Role of NO and Endothelin in Human NEC

描述（由申请人提供）：该项目的目标是在人类肠道中测试一个关于坏死性小肠结肠炎（NEC）发展之前缺血发病机制的新假设，NEC是婴儿最常见的获得性胃肠道疾病，也是新生儿发病率和死亡率的主要因素。本研究假设：NEC发生前的相关微血管缺血是由内皮源性血管扩张剂一氧化氮（NO）和内皮源性收缩剂内皮素-1 （ET-1）分泌失衡引起的。我们认为，内皮型一氧化氮合酶（eNOS）的表达和活性在NEC的肠道中降低，而ET-1的表达增加；此外，我们认为介导et -1血管收缩的内皮素ETA受体在NEC中表达增加。这一假设最初是基于在围产期猪身上开展的工作而提出的，在这项工作中，我们证明了NO和ET-1在新生儿肠道血管调节中的作用明显大于幼年猪；此外，我们证明，在中度缺血-再灌注发作后，NO和ET-1之间存在明显和持续的失衡。在此动物实验的基础上，我们对NEC和非NEC患者切除后恢复的人类肠道进行了初步研究。该初步研究显示，NEC患者肠道中eNOS表达减少，ET-1表达增加；此外，从NEC肠中获取的肠系膜小动脉显示内源性NO在血管调节中的作用降低，但内源性ET-1的作用增加。本应用建议通过仅在人类肠道中进行前瞻性研究来扩大这些观察结果。提出了四个具体目标：Aim[1]将扩大对NEC和非NEC病例肠中eNOS表达的观察；进一步的研究将通过免疫组织化学来评估eNOS的活性和定位。还将评估非政府组织的假定贡献。Aim[2]将扩大对NEC和非NEC病例肠中ET-1表达的观察。我们将增加免疫组织化学对ET-1定位的评估，并评估内皮素受体的存在。Aim[3]将扩大从NEC和非NEC患者切除的肠系膜残余肠中收集的小动脉的血流动力学调节的观察。研究旨在评估NO和ET-1在这一调节中的作用，寻找证据表明，NEC小肠小动脉中NO的作用降低，ET-1的作用增加。目的[4]将验证NO通过与细胞色素氧化酶相互作用的能力直接调节肠道耗氧量的假设，具体而言，我们将确定NO是否以生理相关的方式抑制线粒体呼吸活动从而减弱肠道耗氧量。这项研究的独特之处在于，它将严格检验一个关于NEC在人类肠道而不是在动物模型中发病的新假设。如果成功，这些数据可以为NEC新的药物治疗策略的前瞻性随机试验提供一个平台，例如，内皮素受体的药理学操作。

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.