Exploration of serum- and cell culture-derived exosomes from dogs

Exosomes are defined as extracellular membrane vesicles, 30–150 nm in diameter, derived from all types of cells. They originate via endocytosis and then they are released through exocytosis to the extracellular space, being found in various biological fluids as well as in cell culture medium. In the last few years, exosomes have gained considerable scientific interest due to their potential use as biomarkers, especially in the field of cancer research. This report describes a method to isolate, quantify and identify serum- and cell culture-derived exosomes from dog samples, using small volumes (100 μL and 1 mL, respectively)

Large-scale mapping of mutations affecting zebrafish development

BACKGROUND: Large-scale mutagenesis screens in the zebrafish employing the mutagen ENU have isolated several hundred mutant loci that represent putative developmental control genes. In order to realize the potential of such screens, systematic genetic mapping of the mutations is necessary. Here we report on a large-scale effort to map the mutations generated in mutagenesis screening at the Max Planck Institute for Developmental Biology by genome scanning with microsatellite markers. RESULTS: We have selected a set of microsatellite markers and developed methods and scoring criteria suitable for efficient, high-throughput genome scanning. We have used these methods to successfully obtain a rough map position for 319 mutant loci from the Tübingen I mutagenesis screen and subsequent screening of the mutant collection. For 277 of these the corresponding gene is not yet identified. Mapping was successful for 80 % of the tested loci. By comparing 21 mutation and gene positions of cloned mutations we have validated the correctness of our linkage group assignments and estimated the standard error of our map positions to be approximately 6 cM. CONCLUSION: By obtaining rough map positions for over 300 zebrafish loci with developmental phenotypes, we have generated a dataset that will be useful not only for cloning of the affected genes, but also to suggest allelism of mutations with similar phenotypes that will be identified in future screens. Furthermore this work validates the usefulness of our methodology for rapid, systematic and inexpensive microsatellite mapping of zebrafish mutations

Investigations of the function of ACE2 and Angiotensin-(1-7) in smooth muscle cells.

