Les cellules endothéliales progénitrices («Endothelial Progenitor Cells», EPCs) sont des précurseurs endothéliaux qui possèdent un potentiel considérable dans la réparation et la régénération vasculaire. Dans le contexte des maladies cardiovasculaires, la compréhension du rôle des EPCs dans la régulation de la thrombogenèse et la réparation endothéliale est pertinente et nécessaire pour comprendre leur potentiel thérapeutique. Nous avons rapporté que les EPCs interagissent avec les plaquettes via la P-sélectine et inhibent l’adhésion, l’activation et l’agrégation des plaquettes ainsi que la formation de thrombus. Plus récemment, nous avons démontré que les EPCs expriment le récepteur inflammatoire CD40 et il est bien connu que les plaquettes constituent la source principale de la forme soluble de son agoniste le CD40L («soluble CD40 Ligand», sCD40L). Ainsi, nous avons émis l’hypothèse principale que l’axe CD40L/CD40 dans les EPCs influence leurs fonctions anti-thrombotique et pro-angiogénique. Pour vérifier cette hypothèse, nous avons réussi à générer des «early» et «late» EPCs à partir de cellules mononucléaires du sang périphérique («Peripheral Blood Mononuclear Cells», PBMCs) en culture. Nous avons mis en évidence l’existence de l’axe CD40L/CD40 dans ces EPCs en démontrant l’expression des protéines adaptatrices, nommées les facteurs associés au récepteur du facteur de nécrose tumorale («TNF Receptor Associated Factors», TRAFs). Dans une première étude, nous avons investigué l’effet du sCD40L sur la fonction des «early» EPCs dans l’agrégation plaquettaire. En effet, nous avons démontré que le sCD40L renverse leur effet inhibiteur sur l’agrégation plaquettaire, et ce sans avoir un effet significatif sur la sécrétion de prostacycline (PGI2) et d’oxyde nitrique («Nitric Oxide», NO) par ces cellules. De plus, aucun effet du sCD40L n’a été noté sur l’apoptose et la viabilité de ces cellules. Par contre, nous avons noté une augmentation importante du stress oxydatif dans les «early» EPCs suite à leur stimulation avec le sCD40L. L’inhibition du stress oxydatif renverse l’effet du sCD40L sur les «early» EPCs dans l’agrégation plaquettaire. Ces résultats pourraient expliquer, en partie, la fonction réduite des EPCs chez les individus présentant des niveaux élevés de sCD40L en circulation. Dans une deuxième étude, nous avons étudié l’effet de sCD40L dans la fonction des «early» EPCs en relation avec l’angiogenèse. Nous avons identifié, dans un premier temps,les métalloprotéinases de la matrice («Matrix Metalloproteinases», MMPs) qui sont sécrétées par ces cellules. Nous avons trouvé que les «early» EPCs relâchent principalement la MMP-9 et que cette relâche est augmentée par le sCD40L. Le sCD40L induit aussi la phosphorylation de la p38 MAPK qui contribue à augmenter la sécrétion de MMP-9. Des études fonctionnelles ont démontré que le prétraitement des «early» EPCs au sCD40L potentialise la réparation endothéliale des HUVECs. En conclusion, l’ensemble de nos travaux, dans le cadre de ce projet de doctorat, nous a permis d’élucider les mécanismes responsables de l’action du sCD40L sur les effets inhibiteur et angiogénique des «early» EPCs dans l’agrégation plaquettaire et l’angiogenèse, respectivement. Ces résultats ajoutent de nouvelles connaissances sur le rôle des EPCs et pourront constituer la base pour des études futures permettant de corréler les niveaux élevés du sCD40L circulant et l’incidence des maladies cardiovasculaires, particulièrement l’athérothrombose. / Endothelial progenitor cells (EPCs) are endothelial precursors which possess a considerable therapeutic potential in vascular repair and regeneration. In the context of cardiovascular diseases, the understanding of the role of EPCs in the regulation of thrombogenesis and endothelial repair is relevant and necessary to the understanding of their therapeutic potential. We have shown that EPCs interact with platelets via P-selectin and inhibit the adhesion, activation and aggregation of platelets as well as thrombus formation. Recently, we have shown that EPCs express the inflammatory receptor CD40 and it is well known that platelets are the main source of the soluble form of its agonist CD40L («soluble CD40 ligand», sCD40L). Hence, we have hypothesized that the CD40L/CD40 axis in EPCs influences the anti-thrombotic and pro-angiogenic functions of EPCs. To verify this hypothesis, we have successfully generated early and late EPCs from peripheral blood mononuclear cells in culture. We have demonstrated the existence of the CD40L/CD40 axis in EPCs by showing the expression of adaptor proteins, named tumor necrosis factor associated factors (TRAFs). In our first study, we investigated the effect of sCD40L on the function of early EPCs in platelet aggregation. Indeed, we have shown that sCD40L reverses their inhibitory effect on platelet aggregation without having an effect on prostacyclin (PGI2) and nitric oxide (NO) secretion by these cells. Moreover, no effect of sCD40L has been noted on the apoptosis and viability of these cells. However, we have shown a significant increase in oxidative stress in early EPCs following sCD40L stimulation. The inhibition of oxidative stress reverses the effect of sCD40L on early EPCs in platelet aggregation. These results could partially explain the decreased function of EPCs in individuals displaying higher levels of sCD40L in circulation. In our second study, we have studied the effect of sCD40L on the function of early EPCs in relation to angiogenesis. First, we have identified the matrix metalloproteinases (MMPs) which are secreted by these cells. We have found that early EPCs mainly release MMP-9 and that this release is increased by sCD40L. The sCD40L also induces the phosphorylation of p38 MAPK which contributes to increase the secretion of MMP-9. In functional studies, we have shown that pretreatment of early EPCs with sCD40L can potentialize HUVEC endothelial repair. In conclusion, our work in the context of this doctoral research project has allowed us to study the mechanisms involved in the role of sCD40L in the inhibitory and angiogenic function of early EPCs in platelet aggregation and angiogenesis, respectively. These results add new insights to the role of EPCs and could constitute the basis for future studies allowing for the correlation between high levels of sCD40L and the incidence of cardiovascular disease, particularly atherothrombosis.

