20 research outputs found
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\. LiteraturĂŒbersicht
3\. Materialien
4\. Methoden
5.1 Ergebnisse Teil 1
5.2 Ergebnisse Teil 2
6\. Diskussion
7\. Zusammenfassung
8\. Summary
LiteraturverzeichnisAngiogenese, die Neubildung von BlutgefÀssen, kommt physiologischerweise nur
im Embryo und Fetus sowie beim Adulten im Rahmen zyklischer Prozesse im Ovar,
in der Plazenta und bei der Entwicklung der MilchdrĂŒse 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, nÀmlich die Etablierung und Charakterisierung von in vitro-
Modellen der Angiogenese muriner und humaner mikrovaskulÀrer Endothelzellen
sowie die Charakterisierung verschiedener Plasmidkonstrukte auf Effizienz in
diesen Modellen. Im Rahmen der Transfektionsversuche sollten zusÀtzlich
morphologische VerÀnderungen transfizierter Endothelzellen auf licht- und
elektronenmikroskopischer Basis untersucht werden, um einerseits Aussagen ĂŒber
die Aufnahme der Transfektionskomplexe in die Zellen sowie deren Weg in den
Zellkern zu machen und andererseits mögliche morphologische SchÀdigungen der
Zellen infolge der Transfektion zu beurteilen und abzuschÀtzen.
Analog zur in vivo-Angiogenese durchliefen die eingesetzten mikrovaskulÀren
Endothelzellen, isoliert aus der Vorhaut von Neugeborenen sowie aus dem
Myokard zwei Wochen alter MĂ€use, durch Stimulation mit pro-angiogenen Faktoren
verschiedene Stadien der angiogenen Kaskade in vitro. Initiiert wurde die
angiogene Kaskade morphologisch durch eine vollstÀndige bzw. partielle
Konfluenz der Endothelzellen (Stadium 1). Es kam zur linearen und zirkulÀren
Aneinanderreihung von Endothelzellen (Stadium 2) und schliesslich zur
Ausbildung kapillarÀhnlicher Strukturen mit einem zentralen Lumen (Stadium 3
und 4). Die Endothelzellen bildeten ein basalmembranÀhnliches Material,
welches interzellulÀr sowie in intrazellulÀren Vakuolen und im Lumen
kapillarÀhnlicher Strukturen detektiert werden konnte. Die Lumenbildung
erfolgte durch Ausbildung intrazellulÀrer Vakuolen sowie durch Apoptose.
WĂœhrend die eingesetzten murinen Endothelzellen planar zur
KulturschalenoberflÀche kapillarÀhnliche Strukturen ausbildeten
(zweidimensionales in vitro-Modell der Angiogenese), konnte mit den humanen
Endothelzellen erstmals ein realitÀtsnahes, dreidimensionales in vitro-Modell
der Angiogenese etabliert werden, in dem analog zur in vivo-Angiogenese die
Ausbildung kapillarÀhnlicher Strukturen durch Degradation eines von den Zellen
selbst sezernierten basalmembranÀhnlichen Substrates sowie Migration bzw.
Invasion, Proliferation und Differenzierung erfolgte. Im basalmembranÀhnlichen
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
âLatenzzeit". Nach dem Ablösen der kapillarĂ€hnlichen 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
kapillarÀhnliche Strukturen ausbildeten, welche zuvor unbeteiligt an der
Ausbildung dieser Strukturen waren. Die Ergebnisse indizieren, dass wÀhrend
der âLatenzzeit" eine Differenzierung der Endothelzellen in einen angiogenen
PhÀnotyp erfolgte.
