Aki Hirayama

PPAR-γ Activator Pioglitazone Prevents Age-Related Atrial Fibrillation Susceptibility by Improving Antioxidant Capacity and Reducing Apoptosis in a Rat Model

PPAR‐γ Activator as Upstream Therapy for Atrial Fibrillation in Rat. Introduction: The in vivo role of peroxisome proliferator‐activated receptor (PPAR)‐γ, an essential transcriptional mediator of lipid and glucose metabolism, in atrial fibrillation (AF) remains to be fully elucidated. We investigated the effects of pioglitazone, a PPAR‐γ activator, in an in vivo AF rat model.

Hyperglycemia induces oxidative and nitrosative stress and increases renal functional impairment in Nrf2-deficient mice

The transcription factor Nrf2 regulates the expression of antioxidant genes. Hyperglycemia‐induced oxidative stress is involved in the pathogenesis of diabetes and its complications. However, little is known about the protective role of Nrf2 in diabetes. To gain insight into the protective role of Nrf2 in diabetes we treated Nrf2 knockout (Nrf2 KO) mice with streptozotocin (STZ). The STZ Nrf2 KO mice did not develop renal hyperfiltration, which was observed in the STZ‐treated wild‐type (STZ WT) mice, but renal function gradually deteriorated over the 10‐week observation period. Urinary excretion of nitric oxide metabolites and the occurrence of 8‐nitroguanosine, which was detected in glomerular lesions, were increased in STZ Nrf2 KO mice during the early stages after treatment. In vivo electron paramagnetic resonance analysis revealed an accelerated rate of decay of the 3‐carbamoyl‐2,2,5,5‐tetramethylpyrrolidine‐1‐oxyl spin probe signal in STZ Nrf2 KO mice. The addition of superoxide dismutase prolonged the half‐life of the signal, which suggested that increased oxygen radical formation occurred in the STZ Nrf2 KO mice. These results suggested that hyperglycemia increased oxidative and nitrosative stress and accelerated renal injury in the Nrf2 KO mice and that Nrf2 serves as a defense factor against some diabetic complications.

Nrf2 regulates the sensitivity of death receptor signals by affecting intracellular glutathione levels

Nrf2 is a basic leucine zipper transcriptional activator that is essential for the coordinate transcriptional induction of various antioxidant drug-metabolizing enzymes. Numerous studies have firmly established Nrf2s importance in protection from oxidative stress and certain chemical insults. Given the protective function of Nrf2, surprisingly few studies have focused on the relationship between Nrf2 and apoptosis. Therefore, we analysed how Nrf2 influences Fas signaling using Nrf2-deficient T cells. At a concentration of 1 μg/ml, the anti-Fas antibody induced 60% of cell death in Nrf2-deficient cultured thymocytes while, using the same treatment, only 40% of Nrf2 wild-type thymocytes died (P<0.05). Nrf2 deficiency enhances the sensitivity of Fas-mediated apoptosis in T cells. Next we examined the effect of Nrf2 deficiency during hepatocellular apoptosis in vivo. In comparison to wild-type mice, Nrf2-deficient mice displayed more severe hepatitis after induction with the anti-Fas antibody or tumor necrosis factor (TNF)-α. The enhanced sensitivity to anti-Fas or TNF-α stimulation was restored by preadministration of glutathione ethyl monoester, a compound capable of passing the cell membrane and upregulating the intracellular levels of glutathione. The results indicated that Nrf2 activity regulates the sensitivity of death signals by means of intracellular glutathione levels.

pH-sensitive radical-containing-nanoparticle (RNP) for the L-band-EPR imaging of low pH circumstances.

