Role of Staphylococcus aureus surface proteins in experimental endocarditis

Résumé Le staphylocoque doré est un pathogène responsable d'une grande variété de maladies chez l'être humain. Il est extrêmement bien équipé de facteurs de virulence, dont les adhésines. Jusqu'à présent, 21 protéines liant des composants de tissus de l'hôte ("microbial surface components reacting with adherence matrix molecules, MSCRAMM") ont été identifiées, par exemple le "clumping factor" A (CIfA) ou la "fibronectin-binding protein" A (FnBPA). Néanmoins, pour la plupart de ces protéines, leur rôle dans la pathogénie des infections à staphylocoque doré reste à être élucidé. Le but de cette thèse est de contribuer à ce processus. Premièrement, les "MSCRAMM" CIfA, CIfB, FnBPA, FnBPB, Cna, SpA, Pls, SdrC, SdrD, SdrE, SasD, SasE, SasF, SasG, Sasl, SasJ et SasK ont été exprimés dans une bactérie substitut, Lactococcus lactis, et testés pour leurs propriétés adhésives et leur pathogénicité dans un modèle d'endocardite expérimentale (voir chapitre 1). Cette technique a préalablement été utilisée avec succès et a l'avantage d'éviter le contexte complexe des redondances et systèmes de régulations propres au staphylocoque doré. Les résultats montrent que, de tous les facteurs de virulence testés, seuls CIfA et FnBPA sont d'importance primordiale dans le développement d'endocardite expérimentale. En ce qui concerne l'internalisation dans les cellules endothéliales, seulement FnPBA et FnBPB en sont capables. En outre, l'adhérence à chacun des ligands testés (fibrinogène, fibronectine, kératine, élastine, collagène, et les caillots de fibrine et plaquettes) est très spécifique et est médiée par une ou plusieures adhésines provenant du staphylocoque doré. Par conséquence, ces protéines pourraient représenter des cibles potentielles pour de futures thérapies anti-adhésives contre le staphylocoque doré. Deuxièmement, l'expression des facteurs de virulence décrits dans le chapitre 1 par les souches recombinantes de lactocoques a été vérifiée par une nouvelle méthode utilisant la spectrométrie de masse (voir chapitre 2). L'expression de toutes ces protéines par les souches recombinantes a pu être confirmée. Cette méthode pourrait être de grande valeur dans la vérification de la présence de protéines quelconques dans toutes sortes d'applications. Troisièmement, deux facteurs de virulence du staphylocoque, CIfA et une forme tronquée de FnBPA, ont été exprimés de façon simultanée dans une souche recombinante de lactocoque (voir chapitre 3}. Contrairement à une souche exprimant la FnBPA entière, une souche exprimant la forme tronquée de FnPBA, qui ne contient plus le domaine capable de lier le fibrinogène, perd complètement sa capacité d'infecter dans le modèle d'endocardite expérimentale. Par contre, il est montré que, en cas de complémentation de la forme tronquée de FnPBA avec le domaine de liaison au fibrinogène de CIfA dans la souche double recombinante, le phénotype intégral de FnBPA est récupéré. En conséquence, les facteurs de virulence sont capables de coopérer dans le but de la pathogénie des infections à staphylocoque doré. Summary Staphylococcus aureus is a human pathogen causing a wide variety of disease. It is extremely well equipped with both secreted and surface-attached virulence factors, which can act as adhesins to host tissues. In total, twenty-one microbial surface components reacting with adherence matrix molecules (MSCRAMMs) have been identified, so far. These include well-characterized adhesins such as clumping factor A (CIfA) or fibronectin-binding protein A (FnBPA). However, for most of them their potential role in the pathogenesis of staphylococcal infections remains to be elucidated. This has been attempted in this thesis work. Firstly, the staphylococcal MSCRAMMs CIfA, CIfB, FnBPA, FnBPB, Cna, SpA, Pls, SdrC, SdrD, SdrE, SasD, SasE, SasF, SasG, Sasl, SasJ, and SasK have been expressed in a surrogate bacterium, Lactococcus lactis, and tested for their in vitro adherence properties and their pathogenicity in the rat model of experimental endocarditis (see chapter 1). This model has successfully been used previously, and has the advantage of bypassing the complex S. aureus background of redundancies and differential regulation. Here, it is shown that of the seventeen tested potential virulence factors, only CIfA and FnBPA are critical for the pathogenesis of experimental endocarditis in rats, while internalization into bovine endothelial cells is mediated exclusively by FnBPA and FnBPB. In addition, the adherence to specific host ligands (fibrinogen, fibronectin, keratin, elastin, collagen, and fibrin-platelet clots) is highly specific and mediated by one or few staphylococcal adhesins, respectively. Thus, these surface proteins may represent potential targets for an anti-adhesive strategy against S. aureus infections. Secondly, the expression of the staphylococcal proteins by L. lactis recombinants described in chapter 1 was tested by a novel method using mass spectrometry (see chapter 2). The expression of all the staphylococcal proteins by the respective recombinant lactococcal strain could be confirmed. This method may prove to be of great value in the confirmation of the presence of any given protein in various experimental settings. Thirdly, two staphylococcal virulence factors, CIfA and a truncated form of FnBPA, were expressed simultaneously in one recombinant lactococcal strain (see chapter 3). In contrast to a recombinant strain expressing full-length FnPBA, a recombinant strain expressing a truncated FnPBA, lacking the domain capable of binding fibrinogen, completely lost infectivity in experimental endocarditis. However, it is shown that the complementation of the truncated form of FnBPA with the fibrinogenbinding domain of CIfA in a double recombinant strain results in the recovery of the complete phenotype of full-length FnBPA. Thus, virulence factors can cooperate in the pathogenesis of staphylococcal infections.