Die Polyfektion mittels aktivierten Dendrimeren erfolgte vergleichend im
Stadium der endohelialen Proliferation (Stadium 0) sowie der Bildung
kapillarÀhnlicher Strukturen (Stadium 3 bzw. 4). Die Transfektion
proliferierender Endothelzellen fĂŒhrte 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 AdhĂ€sionsmolekĂŒl 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 kapillarÀhnlicher
Strukturen spiegelten die angiogenetische Situation beider in vitro-Modelle
wider. WĂ€hrend die Transfektion muriner Endothelzellen im Stadium 3,
unabhÀngig vom eingesetzten Vektor, zu keiner Expression der Luciferase
fĂŒhrte, wurde nach Transfektion humaner Endothelzellen im Stadium der
dreidimensionalen Organisation kapillarÀhnlicher 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 spÀteren Zeitpunkten des Stadiums 4
transfiziert wurden. Dies ist durch die unterschiedliche Proliferationsrate
der Endothelzellen in diesem Stadium zu erklÀren. Diese Ergebnisse indizieren
eine ZellzyklusabhÀngigkeit 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 primÀren Lysosomen aus den Endosomen
entkommen, da freie DNA-Dendrimer-Komplexe an der Kernmembran vor dem
Erscheinen multivesikulĂœrer Körper (spĂ€te Endosomen) in humanen Endothelzellen
detektiert werden konnten. In den murinen Endothelzellen schien sich der
intrazellulÀre Abbau der Komplexe im Vergleich zu den humanen Endothelzellen
langsamer zu vollziehen, welches die allgemein höhere Effzienz des
Gentransfers in diesen Zellen zumindest partiell erklÀren könnte.
In einer weiteren Versuchsreihe wurde die Steigerung der E-sel-
PromotoraktivitÀt (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 AktivitÀt des E-sel-
Promotors um ein Vielfaches fĂŒhrte, konnte die PromotoraktivitĂ€t 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 hierĂŒber Aufschluss geben.
Im Hinblick auf eine Reduktion von Tierversuchen durch Ersatz- und
ErgÀnzungsmethoden kommt den in der vorliegenden Arbeit etablierten in vitro-
Modellen der Angiogenese eine besondere Bedeutung zu. Ăber den
Tierschutzaspekt hinaus stellen sie kostengĂŒnstige, sensitive, einfache
experimentelle Systeme fĂŒr weiterfĂŒhrende Untersuchungen dar. WĂ€hrend das
murine Zellkulturmodell insbesondere fĂŒr Gentransferstudien geeignet ist, da
sich die murinen Endothelzellen effizient transfizieren lassen, steht mit dem
realitÀtsnahen, dreidimensionalen in vitro-Modell der Angiogenese humaner
Endothelzellen ein experimentelles System zur VerfĂŒgung, welches sich in der
Hauptsache fĂŒr Untersuchungen der in vitro-Angiogenese eignet. Aufgrund des
fehlenden Einsatzes einer dreidimensionalen extrazellulÀren Matrix und der
damit verbundenen Reduktion nicht bzw. schwierig standardisierbarer Faktoren
ist dieses Modell geeignet fĂŒr Untersuchungen von beispielsweise
ZelladhĂ€sionsmolekĂŒlen, Zell-Matrix-Interaktionen oder auch zur
Identifizierung spezifischer Inhibitoren der Lumenbildung. Die etablierten
Endothelzellkulturen werden derzeit bereits in anderen Laboratorien fĂŒr
Ă€hnliche 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 âlatency 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 âlatency 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
BACKGROUND: The sheep is an important model animal for testing novel fracture treatments and other medical applications. Despite these medical uses and the well known economic and cultural importance of the sheep, relatively little research has been performed into sheep genetics, and DNA sequences are available for only a small number of sheep genes. RESULTS: In this work we have sequenced over 47 thousand expressed sequence tags (ESTs) from libraries developed from healing bone in a sheep model of fracture healing. These ESTs were clustered with the previously available 10 thousand sheep ESTs to a total of 19087 contigs with an average length of 603 nucleotides. We used the newly identified sequences to develop RT-PCR assays for 78 sheep genes and measured differential expression during the course of fracture healing between days 7 and 42 postfracture. All genes showed significant shifts at one or more time points. 23 of the genes were differentially expressed between postfracture days 7 and 10, which could reflect an important role for these genes for the initiation of osteogenesis. CONCLUSION: The sequences we have identified in this work are a valuable resource for future studies on musculoskeletal healing and regeneration using sheep and represent an important head-start for genomic sequencing projects for Ovis aries, with partial or complete sequences being made available for over 5,800 previously unsequenced sheep genes
