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Hematopoietic stem and progenitor cells (HSPCs) repopulate the blood system upon transplantation. A large-scale genetic approach to understand the factors that participate in successful engraftment has not been undertaken. In this thesis, I present the development of a novel live imaging-based competitive marrow repopulation assay in adult zebrafish, which allows fast and quantitative measurement of HSPC engraftment capability. Using this assay, a transplantation-based chemical screen was performed, which led to the discovery of 10 compounds that can enhance the marrow engraftment capability in zebrafish. Among them, the arachidonic acid-derived epoxyeicosatrienoic acids (EET), had conserved effects on both short- and long-term bone marrow engraftment in mice. Genetic analysis in zebrafish embryos demonstrated that EET acts through a \(G\alpha12/13\)-mediated receptor, which activates PI3K and induces transcription factors of the AP-1 family. This PI3K/AP-1 pathway directly induced the transcription of HSC marker, runx1, in embryos. The activation of PI3K by EET promoted HSPC migration and interactions with niche cells. Our studies define a role for EETs in the development of blood stem cells during embryogenesis, and in engraftment in adult vertebrates. The other compounds discovered in the screen implicate additional novel signaling pathways involved in the HSPC engraftment process, which require further investigation. In summary, this thesis elucidated an important role of bioactive lipids in regulating HSC engraftment in adults and during embryo development. Systematically mapping out the regulatory network will tremendously benefit both the basic understanding of stem cell biology and the clinical manipulation to generate better stem cells for transplantation.

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Ischemic heart disease and resulting heart failure (HF) belong to the leading causes of death in developed countries. In order to prevent HF and improve clinical outcome in patients with myocardial infarction, novel therapies are required to protect the heart against the detrimental effect of ischemic injury. Due to the failure to translate numerous available experimental cardioprotective strategies into clinical practice, the need for novel protective treatments persists. We have, therefore, tried to apply a novel approach to cardiac protection against the postischemic HF induced in rats by ligation of the coronary artery. For this purpose, we have studied (i) the preventive and therapeutic effects of adaptation to continuous normobaric hypoxia (CNH; 12% O2) and exercise training (ExT; treadmill running), and (ii) the possible cardioprotective potential of epoxyeicosatrienoic acid (EET)-based therapy in order to attenuate the postischemic HF in rats. Adaptation to CNH and ExT is known for their cardioprotection in acute ischemia/reperfusion (I/R) injury manifested as reduction of infarct size. EETs exert antihypertensive effects and thus seem to be perspective for the research in clinically relevant models of cardioprotection in hypertensive animals. Our results have revealed that: - CNH prior to...

Introduction: Epoxyeicosatrienoic acids (EETs) are converted by the enzyme soluble epoxid hydrolase (sEH) to the biologically inactive dihydroxyeicosatrienoic acids (DHETs). EETs are significantly involved in the control of blood pressure, they influence vascular tone and renal transport mechanism. sEH inhibitor reduce blood pressure by increasing the bioavailability of EETs in many models of hypertension. Aim of the study: To determine that sEH inhibitor decreases blood pressure and improves the renal function during the development of malignant hypertension in transgenic rats after the induction of the mouse renin gene. Methods: Hypertension in Cyp1a1-Ren-2 transgenic rats was induced through a dietary administration of the natural xenobiotic indole-3-carbinol (I3C, 0.3 %) for 3 and 11 days. I3C activates the renin gene. At the same time, during a three-day induction of hypertension, the inhibitor of nitric oxide synthase L-NAME (600 mg/l) was administered in drinking water. The sEH inhibitor c-AUCB was given in drinking water at a dose of 13 or 26 mg/l, starting 48 hours before the initiation of I3C and L-NAME administration. Radiotelemetric measurement of blood pressure was performed and renal excretory parameters were monitored in the conscious animals. The effects on renal hemodynamics and...

Introduction: Two major product groups originate from the arachidonic acid metabolic pathway of cytochromes P450: epoxyeicosatrienoic acid (EETs) and 19 and 20-hydroxyeicosatetraenoic acid (19- and 20-HETE). These metabolites play an important role in the regulation of blood pressure, inflammatory responses, regulation of sodium excretion and other crucial physiological processes. Hypothesis: Our studies were based on the hypothesis that abnormalities in the production and function of these cytochrome P450 metabolites significantly contribute to the pathophysiology of hypertension development, in particular in the angiotensin II-dependent models. Objective: To investigate if the increased bioavailability of the above-mentioned metabolites in the kidney tissue will result in blood pressure reduction in the ANG II - dependent rat model of hypertension. Methods: The two methods to increase the concentration of EETs was chosen. In the first part of the study, we administered a soluble epoxide hydrolase inhibitor cAUCB [cis-4- [4- (3-adamantan-1-yl- ureido) cyclohexyloxy] benzoic acid, at a dose of 26 mg.l-1 administered in drinking water], an enzyme responsible for inactivation of biologically active forms of EETs. In the second series of the experiments we applied a synthetic EET analogue, called...