Liver progenitor cells (LPCs) play a major role in the regeneration process following chronic liver damage. LPCs can differentiate into hepatocytes and cholangiocytes and thus are capable of replenishing the damaged liver. Due to their plasticity and robust nature in culture systems, they are promising candidates for use in cell therapy. However, to be able to use LPCs as tissue regenerating stem cell-like cells in the clinic, we need to fully understand how they are controlled. Although a strong association between LPCs and inflammation has been shown in many chronic liver diseases, the role of the immune system in LPC-mediated hepatic regeneration is poorly understood. We hypothesise that specific immune cells and mediators are needed to induce the LPC compartment, and that these are common to the LPC response in different injury settings. Therefore, the present study focused on the characterisation of the inflammatory environment in the LPC response, which generates this niche. The aims of this study were (i) to identify the immune cells that are important for the LPC response, (ii) to define the cytokine profile and (iii) to determine the role of the cytokine producing cells during liver regeneration. To study hepatic inflammation following liver injury, a diet-induced model of liver injury (choline-deficient, ethionine-supplemented diet, CDE diet) was compared to two transgenic mouse models of immune-mediated hepatitis (Met-Kb, 178.3). Although all three models are characterised by hepatitis, histological analysis revealed that LPCs were only detectable in the CDE and Met-Kb livers. In the 178.3 model, livers regenerated from proliferating hepatocytes. An LPC response could not be induced in these mice even when liver damage was made more severe. In the other two models, LPC numbers increased over time showing the highest numbers one week after the peak of liver injury. LPCs were often found in close proximity to inflammatory cells, in particular macrophages.

Thesis (Ph. D.)--University of Oklahoma. / Bibliography: leaves 137-158.

Κατά την ανάπτυξη ενός οργανισμού η απόκτηση εξειδικευμένων κυτταρικών λειτουργιών είναι μια προοδευτική διαδικασία η οποία περιλαμβάνει την ασύμμετρη διαίρεση των βλαστικών κυττάρων για την παραγωγή προγονικών κυττάρων τα οποία σταδιακώς εξέρχονται από τον κυτταρικό κύκλο και διαφοροποιούνται μέσω της εγκαθίδρυσης του κατάλληλου μεταγραφικού προγράμματος. Προκειμένου να κατανοήσουμε τη ρύθμιση αυτών των γεγονότων μελετήσαμε την Geminin, ένα κεντρικό ρυθμιστή του κυτταρικού κύκλου, στο ανοσοποιητικό σύστημα. Δημιουργήσαμε ζωικά μοντέλα στα οποία απενεργοποιήσαμε το γονίδιο της Geminin στα λεμφοκύτταρα. Τα αποτελέσματα μας έδειξαν ότι η απενεργοποίηση της Geminin στα λεμφοκύτταρα δεν επηρεάζει σημαντικά τη διαφοροποίηση των προγονικών Τ κυττάρων στο θύμο. Απουσία της Geminin τα προγονικά θυμοκύτταρα δεσμεύονται προς διαφοροποίηση στην Τ κυτταρική σειρά και παράγουν διαφοροποιημένα θυμοκύτταρα. Παρατηρήθηκαν μικρές μειώσεις στον αριθμό των DN1, DN4 και DP κυττάρων. Σε αντίθεση τα αθώα (naïve), ρυθμιστικά (regulatory) και Τ κύτταρα μνήμης (memory T cells), παρουσίασαν σημαντικές μειώσεις απουσία της Geminin. Επιπλέον βρήκαμε ότι ο πολλαπλασιασμός των περιφερικών Τ κυττάρων ύστερα από την ενεργοποίηση τους μέσω του TCR υποδοχέα παρουσίασε σημαντικές ανωμαλίες ενώ παρατηρήθηκαν και σημαντικές διαταραχές της προόδου του κυτταρικού κύκλου απουσία της Geminin. Οι μεταβολές που παρατηρήθηκαν στην έκφραση του Cdt1 και σε κυκλίνες των ενεργοποιημένων περιφερικών Τ κυττάρων μπορεί να εμπλέκονται στο μηχανισμό που εξηγεί τις διαταραχές των περιφερικών Τ κυττάρων απουσία της Geminin. Επίσης Τ κύτταρα από τα οποία είχε απενεργοποιηθεί η Geminin δεν είναι ικανά να αποικίσουν τα λεμφοειδή όργανα μυών από τους οποίους απουσιάζουν τα λεμφοκύτταρα, αποτέλεσμα το οποίο δείχνει διαταραχές του ομοιοστατικού πολλαπλασιασμού αυτών των κυττάρων. Συμπερασματικά η Geminin είναι απαραίτητη για την αυστηρή ρύθμιση των επαναλαμβανόμενων κυτταρικών διαιρέσεων των περιφερικών Τ κυττάρων αλλά δεν επηρεάζει σημαντικά την διαφοροποίηση των προγονικών Τ κυττάρων. Επιπλέον τα αποτελέσματα αυτά προτείνουν ότι υπάρχουν εγγενείς διαφορές στην ρύθμιση του κυτταρικού κύκλου μεταξύ θυμοκυττάρων και περιφερικών Τ κυττάρων. / During development, acquisition of specialized function is a progressive, gradual process that involves the asymmetric divisions of stem cells to generate progeny that will exit the cell cycle and terminally differentiate through the establishment of an appropriate transcriptional program. In order to understand this process we studied Geminin, a key cell cycle regulator, that