Inhibitor Of Mitochondrial Permeability Transition Pore Research Articles

The effect of the mitochondrial permeability transition pore on apoptosis in Eimeria tenella host cells

Although the mitochondrial permeability transition pore (MPTP) is associated with cellular apoptosis and necrosis, its effect in host response to Eimeria infections is not well understood. In an effort to better understand the effect of MPTP on apoptosis in Eimeria tenella host cells, an MPTP inhibitor (cyclosporin A) was used to inhibit MPTP opening in vitro. Cecal epithelial cells from chick embryos, which were either treated or non-treated with cyclosporin A, were used as Eimeria tenella host cells. In addition, primary chick embryo cecum epithelial cell culture techniques and flow cytometry were used to detect the dynamic changes in MPTP opening, mitochondrial transmembrane potential, and cell apoptosis rate of Eimeria tenella host cells. Compared with the control group, cytometric techniques showed that untreated host cells exhibited a significantly higher (P < 0.01) degree of MPTP opening but lower (P < 0.01 or P < 0.05) mitochondrial transmembrane potential. Moreover, untreated group cells had less apoptosis (P < 0.01) at 4 h and more apoptosis (P < 0.05 or P < 0.01) at 24 to 120 h as compared with control group cells. After the application of cyclosporin A, the degree of MPTP opening in the treated group was significantly lower (P < 0.01) at 4 to 120 h compared to the untreated group, whereas the treated group had higher (P < 0.05 or P < 0.01) mitochondrial transmembrane potentials at 24 to 120 h. Flow cytometry assays also showed that there was less (P < 0.05 or P < 0.01) apoptosis after 24 h in the treated group than in the untreated group. Taken together, these observations indicate that MPTP is a key node that plays a predominant role in the mitochondrial apoptosis pathway in the host cell induced by Eimeria tenella.

Mitochondrial-dependent mechanisms are involved in angiotensin II-induced apoptosis in dopaminergic neurons.

Introduction:We recently demonstrated that angiotensin II (Ang II) was involved in the etiology of Parkinson’s disease (PD) via induction of apoptosis of dopaminergic neurons, but the mechanisms are not completely elucidated. Here, we asked whether mitochondrial-dependent mechanisms contributed to the Ang II-induced dopaminergic neuronal apoptosis.Materials and methods:CATH.a cells were incubated with Ang II in combination with mitochondrial permeability transition pore (mPTP) inhibitors or angiotensin receptor antagonists, and apoptosis rate, caspase-3 activity, cytochrome c levels, and mPTP opening were assessed.Results:We showed that Ang II triggered apoptosis via a mitochondrial-dependent pathway, as elevated cytochrome c levels were observed in the cytosol. By employing cyclosporin A and sanglifehrin A, two specific mPTP inhibitors, we revealed that cytochrome c release from mitochondria into cytoplasm was ascribed to mPTP opening. Meanwhile, the aforementioned effects could be abrogated by an AT1 receptor antagonist losartan rather than an AT2 receptor antagonist PD123319.Conclusion:This study demonstrates that Ang II triggers mitochondrial-dependent apoptosis via facilitating mPTP opening through an AT1 receptor-mediated fashion in dopaminergic neurons. These findings give insight into the effect of Ang II in the etiology of PD, and reinforce the application of AT1 receptor antagonists for PD treatment.

Mitochondrial permeability transition pore (MPTP) desensitization increases sea urchin spermatozoa fertilization rate.

Mitochondrial permeability transition pore (MPTP) is a protein complex whose opening promotes an abrupt increase in mitochondrial inner membrane permeability. Calcium signaling pathways are described in gametes and are involved in the fertilization process. Although mitochondria may act as Ca(2+) store and have a fast calcium-releasing mechanism through MPTP, its contribution to fertilization remains unclear. The work aimed to investigate the MPTP phenomenon in sea urchin spermatozoa and its role on the fertilization. Several pharmacological tools were used to evaluate the MPTP's physiology. Our results demonstrated that MPTP occurs in male gametes in a Ca(2+) - and voltage-dependent manner and it is sensitive to cyclosporine A. Additionally, our data show that MPTP opening does not alter ROS generation in sperm cells. Inhibition of MPTP in spermatozoa strongly improved the fertilization rate, which may involve mechanisms that increase the spermatozoa lifespan. The present work is the first report of the presence of a voltage- and Ca(2+) -dependent MPTP in gametes of invertebrates and indicates MPTP opening as another evolutionary feature shared by sea urchins and mammals. Studies about MPTP in sea urchin male gametes may contribute to the elucidation of several mechanisms involved in sperm infertility.

