Carbonic anhydrase IX proteoglycan-like and intracellular domains mediate pulmonary microvascular endothelial cell repair and angiogenesis

Abstract

The lungs of patients with acute respiratory distress syndrome (ARDS) have hyperpermeable capillaries that must undergo repair in an acidic microenvironment. Pulmonary microvascular endothelial cells (PMVECs) have an acid-resistant phenotype, in part due to carbonic anhydrase IX (CA IX). CA IX also facilitates PMVEC repair by promoting aerobic glycolysis, migration, and network formation. Molecular mechanisms of how CA IX performs such a wide range of functions are unknown. CA IX is composed of four domains known as the proteoglycan-like (PG), catalytic (CA), transmembrane (TM), and intracellular (IC) domains. We hypothesized that the PG and CA domains mediate PMVEC pH homeostasis and repair, and the IC domain regulates aerobic glycolysis and PI3k/Akt signaling. The functions of each CA IX domain were investigated using PMVEC cell lines that express either a full-length CA IX protein or a CA IX protein harboring a domain deletion. We found that the PG domain promotes intracellular pH homeostasis, migration, and network formation. The CA and IC domains mediate Akt activation but negatively regulate aerobic glycolysis. The IC domain also supports migration while inhibiting network formation. Finally, we show that exposure to acidosis suppresses aerobic glycolysis and migration, even though intracellular pH is maintained in PMVECs. Thus, we report that 1) the PG and IC domains mediate PMVEC migration and network formation, 2) the CA and IC domains support PI3K/Akt signaling, and 3) acidosis impairs PMVEC metabolism and migration independent of intracellular pH homeostasis.

Keywords: acidic microenvironment; acidosis; acute respiratory distress syndrome (ARDS); aerobic glycolysis; lung capillaries.

Figure 1.

Pulmonary microvascular endothelial cell (PMVEC) mutant cell lines. CRISPR-Cas9 and a retro-lentivirus method was used to generate four PMVEC cell lines expressing either a full-length carbonic anhydrase IX (CA IX) (rWT) or a CA IX protein harboring a specific domain deletion [Δproteoglycan-like (ΔPG), Δcatalytic (ΔCA), and Δintracellular (ΔIC)] (12, 13). In addition, these cell lines were engineered to have a Tet-Off system that allows conditional knockdown of the CA IX protein in the presence of doxycycline (13, 27). Domain deletions and the doxycycline Tet-Off system were verified through Western blotting and immunocytochemistry (13). ΔIC cells did not localize the CA IX mutant protein to the cell membrane (13, 23).

Figure 2.

The catalytic (CA) and intracellular (IC) domains negatively regulate aerobic glycolysis. rWT, Δproteoglycan-like (ΔPG), ΔCA, and ΔIC cell lines were seeded at 4.0 × 10⁵ cells per well in six-well plates and grown for 2 days under bicarbonate-buffered media. Media were replaced with 7.4 or 6.4 pH bicarbonate-free HEPES-buffered media and glucose-lactate measurements were made 24 h later. A: media lactate measurements. B: media glucose measurements. ΔPG cells had reduced media glucose but no difference in media lactate. ΔCA cells showed increased lactate production and a trend toward higher glucose consumption in 7.4 pH media. ΔIC cells also had elevated lactate production and glucose consumption in 7.4 pH media. Acidosis decreased media lactate and increased media glucose. No difference was seen between cell lines under 6.4 pH media. Data represent means ± SD. One-way ANOVA and a Tukey’s post hoc test were used to compare groups. *Significant difference (P < 0.05) from 7.4 pH rWT.

Figure 3.

The proteoglycan-like (PG) domain supports intracellular pH homeostasis. rWT, ΔPG, Δcatalytic (ΔCA), and Δintracellular (ΔIC) cell lines were seeded at 4.0 × 10⁵ cells per well in six-well plates and grown for 2 days under bicarbonate-buffered media. Media were replaced with 7.4 or 6.4 pH bicarbonate-free HEPES-buffered media for 24 h before measuring intracellular pH with BCECF-AM. ΔPG cells had lower intracellular pH under 7.4 pH media, but no drop in intracellular pH was seen in rWT, ΔCA, and ΔIC cells. No difference in intracellular pH was seen between cell lines during acidosis conditions. Data represent means ± SD. One-way ANOVA and a Tukey’s post hoc test were used to compare groups. *Significant difference (P < 0.05) from 7.4 pH rWT.

