Pathways and pathogens affecting gastric epithelial repair in vivo

描述(申请人提供)：消化性溃疡疾病仍然是美国医疗保健系统和世界范围内的重大负担，主要由胃部病原体幽门螺杆菌的存在或摄入的治疗药物的上消化道毒性引起。我们工作的基本原理是，改进的或替代的治疗策略可以建立在理解胃防御机制的基础上，以应对这些挑战。根据初步研究，我们假设粘膜旁环境，包括细胞内和细胞外钙离子的作用，是胃表面上皮修复的中心调节因素。这项应用的目的是确定胃肠道毒性双膦酸类药物、幽门螺杆菌病原体或这些应激源的组合对胃上皮修复抑制的潜在机制。我们特别询问急性幽门螺杆菌和双膦酸类药物的发病机制是否通过它们对宿主钙信号的干扰来实现，我们证明这对修复是重要的。我们开创了光学技术的先河，可以实时产生局部损伤并持续量化修复。我们专注于体内局部病变修复的工作将外推到评估胃炎和溃疡的实验，以增强与临床观察到的胃病理的相关性。在这一应用中，我们介绍了在胃表面上皮细胞内钙动员和在胃腔粘膜旁微环境中细胞外钙动员的成像新能力。我们的第一个目标是研究钙依赖信号在胃局灶性病变修复过程中的作用。实验基于初步发现，磷脂酶C、蛋白激酶C、前列腺素以及细胞内和细胞外钙动员都是完整胃有效修复所必需的。我们将确定这些促进胃修复的钙依赖信号通路的层次和顺序，并询问GI毒性双膦酸类药物是否通过干扰这种钙信号通路来干扰胃上皮修复。我们的第二个目标是研究幽门螺杆菌的存在如何影响胃修复。实验是基于初步观察，即荧光标记的幽门螺杆菌在几分钟内专门聚集在局灶性病变的部位，并减缓该病变的修复。我们还观察到幽门螺杆菌在肉眼可见的溃疡部位选择性积聚。我们将利用化学传感基因缺陷的幽门螺杆菌突变体，并操纵邻近粘膜间隙中潜在的趋化物质的水平，来询问幽门螺杆菌是否由于细菌化学传感而在损伤部位聚集。我们将评估这种堆积对溃疡愈合和双磷酸盐胃炎进展的影响。这些结果将为理解发病机制中的早期因素提供一个独特的窗口，这些因素有可能导致预防性干预。 公共卫生相关性：胃炎和消化性溃疡疾病是美国医疗保健系统的重大负担，在很大程度上是由胃部病原体幽门螺杆菌的存在或摄入的治疗药物(如阿司匹林或用于治疗骨质疏松症的双膦酸盐药物)的毒性引起的。这个项目探索了最初暴露在幽门螺杆菌或双膦酸类物质中引起胃刺激的细胞和分子机制，以及胃对这种侮辱迅速愈合的非凡能力。

DESCRIPTION (provided by applicant): Peptic ulcer disease remains a significant burden in the US healthcare system and worldwide, caused in large part by the presence of the gastric pathogen Helicobacter pylori or the upper GI toxicity of ingested therapeutic drugs. The rationale for our work is that improved or alternative therapeutic strategies can be based on understanding the mechanisms of gastric defense rallied against such challenges. Based on preliminary studies, we hypothesize that the juxtamucosal environment, including a role for both intracellular and extracellular Ca2+, is a central regulator of gastric repair of the surface epithelium. The objective of this application is to identify the underlying mechanisms for inhibited gastric epithelial repair in response to the GI- toxic bisphosphonate drugs, the H. pylori pathogen, or a combination of these stressors. We specifically ask if the pathogenesis from acute H. pylori and bisphosphonate drugs is via their interference with host Ca2+ signaling, which we show to be important for repair. We have pioneered optical technologies that allow real- time creation of focal damage and continual quantification of repair. Our work focusing on the repair of focal lesions in vivo will be extrapolated to experiments evaluating gastritis and ulceration to enhance relevance to gastric pathologies observed clinically. In this application, we introduce the novel ability to image intracellular calcium mobilization in vivo in the gastric surface epithelium, and extracellular calcium mobilization in the juxtamucosal microenvironment of the gastric lumen. Our first aim is to examine the role of calcium-dependent signaling during the repair of focal gastric lesions. Experiments are based on preliminary findings that show phospholipase C, protein kinase C, prostaglandins, and both intracellular and extracellular calcium mobilization are required for efficient gastric repair in the intact stomach. We will define the hierarchy and sequence among these calcium-dependent signaling routes that promote gastric repair and ask if the GI-toxic bisphosphonate drugs interfere with gastric epithelial repair by interrupting this Ca2+ signaling. Our second aim examines how the presence of H. pylori affects gastric repair. Experiments are based on preliminary observations that fluorescently-labeled H. pylori accumulate specifically at the site of a focal lesion within minutes, and slow the repair of that lesion. We also observe selective accumulation of H. pylori at sites of macroscopic ulceration. We will ask if H. pylori accumulates at damage sites due to bacterial chemosensing, using H. pylori mutants defective in chemosensing genes and manipulating the level of potential chemoattractants in the juxtamucosal space. We will evaluate the impact of this accumulation on ulcer healing and on progression of bisphosphonate gastritis. The outcomes will provide a unique window into understanding early factors in pathogenesis, which have potential to lead to preventive interventions. Public Health Relevance: Gastritis and peptic ulcer disease are a significant burden in the US healthcare system, caused in large part by the presence of the stomach pathogen Helicobacter pylori or the toxicity of ingested therapeutic drugs (such as aspirin, or the bisphosphonate drugs used to treat osteoporosis). This project explores the cellular and molecular mechanisms underlying the stomach irritation caused by initial exposure to H. pylori or bisphosphonates, and the remarkable ability of the stomach to heal itself quickly in response to such insults.