen-I matrix (shown as a block), macrophages form protrusions with globular-shaped podosomes at their ends. These so-called 3D podosomes do not present the core/ring organization that characterizes podosomes in a 2D situation (Figure1A). Matrix proteolysis by podosomes combined with the dynamic of protrusions drills a tunnel which, in vivo, may allow trans-tissular migration of macrophages and other cells. (B) iDC podosomes participate in the recognition and treatment of antigens. Side view of a Boyden chamber showing iDCs seeded on a gelatin-coated polycarbonate filter. Podosomes form on top of the pores, as the topographical changes initiate podosome construction and shape its morphology: the adhesive ring remains positioned at the level of the filter but the actin core extends into the pore. Extension of the actin core is dependent on myosin-II activity.The protrusion that now exposes MT1-MMP (not shown in the figure) and PRR becomes a prime site for matrix proteolysis, antigen capture and endocytosis. This podosome architecture could allow iDCs to detect distant pathogens. (C) Endothelial cell podosomes locally digest the BM during angiogenesis.

( 3 )

 3 Immunofluorescentstaining of the boxed area in[START_REF] Rowe | Breaching the basement membrane: who, when and how?[END_REF] showing a region of the distal and mature vasculature at higher magnification: in the absence of pro-angiogenic stimulus, the basement membrane wraps the tube composed of endothelial cells and provides them with survival and quiescence signals. (4) Zoom of the boxed area in[START_REF] Pozzi | The nature and biology of basement membranes[END_REF]; in response to VEGF, some cells become tip cells and assemble podosomes that locally degrade the underlying BM. (5) Zoom of the boxed area in (2); filopodia emerge through the gaps thus created. (6) Zoom of the boxed area in (2); the cell buds and the filopodia grip the ECM by engaging integrins, allowing this cell to extrude from the parent vessel to give rise to vascular branching. A sketch depicting each situation is presented in the bottom panel.Table I: Different types of signals regulate podosome formation in endothelial cells. Many factorsare able to modulate podosome formation, especially in cells where the structures are inducible such as endothelial cells. The signals are often of extracellular origin such as cytokines, nature and properties of the matrix, but may also result from internal changes concerning the metabolism and microRNAs. These regulations are dynamic as podosomes in turn act upon the microenvironment. Up and down arrows indicate if the assembly/formation of podosomes in enhanced (green) or reduced (red), respectively.

Acknowledgements

The authors apologize to the many scientists whose work could not be properly discussed and referenced due to limited space. The laboratory (http://genot-lab.org/ ) is funded by the Ligue contre le Cancer, committee of the Gironde, the University of Bordeaux (transversal project HYPOXCELL), the national research agency ANR-17-CE11-0033-01 and the "Marfans" Association. FA is funded by the Berthe Fouassier-Eye Disease Foundation, under the auspices of the Fondation de France. The Invadosome Consortium is an international network of laboratories interested in adhesion structures involved in invasive processes. It is open to the entire scientific community (http://www.invadosomes.org/)

lifespan, composition and functions.

Competing interest

The authors declare no competing interests

Mechanical

confinement aorta [START_REF] Spuul | Microfluidic devices for the study of actin cytoskeleton in constricted environments: Evidence for podosome formation in endothelial cells exposed to a confined environment[END_REF] stiffness liver sinusoids [START_REF] Juin | Extracellular matrix rigidity controls podosome induction in microvascular endothelial cells[END_REF]


MD Yohann Bohbot

MD Christophe De Meester De Ravenstein

MD Gagandeep Chadha

MD Dan Rusinaru

MD Khadija Belkhir

MSc Camille Trouillet

MD Agnes Pasquet

MD, PhD Sylvestre Marechaux

MD Jean-Louis Vanoverschelde

Professor Christophe Tribouilloy
email: tribouilloy.christophe@chu-amiens.fr

Relationship Between Left Ventricular Ejection Fraction and Mortality in Asymptomatic and Minimally Symptomatic Patients With Severe Aortic Stenosis

Keywords: Aortic stenosis, left ventricular ejection fraction, prognosis, mortality, surgery, conservative management. ABBREVIATION LIST AF: atrial fibrillation AS: aortic stenosis AVA: aortic valve area AVR: aortic valve replacement BSA: body surface area HR: hazard ratio LV: left ventricular LVEF: left ventricular ejection fraction

