Atrial fibrillation (AF) is the most common and relevant arrhythmia in the clinical routine. AF is predicted to affect 6–12 million patients in the USA by 2050 and 18 million patients in Europe by 2060. Cardiac fibrosis and inflammation decisively determine the course of the disease and the clinical outcome of the patients. Despite the tremendous impact on human health, detailed understanding on the molecular mechanisms that contribute to fibrosis in AF is limited. This study provides further evidence for the current paradigm shift that atrial fibrillation is more of a systemic-inflammatory disease than a mere ion-channel dysfunction. The aim of this study was to investigate the role of polo-like kinase 2 (PLK2) and the pro-inflammatory cytokine osteopontin (OPN) with respect to fibroblast (dys)function and fibrosis formation in order to derive novel targets for targeted, fibroblast-specific pharmacotherapy. All patients who participated in this study gave their written informed consent in accordance to the declaration of Helsinki. The study was approved by the local bioethics committee (Ethikkommission an der Technischen Universität Dresden). Human fibroblasts were isolated by outgrowth culture from right atrial biopsies of patients suffering from sinus rhythm (SR) and AF. Murine fibroblasts were isolated by whole-heart Langendorff-perfusion. Quantitative PCR and western blot were used to detect PLK2 transcript expression and protein abundance, respectively. Functional assessment of cardiac function was done with transthoracic echocardiography and surface ECG recordings. Cell culture experiments were performed to evaluate the effects on functional fibroblast properties such as proliferation and differentiation after changing PLK2 activity by genetic knockout (KO) or pharmacological inhibition. A mass spectrometry based secretome analysis was performed by our collaborating laboratory of Prof. Manuel Mayr at King’s College London. An enzyme-linked immunosorbent assay (ELISA) was done to measure OPN in the peripheral blood of patients in SR and with permanent AF. Right atrial appendage tissue and fibroblasts from AF patients displayed significantly lower expression of PLK2 mRNA and protein due to increased DNA-methylation of the PLK2 promotor when compared to sinus rhythm (SR) control patient atria or fibroblasts. This methylation was induced in cardiac fibroblasts by chronic hypoxia (1% O2) exposure for 96 h. Pharmacological inhibition as well as global KO of PLK2 in cardiac fibroblasts resulted in elevated myofibroblast differentiation and reduced fibroblast proliferation. PLK2 KO mice displayed vast interstitial fibrosis areas as observed from histological cross sections stained either with Sirius red or with Masson’s trichrome staining. Transthoracic echocardiography revealed systolic and diastolic dysfunction in PLK2 KO mice and AF-typical ECG alterations such as prolonged PQ interval and QRS duration. Mass spectrometry proteomics revealed de novo expression of OPN in the PLK2-KO-fibroblast secretome. Furthermore, we found higher OPN plasma levels in AF patients correlated with electrophysiologically determined fibrosis compared to non-fibrosis control patients. Finally, we identified that the p42/44 MAPK signal transduction cascade is linking to reduced PLK2 expression and enhanced OPN release. Specifically, we found that KO of PLK2 increase p42 MAPK phosphorylation which