Cyclosporine-A mimicked the ischemic pre- and postconditioning-mediated cardioprotection in hypertensive rats: Role of PKCε

Our aim was to assess the action of cyclosporine-A (CsA) against reperfusion injury in spontaneously hypertensive rats (SHR) compared to the effects of ischemic pre- (IP) and postconditioning (IPC), examining the role played by PKCε. Isolated hearts were submitted to the following protocols: IC: 45min global ischemia (GI) and 1h reperfusion (R); IP: a cycle of 5min GI and 10min of R prior to 45min-GI; and IPC: three cycles of 30s-GI/30s-R at the start of R. Other hearts of the IC, IP and IPC groups received CsA (mitochondrial permeability transition pore inhibitor) or chelerythrine (Che, non-selective PKC inhibitor). Infarct size (IS) was assessed. TBARS and reduced glutathione (GSH) content — as parameters of oxidative damage, the expression of P-Akt, P-GSK-3β, P-PKCε and cytochrome c (Cyc) release — as an index of mitochondrial permeability and the response of isolated mitochondria to Ca2+ were also measured. IS similarly decreased in preconditioned, postconditioned and CsA treated heart showing the highest values in the combinations IP+CsA and IPC+CsA. TBARS decreased and GSH was partially preserved after all interventions. The content of P-Akt, P-GSK-3β and P-PKCε increased in cytosol and decreased in mitochondria after IP and IPC. In CsA treated hearts these enzymes increased in both fractions reaching the highest values. Cyc release was attenuated and the response of mitochondria to Ca2+ was improved by the interventions. The beneficial effects of IP and IPC were annulled when PKC was inhibited with Che. A PKCε/VDAC association was also detected. These data show that, in SHR, the CsA treatment mimicked and reinforced the cardioprotective action afforded by IP and IPC in which PKCε-mediated attenuation of mitochondrial permeability appears as the main mechanism involved.

Expression level of P2X7 receptor is a determinant of ATP-induced death of mouse cultured neurons

Activation of P2X7 receptor (P2X7R), a purinergic receptor, expressed by neurons is well-known to induce their death, but whether or not their sensitivity to ATP depends on its expression levels remains unclear. Here, we examined the effect of the expression level of P2X7Rs on cell viability using pure neuron cultures, co-cultures with astrocytes derived from SJL- and ddY-strain mice, and mouse P2X7R-expressing HEK293T cell systems. Treatment of pure neuron cultures with 5mM ATP for 2h, followed by 3-h incubation in fresh medium, resulted in death of both types of neurons, and their death was prevented by administration of P2X7R-specific antagonists. In both SJL- and ddY-neurons, ATP-induced neuronal death was inhibited by a mitochondrial permeability transition pore inhibitor cyclosporine A, mitochondrial dysfunction being involved in their death. The ATP-induced neuronal death was greater for SJL-neurons than for ddY-ones, this being correlated with the expression level of P2X7R in them, and the same results were obtained for the HEK293T cell systems. Co-culture of neurons with astrocytes increased the ATP-induced neuronal death compared to the case of pure neuron cultures. Overall, we reveal that neuronal vulnerability to ATP depends on the expression level of P2X7R, and co-existence of astrocytes exacerbates ATP-induced neuronal death.

HERPUD1 protects against oxidative stress-induced apoptosis through downregulation of the inositol 1,4,5-trisphosphate receptor

