Effect of ethylisopropyl amiloride,a Na+-H+ exchange inhibitor,on cardioprotective effect of ischaemic and angiotensin preconditioning

The present study is designed to investigate the role of Na⁺-H⁺ exchanger in the cardioprotective effect of ischaemic and angiotensin (Ang II) preconditioning. Isolated perfused rat heart was subjected to global ischaemia for 30 min followed by reperfusion for 120 min. Coronary effluent was analysed for LDH and CK release to assess the degree of cardiac injury. Myocardial infarct size was estimated macroscopically using TTC staining. Left ventricular developed pressure (LVDP) and dp/dt were recorded to evaluate myocardial contractility. Four episodes of ischaemic or Ang II preconditioning markedly reduced LDH and CK release in coronary effluent and decreased myocardial infarct size. 5-(N-ethyl-N-isopropyl)amiloride (EIPA), a Na⁺-H⁺ exchange inhibitor, produced no marked effect on ischaemic preconditioning and Ang II preconditioning induced cardioprotection. On the other hand, EIPA administration prior to global ischaemia produced a similar reduction in myocardial injury as was noted with ischaemic preconditioning or Ang II preconditioning. On the basis of these results, it may be concluded that inhibition of Na⁺-H⁺ exchanger protects against ischaemia-reperfusion induced myocardial injury whereas activation of Na⁺-H⁺ exchanger may not mediate the cardioprotective effect of ischaemic and Ang II preconditioning.     

Metabolic basis for contractile dysfunction following chronic partial bladder outlet obstruction in rabbits

Our study is designed to correlate nitrite concentration, an index of nitric oxide (NO) release with mast cell peroxidase (MPO), a marker of cardiac mast cell degranulation and cardioprotective effect of ischaemic preconditioning in isolated perfused rat heart subjected to 30 min of global ischaemia and 30 min of reperfusion. Ischaemic preconditioning, comprised of four episodes of 5 min global ischaemia and 5 min of reperfusion, markedly reduced the release of lactate dehydrogenase (LDH) and creatine kinase (CK) in coronary effluent and incidence of ventricular premature beats (VPBs) and ventricular tachycardia and fibrillation (VT/VF) during reperfusion phase. Ischaemia-reperfusion induced release of MPO was markedly reduced in ischaemic preconditioned hearts. Increased release of nitrite was noted during reperfusion phase after sustained ischaemia in preconditioned hearts as compared to control hearts. No alterations in the release of nitrite was observed immediately after ischaemic preconditioning. However, ischaemic preconditioning markedly increased the release of MPO prior to global ischaemia. It is proposed that cardioprotective and antiarrhythmic effect of ischaemic preconditioning may be ascribed to degranulation of cardiac mast cells. Depletion of cytotoxic mediators during ischaemic preconditioning and consequent decreased release of these mediators during sustained ischaemia-reperfusion may be associated with preservation of structures in isolated rat heart responsible for NO release.     

Cardioprotective effect of polydatin against ischemia/reperfusion injury: Roles of protein kinase C and mito KATP activation

Polydatin preconditioning (PPC) has been reported to be protective against brain and intestine ischemia/reperfusion injury (I/R injury), but whether polydatin exerts cardioprotective effect against myocardial ischemia/reperfusion and the underlying mechanisms remain unclear. Previous studies have demonstrated that oxidative stress plays an important role in the process of I/R. Elevation of oxidative agents and decline in anti-oxidant substance would promote I/R. Meanwhile, the activation of PKC signaling seems to mediate the cardioprotective effects of many drugs by alleviating Ca²⁺ influx. In the present study, we reported for the first time that intravenous administration of polydatin before I/R significantly limited the infarct size, creatine phosphokinase (CPK) and lactate dehydrogenase (LDH) leakage from the damaged myocardium after I/R. The activity of SOD and the content of MDA remarkably changed in the presence of polydatin as well. However, the cardiac function-preserving and myocardial enzymes leakage-limiting effects of polydatin vanished in the presence of PKC inhibitors and mito K_ATP channel blockers. But there was not a significant change in the activity of SOD and MDA content. We therefore conclude that PPC exerts cardioprotective effect by the activation of PKC-K_ATP-dependent signaling and the direct anti-oxidative stress mechanisms.     

