Arginine as the main substrate of both isoforms of arginase is also the substrate for the three isoforms of nitric oxide synthase (NOS). Therefore, excessive activity of either NOS or arginase reduces substrate availability for the other arginine consumer. In fact, most cells express arginase and NOS isoforms at the same time. In most cells enzymatic activity of each of these enzymes is tightly regulated by direct protein modification or by induction of enzyme expression. In case of arginase an increased activity of arginase has consequences for both pathways: a loss of NO/cGMP signaling and an improvement of polyamine metabolism. Exam- ples for increased arginase activity and expression are found in artherosclerosis, hypertension, inflammation, aging, stroke, myocardial infarction, and heart failure to name just a few ( Wu and Meininger, 2000; Ryoo et al., 2008; Bagnost et al., 2010; Heusch et al., 2010 ). It is commonly accepted that excessive acti- vation of arginase is associated with disease progress. Whether the final effect of arginase activation depends on the activa- tion of arginase-dependent pathways (polyamine pathways) or on the inhibitory effect on NO/cGMP-dependent pathways or on both pathways remains elusive. Furthermore, at least in case of atherosclerosis, arginase II may activate intracellular signal- ing independent of its enzymatic activity ( Xiong et al., 2013 ). Regarding the role of arginase in endothelial dysfunction, argi- nine metabolism seems to play a relevant role by either reduc- ing arginine uptake ( Martens et al., 2014 ), activation of arginase II ( Pandey et al., 2014 ), or activation of arginase I ( Gao et al., 2007 ). A precise understanding of the regulation and function of arginase and a subsequent translation of these findings into medical therapies will certainly improve clinical outcomes. The current review will focus on the role of arginase during ischemiaand in particular during reperfusion.
during hepatic ischemia/reperfusion injury . In turn, hepatic remote injury was observed after 30 minutes of intestinal ischemiaand 90 minutes of reperfusion, presenting histopathologically with hepatocyte cytoplasmic clod formation, vacuolization and sinusoidal congestion . Mechanistically, circulating inflammatory mediators derived from the primarily injured tissue and provided through a systemic inflammatory response elicit the injury in remote organs . Herein, we found only mild hepatic affection after pulmonary ischemiaandreperfusion, but no histopathological findings were obtained from cardiac and renal specimens. Potential reason is the only mild pulmonary ischemic insult, as discussed above. Nevertheless, despite the inflammatory response, remote organ injury during pulmonary ischemiaandreperfusion may also be elicited hemodynamically. During lung transplantation, for example, clamping of one lung hilum leads to redirection of the entire cardiac output through only “half” of the pulmonary vascular bed. Although the pulmonary arteries are capacity vessels and may compensate volume overload through dilatation quite impressively under normal physiologic conditions, this function may be dramatically impaired in terminal lung diseases, which reason transplantation. In fact, end-stage lung disease patients frequently present with secondary pulmonary hypertension and therefore about 36% of lung transplantation procedures require the intraoperative use of extracorporeal circulation techniques in order to prevent acute right heart dilatation, congestion and ultimately failure .
The results of the present study revealed that K ATP channels also contribute to the regulation of the Ca 2+ homeostasis in EC during ischemic conditions. Treatment with K ATP channel inhibitors glybenclamide or HMR1098 from onset of ischemic conditions to the end of reperfusion reduced the Ca 2+ overload during ischemiaandreperfusion. The basic features of changes in Ca 2+ homeostasis under ischemiaandreperfusion were previously described (Noll et al. 1995, Ladilov et al. 2000, Schäfer et al. 2001): Coronary EC develop a biphasic Ca 2+ overload during ischemia, an initial rise mainly due to Ca 2+ release from the ER and a secondary rise due to Ca 2+ influx from the extracellular space. When applied from onset of ischemia, K ATP channel inhibitors reduced the initial rise of cytosolic Ca 2+ . Our Ca 2+ -free experiments also showed a reduction of the first rise. This observation indicates that the first Ca 2+ rise contains a part of Ca 2+ influx from the extracellular space. This extracellular influx component is dependent on plasmalemmal K ATP channels, probably by their effect on the rapidly developing hyperpolarisation.
