Pathways and pathogens affecting gastric epithelial repair in vivo

描述（由申请人提供）：消化性溃疡疾病在美国和世界范围内仍然是一个重要的负担，在很大程度上是由胃病原体幽门螺杆菌的存在或摄入治疗药物的上消化道毒性引起的。我们工作的基本原理是，改进或替代治疗策略可以基于理解胃防御对抗这些挑战的机制。根据初步研究，我们假设粘膜旁环境，包括细胞内和细胞外Ca2+的作用，是胃表面上皮修复的中心调节因子。本应用的目的是确定胃肠道毒性双膦酸盐药物、幽门螺杆菌病原体或这些应激源的组合对抑制胃上皮修复的潜在机制。我们特别询问急性幽门螺杆菌和双膦酸盐药物的发病机制是否通过它们对宿主Ca2+信号的干扰，我们发现这对修复很重要。我们开创了光学技术，允许实时创建焦点损伤和持续的修复量化。我们的工作重点是在体内局灶性病变的修复，将外推到评估胃炎和溃疡的实验中，以增强与临床观察到的胃病理的相关性。在这个应用中，我们介绍了一种新的成像胃表面上皮细胞内钙动员的能力，以及胃腔粘膜旁微环境中细胞外钙动员的能力。我们的第一个目的是研究钙依赖信号在局灶性胃病变修复中的作用。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是完整胃有效修复所必需的。我们将定义这些促进胃修复的钙依赖性信号通路的层次和顺序，并询问gi毒性双膦酸盐药物是否通过中断这种Ca2+信号通路来干扰胃上皮修复。我们的第二个目的是研究幽门螺旋杆菌的存在如何影响胃修复。实验基于初步观察，荧光标记的幽门螺杆菌在几分钟内特异性地聚集在局灶性病变部位，并减缓病变的修复。我们还观察到幽门螺杆菌在宏观溃疡部位的选择性积累。我们将询问幽门螺杆菌是否由于细菌化学感应而在损伤部位积聚，使用化学感应基因缺陷的幽门螺杆菌突变体并操纵粘膜旁空间中潜在化学引诱剂的水平。我们将评估这种积累对溃疡愈合和双膦酸盐胃炎进展的影响。这些结果将为了解发病机制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。

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.