Photodynamic therapy induces selective extravasation of macromolecules: Insights using intravital microscopy.

Photodynamic therapy (PDT) with Visudyne acts by direct cellular phototoxicity and/or by an indirect vascular-mediated effect. Here, we demonstrate that the vessel integrity interruption by PDT can promote the extravasation of a macromolecular agent in normal tissue. To obtain extravasation in normal tissue PDT conditions were one order of magnitude more intensive than the ones in tissue containing neovessels reported in the literature. Fluorescein isothiocyanate dextran (FITC-D, 2000 kDa), a macromolecular agent, was intravenously injected 10 min before (LK0 group, n=14) or 2h (LK2 group, n=16) after Visudyne-mediated PDT in nude mice bearing a dorsal skin fold chamber. Control animals had no PDT (CTRL group, n=8). The extravasation of FITC-D from blood vessels in striated muscle tissue was observed in both groups in real-time for up to 2500 s after injection. We also monitored PDT-induced leukocyte rolling in vivo and assessed, by histology, the corresponding inflammatory reaction score in the dorsal skin fold chambers. In all animals, at the applied PDT conditions, FITC-D extravasation was significantly enhanced in the PDT-treated areas as compared to the surrounding non-treated areas (p<0.0001). There was no FITC-D leakage in the control animals. Animals from the LK0 group had significantly less FITC-D extravasation than those from the LK2 group (p=0.0002). In the LK0 group FITC-D leakage correlated significantly with the inflammation (p<0.001). At the selected conditions, Visudyne-mediated PDT promotes vascular leakage and FITC-D extravasation into the interstitial space of normal tissue. The intensity of vascular leakage depends on the time interval between PDT and FITC-D injection. This concept could be used to locally modulate the delivery of macromolecules in vivo.

Fate of Hydrophilic Nanoparticles in Biological Environments

The nanoparticles developed are based on chitosan, a biocompatible and biodegradable polysaccharide. The chitosan nanoparticles are formed in an entirely water-based process by electrostatic interactions with other biocompatible molecules. As a prerequisite to understand the fate of such nanoparticles in cells, comprehensive characterization and stability studies serve to identify quantitatively the impact of the raw material characteristics and preparation conditions on the nanoparticle characteristics. Methods included H-1 NMR spectroscopy, dilution viscometry, particle size analysis and electron microscopy. Cytotoxicity and cell uptake experiments on RAW 264.7 murine macrophages and p23 murine endothelial cells were performed to investigate the correlation with nanoparticle characteristics and effect of surface decoration with alginate. Cytotoxicity was assessed by the MTT survival test; cell uptake was monitored by fluorescent microscopy using labeled polymers.

Arginase II Promotes Macrophage Inflammatory Responses Through Mitochondrial Reactive Oxygen Species, Contributing to Insulin Resistance and Atherogenesis.

