Impaired wound healing is a major unsolved clinical problem in aging, diabetes, sickle cell disease (SCD), glucocorticoid (GC) therapy or Cushing syndrome.

First, we investigated the mechanisms underlying SCD ulcers, using SAD transgenic mouse model with mutated human beta globin. Old SAD mice displayed delayed healing correlated with the severity of anemia and hemolysis, as in human SCD, related to impaired cutaneous angiogenesis and poor endothelial progenitor cell (EPC) mobilization from the bone marrow. CXCL12 secretion by keratinocytes and inflammatory cells in SAD wounds was low. Noticeably, local wound therapy with EPCs or with recombinant CXCL12 restored wound angiogenesis and healing in old SAD mice.

Second, we questioned the role of mineralocorticoid receptor (MR) in wound healing and proposed that MR blockade could improve reepithelialization in pathological situations (GC-treated skin, diabetic mouse models); I also participated to the demonstration that MR blockade limits GC-induced epidermal atrophy in human skin.

We used skin explants from mouse and humans, full thickness skin wounds on the back of mice, and analyzed wound closure after skin biopsy in healthy volunteers pre-treated with GC, as a post-hoc study of the SPIREPI clinical trial. We found that MR blockers can rescue the pathological delayed wound healing while they did not modify wound closure in normal condition. We show that the improvement of GCinduced wound healing delay by MR blockade involves restoration of keratinocyte proliferation and down-regulation of the epithelial sodium channel. These results suggest an important and entirely novel role for MR in to improve delayed wound closure.

Acknowledgment:

I am happy to say a thank to the jury members for taking the time to evaluate my thesis. I especially thank to the reviewers have reviewed my manuscript and helping me to improve it through their remarks and proposals. I acknowledge Vietnamese government for supporting me the scholarship during 3 years.

I also acknowledge FRM for giving me the funding for the important fourth year of thesis.

I would like say a huge thank to my supervisor, Sélim for supporting and taking care my life during four years in Paris. I always see you as my father.

I would like to say a big thank to my advisor, Nicolette who always beside me during four years. I particularly thank you for your huge help and putting up me during the months of writing the manuscript and thesis. Thank you for the world formed research, for giving an excellent job and for sending me your knowledge. Karine Raymond, many thanks for teaching me techniques and giving me useful suggestions and comment. But also for the long hours of discussions during manipulations as well as motivation and support you have given me during these years. Especially, I would acknowledge you for your important help in applying the funding for the fourth year of my thesis.

Happy to thank Frederic have mounted the MR and wound healing project and help us in our reflections. Thanks to members from your lab: Basile, Soumaya, Christian.

I would also like to send a thank to my friend, Dany who has always offered extremely useful suggestions and comment for my work.

Thanks to Michele, Marie-Jo and Aude forever taking the time to help me.

Thanks to my colleagues from the laboratory both past and present: Sarah, Thomas, Geoffroy, Zhe, Mathieu, Romain, for taking the time to give me a help for discussion and manipulations in the laboratory. I thank the friends, colleagues in Saint Antoine Research Center for the help you have given me at all levels.

Thanks to my friends in UEVF for the funny time.

Thanks to all my friends and family for your support during these four years.

Finally, I especially thank my wife, Lĩnh for take caring all of house work and never complain each time I leave home from early and come back very late at night. Thanks to my daughter, Thảo Anh who always happy with us in our most difficult time.

Mechanisms of delayed wound healing in various models of human diseases

Résumé:

Le retard de cicatrisation (RETCIC) est un problème clinique majeur, touchant les gens âgés, les diabétiques, drépanocytaires (DR), ou traités par les glucocorticoides (GC). Nous avons abordé les mécanismes des ulcères DR dans un modèle transgénique ayant un gène beta globine humain muté (SAD). Les souris SAD âgées ont un RETCIC corrélé au degré d'anémie et d'hemolyse, comme dans la DR humaine ; le RETCIC est lié à l'altération de l'angiogenèse cutanée et un faible recrutement de cellules endothéliales progénitrices (EPC) depuis la moelle. La sécrétion de CXCL12 dans la plaie par les kératinocytes et les cellules inflammatoires est diminuée. Fait notable, le traitement local de la plaie par des EPC ou du CXCL12 recombinant restaurent l'angiogenèse et une cicatrisation des souris SAD âgées.

Nous avons ensuite abordé le rôle du récepteur minéralocorticoide dans la cicatrisation cutanée, et proposé ses antagonistes (MRA) pour améliorer le RETCIC en pathologie (traitement local GC, diabète murin); j'ai aussi participé à la démonstration de l'effet bénéfique des MRA sur l'atrophie épidermique GC-induite chez l'homme. A l'aide d'explants de peau murine et humaine, de plaies cutanées crées sur le dos de souris, et par l'analyse de la cicatrisation de biopsies chez le volontaire sain après traitement GC (étude post-hoc de l'essai SPIREPI), nous

... I -A -CHAPTER 1 -INTRODUCTION

The primary function of the skin is to serve as a protective barrier against the environment. Loss of the integrity of large portions of this barrier -as a result of injury or disease-may lead to major complications or even death. The ability of the skin to repair any wound is therefore a main skin "duty" in order to keep a proper homeostasis. Wound healing represents therefore a major organ functional ability of the skin in humans as well as in mammals.

Skin repair is a multistep complex biological process that requires a close interaction of multiple cell types in a highly coordinated program. It involves hemostasis, inflammation, angiogenesis, migration and proliferation of progenitor cells, as well as production and remodeling of the extracellular matrix (ECM). As a peculiar example recent studies have allowed a better understanding of the intervention of distant stem cells derived from distant tissues such as bone marrow in order to allow cutaneous healing.

Abnormalities of wound healing are very common. These lead mainly to impaired repair that itself provokes chronic ulcers. The causes of such healing delays are multiple. During the work of this PhD, we chose to focus on the mechanisms implicated in a severe circumstance of wound healing delay, namely sickle cell disease. We also extended my work toward the implication of a new pathway implicated in cutaneous repair, namely mineralocorticoid receptor (MR) activation.

Such pathway may play a beneficial role in important disease with healing delay such as diabetes and skin adverse reactions to glucocorticoid treatments.

Mechanisms implicated in normal skin wound I -Ahealing

The normal mammalian response to cutaneous injury occurs in overlapping stages: detersion, inflammation, new tissue formation, and remodeling [START_REF] Singer | Cutaneous wound healing[END_REF][START_REF] Gurtner | Wound repair and regeneration[END_REF]). Full healing is achieved only after a long lag time that may reach one year in humans. Many cellular and molecular players are involved in wellorchestrated dynamic and very complex processes. These phenomena include phagocytosis, production of enzymes and reactive oxygen species, cell proliferation, cell apoptosis, and migration that occur at different time points. Recent discoveries have led to many attempts to cell and stem cell therapies for pathological wound healing [START_REF] Lau | Exploring the role of stem cells in cutaneous wound healing[END_REF]). The main achievements concerning the four phases of cutaneous wound healing will be summarized here. Schematic representation of the three different phases of wound repair [START_REF] Werner | Regulation of wound healing by growth factors and cytokines[END_REF].

A-Inflammatory phase: 12-24 h after injury the wounded area is filled with a blood clot. Neutrophils have invaded into the clot.

B-New tissue formation: at days 3-7 after injury, the majority of neutrophils have undergone apoptosis. Instead, macrophages are abundant in the wound tissue at this stage of repair. Endothelial cells migrate into the clot; they proliferate and form new blood vessels. Fibroblasts migrate into the wound tissue, where they proliferate and deposit extracellular matrix. The new tissue is called granulation tissue.

Keratinocytes proliferate at the wound edge and migrate down the injured dermis and above the provisional matrix.

C-Tissue remodeling: 1-2 weeks after injury the wound is completely filled with granulation tissue. Fibroblasts have transformed into myofibroblasts, leading to wound contraction and collagen deposition. The wound is completely covered with a neo-epidermis.

I -A -1 -Inflammatory phase

This stage corresponds to the classical early stage of wound repair although all phases are redundant (Figure 2). Components of the coagulation cascade, inflammatory pathways and immune system are needed to prevent ongoing blood and fluid losses, to remove dead and dying tissues and to prevent infection. This stage involves hemostasis first, and mechanisms of inflammatory response as the second step. Schematic representation of main cellular actors involved in wound inflammation [START_REF] Singer | Cutaneous wound healing[END_REF].

1-Hemostasis

Skin injury causes vascular disruption and extravasation of blood constituents. The first step of wound healing is thus hemostasis to stop the bleeding. During this initial phase, three steps occur in a rapid sequence. Vascular spasm is the first response as the blood vessels constrict in the earlier minutes to prevent ongoing blood and fluid losses. In the second step, the activated platelets and endothelial cells from existing but altered dermal capillaries will play a major role. The formation of the platelet plug results from the platelet aggregation at the sites of vessel breaks.

Platelets stick together to form a temporary seal that covers the break in the vessel wall. This plug is fragile. Is strengthening requires the formation of a blood clot which, leading to the formation of a fibrin clot. Such aggregated platelets on altered endothelia provides an important matrix scaffold for neutrophils and monocytes [START_REF] Tonnesen | Angiogenesis in wound healing[END_REF]. Indeed such scaffold reinforced by the fibrin clot also serves as a gradient of cytokines, chemokines and growth factors that are able to direct inflammatory cell recruitment. To do so, platelets secrete several chemokines and growth factors implicated in early wound repair stage such as IL-1, TNF, TGF-α, TGF-β, PDGF and VEGF, platelet factor IV (PF4) and complement proteins [START_REF] Broughton | Wound healing: an overview[END_REF][START_REF] Barrientos | Growth factors and cytokines in wound healing[END_REF]). In addition, activated endothelial cells activate COX-2 and -synthesize products of arachidonic acid degradation, namely various prostaglandins that cause vasodilation and an augmentation of vascular permeability, and leukotrienes, particularly LTB4, which activate and recruit neutrophils and macrophages [START_REF] Broughton | Wound healing: an overview[END_REF].

2-Inflammatory response

Shortly after plug formation, polymorphonuclear leukocytes (neutrophils, PMN) begin to transmigrate into the wound site, followed later by monocytes/macrophages. They are recruited to the wound in response to IL-1, TNF, TGF-β, PF4 mainly coming from platelets, and later from activated endothelial cells.

As said, neutrophils invade the wound in large numbers, within a few minutes of injury, as a result of IL-1, TNF, TGF-β, PF4 secretion locally [START_REF] Singer | Cutaneous wound healing[END_REF]. Neutrophils transmigrate through the vascular wall by diapedesis, this being facilitated by the modifications of permeability induced by secreted prostaglandins.

The primary function of recruited neutrophils is the debridement of devitalized tissue and phagocytosis of foreign particles, as well as bacteria. These neutrophils also secrete proteolytic enzymes such as serine proteases (cathepsin G, proteinase-3, elastase, urokinase-type plasminogen activator) and metalloproteases [START_REF] Weiss | Tissue destruction by neutrophils[END_REF].

As a consequence of the functionality of myeloperoxidase (MPO), PMN generate cationic peptides and oxygen free radicals, which display anti-microbial activity [START_REF] Broughton | Wound healing: an overview[END_REF]Eming et al. 2007). Furthermore, some studies showed that neutrophils are an important source for pro-angiogenic factors, including VEGF-A and IL-8 [START_REF] Li | IL-8 directly enhanced endothelial cell survival, proliferation, and matrix metalloproteinases production and regulated angiogenesis[END_REF][START_REF] Ancelin | Vascular endothelial growth factor VEGF189 induces human neutrophil chemotaxis in extravascular tissue via an autocrine amplification mechanism[END_REF][START_REF] Ohki | Granulocyte colonystimulating factor promotes neovascularization by releasing vascular endothelial growth factor from neutrophils[END_REF][START_REF] Schruefer | The proangiogenic capacity of polymorphonuclear neutrophils delineated by microarray technique and by measurement of neovascularization in wounded skin of CD18-deficient mice[END_REF].

During normal wound healing in mice, the number of PMN is maximal at day 2 of the wound and then gradually decreases and disappears, even before complete reepithelialization [START_REF] Kim | Dynamics of neutrophil infiltration during cutaneous wound healing and infection using fluorescence imaging[END_REF]. Their presence in large numbers is observed in chronic wounds [START_REF] Simpson | The neutrophilic leukocyte in wound repair a study with antineutrophil serum[END_REF]Diegelmann 2003). Neutrophils are eliminated primarily by apoptosis and then by phagocytosis by macrophages [START_REF] Witko-Sarsat | Regulating neutrophil apoptosis: new players enter the game[END_REF].

The influx of neutrophils is quickly followed by the migration of monocytes. These are recruited from the circulating blood and marrow to the wound site in response to chemo-attractants and differentiate locally into macrophages [START_REF] Werner | Regulation of wound healing by growth factors and cytokines[END_REF]. In addition to these major newly recruited monocytes/macrophages, another population of resident skin macrophage is also presented at the wound side, although with a low density and minor role [START_REF] Macdonald | An antibody against the colony-stimulating factor 1 receptor depletes the resident subset of monocytes and tissue-and tumor-associated macrophages but does not inhibit inflammation[END_REF]. The infiltration of macrophages increase during the inflammatory stage, peak at day 2 of the wound and their number gradually decreases during maturation stage, but these cells are massively remained at later time [START_REF] Martin | Inflammatory cells during wound repair: the good, the bad and the ugly[END_REF]. There are several evidences for characteristic of macrophages. Some reports have demonstrated macrophages in wound as the IL4 activated cells since these macrophages express various markers of tissue remodeling after activated by IL4 [START_REF] Raes | FIZZ1 and Ym as tools to discriminate between differentially activated macrophages[END_REF][START_REF] Wynes | Induction of macrophage insulin-like growth factor-I expression by the Th2 cytokines IL-4 and IL-13[END_REF][START_REF] Mosser | Exploring the full spectrum of macrophage activation[END_REF][START_REF] Martinez | Alternative activation of macrophages: an immunologic functional perspective[END_REF]. In contrast, a work on aseptic wounds described wound macrophages as cells bearing the activation markers such as TNFα, mannose receptor [START_REF] Daley | The phenotype of murine wound macrophages[END_REF].

The importance of inflammatory cells in wound healing is a hot topic. Macrophages are the cells that are responsible for ending the local inflammation steps as well as the initiation of the following steps of proliferation, angiogenesis, granulation tissue formation, fibroblasts recruitment, and remodeling of the wound (DiPietro 1995).

However all these tasks relate on two types of macrophages, called the M1 and M2 macrophages. Of note, the M1 macrophages are activated in response to microbial agents like lipopolysaccharide (LPS) and inflammatory cytokines like IFN. In turn, these cells exhibit antimicrobial properties by release of many important proinflammatory cytokines such as TNFα, IL-1β, IL-6, or IL-12, and overexpress MHC class II molecules. On the other hand, M2 macrophages, which seen as a result of activation macrophages by IL4 and IL13, are activated macrophages. They are a much more heterogeneous population, composed of all macrophages that do not correspond to M1 characteristics [START_REF] Gordon | Alternative activation of macrophages[END_REF][START_REF] Mantovani | The chemokine system in diverse forms of macrophage activation and polarization[END_REF]). It has been reported that M2 macrophages play an important role in angiogenesis, wound healing and the protection against parasitic infection [START_REF] Gordon | Alternative activation of macrophages[END_REF]. However, these cells are also involved in several diseases, such as fibrosis, allergy [START_REF] Duffield | The inflammatory macrophage: a story of Jekyll and Hyde[END_REF].

Thus, both M1 and M2 macrophages are pivotal for healing process. Therefore, the balance between the two phenotypes is important in the different stage of wound healing. More M1 macrophages are needed to remove debris and to kill invading pathogens, while M2 macrophages may have more important role in later phases. In this context, study of Sindrilaru et al indicate that an unrestrained proinflammatory M1 macrophage population lead to alteration of wound healing in humans and mice [START_REF] Sindrilaru | An unrestrained proinflammatory M1 macrophage population induced by iron impairs wound healing in humans and mice[END_REF].

The infiltration of macrophages into wound bed is changed during the healing process suggesting different roles of these inflammatory cells in the diverse phases of wound repair. Indeed, it has recently been demonstrated that early depletion of macrophages during the inflammation phase of a surgical wound leads to a reduction of angiogenesis and the formation of granulation tissue together with an alteration of the re-epithelialization [START_REF] Mirza | Selective and specific macrophage ablation is detrimental to wound healing in mice[END_REF][START_REF] Lucas | Differential roles of macrophages in diverse phases of skin repair[END_REF]. In contrast, an invalidation of macrophages during the intermediate stage of proliferation causes a hemorrhage in wound and prevents the transition to the maturation and remodeling phase. Finally, depletion of macrophages during the late phase of remodeling does not affect the final result [START_REF] Goren | A transgenic mouse model of inducible macrophage depletion: effects of diphtheria toxin-driven lysozyme M-specific cell lineage ablation on wound inflammatory, angiogenic, and contractive processes[END_REF][START_REF] Lucas | Differential roles of macrophages in diverse phases of skin repair[END_REF]). However, it has been suggested that inflammatory cells are not needed for wound closure since the study showed that wound healing on PU.1 null mice (which lacked macrophages, mast cells and functional neutrophils) treated with antibiotic is similar to wild types. The actions of macrophages are mediated by the secretion of a large number of cytokines and growth factors including EGF, TGF-α, TGF-β, PDGF and VEGF, IL-1, IL-6 and TNF-α [START_REF] Barrientos | Growth factors and cytokines in wound healing[END_REF]. Macrophages also participate to the debridement of wounds by releasing NO in the extracellular medium by the action of iNOS induced by IL-1 and TNF-α [START_REF] Goldman | Functional age-dependent changes in bronchoalveolar lavage rat cells[END_REF]).

Of note, in addition to neutrophils and macrophages, other innate immune cells, such as mast cells, dendritic cells, eosinophils and basophils can be recruited in the wound bed [START_REF] Noble | Matrix regulation of lung injury, inflammation, and repair: the role of innate immunity[END_REF]. Moreover, a role for T cells and B cells in the wound healing process has also been demonstrated. Indeed, in the absence of dendritic cell-derived T cells, healing is delayed [START_REF] Jameson | A role for skin gammadelta T cells in wound repair[END_REF], and epidermally-derived T cells locally secrete growth factors in the skin during the healing process [START_REF] Toulon | A role for human skin-resident T cells in wound healing[END_REF]. Mice that are deficient in B-1 cells exhibit increased inflammatory cell infiltration, particularly neutrophils, and have a delayed healing response [START_REF] Oliveira | B-1 cells modulate the kinetics of wound-healing process in mice[END_REF].