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
Preclinical Assessment of Bacteriophage Therapy against Experimental Acinetobacter baumannii Lung Infection
Respiratory infections caused by multidrug-resistant Acinetobacter baumannii are difficult to treat and associated with high mortality among critically ill hospitalized patients. Bacteriophages (phages) eliminate pathogens with high host specificity and efficacy. However, the lack of appropriate preclinical experimental models hampers the progress of clinical development of phages as therapeutic agents. Therefore, we tested the efficacy of a purified lytic phage, vB_AbaM_Acibel004, against multidrug-resistant A. baumannii clinical isolate RUH 2037 infection in immunocompetent mice and a human lung tissue model. Sham- and A. baumannii-infected mice received a single-dose of phage or buffer via intratracheal aerosolization. Group-specific differences in bacterial burden, immune and clinical responses were compared. Phage-treated mice not only recovered faster from infection-associated hypothermia but also had lower pulmonary bacterial burden, lower lung permeability, and cytokine release. Histopathological examination revealed less inflammation with unaffected inflammatory cellular recruitment. No phage-specific adverse events were noted. Additionally, the bactericidal effect of the purified phage on A. baumannii was confirmed after single-dose treatment in an ex vivo human lung infection model. Taken together, our data suggest that the investigated phage has significant potential to treat multidrug-resistant A. baumannii infections and further support the development of appropriate methods for preclinical evaluation of antibacterial efficacy of phages
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Dysregulation of the innate immune system drives lung injury and its systemic sequelae due to breakdown of vascular barrier function, harmful hyperinflammation and microcirculatory failure, which contribute to the unfavourable outcome of patients with severe pneumonia. A variety of promising therapeutic targets have been identified and numerous innovative therapeutic approaches demonstrated to improve lung injury in experimental preclinical studies. However, at present specific preventive or curative strategies for the treatment of lung failure in pneumonia in addition to antibiotics are still missing. The aim of this mini-review is to give a short overview of some, but not all, adjuvant therapeutic strategies for pneumonia and its most important complications, sepsis and acute respiratory distress syndrome, and briefly discuss future perspectives
Standard bone healing stages occur during delayed bone healing, albeit with a different temporal onset and spatial distribution of callus tissues
Bone healing is considered as a
recapitulation of a developmental program initiated at
the time of injury. This study tested the hypothesis that
in delayed bone healing the regular cascade of healing
events, including remodeling of woven to lamellar bone,
would be similar compared to standard healing, although
the temporal onset would be delayed.
A tibial osteotomy was performed in sheep and
stabilized with a rotationally unstable fixator leading to
delayed healing. The sheep were sacrificed at 2, 3, 6, 9
weeks and 6 months postoperatively. The temporal and
spatial tissue distributions in the calluses and the bone
microstructure were examined by histology.
Although histological analysis demonstrated
temporal and spatial callus tissue distribution
differences, delayed healing exhibited the same
characteristic stages as those seen during uneventful
standard healing. The delayed healing process was
characterized by a prolonged presence of hematoma, a
different spatial distribution of new bone and delayed
and prolonged endochondral bone formation. A change
in the spatial distribution of callus formation was seen
by week 6 leading to bone formation and resorption of
the cortical bone fragments, dependent on the degree to
which the cortical bone fragments were dislocated. At 6
months, only 5 out of 8 animals showed complete bony
bridging with a continuous periosteum, although
lamellar bone and newly formed woven bone were
present in the other 3 animals.
This study demonstrates that during delayed bone healing all stages of the healing cascade likely take
place, even if bony consolidation does not occur.
Furthermore, the healing outcome might be related to the
periosteumâs regenerative capacity leading to bony union
or absence of bony bridging