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Concentric left ventricular hypertrophy is a compensatory mechanism for the chronic pressure overload observed in aortic stenosis (AS). Although the development of concentric left ventricular (LV) remodeling and LV hypertrophy is a physiological response to increased wall stress in order to maintain left ventricular ejection fraction (LVEF), the adaptive mechanisms to pressure overload finally become insufficient and the LV becomes unable to maintain normal stroke volume in the setting of limited preload reserve, a condition known as "afterload mismatch". Consequently, systolic wall stress becomes markedly elevated, and LVEF decreases, eventually leading to the development of myocardial fibrosis and potentially irreversible myocardial dysfunction if the valvular obstacle is not relieved [START_REF] Dweck | Calcific aortic stenosis: a disease of the valve and the myocardium[END_REF][START_REF] Gaasch | Left ventricular structural remodeling in health and disease: with special emphasis on volume, mass, and geometry[END_REF][START_REF] Hein | Progression from compensated hypertrophy to failure in the pressure-overloaded human heart: structural deterioration and compensatory mechanisms[END_REF].

Several recent studies have suggested increased mortality in symptomatic AS patients with LVEF between 50 and 59% (4-6). However, symptomatic patients with severe AS have a class I indication for surgery because of symptoms, irrespective of the LVEF. According to American College of Cardiology/American Heart Association [START_REF]ACC/AHA guidelines for the management of patients with valvular heart disease. A report of the American College of Cardiology/American Heart Association. Task Force on Practice Guidelines (Committee on Management of Patients with Valvular Heart Disease)[END_REF] and European society of Cardiology [START_REF] Baumgartner | ESC/EACTS Guidelines for the management of valvular heart disease[END_REF] guidelines, aortic valve replacement (AVR) is a class I indication in patients with asymptomatic severe aortic stenosis (AS) and left ventricular systolic dysfunction defined as LVEF < 50%.

However, this guideline is rarely applicable because LV systolic dysfunction in patients with asymptomatic severe AS is uncommon, as it has been previously reported in less than 1% of patients [START_REF] Henkel | Asymptomatic left ventricular systolic dysfunction in patients with severe aortic stenosis: characteristics and outcomes[END_REF]. To our knowledge, no data are available concerning the prognostic value of LVEF in asymptomatic or minimally symptomatic AS patients in order to propose a LVEF threshold value for AVR in these patients. We hypothesized that a LVEF cutoff of 50% for referral to aortic valve replacement (AVR) in asymptomatic AS is too low and that a LVEF cutoff higher than 50% would be associated with poor outcome, as LVEF < 50% usually occurs late in the course of the disease, after onset of symptoms, when myocardial impairment is probably at least partially irreversible [START_REF] Lund | Preoperative risk evaluation and stratification of long-term survival after valve replacement for aortic stenosis. Reasons for earlier operative intervention[END_REF][START_REF] Lund | Cardiac pathology after isolated valve replacement for aortic stenosis in relation to preoperative patient status. Early and late autopsy findings[END_REF]. The objective of this study was therefore to determine the best LVEF cutoff to predict long-term mortality in patients with asymptomatic or minimally symptomatic severe AS and LVEF ≥ 50% under conservative management and after surgical correction of AS.

METHODS

Between 2000 and 2016, patients aged ≥ 18 years diagnosed with at least mild AS (aortic valve calcification with restricted systolic leaflet motion and aortic valve area (AVA) < 2 cm²) in the echocardiography laboratories of 2 French (Amiens and Lille) and one Belgian (Brussels) tertiary centers, were prospectively identified and included in an electronic database. Patients presenting the following criteria were excluded: (i) patients with more than mild aortic and/or mitral regurgitation; (ii) patients with prosthetic valves, congenital heart disease (with the exception of bicuspid aortic valves), supravalvular or subvalvular AS, or dynamic left ventricular outflow tract obstruction, and (iii) patients who declined to participate in the study. The present analysis was based on 1,678 patients with severe AS (defined as AVA ≤ 1 cm² and/or AVA normalized to body surface area [BSA] ≤ 0.6 cm²/m²), LVEF ≥ 50% and no or minimal symptoms at diagnosis. Symptoms were ascertained by each patient's cardiologist. Patients with atypical chest pain and elderly patients with minimal dyspnea not clearly related to AS were considered to be minimally symptomatic.

Patients were retrospectively divided into three groups according to baseline LVEF: LVEF ≥ 60%, LVEF between 55 and 59% and LVEF < 55%. The Charlson comorbidity index, comprising the sum of the individual comorbidities, was calculated for each patient [START_REF] Charlson | A new method of classifying prognostic comorbidity in longitudinal studies: development and validation[END_REF].

Coronary artery disease was defined by the presence of a documented history of acute coronary syndrome, coronary artery disease previously confirmed by coronary angiography or history of coronary revascularization. The study was approved by an independent ethics committee and was conducted in accordance with institutional policies, national legal requirements, and the revised Declaration of Helsinki.

