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.

Extrinsic acidosis suppresses glycolysis and migration while increasing network formation in pulmonary microvascular endothelial cells

“…Generation of CA IX-depleted cells. CA IX knockout PMVECs were generated using CRISPR-Cas9 gene editing technology as previously described (5,27).…”

“…For extrinsic acidosis, media pH was decreased by titrating with 1 N HCl to achieve a pH of 6.8, 6.6, or 6.4. For intrinsic acidosis, cells were either treated with 150 M SLC-0111 (specific CA IX and CA XII inhibitor, MedKoo Biosciences, Morrisville, NC and gift from Dr. Supuran, Italy), which decreases both intra-and extracellular pH (27), or 1% hypoxia. One day after treatments, media pH was measured using a pH meter (Denver Instrument Company, Bohemia, NY).…”

“…After two rinses with HBSS, intracellular pH was assessed by ratiometric measurements using a SpectraMax iD5 Multi-Mode Microplate Reader (Molecular Devices, San Jose, CA) with dual excitation (440 and 490 nm) and single emission (535 nm) wavelengths. For calibration, 10 M nigericin sodium (Adipogen, San Diego, CA) with pH 6.0 to 8.0 solutions containing 120 mM KCl, 2 mM CaCl 2•2H2O, 1 mM MgCl2, and 10 mM glucose in 20 mM HEPES (or MES) was used as previously described (27).…”

“…One day after treatments with different conditions, cells were collected and subjected to immunoblot analysis as previously described (31). Primary antibodies for CA IX (M75, 1:700 dilution) (27) and GAPDH (1:1,000 dilution) were obtained from BioScience Slovakia (Bratislava, Slovakia) and Cell Signaling (Danvers, MA), respectively, and antibodies for Na ϩ /H ϩ exchanger-1 (NHE-1, 1:200 dilution) (3), hexokinase-1 (HXK I, 1:200 dilution) (42), pyruvate kinase M-type (PKM, 1:500 dilution) (44), pyruvate dehydrogenase E1␣ subunit (PDH-E1␣, 1:300 dilution) (45), and N-cadherin (1:200 dilution) (34) were obtained from Santa Cruz Biotechnology (Dallas, TX).…”

Carbonic Anhydrase Inhibitors Targeting Metabolism and Tumor Microenvironment

“…Other groups also used SLC-0111 in various biomedical studies in which selective inhibition of some CA isoforms was needed. These include the effects on prostate cancer cells of SLC-0111 alone or in combination with daunorubicin [ 184 ], radiobiological effects of CA IX inhibition in human breast cancer cells [ 185 ], microvascular endothelial cell pH regulation [ 186 ], glycolysis and migration suppression in pulmonary microvascular endothelial cells [ 187 ], and the involvement of CA isoforms in mitochondrial biogenesis and in the control of lactate production in human Sertoli cells [ 188 ]. All these studies confirm the usefulness of this clinical candidate in tumors and other biomedical conditions.…”