BACKGROUND: Macrophage-mediated chronic inflammation is mechanistically linked to insulin resistance and atherosclerosis. Although arginase I is considered antiinflammatory, the role of arginase II (Arg-II) in macrophage function remains elusive. This study characterizes the role of Arg-II in macrophage inflammatory responses and its impact on obesity-linked type II diabetes mellitus and atherosclerosis. METHODS AND RESULTS: In human monocytes, silencing Arg-II decreases the monocytes' adhesion to endothelial cells and their production of proinflammatory mediators stimulated by oxidized low-density lipoprotein or lipopolysaccharides, as evaluated by real-time quantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. Macrophages differentiated from bone marrow cells of Arg-II-deficient (Arg-II(-/-)) mice express lower levels of lipopolysaccharide-induced proinflammatory mediators than do macrophages of wild-type mice. Importantly, reintroducing Arg-II cDNA into Arg-II(-/-) macrophages restores the inflammatory responses, with concomitant enhancement of mitochondrial reactive oxygen species. Scavenging of reactive oxygen species by N-acetylcysteine prevents the Arg-II-mediated inflammatory responses. Moreover, high-fat diet-induced infiltration of macrophages in various organs and expression of proinflammatory cytokines in adipose tissue are blunted in Arg-II(-/-) mice. Accordingly, Arg-II(-/-) mice reveal lower fasting blood glucose and improved glucose tolerance and insulin sensitivity. Furthermore, apolipoprotein E (ApoE)-deficient mice with Arg-II deficiency (ApoE(-/-)Arg-II(-/-)) display reduced lesion size with characteristics of stable plaques, such as decreased macrophage inflammation and necrotic core. In vivo adoptive transfer experiments reveal that fewer donor ApoE(-/-)Arg-II(-/-) than ApoE(-/-)Arg-II(+/+) monocytes infiltrate into the plaque of ApoE(-/-)Arg-II(+/+) mice. Conversely, recipient ApoE(-/-)Arg-II(-/-) mice accumulate fewer donor monocytes than do recipient ApoE(-/-)Arg-II(+/+) animals. CONCLUSIONS: Arg-II promotes macrophage proinflammatory responses through mitochondrial reactive oxygen species, contributing to insulin resistance and atherogenesis. Targeting Arg-II represents a potential therapeutic strategy in type II diabetes mellitus and atherosclerosis. (J Am Heart Assoc. 2012;1:e000992 doi: 10.1161/JAHA.112.000992.).

On the retinal toxicity of intraocular glucocorticoids.

Corticosteroids are hormones involved in many physiological responses such as stress, immune modulation, protein catabolism and water homeostasis. The subfamily of glucocorticoids is used systemically in the treatment of inflammatory diseases or allergic reactions. In the eye, glucocorticoides are used to treat macular edema, inflammation and neovascularization. The most commonly used glucocorticoid is triamcinolone acetonide (TA). The pharmaceutical formulation of TA is not adapted for intravitreal administration but has been selected by ophthalmologists because its very low intraocular solubility provides sustained effect. Visual benefits of intraocular TA do not clearly correlate with morpho-anatomical improvements, suggesting potential toxicity. We therefore studied, non-common, but deleterious effects of glucocorticoids on the retina. We found that the intravitreal administration of TA is beneficial in the treatment of neovascularization because it triggers cell death of endothelial cells of neovessels by a caspase-independent mechanism. However, this treatment is toxic for the retina because it induces a non-apoptotic, caspase-independent cell death related to paraptosis, mostly in the retinal pigmented epithelium cells and the Müller cells.

Pathogenesis of streptococcal and staphylococcal endocarditis.

Although streptococcal and S. aureus IE share the same primary site of infection, their pathogenesis and clinical evolution present several major differences. Streptococci adhere to cardiac valves with pre-existing endothelial lesions. In contrast, S. aureus can colonize either damaged endothelium or invade physically intact endothelial cells. These interactions are mediated by multiple surface adhesins, some of which have been only partially characterized. Streptococci produce surface glucans (gtf and ftf), ECM adhesins (e.g., fibronectin-binding proteins, FimA), and platelet aggregating factors (phase I and phase II antigens, pblA, pblB, and pblT), all of which have been.

Influence of triamcinolone intravitreal injection on retinochoroidal healing processes.

