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京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第21715号 / 薬科博第106号 / 新制||薬科||11(附属図書館) / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 山下 富義, 教授 髙倉 喜信, 講師 樋口 ゆり子 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

Bacterial meningitis occurs when blood-borne bacteria are able to penetrate highly specialized brain endothelial cells (BECs) and gain access to the meninges. Neisseria meningitidis (Nm) is a human-exclusive pathogen for which suitable in vitro models are severely lacking. Until recently, modeling BEC-Nm interactions has been almost exclusively limited to immortalized human cells that lack proper BEC phenotypes. Specifically, these in vitro models lack barrier properties, and continuous tight junctions. Alternatively, humanized mice have been used, but these must rely on known interactions and have limited translatability. This motivates the need to establish novel human-based in vitro BEC models that have barrier phenotypes to research Nm-BEC interactions. Recently, a human induced pluripotent stem cell (iPSC) model of BECs has been developed that possesses superior BEC phenotypes and closely mimics the in vivo blood vessels present at the blood-meningeal barrier. Here, iPSC-BECs were tested as a novel cellular model to study Nm-host pathogen interactions, with focus on host responses to Nm infection. Two wild type strains and three mutant strains of Nm were used to confirm that these followed similar phenotypes to previously described models. Importantly, the recruitment of the recently published pilus adhesin receptor CD147 underneath meningococcal microcolonies could be verified in iPSC-BECs. Nm was also observed to significantly increase the expression of pro-inflammatory and neutrophil-specific chemokines IL6, CXCL1, CXCL2, CXCL8, and CCL20, at distinct time points of infection, and the secretion of IFN γ and RANTES by iPSC-BECs. Nm was directly observed to disrupt tight junction proteins ZO-1, Occludin, and Claudin-5 at late time points of infection, which became frayed and/or discontinuous upon infection. This destruction is preceded by, and might be dependent on, SNAI1 activation (a transcriptional repressor of tight junction proteins). In accordance with tight junction loss, a sharp loss in trans-endothelial electrical resistance, and an increase in sodium fluorescein permeability was observed at late infection time points. Notably, bacterial transmigration correlated with junctional disruption, indicating that the paracellular route contributes for bacterial crossing of BECs. Finally, RNA-Sequencing (RNA-Seq) of sorted, infected iPSC-BECs was established through the use of fluorescence-activated cell sorting (FACS) techniques following infection. This allowed the detection of expression data of Nm-responsive host genes not previously described thus far to play a role during meningitidis. In conclusion, here the utility of iPSC-BECs in vitro to study Nm infection could be demonstrated. This is the first BEC in vitro model to express all major BEC tight junctions and to display high barrier potential. Altogether, here this model provides novel insights into Nm pathogenesis, including an impact of Nm on barrier properties and tight junction complexes and suggests that the paracellular route contributes to Nm traversal of BECs. / Eine bakterielle Meningitis tritt auf, wenn durch Blut übertragene Bakterien hochspezialisierte Hirnendothelzellen (BEC) durchdringen und Zugang zu den Meningen erhalten. Neisseria meningitidis (Nm) ist ein human-exklusiver Erreger, für dessen Untersuchung es an geeigneten In-vitro-Modellen mangelt. Bis vor kurzem war die Modellierung von BEC-Nm-Wechselwirkungen fast ausschließlich auf immortalisierte humane Zellen beschränkt, denen wichtige BEC-Phänotypen fehlen. Besonders hervorzuheben sind das Fehlen physiologischer Barriereeigenschaften durch unkontinuierliche dichte Zell-Zell-Verbindungen. Als alternative Modellorganismen können humanisierte Mäuse verwendet werden, die sich jedoch auf bekannte Wirt-Erreger-Wechselwirkungen stützen und durch Speziesunterschiede eine eingeschränkte Übersetzbarkeit aufweisen. Dies begründet die Notwendigkeit, neuartige humane In-vitro-BEC-Modelle zu etablieren, die physiologische Barrierephänotypen aufweisen, um Nm-BEC-Wechselwirkungen zu untersuchen. Kürzlich wurde ein humanes Modell entwickelt, welches auf aus induziert pluripotenten Stammzellen (iPSCs) abgeleiteten humanen BECs basiert und sich durch einen physiologischen Blut-Hirn-Schranken-Phänotyp auszeichnet. Die iPSC-BECs wurden in dieser Arbeit als neuartiges zelluläres Modell getestet, um Nm-Wirt-Pathogen-Wechselwirkungen zu untersuchen, wobei der Schwerpunkt auf Wirtsreaktionen auf Nm-Infektionen lag. Zwei Wildtypstämme und drei Mutantenstämme von Nm wurden verwendet, um zu bestätigen, dass diese ähnlichen Phänotypen wie in zuvor beschriebenen Modellen folgten. Hervorzuheben ist, dass die Rekrutierung des kürzlich veröffentlichten Pilus-Adhäsin-Rezeptors CD147 unter