Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 5 uppsatser.

Diss. (sammanfattning) Uppsala : Univ., 2004. / Härtill 4 uppsatser.

Licentiatavhandling (sammanfattning) Linköping : Linköpings universitet, 2009. / Härtill 2 uppsatser.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2008. / Härtill 3 uppsatser.

Η χρήση ενδοφλέβιων καθετήρων είναι πλέον συνώνυμη με τις λοιμώξεις που προκαλούνται από στελέχη S. epidermidis. Η ικανότητα του συγκεκριμένου μικροοργανισμού να σχηματίζει βιομεμβράνες σχετίζεται άμεσα με την παθογένειά του. Ορισμένες ερευνητικές ομάδες έχουν περιγράψει κάποιους από τους μηχανισμούς σχηματισμού βιομεμβράνης καθώς και μόρια που συμμετέχουν σε αυτούς. Η ερευνητική μας ομάδα έχει απομονώσει ένα όξινο, θειωμένο πολυσακχαρίτη (20-kDa PS ή PS) ο οποίος φαίνεται να είναι ο κύριος αντιγονικός καθοριστής της εξωκυττάριας βλεννώδους στιβάδας του S. epidermidis. Αντισώματα έναντι αυτού του πολυσακχαρίτη έχει δειχθεί ότι προστατεύουν από σταφυλοκοκκική κερατίτιδα, από νεογνική βακτηριαιμία ενώ ταυτόχρονα διαχωρίζουν τα στελέχη S. epidermidis από τους υπόλοιπους πηκτάση-αρνητικούς σταφυλοκόκκους. Ένας από τους κύριους στόχους της παρούσας διατριβής ήταν η διερεύνηση του ρόλου του PS στην προσκόλληση του S. epidermidis σε ενδοθηλιακά κύτταρα. Για το λόγο αυτό χρησιμοποιήθηκαν ένα πρότυπο στέλεχος S. epidermidis που παράγει τον PS (ΑΤCC35983) και ένα κλινικό στέλεχος το οποίο έχει δειχθεί ότι δεν παράγει τον συγκεκριμένο πολυσακχαρίτη. Τα αποτελέσματα που ελήφθησαν δείχνουν ότι ο PS διευκολύνει την προσκόλληση του S. epidermidis στα ενδοθηλιακά κύτταρα με δοσοεξαρτώμενο τρόπο. Παρά το γεγονός ότι ο ακριβής μηχανισμός προσκόλλησης δεν έχει βρεθεί, εντούτοις μπορεί να συσχετισθεί με το μηχανισμό πρόσδεσης των σταφυλοκόκκων μέσω των αλυσίδων γλυκοσαμινογλυκανών στα στελέχη που παράγουν τον πολυσακχαρίτη. Σε ό,τι αφορά τα στελέχη που δεν παράγουν τον PS ο προαναφερόμενος μηχανισμός συμμετέχει μερικώς στην προσκόλληση του S. epidermidis. Ο δεύτερος στόχος της παρούσας μελέτης ήταν να διερευνηθεί κατά πόσο μεταβάλλεται η έκφραση ορισμένων μακρομορίων του εξωκυττάριου χώρου διότι είναι γνωστό ότι οι πρωτεογλυκάνες της κυτταρικής επιφάνειας συμμετέχουν στην προσκόλληση του S. epidermidis σε ενδοθηλιακά κύτταρα. Για το σκοπό αυτό χρησιμοποιήθηκαν τέσσερα στελέχη S. epidermidis που διέφεραν τόσο ως προς το φαινότυπο όσο και ως προς τον γονότυπο. Τα αποτελέσματα που ελήφθησαν έδειξαν ότι δεν υπάρχουν σημαντικές μεταβολές στην έκφραση των δύο κύριων πρωτεογλυκανών των ενδοθηλιακών κυττάρων, συνδεκάνης-1 και γλυπικάνης-1, σε κανένα από τα χρονικά διαστήματα που χρησιμοποιήθηκαν. Εντούτοις, τα αποτελέσματα δεν μπορούν να αποκλείσουν τη λειτουργία των πρωτεογλυκανων 202 ως γέφυρες που διευκολύνουν την πρόσδεση και τη μετέπειτα είσοδο του S. epidermidis στα ενδοθηλιακά κύτταρα. Έχει δειχθεί ότι το ενδοθήλιο διαδραματίζει σημαντικό ρόλο σε περιπτώσεις λοιμώξεων καθώς πραγματοποιείται ενεργοποίηση της έκφρασης ενός ευρέως φάσματος μορίων προσκόλλησης και εξωκυττάριου χώρου. Στην παρούσα μελέτη διερευνήθηκαν και αξιολογήθηκαν τα ειδικά προφίλ έκφρασης τέτοιων μορίων μέσω Real-Time PCR, ELISA, ανοσοφθορισμού και ζυμογραφίας. Τα αποτελέσματα που ελήφθησαν δείχνουν ότι αν και η γενική απόκριση των ενδοθηλιακών κυττάρων σε περιπτώσεις λοιμώξεων με στελέχη S. epidermidis είναι να αποικοδομήσουν τον εξωκυττάριο χώρο, τα γονίδια που ενεργοποιούνται είναι διαφορετικά και έχουν σχέση με τα