To analyze the effects of triamcinolone intravitreal injection on the wound healing processes after argon laser retinal photocoagulation, wild type C57BL/6J mice, 8-12 weeks old underwent a standard argon laser photocoagulation protocol. After pentobarbital anesthesia and pupil dilatation, argon laser lesions were induced (50microm, 400mW, 0.05s). Two photocoagulation impacts created two disc diameters from the optic nerve in both eyes. The photocoagulated mice were divided into four groups: Group I (n=12), photocoagulation controls, did not receive any intravitreous injection. Group II (n=12), received an intravitreous injection of 1microl of balanced salt solution (BSS). Group III (n=12), received an intravitreous injection of 1microl containing 15microg of triamcinolone acetonide (TAAC) in BSS. Two mice from each of these three groups were sacrificed at 1, 3, 7, 14 days and 2 and 4 months after photocoagulation. Group IV (n=10) received 1.5, 3, 7.5, 15, or 30microg of TAAC and were all sacrificed on day 14. The enucleated eyes were subjected to systematic analysis of the cellular remodeling processes taking place within the laser lesion and its vicinity. To this purpose, specific antibodies against GFAP, von Willebrand factor, F4/80 and KI67 were used for the detection of astrocytes, activated Müller cells, vascular endothelial cells, infiltrating inflammatory cells and actively proliferating cells. TUNEL reaction was also carried out along with nuclear DAPI staining. Temporal and spatial observations of the created photocoagulation lesions demonstrate that 24h following the argon laser beam, a localized and well-delineated affection of the RPE cells and choroid is observed in mice in Groups I and II. The inner retinal layers in these mice eyes are preserved while TUNEL positive (apoptotic) cells are observed at the retinal outer nuclear layer level. At this stage, intense staining with GFAP is associated with activated retinal astrocytes and Müller cells throughout the laser path. From day 3 after photocoagulation, dilated new choroidal capillaries are detected on the edges of the laser lesion. These processes are accompanied by infiltration of inflammatory cells and the presence of proliferating cells within the lesion site. Mice in Group III treated with 15microg/mul of triamcinolone showed a decreased number of infiltrating inflammatory cells and proliferating cells, which was not statistically significant compared to uninjected laser treated controls. The development of new choroidal capillaries on the edges of the laser lesion was also inhibited during the first 2 months after photocoagulation. However, on month 4 the growth of new vessels was observed in these mice treated with TAAC. Mice of Group IV did not show any development of new capillaries even with small doses. After argon laser photocoagulation of the mouse eye, intravitreal injection of triamcinolone markedly influenced the retina and choroid remodeling and healing processes. Triamcinolone is a powerful inhibitor of the formation of neovessels in this model. However, this inhibition is transient. These observations should provide a practical insight for the mode of TAAC use in patients with wet AMD.

Distribution of neuropeptide Y Y1 receptors in rodent peripheral tissues.

Using a sensitive immunohistochemical technique, the localization of neuropeptide Y (NPY) Y1-receptor (Y1R)-like immunoreactivity (LI) was studied in various peripheral tissues of rat. Wild-type (WT) and Y1R-knockout (KO) mice were also analyzed. Y1R-LI was found in small arteries and arterioles in many tissues, with particularly high levels in the thyroid and parathyroid glands. In the thyroid gland, Y1R-LI was seen in blood vessel walls lacking alpha-smooth muscle actin, i.e., perhaps in endothelial cells of capillaries. Larger arteries lacked detectable Y1R-LI. A distinct Y1R-immunoreactive (IR) reticulum was seen in the WT mouse spleen, but not in Y1R-KO mouse or rat. In the gastrointestinal tract, Y1R-positive neurons were observed in the myenteric plexus, and a few enteroendocrine cells were Y1R-IR. Some cells in islets of Langerhans in the pancreas were Y1R-positive, and double immunostaining showed coexistence with somatostatin in D-cells. In the urogenital tract, Y1R-LI was observed in the collecting tubule cells of the renal papillae and in some epithelial cells of the seminal vesicle. Some chromaffin cells of adrenal medulla were positive for Y1R. The problem of the specificity of the Y1R-LI is evaluated using adsorption tests as well as comparisons among rat, WT mouse, and mouse with deleted Y1R. Our findings support many earlier studies based on other methodologies, showing that Y1Rs on smooth muscle cells of blood vessels mediate NPY-induced vasoconstriction in various organs. In addition, Y1Rs in other cells in parenchymal tissues of several organs suggest nonvascular effects of NPY via the Y1R.

Caveolin expression changes in the neurovascular unit after juvenile traumatic brain injury: signs of blood-brain barrier healing?

Traumatic brain injury (TBI) is one of the major causes of death and disability in pediatrics, and results in a complex cascade of events including the disruption of the blood-brain barrier (BBB). A controlled-cortical impact on post-natal 17 day-old rats induced BBB disruption by IgG extravasation from 1 to 3 days after injury and returned to normal at day 7. In parallel, we characterized the expression of three caveolin isoforms, cav-1, cav-2 and cav-3. While cav-1 and cav-2 are expressed on endothelial cells, both cav-1 and cav-3 were found to be present on reactive astrocytes, in vivo and in vitro. Following TBI, cav-1 expression was increased in blood vessels at 1 and 7 days in the perilesional cortex. An increase of vascular cav-2 expression was observed 7 days after TBI. In contrast, astrocytic cav-3 expression decreased 3 and 7 days after TBI. Activation of eNOS (via its phosphorylation) was detected 1 day after TBI and phospho-eNOS was detected both in association with blood vessels and with astrocytes. The molecular changes involving caveolins occurring in endothelial cells following juvenile-TBI might participate, independently of eNOS activation, to a mechanism of BBB repair while, they might subserve other undefined roles in astrocytes.

A novel cyclosporin a aqueous formulation for dry eye treatment: in vitro and in vivo evaluation.

