Erythropoietin affords additional cardioprotection to preconditioned hearts by enhanced phosphorylation of glycogen synthase kinase-3 beta

The aim of this study was to determine whether erythropoietin (EPO) affords additional cardioprotection to the preconditioned myocardium by enhanced phosphorylation of Akt, STAT3, or glycogen synthase kinase-3beta (GSK-3 beta). Preconditioning (PC) with 5-min ischemia/5-min reperfusion and EPO (5,000 U/kg iv) reduced infarct size (as % of area at risk, %IS/AR) after 20-min ischemia in rat hearts in situ from 56.5 +/- 1.8% to 25.2 +/- 2.1% and to 36.2 +/- 2.8%, respectively. PC-induced protection was significantly inhibited by a protein kinase C inhibitor, chelerythrine (5 mg/kg), and slightly blunted by a phosphatidylinositol-3-kinase inhibitor, wortmannin (15 microg/kg). The opposite pattern of inhibition was observed for EPO-induced protection. The combination of PC and EPO further reduced %IS/AR to 8.9 +/- 1.9%, and this protection was inhibited by chelerythrine and wortmannin. The additive effects of PC and EPO on infarct size were mirrored by their effects on the level of phosphorylated GSK-3 beta at 5 min after reperfusion but not their effects on the level of phospho-Akt or phospho-STAT3. To mimic phosphorylation-induced inhibition of GSK-3 beta activity, SB-216763 (SB), a GSK-3 beta inhibitor, was administered before ischemia or 5 min before reperfusion. Infarct size was significantly reduced by preischemic injection (%IS/AR = 40.4 +/- 2.2% by 0.6 mg/kg SB and 34.0 +/- 1.8% by 1.2 mg/kg SB) and also by prereperfusion injection (%IS/AR = 32.0 +/- 2.0% by 1.2 mg/kg SB). These results suggest that EPO and PC afford additive infarct size-limiting effects by additive phosphorylation of GSK-3beta at the time of reperfusion by Akt-dependent and -independent mechanisms.     

Inconsistent relation of MAPK activation to infarct size reduction by ischemic preconditioning in pigs

The importance of the activation of mitogen-activated protein kinases (MAPK) for the cardioprotection achieved by ischemic preconditioning (IP) is still controversial. We therefore measured infarct size and p38, extracellular signal-regulated kinase (ERK), and c-Jun NH(2)-terminal kinase (JNK) MAPK phosphorylation (by biopsies) in enflurane-anesthetized pigs. After 90 min low-flow ischemia and 120 min reperfusion, infarct size averaged 18.3 +/- 12.4 (SD)% (group 1, n = 14). At similar subendocardial blood flows, IP by 10 min ischemia and 15 min reperfusion (group 2, n = 14) reduced infarct size to 6.2 +/- 5.1% (P < 0.05). An inconsistent increase in p38, ERK, and p54 JNK phosphorylation (by Western blot) was found during IP; p46 JNK phosphorylation increased with the subsequent reperfusion. At 8 min of the sustained ischemia, p38, ERK, and p54 JNK phosphorylation were increased with no difference between groups (medians: p38: 207% of baseline in group 1 vs. 153% in group 2; ERK: 142 vs. 144%; p54 JNK: 171 vs. 155%, respectively). MAPK phosphorylation and reduction of infarct size by IP were not correlated, thus not supporting the concept of a causal role of MAPK in mediating cardioprotection by IP.     

Cystic fibrosis as a bowel cancer syndrome and the potential role of CK2

Thyroid hormones are major regulators of postnatal brain development. Thyroid hormones act through nuclear receptors to modulate the expression of specific genes in the brain. We have used microarray analysis to identify novel responsive genes in 14-day-old hypothyroid rat brains, and discovered that synaptosomal-associated protein of 25 kDa (SNAP-25) was one of the thyroid hormone-responsive genes. SNAP-25 is a presynaptic plasma membrane protein and an integral component of the vesicle docking and fusion machinery mediating secretion of neurotransmitters and is required for neuritic outgrowth and synaptogenesis. Using microarray analysis we have shown that SNAP-25 was down-regulated in the hypothyroid rat brain compared with the age-matched controls. Real-time RT-PCR and western blotting analysis confirmed that SNAP-25 mRNA and protein levels decreased significantly in the developing hypothyroid rat brain. Our data suggest that in the developing rat brain, SNAP-25 expression is regulated by thyroid hormone, and thyroid hormone deficiency can cause decreased expression of SNAP-25 and this may on some level account for the impaired brain development seen in hypothyroidism.     

