HB-EGF Therapy for Necrotizing Entercolitis

描述(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，有望取代肺功能不全成为早产儿的主要死亡原因。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)在保护肠粘膜免受损伤中的作用。我们工作的长期目标是基于这样一个前提，即保护肠粘膜免受伤害是开发预防疾病的新策略的合理目标，并涉及临床应用HB-EGF，包括治疗和预防，以保护新生儿免受NEC的伤害。最近，我们发现HB-EGF降低了NEC大鼠模型的组织学损伤和死亡率。NEC模型是指幼鼠处于反复的低氧/低温、高渗喂养和脂多糖(HHHTF+LPS)暴露的循环中，导致与人类NEC难以区分的肠道损害。我们的中心假设是，患有NEC的组织局部缺乏HB-EGF，使部分肠道容易受到损伤，外源性HB-EGF通过促进肠道修复保护肠道免受损伤，从而保护肠道通透性。为了达到我们的目标，我们提出了三个具体的目标，使我们能够更好地了解HB-EGF在受损肠道中的作用机制，作为开发治疗临床方案的先决条件：目的[1]验证HB-EGF功能增强将导致对NEC耐药的假设，而HB-EGF功能丧失将导致NEC易感性增加。我们将对新生的HB-EGF转基因和基因敲除小鼠进行实验性NEC，将它们暴露在HHHTF中，通过系统吸收氟葡聚糖来评估肠道屏障功能，使用体外小室研究和细菌易位测定；肠道组织学；以及死亡率。目的[2]验证HB-EGF在HHHTF诱导NEC过程中通过诱导肠道修复介质保护肠道的假说。NEC小鼠模型将用于HB-EGF TG、HB-EGF KO和WT小鼠，测量的终点包括修复介质(整合素、FAK、PXILIN、RHO)的mRNA表达和蛋白产生。体外Ussing小室研究也将被用来进一步研究HB-EGF促进肠道重建的机制。目的[3]验证HB-EGF通过影响上皮细胞-基质相互作用促进修复的假说。修复的体外刮伤模型将被用来研究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，它将提供更好的了解HB-EGF在保护肠道免受NEC感染方面的机制，作为开发基于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.