Figure 1.
Prolonged hypoxia exposure increases endothelial cell proliferation.
Seventy two hours of hypoxia exposure significantly stimulates endothelial cell proliferation when compared to all other groups. Human pulmonary artery endothelial cells (HPAEC) were exposed to normoxic or hypoxic (1% O2) conditions for 24-, 48-, or 72 hours (n = 4). Following exposure, cell proliferation was assessed by MTT (Figure 1A) assay and Trypan Blue Dye Exclusion Assay (Figure 1B). * p<0.0001.
Figure 2.
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.
Figure 3.
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.
Figure 4.
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.
Figure 5.
Hypoxia exposure stimulates endothelial ROS release.
Human pulmonary artery endothelial cells were exposed to normoxic or hypoxic (1% O2) conditions for 24-, 48- or 72-hours. Following exposure, HPAEC ROS release was assessed by DCF staining (A, n = 3) and Amplex Red assay (C, n = 4). Results demonstrate that prolonged hypoxia exposure significantly increases endothelial ROS production whereas administration with the antioxidants, PEG- catalase or superoxide dismutase reduces these effects (B). Amplex Red Assay indicates that chronic hypoxia exposure promotes H2O2 release (C). * p<0.0001 when compared to normoxic controls.
Figure 6.
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.