Maria-Giulia Perrelli

Ischemia/reperfusion injury and cardioprotective mechanisms: Role of mitochondria and reactive oxygen species

Reperfusion therapy must be applied as soon as possible to attenuate the ischemic insult of acute myocardial infarction (AMI). However reperfusion is responsible for additional myocardial damage, which likely involves opening of the mitochondrial permeability transition pore (mPTP). In reperfusion injury, mitochondrial damage is a determining factor in causing loss of cardiomyocyte function and viability. Major mechanisms of mitochondrial dysfunction include the long lasting opening of mPTPs and the oxidative stress resulting from formation of reactive oxygen species (ROS). Several signaling cardioprotective pathways are activated by stimuli such as preconditioning and postconditioning, obtained with brief intermittent ischemia or with pharmacological agents. These pathways converge on a common target, the mitochondria, to preserve their function after ischemia/reperfusion. The present review discusses the role of mitochondria in cardioprotection, especially the involvement of adenosine triphosphate-dependent potassium channels, ROS signaling, and the mPTP. Ischemic postconditioning has emerged as a new way to target the mitochondria, and to drastically reduce lethal reperfusion injury. Several clinical studies using ischemic postconditioning during angioplasty now support its protective effects, and an interesting alternative is pharmacological postconditioning. In fact ischemic postconditioning and the mPTP desensitizer, cyclosporine A, have been shown to induce comparable protection in AMI patients.

Microvascular dysfunction induced by reperfusion injury and protective effect of ischemic preconditioning.

Hepatic ischemia/reperfusion injury has immediate and deleterious effects on the outcome of patients after liver surgery. The precise mechanisms leading to the damage have not been completely elucidated. However, there is substantial evidence that the generation of oxygen free radicals and disturbances of the hepatic microcirculation are involved in this clinical syndrome. Microcirculatory dysfunction of the liver seems to be mediated by sinusoidal endothelial cell damage and by the imbalance of vasoconstrictor and vasodilator molecules, such as endothelin (ET), reactive oxygen species (ROS), and nitric oxide (NO). This may lead to no-reflow phenomenon with release of proinflammatory cytokines, sinusoidal plugging of neutrophils, oxidative stress, and as an ultimate consequence, hypoxic cell injury and parenchymal failure. An inducible potent endogenous mechanism against ischemia/reperfusion injury has been termed ischemic preconditioning. It has been suggested that preconditioning could inhibit the effects of different mediators involved in the microcirculatory dysfunction, including endothelin, tumor necrosis factor-alpha, and oxygen free radicals. In this review, we address the mechanisms of liver microcirculatory dysfunction and how ischemic preconditioning could help to provide new surgical and/or pharmacological strategies to protect the liver against reperfusion damage.

Mitochondrial Pathways, Permeability Transition Pore, and Redox Signaling in Cardioprotection: Therapeutic Implications

Reperfusion therapy is the indispensable treatment of acute myocardial infarction (AMI) and must be applied as soon as possible to attenuate the ischemic insult. However, reperfusion is responsible for additional myocardial damage likely involving opening of the mitochondrial permeability transition pore (mPTP). A great part of reperfusion injury occurs during the first minute of reperfusion. The prolonged opening of mPTP is considered one of the endpoints of the cascade to myocardial damage, causing loss of cardiomyocyte function and viability. Opening of mPTP and the consequent oxidative stress due to reactive oxygen and nitrogen species (ROS/RNS) are considered among the major mechanisms of mitochondrial and myocardial dysfunction. Kinases and mitochondrial components constitute an intricate network of signaling molecules and mitochondrial proteins, which interact in response to stressors. Cardioprotective pathways are activated by stimuli such as preconditioning and postconditioning (PostC), obtained with brief intermittent ischemia or with pharmacological agents, which drastically reduce the lethal ischemia/reperfusion injury. The protective pathways converging on mitochondria may preserve their function. Protection involves kinases, adenosine triphosphate-dependent potassium channels, ROS signaling, and the mPTP modulation. Some clinical studies using ischemic PostC during angioplasty support its protective effects, and an interesting alternative is pharmacological PostC. In fact, the mPTP desensitizer, cyclosporine A, has been shown to induce appreciable protections in AMI patients. Several factors and comorbidities that might interfere with cardioprotective signaling are considered. Hence, treatments adapted to the characteristics of the patient (i.e., phenotype oriented) might be feasible in the future.