is known to stimulate OPN transcription. Thus inhibition of p42/44 MAPK resulted in diminished OPN expression. In a dermal fibrosis model the administration of Mesalazine in vitro resulted in reduced p42 MAPK and SMAD2/3 phosphorylation and thereby reduced OPN and αSMA expression. To explore the general validity and relevance of the PLK2 signaling pathway for fibrosis, a dermal model of radiation-induced fibrosis was used. This approach a) confirmed the observations that were made in the heart and b) showed that the use of Mesalazine in vitro led to a reduced p42 MAPK and SMAD2 / 3 phosphorylation and thus to a significantly reduced OPN and αSMA expression. Fibroblasts from patients with permanent AF express less PLK2 than cells from SR control patients. The loss of physiological PLK2 activity coincides with marked changes in the proliferation and differentiation of cardiac fibroblasts. These changes favor fibrosis in the atrial tissues, which is further enhanced by the local and systemic increase of OPN. The present study identifies PLK2 as a novel regulator of cardiac fibroblast function and fibrosis. Restoration of the physiological methylation status of the PLK2 promoter or the inhibition of OPN with Mesalazine could be of particular clinical interest. The tangible clinical and pharmacological feasibility will be subject of future investigations. / Vorhofflimmern (VHF) ist die häufigste und bedeutsamste Arrhythmie in der täglichen klinischen Praxis. VHF wird bis 2050 voraussichtlich 6 -12 Millionen Menschen in den USA und bis 2060 zirka 18 Millionen Menschen in Europa betreffen. Fibrose und Entzündungsprozesse bestimmen entscheidend den Krankheitsverlauf und das klinische Outcome der Patienten. Trotz dieser großen Relevanz sind detaillierte Informationen zu den beteiligten molekularen Pathomechanismen weitgehend unklar. Diese Studie liefert weitere Evidenz für den aktuellen Paradigmenwechsel, dass Vorhofflimmern eher eine systemisch-entzündliche Erkrankung als eine bloße Ionenkanaldysfunktion ist. Ziel dieser Arbeit war es die Rolle der polo-like Kinase 2 (PLK2) und des proinflammatorischen Zytokins Osteopontin (OPN) im Hinblick auf Fibroblasten(dys)funktion und Fibroseentstehung zu untersuchen, um neuartige Angriffspunkte für zielgerichtete, Fibroblasten-spezifische Pharmakotherapie abzuleiten. Alle Patienten wurden über die Teilnahme an der Studie aufgeklärt und gaben ihr schriftliches Einverständnis. Die vorliegende Studie ist konform mit der Deklaration von Helsinki und enthaltene Tierversuche erhielten ein positives Votum der lokalen Tierschutzbehörde. Humane Vorhoffibroblasten wurden mit der „Outgrowth“-Methode aus Gewebeproben von Patienten im Sinusrhythmus (SR) und im permanenten Vorhofflimmern isoliert. Murine Herzfibroblasten wurden aus dem Überstand nach Langendorff-Perfusion durch mehrere Zentrifugationsschritte gewonnen. Zur Detektion von PLK2 und anderen Markerproteinen wurden (quantitative) PCRs und Western Blots durchgeführt. Zur Beurteilung der Herzfunktion in vivo, wurde transthorakale Echokardiografie mit Oberflächen-EKG-Ableitung genutzt. Nachfolgende Zellkulturexperimente beleuchteten die Auswirkungen pharmakologischer PLK2 Inhibition oder genetischen Knock-Outs auf Fibroblasten im Hinblick auf Proliferation, Differenzierung, Seneszenzentwicklung und Sekretion. Eine Massenspektrometrie-basierte Untersuchung des Sekretoms PLK2-defizienter Fibroblasten