Motohide Shimazu

Calcium-induced generation of reactive oxygen species in brain mitochondria is mediated by permeability transition.

Mitochondrial uptake of calcium in excitotoxicity is associated with subsequent increase in reactive oxygen species (ROS) generation and delayed cellular calcium deregulation in ischemic and neurodegenerative insults. The mechanisms linking mitochondrial calcium uptake and ROS production remain unknown but activation of the mitochondrial permeability transition (mPT) may be one such mechanism. In the present study, calcium increased ROS generation in isolated rodent brain and human liver mitochondria undergoing mPT despite an associated loss of membrane potential, NADH and respiration. Unspecific permeabilization of the inner mitochondrial membrane by alamethicin likewise increased ROS independently of calcium, and the ROS increase was further potentiated if NAD(H) was added to the system. Importantly, calcium per se did not induce a ROS increase unless mPT was triggered. Twenty-one cyclosporin A analogs were evaluated for inhibition of calcium-induced ROS and their efficacy clearly paralleled their potency of inhibiting mPT-mediated mitochondrial swelling. We conclude that while intact respiring mitochondria possess powerful antioxidant capability, mPT induces a dysregulated oxidative state with loss of GSH- and NADPH-dependent ROS detoxification. We propose that mPT is a significant cause of pathological ROS generation in excitotoxic cell death.

HIF-1α is necessary to support gluconeogenesis during liver regeneration

Coordinated recovery of hepatic glucose metabolism is prerequisite for normal liver regeneration. To examine roles of hypoxia inducible factor-1alpha (HIF-1alpha) for hepatic glucose homeostasis during the reparative process, we inactivated the gene in hepatocytes in vivo. Following partial hepatectomy (PH), recovery of residual liver weight was initially retarded in the mutant mice by down-regulation of hepatocyte proliferation, but occurred comparably between the mutant and control mice at 72h after PH. At this time point, the mutant mice showed lowered blood glucose levels with enhanced accumulation of glycogen in the liver. The mutant mice exhibited impairment of hepatic gluconeogenesis as assessed by alanine tolerance test. This appeared to result from reduced expression of PGK-1 and PEPCK since 3-PG, PEP and malate were accumulated to greater extents in the regenerated liver. In conclusion, these findings provide evidence for roles of HIF-1alpha in the regulation of gluconeogenesis under liver regeneration.

Cyclophilin D-Sensitive Mitochondrial Permeability Transition in Adult Human Brain and Liver Mitochondria

The mitochondrial permeability transition (mPT) is considered to be a major cause of cell death under a variety of pathophysiological conditions of the central nervous system (CNS) and other organs. Pharmacological inhibition or genetic knockout of the matrix protein cyclophilin D (CypD) prevents mPT and cell degeneration in several models of brain injury. If these findings in animal models are translatable to human disease, pharmacological inhibition of mPT offers a promising therapeutic target. The objective of this study was to validate the presence of a CypD-sensitive mPT in adult human brain and liver mitochondria. In order to perform functional characterization of human mitochondria, fresh tissue samples were obtained during hemorrhage or tumor surgery and mitochondria were rapidly isolated. Mitochondrial calcium retention capacity, a quantitative assay for mPT, was significantly increased by the CypD inhibitor cyclosporin A in both human brain and liver mitochondria, whereas thiol-reactive compounds and oxidants sensitized mitochondria to calcium-induced mPT. Brain mitochondria underwent swelling upon calcium overload, which was reversible upon calcium removal. To further explore mPT of human mitochondria, liver mitochondria were demonstrated to exhibit several classical features of the mPT phenomenon, such as calcium-induced loss of membrane potential and respiratory coupling, as well as release of the pro-apoptotic protein cytochrome c. We concluded that adult viable human brain and liver mitochondria possess an active CypD-sensitive mPT. Our findings support the rationale of CypD and mPT inhibition as pharmacological targets in acute and chronic neurodegeneration.

