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PDF - 3176KB, Characterization of B16F10 wt and msVEGFR2 expressing clones 13.3 and 16.4 tumors in vivo.

PDF - 86KB, Construction maps for the pIFP1.4-HREmsVEGFR2 and pHREmsVEGFR2 vector

PDF - 2157KB, Characterization of the transfected clones for msVEGFR2 and mVEGF-A production.

PDF - 1037KB, Effect of the IFP1.4 and mCherry expression on the B16F10 cells proliferation.

PDF - 2705KB, Expression of VEGFRs on the surface of B16F10-msVEGFR2 melanoma clone 13.3 and 16.4.

PDF - 150KB, Supplementary Materials and Methods and legends for Supplementary Figures 1 through 6.

International audience; Pathologic angiogenesis directly responds to tumour hypoxia and controls the molecular/cellular composition of the tumour microenvironment, increasing both immune tolerance and stromal cooperation with tumour growth. Myo-inositol-trispyrophosphate (ITPP) provides a means to achieve stable normalization of angiogenesis. ITPP increases intratumour oxygen tension (pO2) and stabilizes vessel normalization through activation of endothelial Phosphatase-and-Tensin-homologue (PTEN). Here, we show that the tumour reduction due to the ITPP-induced modification of the tumour microenvironment by elevating pO2 affects the phenotype and properties of the immune infiltrate. Our main observations are as follows: a relative change in the M1 and M2 macrophage-type proportions, increased proportions of NK and CD8+T cells, and a reduction in Tregs and Th2 cells. We also found, in vivo and in vitro, that the impaired access of PD1+NK cells to tumour cells is due to their adhesion to PD-L1+/PD-L2+ endothelial cells in hypoxia. ITPP treatment strongly reduced PD-L1/PD-L2 expression on CD45+/CD31+ cells, and PD1+ cells were more numerous in the tumour mass. CTLA-4+ cell numbers were stable, but level of expression decreased. Similarly, CD47+ cells and expression were reduced. Consequently, angiogenesis normalization induced by ITPP is the mean to revert immunosuppression into an antitumor immune response. This brings a key adjuvant effect to improve the efficacy of chemo/radio/immunotherapeutic strategies for cancer treatment.

The human HEPC-CB.1 cell line with many characteristics of endothelial progenitor cells (EPC) was tested for its proangiogenic properties as a potentially therapeutic compound. HEPC-CB.1 cells’ potential to differentiate into endothelial cells was revealed after treating the cells with a mixture of ATRA, cAMP and VEGF, as shown by the reduced expression levels of CD133, CD271 and CD90 antigens, augmentation of CD146 and CD31, and a decrease in cell clonogenicity. The cooperation of HEPC-CB.1 with the endothelial cell line HSkMEC.2 resulted in the formation of a common network. Tube formation was significantly more effective when resulting from HEPC-CB.1 and HSkMEC.2 cell co-culture as compared to a monoculture of each cell line. The exocrine mechanism of HEPC-CB.1 and HSkMEC.2 cross talk by secreted factors was evidenced using the HEPC-CB.1 supernatant to increase the efficacy of HSkMEC.2 tube formation. The proangiogenic factors produced by HEPC-CB.1 were identified using cytokine antibody array. Out of 120 examined factors, the HEPC-CB.1 cell line produced 63, some with known angiogenic activity. As in vivo the angiogenic process occurs at low oxygen tension, it was observed that in hypoxia, the production of defined factors was augmented. The presented results demonstrate that HEPC-CB.1 cells are able to both cooperate and integrate in a newly formed network and produce factors that help the network formation. The results suggest that HEPC-CB.1 cells are indeed endothelial progenitors and may prove to be an effective tool in regenerative medicine. Electronic supplementary material The online version of this article (10.1007/s11033-020-05662-6) contains supplementary material, which is available to authorized users.

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.

