Chronic hypoxia promotes pulmonary artery endothelial cell proliferation through H2O2-induced 5-lipoxygenase.

Pulmonary Hypertension (PH) is a progressive disorder characterized by endothelial dysfunction and proliferation. Hypoxia induces PH by increasing vascular remodeling. A potential mediator in hypoxia-induced PH development is arachidonate 5-Lipoxygenase (ALOX5). While ALOX5 metabolites have been shown to promote pulmonary vasoconstriction and endothelial cell proliferation, the contribution of ALOX5 to hypoxia-induced proliferation remains unknown. We hypothesize that hypoxia exposure stimulates HPAEC proliferation by increasing ALOX5 expression and activity. To test this, human pulmonary artery endothelial cells (HPAEC) were cultured under normoxic (21% O2) or hypoxic (1% O2) conditions for 24-, 48-, or 72 hours. In a subset of cells, the ALOX5 inhibitor, zileuton, or the 5-lipoxygenase activating protein inhibitor, MK-886, was administered during hypoxia exposure. ALOX5 expression was measured by qRT-PCR and western blot and HPAEC proliferation was assessed. Our results demonstrate that 24 and 48 hours of hypoxia exposure have no effect on HPAEC proliferation or ALOX5 expression. Seventy two hours of hypoxia significantly increases HPAEC ALOX5 expression, hydrogen peroxide (H2O2) release, and HPAEC proliferation. We also demonstrate that targeted ALOX5 gene silencing or inhibition of the ALOX5 pathway by pharmacological blockade attenuates hypoxia-induced HPAEC proliferation. Furthermore, our findings indicate that hypoxia-induced increases in cell proliferation and ALOX5 expression are dependent on H2O2 production, as administration of the antioxidant PEG-catalase blocks these effects and addition of H2O2 to HPAEC promotes proliferation. Overall, these studies indicate that hypoxia exposure induces HPAEC proliferation by activating the ALOX5 pathway via the generation of H2O2

ROS mediate hypoxia-induced increases in endothelial ALOX5 expression and cell proliferation.

<p>Human pulmonary artery endothelial cells (HPAEC) were exposed to 0, 10, 100, and 200 µM hydrogen peroxide (H₂O₂) for 24 hours. Following exposure, supernatants were collected to assess cell toxicity by adenylate kinase release. Results demonstrate no significant changes in cell death as indicated by adenylate kinase release (n = 4–6; data not shown). HPAEC were collected and total RNA was isolated for quantitative real-time PCR gene expression analysis. ALOX5 was normalized to the housekeeping gene β-globin. Relative expression was calculated using the Delta-Delta C_T method and values were expressed as percent of control (A, n = 4–5). * p<0.05 when compared to untreated controls. H₂O₂ exposure stimulates HPAEC ALOX5 protein levels as analyzed by western blot (B, n = 4). PEG-Catalase (10U - 1000 U/ml) administration during the final 24 hours of the 72 hour hypoxia exposure prevents hypoxia-induced elevations in endothelial ALOX5 expression (C, n = 5) and cell proliferation (D, n = 6). * p<0.01 when compared to normoxic groups. ** p<0.05 when compared to untreated hypoxia controls.</p

Hypoxia exposure stimulates endothelial ROS release.

<p>Human pulmonary artery endothelial cells were exposed to normoxic or hypoxic (1% O₂) conditions for 24-, 48- or 72-hours. Following exposure, HPAEC ROS release was assessed by DCF staining (A, n = 3) and Amplex Red assay (C, n = 4). Results demonstrate that prolonged hypoxia exposure significantly increases endothelial ROS production whereas administration with the antioxidants, PEG- catalase or superoxide dismutase reduces these effects (B). Amplex Red Assay indicates that chronic hypoxia exposure promotes H₂O₂ release (C). * p<0.0001 when compared to normoxic controls.</p

Pharmacological inhibition of ALOX5 signaling attenuates hypoxia-induced endothelial proliferation.

<p>ALOX5 blockade by zileuton administration reduces hypoxia-induced endothelial proliferation when measured by MTT Assay (A, n = 4). FLAP inhibition by MK-886 attenuates endothelial proliferation following hypoxia exposure (B, n = 4) Pre-treatment with the cysteinyl leukotriene receptor antagonist, montelukast prevents endothelial proliferation during prolonged hypoxia exposure (C, n = 6) HPAEC were exposed to normoxic or hypoxic conditions for 72 hours. ALOX5 inhibitors, zileuton (10 µM) and MK-886 (0.5 µM) were administered during the final 24 hours of normoxia or hypoxia exposure. Cell proliferation was then assessed by MTT assay. * p<0.05 when compared to normoxic groups. ** p<0.05 when compared to untreated hypoxic groups.</p

Prolonged hypoxia exposure increases endothelial cell proliferation.

<p>Seventy two hours of hypoxia exposure significantly stimulates endothelial cell proliferation when compared to all other groups. Human pulmonary artery endothelial cells (HPAEC) were exposed to normoxic or hypoxic (1% O₂) conditions for 24-, 48-, or 72 hours (n = 4). Following exposure, cell proliferation was assessed by MTT (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098532#pone-0098532-g001" target="_blank">Figure 1A</a>) assay and Trypan Blue Dye Exclusion Assay (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098532#pone-0098532-g001" target="_blank">Figure 1B</a>). * p<0.0001.</p

Chronic hypoxia exposure increases endothelial ALOX5 expression.

<p>Seventy two hours of hypoxia exposure significantly stimulates endothelial ALOX5 expression when compared to all other groups. HPAEC were exposed to normoxic or hypoxic conditions for 24-, 48-, or 72 hours. Following exposure, cells were collected, and total RNA and protein were isolated for expression analyses via quantitative real time PCR and Western blot respectively. Results indicate that ALOX5 mRNA levels are significantly increased following hypoxia exposure (A, n = 5). Chronic hypoxia exposure also causes a 3-fold elevation in ALOX5 protein expression levels (B, n = 4). Endothelial FLAP expression is also increased when compared to all other groups (C, n = 5–7). Values are expressed as percent of control. * p<0.001 when compared to all other groups.</p