στελέχη που χρησιμοποιούνται. Φαίνεται ότι τα ενδοθηλιακά κύτταρα προσπαθούν να μειώσουν τη συνοχή του εξωκυττάριου χώρου έτσι ώστε να διευκολύνουν την μετακίνηση ουδετεροφίλων στο σημείο της λοίμωξης. Μελλοντικές μελέτες στις οποίες θα χρησιμοποιηθούν γενετικά τροποποιημένα βακτηριακά στελέχη σε συν-καλλιέργειες ενδοθηλιακών κυττάρων με μακροφάγα ή μονοκύτταρα θα διευκρινήσουν τις αλληλεπιδράσεις που πραγματοποιούνται in vivo. / The use of intravenous catheters and other medical implanted devices is synonymous to Staphylococcus epidermidis infections. The ability of the specific microorganism to cause infections is directly related to its ability to form biofilms. Some of the mechanisms and molecules participating in the formation of biofilm have been elucidated. Our research group has isolated an acidic, sulfated polysaccharide (20-kDa PS or PS) that appear to be the main antigenic component of S. epidermidis extracellular mucous layer. Antibodies against this polysaccharide protect from staphylococcal keratitis, neonate bacteremia and discriminate S. epidermidis strains from other CoNS. In the present dissertation one of the major goals was to evaluate the role of PS in S. epidermidis’ adherence to human endothelial cells. A PS reference strain (ATCC35983) and a non-PS producing clinical strain were used. Results obtained showed that PS facilitates S. epidermidis’ adherence to endothelial cells in a dose dependent manner. Although the fine mechanism for such a binding is not as yet clear, it may be correlated with the staphylococcal binding through glycasaminoglycan chains at least for the strains that produce this polysaccharide. As long as the non-PS producing strains are concerned the involvement of the above mentioned mechanism has partial contribution to S. epidermidis adherence. Considering that cell surface proteoglycans are involved in S. epidermidis adherence to endothelial cells, the second major goal of this study was to examine whether gene expression of certain matrix biomacromolecules is modified in S. epidermidis infected endothelial cells as compared to non-infected ones. Four different phenotypically and genotypically strains were used. Results show no significant difference in expression of two main cell-surface proteoglycans, syndecan-1 and glypican-1, in all time points and for all strains used. These results, however, cannot exclude that proteoglycans may be the bridge facilitating S. epidermidis adherence and subsequently invasion to endothelial cells. It has been established that endothelium plays a crucial role in inflammation. This is achieved by the consequent expression of a wide variety of adhesion and extracellular matrix molecules. In the present study, the specific expression profile of such molecules was also evaluated using Real-Time PCR, ELISA, immunofluorescence and zymography. Obtained results indicate that, even though the general reaction of endothelial cells is to disintegrate the structure of the extracellular matrix, the genes activated are different with respect to the 204 strains that were used. It seems that the endothelial cells’ effort to decrease the extracellular matrix cohesion is a way to facilitate migration of macrophages at the site of infection. Future studies using genetically modified bacterial strains in cocultures of endothelial cells with macrophages or monocytes will elucidate the interactions that actually happen in vivo.