has been shown to affect cellular decisions of differentiation. Towards this direction we focused on the immune system and investigated the role of Geminin in self-renewal and differentiation of stem and progenitor cells. In order to gain insight into the in vivo role of Geminin in progenitor cell division and differentiation, we have deleted Geminin in cells of the lymphoid lineage. The inactivation of Geminin in the lymphoid lineage does not alter progenitor T cell differentiation in the thymus. In the absence of Geminin progenitor T cells commit, differentiate and generate differentiated thymocytes. Minor reduction in the number of DN1, DN4 and DP progenitor T cells were observed. In contrast naïve, regulatory and memory peripheral T cells show a significant reduction in the absence of Geminin. Moreover, proliferation of Geminin deficient peripheral T cells upon TCR activation is severely compromised, accompanied by cell cycle progression defects. The deregulated protein levels of Cdt1 and cyclins in activated peripheral T cells lacking Geminin, may be involved in the mechanism responsible for the observed phenotype of Geminin deficient peripheral T cells. More importantly Geminin deficient T cells fail to repopulate lymphopenic hosts suggesting defects in homeostatic proliferation. In conclusion Geminin is essential to regulate the repeated divisions of peripheral T cells but does not significantly affect progenitor T cell differentiation. In addition our results suggest that there are intrinsic differences in cell cycle regulation of thymocytes and peripheral T cells.

Most existing chemotherapeutics lack adequate specificity for transformed cells and therefore have high rates of collateral damage to normal tissue. Moreover, such therapies often depend on p53 to induce cell death and are ineffective on the large number of human cancers that have lost p53 function. The discovery of novel p53-independent cancer therapies is therefore of significant interest. The Chicken Anemia Virus protein Apoptin selectively induces apoptosis in transformed cells in a p53-independent manner while leaving normal primary cells unaffected. This selectivity is thought to be largely due to cell type-specific localization: in primary cells Apoptin is cytoplasmic, whereas in transformed cells the protein localizes to the nucleus. The basis for this cell type-specific localization remains to be determined. In this study, Apoptin is revealed to be a nucleo-cytoplasmic shuttling protein whose localization is mediated by an N-terminal nuclear export signal (NES) and a C-terminal nuclear localization signal (NLS). Both signals are required for cell type-specific localization, as Apoptin fragments containing either the NES or NLS fail to localize differently between transformed and primary cells. Significantly, cell type-specific localization can be rescued in trans by co-expression of the two separate fragments, which are able to interact through an Apoptin multimerization domain. Interestingly, this multimerization domain overlaps with the NES suggesting that these two activities may be functionally coupled in cytoplasmic retention in primary cell types. Factors present in transformed cells induce localization of Apoptin to the nucleus where a biochemically distinct, more soluble form of the protein exists. Using affinity-purification and mass spectroscopy it was found that, specifically in transformed cells, Apoptin is associated with APC1, a subunit of the anaphase-promoting complex/cyclosome (APC/C). The APC/C is required to establish a mitotic cell-cycle checkpoint, and its inhibition results in G2/M arrest and apoptosis. Expression of wild type Apoptin in transformed cells inhibits APC/C function and induces G2/M arrest and apoptosis, whereas Apoptin mutants that are unable to associate with APC1 have no effect. In p53 null cells, ablation of APC1 by RNA interference induces a G2/M arrest and apoptosis analogous to that observed following Apoptin expression. Furthermore, Apoptin was found to induce the formation of PML bodies and to recruit APC/C subunits to these nuclear structures suggesting a mechanism involving sequestration and subsequent inhibition of the APC/C. Thus, the results of this study clarify Apoptin cell type-specific localization behavior and explain the ability of Apoptin to induce apoptosis in transformed cells in the absence of p53. This study advances a newly emerging field of viral mechanisms of apoptosis involving G2/M arrest and APC/C modulation. The resultant p53-independent apoptosis suggests that the APC/C may be an attractive target for the development of anti-cancer drugs.