The delta-opioid receptor agonist DADLE at reperfusion protects the heart through activation of pro-survival kinases via EGF receptor transactivation

The specific delta-opioid receptor agonist [D-Ala(2)-D-Leu(5)]enkephalin (DADLE) protects against infarction in the heart when given before ischemia. In rabbit, this protection leads to phosphorylation of the pro-survival kinases Akt and extracellular signal-regulated kinase (ERK) and is dependent on transactivation of the epidermal growth factor receptor (EGFR). DADLE reportedly protects rat hearts at reperfusion. We therefore tested whether DADLE at reperfusion could protect isolated rabbit hearts subjected to 30 min of regional ischemia and 120 min of reperfusion and whether this protection is dependent on Akt, ERK, and EGFR. DADLE (40 nM) was infused for 1 h starting 5 min before reperfusion and reduced infarct size from 31.0 +/- 2.3% in the control group to 14.6 +/- 1.6% (P = 0.01). This protection was abolished by cotreatment of the metalloproteinase inhibitor (MPI) and the EGFR inhibitor AG1478. In contrast, 20 nM DADLE, although known to be protective before ischemia, failed to protect. Western blotting revealed that DADLE's protection was correlated to increase in phosphorylation of the kinases Akt and ERK1 and -2 in reperfused hearts (2.5 +/- 0.5, 1.6 +/- 0.2, and 2.3 +/- 0.7-fold of baseline levels, P < 0.05 vs. control). The DADLE-dependent increases in Akt and ERK1/2 phosphorylation were abolished by either MPI or AG1478, confirming a signaling through the EGFR pathway. Additionally, DADLE treatment increased phosphorylation of EGFR (1.4 +/- 0.2-fold, P = 0.03 vs. control). Thus the delta-opioid agonist DADLE protects rabbit hearts at reperfusion through activation of the pro-survival kinases Akt and ERK and is dependent on the transactivation of the EGFR.     

TNF-alpha antibodies are as effective as ischemic preconditioning in reducing infarct size in rabbits

Pretreatment with tumor necrosis factor-alpha (TNF-alpha) antibodies abolishes myocardial infarct size reduction by late ischemic preconditioning (IP). Whether or not TNF-alpha is also important for myocardial infarct size reduction by classic IP is unknown. Anesthetized rabbits were untreated (group 1, n = 7), classically preconditioned by 5 min left coronary artery occlusion/10 min reperfusion (group 2, n = 6), or pretreated with TNF-alpha antibodies without (group 3, n = 6) or with IP (group 4, n = 6) before undergoing 30 min of occlusion and 180 min of reperfusion. Infarct size in group 1 was 44 +/- 11 (means +/- SD)% of the area at risk. With a comparable area at risk, infarct size was reduced to 13 +/- 7%, 23 +/- 8%, and 19 +/- 12% (all P < 0.05) in groups 2, 3, and 4, respectively. The circulating TNF-alpha concentration was increased during ischemia in group 1 from 752 +/- 403 to 1,542 +/- 482 U/ml (P < 0.05) but remained unchanged in all other groups. Circulating TNF-alpha concentration during ischemia and infarct size correlated in all groups (r = 0.76). IP, TNF-alpha antibodies, and the combined approach reduced infarct size to a comparable extent. Therefore, the question of whether or not TNF-alpha is causally involved in the infarct size reduction by IP in rabbits could not be answered.     

Effect of combined therapy with hyperbaric oxygen and antioxidant on infarct volume after permanent focal cerebral ischemia

The aim of the present study was to evaluate the efficiency of combination of hyperbaric oxygen (HBO) and an antioxidant on permanent focal cerebral ischemia. Male Wistar rats underwent permanent middle cerebral artery occlusion (MCAO). Then, animals were randomly assigned to one of four groups: the control group (n=9) received no treatment, HBO group (n=9) was treated for 90 min at 2.5 absolute atmosphere for 3 days, the U-74389G group (n=8) received single U-74389G injection (3 mg/kg), the HBO + U-74389G group (n=8) received both HBO and U-74389G treatments. Treatments were initiated within the first 10 min after MCAO. After 3 days, the infarct volumes in rat brains were measured. The infarct ratios were 25.6+/-6.5 % for the control group, 21.9+/-6.4 % for the HBO group, 15.7+/-5.7 % for U-74389G group and 12.5+/-3.8 % for HBO + U74389G group. The infarct volumes were significantly reduced in rats treated with U-74389G (p<0.05) and combination therapy (p<0.05). HBO failed to reduce infarct volume significantly. We concluded that 1) U-74389G is more beneficial than HBO on permanent MCAO in rats, and 2) a combined therapy failed to significantly improve infarct volume more than either single treatment.     