Figure 4.

The proteoglycan-like (PG) and intracellular (IC) domains mediate pulmonary microvascular endothelial cell (PMVEC) migration. rWT, ΔPG, ΔCA, and ΔIC cell lines were seeded at 4.0 × 10⁵ cells per well in six-well plates and grown for 2 days under bicarbonate-buffered media. A pipette tip was used to induce a scratch wound across the cell monolayer. Media were replaced with 7.4 or 6.4 pH bicarbonate-free HEPES-buffered media, and 24 h later, wound size was measured using ImageJ software. A: images of the baseline scratch wound. B: quantification of the baseline scratch wound size. Cell lines had similar baseline scratch wound sizes. C: images of the scratch wound 24 h after the injury in 7.4 or 6.4 pH media. D: quantification of the wound size 24 h after the initial scratch in 7.4 or 6.4 pH. ΔPG and ΔIC cells had larger wound sizes than the rWT cells under 7.4 pH media. Acidosis suppressed PMVEC wound healing although no difference was detected between cell lines under 6.4 pH media. Data represent means ± SD. One-way ANOVA and a Tukey’s post hoc test were used to compare groups. *Significant difference (P < 0.05) from 7.4 pH rWT.

Figure 5.

The proteoglycan-like (PG) domain promotes whereas the intracellular (IC) domain inhibits network formation. Ninety-six-well plates were coated with 30 µL of Matrigel per well and incubated in 37°C ambient air for 1 h. rWT, ΔPG, Δcatalytic (CA), and ΔIC cell lines were then seeded at 5.0 × 10⁴ cells per well with 7.4 pH bicarbonate-free HEPES-buffered media. Angiogenic parameters were measured 24 h later using ImageJ software. A: Matrigel network images of rWT, ΔPG, ΔCA, and ΔIC cell lines. B–F: number of nodes (B), junctions (C), total segment length (D), master junctions (E), and branches (F) formed by each cell line. ΔPG cells were unable to form networks, showing fewer nodes, junctions, total segment length, master junctions, and branches. No difference in Matrigel networks was detected between ΔCA and rWT cells. ΔIC cells had increased network formation, showing more nodes, junctions, total segment length, and master junctions than rWT cells. Data represent means ± SD. One-way ANOVA and a Tukey’s post hoc test were used to compare groups. *Significant difference (P < 0.05) from rWT.

Figure 6.

The catalytic (CA) and intracellular (IC) domains mediate Akt phosphorylation in pulmonary microvascular endothelial cells (PMVECs). Cells were seeded at 1.5 × 10⁵ cells per well in a six-well plate and incubated in bicarbonate DMEM with low serum (0.1% FCS) overnight. The next morning, cells were further incubated in FCS-free bicarbonate DMEM for 2 h. After conditioning, cells were rinsed with HBSS and 7.4 pH bicarbonate DMEM with 10% FCS was added for 30 min. Whole cell lysate was then collected and subjected to immunoblotting for Phospho-Akt (Ser473), Pan-Akt, and GAPDH. Western blots were analyzed using ImageJ software. A: Western blot bands for phospho-Akt (Ser473), Pan-Akt, and GAPDH from rWT, Δproteoglycan-like (ΔPG), ΔCA, and ΔIC cell lines. B: the ratiometric measurements of phospho-Akt (Ser473) to Pan-Akt band intensities. ΔCA and ΔIC cells showed lower phospho-Akt (Ser473) compared with rWT during serum stimulation. No difference in Pan-Akt and GAPDH expression was seen between cell lines. Data represent means ± SD. One-way ANOVA and a Tukey’s post hoc test were used to compare groups. *Significant difference (P < 0.05) from rWT.

Figure 7.

The domain specific functions of carbonic anhydrase IX (CA IX) in pulmonary microvascular endothelial cell (PMVEC) integrity and repair. CA IX is critical to PMVEC repair, but the specific function of each CA IX domain is unknown. CA IX is composed of four domains known as the proteoglycan-like domain (PG), catalytic domain (CA), transmembrane domain (TM), and the intracellular domain (IC). The PG domain mediates intracellular pH homeostasis, cell migration, and network formation. The CA and IC domains promote the PI3k/Akt signaling pathway, but negatively regulate aerobic glycolysis. In addition, the IC domain inhibits network formation while supporting cell migration. Acidosis suppresses aerobic glycolysis and migration independent of intracellular pH homeostasis.