Magnetic compression anastomosis for biliary obstruction: review and experience at Tokyo Medical University Hospital

BackgroundMagnetic compression anastomosis (MCA) is a revolutionary, minimally invasive method of performing choledochoenterostomy or choledochocholedochostomy without using surgical techniques in patients with biliary stricture or obstruction. Herein, we describe a case series of MCA for severe biliary stricture or obstruction, which could not be treated with conventional therapies.Patients and methodsTwo patients with biliary obstruction were treated using MCA for choledochocholedochostomy and choledochoenterostomy at Tokyo Medical University Hospital and Tokyo Medical University Hachioji Medical Center. Endoscopically, a samarium–cobalt (Sm–Co) rare-earth magnet was placed at the superior site of obstruction through the percutaneous transhepatic biliary drainage route and another Sm–Co magnet was placed at the inferior site of obstruction. A comprehensive computer-aided literature search for MCA was performed up to September 2009 by using MEDLINE and EMBASE.ResultsMCA techniques enabled complete anastomosis in both cases without procedure-related complications.ConclusionsThe MCA technique is a revolutionary method of performing choledochocholedochostomy and choledochoenterostomy interventionally in patients with biliary obstruction, for whom the conventional endoscopic procedure is not available, or in candidates who are deemed unsuitable for surgery.

Hypoxia-inducible factor-1α expression and gemcitabine chemotherapy for pancreatic cancer

The normal pancreas has an abundant blood flow, in contrast to pancreatic cancer, which is a hypovascular tumor. During hypoxia under a hypovascular environment, the transcription factor hypoxia-inducible factor-1α (HIF-1α) is activated. High HIF-1α expression reduces sensitivity to gemcitabine (GEM) which is used as a treatment for pancreatic cancer. The objective of this study was to clarify HIF-1α expression in pancreatic cancer and the association of its effects to GEM treatment. We used the human pancreatic ductal carcinoma cell lines AsPC-1 and BxPC-3 to evaluate cell proliferation, HIF-1α protein expression and sensitivity to GEM in a hypoxic environment of 1% O2 in 48 pancreatic cancer patients who received adjuvant GEM treatment after pancreatectomy. We divided the patients according to HIF-1α expression and the presence of single nucleotide polymorphisms, and we based our evaluation on the adverse events associated with GEM chemotherapy and patient outcome. The hypoxic environment promoted cell proliferation, induced HIF-1α expression and increased GEM resistance, especially in AsPC-1 cells, which included a mutant homozygote for HIF-1α(C1772T). There were no significant differences between the HIF-1α(-) and HIF-1α(+) groups in either adverse events or patient outcomes. HIF-1α enhanced neo-microvascularity in a hypoxic environment and increased drug resistance. The period until recurrence was shorter in the patients with a strong HIF-1α expression, than that in those with a weak HIF-1α expression.

Evaluation of putative inhibitors of mitochondrial permeability transition for brain disorders ― Specificity vs. toxicity

Inhibition of mitochondrial permeability transition (mPT) has emerged as a promising approach for neuroprotection and development of well-tolerated mPT inhibitors with favorable blood-brain barrier penetration is highly warranted. In a recent study, 28 clinically available drugs with a common heterocyclic structure were identified as mPT inhibitors e.g. trifluoperazine, promethazine and nortriptyline. In addition, neuroprotection by structurally unrelated drugs e.g. neurosteroids, 4-hydroxy-tamoxifen and trimetazidine has been attributed to direct inhibition of mPT. The regulation of mPT is complex and highly dependent on the prevailing experimental conditions. Several features of mPT, such as swelling, depolarization or NADH oxidation, can also occur independently of the mPT phenomenon. Here, in isolated rodent brain-derived and human liver mitochondria, we re-evaluate drugs promoted as potent mPT inhibitors. We address the definition of an mPT inhibitor and present strategies to reliably detect mPT inhibition in vitro. Surprisingly, none of the 12 compounds tested displayed convincing mPT inhibition or effects comparable to cyclophilin D inhibition by the non-immunosuppressive cyclophilin inhibitor D-MeAla(3)-EtVal(4)-Cyclosporin (Debio 025). Propofol and 2-aminoethoxydiphenyl borate (2-APB) inhibited swelling in de-energized mitochondria but did not increase calcium retention capacity (CRC). Progesterone, trifluoperazine, allopregnanolone and 4-hydroxy-tamoxifen dose-dependently reduced CRC and respiratory control and were thus toxic rather than beneficial to mitochondrial function. Interestingly, topiramate increased CRC at high concentrations likely by a mechanism separate from direct mPT inhibition. We conclude that a clinically relevant mPT inhibitor should have a mitochondrial target and increase mitochondrial calcium retention at concentrations which can be translated to human use.