Although there are many enzymes that can potentially produce superoxide anions in the lung, including uncoupled NO synthase, xanthine oxidase, cytochrome P450 and mitochondrial respiratory chain enzymes, 71 the contribution of these enzymes to the vascular generation of ROS is relatively minor compared with NADPH oxidase. Numerous studies have shown that NADPH oxidase−derived ROS can promote and modulate oxygen radical production by other sources, thereby amplifying the total amounts of ROS. 58 Selective inhibition of NADPH oxidase with apocynin attenuated the IR-induced increase in pulmonary vascular permeability in lungs from rabbits and WT mice, demonstrating the role of NADPH oxidase in our model of ischemiaandreperfusion. The selectivity of apocynin for NADPH oxidase has been well characterized. 97 Apocynin impedes the assembly of the p47 phox and p67 phox subunits within the membrane NADPH oxidase complex, thereby blocking formation of an active enzyme and preventing ROS generation. 97 In animal models, apocynin affords protection against IR-induced lung injury. 112, 113 Favorable effects of inhibiting NADPH oxidase were further confirmed by experiments with mice lacking Nox2, the main catalytic subunit of the enzyme, and in in vivo experiments. Reperfusion of isolated lungs from Nox2 KO mice was also associated with suppressed ROS production during the first minutes of reperfusion.
Suitable and reproducible experimental models of translational research in reconstructive surgery that allow in-vivo investigation of diverse molecular and cellular mechanisms are still limited. To this end we created a novel murine model of acute hindlimb ischemia-reperfusion to mimic a microsurgical free flap procedure. Thirty-six C57BL6 mice (n = 6/group) were assigned to one control and five experimental groups (subject to 6, 12, 96, 120 hours and 14 days of reperfusion, respectively) following 4 hours of complete hindlimb ischemia. Ischemiaandreperfusion were monitored using Laser- Doppler Flowmetry. Hindlimb tissue components (skin and muscle) were investigated using histopathology, quantitative immunohistochemistry and immunofluorescence. Despite massive initial tissue damage induced by ischemia-reperfusion injury, the structure of the skin component was restored after 96 hours. During the same time, muscle cells were replaced by young myotubes. In addition, initial neuromuscular dysfunction, edema and swelling resolved by day 4. After two weeks, no functional or neuromuscular deficits were detectable. Furthermore, upregulation of VEGF and tissue infiltration with CD34-positive stem cells led to new capillary formation, which peaked with significantly higher values after two weeks. These data indicate that our model is suitable to investigate cellular and molecular tissue alterations from ischemia- reperfusion such as occur during free flap procedures.
Several antioxidants (e.g. NAC, GSH, α-tocopherol) and direct radical scavengers (e.g. SOD, CAT), as already mentioned above, demonstrate huge potential in different models of hepatic IRI. High dosages of allopurinol demonstrated to have antioxidant potential in hepatic IR, as it inhibits the enzyme xanthine-oxidase which participates in ROS generation. 2 α-Lipoic acid, a well described antioxidant, has shown to regulate several signal-transduction pathways resulting in tissue protection. Selective iNOS inhibitors have proved tissue protection during hepatic IR, 36, 38 as iNOS upregulation goes along with excessive NO production, which is linked to the formation of peroxynitrite and to its systematic impact on vasodilatation (see section 1.2.4). The pretreatment of livers suffering warm or cold ischemia followed by reperfusion, with the hormone atrial natriuretic peptide (ANP) reduced tissue damage and increased liver function, due to direct impact on several mediators of IR (NF-κB, AP-1, ROS, TNF-α and HSP 70). 66, 67
Five mm distal and proximal from the applied ligatures (Fig. 4), two-colored filaments were attached at the anti-mesenterial side of the intestine to mark the entire length of ischemic intestine and especially know to the orientation of proximal/distal along the ischemic intestine when dissecting tissues for histology (see 5.2.7 “Histology and Light Microscopy”). This enabled first direct histological comparability of intestinal injury of ileum among all study groups and also allowed assessment whether the graded intestinal injury (see 5.2.8 “Pathology and Villi Length Analysis”) might not be the same and hence vary along the entire length of the ischemic ileum. Following the ligation, the exposed intestine was then gently relocated into the abdominal cavity including the microvascular clamps. The abdominal wall was closed to create warm ischemia for a 30 min time period. After this the abdominal cavity was reopened, and the vascular clamp and the sutures were removed. The abdominal wall was closed for a second time tightly. Reperfusion was allowed for a 60 min period. Then rats were euthanized by decapitation. Subsequently, individual samples of the terminal ileum, the duodenum, the jejunum, the colon, the pancreas, the liver, kidneys, the lungs, and the brain were obtained in fixation medium as described in detail below (see 5.2.5 “Tissue