I -A -2 -New tissue formation

This proliferative phase includes, angiogenesis, and proliferation and migration of fibroblasts, synthesis of components of the extracellular matrix of the dermis and finally keratinocyte proliferation. It is characterized by the formation of granulation tissue and results in healing of the wound.

1-Angiogenesis and vasculogenesis

Angiogenesis is a vital component of the normal healing process. In addition to angiogenesis which consists a sprouting of capillaries from preexisting blood vessels, several studies suggest that the vasculogenesis process also participates in the vascular regeneration during wound healing. It consists of generation of new vessels, from endothelial progenitor cells (EPCs) mostly derived from the marrow or even resident skin [START_REF] Amoh | Nascent blood vessels in the skin arise from nestin-expressing hairfollicle cells[END_REF]Eming et al. 2007) (figure 4). In setting of injury, resident skin endothelial cells (ECs) migrate into the wound at the leading tip of capillaries, forming tube-like structures (ref) that continue to extend, branch, and creating networks. These events are first promoted by the resident skin progenitors, and of course require a dynamic temporally and spatially regulated interaction between ECs various angiogenic factors, and surrounding ECM proteins [START_REF] Tonnesen | Angiogenesis in wound healing[END_REF]. The angiogenic factors that can stimulate wound angiogenesis are gradually being elucidated. Indeed, FGF-1 and FGF-2 are the first members of FGF family to be discovered that have potent angiogenic activity (ref).

Fibrin and fibrinogen bound FGF-2 could enhance the proliferation of ECs through induction of intergrin v3 and FGF-R1 [START_REF] Sahni | Potentiation of endothelial cell proliferation by fibrin(ogen)-bound fibroblast growth factor-2[END_REF][START_REF] Sahni | Stimulation of endothelial cell proliferation by FGF-2 in the presence of fibrinogen requires alphavbeta3[END_REF].

In addition, induction of v3 by FGF-2 can also facilitate localization of MMP-2 to ECs that can subsequent promote EC migration [START_REF] Brooks | Localization of matrix metalloproteinase MMP-2 to the surface of invasive cells by interaction with integrin alpha v beta 3[END_REF]. Furthermore, FGF-2 together with VEGF, TNF-a and hypoxia could promote increase of plasmin levels, which activates invasion of ECs into fibrin matrix, modulating EC migration and angiogenesis [START_REF] Koolwijk | Cooperative effect of TNFalpha, bFGF, and VEGF on the formation of tubular structures of human microvascular endothelial cells in a fibrin matrix. Role of urokinase activity[END_REF].

In addition to FGF, VEGF is vital angiogenic factor in wound angiogenesis. This protein is secreted by numerous cell types including keratinocytes, macrophages, ECs, fibroblasts. Regarding to functional activity, it has been demonstrated that VEGF can bind to fibrin or fibrinogen, that in turn facilitates localization and mitogenic activity of VEGF at wound sites in modulating EC proliferation [START_REF] Sahni | Vascular endothelial growth factor binds to fibrinogen and fibrin and stimulates endothelial cell proliferation[END_REF]. Of note, functional activity of VEGF is also enhanced through the interaction with it receptor VEGFR. Indeed, VEGFR2 that express on surface of ECs, interacts with VEGF and induce the activity of ECs, stimulate the formation of new capillary.

Moreover, the activity of VEGF is modulated by up-regulation of several intergrin receptors (v3, 11, and 21) and enhanced expression of the anti-apoptotic protein Bcl-2 [START_REF] Senger | Stimulation of endothelial cell migration by vascular permeability factor/vascular endothelial growth factor through cooperative mechanisms involving the alphavbeta3 integrin, osteopontin, and thrombin[END_REF][START_REF] Senger | Angiogenesis promoted by vascular endothelial growth factor: regulation through alpha1beta1 and alpha2beta1 integrins[END_REF]. The expression of VEGF during wound healing is variety and is regulated by many factors including hypoxia, eNOS derived NO, and inflammatory cytokines (Murohara et al. 1998;Murohara et al. 1998). Of note, VEGF is highly expressed during granulation tissue formation from day 4 through 7 after wounding, promoting the EC proliferation, migration and modulate angiogenesis, while FGF-2 expression peak after initial wound and declines towards day 7, suggesting the different role of these factors in wound angiogenesis [START_REF] Nissen | Vascular endothelial growth factor mediates angiogenic activity during the proliferative phase of wound healing[END_REF].

Furthermore, there are many other molecules that are known to be involved in angiogenesis during wound healing, such as Angiopoietins (Angs), Tie-1, Tie-2, TGF-. Angs comprise a family of four ligands that are EC-specific growth factors, exhibiting separate but synergistic effects with VEGF on angiogenesis. They act by means of the Tie2 receptor, which is mainly expressed on surface of ECs [START_REF] Papapetropoulos | Angiopoietin-1 inhibits endothelial cell apoptosis via the Akt/survivin pathway[END_REF][START_REF] Tonnesen | Angiogenesis in wound healing[END_REF]. Ang-1 induces the association of new vessels with pericytes and vascular smooth muscle cells, resulting in their stabilization. In contrast, Ang-2 acts as an antagonistic ligand of Tie-2 receptor and induces destabilization and remodeling vessels [START_REF] Saharinen | VEGF and angiopoietin signaling in tumor angiogenesis and metastasis[END_REF]). Both Ang-1 and Ang-2 as well as Tie-2 are expressed in wound tissue, especially the upregulation of Ang-2 and Tie-2 was observed upon injury. Interestingly, overexpression of Ang-1 in wound of diabetic mice stimulated various events of wound healing including angiogenesis [START_REF] Bitto | Angiopoietin-1 gene transfer improves impaired wound healing in genetically diabetic mice without increasing VEGF expression[END_REF]. TGF-b could promotes angiogenesis through stimulate the recruitment of macrophages that then would produce other active angiogenesis factors [START_REF] Tonnesen | Angiogenesis in wound healing[END_REF].

In addition to angiogenesis and vasculogenesis derived from local prognenitors, several studies have shown the recruitment of bone marrow-originated cells into cutaneous wounds (Asahara et al. 1999;[START_REF] Fathke | Contribution of bone marrow-derived cells to skin: collagen deposition and wound repair[END_REF][START_REF] Galiano | Topical vascular endothelial growth factor accelerates diabetic wound healing through increased angiogenesis and by mobilizing and recruiting bone marrow-derived cells[END_REF][START_REF] Bluff | Bone marrow-derived endothelial progenitor cells do not contribute significantly to new vessels during incisional wound healing[END_REF][START_REF] Okuno | Bone marrow-derived cells serve as proangiogenic macrophages but not endothelial cells in wound healing[END_REF]). Fathke et al. using marrow chimeric mice demonstrated the presence, of endothelial cells deriving from the marrow in healing skin specimens. These authors reported that less than 0.1% of total cells from the marrow are present in wounds and co-express CD31 by day 28 after wounding [START_REF] Fathke | Contribution of bone marrow-derived cells to skin: collagen deposition and wound repair[END_REF]). Asahara et al. studied the bone marrow-derived cells (BMDCs)

in the skin wounds of wild-type (WT) mice transplanted with bone marrow of transgenic lacZ protein, which was under the control of an endothelial-specific promoter flk-1 or tie-2. The number of endothelial progenitor cells (EPCs) CD34+

CD31+ from the marrow was maximal at day 4 after surgical wound and decreased to a very low level after 4 weeks once the healing has ended (Asahara et al. 1999). In the same way, using the Tie2/lacZ bone marrow transplant (BMT) model, Bluff et al.

also found BM-derived EPCs in skin wounds. The kinetics was not the same, the number of marrow derived EPC increased gradually from day 5 and peaked at 14 days post wounding [START_REF] Bluff | Bone marrow-derived endothelial progenitor cells do not contribute significantly to new vessels during incisional wound healing[END_REF]). This may relate to wounding differences. In contrast, using a GFP-bone marrow chimeric mice, Okuno et al. showed that BMDCs found in acute and chronic wounds did not express CD31, but rather the leukocyte antigens CD45 (70%) and CD11b (45%) by flow cytometry, and F4/80 by immunohistochemistry. These wound recruited marrow-cells appear to be M2 macrophages, recruited under the chemotactic action of CSF. They express metalloproteinases. Indeed, pharmacological inhibition of CSF or invalidation of CSF-1 gene led to a delayed angiogenesis and wound healing [START_REF] Okuno | Bone marrow-derived cells serve as proangiogenic macrophages but not endothelial cells in wound healing[END_REF]). In the same study, when the recipient mice were grafted with full VEGFR2-GFP bone marrow cells, GFP cells co-expressing CD31 were detected in the skin wounds. In contrast, when mice were grafted with the LysM-GFP bone marrow, GFP cells detected in the wounds expressed F4/80 [START_REF] Okuno | Bone marrow-derived cells serve as proangiogenic macrophages but not endothelial cells in wound healing[END_REF]. Therefore there seem to be in the marrow a population of progenitors able to form vessels in the wounded skin as well as another population of myeloid cells that secrete VEGFa and stimulate paracrine angiogenesis. In addition, bone marrow-derived mesenchymal stem cells (MSCs) are capable of differentiating into endothelial cells [START_REF] Wu | Mesenchymal stem cells enhance wound healing through differentiation and angiogenesis[END_REF]). These reports are therefore at least partly contradictory and open the question of the degree of involvement of bone marrow-derived EPCs in angiogenesis and vasculogenesis during skin wound healing.

Several recent studies have demonstrated the impact of EPCs during wound healing process [START_REF] Montesinos | Adenosine A(2A) receptor activation promotes wound neovascularization by stimulating angiogenesis and vasculogenesis[END_REF][START_REF] Bauer | The bone marrow-derived endothelial progenitor cell response is impaired in delayed wound healing from ischemia[END_REF][START_REF] Bluff | Bone marrow-derived endothelial progenitor cells do not contribute significantly to new vessels during incisional wound healing[END_REF] Indeed, EPCs contributed only 4.4% ± 1.5% of total ECs in the granulation tissue [START_REF] Bluff | Bone marrow-derived endothelial progenitor cells do not contribute significantly to new vessels during incisional wound healing[END_REF]). In contrast, other investigators have shown a larger EPC recruitment to this wound type [START_REF] Bauer | The bone marrow-derived endothelial progenitor cell response is impaired in delayed wound healing from ischemia[END_REF]) In response to platelet, endothelial and macrophages cytokines, fibroblasts synthesize initially a provisional extracellular matrix composed of collagen type III glycosamicoglycanes, elastin, and fibronectin [START_REF] Broughton | Wound healing: an overview[END_REF]. During this process, cellularity at the wound site increases and this proliferative phase lasts for several days until the wound area is filled to restore tissue integrity. The dermalepidermal interaction is crucial during this phase and during all the remaining healing process. In incisional wounds, TGF-β expression is maximal at day 7 and modulates remodeling of the extracellular matrix. TGF-β stimulates the synthesis of type I collagen by fibroblasts, inhibits the production of matrix metalloproteases (MMPs)

2-Migration and proliferation of fibroblasts

and increases the production of tissue inhibitors of metalloproteinases (TIMPs) [START_REF] Barrientos | Growth factors and cytokines in wound healing[END_REF]. In excisional wounds, TGF-β stimulates the differentiation of fibroblasts into myofibroblasts. Such cells express smooth muscle actin (α-SMA), have contractile activity and cause contraction of the wound [START_REF] Darby | Alpha-smooth muscle actin is transiently expressed by myofibroblasts during experimental wound healing[END_REF][START_REF] Sethi | Evidence for sequential utilization of fibronectin, vitronectin, and collagen during fibroblast-mediated collagen contraction[END_REF].

There are conflicting data on the origin of fibroblasts repopulating skin wounds. In their model of chimeric transplanted GFP marrow mouse, Fathke et al. showed the presence of BMDCs in the skin wounds at a late time (day 42), having a fusiform fibroblast-like appearance, capable of contracting collagen matrix in vitro and expressing collagen I and III. They also showed that collagen I was also expressed by dermal non-bone marrow-derived cells (GFP-cells) [START_REF] Fathke | Contribution of bone marrow-derived cells to skin: collagen deposition and wound repair[END_REF]). These results suggest that at least part of the dermal cells secreting collagen I derived from bone marrow. In contrast to these results, it was recently shown using another model, that wound fibroblasts expressing collagen type I were never of marrow origin [START_REF] Higashiyama | Differential contribution of dermal resident and bone marrowderived cells to collagen production during wound healing and fibrogenesis in mice[END_REF]. All these data leaves the subject of the origin of fibroblasts involved in wound healing pending for further works.

3-Re-epithelialization

Re-epithelialization of injured skin is a major and later step in the formation of new tissue. It generally involves the migration of adjacent epidermal keratinocytes into center of wound, the proliferation of keratinocytes from wound edges and or hair follicles remnants. The wound re-epithelialization is ensured by local epidermal progenitoes from the wound edges as well as by epithelial stem cells from hair follicles or sweat glands [START_REF] Martin | Wound healing--aiming for perfect skin regeneration[END_REF][START_REF] Miller | Reepithelialization of porcine skin by the sweat apparatus[END_REF][START_REF] Roh | Cutaneous stem cells and wound healing[END_REF][START_REF] Lau | Exploring the role of stem cells in cutaneous wound healing[END_REF]). This process is activated by signaling pathways of epithelial and nonepithelial cells at the wound edges, which release a myriad of different cytokines and growth factors, e.g. hepatocyte growth factor (HGF), fibroblast growth factor (FGF), epidermal growth factors (EGF), insulin-like growth factor-1 (IGF-1) [START_REF] Werner | Regulation of wound healing by growth factors and cytokines[END_REF]. Wound re-epithelialization is also promoted by various receptors such as G-protein coupled receptors, tyrosine kinase receptors [START_REF] Sano | Keratinocyte-specific ablation of Stat3 exhibits impaired skin remodeling, but does not affect skin morphogenesis[END_REF][START_REF] Amendt | Resistance of keratinocytes to TGFbeta-mediated growth restriction and apoptosis induction accelerates re-epithelialization in skin wounds[END_REF]Ashcroft et al. 2003;[START_REF] Chmielowiec | c-Met is essential for wound healing in the skin[END_REF][START_REF] Raja | Wound reepithelialization: modulating keratinocyte migration in wound healing[END_REF]), or nuclear receptors as peroxysome-proliferator-activated receptors, androgen, estrogen and glucocorticoid receptors [START_REF] Icre | Functions of the peroxisome proliferatoractivated receptor (PPAR) alpha and beta in skin homeostasis, epithelial repair, and morphogenesis[END_REF][START_REF] Michalik | Involvement of PPAR nuclear receptors in tissue injury and wound repair[END_REF][START_REF] Pullar | beta-Adrenergic receptor antagonists accelerate skin wound healing: evidence for a catecholamine synthesis network in the epidermis[END_REF]) they become flattened and desmosomes, are altered [START_REF] Matsuzaki | Re-epithelialisation and the possible involvement of the transcription factor, basonuclin[END_REF]). As a result of the morphological changes, epidermal cells at the edge of a wound display lateral mobility and an ability to migrate. Hemidesmosomes, are junctions that link the epidermal cells to the basement membrane. They becomes also altered in order to allow keratinocytes to migrate [START_REF] Matsuzaki | Re-epithelialisation and the possible involvement of the transcription factor, basonuclin[END_REF]). In addition to wound edges, the keratinocyte migration occurs also from remaining skin appendages.

Multiple elements are involved in keratinocyte migration and re-epithelialization, including the ECM, MMPs, integrin receptors, and growth factors. The effect of the keratinocyte growth factor (KGF) on re-epithelialization and wound healing is still discussed. KGF mRNA is highly expressed during wound healing [START_REF] Werner | Large induction of keratinocyte growth factor expression in the dermis during wound healing[END_REF]) and KGF stimulates keratinocyte proliferation and migration [START_REF] Tsuboi | Keratinocyte growth factor (FGF-7) stimulates migration and plasminogen activator activity of normal human keratinocytes[END_REF]. Sanz Garcia et al. identified that EGF, KGF and basic FGF (bFGF) all promote wound re-epithelialisation (Sanz [START_REF] Garcia | Experimental model for local application of growth factors in skin re-epithelialisation[END_REF]. However, Guo et al.

reported that mice null for the KGF gene showed no delay in healing of dorsal incisional wounds and tail wounds [START_REF] Guo | Keratinocyte growth factor is required for hair development but not for wound healing[END_REF]. In mice where FGF function had been blocked in the epidermis using a truncated FGFR2-IIIb, Werner et al. reported healing of incisional dorsal wounds was delayed (Werner et al. 1994).

TGF-1 is also one of the most important players for epithelial cell migration during re-epithelialization (O'Kane and Ferguson 1997), as it stimulates the expression of integrin subunits that promote keratinocyte migration on the provisional ECM.

Keratinocytes use their surface integrin receptors to interact with a fibronectin-rich provisional matrix. The direction of migration is also regulated by the binding of keratinocytes integrin receptors on the newly formed collagen molecules in the wound bed. Dissociation of this binding allows the keratinocytes to migrate from the edges toward the wound center. MMPs also play an important role in keratinocyte migration by their involvement in this dissociation. Migrating keratinocytes produce MMPs, such as MMP-9, which specifically degrades type IV collagen and laminins in the basement membrane. This allows cells to leave the basement membrane and migrate into the wound [START_REF] Parks | Matrix metalloproteinases in repair[END_REF].

It has been shown that the decrease in caspase-8 expression in keratinocytes of wound edges raises keratinocyte proliferation [START_REF] Lee | Dynamic expression of epidermal caspase 8 simulates a wound healing response[END_REF]). Migrating keratinocytes express keratins 6 and 16, loose the expression of gap junction proteins as connexin 43 [START_REF] Brandner | Connexins 26, 30, and 43: differences among spontaneous, chronic, and accelerated human wound healing[END_REF], and overexpress β1 integrin, allowing them to interact with components of the extracellular matrix (ECM) of granulation tissue and to migrate to cover the wound [START_REF] Margadant | Unique and redundant functions of integrins in the epidermis[END_REF]. Once keratinocytes cover all the granulation tissue, they loose the expression of keratin 6

and 16 and start to express keratins 14 and 5 of the basal layer. These keratinocytes synthetize proteases such as MMP-1 and basement membrane proteins such as laminin-5, which allow them to re-epithelialize the wound (Eming et al. 2007). Basal keratinocytes differentiate thereafter following the physiological epidermal program, giving suprabasal layers expressing keratins 1 and 10, and subsequently the terminal differentiation markers including fillagrin, loricrin and involucrin [START_REF] Singer | Cutaneous wound healing[END_REF]. It has been nicely shown that in addition to interfollicular epidermal progenitors, hair follicle stem cells were also involved in wound re-epidermization [START_REF] Ito | Stem cells in the hair follicle bulge contribute to wound repair but not to homeostasis of the epidermis[END_REF].