Neutralization of Blood Group A-Antigen by a Novel Anti-A Antibody: Overcoming ABO-Incompatible Solid-Organ Transplantation

Background. The major barrier to ABO-incompatible solid-organ transplantation is acute humoral rejection. It is known to be triggered by antidonor blood group A/B antibodies, which might bind to A/B-antigen on the endothelium of the graft. Various strategies to reduce antiblood group antibody by overcoming ABO-incompatible transplantation have been tried. However, antigen-suppressing procedures have not been performed. Methods. We produced a novel anti-A antibody (K7508) by immunizing mice with salivary mucin of a blood group A individual, thereby clarifying that blood group A-antigen is expressed in endothelial cells of the liver. We investigated whether K7508 can mask A-antigen on the cells in vitro. Next, we immunized mice with A-antigen-expressing cells coated with K7508 or its Fab fragment, and measured anti-A antibody production in the mice. Results. Blood group A-antigen-expressing cells, such as blood group A-red blood cells (A-RBCs) and A431 cells, coated with K7508 were not recognized by another anti-A antibody in flow cytometry, indicating that A-antigen was masked by K7508 in vitro. The A-antigen on the paraffin-embedded liver tissue was also masked by K7508. Furthermore, the production of anti-A antibody in mice immunized with A-antigen-expressing cells coated with K7508 or its Fab fragment was significantly suppressed compared to that in mice immunized with non-coated cells alone, indicating that A-antigen was neutralized by K7508 in vivo. Conclusions. The neutralization of blood group antigen by antiblood group antibody and especially its Fab fragment might represent one strategy to overcome ABO-incompatible organ transplantation.

Prediction of a side effect and efficacy of adjuvant chemotherapy with gemcitabine for post operative patient of pancreatic cancer by a genetic polymorphism analysis

BACKGROUND/AIMS Single nucleotide polymorphism (SNP) of the genes for ATP-binding cassette transporters is related to the side effects of anticancer drugs and that of drug metabolism-related enzyme genes is involved in the activation of gemcitabine (GEM). METHODOLOGY Forty eight patients treated with adjuvant GEM chemotherapy after pancreatic cancer resection was examined for the SNP of multidrug-resistance 1 (MDR1) 2677, MDR1 3435, breast cancer resistance protein (BCRP) 421, ribonucleotide reductase M1 (RRM1)(-)524, RRM1(-)37 and deoxycytidine deaminase (CDA) 208. We divided the patients according to normal group: patients homozygous for a wild-type allele or heterozygous for a mutant allele and mutant group: those homozygous for a mutant allele. Both groups were compared regarding the outcome and the occurrence and severity of side effects. RESULTS MDR1 2677, MDR1 3435, BCRP421, RRM1(-) 524, RRM1(-) 37 and CDA mutant groups comprised 37.5, 31.3, 0, 12.5, 4.2 and 4.2%, respectively. The occurrence of >G3 side effects was the most frequent in the MDR1 2677 mutant group at 39%. The disease-free survival and overall survival tended to be longer in the MDR1 2677 mutant group. CONCLUSIONS A correlation between the SNP of MDR1 2677 and drug response in patients receiving GEM chemotherapy.