PURPOSE: The aim of the present study was the in vitro and in vivo evaluation of a novel aqueous formulation based on polymeric micelles for the topical delivery of cyclosporine A for dry eye treatment. METHODS: In vitro experiments were carried out on primary rabbit corneal cells, which were characterized by immunocytochemistry using fluorescein-labeled lectin I/isolectin B4 for the endothelial cells and mouse monoclonal antibody to cytokeratin 3+12 for the epithelial ones. Living cells were incubated for 1 hour or 24 hours with a fluorescently labeled micelle formulation and analyzed by fluorescence microscopy. In vivo evaluations were done by Schirmer test, osmolarity measurement, CyA kinetics in tears, and CyA ocular distribution after topical instillation. A 0.05% CyA micelle formulation was compared to a marketed emulsion (Restasis). RESULTS: The in vitro experiments showed the internalization of micelles in the living cells. The Schirmer test and osmolarity measurements demonstrated that micelles did not alter the ocular surface properties. The evaluation of the tear fluid gave similar CyA kinetics values: AUC = 2339 ± 1032 min*μg/mL and 2321 ± 881.63; Cmax = 478 ± 111 μg/mL and 451 ± 74; half-life = 36 ± 9 min and 28 ± 9 for the micelle formulation and Restasis, respectively. The ocular distribution investigation revealed that the novel formulation delivered 1540 ± 400 ng CyA/g tissue to the cornea. CONCLUSIONS: The micelle formulation delivered active CyA into the cornea without evident negative influence on the ocular surface properties. This formulation could be applied for immune-related ocular surface diseases.

Vascular Integrins: Therapeutic and Imaging Targets of Tumor Angiogenesis

Cells, including endothelial cells, continuously sense their surrounding environment and rapidly adapt to changes in order to assure tissues and organs homeostasis. The extracellular matrix (ECM) provides a physical scaffold for cell positioning and represents an instructive interface allowing cells to communicate over short distances. Cell surface receptors of the integrin family emerged through evolution as essential mediators and integrators of ECM-dependent communication. In preclinical studies, pharmacological inhibition of vascular integrins suppressed angiogenesis and inhibited tumor progression. alpha(V)beta(3) and alpha(V)beta(5) were the first integrins targeted to suppress tumor angiogenesis. Subsequently, additional integrins, in particular alpha(1)beta(1), alpha(2)beta(1), alpha(5)beta(1), and alpha(6)beta(4), emerged as potential therapeutic targets. Integrin inhibitors are currently tested in clinical trials for their safety and antiangiogenic/antitumor activity. In this chapter, we review the role of integrins in angiogenesis and present recent advances in the use of integrin antagonists as potential therapeutics in cancer and discuss future perspectives.

Recombinant human tumor necrosis factor: an efficient agent for cancer treatment.

Recombinant human TNF (rhTNF) has a selective effect on endothelial cells in tumour angiogenic vessels. Its clinical use has been limited because of its property to induce vascular collapsus. TNF administration through isolated limb perfusion (ILP) for regionally advanced melanomas and soft tissue sarcomas of the limbs was shown to be safe and efficient. When combined to the alkylating agent melphalan, a single ILP produces a very high objective response rate. ILP with TNF and melphalan provided the proof of concept that a vasculotoxic strategy combined to chemotherapy may produce a strong anti-tumour effect. The registered indication of TNF-based ILP is a regional therapy for regionally spread tumours. In soft tissue sarcomas, it is a limb sparing neoadjuvant treatment and, in melanoma in-transit metastases, a curative treatment. Despite its demonstrated regional efficiency TNF-based ILP is unlikely to have any impact on survival. High TNF dosages induce endothelial cells apoptosis, leading to vascular destruction. However, lower TNF dosage produces a very strong effect that is to increase the drug penetration into the tumour, presumably by decreasing the intratumoural hypertension resulting in better tumour uptake. TNF-ILP allowed the identification of the role of alphaVbeta3 integrin deactivation as an important mechanism of antiangiogenesis. Several recent studies have shown that TNF targeting is possible, paving the way to a new opportunity to administer TNF systemically for improving cancer drug penetration. TNF was the first agent registered for the treatment of cancer that improves drug penetration in tumours and selectively destroys angiogenic vessels.

