Oxidized low-density lipoprotein (oxLDL) triggers hypoxia-inducible factor-1α (HIF-1α) accumulation via redox-dependent mechanisms

Oxidized low-density lipoprotein (oxLDL) and macrophages play a central role in atherosclerosis. Here, we obtained evidence that oxLDL induced hypoxia-inducible factor-1α (HIF-1α) protein accumulation in human macrophages (Mono-Mac-6) under normoxia. HIF-1α accumulation was attenuated by pretreatment with the antioxidant N-acetyl-L-cysteine (NAC), the nitric oxide (NO) donor S-nitrosoglutathione (GSNO), and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitors such as diphenyleniodonium (DPI) or 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), thus implicating the contribution of oxLDL-generated reactive oxygen species (ROS). Whereas oxLDL did not modulate HIF-1α mRNA levels, experiments with cycloheximide pointed to a translational mechanism in oxLDL action. HIF-1–dependent luciferase reporter gene analysis underscored HIF-1 transactivation. Our results indicate that oxLDL induced HIF-1α accumulation and HIF-1–dependent reporter gene activation in human macrophages via a redox-mediated pathway. This finding may suggest a role of HIF-1 in atherosclerosis and oxLDL-induced pathogenesis.

Oxidatively modified low-density lipoprotein (oxLDL) emerged as a pathogenetic factor in atherosclerosis.¹  There is accumulating evidence for oxidative stress in vascular dysfunction/atherogenesis and a significant contribution of macrophages in oxLDL uptake, foam cell formation, and disease progression.¹  Considering the role of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases in generating (reactive oxygen species) ROS, we recently demonstrated that NADPH oxidase is a major source of ROS in response to oxLDL with the further notion that diphenyleniodonium (DPI), a NADPH oxidase inhibitor, eliminated ROS generation in macrophages.² 

Hypoxia-inducible factor-1 (HIF-1) is a heterodimeric transcription factor composed of α and β subunits.³  Although HIF-1β is constitutively expressed in many cell types, HIF-1α is present at undetectable amounts under normoxia because of rapid proteasomeal degradation and is stabilized on hypoxia.^4,5  Following HIF-1α/HIF-1β heterodimerization and translocation to the nucleus, binding to promoter-specific HRE (hypoxia response element) sites drives classical hypoxia responsive genes.^6,7  Studies suggested that ROS provides a redox signal for hypoxic HIF-1 induction.⁸  Moreover, ROS appears to regulate HIF-1 under normoxia. In some cell types, increased ROS production has been associated with HIF-1 activation by hormones, growth factors, and transition metals.^8,9  In addition, direct exposure of cells to ROS may stabilize HIF-1α and promote vascular endothelial growth factor (VEGF) expression.^8,10  Along that line cytokine-mediated regulation of HIF-1 may require a nonhypoxic but ROS-sensitive pathway.¹¹ 

Here, we showed that oxLDL provoked HIF-1α accumulation and HIF-1–dependent transcriptional activation in human Mono-Mac-6 (MM6) macrophages through a ROS-dependent pathway.

MM6 cells were grown in RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 1% nonessential amino acids, 1 mM sodium pyruvate, 9 μg/mL bovine insulin and antibiotics in a humidified 5% CO₂ atmosphere at 37°C. Cell dishes at 80% to 90% confluence were exposed to native low-density lipoprotein (nLDL), oxLDL, and/or inhibitors. RCC4 cells, which constitutively express HIF-1α were kindly provided by Prof Ratcliffe, Wellcome Trust Centre for Human Genetics, University of Oxford, United Kingdom.^12,13  nLDL was purchased from Sigma (Taufkirchen, Germany) and oxidized as described.² 

HIF-1α (antibody from Becton Dickinson, Heidelberg, Germany) as well as actin (to confirm equal protein loading) was determined by Western analysis as described.¹⁴ 

MM6 cells (5 × 10⁵) were seeded in 6-cm dishes 1 day before transfection with 3 μg plasmid pGL3-EPO-HRE-SV40-LUC, which harbors 3 copies of the erythropoietin hypoxia responsive element in front of the SV 40 promoter or a VEGF reporter plasmid (pGL3-1.5kbVEGFprom, obtained from Prof Maity, University of Pennsylvania Medical Center, Philadelphia), for 8 hours, using dioleoyl-1,2-diacyl-3-trimethylammonium-propane (DOTAP) for transfection according to company instructions. Thereafter, medium was changed and cells were stimulated.

MM6 cells (5 × 10⁶) were incubated with or without 200 μg/mL oxLDL for 5 hours. Total RNA was isolated using the peqGOLD RNAPure kit, followed by HIF-1α mRNA reverse transcriptase–polymerase chain reaction (RT-PCR). Primer selection was as follows: HIF-1α,5′-CTCAAAGTCGGACAGCCTCA-3′,5′-CCCTGCAGTAGGTTTCTGCT-3′; actin, 5′-TGACGGGGTCACCCACACTGTGCCCATCTA-3′, 5′-CTAGAAGCATTTGCGGTCGACGATGGAGGG-3′. Amplification program was as follows: 95°C, 30 seconds; 56°C, 30 seconds; 72°C, 1 minute; 20 cycles; 72°C, 10 minutes. Products were separated on 2% agarose gels and visualized with ethidium bromide.