Minocycline sensitizes rodent and human liver mitochondria to the permeability transition: Implications for toxicity in liver transplantation

The antibiotic minocycline exerts cytoprotection in animal disease models. One proposed mechanism is modulation of the mitochondrial permeability transition (mPT), a Ca2 -dependent pathogenic event leading to necrotic and/or apoptotic cell death.1–5 A recent study in HEPATOLOGY by Theruvath et al.,6 investigating storage/ reperfusion injury following rat liver transplantation, concluded that minocycline prevented mPT and mitigated liver injury by decreasing mitochondrial Ca2 uptake without affecting mitochondrial respiration. Further, the authors argue that it could be consistent with clinical practice to (pre)treat stored livers and graft recipients with minocycline. The driving force for mitochondrial Ca2 transport is the mitochondrial membrane potential and the amount of Ca2 retained is dependent on the proton gradient and the matrix pH.7 Respiratory inhibition will decrease Ca2 retention capacity and sensitize mitochondria toward mPT.5,7 Further, endogenous inhibitors of mPT such as adenine nucleotides and Mg2 will influence the amount of Ca2 sequestered prior to mPT. In Theruvath et al., the effect of minocyline on mPT was determined in two classical assays, both using bolus additions of calcium chloride: (1) the swelling assay and (2) the Ca2 retention capacity assay. In both assays, the endpoint is Ca2 overload and induction of mPT. The authors found that minocycline prevented Ca2 induced swelling and decreased Ca2 retention and interpreted this as a specific inhibitory effect on Ca2 uptake. They excluded respiratory inhibition as the explanation to their findings by determining the respiration of mitochondria exposed to minocycline with and without Ca2 addition. However, the buffer used in the respiration assay was different from the one used in the Ca2 bolus assays, with high Mg2 concentration (Mg2 is a known endogenous inhibitor of mPT) and with the presence of the potent pharmacological mPT inhibitor cyclosporin A during Ca2 addition. We argue that minocycline at moderate to high dosing, similar to what we have shown in brain mitochondria, prevents Ca2 -uptake and mPT-induced swelling by respiratory inhibition.1,5 Further, depending on the buffer system used, the decreased Ca2 retention can be explained by minocycline-induced increase of mPT sensitivity related to (1) inhibited respiration1,5 and (2) chelating of Mg2 ,8 or (3) direct activation of mPT (even during concurrent cyclosporin A treatment) by adding Ca2 or in Ca2 loaded mitochondria, as recently shown by Kupsch et al.8 To stringently evaluate effects of minocycline during the process of Ca2 uptake, retention, and mPT, mitochondrial oxygen consumption can be monitored during a continuous Ca2 infusion (Fig. 1A,B). This assay provides information of the bioenergetic demand on mitochondria caused by Ca2 uptake as well as the respiratory inhibition triggered by mitochondrial Ca2 overload and mPT.5,7 Alternatively, the effect of minocycline on isolated mitochondria can be displayed by following changes of extramitochondrial Ca2 during a slow infusion of the cation. In these more physiologically relevant models, minocycline dose-dependently reduces Ca2 retention capacity and sensitizes rat and, importantly, human liver mitochondria to the mPT in the dose range used by Theruvath et al. (0-100 nmol/mg mitochondria; Fig. 1). In conclusion, minocycline may be a promising agent for cytoprotection at relevant dosing through mechanisms other than mPT inhibition. In the clinical setting, prevention of mitochondrial Ca2 uptake via respiratory inhibition is likely not beneficial to the organism. Further, to sensitize mitochondria to mPT by chelating Mg2 is not a viable strategy for cytoprotection. This must be kept in mind when considering the use of minocycline, even at moderate dosing, to mitigate storage/reperfusion injury during liver transplantation.

Discontinuation of Living Donor Liver Transplantation for PSC Due to Histological Abnormalities in Intraoperative Donor Liver Biopsy

Liver transplantation is the only curative treatment known to date for end‐stage liver disease occurring as a result of primary sclerosing cholangitis (PSC). Here, we report a case in which living donor liver transplantation (LDLT) for PSC was cancelled because of histological abnormalities in intraoperative biopsy of the donor liver. The donor was the mother of the recipient, and her preoperative evaluation revealed no abnormalities. In the donor operation, the donor liver biopsy revealed expansion of the portal zone with lymphocytic infiltration and dense concentric fibrosis developed around a bile duct. These histological findings were identical to those of early‐stage PSC; therefore, the LDLT was called off. The experience in this case suggests that preoperative liver biopsy may be useful to exclude first‐degree relative donors with potential PSC prior to LDLT for PSC.