Hyperoxia downregulates angiotensin-converting enzyme-2 in human fetal lung fibroblasts

Abstract

Background: Angiotensin (ANG) II is involved in experimental hyperoxia-induced lung fibrosis. Angiotensin-converting enzyme-2 (ACE-2) degrades ANG II and is thus protective, but is downregulated in adult human and experimental lung fibrosis. Hyperoxia is a known cause of chronic fibrotic lung disease in neonates, but the role of ACE-2 in neonatal lung fibrosis is unknown. We hypothesized that ACE-2 in human fetal lung cells might be downregulated by hyperoxic gas.

Methods: Fetal human lung fibroblast IMR90 cells were exposed to hyperoxic (95% O2/5% CO2) or normoxic (21% O2/5% CO2) gas in vitro. Cells and culture media were recovered separately for assays of ACE-2 enzymatic activity, mRNA, and immunoreactive protein.

Results: Hyperoxia decreased ACE-2 immunoreactive protein and enzyme activity in IMR90 cells (both P < 0.01), but did not change ACE-2 mRNA. ACE-2 protein was increased in the cell supernatant, suggesting protease-mediated ectodomain shedding. TAPI-2, an inhibitor of TNF-α-converting enzyme (TACE/ADAM17), prevented both the decrease in cellular ACE-2 and the increase in soluble ACE-2 (both P < 0.05).

Conclusion: These data show that ACE-2 is expressed in fetal human lung fibroblasts but is significantly decreased by hyperoxic gas. They also suggest that hyperoxia decreases ACE-2 through a shedding mechanism mediated by ADAM17/TACE.

Figure 1

Caspase 9 in fetal lung fibroblasts exposed to hyperoxic or normoxic gas. IMR 90 cells in confluent culture were exposed for 72 h to 95% oxygen with 5% CO₂. Control cells were incubated in 21% O₂ with 5% CO₂ for 72 h. At the end of 72 h, cells were either harvested or incubated for an additional “recovery” phase in serum free media for 24 h. Western blotting for caspase 9 was then performed as described in Materials and Methods. Despite the slight decrease in band intensity in hyperoxia-treated cells, quantitation by densitometry demonstrated no statistically significant difference in the caspase 9/β-actin ratio (not shown). PowerPoint slide

Figure 2

Hyperoxia with normoxic recovery downregulates angiotensin-converting enzyme-2 (ACE-2) in human fetal lung fibroblasts. IMR90 cells were exposed to hyperoxic or normoxic gas as described in Figure 1. (a) Western blotting for ACE-2 protein was then performed and normalized to β-actin. (b) There was no change in ACE-2 without recovery but with recovery hyperoxia caused a significant decrease in ACE-2 immunoreactive protein (74.6%, *P < 0.01 vs. 21% O₂ by Student’s t-test). PowerPoint slide

Figure 3

Hyperoxia with normoxic recovery reduces angiotensin-converting enzyme-2 (ACE-2) enzymatic activity but not ACE-2 mRNA. IMR90 cells were exposed to hyperoxic or normoxic gas as described in Figure 1 (with recovery only), but cells were harvested for assay of ACE-2 enzyme activity (11) with and without inclusion of the ACE-2 competitive inhibitor DX600 at 1 µmol/l in the enzyme assay vessel. (a) ACE-2 enzymatic activity showed a statistically significant reduction by hyperoxia (with recovery) and complete blockage by DX₆₀₀ (*P < 0.01 vs. 21% O₂ by ANOVA and Dunnett’s test). The (b) ACE-2 mRNA quantification by qRTPCR showed no significant change. See Methods for details. PowerPoint slide

Figure 4

Hyperoxia with normoxic recovery reduces cellular angiotensin-converting enzyme-2 (ACE-2) by a TAPI-2-sensitive mechanism. IMR90 cells were exposed to hyperoxic or normoxic gas as described in Figure 1 (with recovery), but in the presence or absence of the ADAM17/TACE inhibitor TAPI-2. Cell monolayers were then harvested for ACE-2 western blotting (upper panel). Densitometry (lower panel) demonstrated a significant reduction in cell monolayer-associated ACE-2 in hyperoxia treated cells compared to the control (*P < 0.05 vs. CTL by ANOVA and Tukey’s multiple comparison test). Addition of TAPI-2 prevented the decrease. PowerPoint slide

Figure 5

Hyperoxia with normoxic recovery increases soluble angiotensin-converting enzyme-2 (ACE-2) by A TAPI-2-sensitive mechanism. IMR90 cells were exposed to hyperoxic or normoxic gas as described in Figure 1 (with recovery), but in the presence or absence of the ADAM17/TACE inhibitor TAPI-2. Cell-free culture supernatants were then harvested separately from the cell monolayer and were concentrated and subjected to western blotting for ACE-2 (upper panel). Densitometry (lower panel) demonstrated a significant increase in soluble ACE-2 in hyperoxia-treated cell supernatants compared to the control (*P < 0.01 vs. CTL by ANOVA and Student-Newman-Keuls multiple comparisons test). Addition of TAPI-2 prevented the increase (**P < 0.01 vs. 95% O₂ by ANOVA and Student-Newman-Keuls multiple comparisons test). PowerPoint slide

Figure 6

Hyperoxia with normoxic recovery increases ADAM17/TACE in fetal human lung fibroblasts. IMR90 cells were exposed to hyperoxic or normoxic gas as described in Figure 1 (with recovery), then were recovered for western blotting of ADAM17/TACE as described in Materials and Methods. (a) Western blotting for ADAM17/TACE protein was then performed and normalized to β-actin. (b) By densitometry, hyperoxia with normoxic recovery caused a significant increase in ADAM17/TACE (3.1-fold, *P < 0.05 by Student’s t-test). (c) Replicate wells were harvested for recovery of total RNA followed by qRTPCR as described in Methods; hyperoxia with normoxic recovery also caused a significant increase in ADAM17/TACE mRNA (2.2-fold, *P < 0.05 by Student’s t-test). PowerPoint slide

Figure 7

Proposed role of angiotensin-converting enzyme-2 (ACE-2) in hyperoxia-induced lung fibrosis. Angiotensinogen (AGT) produced by either alveolar epithelial cells or myofibroblasts (3) can be cleaved by a variety of pulmonary aspartyl proteases (3) to yield angiotensin (ANG) I, which in turn is cleaved by various lung enzymes to yield the profibrotic ANGII. ANGII is degraded by ACE-2 to yield the antifibrotic peptide ANG1-7, which acts through its receptor mas. Hyperoxia causes, by mechanisms yet unknown, the release of ACE-2 from the cell through ectodomain cleavage mediated by increased ADAM-17/TACE. Through this mechanism, hyperoxia is theorized to promote fibrosis through both (a) promoting accumulation of ANGII (8,9) and (b) reducing production of the antifibrotic peptide ANG1-7 (21). See text for Discussion. Processes depicted here as intracellular (dark shaded areas) or extracellular (light shading) may well occur in either or both compartments. PowerPoint slide