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The 'normobaric oxygen paradox': Another potential way to use oxygen. CME activity 2013/1
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Hypoxia is a key determinant of tissue pathology during tumor development and organ ischemia. However, little is known regarding
hypoxic regulation of genes that are directly involved in cell death or death resistance. Here we report the striking induction
by severe hypoxia of the anti-apoptotic protein IAP-2. Hypoxic cells with IAP-2 up-regulation became resistant to apoptosis.
IAP-2 was induced by hypoxia per se rather than by the secondary effects of hypoxia, including ATP depletion and cell injury. The inductive response did not
relate to alterations of cellular redox status or arrest of mitochondrial respiration. On the other hand, IAP-2 induction
was attenuated by actinomycin D, suggesting a role for gene transcription. In vitro nuclear run-on assays demonstrated specific increases in IAP-2 transcriptional activity after hypoxia exposure. HIF-1, the
primary transcription factor that is responsible for multiple gene activation under hypoxia, does not have a role in IAP-2
expression. HIF-1 and IAP-2 were induced by different degrees of hypoxia; severe hypoxia or anoxia was required for IAP-2
induction. Moreover, cobalt chloride and desferrioxamine activated HIF-1 but not IAP-2. Finally, IAP-2 was induced by severe
hypoxia in mouse embryonic stem cells that were deficient of HIF-1. Thus, this study not only provides the first demonstration
of hypoxic regulation of an anti-apoptotic gene but also suggests the participation of novel hypoxia-responsive transcription
mechanisms.
It has been proposed that relative changes of oxygen availability rather than steady state hypoxic or hyperoxic conditions, play an important role in HIF transcriptional effects. According to this hypothesis describing the "normobaric oxygen paradox", normoxia following a hyperoxic event is sensed by tissues as an oxygen shortage, upregulating HIF-1 activity. With the aim of confirming at cellular and at functional level that normoxia following an hyperoxic event is "interpreted" as a hypoxic event, we report a combination of experiments addressing the effects of an intermittent increase of oxygen concentration on HIF-1 levels and the activity level of specific oxygen-modulated proteins in cultured human umbilical vein endothelial cells (HUVECs), and the effects hemoglobin (Hb) levels after intermittent breathing normobaric high (100%) and low (15%) oxygen in vivo in humans. Our experiments confirm that during recovery after hyperoxia, an increase of HIF expression occurs in HUVECs, associated to an increase of matrix metalloproteinases activity. These data suggest that endothelial cells "interpret" the return to normoxia after hyperoxia as a hypoxic stimulus. At functional level, our data show that both breathing 15% and 100% oxygen 30 minutes every other day for a period of 10 days, induces an increase of Hb levels in humans. This effect was enhanced after the cessation of the oxygen breathing. These results indicate that a sudden decrease in tissue oxygen tension after hyperoxia, may act as a trigger for EPO synthesis so corroborating the hypothesis that "relative" hypoxia is a potent stimulator of HIF mediated gene expressions.
Introduction
Hypoxia-inducible factor-1 (HIF1) controls the expression of genes involved in the cellular response to hypoxia. No information is available on its expression in critically ill patients. Thus, we designed the first clinical study in order to evaluate the role of HIF1α as a prognosis marker in patients suffering from shock.
Methods
Fifty consecutive adult patients with shock and 11 healthy volunteers were prospectively enrolled in the study. RNA was extracted from whole blood samples and expression of HIF1α was assessed over the first four hours of shock. The primary objective was to assess HIF1α as a prognostic marker in shock. Secondary objectives were to evaluate the role of HIF1α as a diagnostic and follow-up marker. Patient survival was evaluated at day 28.
Results
The causes of shock were sepsis (78%), hemorrhage (18%), and cardiac dysfunction (4%). HIF1α expression was significantly higher in the shock patients than in the healthy volunteers (121 (range: 72-168) versus 48 (range: 38-54) normalized copies, P <0.01), whatever the measured isoforms. It was similar in non-survivors and survivors (108 (range 84-183) versus 121(range 72-185) normalized copies, P = 0.92), and did not significantly change within the study period.
Conclusions
The present study is the first to demonstrate an increased expression of HIF1α in patients with shock. Further studies are needed to clarify the potential association with outcome. Our findings reinforce the value of monitoring plasma lactate levels to guide the treatment of shock.
Recent publications reflect the anti-doping authorities’ concern about the use of altitude simulator systems as violating
the spirit of sport criterion (Levine 2006; Loland and Murray 2007; Spriggs 2005). The aim of our study was to determine whether intermittent hypoxic treatments could modify the hemoglobin, hematocrit,
reticulocytes, and erythropoietic stimulation index (OFF-Hr Score) values after administration of rHuEPO-alpha. Although these
hematological parameters are of secondary nature some international sport federations currently exclude athletes who show
aberrant values of these parameters from competition. Ten young male Wistar rats were treated, three times a week for 2weeks,
with 500IU of rHuEPO-alpha. After the treatment, the animals were randomly divided into two groups: normoxic and hypoxic.
The normoxic group was maintained at 21% O2 24h a day for 23days. The hypoxic group was maintained 12h at 21% O2 and 12h at 12% O2 (~4,000m) the same time period. After the rHuEPO-alpha treatment, the hypoxic group of animals had a faster recovery rate
in the reticulocyte count, elevated concentrations of hemoglobin and hematocrit and a significant increase in the endogenous
EPO levels when compared with the normoxic group of animals. These changes led to significant modifications in the OFF-Hr
Score between the hypoxic and normoxic animals. Intermittent hypoxic treatments after rHuEPO administration can significantly
modify the main hematological parameters tested by the anti-doping authorities. Our results in an animal model suggest checking
the described phenomena in humans in order to reach major conclusions.