The composition and conformational state of biological molecules have a profound influence on cell behavior and large-scale processes including development and disease progression. Fibronectin fibers are a prevalent component of the extracellular matrix that are believed to adopt a wide array conformations with different functions. Two factors that are hypothesized to regulate fibronectin conformation, and hence fibronectin biological function, are allosteric regulators, such as heparan sulfates, and mechanical strain. However, the relative influence of allosteric regulators and mechanical forces on fibronectin conformation has not been determined. This conformational regulation is especially important in the context of the heparin 2 binding domain (modules III12 to III14), which is known to bind and present numerous growth factors, such as vascular endothelial growth factor, to cells. This thesis will highlight three contributions to this field. First, a new, and remarkably simple technique was developed that permits the detection of the non-equilibrium fibronectin conformations. This technique is founded on the identification of monoclonal antibodies that have altered affinities for fibronectin based on heparin treatment or mechanical strain dependence, or that bind fibronectin equally well in all conditions. Second, the impact of both heparin and mechanical strain on the binding of VEGF to the hep2 region of fibronectin was investigated. It was discovered that both strain and heparin co-regulate VEGF binding. Finally, studies of cell attachment and migration on single fibers of fibronectin with controlled strain states provided the first direct evidence that mechanical strain regulates cell attachment, spreading, and migration on a fibronectin matrix. This body of work demonstrating that the conformational changes in fibronectin lead to altered biological activity has broad impact in a number of fields due to the ubiquitous presence and requirement of fibronectin in cell and tissue function.

Polydimethylsiloxane (PDMS) microfluidic channels, fabricated using low cost and simple soft lithography methods, conventionally have rectangular cross-sections. Despite being often used for organs-on-a-chip and cardiovascular research, these devices do not mimic the circular cross-sections of blood vessels in the human body, creating potential inaccuracies in observed flow conditions and cell behaviours. The purpose of this thesis is to (i) compare and optimize fabrication techniques for microchannels with circular cross-sections, (ii) assess biocompatibility of different surface functionalization approaches for Human Umbilical Vein Endothelial Cell (HUVEC) adhesion and growth, (iii) culture HUVECs within circular microchannels to mimic blood vessel features, and (iv) compare gene expression of HUVECs cultured in 3D circular microchannels to those cultured on 2D surfaces. We show that wire molding is superior to the gas stream technique for producing circular cross-section microchannels with high aspect ratios, circularity, and channel geometry precision. Fibronectin (FN) and polydopamine (PD) surface coatings on PDMS, as well as alternative collagen substrates, were tested for biocompatibility with HUVECs in 2D cultures; fibronectin coated PDMS (PDMS-FN) substrates facilitated cell attachment, spreading and growth. We demonstrate the capability of growing HUVECs on the inner surface of circular PDMS microchannels created using the wire-mold method and treated with fibronectin. A syringe pump was used to induce shear stress on the HUVECs grown in circular microchannels. Relative to static growth conditions, longer cell culture growth periods were more feasible under flow and altered cell morphology was observed. Finally, Microarray analysis revealed significantly different gene expression profiles for HUVECs cultured within PDMS-FN circular cross-section microchannels as compared to HUVECs cultured on PDMS-FN in a 2D environment, thereby highlighting the critical importance of in vitro conditions for mimicking the in vivo reality.