Das Renin - Angiotensin - System (RAS) spielt eine entscheidende Rolle in der Entstehung einer Hypertonie sowie von assoziierten kardiovaskulären Erkrankungen, wie Schlaganfall, Myokardinfarkt, Herz- und Niereninsuffizienz. Als Haupteffektor des RAS wurde lange Zeit der Vasokonstriktor Angiotensin II (Ang II) angesehen, welcher durch die Carboxydipeptidase ACE (Angiotensin converting enzyme) erzeugt wird. In den letzten Jahren hat sich aber das Bild des RAS grundlegend geändert, wobei die Identifizierung von ACE2 (Angiotensin converting enzyme 2) eine ausschlaggebende Rolle spielte. Diese Carboxypeptidase ist unter anderem verantwortlich für den Abbau des Vasokonstriktors Ang II sowie für die Erzeugung von Angiotensin-(1-7) (Ang-(1-7)). Das Heptapeptid wiederum agiert über seinen Rezeptor Mas und scheint den Effekten von Ang II entgegenzuwirken. So rückte in den letzten Jahren die ACE2 / Ang-(1-7) / Mas Achse in den Fokus der Forschung. Im Rahmen dieser Doktorarbeit sollte nun die Rolle von ACE2 in der Pathogenese von spontan hypertensiven Ratten mit Neigung zum Schlaganfall (SHRSP) näher untersucht werden. Dieses Modell war insofern interessant, da hier das ACE2 - Gen, welches sich auf dem X-Chromosom befindet, zu einem quantitativen Merkmalslokus für Hypertonie kartiert wurde. Überdies wiesen die SHRSP Ratten eine erniedrigte ACE2 Expression auf. Folglich wurden in dieser Arbeit transgene SHRSP Ratten mit Überexpression von humanem ACE2 in den vaskulären glatten Muskelzellen charakterisiert. Dabei führte die Überexpression von ACE2 zu einem signifikant erniedrigten Blutdruck, einer verminderten Reaktion auf den Vasokonstriktor Ang II sowie zu einer verbesserten Endothelfunktion. Des Weiterem zeigten die transgenen Tiere im Vergleich zu ihren SHRSP Kontrollen eine herabgesetzte Bildung von kardialer Hypertrophie und Fibrose sowie eine verbesserte Nierenfunktion. Überdies konnte auch in den normotensiven Sprague Dawley (SD) Ratten mit Überexpression von ACE2 in den vaskulären glatten Muskelzellen eine verbesserte endothelabhängige Vasodilatation detektiert werden. Somit wurde nachgewiesen, dass ACE2 eine bedeutende Rolle in der Aufrechterhaltung der Endothelfunktion besitzt, wobei dies wiederum einen kardiovaskulären sowie renalen Schutz auszuüben scheint. Um insbesondere die Rolle des Heptapeptids Ang-(1-7) zu klären, welches bei einer ACE2 Überexpression in den SHRSP Tieren signifikant erhöht war, wurden ferner transgene Ratten generiert, welche in den glatten Muskelzellen vermehrt Ang (1 7) erzeugen. Erste Ergebnisse zeigten aber keine Unterschiede zwischen transgenen Ratten und Kontrolltieren. Eine weitere Aufgabe bestand in der Etablierung einer Methode zur Verbesserung einer Transgenexpression in den glatten Muskelzellen von Ratten. Hierbei fand die Cre-loxP Technologie Anwendung. Diese ermöglichte die Generierung von Ratten mit einer starken Expression des gewünschten Gens, wobei aber die Gewebespezifität noch besser untersucht werden muss. Ein weiterer Teil dieser Arbeit bestand in einem in vitro Projekt zur näheren Charakterisierung der erst kürzlich beschriebenen Rezeptor-ähnlichen Funktion von ACE. Dabei wurden mögliche Liganden gesucht, wobei vor allem Ang-(1-7) geprüft werden sollte. Des Weiteren wurde die Rolle der zwei katalytischen Domänen von ACE in Bezug auf die Induktion des intrazellulären Signalweges untersucht. Dabei konnte gezeigt werden, dass ACE- Inhibitoren sowie Bradykinin und Angiotensin I eine Signaltransduktion über ACE auslösen. Andere Substrate hingegen, so auch Ang-(1-7), konnten eine solche Wirkung ausschließlich bei ACE-Formen mit nur einer aktiven Domäne auslösen. Die gleichzeitige Bindung dieser Substrate an beide katalytischen Domänen von ACE schien die Auslösung einer Signaltransduktion eher zu hemmen. Die Aktivierung der ACE2/ Ang-(1-7) / Mas Achse könnte somit als neue therapeutische Strategie bei der Bekämpfung von essentieller Hypertonie sowie anderer kardiovaskulärer Erkrankungen angestrebt werden. Ferner deutet die Identifizierung des ACE-abhängigen intrazellulären Signalweges an, dass das RAS noch unerwartete Aspekte mit klinischen Auswirkungen besitzt.The renin – angiotensin - system (RAS) plays a crucial role in the development of hypertension as well as associated cardiovascular diseases, such as stroke, myocardial infarction, heart and renal failure. For long time the vasoconstrictive angiotensin II (Ang II), which is generated by ACE (angiotensin converting enzyme), was considered as main active peptide in the RAS. Over the past years the view of the RAS changed dramatically, and the identification of ACE2 (angiotensin converting enzyme 2) played a pivotal role in this process. Amongst others, this carboxypeptidase is responsible for the reduction of Ang II as well as the generation of angiotensin-(1-7) (Ang-(1-7)). In turn, the heptapeptide acts on Mas and counteracts the effects of Ang II. Consequently, over the past years the ACE2 / Ang-(1-7) / Mas axis moved into the centre of interest. In this thesis the role of ACE2 in the pathogenesis of hypertension in spontaneous hypertensive stroke-prone rats (SHRSP) was investigated. This animal model was interesting, since the ACE2 gene could be mapped to a quantitative trait locus for hypertension on the X chromosome, and furthermore SHRSP rats display reduced ACE2 expression. Therefore, transgenic rats on a SHRSP genetic background expressing the human ACE2 in vascular smooth muscle cells were characterized. The vascular overexpression of ACE2 reduced high blood pressure, attenuated the vasoconstrictive response to Ang II, and improved endothelial function. In addition, the transgenic rats displayed a reduced development of cardiac hypertrophy and fibrosis as well as an improved kidney function in comparison to the controls. Also the overexpression of ACE2 in vascular smooth muscle cells of normotensive Sprague Dawley (SD) rats led to an improved endothelial dependent vasodilation. Therefore it could be shown that ACE2 exhibits a crucial role in the maintainance of endothelial function through which it apparently exerts cardiovascular and renal protection. In order to investigate further the role of the heptapeptide Ang-(1-7), which is significantly increased in the ACE2 overexpressing SHRSP rats, transgenic rats were generated with enhanced secretion of Ang-(1-7) from smooth muscle cells. The first results of their characterization did not reveal any differences between transgenic and control rats. One further project aimed at the establishment of a method for the improvement of transgene expression in smooth muscle cells of rats. In order to achieve this, a method based on the Cre-loxP technology was used. This allowed the generation of transgenic rats with strong expression of the desired transgene, although the extent of tissue specificity still needs to be shown. Another part of this thesis was an in vitro project concerning the characterization of the recently published receptor-like function of ACE in more detail. Looking for possible ligands in particular Ang-(1-7) was of interest and the role of the two catalytic ACE domains. It could be shown that ACE inhibitors as well as bradykinin and angiotensin I induce a outside-in signaling via ACE. In contrast, other substrates like Ang-(1-7) could induce such signal transduction only in ACE forms with one catalytic domain. The simultaneous binding of these substrates on both domains of ACE appears to inhibit this outside-in signaling. The activation of the ACE2 / angiotensin-(1-7) / Mas axis may be a novel therapeutic strategy in primary hypertension as well as other cardiovascular diseases. In addition, the ACE- dependent signaling pathway implies that the RAS has still unexpected facets with clinical implication

Genetically altered animal models for Mas and Angiotensin-(1-7)

Mas is the receptor for angiotensin-(1-7) and involved in cardiovascular and neuronal regulation, in which also the heptapeptide plays a major role. Mas-deficient mice were previously generated by us and their characterization showed that Mas has important functions in behaviour and cardiovascular regulation. These mice exhibit increased anxiety but despite an enhanced long-term potentiation in the hippocampus are not performing better in learning paradigms. When Mas-deficient mice are backcrossed to the FVB/N genetic background a cardiovascular phenotype is uncovered: the backcrossed animals become hypertensive. Concordant with our detection by fluorescent in-situ hybridization of Mas-mRNA in mouse endothelium, this phenotype is caused by endothelial dysfunction based on a dysbalance between nitric oxide and reactive oxygen species in the vessel wall. In agreement with these data, transgenic SHR-SP-rats overexpressing ACE2 in the vessel wall exhibit reduced blood pressure due to an improved endothelial function. Moreover, angiotensin-(1-7) overexpression in transgenic rats has cardioprotective and hemodynamic affects. In conclusion, the angiotensin-(1-7) / Mas axis has important functional implications in vascular regulation and blood pressure control in particular in pathophysiological situations