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.

项目摘要/摘要 这位露丝·L Kirschstein国家研究服务奖个人博士前奖学金（父母F31） 提案描述了一个为期2.5年的培训计划，准备里斯史蒂文斯，博士候选人，为研究- 在肺生物学领域的密集职业生涯。主要导师Ji Young Lee博士是一名肺病学家/重症监护医师 在肺内皮方面有专长罗恩Balczon也将作为一个支持共同导师，提供 在分子生物学方面的专业知识，并有丰富的指导经验。拟议的研究和培训 计划将在跨学科和资金充足的肺部生物学中心进行，该中心拥有相当大的 在肺生物学领域开展博士前学生职业生涯的历史。在整个过程中， 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，候选人将在1）内皮蛋白纯化和分析，2） 着陆台技术和腺相关病毒载体，用于肺内皮细胞的基因编辑，以及3） 转化体内酸中毒和肺炎模型。该研究计划旨在确定是否 碳酸酐酶IX（CA IX）作为一种新型的CO2/HCO 3-传感器并维持肺内皮屏障 在酸中毒期间保持完整性。了解肺内皮细胞如何感知和响应血气 这种紊乱与肺炎和急性呼吸窘迫综合征（ARDS）高度相关。人类 患有细菌感染的肺具有高度酸性的组织pH值，范围为6.80至6.15， 被鉴定为严重ARDS亚表型的主要特征。肺微血管内皮 细胞（PMVEC）通过表达碳酸酐酶IX（CA IX）来适应ARDS肺的酸性环境， 独特的跨膜同种型。CA IX对于PMVEC的耐酸性表型是必需的， 细胞内pH稳态、修复和酸应激下的血管生成。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PI 3 K/Akt信号传导的激活 信号通路是内皮细胞存活和修复的主要调节因子。这些结果表明， 的CA IX可以调节IC域的信令功能。CA IX在结构上也与 CO2/HCO 3感受器受体蛋白酪氨酸磷酸酶-β（RPTPβ）。因此，我们的目标是测试假设 CA IX作为一种新型的CO2/HCO 3传感器，协调细胞对酸中毒的反应，保护细胞免受CO2/HCO 3的伤害。 肺血管内皮屏障的损害。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， 系统来研究CA IX信号传导对肺内皮屏障完整性的作用。解释信号 CA IX的机制将导致开发针对肺微循环的治疗方法， 肺炎和代谢性酸中毒。