Homocysteine-inducible, endoplasmic reticulum (ER) stress-inducible, ubiquitin-like domain member 1 (HERPUD1), an ER resident protein, is upregulated in response to ER stress and Ca2+ homeostasis deregulation. HERPUD1 exerts cytoprotective effects in various models, but its role during oxidative insult remains unknown. The aim of this study was to investigate whether HERPUD1 contributes to cytoprotection in response to redox stress and participates in mediating stress-dependent signaling pathways. Our data showed that HERPUD1 protein levels increased in HeLa cells treated for 30min with H2O2 or angiotensin II and in aortic tissue isolated from mice treated with angiotensin II for 3 weeks. Cell death was higher in HERPUD1 knockdown (sh-HERPUD1) HeLa cells treated with H2O2 in comparison with control (sh-Luc) HeLa cells. This effect was abolished by the intracellular Ca2+ chelating agent BAPTA-AM or the inositol 1,4,5-trisphosphate receptor (ITPR) antagonist xestospongin B, suggesting that the response to H2O2 was dependent on intracellular Ca2+ stores and the ITPR. Ca2+ kinetics showed that sh-HERPUD1 HeLa cells exhibited greater and more sustained cytosolic and mitochondrial Ca2+ increases than sh-Luc HeLa cells. This higher sensitivity of sh-HERPUD1 HeLa cells to H2O2 was prevented with the mitochondrial permeability transition pore inhibitor cyclosporine A. We concluded that the HERPUD1-mediated cytoprotective effect against oxidative stress depends on the ITPR and Ca2+ transfer from the ER to mitochondria.

Diamide accelerates opening of the Tl+-induced permeability transition pore in Ca2+-loaded rat liver mitochondria

Opening of the mitochondrial permeability transition pore (MPTP) in the inner membrane is due to matrix Ca2+ overload and matrix glutathione loss. Fixing the ‘m’ conformation of the adenine nucleotide translocase (ANT) by ADP or N-ethylmaleimide (NEM) inhibits opening of the MPTP. Oxidants (diamide or tert-butylhydroperoxide (tBHP)) fix the ANT in ‘c’ conformation, and the ability of ADP to inhibit the MPTP is thus attenuated. Earlier we found (Korotkov and Saris, 2011) that calcium load of rat liver mitochondria resulted in Tl+-induced MPTP opening, which was accompanied by a decrease in state 3, state 4, and 2,4-dinitrophenol-uncoupled respiration, as well as increased swelling and membrane potential dissipation. These effects, which were increased by diamide and tBHP, were visibly reduced in the presence of the MPTP inhibitors (ADP, NEM, and cyclosporine A). Our data suggest that conformational changes of the ANT and matrix glutathione loss may be directly involved in opening the Tl+-induced MPTP in the inner membrane of Ca2+-loaded rat liver mitochondria.

IL-17 Induces MPTP opening through ERK2 and P53 signaling pathway in human platelets

The opening of mitochondrial permeability transition pore (MPTP) plays a critical role in platelet activation. However, the potential trigger of the MPTP opening in platelet activation remains unknown. Inflammation is the crucial trigger of platelet activation. In this study, we aimed to explore whether and how the important inflammatory cytokine IL-17 is associated with MPTP opening in platelets activation by using MPTP inhibitor cyclosporine-A (CsA). The mitochondrial membrane potential (ΔΨm) was detected to reflect MPTP opening levels. And the platelet aggregation, activation, and the primary signaling pathway were also tested. The results showed that the MPTP opening levels were increased and Δψm reduced in platelets administrated with IL-17. Moreover, the levels of aggregation, CD62P, PAC-1, P53 and the phosphorylation of ERK2 were enhanced along with the MPTP opening in platelets pre-stimulated with IL-17. However, CsA attenuated these effects triggered by IL-17. It was suggested that IL-17 could induce MPTP opening through ERK2 and P53 signaling pathway in platelet activation and aggregation.

A New Therapeutic Modality for Acute Myocardial Infarction: Nanoparticle-Mediated Delivery of Pitavastatin Induces Cardioprotection from Ischemia-Reperfusion Injury via Activation of PI3K/Akt Pathway and Anti-Inflammation in a Rat Model.