Cytochrom P450 (CYP)-Enzyme hydroxylieren und epoxydieren Arachidonsäure (AA) zu bioaktiven Metaboliten wie 20-Hydroxyeicosatetraensäure (20-HETE) und Epoxyeicosatriensäuren (EETs). Diese CYP-abhängigen Eicosanoide fungieren als Mediatoren bei der Regulation der Gefäß-, Nieren- und Herzfunktion. Hauptziel der vorliegenden Arbeit war es, die Identität der 20-HETE bildenden CYP-Isoformen in der Mausniere aufzuklären. Ein weiterer Schwerpunkt war die Bestimmung von Veränderungen im Metabolismus CYP-abhängiger Eicosanoide in Tiermodellen des akuten Nieren- und Herzversagens. Zur Identifizierung der 20-HETE bildenden CYP-Isoform wurde die Substrat- und Wirkungsspezifität von Cyp4a10, Cyp4a12a, Cyp4a12b und Cyp4a14, sowie ihre geschlechts- und stammspezifische Expression charakterisiert. Die Ergebnisse dieser Arbeit zeigen, dass Cyp4a12a die 20-HETE Synthase der Mausniere ist. Cyp4a12a wird durch Androgene induziert und seine Expressionshöhe ist für geschlechts- und stammspezifische Unterschiede in der 20-HETE Bildung verantwortlich. Im Rattenmodell des Ischämie/Reperfusions (I/R)-induzierten Nierenschadens wird eine 20-HETE Freisetzung durch I/R induziert. Wir konnten zeigen, dass der I/R-Schaden durch Hemmung der 20-HETE Bildung signifikant reduziert wird. Im Rattenmodell der Herzinsuffizienz (SHHF) ist das Herzversagen mit einer Variante des EPHX2 Gens assoziiert. EPHX2 kodiert für die lösliche Epoxidhydrolase (sEH), die den Abbau von EETs katalysiert. Wir konnten zeigen, dass die Genvariation zu signifikant höheren sEH-Aktivitäten im Herzen (3-fachen) und in der Niere (30-fachen) führt, im Vergleich zu Rattenstämmen, die keine Herzinsuffizienz entwickeln. Die vorliegende Arbeit unterstreicht die pathophysiologische Bedeutung von Veränderungen im Metabolismus von 20-HETE und EETs. Daher erscheint es vielversprechend, den CYP-Eicosanoid Stoffwechsel als neuen Angriffspunkt für die pharmakologische Behandlung kardiovaskulärer Erkrankungen zu erschließen. / Cytochrome P450 (CYP) enzymes hydroxylate and epoxidize arachidonic acid (AA) to bioactive metabolites such as 20-hydroxyeicosatetraenoic acid (20-HETE) and epoxyeicosatrienoic acids (EETs). These CYP-dependent eicosanoids serve as mediators in the regulation of vascular, renal and cardiac function. The main objective of the present study was to identify the 20-HETE producing CYP isoforms in the mouse kidney. Another focus was to determine changes in the metabolism of CYP-dependent eicosanoids in animal models of acute renal and heart failure. To identify the 20-HETE producing CYP-isoform the substrate and reaction specificity of Cyp4a10, Cyp4a12a, Cyp4a12b and Cyp4a14, as well as their sex- and strain-specific expression were characterized. The present study shows that Cyp4a12a is the predominant AA hydroxylase in the mouse kidney. Cyp4a12a is induced by androgens and its expression determines the sex and strain-specific differences in 20-HETE generation. In a rat model of renal ischemia/reperfusion (I/R) injury, I/R triggered the release of 20-HETE and we were able to ameliorate renal injury by pharmacological inhibition of 20-HETE production. In a rat model of heart failure (spontaneously hypertensive heart failure rats, SHHF) the heart failure phenotype is associated with a variant of the EPHX2 gene. EPHX2 is coding for the soluble epoxide hydrolase (sEH) which catalyze the degradation of EETs. We found that the gene variation leads to significantly higher sEH activities in the heart (3-fold) and in the kidney (30-fold) compared to rat strains not prone to the development of heart failure. The present study emphasizes the pathophysiological relevance of changes in the biosynthesis and degradation of 20-HETE and EETs. Therefore, it appears promising to develop the CYP-eicosanoid pathway as a novel clinical target for the treatment of cardiovascular diseases.