Each experiment was performed at least 3 times, and representative data are shown. Data are presented as means ± SD. The unpaired Student t test was used to evaluate the significance of differences between groups, accepting P < .05 as the level of significance.

To examine the effect of oxLDL on HIF-1α accumulation, MM6 cells were treated with oxLDL or nLDL. As shown in Figure 1A, oxLDL dose-dependently (25-200 μg/mL) induced HIF-1α accumulation. oxLDL was obtained by the CuSO₄/EDTA oxidation protocol. Therefore, we ruled out that neither CuSO₄ nor EDTA at the relevant concentrations promoted HIF-1α accumulation (Figure 1A). As a positive control, to unequivocally localize HIF-1α, we used an RCC4 protein lysate with constitutive HIF-1α expression (Figure 1A). Furthermore, we established that oxLDL but not nLDL, induced HIF-1α accumulation (Figure 1B). The most efficient response was noticed between 4 and 8 hours. Under these experimental conditions oxLDL was nontoxic (data not shown).

Reports from several groups suggested that ROS stabilizes HIF-1α and activates HIF-1.^8-10  Therefore, we investigated whether ROS, produced in response to oxLDL, contributed to HIF-1α accumulation (Figure 1C). N-acetyl-L-cysteine (NAC) is an antioxidant, known to decrease ROS. The addition of 3 mM NAC to MM6 cells for 2 hours prior to the stimulation with 200 μg/mL oxLDL for 8 hours completely blocked HIF-1α accumulation. DPI inhibits oxLDL-mediated ROS formation in macrophages by blocking NADPH oxidase.²  When cells were pretreated with 30 μM DPI for 2 hours prior to oxLDL stimulation, HIF-1α accumulation was suppressed. Because DPI may inhibit mitochondrial complex I, we studied the effect of the structurally unrelated NADPH oxidase inhibitor AEBSF in parallel, which again prevented HIF-1α accumulation. The effect of nitric oxide (NO) derived from the most physiologic donor GSNO may be rationalized by the diffusion controlled interaction of

with NO, thus allowing NO to attenuate HIF-1α accumulation. Our experiments with NADPH oxidase inhibitors (DPI, AEBSF) and GSNO suggest that

\(\mathrm{O}_{2}^{-}\)

might play a role in oxLDL-induced HIF-1α accumulation. Previously, it was demonstrated that mitogen-activated protein kinase (MAPK) family members such as p38 or ERK1/2 attenuated transactivation of HIF-1α in hypoxic cells. In MM6 cells, neither p38 (SB203580) nor ERK (PD98059) inhibitors affect oxLDL-induced HIF-1α accumulation (Figure 1D). Both inhibitors did not significantly alter HIF-1 transactivation in response to oxLDL (data not shown). Next, we examined the time-dependent disappearance of HIF-1α in the presence of the translational inhibitor cycloheximide (Figure 1E). Cells were treated with oxLDL for 4 hours followed by the subsequent addition of cycloheximide versus vehicle for 15 to 60 minutes. Cycloheximide decreased HIF-1α expression starting at 15 minutes, suggesting that oxLDL increased HIF-1α through a translational mechanism. The persistence of detectable HIF-1α protein 15 minutes following cycloheximide treatment could represent a delay in the onset of the cycloheximide effect but implies that the mode of action of oxLDL leaves the degradative pathway largely intact. Thus, it appeared advised to examine HIF-1α mRNA expression. RT-PCR (Figure 1F) revealed that oxLDL left HIF-1α mRNA levels unaltered. We then tested the ability of oxLDL to activate a HIF-1–dependent luciferase reporter gene construct, containing 3 copies of a hypoxia-response element derived from the human erythropoietin gene.

Treatment of MM6 cells with oxLDL resulted in significant reporter gene activation that was largely abrogated by DPI (Figure 2A). nLDL showed no reporter signal, whereas hypoxia elicited a luciferase activity comparable to oxLDL. Similarly, treatment of cells with oxLDL led to an increase in VEGF promoter activity (Figure 2B). Our results are consistent with previous studies, showing a 2- to 3-fold increase in reporter activity by hypoxia^14,15  and further studies reporting hypoxic VEGF production in macrophages.¹⁶  Atherosclerosis is a progressive disease and a common cause of morbidity and mortality in the Western world. Therefore, our results on HIF-1α accumulation by oxLDL appear of clinical relevance. It is well established that HIF-1α plays a major role in VEGF expression and angiogenesis⁹  with the notion that VEGF mediates important alterations associated with atherogenesis and the angiogenic activity of macrophages.¹⁶  Recent findings suggest that neovascularization within atherosclerotic plaques is a sign of advanced atherosclerosis/restenosis.¹⁷  It is tempting to speculate that oxLDL-induced HIF-1α accumulation and VEGF up-regulation are important in these pathophysiologic settings. A series of molecules and molecular pathways emerged that contribute to the pathogenesis and progression of both atherosclerosis and cancer.^18,19  HIF-1 may be added to that list, considering the important tumor angiogenic role of HIF-1α. Our observations propose a new signaling quality of oxLDL in HIF-1 accumulation and demonstrate ROS involvement. This appears of considerable importance to fully understand progression of atherosclerosis and remodeling of the arterial wall by oxLDL.