Mitochondrial K<Subscript>ATP</Subscript> channels-derived reactive oxygen species activate pro-survival pathway in pravastatin-induced cardioprotection

Reactive oxygen species (ROS) are important intracellular signaling molecules and are implicated in cardioprotective pathways including ischemic preconditioning. Statins have been shown to have cardioprotective effects against ischemia/reperfusion injury, however, the precise mechanisms remain to be elucidated. We hypothesized that ROS-mediated signaling cascade may be involved in pravastatin-induced cardioprotection. Cultured rat cardiomyocytes were exposed to H₂O₂ for 30 min to induce cell injury. Pravastatin significantly suppressed H₂O₂-induced cell death evaluated by propidium iodide staining and the MTT assay. Incubation with pravastatin activated catalase, and prevented a ROS burst induced by H₂O₂, which preserved mitochondrial membrane potential. Protective effects were induced very rapidly within 10 min, which was concordant with the up-regulation of phosphorylated ERK1/2. L-NAME, 5HD, N-acetylcysteine (NAC) and staurosporine inhibited ERK1/2 phosphorylation and also reduced pravastatin-induced cardioprotection, suggesting NO, mitochondrial K_ATP (mitoK_ATP) channels, ROS and PKC should be involved in the cardioprotective signaling. We also demonstrated that pravastatin moderately up-regulated ROS generation in a 5HD-inhibitable manner. In isolated perfused rat heart experiments, pravastatin administered 10 min prior to no-flow global ischemia significantly improved left ventricular functional recovery, and also reduced infarct size, which were attenuated by the treatment with NAC, 5HD, L-NAME or staurosporine. Administration of pravastatin from the beginning of reperfusion also conferred cardioprotection. Pravastatin protected the cardiomyocytes against oxidative stress by preventing the ROS burst and preserving mitochondrial function. Moderately up-regulated ROS production by mitoK_ATP channels opening is involved in the pro-survival signaling cascade activated by pravastatin.     

Epidermal growth factor protects against myocardial ischaemia reperfusion injury through activating Nrf2 signalling pathway

Alleviating the oxidant stress associated with myocardial ischaemia reperfusion has been demonstrated as a potential therapeutic approach to limit ischaemia reperfusion (I/R)-induced cardiac damage. It is reported that EGFR/erbB2 signalling is an important cardiac survival pathway in cardiac function and activation of EGFR has a cardiovascular effect in global ischaemia. Epidermal growth factor (EGF), a typical EGFR ligand, was considered to have a significant role in activating EGFR. However, no evidence has been published whether exogenous EGF has protective effects on myocardial ischaemia reperfusion. This study aims to investigate the effects of EGF in I/R-induced heart injury and to demonstrate its mechanisms. H9c2 cells challenged with H₂O₂ were used for in vitro biological activity and mechanistic studies. The malondialdehyde (MDA) and Superoxide Dismutase (SOD) levels in H9c2 cells were determined, and the cell viability was assessed by MTT assay. Myocardial I/R mouse administrated with or without EGF were used for in vivo studies. Pretreatment of H9c2 cells with EGF activated Nrf2 signalling pathway, attenuated H₂O₂-increased MDA and H₂O₂-reduced SOD level, followed by the inhibition of H₂O₂-induced cell death. In in vivo animal models of myocardial I/R, administration of EGF reduced infarct size and myocardial apoptosis. These data support that EGF decreases oxidative stress and attenuates myocardial ischaemia reperfusion injury via activating Nrf2.     

Knockdown of endogenous RNF4 exacerbates ischaemia‐induced cardiomyocyte apoptosis in mice

RNF4, a poly‐SUMO‐specific E3 ubiquitin ligase, is associated with protein degradation, DNA damage repair and tumour progression. However, the effect of RNF4 in cardiomyocytes remains to be explored. Here, we identified the alteration of RNF4 from ischaemic hearts and oxidative stress‐induced apoptotic cardiomyocytes. Upon myocardial infarction (MI) or H₂O₂/ATO treatment, RNF4 increased rapidly and then decreased gradually. PML SUMOylation and PML nuclear body (PML‐NB) formation first enhanced and then degraded upon oxidative stress. Reactive oxygen species (ROS) inhibitor was able to attenuate the elevation of RNF4 expression and PML SUMOylation. PML overexpression and RNF4 knockdown by small interfering RNA (siRNA) enhanced PML SUMOylation, promoted p53 recruitment and activation and exacerbated H₂O₂/ATO‐induced cardiomyocyte apoptosis which could be partially reversed by knockdown of p53. In vivo, knockdown of endogenous RNF4 via in vivo adeno‐associated virus infection deteriorated post‐MI structure remodelling including more extensive interstitial fibrosis and severely fractured and disordered structure. Furthermore, knockdown of RNF4 worsened ischaemia‐induced cardiac dysfunction of MI models. Our results reveal a novel myocardial apoptosis regulation model that is composed of RNF4, PML and p53. The modulation of these proteins may provide a new approach to tackling cardiac ischaemia.     