Fig. 1. Impact of NO synthase inhibition on capillary filtration coefficient (Kfc) and lung weight gain (LWG) in isolated perfused rabbit lung. I/R lungs underwent 4 hour ischemia with following reperfusion, no treatment was administered. Time matched nonischemic control (NIC) lungs were perfused and normoxically ventilated throughout 6 hrs. NOS inhibitors L-NMMA 400 µM, 1400W 10 µM, BYK 191023 20 µM, or VNIO 1 µM were applied 5 min before ischemia according to protocol. Kfc was determined gravimetrically from the slope of the lung weight gain curve induced by a 7.5-mmHg step elevation of the venous pressure for 8 min. LWG was calculated as the difference in organ weight measured directly before and 5 min after each of these pressure elevation maneuvers.
Acute myocardial infarction (AMI) remains one of the leading causes of hospitalization and cardiovascular mortality worldwide. 1 Although mortality attributable to the acute coronary event has declined substantially with improved reperfusion therapies, long- term morbidity has increased because of secondary heart failure in survivors of AMI. The major cause of MI is atherosclerosis of coronary arteries. In particular atherosclerotic plaques acquiring an unstable phenotype are prone to rupture and subsequent thrombus formation, resulting in occlusion of the coronary artery. 2 The prolonged occlusion of a coronary artery results in ischemic damage of the cardiac tissue. While reperfusion of the diseased blood vessel is the therapeutic mainstay and improves survival substantially, the restoration of blood flow to previously ischemic tissue can itself induce further cardiac damage, a phenomenon known as myocardial ischemiareperfusion injury (IRI). 3 IRI triggers pronounced tissue-disruptive and sterile pro-inflammatory responses, which compromise the cardiac functional outcome. 4 Current treatment strategies for AMI are based on restoring blood flow in the coronary artery (reperfusion) by dissolving the thrombus with fibrinolytic agents and/or mechanical, baloon-mediated stretching of the occluded artery and implantation of an intravascular stent. Although reperfusion strategies are successful in limiting injury to the heart, reducing infarct size and improving overall prognosis, patients with AMI have an increased short-term and long-term risk of heart failure, which is at least partially attributed to the ensuing IRI. Therefore, better understanding of the pathophysiology of myocardial IRI is a key to devise better treatment strategies for the prevention and treatment of adverse cardiac remodeling and subsequent heart failure following myocardial IRI.
Normothermic machine perfusion (NMP) of kidney grafts is a promising new preservation method to improve graft quality and clinical outcome. Routinely, kidneys are washed out of blood remnants and cooled using organ preservation solutions prior to NMP. Here we assessed the effect of cold preflush compared to direct NMP. After 30 min of warm ischemia, porcine kidneys were either preflushed with cold histidine-tryptophan-ketoglutarate solution (PFNMP group) prior to NMP or directly subjected to NMP (DNMP group) using a blood/buffer solution. NMP was performed at a perfusion pressure of 75 mmHg for 6 h. Functional parameters were assessed as well as histopathological and biochemical analyses. Renal function as expressed by creatinine clearance, fractional excretion of sodium and total output of urine was inferior in PFNMP. Urine protein and neutrophil gelatinase-associated lipocalin (NGAL) concentrations as markers for kidney damage were significantly higher in the PFNMP group. Additionally, increased osmotic nephropathy was found after PFNMP. This study demonstrated that cold preflush prior to NMP aggravates ischemiareperfusion injury in comparison to direct NMP of warm ischemia-damaged kidney grafts. With increasing use of NMP systems for kidneys and other organs, further research into graft flushing during retrieval is warranted.