The skin constitutes with its microbiota the first line of body defense against exogenous stress including air pollution. Especially in urban or sub-urban areas, it is continuously exposed to many environmental pollutants including gaseous nitrogen dioxide (gNO2). Nowadays, it is well established that air pollution has major effects on the human skin, inducing various diseases often associated with microbial dysbiosis. However, very few is known about the impact of pollutants on skin microbiota. In this study, a new approach was adopted, by considering the alteration of the cutaneous microbiota by air pollutants as an indirect action of the harmful molecules on the skin. The effects of gNO2 on this bacterial skin microbiota was investigated using a device developed to mimic the real-life contact of the gNO2 with bacteria on the surface of the skin. Five strains of human skin commensal bacteria were considered, namely Staphylococcus aureus MFP03, Staphylococcus epidermidis MFP04, Staphylococcus capitis MFP08, Pseudomonas fluorescens MFP05, and Corynebacterium tuberculostearicum CIP102622. Bacteria were exposed to high concentration of gNO2 (10 or 80 ppm) over a short period of 2 h inside the gas exposure device. The physiological, morphological, and molecular responses of the bacteria after the gas exposure were assessed and compared between the different strains and the two gNO2 concentrations. A highly significant deleterious effect of gNO2 was highlighted, particularly for S. capitis MFP08 and C. tuberculostearicum CIP102622, while S. aureus MFP03 seems to be the less sensitive strain. It appeared that the impact of this nitrosative stress differs according to the bacterial species and the gNO2 concentration. Thus the exposition to gNO2 as an air pollutant could contribute to dysbiosis, which would affect skin homeostasis. The response of the microbiota to the nitrosative stress could be involved in some pathologies such as atopic dermatitis.

International audience; Microvascular endothelial cells constitute potential targets for exogenous microorganisms, in particular for vector-borne pathogens. Their phenotypic and functional variations according to the organs they are coming from provide an explanation of the organ selectivity expressed in vivo by pathogens. In order to make available relevant tools for in vitro studies of infection mechanisms, our aim was to immortalize bovine organospecific endothelial cells but also to assess their permissivity to viral infection. Using transfection with SV40 large T antigen, six bovine microvascular endothelial cell lines from various organs and one macrovascular cell line from an umbilical cord were established. They display their own panel of endothelial progenitor/mature markers, as assessed by flow cytometry and RT-qPCR, as well as the typical angiogenesis capacity. Using both Bluetongue and foot-and-mouth disease viruses, we demonstrate that some cell lines are preferentially infected. In addition, they can be transfected and are able to express viral proteins such as BTV8-NS3. Such microvascular endothelial cell lines bring innovative tools for in vitro studies of infection by viruses or bacteria, allowing for the study of host-pathogen interaction mechanisms with the actual in vivo target cells. They are also suitable for applications linked to microvascularization, such as anti-angiogenic and anti-tumor research, growing fields in veterinary medicine.

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

In the perfect world—an ideal laboratory grown vascular graft must be composed of viable tissue elements with potentials for repair and regeneration, an acceptable range of resistance to thrombosis—infections, sensitivity to vasoactive mediators, secretory and contractile properties, mimicking natural blood vessels. Three key components of generating such custom-made vessels are biocompatible polymer of natural structural proteins for building a scaffold, healthy primary vascular cells grown in the scaffold maintaining close anatomical and functional integrity, and provision for a microenvironment for providing balanced metabolic support. In addition, maintenance of a pulsatile flow of nutrients and polarization of cellular components in the synthetic vessels are also important. Obtaining decellularized vascular grafts from cadaveric donors or xenografts, populating with recipient’s primary vascular cells, and maintaining them in bioreactors or recipients own body tissue are other potential alternative approaches. Here, we will discuss some central aspects and updated information on authentic artificial blood vessel.

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.

Heparanase is overexpressed by tumor cells and degrades the extracellular matrix proteoglycans through cleavage of heparan sulfates (HS), allowing pro-angiogenic factor release and thus playing a key role in tumor angiogenesis and metastasis. Here we propose new HS analogs as potent heparanase inhibitors: Heparin as a positive control, Dextran Sulfate, λ-Carrageenan, and modified forms of them obtained by depolymerization associated to glycol splitting (RD-GS). After heparanase activity assessment, 11 kDa RD-GS-λ-Carrageenan emerged as the most effective heparanase inhibitor with an IC50 of 7.32 ng/mL compared to 10.7 ng/mL for the 16 kDa unfractionated heparin. The fractionated polysaccharides were then tested in a heparanase-rich medium-based in vitro model, mimicking tumor microenvironment, to determine their effect on microvascular endothelial cells (HSkMEC) angiogenesis. As a preliminary study, we identified that under hypoxic and nutrient poor conditions, MCF-7 cancer cells released much more mature heparanase in their supernatant than in normal conditions. Then a MatrigelTM assay using HSkMEC cultured under hypoxic conditions in the presence (or not) of this heparanase-rich supernatant was realized. Adding heparanase-rich media strongly enhanced angiogenic network formation with a production of twice more pseudo-vessels than with the control. When sulfated polysaccharides were tested in this angiogenesis assay, RD-GS-λ-Carrageenan was identified as a promising anti-angiogenic agent.

