Search

International audience; The melanocortin 1 receptor (MC1R) is a G-protein coupled receptor (GPCR) which plays a major role in controlling melanogenesis. A large body of evidence indicates that GPCRs are part of large protein complexes that are critical for their signal transduction properties. Among proteins which may affect MC1R signaling, neurofibromin (Nf1), a GTPase activating protein (GAP) for Ras, is of special interest as it regulates adenylyl cyclase activity and ERK signaling, two pathways involved in MC1R signaling. Moreover, mutations in this gene encoding Nf1 are responsible for neurofibromatosis type I, a disease inducing hyperpigmented flat skin lesions. Using co-immunoprecipitation and Bioluminescence Resonance Energy Transfer experiments we demonstrated a physical interaction of Nf1 with MC1R. In particular, the GAP domain of Nf1 directly and constitutively interacts with MC1R in melanocytes. Pharmacologic and genetic approaches revealed that the GAP activity of Nf1 is important to regulate intracellular signaling pathways involved in melanogenesis and, consequently, melanogenic enzyme expression and melanin production. These finding shed new light on the understanding and cure of skin pigmentation disorders.

Several attempts have been made to deliver reactive oxygen and nitrogen species (RONS) produced by non-thermal plasma onto large surfaces in a controllable way compatible with skin treatments. In this paper, the effect of pulse frequency, in the range 0.5–20 kHz, on the discharge behavior was studied on skin tissue models to evaluate potential treatment changes. This has been done through electrical characterization, visualization of the helium flow (by Schlieren technique), produced plasma jet modifications (ICCD imaging) and RONS measurements. The results show that, in addition to its well known important role in the production of the chemical species, the applied discharge frequency plays a very significant role in the size of the treated surface. An enhancement of NO*, OH* and O* production in the gas phase at the higher frequency is reported and assigned to the stronger mixing of the helium flow with ambient air. The efficacy of plasma jet on transporting RONS on/into agarose gel and pig skin has been evaluated. The distribution of the reactive species on the target, or passing through, is strongly dependent on the discharge frequency and consequently induces pH variations. The present study supports a new way for enlarging the treated surface by using a simple jet at high frequency in the 20 kHz range, leading, with appropriate gas flow and distance to target, to conditions of RONS production that are compatible with potential uses for biomedical or cosmetic applications.

International audience; Soluble guanylate cyclase (sGC) is the human receptor of nitric oxide (NO) in numerous kinds of cells and produces the second messenger 3',5'-cyclic guanosine monophosphate (cGMP) upon NO binding to its heme. sGC is involved in many cell signaling pathways both under healthy conditions and under pathological conditions, such as angiogenesis associated with tumor growth. Addressing the selective inhibition of the NO/cGMP pathway is a strategy worthwhile to be investigated for slowing down tumoral angiogenesis or for curing vasoplegia. However, sGC inhibitors are lacking investigation. We have explored a chemical library of various natural compounds and have discovered inhibitors of sGC. The selected compounds were evaluated for their inhibition of purified sGC in vitro and sGC in endothelial cells. Six natural compounds, from various organisms, have IC50 in the range 0.2-1.5 μM for inhibiting the NO-activated synthesis of cGMP by sGC, and selected compounds exhibit a quantified antiangiogenic activity using an endothelial cell line. These sGC inhibitors can be used directly as tools to investigate angiogenesis and cell signaling or as templates for drug design.

International audience; Leucine‐rich repeat and immunoglobin‐domain containing (LRRIG) proteins that are commonly involved in protein‐protein interactions play important roles in nervous system development and maintenance. LINGO‐1, one of this family members, is characterized as a negative regulator of neuronal survival, axonal regeneration, and oligodendrocyte precursor cell (OPC) differentiation into mature myelinating oligodendrocytes. Three LINGO‐1 homologs named LINGO‐2, LINGO‐3, and LINGO‐4 have been described. However, their relative expression and functions remain unexplored. Here, we show by in situ hybridization and quantitative polymerase chain reaction that the transcripts of LINGO homologs are differentially expressed in the central nervous system. The immunostaining of brain slices confirmed this observation and showed the co‐expression of LINGO‐1 with its homologs. Using BRET (bioluminescence resonance energy transfer) analysis, we demonstrate that LINGO proteins can physically interact with each of the other ones with comparable affinities and thus form the oligomeric states. Furthermore, co‐immunoprecipitation experiments indicate that LINGO proteins form heterocomplexes in both heterologous systems and cortical neurons. Since LINGO‐1 is a promising target for the treatment of demyelinating diseases, its ability to form heteromeric complexes reveals a new level of complexity in its functioning and opens the way for new strategies to achieve diverse and nuanced LINGO‐1 regulation.

International audience; The maintenance of skin integrity is crucial to ensure the physiological barrier against exogenous compounds, microorganisms and dehydration but also to fulfill social and aesthetic purposes. Besides the development of new actives intended to enter a formulation, innovative technologies based on physical principles have been proposed in the last years. Among them, Cold Atmospheric Plasma (CAP) technology, which already showed interesting results in dermatology, is currently being studied for its potential in skin treatments and cares. CAP bio-medical studies gather several different expertise ranging from physics to biology through chemistry and biochemistry, making this topic hard to pin. In this review we provide a broad survey of the interactions between CAP and skin. In the first section, we tried to give some fundamentals on skin structure and physiology, related to its essential functions, together with the main bases on cold plasma and its physicochemical properties. In the following parts we dissected and analyzed each CAP parameter to highlight the already known and the possible effects they can play on skin. This overview aims to get an idea of the potential of cold atmospheric plasma technology in skin biology for the future developments of dermo-cosmetic treatments, for example in aging prevention.