Protective effect of total phenylethanoid glycosides from Monochasma savatieri Franch on myocardial ischemia injury

The present study was designed to investigate the cardioprotective effect of total phenylethanoid glycosides from Monochasma savatieri Franch (TPG). The data showed that there were mainly four phenylethanoid glycosides isolated and identified from TPG. TPG significantly increased cells viability and inhibited morphological changes on H9c2 cardiomyocytes induced by H₂O₂ or Na₂S₂O₄. In addition, TPG significantly decreased T-wave elevation and histopathological changes of heart tissues in myocardial infracted rats induced by isoproterenol. It also significantly reduced the infarct size induced by ligating the coronary artery in rats, increased the activities of antioxidative enzymes superoxide dismutase (SOD), the content of glutathione (GSH), and decreased the leakage of lactic dehydrogenase (LDH), the activities of creatine kinase (CK) and the content of maleic dialdehyde (MDA). In conclusion, these results suggested that TPG from Monochasma savatieri Franch might be developed as new natural medicine or food additives with effects of prevention of coronary artery disease due to its significant antioxidant activity.     

Hypertrophic Preconditioning Attenuates Myocardial Ischaemia‐Reperfusion Injury by Modulating SIRT3‐SOD2‐mROS‐Dependent Autophagy

BackgroundIschaemic preconditioning elicited by brief periods of coronary occlusion and reperfusion protects the heart from a subsequent prolonged ischaemic insult. Here, we test the hypothesis that short‐term non‐ischaemic stimulation of hypertrophy renders the heart resistant to subsequent ischaemic injury.Methods and ResultsTransient transverse aortic constriction (TAC) was performed for 3 days in mice and then withdrawn for 4 days by aortic debanding, followed by subsequent exposure to myocardial ischaemia‐reperfusion (I/R) injury. Following I/R injury, myocardial infarct size and apoptosis were significantly decreased, and cardiac dysfunction was markedly improved in the TAC preconditioning group compared with the control group. Mechanistically, TAC preconditioning markedly suppressed I/R‐induced autophagy and preserved autophagic flux by deacetylating SOD2 via a SIRT3‐dependent mechanism. Moreover, treatment with an adenovirus encoding SIRT3 partially mimicked the effects of hypertrophic preconditioning, whereas genetic ablation of SIRT3 in mice blocked the cardioprotective effects of hypertrophic preconditioning. Furthermore, in vivo lentiviral‐mediated knockdown of Beclin 1 in the myocardium ameliorated the I/R‐induced impairment of autophagic flux and was associated with a reduction in cell death, whereas treatment with a lentivirus encoding Beclin 1 abolished the cardioprotective effect of TAC preconditioning.ConclusionsThe present study identifies TAC preconditioning as a novel strategy for induction of an endogenous self‐defensive and cardioprotective mechanism against cardiac injury. Specifically, TAC preconditioning reduced myocardial autophagic cell death in a SIRT3/SOD2 pathway‐dependent manner.     

Role of the JAK-STAT pathway in protection of hydrogen peroxide preconditioning against apoptosis induced by oxidative stress in PC12 cells

The aim of this study was to investigate the role of JAK-STAT pathway in the cytoprotection afforded by preconditioning with H₂O₂. It was shown that (1) Preconditioning with 100 μmol/L H₂O₂ can markedly protect PC12 cells against apoptosis and cytotoxicity induced by 300 μmol/L H₂O₂; (2) The expression and tyrosine phosphorylation of JAK2, not JAK1 were rapidly increased at 5 min after H₂O₂ preconditioning; (3) The expression of STAT1 and STAT3 were significantly increased at 15 min after H₂O₂ preconditioning, and the pTyr-STAT1 and pTyr-STAT3 were markedly increased at 60 min after H₂O₂ preconditioning; (4) Pretreatment with the JAK inhibitor AG-490 (10 μmol/L) 20 min before H₂O₂ preconditioning blocked not only the activation of JAK2, STAT1 and STAT3, but also the cytoprotection of H₂O₂ preconditioning against apoptosis and cytotoxicity induced by oxidative stress. These findings suggested that preconditioning with H₂O₂ activated the JAK-STAT pathway that played an important role in the cytoprotection induced by H₂O₂ preconditioning.     

