That PAF may be an important endogenous mediator in NEC is suggested by the following: (a) PAF induces in rats and mice intestinal necrosis which is morphologically similar to human NEC. (b) Most of the gut injury induced by agents or conditions that are known to cause bowel injury, e.g., LPS, TNF, endothelia, and hypoxia, can be blocked by PAF antagonists; (c) plasma PAF levels are elevated in patients with NEC; and (d) acetylhydrolase, the PAF-degrading enzyme, is deficient in the neonate. By using the model of PAF-induce NEC we found that the pathophysiological changes of PAF-induced gut injury are complex and involve secondary events such as leukocyte activation and formation of mediators such as TNF, leukotrienes (LT), and oxygen radicals. Although the signal transduction pathway of PAF has been outlined recently, the molecular mechanism of PAF action is still unclear. In the first part of the proposal we will examine the regulation and role of transcriptional factors (TFs) NF-kappaV and AP-1 in PAF- induced intestinal injury. These TFs are nuclear messengers involved in the inflammatory process, since they up-regulated gene expression of cell adhesion molecules and other pro-inflammatory mediators. We hypothesize that PAF activates PKC and stimulates oxygen radical release, which in turn activates and induces gene expression of NF-kappaB and AP-1. The activated NF-kappaB and AP-1 up regulated adhesion molecules and cytokines such as TNF. In the second part of the study, we will examine if PAF activates phospholipase (PL) A2, 5-lipoxygenase (LO) activating protein (FLAP),and PLA2 activating protein (PLAP), resulting in formation of LTs, which may aggravate injury, up-regulate AP-1 and NF-kappaB activity. Preliminary results show that PAF stimulated mRNA formation of NF-kappaB and AP-1), MK-886 (FLAP inhibitor) and anti-CD18 prevented PAF-induced injury.
Our specific aims are: (1) To examine the effect of PAF on these TFs. (2) To investigate the mechanism of PAF-induced expression and activation of TFs, and examine the consequence of blocking these TFs. (3) To examine the effect of PAF on the expression and activation of proteins responsible for LT production and the role of 5-LO products on PAF-induced injury and TF regulation. (4) To localize NF- kappaB and AP-1 and their mRNA in the intestine. (5) To examine the role of leukocytes in PAF-induced expression and activation of TFs, PLA2, and FLAP. This study will shed light on the molecular mechanism of PAF action and PAF-induced intestinal injury.
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