Echocardiography

All patients underwent a comprehensive Doppler-echocardiographic assessment using commercially available ultrasound systems. Aortic flow was systematically recorded using continuous wave Doppler on several views (apical five-chamber, right parasternal, suprasternal, and epigastric). The view identifying the highest velocities was used to determine Vmax. Three consecutive measurements in patients in sinus rhythm or 5 consecutive measurements in patients in atrial fibrillation (AF) in this view were systematically averaged. The alignment of both pulsed and continuous-wave Doppler was optimized to be parallel with flow. Pressure gradients were calculated using the simplified Bernoulli equation [START_REF] Currie | Continuous-wave Doppler echocardiographic assessment of severity of calcific aortic stenosis: a simultaneous Doppler-catheter correlative study in 100 adult patients[END_REF]. AVA was calculated using the continuity equation [START_REF] Skjaerpe | Noninvasive estimation of valve area in patients with aortic stenosis by Doppler ultrasound and two-dimensional echocardiography[END_REF]. Left ventricular (LV) dimensions were assessed from parasternal longaxis views by 2-dimensional-guided M-mode using the leading-edge methodology at enddiastole and end-systole. LVEF was calculated using Simpson's biplane method tracings of the interface between the compacted myocardium and the LV cavity in the apical four-and twochamber views [START_REF] Lang | Recommendations for cardiac chamber quantification by echocardiography in adults: an update from the American Society of Echocardiography and the European Association of Cardiovascular Imaging[END_REF]. LV mass was estimated by an equation based on linear measurements normalized to BSA [START_REF] Lang | Recommendations for cardiac chamber quantification by echocardiography in adults: an update from the American Society of Echocardiography and the European Association of Cardiovascular Imaging[END_REF]. Systolic pulmonary artery pressure was calculated from the maximum peak tricuspid regurgitation velocity, using the simplified Bernoulli equation.

Follow-up

Median [interquartile range] follow-up was 43.0 months. Patients were followed by clinical consultations and echocardiography in the outpatient clinics of the 3 tertiary centers. A few patients were followed in public hospitals or private practices by referring cardiologists working in collaboration with the tertiary centers. Ninety-four percent of patients were followed for at least 2 years or until death. Follow-up was complete up until death or the end of the study for 1,510 patients (90%). Events were ascertained by direct patient interview and clinical examination and/or by repeated follow-up letters, questionnaires, and telephone calls to physicians, patients, and (if necessary) next of kin. The study endpoint was overall survival in medically and surgically managed patients after diagnosis at baseline echocardiography. Survival in medically and surgically managed patients encompassed all modalities of medical and surgical management. Survival was also analyzed separately in the subgroup of patients operated within 3 months after the baseline echocardiography (surgical management group) and in the subgroup of patients not operated on during the first three months after baseline echocardiography (conservative management group). Survival analysis in the conservative management group continued until last follow-up on medical management (censored at surgery).

Statistical analysis

SPSS software (version 18.0, IBM) and R (R project for Statistical Computing, version 3.3.3) were used for statistical analysis. Continuous variables were expressed as mean ± standard deviation or median [interquartile range], and categorical variables were expressed as numbers and percentages. The relationship between continuous baseline variables and the various groups was explored using one-way ANOVA (for normally distributed variables) or Kruskal-Wallis tests (for non-normally distributed variables). Pearson's chi-squared statistic or Fisher's exact test was used to examine the association between categorical baseline variables and the various groups. The significance of the difference between the reference group (LVEF ≥ 60%) and the other groups was examined when a significant intergroup difference was observed. Inter-individual differences were compared with a Mann-Whitney U test (with Bonferroni correction for multiple comparisons) and a Tukey test for normally distributed data. The start of follow-up for survival analysis was baseline echocardiography. The intraobserver, interobserver and intercenter reproducibility were evaluated using Pearson correlation coefficient, intraclass correlation coefficient (ICC) and coefficient of variation (CV). To assess intraobserver reproducibility, the main investigator of Amiens (YB) repeated the analysis in a randomly chosen subset of 20 patients with severe AS, ≥1 month after the first analysis. To assess the interobserver reproducibility, those 20 randomly chosen exams were analyzed by 2 investigators of Amiens (YB and CT). To assess intercenter reproducibility, those 20 randomly chosen exams were delivered to the other 2 centers and analyzed independently by one investigator of each center (YB for Amiens, SM and JLV for Lille and Brussels respectively). Event rates ± standard errors in the 3 groups were estimated using the Kaplan-Meier method and compared by twosided log-rank tests. Univariate and multivariable analyses of times to events were performed using Cox proportional hazards models with LVEF as an independent variable in a categorical format without the use of model-building techniques. The following covariates considered to have a potential prognostic impact (on the basis of epidemiological data) were included: age, sex, BSA, hypertension, coronary artery disease, history of myocardial infarction, history of AF, comorbidity index and aortic valve area. Hazard ratios (HR) and their 95% confidence intervals (CI) were estimated for all-cause mortality. The proportional hazards assumption was confirmed using statistics and graphs based on Schoenfeld residuals. For continuous variables, the assumption of linearity was assessed by plotting residuals against independent variables. Penalized smoothing splines (P-splines) were used to illustrate the association between LVEF as a continuous variable and the risk of mortality. Subgroup analyses (stratified by age, sex, coronary artery disease, hypertension, history of myocardial infarction and AF) were performed to determine the homogeneity of the association between low LVEF (<55%) and the outcome variable. The effect of LVEF on the risk of overall mortality was first estimated in each subgroup using a Cox univariate model, and then formally tested for first-order interactions in Cox models by entering interaction terms separately for each subgroup. The limit of statistical significance was p<0.05. All tests were two-tailed.