AimThere is an unmet need to develop an innovative cardioprotective modality for acute myocardial infarction (AMI), for which the effectiveness of interventional reperfusion therapy is hampered by myocardial ischemia-reperfusion (IR) injury. Pretreatment with statins before ischemia is shown to reduce MI size in animals. However, no benefit was found in animals and patients with AMI when administered at the time of reperfusion, suggesting insufficient drug targeting into the IR myocardium. Here we tested the hypothesis that nanoparticle-mediated targeting of pitavastatin protects the heart from IR injury.Methods and ResultsIn a rat IR model, poly(lactic acid/glycolic acid) (PLGA) nanoparticle incorporating FITC accumulated in the IR myocardium through enhanced vascular permeability, and in CD11b-positive leukocytes in the IR myocardium and peripheral blood after intravenous treatment. Intravenous treatment with PLGA nanoparticle containing pitavastatin (Pitavastatin-NP, 1 mg/kg) at reperfusion reduced MI size after 24 hours and ameliorated left ventricular dysfunction 4-week after reperfusion; by contrast, pitavastatin alone (as high as 10 mg/kg) showed no therapeutic effects. The therapeutic effects of Pitavastatin-NP were blunted by a PI3K inhibitor wortmannin, but not by a mitochondrial permeability transition pore inhibitor cyclosporine A. Pitavastatin-NP induced phosphorylation of Akt and GSK3β, and inhibited inflammation and cardiomyocyte apoptosis in the IR myocardium.ConclusionsNanoparticle-mediated targeting of pitavastatin induced cardioprotection from IR injury by activation of PI3K/Akt pathway and inhibition of inflammation and cardiomyocyte death in this model. This strategy can be developed as an innovative cardioprotective modality that may advance currently unsatisfactory reperfusion therapy for AMI.

Targeting pancreatic cancer cells by a novel hydroxamate-based histone deacetylase (HDAC) inhibitor ST-3595.

In the current study, we tested the potential anti-pancreatic cancer activity of a novel hydroxamate-based histone deacetylase (HDAC) inhibitor ST-3595. We showed that ST-3595 exerted potent anti-proliferative and cytotoxic activities against both established pancreatic cancer cell lines (PANC-1, AsPC-1, and Mia-PaCa-2), and patient-derived primary cancer cells. It was, however, generally safe to non-cancerous pancreatic epithelial HPDE6c7 cells. ST-3595-induced cytotoxicity to pancreatic cancer cells was associated with significant apoptosis activation. Reversely, the pan caspase inhibitor z-VAD-fmk and the caspase-8 inhibitor z-ITED-fmk alleviated ST-3595-mediated anti-pancreatic cancer activity in vitro. For the mechanism study, ST-3595 inhibited HDAC activity, and induced mitochondrial permeability transition pore (MPTP) opening in pancreatic cancer cells. Inhibition of MPTP, by cyclosporin A, sanglifehrin A, or by cyclophilin-D (Cyp-D) siRNA knockdown, dramatically inhibited ST-3595-induced pancreatic cancer cell apoptosis. Meanwhile, we found that a low concentration of ST-3595 dramatically sensitized gemcitabine-induced anti-pancreatic cancer cell activity in vitro. In vivo, ST-3595 oral administration inhibited PANC-1 xenograft growth in nude mice, and this activity was further enhanced when in combination with gemcitabine. In summary, the results of this study suggest that targeting HDACs by ST-3595 might represent as a novel and promising anti-pancreatic cancer strategy.

Mitochondrial permeability transition and its regulatory components are implicated in apoptosis of primary cultures of rat proximal tubular cells exposed to lead.

Previous studies have already demonstrated that mitochondria play a key role in Pb-induced apoptosis in primary cultures of rat proximal tubular (rPT) cells. To further clarify the underlying mechanism of Pb-induced mitochondrial apoptosis, this study was designed to investigate the role of mitochondrial permeability transition (MPT) and its regulatory components in Pb-induced apoptosis in rPT cells. Mitochondrial permeability transition pore (MPTP) opening together with disruption of mitochondrial ultrastructure, translocation of cytochrome c from mitochondria to cytoplasm and subsequent caspase-3 activation were observed in this study, suggesting that MPT is involved in Pb-induced apoptosis in rPT cells. Simultaneously, Pb-induced caspase-3 activation and apoptosis can be significantly inhibited by three MPTP inhibitors (CsA, DIDS, BA), which target different regulatory components of MPTP (Cyp-D, VDAC, ANT), respectively, demonstrating that Cyp-D, VDAC and ANT participate in MPTP regulation during lead exposure. Moreover, decreased ATP levels and increased ADP/ATP ratio induced by lead treatment can be significantly reversed by BA, indicating that Pb-mediated ANT dysfunction resulted in ATP depletion. In addition, up-regulation of VDAC-1, ANT-1 together with down-regulation of Cyp-D, VDAC-2 and ANT-2 at both the levels of transcription and translation were revealed in rPT cells under lead exposure conditions. In conclusion, Pb-mediated mitochondrial apoptosis in rPT cells is dependent on MPTP opening. Different expression levels in each isoform of three regulatory components contribute to alteration in their functions, which may promote the MPTP opening.