Signalling pathways in skin homeostasis

SUMMARY The results presented here contribute to a better understanding of the crucial molecular relationships and signalling cues exchanged by several fundamental cell types (epidermal keratinocytes, dermal fibroblasts, immune and endothelial cells) of the skin. Importantly we provide evidence to directly implicate Wnt/ß-catenin signalling as a putative player in different cell types (keratinocytes and neutrophils) in mediation of the cutaneous inflammatory response (Fart A). Finally we highlight the importance of several molecules, specifically expressed in the hair follicle stem cell niche to the morphogenesis and homeostasis of the hair follicle (Part B). PART A Currently the body of work pertaining to Wnt signalling and immune cells largely focuses on Wnt signalling in the development of these cells. The data presented here suggests a novel mechanism in which Wnt signalling appears to modulate immune cell recruitment to the skin. Keratinocytes are major contributors to early inflammatory responses by the release of chemokines which recruit immune cells. The resultant inflammatory response is a dynamic process of sequentially infiltrating immune cells governed by a network of growth factors, chemokines and cytokines. In wild type mice the response is typified by a rapid and substantial infiltration of neutrophils followed at later time points by macrophages and Tcells. The expression of the canonical Wnt pathway activating ligand, Wnt3a, is able to induce a strong neutrophil infiltration in the dermis. This response originates in keratinocytes, as it is abrogated upon keratinocyte-specific ablation of ß-catenin. Notably, this suggests that the crucial cross talk between these resident cells and recruited immune cells is, in part, mediated by Wnt signalling. In corroboration of this role of Wnt-mediated recruitment of neutrophils, expression of the Wnt inhibitory ligand sFRPI during acute inflammation results in a dramatic 'dampening' of immune cell infiltration in particular of neutrophil chemoattraction. Importantly, an intrinsic Wnt signalling pathway is essential for neutrophil chemoattraction in response to inflammatory stimuli. There is a marked reduction of neutrophil infiltration in mice grafted with a ß-catenin deficient bone marrow upon TPA induced cutaneous inflammation. Additionally, neutrophils lacking Wnt/ß-catenin fail to respond to IFNγ, an early inflammatory cue, in vitro. In combination, these data indicate a potent function of Wnt signalling in immune cell recruitment and the modulation of the inflammatory response. PART B Tissue specific stem cells form the cellular base on which tissue homeostasis and repair of adult tissue relies. The maintenance of this stem cell pool is highly dependent on the immediate environment or niche. We have identified three genes, the fibroblast growth factor receptor 1 (FGFR1), serpin protease inhibitor (serpin F1) and the haematopoietic cell phosphatase (Hcph) to be specifically expressed in a small population of stromal cells which are in close contact to bulge stem cells. These specialized stromal cells might represent an essential mesenchymal component of the skin stem cell niche and may regulate stem cell proliferation and differentiation. Multiple FGFR1 isoforms are generated through alternate transcript splicing and are able to interact with both FGFs and cell adhesion molecules. Two predominant forms of the receptor are FGFR1-α and FGFR1-ß. Expression of a dominant negative form of the alpha isoform prevents hair follicle morphogenesis altogether. Given that FGFR1-ß signals principally through the FGF ligands, this data indicates that FGF signalling is dispensable for follicle morphogenesis. Moreover the loss of follicular morphogenesis upon suggests a requirement for signalling via cell adhesion molecule association with the receptor as FGFR1 α has a greater affinity for these molecules. The expression of the second candidate niche gene serpin f1, lead to the complete ablation of hair follicle morphogenesis. The serpin f1 product, pigment-epithelial derived factor (PEDF) has potent anti-angiogenic effects. Immunohistochemical analysis using CD31, a endothelial cell marker, revealed that although these cells are present, they have are disorganised and do not form vessels. Interestingly, endothelial cells have been found to contribute to the neuronal stem cell niche and our results suggest a similar mechanism in the skin. SHP1, the Hcph gene product, is a phosphatase which acts in the haematopoetic system. Motheaten mice carrying spontaneous mutations in the Hcph gene have patchy alopecia in their skin and severe defects in their haematopoietic system. However the haematopoietic rescue of the mouse does not result in normal follicular homeostasis. Additionally, ablation of Hcph in either the dermal or keratinocyte compartments of the skin produces hair follicles with abberant morphologies. This data indicates that although SHP1 is not essential for hair follicle morphogenesis it is required in both epidermal and dermal compartments to maintain follicular morphology. RÉSUMÉ PARTIE A Jusqu'à présent, les travaux dédiés à l'étude de la voie de signalisation Wnt dans le système immunitaire se sont essentiellement concentrés sur son rôle dans le développement des cellules immunitaires. Les données présentées ici suggèrent fortement et de manière nouvelle, l'existence d'un mécanisme par lequel la voie de signalisation Wnt/ß-caténine module le recrutement de cellules immunitaires dans un tissu périphérique, la peau, et ainsi la réponse inflammatoire cutanée. La réponse inflammatoire cutanée est un processus dynamique d'infiltration séquentielle de diverses cellules immunitaires, orchestré par un réseau de facteurs de croissance, chémokines et cytokines. Les kératinocytes sont des contributeurs majeurs à la réponse inflammatoire précoce par la libération de chémokines qui permettent ensuite de recruter les cellules immunitaires. Dans des souris sauvages, la réponse est d'abord caractérisée par une infiltration rapide et substantielle de neutrophiles, suivie par celle des macrophages et des lymphocytes T. L'expression d'un ligand activateur de le voie canonique de signalisation Wnt (après injection infra-dermique de fibroblastes sur-exprimant Wnt-3a) induit une infiltration dermique très marquée