Le carcinome hépatocellulaire (CHC) est un cancer de mauvais pronostic. Les maladies chroniques du foie provoquent le développement d'une cirrhose qui évoluera éventuellement en CHC. Le récepteur de l'insuline (IR) est exprimé sous deux isoformes, IR-A et IR-B, qui résultent de l'épissage alternatif de l'ARNm. IR-B est l'isoforme majoritairement exprimé dans l'hépatocyte adulte et IR-A dans l'hépatocyte fœtal. Plusieurs travaux montrent une surexpression de IR-A dans différents cancers, associée à une signalisation oncogénique en réponse à l'IGF-II. Le rôle de la signalisation IGF-II/IR-A a peu été investigué dans le CHC. Notre étude porte sur le rôle biologique de la surexpression de IR-A dans les cellules de CHC au regard de la surexpression d'IGF-II. Une augmentation du ratio d'expression IR-A/IR-B est observée dans 70% des tumeurs de CHC par rapport au foie adjacent non tumoral, qui est significativement associée à l'expression de marqueurs clinicopathologiques de mauvais pronostic dont des marqueurs de cellules souches/progénitrices (CSP) et à une faible survie post-hépatectomie. Seules 9,4% des tumeurs surexpriment IGF-II. In vivo, la surexpression stable de IR-A, et non de IR-B, augmente la tumorigénicité de deux lignées de CHC disposant (Huh7) ou non (PLC/PRF5) d'une boucle autocrine d'IGF-II, et est associée à l'induction de différentes signatures pro-inflammatoires. Sans effet sur la prolifération, la surexpression de IR-A stimule la migration et l'invasion in vitro et augmente l'expression de marqueurs CSP. En conclusion, ces résultats identifient IR-A comme un nouvel acteur de la progression du CHC de façon indépendante d'une boucle autocrine d'IGF-II. / Hepatocellular carcinoma (HCC) is a poor prognosis cancer. Chronic liver diseases induce cirrhosis which will eventually evolve into HCC. The insulin receptor (IR) exists in two isoforms, IR-A and IR-B, resulting from mRNA alternative splicing. Adult hepatocytes predominantly express IR-B while fœtal hepatocytes mainly express IR-A. The overexpression of IR-A associated with IGF2 oncogenic signaling has been abundantly described in cancer cells. Little is known about IGF2/IR-A signaling in HCC. Our study aims to evaluate the biological functions associated to IR-A overexpression in HCC in relation to IGF2 overexpression. We observed that 70% of 85 HCC tumors showed upregulation of IR-A/IR-B ratio compared to adjacent nontumor tissue, which was significantly associated with clinicopathological markers of aggressive tumours such as stem/progenitor cell (SPC) markers and correlated with reduced patient survival after surgery. IGF2 upregulation was observed in only 9.4% of HCC. Stable overexpression of IR-A, but not IR-B, increased in vivo tumorigenicity in two HCC cell lines presenting (Huh7) or not (PLC/PRF5) an autocrine IGF2 secretion loop and was associated with the induction of different pro-inflammatory gene signatures. While IR-A overexpression did not promote cell proliferation in vitro, it stimulated migration and invasion and increased some SPC markers expression. Altogether these results highlight IR-A as a novel player in HCC progression irrespective of an IGF2 autocrine loop.

Submitted by Biblioteca do Instituto Biomédico BIB (uffbib@gmail.com) on 2017-06-05T19:47:11Z No. of bitstreams: 1 Helena Naly Miguens Rocha.pdf: 1130470 bytes, checksum: 43146823dc28af52b34aa41dd863ff95 (MD5) / Made available in DSpace on 2017-06-05T19:47:11Z (GMT). No. of bitstreams: 1 Helena Naly Miguens Rocha.pdf: 1130470 bytes, checksum: 43146823dc28af52b34aa41dd863ff95 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Mecanismos de ativação e reparo endoteliais em resposta ao fluxo sanguíneo retrógrado (FSR) exacerbado ainda não foram completamente elucidados, nem em condições fisiológicas, nem na hipertensão arterial sistêmica (HAS). O objetivo deste estudo foi determinar os efeitos do FSR exacerbado sobre biomarcadores endoteliaisem indivíduos saudáveis e com HAS. Oito homens saudáveis (grupo CT; 36±3) e oito pacientes com HAS(grupo HAS;39±5) foram submetidos a manobra de indução de FSR em um dos braços, através da insuflação de dois manguitos, um no antebraço a 75mmHg e outro manguito próximo ao ombro a 40 mmHg, por 30 minutos. A avaliação do fluxo sanguíneo (ultrassom vascular) e a coleta de sangue foram realizadas no momento basal e no 30º minuto de manobra em ambos os braços (contralateral e ipsilateral). Ativação endotelial, micropartículas endoteliais (MPE) e células progenitoras endoteliais (CPE) foram mensuradas por citometria de fluxo. Nitrito foi mensurado por NOA Sievers. Em condições basais, fluxo sanguíneo médio, condutância vascular, taxa de cisalhamento média (p<0,01) e MPE (p=0,03) foram maiores no grupo HAS quando comparado ao grupo CT. Níveis basais de CPE estavam reduzidos no grupo HAS, permanecendo assim durante a manobra (p<0,01). Ambos os grupos apresentaram redução no fluxo sanguíneo médio e na condutância vascular (p≤0,01), bem como aumento na taxa de cisalhamento retrógrado (p<0,01) e no índice de cisalhamento oscilatório (p<0,01) durante a manobra. Somente o grupo HAS aumentou o número de MPE (p=0,02) e a ativação endotelial (p=0,04) durante a manobra. A razão MPE/CPE foi maior em ambos os momentos no grupo HAS (p<0,02). A resposta dos níveis séricos de nitrito a manobra foi menor no grupo HAS (p=0,03). Conclui-se que pacientes com HAS apresentam um quadro subclínico de disfunção endotelial com comprometimento no reparo vascular, o que foi agravado pela indução de FSR. / Endothelial activation and repair mechanism in response to increased retrograde blood flow (RBF) have not beenfully elucidated, neither in physiological conditions nor in hypertension. We aimed to determinethe effects of increased RBF on endothelial biomarkers in healthy individuals and hypertensive patients. Eight healthy subjects (CT group; 36±3) and eight hypertensive men (HT group; 39±5) underwent a maneuver to increase RBF, using two pneumatic cuffs: one in the forearm, inflated to 75 mmHg and one near the shoulder, inflated to 40 mmHg, for 30 minutes.Blood flow measures (ultrasound doppler) and blood samples were obtained at baseline and during the last minute of the maneuver from both arms (ipsilateral and contralateral). Endothelial activation, endothelial microparticle (EMP) and endothelial progenitor cell (EPC) were measured by flow cytometry. Nitrite was measured through NOA Sievers.At baseline, mean blood flow, vascular conductance, mean shear rate (p<0.01) and EMP (p=0.03) were higher in HT group than CT group. Baseline EPCs levels were reduced in HT group, which was sustained during the maneuver (p<0.01). Both groups presented decreased mean blood flow and vascular conductance (p<0.01), along with increased retrograde shear rateand oscillatory shear index (p<0.01), during the maneuver. Only the HT group showed increased EMP (p=0.02) and endothelial activation levels (p=0.04). EMP/EPC was higher in HT group in both moments (p<0.02).Nitrite levels in response to the maneuver were lower in HT group (p=0.03).Hypertensive patients present a subclinical endothelial dysfunction along with impaired endothelial repair, which was worsened by the RBF induction.