Enhancing Hematopoietic Stem Cell Transplantation Efficacy by Mitigating Oxygen Shock

Hematopoietic stem cells (HSCs) reside in hypoxic niches within bone marrow and cord blood. Yet, essentially all HSC studies have been performed with cells isolated and processed in non-physiologic ambient air. By collecting and manipulating bone marrow and cord blood in native conditions of hypoxia, we demonstrate that brief exposure to ambient oxygen decreases recovery of long-term repopulating HSCs and increases progenitor cells, a phenomenon we term extraphysiologic oxygen shock/stress (EPHOSS). Thus, true numbers of HSCs in the bone marrow and cord blood are routinely underestimated. We linked ROS production and induction of the mitochondrial permeability transition pore (MPTP) via cyclophilin D and p53 as mechanisms of EPHOSS. The MPTP inhibitor cyclosporin A protects mouse bone marrow and human cord blood HSCs from EPHOSS during collection in air, resulting in increased recovery of transplantable HSCs. Mitigating EPHOSS during cell collection and processing by pharmacological means may be clinically advantageous for transplantation.

Vasopressin attenuates ischemia–reperfusion injury via reduction of oxidative stress and inhibition of mitochondrial permeability transition pore opening in rat hearts

Aim of this study was to investigate the involvement of the mitochondrial permeability transition pore (MPTP) and oxidative stress in the cardioprotective effect of vasopressin (AVP) on ischemia/reperfusion (I/R) injury. Anesthetized male wistar rats were subjected to regional 30min ischemia and 120min reperfusion and randomly divided into nine groups: (1) Control; saline was administered intravenously before ischemia, (2) vasopressin was administrated 10min prior to ischemia, (3, 4) Atractyloside as MPTP opener, was injected 5min prior to reperfusion without and with vasopressin, (5, 6) Cyclosporine A as a MPTP closer, was injected 5min prior to reperfusion without and with vasopressin, (7) mitochondria were isolated from control group and CaCl2 was added as MPTP opener and swelling inducer, (8) isolated mitochondria from Control hearts was incubated with Cyclosporine A before adding the CaCl2 (9) CaCl2 was added to isolated mitochondria from vasopressin group. Infusion of vasopressin decreased infarct size (18.6±1.7% vs. control group 37.6±2.4%), biochemical parameters [LDH (Lactate Dehydrogenase), CK-MB (Creatine Kinase-MB) and MDA (Malondialdehyde) plasma levels, PAB (Prooxidant–antioxidant balance)] compared to control group. Atactyloside suppressed the cardioprotective effect of vasopressin (32.5±1.9% vs. 18.6±1.7%) but administration of the Cyclosporine A without and with vasopressin significantly reduced infarct size to 17.7±4% (P<0.001) and 22.7±3% (P<0.01) respectively, vs. 37.6±2.4% in control group. Also, vasopressin, similar to Cyclosporine A, led to decrease in CaCl2-induced swelling. It seems that vasopressin through antioxidant effect and MPTP inhibition has created a cardioprotection against ischemia/reperfusion injuries.

Mitochondrial dysfunction confers albumin-induced NLRP3 inflammasome activation and renal tubular injury.

Proteinuria is involved in the development of tubular lesions and in the progressive loss of renal function in chronic kidney diseases via uncertain mechanisms. Growing evidence suggests a pathogenic role of mitochondrial dysfunction in chronic kidney diseases. Therefore, the present study aimed to define the roles of mitochondria in proteinuria-induced renal tubular injury and their underlying mechanisms. Using the albumin-overload mouse model, we observed severe tubular structure damage and striking tubular cell apoptosis. Furthermore, tubular epithelial cells displayed a loss of E-cadherin expression and gained expression of α-smooth muscle actin and vimentin, indicating a cellular phenotypic alteration. Strikingly, these albumin overload-induced abnormalities were robustly blocked by a mitochondrial SOD2 mimic, Mn(III) tetrakis (4-benzoic acid)porphyrin chloride (MnTBAP). In agreement with these results, we observed a marked change in mitochondrial morphology accompanied by mitochondrial cytochrome c release and a copy number reduction of mitochondrial DNA. These alterations were largely reversed by MnTBAP, suggesting a key role for mitochondria-derived oxidative stress in mediating the albumin effect on mitochondrial dysfunction and subsequent tubular injury. Moreover, the NOD-like receptor family, pyrin domain-containing 3 (NLRP3)/caspase-1/cytokine cascade was activated in the kidney by albumin overload and was entirely abolished by MnTBAP. In albumin-treated mouse proximal tubular cells, albumin directly induced ROS production, mitochondrial dysfunction, NLRP3/caspase-1/cytokine cascade activation, cell apoptosis, and cellular phenotypic transition. Similar to our in vivo results, treatment with either MnTBAP or cyclosporin A, a mitochondrial permeability transition pore inhibitor, remarkably attenuated these abnormalities in cells. Taken together, these novel findings demonstrate a potential role for the mitochondrial dysfunction/NLRP3 inflammasome axis in the pathogenesis of proteinuria-induced renal tubular injury.