Cardioprotective pathways during reperfusion: focus on redox signaling and other modalities of cell signaling.

Post-ischemic reperfusion may result in reactive oxygen species (ROS) generation, reduced availability of nitric oxide (NO•), Ca(2+)overload, prolonged opening of mitochondrial permeability transition pore, and other processes contributing to cell death, myocardial infarction, stunning, and arrhythmias. With the discovery of the preconditioning and postconditioning phenomena, reperfusion injury has been appreciated as a reality from which protection is feasible, especially with postconditioning, which is under the control of physicians. Potentially cooperative protective signaling cascades are recruited by both pre- and postconditioning. In these pathways, phosphorylative/dephosphorylative processes are widely represented. However, cardioprotective modalities of signal transduction also include redox signaling by ROS, S-nitrosylation by NO• and derivative, S-sulfhydration by hydrogen sulfide, and O-linked glycosylation with beta-N-acetylglucosamine. All these modalities can interact and regulate an entire pathway, thus influencing each other. For instance, enzymes can be phosphorylated and/or nitrosylated in specific and/or different site(s) with consequent increase or decrease of their specific activity. The cardioprotective signaling pathways are thought to converge on mitochondria, and various mitochondrial proteins have been identified as targets of these post-transitional modifications in both pre- and postconditioning.

Redox balance and cardioprotection

Coronary artery disease is a major cause of morbidity and mortality in the Western countries. Acute myocardial infarction is a serious and often lethal consequence of coronary artery disease, resulting in contractile dysfunction and cell death. It is well known that unbalanced and high steady state levels of reactive oxygen and nitrogen species (ROS/RNS) are responsible for cytotoxicity, which in heart leads to contractile dysfunction and cell death. Pre- and post-conditioning of the myocardium are two treatment strategies that reduce contractile dysfunction and the amount of cell death considerably. Paradoxically, ROS and RNS have been identified as a part of cardioprotective signaling molecules, which are essential in pre- and post-conditioning processes. S-nitrosylation of proteins is a specific posttranslational modification that plays an important role in cardioprotection, especially within mitochondria. In fact, mitochondria are of paramount importance in either promoting or limiting ROS/RNS generation and reperfusion injury, and in triggering kinase activation by ROS/RNS signaling in cardioprotection. These organelles are also the targets of acidosis, which prevents mitochondrial transition pore opening, thus avoiding ROS-induced ROS release. Therefore, we will consider mitochondria as either targets of damage or protection from it. The origin of ROS/RNS and the cardioprotective signaling pathways involved in ROS/RNS-based pre- and post-conditioning will be explored in this article. A particular emphasis will be given to new aspects concerning the processes of S-nitrosylation in the cardioprotective scenario.

Postconditioning induces an anti-apoptotic effect and preserves mitochondrial integrity in isolated rat hearts.

Postconditioning (PostC) may limit mitochondrial damage and apoptotic signaling. We studied markers of apoptosis and mitochondrial protection in isolated rat hearts, which underwent a) perfusion without ischemia (Sham), b) 30-min ischemia (I) plus 2-hour reperfusion (R), or c) PostC protocol (5 intermittent cycles of 10-s reperfusion and 10-s ischemia immediately after the 30-min ischemia). Markers were studied in cytosolic (CF) and/or mitochondrial (MF) fractions. In CF, while pro-apoptotic factors (cytochrome c and caspase-3) were reduced, the anti-apoptotic markers (Bcl-2 and Pim-1) were increased by PostC, compared to the I/R group. Accordingly, phospho-GSK-3beta and Bcl-2 levels increased in mitochondria of PostC group. Moreover, I/R reduced the level of mitochondrial structural protein (HSP-60) in MF and increased in CF, thus suggesting mitochondrial damage and HSP-60 release in cytosol, which were prevented by PostC. Electron microscopy confirmed that I/R markedly damaged cristae and mitochondrial membranes; damage was markedly reduced by PostC. Finally, total connexin-43 (Cx43) levels were reduced in the CF of the I/R group, whereas phospho-Cx43 level resulted in higher levels in the MF of the I/R group than the Sham group. PostC limited the I/R-induced increase of mitochondrial phospho-Cx43. Data suggest that PostC i) increases the levels of anti-apoptotic markers, including the cardioprotective kinase Pim-1, ii) decreases the pro-apoptotic markers, e.g. cytochrome c, iii) preserves the mitochondrial structure, and iv) limits the migration of phospho-Cx43 to mitochondria.

