Specific inhibition of hypoxia inducible factor 1 exaggerates cell injury induced by in vitro ischemia through deteriorating cellular redox environment

Hypoxia inducible factor 1 (HIF-1) has been suggested to play a critical role in the fate of cells exposed to hypoxic stress. However, the mechanism of HIF-1-regulated cell survival is still not fully understood in ischemic conditions. Redox status is critical for decisions of cell survival, death and differentiation. We investigated the effects of inhibiting HIF-1 on cellular redox status in SH-SY5Y cells exposed to hypoxia or oxygen and glucose deprivation (OGD), coupled with cell death analyses. Our results demonstrated that inhibiting HIF-1α expression by HIF-1α specific small interfering RNA (siRNA) transfection increased reactive oxygen species generation, and transformed the cells to more oxidizing environments (low GSH/GSSG ratio, low NADPH level) under either hypoxic or OGD exposure. Cell death increased dramatically in the siRNA transfected cells, compared to non-transfected cells after hypoxic/OGD exposures. In contrast, increasing HIF-1α expression by desferrioxamine, a metal chelator and hydroxylase inhibitor, induced a more reducing environment (high GSH/GSSG ratio, high NADPH level) and reduced cell death. Further studies showed that HIF-1 regulated not only glucose transporter-1 expression, but also the key enzymes of the pentose phosphate pathway such as glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. These enzymes are important in maintaining cellular redox homeostasis by generating NADPH, the primary reducing agent in cells. Moreover, catalase significantly decreased cell death in the siRNA-transfected cells induced by hypoxia and OGD. These results suggest that maintenance of cellular redox status by HIF-1 protects cells from hypoxia and ischemia mediated injuries.     

Novel role for the δ-opioid receptor in hypoxic preconditioning in rat retinas

δ-Opioid receptor (DOR) is an oxygen-sensitive protein whose function in the rat retina is unknown. We examined whether DOR is involved in hypoxic preconditioning (HPC)-mediated retinoprotection following intraocular pressure (IOP) elevation. Rats were exposed to intermittent hypoxia (10% oxygen) to induce HPC. Unilateral retinal ischemia/reperfusion injury was induced by elevating IOP to 100 mmHg for 1 h. HPC attenuated the loss of neuronal marker expression and increased pro-apoptotic caspase 3 activity in the IOP retina. Excess superoxide production and 8-iso-prostaglandin F2α accumulation caused by enhanced oxidant protein expression and reduced antioxidant enzyme level after IOP elevation were largely abrogated by HPC. HPC markedly increased the expression of hypoxia-inducible factor-1α (HIF-1α) and DOR, but intravitreal administration of HIF-1α-specific small interfering RNA abrogated the up-regulation of DOR. This suggested that DOR functions downstream of HIF-1α. However, the endogenous content of leucine enkephalin in retinas was not affected by HPC or IOP. Treatment of retinas with the DOR antagonist naltrindole attenuated the HPC-induced protection and activation of extracellular signal-regulated kinase. These results suggest a novel mechanism of HPC-mediated retinoprotection whereby HIF-1α induces the expression of DOR, and DOR-mediated activation of extracellular signal-regulated kinase triggers cellular events that correct the redox imbalance in the post-ischemic retina.     

The Role of Hypoxia in the Differentiation of P19 Embryonal Carcinoma Cells into Dopaminergic Neurons

Nervous system development at early stage is in hypoxic environment. Very little is known about the role of hypoxia in neuronal development. P19 embryonal carcinoma (EC) cells are a widely used model for studying early neuronal development. In this study we investigated the roles of hypoxia in differentiation of dopaminergic neurons derived from P19 EC cells. Results demonstrate that hypoxia increases the percentage of differentiated neurons, especially neurons of dopaminergic phenotype. To investigate the potential mechanism involved in hypoxia promoted differentiation of dopaminergic neurons, we measured the expression of hypoxia-inducible factor 1α (HIF-1α), based on its characteristic response to hypoxia. The result shows that HIF-1α mRNA level in P19 EC cells increases after hypoxia treatment. It is known that HIF-1α regulates the expression of tyrosine hydroxylase (TH) gene through binding to its promoter. Therefore, we propose that the underlying mechanism for hypoxia promoted differentiation of dopaminergic neurons was mediated by HIF-1α up-regulation under hypoxia. Yue Wang—Co-first author. Special Issue in honor of Dr. Ji-Sheng Han.     

