Surfactant deficiency plays an integral role in the pathogenesis of neonatal lung diseases such as RDS and acute lung injury. Although surfactant replacement therapy has made an impact in RDS, it is not totally effective. An alternative strategy to increase the alveolar surfactant pool is by stimulating biosynthesis. Surfactant synthesis is tightly controlled by the rate-limiting enzyme, cytidylyltransferase (CT). CT activity is inhibited by sphingolipids and stimulated by fatty acids. However, prior studies administering fatty acids in vivo to stimulate surfactant production have had mixed success with associated toxicity. Thus, the goal of this proposal is to develop a novel and safe approach to stimulating surfactant synthesis in fetal lung by the use of very low density lipoproteins (VLDL) and lipoprotein lipase (LPL). This revised competing renewal expands from recent advances in our laboratory made through support of the existing grant showing that 1) fatty acids carried within very low density lipoproteins (VLDL) are potent stimulators of CT activity in vitro in the presence of LPL and 2) oxidized lipoproteins inhibit surfactant synthesis, . These observations led to the hypothesis that native lipoprotein loading and oxidized lipoproteins differentially regulate the CT enzyme. We will investigate whether loading with native lipoproteins (VLDL) increases CT activity by altering fatty acids and sphingolipids associated with the enzyme (AIM 1). We will also investigate how modified (oxidized) lipoproteins acutely down-regulate surfactant synthesis by inducing CT proteolysis (AIM 2). Our hypothesis will be tested, in vivo, by maternal administration of lipoproteins in pregnant rats and with analysis conducted in primary fetal type II cells. These in vivo studies will be supplemented with use of a lipoprotein-responsive type II (MLE12) cell line. The unique contributions of this proposal impacting the field of surfactant metabolism include: 1) mechanistic studies with potential clinical application by which native lipoproteins control CT function post-translationally (AIM 1) and 2) studies investigating CT regulation at the level of protein stability (AIM 2) .
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