Upregulation of Aquaporin 1 Mediates Increased Migration and Proliferation in Pulmonary Vascular Cells From the Rat SU5416/Hypoxia Model of Pulmonary Hypertension

Figures

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  Figure 1. Validation of model. Bar graphs showing mean ± SD values for (A) right ventricular systolic pressure (RVSP) and (B) right ventricle (RV) to left ventricle plus septum (LV + S) weight ratio in control and SuHx rats. Each dot represents a single animal. Value of p determined by t-test. (C) Representative histology images (H&E staining) illustrating vascular remodeling in SuHx rats. Dark red blood cells are clearly evident within vessels. Arrows point to arteries (top) and small vessels (bottom); bar indicates 200 μm. (D) Representative images showing immunofluorescence for DAPI (nuclei), smooth muscle-specific α-actin (SMA), von Willebrand factor (vWF), and the merged image for pulmonary arterial smooth muscle cells (PASMCs) and MVECs isolated from control rats. Scale bar indicates 50 μm. Results representative of those obtained in at least 10 isolates.

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  Figure 2. Effect of exposure to in vitro hypoxia on aquaporin 1 (AQP1) protein levels in MVECs. Representative immunoblot showing AQP1 and β-tubulin protein expression in MVECs exposed to 4% O2 for 48 h. Cells were treated with vehicle (Veh; 1:1,000 DMSO) or TC-S 7009 (10 μM), a hypoxia-inducible factor-2 (HIF-2) inhibitor (iHIF-2) prior to hypoxic challenge. Bar graphs show mean ± SD values for the ratio of AQP1 to β-tubulin protein levels, normalized to Control + Veh group. Value of p determined from two-way ANOVA with Holm-Sidak post-test. Each dot represents samples from different animals.

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  Figure 3. Pulmonary vascular AQP1 expression. (A) Immunoblot image showing AQP1 protein levels in pulmonary arteries (PA) from control and SuHx rats. (B) Representative images and bar graphs (mean ± SD values) for AQP1 protein levels in microvascular endothelial cell (MVEC) and PASMCs isolated from control and SuHx rats. (C) Bar graphs (mean ± SD values) for mRNA levels of AQP1 relative to cyclophilin B (CpB; housekeeping) and normalized to values in Control rat samples. For all graphs, each dot represents samples from different animals and values of p determined by t-test.

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  Figure 4. Proliferation and migration in cells from Control and SuHx rats. (A) Bar graphs (mean ± SD values) for proliferation, measured by BrdU incorporation normalized to Control value, in PASMC and MVEC isolated from control and SuHx rats. Values of p were determined from Mann-Whitney Rank Sum test. (B) Bar graphs (mean ± SD values) for migrated cells (% of total) were assessed by transwell assay using PASMCs and MVECs from control and SuHx rats. Values of p determined by t-test. For all graphs, each dot represents samples from different animals.

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  Figure 5. Effect of depleting AQP1 on proliferation and migration. (A) Representative images demonstrating level of protein knockdown achieved using siRNA directed against AQP1 (siAQP1) compared to non-targeting siRNA (siNT). Result representative of at least five experiments for each cell type performed on cells from different animals. (B) Bar graphs (mean ± SD values) for proliferation, measured by BrdU incorporation and normalized to control, and migration, measured via transwell assay, in PASMCs isolated from control and SuHx rats. Values of p for proliferation and migration in PASMCs were determined from two-way ANOVA with Holm-Sidak post test to determine differences between specific groups. (C) Bar graphs (mean ± SD values) for proliferation and migrated cells in MVEC from SuHx rats. Values of p determined by t-test (migration) or one-sample t-test (proliferation) against a mean of 100. For all graphs, each dot represents samples from different animals.

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  Figure 6. Effect of increasing AQP1 on proliferation and migration. (A) Representative images demonstrate a level of HA-tagged AQP1 protein expressed following infection with an adenovirus containing GFP or AQP1 compared to β-tubulin (loading control). (B) Bar graph (mean ± SD values) for proliferation, measured by BrdU incorporation and normalized to control, in pulmonary MVECs isolated from control rats and infected with GFP- or AQP1-containing virus. Value of p determined from Mann Whitney Rank Sum Test. (C) Bar graph (mean ± SD values) for migrated cells in MVECs from control rats infected with virus-containing GFP or AQP1. Values of p determined by t-test. For all graphs, each dot represents samples from different animals.

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  Figure 7. Effect of increasing AQP1 expression on endothelial barrier function. Bar graphs show mean ± SD values for baseline transendothelial resistance measured by electric cell-substrate impedance sensing (ECIS) in MVECs from (A) Control and SuHx rats and (B) from normal rats infected with adenovirus containing GFP (control) or AQP1. Values of p determined by t-test. For all graphs, each dot represents independent experiments with cells from different animals.