Hiroki Daijo

Hydrogen Sulfide Inhibits Hypoxia- But Not Anoxia-Induced Hypoxia-Inducible Factor 1 Activation in a von Hippel-Lindau- and Mitochondria-Dependent Manner

AIMS In addition to nitric oxide and carbon monoxide, hydrogen sulfide (H(2)S) is an endogenously synthesized gaseous molecule that acts as an important signaling molecule in the living body. Transcription factor hypoxia-inducible factor 1 (HIF-1) is known to respond to intracellular reduced oxygen (O(2)) availability, which is regulated by an elaborate balance between O(2) supply and demand. However, the effect of H(2)S on HIF-1 activity under hypoxic conditions is largely unknown in mammalian cells. In this study, we tried to elucidate the effect of H(2)S on hypoxia-induced HIF-1 activation adopting cultured cells and mice. RESULTS The H(2)S donors sodium hydrosulfide and sodium sulfide in pharmacological concentrations reversibly reduced cellular O(2) consumption and inhibited hypoxia- but not anoxia-induced HIF-1α protein accumulation and expression of genes downstream of HIF-1 in established cell lines. H(2)S did not affect HIF-1 activation induced by the HIF-α hydroxylases inhibitors desferrioxamine or CoCl(2). Experimental evidence adopting von Hippel-Lindau (VHL)- or mitochondria-deficient cells indicated that H(2)S did not affect neosynthesis of HIF-1α protein but destabilized HIF-1α in a VHL- and mitochondria-dependent manner. We also demonstrate that exogenously administered H(2)S inhibited HIF-1-dependent gene expression in mice. INNOVATION For the first time, we show that H(2)S modulates intracellular O(2) homeostasis and regulates activation of HIF-1 and the subsequent gene expression induced by hypoxia by using an in vitro system with established cell lines and an in vivo system in mice. CONCLUSIONS We demonstrate that H(2)S inhibits hypoxia-induced HIF-1 activation in a VHL- and mitochondria-dependent manner.

Persisting mild hypothermia suppresses hypoxia-inducible factor-1alpha protein synthesis and hypoxia-inducible factor-1-mediated gene expression.

The transcription factor hypoxia-inducible factor-1 (HIF-1) plays an essential role in regulating gene expression in response to hypoxia-ischemia. Ischemia causes the tissue not only to be hypoxic but also to be hypothermic because of the hypoperfusion under certain circumstances. On the other hand, the induced hypothermia is one of the most common therapeutic modalities to extend tolerance to hypoxia. Although hypoxia elicits a variety of cellular and systemic responses at different organizational levels in the body, little is known about how hypoxia-induced responses are affected by low temperature. We examined the influence of mild hypothermic conditions (28-32 degrees C) on HIF-1 in both in vitro and in vivo settings. In vitro experiments adopting cultured cells elucidated that hypoxia-induced HIF-1 activation was resistant to 4-h exposure to the low temperature. In contrast, exposure to the low temperature as long as 24 h suppressed HIF-1 activation and the subsequent upregulation of HIF-1 target genes such as VEGF or GLUT-1. HIF-1alpha protein stability in the cell was not affected by hypothermic treatment. Furthermore, intracellular ATP content was reduced under 1% O(2) conditions but was not largely affected by hypothermic treatment. The evidence indicates that reduction of oxygen consumption is not largely involved in suppression of HIF-1. In addition, we demonstrated that HIF-1 DNA-binding activity and HIF-1-dependent gene expressions induced under 10% O(2) atmosphere in mouse brain were not influenced by treatment under 3-h hypothermic temperature but were inhibited under 5-h treatment. On the other hand, we indicated that warming ischemic legs of mice for 24 h preserved HIF-1 activity. In this report we describe for the first time that persisting low temperature significantly reduced HIF-1alpha neosynthesis under hypoxic conditions, leading to a decrease in gene expression for adaptation to hypoxia in both in vitro and in vivo settings.

General anesthetics inhibit erythropoietin induction under hypoxic conditions in the mouse brain.

