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

描述(申请人提供)：Barrett‘s食道(BE)是用肠化的柱状上皮替代正常的鳞状食道上皮。它是对慢性酸和胆汁反流的反应，是食管腺癌(EAC)发生的重要危险因素。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，BE发病机制仍然知之甚少，部分原因是缺乏实验动物模型。为BE开发创新的、基于遗传的和生理上相关的小鼠模型是我的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的限速酶。在大鼠胆汁反流模型中，COX-2的表达是由酸反流诱导的，抑制COX-2可以减少BE和癌症的进展。众所周知，COX-2还会增加细胞内的氧化应激并损伤DNA。然而，环氧合酶-2在BE和EAC的发病机制中的作用还没有得到检验。我们使用了一种新的三维体外细胞培养系统来模拟BE的发病机制。当我们在正常的人食道角质形成细胞中表达COX-2时，我们观察到了肠粘蛋白充满的囊变的发展。这表明，COX-2的表达足以诱导角质形成细胞发生改变，这种细胞具有与BE细胞一致的粘蛋白分泌特征。因此，我们假设COX-2在小鼠食道中的表达导致慢性食管炎，这种慢性食管炎通过引发氧化应激、DNA损伤以及化生和异型增生的发展而建立GERD食管炎模型。提出这项研究的理由是，虽然临床观察数据表明COX-2在Barrett‘s食道的发病机制中发挥了作用，但这一点尚未在动物模型中得到证实。一旦它被确立，就可以更多地考虑药物方法来预防BE的发生并限制向癌症的进展。在强大的初步数据的指导下，这一假说将通过以下相互关联的特定目标进行检验：1)K14-COX2小鼠食道中的慢性COX-2活性是否会导致炎症、氧化应激、DNA损伤和采用改变的分化程序？2)抗氧化反应减弱或DNA修复缺陷是否与食道COX-2的表达协同作用，加速DNA损伤、化生和异型增生的发生？ 摘要BE是一种越来越常见的癌症前期疾病，也是美国新出现的健康问题。我们的研究意义重大，因为他们从机制上探索了COX2对BE发病机制的贡献，我们预计我们的方法将产生改进的BE小鼠模型。此外，作为职业生涯中期研究人员奖，这项研究将为PI提供一个突出的重点：1)提高他在小鼠病理生物学研究和综合表型方面的技能；2)作为这些领域初级研究人员的指导基础；以及3)在小鼠病理生物学方面进行最先进的生物医学研究。

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.