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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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Prolonged hypoxia exposure increases endothelial cell proliferation.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 (Figure 1A) assay and Trypan Blue Dye Exclusion Assay (Figure 1B). * p<0.0001.
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pone-0098532-g001: Prolonged hypoxia exposure increases endothelial cell proliferation.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 (Figure 1A) assay and Trypan Blue Dye Exclusion Assay (Figure 1B). * p<0.0001.

Mentions: Hypoxia is associated with significant endothelial alterations which are thought to contribute to PH development and progression [3], [20]. Pulmonary arteries [4] and lung sections of PH patients demonstrate abnormal endothelial cell growth [5], [6]. To determine whether hypoxia alters HPAEC function in vitro, we assessed HPAEC proliferation following 24, 48, and 72 hours of hypoxia exposure. While 24 and 48 hours of hypoxia exposure had no effect on cellular proliferation, 72 hours of hypoxia increases cellular proliferation when measured by MTT assay (Figure 1A) and Trypan Blue Dye Exclusion Assay (Figure 1B).


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)

Prolonged hypoxia exposure increases endothelial cell proliferation.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 (Figure 1A) assay and Trypan Blue Dye Exclusion Assay (Figure 1B). * p<0.0001.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0098532-g001: Prolonged hypoxia exposure increases endothelial cell proliferation.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 (Figure 1A) assay and Trypan Blue Dye Exclusion Assay (Figure 1B). * p<0.0001.
Mentions: Hypoxia is associated with significant endothelial alterations which are thought to contribute to PH development and progression [3], [20]. Pulmonary arteries [4] and lung sections of PH patients demonstrate abnormal endothelial cell growth [5], [6]. To determine whether hypoxia alters HPAEC function in vitro, we assessed HPAEC proliferation following 24, 48, and 72 hours of hypoxia exposure. While 24 and 48 hours of hypoxia exposure had no effect on cellular proliferation, 72 hours of hypoxia increases cellular proliferation when measured by MTT assay (Figure 1A) and Trypan Blue Dye Exclusion Assay (Figure 1B).

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