To Live and to Die with Reactive Oxygen Species

Cardiovascular diseases represent the main cause of death worldwide, mostly caused by myocardial infarction, stroke, or heart failure. These diseases involve the heart or blood vessels and are associated with an imbalance of reactive oxygen species (ROS) in cells. Endothelial cells form the inner monolayer of the vasculature and are directly exposed to biochemical and biomechanical influences in the blood. These stimuli regulate both, the healthy and harmful formation of ROS as well as vasodilation and permeability. Hydrogen peroxide (H2O2) is the most abundant intracellular ROS. Beside H2O2, calcium ions (Ca 2+) and nitric oxide (NO) are important second messengers in endothelial cells that are largely responsible for adjusting the vascular tone. In the present project, the interaction of these three mediators and their effects on the cardiovascular system will be examined. The use of luminescent sensors for H2O2, Ca2+ and NO allows their quantification on a fluorescence microscope in response to certain stimuli and in different locations of endothelial cells. In addition, a newly developed method enables the formation of H2O2 within these cell locations. This approach uses an enzyme derived from yeast, a D-amino acid oxidase (DAAO) that converts D-amino acids into H2O2. Thus, the influence of locally generated H2O2 on NO and Ca 2+ will be mimicked within cells and the correlation can be observed. In parallel to these cellular measurements, this approach is also pursued in a living mouse model by incorporating DAAO into their genetic information. Recently such a transgenic mouse was developed by professor Dr. Michel at my host laboratory. If these mice are fed with D-amino acids, H2O2 is formed in their cells. The development of another mouse model that exclusively expresses DAAO in its endothelial cells will enable to study the influence of H2O2-mediated effects on blood vessels. Thus, this informative model system will enable to characterize effects of oxidative stress in the vessel wall in vivo and to analyze H2O2-modulated vasorelaxation responses in blood vessels isolated from this mouse strand. This proposal uses state-of-the-art imaging approaches, powerful biosensors, and entirely novel chemogenetic approaches to define the roles of H2O2 in the vasculature. The proposed studies use a combination of in vitro, in vivo, and ex vivo approaches that will extend my strong background in cell imaging and biophysics into vascular cells and tissues. I will lead the project guided by my supervisor of Prof. Thomas Michel at Brighams and Womens Hospital in Boston and in close collaboration with Prof. Wolfgang F. Graier from my home laboratory.

Oxidative stress is a common risk factor for cardiovascular and neurological diseases. Hydrogen peroxide (H2O2) is the most stable reactive oxygen species, which is required at low concentrations for physiological processes, but at high concentrations leads to oxidative stress. During my stay in the lab of Dr. Thomas Michel at the Brigham and Women's Hospital, Harvard Medical School, we developed in vitro and in vivo models to elucidate the effects of (patho)physiological H2O2: We performed live cell imaging measurement in human umbilical vein endothelial cells (HUVEC) expressing the novel ultrasensitive H2O2 biosensor HyPer7.2 and detected robust H2O2 signals in response to the G-protein coupled receptor (GPCR) agonist histamine and to the receptor tyrosine kinase (RTK) agonist VEGF. NADPH oxidases (Nox) isoforms are potential sources of cellular H2O2 generation. Using siRNA-mediated knockdown experiments we found that receptor-mediated H2O2 signaling is differentially dependent on distinct NOX isoforms 2 and 4 as well as on the small GTPase Rac1, a major constituent of NOX2. We used this model to investigate the incompletely understood antioxidant effects of statins. Statins are HMG-CoA inhibitors that in addition to lowering LDL cholesterol also block the synthesis of isoprenoid lipids necessary for the post-translational modification of signaling proteins, including Rac1. We observed that simvastatin treatment promotes Rac1 translocation from the cellular membrane to the nucleus accompanied by the loss of endothelial H2O2 responses to histamine and VEGF. These simvastatin effects were reversible by treatment with mevalonic acid, the enzymatic product of the HMG-CoA reductase. Our studies establish that intracellular H2O2 responses to histamine and VEGF involve distinct Nox isoforms and are dependent on Rac1. We also developed new tissue-specific transgenic mouse models for oxidative stress using the yeast-derived enzyme D-amino acid oxidase (DAAO), which generates H2O2 in the presence of D-amino acids. By using the putatively endothelium-specific Cdh5 promoter, we introduced DAAO into mouse endothelial cells, expecting a vascular defect after induction of oxidative stress by feeding the mice D-alanine. However, these mice quickly developed an unexpected ataxic phenotype 2 days after providing D-alanine in the drinking water; control mice were entirely unaffected. Necropsies of the transgenic mice showed selective neurodegeneration in the spinal cord affecting the dorsal sensory column only. Immunohistochemical analysis revealed robust transgene expression in dorsal root ganglia in addition to the endothelium. Importantly, echocardiography of chronically treated mice establishes the presence of hypertrophic cardiomyopathy. Taken together, this pathology is similar to the phenotype of patients with Friedreich's ataxia, the most common form of hereditary ataxia in humans caused by mitochondrial dysfunction and oxidative stress. This new transgenic mouse line represents a new and potentially important mouse model for Friedreich's Ataxia and affirms the importance of oxidative stress in neurodegeneration.