For the imaging of low pH circumstances in vivo, a pH-sensitive radical-containing-nanoparticle (RNP), which has an intense electron paramagnetic resonance (EPR) signal, was designed and developed using a self-assembling amphiphilic block copolymer (PEG-b-PCTEMPO) composed of a hydrophilic poly(ethylene glycol) (PEG) segment and a hydrophobic poly(chloromethylstyrene) (PCMS) segment in which the chloromethyl groups were converted to 2,2,6,6-tetramethylpiperidinyloxys (TEMPOs) via the amination of PEG-b-PCMS block copolymer with 4-amino-TEMPO. This RNP formed core-shell-type micelles in the physiological environment, and the cumulant average diameter of the RNP was about 50 nm. The cytotoxicity and acute toxicity studies for the RNP revealed that the median inhibitory concentration (IC(50)) of TEMPO radicals in RNP core and median lethal dose (LD(50)) of RNP were >8 mmol N(TEMPO)/L and >600 mg/kg (>960 mumol N(TEMPO)/kg), respectively, indicating fairly low toxicity. The blood circulation of the RNP was evaluated using ICR mice. Contrary to the rapid clearance of low-molecular-weight TEMPO derivatives such as 4-hydroxy-TEMPO (TEMPOL) from the bloodstream, the EPR signal of the RNP remained for a fairly long period of time. Actually, the signal was observed in the blood for more than 2 h, as monitored by EPR spectroscopy. The compartmentalization of the TEMPO radicals in the RNP core improved the stability in the bloodstream. Since an amino group was introduced in each repeating unit of the PCTEMPO segment, the disintegration of the RNP was caused by the protonation of the amino groups in response to the acidic pH environment (pH < 6.0), as confirmed by the dynamic light scattering (DLS) measurements. In addition, a drastic change in the EPR spectra from broad to sharp triplet was observed, accompanying the disintegration. This change was based upon the mobility of the TEMPO moieties covalently conjugated in the hydrophobic segment, which was confirmed by the rotational correlation time of the TEMPO moieties on the PCTEMPO segment. Note that the peak intensity of the EPR signal increased at around the phase transition point (ca. pH = 6.0). When pH-sensitive RNP solutions at pH values 5.6 and 7.4 were visualized using an L-band EPR imaging system, the phantom images showed a remarkable on-off regulation in response to the acidic pH environment. These results demonstrate that pH-sensitive RNPs are expected to serve as nanoprobes for the in vivo EPR imaging of low pH circumstances.

EPR IMAGING OF REDUCING ACTIVITY IN Nrf2 TRANSCRIPTIONAL FACTOR-DEFICIENT MICE

Mice that lack the Nrf2 (NF-E2-related factor 2) transcription factor develop a lupus-like autoimmune nephritis. The tissue-reducing activity of Nrf2-deficient mice was evaluated using a combination of real-time EPR imaging and spin probe kinetic analysis. Substantial delay in the spin probe 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (Carbamoyl-PROXYL) disappearance in the liver and kidneys of Nrf2-deficient mice was observed by EPR imaging. The half-life of the spin probe in the upper abdominal area was prolonged in both the Nrf2-deficient mice and in aged mice. The combination of Nrf2 deficiency and aging in female mice resulted in the most prolonged half-life of disappearance, which was four times longer than that of juvenile female mice with a wild-type genotype. These results indicate that the low reducing activity in these organs is brought about by both Nrf2 deficiency and the aging process, and it may play a key role in the onset of autoimmune nephritis. This combination of the EPR imaging and half-life analysis appears to be a very powerful tool in the real-time analysis of reducing activity.

Redox regulation in radiation-induced cytochrome c release from mitochondria of human lung carcinoma A549 cells.