de neutrophiles. De plus, la réponse est éliminée en l'absence de ß-caténine spécifiquement dans les kératinocytes, indiquant que ces cellules sont à l'origine de la réponse. De manière remarquable, ceci suggère qu'une signalisation cruciale entre ces cellules résidentes de la peau et les cellules immunitaires recrutées est, au moins en partie, médiée par la voie Wnt. Corroborant ce rôle de la voie Wnt/ß-caténine dans le recrutement des neutrophiles, l'expression d'un ligand inhibiteur de la voie (sFRP1) résulte au cours d'une inflammation aigüe en une réduction spectaculaire de l'infiltration des cellules immunitaires en général, et des neutrophiles en particulier. De manière importante, la voie de signalisation Wnt est intrinsèquement requise pour la chémoattraction des neutrophiles en réponse à un stimulus inflammatoire. En effet, suite à une inflammation cutanée induite par un ester de phorbol (TPA), une réduction notable de l'infiltration des neutrophiles est observée dans des souris préalablement greffées avec de la moelle osseuse constituée de cellules déficientes en ß-caténine. De plus, in vitro, les neutrophiles sans ß-caténine ne répondent pas à une stimulation par l'interféron γ, qui est pourtant un signal inflammatoire établi in vivo. En conclusion, nos données indiquent que la voie de signalisation Wnt/ß-caténine joue une fonction active dans le recrutement des cellules immunitaires vers un organe périphérique, la peau, ainsi que dans la modulation, à plusieurs niveaux, de la réponse inflammatoire cutanée. PARTIE B Les cellules souches tissu-spécifiques forment la base cellulaire sur laquelle repose l'homéostase et la réparation tissulaires chez l'adulte. La maintenance de ce réservoir de cellules souches est hautement dépendante de leur environnement cellulaire immédiat, encore appelé «niche des cellules souches». Dans la peau, ces cellules stromales spécialisées représentent un compartiment mésenchymateux essentiel de la niche des cellules souches en régulant leurs prolifération et différentiation. Nous avons identifié trois gènes, le «récepteur 1 àux facteurs de croissance des fibroblastes » (Fgfr1 ), l' «inhibiteur de protéase à sérine » (serpinf1 ou pedf) et la « phosphatase des cellules hématopoiétiques » (Hcph ou Ptpn6), comme spécifiquement exprimés par une petite population de cellules stromales qui sont étroitement associées aux cellules souches de la peau (localisées au niveau du bombement du follicule pileux). Pour analyser leur fonction dans ce contexte, nous avons utilisé un test de reconstitution complète de peau murine en combinaison à des. transductions géniques basées sur l'utilisation de lentivirus. Ce test repose sur le mélange de deux compartiments cellulaires, épidermique (kératinocytes) et dermique (fibroblastes), greffés sur une zone ouverte de peau du dos d'une souris pour ensemble reconstituer la peau. Des isoformes multiples de FGFR1 sont générées par épissage alternatif de transcrits et sont capables d'interagir à la fois avec les FGFs (facteurs de croissance des fibroblastes) et les molécules d'adhésion cellulaires. Les deux formes prédominantes du récepteur, FGFR1-α et FGFR1-ß, ne différent que par le «domaine ressemblant aux immunoglobulines 1 » (immunoglobulin-like 1 domain), absent de FGFR1-ß. De plus, FGFR1-ß a une affinité plus grande pour les FGFs et plus faible pour les molécules d'adhésion cellulaires telles que la Ncadhérine (connue pour activer FGFR). La sur-expression de l'une ou l'autre des formes n'empêche pas la morphogenèse folliculaire mais conduit à la formation de follicules aberrants. Toutefois, une différence phénotypique majeure est observée lorsqu'une forme «Dominant-Négatif » (DN) est exprimée dans le compartiment dermique. La sur-expression de FGFR1-ß DN conduit en effet à la formation de follicules petits et tronqués, avec des gaines épithéliales et un bulbe élargis ainsi qu'une petite papille dermique. Par contre, l'expression de FGFR1-α DN abolit complètement la morphogenèse folliculaire. Etant donné que la signalisation par FGFR1-ß est principalement dépendante des ligands FGFs, ces données indiquent que la signalisation par ceux-cì est non-nécessaire à la morphogenèse folliculaire. De plus, l'abolition du processus par la sur-expression de FGFR1-a DN suggëre une signalisation nécessaire entre le récepteur FGFR1 et une ou des molécules d'adhésion cellulaire. L'expression de notre second candidat comme gène spécifique de la niche des cellules souches de la peau, serpinf1, prévient la morphogenèse folliculaire. Seules de petites structures ressemblant à des cystes sont observées après reconstitution de la peau. De plus, dans ces transplants, aucune cellule CD34-positive (marqueur des cellules souches) n'est retrouvée associé à ces cystes. Le produit du gène serpin f1, le «facteur dérivé d'épithélium pigmentaire » (PEDF) est un puissant facteur anti-angiogénique. Nous avons donc analysé la vascularisation des transplants par immunohistochirnies utilisant CD31, un marqueur des cellules endothéliales. Nos résultats révèlent que les cellules endothéliales sont bien présentes, mais de manière désorganisée et ne formant pas de vaisseaux. De manière intéressante, les cellules endothéliales contribuent activement à la niche des cellules souches neuronales, et nos résultats suggèrent donc l'existence possible d'un mécanisme similaire dans la peau. SHP1, le produit du gène Hcph, est une phosphatase quì agit dans le système hématopoiétique. Les souris « motheaten »qui portent des mutations spontanées du gène ont une alopécie inégale au niveau de la peau et de sévères troubles du système hématopoiétique. Pour s'assurer que le phénotype observé au niveau de la peau n'est pas une conséquence d'un défaut du système hématopoiétique, nous avons transplanté des souris Hcph -/- avec de la moelle osseuse sauvage afin de restaurer la fonction de SHP 1 dans le système hématopoiétique. Toutefois, le défaut de morphologie folliculaire est maintenu. De plus, l'ablation d'Hcph dans le compartiment dermique ou épidermique d'essais de reconstitution de peau conduit à la production de follicules pileux avec des morphologies aberrantes. Ces données indiquent que SHP1 n'est pas essentiel à la morphogenèse folliculaire mais est toutefois requis à la fois dans les compartiments épidermiques et dermiques pour la maintenance de la forme du follicule.

