The renin-angiotensin system (RAS) is of central importance in blood pressure regulation and in the initiation of target organ damage. In particular, local angiotensin-II generating systems in tissues such as brain, heart, vessels, and kidney are involved in these processes. Therefore, transgenic rats with local up- or downregulation of RAS components in these organs, e.g. by the local expression of antisense-RNA or of a peptide-liberating protein, were produced and analyzed to clarify the local functions of angiotensin II. Other genetically altered mouse and rat models for non-classical RAS components such as ACE2, the renin receptor, angiotensin(1-7) and its receptor Mas, have elucidated the physiological function of these factors. Together with transgenic rats overexpressing this peptide, Mas-knockout mice characterized the angiotensin(1-7)/Mas system as a cardioprotective axis that counteracts the classical RAS effects in particular improving endothelial function. Furthermore, these animals showed that angiotensin(1-7) and Mas are important for insulin sensitivity and the pathogenesis of metabolic syndrome.
The kallikrein-kinin system (KKS) is an important hormone system for cardiovascular regulation also mostly counteracting the effects of the RAS. As models for the functional analysis of the KKS in intact animals, transgenic rats were generated expressing different components of the system, such as tissue kallikrein, the kinin B1 or the B2 receptor either ubiquitously or specifically in cardiovascular organs. These animals supported the protective role of the KKS in kidney and heart against ischemic, diabetic, and hypertrophic injury. Knockout mice for the kinin B1 receptor were generated and revealed important functions of this protein in pain perception and inflammation. Moreover, the B1 receptor turned out to be involved in sepsis, stroke, multiple sclerosis and high-fat diet induced obesity. Mice lacking both kinin receptors and thereby being devoid of a functional KKS were also generated and shown to be completely normal at baseline suggesting that the KKS is irrelevant for development and basic regulation of the cardiovascular system but is involved in the pathogenesis of multiple diseases and, thus, a relevant drug target.
Natriuretic peptide system
There are 3 natriuretic peptides (NP), ANP, BNP, and CNP, which interact with two natriuretic peptide receptors, NPR-A and NPR-B, to induce a multitude of actions in heart, kidney, vessels, brain and other tissues. The receptors are dimeric molecules, which after activation synthesize cyclic GMP. We have shown that dimerization is essential for the activation of the receptors and have designed dominant negative mutants to downregulate the activity of the receptors in cells and transgenic animals. Transgenic rat models expressing a dominant negative mutant for NPR-B exhibit sympathetic activation and develop cardiac hypertrophy supporting a cardioprotective action of this receptor and its ligand CNP. Moreover, these animals show an impaired bone growth in accordance with the phenotype of knockout mice for NPR-B and CNP and humans with mutations in the NPR-B gene.
Serotonin is a monoamine which functions as an important neurotransmitter in the central nervous system and as a major peripheral mediator produced by enterochromaffin cells of the gut and transported and released by platelets in the circulation. We discovered that vertebrates have two tryptophan hydroxylases, the rate limiting enzymes in serotonin synthesis, TPH1 and TPH2. Mice deficient in TPH1, the isoform responsible for the synthesis of serotonin in the gut, showed that peripheral serotonin is involved in thrombosis, pulmonary hypertension, remodelling of mammary glands, tumor angiogenesis, liver regeneration, and hepatitis. Mice deficient in TPH2, the isoform responsible for the synthesis of serotonin in the brain, were surprisingly viable and fertile, despite a near complete lack of serotonin in the brain, and showed growth retardation and altered autonomic control leading to impairment of sleep, respiration, and cardiovascular parameters. In addition, these mice exhibit increased aggression and maternal neglect. Furthermore, searching for molecules, which are crucial for the development of serotonergic neurons, we developed protocols for the differentiation of embryonic stem (ES) cells into serotonin-producing neurons. Based on genetic modifications of the ES cells, the method allows to select this neuronal population and to monitor their development in in-vitro and in-vivo studies.
In a collaborative work with the Charité studying gender effects in cardiac hypertrophy and failure, the group generates and characterizes animal models with altered androgen receptor expression in distinct cell types of the heart.
Importins are essential components of the machinery that transports proteins into the nucleus of eukaryotic cells. In an approach to study the physiological function of alpha importins we have generated knockout mice for all five known paralogs in the mouse. The most obvious phenotype was discovered in mice lacking importin alpha7: Both sexes of these animals are infertile. The molecular basis of this phenotype is currently analyzed. In addition, we could show that the absence of importin alpha5 during mouse development does not significantly interfere with neuronal differentiation and proper brain development, in contrast to the prediction based on a study in cell culture. Comparative studies on alpha-importin deficient mice and cells derived from them will allow to discover novel redundancies and specificities in nuclear transport.
Transgenic and stem cell technology
The group has also a strong emphasis in the field of rat embryology and stem cell research. The rat is the preferred animal in physiological and behavioural studies. However so far, there is no reliable method of generating targeted genetic alterations in these species. In order to obtain rat pluripotent stem cells two methodologies were applied in our group: isolation of ES (embryonic stem) from rat preimplantation embryos and generation of induced pluripotent stem (iPS) cells from fibroblasts upon infection with lentiviruses carrying pluripotency genes. These cells are used to explore the signalling cascades underlying mechanisms of pluripotency in the rat, to develop protocols in regenerative therapy, and to establish homologous recombination and thereby allowing gene targeting in the rat. Furthermore, transgenic rats have been produced carrying constructs, which express small interference RNAs suited to downregulate specific genes. The first target gene was the insulin receptor yielding a unique inducible rat model for diabetes mellitus type 2.