RESULTS

Baseline characteristics and management

The study population consisted of 1,678 patients (839 males: 49.8%; mean age: 76 years, mean LVEF: 64%) divided into 1,108 patients with LVEF ≥ 60% (66%), 331 patients with LVEF between 55 and 59% (20%) and 239 patients with LVEF < 55% (14%). Seven hundred and fiftyeight patients were managed medically: 469 with LVEF ≥ 60% (62%), 167 with LVEF between 55 and 59% (22%), and 122 with LVEF < 55% (16%) and 920 patients were managed surgically (patients operated within 3 months after baseline echocardiography): 639 with LVEF ≥ 60% (69%), 164 with LVEF between 55 and 59% (18%), and 117 with LVEF < 55% (13%).

No differences in terms of age, BSA, hypertension, diabetes mellitus, history of coronary artery disease and Charlson comorbidity index were observed between the groups. Patients with LVEF between 55 and 59% were more often men (p<0.05) and had greater LV end-diastolic (p<0.05) and end-systolic diameters (p<0.001) and volumes (both p<0.001) compared to the reference group (LVEF ≥ 60%). Patients with LVEF < 55% were mostly men (p<0.05), had a higher frequency of AF (p<0.05) and more frequently a history of myocardial infarction compared to patients with LVEF ≥ 60%. They had lower peak aortic jet velocities (p<0.001), mean pressure gradients and indexed stroke volumes (both p<0.05), greater LV end-systolic and end-diastolic diameters and volumes (all p<0.001) and lower global longitudinal strain values (p<0.05) compared to the reference group (Table 1).

Reproducibility

Intraobserver, interobserver and intercenter reproducibility for LVEF values are summarized in Supplemental Table 1. Intraobserver (R=0.96; ICC: 0.95; CV= 3%), interobserver (R=0.90; ICC=0.87, CV= 4.5%) and intercenter (R between 0.88 and 0.90; ICC between 0.83 and 0.89 and CV between 4.2% and 4.6%) analyzes showed good reproducibility.

Outcome analyses

Outcome with medical and surgical treatment.

Five-year survival rate was 72±2% for patients with LVEF ≥60%, 74±2% for patients with LVEF between 55 and 59% and 59±4% for patients with LVEF <55% (log rank p value<0.001).

Univariate and multivariable Cox analyses revealed between-group differences for overall mortality (both p<0.001) (Table 2). No significant difference was observed between the LVEF ≥ 60% and LVEF 55 to 59% groups, even after covariate adjustment (p=0.199) (Table 2) and further adjustment for surgery treated as a time dependent variable (p=0.48). Patients with LVEF<55% displayed significant excess mortality even after covariate adjustment (Adjusted HR=2.29[1.68-3.17]; p<0.001) (Table 2) and further adjustment for surgery treated as a time dependent variable (Adjusted HR=2.77[2.13-3.61]; p<0.001) (Figure 1A) compared to the reference group of patients with LVEF ≥ 60%.

As regard to gender, LVEF<55% was associated with increased mortality in men (HR 2.19 [1.54-3.11]; p<0.001) and women (HR 2.08 [1.41-3.06]; p<0.001) with no interaction between sex and LVEF<55% (p for interaction= 0.85). As illustrated by the supplemental figure 2, on multivariable analysis, LVEF<55% was associated with excess mortality compared to LVEF≥60% irrespective of gender (adjusted HR=2.29[1.47-3.56]; p<0.001 for men and adjusted HR=2.13[1.39-3.24]; p<0.001 for women). Further adjustment for surgery treated as a time dependent variable showed comparable results (adjusted HR=2.65[1.91-3.77]; p<0.001 for men and adjusted HR=2.38[1.52-3.51]; p<0.001 for women).