Delayed erythropoietin therapy reduces post-MI cardiac remodeling only at a dose that mobilizes endothelial progenitor cells

We examined the cardiac effects of chronic erythropoietin (EPO) therapy initiated 7 days after myocardial infarction (MI) in rats. A single high dose of EPO has been shown to reduce infarct size by preventing apoptosis when injected immediately after myocardial ischemia. The proangiogenic potential of EPO has also been reported, but the effects of chronic treatment with standard doses after MI are unknown. In this study, rats underwent coronary occlusion followed by reperfusion or a sham procedure. Infarcted rats were assigned to one of three treatment groups: 1) 0.75 microg/kg darbepoetin (MI+darb 0.75, n = 12); 2) 1.5 microg/kg darbepoetin (MI+darb 1.5, n = 12); 3) vehicle (MI+PBS, n = 16), once a week from day 7 postsurgery. Sham rats received the vehicle alone (n = 10). After 8 wk of treatment, the animals underwent echocardiography, left ventricular pressure-volume measurements, and peripheral blood endothelial progenitor cell (EPC) counting. MI size and capillary density in the border zone and the area at risk (AAR) were measured postmortem. The AAR was similar in the three MI groups. Compared with MI+PBS, the MI+darb 1.5 group showed a reduction in the MI-to-AAR ratio (20.8% vs. 38.7%; P < 0.05), as well as significantly reduced left ventricle dilatation and improved cardiac function. This reduction in post-MI remodeling was accompanied by increased capillary density (P < 0.05) and by a higher number of EPC (P < 0.05). Both darbepoetin doses increased the hematocrit, whereas MI+darb 0.75 did not increase EPC numbers or capillary density and had no functional effect. We found that chronic EPO treatment reduces MI size and improves cardiac function only at a dose that induces EPC mobilization in blood and that increases capillary density in the infarct border zone.     

Differential Expression of SNAP-25 Isoforms and SNAP-23 in the Adrenal Gland

Abstract : In the rat adrenal gland, we previously observed that SNAP-25 is not restricted to the plasmalemma in noradrenergic cells as it is in adrenergic cells, and hypothesized that SNAP-25 isoform expression is different in the two phenotypes. Expression of SNAP-25 isoforms and SNAP-23 was examined by immunoblotting, immunofluorescence, and RT-PCR. Amplifications of SNAP-25 mRNAs were combined with Southern hybridization, restriction enzyme analysis, and sequencing of cloned PCR products to compare SNAP-25 isoform expression in rat and bovine adrenal glands. SNAP-25 and SNAP-23 mRNA and protein are expressed in the glands ; SNAP-23 is enriched in the adrenal cortex, whereas SNAP-25 is restricted to the adrenal medulla. Furthermore, high levels of SNAP-25 and low levels of SNAP-23 are observed in the PC12 cells, whereas both SNAP-25 and SNAP-23 are expressed in adrenal medullary cultures. In all extracts, the SNAP-23 mRNA corresponded to SNAP-23a. SNAP-25a is the major form expressed in rat adrenal glands (75%), as it is in PC12 cells (80%), but both SNAP-25a and SNAP-25b (40% vs. 60%) are expressed in bovine adrenal medulla in situ and in culture. In addition, an enriched population of adrenergic cells (93%) expressed a higher level of SNAP-25b (70%), suggesting that this isoform may not be restricted to fast neurotransmission.     

Multi-Modal Assessment of Long-Term Erythropoietin Treatment after Neonatal Hypoxic-Ischemic Injury in Rat Brain

Erythropoietin (EPO) has been recognized as a neuroprotective agent. In animal models of neonatal brain injury, exogenous EPO has been shown to reduce lesion size, improve structure and function. Experimental studies have focused on short course treatment after injury. Timing, dose and length of treatment in preterm brain damage remain to be defined. We have evaluated the effects of high dose and long-term EPO treatment in hypoxic-ischemic (HI) injury in 3 days old (P3) rat pups using histopathology, magnetic resonance imaging (MRI) and spectroscopy (MRS) as well as functional assessment with somatosensory-evoked potentials (SEP). After HI, rat pups were assessed by MRI for initial damage and were randomized to receive EPO or vehicle. At the end of treatment period (P25) the size of resulting cortical damage and white matter (WM) microstructure integrity were assessed by MRI and cortical metabolism by MRS. Whisker elicited SEP were recorded to evaluate somatosensory function. Brains were collected for neuropathological assessment. The EPO treated animals did not show significant decrease of the HI induced cortical loss at P25. WM microstructure measured by diffusion tensor imaging was improved and SEP response in the injured cortex was recovered in the EPO treated animals compared to vehicle treated animals. In addition, the metabolic profile was less altered in the EPO group. Long-term treatment with high dose EPO after HI injury in the very immature rat brain induced recovery of WM microstructure and connectivity as well as somatosensory cortical function despite no effects on volume of cortical damage. This indicates that long-term high-dose EPO induces recovery of structural and functional connectivity despite persisting gross anatomical cortical alteration resulting from HI.     