Ischemic preconditioning (IPC) is an established method to avoid ischemia- reperfusion injury in different vascular beds (12, 33, 34). It was first described by Murry et al. in an experimental setting using the ligation of a dog’s coronary artery for three periods of 5 minute ischemia prior to a prolonged ischemia (12). A significant amount of myocardial tissue could be protected using this approach. Until now, it has proved beneficial on different surrogate endpoints in several smaller human trials applying IPC during coronary angioplasty or cardiac surgery (35). The effects of IPC can be classified into an early phase of protection, which occurs during the first hours after IPC, and a late phase of protection, which is observed approximately 24 hours after IPC (36, 37). Previous data suggest that there is great variation in the amount of protection conferred by this mechanical intervention (36, 38, 39), and different
Not only hypoxia, but also cardiac ischemia-reperfusion introduces subsequent epigenetic changes, which influence the resulting tissue damage. In correlation with gene expression, alterations in histone modifications and DNA methylation were reported for specific promoter regions, like for nitric oxide synthase (NOS) and VEGF (reviewed in Webster et al., 2013). HDAC activity seems to be highly involved in myocardial infarction. In a murine model of IR, a global loss of pan-acetylation of H3 and H4 was reported. Administration of group I and II HDAC inhibitors, TSA or Scriptaid, minimized the resulting infarct by half, prevented the induction of HIF-1α, and reduced cell death and vascular permeability. Treatment with inhibitors 12 hours post-reperfusion revealed no effect, arguing for an earlier epigenetic treatment window (Granger et al., 2008). Pre- administration of inhibitors of histone demethylases, like deferoxamine mesylate salt (DFOM), and dimethyloxaloylglycine (DMOG) were shown to reduce the infarct size in stroke in mice, independent of their effect on HIF-1α stability (Zeynalov et al., 2009). These studies were the first to prove a general epigenetic component in the pathogenesis of cardiac IRI and stroke, and might pave the way for novel therapeutic approaches. It is speculated, that the tolerosome upon ischemic conditioning is mediated by epigenetic mechanisms, which have the power to transduce sub-lethal stresses in durable gene expression patters, triggering an adaptive programme (Gidday et al., 2015).
Kirkby et al. clamped in their model of renal IRI the pedicles of both kidneys for 30 minutes followed by a 6h reperfusion period. Bilirubin was administered intravenously at a dose of 1.5mg/Kg 1h before reperfusionand continuously during reperfusion respectively at a dose of 20mg/Kg 1h before and during ischemia (bolus administration). After 6h of reperfusion animals where sacrificed and tissue samples as well as serum samples where collected for further analyses. This experimental design did not show any significant protective effects of bilirubin administration on renal IRI. Only serum creatinine levels in the 20mg/Kg treatment group showed a significant decrease if compared to the vehicle control. However, neither the intravenous bolus nor continuous infusion of bilirubin led to a significant protection to the renal medulla (56).
can lead to cardiomyocyte death due to hyper-contracture . 1.2.1 Pathophysiology of ischemia/reperfusion-injuries
The occurring ischemia is accompanied by a lack of oxygen in the respective tissue or organ. Within minutes, the absence of oxygen in the heart leads to a complete catabolism of adenosine triphosphate (ATP) to hypoxanthine, adenosine, inosine, xanthine, and uric acid . At the same time, ATP-dependent ion transporters stop working, which leads to an influx of calcium and an efflux of potassium . This and the rising level of hypoxanthine are responsible for the transformation of the NAD-reducing enzyme xanthine dehydrogenase to the oxygen radical-producing xanthine oxidase, which oxidizes hypoxanthine to xanthine . This reaction leads to a massive release of many ROS (for example superoxide, hydrogen peroxide, or hydroxyl radicals) . This process is amplified by the reestablished molecular oxygen into the tissue after reperfusion, that further reacts with hypoxanthine and xanthine oxidase to produce more superoxide anions and hydrogen peroxides. Together with iron as a catalyst they react via the Haber-Weiss reaction to form hydroxyl radicals. This species can destroy the cell membrane by lipid peroxidation . Besides those direct influences on cell damage, ROS also stimulate neutrophils and the upregulation of adhesion molecules (especially vascular cell adhesion molecule-1 (VCAM- 1), intercellular adhesion molecule-1 (ICAM-1)) [15-17]. This further contributes to an increased binding of white blood cells to the endothelium and their migration into the surrounding tissue, where they can distribute ROS and other enzymes as well as aggressive enzymes and mediators (myeloperoxidase (MPO), elastase, platelet-activating factor (PAF)) that can injure cells and the microvasculature [17-19]. Accordingly, it is possible that there are constantly new created ROS, which could lead to further injuries.