Tat‐antioxidant 1 protects against stress‐induced hippocampal HT‐22 cells death and attenuate ischaemic insult in animal model

Oxidative stress‐induced reactive oxygen species (ROS) are responsible for various neuronal diseases. Antioxidant 1 (Atox1) regulates copper homoeostasis and promotes cellular antioxidant defence against toxins generated by ROS. The roles of Atox1 protein in ischaemia, however, remain unclear. In this study, we generated a protein transduction domain fused Tat‐Atox1 and examined the roles of Tat‐Atox1 in oxidative stress‐induced hippocampal HT‐22 cell death and an ischaemic injury animal model. Tat‐Atox1 effectively transduced into HT‐22 cells and it protected cells against the effects of hydrogen peroxide (H₂O₂)‐induced toxicity including increasing of ROS levels and DNA fragmentation. At the same time, Tat‐Atox1 regulated cellular survival signalling such as p53, Bad/Bcl‐2, Akt and mitogen‐activate protein kinases (MAPKs). In the animal ischaemia model, transduced Tat‐Atox1 protected against neuronal cell death in the hippocampal CA1 region. In addition, Tat‐Atox1 significantly decreased the activation of astrocytes and microglia as well as lipid peroxidation in the CA1 region after ischaemic insult. Taken together, these results indicate that transduced Tat‐Atox1 protects against oxidative stress‐induced HT‐22 cell death and against neuronal damage in animal ischaemia model. Therefore, we suggest that Tat‐Atox1 has potential as a therapeutic agent for the treatment of oxidative stress‐induced ischaemic damage.     

Combined pre‐conditioning with salidroside and hypoxia improves proliferation,migration and stress tolerance of adipose‐derived stem cells

Oxidative stress after ischaemia impairs the function of transplanted stem cells. Increasing evidence has suggested that either salidroside (SAL) or hypoxia regulates growth of stem cells. However, the role of SAL in regulating function of hypoxia‐pre–conditioned stem cells remains elusive. Thus, this study aimed to determine the effect of SAL and hypoxia pre‐conditionings on the proliferation, migration and tolerance against oxidative stress in rat adipose‐derived stem cells (rASCs). rASCs treated with SAL under normoxia (20% O₂) or hypoxia (5% O₂) were analysed for the cell viability, proliferation, migration and resistance against H₂O₂‐induced oxidative stress. In addition, the activation of Akt, Erk1/2, LC3, NF‐κB and apoptosis‐associated pathways was assayed by Western blot. The results showed that SAL and hypoxia treatments synergistically enhanced the viability (fold) and proliferation of rASCs under non‐stressed conditions in association with increased autophagic flux and activation of Akt, Erk1/2 and LC3. H₂O₂‐induced oxidative stress, cytotoxicity, apoptosis, autophagic cell death and NF‐κB activation were inhibited by SAL or hypoxia, and further attenuated by the combined SAL and hypoxia pre‐treatment. The SAL and hypoxia pre‐treatment also enhanced the proliferation and migration of rASCs under oxidative stress in association with Akt and Erk1/2 activation; however, the combined pre‐treatment exhibited a more profound enhancement in the migration than proliferation. Our data suggest that SAL combined with hypoxia pre‐conditioning may enhance the therapeutic capacity of ASCs in post‐ischaemic repair.     