Resveratrol alters human endothelial cells redox state and causes mitochondrial-dependent cell death

Studies analyzing the impact of natural antioxidants (NA) on Endothelial Cells (ECs) have dramatically increased during the last years, since a deregulated ECs redox state is at the base of the onset and progression of several cardiovascular diseases. However, whether NA can provide cardiovascular benefits is still a controversial area of debate. Resveratrol (RES), a natural polyphenol found in grapes, is believed to provide cardiovascular benefits by virtue of its antioxidant effect on the endothelium. Here, we report that tissue-attainable doses of resveratrol increased the intracellular oxidative state, thus affecting mitochondrial membrane depolarization and inducing EC death. Cyclosporine A, a mitochondrial permeability transition pore inhibitor, prevented oxidative-mediated cell death, thus implicating mitochondria in resveratrol-induced EC impairment. The specific cytochrome P450 (CYP) 2C9 inhibitor, sulfaphenazole, counteracted both oxidative stress and mitochondrial membrane depolarization, providing EC protection against resveratrol-elicited pro-oxidant effects. Our findings strongly suggest that CYP2C9 mediates resveratrol-induced oxidative stress leading to mitochondria impairment and EC death.

Reactive oxygen species-provoked mitochondria-dependent cell death during ageing of elm (Ulmus pumila L.) seeds.

Previous studies have shown that controlled deterioration treatment (CDT) induces programmed cell death in elm (Ulmus pumila L.) seeds, which undergo certain fundamental processes that are comparable to apoptosis in animals. In this study, the essential characteristics of mitochondrial physiology in elm seeds during CDT were identified by cellular ultrastructural analysis, whole-body optical imaging, Western blotting and semi-quantitative RT-PCR. The alteration in mitochondrial morphology was an early event during CDT, as indicated by progressive dynamic mitochondrial changes and rupture of the mitochondrial outer membrane; loss of mitochondrial transmembrane potential (Δψ(m)) ensued, and mitochondrial ATP levels decreased. The mitochondrial permeability transition pore inhibitor cyclosporine A effectively suppressed these changes during ageing. The in situ localization of production of reactive oxygen species (ROS), and evaluation of the expression of voltage-dependent anion-selective channel and cyclophilin D indicated that the levels of mitochondrial permeability transition pore components were positively correlated with ROS production, leading to an imbalance of the cellular redox potential and ultimately to programmed cell death. Pre-incubation with ascorbic acid slowed loss of mitochondrial Δψ(m), and decreased the effect of CDT on seed viability. However, there were no significant changes in multiple antioxidant elements or chaperones in the mitochondria during early stages of ageing. Our results indicate that CDT induces dynamic changes in mitochondrial physiology via increased ROS production, ultimately resulting in an irreversible loss of seed viability.

Role of the MPTP in conditioning the heart - translatability and mechanism.