wurde im Labor unseres Kollaborationspartners Prof. Manuel Mayr am King’s College London durchgeführt. OPN im peripheren Blut von Vorhofflimmerpatienten wurde mittels eines ELISAs gemessen. Ob die betreffenden Patienten Fibrose der Vorhöfe aufwiesen oder nicht, wurde in klinisch-elektrophysiologischen Untersuchungen, dem sogenannten Mapping, bestimmt. Im Vergleich zu SR-Kontrollen, war die PLK2 mRNA- beziehungsweise Protein-Expression in isolierten Fibroblasten und auf Gewebeebene in VHF-Proben signifikant erniedrigt. Dies korrelierte mit PLK2-Promotermethylierung in der Hälfte der VHF-Proben. In SR-Kontrollen konnten wir keine Methylierung des PLK2 Promoters nachweisen. Die Herunterregulation der PLK2 mRNA-Expression bzw. die Induktion der Promotermethylierung konnten in humanen kardialen Fibroblasten durch Exposition gegenüber chronischer Hypoxie (1% O2) experimentell herbeigeführt werden. Pharmakologische Inhibition und der genetische Knockout (KO) von PLK2 gingen in vitro mit erniedrigter Proliferation aber gesteigerter Differenzierung in Myofibroblasten einher. PLK2-KO-Mäuse entwickelten im Gegensatz zu ihren Wildtyp-Geschwistertieren ausgeprägte Areale interstitieller ventrikulärer Fibrose. Dies spiegelte sich in einer ausgeprägten systolischen und Diastolischen Funktionsstörung des Herzens bei 4 Monate-alten PLK2 KO Tieren wider. Die Sekretomanalyse deckte eine de novo Sekretion von OPN in PLK2-KO-Fibroblasten auf. Im Einklang mit diesem Ergebnis konnten wir höhere OPN-Plasmaspiegel auch bei VHF-Patienten messen, die mit dem Vorhandensein von elektrophysiologisch bestimmten Fibrosearealen korrelierte. Abschließend konnte der p42/44-MAPK-Signalweg als Bindeglied zwischen erniedrigter PLK2-Expression und erhöhter OPN-Freisetzung identifiziert werden. Verminderte PLK2 Expression beziehungsweise Aktivität gehen mit einer gesteigerten Proteinexpression und Phosphorylierung von p42/44 MAPK einher. P42/44 MAPK wiederum stimuliert dann die OPN-Transkription. Folgerichtig führte die Inhibition von p42/44 MAPK zu einer signifikant verminderten OPN-Expression. Um die Allgemeingültigkeit des PLK2-Signalweges für die Entstehung von Fibrose zu erforschen, wurde ein dermales Modell strahleninduzierter Fibrose benutzt. Darin bestätigten sich zum einen die Beobachtungen, die am Herzen gemacht wurden, und zum anderen führte der Einsatz von Mesalazin in vitro zu einer reduzierten p42 MAPK- und SMAD2 / 3-Phosphorylierung und damit zu einer deutlich verringerten OPN- und αSMA-Expression. Fibroblasten von Patienten im permanenten Vorhofflimmern exprimieren weniger PLK2 als Fibroblasten aus SR-Kontrollpatienten. Der Verlust der physiologischen PLK2-Aktivität geht mit ausgeprägten Veränderungen der Proliferation und Differenzierung von Fibroblasten im Herzen einher. Diese Veränderungen begünstigen eine profibrotische Situation auf Gewebeebene, welche durch die lokale als auch systemische Erhöhung des Plasmaosteopontins weiter begünstigt wird. Die vorliegende Studie identifiziert erstmalig PLK2 als neuen Regulator der Fibroblastenfunktion und Fibrose. Gleichzeitig stellen die Wiederherstellung des physiologischen Methylierungstatuses des PLK2 Promoters oder die Inhibition von OPN mittels Mesalazin vielversprechende therapeutische Optionen im Kampf gegen die Fibrosierung des Herzmuskels dar. Die konkrete pharmakotherapeutische Umsetzbarkeit muss in künftigen Forschungsvorhaben überprüft werden.