Expression of Kv1.2 in microglia and its putative roles in modulating production of proinflammatory cytokines and reactive oxygen species

Microglial cells are endowed with different potassium ion channels but their expression and specific functions have remained to be fully clarified. This study has shown Kv1.2 expression in the amoeboid microglia in the rat brain between 1 (P1) and 10 (P10) days of age. Kv1.2 expression was localized in the ramified microglia at P14 and was hardly detected at P21. In postnatal rats exposed to hypoxia, Kv1.2 immunoreactivity in microglia was markedly enhanced. Quantitative RT-PCR analysis confirmed Kv1.2 mRNA expression in microglial cells in vitro . It was further shown that Kv1.2 and protein expression coupled with that of interleukin 1β (IL-1β) and tumor necrosis factor-α (TNF-α) was significantly increased when the cells were subjected to hypoxia. The same increase was observed in cells exposed to adenosine 5'-triphosphate (ATP) and lipopolysaccharide (LPS). Concomitantly, the intracellular potassium concentration decreased significantly. Blockade of Kv1.2 channel with rTityustoxin-Kα (TsTx) resulted in partial recovery of intracellular potassium concentration accompanied by a reduced expression of IL-1β and TNF-α mRNA and protein expression and intracellular reactive oxygen species (ROS) production. We conclude that Kv1.2 in microglia modulates IL-1β and TNF-α expression and ROS production probably by regulating the intracellular potassium concentration.     

HIF-1 alpha is an essential effector for purine nucleoside-mediated neuroprotection against hypoxia in PC12 cells and primary cerebellar granule neurons

Hypoxia-inducible factor-1 alpha (HIF-1α) and purine nucleosides adenosine and inosine are critical mediators of physiological responses to acute and chronic hypoxia. The specific aim of this paper was to evaluate the potential role of HIF-1α in purine-mediated neuroprotection. We show that adenosine and inosine efficiently rescued clonal rat pheochromocytoma (PC12) cells (up to 43.6%) as well as primary cerebellar granule neurons (up to 25.1%) from hypoxic insult, and furthermore, that HIF-1α is critical for purine-mediated neuroprotection. Next, we studied hypoxia or purine nucleoside increased nuclear accumulation of HIF-1α in PC12 cells. As a possible result of increased protein stabilization or synthesis an up to 2.5-fold induction of HIF-1α accumulation was detected. In cerebellar granule neurons, purine nucleosides induced an up to 3.1-fold HIF-1α accumulation in cell lysates. Concomitant with these results, small interfering RNA-mediated reduction of HIF-1α completely abolished adenosine- and inosine-mediated protection in PC12 cells and severely hampered purine nucleoside-mediated protection in primary neurons (up to 94.2%). Data presented in this paper thus clearly demonstrate that HIF-1α is a key regulator of purine nucleoside-mediated rescue of hypoxic neuronal cells.     

Detection of reactive oxygen species via endogenous oxidative pentose phosphate cycle activity in response to oxygen concentration: implications for the mechanism of HIF-1alpha stabilization under moderate hypoxia