Mitochondria have long been known to be the gatekeepers of cell fate. This is particularly so in the response to acute ischaemia‐reperfusion injury (IRI). Following an acute episode of sustained myocardial ischaemia, the opening of the mitochondrial permeability transition pore (MPTP) in the first few minutes of reperfusion, mediates cell death. Preventing MPTP opening at the onset of reperfusion using either pharmacological inhibitors [such as cyclosporin A (CsA) ] or genetic ablation has been reported to reduce myocardial infarct (MI) size in animal models of acute IRI. Interestingly, the endogenous cardioprotective intervention of ischaemic conditioning, in which the heart is protected against MI by applying cycles of brief ischaemia and reperfusion to either the heart itself or a remote organ or tissue, appears to be mediated through the inhibition of MPTP opening at reperfusion. Small proof‐of‐concept clinical studies have demonstrated the translatability of this therapeutic approach to target MPTP opening using CsA in clinical settings of acute myocardial IRI. However, given that CsA is a not a specific MPTP inhibitor, more novel and specific inhibitors of the MPTP need to be discovered – the molecular identification of the MPTP should facilitate this. In this paper, we review the role of the MPTP as a target for cardioprotection, the potential mechanisms underlying MPTP inhibition in the setting of ischaemic conditioning, and the translatability of MPTP inhibition as a therapeutic approach in the clinical setting.Linked ArticlesThis article is part of a themed section on Conditioning the Heart – Pathways to Translation. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue‐8

The 18-kDa Translocator Protein Inhibits Vascular Cell Adhesion Molecule-1 Expression via Inhibition of Mitochondrial Reactive Oxygen Species.

Translocator protein 18 kDa (TSPO) is a mitochondrial outer membrane protein and is abundantly expressed in a variety of organ and tissues. To date, the functional role of TSPO on vascular endothelial cell activation has yet to be fully elucidated. In the present study, the phorbol 12-myristate 13-acetate (PMA, 250 nM), an activator of protein kinase C (PKC), was used to induce vascular endothelial activation. Adenoviral TSPO overexpression (10–100 MOI) inhibited PMA-induced vascular cell adhesion molecule-1 (VCAM-1) and intracellular cell adhesion molecule-1 (ICAM-1) expression in a dose dependent manner. PMA-induced VCAM-1 expressions were inhibited by Mito-TEMPO (0.1–0.5 μM), a specific mitochondrial antioxidants, and cyclosporin A (1–5 μM), a mitochondrial permeability transition pore inhibitor, implying on an important role of mitochondrial reactive oxygen species (ROS) on the endothelial activation. Moreover, adenoviral TSPO overexpression inhibited mitochondrial ROS production and manganese superoxide dismutase expression. On contrasts, gene silencing of TSPO with siRNA increased PMA-induced VCAM-1 expression and mitochondrial ROS production. Midazolam (1–50 μM), TSPO ligands, inhibited PMA-induced VCAM-1 and mitochondrial ROS production in endothelial cells. These results suggest that mitochondrial TSPO can inhibit PMA-induced endothelial inflammation via suppression of VCAM-1 and mitochondrial ROS production in endothelial cells.

Eicosapentaenoic acid (EPA) induced apoptosis in HepG2 cells through ROS–Ca2+–JNK mitochondrial pathways

Eicosapentaenoic acid (EPA), a well-known dietary n−3 PUFAS, has been considered to inhibit proliferation of tumor cells. However, the molecular mechanism related to EPA-induced liver cancer cells apoptosis has not been reported. In this study, we investigated the effect of EPA on HepG2 cells proliferation and apoptosis mechanism through mitochondrial pathways. EPA inhibited proliferation of HepG2 cells in a dose-dependent manner and had no significant effect on the cell viability of humor normal liver L-02 cells. It was found that EPA initially evoked ROS formation, leading to [Ca2+]c accumulation and the mitochondrial permeability transition pore (MPTP) opening; EPA-induced HepG2 cells apoptosis was inhibited by N-acetylcysteine (NAC, an inhibitor of ROS), 1,2-bis (2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA-AM, a chelator of calcium) and CsA (inhibitor of MPTP). The relationship between ROS production, the increase of cytoplasmic Ca and MPTP opening was detected. It seems that ROS may act as an upstream regulator of EPA-induced [Ca2+]c generation, moreover, generation of ROS, overload of mitochondrial [Ca2+]c, and JNK activated cause the opening of MPTP. Western blotting results showed that EPA elevated the phosphorylation status of JNK, processes associated with the ROS generation. Simultaneously, the apoptosis induced by EPA was related to release of cytochrome C from mitochondria to cytoplasm through the MPTP and activation of caspase-9 and caspase-3. These results suggest that EPA induces apoptosis through ROS–Ca2+–JNK mitochondrial pathways.

