An in vitro model of angiogenesis of endothelial cells from the murine myocardium and from the human neonatal foreskin and transfection of endothelial cells with different plasmid constructs

Titelblatt, Inhaltsverzeichnis, Lebenslauf 1\. Einleitung 2\. Literaturübersicht 3\. Materialien 4\. Methoden 5.1 Ergebnisse Teil 1 5.2 Ergebnisse Teil 2 6\. Diskussion 7\. Zusammenfassung 8\. Summary LiteraturverzeichnisAngiogenese, die Neubildung von Blutgefässen, kommt physiologischerweise nur im Embryo und Fetus sowie beim Adulten im Rahmen zyklischer Prozesse im Ovar, in der Plazenta und bei der Entwicklung der Milchdrüse vor (Risau, 1997). Alle anderen Formen der Angiogenese sind mit pathologischen Prozessen, insbesondere dem Tumorwachstum, verbunden. Eine hoffnungsvolle und Erfolg versprechende Alternative zu bisherigen Strategien der Tumortherapie ist die gentherapeutische Anti-Angiogenese. Eine selektive Expression des Transgens von Endothelzellen kann dabei durch Einsatz von Endothelzell-spezifischen genregulatorischen Elementen erzielt werden. Die vorliegende Arbeit verfolgte zwei Hauptziele, nämlich die Etablierung und Charakterisierung von in vitro- Modellen der Angiogenese muriner und humaner mikrovaskulärer Endothelzellen sowie die Charakterisierung verschiedener Plasmidkonstrukte auf Effizienz in diesen Modellen. Im Rahmen der Transfektionsversuche sollten zusätzlich morphologische Veränderungen transfizierter Endothelzellen auf licht- und elektronenmikroskopischer Basis untersucht werden, um einerseits Aussagen über die Aufnahme der Transfektionskomplexe in die Zellen sowie deren Weg in den Zellkern zu machen und andererseits mögliche morphologische Schädigungen der Zellen infolge der Transfektion zu beurteilen und abzuschätzen. Analog zur in vivo-Angiogenese durchliefen die eingesetzten mikrovaskulären Endothelzellen, isoliert aus der Vorhaut von Neugeborenen sowie aus dem Myokard zwei Wochen alter Mäuse, durch Stimulation mit pro-angiogenen Faktoren verschiedene Stadien der angiogenen Kaskade in vitro. Initiiert wurde die angiogene Kaskade morphologisch durch eine vollständige bzw. partielle Konfluenz der Endothelzellen (Stadium 1). Es kam zur linearen und zirkulären Aneinanderreihung von Endothelzellen (Stadium 2) und schliesslich zur Ausbildung kapillarähnlicher Strukturen mit einem zentralen Lumen (Stadium 3 und 4). Die Endothelzellen bildeten ein basalmembranähnliches Material, welches interzellulär sowie in intrazellulären Vakuolen und im Lumen kapillarähnlicher Strukturen detektiert werden konnte. Die Lumenbildung erfolgte durch Ausbildung intrazellulärer Vakuolen sowie durch Apoptose. Wýhrend die eingesetzten murinen Endothelzellen planar zur Kulturschalenoberfläche kapillarähnliche Strukturen ausbildeten (zweidimensionales in vitro-Modell der Angiogenese), konnte mit den humanen Endothelzellen erstmals ein realitätsnahes, dreidimensionales in vitro-Modell der Angiogenese etabliert werden, in dem analog zur in vivo-Angiogenese die Ausbildung kapillarähnlicher Strukturen durch Degradation eines von den Zellen selbst sezernierten basalmembranähnlichen Substrates sowie Migration bzw. Invasion, Proliferation und Differenzierung erfolgte. Im basalmembranähnlichen Material wurde immunhistochemisch Kollagen IV identifiziert. Ein besonderer Befund dieser Arbeit, beobachtet im murinen Zellkulturmodell, war der zyklische Verlauf der in vitro-Angiogenese mit dazwischen liegender „Latenzzeit". Nach dem Ablösen der kapillarähnlichen Strukturen von der Kulturschale konnte nach ca. 2 Monaten Kultivierung der auf der Kulturschale verbliebenen Endothelzellen ein erneutes Auftreten dieser Strukturen beobachtet werden. Interessant war, dass im zweiten Zyklus gerade die Zellen kapillarähnliche Strukturen ausbildeten, welche zuvor unbeteiligt an der Ausbildung dieser Strukturen waren. Die Ergebnisse indizieren, dass während der „Latenzzeit" eine Differenzierung der Endothelzellen in einen angiogenen Phänotyp erfolgte. Die Polyfektion mittels aktivierten Dendrimeren erfolgte vergleichend im Stadium der endohelialen Proliferation (Stadium 0) sowie der Bildung kapillarähnlicher Strukturen (Stadium 3 bzw. 4). Die Transfektion proliferierender Endothelzellen führte mit allen untersuchten Vektoren pJWM115 (CMV-luc), pCK5 (Ets-1l-luc), pPS12 (Ets-1k-luc) und pPS6 (E-sel-luc) zur Expression des Reportergens Luciferase, woraus zu schliessen ist, dass sowohl das Adhäsionsmolekül E-Selektin als auch der Transkriptionsfaktor Ets-1 in proliferierenden murinen und humanen Endothelzellen exprimiert werden. Insgesamt wurden mit den Vektoren pCK5 und pPS12 höhere Expressionsraten der Luciferase erzielt als mit dem pPS6-Vektor. Die Ergebnisse der Transfektion muriner und humaner Endothelzellen im Stadium der Bildung kapillarähnlicher Strukturen spiegelten die angiogenetische Situation beider in vitro-Modelle wider. Während die Transfektion muriner Endothelzellen im Stadium 3, unabhängig vom eingesetzten Vektor, zu keiner Expression der Luciferase führte, wurde nach Transfektion humaner Endothelzellen im Stadium der dreidimensionalen Organisation kapillarähnlicher Strukturen (Stadium 4) eine