A restoration of an intact basement membrane that connects the epidermis and dermis occurs through proteins secretion by keratinocytes and fibroblasts [START_REF] Li | Pathophysiology of acute wound healing[END_REF][START_REF] Gurtner | Wound repair and regeneration[END_REF]) (Figure 3).

I -A -3 -Tissue remodelling phase

After the recruitment of inflammatory cells, the angiogenesis, cell proliferation, and re-epithelialization steps, have finished; we are facing healed skin but that still is far from a normal skin. At that time point, begins the remodeling phase. This stage aims to restore a better tissue structural integrity and functional competence. This last phase consists in the synthesis of the dermal extra cellular matrix and its subsequent changes over time. During this period, there is a balance between the synthesis of new components of the extracellular matrix and their degradation by proteases. The processes of regression of granulation tissue are not well known. Key elements of this stage are the regression of vascular structures, decrease of leukocyte infiltrate which returns progressively to a normal level and substitution of transient components of the extracellular matrix as vitronectin, fibronectin and type III collagen by the permanent collagenous fibrous components such as type I collagen [START_REF] Li | Pathophysiology of acute wound healing[END_REF]). The apoptosis of leukocytes and endothelial cells is a major mechanism, responsible for the decrease of their number in the granulation tissue [START_REF] Desmouliere | Apoptosis mediates the decrease in cellularity during the transition between granulation tissue and scar[END_REF]. Other mechanisms involve the reduction of local expression of growth factors and chemokines, decreasing the recruitment and proliferation, and the augmentation of expression of anti-inflammatory and anti-angiogenic cytokines [START_REF] Dipietro | Wound healing: the role of the macrophage and other immune cells[END_REF].

The formation of the extracellular matrix follows a specific process during wound healing. Initially, the matrix is composed of fibrin and fibronectin as a result from hemostasis. Glycosaminoglycans and proteoglycans are then synthesized by fibroblasts [START_REF] Broughton | Wound healing: an overview[END_REF]. This primary matrix will then be replaced by a more resistant and organized matrix composed of collagen. In healthy skin, collagen in the dermis consists of 80-90% of type I and 10-20% type III. However, during early steps of wound healing, type III collagen becomes the predominant collagen synthesized by fibroblast in granulation tissue. Col III first appears after 48 to 72 hours (30%) and is maximally secreted between 5 and 7 days. The peak of type III collagen is precocious and matches with a peak of fibronectin [START_REF] Ehrlich | Regulation of wound healing from a connective tissue perspective[END_REF][START_REF] Li | Pathophysiology of acute wound healing[END_REF]). MMPs and their inhibitors (TIMPs) are responsible for the remodeling of extracellular matrix components. Their activity and synthesis is influenced by changes in local concentrations of TGF, PDGF, IL-1 and EGF [START_REF] Henry | Inflammatory mediators in wound healing[END_REF]. Collagen synthesis is continued for 4 to 5 weeks after wound. The initially deposited collagen is thinner than that in a non-injured normal skin and oriented parallel to the skin surface. It will be subsequently digested, and new, thicker and organized collagen is deposited in bunches along the skin tension lines.

Nevertheless, the dermis will never recover its original structure and the wound will not recover its strength at 100%: at one week, the healing skin displays 3% of its original strength. It increases progressively to 30% in 3 weeks, and 80% after 3 months (Diegelmann 2003;[START_REF] Broughton | Wound healing: an overview[END_REF].

Endothelial progenitor cell-a key actor of I -Bangiogenesis and wound repair

EPCs are bone marrow-derived progenitor cells that were first discovered as significant contributors to neovascularization by Asahara et al. [START_REF] Asahara | Isolation of putative progenitor endothelial cells for angiogenesis[END_REF].

In response to injury, these cells are recruited into circulation and homed to ischemia sites; these cells undergo in situ differentiation, and ultimately participate to the formation of new vessels (Asahara et al. 1999;[START_REF] Carmeliet | Manipulating angiogenesis in medicine[END_REF]) (Figure 7). Although the molecular identification of EPCs remains a topic of debate, studies suggest that two functionally distinct subpopulations exist based on in vitro isolation techniques: early outgrowth EPCs and late outgrowth EPCs [START_REF] Sieveking | Strikingly different angiogenic properties of endothelial progenitor cell subpopulations: insights from a novel human angiogenesis assay[END_REF][START_REF] Asahara | Concise review: Circulating endothelial progenitor cells for vascular medicine[END_REF]. Specifically, early outgrowth EPCs appear to function in a paracrine role in promoting neovascularization whereas late growth EPCs directly differentiate into endothelial tubes [START_REF] Sieveking | Strikingly different angiogenic properties of endothelial progenitor cell subpopulations: insights from a novel human angiogenesis assay[END_REF]. EPCs have been characterized and classified based on multiple markers. However, none of the markers used are specific for EPCs [START_REF] Timmermans | Endothelial progenitor cells: identity defined?[END_REF]). However, a common feature of EPCs is their functional ability to mobilize and home to injured areas and promote vessel formation [START_REF] Lamping | Endothelial progenitor cells: sowing the seeds for vascular repair[END_REF]. Although the mobilization of EPCs is a complex event and that the mechanisms that specifically recruit these cells to wound sites remain under investigation, numerous molecules and signaling pathways have already been addressed and showed to be implicated in this process. EPCs are thought to mobilize from BM and home to areas of endothelial damage via adhesion molecules. Alternatively, resident EPC may be present at low levels in tissues (ref).

The secreted proteases cathepsin L and MMP2 regulate the transmigration of EPCs.

These cells may subsequently mature and differentiate towards the endothelial lineage [START_REF] Urbich | Cathepsin L is required for endothelial progenitor cell-induced neovascularization[END_REF][START_REF] Cheng | Mechanisms underlying the impairment of ischemia-induced neovascularization in matrix metalloproteinase 2-deficient mice[END_REF]).

Of note, nitric oxide (NO) signaling and reactive oxygen species (ROS) have also been implicated in EPC mobilization and activity [START_REF] Gallagher | Diabetic impairments in NOmediated endothelial progenitor cell mobilization and homing are reversed by hyperoxia and SDF-1 alpha[END_REF][START_REF] Hamed | Nitric oxide and superoxide dismutase modulate endothelial progenitor cell function in type 2 diabetes mellitus[END_REF]. Moreover, previous studies indicate that EPCs are mobilized in response to tissue ischemia. This probably results through cytokine and/or chemokine secretion from ischemic tissue, thereby augmenting neovascularization of ischemic tissues (Takahashi, T 1999;[START_REF] Ishida | Pivotal role of the CCL5/CCR5 interaction for recruitment of endothelial progenitor cells in mouse wound healing[END_REF]).

Among cytokines implicated in EPC mobilization and angiogenesis, the family of vascular endothelial growth factors (VEGF) and their receptors have been identified

as the most potent and predominant ones [START_REF] Roskoski | VEGF receptor protein-tyrosine kinases: structure and regulation[END_REF][START_REF] Carmeliet | Molecular mechanisms and clinical applications of angiogenesis[END_REF].

VEGFs are endothelial cell specific mitogens and potent inducers of vascular permeability. They play a crucial role in endothelial cell motility; stimulate vascular endothelial cell growth, survival, and proliferation and eventually increase the number of capillaries. This wide range of effects is mediated in part by the multiple VEGF members, VEGF-A through VEGF-D and in associated with three VEGF receptortyrosine kinases, known as VEGFR-1, -2 and -3, as well as with co-receptors including neuropilins (Nrp) (Figure 8) [START_REF] Ferrara | Vascular endothelial growth factor: basic science and clinical progress[END_REF][START_REF] Olsson | VEGF receptor signalling -in control of vascular function[END_REF]Eming et al. 2007). and delayed wound healing [START_REF] Frank | Regulation of vascular endothelial growth factor expression in cultured keratinocytes. Implications for normal and impaired wound healing[END_REF]. Since then, several studies have shown that VEGF levels are indeed increased following tissue injury, and that this level correlated with the migration of EPCs to ischemic area (Asahara et al. 1999;[START_REF] Tepper | Adult vasculogenesis occurs through in situ recruitment, proliferation, and tubulization of circulating bone marrow-derived cells[END_REF]. In addition, it has been shown that topical VEGF-A application improves the delayed wound healing in diabetic mice through local upregulation of other angiogenic cytokines and recruitment of BMDCs [START_REF] Galiano | Topical vascular endothelial growth factor accelerates diabetic wound healing through increased angiogenesis and by mobilizing and recruiting bone marrow-derived cells[END_REF]). VEGF-A has also been reported to regulate the expression of endothelial cell surface proteins, known as integrins, that link cells with various members of the ECM. Integrins, which comprise a family of transmembrane heterodimeric proteins, play a major role in controlling EPC mobilization and homing to injury and ischemic areas [START_REF] Caiado | Endothelial progenitor cells and integrins: adhesive needs[END_REF]. According to its powerful angiogenic ability, VEGF-A even has been applied in clinical therapy for some human ischemic diseases (you need reference here).

There are various sources for VEGF-A secretion during wound healing. These include keratinocytes, macrophages, platelets, neutrophils and mast cells. The expression of these proteins can be regulated by numerous factors including growth factors, pro-inflammatory cytokines, hormones and cellular stress [START_REF] Fukumura | Tumor induction of VEGF promoter activity in stromal cells[END_REF][START_REF] Kishimoto | In vivo detection of human vascular endothelial growth factor promoter activity in transgenic mouse skin[END_REF][START_REF] Pages | Transcriptional regulation of the Vascular Endothelial Growth Factor gene--a concert of activating factors[END_REF]. One of the best characterized mechanisms of VEGF-A synthesis in wound healing as well as other settings is hypoxia-inducible factor-1 (HIF-1). HIF-1 is a transcription factor that exists as a dimeric complex, consisting of a cytoplasmic 𝛼 subunit and a nuclear  subunit [START_REF] Ceradini | Homing to hypoxia: HIF-1 as a mediator of progenitor cell recruitment to injured tissue[END_REF]. During hypoxia, HIF-1 translocates into the nucleus to form a complex with HIF-1, initiating the transcription of genes inducing angiogenesis including VEGF and chemokine stromal cell-derived factor-1 (SDF-1. Thus, strategies to stabilize HIF-1 may enhance EPC mobilization and function [START_REF] Ceradini | Progenitor cell trafficking is regulated by hypoxic gradients through HIF-1 induction of SDF-1[END_REF][START_REF] Hoenig | Hypoxia inducible factor-1 alpha, endothelial progenitor cells, monocytes, cardiovascular risk, wound healing, cobalt and hydralazine: a unifying hypothesis[END_REF]) and therefore improve delayed wound healing [START_REF] Botusan | Stabilization of HIF-1alpha is critical to improve wound healing in diabetic mice[END_REF][START_REF] Thangarajah | The molecular basis for impaired hypoxia-induced VEGF expression in diabetic tissues[END_REF]).

Besides VEGF, other peptides have been shown to modulate EPC recruitement during wound healing. Chemokines also crucial players in EPC and hematopoietic stem cell (HSC) trafficking and angiogenesis during wound healing process.CCL5 is one of these. An elegant study by Ishida et al. was able to demonstrate that CCL5/CCR5 interaction is an important molecular pathway for modulation of EPCs and eventual neovascularization during wound healing process. Indeed, CCL5 is secreted at day 1 post wounding by F480 infiltrating macrophages and allows mobilization of CCR5+ EPC from marrow to granulation tissue. [START_REF] Ishida | Pivotal role of the CCL5/CCR5 interaction for recruitment of endothelial progenitor cells in mouse wound healing[END_REF]).

This study also demonstrated that EPCs are important not only as the progenitors of endothelial cells, but also as the source of growth factors during tissue repair important for other healing steps [START_REF] Ishida | Pivotal role of the CCL5/CCR5 interaction for recruitment of endothelial progenitor cells in mouse wound healing[END_REF].

The chemokine CXCL12 (also known as SDF-1) and its specific receptor CXCR4 is another important factor mediating recruitment of various stem/progenitor cells like EPCs or Hematopoietic stem cells. Indeed, CXCL12 that express in different tissues can stimulate the adhesion, migration and homing of progenitor cells from circulation to ischemic tissues. In contrast, blockade of this chemokine in ischemic tissue or CXCR4 on circulating cells reduce the mobilization of progenitors to sites of injury [START_REF] Ceradini | Progenitor cell trafficking is regulated by hypoxic gradients through HIF-1 induction of SDF-1[END_REF]. Importantly, the level of SDF-1 expression in injured tissue correlates with stem cell recruitment and healing. The EPC recruitment into tissue depends on the upregulation of SDF-1 [START_REF] Askari | Effect of stromal-cell-derived factor 1 on stem-cell homing and tissue regeneration in ischaemic cardiomyopathy[END_REF][START_REF] Yamaguchi | Stromal cell-derived factor-1 effects on ex vivo expanded endothelial progenitor cell recruitment for ischemic neovascularization[END_REF][START_REF] Ceradini | Progenitor cell trafficking is regulated by hypoxic gradients through HIF-1 induction of SDF-1[END_REF]). Consistent with this view, it has been reported that the impaired EPC homing and poor angiogenesis in diabetic wounds are due to decreased level of SDF-1. And this defect could be reversed by therapy of this chemokine into wounds [START_REF] Gallagher | Diabetic impairments in NOmediated endothelial progenitor cell mobilization and homing are reversed by hyperoxia and SDF-1 alpha[END_REF]). In contrast, inhibition of SDF-1 leads to further impairment of wound healing, decrease angiogenesis in the diabetic mice [START_REF] Bermudez | Inhibition of stromal cell-derived factor-1alpha further impairs diabetic wound healing[END_REF].

Of note, the expression of SDF-1 is regulated by many factors, such as hypoxia or the activity of eNOS [START_REF] Ceradini | Progenitor cell trafficking is regulated by hypoxic gradients through HIF-1 induction of SDF-1[END_REF][START_REF] Gallagher | Diabetic impairments in NOmediated endothelial progenitor cell mobilization and homing are reversed by hyperoxia and SDF-1 alpha[END_REF]).

Victor W. Wong and Jeffrey D. Crawford 2013

Re-epithelialization: role of nuclear receptors I -Cand ligands

Re-epithelialization is an important step in the formation of new tissue after wounding, involving both proliferation and migration of keratinocytes from the edges of wound to reconstitute a complete barrier. This process is promoted by many signaling pathways with the critical involvement of various families of receptors including nuclear receptors (NR), such as peroxysome-proliferator-activated receptors (PPAR), androgen (AR), estrogen (ER) and glucocorticoid (GR) receptors and their ligands [START_REF] Icre | Functions of the peroxisome proliferatoractivated receptor (PPAR) alpha and beta in skin homeostasis, epithelial repair, and morphogenesis[END_REF][START_REF] Michalik | Involvement of PPAR nuclear receptors in tissue injury and wound repair[END_REF][START_REF] Pullar | beta-Adrenergic receptor antagonists accelerate skin wound healing: evidence for a catecholamine synthesis network in the epidermis[END_REF]. It is generally considered that estrogens enhance wound repair, while androgens impair wound healing. Role of estrogens and its receptor are different between male and female. Activation of estrogen receptor by its ligand estrogen markedly delays wound re-epithelialization in male mice [START_REF] Gilliver | 17beta-estradiol inhibits wound healing in male mice via estrogen receptor-alpha[END_REF], while systemic or topical estrogen treatment can reverse age-related impaired healing in animal models and humans females, in association with accelerated re-epithelialization, reduced local inflammation and enhanced matrix deposition [START_REF] Ashcroft | Estrogen accelerates cutaneous wound healing associated with an increase in TGF-beta1 levels[END_REF][START_REF] Ashcroft | Topical estrogen accelerates cutaneous wound healing in aged humans associated with an altered inflammatory response[END_REF]Hardman et al. 2008). The sex difference in the effects of systemic estrogen treatment on wound re-epithelialization may partly be explained by a fundamental difference in the response of keratinocytes to estrogens: cells cultured from female mice migrate more rapidly following treatment with an estrogen 17--estradiol [START_REF] Emmerson | Unique and synergistic roles for 17beta-estradiol and macrophage migration inhibitory factor during cutaneous wound closure are cell type specific[END_REF]; those from male donors were unresponsive to estrogen in the same assay. Another critical actor in wound re-epithelialization is AR. Lai and colleagues have demonstrated that monocytes/macrophages-specific deletion of AR lead to improvement of wound healing, whereas keratinocyte-and fibroblast-specific ARKO mice do not. Even though AR in keratinocytes and fibroblasts is dispensable for overall wound healing, AR in these 2 cell types plays key but opposing roles to regulate re-epithelialization. Keratinocyte AR promoted re-epithelialization, while fibroblast AR suppressed it [START_REF] Lai | Monocyte/macrophage androgen receptor suppresses cutaneous wound healing in mice by enhancing local TNF-alpha expression[END_REF]). Among three subtypes of PPAR, PPAR and  are important modulators for the re-epithelialization during wound healing and each of them plays a specific role in this process. PPAR is mainly involved in the early inflammation phase of the healing, whereas PPAR is implicated in the control of keratinocyte proliferation [START_REF] Michalik | Impaired skin wound healing in peroxisome proliferator-activated receptor (PPAR)alpha and PPARbeta mutant mice[END_REF]). In contrast, PPARhas been reported to contribute to the wound healing, not only by accelerating the migration of keratinocytes but also by promoting extracellular matrix-mediated cellular interactions at the wound edge through TGF-1/Smad3 signaling-dependent or -independent pathway [START_REF] Ham | PPARdelta promotes wound healing by up-regulating TGF-beta1-dependent or -independent expression of extracellular matrix proteins[END_REF]. Moreover, according to Nakamura et al. this receptor is also involved in the corneal epithelial wound healing [START_REF] Nakamura | Functional role of PPARdelta in corneal epithelial wound healing[END_REF]. Vitamin D hormone metabolites and analogues inhibit the proliferation of cultured keratinocytes that may bring benefits in human hyperproliferative skin diseases such as psoriasis [START_REF] Lehmann | Role of the vitamin D3 pathway in healthy and diseased skin-facts, contradictions and hypotheses[END_REF]. In contrast, the thyroid hormones stimulate keratinocyte proliferation, and therefore accelerate wound healing [START_REF] Safer | Topical thyroid hormone accelerates wound healing in mice[END_REF].