Mitigation of a Newly Discovered Phenomenon, Extra Physiologic Oxygen Shock/Stress (EPHOSS), Mediated By the Mitochondria Permeability Transition Pore, Greatly Improves Stem Cell Collection and Transplantation

Hematopoietic stem cells (HSCs) reside in hypoxic niches within the bone marrow (BM). Yet, all HSC studies have been performed to date with cells immediately isolated in non-physiologic ambient air, whether or not they are subsequently processed in low oxygen tension. By collecting/manipulating BM in physiologically native conditions of hypoxia where all procedures are performed inside a hypoxic chamber, we demonstrate that brief exposure of mouse BM or human cord blood (CB) to ambient oxygen decreases recovery of phenotypically-defined and functional self-renewing long-term repopulating HSC and concomitantly increases numbers of progenitor cells, a phenomenon we term Extra Physiologic Oxygen Shock/Stress (EPHOSS). This new phenomenon is exquisitely sensitive to oxygen and great care must be taken to ensure all reagents, solutions, plastics, and anything that will come into contact with the cell suspension, is extensively pre-equilibrated in hypoxia. Up to 5-fold greater numbers of long-term (LT)-HSCs (CD34-CD150+Lin-Sca1+c-kit+CD41-CD48- or CD34-CD135-Lin-Sca1+c-kit+) could be recovered from mouse BM harvested in 3% O2 compared to BM harvested in air, or even BM harvested in 3% O2 and then exposed to air for as little as 30 min before analysis, even if subsequently returned to hypoxia. There was a concomitant decrease in short term-HSCs and multipotent progenitors when BM was harvested in hypoxia, an effect associated with decreased functional cytokine-stimulated colony formation of hematopoietic progenitor cells (HPC: CFU-GM, BFU-E, and CFU-GEMM). Similarly, if human CB was harvested under similar low oxygen conditions, a 3-fold increase in recovered HSCs (Lin-CD34+CD38-CD45RA+CD90+CD49f+) could be achieved compared to CB harvested in air. Using a custom mouse respirator to conduct competitive repopulating transplant experiments completely in a 3% O2 environment revealed an increase in competitive repopulating units (CRUs) up to more than 42 fold was recovered when BM is harvested in hypoxic conditions compared to air harvested BM in primary recipients, thus demonstrating the beneficial effects of hypoxic harvest on functional/transplantable HSCs with secondary transplant capability. These data strongly support the surprising conclusion that, until now, the true numbers of HSCs and the transplantation potency of BM and CB has been routinely and consistently underestimated because of rapid initiation of differentiation of LT-HSCs in ambient air. We present evidence linking mitochondrial function and cyclophilin D to EPHOSS. Genetically or pharmacologically suppressing cyclophilin D function, or p53 gene deletion link production of reactive oxygen species (ROS) to induction of the mitochondrial permeability transition pore (MPTP) as a molecular mechanism of EPHOSS, where rapid ROS generation in HSCs after exposure to “hyperoxic” room air initiates an irreversible cascade of differentiation signals (see illustration). We present additional evidence from gene knock-out model studies implicating roles for miR210 and Hif-1a in EPHOSS. The MPTP inhibitor, cyclosporine A, protects phenotypically-defined as well as functional and transplantable HSCs from EPHOSS during collection in air resulting in at least a 3-fold increase in HSC recovery as well as increased transplantation potency. Thus, pharmacological mitigation of EPHOSS during HSC collections for use in patient transplantation procedures may be clinically advantageous. Because cyclosporine A is already in use clinically, this EPHOSS-reducing strategy may be readily and easily tested for efficacy in a hospital setting. Because many different adult stem cells exist naturally in hypoxic niches, EPHOSS is likely relevant to other stem cells routinely harvested in air. Evidence suggests that aged HSCs may be more sensitive to the deleterious effects of EPHOSS than young HSCs. We propose that metabolic profiling of stem cells, including cancer stem cells, may not accurately represent the metabolism, behavior, and responses of these cells as they exist in their native hypoxic environments because they are harvested and studied in air. Thus, experimental designs that include a consideration of EPHOSS effects may be required to obtain a more complete understanding of stem cell metabolism and biology especially as it relates to stem cell aging or responses of cancer stem cells to chemotherapy. [Display omitted] DisclosuresBroxmeyer:CordUse: Membership on an entity’s Board of Directors or advisory committees.