HB-EGF Therapy for Necrotizing Entercolitis

描述（由申请人提供）：坏死性小肠结肠炎（NEC）是新生儿最常见的胃肠道疾病，预计将取代肺功能不全成为早产儿死亡的主要原因。我们已经积累了多条证据，支持肝素结合的egf样生长因子（HB-EGF）通过保存肠粘膜来保护肠道免受损伤。我们工作的长期目标是基于这样一个前提，即保护肠黏膜免受损伤代表了开发预防该疾病的新策略的逻辑目标，并涉及临床给药HB- EGF，治疗和预防，以保护新生儿免受NEC的侵害。最近，我们已经证明HB-EGF可以降低NEC大鼠模型的组织学损伤和死亡率，在这种模型中，大鼠幼仔暴露于缺氧/低体温、高高压喂养和暴露于LPS （HHHTF+LPS）的反复循环中，导致与人类NEC没有区别的肠道病变。我们的中心假设是，受NEC影响的组织存在HB-EGF的局部缺乏，这使得肠道的一部分容易受到损伤，并且外源性HB-EGF的管理通过促进肠道恢复来保护肠道免受损伤，从而保持肠道通透性。为了实现我们的目标，我们提出了三个具体目标，以便更好地理解HB-EGF在受损肠道中功能的机制基础，作为制定治疗性临床方案的先决条件：目的b[1]验证HB-EGF功能获得将导致对NEC的抗性，而HB-EGF功能丧失将导致对NEC的易感性增加的假设。我们将新生儿HB-EGF转基因和基因敲除小鼠暴露于HHHTF，进行实验性NEC实验，通过肠内FICT-葡聚糖的全身吸收来评估肠道屏障功能，使用室内离体研究和测定细菌易位；肠道组织学;和死亡率。目的验证HB-EGF通过诱导肠道修复介质在体内保护肠道的假说。小鼠NEC模型将用于HB-EGF TG， HB-EGF KO和WT小鼠，测量终点包括mRNA表达和恢复介质（整合素，FAK, paxillin, Rho）的蛋白质产生。体外实验还将进一步研究HB-EGF促进肠道恢复的机制。目的验证HB-EGF通过影响上皮细胞-基质相互作用促进细胞修复的假说。我们将利用体外刮伤修复模型来研究HB-EGF促进修复的分子机制。这项研究与公共卫生的相关性在于，它将更好地了解HB-EGF保护肠道免受NEC感染的机制，这是开发基于HB-EGF的治疗方案的先决条件。

DESCRIPTION (provided by applicant): Necrotizing enterocolitis (NEC) is the most common gastrointestinal disease of newborns, and is anticipated to replace pulmonary insufficiency as the leading cause of death in premature babies. We have accumulated multiple lines of evidence supporting a role for heparin-binding EGF-like growth factor (HB-EGF) in protection of the intestines from injury via preservation of the intestinal mucosa. The long term goal of our work is based on the premise that protection of the intestinal mucosa from injury represents a logical target for the development of novel strategies to prevent the disease, and involves the clinical administration of HB- EGF, both therapeutically and prophylactically, to protect neonates from NEC. Recently, we have shown that HB-EGF decreases histologic injury and mortality in a rat model of NEC whereby rat pups are exposed to repeated cycles of hypoxia/hypothermia, hypertonic feedings and exposure to LPS (HHHTF+LPS), leading to intestinal lesions indistinguishable from those of human NEC. Our central hypothesis is that tissues afflicted with NEC have a local deficiency of HB-EGF which leaves a portion of the intestine susceptible to injury, and that administration of exogenous HB-EGF protects the intestine from injury via promotion of intestinal restitution, thereby preserving intestinal permeability. Towards our goal, we have proposed three specific aims that will allow a better understanding of the mechanistic basis of HB-EGF function in injured intestine, as a prerequisite to developing therapeutic clinical protocols: Aim [1] To test the hypothesis that HB-EGF gain-of-function will lead to resistance to NEC whereas HB-EGF loss-of-function will lead to increased susceptibility to NEC. We will subject neonatal HB-EGF transgenic and knockout mice to experimental NEC by exposing them to HHHTF, with evaluation of gut barrier function by systemic absorption of enteral FICT- dextran, Ussing chamber ex vivo studies and determination of bacterial translocation; intestinal histology; and mortality. Aim [2] To test the hypothesis that HB-EGF protects the intestine in vivo by inducing mediators of intestinal restitution during HHHTF-induced NEC. The mouse model of NEC will be used in HB- EGF TG, HB-EGF KO and WT mice, with measured endpoints including mRNA expression and protein production of mediators of restitution (integrins, FAK, paxillin, Rho). Ex vivo Ussing chamber studies will also be performed to further examine the mechanisms by which HB-EGF promotes intestinal restitution. Aim [3] To test the hypothesis that HB-EGF promotes restitution by affecting epithelial cell-matrix interactions. The in vitro scrape-wounding model of restitution will be utilized to examine the molecular mechanisms utilized by HB-EGF to promote restitution. The relevance of this research to public health is that it will provide a better understanding of the mechanisms utilized by HB-EGF in protection of the intestines from NEC, as a prerequisite for the development of HB-EGF-based therapeutic regimens.