Traumatic brain injury (TBI) is a worldwide epidemic as the number of victims living with the resulting cognitive and physical impairment continues to rise, principally due to limited treatment options which fail to address its multifaceted sequelae. By approaching TBI therapy from a molecular standpoint, we have the opportunity to develop a better understanding of the mechanisms which prevent effective recovery. With this information, we can move toward the identification of novel therapeutic treatments which target specific molecules to improve patient outcome following TBI. Here, we have focused on the therapeutic potential of osteopontin (OPN), an extracellular matrix (ECM) protein which is a substrate of several matrix metalloproteinases (MMPs), and capable of acting as both a cytokine and modulator of axonal outgrowth during synaptic recovery. The ECM and its components are of particular interest with respect to selecting novel TBI therapeutics since this network has been implicated in neuronal plasticity during both development and following central nervous system (CNS) insult. In this dissertation study, the temporal and spatial profile of OPN expression, its protein and transcript localization within reactive glia (IBA1 positive microglia or GFAP positive astroglia), and its interaction with the cytoarchitectural protein (microtubule associated protein 1B, MAP1B) after injury were each compared under conditions of deafferentation induced synaptogenesis. Two TBI models were employed: one exhibiting adaptive synaptic plasticity (unilateral entorhinal cortex lesion, UEC), and the other generating maladaptive synaptic plasticity (central fluid percussion injury followed by bilateral entorhinal cortex lesions, TBI+BEC), in each case targeting 1, 2, and 7d postinjury intervals. In addition, we examined the potential for converting the adaptive response to one of maladaptive plasticity by attenuating immune reactivity through acute administration of the tricyclic antibiotic minocycline, utilizing a dosing paradigm previously demonstrated to reduce inflammation. To more clearly confirm that OPN has a role in successful synaptic regeneration, we developed a colony of OPN knockout (KO) mice which were used to profile synaptic structure and functional outcome under conditions of UEC-induced synaptogenesis. In Chapter 2, we report that full length OPN responds robustly in the acute (1-2d postinjury) degenerative period following UEC and TBI+BEC. After UEC, time-dependent differences were observed for two alternative, MMP-processed OPN forms, including early increase in a RGD 45 kD, integrin binding fragment (1d), and delayed increase in a C-terminal 32 kD OPN peptide (7d). OPN transcript was also elevated acutely after UEC, a finding which was pronounced in enriched dentate molecular layer (ML) fractions. Parallel immunohistochemistry (IHC) and in situ hybridization localized OPN protein and transcript to reactive glia following UEC. This localization was concentrated within microglia which delineated the border between the intact and deafferented ML, a pattern which was less pronounced in maladaptive TBI+BEC animals. The timing of this glial movement suggests that OPN regulates microglial migration and, potentially, could act as an astrokine to recruit activated astrocytes for influencing subsequent synaptic regeneration. MAP1B staining confirmed dendritic loss during axonal degeneration and dendritic atrophy, with a reemergence during collateral axonal sprouting. However, OPN colocalization with MAP1B was minimal, suggesting a minor role for OPN in reorganization of dendritic/axonal cytoarchitecture in this model of deafferentation. Minocycline reduced acute OPN protein response 2d after UEC, and caused a more random OPN positive glial distribution, similar to that of the maladaptive TBI+BEC. The role of OPN in the inflammation-directed degeneration of terminals is supported by reduced MMP-9 activity, which is temporally correlated with the reduction of MMP-generated OPN lytic fragments (45 kD). Interestingly, this reduction of integrin-binding OPN peptide also matched the impaired removal of presynaptic terminals, evidenced by diminished synapsin 1 clearance in animals which received postinjury minocycline. In Chapter 3, we sought to more precisely evaluate the role of OPN following deafferentation, utilizing wild type (WT) C57BL/6 and OPN KO mice subjected to UEC, comparing the spatio-temporal injury response between WT and KO. To do this we profiled several