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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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ALOX5 gene silencing prevents hypoxia-induced endothelial cell proliferation.HPAEC transfected with scrambled- and ALOX5-siRNA were exposed to normoxic or hypoxic conditions for 72 hours. Following exposure, cells were collected for ALOX5 expression analysis. Transfection with ALOX5 siRNA produces a 1.5 fold decrease in ALOX5 protein expression following hypoxia exposure (A, n = 5). Proliferation of control and transfected cells was also assessed following 72-hour exposure to normoxic and hypoxic conditions. ALOX5 gene silencing prevents endothelial cell proliferation during chronic hypoxia exposure (B, n = 5). Values are expressed as fold change. * p<0.01 when compared to normoxic groups, ** p<0.05 when compared to hypoxic controls.
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pone-0098532-g003: ALOX5 gene silencing prevents hypoxia-induced endothelial cell proliferation.HPAEC transfected with scrambled- and ALOX5-siRNA were exposed to normoxic or hypoxic conditions for 72 hours. Following exposure, cells were collected for ALOX5 expression analysis. Transfection with ALOX5 siRNA produces a 1.5 fold decrease in ALOX5 protein expression following hypoxia exposure (A, n = 5). Proliferation of control and transfected cells was also assessed following 72-hour exposure to normoxic and hypoxic conditions. ALOX5 gene silencing prevents endothelial cell proliferation during chronic hypoxia exposure (B, n = 5). Values are expressed as fold change. * p<0.01 when compared to normoxic groups, ** p<0.05 when compared to hypoxic controls.

Mentions: Recent studies demonstrate that ALOX5 contributes to HPAEC proliferation [11]. Additionally, concomitant increases in ALOX5 and HPAEC proliferation following hypoxia exposure suggest a potential association between ALOX5 activity and hypoxia-induced endothelial proliferation. To determine whether ALOX5 mediates hypoxia-induced endothelial proliferation, human pulmonary artery endothelial cells (HPAEC) were transfected with control (scrambled) or ALOX5 silencing RNA. Transfection with ALOX5 siRNA reduces ALOX5 protein expression when compared to normoxic and hypoxic controls (Figure 3A). Densitometry reveals that ALOX5 siRNA transfection decreases hypoxia-induced ALOX5 protein levels by greater than 50% when compared to untreated hypoxic groups (Figure 3A). Following transfection, control- and siALOX5 human pulmonary artery endothelial cells were placed in normoxic or hypoxic conditions for 72 hours then collected for cell proliferation analyses. Transfection with ALOX5 siRNA significantly decreases hypoxia-induced HPAEC proliferation when compared to hypoxia scrambled-controls as measured by trypan blue dye exclusion assay (Figure 3B). These results indicate that ALOX5 is a significant contributor to hypoxia-induced increases in endothelial cell proliferation.


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)

ALOX5 gene silencing prevents hypoxia-induced endothelial cell proliferation.HPAEC transfected with scrambled- and ALOX5-siRNA were exposed to normoxic or hypoxic conditions for 72 hours. Following exposure, cells were collected for ALOX5 expression analysis. Transfection with ALOX5 siRNA produces a 1.5 fold decrease in ALOX5 protein expression following hypoxia exposure (A, n = 5). Proliferation of control and transfected cells was also assessed following 72-hour exposure to normoxic and hypoxic conditions. ALOX5 gene silencing prevents endothelial cell proliferation during chronic hypoxia exposure (B, n = 5). Values are expressed as fold change. * p<0.01 when compared to normoxic groups, ** p<0.05 when compared to hypoxic controls.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0098532-g003: ALOX5 gene silencing prevents hypoxia-induced endothelial cell proliferation.HPAEC transfected with scrambled- and ALOX5-siRNA were exposed to normoxic or hypoxic conditions for 72 hours. Following exposure, cells were collected for ALOX5 expression analysis. Transfection with ALOX5 siRNA produces a 1.5 fold decrease in ALOX5 protein expression following hypoxia exposure (A, n = 5). Proliferation of control and transfected cells was also assessed following 72-hour exposure to normoxic and hypoxic conditions. ALOX5 gene silencing prevents endothelial cell proliferation during chronic hypoxia exposure (B, n = 5). Values are expressed as fold change. * p<0.01 when compared to normoxic groups, ** p<0.05 when compared to hypoxic controls.
Mentions: Recent studies demonstrate that ALOX5 contributes to HPAEC proliferation [11]. Additionally, concomitant increases in ALOX5 and HPAEC proliferation following hypoxia exposure suggest a potential association between ALOX5 activity and hypoxia-induced endothelial proliferation. To determine whether ALOX5 mediates hypoxia-induced endothelial proliferation, human pulmonary artery endothelial cells (HPAEC) were transfected with control (scrambled) or ALOX5 silencing RNA. Transfection with ALOX5 siRNA reduces ALOX5 protein expression when compared to normoxic and hypoxic controls (Figure 3A). Densitometry reveals that ALOX5 siRNA transfection decreases hypoxia-induced ALOX5 protein levels by greater than 50% when compared to untreated hypoxic groups (Figure 3A). Following transfection, control- and siALOX5 human pulmonary artery endothelial cells were placed in normoxic or hypoxic conditions for 72 hours then collected for cell proliferation analyses. Transfection with ALOX5 siRNA significantly decreases hypoxia-induced HPAEC proliferation when compared to hypoxia scrambled-controls as measured by trypan blue dye exclusion assay (Figure 3B). These results indicate that ALOX5 is a significant contributor to hypoxia-induced increases in endothelial cell proliferation.

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