A. Background<p><p>Skin wound healing (WH) is a dynamic and extremely determinate process of cellular, humoral and molecular mechanisms which begins directly after wounding and can last for years. WH is described as is an intricate process in which the skin (or another organ-tissue) repairs itself after injury. The process of skin WH occurs through the actions of an interplay of cells, growth factors and cytokines leading to wound closure.<p><p>WH occurs in three precisely and highly programmed phases: the inflammatory phase (day 0 to day 7) followed by the proliferative phase or vasculogenic phase (day 7 to day 21) and finally the remodeling phase (2 days - up to 2 years). For a successful healing, all three phases must occur in the proper sequence and time frame.<p><p>Many factors can interfere with one or more phases of the WH process, thus causing improper or impaired healing. The proliferation phase, in particular, requires the participation of various cells types such as fibroblasts, endothelial cells (ECs) and endothelial progenitor cells (EPCs), to produce a healthy well-vascularized granulation tissue for epithelization and wound closure.<p><p>A.1 Wound Healing And Obesity<p><p>In 2008, over 1.4 billion adults, 20 and older, were overweight. Of these, obesity has been shown to affect over 500 million people (OMS website). Moreover, the prevalence of obesity continues to rise, and by 2018, it is estimated that obesity will cost $ 347 billion annually.<p><p>Each year, in the US, approximately 33 million overweight and obese patients undergo surgery. Obesity causes a number of known health problems and increased post-surgical complications such as wound infection, dehiscence, hematoma and seroma. Surgeons anecdotally report WH complications among obese patients; however, little research has been conducted to investigate the mechanisms mediating impaired obesity-related WH. <p><p>Some previous work on diabetic patients and diabetic mice showed an imbalance between pro-oxydant and anti-oxydant genes as well as impaired EPCs proliferation and tube formation during the WH process. More then a hundred cytologic factors have been found to impair WH in the type 2 diabetic patient. It is a very complex and multifactorial problem involving decreased growth factors secretion, impaired keratinocyte and fibroblast functions, impaired EPs function, alteration of the macrophage function and granulation tissue synthesis, etc. <p><p>Based on these findings and because obesity is associated with the development of type 2 diabetes, we hypothetize that, impaired balance between pro-apoptotic/anti-apoptotic and pro- oxydant /anti-oxydant genes is involved in impaired WH. Furthermore, we hypothetize that impaired EPCs function leads to the perturbation of the proliferation phase of obesity impaired WH.<p><p>A.2. Wound Healing and Age<p><p>The world population is aging; by 2030, nearly 20% of Americans, (± 72 million people), will be 65 years old and older. In 2010, 17% of the European population was over the age of 65. By 2060, it is projected that the share of those aged 65 and over will rise to 30%, accounting for more then 150 million people. (ec.europa.eu) These aging subjects undergo an increasing number of surgical procedures: in the past two decades, the percentage of surgeries in patients over 65 has doubled to nearly 40%.<p>As a corollary, it is well established knowledge that elderly WH is impaired. However, little is known about the underlying mechanisms of age-related impaired WH.<p><p>As previously mentioned, adult BM-derived EPCs contribute to peripheral tissue repair and regeneration. In light of the abundant literature suggesting that neovascularization is impaired in the elderly, we characterize a novel model of senile cutaneous WH and investigate the role that vasculogenesis plays in the pathogenesis of age related impaired WH.<p>Aged mice colonies have traditionally been the model for aged small mammalian research, however, the ability to use a readily-available transgenic mouse model with features of accelerated aging would aid in the exploration of targeted therapies and a great number of age-related investigations.<p><p>We hypothesize that the Hutchinson-Gilford Progeria Syndrome (HGPS) Zmpste24 deficient (Zmpste24-/-) mouse mimics physiological ageing and can be used as a novel model for the study of senescent WH. We further hypothetized that impaired balance between pro-apoptotic/anti-apoptotic and pro-oxydant /anti-oxydant genes as well as impaired EPCs function are responsible for the impairment of the proliferative phase, leading to overall impaired WH.<p><p>A.3 Aims<p><p>Recently, a great deal of research has been directed at understanding the critical factors inducing poorly healing wounds. However, a lot remains unclear.<p><p>It is now well accepted that new blood vessel formation occurs not only by angiogenesis (blood vessels formation from a preexisting network of capillaries), but also by vasculogenesis (blood vessels formation from BM SCs recruitment) and that EPCs contribute to as much as 25% of new blood vessels formed in healing tissues4. They are mobilized from the BM in response to injury and production of local cytokines, are incorporate into wounds and play an integral role in systemic tissue repair. <p><p>Based on this finding, we hypothesized that co-morbidities related impaired WH may be due, in part, to decreased EPCs number, migration/homing, and/or function resulting in impaired vasculogenesis. Because age and/or obesity have been shown to be one of the most common predictors of altered WH, we decided to focus on these two parameters.<p><p>Following a bedside to bench approach the purpose of this work was to 1) develop coherent and translatable models of co-morbidity digging in the physiologic/pathologic mechanisms underlying altered healing in obese and senile mice; 2) develop targeted therapeutics to improve impaired WH.