Hemin with Peroxidase Activity Can Inhibit the Oxidative Damage Induced by Ultraviolet A

Excessive reactive oxygen species (ROS), a highly reactive substance that contains oxygen, induced by ultraviolet A (UVA) cause oxidative damage to skin. We confirmed that hemin can catalyze the reaction of tyrosine (Tyr) and hydrogen peroxide (H₂O₂). Catalysis was found to effectively reduce or eliminate oxidative damage to cells induced by H₂O₂ or UVA. The scavenging effects of hemin for other free-radical ROS were also evaluated through pyrogallol autoxidation, 1,1-diphenyl-2-picrylhydrazyl radical (DPPH·)-scavenging assays, and phenanthroline–Fe²⁺ assays. The results show that a mixture of hemin and tyrosine exhibits strong scavenging activities for H₂O₂, superoxide anion (O₂⁻·), DPPH·, and the hydroxyl radical (·OH). Furthermore, the inhibition of oxidative damage to human skin keratinocyte (HaCaT) cells induced by H₂O₂ or UVA was evaluated. The results show that catalysis can significantly reduce the ratio of cell apoptosis and death and inhibit the release of lactate dehydrogenase (LDH), as well as accumulation of malondialdehyde (MDA). Furthermore, the resistance to apoptosis was found to be enhanced. These results show that the mixture of hemin and tyrosine has a significantly protective effect against oxidative damage to HaCaT cells caused by UVA, suggesting it as a protective agent for combating UVA damage.     

TNFalpha is required to confer protection in an in vivo model of classical ischaemic preconditioning

Although Tumor Necrosis Factor alpha (TNFα) is used as a preconditioning mimetic in vitro, its role in ischaemic preconditioning (IPC) has not been clearly defined. Here, we propose to use an in vivo model (that takes into account the activation of leukocytes which may affect levels of TNFα) to demonstrate that i) TNFα acts as a trigger in IPC and ii) the dose-dependent nature of this cardioprotective effect of TNFα. Male Wistar rats were subjected to 30 min of left coronary artery occlusion (index ischaemia), followed by 24 h reperfusion. In the presence or absence of a soluble TNFα receptor (sTNFα-R), preconditioning was induced by 3 cycles of ischaemia (3 min)/reperfusion (5 min) (IPC) or various doses (0.05-4 μg/kg) of exogenous TNFα. Following 24 h reperfusion, infarct size (IS, expressed as % of the area at risk (AAR)) was assessed. Tissue levels of TNFα from the AAR, following IPC and TNFα stimulus were determined using Western Blot. IPC caused decrease in IS (4.5 ± 1.3% vs 30.8 ± 4.3% in ischaemic rats; P < 0.001) and increase of TNFα levels following the IPC stimulus. The protective effect of IPC was abrogated in the presence of the sTNFα-R. In addition, exogenous TNFα dose-dependently reduced IS with maximal protection at a dose of 0.1 μg/kg (IS = 12.6%, P < 0.01 vs ischaemic). In conclusion our data provide strong evidence for a role of TNFα during the trigger phase of IPC. In addition, exogenous TNFα mimics IPC by providing a dose-dependent cardioprotective effect against ischaemia-reperfusion injury in vivo.     

Low concentration of GA activates a preconditioning response in HepG2 cells during oxidative stress—roles of Hsp90 and vimentin

Oxidative stress can be a significant cause of cell death and apoptosis. We performed studies in HepG2 cells to explore whether prior exposure to oxidative stress (“oxidative preconditioning”) and geldanamycin (GA) treatment can protect the cell from damage caused by subsequent oxidative insults. The cells were treated with 10 nM GA for 24 h before oxidative stress. Oxidative preconditioning was achieved by 2 h exposures to H₂O₂ (50 μM) separated by a 10-h recovery period in normal culture medium. Oxidative stress was induced by exposure to 500 μM H₂O₂ for 24 h. The effects of GA and oxidative preconditioning were investigated on the formation of Hsp90, vimentin, insoluble vimentin aggregates, and cleavage of vimentin in a cell culture model of oxidative stress. GA treatment leads to enhanced expression of Hsp90 and vimentin and to inhibition of vimentin protein aggregation. Similar results were obtained by oxidative preconditioning. It is confirmed that low concentrations of GA protected HepG2 cells from subsequent oxidative stress by increasing the levels of Hsp90 and by alleviating the extent of cell apoptosis induced by oxidative stress, which is similar to oxidative preconditioning. However, in contrast to preconditioning, GA treatment obviously changed binding activity of Hsp90 to vimentin cleavages. All the above indicated that low concentrations of GA treatment triggered cell protection from oxidative stress. Both the level of Hsp90 and its ability to bind with vimentin were changed by low concentrations of GA and might contribute to oxidative stress protection.     

