Role of NO and Endothelin in Human NEC

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DESCRIPTION (provided by applicant): The goal of this project is to test, in human intestine, a novel hypothesis regarding the pathogenesis of the ischemia which precedes the development of necrotizing enterocolitis (NEC), the most common acquired gastrointestinal disease of infancy and a major contributor to neonatal morbidity and mortality. The study hypothesis is as follows: the relevant microvascular ischemia preceding NEC is caused by an imbalance between the production of the potent, endothelium-derived vasodilator nitric oxide (NO) and the potent, endothelium-derived constrictor endothelin-1 (ET-1). We contend that expression and activity of the endothelial isoform of nitric oxide synthase (eNOS) is decreased while the expression of ET-1 is increased in intestine afflicted with NEC; as well, we believe that expression of the endothelin ETA receptor, which mediates ET-1-based vasoconstriction, is increased in NEC. This hypothesis was initially developed based on work carried out in perinatal swine, in which we demonstrated that the roles of NO and ET-1 in intestinal vascular regulation are substantially greater in newborn than juvenile swine; furthermore, we demonstrated that a pro-found and sustained imbalance between NO and ET-1 occurs after modest episodes of ischemia-reperfusion. Based on this animal work, we carried out a pilot study in human intestine recovered from resections in NEC and non-NEC patients. This pilot study revealed a decreased expression of eNOS and an increased expression of ET-1 in intestine from NEC patients; as well, mesenteric arterioles harvested from NEC intestine demonstrated a reduced role for endogenous NO in vascular regulation, but an increased role for endogenous ET-1. This application proposes to expand these observations by carrying out a prospective study solely in human intestine. Four specific aims are proposed: Aim [1] will expand observations regarding the expression of eNOS in intestine from NEC and non-NEC cases; additional studies will be carried out to evaluate eNOS activity and localization by immunohistochemistry. The putative contribution of iNOS will also be assessed. Aim [2] will expand observations regarding expression of ET-1 in intestine from NEC and non-NEC cases. We will add the evaluation of ET-1 localization per immunohistochemistry and also evaluate the presence of endothelin receptors. Aim [3] will expand observations regarding the hemodynamic regulation of arterioles harvested from the mesenteric remnants of intestine resected from NEC and non-NEC patients. Studies are designed to evaluate the roles of NO and ET-1 in this regulation, looking for evidence that the role of NO is decreased and of ET-1 is increased in arterioles harvested from NEC intestine. Aim [4] will test the hypothesis that NO directly regulates intestinal oxygen consumption by means of its ability to interact with cytochrome oxidase, specifically, we will determine if NO acts to inhibit mitochondrial respiratory activity and so attenuate intestinal oxygen consumption in a physiological relevant manner. This study is unique in that it will rigorously test a novel hypothesis regarding NEC pathogenesis in human intestine rather than in an animal model. If successful, these data could provide a platform for a prospective, randomized trial of new medical therapeutic strategies for NEC, e.g., the pharmacological manipulation of endothelin receptors.