Xanthine oxidase-induced oxidative stress causes activation of NF-κB and inflammation in the liver of type I diabetic rats

We previously showed that xanthine oxidase activity increases in type I diabetic animals and that this is a significant cause of the oxidative stress which occurs in the disease. The aim of this work was to search for molecular links between xanthine oxidase-induced oxidative stress and inflammation in Type I diabetes and to assess the ability of allopurinol, a drug widely used in clinical practice, to prevent both processes. 3-month-old male Wistar rats were made diabetic by injection (i.p.) of either streptozotocin or alloxan. Allopurinol (32 mg/Kg) was administered (i.p) to diabetic rats after they had shown clear signs of diabetes such as glucosuria and polyuria. Hepatic phospho-IKKbeta and phospho-IkappaBalpha contents were increased in diabetic animals. This was accompanied by increased levels of NF-kappaB (p65 protein content) in liver nuclear extracts. Hepatic expression of NF-kappaB dependent inflammatory cytokines and enzymes, namely interleukin 1beta, iNOS and interleukin 6 were markedly increased. Both diabetes-induced activation of NF-kappaB signalling cascade and subsequent over expression of inflammatory cytokines and enzymes were abolished by administration of allopurinol. Moreover, we found a significant neutrophil infiltration in the liver of diabetic animals. These events were also prevented by administration of allopurinol.

Catestatin reduces myocardial ischaemia/reperfusion injury: involvement of PI3K/Akt, PKCs, mitochondrial KATP channels and ROS signalling.

Catestatin (CST) limits myocardial ischaemia/reperfusion (I/R) injury with unknown mechanisms. Clearly phosphoinositide-3-kinase (PI3K), protein kinase C (PKC) isoforms, including intra-mitochondrial PKCε, mitochondrial KATP (mitoKATP) channels and subsequent reactive oxygen species (ROS)-signalling play important roles in postconditioning cardioprotection, preventing mitochondrial permeability transition pore (mPTP) opening. Therefore, we studied the role of these extra- and intra-mitochondrial factors in CST-induced protection. Isolated rat hearts and H9c2 cells underwent I/R and oxidative stress, respectively. In isolated hearts CST (75nM, CST-Post) given in early-reperfusion significantly reduced infarct size, limited post-ischaemic contracture, and improved recovery of developed left ventricular pressure. PI3K inhibitor, LY-294002 (LY), large spectrum PKC inhibitor, Chelerythrine (CHE), specific PKCε inhibitor (εV1-2), mitoKATP channel blocker, 5-Hydroxydecanoate (5HD) or ROS scavenger, 2-mercaptopropionylglycine (MPG) abolished the infarct-sparing effect of CST. Notably the CST-induced contracture limitation was maintained during co-infusion of 5HD, MPG or εV1-2, but it was lost during co-infusion of LY or CHE. In H9c2 cells challenged with H2O2, mitochondrial depolarization (an index of mPTP opening studied with JC1-probe) was drastically limited by CST (75nM). Our results suggest that the protective signalling pathway activated by CST includes mitoKATP channels, ROS signalling and prevention of mPTP opening, with a central role for upstream PI3K/Akt and PKCs. In fact, all inhibitors completely abolished CST-infarct-sparing effect. Since CST-anti-contracture effect cannot be explained by intra-mitochondrial mechanisms (PKCε activation and mitoKATP channel opening) or ROS signalling, it is proposed that these downstream signals are part of a reverberant loop which re-activates upstream PKCs, which therefore play a pivotal role in CST-induced protection.