outcome measures which assessed OPN role in different aspects of recovery: 1) expression of select proteins important in various stages of synaptic recovery, 2) glial response, 3) cognitive recovery, and 4) MMP enzymatic activity. Compared to WT mice, OPN KO mice did not show significant differences in the acute injury-induced alteration of proteins important to cytoarchitectural reorganization (MAP1B) or stabilization of the synaptic junction (N-cadherin). However, both Western blot and IHC analyses showed OPN KO mice had impaired presynaptic terminal clearance, supported by attenuated synapsin 1 breakdown, a result quite similar to that of the minocycline-treated rats with OPN reduction in Chapter 2. This impaired degeneration in OPN KO mice at 2d postinjury correlated with IHC evidence for altered microglial morphology, and hippocampal function assessed by the novel object recognition (NOR) task. Our NOR results confirmed cognitive dysfunction in OPN KO mice during the 4-21d period of synapse reorganization after UEC. In addition, OPN KO decreased MMP-9 activity, an effect associated with reduced MMP-9 bound lipocalin 2 (LCN2), a persistently activated form of that MMP. These latter findings further support the hypothesis that MMP processing of OPN contributes to effective regenerative response after injury. Collectively, the studies presented in the two chapters of this dissertation provide evidence that OPN is a critical element in the acute immune response following injury-induced CNS deafferentation. They suggest that the cytokine can be produced by reactive microglia, may mediate cell migration and acute degenerative clearance, potentially serves as an astrokine to recruit those glia to sites of synaptic repair, and that these processes are disrupted when OPN is either reduced or ablated. Interestingly, this OPN role in synaptogenesis appears to involve ECM interaction with MMP-9, possibly regulated by LCN2. Most importantly, OPN involvement seems to affect the time-dependent progression of synaptic repair, an effect which can be measured by efficacy of functional outcome

Traumatic brain injury (TBI) is a serious health concern, causing cognitive, motor, and sensory deficits, including olfactory dysfunction. This dissertation explores the effects of TBI on synaptic plasticity within the olfactory system, seeking to define mechanisms guiding postinjury sensory reinnervation. Physical forces induced by TBI can axotomize olfactory receptor neurons (ORNs), which innervate olfactory bulb (OB). These axons regenerate OB projections after injury, a process involving growth through a complex extracellular matrix (ECM). As such, we investigated a potential molecular mechanism capable of modifying local OB ECM to support postinjury synaptogenesis. Since matrix metalloproteinases (MMPs) and their ECM substrates are recognized for TBI therapeutic potential, we explored the role of MMP9 and its substrate osteopontin (OPN) in promoting ORN reinnervation of the OB after mild fluid percussion injury (FPI). First, we confirmed that FPI deafferented the mouse OB. In Chapter 2, we showed concurrent activation of neuroglia, elevated spectrin proteolysis and reduction in ORN-specific olfactory marker protein (OMP). As OMP normalized during regeneration, growth associated protein-43kD (GAP-43) peaked, marking OB entry of ORN growth cones. Ultrastructural analysis revealed ongoing ORN axon shrinkage and degeneration, glial phagocytosis of cellular debris, and a reorganization of synaptic structure. To explore ECM role in mediating postinjury OB reinnervation, we defined the time course of MMP9 activity and several downstream targets. Chapter 3 reports biphasic MMP9 activity increase during acute/subacute degeneration, accompanied by robust generation of 48kD OPN cell signaling peptide. OPN receptor CD44 also increased during the acute/subacute interval, suggesting potential interaction of the two proteins. Finally, we utilized MMP9 knockout (MMP9KO) mice to confirm MMP9 role in OB synaptogenesis. In Chapter 4, MMP9KO reversed FPI-induced lysis of 49kD OPN and altered postinjury expression of ORN axon degeneration marker OMP. Additional ultrastructural analysis verified delayed recovery of OB synaptic features within the injured MMP9KO. Overall, we demonstrated that mild FPI elicits ORN axotomy to induce OB reactive synaptogenesis, and that MMP9 supports reinnervation by processing OPN for activation of local glia, cells which reorganize the ECM for synapse regeneration.