<p><p>B. Material and Methods<p><p>B.1 Human Model<p><p>Since obesity impairs WH and BM EPCs are important for tissue repair, we hypothesize that obesity- impaired WH is due, in part, to impaired EPCs mobilization, trafficking, and function. Peripheral blood was obtained from non diabetic, obese (BMI > 30, n = 25), and non obese (BMI < 30, n = 17) subjects. Peripheral blood human EPCs were isolated, quantified, and functionally assessed.<p>As for aged impaired WH, EPCs of aged subjects have already been found to have decreased adhesion, migration and proliferative properties as well as being decreased in number in elderly patients undergoing surgery compared to younger patients.<p><p>B.2. Mice Models<p><p>Two models of WH were developed and characterized.<p>In order to isolate the effect of obesity on EPCs and WH, OB non-diabetic female TallyHo/JngJ mouse were selected (Female mice don’t express hyperglycemia and hyperinsulinemia). Female SWR/J non-OB mice were used as control mice. In order to limit variables, TallyHO/JngJ obese mice were selected over other OB mice that exhibit a polygenic type of obesity (Jackson Laboratory Website). By selecting this mouse model, we have excluded in our selection of the ideal model common confounding factors such as hyperglycemia, hyperinsulinemia, immune disorders.<p><p>Zmpste24 is a metalloproteinase involved in the maturation of lamin A (LmnA), an essential component of the nuclear envelope. When Zmpste24 or LmnA are knocked-out, mice exhibit profound nuclear architectural abnormalities and histopathological defects that phenocopy an accelerated aging process. Of crucial importance, the lamin-A dependent nuclear alterations seen in Zmpste24-deficient mice have also been found in human physiological aging. We defined the utilization of the Hutchinson-Gilford Progeria Syndrome (HGPS) Zmpste24 deficient (Zmpste24- /-) mouse as a novel model for the study of senescent WH (controls used were C57BL/6J mice).<p><p>B.3. Wounding Model and Data Collection<p><p>All mice group underwent wounding using a stented wound model developed in our laboratory and previously published. Briefly, paired 6-mm circular, full-thickness wounds extending through the panniculus carnosus were made on the dorsal skin of the mouse. An O-ring, 12-mm splint made of silicone sheeting was then sutured to the skin around the wound. To minimize wound contraction and reliably recapitulated the granulation and re-epithelialization seen in human WH by secondary intention. Time to wound closure was measured using standardized digital photographs taken on days 0, 7, 14, and 21. Wound closure was calculated as a percentage of the original wound.<p><p>For each model, EPCs were harvested, quantified by flow-cytometry and their function tested. Wounds were harvested at various time points and RNA, DNA and protein analysis were conducted. Finally immunohistochemistry to assess epidermal thickness, vascularity and WH were also realized.<p><p>In a second step, after characterization of the models, local (using targeted siRNA gel) and systemic therapies (using AMD3100, a PC mobilizer) were applied on the wounds and compared to controls. WH was monitored. We conducted the previously mentioned analysis (RT-PCR, ELISA and DNA analysis) on the harvested samples.<p><p>All values are expressed as a mean ± standard error of mean (SEM). The number of mice per treatment group was determined using G*Power (G*Power©, Melbourne, Australia) to provide a power greater than 0.80. Student T test was realized to compare two groups among each other.<p><p>C. Results<p><p>C.1. Human EPCs Have Impaired Function<p><p>There was no difference in the number of baseline circulating human EPCs in non-diabetic OB and non-OB<p>subjects, but EPCs from OB subjects had impaired adhesion (p<0.05), migration (p<0.01), and proliferation (p<0.001).<p><p>C.2. Obesity and Wound Healing<p><p>TallyHo/JgnJ OB mice demonstrated significantly impaired healing when compared to SWR/J control mice. They healed at an average of 28 ± 2 days (p<0.05). Post-wounding circulating EPCs were quantified and wounds were analyzed. Circulating EPCs recruitment is impaired in wounded TallyHo/JngJ mice and their wounds shown significantly decreased new blood vessel formation through decreased HIF-1α/SDF-1α signaling (p<0.05). Their wounds are characterized by increased apoptosis, increased DNA damage and impaired pro-/anti-oxydant balance. Immunonistochemistry and histology showed decreased vascular vessels in TallyHo/JngJ wounds and thinner epidermal thickness.<p><p>In the local treatment phase, local p53 silencing consistently improved WH to a nearly normal healing time (wounds healed in 18 ± 2 days, p<0.05). sip53 treatment showed a significant decrease in pro-apoptotic markers (p53, Bax, PUMA p<0.05) and a significant increase in angiogenic markers (VEGF, SDF-1α, HIF-1α) with increased blood vessel formation and decreased DNA damage.<p><p>C.3. Age and Wound Healing<p><p>In these experiments, we show that not only is Zmpste24-/- WH impaired when compared to C57BL/6J mice (Zmpste24-/- mice healed at average 40 days ± 2 days p<0.05) at all time points but that they also showed decreased vascularity and proliferation in the wound bed (p<0.05).<p><p>Histological analysis was performed utilizing hematoxylin and eosin staining to assess epidermal thickness, CD31 immunofluorescence to assess vascular density, p53 and caspase 3 to assess apoptosis, 8’OHdG staining to assess DNA damage and PCNA to assess proliferation. Epidermal thickness was significantly decreased in Zmpste24-/- animals compared to WT as well as vascular density, and proliferation in Zmpste24-/- wound tissue (p<0.05). <p><p>Circulating vasculogenic EPCs recruitment was impaired in Zmpste24-/- mice and their wounds showed significantly decreased new blood vessel formation through decreased HIF-1α/SDF-1α signaling (p<0.05). Zmpste24-/- wounds are characterized by increased apoptosis and an abnormal rise in ROS.<p>In the treatment phase, local p53 silencing consistently improved healing by more then a two fold (18 ± 2 days). VEGF production was significantly increased and pro-apoptotic factors were significantly downregulated in siRNA-treated Zmpste24-/- mice (p<0.05). DNA damage due to ROS production was also shown to be significantly decreased following treatment. Our results suggest a vasculogenic dysfunction in wound closure and showed that the specific knock down of p53 significantly improves WH.