Carbonic Anhydrase IX Acts as a Novel CO2/HCO3- Sensor and Protects the Pulmonary Endothelial Barrier from Acidosis

项目摘要/摘要 这是Ruth L.Kirschstein国家研究服务奖个人博士后奖学金(家长F31) 该提案描述了一项为期2.5年的培训计划，为博士候选人里斯·史蒂文斯的一项研究做准备。 在肺部生物学领域有丰富的职业生涯。主要导师李智英博士是一名肺科医生/重症医生。 在肺内皮细胞方面有专业知识。Ron Balczon博士还将担任支持联合导师，提供 具有分子生物学方面的专业知识，并拥有丰富的指导经验。拟议的研究和培训 计划将在跨学科和资金充足的肺生物学中心进行，该中心有相当大的 在肺部生物学领域开始博士后生涯的历史。在整个过程中 提议，候选人将学习分子和生理学方法来研究肺内皮细胞。 具体地说，候选人将发展1)内皮蛋白纯化和分析方面的高级技能，2) 用于肺内皮细胞基因编辑的着陆板技术和腺相关病毒载体，以及3) 翻译的体内酸中毒和肺炎模型。拟议的研究计划旨在确定 碳酸酐酶IX(CA IX)作为一种新型的CO2/HCO3-感受器维持肺内皮细胞屏障 酸中毒期间的完整性。了解肺内皮细胞对血气的感觉和反应 精神错乱与肺炎和急性呼吸窘迫综合征(ARDS)高度相关。人类 有细菌感染的肺部组织有高度酸性的组织，范围从6.80到6.15，以及代谢性酸中毒 被确认为严重ARDS亚型的主要特征。肺微血管内皮细胞 肺微血管内皮细胞(PMVECs)通过表达碳酸氢酶IX(CAIX)，a，a来适应ARDS肺的酸性环境 独特的跨膜异构体。CA IX对PMVECs的耐酸表型是必不可少的，促进 酸胁迫下细胞内pH的动态平衡、修复和血管生成。最近，我们发现CA IX的 细胞外催化(CA)结构域和细胞内(IC)结构域介导PI3K/Akt信号的激活 通路，内皮细胞存活和修复的主要调节器。这些结果表明，该催化剂的活性 可能调节IC域的信号转导功能。CA IX在结构上也与 二氧化碳/碳酸氢受体酪氨酸磷酸酶-𝛾(rptp-𝛾)。因此，我们的目标是检验这一假设 CA IX作为一种新型的CO2/HCO3传感器，协调细胞对酸中毒的反应，保护 防止酸中毒和感染的肺内皮细胞屏障。作为我项目的一部分，我将使用着陆台 技术和AAV载体在体外和体内对肺内皮细胞进行基因编辑，创造整合的 研究CA IX信号在肺内皮屏障完整性中的作用的系统。阐明信号转导 CA IX的作用机制将导致针对肺微循环的治疗方法的发展 肺炎和代谢性酸中毒。

PROJECT SUMMARY/ABSTRACT This Ruth L. Kirschstein National Research Service Award Individual Predoctoral fellowship (Parent F31) proposal describes a 2.5-year training program to prepare Reece Stevens, a PhD candidate, for a research- intensive career in the field of lung biology. The primary mentor, Dr. Ji Young, Lee is a pulmonologist/intensivist with expertise on the pulmonary endothelium. Dr. Ron Balczon will also act as a supporting co-mentor, providing expertise in molecular biology and has extensive mentoring experience. The proposed research and training plan will be carried out at the interdisciplinary and well-funded Center for Lung Biology which has a considerable history for launching the careers of predoctoral students in the field of lung biology. Throughout the course of the proposal, the candidate will learn molecular and physiological approaches to study the pulmonary endothelium. Specifically, the candidate will develop advanced skills in 1) endothelial protein purification and analysis, 2) landing pad technology and adeno-associated virus vectors for genetic editing of lung endothelial cells, and 3) translational in vivo acidosis and pneumonia models. The proposed research plan aims to determine whether carbonic anhydrase IX (CA IX) acts as a novel CO2/HCO3- sensor and maintains pulmonary endothelial barrier integrity during acidosis. Understanding how the pulmonary endothelium senses and responds to blood gas derangements is highly relevant to pneumonia and the Acute Respiratory Distress Syndrome (ARDS). Human lungs with bacterial infections have highly acidic tissue pH that ranges from 6.80 to 6.15, and metabolic acidosis was identified as a major characteristic of a severe ARDS subphenotype. Pulmonary microvascular endothelial cells (PMVECs) adapt to the acidic environment of ARDS lungs by expressing carbonic anhydrase IX (CA IX), a unique transmembrane isoform. CA IX is essential for the acid resistant phenotype of PMVECs, facilitating intracellular pH homeostasis, repair, and angiogenesis under acid stress. Recently, we found that CA IX’s extracellular catalytic (CA) domain and intracellular (IC) domain mediate activation of the PI3K/Akt signaling pathway, a major modulator of endothelial cell survival and repair. These results suggest that the catalytic activity of CA IX may regulate the signaling function of the IC domain. CA IX is also structurally homologous to the CO2/HCO3- sensor receptor protein tyrosine phosphatases-𝛾 (RPTP𝛾). Therefore, we aim to test the hypothesis that CA IX acts as a novel CO2/HCO3- sensor, coordinating the cellular response to acidosis and protecting the pulmonary endothelial barrier from acidosis and infection. As part of my project, I will use the landing pad technology and AAV vectors to genetically edit lung endothelial cells in vitro and in vivo, creating integrative systems to study the role of CA IX signaling on pulmonary endothelial barrier integrity. Elucidating the signaling mechanism of CA IX will lead to the development of therapies targeting the lung microcirculation in patients with pneumonia and metabolic acidosis.