Post-natal development of enteric glial cell-epithelial interactions in repair of ischemic-injured intestine

项目总结/摘要 如果上皮覆盖不能迅速恢复，肠粘膜屏障的破坏会导致败血症和死亡， 特别是在新生儿中。婴儿死亡率高于成年人的原因是， 肠损伤尚未得到解释。高婴儿死亡率是由与局部缺血有关的疾病引起的。 再灌注（I/R）损伤，包括坏死性小肠结肠炎和肠扭转。在幼年猪（6-8周龄）中，我们 已经广泛地研究了以上皮细胞迁移（恢复）为标志的显著快速的肠修复。 然而，在最近发表的一项研究中，该提案的修订版中，我们已经表明， 缺血性损伤后粘膜修复恢复中的年龄依赖性缺陷， 新生仔猪缺乏恢复。有趣的是，这种修复缺陷可以通过 缺血损伤的幼年匀浆，表明更成熟的细胞群刺激恢复。一个小区 特别感兴趣的群体是肠神经胶质细胞（EGC）网络，我们已经显示出成熟 出生后的猪在修订后的提案中，我们已经表明，猪EGC条件培养基 在受伤的新生猪IPEC-J2细胞中刺激细胞迁移（恢复）。然而，差距仍然存在， 我们对上皮细胞和潜在EGC之间的信号传导机制的了解， 赔偿为了解决这一差距，我们对缺血损伤的新生儿进行了RNAseq分析， 青少年粘膜，并创造了一个公正的发现管道的方法，专注于显着不足 新生儿的信号通路。其中一种途径包括EGF，它由EGC分泌， proEGF和上皮膜联蛋白A2，其通过β1-整联蛋白的信号内化启动细胞迁移。我们 中心假设是缺血损伤粘膜的修复依赖于EGC的出生后迁移 进入固有层，以便向受伤的上皮发出信号以有效地恢复。我们将测试这个 有两个具体目标的假设：1）确定年龄依赖性缺陷的迁移信号机制， 伤口附近的肠上皮细胞。我们的工作假设是，有一个年龄依赖性 至少部分地通过膜联蛋白A2信号传导响应损伤而发展上皮恢复。2)确定 围产期粘膜EGC网络结构、密度和分泌组的变化。我们的假设是 在接近上皮的地方，分泌屏障促进因子（包括proEGF）的EGCs不足 来刺激新生儿的恢复为了研究这些特定的目标，我们将使用我们独特的年龄依赖性 猪粘膜修复模型、受伤的猪新生IPEC-J2细胞模型、猪原代培养 EGC、通过质谱进行的EGC分泌组分析，以及神经胶质细胞的最新成像技术， 包括溶剂清除器官的免疫标记三维成像（iDISCO）。使用 无偏见的发现管道方法，我们希望能够定义信令机制，是有缺陷的 在新生儿损伤的肠道中，最终可以靶向提高肠衰竭患者的生存率

PROJECT SUMMARY/ ABSTRACT Breaches in the intestinal mucosal barrier lead to sepsis and death if epithelial coverage is not rapidly restored, particularly in neonates. The reason for higher mortality in infants as compared to more mature individuals with intestinal injury has not been explained. High infant mortality results from diseases associated with ischemia/ reperfusion (I/R) injury, including necrotizing enterocolitis and volvulus. In juvenile pigs (6-8-weeks of age), we have extensively studied the remarkably rapid intestinal repair marked by epithelial cell migration (restitution). However, in a recently published study featured in the revised version of this proposal, we have shown an intriguing age-dependent deficiency in the recovery of mucosal repair following ischemic injury with a near-total lack of restitution in neonatal piglets. Interestingly, this defect in repair can be rescued by a homogenate of ischemic injured juvenile homogenate, suggesting more mature cell populations stimulate restitution. One cell population of particular interest is the enteric glial cell (EGC) network, which we have shown matures postnatally in pigs. In the revised proposal, we have now shown that porcine EGC-conditioned media stimulates cell migration (restitution) in wounded neonatal porcine IPEC-J2 cells. However, a gap remains in our knowledge regarding the signaling mechanisms between epithelium and underlying EGC that results in restitution. To address this gap, we have performed RNAseq analyses of ischemic-injured neonatal and juvenile mucosa, and created an unbiased discovery pipeline approach to focus on significantly deficient signaling pathways in the neonate. One such pathway includes EGF, which is secreted by the EGC as proEGF, and epithelial annexin A2, which initiates cell migration by signaling internalization of β1-integrin. Our central hypothesis is that repair of ischemic-injured mucosa is reliant on postnatal migration of EGC into the lamina propria in order to signal wounded epithelium to efficiently restitute. We will test this hypothesis with two specific aims: 1) Determine age-dependent defects in migration signaling mechanisms in wound-adjacent intestinal epithelial cells. Our working hypothesis is that there is an age-dependent development of epithelial restitution in response to injury at least in part via annexin A2 signaling. 2) Determine perinatal changes in mucosal EGC network structure, density and secretome. Our working hypothesis is that there are insufficient EGCs secreting barrier-promoting factors, including proEGF, in proximity to the epithelium to stimulate restitution in neonates. To examine these specific aims, we will use our unique age-dependent porcine model of mucosal repair, a wounded porcine neonatal IPEC-J2 cell model, primary culture of porcine EGC, EGC secretome analyses via mass spectrometry, and state of the art imaging techniques for glial cells, including immunolabeling-enabled three-dimensional imaging of solvent-cleared organs (iDISCO). Using an unbiased discovery pipeline approach, we expect to be able to define signaling mechanisms that are deficient in neonatal injured intestine that can ultimately be targeted to enhance survival of patients with intestinal failure