International audience

Ticks are the most important vectors of pathogens affecting both domestic and wild animals worldwide. Hard tick feeding is a slow process&mdash, taking up to several days&mdash, and necessitates extended control over the host response. The success of the feeding process depends upon injection of tick saliva, which not only controls host hemostasis and wound healing, but also subverts the host immune response to avoid tick rejection that creates a favorable niche for the survival and propagation of diverse tick-borne pathogens. Here, we report on the molecular and biochemical features and functions of an Ixodes ricinus serine protease inhibitor (IrSPI). We characterize IrSPI as a Kunitz elastase inhibitor that is overexpressed in several tick organs&mdash, especially salivary glands&mdash, during blood-feeding. We also demonstrated that when IrSPI is injected into the host through saliva, it had no impact on tissue factor pathway-induced coagulation, fibrinolysis, endothelial cell angiogenesis or apoptosis, but the protein exhibits immunomodulatory activity. In particular, IrSPI represses proliferation of CD4+ T lymphocytes and proinflammatory cytokine secretion from both splenocytes and macrophages. Our study contributes valuable knowledge to tick-host interactions and provides insights that could be further exploited to design anti-tick vaccines targeting this immunomodulator implicated in I. ricinus feeding.

International audience; Cold atmospheric plasmas (CAPs) have become great challenge due to their vast potentialfor biomedical applications. Theresearch activities in the biological field range from the study of wound healing, cancer treatment, dentistry, sterilization and decontamination,treatment of implants for biocompatibility, dermatology and cosmetics. The biological effects of CAP are strongly linked to generation of reactive oxygen and nitrogen species (RONS). An important issue in application of CAP for tissue treatment concerned the penetration of RONS into biological tissue. RONS distribution and penetration can vary from tissue to tissue and, depending on their concentration, the biological effect can be beneficial or deleterious.In the present work, an agarose gel phantom is used as surrogates of human skin tissue to study its barrier effects on transportation and distribution of RONS after CAP treatment. We showed how the plasma conditions such as the gap distance, the treatment time and thehigh voltage frequency influence the RONS deposition, distribution and diffusion through the target. We also developed a new technique to visualize and quantify the local acidification induced by CAP treatment. These data together with our plasma source characterizationhelp us to determine safe treatment parameters for cosmetic application of CAP on the skin. This work was supportedby Cosmetosciences, a global training and research program dedicated to the cosmetic industry, located in the heart of the Cosmetic Valley, this program led by University of Orléans through the Région Centre-Val de Loire, France

National audience

Skin injury leading to chronic wounds is of high interest due to the increasing number of patients suffering from this symptom. Proliferation, migration, and angiogenesis are key factors in the wound healing processes. For that reason, controlled promotion of these processes is required. In this work, we present the portable helium-dielectric barrier discharge (He-DBD)-based reaction-discharge system of controlled gas temperature for biological activities. To make this He-DBD-based reaction-discharge system safe for biological purposes, a multivariate optimization of the operating parameters was performed. To evaluate the effect of the He-DBD operating parameters on the rotational gas temperature Trot(OH), a design of experiment followed by a Response Surface Methodology was applied. Based on the suggested statistical model, the optimal operating conditions under which the Trot(OH) is less than 37 °C (310 K) were estimated. Then, the resulted model was validated in order to confirm its accuracy. After estimation the optical operating conditions of He-DBD operation, the spectroscopic characteristic of the He-DBD-based reaction-discharge system in relevance to the several optical temperatures in addition to electron number density has been carried out. Additionally, the qualitative and quantitative analyses of the reactive oxygen species and reactive nitrogen species were performed in order to investigate of reactions and processes running in the He-DBD-gaseous phase and in the He-DBD-treated liquid. Next, the developed portable He-DBD-based reaction-discharge system, working under the optimal operating conditions, was used to stimulate the wound healing process. It was found that a 30 s He-DBD treatment significantly increased the proliferation, migration, and angiogenesis of keratinocytes (HaCaT) and fibroblasts (MSU-1.1) cell lines, as well as human skin microvascular endothelial cells (HSkMEC.2). Hence, the application of the cold atmospheric pressure plasma generated in this He-DBD-based reaction-discharge system might be an alternative therapy for patient suffering from chronic wounds.

The biological effects induced by cold atmospheric plasmas (CAPs) on human tissues are mainly due to the production of reactive oxygen and nitrogen species (RONS). Some RONS are also responsible for pH lowering of the treated medium. The CAP-induced acidification has beneficial effect on biological tissues, contributing to the anti-bacterial effect and to the healing improvement observed in treated wound. In this work we investigated the local acidification induced by a helium CAP treatment using tissue models made of agarose gels with adjusted pH around 7.4 to mimic generic organs or around 5.5 to simulate skin surface pH. Using fluorescein as a pH-sensitive fluorescent marker, we developed a useful technique to visualize and quantify the local acidification induced by CAP exposure of tissue surface. The different capillaries used to produce the plasma jet, the treatment time, the initial pH of the surface and the buffer capacity of the tissue model were shown to modulate both the size of the impacted surface and the intensity of the pH decrease. The proposed technique can be advantageous to study the acidifying effect induced by plasma. This method can help to plan safe and controlled plasma treatments in order to avoid hyper-acidification of the tissue, especially when a localized treatment is administered.

