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.
Highlights
Pulmonary Hypertension (PH) is a progressive disorder characterized by sustained increases in pulmonary arterial pressures and vascular remodeling
Hypoxia is associated with significant endothelial alterations which are thought to contribute to PH development and progression [3,20]
Our results indicate that 72 hours of hypoxia exposure significantly increases human pulmonary artery endothelial cells (HPAEC) ALOX5 mRNA (Figure 2A) when analyzed by quantitative real-time PCR (qRT-PCR) and ALOX5 protein expression (Figure 2B) as measured by western blot
Summary
Pulmonary Hypertension (PH) is a progressive disorder characterized by sustained increases in pulmonary arterial pressures and vascular remodeling. The mechanisms underlying PH remain unknown, hypoxia induces PH in experimental models and is believed to contribute to disease development [1,2] by impairing endothelial cell function [3,4] as evidenced by abnormal endothelial cell growth in lung sections and pulmonary artery endothelial cells from PH patients [5,6]. Altered expression of arachidonate 5-lipoxygenase (ALOX5), the enzyme that catalyzes the production of vasoactive leukotrienes from arachidonic acid, is associated with endothelial proliferation and PH development. Previous studies demonstrate that patients with idiopathic pulmonary hypertension exhibit increased ALOX5 expression in lung tissue, in small pulmonary artery endothelial cells. Inhibitors of CysLT production attenuate proliferation of pulmonary artery endothelial cells [11]
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