Elucidation of Signal Transduction Mechanisms with Pathological Relevance for Cardiovascular Diseases in-vitro and in-vivo

Cardiovascular diseases continue to represent the leading cause of morbidity and mortality in developed countries. Although major advances have been made in the prevention and treatment of these conditions, there is still a great demand for novel treatment options due to their high prevalence and degree of impairment in a constantly aging society. Therefore, one of the main areas of pharmaceutical research focuses on the identification and validation of novel drug targets and underlying signaling pathways in cardiovascular diseases both in vitro and in-vivo. In this regard, the emphasis of my PhD thesis lies on three distinct areas within the field of cardiovascular research: (i) Identification of DCHS1 as a novel potential target of beta-arrestin in cells Beta-arrestins are cytosolic proteins which were first identified for their function in the desensitization process of G Protein-coupled receptors. However, since then many other proteins and signaling pathways affected by beta-arrestins were discovered. In my PhD thesis I describe the identification of a novel potential target protein of beta-arrestin in-vitro, the protocadherin DCHS1, a protein which was very recently found to be involved in the pathogenesis of a valvular heart disease termed Mitral Valve Prolapse (MVP). Cellular experiments were performed in HEK293A cells to evaluate protein steady state levels and protein stability of DCHS1 upon co-expression with beta arrestin because protein down-regulation is a typical beta-arrestin-mediated effect and reduced DCHS1 protein stability was found to be causative for MVP. Therefore, in order to evaluate alterations in protein steady-state levels, DCHS1 was fused to yellow fluorescence protein (DCHS1-eYFP) followed by quantification of protein levels by fluorescence measurement in the absence and presence of beta-arrestin. Interestingly, co-transfection with beta-arrestin revealed a significantly higher fluorescence level of DCHS1-eYFP compared to the control without transfection of beta-arrestin. This up-regulation of DCHS1-eYFP by beta arrestin was subsequently verified by western blot analysis of cell lysates using anti-GFP and anti-DCHS1 antibodies. Furthermore, the observed beta arrestin effect could be significantly reduced by C-terminal truncation of DCHS1-eYFP. In conclusion, I provide results that beta-arrestin shows an increasing effect on the protein steady-state levels of DCHS1 in cells. (ii) Cloning and in-vitro characterization of a genetically encoded EPAC-based FRET biosensor for real-time measurement of cAMP levels in living cells Cyclic adenosine monophosphate (cAMP) is the major second messenger involved in transduction of physiological and pathological effects upon beta-adrenergic receptor (beta-AR) stimulation in living cells. Overstimulation and resulting down regulation of beta-AR is one of the major hallmarks in the pathogenesis of chronic heart failure. Therefore, the search for novel drug targets and the development of novel drugs affecting the beta-adrenergic signaling events through cAMP, either directly or indirectly, is of great interest for pharmaceutical research. In my PhD thesis I describe the development and characterization of a robust in vitro assay for rapid and quantitative measurement of intracellular cAMP levels in living cells in real-time using an EPAC-based FRET biosensor. For molecular cloning of the FRET biosensor, I first optimized the FRET fluorophore pair CFP/YFP as well as the cAMP-sensing EPAC1 protein by mutagenesis to enhance the performance and dynamic range of the resulting sensor. As a next step, I generated a stable HEK293A cell line expressing the FRET biosensor and pharmacologically characterized it by treatment of the cells with different substances affecting intracellular cAMP levels. Thereby, I was able to determine concentration-response relationships for the cAMP-increasing agents forskolin, a direct stimulator of adenylate cyclase, as well as for the beta-adrenergic receptor agonist, (-)-isoproterenol. Furthermore, I verified the specificity of the ( ) isoproterenol effect by determination of concentration-response relationship of the beta-adrenergic receptor antagonist propranolol. With this characterization, I proved that the generated biosensor is able to quantitatively measure changes in cAMP levels in a rapid and robust manner while offering the possibility to directly measure the cAMP level of living cells in suspension in real-time. With this biosensor in hands it is now possible to both identify novel targets affecting cAMP signaling by transfection of the stable cell line, as well as to rapidly assay new pharmacological substances for their influence on any point within the cAMP signaling cascade. (iii) Identification of a role of the bradykinin B2 receptor in the pathology of atherosclerosis in-vivo The bradykinin B2 receptor (B2R) represents an important vascular receptor primarily associated with regulation of blood pressure and blood vessel permeability. However, its exact role in the development and progression of atherosclerosis is not known to date. In my PhD thesis I investigated the role of the B2R in-vivo in different transgenic mouse lines. Therefore, ApoE-deficient transgenic mice expressing different levels of B2R were generated and atherosclerotic plaque formation in murine aortas was determined by Oil-red-O staining. The quantification of the lesion areas revealed a pronounced pro-atherogenic role of the B2R. To further investigate the underlying pathomechanism of this pro-atherogenic effect, reactive oxygen species (ROS) content in aortas were determined by DHE staining. ROS content were significantly elevated in aortas from mice with higher levels of B2R. This increased ROS content was found to be due to reduced levels of eNOS cofactor tetrahydrobiopterin (BH4) leading to an uncoupling of eNOS. Furthermore, downregulation of the major BH4-synthesizing enzyme, GTP-cyclohydrolase I (GchI), was found in aortas with higher levels of B2R. To prove the causality of this pathomechanism, transgenic mice were treated with the BH4 analogue sapropterin orally, which was able to significantly retard atherosclerotic lesion formation. Taken together, the work in my PhD thesis provides further insights into signaling pathways involved in the development and progression of important cardiovascular diseases. Furthermore, it validates a biosensor approach for investigation of cAMP signaling processes in real-time and the possibility for rapid screening of new pharmacological substances.