Finally, GR and its ligands are classical modulators of skin proliferation and differentiation. Glucocorticoids derivatives are frequently used in acute and chronic inflammatory diseases; however, unwanted side-effects, such as epidermal atrophy or delayed wound healing are common, limiting their long-term use (

Abnormal wound healing in human and/or mice I -Dcontext

As detailed above, normal wound healing is crucial in restoring the cutaneous barrier in order to allow the skin to perform its dedicated functions, such as preventing infection by foreign agents and fluid loss. The healing process is not only complex but also fragile. The kinetics and quality of skin healing are major parameters in the clinical practice. They are under the influence of several factors. Various abnormalities in any of these may alter normal healing leading to chronic wounds.

Again, such situation can be responsible for a significant morbidity and mortality that place chronic wounds as a major public health problems that we will discuss below.

I -D -1 -Age-related delayed wound healing

The ageing process in humans is well known to be detrimental to wound healing.

Although the exact mechanisms of this age related process are remained unclear, it has been demonstrated that an increase in fragility of aged skin together with impaired immune response and cellular ageing contribute to impaired wound healing in the elderly, associated to disruption of the normal phases of repair, leading to an increase in healing time following injury [START_REF] Thomas | Age-related changes in wound healing[END_REF].

Archival studies highlight several events which contribute to age-related delayed healing. First, platelet function was found to be impaired in the elderly patients, indicated by a significantly longer "closure time", and assessed by measuring the time taken for platelets to occlude a Platelet Function Analyser-PFA 100™ [START_REF] Boldt | Changes of haemostasis in patients undergoing major abdominal surgery--is there a difference between elderly and younger patients?[END_REF]). In addition, Fukaya et al. determined that platelet aggregation is increased with age, indicating a delayed wound hemostasis in aged individuals [START_REF] Fukaya | Factors influencing platelet function in elderly patients with chronic phase thrombotic diseases][END_REF]. Second, the impaired healing of the elderly has been strongly linked to excessive inflammation; especially neutrophil recruitment and protease production in response to the increased pro-inflammatory cytokine production [START_REF] Ashcroft | Aging alters the inflammatory and endothelial cell adhesion molecule profiles during human cutaneous wound healing[END_REF][START_REF] Ashcroft | Potential role of estrogens in wound healing[END_REF]. Moreover, study of Reed et al. showed that together with aging, dermal fibroblast proliferation and migration are impaired [START_REF] Reed | Impaired migration, integrin function, and actin cytoskeletal organization in dermal fibroblasts from a subset of aged human donors[END_REF]). The proliferation and migration of epithelial cells during wound healing is also delayed with intrinsic ageing in both healthy human subjects and animal models [START_REF] Holt | Effect of age on wound healing in healthy human beings[END_REF][START_REF] Ashcroft | Estrogen accelerates cutaneous wound healing associated with an increase in TGF-beta1 levels[END_REF]). More interestingly, the majority of studies have associated ageing with a decreased angiogenesis in relation with a defect in the production of VEGF [START_REF] Swift | Impaired wound repair and delayed angiogenesis in aged mice[END_REF]) and TGF-β [START_REF] Rivard | Age-dependent impairment of angiogenesis[END_REF] or with an alteration in matrix synthesis and reduced proliferation of progenitor endothelial cells [START_REF] Reed | Impaired angiogenesis in the aged[END_REF].

I -D -2 -Chronic wounds

Chronic wounds are wounds that have failed to progress through the normal stages of healing leading to a prolonged loss of substance. In the human setting, chronic wounds are usually defined by a persistence of an unhealed wound for at least 3 months. However, such classification does not fit with all situation since normal healing depends also of the age, surface of initial wound as well as the medical care that has been given.. . In Western countries, the most common causes of chronic ulcers are vascular diseases that are responsible for up to 70% affected patients [START_REF] Nwomeh | Physiology of the chronic wound[END_REF] (Figure 10). The vascular disorders causing chronic wounds can either derive from large diameter vessels diseases or in contrast small diameter vessels diseases.

Disorders of lower limbs large diameter arteries or veins can indeed lead to ischemia. standing at work. More rarely, ischemia can result from microangiopathies which are small vessels diseases. These are found mainly in diabetes and more rarely in neuropathies, nutritional deficiencies, some genetic diseases, systemic auto-immune diseases [START_REF] Fonder | Treating the chronic wound: A practical approach to the care of nonhealing wounds and wound care dressings[END_REF]. In developing countries, the most common causes of chronic wounds are specific infections, arterial disease and traumatisms [START_REF] Sibanda | A prospective evaluation of lower extremity ulcers in a Zimbabwean population[END_REF]).

As mentioned above, diabetes is responsible for frequent delayed healing. Diabetes can induce tissue hypoxemia through the induction of large vessels atheroma, neuropathy induced pressure ulcers, increased susceptibility to infections or microangiopathy. The mechanisms of microangiopathy have been addressed by animal model studies or in humans. Microangiopathy has been shown in db/db mice to result from an alteration of EPC mobilization from marrow to wounds [START_REF] Gallagher | Diabetic impairments in NOmediated endothelial progenitor cell mobilization and homing are reversed by hyperoxia and SDF-1 alpha[END_REF][START_REF] Marrotte | Manganese superoxide dismutase expression in endothelial progenitor cells accelerates wound healing in diabetic mice[END_REF]. Therefore the importance of these EPC mobilization is illustrated by its implication in diabetic wounds.

In addition to the cases of the chronic wounds mentioned above, many issues of delayed wound healing have been reported are steroids related. Indeed, glucocorticoids, that are wide used to treat chronic inflammatory or auto-immune diseases, usually accompanied by side-effects including skin atrophy and delayed wound healing [START_REF] Hengge | Adverse effects of topical glucocorticosteroids[END_REF]). In the same context of wound healing, while estrogen is considered to enhance wound repair and therefore the deprivation of this steroid as the major factor controlling delayed healing in elderly subjects; androgen, incontrast, is important factor of the delayed wound healing. Indeed, many genes, that are known to be associated with delayed healing, such as growth factor alpha (TGFα), arginase 1 (ARG1) have been found to be altered in elderly male subjects, are also known to be estrogen regulated (Hardman and Ashcroft 2008), while the deletion of androgen receptor in monocytes/macrophages lead to improvement of wound healing [START_REF] Lai | Monocyte/macrophage androgen receptor suppresses cutaneous wound healing in mice by enhancing local TNF-alpha expression[END_REF]).

Chronic ulcers are sources of various and sometimes serious complications in patients. These may include skin infections, contact dermatitis, subcutaneous fibrosis with secondary functional limitation of the adjacent joints (ankylosis), and moreover chronic pain with risk of depression or mood disorders. These complications, in addition to functional impairment, have psychological impact and lead to a worsening in the quality of life of affected patients.

When chronic wounds are severe and worsen despite adapted treatment, amputation may be necessary. In the United States, the incidence of secondary amputation to a diabetic leg ulcer is estimated to reach 11.3/1000 patient-years [START_REF] Adler | Lower-extremity amputation in diabetes. The independent effects of peripheral vascular disease, sensory neuropathy, and foot ulcers[END_REF].

Importantly, prolonged ulcers may be complicated by the development of squamous cell carcinoma arising on one part of the wound [START_REF] Menke | Impaired wound healing[END_REF]). The incidence of burn scars undergoing malignant transformation has been reported to be 0.77 to 2% of affected cases. All parts of the body can be concerned, but the extremities and the scalp are most frequently affected [START_REF] Copcu | Marjolin's ulcer: a preventable complication of burns?[END_REF].

In USA currently 1 to 2% of individuals will be affected by leg ulceration during their lifetime, and this figure will likely increase with the aging population [START_REF] Fonder | Treating the chronic wound: A practical approach to the care of nonhealing wounds and wound care dressings[END_REF]Piel et al. 2013). Every year in the United States, about 6.5 million people present one or several chronic skin ulcers secondary to venous insufficiency, diabetes or pressure ulcers [START_REF] Singer | Cutaneous wound healing[END_REF]. In Europe, 5-15% of individuals between 30 and 70 years suffer from venous insufficiency, including at least 1% with varicose ulcer [START_REF] Welt | Skin diseases in Europe. Wound healing[END_REF]. Similarly, the prevalence of type II diabetes in the United States is 2% and the incidence of leg ulcers in these diabetic patients is 15% [START_REF] Reiber | The epidemiology of diabetic foot problems[END_REF]. In addition, an increase of these numbers is expected due to the aging of the population and the high prevalence of the causes of poor healing in the elderly [START_REF] Nelzen | The prevalence of chronic lowerlimb ulceration has been underestimated: results of a validated population questionnaire[END_REF]. Indeed, healing mechanisms are modified in aging skin.

Even, in the absence of any specific disorder, skin in old patients will heal in a longer kinetics [START_REF] Taylor | Extent of iron pick-up in deforoxaminecoupled polyurethane materials for therapy of chronic wounds[END_REF]. This relates with various mechanisms including a reduction in stem cell compartment.

Medical care for chronic ulcers has a very high cost from society. In the US, the total cost of care and morbidity of skin wounds is estimated 13 to 15 billion USD per year and the treatment of venous ulcers alone amounted to 3 billion [START_REF] Bergan | Chronic venous disease[END_REF][START_REF] Fonder | Treating the chronic wound: A practical approach to the care of nonhealing wounds and wound care dressings[END_REF]. It has become mandatory to develop new treatments that are effective and easily accessible for wound healing problems. This makes it crucial to understand the cellular and molecular mechanisms involved in this phenomenon.

II -Aims of work

As detailed above, impaired wound healing is a major clinical problem in various situations including in subjects treated by corticosteroids/dcorticoids, in the elderly and in pathological conditions, such as diabetes, sickle cell disease or Cushing syndrome. Efforts towards improving wound healing represent a major challenge, to improve quality of life. It is therefore important to further improve the understanding about the mechanisms underlying the normal and abnormal wound healing in those situations. In this work, we aimed to address two questions :

1. Develop a murine model for SCD ulcers to improve the understanding of the cellular and molecular pathways responsible for a long-lasting and severe sickle cell leg ulcers.

2. Investigate specific targets in delayed cutaneous wound healing in various pathological situations such as glucocorticoid treated patients, diabetes) and more specifically the implication of the mineralocorticoid receptor inhibition.

CHAPTER 2 -DELAYED WOUND HEALING IN MURINE MODEL OF SICKLE CELL DISEASE

I -Introduction

Sickle cell disease I -A -

I -A -1 -Genetics

Sickle cell disease (SCD) is a recessive autosomic disorder of the b globin chain of hemoglobin. It is one the most common severe genetic disease reported in many countries worldwide [START_REF] Weatherall | A case for developing North-South partnerships for research in sickle cell disease[END_REF][START_REF] Piel | Global distribution of the sickle cell gene and geographical confirmation of the malaria hypothesis[END_REF]. It results from a single mutation at the -globin gene in which the 17th nucleotide thymine is replaced by adenine and as a consequence, the sixth amino acid in the β-globin chain changed to valine instead of glutamic acid [START_REF] Pauling | Sickle cell anemia a molecular disease[END_REF][START_REF] Bunn | Pathogenesis and treatment of sickle cell disease[END_REF]. Hemoglobin S (HbS) produced as a result of this defect, is poorly soluble and polymerizes under deoxygenated condition, giving sickle-shaped red blood cells (RBCs) [START_REF] Bunn | Pathogenesis and treatment of sickle cell disease[END_REF]).

I -A -2 -Epidemiology

Sickle cell anemia (SCA) occurs with the highest prevalence in low-and middle income countries. Especially, the greatest burden of the disease lies in sub-Saharan Africa, with the prevalence of sickle cell trait ranges between 10 and 45% [START_REF] Okwi | An up-date on the prevalence of sickle cell trait in Eastern and Western Uganda[END_REF]Piel et al. 2013). According to a recent report, more than 230 000 children with SCA were born in this region every year, that represents about 79.2% of the global total SCA world wideband this proportion is expected to increase to 88% by 2050 [START_REF] Modell | Global epidemiology of haemoglobin disorders and derived service indicators[END_REF]Piel et al. 2013). Moreover, this disorder is of growing importance. Indeed, beyond its old geographical distribution, and due to population migrations, it became recently the most frequent genetic disease diagnosed in Western countries such as the United Kingdom and France [START_REF] Piel | Global distribution of the sickle cell gene and geographical confirmation of the malaria hypothesis[END_REF]Piel et al. 2013). In these two countries, the incidence of SCD is around 1/1500 to 1/3800 newborns per year [START_REF] Piel | Global distribution of the sickle cell gene and geographical confirmation of the malaria hypothesis[END_REF][START_REF] Thuret | Neonatal screening for sickle cell disease in France: evaluation of the selective process[END_REF].

I -A -3 -Clinical presentation

SCD begins usually in infancy. It is characterized by different signs and symptoms.

These include many life-threatening complications including acute chest syndrome, but also various tissue damage such as stroke, pulmonary hypertension, renal damage, and bone infarct. These complications of SCD have long been as driven by any of two major pathophysiological processes: micro-vascular occlusion and hemolysis (figure 12) [START_REF] Lane | Sickle cell disease[END_REF][START_REF] Rees | Sickle-cell disease[END_REF]

Figure 12. Pathophysiology of sickle cell disease

The roles of HbS polymerisation, hyperviscosity, vaso-occlusion, haemolysis, and endothelial dysfunction are shown. Deoxygenation causes HbS to polymerise, leading to sickled erythrocytes. Vaso-occlusion results from the interaction of sickled erythrocytes with leucocytes and the vascular endothelium. Vaso-occlusion then leads to infarction, haemolysis, and inflammation; inflammation enhances the expression of adhesion molecules, further increasing the tendency of sickled erythrocytes to adhere to the vascular endothelium and to worsen vaso-occlusion.

Reperfusion of the ischaemic tissue generates free radicals and oxidative damage.

The damaged erythrocytes release free haemoglobin in to the plasma, which strongly bind to nitric oxide, causing functional nitric oxide defi ciency and contributing to the Sickle cell vascular occlusion: under deoxygenated condition, HbS Hb loose their solubility and become polymerized. Such polymerized b chains lead to the sickle rigid, RBCs. These are stiff cells that cannot easily circulate in narrow capillary beds and therefore they occlude microvessels in various tissues. Progressively, the entrapment of erythrocytes and leucocytes in the microcirculation increases. This, occlusion induces an interaction between these sickle erythrocytes and endothelial cells of vessel wall that lead to vascular adhesion of erythrocytes and leukocytes as well as tissue ischemia. HERE you should speak about the adhesion molecules that play a role There is no enough molecular data although there are lot of papers. It is important to note that beside the polymerization of HbS, the inflammatory trapped cells play a role to enhance the vascular obstruction. Indeed, in transgenic mice expressing HbS, cycles of experimental hypoxia but also treatment with proinflammatory drugs lead both to enhance endothelial leucocyte-erythrocyte adhesive interactions in the postcapillary venules leading to vascular occlusion [START_REF] Osarogiagbon | Reperfusion injury pathophysiology in sickle transgenic mice[END_REF][START_REF] Frenette | Sickle cell vaso-occlusion: multistep and multicellular paradigm[END_REF][START_REF] Turhan | Primary role for adherent leukocytes in sickle cell vascular occlusion: a new paradigm[END_REF][START_REF] Belcher | Transgenic sickle mice have vascular inflammation[END_REF]. Such ischemia events are followed by reperfusion. These ischemia reperfusion cycles induce oxidant stress, with activation of vascular oxidases [START_REF] Wood | Endothelial cell NADPH oxidase mediates the cerebral microvascular dysfunction in sickle cell transgenic mice[END_REF]) and inflammatory stress [START_REF] Frenette | Sickle cell vaso-occlusion: multistep and multicellular paradigm[END_REF][START_REF] Belcher | Transgenic sickle mice have vascular inflammation[END_REF][START_REF] Belcher | Critical role of endothelial cell activation in hypoxia-induced vasoocclusion in transgenic sickle mice[END_REF].

Hemolysis: Apart from vaso-occlusion, ischemia and reperfusion, hemolytic anemia with hemolysis is another consequence of polymerization of HbS. Several data indicate that hemolysis is another pathophysiological mechanism in SCD tissue damage. However, controversial data are still found. Hemolysis has been known to be a causative reason of anemia, fatigue, and cholelithiasis, but there is now evidence that it also contributes to the development of vasculopathy and tissue damage. Patients with SCD have a risk of vasculopathy, characterized by systemic and pulmonary hypertension, endothelial dysfunction, and proliferative changes in the intima and smooth muscle of blood vessels [START_REF] Pegelow | Natural history of blood pressure in sickle cell disease: risks for stroke and death associated with relative hypertension in sickle cell anemia[END_REF][START_REF] Ohene-Frempong | Cerebrovascular accidents in sickle cell disease: rates and risk factors[END_REF][START_REF] Gladwin | Pulmonary hypertension as a risk factor for death in patients with sickle cell disease[END_REF]Kato et al. 2006). Studies have suggested that some complications of SCD namely priapism, pulmonary hypertension and leg ulceration were associated with low concentration of steady state hemoglobin and high level of intravascular hemolysis [START_REF] Ataga | Pulmonary hypertension in patients with sickle cell disease: a longitudinal study[END_REF][START_REF] Kato | Deconstructing sickle cell disease: reappraisal of the role of hemolysis in the development of clinical subphenotypes[END_REF]). An association between the development of pulmonary hypertension and the level of hemolytic anemia was noted in several studies of SCD patients [START_REF] Gladwin | Pulmonary hypertension as a risk factor for death in patients with sickle cell disease[END_REF][START_REF] Ataga | Pulmonary hypertension in patients with sickle cell disease: a longitudinal study[END_REF][START_REF] Castro | Pulmonary hypertension associated with sickle cell disease: clinical and laboratory endpoints and disease outcomes[END_REF][START_REF] Minniti | Elevated tricuspid regurgitant jet velocity in children and adolescents with sickle cell disease: association with hemolysis and hemoglobin oxygen desaturation[END_REF].

How hemolysis leads to tissue damage? Briefly, high level of free hemoglobin is released after intravascular hemolysis in capillaries. Such free Hb quenches Nitric Oxide (NO) and leads to the generation of reactive oxygen species. This may happen either directly or indirectly via NADP oxidase or via xanthine oxidase. Decreased NO bioavailability is responsible for platelet and tissue factor activation leading to the activation of the coagulation cascade (Figure 13) [START_REF] Gladwin | Hemolysis-associated hypercoagulability in sickle cell disease: the plot (and blood) thickens![END_REF].

