Following acute lung injury, the alveolar epithelium is severely injured and an inflammatory response within the alveolar wall ensures. In many patients effective restoration of the epithelium fails to occur, and there is a rapid increase in the number of mesenchymal cells and mesenchymal cell connective tissue products within the alveolar wall and airspace. This results in a severe impairment of gas exchange often leading to death. Therefore, to understand that pathogenesis of acute fibrosis, it is necessary to examine the phenotypic properties of the mesenchymal cell subpopulations present. Our hypothesis is that chemotaxis, growth factor responsiveness, and connective tissue production rate are phenotypic properties important in the evolution of fibrosis. Our fundamental methodologic assumption is as follows: to optimally test this hypothesis, defined mesenchymal cell subpopulations freshly isolated from the lungs of patients dying with acute lung injury and control lungs must be studied ex vivo, ie. rapidly and with as little in vitro manipulation as possible. Since currently available methods for stratifying freshly isolated mesenchymal cells into subpopulations are not sufficient, this investigation will be divided into two parts: 1. Ultrastructural analysis indicates that the vast majority of mesenchymal cells in the alveolar wall and airspace following acute lung injury are myofibroblasts. Therefore, routine enzymatic dispersion techniques can be utilized to obtain a population of mesenchymal cells highly enriched in myofibroblasts. 2. Methods will be developed to obtain defined mesenchymal cell subpopulations based on three general properties of the cell: a) surface moieties amenable to affinity separation (rosetting, panning, preparative FACS), b) rate of chemotaxis, c) buoyant density. Myofibroblasts (part 1) and defined subpopulations (part 2) will be examined for their chemotactic and replication response to purified growth factors of platelet and macrophage origin (fibronectin, platelet derived growth factor) as well as for their connective tissue production (collagens I, III fibronectin). Comparing the phenotypic characteristics of defined subpopulations in acutely injured lungs to their counterparts in control lungs may provide important insights into the pathogenesis of acute fibrosis. It is our goal to use this improved understanding of the pathogenesis of acute lung injury to devise rational therapeutic strategies designed to interrupt the fibrotic process.
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