geringe Expression detektiert. Dabei konnte ein wichtiger Unterschied zwischen den einzelnen Versuchen im humanen Zellkulturmodell festgestellt werden. Humane Endothelzellen, die zu Beginn des Stadiums 4 in die Transfektionsexperimente einbezogen wurden, zeigten eine höhere Expression der Luciferase als Endothelzellen, die zu späteren Zeitpunkten des Stadiums 4 transfiziert wurden. Dies ist durch die unterschiedliche Proliferationsrate der Endothelzellen in diesem Stadium zu erklären. Diese Ergebnisse indizieren eine Zellzyklusabhängigkeit des verwendeten Gentransfersystems. Ein effizienter Gentransfer konnte nur in proliferierenden Endothelzellen beobachtet werden, als Resultat einer gesteigerten Aufnahme der Komplexe sowie eines effizienteren Eintritts der Komplexe bzw. Plasmid-DNA in den Kern. Die ultrastrukturelle Untersuchung liess vermuten, dass die durch Endozytose aufgenommenen Komplexe vor der Fusion mit primären Lysosomen aus den Endosomen entkommen, da freie DNA-Dendrimer-Komplexe an der Kernmembran vor dem Erscheinen multivesikulýrer Körper (späte Endosomen) in humanen Endothelzellen detektiert werden konnten. In den murinen Endothelzellen schien sich der intrazelluläre Abbau der Komplexe im Vergleich zu den humanen Endothelzellen langsamer zu vollziehen, welches die allgemein höhere Effzienz des Gentransfers in diesen Zellen zumindest partiell erklären könnte. In einer weiteren Versuchsreihe wurde die Steigerung der E-sel- Promotoraktivität (pPS6) durch die Endothelzell-spezifischen Enhancer 5xebs (pPO18) und Flk-1 (pPO14) sowie durch das HLA-Intron (pPO12) untersucht. Im Gegensatz zum HLA-Intron, welches zu einer Erhöhung der Aktivität des E-sel- Promotors um ein Vielfaches führte, konnte die Promotoraktivität durch die Endothelzell-spezifischen Enhancer nicht gesteigert werden. Dies deutet darauf hin, dass die entsprechenden Transkriptionsfaktoren wie z.B. Ets-1 in den untersuchten Zellkulturmodellen nicht in ausreichender Menge exprimiert werden. Genexpressionsanalysen oder immunhistochemische Untersuchungen der Endothelzellen in verschiedenen Stadien der angiogenen Kaskade in vitro könnten hierüber Aufschluss geben. Im Hinblick auf eine Reduktion von Tierversuchen durch Ersatz- und Ergänzungsmethoden kommt den in der vorliegenden Arbeit etablierten in vitro- Modellen der Angiogenese eine besondere Bedeutung zu. Über den Tierschutzaspekt hinaus stellen sie kostengünstige, sensitive, einfache experimentelle Systeme für weiterführende Untersuchungen dar. Während das murine Zellkulturmodell insbesondere für Gentransferstudien geeignet ist, da sich die murinen Endothelzellen effizient transfizieren lassen, steht mit dem realitätsnahen, dreidimensionalen in vitro-Modell der Angiogenese humaner Endothelzellen ein experimentelles System zur Verfügung, welches sich in der Hauptsache für Untersuchungen der in vitro-Angiogenese eignet. Aufgrund des fehlenden Einsatzes einer dreidimensionalen extrazellulären Matrix und der damit verbundenen Reduktion nicht bzw. schwierig standardisierbarer Faktoren ist dieses Modell geeignet für Untersuchungen von beispielsweise Zelladhäsionsmolekülen, Zell-Matrix-Interaktionen oder auch zur Identifizierung spezifischer Inhibitoren der Lumenbildung. Die etablierten Endothelzellkulturen werden derzeit bereits in anderen Laboratorien für ähnliche Experimente eingesetzt.Angiogenesis, the formation of new blood vessels by endothelial cells, plays an important role during prenatal growth as well as postnataly in diseases such as tumor growth. Targeting endothelial cells by gene transfer to inhibit angiogenesis offers an attractive anticancer approach. A selective expression of the transgene by endothelial cells can be achieved by use of endothelial- specific gene regulatory elements. The present work pursued two main aims, namely the establishment and characterization of in vitro-models of angiogenesis of murine and human microvascular endothelial cells as well as the characterization of different plasmid constructs on efficiency in these models. Furthermore morphological alterations of transfected endothelial cells should be examined on light and electron microscopic basis in order to make statements about the uptake of the complexes by the cells and their way to the nucleus as well as to appraise possible morphological damages of the cells caused by transfection. The angiogenic cascade of endothelial cells, isolated from the human neonatal foreskin and from the myocardium of two weeks old mice, was induced by pro- angiogenic factors. The beginning of the angiogenic cascade was initiated morphologically by a complete as well as partial confluence of the endothelial cells (stage 1). A linear and circular side by side arrangement of cells (stage 2) and the formation of capillary-like structures with an internal lumen (stage 3 and 4) could be observed. The endothelial cells produced a basement membrane-like material, which was found intercellularly as well as in intracellular vacuoles and in the lumen of capillary-like structures. In lumen formation vacuolization as well as apoptosis were involved. While the invested murine endothelial