Sepsis represents the systemic response to infection, and the ensuing inflammatory cascade leads to the development of multi-organ failure and high mortality. The acute respiratory distress syndrome (ARDS) represents a process of devastating lung injury that complicates sepsis and contributes to propagation of the host inflammatory response long after the initial infection has been treated. ARDS results in loss of surfactant, alveolar collapse, and refractory hypoxemia. While mechanical ventilation is necessary to support patients with ARDS, ongoing damage to the lung is inevitable with delivery of high airway pressures to injured lung parenchyma. Thus, novel molecular strategies that target amelioration of lung injury through preservation of surfactant expression hold promise for improving overall outcomes from sepsis. It has long been proposed that significant surfactant loss occurs through protein destruction by nitric oxide (NO) and its metabolites. Excessive production of NO during sepsis and ARDS results from upregulation of NOS2 expression in parenchymal and inflammatory cells. We and other investigators reported a novel mechanism by which NOS2- derived NO interferes with surfactant expression and propagates lung dysfunction. In a murine model of endotoxemic lung injury, we demonstrated that NOS2-derived NO downregulates expression of surfactant protein-B in lung epithelial cells. Although systemic NOS2 inhibitors have not improved survival in human sepsis, and exogenous surfactant replacement has not shown benefit in ARDS, we postulate that the ability to modulate localized NOS2 expression in the lung represents an exciting approach toward restoring """"""""renewable"""""""" surfactant expression during ARDS. Our overall hypothesis is that NOS2-derived NO in lung epithelial cells plays a critical role in propagation of lung injury during sepsis through modulating expression of surfactant proteins. To test our hypothesis, we propose three Specific Aims.
In AIM 1, we will determine the role of NOS2 in a murine model of sepsis-induced lung injury that mirrors human ARDS. Wild type and NOS2-deficient mice will be subjected to cecal-ligation and puncture (CLP) and antibiotic treatment, followed by low tidal volume mechanical ventilation. We will assess NOS2 expression, lung physiology, cellular inflammation, vascular leak, cytokine levels, surfactant function and expression of surfactant proteins.
In AIM 2, we will examine the direct effect of NOS2 expression in the lung epithelial cell on surfactant expression and function during acute lung injury. We will selectively modify SPB and/or NOS2 expression within the lung epithelial cell using two transgenic animals and appropriate controls.
In AIM 3, we will explore the role of NOS2-derived NO on regulation of human surfactant protein-B expression. We will perform transfection analyses, in vivo binding studies (chromatin immunoprecipitation assays), and S-nitrosylation assays in human and murine transformed cell lines and in primary murine distal lung epithelial cells isolated from wild-type and NOS2-deficient mice.
Sepsis represents the systemic response to infection, and the ensuing inflammatory cascade leads to the development of multi-organ failure and high mortality. The acute respiratory distress syndrome (ARDS) represents a process of devastating lung injury that complicates sepsis and contributes to propagation of the host inflammatory response long after the initial infection has been treated. Achievement of the AIMS of this proposal hold significant promise for gaining novel insights into the pathobiology of sepsis-induced ARDS that can inform the development of new treatment strategies for these devastating syndromes.
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