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

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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.