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

碳酸酐酶 IX 作为新型 CO2/HCO3- 传感器并保护肺内皮屏障免受酸中毒的影响

• 批准号: 10678442
• 负责人: Reece P Stevens
• 金额: $ 3.17万
• 依托单位: UNIVERSITY OF SOUTH ALABAMA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-01 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10678442
• 关键词: AKT Signaling Pathway; Acidosis; Acids; Acute Respiratory Distress Syndrome; Bacterial Infections; Bicarbonates; Biology; Blood; Blood capillaries; Blood gas; Buffers; Carbon Dioxide; Catalytic Domain; Cell Survival; Cells; Characteristics; Communication; Disease; Doctor of Philosophy; Endothelial Cells; Endothelium; Environment; Enzymes; Extracellular Space; Family; Fellowship; Funding; Gases; Genetic Vectors; Glycolysis; Human; Hypoxia; In Vitro; Individual National Research Service Award; Infection; Injury; Learning; Lung; Manuscripts; Mediating; Membrane; Mentors; Metabolic acidosis; Microcirculation; Molecular; Molecular Biology; Molecular Conformation; PIK3CG gene; Parents; Patients; Phenotype; Physiological; Pneumonia; Production; Protein Analysis; Protein Isoforms; Protein Tyrosine Phosphatase; Proteoglycan; Protons; Recording of previous events; Research; Resistance; Role; Severities; Signal Transduction; System; Tail; Technology; Testing; Tissues; Training; Training Programs; Ventilator; Work; acid stress; adeno-associated viral vector; angiogenesis; base; carbonate dehydratase; career; doctoral student; experience; extracellular; in vivo; injured; kidney cell; land use; lung microvascular endothelial cells; new therapeutic target; novel; pH Homeostasis; pneumonia model; pre-doctoral; preservation; protein purification; receptor; repaired; response; sensor; skills; targeted treatment; therapy development

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.

项目概要/摘要 Ruth L. Kirschstein 国家研究服务奖个人博士前奖学金（家长 F31） 提案描述了一个为期 2.5 年的培训计划，旨在为博士生里斯·史蒂文斯 (Reece Stevens) 的研究做好准备—— 主要导师 Ji Young, Lee 博士是一名肺病专家/重症监护医师。 拥有肺内皮细胞专业知识的 Ron Balczon 博士也将担任辅助导师，提供帮助。 分子生物学方面的专业知识，并具有丰富的指导研究和培训经验。 该计划将在跨学科且资金充足的肺生物学中心进行，该中心拥有大量的 在整个过程中开展博士前学生在肺生物学领域的职业生涯的历史。 根据建议，候选人将学习分子和生理学方法来研究肺内皮。 具体来说，候选人将培养以下方面的高级技能：1) 内皮蛋白纯化和分析，2) 用于肺内皮细胞基因编辑的着陆垫技术和腺相关病毒载体，以及3) 拟议的研究计划旨在确定是否存在转化体内酸中毒和肺炎模型。 碳酸酐酶 IX (CA IX) 作为一种新型 CO2/HCO3- 传感器并维持肺内皮屏障 了解酸中毒期间的完整性。了解肺内皮如何感知血气并做出反应。 精神错乱与肺炎和人类急性呼吸窘迫综合征（ARDS）高度相关。 细菌感染的肺部组织 pH 值呈高酸性，范围为 6.80 至 6.15，并且存在代谢性酸中毒 被确定为严重 ARDS 亚表型的主要特征。 细胞 (PMVEC) 通过表达碳酸酐酶 IX (CA IX) 来适应 ARDS 肺的酸性环境，碳酸酐酶 IX 是一种 独特的跨膜亚型对于 PMVEC 的耐酸表型至关重要。 最近，我们发现 CA IX 在酸应激下的细胞内 pH 稳态、修复和血管生成。 胞外催化 (CA) 结构域和胞内 (IC) 结构域介导 PI3K/Akt 信号传导的激活 途径，内皮细胞存活和修复的主要调节剂。这些结果表明催化活性。 CA IX 可以调节 IC 结构域的信号传导功能，其结构也与 IC 结构域同源。 CO2/HCO3-传感器受体蛋白酪氨酸磷酸酶-𝛾（RPTP𝛾）因此，我们的目的是检验这一假设。 CA IX 作为一种新型 CO2/HCO3- 传感器，协调细胞对酸中毒的反应并保护 肺内皮屏障免受酸中毒和感染的影响作为我项目的一部分，我将使用着陆垫。 技术和 AAV 载体在体外和体内对肺内皮细胞进行基因编辑，形成整合 系统研究 CA IX 信号传导对肺内皮屏障完整性的作用。 CA IX 的机制将导致针对肺微循环患者的治疗方法的开发 肺炎和代谢性酸中毒。