The main feature of the ischemic phase is the loss of mitochondrial respiration. Reduced energy status leads to a breakdown of energy-dependent metabolic pathways and transport processes, finally resulting in perturbation of ion homeostasis and activation of proteases (Clavien et al., 1992; Rosser et al., 1995). These include aspartate proteases, matrix metalloproteases, and Ca 2+ -requiring calpains (Takei et al., 1991; Upadhya et al., 1997; Calmus et al., 1995), which proteolytically cleave and disrupt membrane and cytoskeletal proteins. Additionally, endonucleases cause nuclear chromatin damage, and Ca 2+ -dependent phospholipases alter membrane fluidity and function (Trump et al., 1992). The activation of ATPases by increased cytosolic calcium hastens ATP depletion, even worsening the low cellular energy state during ischemia (Rosser et al., 1995). In organ transplantation, hypothermic storage itself leads to negative effects like cell swelling and calcium alterations despite the beneficial properties of low temperatures to prolong the possible storage time by reducing the metabolic rate (Hansen et al., 1994; Marsh et al., 1989). Sinusoidal endothelial cells (SEC) seem to be more susceptible to cold ischemia than hepatocytes (Otto et al., 1984) and contribute to reduced organ viability by detaching from their cellular matrix at the beginning of the reperfusion process (McKeown et al., 1988).
1. Introduction Chemokines play several important roles. They do not only maintain migration, but also influence T-cell differentiation (Luther and Cyster, 2001), angiogenesis, and the maturation of T-, B- and Dendritic Cells (Rossi and Zlotnik, 2000). There is evidence that chemokines are involved in ischemia-reperfusion-models, for example in hepatic ischemia-reperfusion where hepatocytes produce chemotactic signals which then lead to increased tissue damage (Jaeschke, 2006). Although there is only few chemokines constitutively present in the CNS, in pathologies like ischemic stroke they are suggested to be upregulated. CXCL1 (chemokine C-X-C motif ligand), which is also known as growth related oncogene (GRO) alpha is one of the best examined chemokines in inflammatory processes. Its target cells are mainly neutrophils and its function was detected in bacterial peritonitis (Giron-Gonzalez et al., 2001), HIV-infection (Lane et al., 2001) and other inflammatory processes. Losy et al. (Losy et al., 2005) detected elevated CXCL1 levels in CSF in patients with cerebral ischemia. Especially in the early phase of stroke, a chemokine upregulation was shown and a positive correlation between CSF CXCL1 levels and the stroke area in CT scans after 24 hours suggest that CXCL1 contributes to the increased tissue injury after cerebral ischemia. Other publications in experimental stroke also showed the upregulation of CXCL1 as a specific inflammatory marker after 4 hours in plasma and peripheral tissue as well as after 24 hours in the brain (Chapman et al., 2009). The role of CXCL2, a different chemokine still remains unclear: on one hand CXCL2 is increased 24-72 hours after cerebral ischemia, but the administration of an antibody against CXCR2, the corresponding receptor, does not improve the outcome after 72 hours of ischemia (Brait et al., 2011). CCL2 as a monocyte chemoattractant protein of the CC-chemokine subfamily is the most potent out of this group in activating signal transduction pathways leading to monocyte migration (Sozzani et al., 1994). As it can also be produced by neurons, microglia and astrocytes, its role in neuroinflammatory processes is even more important. Although there is evidence that CCL2 is involved in ischemic stroke, its role is controversial. On one hand it is supposed to support endogenous neurovascular protection (Stowe et al., 2012) and on the other hand CCL2 overexpression leads to increased infarct volumes and monocyte and macrophage invasion (Chen et al., 2003).
