Mouse models for esophageal Cox-2 oxidative stress and DNA damage

描述（由申请人提供）：Barrett食管（BE）是用无肠化柱状上皮代替正常的鳞状食管上皮。它是对慢性酸和胆汁反流的反应，是发展为食管腺癌（EAC）的重要危险因素。BE被认为是对慢性组织损伤和促炎前列腺素和细胞因子释放的适应性反应。然而，由于实验动物模型的缺乏，对BE发病机制的理解仍然很差。开发创新的、基于基因的和生理相关的BE小鼠模型是我实验室的一个重要的长期目标。Cox-1和Cox-2是前列腺素生物合成中的限速酶。在大鼠胆汁反流模型中，酸反流诱导食管中Cox-2表达，抑制Cox-2可减少BE和癌症的进展。Cox-2还会增加细胞内氧化应激和损伤DNA。然而，Cox-2在BE和EAC发病机制中的作用尚未得到证实。我们利用一种新的3D体外细胞培养系统来模拟BE的发病机制。当我们在正常人食管角化细胞中表达Cox-2时，我们观察到肠粘液囊肿的发展。这表明Cox-2的表达足以诱导角质形成细胞的细胞谱系发生改变，这种细胞谱系具有与BE细胞一致的粘液分泌特征。因此，我们假设Cox-2在小鼠食管中的表达导致慢性食管炎，通过引发氧化应激、DNA损伤以及化生和不典型增生的发展来模拟胃食管反流性食管炎。提出这项研究的基本原理是，虽然临床观察数据表明Cox-2在Barrett食管发病机制中的作用，但这尚未在动物模型中得到证实。一旦确定，可以更多地考虑药理学方法来预防be的发作和限制癌症的进展。在强有力的初步数据的指导下，这一假设将通过以下相互关联的特定目的来验证：1)K14-Cox2小鼠食道中慢性Cox-2活性是否导致炎症、氧化应激、DNA损伤和分化程序改变？2)抗氧化反应减弱或DNA修复缺陷是否与食管Cox-2表达协同加速DNA损伤、化生和发育不良的发生？

DESCRIPTION (provided by applicant): Barrett's esophagus (BE) is the replacement of the normal squamous esophageal epithelium with an intestinalized columnar epithelium. It occurs in response to chronic acid and bile reflux and is an important risk factor for the development of esophageal adenocarcinoma (EAC). BE is thought to be an adaptive response to chronic tissue injury and the release of pro-inflammatory prostaglandins and cytokines. However, the mechanisms underpinning BE pathogenesis remain poorly understood in part due to the paucity of experimental animal models. The development of innovative, genetically based and physiologically relevant mouse models for BE is an important long-term objective of my lab. Cox-1 and Cox-2 are the rate-limiting enzymes in prostaglandin biosynthesis. Cox-2 expression is induced in the esophagus by acid reflux, and the inhibition of Cox-2 reduces the progression to BE and cancer in a rat bile reflux model. Cox-2 is also known to increase intracellular oxidative stress and damage DNA. Nevertheless, a role for Cox-2 in the pathogenesis of BE and EAC has not been tested. We have utilized a novel 3D in vitro cell culture system to model BE pathogenesis. When we express Cox-2 in normal human esophageal keratinocytes we observe the development of intestinal mucin-filled cysts. This suggests that Cox-2 expression is sufficient to induce an altered cell lineage from keratinocytes, one that has mucin-secretory features consistent with BE cells. We therefore hypothesize that Cox-2 expression in the murine esophagus results in a chronic esophagitis that models GERD esophagitis by provoking oxidative stress, DNA damage, and the development of metaplasia and dysplasia. The rationale for the proposed research is that while a role for Cox-2 in the pathogenesis of Barrett's esophagus is suggested by clinical observational data, this has not been proven in animal models. Once it is established, greater consideration can be given to pharmacological approaches to prevent the onset of BE and limit progression to cancer. Guided by strong preliminary data, this hypothesis will be tested by the following inter-related Specific Aims: 1) Does chronic Cox-2 activity in the esophagus of K14-Cox2 mice result in inflammation, oxidative stress, DNA damage, and the adoption of an altered differentiation program? 2) Can a diminished antioxidant response or defective DNA repair synergize with esophageal Cox-2 expression to accelerate the onset of DNA damage, metaplasia, and dysplasia? Summary BE is an increasingly common precancerous condition and an emerging U.S. health problem. Our studies are significant because they mechanistically explore the contributions of Cox2 to BE pathogenesis, and we anticipate our approaches will yield improved mouse models for BE. Additionally, as a Midcareer Investigator Award, this study will provide an outstanding focus for the PI to: 1) advance his skills in mouse pathobiology research and comprehensive phenotyping; 2) serve as a basis for mentoring of junior investigators in these areas; and 3) Conduct state-of-the-art biomedical research in mouse pathobiology.