Background Erythropoietin (EPO), originally identified as a hematopoietic growth factor produced in the kidney and fetal liver, is also endogenously expressed in the central nervous system (CNS). EPO in the CNS, mainly produced in astrocytes, is induced under hypoxic conditions in a hypoxia-inducible factor (HIF)-dependent manner and plays a dominant role in neuroprotection and neurogenesis. We investigated the effect of general anesthetics on EPO expression in the mouse brain and primary cultured astrocytes. Methodology/Principal Findings BALB/c mice were exposed to 10% oxygen with isoflurane at various concentrations (0.10–1.0%). Expression of EPO mRNA in the brain was studied, and the effects of sevoflurane, halothane, nitrous oxide, pentobarbital, ketamine, and propofol were investigated. In addition, expression of HIF-2α protein was studied by immunoblotting. Hypoxia-induced EPO mRNA expression in the brain was significantly suppressed by isoflurane in a concentration-dependent manner. A similar effect was confirmed for all other general anesthetics. Hypoxia-inducible expression of HIF-2α protein was also significantly suppressed with isoflurane. In the experiments using primary cultured astrocytes, isoflurane, pentobarbital, and ketamine suppressed hypoxia-inducible expression of HIF-2α protein and EPO mRNA. Conclusions/Significance Taken together, our results indicate that general anesthetics suppress activation of HIF-2 and inhibit hypoxia-induced EPO upregulation in the mouse brain through a direct effect on astrocytes.

The intravenous anesthetic propofol inhibits lipopolysaccharide-induced hypoxia-inducible factor 1 activation and suppresses the glucose metabolism in macrophages

PurposeHypoxia-inducible factor 1 (HIF-1) is a master transcription factor of hypoxia-induced gene expression. Anesthetics and perioperative drugs have been reported to affect HIF-1 activity. However, the effect of propofol on HIF-1 activity is not well documented. In this study, we investigated the effect of propofol on HIF-1 activation using macrophage-differentiated THP-1 cells.MethodsCells were exposed to lipopolysaccharide (LPS) under 20 or 1% O2 conditions with or without propofol treatment. The cell lysate was subjected to Western blot analysis using anti-HIF-1α and HIF-1β antibodies. HIF-1-dependent gene expression was investigated by quantitative real-time reverse-transcriptase PCR analysis and luciferase assay. The amount of cellular lactate and ATP was assayed.ResultsPropofol suppressed HIF-1α protein accumulation induced by LPS, but not by hypoxia in the THP-1 cells in a dose-dependent manner by inhibiting the neo-synthesis of HIF-1α protein. Induction of the HIF-1 downstream gene expression including glucose transporter 1, enolase 1, lactate dehydrogenase A, pyruvate dehydrogenase kinase-1 and vascular endothelial growth factor was inhibited by propofol. Propofol suppressed LPS-induced lactate accumulation and ATP content in THP-1 cells.ConclusionOur experimental results indicate that propofol inhibits HIF-1 activation and downstream gene expression induced by LPS and suppressed HIF-1-dependent glucose metabolic reprogramming. HIF-1 suppression by propofol in macrophages may explain molecular mechanisms behind the inhibitory effect of propofol on cellular inflammatory responses.

The intravenous anesthetics barbiturates inhibit hypoxia-inducible factor 1 activation