The oxidative pentose phosphate cycle (OPPC) is necessary to maintain cellular reducing capacity during periods of increased oxidative stress. Metabolic flux through the OPPC increases stoichiometrically in response to a broad range of chemical oxidants, including those that generate reactive oxygen species (ROS). Here we show that OPPC sensitivity is sufficient to detect low levels of ROS produced metabolically as a function of the percentage of O2. We observe a significant decrease in OPPC activity in cells incubated under severe and moderate hypoxia (ranging from <0.01 to 4% O2), whereas hyperoxia (95% O2) results in a significant increase in OPPC activity. These data indicate that metabolic ROS production is directly dependent on oxygen concentration. Moreover, we have found no evidence to suggest that ROS, produced by mitochondria, are needed to stabilize hypoxia-inducible factor 1alpha (HIF-1alpha) under moderate hypoxia. Myxothiazol, an inhibitor of mitochondrial electron transfer, did not prevent HIF-1alpha stabilization under moderate hypoxia. Moreover, the levels of HIF-1alpha that we observed after exposure to moderate hypoxia were comparable between rho0 cells, which lack functional mitochondria, and the wild-type cells. Finally, we find no evidence for stabilization of HIF-1alpha in response to the non-toxic levels of H2O2 generated by the enzyme glucose oxidase. Therefore, we conclude that the oxygen dependence of the prolyl hydroxylase reaction is sufficient to mediate HIF-1alpha stability under moderate as well as severe hypoxia.     

Reactive oxygen species attenuate nitric-oxide-mediated hypoxia-inducible factor-1alpha stabilization

Tissue hypoxia/ischemia are major pathophysiological determinants. Conditions of decreased oxygen availability provoke accumulation and activation of hypoxia-inducible factor-1 (HIF-1). Recent reports demonstrate a crucial role of HIF-1 for inflammatory events. Regulation of hypoxic responses by the inflammatory mediators nitric oxide (NO) and reactive oxygen species (ROS) is believed to be of pathophysiolgical relevance. It is reported that hypoxic stabilization of HIF-1alpha can be antagonized by NO due to its ability to attenuate mitochondrial electron transport. Likely, the formation of ROS could contribute to this effect. As conflicting results emerged from several studies showing either decreased or increased ROS production during hypoxia, we used experiments mimicking hypoxic intracellular ROS changes by using the redox cycling agent 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), which generates superoxide inside cells. Treatment of A549, HEK293, HepG2, and COS cells with DMNQ resulted in a concentration-dependent raise in ROS which correlated with HIF-1alpha accumulation. By using a HIF-1alpha-von Hippel-Lindau tumor suppressor protein binding assay, we show that ROS produced by DMNQ impaired prolyl hydroxylase activity. When HIF-1alpha is stabilized by NO, low concentrations of DMNQ (<1 microM) revealed no effect, intermediate concentrations of 1 to 40 microM DMNQ attenuated HIF-1alpha accumulation and higher concentrations of DMNQ promoted HIF-1alpha stability. Attenuation of NO-induced HIF-1alpha stability regulation by ROS was mediated by an active proteasomal degradation pathway. In conclusion, we propose that scavenging of NO by ROS and vice versa attenuate HIF-1alpha accumulation in a concentration-dependent manner. This is important to fully elucidate HIF-1alpha regulation under inflammatory conditions.     

The overexpression and nuclear translocation of Trx-1 during hypoxia confers on HepG2 cells resistance to DDP,and GL-V9 reverses the resistance by suppressing the Trx-1/Ref-1 axis

Microenvironmental hypoxia gives many tumor cells the capacity for drug resistance. Thioredoxin family members play critical roles in the regulation of cellular redox homeostasis in a stressed environment. In this study, we established a hypoxia–drug resistance (hypoxia-DR) model using HepG2 cells and discovered that the overexpression and nuclear translocation of thioredoxin-1 (Trx-1) are closely associated with this resistance through the regulation of the metabolism by the oxidative stress response to glycolysis. Intranuclear Trx-1 enhances the DNA-binding activity of HIF-1α via its interaction with and reducing action on Ref-1, resulting in increased expression of glycolysis-related proteins (PDHK1, HKII, and LDHA), glucose uptake, and lactate generation under hypoxia. Meanwhile, we found that GL-V9, a newly synthesized flavonoid derivative, shows an ability to reverse the hypoxia-DR and has low toxicity both in vivo and in vitro. GL-V9 could inhibit the expression and nuclear translocation of Trx-1 and then suppress HIF-1α DNA-binding activity by inhibiting the Trx-1/Ref-1 axis. As a result, glycolysis is weakened and oxidative phosphorylation is enhanced. Thus, GL-V9 leads to an increment in intracellular ROS generation and consequently intensified apoptosis induced by DDP.     

