Carine Michiels

IMPORTANCE OF SE-GLUTATHIONE PEROXIDASE, CATALASE, AND CU/ZN-SOD FOR CELL SURVIVAL AGAINST OXIDATIVE STRESS

Eukaryotic cells have to constantly cope with highly reactive oxygen-derived free radicals. Their defense against these free radicals is achieved by natural antioxidant molecules but also by antioxidant enzymes. In this paper, we review some of the data comparing the efficiency of three different antioxidant enzymes: Cu/Zn-superoxide dismutase (Cu/Zn-SOD), catalase, and selenium-glutathione peroxidase. We perform our comparison on one experimental model (human fibroblasts) where the activities of these three antioxidant enzymes have been modulated inside the cells, and the repercussion of these changes was investigated in different conditions. We also focus our attention on the protecting role of selenium-glutathione peroxidase, because this enzyme is very rarely studied due to the difficulties linked to its biochemical properties. These studies evidenced that all three antioxidant enzymes give protection for the cells. They show a high efficiency for selenium-glutathione peroxidase and emphasize the fact that each enzyme has a specific as well as an irreplaceable function. They are all necessary for the survival of the cell even in normal conditions. In addition, these three enzymes act in a cooperative or synergistic way to ensure a global cell protection. However, optimal protection is achieved only when an appropriate balance between the activities of these enzymes is maintained. Interpretation of the deleterious effects of free radicals has to be analyzed not only as a function of the amount of free radicals produced but also relative to the efficiency and to the activities of these enzymatic and chemical antioxidant systems. The threshold of protection can indeed vary dramatically as a function of the level of activity of these enzymes.

Glutathione peroxidase, superoxide dismutase, and catalase inactivation by peroxides and oxygen derived free radicals

Glutathione peroxidase (GPX), superoxide dismutase (SOD) and catalase are the most important enzymes of the cell antioxidant defense system. However, these molecules are themselves susceptible to oxidation. The aim of this work was to estimate to what extent this system could be inactivated by its own substrates. We tested the effect of hydrogen peroxide, cumene hydroperoxide, t-butyl hydroperoxide and hydroxyl and superoxide radicals on GPX, SOD and catalase. For GPX, a 50% inactivation was observed at 10(-1) M (30 min, 37 degrees C) for hydrogen peroxide, 3 x 10(-4) M (15 min, 37 degrees C) for cumene hydroperoxide and 5 x 10(-5) M (11 min, 37 degrees C) for t-butyl hydroperoxide. Unlike the hydroxyl radicals, superoxide anions did not inactivate this enzyme. Catalase was inactivated by hydroxyl radicals and by superoxide anions but organic peroxides had no effect. SOD was inactivated by 50% by hydrogen peroxide at 4 x 10(-4) M (20 min, 37 degrees C), but organic peroxides and hydroxyl radicals were ineffective on this enzyme. Since the three enzymes of the antioxidant system are susceptible to at least one of the oxidative reactive molecules, in the case of high oxidative stresses such an inhibition could take place, leading to an irreversible autocatalytical process in which the production rate of the oxidants will continuously increase, leading to cell death.

Endothelial cell functions

Endothelial cells play a wide variety of critical roles in the control of vascular function. Indeed, since the early 1980s, the accumulating knowledge of the endothelial cell structure as well as of the functional properties of the endothelial cells shifted their role from a passive membrane or barrier to a complex tissue with complex functions adaptable to needs specific in time and location. Hence, it participates to all aspects of the vascular homeostasis but also to physiological or pathological processes like thrombosis, inflammation, or vascular wall remodeling. Some of the most important endothelial functions will be described in the following review and more specifically, their role in blood vessel formation, in coagulation and fibribolysis, in the regulation of vascular tone as well as their participation in inflammatory reactions and in tumor neoangiogenesis. J. Cell. Physiol. 196: 430–443, 2003.