<p><p>Because EPCs showed impaired function, lower peripheric blood counts and impaired SDF-1α/HIF-1α signaling, we hypothesized that improving their mobilization by using a progenitor cell mobilizer, AMD3100, known to mobilize SCs from the BM, in a systemic treatment phase will improve WH. Peripheral blood counts were significantly increased and time to wound closure significantly decreased (20 days ± 2, p<0.05). Vasculogenic markers and anti- apoptotic molecules were upregulated compare to non-treated animals.<p><p>D. Conclusions<p><p>Obesity impaired wound closure is a complex problem with many contributory factors. Our results suggest that obesity impairs the BM-derived EPCs response to peripheral injury and this, in turn, impairs wound closure. This impairment is associated with decreased new blood vessel formation and increased DNA damage leading to an increase in the p53 pathway. We also demonstrate that targeted siRNA therapy can partially rescue impaired WH due to obesity. Based on these results we support the encouraging argument that, WH and closure has the potential be improved through specific local and systemic therapies in vivo in our rodent model and that further studies are needed to support this in a clinical environment.<p><p>Impaired WH due to ageing is a complex phenomenon that is partially understood. We demonstrate that the Zmpste24-/- transgenic knockout mouse provides a model for age-related WH investigation. Zmpste24-/- animals heals their wounds with significant delays, showed impaired EPCs mobilization following wounding through an impaired HIF-1α/SDF-1α pathway and increased apoptosis. Furthermore, WH can be improved through specific local siRNA therapy and systemic stem cell mobilization therapies.<p><p>Our results suggest strong similar patterns between obesity and ageing in the way they mediate WH impairments trough (premature) ageing. Our encouraging endeavor to bring WH back to baseline in these diseased models underlines the possibility to reverse the microenvironment alterations and improves EPCs contribution to the WH process. Because EPCs are involved in virtually every tissue repair process happening in the human body, we hope that this work will lead the way for new research in various fields in medicine to improve wound care and quality of life of patients. / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

L’endothélium joue un rôle primordial dans le développement et le maintien des multiples fonctions vasculaires. Il est ainsi largement impliqué dans des situations pathologiques comme les maladies cardio-vasculaires. La description de marqueurs endothéliaux circulants a permis une exploration non invasive de l'endothélium. Notre équipe s’est intéressée principalement aux cellules endothéliales circulantes (CEC), dont le taux reflète la lésion ou l’activation de l’endothélium, et aux progéniteurs endothéliaux circulants (PEC), marqueurs de régénération endothéliale. La découverte en 1997 par Asahara de la présence chez l’adulte de ces PEC, participant à la formation de nouveaux vaisseaux par vasculogenèse, a ouvert de nouvelles perspectives, notamment pour la thérapie cellulaire des pathologies ischémiques. Ce travail a consisté à développer les méthodes d’étude de ces cellules dans plusieurs contextes. Tout d’abord, nous avons exploré l’utilité de ces marqueurs dans la physiopathologie de l’hypertension artérielle pulmonaire (HTAP). Puis nous avons analysé le potentiel de mobilisation des progéniteurs endothéliaux à partir de la paroi vasculaire lors d’une ischémie locale chez des volontaires sains dans le cadre du développement d’un produit de thérapie cellulaire autologue. Une partie de ce projet a été de mettre en place et d’optimiser les techniques d’étude de ces marqueurs. Les CEC ont été quantifiées par immunoséparation magnétique (IMS), technique mise au point en 1992 (Dignat-George 1992) et transférée dans notre laboratoire. La quantification des PEC a été réalisée par cytométrie en flux et par culture cellulaire. En culture, deux types de PEC sont décrits : les PEC précoces, dont l’origine est monocytaire et pour lesquels la culture est déjà standardisée, et les « Endothelial Colony Forming Cells » (ECFC), seules cellules présentant des caractéristiques de cellules endothéliales progénitrices et pouvant être proposées comme produit de thérapie cellulaire. Nous avons optimisé la quantification des ECFC en culture en étudiant l’effet de diverses matrices et de la densité d’ensemencement des cellules mononucléées issues du sang total sur l’obtention de ces cellules et leurs propriétés angiogènes. La dysfonction endothéliale a été décrite comme un élément central dans le développement de l’HTAP dont le diagnostic repose sur la mesure de la pression artérielle pulmonaire par cathétérisme cardiaque droit. En l’absence de marqueur biologique non invasif dans cette maladie, nous avons quantifié les CEC et les progéniteurs circulants dans deux études. Une étude réalisée chez des patients adultes a montré une augmentation spécifique des CEC dans l’HTAP et non dans l’hypertension pulmonaire thromboembolique chronique. Ainsi les CEC semblent être le reflet des lésions endothéliales pulmonaires et non de la sévérité clinique des patients. L’autre étude a montré l’intérêt de la quantification des CEC dans la prise en charge thérapeutique des enfants souffrant d’HTAP secondaire à une cardiopathie congénitale, dont les formes irréversibles présentaient des taux élevés de CEC. Nous avons ainsi défini un nouveau marqueur non invasif à utilité diagnostique et pronostique. Les PEC sont des cellules rares dans le sang circulant, difficiles à expandre, et dont les essais de mobilisation médullaire se sont révélés insuffisants. L’hypothèse récente d’une réserve vasculaire des progéniteurs endothéliaux nous a conduits à étudier l’effet d’un processus d’ischémie locale sur la mobilisation de ces cellules chez des volontaires sains. Deux groupes d'âge ont été