International audience; Active cellular transporters of harmful agents-multidrug resistance (mdr) proteins-are present in tumor, stem and endothelial cells, among others. While mdr proteins are broadly studied in tumor cells, their role in non-tumor cells and the significance of their action not connected with removal of harmful xenobiotics is less extensively documented. Proper assessment of mdr proteins expression is difficult. Mdr mRNA presence is most often evaluated but that does not necessarily correlate with the protein level. The protein expression itself is difficult to determine; usually cells with mdr overexpression are studied, not cells under physiological conditions, in which a low expression level of mdr protein is often insufficient for detection in vitro. Various methods are used to identify mdr mRNA and protein expression, together with functional tests demonstrating their biological drug transporting activities. Data comparing different methods of investigating expression of mdr mRNAs and their corresponding proteins are still scarce. In this article we present the results of a study concerning mdr mRNA and protein expression. Our goal was to search for the best method to investigate the expression level and functional activity of five selected mdr proteins-MDR1, BCRP, MRP1, MRP4 and MRP5-in established in vitro cell lines of human endothelial cells (ECs) and their progenitors. Endothelial cells demonstrated mdr presence at the mRNA level, which was not always confirmed at the protein level or in functional tests. Therefore, several different assays had to be applied for evaluation of mdr proteins expression and functions in endothelial cells. Among them functional tests seemed to be the most conclusive, although not very specific.

Tumour microenvironment determines the fate of treatments. Reconstitution of tumour conditions is mandatory for alternative in vitro methods devoted to cancer development and the selection of therapeutic strategies. This work describes a 3D model of melanoma growth in its environment. Introducing means to mimic tumour angiogenesis, which turns on tumour progression, the model shows that melanoma tumour spheroids allow reconstitution of solid tumours with stromal cells. Angiogenesis evidenced the differential recruitment of endothelial cells (EC) from early progenitors (EEPCs) to mature ECs. Hypoxia was the key parameter that selected and stabilized melanoma cancer stem like cells (CSCs) phenotype based on aldehyde dehydrogenase expression as the best criterion. The 3D-tumour-model demonstrated the distinct reactivity of ECs toward tumour cells in terms of cellular cross-talk and humoral response. Intra-spheroid cell-to-cell membrane dye exchanges, mediated by intercellular interactions, uncovered the melanoma-to-EEPC cooperation. The resulting changes in tumour milieu were evidenced by the chemokinic composition and hypoxia-related variations in microRNA expression assessed in each cellular component of the spheroids. This method brings new tools to decipher the molecular mechanism of tumour-mediated cell recruitment and for in vitro assessment of therapeutic approaches.

National audience

VEGFs are found at high levels in hypoxic tumors. As major components directing pathologic neovascularization, they regulate stromal reactions. Consequently, novel strategies targeting and inhibiting VEGF overproduction upon hypoxia offer considerable potential for modern anticancer therapies controlling rather than destroying tumor angiogenesis. Here, we report the design of a vector expressing the soluble form of VEGF receptor-2 (sVEGFR2) driven by a hypoxia-responsive element (HRE)-regulated promoter. To enable in vivo imaging by infrared visualization, mCherry and IFP1.4 coding sequences were built into the vector. Plasmid construction was validated through transfection into embryonic human kidney HEK293 and murine B16F10 melanoma cells. sVEGFR2 was expressed in hypoxic conditions only, confirming that the gene was regulated by the HRE promoter. sVEGFR2 was found to bind efficiently and specifically to murine and human VEGF-A, reducing the growth of tumor and endothelial cells as well as impacting angiogenesis in vitro. The hypoxia-conditioned sVEGFR2 expression was shown to be functional in vivo: Tumor angiogenesis was inhibited and, on stable transfection of B16F10 melanoma cells, tumor growth was reduced. Enhanced expression of sVEGFR2 was accompanied by a modulation in levels of VEGF-A. The resulting balance reflected the effect on tumor growth and on control of angiogenesis. A concomitant increase of intratumor oxygen tension also suggested an influence on vessel normalization. The possibility to express an angiogenesis regulator as sVEGFR2, in a hypoxia-conditioned manner, significantly opens new strategies for tumor vessel–controlled normalization and the design of adjuvants for combined cancer therapies. Mol Cancer Ther; 13(1); 165–78. ©2013 AACR.

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.