Figure 13. Hemolysis-associated hemostatic activation in SCD.

Intravascular hemolysis releases hemoglobin into plasma which quenches NO and generates ROS (directly via fenton chemistry or via induction of xanthine oxidase and NADP oxidase). In addition, arginase I is released from the red blood cell during hemolysis and metabolizes arginine, the substrate for NO synthesis, further impairing NO synthesis. The depletion of NO is associated with pathological platelet activation and tissue factor expression. Hemolysis and splenectomy are also associated with phosphatidylserine exposure on red cells which can activate tissue factor and form a platform for coagulation [START_REF] Gladwin | Hemolysis-associated hypercoagulability in sickle cell disease: the plot (and blood) thickens![END_REF].

Besides, low NO availability induces the release of Endothelin-1 (ET-1), -a potent vasoconstrictive peptide-by activated endothelial cells: ET-1 plasma levels have been reported abnormally high in SCD patients and murine models while the levels of ET-1 were found to be elevated in the kidney and lung after vaso-occlusive crisis [START_REF] Rybicki | Increased levels of endothelin-1 in plasma of sickle cell anemia patients[END_REF][START_REF] Tharaux | Urinary endothelin-1 as a marker of renal damage in sickle cell disease[END_REF][START_REF] Sabaa | Endothelin receptor antagonism prevents hypoxiainduced mortality and morbidity in a mouse model of sickle-cell disease[END_REF]. Finally, endothelin receptor antagonist (bosentan) treatment was shown to prevent kidney and lung damage in hypoxia/reoxygenation induced SCD events in a transgenic mouse model of sickle cell disease [START_REF] Sabaa | Endothelin receptor antagonism prevents hypoxiainduced mortality and morbidity in a mouse model of sickle-cell disease[END_REF].

Sickle cell Leg ulcers I -B -

Leg ulcers are a severe and poorly known complication of SCD. It affect between 2.5 and 40% of patients depending on the countries evaluated [START_REF] Halabi-Tawil | Sickle cell leg ulcers: a frequently disabling complication and a marker of severity[END_REF]).

The peak is observed in Jamaica while the lowest level is in Saudi Arabia. SCD leg ulcers is a very chronic manifestation of SCD (Figure 14) that develops on lower limbs after the age of 15 years. Such ulcers are the longest-lasting type of ulcer encountered in adults, with one study reporting ulcers lasting up to 300 months [START_REF] Halabi-Tawil | Sickle cell leg ulcers: a frequently disabling complication and a marker of severity[END_REF][START_REF] Minniti | Vasculopathy, inflammation, and blood flow in leg ulcers of patients with sickle cell anemia[END_REF]). These ulcers are unique or multiple.

They occur usually after a triggering event such as a traumatism, an insect bite or an itch but fail to heal afterwards. In contrast, the pathogenesis of sickle cell associated leg ulcers remains poorly known. Indeed, it has been considered that SCD manifestations including leg ulcers are associated with the elevated blood viscosity-related vaso-occlusion or with the hemolysis and endothelial dysfunction alternative subphenotype [START_REF] Aslan | Oxygen radical inhibition of nitric oxide-dependent vascular function in sickle cell disease[END_REF][START_REF] Bowers | SKIP3, a novel Drosophila tribbles ortholog, is overexpressed in human tumors and is regulated by hypoxia[END_REF][START_REF] Gladwin | Pulmonary hypertension as a risk factor for death in patients with sickle cell disease[END_REF][START_REF] Wang | Biological activity of nitric oxide in the plasmatic compartment[END_REF][START_REF] Ataga | Pulmonary hypertension in patients with sickle cell disease: a longitudinal study[END_REF][START_REF] Bowers | Blood viscosity and the expression of inflammatory and adhesion markers in homozygous sickle cell disease subjects with chronic leg ulcers[END_REF][START_REF] Connes | Decreased hematocrit-to-viscosity ratio and increased lactate dehydrogenase level in patients with sickle cell anemia and recurrent leg ulcers[END_REF]). However, these features are not consistent with other series . In addition, leg ulcers are not seen in other chronic haemolytic diseases such as paroxystic nocturnal hemoglobinuria [START_REF] Bunn | Pulmonary hypertension and nitric oxide depletion in sickle cell disease[END_REF][START_REF] Yoshino | Venous hemangioma of the posterior mediastinum[END_REF].

Finally, a recent well conducted study did not find hypoxia in skin ulcers in SCD patients as compared to various controls [START_REF] Bowers | Blood viscosity and the expression of inflammatory and adhesion markers in homozygous sickle cell disease subjects with chronic leg ulcers[END_REF]. At this point, hemolysis-induced vasoconstriction does not appear to constitute the unique mechanism of prolonged ulcers in SCD patients. Therefore, other skin wound healing pathways are probably altered in these recipients.

Only a few studies have investigated sickle cell ulcers in human. In addition to the association studies cited above, two papers have reported biopsies of ulcers [START_REF] Francillon | Histochemical analysis of growth factor, fibronectin, and iron content of sickle cell leg ulcers[END_REF][START_REF] Minniti | Vasculopathy, inflammation, and blood flow in leg ulcers of patients with sickle cell anemia[END_REF]. In these studies with a small number of cases, authors found the mural fibrin thrombi, luminal narrowing and progressive vascular occlusion in the ulcer beds. There was also unusual proliferation of abnormal blood vessels with microangiopathic changes including fibrinoid necrosis in the vessel walls, and fragmented RBCs within vessel walls [START_REF] Minniti | Vasculopathy, inflammation, and blood flow in leg ulcers of patients with sickle cell anemia[END_REF].

These interesting data thus reveal changes to cutaneous vessels in SCD ulcers. However, biopsies were obtained from ulcers that were several months old.

Therefore it is impossible to determine whether these changes are primary events or

Murine model of sickle cell disease I -C -

To date, two murine models of SCD have indeed been developed. SAD mice harbor a recombinant hβ-globin transgene expressing human hemoglobin SAD (α2β2SAD), which contains 2 mutations (Antilles [β 231 ] and D Punjab [β 121Q ] in addition to the β S6V mutation, the most frequently found mutation in human SCD. SAD mice express 19% of human sickle cell hemoglobin (HbS) along with mouse hemoglobin. Under hypoxic conditions, SAD red blood cells show sickling properties and dehydration in vitro, typical of SCD. Homozygous SAD mice die in utero, but hemizygous SAD have reduced survival, priapism and kidney defects. They are used as a SCD murine model [START_REF] Sabaa | Endothelin receptor antagonism prevents hypoxiainduced mortality and morbidity in a mouse model of sickle-cell disease[END_REF].

The Berkeley SCD mice have targeted deletions of murine hemoglobin (α -/-, β -/-) with a knockintransgene containing human sickle cell hemoglobin (α, β S ; A Υ, G Υ, β).

Berkeley mice express human mutated hemoglobin almost exclusively and harbor several hematological and pathological similarities with human SCD including erythrocyte sickling, intravascular hemolysis, severe anemia, leukocytosis, multiorgan infarct, and glomerulosclerosis [START_REF] Manci | Pathology of Berkeley sickle cell mice: similarities and differences with human sickle cell disease[END_REF].

CHAPTER 3 -Role of mineralocorticoid receptor in skin wound healing I -Introduction: Biology of mineralocorticoid receptor

The steroid aldosterone is the main mineralocorticoid hormone; it is synthetized in the glomerular zone of the adrenal cortex in response to hyperkaliemia or sodium depletion, as the end-point of activation of the renin-angiotensin system [START_REF] Rossier | Epithelial sodium transport and its control by aldosterone: the story of our internal environment revisited[END_REF]. Aldosterone stimulates renal sodium reabsorption and potassium excretion, therefore playing a major role in the control of blood pressure and extracellular volume homeostasia.

The molecular and cellular events involved in aldosterone effects in the distal tubule, the connecting tubule and the collecting duct (referred as aldosterone-sensitive distal nephron) have been elucidated [START_REF] Pearce | Collecting duct principal cell transport processes and their regulation[END_REF][START_REF] Penton | Dietary potassium and the renal control of salt balance and blood pressure[END_REF][START_REF] Rossier | Epithelial sodium transport and its control by aldosterone: the story of our internal environment revisited[END_REF]. Aldosterone binds to the mineralocorticoid receptor (MR), a ligand-dependent transcription factor belonging to the nuclear receptor superfamily [START_REF] Viengchareun | The mineralocorticoid receptor: insights into its molecular and (patho)physiological biology[END_REF][START_REF] Zennaro | Mineralocorticoid receptors in the metabolic syndrome[END_REF]. After nuclear translocation of the hormone-receptor complex, it binds to glucocorticoid response elements (GRE) within the promotor region of aldosterone-induced genes to modulate their transcription (there is no mineralocorticoid-specific response element; MR, GR and AR as well as progesterone receptors bind to the same GREs). This will trigger an increase in the activity and number of sodium transporters or channels. In a typical mineralocorticoid target cell, as the renal collecting duct principal cell, the sodium enters the cell through the amiloride-sensitive apical sodium channel (ENaC, for Epithelial Sodium Channel), and is extruded from the cell to the peritubular space by the sodium pump Na-K-ATPase located in the basolateral membrane [START_REF] Pearce | Collecting duct principal cell transport processes and their regulation[END_REF][START_REF] Rossier | Epithelial sodium transport and its control by aldosterone: the story of our internal environment revisited[END_REF]. ENaC and Na-K-ATPase are coordinately activated and up-regulated in the presence of aldosterone, leading to an increase in transepithelial sodium reabsorption via the transcellular route. Aldosterone also stimulates sodium reabsorption in the distal nephron through enhanced expression of the thiazidesensitive NaCl cotransporter and potassium secretion through K channels as ROMK [START_REF] Pearce | Collecting duct principal cell transport processes and their regulation[END_REF][START_REF] Penton | Dietary potassium and the renal control of salt balance and blood pressure[END_REF][START_REF] Rossier | Epithelial sodium transport and its control by aldosterone: the story of our internal environment revisited[END_REF].

Structure of mineralocorticoid receptor I -A -

The mineralocorticoid receptor (MR) is a member of the nuclear receptor (NR) superfamily of ligand-dependent transcription factors, together with the glucocorticoid receptor (GR), the androgen receptor (AR), the progesterone receptor, the estrogen receptor, the thyroid hormone receptor, the retinoic acid receptor(RAR), the peroxisome proliferator-activated receptor (PPAR), the vitamin D receptor (VDR) and numerous orphan receptors [START_REF] Gronemeyer | Principles for modulation of the nuclear receptor superfamily[END_REF][START_REF] Bookout | Anatomical profiling of nuclear receptor expression reveals a hierarchical transcriptional network[END_REF][START_REF] Lu | International Union of Pharmacology. LXV. The pharmacology and classification of the nuclear receptor superfamily: glucocorticoid, mineralocorticoid, progesterone, and androgen receptors[END_REF]. In human, the MR has 984 amino acids (aa), encoded by the NR3C2 gene located on chromosome 4, with 10 exons (the first two exons, 1α and 1β, are untranslated) [START_REF] Morrison | Regional chromosomal assignment of the human mineralocorticoid receptor gene to 4q31.1[END_REF][START_REF] Zennaro | Human mineralocorticoid receptor genomic structure and identification of expressed isoforms[END_REF], while the rat and mouse MR gene is located on chromosome 19, and differs slightly in having three un-translated exons, encoding a 981 aa protein [START_REF] Kwak | 5'-Heterogeneity of the mineralocorticoid receptor messenger ribonucleic acid: differential expression and regulation of splice variants within the rat hippocampus[END_REF]. MR protein has a classical structure of nuclear receptor, with three main domains: a N-terminal domain, followed by a central DNA-binding domain (DBD), and a C-terminal ligand-binding domain (LBD) (Figure 15) [START_REF] Viengchareun | The mineralocorticoid receptor: insights into its molecular and (patho)physiological biology[END_REF][START_REF] Pippal | Structure-function relationships in the mineralocorticoid receptor[END_REF] -The N-terminal domain, consists of 602 aa, is the longest among all the steroid receptors. It possesses several functional domains responsible for ligandindependent transactivation or transrepression including two distinct activation functions-1 regions (AF-1a and AF-1b) and a central inhibitory region. These different regions of the N-terminal domain recruit co-regulators responsible for modulating the transcriptional activity of MR in a highly selective manner and have been considered to be important determinants of mineralocorticoid selectivity (Pascual-Le Tallec and Lombes 2005).

-The DBD contains two motifs of zinc finger's structure that permit binding to DNA and dimerization of receptors. The MR can form homodimers (MR-MR) or can heterodimerize with the GR (MR-GR). The two zinc finger's structures are necessary for binding to hormone responsive elements (HRE). Thus, this domain has the ability to recognize specific target DNA sequences. The MR binds to GRE; there is no MRspecific HRE.

-The LBD is a complex and multifunctional domain that is conserved between species. This domain possesses a ligand-dependent AF-2 and participates in multiple functions, including ligand binding, nuclear localization, dimerization, interaction with heat shock proteins (HSPs), and transcriptional co-activators and ligand-dependent transactivation via AF-2 domain [START_REF] Viengchareun | The mineralocorticoid receptor: insights into its molecular and (patho)physiological biology[END_REF].

The ligands of mineralocorticoid receptor I -B -

Aldosterone and glucocorticoids (cortisol in humans, corticosterone in rodents) are the physiological ligands of MR. These two hormones bind with the same high affinity to MR, even though the dynamic binding parameters and the functional properties of glucocorticoid -MR complexes are somehow different from that of aldosterone -MR [START_REF] Lombes | The mineralocorticoid receptor discriminates aldosterone from glucocorticoids independently of the 11 beta-hydroxysteroid dehydrogenase[END_REF][START_REF] Hellal-Levy | Specific hydroxylations determine selective corticosteroid recognition by human glucocorticoid and mineralocorticoid receptors[END_REF]. According to Hellal-Levy, the aldosterone -MR complex is more effective in terms of transcriptional activity than glucocorticoid -MR complex [START_REF] Hellal-Levy | Specific hydroxylations determine selective corticosteroid recognition by human glucocorticoid and mineralocorticoid receptors[END_REF]). In addition, progesterone and androgens can also bind to the MR and exert partial agonist or antagonist effects depending on the cellular context [START_REF] Quinkler | Renal inactivation, mineralocorticoid generation, and 11beta-hydroxysteroid dehydrogenase inhibition ameliorate the antimineralocorticoid effect of progesterone in vivo[END_REF][START_REF] Marissal-Arvy | Gain of function mutation in the mineralocorticoid receptor of the Brown Norway rat[END_REF][START_REF] Takeda | Effects of aldosterone and angiotensin II receptor blockade on cardiac angiotensinogen and angiotensin-converting enzyme 2 expression in Dahl salt-sensitive hypertensive rats[END_REF].

MR antagonists (MRA) have been developed and are currently used in human clinical care [START_REF] Maron | Aldosterone receptor antagonists: effective but often forgotten[END_REF]. Spironolactone, the most widely used MRA, was approved as a diuretic and natriuretic drug for the management of hypertension and primary aldosteronism, and later on to treat heart failure [START_REF] Menard | The 45-year story of the development of an anti-aldosterone more specific than spironolactone[END_REF]. This is a potent competitive MRA but poorly selective, as it also inhibits the androgen and progesterone receptors, leading to side effects such as gynecomastia, impotence and menstrual disorders. At high concentrations it may also interfere with the GR [START_REF] Kolkhof | Molecular pharmacology of the mineralocorticoid receptor: prospects for novel therapeutics[END_REF]. Eplerenone, a second general MRA, is a very specific antagonist but less effective than spironolactone (MR has about 100 to 500 times less affinity for eplerenone than for spironolactone) [START_REF] Colussi | Spironolactone, eplerenone and the new aldosterone blockers in endocrine and primary hypertension[END_REF]. Of note, the use of MRAs that do not need to be metabolized into active moieties for full efficacy, such as eplerenone may be preferred when effects are expected relatively rapidly after treatment; short half-life is important to consider for safety concerns to yield rapid decrease of active moieties when stopping therapy in case of toxic effect.

In addition to spironolactone and eplerenone, other MRA molecules are used for research on animal or in vitro models including canrenone, RU28318 or RU26752.

Canrenone, an active metabolite of spironolactone, is also used in some countries for human use. An injectable preparation (potassium canrenoate) is available [START_REF] Armani | Mineralocorticoid receptor antagonism induces browning of white adipose tissue through impairment of autophagy and prevents adipocyte dysfunction in high-fat-diet-fed mice[END_REF]. Moreover, novel non-steroidal compounds were also identified or generated recently as potent and selective MRAs, such as BR-4628, BAY 94-8862 (Finerenone) (belonging to the dihydropyridines: L-type calcium channel antagonists), PF-03882845 and SM-368229 [START_REF] Pitt | Safety and tolerability of the novel non-steroidal mineralocorticoid receptor antagonist BAY 94-8862 in patients with chronic heart failure and mild or moderate chronic kidney disease: a randomized, double-blind trial[END_REF][START_REF] Niemann | Mineralocorticoid receptor modulators: a patent review (2007 -2012)[END_REF][START_REF] Pitt | Rationale and design of MinerAlocorticoid Receptor antagonist Tolerability Study-Heart Failure (ARTS-HF): a randomized study of finerenone vs. eplerenone in patients who have worsening chronic heart failure with diabetes and/or chronic kidney disease[END_REF].

Mineralocorticoid receptor specificity I -C -

It has been shown that the MR can bind cortisol and aldosterone with equal high affinity (Kd = 0.5nM) [START_REF] Hellal-Levy | Specific hydroxylations determine selective corticosteroid recognition by human glucocorticoid and mineralocorticoid receptors[END_REF][START_REF] Farman | Multiple aspects of mineralocorticoid selectivity[END_REF]. The mechanisms of differential activation of the MR by the two corticosteroid hormones allowing tissue-specific effects can be considered at the pre-receptor, receptor, and post-receptor levels (Pascual-Le [START_REF] Tallec | The mineralocorticoid receptor: a journey exploring its diversity and specificity of action[END_REF].

