Role of NO and Endothelin in Human NEC

描述（由申请人提供）：本项目的目的是在人体肠道中测试关于坏死性小肠结肠炎（NEC）发生前缺血发病机制的新假设，NEC是婴儿期最常见的获得性胃肠道疾病，是新生儿发病率和死亡率的主要因素。研究假设如下：NEC之前的相关微血管缺血是由有效的内皮源性血管扩张剂一氧化氮（NO）和有效的内皮源性收缩剂内皮素-1（ET-1）之间的不平衡引起的。我们认为，表达和活性的内皮型一氧化氮合酶（eNOS）的减少，而ET-1的表达增加，在肠与NEC折磨，以及，我们认为，内皮素ETA受体的表达，它介导ET-1为基础的血管收缩，增加在NEC。这一假设最初是基于在围产期猪中进行的工作而开发的，在围产期猪中，我们证明了NO和ET-1在肠血管调节中的作用在新生猪中比幼猪大得多;此外，我们证明了在适度的缺血再灌注发作后，NO和ET-1之间发生了预先发现的持续不平衡。基于这种动物实验，我们在NEC和非NEC患者的切除后恢复的人肠中进行了初步研究。这项初步研究揭示了减少eNOS的表达和增加的表达ET-1在肠从NEC患者，以及，肠系膜小动脉收获从NEC肠表现出减少的作用，内源性NO在血管调节，但增加的作用，内源性ET-1。本申请提出通过仅在人肠中进行前瞻性研究来扩展这些观察。提出了四个具体目标：目标[1]将扩大关于NEC和非NEC病例肠中eNOS表达的观察;将进行额外的研究以通过免疫组织化学评价eNOS活性和定位。还将评估iNOS的假定贡献。目的[2]将扩大关于NEC和非NEC病例肠中ET-1表达的观察。我们将通过免疫组织化学增加ET-1定位评价，并评价内皮素受体的存在。目的[3]将扩大关于从NEC和非NEC患者切除的肠肠系膜残余物中采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在这种调节中的作用，寻找证据表明，在从NEC肠收获的小动脉中，NO的作用降低，ET-1的作用增加。目的[4]将检验NO通过其与细胞色素氧化酶相互作用的能力直接调节肠氧消耗的假设，具体地，我们将确定NO是否以生理相关的方式抑制线粒体呼吸活性并因此减弱肠氧消耗。这项研究的独特之处在于，它将严格测试关于NEC在人类肠道而不是动物模型中发病机制的新假设。如果成功的话，这些数据可以为NEC的新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.