International audience

Biosensors and whole cell biosensors consisting of biological molecules and living cells can sense a special stimulus on a living system and convert it to a measurable signal. A major group of them are the bioluminescent sensors derived from luciferases. This type of biosensors has a broad application in molecular biology and imaging systems. In this project, a luciferase-based biosensor for detecting and measuring caspase-9 activity is designed and constructed using the circular permutation strategy. The spectroscopic method results reveal changes in the biosensor structure. Additionally, its activity is examined in a cell-free coupled assay system. Afterward, the biosensor is utilized for measuring the cellular caspase-9 activity upon apoptosis induction in a cancer cell line. In following the gene of biosensor is sub-cloned into a eukaryotic vector and transfected to HEK293T cell line and then its activity is measured upon apoptosis induction in the presence and absence of a caspase-9 inhibitor. The obtained results show that the designed biosensor detects the caspase-9 activity in the cell-free and cell-based systems.

Despite the large number of papers showing the biological effect of cold atmospheric plasmas (CAPs) on skin treatment and although their ability to produce a cocktail of reactive oxygen and nitrogen species (RONS) is largely known, the deep knowledge of these ionized gas sources is far to be uncovered. Many parameters take part in the complex mechanisms induced by plasma, such as voltage, frequency, feeding gas flow rate, temperature, air humidity, and atmospheric gas composition. In this paper, we focus our attention on the influence of oxygen dissolved in the medium in RONS generation during CAP treatment. We evaluate RONS production in Dulbecco’s phosphate-buffered saline equilibrated with two different percentages of oxygen: 18% and 3% O2. The former oxygen concentration is that usually used in most of cell culture and treatment (normoxia) while the latter is close to the physiological level measured inside human skin (physioxia). Intracellular RONS generation upon CAP treatment was also measured in two human skin cell lines maintained either in normoxic or in physioxic conditions. Our data suggest that RONS generation induced by plasma either in the medium or inside cells is influenced by oxygen dissolved in the treated target.

International audience; With the increase of human span life people demand to live and look better. In a world whereappearance is synonymous of health, the demand of new cosmetic has literally exploded. Amongcosmetic treatments, skin care represents a large part of the business of beauty. From 2017 to2023, world-wide non-surgical skin cosmetic treatments business will have a compound annualgrowth rate between 4.7% and 5.3% [1]. The increased demand of non-surgical beautytreatments pushed cosmetic research during the last years. In Europe since 2013 cosmetic testson animal model are definitively banned. This encouraged researchers to develop newapproaches to study skin “in-vitro” using either re-constructed tissue structure or attempting tomimic skin microenvironment parameters. Cell culture has been used since a long time to studyorgan functions in laboratory. In the last decades cell culture has greatly evolved in order tomimic the real tissue structure and microenvironment. Cells are cultivated in three dimensionsand media are adapted to better simulate the extracellular environment. Attempting to get closerto the physiological micro-environment of a cell, scientists underestimated often a keyparameter: the oxygen level [2]. While oxygen represents the 21% of the air gases, in humantissues, its percentage is significantly lower. In skin, it can vary from 7% to 1%. To be as closeas possible to skin microenvironment, in our lab we grow human skin cells either in classicnormoxic condition (18% O2) or in physioxic condition (3% O2). Oxygen has a key role in cellrespiration and in reactive oxygen species (ROS) production. We have already demonstrated thatskin cells raised in physioxia produce lower quantity of reactive oxygen and nitrogen species(RONS) even when exposed to plasma treatment [3]. In this study we evaluated the effect of aCold Atmospheric Plasma (CAP) treatment on human skin cell raised either in normoxia or inphysioxia. In particular, modulating plasma parameters from our helium Plasma Gun, weinvestigated the ability of CAP to improve cell viability and extracellular matrix production suchas collagen, hyaluronic acid and elastin, macromolecules involved in maintaining the health andbeauty of the skin.

National audience

International audience; Nowadays the increases of life expectation in developed world has radically changed people demand and their shopping basket. Since medical research has assuring physical wellness until old age, today people ask not only to stay healthy but also to look young. For this reason in the last decades cosmetic research has literally exploded. New generation cosmetics has appeared on the market and small companies started to develop plasma devices for skin care. Although cold plasma is already used in cosmetics, very little is known about the beneficial mechanism that lead to skin renewing. Cold plasma in fact could produce opposite effects on cells. It could either lead to cell death or stimulate their growth. We have also demonstrated that respecting skin microenvironment and particularly its physiological oxygen level (called physioxia) in in vitro experiments is crucial to be as close as possible to in situ skin [1, 2]. It is especially important for cold plasma treatments as the level of oxygen is closely related to the production of reactive oxygen and nitrogen species (RONS), components of the produced plasma. In this work, we characterized the chemico-physical properties of a He plasma gun [3, 4] (run at 14kV, 2 kHz, He flow of 0.5 sml at 10 mm above target) and its effects on culture media and on skin cells. In particular we analyzed O 2 partial pressure (see Fig. 1), RONS species produced, pH modification, effects on dissolved O 2 levels and electric field generation in order to understand plasma treatment consequences and to define the best settings to achieve a stimulating effect on skin cells. This will be first evaluated in physioxia versus normoxia, by assessing cell viability and motility on human keratinocytes and dermal fibroblasts. These results bring new elements to better understand the physiological response of skin cells to plasma treatment and will help to develop new strategies for cosmetical use. Fig. 1: O 2 pressure and temperature variations versus time in medium (7mm high) in a well of 96 well plate This work was supported by ARD 2020 Cosmetoscience PLASMACOSM project.

