Fig. 5.

Carbonic anhydrase IX (CA IX) activity controls pulmonary microvascular endothelial cell (PMVEC) angiogenesis. A: wild-type and CA IX knockout (KO) PMVECs were seeded on Matrigel-coated 96-well plates at 4.0 × 10⁴ cells/well in pH 6.2 media. Images were obtained 24 h after seeding at ×10 magnification. Representative images are shown for each group. Networks were quantified by ImageJ software. Mean values of wild-type PMVEC network quantification in pH 7.4 media from our previous study (34) are depicted with dashed lines for reference. Compared with the reference data, acidic media decreased network formation in PMVECs and more so in CA IX KO. B–E: PMVECs and pulmonary arterial endothelial cells (PAECs) were seeded at 5.0 × 10⁵ cells/well on 6-well plates on bicarbonate-buffered media at pH 7.4. The next day, cells were treated with DMSO (0.15%), acetazolamide (AZ) (150 µM), or SLC-0111 (150 µM). 3 days after the inhibitor treatment, images were obtained, cells were counted, and lactate dehydrogenase (LDH) release and extracellular pH were measured. SLC-0111 causes significant PMVEC (B) cytotoxicity, whereas it has minimal effect on PAECs. SLC-0111 decreased cell counts (C), increased LDH release (D), and decreased extracellular pH in PMVECs (E), whereas it had no effect on PAECs. Data represent means ± SD. 5 or more separate experiments were performed. Unpaired 2-tailed t-test was used to compare wild-type vs. CA IX KO. 1-way ANOVA was used to compare DMSO vs. AZ vs. SLC-0111 groups. ^Significant difference (P < 0.05) from wild-type PMVECs. *Significant difference (P < 0.05) from PMVEC DMSO group.