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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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ROS mediate hypoxia-induced increases in endothelial ALOX5 expression and cell proliferation.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 CT 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.
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pone-0098532-g006: ROS mediate hypoxia-induced increases in endothelial ALOX5 expression and cell proliferation.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 CT 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.

Mentions: Since elevated ROS production is associated with increased ALOX5 activity [14], [15], [17], we next sought to determine whether H2O2 mediates hypoxia-induced increases in endothelial ALOX5. HPAECs exposed to 0–200 µM concentrations of H2O2 for 24 hours revealed that 100 µM and 200 µM H2O2 concentrations increase endothelial ALOX5 mRNA levels (Figure 6A). Similarly, H2O2 exposure stimulates ALOX5 protein expression when assessed by Western blot (Figure 6B). These data indicate that ALOX5 expression levels are altered by increased ROS. To determine whether hypoxia-induced ROS mediate alterations in HPAEC ALOX5 levels and cell proliferation, we administered the antioxidant, PEG-catalase during the final 24 hours of the 72 hour hypoxia exposure period and determined ALOX5 mRNA levels by qRT-PCR and cell proliferation by MTT Assay. Results demonstrate that catalase administration significantly reduces hypoxia-induced elevations in endothelial ALOX5 gene expression (Figure 6C). Catalase treatment during the final 24 hours of the 72-hour hypoxia exposure also prevents HPAEC proliferation (Figure 6D) in a concentration-dependent manner when compared to untreated hypoxic groups.


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)

ROS mediate hypoxia-induced increases in endothelial ALOX5 expression and cell proliferation.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 CT 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.
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pone-0098532-g006: ROS mediate hypoxia-induced increases in endothelial ALOX5 expression and cell proliferation.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 CT 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.
Mentions: Since elevated ROS production is associated with increased ALOX5 activity [14], [15], [17], we next sought to determine whether H2O2 mediates hypoxia-induced increases in endothelial ALOX5. HPAECs exposed to 0–200 µM concentrations of H2O2 for 24 hours revealed that 100 µM and 200 µM H2O2 concentrations increase endothelial ALOX5 mRNA levels (Figure 6A). Similarly, H2O2 exposure stimulates ALOX5 protein expression when assessed by Western blot (Figure 6B). These data indicate that ALOX5 expression levels are altered by increased ROS. To determine whether hypoxia-induced ROS mediate alterations in HPAEC ALOX5 levels and cell proliferation, we administered the antioxidant, PEG-catalase during the final 24 hours of the 72 hour hypoxia exposure period and determined ALOX5 mRNA levels by qRT-PCR and cell proliferation by MTT Assay. Results demonstrate that catalase administration significantly reduces hypoxia-induced elevations in endothelial ALOX5 gene expression (Figure 6C). Catalase treatment during the final 24 hours of the 72-hour hypoxia exposure also prevents HPAEC proliferation (Figure 6D) in a concentration-dependent manner when compared to untreated hypoxic groups.

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