Cardioprotection of ischaemic preconditioning is associated with inhibition of translocation of MLKL within the plasma membrane

Necroptosis, a form of cell loss involving the RIP1‐RIP3‐MLKL axis, has been identified in cardiac pathologies while its inhibition is cardioprotective. We investigated whether the improvement of heart function because of ischaemic preconditioning is associated with mitigation of necroptotic signaling, and these effects were compared with a pharmacological antinecroptotic approach targeting RIP1. Langendorff‐perfused rat hearts were subjected to ischaemic preconditioning with or without a RIP1 inhibitor (Nec‐1s). Necroptotic signaling and the assessment of oxidative damage and a putative involvement of CaMKII in this process were analysed in whole tissue and subcellular fractions. Ischaemic preconditioning, Nec‐1s and their combination improved postischaemic heart function recovery and reduced infarct size to a similar degree what was in line with the prevention of MLKL oligomerization and translocation to the membrane. On the other hand, membrane peroxidation and apoptosis were unchanged by either approach. Ischaemic preconditioning failed to ameliorate ischaemia–reperfusion‐induced increase in RIP1 and RIP3 while pSer229‐RIP3 levels were reduced only by Nec‐1s. In spite of the additive phosphorylation of CaMKII and PLN because of ditherapy, the postischaemic contractile force and relaxation was comparably improved in all the intervention groups while antiarrhythmic effects were observed in the ischaemic preconditioning group only. Necroptosis inhibition seems to be involved in cardioprotection of ischaemic preconditioning and is comparable but not intensified by an anti‐RIP1 agent. Changes in oxidative stress nor CaMKII signaling are unlikely to explain the beneficial effects.     

Remote renal preconditioning-induced cardioprotection: a key role of hypoxia inducible factor-prolyl 4-hydroxylases

Remote preconditioning is a unique phenomenon in which brief episodes of ischemia and reperfusion to remote organ protect the target organ against sustained ischemia–reperfusion (I/R)-induced injury. Protective effects of remote renal preconditioning (RRPC) are well established in heart, but their mechanisms still remain to be elucidated. So, the present study was designed to investigate the possible role of oxygen-sensing hypoxia inducible factor-prolyl 4-hydroxylases (HIF-P4Hs) in RRPC-induced cardioprotection in rats. Remote renal preconditioning was performed by four episodes of 5 min renal artery occlusion and reperfusion. Isolated rat hearts were perfused on Langendorff apparatus and were subjected to global ischemia for 30 min followed by 120 min reperfusion. The levels of lactate dehydrogenase (LDH) and creatine kinase (CK) were measured in coronary effluent to assess the degree of myocardial injury. Extent of myocardial infarct size and coronary flow rate was also measured. Ethyl 3,4-dihydroxybenzoate (EDHB) and α-ketoglutarate (α-KG) were employed as HIF-P4Hs inhibitor and activator, respectively. Diethyldithiocarbamic acid (DDCA) was employed as NFkB inhibitor. Remote renal preconditioning prevented I/R-induced myocardial injury and produced cardioprotective effects. Pharmacological preconditioning with EDHB (100 mg kg⁻¹ i.p.) mimicked the cardioprotective effects of RRPC. However, α-KG (200 mg kg⁻¹ i.p.) and DDCA (150 mg kg⁻¹ i.p.) abolished cardioprotective effects of RRPC and EDHB. So, it may be concluded that inhibition of HIF-P4H has a key role in RRPC-induced cardioprotection. Further, remote preconditioning-induced HIF-P4H inhibition may have triggered a transduction pathway involving activation of NFkB.     

Hypoxic preconditioning‐induced autophagy enhances survival of engrafted endothelial progenitor cells in ischaemic limb