By dividing the whole study population into two groups with a 55% LVEF cut-off, the 5-year survival rate was 72±2% for patients with LVEF ≥55% and 59±4% for patients with LVEF <55% (log rank p value <0.001). On univariate Cox analysis, overall mortality was significantly higher for the LVEF<55%group (Table 2); this excess risk persisted after covariate adjustment (adjusted HR=2.18 [1.60-2.96]; p=<0.001) (Table 2) and further adjustment for surgery treated as a time dependent variable (Adjusted HR=2.86[2.21-3.68]; p<0.001) (Figure 1B). AVR was associated with better survival in patients with LVEF≥55% than in patients with LVEF<55% (adjusted HR 4.16 [3.33-5.26) vs. adjusted HR 2.32 [1.53-3.57]; p for interaction 0.017).

The shape of the relationship between LVEF as continuous variable and the risk of death was estimated using spline functions for LVEF (Figure 2). After adjustment, there was no increase in mortality risk with decreasing LVEF when it remained ≥55% (adjusted HR, 0.98 [0.96-1.04] per 1% LVEF decrement; P=0.22). With LVEF <55%, there was important increase in mortality risk with decreasing LVEF (adjusted HR, 0.86 [0.75-0.97] per 1% LVEF decrement; P=0.002). As regard to gender, on multivariable analysis, there was no increase in mortality risk with decreasing LVEF when it remained ≥55% (p=0.44 for men and p=0.11 for women) while there was important increase in mortality risk with decreasing LVEF when LVEF was <55% for both, men (p<0.001) and women (p=0.020).

The association between LVEF<55% and risk of death was consistent in subgroups of patients with no interactions between LVEF<55% and any of the subgroups (Figure 3). LVEF<55% was independently associated with mortality in each center compared to LVEF ≥55% (all p<0.05) with no interaction between LVEF<55% and centers (p for interaction=0.21).

Above the 60% cutoff, outcome was not influenced by further LVEF stratification, as no significant difference was observed between patients with LVEF between 60 and 64% and patients with LVEF ≥ 70% (p=0. [START_REF] Currie | Continuous-wave Doppler echocardiographic assessment of severity of calcific aortic stenosis: a simultaneous Doppler-catheter correlative study in 100 adult patients[END_REF], or between patients with LVEF between 65 and 69% and ≥ 70% (p=0.32) after covariate adjustment. No significant difference was observed between patients with LVEF between 55 and 59% and patients with LVEF ≥ 70% (p=0.07), but patients with LVEF < 55% displayed excess mortality compared to patients with LVEF ≥ 70% (adjusted HR=2.74 [1.85-4.01]; p=<0.001) (supplemental figure 2).

Comparison of patients with LVEF < 55% according to the type of management (i.e. initially conservative or surgical) demonstrated excess mortality in medically managed patients, even after covariate adjustment (Adjusted HR: 2.70[1.98-3.67]; p<0.001) (Supplemental figure 3).

Outcome in the conservative management subgroup

Patients were mostly women (p<0.001), more often diagnosed with hypertension (p=0.002) and AF (p<0.001) and more often had a history of myocardial infarction (p=0.016) compared to patients who underwent AVR within 3 months after baseline echocardiography. These patients had less severe AS at baseline with higher AVA, lower peak aortic jet velocity and mean pressure gradients (all p<0.001). They had higher end-systolic diameters (p=0.025) and volumes (p<0.001) and lower LVEF (p<0.001) compared to patients initially managed surgically (Table 3).

In patients initially managed conservatively, 249 (33%) deaths were recorded and 329 (43%) underwent surgery during follow up. Five-year survival rate was 54±3% for patients with LVEF ≥60%, 46±6% for patients with LVEF between 55 and 59% and 38±7% for patients with LVEF <55% (log rank p value<0.001). No significant difference was observed between the LVEF ≥ 60% and LVEF 55 to 59% groups, even after covariate adjustment (p=0.527) (Table 2). Patients with LVEF < 55% displayed significant excess mortality, even after covariate adjustment (Adjusted HR=2.44 [1.51-3.94]; p<0.001) compared to the reference group (Table 2, Figure 4A).

By dividing the whole study population into two groups with a 55% LVEF cutoff, the 5-year survival rate was 52±3% for patients with LVEF ≥ 55% and 38±7% for patients with LVEF <55% (log rank p value <0.001). On univariate Cox analysis, overall mortality was significantly higher for the LVEF < 55% group (Table 2); this excess risk persisted after covariate adjustment (adjusted HR=2.34 [1.49-3.67]; p=<0.001) (Table 2, Figure 4B).

