Pulmonary endothelium is an early locus of functional and structural changes that contribute to the genesis and/or maintenance of acute lung injury(ALl). We will define the mechanisms of transport and the vascular actions of key inflammatory-modulatory macromolecules [e.g. S-nitroso-albumin (SNO-AIb) and myeloperoxidase (MPO)] that are elevated in the circulation after resuscitation from hemorrhagic shock (HS). Accordingly specific aims are to:
Aim #1. Define the physiological mechanism of transcytosis of SNO-AIb in cultured pulmonary endothelium. We will use real time multimode imaging (confocal and multiphoton scanning laser microscopy and total internal reflection fluorescence microscopy, FRET) to define molecular events associated with vascular cell binding, transcytosis and SNO-AIb mediatied signaling via S-nitrosation and/or activation of guanylyl cyclase.
Aim #2. Reveal the physiological mechanisms of MPO transcytosis in live intact pulmonary endothelium Aim #3. Explore the biochemical mechanisms by which MPO affects SNO-albumin signaling in pulmonary vascular cells and intact lung. To reveal the central pathways for intra- and extracellular regulatory MPO-dependent modulation of vascular NO signaling, we will examine the actions of MPO on SNO-albumin transfer and decomposition kinetics in increasingly integrated biological systems including: a) cell free models in vitro; b) rat lung microvacular endothelial cells (RLMVEC); and c) rat lung microvasccular smooth muscle cell and RLMVEC co-culture system.
Specific Aim #4. Place the pathophysiological contributions of SNO-albumin transport, redox status and MPO into the context of ALl after resuscitation from HS. We will contrast the extent of ALl (permeability index, neutrophil accumulation, morphological changes,pulmonary vasoregulation) in isolated perfused lungs and intact mice of wildtype and caveolin-1 and MPO homozygous null mutants.
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