Recent clinical studies have suggested that endothelial progenitor cells (EPCs) transplantation provides a modest benefit for treatment of the ischaemic diseases such as limb ischaemia. However, cell‐based therapies have been limited by poor survival of the engrafted cells. This investigation was designed to establish optimal hypoxia preconditioning and evaluate effects of hypoxic preconditioning‐induced autophagy on survival of the engrafted EPCs. Autophagy of CD34⁺VEGFR‐2⁺ EPCs isolated from rat bone marrow increased after treatment with 1% O₂. The number of the apoptotic cells in the hypoxic cells increased significantly after autophagy was inhibited with 3‐methyladenine. According to balance of autophagy and apoptosis, treatment with 1% O₂ for 2 hrs was determined as optimal preconditioning for EPC transplantation. To examine survival of the hypoxic cells, the cells were implanted into the ischaemic pouch of the abdominal wall in rats. The number of the survived cells was greater in the hypoxic group. After the cells loaded with fibrin were transplanted with intramuscular injection, blood perfusion, arteriogenesis and angiogenesis in the ischaemic hindlimb were analysed with laser Doppler‐based perfusion measurement, angiogram and the density of the microvessels in histological sections, respectively. Repair of the ischaemic tissue was improved significantly in the hypoxic preconditioning group. Loading the cells with fibrin has cytoprotective effect on survival of the engrafted cells. These results suggest that activation of autophagy with hypoxic preconditioning is an optimizing strategy for EPC therapy of limb ischaemia.     

Signal mechanism activated by erythropoietin preconditioning and remote renal preconditioning-induced cardioprotection

It has been recently reported that release of erythropoietin could mediate the cardioprotective effects of remote renal preconditioning. However, the mechanism of erythropoietin-mediated cardioprotection in remote preconditioning is still unexplored. Therefore, the present study was designed to investigate the possible signal transduction pathway of erythropoietin-mediated cardioprotection in remote preconditioning in rats. Remote renal preconditioning was performed by four episodes of 5 min renal artery occlusion followed by 5 min reperfusion. Isolated rat hearts were perfused on Langendorff apparatus and were subjected to global ischemia for 30 min followed by 120 min reperfusion. The levels of lactate dehydrogenase (LDH) and creatine kinase (CK) were measured in coronary effluent to assess the degree of myocardial injury. Extent of myocardial infarct size and coronary flow rate was also measured. Remote renal preconditioning and erythropoietin preconditioning (5,000 IUkg(-1), i.p.) attenuated ischemia-reperfusion-induced myocardial injury and produced cardioprotective effects. However, administration of diethyldithiocarbamic acid (150 mg kg(-1) i.p.), a selective NFkB inhibitor, and glibenclamide (5 mg kg(-1) i.p.), a selective K(ATP) channel blocker, attenuated cardioprotective effects of remote preconditioning and erythropoietin preconditioning. However, administration of minoxidil (1 mg kg(-1) i.v.), a selective K(ATP) channel opener, restored the attenuated cardioprotective effects of remote preconditioning and erythropoietin preconditioning in diethyldithiocarbamic acid pretreated rats. These results suggest that K(ATP) channel is a downstream mediator of NFkB activation in remote preconditioning and erythropoietin preconditioning. Therefore, it may be concluded that erythropoietin preconditioning and remote renal preconditioning trigger similar signaling mechanisms for cardioprotection, i.e., NFkB activation followed by opening of K(ATP) channels.     

Specific antioxidant selenoproteins are induced in the heart during hypertrophy

Selenium (Se) is thought to confer cardioprotective effects through the actions of antioxidant selenoprotein enzymes that directly limit levels of ROS such as hydrogen peroxide (H₂O₂) or that reverse oxidative damage to lipids and proteins. To determine how the selenoproteome responds to myocardial hypertrophy, two mouse models were employed: triidothyronine (T3)- or isoproterenol (ISO)-treatment. After 7 days of T3- and ISO-treatment, cardiac stress was demonstrated by increased H₂O₂ and caspase-3 activity. Neither treatment produced significant increases in phospholipid peroxidation or TUNEL-positive cells, suggesting that antioxidant systems were protecting the cardiomyocytes from damage. Many selenoprotein mRNAs were induced by T3- and ISO-treatment, with levels of methionine sulfoxide reductase 1 (MsrB1, also called SelR) mRNA showing the largest increases. MsrB enzymatic activity was also elevated in both models of cardiac stress, while glutathione peroxidase (GPx) activity and thioredoxin reductase (Trxrd) activity were moderately and nonsignificantly increased, respectively. Western blot assays revealed a marked increase in MsrB1 and moderate increases in GPx3, GPx4, and Trxrd1, particularly in T3-treated hearts. Thus, the main response of the selenoproteome during hypertrophy does not involve increased GPx1, but increased GPx3 for reducing extracellular H₂O₂ and increased GPx4, Trxrd1, and MsrB1 for minimizing intracellular oxidative damage.