Involvement of Tumor Macrophage HIFs in Chemotherapy Effectiveness: Mathematical Modeling of Oxygen,pH, and Glutathione

The four variables, hypoxia, acidity, high glutathione (GSH) concentration and fast reducing rate (redox) are distinct and varied characteristics of solid tumors compared to normal tissue. These parameters are among the most significant factors underlying the metabolism and physiology of solid tumors, regardless of their type or origin. Low oxygen tension contributes to both inhibition of cancer cell proliferation and therapeutic resistance of tumors; low extracellular pH, the reverse of normal cells, mainly enhances tumor invasion; and dysregulated GSH and redox potential within cancer cells favor their proliferation. In fact, cancer cells under these microenvironmental conditions appreciably alter tumor response to cytotoxic anti-cancer treatments. Recent experiments measured the in vivo longitudinal data of these four parameters with tumor development and the corresponding presence and absence of tumor macrophage HIF-1α or HIF-2α in a mouse model of breast cancer. In the current paper, we present a mathematical model-based system of (ordinary and partial) differential equations to monitor tumor growth and susceptibility to standard chemotherapy with oxygen level, pH, and intracellular GSH concentration. We first show that our model simulations agree with the corresponding experiments, and then we use our model to suggest treatments of tumors by altering these four parameters in tumor microenvironment. For example, the model qualitatively predicts that GSH depletion can raise the level of reactive oxygen species (ROS) above a toxic threshold and result in inhibition of tumor growth.     

Comparison between the effects of normoxia and hypoxia on antioxidant enzymes and glutathione redox state in ex vivo culture of CD34(+) cells

Hypoxia maintained biological characteristics of CD34(+) cells through keeping lower intracellular reactive oxygen specials (ROS) levels. The effects of normoxia and hypoxia on antioxidant enzymes and glutathione redox state were compared in this study. Hypoxia decreased the mRNA expression of both catalase (CAT) and glutathione peroxidase (GPX), but not affected mRNAs expression of superoxide dismutase (SOD). While the cellular GPX activities under hypoxia were apparently less than those under normoxia, neither SOD activities nor CAT activities were affected by hypoxia. The analysis of glutathione redox status and ROS products showed the lower oxidized glutathione (GSSG) levels, the higher reduced glutathione (GSH) levels, the higher GSH/GSSG ratios, and the less O(2)- and H(2)O(2) generation under hypoxia (versus normoxia). Meanwhile more primary CD34(+)CD38(-) cells were obtained when cultivation was performed under hypoxia or with N-acetyl cysteine (the precursor of GSH) under normoxia. These results demonstrated the different responses of anti-oxidative mechanism between normoxia and hypoxia. Additionally, the present study suggested that the GSH-GPX antioxidant system played an important role in HSPCs preservation by reducing peroxidation.     

HIF-1 prevents the overproduction of mitochondrial ROS after cytokine stimulation through induction of PDK-1

Excessive reactive oxygen species (ROS) are toxic to hematopoietic cells. The majority of cellular ROS are derived from mitochondria and glucose metabolism, and cytokines stimulate this process. During hypoxia, hypoxia inducible factor-1 (HIF-1) attenuates hypoxia-induced mitochondrial ROS production through the induction of pyruvate dehydrogenase kinase-1 (PDK-1). Previously, we found that thrombopoietin (TPO) induces the generation of mitochondrial ROS. Interestingly, the TPO-induced production of mitochondrial ROS promotes the activation of HIF-1. Based on these findings, we speculated that TPO-activated HIF-1 functions as a feedback mechanism to block the overproduction of ROS following TPO stimulation. We found that TPO induces the expression of PDK-1 in a TPO-dependent cell line, UT-7/TPO, in a HIF-1-dependent manner. Inhibition of either HIF-1 or PDK-1 resulted in the increased production of ROS following TPO stimulation. Our observations suggest that HIF-1 functions as a ROS sensor to prevent the overproduction of mitochondrial ROS following cytokine stimulation.