Hypoxia-inducible factor 1 (HIF-1) is a master transcription factor of hypoxia-induced gene expression. Anesthetics and perioperative drugs have been reported to affect HIF-1 activity. However, the effect of barbiturates on HIF-1 activity has not been reported. In this study, we investigated the effect of thiopental and thiamylal on HIF-1 activity using the neuronal SH-SY5Y cells, the non-neuronal HEK293 cells, and the macrophage-differentiated THP-1 cells. Cells were exposed to 20% or 1% O(2) conditions with or without thiopental or thiamylal treatment. The cell lysate were subjected to Western blot analysis using anti-HIF-1alpha and -HIF-1beta antibodies. HIF-1-dependent gene expression was investigated by semi-quantitative real-time RT-PCR and luciferase assay. Hydroxylation of HIF-1alpha protein was evaluated by in vitro pulldown assay using recombinant protein. Both thiopental and thiamylal reversibly suppressed hypoxia-induced HIF-1 activation in the neuronal and the non-neuronal cells in a dose-dependent manner. Moreover, the barbiturates inhibited lipopolysaccharide-induced HIF-1alpha expression in THP-1 cells. The HIF-1-downstream gene expression was also inhibited by the barbiturates. HIFalpha-hydroxylases activity and HIF-1alpha stability were not affected but the HIF-1alpha protein neosynthesis was inhibited by the barbiturates. Our experimental results indicate that barbiturates inhibit induced HIF-1 activation and downstream genes expression.

Fentanyl activates hypoxia-inducible factor 1 in neuronal SH-SY5Y cells and mice under non-hypoxic conditions in a μ-opioid receptor-dependent manner

Hypoxia-inducible factor 1 (HIF-1) is the main transcription factor responsible for hypoxia-induced gene expression. Perioperative drugs including anesthetics have been reported to affect HIF-1 activity. However, the effect of fentanyl on HIF-1 activity is not well documented. In this study, we investigated the effect of fentanyl and other opioids on HIF-1 activity in human SH-SY5Y neuroblastoma cells, hepatoma Hep3B cells, lung adenocarcinoma A549 cells and mice. Cells were exposed to fentanyl, and HIF-1 protein expression was examined by Western blot analysis using anti-HIF-1α and β antibodies. HIF-1-dependent gene expression was investigated by semi-quantitative real-time reverse transcriptase (RT)-PCR (qRT-PCR) and luciferase assay. Furthermore, fentanyl was administered intraperitoneally and HIF-1-dependent gene expression was investigated by qRT-PCR in the brains and kidneys of mice. A 10-μM concentration of fentanyl and other opioids, including 1 μM morphine and 4 μM remifentanil, induced HIF-1α protein expression and HIF-1 target gene expression in an opioid receptor-dependent manner in SH-SY5Y cells with activity peaking at 24h. Fentanyl did not augment HIF-1α expression during hypoxia-induced induction. HIF-1α stabilization assays and experiments with cycloheximide revealed that fentanyl increased translation from HIF-1α mRNA but did not stabilize the HIF-1α protein. Furthermore, fentanyl induced HIF-1 target gene expression in the brains of mice but not in their kidneys in a naloxone-sensitive manner. In this report, we describe for the first time that fentanyl, both in vitro and in vivo, induces HIF-1 activation under non-hypoxic conditions, leading to increases in expression of genes associated with adaptation to hypoxia.

Cigarette smoke reversibly activates hypoxia-inducible factor 1 in a reactive oxygen species-dependent manner

Cigarette smoke (CS) is a major contributor to the development of a large number of fatal and debilitating disorders. However, the precise molecular mechanisms underlying the effects of CS in lung disease are largely unknown. To elucidate these pathophysiological processes, we examined the in vitro and in vivo effects of CS extract (CSE) and CS on the transcription factor, hypoxia-inducible factor 1 (HIF-1). CSE induced concentration- and time-dependent accumulation of HIF-1α protein in human lung epithelial-like cells under non-hypoxic conditions. Genes upregulated by HIF-1, including vascular endothelial growth factor and regulated in development and DNA damage response 1, both of which are involved in smoking-induced emphysematous changes, were increased by CSE treatment under non-hypoxic conditions in vitro and in vivo. Further investigation revealed that reactive oxygen species were generated in cells exposed to CSE and were required for CSE-mediated induction of HIF-1α protein, as was activation of phosphoinositide 3-kinase and mitogen-activated protein kinase pathways. In conclusion, we demonstrated that CSE and CS induced HIF-1 activation in vitro and in vivo, respectively. The evidence warrants further investigation to indicate that HIF-1 plays an important role in CS-induced gene expression, which is deeply involved in pulmonary cellular stress and small airway remodelling.

