Carbonic anhydrase IX and pulmonary endothelial cell acidosis during infection

项目总结 这份指导临床科学家研究职业发展奖(K08)的提案描述了一个为期五年的 培训计划，以支持医学/博士肺病专家/强化医生李智英的密集研究 独立的医生-科学家职业生涯。主要导师特洛伊·史蒂文斯博士，肺科领域的世界领先者 生物学在培训初级教师方面的卓越记录，以及由以下成员组成的咨询委员会 具有不同专业知识的资深研究人员将提供科学反馈和职业指导 在整个颁奖期间。拟议的工作将在高度跨学科和资金充足的 肺生物学中心，南阿拉巴马大学的优势所在。这位候选人是最近任命的 作为生理学、细胞生物学和内科学助理教授的终身教研员 利用75%的受保护研究时间进行跟踪。25%的时间被分配给该部门的临床活动 在肺部和重症监护医学方面，她正在促进转化性研究。职业生涯发展 计划列出了授课课程、科学会议和职业发展机会，这将有助于 候选人获得核心知识、科学沟通和学术领导技能，并发展 R01基金。拟议的研究计划侧重于提高我们对酸中毒影响的了解 肺炎，并为诊断和治疗翻译奠定了概念基础。酸中毒很常见 在重症肺炎患者中，并与高死亡率有关。老年人酸中毒的病理生理学研究 人们对肺炎知之甚少，目前的治疗方法未能改善主要结果。我们的研究表明 肺微血管内皮细胞(PMVECs)利用碳酸酐酶IX(CA IX)亚型 调节pH值、新陈代谢和迁移。我们还证明了铜绿假单胞菌感染 PMVECs诱导细胞毒性淀粉样蛋白的释放，破坏肺泡毛细血管膜。这些 细胞毒性淀粉样蛋白诱导PMVECs释放可溶性CA IX，从而影响其修复能力。 在这些初步研究的基础上，我们检验了铜绿假单胞菌感染诱导细胞毒性淀粉样蛋白的假设。 产生导致PMVECs中可溶性CA IX脱落，增加肺损伤。特定的目标考验着 假设：1)CA IX在PMVECs和肺组织的酸性调节、代谢和迁移中起关键作用 内皮屏障的完整性；以及，2)铜绿假单胞菌感染引起细胞毒性淀粉样蛋白的产生，导致CA IX PMVECs脱落，增加肺损伤。在体外，我们将使用基因方法和内皮细胞 功能分析评估酸中毒的影响和特定的CA IX功能结构域在 生理和感染条件。在体内和体外，我们将使用酸中毒、肺炎和离体肺 灌流小鼠模型翻译体外研究结果。这项研究的成功完成将提供新的见解 探讨肺炎酸中毒的机制，并帮助确定CA IX和细胞毒性淀粉样蛋白 肺炎的生物标志物和治疗靶点。

PROJECT SUMMARY This Mentored Clinical Scientist Research Career Development Award (K08) proposal describes a five-year training program to support Ji Young Lee, an MD/PhD pulmonologist/intensivist, for her research-intensive independent physician-scientist career. The primary mentor, Dr. Troy Stevens, a world leader in the field of lung biology with a track record of excellence in training junior faculty, and the advisory committee consisting of accomplished researchers with diverse expertise, will provide scientific feedback and career guidance throughout the award period. The proposed work will be carried out at the highly interdisciplinary and well-funded Center for Lung Biology, a strength of the University of South Alabama. The candidate was recently appointed as assistant professor of Physiology and Cell Biology and Internal Medicine on the tenure-accruing investigator track with 75% of protected research time. Twenty five percent time is allocated for clinical activities in the division of pulmonary and critical care medicine, where she is facilitating translational research. The career development plan lays out didactic coursework, scientific meetings and professional development opportunities that will help the candidate obtain core knowledge and scientific communication and academic leadership skills, and develop R01 funding. The proposed research plan focuses on improving our understanding of the effects of acidosis on pneumonia, and establishing the conceptual basis for diagnostic and therapeutic translation. Acidosis is common in critically ill pneumonia patients, and is associated with high mortality. The pathophysiology of acidosis in pneumonia is poorly understood, and current therapies fail to improve major outcomes. Our studies have shown that pulmonary microvascular endothelial cells (PMVECs) utilize the carbonic anhydrase IX (CA IX) isoform to regulate pH, metabolism and migration. We also demonstrated that Pseudomonas aeruginosa infection of PMVECs induces release of cytotoxic amyloid proteins, which disrupts the alveolar capillary membrane. These cytotoxic amyloids induce soluble CA IX shedding from PMVECs which compromises their repair potential. Based on these preliminary studies, we test the hypothesis that P. aeruginosa infection induces cytotoxic amyloid production that leads to shedding of soluble CA IX in PMVECs, increasing lung injury. Specific aims test the hypotheses that: 1) CA IX is critical to the acid regulation, metabolism and migration of PMVECs and pulmonary endothelial barrier integrity; and, 2) P. aeruginosa infection elicits cytotoxic amyloid production, causing CA IX shedding in PMVECs, which increases lung injury. In vitro, we will use genetic approaches and endothelial cell functional assays to evaluate the effects of acidosis and the role of specific CA IX functional domains during physiologic and infectious conditions. In vivo and ex vivo, we will use acidosis, pneumonia and isolated lung perfusion mouse models to translate in vitro findings. Successful completion of this study will provide new insights into the mechanisms underlying acidosis in pneumonia and help identify CA IX and cytotoxic amyloids as biomarkers and therapeutic targets for pneumonia.