O hormônio melatonina produzido pela glândula pineal no período de escuro, participa na regulação circadiana de processos, fisiológicos e fisiopatológicos envolvendo vasos sanguíneos. Alguns destes estudos sugerem que as células endoteliais, que revestem os vasos sanguíneos são alvo para a melatonina circulante e medeiam a regulação do tônus vascular, em condições fisiológicas, e da interação neutrófilo-endotélio, em resposta a um estímulo injuriante. O óxido nítrico produzido pelas células endoteliais é um dos responsáveis por grande parte dos eventos vasculares, e a melatonina inibe a produção de óxido nítrico em diversos modelos. O objetivo deste estudo foi verificar o efeito da melatonina na produção de óxido nítrico induzido por bradicinina em células endoteliais em cultura. Para tanto, utilizamos uma técnica de cultura primária de células endoteliais de rato e através de um marcador fluorescente de óxido nítrico intracelular, mensuramos a fluorescência em microscópio confocal. Foi verificado que a melatonina e seu precursor N-acetilserotonina inibem a produção de óxido nítrico induzido por bradicinina e este efeito não ocorre pela inibição do aumento de cálcio que induz a produção de óxido nítrico. O análogo de receptores MT2 (4P-PDOT) e MT3 (5-MCA-NAT) não provocaram qualquer alteração sobre o aumento de óxido nítrico induzido por bradicinina, e a utilização do antagonista de receptores MT1 e MT2 (luzindol) não reverteu o efeito inibitório da melatonina. Portanto, nossos dados indicam que o efeito da melatonina sobre a atividade da NOS constitutiva não é mediado por receptores de membrana. Considerando que a melatonina é capaz de ligar-se à calmodulina, inibindo desta maneira a atividade da NOS endotelial constitutiva, poderíamos sugerir que este seria o mecanismo de ação. No entanto, é preciso ressaltar que tal atividade não é comprovada para a N-acetilserotonina, assim, apesar de ser este um possível mecanismo de ação, há a necessidade de demonstrar que a N-acetilserotonina está se ligando a calmodulina extraída de células endoteliais. Em resumo, neste trabalho mostramos que a melatonina em concentrações compatíveis com o pico noturno encontrado na circulação, pode modular eventos vasculares no organismo, através da inibição da produção de óxido nítrico em células endoteliais induzida por bradicinina. / Melatonin, the hormone synthesized by the pineal gland at night, signalizes darkness and modulates, in a circadian basis, blood vessels activity. Previous studies suggest that endothelial cells are the target for circulating melatonin and mediate changes in vascular tone and leukocyte-endothelial adherence properties. Melatonin effects can be mediated by several pathways, such as G protein-coupled receptors (MT1 and MT2 receptors), a putative membrane receptor, most probably an enzyme-binding site (MT3 receptor), and several intracellular mechanisms, including calmodulin binding and inhibition of constitutive and induced nitric oxide synthase. The aim of the present study was to characterize melatonin effect on the production of nitric oxide by bradykinin-stimulated endothelial cells in culture. Nitric oxide production was measured in real time at cellular level by detecting fluorescent stimulation of the probe DAF by confocal microscopy. After determining the ideal conditions for recording cumulative dose-response curves for bradykinin (1 – 100 nM) the effect of pre-incubated (1 min) melatonin and analogs was evaluated. Melatonin and its precursor, N-acetylserotonin, but not the selective ligands for receptors MT2 (4P-PDOT) and MT3 (5-MCA-NAT) receptors inhibited bradykinin-stimulated nitric oxide production. This effect was not blocked by the classical antagonist of MT1 and MT2 receptors, luzindol; excluding therefore the participation of membrane receptors. Taking into account that melatonin inhibits calmodulin activation of several enzymes, including constitutive nitric oxide synthase in brain and cerebellum, it could be suggested a similar mechanism for endothelial cells. However, this hypothesis is discussed taking into account that N-acetylserotonin was shown to do not bind neural cells calmodulin. In addition, here we discuss the relevance of the present finding according to physiological and physiopathological roles of endothelial nitridergic system. This analysis point melatonin modulation of constitutive nitric oxide synthase activity as a putative mechanism for explaining melatonin control of vascular tone.