International audience; The last decade has seen an impressive development of the research dedicated to the biomedical applications of Low Temperature Non Thermal Plasmas (LTNTP), ionized gases at temperature close to the ambient one. This is especially true with plasma sources working at atmospheric pressure allowing direct applications on human. Medical applications of LTNTP now concern a very wide range of domains from wound healing [1] to cancer treatment [2,3] or transdermal drug delivery [4] . These applications also include dermatology closely linked to cosmetic. At atmospheric pressure, LTNTP are mainly produced by Dielectric Barrier Discharges (DBD) or by single/multi plasma jets, both types based on electrical discharges at very low power. The produced plasmas emit light in wide wavelength range and contain charged species and reactive species which concentrations depend on discharge configuration and electrical parameters. In addition, LTNTP produce external electric field which can, not only, be applied locally in combination with the species mentioned above, but also alone. All of their characteristics render them very attractive for potential applications in cosmetic especially anti-aging through skin structure modifications, hydration, and oxygenation or skin care product delivery. In this presentation, after a very brief reminder on the plasma state, we will first present the different ways to produce atmospheric LTNTP and discuss about their properties (including those related to transient electric field), in particular the ones of clear interest for cosmetic. Then we will give a brief review on the ongoing biomedical plasma research and applications, with a special focus on the ones related to dermatology treatments. We will end by a survey of possible developments and use in cosmetic.AcknowledgmentsThis work is supported by the ARD2020 COSMETSCIENCES Project “PLASMACOSM”References[1] J Heinlin, G Isbary, et a,l JEADV (2011) 25, 1–11[2] H.R. Metelmann et al Clin. Plas Med. doi.org/10.1016/j.cpme.2015.02.001[3] L. Brullé et al, PLOS ONE, 7, DOI: 10.1371/journal.pone.0052653 (2012)[4] S. Khalgathi, ISPC 22nd proceedings, O-22-6 (2015)

L'action des plasmas froids hors équilibre est désormais largement démontrée à la fois in vitro et in vivo (sur des modèles animaux [1,2] et sur l’homme [3,4]) sur de très nombreuses lignées cellulaires, divers types de tumeurs et de maladies. Néanmoins, en raison de la grande variété des composants du plasma capables de jouer un rôle, la compréhension précise de la chaîne de processus conduisant aux effets observés est loin d'être atteinte. Il est difficile d'isoler ou de mesurer l’action individuelle de chaque composante du plasma ou, au contraire, d'évaluer le bénéfice ou la synergie potentielle de leur combinaison. Jusqu'à présent, de nombreuses expériences in vitro ont été réalisées avec cette intention, mais leur transposition in vivo est très compliquée, même, dans de nombreux cas, impossible en raison de l'environnement complexe des cellules in vivo, notamment la vascularisation (impliquant le flux sanguin et l'oxygénation/physioxie) et la réponse du corps (signalisation, recrutement du système immunitaire). En général, la première étape concerne les études consacrées à la viabilité des cellules in vitro ou des études de toxicité dans un milieu de culture avec l'analyse à la fois des changements induits dans les cellules et dans le milieu. Il faut souligner que les conditions expérimentales utilisées, ainsi que le fait de traiter les cellules dans des plaques multipuits en dehors de leur chambre d'incubation peut générer un stress cellulaire qui peut affecter leur comportement et conduire à des résultats biaisés. De plus, même si les jets de plasmas, très largement utilisés dans les applications biomédicales, sont des systèmes relativement simples dans leur conception (principalement à base de réacteur DBD), il n’en reste pas moins que leur étude est complexe, particulièrement dans l’environnement des cibles biologiques. Les interactions entre le plasma lui-même, le débit de gaz et la cible (liquide, tissu, surface inerte) peuvent changer radicalement la production et les interactions des espèces réactives et des espèces chargées pendant les traitements. Enfin, le champ électrique transitoire généré par le jet de plasma peut interagir avec le milieu environnant et, éventuellement, agir en synergie avec les autres composants actifs du plasma délivrés sur la cible.

