Rapid Control and Assessment of Dissolved Gas Signals on Tissue Function andDisease.

Biologically important gases (in particular, the three trace gas signals H2S, NO and CO as well as O2) mediate many regulatory mechanisms of cell function in mammalian tissues and are involved in the progression of multiple diseases including cancer, blindness, diabetes, wound healing, stroke and cardiovascular disease. However, most research laboratories lack the instrumentation to adequately and safely assess the effects of signaling gases on biological samples, greatly hindering progress in research. Since gases regulate processes that respond rapidly to changes in short-lived signals on the order of seconds to minutes, researchers need the ability to deliver rapid changes in dissolved gas to biological samples and assess acute responses with respect to both secreted factors and changes in intracellular content. Previous work by Dr. Ian Sweet (co-principal investigator at STTR research institution University of Washington) using a trace gas delivery system developed under a NIGMS Technology Development Program R01 grant shows paradigm shifting data where 1) effects of H2S gas differed from the commonly used aqueous donor HS-, 2) rapid control of VEGF release that is independent of the known regulating enzyme (HIF-1), and new mechanisms mediating hormone secretion. The results establish that there is a critical need to test results obtained with donor molecules against the physiologic gaseous form of the signaling molecule. In addition, concentration dependencies were very steep with complex time-courses, and in cells all three gas signals (H2S, NO and CO) simultaneously compete with O2 for metalloproteins highlighting the need for precise control of multiple gases. Responses to changes in O2 have revealed that regulation of VEGF secretory response is very tissue-specific, where retinal response is much faster than islets, liver and kidney so we will design a system that accommodates many tissue/cell models. An automated system controlling dissolved gas for in-vitro tissue enables widespread use and leads to a broad range of scientific findings. Three aims will be carried out 1) building a prototype tissue culture system that delivers rapid, precise changes in trace signaling gases to tissue/cells maintained in vitro, and that enables addition of test compounds and sampling of cellular secretions, 2) demonstrating that the prototype can resolve biologically and clinically relevant effects in response to acute and chronic exposure to trace gas signals, and 3) evaluating and optimizing methods for extracting intracellular components, i.e. metabolites, proteins, mRNA etc., at a specific time during the experiment. The end-product in Phase II will incorporate optical analysis/imaging capabilities, and software control for a fully automated experimental platform that resolves rapid, acute biological processes driven by effects of multiple trace signaling gasses and test compounds. In the existing $20B cell culture automation market, no products provide the functionality of our gas control/tissue assessment system, which will accelerate the development of therapeutics for a wide range of disease states including cancer, regenerative medicine, and metabolic diseases.