Outcome in the surgical management subgroup

In patients who underwent surgery within 3 months after baseline echocardiography, 151 (16%) deaths were recorded during follow-up. Five-year survival rate was 83±2% for patients with LVEF ≥ 60%, 81±4% for patients with LVEF between 55 and 59% and 68±6% for patients with LVEF < 55% (log rank p value<0.001). No significant difference was observed between patients with preoperative LVEF ≥ 60% and patients with preoperative LVEF between 55 and 59%, even after covariate adjustment (p=0.360) (Table 2). Patients with preoperative LVEF < 55% displayed significant excess mortality, even after covariate adjustment (Adjusted HR=2.51 [1.58-4.00]; p<0.001) compared to those with preoperative LVEF ≥ 60% (Table 2, Figure 5A). By dividing the whole study population into two groups with a 55% preoperative LVEF cutoff, the 5-year survival rate was 83±2% for patients with LVEF ≥ 55% and 68±6% for patients with LVEF < 55% (log rank p value <0.001). On univariate Cox analysis, overall mortality was significantly higher for the LVEF < 55% group (Table 2); this excess risk persisted after covariate adjustment (adjusted HR=2.38 [1.52-3.72]; p=<0.001) (Table 2, Figure 5B). We did an analysis integrating the time after recoding the surgery variable (no surgery, surgery performed within 3 months after baseline echocardiography and surgery performed after 3 months). The interaction between this new surgery variable and LVEF was significant (p for interaction= 0.021).

DISCUSSION

The present study, based on a large registry of patients with severe AS and no or minimal symptoms at diagnosis and LVEF ≥ 50% managed in routine clinical practice, demonstrates that baseline LVEF assessed by Doppler-echocardiography is a major independent determinant of long-term outcome in response to conservative or surgical management. LVEF has a major impact on mortality that persists after adjustment for factors known to be major determinants of outcome, such as age, comorbidity, atrial fibrillation, coronary artery disease, history of myocardial infarction and aortic valve area. LVEF < 55% (observed in 14% of the study population) was associated with a greater than 2-fold increase in the risk of all-cause mortality. Moreover, our results show that LVEF < 55% is not simply predictive of excess mortality in medically managed patients but is also an independent determinant of poorer survival after surgical correction of AS, despite the marked reduction of mortality associated with surgery.

While surgery is currently a class I indication in patients with LVEF < 50%, this is a very rare situation in asymptomatic severe AS. These results suggest that AVR could be discussed before reaching an LVEF of 55%.

Concentric left ventricular hypertrophy is a compensatory mechanism for the chronic pressure overload observed in AS. As a result of impaired LV relaxation and compliance, this adaptive mechanism initially leads to diastolic dysfunction, constituting an established prognostic marker in AS [START_REF] Mandinov | Impact of systolic and diastolic dysfunction on postoperative outcome in patients with aortic stenosis[END_REF][START_REF] Rusinaru | Left Atrial Volume and Mortality in Patients With Aortic Stenosis[END_REF][START_REF] Debry | Prognostic significance of left ventricular concentric remodelling in patients with aortic stenosis[END_REF][START_REF] Christensen | Relation of Left Atrial Size, Cardiac Morphology, and Clinical Outcome in Asymptomatic Aortic Stenosis[END_REF] and subsequently systolic dysfunction. Some studies [START_REF] Kearney | Global longitudinal strain is a strong independent predictor of all-cause mortality in patients with aortic stenosis[END_REF][START_REF] Lancellotti | Risk stratification in asymptomatic moderate to severe aortic stenosis: the importance of the valvular, arterial and ventricular interplay[END_REF][START_REF] Salaun | Apical four-chamber longitudinal left ventricular strain in patients with aortic stenosis and preserved left ventricular ejection fraction: analysis related with flow/gradient pattern and association with outcome[END_REF] have shown that a decrease in myocardial contractility of longitudinal fibers occurs earlier and can be detected in many patients with AS despite a normal LVEF. This longitudinal fiber dysfunction, evaluated by studying myocardial deformation in 2-dimensional strain analysis, is associated with poor outcomes. Although the development of concentric LV hypertrophy is a physiological response to maintain LVEF, the adaptive mechanisms to pressure overload subsequently become insufficient. Systolic wall stress becomes markedly elevated, and LVEF decreases, eventually leading to the development of myocardial fibrosis and potentially irreversible myocardial dysfunction if the valvular obstacle is not relieved (1-3). Myocardial fibrosis can also be observed prior to onset of LV dysfunction and is associated with increased morbidity and mortality even after aortic valve replacement [START_REF] Yarbrough | Myocardial remodeling with aortic stenosis and after aortic valve replacement: mechanisms and future prognostic implications[END_REF]. Consequently, the 50% LVEF cutoff for referral to AVR in asymptomatic AS appears to be too low because this is an uncommon situation that occurs late in the course of the disease, when myocardial impairment is probably (at least partially) irreversible [START_REF] Capoulade | Echocardiographic predictors of outcomes in adults with aortic stenosis[END_REF][START_REF] Dahl | Effect of left ventricular ejection fraction on postoperative outcome in patients with severe aortic stenosis undergoing aortic valve replacement[END_REF].