The relationship between erythropoietin pretreatment with blood-brain barrier and lipid peroxidation after ischemia/reperfusion in rats

Blood-brain barrier (BBB) leakage plays a role in the pathogenesis of many pathological states of the brain including ischemia and some neurodegenerative disorders. In recent years, erythropoietin (EPO) has been shown to exert neuroprotection in many pathological conditions including ischemia in the brain. This study aimed to investigate the effects of EPO on BBB integrity, infarct size and lipid peroxidation following global brain ischemia/reperfusion in rats. Wistar male rats were divided into four groups (each group n=8); Group I; control group (sham-operated), Group II; ischemia/reperfusion group, Group III; EPO treated group (24 h before decapitation--000 U/kg r-Hu EPO i.p.), Group IV; EPO+ ischemia/reperfusion group (24 h before ischemia/reperfusion--3000 U/kg r-Hu EPO i.p.). Global brain ischemia was produced by the combination of bilateral common carotid arteries occlusion and hemorrhagic hypotension. Macroscopical and spectrophotometrical measurement of Evans Blue (EB) leakage was observed for BBB integrity. Infarct size was calculated based on 2,3,5-triphenyltetrazolium chloride (TTC) staining. Lipid peroxidation in the brain tissue was determined as the concentration of thiobarbituric acid-reactive substances (TBARS) for each group. Ischemic insult caused bilateral and regional BBB breakdown (hippocampus, cortex, corpus striatum, midbrain, brain stem and thalamus). EPO pretreatment reduced BBB disruption, infarct size and lipid peroxide levels in brain tissue with 20 min ischemia and 20 min reperfusion. These results suggest that EPO plays an important role in protecting against brain ischemia/reperfusion through inhibiting lipid peroxidation and decreasing BBB disruption.     