cells formed capillary-like structures planar to the culture dish surface (two-dimensional in vitro-model of angiogenesis), a realistic three-dimensional in vitro-model of angiogenesis could be established with the human endothelial cells with strong similarity with angiogenesis in vivo. Collagen IV, a basement membrane component, was identified by immunolabeling. An especial result of this work observed in the murine cell culture model was the cyclic course of in vitro-angiogenesis with a „latency time" between. After detachment of the capillary-like structures of the culture dish a reappearance of these structures could be observed after approximately 2 months cultivation of the remained cells. Interesting was, that the capillary-like structures in the second cycle were formed by endothelial cells, which were uninvolved at the formation of these structures before. The results indicate that a differentiation of the endothelial cells to an angiogenic phenotype took place during the „latency time". Endothelial cells at different stages of the angiogenic cascade (stage 0 versus 3 respectively 4) were transfected by polyfection with activated dendrimers. Transfection of proliferative endothelial cells (stage 0) led to expression of the reporter gene (luciferase) with all vectors invested pJWM115 (CMV-luc), pCK5 (Ets-1l-luc), pPS12 (Ets-1k-luc), pPS6 (E-sel-luc). This indicates the expression of the adhesion molecule E-selectin as well as of the transcription factor Ets-1 by proliferative murine and human endothelial cells. Highest transfection efficiencies were found by using the pJWM115-, pCK5- and the pPS12-vector. The results of transfection of murine and human endothelial cells forming capillary-like structures (stage 3 resp. 4) reflected the angiogenic situation in both in vitro-models. No reporter gene expression, independently from the vector used, could be detected after transfection of murine endothelial cells at stage 3. However, transfection of human endothelial cells at stage 4 led to an expression, marginal higher than control expression. An important difference between the individual experiments in the human cell culture model could be determined on that occasion. Human endothelial cells that were transfected at beginning of stage 4, showed a higher expression of the reporter gene than cells that were transfected at later times of stage 4. This is to be explained by the different proliferative rate of endothelial cells at this stage of the angiogenic cascade. These results show a cell cycle dependence of the gene transfer system used in this work. An efficient gene transfer could be observed in proliferative endothelial cells only as result of a raised uptake of complexes as well as a more efficient entry of complexes resp. plasmid DNA into the nucleus. Ultrastructural examination of transfected cells let suspect that the complexes uptaken by endocytosis escape from the endosomes before fusion with primary lysosomes since free DNA-dendrimer-complexes at the nuclear membrane could be detected before appearance of multivesicular bodies (late endosomes) in human endothelial cells. In the murine endothelial cells the intracellular degradation of the complexes seemed to take place more slowly in comparison to human cells which could at least partially explain the broadly higher efficiency of gene transfer in murine cells. In another experiment the increase of the E-sel promoter activity (pPS6) by two endothelial-specific enhancers 5xebs (pPO18) and flk-1 (pPO14) as well as by the HLA-intron (pPO12) was examined. In contrast to the HLA-intron, which led to a multiple increase of the activity of the E-sel promoter, promoter activity could not be raised by the endothelial-specific enhancers. This indicates that the corresponding transcription factors like for example Ets-1 are not expressed in the examined cell culture models in sufficient quantity. Gene expression analyses or immunohistochemical examinations of the endothelial cells at different stages of the angiogenic cascade in vitro could give more information. In view of a reduction of animal experiments through substitute and supplement methods, a special meaning comes up to the established in vitro-models of angiogenesis. In addition to the animal protection aspect, they represent cost-effective, sensitive, simple experimental systems for continuing examinations. While the murine cell culture model is suitable for gene transfer studies since murine endothelial cells can efficiently be transfected, an experimental system is available with the realistic three- dimensional in vitro-model of angiogenesis of human endothelial cells which is suitable for examinations of in vitro-angiogenesis. On the basis of the lacking use of a three-dimensional extracellular matrix and the connected reduction of not resp. difficult to standardize factors this model is suitable for examinations of cell adhesion molecules, cell matrix interactions or also for identification of specific inhibitors of lumen formation. The established endothelial cell cultures already are used for similar experiments in other laboratories at present