O endotélio é a camada celular interna dos vasos sanguíneos, responsável pela homeostase vascular. Além disso, é a porta de entrada para as células de defesa frente a um quadro de inflamação. A camada endotelial é alvo de diversos estudos devido ao seu caráter de fácil expansão em cultura, porém sua biologia ainda não é completamente compreendida. Devido à sua localização privilegiada, o endotélio está susceptível a alterações da composição da corrente sanguínea. A melatonina, hormônio produzido ritmicamente pela glândula pineal e de forma não rítmica em diversos outros tecidos, tem propriedade citoprotetora. Diversos estudos já demonstraram que ela atua, por diferentes mecanismos de ação e faixas de concentração, como um mediador anti-inflamatório sobre o endotélio. Segundo a hipótese de trabalho de nosso grupo, o eixo imune-pineal, a glândula pineal e o sistema imunológico se interligam em uma comunicação bidirecional, na qual a produção rítmica de melatonina é inibida frente a um quadro de inflamação para que ocorra a montagem da resposta inflamatória independente da hora do dia. Assim sendo, esta dissertação se baseou na hipótese de que as células endoteliais apresentam um ritmo em sua maquinaria que altere a intensidade de resposta frente a um estímulo inflamatório, e propôs avaliar como a melatonina agiria na regulação desse ritmo. Dessa forma, avaliamos como o tratamento sistêmico com LPS afetaria a produção noturna de melatonina, modulando a reatividade de células endoteliais da microcirculação. Demonstramos que o tratamento com LPS diminui os níveis circulantes deste hormônio e inibe a transcrição gênica da enzima chave, AA-NAT. Na periferia, o tratamento com LPS aumenta a reatividade de células endoteliais independente da hora do dia de administração mesmo após 18 dias em cultura. Este efeito é transiente, pois quando o tratamento é realizado seis horas antes da morte, os parâmetros analisados retornam aos níveis basais. A melatonina, administrada juntamente com LPS, reverte o efeito do LPS sobre as células endoteliais, além de reduzir a concentração plasmática de TNF. Além disso, células endoteliais obtidas de animais mortos à noite possuem menor estado de ativação que células provenientes de animais mortos de dia. De maneira geral, o efeito observado sobre as células endoteliais é inversamente correlacionado com a concentração plasmática de melatonina. Esses dados sugerem que as células endoteliais possuem uma espécie de \"memória celular\", pois armazenam as informações do estado do animal doador mesmo após todos os procedimentos em cultura. Adicionalmente, demonstramos a dinâmica do eixo imune-pineal in vivo. Juntamente, nossos dados permitem concluir que a melatonina prima as células endoteliais, modulando sua reatividade de acordo com a hora do dia e o estado de saúde do animal. / The endothelium is the vascular internal cellular layer, responsible for vascular homeostasis. Additionally, it regulates immune cells entrance during an inflammatory response. The endothelial layer is the focus of many studies due to its facility of culture expansion, but its biology is not yet totally understood. Because of its privileged localization, the endothelium is susceptible to plasma compounds changes. Melatonin, rhythmically produced by pineal gland and in a non rhythm way in others tissues, has citoprotector properties. Many studies have already shown that melatonin acts on endothelium as an anti-inflammatory mediator, through different mechanisms of action and concentrations ranges. Considering our work hypothesis, the immune-pineal axis, that suggests that the pineal gland and immune system are integrated through a bidirectional communication, melatonin rhythm production is inhibited during an injury, permitting the mounting of immune response independently of the hour of the day. This dissertation is based on the hypothesis that endothelial cells presents a rhythm in its machinery that alters the response intensity due to an inflammatory stimuli. We analyzed how LPS systemic treatment affects the melatonin nocturnal production, modulating the endothelial cells reactivity of microcirculation. We demonstrated that LPS treatment reduced plasma melatonin level and inhibited gene transcription of key enzyme, AA-NAT. On the periphery, LPS treatment increased endothelial cells reactivity independently of the hour of the day even after 18 days in culture. This effect was transient, once the parameters analyzed returned to basal levels when the treatment was done six hours before the death. Melatonin administrated together with LPS, reverted LPS effects on the endothelial cells, and also reduced plasma TNF concentration. Endothelial cells obtained from animal killed at nighttime are more activated than cells obtained from animals killed at daytime. Generally, the endothelial cells effects are inversely correlated with plasma melatonin level. These data suggests that endothelial cells have a \"cellular memory\", because they are capable to retain the information of donor animal state even after all culture proceedings. Additionally, we demonstrated the immune-pineal axis dynamics in vivo. All together, our results permit to conclude that melatonin primes the endothelial cells, modulating their reactivity according to the hour of the day and donor animal health.