Synopsis Objective Polyphenols are strong antioxidant molecules allowing prevention of skin photo-ageing damages, but their use is limited due to low solubility and toxicity towards skin cells. We postulated that enzymatic glucosylation could improve their solubility, stability and, consequently, their efficacy. The aim of this work was to study changes induced by addition of a glucose moiety on two polyphenols displaying very different chemical structures [caffeic acid (CA), epigallocatechin-3-gallate (EGCG) and there glucosylated form, Glc-CA and Glc-EGCG] by assessing their cytotoxic properties and their antioxidant and anti-inflammatory activities. Methods Their antioxidant effect was assessed first by the classical DPPH radical-scavenging method. Then, a panel of human skin cells (keratinocytes, melanocytes, fibroblasts and endothelial cells) was used to evaluate their effect on cell toxicity and their antioxidant activities. With this aim, a photo-ageing model based on UV irradiation of skin cells was established. Molecule activity was assessed on reactive oxygen species (ROS) production, on superoxide dismutase (SOD) and catalase activities and, finally, on inflammatory factor production IL-6, IL-8 and IL-1β. Results In an acellular model, antioxidant activity assessed by DPPH method was strongly reduced for Glc-CA compared to CA, whereas it remained the same for Glc-EGCG compared to EGCG. Glucosylated derivatives did not display more toxic effect on various skin cells. Moreover, toxicity was even strongly reduced for caffeic acid upon glucosylation. The efficacy of glucosyl-compounds against UV-induced ROS production was preserved, both with pre- and post-UV treatments. Particularly, a better antioxidant efficacy was shown by Glc-EGCG, vs. EGCG, on keratinocytes. In addition, an induction of SOD and catalase activity was clearly observed for Glc-CA. Both glucosyl-polyphenols display the same activity as their parent molecule in decreasing inflammatory factor production. Conclusion Our results demonstrated that enzymatic glucosylation of CA and EGCG led to an improved or preserved antioxidant activity in a cellular model of UV-induced skin ageing, despite the decrease in instantaneous antioxidant properties observed for Glc-CA. Glc-EGCG is specifically more active on keratinocytes, suggesting a specific targeting. Such glucosylated polyphenols displaying improved physicochemical and biological properties should be better candidates than natural ones for use in food additives and cosmetics. Resume Objectifs Les polyphenols sont des molecules ayant un fort pouvoir antioxydant permettant de prevenir les dommages dus au photovieillissement de la peau mais leur utilisation est limitee de par leur faible solubilite et leur toxicite envers les cellules de la peau. Nous avons postule que la glucosylation enzymatique pourrait ameliorer leurs solubilite, stabilite et, en consequence, leur efficacite. Le but de ce travail est d'etudier les changements induits par l'addition d'un glucose sur deux polyphenols ayant une structure chimique tres differente [acide cafeique (CA), epigallocatechin-3-gallate (EGCG) et leurs formes glucosylees, Glc-CA et Glc-EGCG] en evaluant leurs proprietes cytotoxiques et leurs activites antioxydante et anti-inflammatoire. Methodes Leur pouvoir antioxydant a d'abord ete evalue par la methode classique de reduction du radical DPPH. Puis, un ensemble de cellules de peau humaine (keratinocytes, melanocytes, fibroblastes et cellules endotheliales) a ete utilise pour evaluer leur effet sur la toxicite cellulaire et leurs activites anti-oxydantes. Pour cela, un modele de photovieillissement base sur l'irradiation UV de cellules de peau a ete etabli. L'activite des molecules a ete evaluee sur la production d'especes reactives de l'oxygene (ROS), sur les activites superoxyde dismutase (SOD) et catalase et, enfin, sur la production de facteurs anti-inflammatoires IL-6, IL-8 et IL1β. Resultats Dans un modele acellulaire, l'activite antioxydante mesuree par la methode au DPPH etait fortement reduite pour le Glc-CA compare au CA, alors qu'elle restait la meme pour le Glc-EGCG compare a l'EGCG. Les derives glucosyles ne presentaient pas plus d'effet toxique sur les differentes cellules de peau. De plus, la toxicite etait meme fortement reduite pour l'acide cafeique suite a sa glucosylation. L'efficacite des composes glucosyles contre la production de ROS induite par les UV etait preservee, a la fois lors des traitements pre et post-UV. En particulier, une meilleure efficacite antioxydante a ete demontree avec le Glc-EGCG, vs. EGCG, sur les keratinocytes. Une augmentation des activites SOD et catalase a ete nettement observee pour le Glc-CA. Les deux polyphenols glucosyles montrent la meme activite que leur molecule parente en diminuant la production de facteurs anti-inflammatoires. Conclusions Nos resultats demontrent que la glucosylation enzymatique de CA et EGCG conduit a la preservation de leur activite antioxydante dans un modele cellulaire de vieillissement de la peau par les UV, en depit de la diminution de l'activite antioxydante instantanee observe pour Glc-CA. Glc-EGCC est specifiquement plus actif que l'EGCG sur les keratinocytes, suggerant un ciblage specifique. Ces antioxydants glucosyles presentant une amelioration de leurs proprietes physicochimiques et biologiques devraient etre de meilleurs candidats que les molecules naturelles pour etre utilises comme complements alimentaires ou en cosmetique.

International audience; This chapter describes a short historical overview of the progress in endothelium research and point the importance of organ-selective characteristics according to the present knowledge about endothelium biology. Uncovering the advantages that the endothelial cell properties and characteristics provide for the development of future targeted therapies, the review describes why mature endothelial cells due to their organ-specificity can be useful to target diseased organs. In the same line, endothelium properties will be exploited to make the endothelial cells a disease marker, e.g., in diabetes, stroke, cancer, inflammation, or ischemia and to provide a potential diagnostic indicator for the estimation of metastatic progression. New perspectives are thus opened by endothelial cells that can be considered both as a reporter and a target. These features can be combined with new cell-mediated and cell-targeted therapeutics designed to correct angiogenesis. Examples of such possible applications are detailed in the repair of tumor angiogenesis with help of endothelial cell precursors through their ability to target the pathologic angiogenesis and participate to normalization of the pathologic vasculature. The hypothesis that normalized angiogenesis may provide an efficient treatment, working as adjuvant to classical therapies, is being developed. The objective is to reach a mechanical stabilization that should result in an advantageous change of the tumor microenvironment.