inclus afin d'évaluer l'impact du vieillissement sur la méthode de mobilisation étudiée. Malgré un effet de cette ischémie sur la dilatation endothéliale cette méthode n’a pas permis de mobiliser significativement les PEC issus de la paroi endothéliale, quel que soit l'âge des sujets. A l’inverse, l’hypoxie a eu un effet délétère sur les capacités angiogènes des ECFC. / The endothelium plays a key role in the development and the homeostasis of vascular functions. It is also well involved in pathological situations like cardiovascular diseases. Thanks to the description of circulating endothelial markers, non invasive study of the endothelium is now possible. Our group was particularly interested in circulating endothelial cells (CECs), the level of which reflects an endothelial activation or lesion, and to circulating endothelial progenitors cells (EPCs), markers of endothelial repair. EPC description by Asahara in 1997 in adult blood, involved in new blood vessel formation by vasculogenesis, offered new perspectives, specially for cell therapy in ischemic diseases. This work consisted in the development of methods to study these markers in different contexts. First, we explored the interest of these markers in the physiopathology of pulmonary arterial hypertension (PAH). Then we evaluated endothelial progenitors mobilization from the vascular wall by a local ischemia process in healthy volunteers, in the perspective of an autologous cell therapy product development. One part of this project was the implementation and optimization of the methods to study CEC and EPC. CEC were quantified by magnetic immunoseparation. This technique was developped in 1992 by F. Dignat-George's group and transferred in our laboratory. EPC were quantified by flow cytometry and cell culture. Two types of EPC are described in culture: the early EPC, which originate from monocyte lineage and which culture is standardized, and the « Endothelial Colony Forming Cells » (ECFC), the only cells presenting endothelial progenitor cell properties and which use as a cell therapy product can be considered. ECFC quantification by culture was optimized by assessment of the impact of diverse matrices and seeding concentrations of mononuclear cells isolated from whole blood, on ECFC commitment and their angiogenic properties. Endothelial dysfunction was described as a central element in the development of PAH, which diagnosis is based on the use of right heart catheterization. Due to the lack of noninvasive marker for this disease, CEC and circulating progenitors were quantified in two studies. One of them realized in adult patients showed a specific increase of CEC in PAH and not in post-embolic PH. CEC would then reflect the presence of specific endothelial lesions and not the clinical state of the patients. The other study demonstrated the interest of CEC quantification in the therapeutic care of children with PAH secondary to congenital heart disease, for whom patients in irreversible state had a higher level of CEC. We then defined a new noninvasive biomarker.that can be used for the diagnosis and prognosis of PAH. EPC are rare events in whole blood, difficult to expand and for which, mobilization protocols revealed insufficient. The recent hypothesis of a vascular reservoir for endothelial progenitor led us to study the effect of a local ischemia procedure on the mobilization of these cells in healthy volunteers. Two age groups were included to assess the impact of aging on this procedure. Despite a significant endothelial dilation with the local ischemia, no EPC were mobilized, whatever the age group. Ischemia even altered ECFC angiogenic properties.

Type 1 diabetes develops through the progressive destruction of the insulin-producing beta-cells. Regeneration or replacement of beta-cells is therefore needed to restore normal glucose homeostasis. Presently, normoglycemia can be achieved by the transplantation of whole pancreas or isolated islets of Langerhans. Islet transplantation can be performed through a simple laparoscopic procedure, but the long-term graft survival is low due to poor revascularization and early cell death. This thesis examined the possibility of using different auxiliary cells (Schwann cells, endothelial progenitor cells, and neural crest stem cells) to improve the engraftment and function of endogenous and transplanted islets. Co-transplantation of Schwann cells with islets improved islet graft function early after transplantation, and caused an increased islet mass at one month posttransplantation. However, the vascular densities of these grafts were decreased, which also related to an impaired graft function. Islet grafts containing endothelial progenitor cells had a superior vascular density, with functional chimeric blood vessels and substantially higher blood perfusion and oxygen tension than control transplants. By culturing and transplanting islets together with neural crest stem cells it was found that islets exposed to these cells had a higher beta-cell proliferation compared with control islets. At one month posttransplantation, the grafts with neural crest stem cells also had a superior vascular- and neural density. The potential of intracardially injected neural crest stem cells to home to the pancreas and ameliorate hyperglycemia in diabetic mice was investigated. During a three-week period after such cell treatment blood glucose concentrations decreased, but were not fully normalized. Neural crest stem cells were present in more than 10% of the pancreatic islets at two days postinjection, at which time the beta-cell proliferation was markedly increased when compared with islets of saline-treated diabetic animals. Three weeks later, a doubled beta-cell mass was observed in animals receiving neural crest stem cells. In summary, islets can easily be transplanted together with different auxiliary cells. Some of these cells provide the possibility of improving vascular- and neural engraftment, as well as beta-cell growth and survival. Systemic administration of neural crest stem cells holds the potential of regenerating the endogenous beta-cells.