A melatonina é produzida pela glândula pineal somente durante o escuro e atinge rapidamente a circulação, além disso, outros tecidos e células são capazes de produzir melatonina. As células endoteliais, devido a sua localização, são excelentes alvos para as ações da melatonina. O entendimento dos mecanismos de ação pelos quais a melatonina desenvolve seus efeitos sobre as células endoteliais, possibilitaria o uso desta indolamina e de seus análogos como uma importante ferramenta farmacológica. No presente trabalho, demonstramos que a melatonina em concentrações compatíveis com as encontradas na circulação durante o pico noturno de produção pela pineal, atua sobre as células endoteliais inibindo a produção de NO proveniente da enzima constitutiva (eNOS), enquanto altas concentrações de melatonina, que podem ser atingidas por exemplo pela produção por células imunocompetentes ativadas, inibem a produção induzida de NO mediada pela iNOS. A melatonina (1 nM) inibe a produção constitutiva de NO induzida por agonistas que atuam através da ativação de receptores acoplados à proteína G (histamina, carbacol e ATP/P2Y), e este efeito deve-se à inibição do aumento de [Ca2+]i por liberação de estoques intracelulares, sendo independente da ativação de receptores de melatonina. A melatonina inibe os efeitos decorrentes da produção de NO induzida por bradicinina como a produção de GMPc por células endoteliais e a vasodilatação de arteríolas \"in vivo\". A melatonina inibe a produção de NO induzida por LPS também de maneira independente da ativação de seus receptores, porém, em concentrações muito maiores (1-10 µM) do que a necessária para inibir a produção constitutiva. Estes efeitos devem-se à inibição da expressão da enzima iNOS por impedir a translocação do NF-kB ao núcleo. A vasodilatação de aortas induzida por LPS também é inibida por melatonina. Podemos concluir até o momento que as células endoteliais, devido a sua localização, são excelentes sensores para as ações da melatonina e podem auxiliar no melhor entendimento do conceito \"eixo imune-pineal\". Os estudos sobre os mecanismos pelos quais a melatonina atua em condições fisiológicas e fisiopatológicas são essenciais para se conhecer o potencial terapêutico da melatonina. / Melatonin, the darkness hormone, produced at night by the pineal gland, is also synthesized in a non-rhythmic manner by other cells. Pineal and extra-pineal melatonin reaches endothelial layer, and the understanding of its mechanism of action will improve the possibilities of using this indolamine and derivates as pharmacological tools. Here we showed that melatonin, in concentrations compatible to nocturnal melatonin surge impairs the activity of eNOS, while much higher concentrations, which can be attained by activated immune competent cells, impair the induction of iNOS synthesis. As a consequence of inhibiting eNOS we showed that melatonin inhibits vasodilation of the microcirculation induced by bradykinin. The inhibitory effect of melatonin is observed only when eNOS is activated by triggering G protein-coupled receptors (bradykinin B2, muscarinic and P2Y purine receptors). Activation of eNOS by calcium-channel operated receptors (P2X) is not blocked by melatonin. Inhibition of the transcription of iNOS results in inhibition of the LPS-induced vasodilation of rat aorta. As a matter of fact, here we show that LPS effect is dependent on the endothelial layer. The mechanism of action of melatonin in inhibiting iNOS transcription is due to block of the NF-kB pathway. Our work contributed to unravel the role of endothelium cells as targets for melatonin and as a key player in the \"immune-pineal axis\". The understanding of the concentrations ranges reached by endogenous production, i.e., the discrimination between the levels achieved during physiological and physiopathological responses, are essential for using these substances as analogous therapeutical tools.