International audience; Among innovative strategies developed for cancer treatments, gene therapies stand of great interest despite their well-known limitations in targeting, delivery, toxicity or stability. The success of any given gene-therapy is highly dependent on the carrier efficiency. New approaches are often revisiting the mythic trojan horse concept to carry therapeutic nucleic acid, i.e. DNAs, RNAs or small interfering RNAs, to pathologic tumor site. Recent investigations are focusing on engineering carrying modalities to overtake the above limitations bringing new promise to cancer patients. This review describes recent advances and perspectives for gene therapies devoted to tumor treatment, taking advantage of available knowledge in biotechnology and medicine.

International audience; nefficient immune response is a major glitch during tumor growth and progression. Chaotic and leaky blood vessels created in the process of angiogenesis allow tumor cells to escape and extricate anti-cancer immunity. Proangiogenic characteristics of hypoxic tumor microenvironment maintained by low oxygen tension attract endothelial progenitor cells, drive expansion of cancer stem cells, and deviantly differentiate monocyte descendants. Such cellular milieu further boosts immune tolerance and eventually appoint immunity for cancer advantage. Blood vessel normalization strategies that equilibrate oxygen levels within tumor and fix abnormal vasculature bring exciting promises to future anticancer therapies especially when combined with conventional chemotherapy. Recently, a new group of microRNAs (miRs) engaged in angiogenesis, called angiomiRs and hypoxamiRs, emerged as new therapeutic targets in cancer. Some of those miRs were found to efficiently regulate cancer immunity and their dysregulation efficiently programs aberrant angiogenesis and cancer metastasis. The present review highlights new findings in the field of miRs proficiency to normalize aberrant angiogenesis and to restore anti-tumor immune responses.

International audience; Tumor microenvironment is a complex and highly dynamic milieu that provides very important clues on tumor development and progression mechanisms. Tumor-associated endothelial cells play a key role in stroma organization. They achieve tumor angiogenesis, a formation of tumor-associated (angiogenic) vessels mainly through sprouting from locally preexisting vessels and/or recruitment of bone marrow-derived endothelial progenitor cells. This process participates to supply nutritional support and oxygen to the growing tumor. Endothelial cells constitute the interface between circulating blood cells, tumor cells and the extracellular matrix, thereby controlling leukocyte recruitment, tumor cell behavior and metastasis formation. Hypoxia, a critical parameter of the tumor microenvironment, controls endothelial/tumor cell interactions and is the key to tumor angiogenesis development. Under hypoxic stress, tumor cells produce factors that promote angiogenesis, vasculogenesis, tumor cell motility, metastasis and cancer stem cell selection. Targeting tumor vessels is a therapeutic strategy that has lately been fast evolving from antiangiogenesis to vessel normalization as discussed in this review. We shall focus on the pivotal role of endothelial cells within the tumor microenvironment, the specific features and the part played by circulating endothelial precursors cells. Attention is stressed on their recruitment to the tumor site and their role in tumor angiogenesis where they are submitted to miRNAs-mediated de/regulation. Here the compensation of the tumor deregulated angiogenic miRNAs - angiomiRs - is emphasized as a potential therapeutic approach. The strategy is to over express anti-angiomiRs in the tumor angiogenesis site upon selective delivery by precursor endothelial cells as miRs carriers.

Oxygen supply and diffusion into tissues are necessary for survival. The oxygen partial pressure (pO(2)), which is a key component of the physiological state of an organ, results from the balance between oxygen delivery and its consumption. In mammals, oxygen is transported by red blood cells circulating in a well-organized vasculature. Oxygen delivery is dependent on the metabolic requirements and functional status of each organ. Consequently, in a physiological condition, organ and tissue are characterized by their own unique 'tissue normoxia' or 'physioxia' status. Tissue oxygenation is severely disturbed during pathological conditions such as cancer, diabetes, coronary heart disease, stroke, etc., which are associated with decrease in pO(2), i.e. 'hypoxia'. In this review, we present an array of methods currently used for assessing tissue oxygenation. We show that hypoxia is marked during tumour development and has strong consequences for oxygenation and its influence upon chemotherapy efficiency. Then we compare this to physiological pO(2) values of human organs. Finally we evaluate consequences of physioxia on cell activity and its molecular modulations. More importantly we emphasize the discrepancy between in vivo and in vitro tissue and cells oxygen status which can have detrimental effects on experimental outcome. It appears that the values corresponding to the physioxia are ranging between 11% and 1% O(2) whereas current in vitro experimentations are usually performed in 19.95% O(2), an artificial context as far as oxygen balance is concerned. It is important to realize that most of the experiments performed in so-called normoxia might be dangerously misleading.