The metabolism of cortisol is a key mechanism for maintaining MR selectivity in some target tissues. Indeed, the concentration of cortisol in plasma is 100 to 1000 times higher than that of aldosterone; the mineralocorticoid selectivity mainly depends, at a cellular level, on the enzymatic activity of 11-beta-hydroxysteroid dehydrogenase type 2 (11HSD2), which converts cortisol into inactive cortisone, resulting in the preferential aldosterone-dependent MR activity (Farman and Rafestin-Oblin 2001) (Figure 16). However, the mineralocorticoid specificity is not only ensured by 11HSD2 because its expression is mainly restricted to sodiumtransporting epithelial cells. HSD2 is very low or absent in some MR-expressing tissues such as the myocardium and hippocampus. This is a strong indication that other molecular mechanisms are important determinants of receptor specificity and action. At the receptor level, mineralocorticoid selectivity is conferred by ligand-induced conformational changes of MR, which differ in presence of gluco-and mineralocorticoid ligands leading to differential transactivation activity. It has been shown that aldosterone dissociates more slowly from the receptor and induces more efficient transactivation than cortisol [START_REF] Lombes | The mineralocorticoid receptor discriminates aldosterone from glucocorticoids independently of the 11 beta-hydroxysteroid dehydrogenase[END_REF][START_REF] Farman | Multiple aspects of mineralocorticoid selectivity[END_REF]. The N-/C-terminal interaction of the MR, which may serve to stabilize ligand binding to the receptor [START_REF] Li | A role of the aminoterminal (N) and carboxyl-terminal (C) interaction in binding of androgen receptor to chromatin[END_REF][START_REF] Centenera | The contribution of different androgen receptor domains to receptor dimerization and signaling[END_REF], is stronger in the presence of aldosterone than cortisol [START_REF] Rogerson | Interdomain interactions in the mineralocorticoid receptor[END_REF], suggesting another important step towards ligand specificity of the MR.

At the post-receptor level, several post-translational modifications, such as phosphorylation, ubiquitination, may occur and modify MR activity [START_REF] Viengchareun | The mineralocorticoid receptor: insights into its molecular and (patho)physiological biology[END_REF].

Mineralocorticoid receptor and pathology of skin I -D -

The notion of excessive/inappropriate MR activity in diseases originates from several pathological situations, in animal models and human subjects, revealing the benefit of treatments with MR blockers. Indeed, efficiency of MR antagonists to reduce excessive/inappropriate MR activation in cardio-vascular pathology was provided in experimental and clinical situations. Pionneer clinical trials showed that spironolactone or eplerenone added on top of standard care limits morbidity and mortality in patients with cardiac insufficiency or following myocardial infarction [START_REF] Pitt | The effect of spironolactone on morbidity and mortality in patients with severe heart failure. Randomized Aldactone Evaluation Study Investigators[END_REF][START_REF] Pitt | Eplerenone, a selective aldosterone blocker, in patients with left ventricular dysfunction after myocardial infarction[END_REF]. MR blockade improves vascular dysfunction [START_REF] Leopold | Aldosterone impairs vascular reactivity by decreasing glucose-6-phosphate dehydrogenase activity[END_REF][START_REF] Koenig | Direct role for smooth muscle cell mineralocorticoid receptors in vascular remodeling: novel mechanisms and clinical implications[END_REF], limits progression towards renal failure [START_REF] Shavit | Aldosterone blockade and the mineralocorticoid receptor in the management of chronic kidney disease: current concepts and emerging treatment paradigms[END_REF], or restores vision in patients with chronic serous chorioretinopathy, a vision-threatening eye disease [START_REF] Zhao | Mineralocorticoid receptor is involved in rat and human ocular chorioretinopathy[END_REF]. MR antagonism was also shown to be beneficial in diabetes and metabolic syndrome; it improves glucose tolerance, decreases insulin resistance, plasma levels of trigglycerides and pro-inflammatory cytokines [START_REF] Guo | Mineralocorticoid receptor blockade reverses obesity-related changes in expression of adiponectin, peroxisome proliferatoractivated receptor-gamma, and proinflammatory adipokines[END_REF][START_REF] Hirata | Blockade of mineralocorticoid receptor reverses adipocyte dysfunction and insulin resistance in obese mice[END_REF][START_REF] Wada | Spironolactone improves glucose and lipid metabolism by ameliorating hepatic steatosis and inflammation and suppressing enhanced gluconeogenesis induced by high-fat and high-fructose diet[END_REF].

MR is expressed in different part of skin such as epidermis, hair follicle, sweat and sebaceous glands, vessels, suggesting its multiple functions in cutaneous tissue [START_REF] Kenouch | Human skin as target for aldosterone: coexpression of mineralocorticoid receptors and 11 beta-hydroxysteroid dehydrogenase[END_REF][START_REF] Zennaro | Tissue-specific expression of alpha and beta messenger ribonucleic acid isoforms of the human mineralocorticoid receptor in normal and pathological states[END_REF][START_REF] Farman | The mineralocorticoid receptor as a novel player in skin biology: beyond the renal horizon?[END_REF]. Glucocorticoids are widely used in clinical treatments of chronic inflammatory or auto-immune skin diseases. However, aside the great efficacy, treatments with glucocorticoids are often accompanied by unwanted side-effects including skin atrophy and delayed wound healing, of great concern for patients [START_REF] Hengge | Adverse effects of topical glucocorticosteroids[END_REF]). Glucocorticoids classically act through binding to the glucocorticoid receptor (GR); they can also bind, even with higher affinity, to the MR; MR-glucocorticoid complexes may contribute to glucocorticoid-induced side-effects in the skin (Sainte [START_REF] Marie | Targeted skin overexpression of the mineralocorticoid receptor in mice causes epidermal atrophy, premature skin barrier formation, eye abnormalities, and alopecia[END_REF][START_REF] Farman | The mineralocorticoid receptor as a novel player in skin biology: beyond the renal horizon?[END_REF].

Firstly, unexpected roles of MR in epidermal keratinocytes have been highlighted by Sainte Marie et al, by using a mouse model expressing the human MR under the control of a keratinocyte-specific promoter (K5-MR mice) (Sainte [START_REF] Marie | Targeted skin overexpression of the mineralocorticoid receptor in mice causes epidermal atrophy, premature skin barrier formation, eye abnormalities, and alopecia[END_REF]).

Post-natal induction of MR was associated with alopecia and dysmorphy of hair follicles, whereas gestational expression of the MR led to premature skin barrier formation and epidermal atrophy (Sainte [START_REF] Marie | Targeted skin overexpression of the mineralocorticoid receptor in mice causes epidermal atrophy, premature skin barrier formation, eye abnormalities, and alopecia[END_REF]). The pathways regulated by epidermal MR are still poorly identfied. Of note, we have recently reported that the 3 subunits of the epithelial sodium channel (ENaC) are overexpressed in the skin of K5-MR mice at birth [START_REF] Maubec | Topical Mineralocorticoid Receptor Blockade Limits Glucocorticoid-Induced Epidermal Atrophy in Human Skin[END_REF]. The enhanced ENaC expression may depend on glucocorticoid-activated MR, because of the perinatal rise in plasma glucocorticoid concentration. The blockade of MR could limit the glucocorticoidinduced epidermal atrophy and and it is accompanied by reduced ENaC expression (Sainte [START_REF] Marie | Targeted skin overexpression of the mineralocorticoid receptor in mice causes epidermal atrophy, premature skin barrier formation, eye abnormalities, and alopecia[END_REF][START_REF] Maubec | Topical Mineralocorticoid Receptor Blockade Limits Glucocorticoid-Induced Epidermal Atrophy in Human Skin[END_REF]. The blockade of the activity of ENaC also rescued the reduced keratinocyte proliferation and epidermal atrophy, suggesting an involvement of ENaC activation in the glucocorticoid-stimulated epidermal MR cascade [START_REF] Maubec | Topical Mineralocorticoid Receptor Blockade Limits Glucocorticoid-Induced Epidermal Atrophy in Human Skin[END_REF], and pointing on mineralocorticoid signaling as a novel epidermal modulator.

In addition to their effects on epidermis, aldosterone and MR antagonists have been reported as modulators of elastin and collagen production in dermis of human skin [START_REF] Mitts | Aldosterone and mineralocorticoid receptor antagonists modulate elastin and collagen deposition in human skin[END_REF]. Restoration of dermal homeostasis by MR antagonism may rely on anti-fibrotic and anti-inflammatory actions, leading to improve collagen structural organization and remodeling and cellularity, a mechanism also occuring in heart diseases. In a mouse model of aging skin, associating UV irradiation and metabolic syndrome, Nagase et al. revealed beneficial action of MR blockade in some aginglike features [START_REF] Nagase | Aging-like skin changes in metabolic syndrome model mice are mediated by mineralocorticoid receptor signaling[END_REF]. Recently, Tiganescu et al. showed that the agingdependent dermal atrophy and delayed wound healing in mice were restored by inactivation of the glucocorticoid-activating enzyme 11HSD1 (a reductase that converts inactive glucocorticoids as cortisone into active ones, as cortisol) ); 11HSD1 is expressed in epidermis, and its inactivation leads to reduced local glucocorticoid production, thus limiting the possibility of MR occupancy [START_REF] Tiganescu | 11beta-Hydroxysteroid dehydrogenase blockade prevents age-induced skin structure and function defects[END_REF].

Since the activity of the MR-protecting enzyme 11HSD2 is minimal in human epidermis [START_REF] Kenouch | Human skin as target for aldosterone: coexpression of mineralocorticoid receptors and 11 beta-hydroxysteroid dehydrogenase[END_REF], it permits MR occupancy by glucocorticoids.

Glucocorticoid activation of the MR is most likely to occur in face of local application of dermocorticoids. Whether and how this mechanism could participate to some of the deleterious side-effects of dermocorticoids, as skin atrophy and delayed wound healing, is an important therapeutical issue, as it could be limited by local coadministration of MR antagonists.

GLUCOCORTICOID EXCESS

GR Anti-inflammatory beneficial effects MR Deleterious effects ? MR antagonists

III -Article 3:

I also participated to the work documenting the benefit of MR blockade to limit the glucocorticoid-induced epidermal atrophy [START_REF] Maubec | Topical Mineralocorticoid Receptor Blockade Limits Glucocorticoid-Induced Epidermal Atrophy in Human Skin[END_REF]. In this study, we first reproduced a classical and major deleterious side effect of glucocorticoids, by showing that the potent glucocorticoid clobetasol induces epidermal atrophy in cultured human skin explants and in subjects with local treatment of the glucocorticoid as a gel (in 23 healthy volunteers from the SPIREPI clinical trial). We asked whether glucocorticoid-induced epidermal atrophy could be related to inappropriate activation of cutaneous MR by glucocorticoids. Interestingly, we found that the epidermal atrophy could be limited when glucocorticoid was co-administrated with a MRA (canrenoate or eplerenone), ex vivo in cultured human skin explants and in vivo in the SPIREPI study. Although the mechanisms of this beneficial effect are far from being elucidated, we showed that addition of MRA was associated with the restoration of impaired keratinocyte proliferation. Furthermore, the blockade of the activity of ENaC by phenamil also improved the epidermal atrophy in skin explants and fully restored the impaired keratinocyte proliferation. These results suggest that ENaC activity may participate to the glucocorticoid-stimulated epidermal MR cascade. Altogether, these findings identify MR as a factor regulating epidermal homeostasis and suggest that topical MR blockade could limit glucocorticoid-induced epidermal atrophy.

General discussion, conclusions and perspectives

With increasing life expectancy, the elderly populations are expanding at a high rate, with an escalating incidence of relative pathologies, such as dementia, osteoporosis, stroke, and a risk of delayed healing which in turn leads to a higher incidence of chronic wounds. Management of chronic wounds is prolonged, time consuming and therefore is responsible for a very high cost for many countries, especially in western ones. Importantly, prolonged wounds are responsible for a high rate of complications. Some of these such as severe infection or carcinoma are life threatening. In the same way, as detailed before, chronic resistant and progressive wounds may need amputation. In view of all that consequences, chronic wounds have a public health impact.

The current "gold standard" of wound management comprises regular assessment for infection and risk factors, and daily or multi weekly care of the wound with dressings and/or debridement of necrotic or fibrinous tissue. Other measures focus on secondary complications, such as reducing infection, using silver-impregnated dressings [START_REF] White | Topical therapies for diabetic foot ulcers: standard treatments[END_REF] or maggot debridement therapy [START_REF] Sherman | Maggot therapy for treating diabetic foot ulcers unresponsive to conventional therapy[END_REF][START_REF] Bowling | Larval therapy: a novel treatment in eliminating methicillin-resistant Staphylococcus aureus from diabetic foot ulcers[END_REF]. Besides the symptomatic measures, it is mandatory to consider etiological and or mechanistic treatments measures. The most frequent causes of ulcers are arterial or venous disease. Thereof, restorations of arterial flux through exercise, avoidance of atheroma risk factors, are necessary in such patients.

In the same way, compression dressings for venous ulcers are needed each day [START_REF] Grey | ABC of wound healing. Non-surgical and drug treatments[END_REF]). Offloading therapy is recommended for diabetic ulcers [START_REF] Edmonds | Diabetic foot ulcers: practical treatment recommendations[END_REF]). However, there are no treatment that stimulates wound healing. In the past decade, topical PDGF has been approved for diabetic ulcers but was afterwards withdrawn from the market, possibly because of low efficiency.

Therefore, a better understanding of the molecular and cellular mechanisms underlying chronic non-healing wounds is still needed. While there are differences between the mechanisms of each type of delayed wound healing, there are also several common factors that may be found in various diseases. These include for example prolonged and excessive inflammation, ineffective reepithelialisation, impaired angiogenesis [START_REF] Menke | Impaired wound healing[END_REF]).

In the first part of the thesis work, we focused on the mechanisms involved in sickle cell ulcers. The reason why we chose this disease comes from the clinical situation of affected patients. Indeed, although Sickle cell disease (SCD) is an orphan disease, the course of ulcers is very intriguing. The first peculiar fact is the variation in SS patients of the ulcer incidence. This varies between 0 and 40% depending on the countries, although there are no any linkage between ulcers and the SCD haplotype [START_REF] Halabi-Tawil | Sickle cell leg ulcers: a frequently disabling complication and a marker of severity[END_REF]. However, the unique genetic study that was done has demonstrated polymorphisms in genes intervening in angiogenesis [START_REF] Nolan | Sickle cell leg ulcers: associations with haemolysis and SNPs in Klotho, TEK and genes of the TGFbeta/BMP pathway[END_REF]. SCD ulcers most usually develops after a triggering event: traumatisms mainly, but also insect bite or itching (ref). Once the wound has developed, the course is going to be prolonged in a subset of affected patients. Very impressively, the duration of SCD ulcers is really the most prolonged that may concern human beings, especially that these are still young adults. Indeed, in the series of our department the duration with ulcerated skin was 240 months [START_REF] Halabi-Tawil | Sickle cell leg ulcers: a frequently disabling complication and a marker of severity[END_REF]. In accordance, another most recent study has even found higer duration that are 300 months [START_REF] Minniti | Vasculopathy, inflammation, and blood flow in leg ulcers of patients with sickle cell anemia[END_REF]). Thinking about that duration of ulcer at the same site is a main point in discussing the pathogenesis of SCD ulcers. Indeed, in other thrombotic diseases such as cryoglobulinemia or antiphospholipid syndromes, the ulcers develop due to hypoxia. But afterwards, and even without any specific treatments, each ulcerated lesion will usually heal. This is not the case in SCD patients: there skin wounds persist. Even red blood cell transfusions and/or autologous skin grafting can induce at the best transient improvements with frequent recurrences at the same site (ref). At that point, we postulated that besides and whatever are the ulcer inducing mechanisms, these patients may have a severe abnormality in healing mechanisms.

Another close reason for performing the first work is the absence of availability of tissues to study the healing pathways in humans. Indeed, these patients are affected with very painful ulcers. All procedures, even wound care are very painful. Performing biopsies will be therefore difficult. In addition, since interpretation of the changes found depends on the stage of ulcers, several repeted biopsies will be needed. As an example the study of Marti Cavajal, the most powerful in vivo human study in SCD, has collected samples with a real variability in time course of the ulcers [START_REF] Marti-Carvajal | Interventions for treating leg ulcers in people with sickle cell disease[END_REF]. Finally, biopsies may worsen the course of SCD ulcers as any traumatism.

Therefore, at the beginning of this work, we were facing the challenge of finding the best way to address the question of poor wound healing in SCD. There were no animal model for SCD ulcers and still no recognized one. There were however mice with genetic engineering that were used for other tissue disorders of the disease. This is why we decided to put our efforts in developing a model for SCD skin delayed healing that could allow us to study the mechanisms of this pathogenic phenomenon.

Here, we were able to show that cutaneous wound healing is severely altered in old SAD mice, whereas young animals do not show any abnormality. Importantly, the healing delay in old SAD mice correlated with the degree of anemia and hemolysis.

These features of SAD mice parallel the characteristics of ulcers in SCD patients. Indeed, leg ulcers develop only after 15 years in sickle cell anemia patients, at a median age of 21 years, when the features of SCD have been fully developed [START_REF] Halabi-Tawil | Sickle cell leg ulcers: a frequently disabling complication and a marker of severity[END_REF]). In addition, most of these ulcers present in correlation with a hyper-hemolytic phenotype with increased reticulocytosis (Kato et al. 2006;[START_REF] Taylor | Chronic hyper-hemolysis in sickle cell anemia: association of vascular complications and mortality with less frequent vasoocclusive pain[END_REF][START_REF] Minniti | Laboratory and echocardiography markers in sickle cell patients with leg ulcers[END_REF][START_REF] Minniti | Vasculopathy, inflammation, and blood flow in leg ulcers of patients with sickle cell anemia[END_REF]. Therefore, the poor healing of wounds in old SAD mice appears pertinent to mimic the human SCD leg ulcers and these animals are therefore a new and original model for the study of sickle cell ulcers.

Crucially wound angiogenesis was altered in SAD mice. This reduction appeared to be secondary, at least partly, to a decreased EPC proliferation and mobilization from the bone marrow. Furthermore, a decreased CXCL12 expression by keratinocytes and leukocytes was found in SAD wounds. This was corroborated by the fact that local injection of extra EPCs or better the recombinant CXCL12 into SAD wounds restored the angiogenesis impairment and rescued the healing defect, with a paralell enhanced EPC mobilization. This is the first study of the cellular and molecular mechanisms implicated in sickle cell ulcers in a murine model that provides promising therapeutic perspectives for clinical trials.