Pharmacologically active microcarriers influence VEGF-A effects on mesenchymal stem cell survival

Resistance of transplanted mesenchymal stem cells (MSCs) in post‐ischemic heart is limited by their poor vitality. Vascular‐endothelial‐growth‐factor‐A (VEGF‐A) as such or slowly released by fibronectin‐coated pharmacologically‐active‐microcarriers (FN‐PAM‐VEGF) could differently affect survival kinases and anti‐apoptotic mediator (e.g. Bcl‐2). Therefore VEGF‐A or FN‐PAM‐VEGF could differently enhance cell proliferation, and/or resistance to hypoxia/reoxygenation (H/R) of MSCs. To test these hypotheses MSCs were incubated for 6‐days with VEGF‐A alone or with FN‐PAM‐VEGF. In addition, MSCs pre‐treated for 24‐hrs with VEGF‐A or FN‐PAM‐VEGF were subsequently exposed to H/R (72‐hrs 3% O2 and 3‐hrs of reoxygenation). Cell‐proliferation and post‐hypoxic vitality were determined. Kinases were studied at 30‐min., 1‐ and 3‐days of treatment. Cell‐proliferation increased about twofold (P < 0.01) 6‐days after VEGF‐A treatment, but by a lesser extent (55% increase) with FN‐PAM‐VEGF (P < 0.05). While MSC pre‐treatment with VEGF‐A confirmed cell‐proliferation, pre‐treatment with FN‐PAM‐VEGF protected MSCs against H/R. In the early phase of treatments, VEGF‐A increased phospho‐Akt, phospho‐ERK‐1/2 and phospho‐PKCε compared to the untreated cells or FN‐PAM‐VEGF. Afterword, kinase phosphorylations were higher with VGEF, except for ERK‐1/2, which was similarly increased by both treatments at 3 days. Only FN‐PAM‐VEGF significantly increased Bcl‐2 levels. After H/R, lactate dehydrogenase release and cleaved Caspase‐3 levels were mainly reduced by FN‐PAM‐VEGF. While VEGF‐A enhances MSC proliferation in normoxia, FN‐PAM‐VEGF mainly hampers post‐hypoxic MSC death. These different effects underscore the necessity of approaches suited to the various conditions. The use of FN‐PAM‐VEGF could be considered as a novel approach for enhancing MSC survival and regeneration in hostile environment of post‐ischemic tissues.

Id2 leaves the chromatin of the E2F4–p130-controlled c-myc promoter during hepatocyte priming for liver regeneration

The Id (inhibitor of DNA binding or inhibitor of differentiation) helix-loop-helix proteins are involved in the regulation of cell growth, differentiation and cancer. The fact that the molecular mechanisms of liver regeneration are not completely understood prompted us to study the fate of Id2 in proliferating liver. Id2 increases in liver regeneration after partial hepatectomy, following the early induction of its gene. Co-immunoprecipitation shows that Id2 forms a complex with E2F4, p130 and mSin3A in quiescent liver and all these components are present at the c-myc promoter as shown using ChIP (chromatin immunoprecipitation). Activation of c-myc during hepatocyte priming (G0-G1 transition) correlates with the dissociation of Id2 and HDAC (histone deacetylase), albeit p130 remains bound at least until 6 h. Moreover, as the G0-G1 transition progresses, Id2 and HDAC again bind the c-myc promoter concomitantly with the repression of this gene. The time course of c-myc binding to the Id2 promoter, as determined by ChIP assays is compatible with a role of the oncoprotein as a transcriptional inducer of Id2 in liver regeneration. Immunohistochemical analysis shows that Id2 also increases in proliferating hepatocytes after bile duct ligation. In this case, the pattern of Id2 presence in the c-myc promoter parallels that found in regenerating liver. Our results may suggest a control role for Id2 in hepatocyte priming, through a p130 dissociation-independent regulation of c-myc.