Reactive Oxygen Species Activate the HIF-1α Promoter Via a Functional NFκB Site

Objective—Reactive oxygen species have been implicated as signaling molecules modulating the activity of redox-sensitive transcription factors such as nuclear factor kappa B (NF-&kgr;B). Recently, the transcription factor hypoxia-inducible factor-1 (HIF-1), known to mediate gene expression by hypoxia, has been found to be also activated by nonhypoxic factors in a redox-sensitive manner. We therefore aimed to elucidate the link between these 2 important redox-sensitive transcription factors. Methods and Results—In pulmonary artery smooth muscle cells, reactive oxygen species generated either by exogenous H2O2 or by a NOX4-containing NADPH oxidase stimulated by thrombin activated or induced NF-&kgr;B and HIF-1&agr;. The reactive oxygen species-mediated HIF-1&agr; induction occurred on the transcriptional level and was dependent on NF-&kgr;B. Transfection experiments with wild-type or mutant HIF-1&agr; promoter constructs revealed the presence of a yet unidentified NF-&kgr;B binding element. Gel shift analyses and chromatin immunoprecipitation verified binding of NF-&kgr;B to this site. Furthermore, reactive oxygen species enhanced expression of plasminogen activator inhibitor-1, which was prevented by dominant-negative I&kgr;B or mutation of the HIF-1 binding site within the plasminogen activator inhibitor-1 promoter. Conclusion—These findings show for the first time to our knowledge that reactive oxygen species directly link HIF-1&agr; and NF-&kgr;B, implicating an important pathophysiological role of this novel pathway in disorders associated with elevated levels of reactive oxygen species.

Lactate Influx through the Endothelial Cell Monocarboxylate Transporter MCT1 Supports an NF-κB/IL-8 Pathway that Drives Tumor Angiogenesis

Lactate generated from pyruvate fuels production of intracellular NAD(+) as an end result of the glycolytic process in tumors. Elevated lactate concentration represents a good indicator of the metabolic adaptation of tumors and is actually correlated to clinical outcome in a variety of human cancers. In this study, we investigated whether lactate could directly modulate the endothelial phenotype and thereby tumor vascular morphogenesis and perfusion. We found that lactate could enter endothelial cells through the monocarboxylate transporter MCT-1, trigger the phosphorylation/degradation of IκBα, and then stimulate an autocrine NF-κB/IL-8 (CXCL8) pathway driving cell migration and tube formation. These effects were prevented by 2-oxoglutarate and reactive oxygen species (ROS) inhibitors, pointing to a role for prolyl-hydroxylase and ROS in the integration of lactate signaling in endothelial cells. PHD2 silencing in endothelial cells recapitulated the proangiogenic effects of lactate, whereas a blocking IL-8 antibody or IL-8-targeting siRNA prevented them. Finally, we documented in mouse xenograft models of human colorectal and breast cancer that lactate release from tumor cells through the MCT4 (and not MCT1) transporter is sufficient to stimulate IL-8-dependent angiogenesis and tumor growth. In conclusion, our findings establish a signaling role for lactate in endothelial cells and they identify the lactate/NF-κB/IL-8 pathway as an important link between tumor metabolism and angiogenesis.

ERK activation upon hypoxia: involvement in HIF-1 activation.

Hypoxia‐inducible factor‐1 (HIF‐1) is a transcription factor activated by hypoxia. The HIF‐1 activation transduction pathway is poorly understood. In this report, we investigated the activation of extracellular regulated kinases (ERK) in hypoxia and their involvement in HIF‐1 activation. We demonstrated that in human microvascular endothelial cells‐1 (HMEC‐1), ERK kinases are activated during hypoxia. Using dominant negative mutants, we showed that ERK1 is needed for hypoxia‐induced HIF‐1 transactivation activity. Moreover, using a kinase assay and Western blot experiments, we showed that HIF‐1α is phosphorylated in hypoxia by an ERK‐dependent pathway. These results evidence the role of mitogen‐activated protein kinase in the transcriptional response to hypoxia.

Hypoxia-induced activation of HIF-1: role of HIF-1α-Hsp90 interaction

The protein chaperone heat shock protein 90 (Hsp90) is a major regulator of different transcription factors such as MyoD, a basic helix loop helix (bHLH) protein, and the bHLH‐Per‐aryl hydrocarbon nuclear translocator (ARNT)‐Sim (PAS) factors Sim and aryl hydrocarbon receptor (Ahr). The transcription factor hypoxia‐inducible factor‐1α (HIF‐1α), involved in the response to hypoxia, also belongs to the bHLH‐PAS family. This work was aimed to investigate the putative role of Hsp90 in HIF‐1 activation by hypoxia. Using a EGFP‐HIF‐1α fusion protein, co‐immunoprecipitation experiments evidenced that the chimeric protein expressed in COS‐7 cells interacts with Hsp90 in normoxia but not in hypoxia. We also demonstrated that Hsp90 interacts with the bHLH‐PAS domain of HIF‐1α. Moreover, Hsp90 is not co‐translocated with HIF‐1α into the nucleus. At last, we showed that Hsp90 activity is essential for HIF‐1 activation in hypoxia since it is inhibited in the presence of geldanamycin. These results indicate that Hsp90 is a major regulator in HIF‐1α activation.