The prognostic value of LVEF has been studied in symptomatic AS patients. LVEF < 60% has been reported to be associated with poorer long-term outcomes in conservatively managed patients [START_REF] Taniguchi | Prognostic Impact of Left Ventricular Ejection Fraction in Patients With Severe Aortic Stenosis[END_REF] and in patients undergoing surgical AVR [START_REF] Taniguchi | Prognostic Impact of Left Ventricular Ejection Fraction in Patients With Severe Aortic Stenosis[END_REF][START_REF] Lund | Preoperative risk evaluation and stratification of long-term survival after valve replacement for aortic stenosis. Reasons for earlier operative intervention[END_REF][START_REF] Lund | Cardiac pathology after isolated valve replacement for aortic stenosis in relation to preoperative patient status. Early and late autopsy findings[END_REF]. Nevertheless, these symptomatic patients already present a class I indication for surgery irrespective of the LVEF, and, to the best of our knowledge, no study has yet specifically focused on LVEF in asymptomatic and minimally symptomatic patients, excluding those with LVEF < 50% who already have a class I indication for AVR in AHA/ACC and European guidelines [START_REF]ACC/AHA guidelines for the management of patients with valvular heart disease. A report of the American College of Cardiology/American Heart Association. Task Force on Practice Guidelines (Committee on Management of Patients with Valvular Heart Disease)[END_REF][START_REF] Baumgartner | ESC/EACTS Guidelines for the management of valvular heart disease[END_REF]. While surgery is currently a class I indication in asymptomatic patients with severe AS with LVEF < 50% [START_REF]ACC/AHA guidelines for the management of patients with valvular heart disease. A report of the American College of Cardiology/American Heart Association. Task Force on Practice Guidelines (Committee on Management of Patients with Valvular Heart Disease)[END_REF][START_REF] Baumgartner | ESC/EACTS Guidelines for the management of valvular heart disease[END_REF], this is an exceptional situation. The results of the present study show that, among asymptomatic or minimally symptomatic patients with severe AS and LVEF ≥ 50%, LVEF remains a strong predictor of survival during long-term follow-up on conservative and surgical management. LVEF <55% (observed in 14% of patients in the study population) is associated with a greater than 2-fold increase in the risk of all-cause mortality in medically or surgically managed patients after adjustment for factors known to be major determinants of outcome. Although surgery is associated with a reduction of mortality in this setting compared to conservative management, excess mortality persists after AVR. These results suggests that AVR could be discussed before reaching an LVEF of 55%

Limitations

As this study was based on retrospective follow-up data, it presents the inherent limitations of this type of analysis. The specific indications for surgery during follow-up were not recorded in our database. Diagnosis and follow-up were performed by cardiologists with expertise in valvular heart disease, and surgical decisions were taken by the cardiology team with the approval of the patient's physician in accordance with current practice guidelines and the operative risk. We chose to analyze a subgroup of patients with no or minimal symptoms (i.e. patients with atypical chest pain and elderly patients with minimal dyspnea not clearly related to AS) because it is often difficult to differentiate between asymptomatic individuals and those with minimal symptoms in populations of elderly patients. Although previous guidelines have used LVEF of <50% as a cutoff for LV dysfunction in AS, recent ASE guidelines state that a normal LVEF in men is 52% and above whereas for women it is 54% and above [START_REF] Lang | Recommendations for cardiac chamber quantification by echocardiography in adults: an update from the American Society of Echocardiography and the European Association of Cardiovascular Imaging[END_REF]. In our study, on multivariable analysis, there was no increase in mortality risk with decreasing LVEF when it remained ≥55% in men and women respectively, while there was important increase in mortality risk with decreasing LVEF when LVEF was <55% for both genders.

Limitations of LVEF are its operator and acoustic window dependency. In our opinion, these inherent limitations of LVEF do not hamper its ability to predict mortality. Elsewhere, precise LVEF thresholds are used when discussing surgery for patients with heart valve disease as well as in heart failure patients before cardiac resynchronization therapy or when considering intracardiac defibrillator implantation in patients with systolic LV dysfunction in primary prevention. Lastly, this study exclusively concerned patients with severe AS and no significant valve regurgitation. Further studies are needed to evaluate the role of LVEF in other subsets of patients with severe AS.