A dual role of SNAP‐25 as carrier and guardian of synaptic transmission

In this issue, Matteoli and colleagues show that SNAP-25 levels regulate the efficacy of presynaptic glutamate release and thereby alter short-term plasticity, with potential relevance for psychiatric diseases.EMBO reports(2013) 14 7, 645–651 doi:10.1038/embor.2013.75Control of exocytotic neurotransmitter release is essential for communication in the nervous system and for preventing synaptic abnormalities. The function of synaptosomal-associated protein of 25 kDa (SNAP-25) as a crucial component of the core machinery required for synaptic vesicle fusion is well established, but evidence is growing to suggest an additional modulatory role in neurotransmission. In this issue of EMBO reports, Antonucci et al show that the efficacy of evoked glutamate release is modulated by the expression levels of SNAP-25—a function that might relate to the ability of SNAP-25 to modulate voltage-gated calcium channels and presynaptic calcium ion concentration [1]. Altered synaptic transmission and short-term plasticity due to changes in SNAP-25 expression might have direct consequences for brain function and for the development of neuropsychiatric disorders.Communication between neurons is essential for brain function and occurs through chemical neurotransmission at specialized cell–cell contacts termed ‘synapses''. Within the nerve terminal of the presynaptic neuron electrical stimuli cause the opening of voltage-gated calcium channels (VGCCs), which results in the influx of calcium ions. This triggers the exocytic release of neurotransmitter by fusion of synaptic vesicles with the presynaptic membrane. Released neurotransmitter molecules are detected by specific receptors expressed by the postsynaptic neuron.Calcium-induced synaptic vesicle fusion requires complex assembly between the soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) synaptobrevin 2, located on the synaptic vesicle, and the abundant plasma membrane SNAREs SNAP-25 and syntaxin 1, on the opposing presynaptic plasma membrane. SNARE complex assembly is tightly regulated by Sec1/Munc18-like proteins [2]. Further regulatory factors such as the synaptic vesicle calcium-sensing protein synaptotagmin 1 couple the SNARE machinery to presynaptic calcium influx. SNARE-mediated neurotransmitter release occurs preferentially at the active zone—a presynaptic membrane domain specialized for exocytosis within which VGCCs are positioned close to docked synaptic vesicles through a proteinaceous cytomatrix and associated cell adhesion molecules [3,4].Altered short-term plasticity due to changes in SNAP-25 expression might have direct consequences for brain function and for the development of neuropsychiatric disordersAn unresolved conundrum in synaptic transmission remains—the observation that SNARE proteins, such as SNAP-25, are among the most highly expressed, in copy number, presynaptic proteins, whilst only a handful of SNARE complexes are needed to drive the fusion of a single synaptic vesicle [5]. Why, then, are SNAREs such as SNAP-25 so abundant? One possible explanation might be that SNARE proteins, in addition to forming trans-SNARE complexes, assemble with other proteins, and such partitioning might regulate neurotransmission. For example, SNAP-25 has been shown to negatively regulate VGCCs in glutamatergic but not in GABAergic neurons [6]. A secondary regulatory function of SNAP-25 is also supported by its genetic association with synaptic abnormalities such as schizophrenia and attention deficit hyperactivity disorder (ADHD) in humans [7]. SNAP-25 expression is reduced twofold in the hippocampus and frontal lobe from schizophrenic patients [8] and in animal models for ADHD [9]. Thus, SNAP-25 expression levels might crucially regulate normal synaptic function.A new study in this issue of EMBO reports by Antonucci and colleagues investigates the consequences of reduced SNAP-25 expression on synaptic function in SNAP-25^+/− heterozygous (Het) mutant mice. By using patch clamp electrophysiology, Antonucci et al revealed a selective enhancement of glutamatergic but not GABAergic neurotransmission as a result of reduced SNAP-25 expression. Several other parameters including the amplitude and frequency of miniature excitatory and inhibitory currents were unaffected. These data indicate that reduced levels of SNAP-25, an essential component of the fusion machinery, selectively enhance evoked release of glutamate whilst synaptic connectivity and postsynaptic glutamate receptor sensitivity remain unaltered. Further electrophysiological experiments in hippocampal neurons in culture showed that elevated glutamatergic transmission was probably due to increased release probability rather than changes in the number of fusion-prone, so-called ‘readily releasable synaptic vesicles''. This effect was occluded by pharmacologically induced calcium entry bypassing VGCCs, suggesting that altered calcium influx might underlie the differences in evoked glutamate release between wild-type and SNAP-25 Het neurons. As schizophrenia and ADHD are associated with changes in short-term plasticity, a paradigm reflecting presynaptic function, Antonucci et al analysed neurotransmission by paired-pulse stimulation—a protocol whereby two closely paired stimuli are applied within a 50 ms time interval. Wild-type neurons showed significant short-term facilitation, that is, a stronger response to the second stimulus as a result of increased calcium levels in the presynaptic compartment. By contrast, Het neurons had a reduced response to the second stimulus. Such paired-pulse depression is commonly viewed as a sign of increased release probability, which occurs when the first stimulus induces a partial depletion of release-ready synaptic vesicles during paired stimulation. As a consequence, the second stimulus evokes a comparably reduced response [3]. The switch from paired-pulse facilitation to depression was not fully reproduced in hippocampal slices from wild-type and Het mice, although facilitation seemed to be attenuated in SNAP-25 Het slices. One possible explanation for the apparent discrepancy between cultured neurons taken from newborn animals and acute slices from adult mice is the constant postnatal increase in SNAP-25 expression in SNAP-25 Het mice [10], which might partly counteract the defects caused by heterozygosity. Consistent with this explanation are data from rescue experiments by Antonucci et al, which showed that altered neurotransmission and defects in short-term plasticity in Het neurons can be gradually recovered in parallel with increased SNAP-25 expression. Moreover, cultured neurons show substantially higher levels of endogenous activity compared with acute slice preparations, leading to possible changes in the partitioning of SNAP-25 between SNARE complexes and association with VGCCs. Further experiments are clearly required to resolve these issues. Irrespective of these potential caveats, the combined data support the hypothesis that alterations in SNAP-25 expression underlie regulatory changes in neurotransmission, resulting in altered short-term plasticity and possibly disease.Many open questions remain. In particular, the precise mechanisms underlying elevated glutamatergic transmission and presynaptic plasticity under conditions of reduced SNAP-25 expression remain elusive. It has been shown before that free SNAP-25 inhibits Cav2.1-type VGCCs [6], an effect reversed by overexpression of synaptotagmin 1, which might associate with SNAP-25. Conversely, SNAP-25 occludes negative regulation of Cav2.2 VGCCs by free syntaxin 1 [3]. Hence, it is tempting to speculate that differential partitioning of SNAP-25 between free, SNARE-, synaptotagmin 1- and VGCC-complexed forms could regulate evoked neurotransmission (Fig 1). In this scenario, reduced SNAP-25 expression in Het animals and in schizophrenic and ADHD patients would be sufficient to sustain SNARE-mediated synaptic vesicle fusion but partially releases VGCCS from SNAP-25-mediated inhibition. This would result in elevated calcium influx and facilitated neurotransmission. Additional levels of regulation could be imposed by developmental switching between alternatively spliced ‘a'' and ‘b'' isoforms of SNAP-25 [11], age-dependent alterations in presynaptic protein turnover and post-translational modifications.Open in a separate windowFigure 1Effect of presynaptic SNAP-25 levels on calcium-induced glutamate release. Top: in wild-type (WT) neurons, SNARE-mediated calcium-triggered synaptic vesicle fusion is negatively regulated by complex formation between SNAP-25 and VGCCs. Bottom: reduced SNAP-25 expression in heterozygotes (Het;^+/−) partly releases VGCCs from SNAP-25-mediated clamping, resulting in elevated calcium influx through VGCCs and increased glutamate release through SNARE-mediated calcium-triggered synaptic vesicle fusion. Note that many key exocytotic proteins have been omitted for clarity. SNAP-25, synaptosomal-associated protein of 25 kDa; SNARE, soluble NSF attachment protein receptor; VGCC. voltage-gated calcium channel.Future studies need to address these possibilities, and their relationship to cognitive impairments and synaptic diseases, such as schizophrenia and ADHD.     