International audience; Endothelial progenitor cells (EPCs) modulate postnatal vascularization and contribute to vessel regeneration in adults. Stem cells and progenitor cells were found in umbilical cord blood, bone marrow, and mobilized peripheral blood cells, from where they were isolated and cultured. However, the yield of progenitor cells is usually not sufficient for clinical application and the quality of progenitor cells varies. The aim of the study was the immortalization of early progenitor cells with high proliferative potential, capable to differentiate to EPCs and, further, toward endothelial cells. Two cell lines, namely HEPC-CB.1 and HEPC-CB.2 (human endothelial progenitor cells-cord blood) were isolated. As assessed by specific antibody labeling and flow cytometric analysis, they express a panel of stem cell markers: CD133, CD13, CD271, CD90 and also endothelial cell markers: CD202b, CD309 (VEGFR2), CD146, CD105, and CD143 but they do not present markers of finally differentiated endothelial cells: CD31, vWf, nor CD45 which is a specific hematopoietic cell marker. Using the multiplex Cytometric Bead Assay, the simultaneous production of proangiogenic cytokines IL8, angiogenin, and VEGF was demonstrated in normoxia and was shown to be increased by hypoxia. Both cell lines, similarly as mature endothelial cells, underwent in vitro pre-angiogenic process, formed pseudovessel structures and present an accelerated angiogenesis in hypoxic conditions. To date, these are the first CD133 positive established cell lines from human cord blood cells.

International audience; Serotonin (5HT) was shown to induce in vitro the production of ROS in the presence of neuronal nitric oxide synthase (nNOS) in addition to the basal NO(+) formation. With the aim of understanding this mechanism, this study investigated the potential binding of 5HT to nNOS. By using [(3)H]5HT, it is reported here that 5HT binds to nNOS, but only when the enzyme is active and in a superoxide-dependent manner. This binding is prevented by DPI but not by L-NAME. The formation of 5HT-nNOS complex was shown to be very well correlated with the production of ROS by 5HT in the presence of nNOS. A mechanism involving nNOS only in its initial step is proposed to explain both the formation of 5HT-nNOS complex and the production of ROS observed in the presence of nNOS and 5HT.

International audience; Acetyl-N-SerAspLysPro (AcSDKP), known as a negative regulator of haematopoiesis, has been principally reported as an inhibitor of haematopoietic pluripotent stem cell proliferation. The tetrapeptide sequence is identical to the N-terminus of thymosin beta 4 (T beta 4), from which it has been suggested that it may be derived. Recently, evidence was shown that T beta 4 plays a role as a negative regulator of actin polymerization leading to the sequestration of its monomeric form. The structural similarity between the N-terminus of T beta 4 and AcSDKP has raised the possibility that AcSDKP may also participate in intracellular events leading to actin sequestration. The effect of T beta 4 on the proliferation of haematopoietic cells was compared to that of AcSDKP. The results revealed that T beta 4, like AcSDKP, exerts an inhibitory effect on the entry of murine primitive bone marrow cells into cell cycle in vitro. Qualitative electrophoretic analysis and quantitative polymerization assays were used to investigate the role of AcSDKP in actin polymerization. AcSDKP does not affect actin assembly at concentrations up to 50 microM, and does not compete with T beta 4 for binding to G-actin. These results suggest that AcSDKP is not involved in cell cycle regulation via an effect on the process of actin polymerization.

International audience; The comparative degradation of N-Ac-Ser-Asp-Lys-Pro (AcSDKP), a negative regulator controlling the proliferation of the hematopoietic pluripotent stem cell, was investigated following incubation with plasma, bone marrow and spleen cells from normal mice and mice bearing a transplantable myeloid leukemia. Using the tetrapeptide, specifically radiolabelled in the lysyl residue, degradation of [3H]AcSDKP was followed by measurement of [3H]Lys formation resulting from its catabolism. It was shown that already after 1 h the degradation of AcSDKP in plasma from leukemic mice was higher compared to that following incubation in plasma from normal mice, whereas incubation with bone marrow cells exhibits a small difference only after 4 hours incubation. However, no increase of AcSDKP catabolic activity was observed following incubation with spleen cells from leukemic animals when compared with incubation of normal spleen cells.

International audience; Surface lectins, specific for given sugar structures, are expressed on human T cells, as shown by flow cytofluorometry using F-neoglycoproteins bearing either beta- and alpha-D-galactosyl, beta-D-galactosyl 6-phosphate, or alpha-L-rhamnosyl groups, but not by F-neoglycoproteins bearing other sugar groups (such as alpha-D-mannosyl groups). After stimulation with Phaseolus vulgaris mitogen, the number of cells that bind beta-D-galactosyl 6-phosphate groups (6-P-beta-D-Galp lec+ cells) increased fourfold during the first five days; these cells are helper (CD4+) T cells. Conversely, cells that bind alpha-L-Rha groups belong to the T suppressor (CD8+) family and their number moderately increased. Upon stimulation by concanavalin A, the number of cells expressing the lectin recognizing alpha-L-Rha groups increased during the first two days and then decreased within the next two days. These results are discussed with regard to the implication of lymphocyte membrane lectins in the suppressor mechanism and in the homing process.