La paraparésie spastique tropicale ou la myélopathie associée à HTLV-1 (TSP/HAM) est une maladie neurologique chronique caractérisée par le développement de paralysies spastiques des membres inférieurs et de déficits sensoriels divers. Une infiltration périvasculaire souvent observée dans le système nerveux central des patients atteints de TSP/HAM correspondant essentiellement à des lymphocytes T CD4+, cibles préférentielles du HTLV-1 in vivo. Cependant, le facteur déclencheur du processus inflammatoire de la TSP/HAM est toujours méconnu. De ce fait, nous nous sommes intéressés à l'étude de l'implication d'une nouvelle population inflammatoire des T CD4+, les TH17, dans cette pathologie. Une quantification de l'expression de l'ARNm d'IL-17, la cytokine inflammatoire sécrétée par les TH17, a été menée sur les cellules du sang périphérique issues de patients infectés ainsi que sur des lignées cellulaires chroniquement infectées par HTLV-1. L'expression élevée de l'IL-17 détectée dans les lignées cellulaires est corrélée avec celle de la protéine transactivatrice du HTLV-1, Tax. Par ailleurs, Tax induit l'expression du régulateur transcriptionnel clé des TH17, RORγ, par l'intermédiaire de la cytokine pro-inflammatoire Ostéopontine. Finalement, nous avons pu montrer l'existence d'une relation dynamique entre l'expression de l'ARNm de Tax, OPN, RORγ, IL-17 et IL-22 chez les patients asymptomatiques et TSP/HAM avec une expression d'IL-17 et d'IL-22 plus élevée chez le groupe des TSP/HAM. Nos résultats suggèrent que l'infection par HTLV-1 in vivo induirait une réponse TH17 qui pourrait avoir un rôle majeur dans la pathogenèse de la TSP/HAM. / HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a neurological inflammatory disease of the central nervous system characterized by a chronic, progressive inflammatory demyelinating myelopathy. It is thought that the pathogenesis of this disease involves a predominant infiltration of CD4+ T cells which are the main subset of in vivo infected cells with HTLV-1. However, until now, the identity of the triggering factor which promotes the inflammatory process in HAM/TSP remains unclear. Therefore, we investigated the implication of the new CD4+ T cells inflammatory lineage TH17 in this disease. We quantified the mRNA expression levels of IL-17, a cytokine associated with the TH17 response, in peripheral blood from 10 HAM/TSP patients, 6 healthy asymotomatic carriers (HCs) and 4 normal uninfected controls as well as in HTLV-1-infected T-cell lines. Elevated production of IL-17 observed in HTLV-1-infected T-cell lines was correlated with the expression of Tax, the major HTLV-1 regulatory protein. Thus, we established that Tax increases the expression of RORγ, the TH17-lineage specific transactivator, by inducing Osteopontin expression, an inflammatory cytokine known to promote TH17 response. Finally, we demonstrated a dynamic relationship between the expression of Tax, Osteopontin, RORγ, IL-17 and IL-22 mRNAs in HCs and HAM/TSP patients, where higher expression of IL-17 and IL-22 were observed in HAM/TSP cases. These findings suggest that in vivo infection by HTLV-1 may lead to a deleterious deviation of CD4+ T Helper response to TH17, that could play a major role in HAM/TSP pathogenesis.

Os bisfosfonatos são drogas capazes de inibir a reabsorção óssea por meio de seu efeito direto sobre as células ósseas, interferindo na dinâmica dos tecidos mineralizados. O alendronato (ALN), um tipo de bisfosfonato nitrogenado, foi utilizado com o objetivo de investigar os seus efeitos sobre os tecidos dentários e periodontais após luxação lateral de molares com as raízes em desenvolvimento. Ratos Wistar com 21 dias de idade tiveram os segundos molares superiores luxados lateralmente. Doses diárias de 2,5 mg / kg de ALN começaram no dia seguinte à luxação; os controles receberam solução salina estéril. As maxilas foram fixadas, descalcificadas e incluídas em parafina ou em resina Spurr 7, 14 e 21 dias pós-luxação. Os cortes foram corados com H & E, incubados por histoquímica TRAP e imuno marcados para osteopontina (OPN), bem como para análise ultraestrutural. Após 21 dias, o ápice dos molares luxados sem ALN estava aberto e desorganizado, coberto por uma camada irregular de cemento celular. Os molares luxados dos animais tratados com ALN apresentaram alguns locais de anquilose, bem como lacunas de reabsorção na superfície do cemento. Os osteoclastos TRAP positivos foram mais numerosos no grupo ALN, apesar de sua aparência latente e sua localização, afastados das trabéculas ósseas, em relação aos controles, achado que foi confirmado com a análise ultraestrutural. A imunomarcação de OPN revelou uma linha grossa imunopositiva na dentina, que deve ter surgido a partir do momento da luxação, enquanto que as amostras tratadas com ALN não apresentaram alterações na dentina. Os