Ischemiareperfusion injury (IRI) is a major cause of acute renal failure, which is common kidney disorder with still high mortality. Its strong inflammatory and oxidative stress reaction of hypoxia andreperfusion frequently occurs in medical state of shock, sepsis or transplantation. During IR Phase, renal proximal tubules are seemed to be the most vulnerable. Subsequently, involve various cellular factor in the post IRI events and these lead to acute kidney injury. 12 The important parts of cell biological outcome of IRI have been researched, but still just few therapies are available. 13
The data of the present study are in accordance to previous work from Partridge (1995). She has reported that hypoxia-reoxygenation induces rearrangement of the actin cytoskeleton, opening of intercellular gaps and an increase in endothelial monolayer permeability. The essential role of F-actin fibers in maintaining cell integrity has also been shown in epithelial cells during ischemia (Molitoris, 1991; Schwartz et al., 1999), indicating that this causal link between cytoskeleton and barrier function is not only confined to endothelial, but also on other epithelial cells. Kuhne et al. (1993) and Watanabe et al. (1991) have shown that even under normoxic conditions, energy depletion alone causes disintegration of actin bundles at the cell periphery and endothelial cell retraction, which coincides withan increase in macromolecule permeability. Disruption of microfilaments and depolymerization of microtubules has also been observed under ATP depletion conditions by other laboratories (Hinshaw et al., 1989), indicating that the disintegration of actin cytoskeleton and cell adhesion structures result, at least in part, from the loss of energy during ischemia, wherease, reorganization of the cell cytoskeleton starts again, when ATP synthesis starts again with the supply of oxygen at the onset of reperfusion.
Before we were able to investigate the impact of the decoy nanoparticles in another inflammatory liver setting, we had to set up a working model of warm ischemia/reperfusionand characterize its features. A stable anesthesia without affecting blood pressure or breathing rate was established using midazolam-fentanyl- injection and isoflurane-inhalation. By placing an atraumatic clip to the Arteria hepatica and the portal vein, the upper lobes of the liver were completely drained from blood and remained entirely ischemic until the clip-removal (visual supervision). Upon restoration of blood supply, the blood pressure decreased about 20 – 40 mm Hg column for 15 – 20 minutes, but recovered soon thereafter. Mucous obstruction of airways could be observed rarely after 2 hours reperfusion. Animal body temperature was controlled rectally and maintained at 36.5°C with a red light heating lamp. Thus, constant conditions for all animal operations could be kept to ensure comparability of experiments.
control cultures that were not subjected to OGD, indicating that melatonin exerts its effect only when pathological conditions like mtPTP formation prevail. It has been shown that CsA in addition to its effect on the mtPTP has the potential to inhibit also the multi drug resistance pump (MDR). This may cause changes in attaining the mitochondrial TMRM fluorescence in the cells that is independent of mtPTP action (for review, see ). Previously it was shown that the MDR inhibition by verapamil, that is also an MDR inhibitor, did not cause any alteration in the CsA-induced mitochondrial hyperpolarization . If the component of MDR inhibition plays a role in our experimental model, one would assume that a treatment with verapamil results in a higher mitochondrial TMRM- uptake. Our results show that MDR inhibition by verapamil did not produce any change in attaining the mitochondrial TMRM-uptake. Furthermore, using different concentrations (50-200 nM, concentrations that are below the self-quenching threshold) of TMRM, showed no changes in mitochondrial fluorescence (data not shown), indicating, that an increased intracellular availability of TMRM as it would result from a MDR blockade, does not alter the baseline fluorescence in our model. Thus, our results show that a possible MDR inhibition in our model does not affect the mitochondrial TMRM uptake using our loading protocol. A large body of evidence suggests that the mtPTP is causally involved in the pathological changes following ischemia/reperfusion [59, 158]. Hence, a blockade of the mtPTP by melatonin may comprise a pharmacological strategy for the treatment of such pathological conditions.