Cells and tissues sense and respond to changes in the levels of gaseous molecules in their environment through evolutionarily conserved pathways. However, the mechanisms by which non-excitable cells respond to changes in carbon dioxide (CO2) concentrations are poorly understood. High partial pressure of CO2 in blood (hypercapnia) leading to an increase in the CO2 tissue level is often a consequence of diseases that impair gas exchange, including the acute respiratory distress syndrome (ARDS) and chronic obstructive pulmonary disease (COPD). It has been shown that high CO2 levels (independent of pH or hypoxia) activate specific signaling pathways that impair alveolar epithelial function by inhibiting migration and proliferation. During the repair process, pathways that promote the repopulation of lost cells in the lung epithelium can be dysregulated and undergo an aberrant
remodeling. We have demonstrated that in patients with bronchopleural fistulae, hypercapnia is associated with prolonged air leaks and delayed lung repair. We  hypothesized that hypercapnia limits the proliferative capacity of AT2 cells and alters the normal AT2/AT1 cell migration and differentiation programs, thus hindering repair after lung injury. C57BL/6 WT mice were exposed to room-air (RA) or hypercapnia (10% CO2) (HC) for 21 days. In isolated AT2 using fluorescenceactivated cell sorting (FACS), RNA sequencing analysis revealed that RA and HC AT2 cells cluster differently, showing heterogeneity in the transcriptome as well as the presence of differentiated processes. Among the differentially expressed genes that become upregulated in AT2 cells after CO2 exposure, was the transcription factor homeodomain-only protein homeobox (HOPX), which is critically involved in the regulation of cell proliferation and differentiation. HOPX regulates alveolar maturation by suppressing surfactant protein production in AT2 cells. Supporting this notion when lung peripheral tissue from mice exposed to HC was assessed we observed a decreased pro-SP-C and NKX2.1 levels which are markers of AT2 cells and increased levels of the AT1 cells markers HOPX, podoplanin (PODO), and aquaporin 5 (AQP5). Cultured 3-dimensional organoids formed in vitro during exposure to normocapnia (NC) for 21 days are typically organized with AT2 at the periphery and AT1 at the center. Analysis of HC 21 days organoids showed size decrease and an increase i n cells that express both AT2 and AT1 markers. These results were also evident when the lung ultrastructure of mice exposed to HC was analyzed by transmission electron microscopy (TEM); AT2 have abnormal lamellar bodies (LB) and significant changes in mitochondrial architecture. HC caused a mild
increase in lung epithelial permeability without leading to significant lung injury or mortality. Exposure to HC impairs AT2 cell proliferation and migration, promoting the differentiation of AT2 cells into AT1 cells through premature activation of HOPX. The HC-induced alveolar epithelial dysfunction observed impairs lung repair. Understanding the molecular signaling pathways involved will be of key importance in the identification of new approaches to improve outcomes in patients with ARDS and COPD.