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

Porter KM, Kang BY, Adesina SE, Murphy TC, Hart CM, Sutliff RL - PLoS ONE (2014)

Bottom Line: A potential mediator in hypoxia-induced PH development is arachidonate 5-Lipoxygenase (ALOX5).Our results demonstrate that 24 and 48 hours of hypoxia exposure have no effect on HPAEC proliferation or ALOX5 expression.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.

View Article: PubMed Central - PubMed

Affiliation: Emory University School of Medicine/Atlanta Veterans Affairs Medical Center, Department of Pulmonary, Allergy and Critical Care Medicine, Atlanta, Georgia, United States of America.

ABSTRACT
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.

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Hypoxia exposure stimulates endothelial ROS release.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.
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pone-0098532-g005: Hypoxia exposure stimulates endothelial ROS release.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.

Mentions: Previous studies demonstrate that hypoxia promotes ROS generation in PH models [22]–[25]. To investigate whether hypoxia exposure increases endothelial ROS release, ROS release was analyzed following 24, 48, and 72-hours of hypoxia exposure. DCF staining indicates that chronic hypoxia exposure increases ROS production in endothelial cells (Figure 5A). This increase in ROS release was attenuated by the administration of PEG-catalase and superoxide dismutase (Figure 5B). Furthermore, Amplex Red assay analysis of hydrogen peroxide (H2O2) release also demonstrates that 72 hours of hypoxia exposure is required for increased in H2O2 (Figure 5C). These data suggest that chronic exposure to hypoxic conditions increases endothelial H2O2.


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

Porter KM, Kang BY, Adesina SE, Murphy TC, Hart CM, Sutliff RL - PLoS ONE (2014)

Hypoxia exposure stimulates endothelial ROS release.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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4048210&req=5

pone-0098532-g005: Hypoxia exposure stimulates endothelial ROS release.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.
Mentions: Previous studies demonstrate that hypoxia promotes ROS generation in PH models [22]–[25]. To investigate whether hypoxia exposure increases endothelial ROS release, ROS release was analyzed following 24, 48, and 72-hours of hypoxia exposure. DCF staining indicates that chronic hypoxia exposure increases ROS production in endothelial cells (Figure 5A). This increase in ROS release was attenuated by the administration of PEG-catalase and superoxide dismutase (Figure 5B). Furthermore, Amplex Red assay analysis of hydrogen peroxide (H2O2) release also demonstrates that 72 hours of hypoxia exposure is required for increased in H2O2 (Figure 5C). These data suggest that chronic exposure to hypoxic conditions increases endothelial H2O2.

Bottom Line: A potential mediator in hypoxia-induced PH development is arachidonate 5-Lipoxygenase (ALOX5).Our results demonstrate that 24 and 48 hours of hypoxia exposure have no effect on HPAEC proliferation or ALOX5 expression.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.

View Article: PubMed Central - PubMed

Affiliation: Emory University School of Medicine/Atlanta Veterans Affairs Medical Center, Department of Pulmonary, Allergy and Critical Care Medicine, Atlanta, Georgia, United States of America.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus