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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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Pharmacological inhibition of ALOX5 signaling attenuates hypoxia-induced endothelial proliferation.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.
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pone-0098532-g004: Pharmacological inhibition of ALOX5 signaling attenuates hypoxia-induced endothelial proliferation.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.

Mentions: We next sought to determine whether alterations in ALOX5 activity contribute to hypoxia-induced endothelial proliferation. To confirm that ALOX5 promotes hypoxia-induced HPAEC proliferation, we measured cellular proliferation in response to hypoxia in the presence or absence of the ALOX5 inhibitor, zileuton. Inhibition of ALOX5 enzyme activity by zileuton administration during the final 24 hours of hypoxia exposure attenuates hypoxia-induced increases in endothelial proliferation (Figure 4A). Similarly, inhibition of the ALOX5 cofactor, FLAP by MK-886 reduces endothelial cell proliferation following hypoxia exposure (Figure 4B). Moreover, pre-treatment with the cysteinyl leukotriene receptor antagonist, montelukast prevents increases in endothelial proliferation during chronic hypoxia exposure (Figure 4C). These data demonstrate that ALOX5 and its LT metabolites mediate hypoxia-induced endothelial proliferation. Additionally, alterations in endothelial cell proliferation caused by hypoxia exposure are in part dependent on activation of CysLT receptors.


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)

Pharmacological inhibition of ALOX5 signaling attenuates hypoxia-induced endothelial proliferation.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.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0098532-g004: Pharmacological inhibition of ALOX5 signaling attenuates hypoxia-induced endothelial proliferation.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.
Mentions: We next sought to determine whether alterations in ALOX5 activity contribute to hypoxia-induced endothelial proliferation. To confirm that ALOX5 promotes hypoxia-induced HPAEC proliferation, we measured cellular proliferation in response to hypoxia in the presence or absence of the ALOX5 inhibitor, zileuton. Inhibition of ALOX5 enzyme activity by zileuton administration during the final 24 hours of hypoxia exposure attenuates hypoxia-induced increases in endothelial proliferation (Figure 4A). Similarly, inhibition of the ALOX5 cofactor, FLAP by MK-886 reduces endothelial cell proliferation following hypoxia exposure (Figure 4B). Moreover, pre-treatment with the cysteinyl leukotriene receptor antagonist, montelukast prevents increases in endothelial proliferation during chronic hypoxia exposure (Figure 4C). These data demonstrate that ALOX5 and its LT metabolites mediate hypoxia-induced endothelial proliferation. Additionally, alterations in endothelial cell proliferation caused by hypoxia exposure are in part dependent on activation of CysLT receptors.

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