Angiostatic kinase inhibitors to sustain photodynamic angio-occlusion.

Targeted angiostatic therapy receives major attention for the treatment of cancer and exudative age-related macular degeneration (AMD). Photodynamic therapy (PDT) has been used as an effective clinical approach for these diseases. As PDT can cause an angiogenic response in the treated tissue, combination of PDT with anti-angiogenic compounds should lead to improved therapy. This study was undertaken to test the clinically used small molecule kinase inhibitors Nexavar® (sorafenib), Tarceva® (erlotinib) and Sutent® (sunitinib) for this purpose, and to compare the results to the combination of Visudyne®-PDT with Avastin® (bevacizumab) treatment. When topically applied to the chicken chorioallantoic membrane at embryo development day (EDD) 7, a clear inhibition of blood vessel development was observed, with sorafenib being most efficient. To investigate the combination with phototherapy, Visudyne®-PDT was first applied on EDD11 to close all <100 μm vessels. Application of angiostatics after PDT resulted in a significant decrease in vessel regrowth in terms of reduced vessel density and number of branching points/mm(2) . As the 50% effective dose (ED50) for all compounds was approximately 10-fold lower, Sorafenib outperformed the other compounds. In vitro, all kinase inhibitors decreased the viability of human umbilical vein endothelial cells. Sunitinib convincingly inhibited the in vitro migration of endothelial cells. These results suggest the therapeutic potential of these compounds for application in combination with PDT in anti-cancer approaches, and possibly also in the treatment of other diseases where angiogenesis plays an important role.

Targeting vascular NADPH oxidase 1 blocks tumor angiogenesis through a PPARα mediated mechanism.

Reactive oxygen species, ROS, are regulators of endothelial cell migration, proliferation and survival, events critically involved in angiogenesis. Different isoforms of ROS-generating NOX enzymes are expressed in the vasculature and provide distinct signaling cues through differential localization and activation. We show that mice deficient in NOX1, but not NOX2 or NOX4, have impaired angiogenesis. NOX1 expression and activity is increased in primary mouse and human endothelial cells upon angiogenic stimulation. NOX1 silencing decreases endothelial cell migration and tube-like structure formation, through the inhibition of PPARα, a regulator of NF-κB. Administration of a novel NOX-specific inhibitor reduced angiogenesis and tumor growth in vivo in a PPARα dependent manner. In conclusion, vascular NOX1 is a critical mediator of angiogenesis and an attractive target for anti-angiogenic therapies.