resultados indicam que o alendronato inibe algumas alterações na dentina e na formação do cemento, induzidas pelo trauma dental de luxação. / Bisphosphonates are drugs that inhibit bone resorption through its direct effect on bone cells, interfering with the dynamics of mineralized tissues. Alendronate (ALN), a nitrogenated bisphosphonate, was used in order to investigate their effects on dental and periodontal tissues after lateral dislocation of molars with developing roots. Twenty one days old Wistar rats had their second molars laterally l. Daily doses of 2.5 mg / kg ALN started the day following the dislocation, while controls received saline solution. The maxillae were fixed, decalcified and embedded in paraffin or in Spurr resin after 7, 14 and 21 days post-dislocation. The sections were stained with H & E, incubated for TRAP, immunolabeled for osteopontin (OPN), and ultrastructurally analyzed by transmission electron microscopy. After 21 days, the apex of the luxated molar without ALN was open and disorganized, covered by an irregular layer of cellular cementum. The luxated molar from ALN-treated animals showed some areas of ankylosis and resorption lacunae on the cementum surface. TRAP-positive osteoclasts were more numerous in the ALN group, despite their latent appearance compared to controls, a finding that was ultrastructurally confirmed. OPN immunostaining revealed a thick immunopositive line in dentin, which must be resultant from the moment of dislocation, while the samples treated with ALN showed no changes in dentin. The results indicate that alendronate inhibits some changes in dentin and cementum formation induced by dental trauma of lateral luxation.

L’ostéopontine (OPN) est une protéine synthétisée et sécrétée par de nombreux types cellulaires. Elle joue un rôle important dans la régulation de la réponse inflammatoire et immune. Elle est également pro-fibrogénique, et présente des propriétés anti-apoptiques. Les NAFLD et ALD sont les premières causes d’ hépatopathies en France. Le spectre de ces complications va de la stéatose à la stéatohépatite, la fibrose, la cirrhose voire le carcinome hépatocellulaire. Le tissu adipeux joue un rôle important dans la survenue et l’évolution des NAFLD. Nous montrons que l'OPN favorise l'inflammation du foie et du tissu adipeux dans les NAFLD, en favorisant le recrutement de macrophages, de cellules dendritiques et de lymphocytes T et en modulant la polarisation de ces cellules immunes. Chez les patients alcooliques, nous rapportons que l’OPN constitue un marqueur prédictif de la fibrose hépatique. Les lésions induites par l'IR hépatique sont la principale cause de dommages survenant au cours des chirurgies du foie. Le rôle de l'OPN lors de l’IR n’a pas été étudié dans le foie. Mes études ont mis en évidence que l'OPN pourrait jouer un rôle protecteur. Son invalidation (OPN-/-) aggrave les lésions hépatiques (inflammation, souffrance et nécrose hépatocytaire) induites par l’IR chez la souris. Ce rôle protecteur de l’OPN pourrait être dû à sa capacité à prévenir la mort hépatocytaire et à limiter la production toxique de NO dans les macrophages. L’ensemble de ces travaux a permis de mettre en évidence de nouveaux rôles de l'OPN dans les lésions induites par l'IR hépatique et pourrait constituer une cible thérapeutique pour les maladies chroniques du foie. / Osteopontin (OPN) is a protein synthesized and secreted by many different types of cells. It plays an important part in the regulation of the inflammatory and immune response. OPN is also pro-fibrogenic, and has anti-apoptotic properties. The nonalcoholic fatty liver disease (NAFLD) and alcoholic liver disease (ALD) are the leading causes of liver disease in France. The range of these complications goes from steatosis to steatohepatitis, fibrosis, cirrhosis and even hepatocellular carcinoma. Adipose tissue plays a significant part on the occurrence and evolution of the NAFLD. We show that OPN facilitates liver’s and adipose tissue’s inflammation in the NAFLD, by facilitating the intake of macrophages, dendritic cells and T cells, and by modulating the polarization of these immune cells. For alcoholic patients, we show that OPN is one of the predictive markers of liver fibrosis. The lesions induced by ischemia-reperfusion (IR) are the main cause of damages occurring during liver’s surgery. The role of OPN in hepatic injury induced by IR has not yet been investigated. My studies demonstrate that OPN could have a protecting role. OPN deficiency in mice (OPN-/-) increases hepatic lesions caused by IR (inflammation, and cell death). OPN could thus partially prevent hepatic injury and inflammation induced by IR. This could be due to its ability to prevent hepatocyte death and production of toxic NO by macrophages. OPN could thus be an important actor in the pathogenesis of chronic liver disease.