Mitochondria in mammalian cells are well-known to play an important role in the intrinsic pathway of genotoxic-agent-induced apoptosis by releasing cytochrome c into cytosol and to be a major source of reactive oxygen species (ROS). The aim of this study was to examine whether mitochondrial ROS are involved in radiation-induced apoptotic signaling in A549 cells. Post-irradiation treatment with N-acetyl-L-cysteine (NAC) inhibited cytochrome c release from mitochondria but did not affect expression levels of Bcl-2, Bcl-X(L) and Bax, suggesting that late production of ROS triggered cytochrome c release. Experiments using DCFDA (a classical ROS fluorescence probe) and MitoAR (a novel mitochondrial ROS probe) demonstrated that intracellular and mitochondrial ROS were enhanced 6h after X irradiation. Furthermore, the O(2)(-*) production ability of mitochondria isolated from A549 cells was evaluated by ESR spectroscopy combined with a spin-trapping reagent (CYPMPO). When isolated mitochondria were incubated with NADH, succinate and CYPMPO, an ESR spectrum due to CYPMPO-OOH was detected. This NADH/succinate-dependent O(2)(-*) production from mitochondria of irradiated cells was significantly increased in comparison with that of unirradiated cells. These results indicate that ionizing radiation enhances O(2)(-*) production from mitochondria to trigger cytochrome c release in A549 cells.

Association of ecNOS gene polymorphisms with end stage renal diseases

Nitric oxide (NO) is a very potent regulator of intrarenal hemodynamics and is thought to be an important factor in the deterioration of renal function. Several polymorphisms of the endothelial NO synthase (eNOS) gene have been reported. For instance, tandem 27-bp repeats in intron 4 of the eNOS gene are polymorphic, i.e. eNOS4a allele has 4 and eNOS4b has 5 tandem repeats, and the association between eNOS4a and myocardial infarction has been reported. In addition, a missense Glu298Asp mutation in exon 7 of the eNOS gene is reported to be a risk factor for hypertension or myocardial infarction. In this study, we investigated the frequencies of these 2 polymorphisms of eNOS gene in patients with end-stage renal diseases (ESRD), and compared them with those of healthy subjects.Genomic DNA was obtained from regularly hemodialyzed patients and healthy volunteers. The allele frequencies of eNOS4a and eNOS4b in intron 4 were analyzed by PCR and the missense Glu298Asp mutation in exon 7 were determined by PCR FMLP analysis.The allele frequency of eNOS4a (eNOS4a/b and eNOS4a/a) in non-diabetic group is significantly higher than that in healthy controls (27.3% vs. 19.0%, p = 0.01) though there is no significant difference between diabetic group and healthy controls. On the other hand, the frequencies of missense Glu298Asp mutation in both non-diabetic and diabetic groups are significantly higher than that in healthy controls (22.5% in non-diabetic, 20.8% in diabetic and 7.4% in control group, p = 0.002: non-diabetic vs. control, p = 0.01: diabetic vs. control).This study clarified that the polymorphisms in intron 4 and exon 7 of eNOS gene are the genetic risk factors for ESRD. The polymorphisms in intron may change the transcriptional activity and those in exon may alter the 3 dimensional structure of the enzyme, and may affect the progression of renal diseases via decreased NO synthesis. Further study is required to clarify the detailed mechanisms.

Newly Synthesized Radical-Containing Nanoparticles Enhance Neuroprotection After Cerebral Ischemia-Reperfusion Injury

BACKGROUND:Antioxidant nitroxyl radicals such as 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) have been investigated for their ability to scavenge free radicals produced by ischemia-reperfusion injury. However, the short in vivo half-life and toxicity of TEMPO have limited their clinical application. OBJECTIVE:We developed a core-shell-type nanoparticle, termed a radical-containing nanoparticle (RNP), to deliver nitroxyl radicals with prolonged in vivo half-life and pH-sensitivity. We evaluated the ability of RNP to deliver TEMPO radicals to the ischemic brain and scavenge free radicals in cerebral ischemia-reperfusion injury using rats. METHODS:When RNPs were administrated to middle cerebral artery occlusion rats, the delivery and clearance of RNPs were detected using electron paramagnetic resonance (EPR) assay. The production of superoxide anion in neuronal cells was observed with dihydroethidium staining. The treatment effects were evaluated by measuring the cerebral infarction volumes, lipid peroxidation and protein oxidation, and neurological symptom scoring. RESULTS:The TEMPO radicals contained in RNPs were detected for 6 hours after intravenous administration as a triplet EPR signal in the ischemic brain, and RNPs significantly reduced the production of superoxide anion in neuronal cells compared with saline and 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyls (TEMPOL). The infarction volumes of rats treated by RNPs were significantly lower than those of rats treated by saline, micelles, and TEMPOL. In addition, RNP treatment suppressed lipid peroxidation and protein oxidation, and limited the adverse effects of TEMPO radicals such as hypotension. CONCLUSION:RNPs could be a promising neuroprotective agent with their enhanced ability to scavenge free radicals and reduced toxicity.