Tempol reduces infarct size in rodent models of regional myocardial ischemia and reperfusion.

Reactive oxygen species (ROS) contribute to ischemia-reperfusion injury of the heart. This study investigates the effects of tempol, a membrane-permeable radical scavenger on (i) the infarct size caused by regional myocardial ischemia and reperfusion of the heart in vivo (rat, rabbit) and in vitro (rat), and (ii) the cell injury caused by hydrogen peroxide (H2O2) in rat cardiac myoblasts (H9c2 cells). In the anesthetized rat, tempol reduced the infarct size caused by regional myocardial ischemia (25 min) and reperfusion (2 h) from 60 +/- 3% (control, n = 8) to 24 +/- 5% (n = 6, p < .05). In the anesthetized rabbit, tempol also attenuated the infarct size caused by myocardial ischemia (45 min) and reperfusion (2 h) from 59 +/- 3% (control, n = 6) to 39 +/- 5% (n = 5, p < .05). Regional ischemia (35 min) and reperfusion (2 h) of the isolated, buffer-perfused heart of the rat resulted in an infarct size of 54 +/- 4% (control n = 7). Reperfusion of hearts with buffer containing tempol (n = 6) caused a 37% reduction in infarct size (n = 6, p < .05). Pretreatment of rat cardiac myoblasts with tempol attenuated the impairment in mitochondrial respiration caused by H2O2 (1 mM for 4 h). Thus, the membrane-permeable radical scavenger tempol reduces myocardial infarct size in rodents.     

Differential effects of hypoxia-ischemia on subunit expression and tyrosine phosphorylation of the NMDA receptor in 7- and 21-day-old rats

The effect of cerebral hypoxia-ischemia (HI) on levels and tyrosine phosphorylation of the NMDA receptor was examined in 7- (P7) and 21 (P21)-day-old rats. Unilateral HI was administered by ligation of the right common carotid artery and exposure to an atmosphere of 8% O2/92% N2 for 2 (P7) or 1.5 (P21) h. This duration of HI produces significant infarction in nearly all of the survivors with damage being largely restricted to the cortex, striatum, and hippocampus of the hemisphere ipsilateral to the carotid artery ligation. NR2A levels in the right hemisphere of P7 pups were markedly reduced after 24 h of recovery, while NR1 and NR2B remained unchanged. In contrast, NR2B, but not NR2A, was reduced after HI at P21. At both ages, HI resulted in a transient increase in tyrosine phosphorylation of a number of forebrain proteins that peaked between 1 and 6 h of recovery. At both P7 and P21, tyrosine phosphorylation of NR2B was enhanced 1 h after HI and had returned to basal levels by 24 h. HI induced an increase in tyrosine phosphorylation of NR2A in 21 day, but not in 7-day-old animals. The differential effects of HI on the NMDA receptor at different post-natal ages may contribute to changing sensitivity to hypoxia-ischemia.     