Towards Inhaled Phage Therapy in Western Europe

The emergence of multidrug-resistant bacteria constitutes a great challenge for modern medicine, recognized by leading medical experts and politicians worldwide. Rediscovery and implementation of bacteriophage therapy by Western medicine might be one solution to the problem of increasing antibiotic failure. In some Eastern European countries phage therapy is used for treating infectious diseases. However, while the European Medicines Agency (EMA) advised that the development of bacteriophage-based therapies should be expedited due to its significant potential, EMA emphasized that phages cannot be recommended for approval before efficacy and safety have been proven by appropriately designed preclinical and clinical trials. More evidence-based data is required, particularly in the areas of pharmacokinetics, repeat applications, immunological reactions to the application of phages as well as the interactions and effects on bacterial biofilms and organ-specific environments. In this brief review we summarize advantages and disadvantages of phage therapy and discuss challenges to the establishment of phage therapy as approved treatment for multidrug-resistant bacteria

Gene identification and analysis of transcripts differentially regulated in fracture healing by EST sequencing in the domestic sheep

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Composite transcriptome assembly of RNA-seq data in a sheep model for delayed bone healing

<p>Abstract</p> <p>Background</p> <p>The sheep is an important model organism for many types of medically relevant research, but molecular genetic experiments in the sheep have been limited by the lack of knowledge about ovine gene sequences.</p> <p>Results</p> <p>Prior to our study, mRNA sequences for only 1,556 partial or complete ovine genes were publicly available. Therefore, we developed a composite <it>de novo </it>transcriptome assembly method for next-generation sequence data to combine known ovine mRNA and EST sequences, mRNA sequences from mouse and cow, and sequences assembled <it>de novo </it>from short read RNA-Seq data into a composite reference transcriptome, and identified transcripts from over 12 thousand previously undescribed ovine genes. Gene expression analysis based on these data revealed substantially different expression profiles in standard versus delayed bone healing in an ovine tibial osteotomy model. Hundreds of transcripts were differentially expressed between standard and delayed healing and between the time points of the standard and delayed healing groups. We used the sheep sequences to design quantitative RT-PCR assays with which we validated the differential expression of 26 genes that had been identified by RNA-seq analysis. A number of clusters of characteristic expression profiles could be identified, some of which showed striking differences between the standard and delayed healing groups. Gene Ontology (GO) analysis showed that the differentially expressed genes were enriched in terms including <it>extracellular matrix</it>, <it>cartilage development</it>, <it>contractile fiber</it>, and <it>chemokine activity</it>.</p> <p>Conclusions</p> <p>Our results provide a first atlas of gene expression profiles and differentially expressed genes in standard and delayed bone healing in a large-animal model and provide a number of clues as to the shifts in gene expression that underlie delayed bone healing. In the course of our study, we identified transcripts of 13,987 ovine genes, including 12,431 genes for which no sequence information was previously available. This information will provide a basis for future molecular research involving the sheep as a model organism.</p

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Standard bone healing stages occur during delayed bone healing, albeit with a different temporal onset and spatial distribution of callus tissues

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