Interestingly, normal skin of old SAD mice shows a decrease in blood vessel diameter while vessel numbers are unmodified. This phenotype may relate to hemolysis with consequent reduced NO concentration and vasoconstriction that are common features of SCD patients [START_REF] Frenette | Sickle cell vaso-occlusion: multistep and multicellular paradigm[END_REF]Kato et al. 2006;[START_REF] Mack | Sickle cell disease and nitric oxide: a paradigm shift?[END_REF]. on later specimens to depict the mechanisms implicated in delayed healing of SAD wounds. At day 7 post wounding, we found a significant decrease in CD31 + blood vessels as well as in Lyve-1 + lymphatic vessels in SAD wounds as compared to WT littermates. These decreases are associated with the fewer recruitment of CD31 + CD45 -endothelial cells in SAD wounds than in WT wounds. These results are consistent with the observations in SCD patients, where the treatment with granulocyte/macrophage colony-stimulating factor (GM-CSF) -that stimulates pericyte-like perivascular mural cells to secrete VEGFa-improves angiogenesis and ulcer healing [START_REF] Pieters | Molgramostim to treat SS-sickle cell leg ulcers[END_REF][START_REF] Alikhan | Topical GM-CSF hastens healing of leg ulcers in sickle cell disease[END_REF][START_REF] Mery | Topical effectiveness of molgramostim (GM-CSF) in sickle cell leg ulcers[END_REF][START_REF] Zhao | Granulocyte/macrophage colony-stimulating factor influences angiogenesis by regulating the coordinated expression of VEGF and the Ang/Tie system[END_REF].

In tissue injury, EPCs -as precursors of endothelial cells-proliferate and are released from bone marrow into circulating peripheral blood and incorporated into the foci of neovascularization at wound sides. They have been demonstrated to be implicated in multiple aspects of angiogenesis occurring during wound healing process as well as tumours. Importantly, impaired EPC recruitment has been shown to be involved in delayed wound healing in several disorders such as diabetes [START_REF] Loomans | Endothelial progenitor cell dysfunction: a novel concept in the pathogenesis of vascular complications of type 1 diabetes[END_REF]Fadini et al. 2005;Fadini et al. 2005;[START_REF] Tepper | Decreased circulating progenitor cell number and failed mechanisms of stromal cellderived factor-1alpha mediated bone marrow mobilization impair diabetic tissue repair[END_REF]. In this context, the impaired wound healing was improved by direct topical application of extra EPCs into the wounds but also through hyperbaric oxygen that stimulates EPC mobilization or injection of factors mediating EPC mobilization and angiogenesis [START_REF] Gallagher | Diabetic impairments in NOmediated endothelial progenitor cell mobilization and homing are reversed by hyperoxia and SDF-1 alpha[END_REF][START_REF] Marrotte | Manganese superoxide dismutase expression in endothelial progenitor cells accelerates wound healing in diabetic mice[END_REF]. Of note, some teams have described improvement of ulcer healing by hyperbaric oxygen in SCD patients. However, the mechanism of such improvement has not been studied. The the impaired wound angiogenesis we found in SAD mice was related to low EPCs content in bone marrow and blood postwounding while this number was similar at baseline. These results suggest an inability to increase the EPC proliferation and mobilization from the bone marrow of SAD mice in response to injury. Moreover, to answer to the question of the functional activity of EPCs in old SAD mice, we have studied the phenotype of these cells in vitro condition without the SCD environment. Our results showed that in vitro, SAD EPCs expressed normal abilities of proliferation and differentiation, indicating the absence of an intrinsic disorder of these cells. This result is equal to in vivo results, showing that SAD EPCs exhibited the same efficiency on wound repair as WT EPCs.

Indeed, both WT and SAD EPCs injected directly into SAD wounds were able to rescue the impaired healing of SAD mice by restoring the defect of angiogenesis,.

These results strongly suggest that defective EPC mobilization in old SAD mice is a consequence of micro-environmental changes in marrow. This situation differs from that of diabetes where db/db EPCs display an intrinsically abnormal phenotype in vitro and in vivo [START_REF] Marrotte | Manganese superoxide dismutase expression in endothelial progenitor cells accelerates wound healing in diabetic mice[END_REF]. Altogether our results strongly suggest that the defect of EPCs in old SAD mice is the defect of mobilization only.

EPC mobilization can be modulated by various intrinsic and extrinsic factors. Here, we found a significant decrease in the expression of several cytokine or chemokine encoding genes including VEGFa, VEGFR2, Tie1, Tie2, IL6, CXCL12 and its receptor CXCR4 in SAD wounds. The CXCL12/CXCR4 axis plays an important role in the homing of progenitor cells in the bone marrow, and in their mobilization to the periphery under various conditions including wound healing [START_REF] Avniel | Involvement of the CXCL12/CXCR4 pathway in the recovery of skin following burns[END_REF][START_REF] Mazo | Hematopoietic stem and progenitor cell trafficking[END_REF][START_REF] Hu | CXCL12/CXCR4 axis promotes mesenchymal stem cell mobilization to burn wounds and contributes to wound repair[END_REF]. Importantly, we found here a correlation between the mRNA level of CXCL12 in wounds and the content of circulating EPCs in the blood.

The injection of CXCL12 into the wounds of SAD mice enabled the wounds to heal as quickly as controls. This improvement of healing is accompanied by the increase in circulating EPCs in CXCL12-treated mice. Importantly, the improvement in EPC mobilization in the SAD mice is positively correlated with the observed increase in wound angiogenesis after CXCL12 injection. These findings strongly suggest that CXCL12-induced increase in EPC mobilization is responsible for the improvement of SAD wound healing. Therefore, the defect in the CXCL12/CXCR4 axis is a major event leading to impaired EPC recruitment and angiogenesis defect to skin wounds in old SAD mice. This is at variance with a previous observation showing an higher CXCL12 levels in plasma of SCD patients than in healthy controls [START_REF] Landburg | Elevated circulating stromal-derived factor-1 levels in sickle cell disease[END_REF]). However, levels of this chemokine did not increase during vaso-occlusive crisis, in contrast with healthy subjects as one should expect. This can indicate a human defect in CXCL12 increase in wound reponse. We believe that this should be addressed in the human setting soonly in view of our results.

The low expression of CXCL12 originates from both keratinocytes and CD45 + leukocytes in healing skin. Our findings are partly in accordance with the observation that the impaired angiogenesis and delayed wound healing in diabetic mice, are associated with the reduction of CXCL12 secretion in wound by keratinocytes and myofibroblasts, and that these defects can be improved, at least in part, by chemokine injection [START_REF] Gallagher | Diabetic impairments in NOmediated endothelial progenitor cell mobilization and homing are reversed by hyperoxia and SDF-1 alpha[END_REF]). In accordance with this notion, inhibition of SDF-1 in the wounds through lentivirus further decreased angiogenesis and healing delay in diabetic mice [START_REF] Bermudez | Inhibition of stromal cell-derived factor-1alpha further impairs diabetic wound healing[END_REF]. Furthermore, inhibition of CXCR4 through topical application of AMD 3100 to diabetic wounds also induces EPC mobilization [START_REF] Nishimura | CXCR4 antagonist AMD3100 accelerates impaired wound healing in diabetic mice[END_REF]. Several studies have shown that beside the effect in inducing EPC mobilization, local injection of some chemokines, such as granulocyte-colony stimulating factor, led to unwanted side-effects with an associated increase in leukocytes resulting in inflammation [START_REF] Morimoto | Leucocytosis induced in rabbits by intravenous or central injection of granulocyte colony stimulating factor[END_REF]. From this context, the questions about the safety and clinical utility of these chemokines were raised. However, our SAD mice did not show any abnormality in skin after CXCL12 injection. Future studies should nevertheless address the question of consequences of CXCL12 injections in wound on other organs as well as on blood cell counts.

During the inflammatory stage of wound healing, neutrophils and macrophages are two main important cell types that infiltrate to wound area to prevent ongoing blood and fluid losses, to phagocytize bacteria and debris to prevent infection. Several reports have demonstrated an up-regulation of inflammatory pathways in SCD patients with ulcers, suggesting an involvement of inflammation in pathogenesis of this disorder [START_REF] Lard | Neutrophil activation in sickle cell disease[END_REF][START_REF] Bowers | Blood viscosity and the expression of inflammatory and adhesion markers in homozygous sickle cell disease subjects with chronic leg ulcers[END_REF][START_REF] Minniti | Vasculopathy, inflammation, and blood flow in leg ulcers of patients with sickle cell anemia[END_REF]). However, using different techniques in repeated experiments, we did not observe any changes in recruitment of neutrophil and macrophage/monocyte at the skin and peripheral blood levels after wounding. These results indicate that there is no implication of neutrophils and macrophages migration in the SCD delayed healing. In clinical series above referenced, the comparisons were made between patients with and without ulcers. While, the power of our study was to compare SAD and WT littermates at the same stage of wound healing where quantitative analysis of cell infiltrate was done.

We believe this is an unbiased tool for addressing inflammatory abnormal changes.

Our observations appear to be different with the previously published data reporting a high content of neutrophils and macrophages in the bloodstream and liver of SAD mice, in association with hepatopathy induced by hypoxia/reperfusion [START_REF] Siciliano | Abnormal modulation of cell protective systems in response to ischemic/reperfusion injury is important in the development of mouse sickle cell hepatopathy[END_REF]). However, our SAD mice -housed in specific pathogen free conditions-did not have higher neutrophil counts after surgical wounding. Altogether, our results indicate that the abnormal healing in SAD mouse appears to differ from other SCD organ damage through the absence of neutrophil and macrophages increase.

In conclusion, old SAD mice present a delayed healing that correlates with anemia and haemolysis and therefore parallel the human situation. This prolonged healing delay relates to the poor EPC proliferation and mobilization from the bone marrow, due to impaired CXCL12 secretion by keratinocytes and leukocytes in wounded skin.

However, the question of why these cells have this defect still remains unanswered.

Future studies should target on exploring the link between sickle cell anemia and the defect of CXCL12 secretion by keratinocytes and leukocytes in the wounding process. Physiologically, CXCL12 expression is known to be induced in a wide variety of cell types in response to stimuli including ischemia and hypoxia [START_REF] Ceradini | Progenitor cell trafficking is regulated by hypoxic gradients through HIF-1 induction of SDF-1[END_REF][START_REF] Miller | The neuroblast and angioblast chemotaxic factor SDF-1 (CXCL12) expression is briefly up regulated by reactive astrocytes in brain following neonatal hypoxic-ischemic injury[END_REF][START_REF] Lee | A subset of human rapidly self-renewing marrow stromal cells preferentially engraft in mice[END_REF]. Interestingly, hypoxia and diminished bioavailability of NO, caused by the hemolysis and vaso-occlusion, are common features that induce mortality and morbidity in SCD patients and animal models, [START_REF] Reiter | Cell-free hemoglobin limits nitric oxide bioavailability in sickle-cell disease[END_REF]Kato et al. 2006;[START_REF] Sabaa | Endothelin receptor antagonism prevents hypoxiainduced mortality and morbidity in a mouse model of sickle-cell disease[END_REF]. From this context, one possible hypothesis for the decreased CXCL12 expression in SAD wounds is that there is an inability to overexpress this chemokine in SAD mice in response to ischemia-induced hypoxia (as occuring in normal situation). This impairement can be explained by the defect of signal connection between bone marrow microenvironment and cutaneous tissue in SAD mice. Future studies are needed to examine more specifically whether there is any abnormality in marrow microenvironment of SAD mice that can affect CXCL12 secretion in skin, and EPC mobilization from bone marrow niches to circulation. One important event should be explored is the production of NO and the reactive oxygen species inside marrow environment of SAD mice in the setting of wounding. Moreover, it is also important to analyse the impact of CXCL12 pathway in patients with SCD ulcers. Since mice did not show significant side effects after CXCL12 injection [START_REF] Gallagher | Diabetic impairments in NOmediated endothelial progenitor cell mobilization and homing are reversed by hyperoxia and SDF-1 alpha[END_REF], and in view of the severity of SCD ulcers, this peptide could be a promising therapy in clinical trials for improving this condition.

In addition to CXCL12/CXCR4 axis, other molecules or pathways that can be responsible for the delayed healing of SAD wounds and sickle cell ulcers should be explored, to get further understanding about the pathogenesis, and to open on new therapies for management of this disorder. Inthis regard, we have performed the RNA sequencing analysis of EPCs isolated from bone marrow of old SAD and WT mice after 5 days of wounding. Interestingly, our preliminary results ipoint on some molecules and pathways that can be involved in the delayed wound healing of SAD mice, since some upstream pathways are significant up-regulated in SAD mice vs WT, together with several down-regulated pathways.

Among activated pathways, histone deacetylase (HDAC) appears to be the most interesting, since the activation score is high, and since HDAC inhibitors have been shown to exhibit multiple modalities of benefit for some disorders including SCD [START_REF] Balasubramanian | A novel histone deacetylase 8 (HDAC8)-specific inhibitor PCI-34051 induces apoptosis in T-cell lymphomas[END_REF][START_REF] Hebbel | The HDAC inhibitors trichostatin A and suberoylanilide hydroxamic acid exhibit multiple modalities of benefit for the vascular pathobiology of sickle transgenic mice[END_REF]. Indeed, HDACs are enzymes that affect gene expression through their influence on chromatin-modifications, by controlling the acetylation of the core histones. The acetylation and deacetylation of histones play a significant role as transcriptional regulators in various cell types.

HDAC inhibitors such as trichostatin A (TSA) have been shown to inhibit vascular cell adhesion molecule-1 and tissue factor expression that are present at high level in SCD [START_REF] Belcher | Critical role of endothelial cell activation in hypoxia-induced vasoocclusion in transgenic sickle mice[END_REF][START_REF] Inoue | Histone deacetylase inhibitor reduces monocyte adhesion to endothelium through the suppression of vascular cell adhesion molecule-1 expression[END_REF][START_REF] Wang | Histone deacetylase inhibitors suppress TF-kappaB-dependent agonist-driven tissue factor expression in endothelial cells and monocytes[END_REF][START_REF] Bowers | Blood viscosity and the expression of inflammatory and adhesion markers in homozygous sickle cell disease subjects with chronic leg ulcers[END_REF].

Consistently, studies of Hebbel and colleagues indicate that TSA and its analog SAHA (suberoylanilide hydroxamic acid) could provide true, multimodality, salubrious therapy for prevention and treatment of chronic vasculopathy of SCD [START_REF] Hebbel | The HDAC inhibitors trichostatin A and suberoylanilide hydroxamic acid exhibit multiple modalities of benefit for the vascular pathobiology of sickle transgenic mice[END_REF]. SAHA is also already approved for clinical human use in other disorder as cutaneous T-cell lymphoma. Moreover, Du and colleagues have recently demonstrated that HDAC inhibition prevents cell death, increase cell survival, and reduces the production of ROS and apoptosis in cardiomyocytes exposed to hypoxia/reoxygenation [START_REF] Du | HDAC4 degradation mediates HDAC inhibition-induced protective effects against hypoxia/reoxygenation injury[END_REF]. More interestingly, class I and III HDAC inhibitors have been shown to accelerate cutaneous wound healing in a mouse model through stimulating keratinocyte proliferation and NO production [START_REF] Spallotta | A nitric oxide-dependent cross-talk between class I and III histone deacetylases accelerates skin repair[END_REF]. Hence, testing the effects of HDAC inhibitors for improving the delayed healing of SAD wounds should be done in order to provide a promising therapy for treatment of sickle cell leg ulcers. In addition to HDAC, several molecules, pathways such as NR1H2, NR1H3, or ABCA1, that are activated in SAD mice, should be interesting to explore.

In contrast to the up-regulated pathways, several genes or pathways apeared to be inhibited in SAD vs WT mice, including upstream of PDGF-BB, CCL2/CCR2, CCL5/CCR5 signalling. These molecules are known to play important roles in multiple aspects of wound healing process. PDGF-BB (Platelet-derived growth factor) is a protein known to generally enhance cell growth, progenitor cell migration and angiogenesis. Therefore, this is a beneficial molecule used for treatment of chronic and non-healing foot ulcer in some diseases, such as diabetes [START_REF] Nagai | Becaplermin: recombinant platelet derived growth factor, a new treatment for healing diabetic foot ulcers[END_REF][START_REF] Chan | Effect of recombinant platelet-derived growth factor (Regranex) on wound closure in genetically diabetic mice[END_REF][START_REF] Allen | Combination therapy accelerates diabetic wound closure[END_REF]. Together with CXCL12/CXCR4 signalling, CCR5-, and CCR2-mediated signalling are major regulators of EPC homing into injured sites. Indeed, the expression of CCR5 and its ligand CCL5 is up-regulated in response to injury. This chemokine is expressed by various cell types including macrophages and endothelial cells. Importantly, the CCL5/CCR5 interaction can directly control EPC recruitment, thereby contributing to neovascularization and subsequent wound healing [START_REF] Ishida | Pivotal role of the CCL5/CCR5 interaction for recruitment of endothelial progenitor cells in mouse wound healing[END_REF]. Hence, the impact of these molecules and signalling in the delayed healing of SAD wounds need to be studied in order to modulate impaired angiogenesis and poor healing of SAD wounds.

A key aim of the second part of this thesis was the identification of a novel pathway that influences the cutaneous wound closure. The objectives were to identify the cellular and molecular effects of MR and its antagonists in the early phase of healing, focusing on re-epithelialization. Our results open on the possibility to develop alternative treatments to accelerate wound repair in pathological situations.

Although it is known that glucocorticoids (endogenous or exogenous) delay wound healing, via binding to the GR [START_REF] Youm | Local blockade of glucocorticoid activation reverses stress-and glucocorticoid-induced delays in cutaneous wound healing[END_REF][START_REF] Tiganescu | Increased glucocorticoid activation during mouse skin wound healing[END_REF], additional mechanisms may exist. Indeed glucocorticoid-induced skin atrophy, another severe deleterious effect of glucocorticoids, is limited by topical administration of MR blockers, as shown in a recent report (where I was involved; see the article 3 in chapter 3) by an experimental ex vivo approach and in vivo test through the SPIREPI clinical trial in healthy volunteers [START_REF] Maubec | Topical Mineralocorticoid Receptor Blockade Limits Glucocorticoid-Induced Epidermal Atrophy in Human Skin[END_REF]. In this part of my thesis, we hypothesized that MR-bound glucocorticoid complexes may be one mechanism for glucocorticoid-induced impaired wound re-epithelialization.

In order to expand our understanding of the role of mineralocorticoid signaling during cutaneous wound healing, the role of the MR was assessed through different approaches: we used skin explants from a transgenic mouse model with keratinocyte-specific MR overexpression (K5-MR mice), local application of different MR antagonists over wounds of adult wild-type mouse back skin and human skin explants in organotypic culture. We focused mostly on the model of impaired wound healing featured by dermocorticoid pretreatment, and also tested the efficiency of MR blockers on impaired wound healing in mouse models of diabetes.