Hemodialysis Does Not Influence the Peroxidative State Already Present in Uremia

Hemodialysis (HD) patients are exposed to high oxidative stress, however, the nature of this stress is still unclear. In this study, we employed a specific lipid peroxidative product, phosphatidylcholine hydroperoxide (PCOOH), and evaluated the peroxidative effect of end stage renal disease by measuring thiobarbituric acid reactive substances (TBARS) and PCOOH in both plasma and erythrocyte membrane. We also surveyed plasma TBARS and PCOOH before and after HD sessions thereby assessing oxidative stress by a single HD procedure. The plasma TBARS level of healthy controls was 2.9 ± 0.4 nmol/ml. Those of HD patients before and after HD session were 5.1 ± 1.4 and 3.1 ± 0.5 nmol/ml, respectively, and the pre-HD plasma TBARS levels were significantly higher than those of controls and after HD. The Plasma PCOOH concentration of patients before HD was 119.7 ± 58.4 pmol/ml and was significantly higher than that of controls which was 88.6 ± 14.3 pmol/ml. After HD, the plasma PCOOH level decreased to 103.2 ± 36.0 pmol/ml, which was still significantly higher than that of controls. In erythrocytes, the PCOOH level of patients was 259.3 ± 105.4 nmol/g RBC and was significantly higher than that of controls with 88.6 ± 32.0 nmol/g RBC. Analyzed with respect to the cause of renal disease, the polycystic kidney disease patients showed significantly lower plasma PCOOH levels than the others. These results suggest that there is an increase of lipid peroxidation in both plasma and erythrocytes of HD patients, though this oxidative stress was not brought about by HD.

FORMATION OF GUANIDINOSUCCINIC ACID, A STABLE NITRIC OXIDE MIMIC, FROM ARGININOSUCCINIC ACID AND NITRIC OXIDE-DERIVED FREE RADICALS

Guanidinosuccinic acid (GSA) is noted for its nitric oxide (NO) mimicking actions such as vasodilatation and activation of the N-methyl-D-aspartate (NMDA) receptor. We have reported that GSA is the product of argininosuccinate (ASA) and some reactive oxygen species, mainly the hydroxyl radical. We tested for GSA synthesis in the presence of NO donors. ASA (1 mM) was incubated with NOR-2, NOC-7 or 3-morpholinosydomine hydrochloride (SIN-1) at 37 degrees C. GSA was determined by HPLC using a cationic resin for separation and phenanthrenequinone as an indicator. Neither NOR-2 or NOC-7 formed GSA. SIN-1, on the other hand, generates NO and the superoxide anion which, in turn, generated peroxynitrite which was then converted to the hydroxyl radical. Incubation of ASA with SIN-1 leads, via this route, to GSA. When ASA was incubated with 1 mM SIN-1, the amount of GSA produced depended on the incubation time and the concentration of ASA. Among the tested SIN-1 concentrations, from 0.5 to 5 mM, GSA synthesis was maximum at 0.5 mM and decreased with increasing concentrations of SIN-1. Carboxy-PTIO, a NO scavenger, completely inhibited GSA synthesis. SOD, a superoxide scavenger, decreased GSA synthesis by 20%, and catalase inhibited GSA synthesis only by 12%; DMSO, a hydroxyl radical scavenger completely inhibited GSA synthesis in the presence of SIN-1. These data suggest that the hydroxyl radical derived from a combination of NO and the superoxide anion generates GSA, a stable NO mimic. Meanwhile, synthesis of GSA by NO produces reactive oxygen and activates the NMDA receptor that generates NO from GSA, suggesting a positive feed back mechanism.