Meningokokken-Mikrokolonien in iPSC-BECs verifiziert werden konnte. Es wurde auch beobachtet, dass Nm die Expression der entzündungsfördernden und neutrophilen spezifischen Chemokine IL6, CXCL1, CXCL2, CXCL8 und CCL20 zu bestimmten Zeitpunkten der Infektion sowie die Sekretion von IFN-γ und RANTES durch iPSC-BECs signifikant erhöht. Es wurde zudem beobachtet, dass Nm die Tight Junction-Proteine ZO-1, Occludin und Claudin-5 zu späten Zeitpunkten der Infektion zerstört, verursacht durch die Infektion wurde ein ausgefranster und/oder diskontinuierlicher Tight Junction-Phänotyp beobachtet. Dieser Zerstörung geht die SNAI1-Aktivierung (ein Transkriptionsrepressor für Tight Junction-Proteine) voraus und könnte von ihr abhängig sein. In Übereinstimmung mit dem Verlust der Tight Junctions wurde zu späten Infektionszeitpunkten ein starker Verlust des transendothelialen elektrischen Widerstands und eine Zunahme der Natriumfluoreszein-Permeabilität beobachtet. Bemerkenswerterweise korrelierte die bakterielle Transmigration mit dem Verlust der Tight Junctions, was darauf hinweist, dass der parazelluläre Weg zur bakteriellen Überwindung von BECs eine entscheidende Rolle spielt. Schließlich wurde die RNA-Sequencing (RNA-Seq) von sortierten, infizierten iPSC-BECs durch die Verwendung von fluoreszenzaktivierten Zellsortiertechniken (FACS) nach der Infektion durchgeführt. Dies ermöglichte erstmalig den Nachweis von Expressionsdaten von Nm-responsiven Wirtsgenen, welche bei der Meningitidis eine Rolle zu spielen scheinen. Zusammenfassend konnte im Rahmen der vorliegenden Arbeit der Nutzen von iPSC-BECs In-Vitro-Modellen zur Untersuchung von Nm-Infektionen gezeigt werden. Dies ist das erste BEC-In-vitro-Modell, das alle wichtigen BEC-Tight Junctions exprimiert und ein hohes Barrierepotential aufweist. Insgesamt liefert das eingesetzte Modell neue Einblicke in die Nm-Pathogenese, einschließlich der Beeinflussung der Barriereeigenschaften und der Tight Junction-Komplexe durch Nm, und gibt erste Hinweise darauf, dass die parazelluläre Route zum Nm-Übertritt von BEC-Barrieren eine entscheidende Rolle spielt.

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Tissue Engineering A Blood Vessel Mimic While Monitoring Contamination Through Sterility Assurance Testing Navid Djassemi Tissue engineering blood vessel mimics has been proposed as a method to analyze the endothelial cell response to intravascular devices that are used in today’s clinical settings for the treatment of cardiovascular disease. Thus, the development of in vitro blood vessel mimics (BVMs) in Cal Poly’s Tissue Engineering Lab has introduced the possibility of assessing the characteristics of cellular response to past, present, and future intravascular devices that aim at treating coronary artery disease. This thesis aimed at improving the methods and procedures utilized in the BVM model. Initial aspects of this project focused on using an expanded polytetrafluoroethylene (ePTFE) scaffold in conjunction with human endothelial cells to replicate the innermost intimal layer of a blood vessel. Human umbilical vein endothelial cells (HUVECs) were pressure sodded onto ePTFE scaffolds through cell sodding techniques in an attempt to effectively and consistently replicate and assess the intimal layer. Through each study ePTFE grafts were subjected to different culture times and steady flow rates to observe and compare the differences in the endothelial cell deposition. Results were inconsistent, although moderate cell adhesion was noted throughout each of the BVM setups. Each study exhibited a range of cell sodding density rates. In the second phase of the thesis, contamination assessment protocols were implemented in the BVM lab. The intent of this part of the project was to assess the relative sterility in the cell culture lab, a critical component involved with the success or hindrance of cell and tissue cultures. Using microbiological validated methods, microbiological tests were conducted to examine the levels of microbial growth in and around the tissue engineering lab. Results were tracked over a two month period in the lab with several observations of aerobic microorganism growth on various counter and lab surfaces. Higher growth trends were found on surfaces outside the cell culture lab, in the general TE lab area. These findings were used to provide overall suggestions on tracking microbes for long-term durations in ongoing BVM setups to directly improve the overall sterility assurance of the studies. As the project reached its conclusion a look back at all the BVM setups and contamination assessments lead to a few suggestions for improving aseptic techniques within the TE lab, such as monitoring microbial growth in the culture processes, creating limit specifications, and creating a standardized way to regulate quality control within the lab environment. Furthermore, as the development BVM evolves, the findings from this report can be used with related research for improving the culture conditions of various BVM studies.