First, we found that MR antagonism does not influence the wound closure in healthy skin. Benefit was observed only on pathological skin. This is important to notice, to avoid excessive extrapolation of our results.

The reduced outgrowth of skin explants from K5-MR mice can be partly reversed by treatment with the MRA canrenoate, suggesting a role of MR in the delayed wound re-epithelialization. MRA also induced wound re-epithelialization in pathological murine models (local glucocorticoid treatment, diabetes), where the wound closure is known to be impaired. The efficiency of the MRAs canrenoate or spironolactone on human skin explants in organotypic culture was also observed, although these compounds are not fully specific for MR, as they can be antagonists for other nuclear receptors such as androgen receptor [START_REF] Kolkhof | Molecular pharmacology of the mineralocorticoid receptor: prospects for novel therapeutics[END_REF]. This is why we also treated human explants with the highly specific MR antagonist eplerenone, that also showed the same efficiency in improving re-epithelialization as spironolactone and canrenoate (because of its very poor solubility, it was not tested over mouse backskin). Thus, we provided evidence that MR blockade improves reepithelialization of pathological wounds. As a preliminary step in human study of effects of MRA in patients, we showed that the healthy volunteers of the SPIREPI study that were treated locally with clobetasol, exhibited delayed closure of the wound following skin biopsy that was slightly improved in the zones where spironolactone was associated with clobetasol.

We identified some of the mechanisms at the origin of the beneficial effect of MRA on the wound re-epithelialization. Firstly, we have shown that the MR is overexpressed in the skin of mice pre-treated with the dermocorticoid clobetasol, and mice with type I and type II diabetes. Such up-regulation provides clues to understand why MRA can be efficient in these pathological situations. Of note, an upregulation of MR in skin of mice with UV irradiation and metabolic syndrome has been previously reported [START_REF] Nagase | Aging-like skin changes in metabolic syndrome model mice are mediated by mineralocorticoid receptor signaling[END_REF]. In this model, considered by the authors as featuring aging skin, MRA treatment (topical spironolactone) reversed the enhanced oxidative stress and inflammation markers, and the histological lesions of the skin [START_REF] Nagase | Aging-like skin changes in metabolic syndrome model mice are mediated by mineralocorticoid receptor signaling[END_REF]. Thus the efficiency of cutaneous MR blockade to improve wound closure in pathological situations may be explained by enhanced MR expression. More generally, benefits of treatment with MR blockers to limit pathology have been reported in experimental and clinical situations including cardiac insufficiency or following myocardial infarction [START_REF] Pitt | The effect of spironolactone on morbidity and mortality in patients with severe heart failure. Randomized Aldactone Evaluation Study Investigators[END_REF][START_REF] Pitt | Eplerenone, a selective aldosterone blocker, in patients with left ventricular dysfunction after myocardial infarction[END_REF], vascular dysfunction [START_REF] Leopold | Aldosterone impairs vascular reactivity by decreasing glucose-6-phosphate dehydrogenase activity[END_REF][START_REF] Adel | Spironolactone improves endothelial dysfunction in streptozotocin-induced diabetic rats[END_REF][START_REF] Koenig | Direct role for smooth muscle cell mineralocorticoid receptors in vascular remodeling: novel mechanisms and clinical implications[END_REF], renal failure [START_REF] Shavit | Aldosterone blockade and the mineralocorticoid receptor in the management of chronic kidney disease: current concepts and emerging treatment paradigms[END_REF], chronic serous chorioretinopathy [START_REF] Zhao | Mineralocorticoid receptor is involved in rat and human ocular chorioretinopathy[END_REF], and metabolic syndrome and diabetes-induced complications [START_REF] Guo | Mineralocorticoid receptor blockade reverses obesity-related changes in expression of adiponectin, peroxisome proliferatoractivated receptor-gamma, and proinflammatory adipokines[END_REF][START_REF] Hirata | Blockade of mineralocorticoid receptor reverses adipocyte dysfunction and insulin resistance in obese mice[END_REF][START_REF] Wada | Spironolactone improves glucose and lipid metabolism by ameliorating hepatic steatosis and inflammation and suppressing enhanced gluconeogenesis induced by high-fat and high-fructose diet[END_REF]). In addition to pathological situations mentioned above, MRA also improved wound closure in a murine model of sickle cell disease with delayed wound healing (SAD mice). Indeed in preliminary experiments, we observed that local canrenoate application over dorsal wounds of 6 SAD mice (2 years old) resulted in enhanced wound closure at day 5, that was twice that of wounds treated with PBS (p= 0.0022). Such observation requires confirmation; it will be also mandatory to investigate MR mRNA levels in this setting. Thus, although the underlying mechanisms are far from being known, blockade of MR may represent a useful therapeutic approach in dermatology. Future studies need to be done on other models of skin pathology to investigate the cellular and molecular pathways implicated in the wound healing modulation of MR.

The question of which cell type express enhanced MR level will be interesting to study. To identify that, the expression of MR should be analyzed in cells sorted from skin and wound, or through immunostaining of skin sections with cell-specific markers. As an illustration of this question, the search for cells involved in delayed wound healing following androgen receptor stimulation revealed that effect of androgens depend on the androgen receptor expressed by macrophages, not by keratinocytes [START_REF] Ashcroft | Androgen receptor-mediated inhibition of cutaneous wound healing[END_REF][START_REF] Lai | Monocyte/macrophage androgen receptor suppresses cutaneous wound healing in mice by enhancing local TNF-alpha expression[END_REF]. Whether this is also the case for MR-sensitive impaired wound closure should be determined.

The MR-dependent cascade in abnormal wound re-epithelialization involves control of keratinocyte proliferation rate: we report here that clobetasol-induced impaired wound closure was associated with reduction in epidermal Ki67 index, that was fully restored when MRA were applied onto the wound. Future experiments should characterize this effect more precisely. Another MR-related event is the modulation of expression of the epithelial sodium channel ENaC. The three subunits of ENaC (alpha, beta and gamma) are expressed in epidermis, and participate to epidermal homeostasis [START_REF] Brouard | Epithelial sodium channel in human epidermal keratinocytes: expression of its subunits and relation to sodium transport and differentiation[END_REF]. The keratinocyte-specific knock out of ENaC alpha subunit in mice results in epidermal hyperplasia [START_REF] Mauro | The ENaC channel is required for normal epidermal differentiation[END_REF]. We found that the glucocorticoid-induced overexpression of ENaC in wounds of mouse back skin was reversed by local administration of canrenoate on the wounds. We also observed, in our ex vivo wound healing assay, that the glucocorticoid-induced delayed epidermal outgrowth of human skin explants in culture was improved by coincubation with phenamil, a blocker of this channel. Our finding that ENaC blockade by phenamil enhances re-epithelialization in human skin explants exposed to glucocorticoids provides functional evidence for the involvement of ENaC in wound closure. It would be interesting to evaluate the wounding capacity of the skin of mice with invalidation of ENaC subunits in keratinocytes.

Another main cutaneous side effect of glucocorticoids is skin atrophy characterised by thin, shiny, and fragile skin with telangiectasia or bruising. I participated to a recent study investigating whether glucocorticoid-induced epidermal atrophy may be caused in part by inappropriate occupancy of MR by glucocorticoids [START_REF] Maubec | Topical Mineralocorticoid Receptor Blockade Limits Glucocorticoid-Induced Epidermal Atrophy in Human Skin[END_REF]. We showed that local MRA treatment limits the epidermal atrophy and the impaired keratinocyte proliferation induced by clobetasol in cultured human skin explants. We also identified ENaC as a novel epidermal MR target, possibly participating to the beneficial effect of MR antagonism, through restoration of keratinocyte proliferation. In the randomized double-blind SPIREPI phase 2 clinical trial, we provided evidence that these findings have clinical relevance, as topical spironolactone administration in healthy volunteers led to improvement of the clobetasol-induced epidermal atrophy [START_REF] Maubec | Topical Mineralocorticoid Receptor Blockade Limits Glucocorticoid-Induced Epidermal Atrophy in Human Skin[END_REF].

In addition to benefit of MR blockade on epidermis (to improve keratinocyte proliferation and re-epithelialization) reported here, MRA may also affect other events in the dermis after wounding, such as inflammation, angiogenesis, and dermal remodeling. In the SPIREPI study, we observed that skin atrophy induced by dermocorticoid was improved at the level of whole skin (measured by echography), i.e. not only in epidermis [START_REF] Maubec | Topical Mineralocorticoid Receptor Blockade Limits Glucocorticoid-Induced Epidermal Atrophy in Human Skin[END_REF]. Endothelial (and smooth muscle) vascular cells, sweat and sebaceous glands, infiltrating monocyte-macrophages, and perhaps EPC recruited at the wound site, all express the MR and may be targets of local application of MRA. It has been reported that aldosterone and MR antagonists modulate elastin and collagen content of human skin or mouse skin [START_REF] Mitts | Aldosterone and mineralocorticoid receptor antagonists modulate elastin and collagen deposition in human skin[END_REF]. MR blockade could reduce oxidative stress and improve endothelial function [START_REF] Rajagopalan | Mineralocorticoid receptor antagonism in experimental atherosclerosis[END_REF][START_REF] Schafer | Addition of the selective aldosterone receptor antagonist eplerenone to ACE inhibition in heart failure: effect on endothelial dysfunction[END_REF]. Adel et al showed that spironolactone improves endothelial dysfunction in streptozotocin-induced diabetic rats, at least partially via its anti-inflammatory and anti-oxidative effects [START_REF] Adel | Spironolactone improves endothelial dysfunction in streptozotocin-induced diabetic rats[END_REF]. Of major interest is the recent report that EPC express the MR and that EPC treatment with aldosterone induces translocation of the MR and impairs functions of EPC, such as differentiation, migration, through increase in reactive oxygen species; in this study, impairment of EPC function was rescued by pharmacological blockade or genetic ablation of the MR [START_REF] Thum | Impairment of endothelial progenitor cell function and vascularization capacity by aldosterone in mice and humans[END_REF]). Although we did not observe changes in the number of the inflammatory cell infiltration in clobetasol-treated skin in the presence or absence of MRA, the effect of MRAs in inflammation or angiogenesis in the wound, as well as the recruitment of bone marrow-originating cells, would be an important issue to investigate. The same questions may arise for impaired wound healing of diabetic mouse models. Since our preliminary results showed that the MRA canrenoate improves delayed wound healing of SAD mice (that show an impaired angiogenesis and poor mobilization of EPCs), the efficiency of MRA in these events should be studied in the next step.

From our findings that MR antagonism improves glucocorticoid-induced epidermal atrophy and impaired wound closure, it is tempting to propose local application of MRA to bring benefit to patients. Local MRA treatment of the abnormal skin should limit systemic effects of these drugs. It will be also important to determine whether the benefits of glucocorticoids are maintained in the presence of MRA. MR blockers should be reformulated to enhance their penetration inside the skin. Additional studies should allow extending our findings to glucocorticoid-treated patients with chronic skin diseases, to evaluate safety of topical spironolactone treatment and its ability for improving tolerance to topical glucocorticoids and quality of life.

In conclusion, my thesis work has been focussed on impaired wound healing, to clarify some of the mechanisms involved and possibly to open new therapeutical possibilities. My contribution to the field will be a strong motivation for my projects.

  montrons que les MRA améliorent le RETCIC pathologique, et n'affectent pas la peau saine normale. Nous avons montré que l'effet bénéfique des MRA topiques implique une augmentation de la prolifération kératinocytaire et le canal sodium épithelial. Ces résultats montrent un rôle inédit et important du MR cutané dans la cicatrisation. my thesis have been done at the "Team of progenitors and endothelial cells during and after pregnancy", led by Professor Sélim Aractingi. Address: Laboratory of progenitors and endothelial cells during and after pregnancy Saint Antoine research center, UPMC/INSERM UMR S Mechanisms implicated in normal skin wound healing ....................................................

Figure 1 .

 1 Figure 1. Stages of wound healing process.

Figure 2 .

 2 Figure 2. The first stage of wound healing -inflammation.

Figure 4 .

 4 Figure 4. Angiogenesis and vasculogenesis: This is part of the proliferative phase. Angiogenesis is predominantly regulated by macrophages, endothelial cells and keratinocytes. The most important mediator during this phase is VEGF (Mahdavian Delavary et al. 2011).

Fibroblasts

  proliferate, migrate to the center of wound and invade the granulation tissue. They are activated essentially by growth factors PDGF, EGF and FGF and inflammatory cytokines IL-1 and TNF-α secreted by platelets, macrophages and keratinocytes[START_REF] Broughton | Wound healing: an overview[END_REF]) (Figure5).

Figure 5 .

 5 Figure 5. Migration and proliferation of fibroblast in wound Source: (Van De Water et al. 2013) (A) In normal tissues, fibroblasts are shielded from "routine" external mechanical perturbations in the skin by ECM. Following a skin injury, the wound is filled with a provisional matrix of fibrin, fibronectin and a complement of growth factors and cytokines. Fibroblasts, along with blood vessels, are stimulated to migrate into center of wound, forming granulation tissue. (B) Tractional forces accompanying fibroblast migration are responsible for local areas of increased stiffness in newly made collagen. Focal adhesion assembly is increased with increasing stiffness and this is accompanied by clustering of integrins within focal adhesions and increased stress fiber assembly resulting in fibroblast acquisition of the proto-myofibroblast phenotype.

Figure 3 .

 3 Figure 3. The new tissue formation phase (including reepithelialization): during this phase active fibroplasia, neovascularization and reepithelization occurs.Dependent on the macrophage phenotype more or less ECM is produced via fibroblasts and myofibroblasts (Mahdavian[START_REF] Delavary | Macrophages in skin injury and repair[END_REF]

Figure 6 .

 6 Figure 6. Tissue remodeling phase: During this phase the balance between ECMbreakdown and -formation is important and determines the eventual scar result. The balance is determined by among others the micro-environment, macrophage phenotype and T-cell response (Mahdavian Delavary et al. 2011).

Figure 7 :

 7 Figure 7 : Implication of EPCs in wound angiogenesis and vasculogenesis Light microscopic detection of EPCs in wound-healing site created in skin of Flk-1/LZ/ BMT and Tie-2/LZ/BMT mice. Flk-1-expressing EPCs in granulation tissue (A) of Flk-1/LZ/BMT and Tie-2-expressing EPCs in blood vessels (D) of Tie-2/LZ/BMT were observed 4 days after wounding (Asahara et al. 1999).

Figure 8 .

 8 Figure 8. Schematic representation of interactions between VEGF family members and their receptors. This is a complex network of interactions of VEGF family members with transmembrane and soluble VEGFR-1 (sVEGFR-1), VEGFR-2, VEGFR-3, and Nrp (Eming et al. 2007) .

  Figure 9. Critical cytokines, chemokines implicated in vasculogenesis. Signaling molecules such as VEGF, SDF-1, FGF, sphingosine-1phosphate (S1P), eNOS, MMP9, and TGF𝛽 regulate the function of EPCs during vasculogenesis. Pericyte activity during vasculogenesis appears to be modulated by PDGF, TGF𝛽, and possibly S1P

Figure 10 .

 10 Figure 10. Types of chronic wounds: A) pressure ulcer (Inui et al. 2010), B)venous leg ulcer[START_REF] Grey | Venous and arterial leg ulcers[END_REF], C) diabetic foot ulcer[START_REF] Olea | First report of myiasis caused by Cochliomyia hominivorax (Diptera: Calliphoridae) in a diabetic foot ulcer patient in Argentina[END_REF]

  Once ischemia having led to tissue necrosis, a complete wound repair cannot occur because of the same ischemic mechanisms. Causes of lower limbs atheroma are mainly (a) tobacco intake, (b) overweight, (c) elevated cholesterol levels, (d) diabetes, (e) hypertension. Causes of venous insufficiency are (a) genetic background, (b) repeated pregnancies in females (c) overweight, (d) prolonged A B C

  These cells are heterogeneity in genotypes and phenotypes as Herrick first described in 1910 (Figure11)[START_REF] Herrick | Peculiar elongated and sickle-shaped red blood corpuscles in a case of severe anemia[END_REF][START_REF] Rees | Sickle-cell disease[END_REF]).

  Figure 11. Peripheral blood smear of a patient with sickle-cell anemia. This blood film shows irreversibly sickled cells (S), a nucleated red blood cell (NR), and a Howell-Jolly Body (HJB) (Rees et al. 2010).

  development of vasculopathy. HbS=sickle haemoglobin. NO=nitric oxide. VCAM=vascular cell-adhesion molecule. (Rees et al. 2010) Oxygenated erythrocyte containing HbS Deoxygenated erythrocyte with polymerization of HbS Dehydrated sickled erythrocyte

  SCD ulcers are resistant to all current treatments including skin grafts (Marti-Carvajal et al. 2014). Probably due, to this long-lasting nature of the ulcers, SCD ulcers display a high level of complications. There are soft tissue infections in %, tibio tarsal ankylosis in 50% of affected patients, soft tissue infections and chronic pain (Halabi-Tawil et al. 2008).

Figure 14 .

 14 Figure 14. Sickle cell leg ulcers Source: (Amini-Adle et al. 2007)

Figure 15 .

 15 Figure 15. Schematic representation of human MR structure (Viengchareun et al. 2007). MR gene, mRNA, protein, functional domains and associated posttranslational modifications are depicted. The human MR (hMR) gene is composed of ten exons, including two untranslated first exons (1α and 1β). The receptor has distinct functional domains (activation function AF-1a, AF-1b and AF-2) and nuclear localization signals (NLS0, NLS1 and NSL2), as well as one nuclear export signal (NES). The positioning of amino acids targeted for phosphorylation, sumoylation, acetylation and ubiquitylation is indicated.

Figure 16 .

 16 Figure 16. Schematic representation of the MR selectivity. Aldosterone target cells express both the MR and the GR. The MR may be occupied by aldosterone (kidney cells) and also by glucocorticoids (that are much more abundant than aldosterone in the plasma) in cells with low activity of the glucocorticoid-metabolizing

  Schacke et al.

	2002; Barnes and Adcock 2009; De Bosscher and Haegeman 2009). Study of
	Sanchis et al. showed that overexpression of GR in keratinocytes lead to a reduced
	re-epithelialization and delayed wound healing, in correlation with repressed KGF
	expression and decreased ERK activity (Sanchis et al. 2012). The role of GR and

glucocorticoids in skin and cutaneous wound healing will be detailed later (MR chapter).