Healthy cells use low levels of reactive oxygen species (ROS) as second messengers in signal transduction pathways. High levels of ROS cause oxidative damage to proteins, lipids and DNA. Oxidant stress has been implicated in the cellular dysfunction associated with ischemia- reperfusion injury, vascular disease, stroke, diabetes, neurodegenerative diseases, liver disease, renal disease, inflammation, cancer, and other disorders. As the awareness of the role of redox stress in health and disease has grown, the demand for new tools to monitor oxidant stress in vivo has increased. Current methods to assess redox events are limited by their inability to provide quantitative data or spatial information on the subcellular sites of oxidant generation. Moreover, existing probes are generally unsuitable for in vivo studies. New methods to monitor intracellular oxidant stress in intact tissues could enhance our understanding of how cell-cell interactions and tissue microenvironments influence the generation of ROS. We propose to create a new system to detect redox status and oxidative stress in specific cells within intact tissues, using a novel combination of existing methods.
In Aim 1 we will create transgenic mice with DNA encoding the redox-sensitive fluorescent protein, RoGFP, inserted at a LoxP-silenced ROSA26 genomic locus. Three lines will be generated, which target the RoGFP sensor to cytosol, mitochondrial matrix, or mitochondrial intermembrane space.
In Aim 2 we will activate expression of the RoGFP genes in primary cells cultured from these mice, using Cre recombinase to delete the stop codon. We will confirm correct targeting of the expressed protein, and confirm its function in response to redox stress.
In Aim 3 we will breed the RoGFP mice with smooth muscle-specific Cre recombinase mice, to elicit RoGFP expression in pulmonary artery smooth muscle cells in the lung. Using that model system to demonstrate efficacy, we will measure redox changes in smooth muscle cells in the intact lung during ventilation with different concentrations of oxygen. Two-photon microscopy will be used to assess the redox status of the subcellular targeted RoGFP proteins in vivo. These animals will therefore provide exciting new tools that will enable us, and other investigators, to monitor subcellular oxidative stress in intact tissue in diverse cell types and disease models. Public Health Relevance Statement: Healthy cells in the body use oxygen free radicals (Reactive Oxygen Species, or ROS) to regulate various cellular functions. Excessive levels of ROS disrupt cell function, and they contribute to cellular injury in a large number of diseases. To understand how ROS affect cells, it is essential to monitor their levels. However, current tools are limited in their ability to assess intracellular ROS. We propose to correct this problem by inserting a gene encoding an ROS-sensitive fluorescent protein into mice. When the gene is turned on, the cell will generate a protein that moves to a known intracellular compartment and signals a change in ROS levels by altering its fluorescence. We will turn this gene on in certain types of cells in the mouse, and measure the fluorescence changes using a form of microscopy that can """"""""see"""""""" deeply into intact tissues. We will test the performance of this sensor in the lungs, where we will measure the ROS response to changes in the concentration of oxygen that the animal is breathing. However, many other investigators will be able to use the same mice where, by turning on the reporter gene in other cell types, it will be possible to monitor ROS in a wide range of different tissues. Hence, this mouse will provide useful information on ROS levels in a wide range of disease models. The successful outcome of this project is supported by extensive preliminary studies demonstrating the feasibility of each step in the process. The end result should significantly extend our ability to assess ROS in intact tissues, in animal models of disease.
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