The effect of estrogen on the vasculature is mediated in part by influences on NO bioavailability. Nitric oxide (NO) is a potent vasodilator which is synthesized in endothelial cell by endothelial nitric oxide synthase (eNOS) catalyzed conversion of L-arginine to L-citrulline. Although estrogen has been shown to increase eNOS expression and/or activity, the mechanism of estrogen-mediated increased eNOS activity in endothelial cells remains elusive. The Ca 2+ /calmodulin complex is known to aid in eNOS activation by dissociating eNOS from the membrane bound protein, caveolin-1. We investigated the role of estrogen on the Ca 2+ homeostasis of the human endothelial cell line, EA.hy926, using thapsigargin (TG), a sarco(endo)plasmic reticulum Ca 2+ -ATPase, or ATP, a purinergic receptor agonist, to evoke increased intracellular calcium concentration ([Ca 2+ ] i ). [Ca 2+ ] i in Fura 2-AM-loaded EA.hy926 cell populations were measured by fluorescence spectrophotometry. Treatment of cells with 17 β-estradiol (E 2 , 1 μM, 24 hours) showed an increased agonist-evoked [Ca 2+ ] i increase due to both higher Ca 2+ release and Ca 2+ influx, which accompanied an increased eNOS protein expression. Both increased [Ca 2+ ] i and eNOS expression were attenuated with the nonselective estrogen receptor (ER) inhibitor, ICI 182,780. We further analyzed the role of ER in E 2 - mediated effects using ERα-knockdown cells. ERα-knockdown was achieved by transfecting the cells with ERα-specific siRNA. E 2 did not influence agonist-evoked [Ca 2+ ] i increase in the ERα-knockdown cells, indicating that the E 2 -mediated effects were ERα-dependent. In the vasculature, both the genomic and nongenomic effects of estrogen are mediated via ERα. In the current study, the effect of E 2 on agonist-evoked [Ca 2+ ] i increase was only observed in chronically treated (1 μM, 24 hours) cells and not acutely treated (1 μM, 5 minutes) cells, suggesting a genomic action of E 2 . The genomic action was verified by treating cells with E 2 in the presence of actinomycin D, a transcription inhibitor. Actinomycin D attenuated the effect of E 2 on agonist-evoked [Ca 2+ ] i increase. The present work revealed a transcription-dependent and ERα-mediated modulation of Ca 2+ homeostasis in human endothelial cells treated with estrogen for the long-term. This data suggests a novel mechanism by which estrogen-mediated NO release may occur in endothelial cells.

Yes / Endothelial dysfunction and damage underlie cerebrovascular disease and ischemic stroke. We undertook corneal confocal microscopy (CCM) to quantify corneal endothelial cell and nerve morphology in 146 patients with an acute ischemic stroke and 18 age-matched healthy control participants. Corneal endothelial cell density was lower (P<0.001) and endothelial cell area (P<0.001) and perimeter (P<0.001) were higher, whilst corneal nerve fbre density (P<0.001), corneal nerve branch density (P<0.001) and corneal nerve fbre length (P=0.001) were lower in patients with acute ischemic stroke compared to controls. Corneal endothelial cell density, cell area and cell perimeter correlated with corneal nerve fber density (P=0.033, P=0.014, P=0.011) and length (P=0.017, P=0.013, P=0.008), respectively. Multiple linear regression analysis showed a signifcant independent association between corneal endothelial cell density, area and perimeter with acute ischemic stroke and triglycerides. CCM is a rapid non-invasive ophthalmic imaging technique, which could be used to identify patients at risk of acute ischemic stroke. / Qatar National Research Fund Grant BMRP20038654