Anesthesia management for emergency laparotomy in a pediatric patient with suspected hereditary angioedema.

Hereditary angioedema (HAE) is caused by complement factor 1 inhibitor (C1-INH) deficiency, and its mode of inheritance is autosomal dominant. We present a case of an 8-year-old patient who required emergency laparotomy after a traffic accident. General anesthesia with tracheal intubation was necessary. The patient’s mother and maternal grandmother had been diagnosed with HAE. HAE is associated with high mortality when airway edema is caused by tracheal intubation. It was impossible to rule out HAE preoperatively in the patient. Therefore, we presumed that he had HAE and treated him with pasteurized C1-INH concentrate. The patient underwent laparotomy uneventfully. Several days after the operation, the laboratory data revealed that the perioperative plasma complement 1 q subunit (C1q) protein level and C1-INH function were not lowered. The diagnosis of HAE was not confirmed, but it was not possible to rule out the diagnosis either. The prophylactic use of a C1-INH in this case may be justified, because the procedure was an emergency and because of the high mortality associated with tracheal intubation in patients with HAE.

Intravascular Ultrasound Observation of an Obstruction of the Left Main Coronary Artery Caused by Displaced Leaflet Calcification and Hematoma After Transcatheter Aortic Valve Implantation

An 89-year-old woman underwent transcatheter aortic valve implantation (TAVI) using a transapical approach for severe senile calcific aortic stenosis (area = 0.39 cm2; peak and mean gradients = 176 mm Hg and 103 mm Hg, respectively; ejection fraction = 62%). Preprocedural computed tomography imaging revealed severe calcification of the left coronary cusp and a small sinus of Valsalva (Figure 1A). The patient was considered high-risk for coronary obstruction after TAVI. Figure 1. Computed tomography (CT) imaging before and after transcatheter aortic valve implantation (TAVI). A , CT imaging before TAVI showing a severe calcification of the left coronary cusp and a small sinus of valsalva. B , CT imaging after TAVI showing the displaced leaflet calcification and …

Persisting mild hypothermia suppresses hypoxia-inducible factor-1α protein synthesis and hypoxia-inducible factor-1-mediated gene expression

The transcription factor hypoxia-inducible factor-1 (HIF-1) plays an essential role in regulating gene expression in response to hypoxia-ischemia. Ischemia causes the tissue not only to be hypoxic but also to be hypothermic because of the hypoperfusion under certain circumstances. On the other hand, the induced hypothermia is one of the most common therapeutic modalities to extend tolerance to hypoxia. Although hypoxia elicits a variety of cellular and systemic responses at different organizational levels in the body, little is known about how hypoxia-induced responses are affected by low temperature. We examined the influence of mild hypothermic conditions (28-32 degrees C) on HIF-1 in both in vitro and in vivo settings. In vitro experiments adopting cultured cells elucidated that hypoxia-induced HIF-1 activation was resistant to 4-h exposure to the low temperature. In contrast, exposure to the low temperature as long as 24 h suppressed HIF-1 activation and the subsequent upregulation of HIF-1 target genes such as VEGF or GLUT-1. HIF-1alpha protein stability in the cell was not affected by hypothermic treatment. Furthermore, intracellular ATP content was reduced under 1% O(2) conditions but was not largely affected by hypothermic treatment. The evidence indicates that reduction of oxygen consumption is not largely involved in suppression of HIF-1. In addition, we demonstrated that HIF-1 DNA-binding activity and HIF-1-dependent gene expressions induced under 10% O(2) atmosphere in mouse brain were not influenced by treatment under 3-h hypothermic temperature but were inhibited under 5-h treatment. On the other hand, we indicated that warming ischemic legs of mice for 24 h preserved HIF-1 activity. In this report we describe for the first time that persisting low temperature significantly reduced HIF-1alpha neosynthesis under hypoxic conditions, leading to a decrease in gene expression for adaptation to hypoxia in both in vitro and in vivo settings.