Physiological and pathological responses to hypoxia

As the average age in many countries steadily rises, heart infarction, stroke, and cancer become the most common causes of death in the 21st century. The causes of these disorders are many and varied and include genetic predisposition and environmental influences, but they all share a common feature in that limitation of oxygen availability participates in the development of these pathological conditions. However, cells and organisms are able to trigger an adaptive response to hypoxic conditions that is aimed to help them to cope with these threatening conditions. This review provides a description of several systems able to sense oxygen concentration and of the responses they initiate both in the acute and also in long-term hypoxia adaptation. The role of hypoxia in three pathological conditions, myocardial and cerebral ischemia as well as tumorigenesis, is briefly discussed.

Adenovirus-Mediated Gene Transfer of Human Platelet-Activating Factor–Acetylhydrolase Prevents Injury-Induced Neointima Formation and Reduces Spontaneous Atherosclerosis in Apolipoprotein E–Deficient Mice

Background—Atherosclerosis is characterized by an early inflammatory response involving proinflammatory mediators such as platelet-activating factor (PAF)-like phospholipids, which are inactivated by PAF-acetylhydrolase (PAF-AH). The effect of adenovirus-mediated expression of PAF-AH on injury-induced neointima formation and spontaneous atherosclerosis was studied in apolipoprotein E–deficient mice. Methods and Results—Intravenous administration of an adenovirus (5×108 plaque-forming units) directing liver-specific expression of human PAF-AH resulted in a 3.5-fold increase of plasma PAF-AH activity at day 7 (P <0.001); this was associated with a 2.4- and 2.3-fold decrease in malondialdehyde-modified LDL autoantibodies and the lysophosphatidylcholine/phosphatidylcholine ratio, respectively (P <0.001 for both). Non-HDL and HDL cholesterol levels in PAF-AH-treated mice were similar to those of control virus-treated mice. Seven days after virus injection, endothelial denudation of the common left carotid artery was induced with a guidewire. Neointima formation was assessed 18 days later. PAF-AH gene transfer reduced oxidized lipoproteins by 82% (P <0.001), macrophages by 69% (P =0.006), and smooth muscle cells by 84% (P =0.002) in the arterial wall. This resulted in a 77% reduction (P <0.001) of neointimal area. Six weeks after adenovirus-mediated gene transfer, spontaneous atherosclerotic lesions in the aortic root were analyzed. PAF-AH gene transfer reduced atherosclerotic lesions by 42% (P =0.02) in male mice, whereas a nonsignificant 14% reduction was observed in female mice. Basal and PAF-AH activity after gene transfer were higher in male mice than in female mice (P =0.01 and P =0.04, respectively). Conclusions—Gene transfer of PAF-AH inhibited injury-induced neointima formation and spontaneous atherosclerosis in apolipoprotein E–deficient mice. Our data indicate that PAF-AH, by reducing oxidized lipoprotein accumulation, is a potent protective enzyme against atherosclerosis.

Is HIF-1α a pro- or an anti-apoptotic protein? ☆

Abstract Hypoxia-inducible factor-1 (HIF-1) is the major transcription factor specifically activated by hypoxia. It induces the expression of different genes whose products play an adaptive role for hypoxic cells and tissues. Besides these protective responses, HIF-1 and/or hypoxia have also been shown to be either anti-apoptotic or pro-apoptotic, according to the cell type and experimental conditions. More severe or prolonged hypoxia rather induces apoptosis that is, at least in part, initiated by the direct association of HIF-1α and p53 and p53-induced gene expression. On the other hand, HIF-1α dimerized with ARNT, as an active transcription factor, can protect cells from apoptosis induced by several conditions. This review is aimed to describe the different mechanisms that account for these opposite effects of HIF-1α.