CONCLUSION

This study demonstrates that LVEF is a powerful predictor of survival in a population of AS patients with no or minimal symptoms at diagnosis and LVEF ≥ 50%. Patients with LVEF < 55% (14%) exhibited excess mortality when managed conservatively and after AVR and should be considered for surgery. To avoid the excess mortality observed after AVR in patients with LVEF < 55%, aortic valve replacement could be discussed before LVEF drops below 55% in asymptomatic patients with severe AS.

Perspectives COMPETENCY IN MEDICAL KNOWLEDGE

Current guidelines recommend aortic valve replacement in asymptomatic severe AS when left ventricular ejection fraction (LVEF) is < 50%. However, LV systolic dysfunction in asymptomatic severe AS is uncommon and data are lacking concerning the prognostic value of higher LVEF cutoffs in asymptomatic or minimally symptomatic AS patients. The present study demonstrated that LVEF < 55% was independently associated with poorer long-term outcomes in conservatively or surgically managed patients.

TRANSLATIONAL OUTLOOK

The optimal cutoff to define normal systolic function is unknown in the setting of AS-associated remodeling. Our findings challenge the current 50% LVEF cut-off value used to define LV systolic dysfunction in severe AS. Further prospective studies are needed to explore the outcome of higher LVEF cutoffs in asymptomatic severe AS. Normally distributed continuous variables are expressed as mean ±1 standard deviation, non-normally distributed continuous variables are expressed as median (25th and 75th percentiles), and categorical variables are expressed as percentages and numbers. Abbreviations: LV: left ventricular and sPAP: systolic pulmonary artery pressure

Figure 1 :

 1 Figure 1: Time dependent adjusted survival curves in medically and surgically managed patients according to left ventricular ejection fraction (LVEF), dividing the population A) into 3 groups (LVEF ≥ 60%, LVEF 55-59% and LVEF < 55%) and B) into 2 groups (LVEF ≥ 55% and LVEF < 55%).

Figure 2 :

 2 Figure 2: Hazard ratio (solid line) and 95% confidence intervals were estimated in a Cox multivariable model with left ventricular ejection fraction (LVEF) represented as a spline function.

Figure 3 :

 3 Figure 3: Hazard ratio (HR) and 95% confidence interval (CI) plot (with a logarithmic scale) for the risk of all-cause mortality associated with left ventricular ejection fraction (LVEF) < 55% in subgroups of patients with asymptomatic and minimally symptomatic severe aortic stenosis.

Figure 4 :

 4 Figure 4: Adjusted survival curves in conservatively managed patients according to left ventricular ejection fraction (LVEF), dividing the population A) into 3 groups (LVEF ≥ 60%, LVEF 55-59% and LVEF < 55%) and B) into 2 groups (LVEF ≥ 55% and LVEF < 55%).

Figure 5 :

 5 Figure 5: Adjusted survival curves in surgically managed patients according to left ventricular ejection fraction (LVEF), dividing the population A) into 3 groups (LVEF ≥ 60%, LVEF 55-59% and LVEF < 55%) and B) into 2 groups (LVEF ≥ 55% and LVEF < 55%).

Table 1 :

 1 Baseline demographic, clinical and echocardiographic characteristics of the study population with asymptomatic and minimally symptomatic severe aortic stenosis according to left ventricular ejection fraction (LVEF)

	Variables	LVEF ≥60%	LVEF 55-59%	LVEF<55%	p value
		(n=1108)	(n=331)	(n=239)
	Demographic data and symptoms Age (years) Male gender (%, n) Body surface area (m²)	75.8±10.3 517 (46.7%) 1.88±0.24
	Medical history and risk factors Hypertension (%,n) Diabetes mellitus (%,n) Coronary artery disease (%, n) History of atrial fibrillation (%, n) History of myocardial infarction (%, n) Charlson comorbidity index	796 (71.8%) 268 (24.2%) 488 (44%) 239 (21.6%) 60 (5.4%) 2.41±1.83
	Echocardiographic parameters
	Aortic valve
	Aortic valve area (cm²)
	Peak aortic jet velocity (m/s)
	Transaortic mean pressure gradient (mmHg)
	Indexed stroke volume (ml/m²)
	Left ventricular function
	LV end-diastolic diameter (mm)
	LV end-systolic diameter (mm)
	LV end-diastolic volume (ml)
	LV end-systolic volume (ml)
	Ejection fraction (%)
	sPAP (mmHg)
	GLS (%) ‡