Reflux-Induced Epithelial-Mesenchymal Transition in Benign Barrett's Esophagus

项目摘要 据估计，5.6%的成年美国人患有巴雷特食管（BE），这是导致食管癌的主要危险因素。 食管腺癌为了预防这种癌症，建议BE患者定期进行内窥镜检查。 监测发育不良，并对发育不良进行射频消融（RFA）治疗。不幸的是， 监测并没有阻止食管癌的死亡，Barrett化生在食管癌后经常复发。 射频消融监测失败和化生复发可能是由于鳞状上皮下肠化生 （SSIM），其中化生腺体位于鳞状上皮层下的固有层中的一种病症 上皮，将它们隐藏起来，使其免受内窥镜检查，并保护它们免受RFA的破坏。SSIM最初是 被认为是内镜消融术的副作用，但最近的研究表明，SSIM存在于绝大多数患者中 没有做过消融手术的患者这种高度流行的SSIM可能是 内镜监测漏诊的肿瘤和RFA后复发性化生的病灶。因此，SSIM似乎 是一个常见的和重要的条件，限制了数百万的内镜监测的疗效， 这阻碍了内窥镜试图根除BE并防止其发展为癌症。 上皮-间质转化（EMT）是上皮细胞获得间质细胞的过程， 包括细胞迁移在内的特征。在BE中，EMT可以使化生的Barrett上皮细胞迁移 进入邻近鳞状上皮下的固有层，导致SSIM。在我们的老鼠模型中， 我们通过建立食管空肠吻合术诱发反流性食管炎，我们的初步数据强烈表明， 邻近溃疡食管鳞状粘膜的空肠上皮细胞发生EMT，这有助于 柱状食管的发展，具有Barrett化生的特征，包括SSIM。 胃食管反流引起食管炎症和活性氧的产生， 可以激活缺氧诱导因子（HIF）的条件。我们的初步数据显示巴雷特的上皮细胞 暴露于酸和胆盐的细胞表现出细胞核HIF-1α和HIF-2α的强烈和持续的增加。在 在BE患者中，我们还发现，当BE患者的Barrett化生中的HIF-1α和HIF-2α水平升高时， 食管灌注有酸或胆汁盐。HIF可以通过引起细胞分泌血管生成素来促进EMT。 内皮生长因子（VEGF），其以自分泌方式结合细胞的VEGF受体，以诱导 触发EMT的VEGF信号传导。我们的初步数据表明，酸和胆汁盐诱导自分泌VEGF 信号传导和EMT特征。因此，我们假设，反流诱导的激活 Barrett上皮细胞中的HIF通过自分泌VEGF信号引起VEGF分泌，从而启动EMT 程序并导致SSIM。本研究的目的是阐明酸和胆汁 盐激活HIF引起VEGF的产生，并探索自分泌VEGF信号在EMT中的作用。 在体外Barrett细胞和反流性食管炎BE患者中，酸和胆汁盐诱导的程序。

Project Summary It has been estimated that 5.6% of adult Americans have Barrett’s esophagus (BE), a major risk factor for esophageal adenocarcinoma. To prevent this cancer, patients with BE are advised to have regular endoscopic surveillance for dysplasia, and to have that dysplasia treated with radiofrequency ablation (RFA). Unfortunately, surveillance has not prevented deaths from esophageal cancer, and Barrett’s metaplasia recurs frequently after RFA. Surveillance failures and metaplasia recurrences might be due to subsquamous intestinal metaplasia (SSIM), a condition in which metaplastic glands are located in the lamina propria under a layer of squamous epithelium that hides them from the endoscopist and shields them from destruction by RFA. SSIM initially was considered a side effect of endoscopic ablation, but recent studies show that SSIM is present in the large majority of Barrett’s patients who have not had ablation procedures. This highly prevalent SSIM might be the source of tumors missed by endoscopic surveillance, and the nidus for recurrent metaplasia after RFA. Thus, SSIM appears to be a frequent and important condition that limits the efficacy of endoscopic surveillance for the millions of patients with BE, and that thwarts endoscopic attempts to eradicate BE and prevent its progression to cancer. Epithelial-mesenchymal transition (EMT) is the process in which epithelial cells acquire mesenchymal characteristics including cell migration. In BE, EMT could enable metaplastic Barrett’s epithelial cells to migrate into the lamina propria underneath adjacent squamous epithelium, resulting in SSIM. In our rat model in which we induce reflux esophagitis by creating an esophago-jejunostomy, our preliminary data strongly suggest that jejunal epithelial cells adjacent to ulcerated esophageal squamous mucosa undergo EMT, which contributes to the development of a columnar-lined esophagus with features of Barrett’s metaplasia including SSIM. Gastroesophageal reflux causes esophageal inflammation and production of reactive oxygen species, conditions that can activate hypoxia inducible factors (HIFs). Our preliminary data show that Barrett’s epithelial cells exposed to acid and bile salts exhibit a strong and sustained increase in nuclear HIF-1α and HIF-2α. In patients with BE, we also show that HIF-1α and HIF-2α levels in their Barrett’s metaplasia rise when the esophagus is perfused with acid or bile salts. HIFs can promote EMT by causing cells to secrete vascular endothelial growth factor (VEGF), which binds the cells’ VEGF receptors in an autocrine fashion to induce VEGF signaling that triggers EMT. Our preliminary data show that acid and bile salts induce autocrine VEGF signaling and EMT features in Barrett’s cell lines. Therefore, we hypothesize that reflux-induced activation of HIFs in Barrett’s epithelial cells causes VEGF secretion with autocrine VEGF signaling, which initiates the EMT program and causes SSIM. The aims of this study are to elucidate the mechanism(s) whereby acid and bile salts activate HIFs to cause VEGF production, and to explore the role of autocrine VEGF signaling in the EMT program induced by acid and bile salts in Barrett’s cells in vitro, and in BE patients with reflux esophagitis.