The broad, long-term objective of this proposal is to characterize the participation of Platelet-activating factor (PAF) acetylhydrolase (PAF-AH) in oxidant-induced and inflammatory responses using both in vitro and in vivo approaches. PAF-AH is a phospholipase A2 with specificity for hydrolysis of lipid messengers such as PAF and oxidatively-fragmented phospholipids. This enzyme has been the subject of intense research in the past two decades owing to its potentially important role in a variety of human syndromes and diseases including vascular disease, bowel necrosis, asthma and sepsis. Despite extensive work, the physiological function of PAF-AH is open for debate and the consequences associated with altered activity levels in various human syndromes are controversial. Here, we will utilize our recently developed PAF-AH knockout mouse in animal models of atherosclerosis and necrotizing enterocolitis to test the hypothesis that PAF-AH plays a key role as a modulator of disease severity. We will complement these in vivo studies with molecular and biochemical approaches to assess the mechanism of PAF-AH inactivation by oxidants, including the identification of domains that confer susceptibility to oxidant attack. Additionally, we will determine the role played by lipoproteins and lipoprotein components in the modulation of oxidant-induced inactivation. Finally, the structural features and posttranslational modifications responsible for observed differences in size and stability of human versus murine PAF-AHs in mammalian cells will be addressed.
The Specific Aims of this proposal are: I. Investigate the mechanism of PAF-AH inactivation by oxidants and test the hypothesis that lipoproteins participate in this process. II. Identify the molecular features in the human and mouse PAF-AH cDNAs that define differences in expression levels, stability and size. III. Determine if deletion of PAF-AH alters the severity of necrotizing enterocolitis using animal models. IV. Test the hypothesis that PAF-AH alters atherosclerosis susceptibility using genetically engineered models.
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