Estrogen status affects sensitivity to focal cerebral ischemia in stroke-prone spontaneously hypertensive rats

Estrogen treatment has been shown to reduce ischemic brain damage. Because endogenous estrogen levels fluctuate markedly during the estrous cycle, we investigated the effect of stage of estrous cycle on ischemic brain damage. Halothane anesthetized 3- to 5-mo-old female Wistar-Kyoto rats (WKY) and stroke-prone spontaneously hypertensive rats (SHRSP) in proestrus (high estradiol levels) or metestrus (low estradiol levels) underwent permanent middle cerebral artery occlusion. In SHRSP, infarct volume at 24 h postocclusion was 24% smaller in proestrus compared with metestrus [208.6 +/- 9.5 mm(3) (n = 7) vs. 272.7 +/- 23.8 mm(3) (n = 7), respectively, means +/- SE; P = 0.0278, unpaired t-test]. In WKY, infarct volumes were similar in proestrus and metestrus [157.0 +/- 5.4 mm(3) (n = 5) and 131.5 +/- 16.5 mm(3) (n = 8), respectively; P = not significant (NS)]. Brain swelling (ipsilateral minus contralateral hemispheric volumes) was similar in proestrus and metestrus for SHRSP [138 +/- 9 mm(3) (n = 6) and 136 +/- 10 mm(3) (n = 7), respectively] and for WKY [103 +/- 15 mm(3) (n = 5) and 90 +/- 11 mm(3) (n = 8), respectively]. Thus the reduction in infarct size in SHRSP is caused by a true attenuation of the infarct volume and not simply by a reduction in brain edema.     

Differential regulation of IL-1beta and TNF-alpha RNA expression by MEK1 inhibitor after focal cerebral ischemia in mice

Activation of the extracellular-signal-responsive kinase (ERK 1/2) by MAP kinase/ERK kinase (MEK1/2) following ischemia/reperfusion in the brain has been associated with cell death since inhibition of MEK1/2 provides neuroprotection in cerebral ischemia injury. Since inflammation has been implicated in ischemic brain injury, the present study investigated whether MEK1/2 modifies expression of two key inflammatory cytokines, IL-1beta and TNFalpha, that have been shown to exacerbate ischemic brain injury. A mouse model of transient cerebral ischemia was deployed to test the effect of selective MEK1/2 inhibitor (SL327) on infarct size and cytokine expression. SL327 (100 mg/kg, i.p.) administered 15 min prior to ischemia resulted in 64% reduction in infarct size over controls (n = 8, P < 0.01). Under the same condition, SL327 significantly reduced peak expression of IL-1beta mRNA (59% reduction compared to vehicle, P < 0.01, n = 4) but not TNF-alpha mRNA. A parallel reduction in IL-1beta protein (67%, P < 0.05, n = 6) was also observed using ELISA analysis. These data suggest that the neuroprotective effect of MEK1/2 inhibition may be mediated by suppression of IL-1beta. The study also demonstrates for the first time that these two cytokines are differentially regulated by kinase mediated signaling pathways.     

Erythropoietin prevents chemotherapy-induced anemia: case report

A thirty-seven year old male patient with heavily pretreated metastatic testicular carcinoma received escalating doses of recombinant human erythropoietin (EPO) before and throughout chemotherapy. Whereas previous chemotherapy regimens repeatedly caused anemic situations in this patient (hemoglobin (HB) 7.0 g/dl requiring multiple transfusions of red blood cells), EPO given as an i.v. bolus injection at escalating doses of 150 to 300 U/kg body weight (BW) twice/week, starting two weeks prior to the identical myelosuppressive treatment protocol, maintained HB at levels above 8.8 g/dl and thus obviated the need for erythrocyte transfusion. EPO was discontinued after 9 weeks of administration when the patient had achieved a hematocrit (HCT) of 41.1% and a HB of 12.7 g/dl. However, erythropoiesis continued to recover for the next 7 weeks reaching a HCT of 42.4% and a HB of 14.3 g/dl, although the next identical chemotherapy cycle had been given within this period. Along with the rise in HB, ferrokinetics changed significantly as measured by serum ferritin, which was reduced to one third at the end of EPO therapy after only 9 weeks (from 979 ng/ml to 320 ng/ml). No side effects due to EPO administration occurred. These data provide first evidence for efficacy of EPO in chemotherapy-induced anemia and may open new avenues for its clinical application.     