Podoplanin (PDPN), an O-glycosylated, transmembrane, mucin-type glycoprotein, is expressed by cancer associated fibroblasts (CAFs). In malignant transformation, PDPN is subjected to changes and its role is yet to be established. Here we show that it is involved in modulating the activity of the CCL21/CCR7 chemokine/receptor axis in a hypoxia-dependent manner. In the present model, breast cancer MDA-MB-231 cells and NKL3 cells express the surface CCR7 receptor for CCL21 chemokine which is a potent chemoattractant able to bind to PDPN. The impact of the CCL21/CCR7 axis in the molecular mechanism of the adhesion of NKL3 cells and of MDA-MB-231 breast cancer cells was reduced in a hypoxic tumor environment. In addition to its known effect on migration, CCL21/CCR7 interaction was shown to allow NK cell adhesion to endothelial cells (ECs) and its reduction by hypoxia. A PDPN expressing model of CAFs made it possible to demonstrate the same CCL21/CCR7 axis involvement in the tumor cells to CAFs recognition mechanism through PDPN binding of CCL21. PDPN was induced by hypoxia and its overexpression undergoes a reduction of adhesion, making it an anti-adhesion molecule in the absence of CCL21, in the tumor. CCL21/CCR7 modulated NK cells/ECs and MDA-MB-231 cells/CAF PDPN-dependent interactions were further shown to be linked to hypoxia-dependent microRNAs as miRs: miR-210 and specifically miR-21, miR-29b which influence PDPN expression.

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.

International audience; The skin is a multifunctional organ and a first line of defense actively protecting from environmental stress caused by injury, microbial treat, UV irradiation and environmental toxins. Diverse cutaneous cell types together with extracellular matrix elements and factors create a dynamic scene for cellular communication crucial in vital processes such as wound healing, inflammation, angiogenesis, immune response. Direct functional success of skin equilibrium depends on its microenvironment settings and particularly the local oxygen tension. Indeed, skin entire milieu is characterized by and highly dependent on its low oxygen tension called physioxia as emphasized in this review. In the context of skin physioxia, we review and propose here new approaches to minimize age-related changes in skin state and function. We particularly emphasize carbohydrate-mediated interactions and new 3D models of engineered skin substitutes. We highlight newly emerged tools and targets including stem cells, miRNAs, matrix metalloproteinases, mitochondria and natural antioxidants that are promising in prevention of skin ageing and disease restraint. In the era of advanced dermatology, new attempts are bringing us closer to 'well being' perception.

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; The tetrapeptide N‐Acetyl‐Ser‐Asp‐Lys‐Pro (AcSDKP), an inhibitor of haematopoietic stem cell proliferation, reduces in vivo and in vitro the damage to the stem cell compartment resulting from treatment with chemotherapeutic agents or ionizing radiations. In order to provide new molecules likely to improve the myeloprotection displayed by this tetrapeptide, we have prepared a set of analogues of AcSDKP. These compounds are derived from the parent peptide by substitution or modification of the N‐ or of the C‐terminus, or substitution of side chains. We report here that almost all investigated analogues retain the antiproliferative activity reducing in vitro the proportion of murine Colony‐Forming Units Granulocyte/Macrophage (CFU‐GM) in S‐phase and inhibiting the entry into cycle of High Proliferative Potential Colony‐Forming Cells (HPP‐CFC). This shows that the polar groups of Ser, Asp or Lys are critical for the expression of biological activity, but that the modification of the N‐ or C‐terminus mostly yielded compounds still retaining antiproliferative activity and devoid of toxicity. The efficacy of AcSDKP analogues in preventing in vitro the primitive haematopoietic cells from entering into cycle makes these molecules new candidates for further in vivo investigations.

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.

The tetrapeptide Acetyl-N-Ser-Asp-Lys-Pro (AcSDKP or Goralatide), a physiological regulator of hematopoiesis, inhibits the entry into the S-phase of murine and human hematopoietic stem cells. It has been shown to reduce the damage to specific compartments in the bone marrow resulting from treatment with chemotherapeutic agents, ionizing radiations, hyperthermy, or phototherapy. The present study was performed to assess the therapeutic potential of AcSDKP in vivo in reducing both the toxicity and the hematopoietic damage induced by fractionated administration of doxorubicin (DOX), a widely used anticancer drug. Here we showed that AcSDKP could reduce DOX-induced mortality in mice and could protect particularly the long-term reconstituting cells (LTRCs) in addition to colony forming units-spleen, high proliferative potential colony-forming cells, and colony-forming units–granulocyte-macrophage (CFU-GM) from DOX toxicity. The protection against DOX-induced mortality in mice was improved when AcSDKP was administered for 3 days, at a dose of 2.4 μg/d, by continuous subcutaneous (SC) infusion or fractionated SC injections starting 48 hours before DOX treatment. Moreover, the recovery of the CFU-GM population in the AcSDKP-DOX–treated mice was optimized by the subsequent administration of granulocyte colony-stimulating factor (G-CSF). The coadministration of AcSDKP with DOX may improve its therapeutic index by reducing both acute hematotoxicity on late stem cells and progenitors and long-term toxicity on LTRCs. Optimization of these treatments combined with G-CSF may provide an additional approach to facilitate hematopoietic recovery after cancer chemotherapy.

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.