Role of NO and Endothelin in Human NEC

描述(申请人提供)：该项目的目标是在人体肠道中测试一种新的假设，即在坏死性小肠结肠炎(NEC)发展之前的缺血的发病机制，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贡献者。研究假设如下：NEC之前的相关微血管缺血是由强大的内皮衍生血管扩张剂一氧化氮(NO)和强大的内皮衍生收缩因子内皮素-1(ET-1)之间的失衡引起的。我们认为，内皮型一氧化氮合酶(ENOS)在NEC中的表达和活性降低，而ET-1的表达增加；同时，我们认为介导ET-1血管收缩的内皮素ETA受体在NEC中的表达增加。这一假说最初是基于在围产期猪身上进行的工作而发展起来的，在这些工作中，我们证明了NO和ET-1在新生猪肠道血管调节中的作用比幼年猪大得多；此外，我们还证明了在适度的缺血再灌注后，NO和ET-1之间出现了预先发现的和持续的失衡。基于这项动物工作，我们在NEC和非NEC患者的切除后恢复的人肠道中开展了一项先导性研究。这项初步研究显示，NEC患者的肠道eNOS表达减少，ET-1表达增加；同时，从NEC肠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。这项申请建议通过仅在人体肠道进行前瞻性研究来扩大这些观察。提出了四个具体的目标：目的[1]将扩大对NEC和非NEC病例肠道eNOS表达的观察；将开展进一步的研究，以评估eNOS的活性和免疫组织化学定位。还将评估iNOS的假定贡献。目的[2]扩大对NEC和非NEC患者肠道组织ET-1表达的观察。我们将根据免疫组织化学添加对ET-1定位的评估，并评估内皮素受体的存在。目的[3]将对NEC和非NEC患者切除的肠系膜残端小动脉的血流动力学调节进行进一步的观察。研究旨在评估NO和ET-1在这一调节中的作用，以寻找从NEC肠收集的小动脉中NO作用降低和ET-1作用增加的证据。目的[4]将验证NO通过与细胞色素氧化酶相互作用直接调节肠道耗氧量的假设，具体地说，我们将确定NO是否以一种生理相关的方式抑制线粒体呼吸活动，从而降低肠道耗氧量。这项研究的独特之处在于，它将严格测试关于NEC在人类肠道而不是在动物模型中发病的新假说。如果成功，这些数据可能为NEC的新药物治疗策略提供一个前瞻性、随机试验的平台，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.