Erythropoietin protects post-ischemic hearts by preventing extracellular matrix degradation: role of Jak2-ERK pathway

Factors predisposing to extracellular matrix degradation associated with myocardial ischemia/reperfusion (IR) usually cause cell death. Recombinant human erythropoietin (EPO) protects the myocardium from IR, but whether it affects extracellular matrix (ECM) degradation is not known. This study examined the effect of the Jak2-ERK pathway, which is triggered by EPO, on the expression of matrix metalloproteinases (MMPs), tissue inhibitor of MMP 4 (TIMP-4), and collagen in post-ischemic hearts. Rat hearts were isolated and perfused in a Langendorff apparatus. IR was induced by 40 min of stopped flow and 120 min of aerobic reperfusion; EPO was added immediately before reperfusion. Compared to untreated controls, poor recovery of the left ventricular developed pressure (LVDP) was seen in IR hearts. IR resulted in myocyte injury measured by creatine kinase MB release and infarction. Western blot analysis showed increased levels of MMP-2 and MMP-9 and reduced levels of TIMP-4 and collagen III. IR rats given 5 IU/ml of EPO showed improved LVDP with reduced injury. EPO increased Jak2 and ERK activity, decreased MMP expression, increased TIMP-4 expression, and prevented collagen degradation in IR hearts. All these effects were blocked by the upstream ERK inhibitor, U0126 (5 microM). These observations show that EPO attenuates extracellular matrix degradation following IR and this may be the basis of the protection from cell death. Jak2-ERK phosphorylation may be an important signal in this process.     

Erythropoietin prevents PC12 cells from 1-methyl-4-phenylpyridinium ion-induced apoptosis via the Akt/GSK-3β/caspase-3 mediated signaling pathway

Apoptosis is a contributing cause of dopaminergic neuron loss in Parkinson disease. Recent work has shown that erythropoietin (EPO) offers protection against apoptosis in a wide variety of tissues. We demonstrate that exposure of PC12 cells to 1-methyl-4-phenylpyridinium ion (MPP⁺) with recombinant human EPO, significantly decreased apoptosis as measured by TUNEL and caspase-3 activity when compared to MPP⁺ treatment alone. EPO induced sustained phosphorylation of Akt and its substrate, GSK-3β, reduced caspase-3 activities in PC12 cells. The anti-apoptotic effect of EPO was abrogated by co-treatment with LY294002, the specific blocker of phosphatidylinositol 3-kinase (PI3K). The effects of EPO on GSK-3β and caspase-3 activities were also blocked by LY294002. LiCl, the inhibitor of GSK-3β, downregulated the caspase-3 activity and blocked the apoptosis induced by MPP⁺. Finally, we determined that EPO transiently activated the ERK signaling pathway, but PD98059, a specific inhibitor of ERK, does not alter the survival effect of EPO in this model system. Thus, these findings indicate that EPO protects against apoptosis in PC12 cells exposed to MPP⁺, through the Akt/GSK-3β/caspase-3 signaling pathway, but the ERK pathway is not involved in the EPO-dependent survival enhancing effect in this model system. The authors Yan Wu and You Shang are equally contributed to this work.     

Ischemic postconditioning during reperfusion activates Akt and ERK without protecting against lethal myocardial ischemia-reperfusion injury in pigs

Transient episodes of ischemic preconditioning (PC) render myocardium protected against subsequent lethal injury after ischemia and reperfusion. Recent studies indicate that application of short, repetitive ischemia only during the onset of reperfusion after the lethal ischemic event may obtain equivalent protection. We assessed whether such ischemic postconditioning (Postcon) is cardioprotective in pigs by limiting lethal injury. Pentobarbital sodium-anesthetized, open-chest pigs underwent 30 min of complete occlusion of the left anterior descending coronary artery and 3-h reflow. PC was elicited by two cycles of 5-min occlusion plus 10-min reperfusion before the 30-min occlusion period. Postcon was elicited by three cycles of 30-s reperfusion, followed by 30-s reocclusion, after the 30-min occlusion period and before the 3-h reflow. Infarct size (%area-at-risk using triphenyltetrazolium chloride macrochemistry; means +/- SE) after 30 min of ischemia was 26.5 +/- 5.2% (n = 7 hearts/treatment group). PC markedly limited myocardial infarct size (2.8 +/- 1.2%, n = 7 hearts/treatment group, P < 0.05 vs. controls). However, Postcon had no effect on infarct size (37.8 +/- 5.1%, n = 7 hearts/treatment group). Within the subendocardium, Postcon increased phosphorylation of Akt (74 +/- 12%) and ERK1/2 (56 +/- 10%) compared with control hearts subjected only to 30-min occlusion and 15-min reperfusion (P < or = 0.05), and these changes were not different from the response triggered by PC (n = 5 hearts/treatment group). Phosphorylation of downstream p70S6K was also equivalent in PC and Postcon groups. These data do not support the hypothesis that application of 30-s cycles of repetitive ischemia during reperfusion exerts a protective effect on pig hearts subjected to lethal ischemia, but this is not due to a failure to phosphorylate ERK and Akt during early reperfusion.