The Interplay between Aquaporin-1 and the Hypoxia-Inducible Factor 1α in a Lipopolysaccharide-Induced Lung Injury Model in Human Pulmonary Microvascular Endothelial Cells

Abstract

Aquaporin-1 (AQP1), a water channel, and the hypoxia-inducible factor 1α (HIF1A) are implicated in acute lung injury responses, modulating among others pulmonary vascular leakage. We hypothesized that the AQP1 and HIF1A systems interact, affecting mRNA, protein levels and function of AQP1 in human pulmonary microvascular endothelial cells (HPMECs) exposed to lipopolysaccharide (LPS). Moreover, the role of AQP1 in apoptosis and wound healing progression was examined. Both AQP1 mRNA and protein expression levels were higher in HPMECs exposed to LPS compared to untreated HPMECs. However, in the LPS-exposed HIF1A-silenced cells, the mRNA and protein expression levels of AQP1 remained unaltered. In the permeability experiments, a statistically significant volume increase was observed at the 360 s time-point in the LPS-exposed HPMECs, while LPS-exposed HIF1A-silenced HPMECs did not exhibit cell swelling, implying a dysfunctional AQP1. AQP1 did not seem to affect cell apoptosis yet could interfere with endothelial migration and/or proliferation. Based on our results, it seems that HIF1A silencing negatively affects AQP1 mRNA and protein expression, as well as AQP1 function, in the setting of lung injury.

Keywords: AQP1; HIF1A; HPMEC; LPS; lung injury.

Figure 1

(A) AQP1 relative mRNA expression, (B) Representative expression of AQP1 and actin proteins in LPS-exposed HPMECs and LPS-exposed HIF1A-silenced HPMECs, (C) AQP1 relative protein expression. (A) AQP1 mRNA expression was measured in untreated HPMECs (ctr), HIF1A-silenced HPMECs, LPS-exposed HPMECs (ctr + LPS), HIF1A-silenced HPMECs exposed to LPS, and in HPMECs transfected with a universal scrambled negative control siRNA duplex (siRNA negative ctr). Data are presented as box plots. Analysis was performed by one-way ANOVA followed by the Kruskal Wallis posthoc test. Line in the middle, median value; lower and upper lines, 25th and 75th centiles; whiskers, range of values; ***, p < 0.001 compared to ctr. Results from 4 independent experiments. (B) Protein expression was analyzed by SDS-PAGE and immunoblotting. Relative protein expression was performed using densitometry with actin as a loading control. (C) AQP1 protein expression was measured in untreated HPMECs (ctr), LPS-exposed HPMECs (ctr + LPS), HIF1A-silenced HPMECs exposed to LPS, and in HPMECs transfected with a universal scrambled negative control siRNA duplex (siRNA negative ctr). Data are presented as box plots. Analysis was performed by one-way ANOVA followed by the Kruskal Wallis posthoc test. Line in the middle, median value; lower and upper lines, 25th and 75th centiles; whiskers, range of values; **, p < 0.01 compared to ctr. Results from 3 independent experiments.

Figure 2

(A,B) Time-course of the osmotic swelling of HPMECs. (A) Representative images of the osmotically challenged untreated HPMECs (ctr), the LPS-exposed HPMECs (ctr + LPS), HIF1A-silenced HPMECs exposed to LPS, and HPMECs exposed to HgCl₂, an AQP1 blocker (B) Time-course representation of the osmotically challenged untreated HPMECs (blue line with circles), HIF1A-silenced HPMECs (yellow line with squares), the LPS-exposed HPMECs (brown line with squares), HIF1A-silenced HPMECs exposed to LPS (green line with triangles), HPMECs exposed to 0.3 mmoL/L HgCl₂ for 5 min (black line with triangles), HPMECs transfected with a universal scrambled negative control siRNA duplex (red line with triangles). Analysis was performed by 2-way ANOVA followed by Tukey’s posthoc test. ****, p < 0.0001 compared to ctr. Results from 4 independent experiments.

Figure 3

(A) Caspase 3 activity, (B,C) Time-course of wound healing of HPMECs. (A) In order to examine apoptosis in HPMECs, caspase 3 activity was measured in untreated HPMECs (ctr), AQP1-silenced HPMECs, HPMECs exposed to 0.3 mmoL/L HgCl₂ for 5 min, HPMECs exposed to 100 nM PMA for 2 h, LPS-exposed HPMECs (ctr + LPS), LPS-exposed HIF1A-silenced HPMECs and HPMECs transfected with a universal scrambled negative control siRNA duplex. Data are presented as boxes. Each box represents the mean values and the bars represent the standard deviation values. Analysis was performed by one-way ANOVA followed by Kruskal Wallis. Results from 3 independent experiments. (B,C) Time-course representation of the percentage of wound closure measured in untreated HPMECs (yellow line with circles), LPS-exposed HPMECs (pink line with squares), HIF1A-silenced HPMECs exposed to LPS (green line with triangles), and HPMECs transfected with a universal scrambled negative control siRNA duplex (blue line with circles). **, p < 0.01 compared to untreated cells. Analysis was performed by 2-way ANOVA followed by the Wilcoxon test. Results from 3 independent experiments.