Postnatal vascular growth is a complex process involving multiple cells types whose functionality is orchestrated by a variety of soluble extracellular growth factors, mechanical stimuli, and matrix derived cues. The central goal for this dissertation project was to elucidate the role of osteopontin, a non-collagenous extracellular matrix protein, in postnatal vascular growth. At the onset, we concluded that the current methods for measurement of vascularity in small animal models were lacking. To address this shortcoming, we pursued micro-CT imaging for analysis of three-dimensional blood vessel architecture. We were able to demonstrate that micro-CT imaging provides an objective, quantitative, and three-dimensional methodology for evaluation of vascular networks that has broad applicability to preclinical studies. Next, we sought to apply the developed imaging techniques, along with other complementary methodologies, to explore the role of osteopontin in postnatal vascular growth. Osteopontin was previously known to elicit survival, migration, and other relevant activities in multiple cell types involved in postnatal vascular growth. Therefore, we sought to determine the in vivo significance of osteopontin in this process. To do so, we compared wild type and Osteopontin-/- mice for (1) their ability to form collateral vessels and functionally recover following acute induction of hind limb ischemia and (2) their capacity for neovascularization, mineralization, remodeling, and the restoration of mechanical properties during fracture healing. Data suggested that OPN is a critical regulator of collateral vessel formation and that this effect is driven by its role in mediating monocyte/macrophage migration and functionality. Secondly, we found that the presence of osteopontin was essential for normal early callus formation, neovascularization, late stage callus remodeling, and restoration of biomechanical strength. Abnormal collagen organization was observed within the remodeling fractures of Osteopontin-/- mice, and we hypothesize that a unifying link between the vascular and bone defects may be related to deficient matrix organization and remodeling. In conclusion, the imaging techniques developed in this thesis provide a novel methodology for quantitative analysis of vascular structures in small animal models. Secondly, this project has yielded an improved understanding of the basic pathophysiological mechanisms that control postnatal blood vessel growth and bone fracture healing.

Mice homozygous for the disrupted renal type 11a sodium/phosphate (Na/Pi) cotransporter gene, Npt2, (Npt2 KO) exhibit renal Pi wasting and hypercalciuria, predisposing factors for renal stone formation. We observed that Npt2 KO mice, but not wild-type littermates form renal stones. The renal stones were evident in newborn, weanling and adult mice and composed of calcium (Ca) and Pi. The presence of renal calcification correlated with the absence of Npt2 gene expression and the presence of genes responsible for the synthesis (1alpha-hydroxylase) and catabolism (24-hydroxylase) of 1,25-dihydroxyvitamin D, whose elevated levels contribute to the hypercalciuria and renal calcification in Npt2 KO mice. The renal calcification was associated with increased osteopontin (OPN) mRNA expression and colocalized with OPN, the latter associates with renal stones in vivo and inhibits Ca mineralization in vitro). These data demonstrate that hyperphosphaturia and hypercalciuria, secondary to Npt2 gene disruption, are sufficient for the development of renal calcification.

Thesis (118-130)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 118-130).

Thesis (Ph. D.)--Rutgers University, 2007. / "Graduate Program in Ceramic and Materials Science and Engineering." Includes bibliographical references.