International audience; In an attempt to identify cell surface molecules involved in recognition phenomena between cells such as keratinocytes and melanocytes and putatively target biological responses modifiers to keratinocytes, we undertook the detection of cell surface sugar specific receptors: membrane lectins. Keratinocyte membrane lectins were found to bind synthetic glycoproteins (neoglycoproteins) carrying either alpha-L-fucosyl or alpha-L-rhamnosyl residues. Fluorescence microscopy observations indicate that cultured keratinocytes are able to bind these two neoglycoproteins while frozen sections of human skin labelled with neoglycoprotein-coated covaspheres show that the selectivity of the binding to keratinocytes is restricted to alpha-L-rhamnosyl-BSA. Keratinocytes were adapted to grow on collagen; harvesting conditions allowing the analysis of keratinocytes by flow cytometry are described. This technique allows the quantification of the binding at 4 degrees C, and the estimation of the endocytosis of F-, neoglycoproteins: F-, alpha-L-Rha-BSA and F-, alpha-L-Fuc-BSA were efficiently internalized. Thereafter, alpha-L-rhamnose-substituted liposomes containing 5-(6)carboxyfluorescein were prepared in order to follow the delivery of the fluorescent dye into cells. This was measured both by flow cytometry and by spectrofluorimetry. The expression of surface lectins was checked upon action of cytokines (IL1 alpha, IL1 beta, IL2 and TNF) which are known as biological response modifiers of keratinocytes.

International audience; The demonstration that AcSDKP, a new regulator of the hematopoietic system, is formed in the bone marrow by a one-step enzymatic maturation processing of thymosin beta 4 (T beta 4) is presented. AcSDKP and T beta 4 were both detected in bone marrow cells (BMC). Incubation of [3H]T beta 4 with either intact or lysed BMC led to the formation of [3H]AcSDKP, whereas the labeled tetrapeptide was not degraded under these conditions. Model enzymatic degradation of T beta 4 carried out with bacterial enzymes suggests that a mammalian endoproteinase Asp-N might be involved in the formation of AcSDKP through the specific cleavage of the Pro4-Asp5 peptidic bond of T beta 4. In contrast, alpha-prolyl-endopeptidase was ineffective in carrying out a similar processing.

International audience; It is shown that AcSDKP a new regulator of the hematopoietic system can be generated from thymosin beta 4 by a one-step enzymatic cleavage in vitro and in vivo. AcSDKP and T beta 4 were both detected in bone marrow cells (BMC). Incubation of [3H]T beta 4 with either intact or lysed BMC led to the formation of [3H]AcSDKP whereas the labelled tetrapeptide was not degraded under these conditions. Model enzymatic degradation of T beta 4 carried out with bacterial enzymes suggests that a mammalian endoproteinase Asp-N might be involved in the formation of AcSDKP through the specific cleavage of the 4Pro-5 Asp peptide bond of T beta 4.

Melanoma is the rarest skin cancer, but one that causes the most of deaths. The critical step of itsprogression is angiogenesis, a physiological tumor-activated process which allows the delivery ofoxygen and nutrients. Tumor cells may then metastasize to the lymph nodes, in particular. Moreover,the tumor inhibits the host immune responses toward itself. All these mechanisms are regulated bynitric oxide (NO ). The aim of the project was to deepen the role of NO in angiogenesis andleukocyte recruitment, two mechanisms based on endothelial cell recognition.We have shown here that NO is necessary for angiogenesis, but that high concentrations are antiangiogenic.This inhibitory effect of NO can be attributed to the decrease of cell-cell interactions andthe inhibition of the PECAM-1/CD31 expression, mainly.Besides, we have demonstrated that leukocyte adhesion on endothelium is inhibited by NO . This canbe explained by the modulation of the expression of molecules able to bind to chemokines:glycosaminoglycans and chemokine receptors, as well as by the down-regulation of the adhesionmolecules CD34 and ICAM-2/CD102 VCAM-1/CD106.In conclusion, NO is able to regulate the main cellular mechanisms of tumor progression bymodulating the expression of surface molecules on endothelial cells. Throughout this work, we haveobserved that these modulations depend on NO concentrations and on the cell type, demonstratingthe pivotal role of NO in cancer progression.; Le mélanome est le cancer de la peau le plus rare, mais celui qui cause le plus de mortalité. L’étapecritique de sa progression est l’angiogenèse, processus détourné par la tumeur pour s’oxygéner et senourrir. Les cellules tumorales peuvent alors former des métastases dans les ganglions lymphatiques,notamment. D’autre part, la tumeur inhibe les réponses immunitaires de l’hôte à son encontre. Tousces mécanismes font intervenir le monoxyde d’azote (NO ). Le but de ce projet a été d’approfondir lerôle du NO dans l’angiogenèse et le recrutement leucocytaire, mécanismes basés sur lareconnaissance cellulaire de l’endothélium.Nous avons montré que le NO est nécessaire à l’angiogenèse, mais que des doses plus fortes sontanti-angiogéniques. Cet effet inhibiteur peut s’expliquer par la diminution des interactions cellules-cellules,et l’inhibition de l’expression de PECAM-1/CD31, principalement.D’autre part, nous avons établi que l’adhésion des leucocytes est inhibée par le NO ce qui a été reliéà la modulation de l’expression des molécules capables de fixer des chimiokines : lesglycosaminoglycannes et les récepteurs de chimiokines, ainsi qu’à la sous-expression des moléculesd’adhésion CD34, ICAM-2/CD102 et VCAM-1/CD106.En conclusion, le NO est capable de réguler des mécanismes cellulaires majeurs de la progressiontumorale, par modulation de l’expression des molécules de surface de l’endothélium. Tout au long dece travail, nous avons observé que les différences de réponse sont dépendantes des doses de NO etdu type cellulaire, ce qui démontre le rôle pivot du NO dans la progression du cancer.