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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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Chronic hypoxia exposure increases endothelial ALOX5 expression.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.
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pone-0098532-g002: Chronic hypoxia exposure increases endothelial ALOX5 expression.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.

Mentions: In vivo studies demonstrate that both hypoxia exposure and MCT administration upregulate 5-lipoxygenase (ALOX5) [9], [21]. To specifically investigate the effect of hypoxia on pulmonary artery endothelial cell ALOX5, HPAEC were exposed to hypoxia for 24-, 48-, or 72-hours. Our results indicate that 72 hours of hypoxia exposure significantly increases HPAEC ALOX5 mRNA (Figure 2A) when analyzed by qRT-PCR and ALOX5 protein expression (Figure 2B) as measured by western blot. Prolonged hypoxia exposure also stimulates a significant increase in the expression of the ALOX5 required cofactor, FLAP when measured by qRT-PCR (Figure 2C).


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)

Chronic hypoxia exposure increases endothelial ALOX5 expression.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.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0098532-g002: Chronic hypoxia exposure increases endothelial ALOX5 expression.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.
Mentions: In vivo studies demonstrate that both hypoxia exposure and MCT administration upregulate 5-lipoxygenase (ALOX5) [9], [21]. To specifically investigate the effect of hypoxia on pulmonary artery endothelial cell ALOX5, HPAEC were exposed to hypoxia for 24-, 48-, or 72-hours. Our results indicate that 72 hours of hypoxia exposure significantly increases HPAEC ALOX5 mRNA (Figure 2A) when analyzed by qRT-PCR and ALOX5 protein expression (Figure 2B) as measured by western blot. Prolonged hypoxia exposure also stimulates a significant increase in the expression of the ALOX5 required cofactor, FLAP when measured by qRT-PCR (Figure 2C).

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