Carbonic anhydrase IX is a critical determinant of pulmonary microvascular endothelial cell pH regulation and angiogenesis during acidosis

Abstract

Carbonic anhydrase IX (CA IX) is highly expressed in rapidly proliferating and highly glycolytic cells, where it serves to enhance acid-regulatory capacity. Pulmonary microvascular endothelial cells (PMVECs) actively utilize aerobic glycolysis and acidify media, whereas pulmonary arterial endothelial cells (PAECs) primarily rely on oxidative phosphorylation and minimally change media pH. Therefore, we hypothesized that CA IX is critical to PMVEC angiogenesis because of its important role in regulating pH. To test this hypothesis, PMVECs and PAECs were isolated from Sprague-Dawley rats. CA IX knockout PMVECs were generated using the CRISPR-Cas9 technique. During serum-stimulated growth, mild acidosis (pH 6.8) did not affect cell counts of PMVECs, but it decreased PAEC cell number. Severe acidosis (pH 6.2) decreased cell counts of PMVECs and elicited an even more pronounced reduction of PAECs. PMVECs had a higher CA IX expression compared with PAECs. CA activity was higher in PMVECs compared with PAECs, and enzyme activity was dependent on the type IX isoform. Pharmacological inhibition and genetic ablation of CA IX caused profound dysregulation of extra- and intracellular pH in PMVECs. Matrigel assays revealed impaired angiogenesis of CA IX knockout PMVECs in acidosis. Lastly, pharmacological CA IX inhibition caused profound cell death in PMVECs, whereas genetic CA IX ablation had little effect on PMVEC cell death in acidosis. Thus CA IX controls PMVEC pH necessary for angiogenesis during acidosis. CA IX may contribute to lung vascular repair during acute lung injury that is accompanied by acidosis within the microenvironment.