The broad, long-term objective is to achieve a deeper understanding of the pathogenesis of acute lung injury (ALI) causing the Acute Respiratory Distress Syndrome (ARDS), and to develop more effective means of its treatment and prevention. Hypotheses to be tested: 1) ALI due to oxidant stress is attributable in large measure to two fundamental and interrelates processes: inflammation and cell death by apoptosis or necrosis; 2) Excitotic mechanisms are a major pathogenic factor in cell death due to oxidant stress; and 3) Vasoactive intestinal peptide (VIP) and the related pituitary adenylate cyclase activating peptide (PACAP) can effectively reduce or prevent at least some forms of ALI, by blocking these critical processes, and by promoting cell survival pathways. Specific Objectives fall into two broad categories: a) Establish the role of inflammatory, apoptotic and excitotoxic mechanisms in the pathogenesis of ALI. This goal is justified by the need to establish criteria for evaluating the second objective; b) Determine the efficacy, potency, and specificity of VIP & PACAP in the protection against ALI, with special reference to the above pathogenic mechanisms, and the receptors and pathways mediating this protection. 1) Document the importance of key inflammatory mechanisms in ALI, including NF- KB activation, an the expression and production of major pro- and anti-inflammatory cytokines. NF-KB activation, will be assessed by evaluating evidence for such activation (nuclear displacement of p50 and p65 subunits) in models of ALI, and correlating the activation with the degree of injury, and the inhibition of activation with protection from injury. Transgenic mice with targeted deletion of NF-KB subunit p50 will be examined for possible resistance to injury. 2) Assess the contribution of apoptotic and necrotic cell death to ALI by: morphologiv evidence, TUNEL-testing, caspase activation, poly (ADP-ribose) polymerase PARP) activation (which often leads to necrosis) or its degradation (a marker of apoptosis), and the degree of protection afforded by anti-apoptotic measures, including caspase inhibitors and upregulation of anti-apoptosis protein bcl2, relative to pro-apoptotic BAX. 3) Validate the concept that endogenous glutamate receptor activation is an important mechanism of oxidant-induced ALI. 4) Determine the mechanisms, receptors, and pathways of lung protection by VIP and PACAP in these experimental models of injury: a) Excitotoxic lung injury due to excessive glutamate receptors due to excessive activation of glutamate receptors, a model that we recently characterized and is uniquely suited to a critical analysis of cell death mechanisms in lung injury; b) oxidant injury due to paraquat and xanthine + xanthine oxidase; and c) endotoxin lipopolysaccharide (LPS)-and Fas- induced apoptotic cell death. Injury will be induced in isolated lungs, cell preparations (alveolar macrophages, alveolar epithelial & pulmonary endothelial cells), and in vivo model of endotoxemia. Whenever available, we will study selected strains of transgenic mice either lacking or overexpressing key factors regulating the inflammatory response, death pathways and survival signals. In searching for the pathways of injury and its modulation by VIP/PACAP, we will use the newly introduced gene microarrays.