Anemia is a frequent complication in oncologic patients. Erythropoietin (EPO) stimulating agents are known as alternatives to transfusion. However, they expose patients to thrombosis and are expensive. Recently, a new phenomenon, the normobaric oxygen paradox (NOP), has been described. In brief, transient hyperoxia followed by a prolonged return to normoxia acts as an effective trigger for EPO production. The mechanism depends on free oxygen radicals and on reduced glutathione (GSH) availabilities. Also, N-acetylcystein (NAC) is known to regenerate the stock of GSH. Very few clinical trials have investigated this phenomenon [1]. The goal of this study was to test the NOP theory on the evolution of hemoglobin and reticulocytes in patients receiving intermittent oxygen with or without NAC compared to a control group.
Splenic contraction associated with apnea causes increased haemoglobin concentration and haematocrit (Hct), an effect that may promote prolonged breath-holding. Hypoxia has been shown to augment this effect, but hypercapnic influences have not been investigated previously.
Eight non-divers performed three series of apneas on separate days after inspiration of oxygen with different carbon dioxide (CO₂) levels. Each series consisted of three apneas 2 minutes apart: one with pre-breathing of 5% CO₂ in oxygen (O₂, 'Hypercapnia'); one with pre-breathing of 100% O₂ ('Normocapnia'); and one with hyperventilation of 100% O₂ ('Hypocapnia'). The apnea durations were repeated identically in all trials, determined from the maximum duration attained in the CO₂ trial. A fourth trial, breathing 5% CO₂ in O₂ for the same duration as these apneas was also performed ('Eupneic hypercapnia'). In three subjects, spleen size was measured using ultrasonic imaging.
Haemoglobin increased by 4% after apneas in the 'Hypercapnia' trial (P = 0.002) and by 3% in the 'Normocapnia' trial (P = 0.011), while the 'Hypocapnia' and 'Eupneic hypercapnia' trials showed no changes. The 'easy' phase of apnea, i.e., the period without involuntary breathing movements, was longest in the 'Hypocapnia' trial and shortest in the 'Hypercapnia' trial. A decrease in spleen size was evident in the hypercapnic trial, whereas in the hypocapnia trial spleen size increased, while only minor changes occurred in the other trials. No differences were observed between trials in the cardiovascular diving response.
There appears to be a dose-response effect of CO₂ on triggering splenic contraction during apnea in the absence of hypoxia.
Many plant antioxidants, intaken through the daily diet or plant-derived dietary supplements, have been shown able to prevent free radical-related diseases by counteracting cell oxidative stress. However, it is now considered that the in vivo beneficial effects of these phytochemicals are unlikely to be explained just by their antioxidant capability. Several plant antioxidants exhibit hormetic properties, by acting as 'low-dose stressors' that may prepare cells to resist more severe stress. In fact, low doses of these phytochemicals activate cell signaling pathways (being the most prominent examples the modulation of the Nrf2/Keap1 pathway, the NF-κB pathway and the Sirtuin-FOXO pathway) but high doses are cytotoxic. Herein we review the adaptive responses induced by the most known plant hormetic antioxidants, which are sulforaphane, resveratrol, curcumin, flavonoids, green tea catechins and diallylsulphides, as well as the molecular mechanisms involved in such responses. Furthermore, this review outlines that the hormetic properties of these bioactive plant antioxidants might be successfully employed for realizing health-promoting dietary interventions especially in the field of neurodegenerative diseases and cancer.
Several interrelated cellular signaling molecules are involved in modulating adaptive compensatory changes elicited by low exposures to toxins and other stressors. The most prominent example of signaling pathway typically involved in this adaptive stress response, is represented by the activation of a redox-sensitive gene regulatory network mediated by the NF-E2-related factor-2 (Nrf2) which is intimately involved in mediating the Antioxidant Responsive Element (ARE)-driven response to oxidative stress and xenobiotics. We investigated if Nrf2 pathway activation following intracellular glutathione depletion through buthionine sulfoximine (BSO) exposure, might be able to alter the response to TNF-α, a proinflammatory cytokine, in cultured human umbilical vein endothelial cells. Herein, we revealed that such a change in the cellular redox status is able to reduce TNF-α induced endothelial activation (as shown by a decreased gene expression of adhesion molecules) by activating an adaptive response mediated by an increased Nrf2 nuclear translocation and overexpression of the ARE genes HO-1 and NQO-1. Furthermore, we have demonstrated the involvement of ERK1/2 kinases in Nrf2 nuclear translocation activated by BSO-induced glutathione depletion. The coordinate induction of endogenous cytoprotective proteins through adaptive activation of Nrf2 pathway is a field of great interest for potential application in prevention and therapy of inflammatory diseases such as atherosclerosis.
Recent findings suggest that besides renal tissue hypoxia, relative decrements in tissue oxygenation, using a transition of the breathing mixture from hyperoxic to normoxic, can also stimulate erythropoietin (EPO) production. To further clarify the importance of the relative change in tissue oxygenation on plasma EPO concentration [EPO], we investigated the effect of a consecutive hyperoxic and hypoxic breathing intervention. Eighteen healthy male subjects were assigned to either IHH (N = 10) or CON (N = 8) group. The IHH group breathed pure oxygen (F(i)O(2) ~ 1.0) for 1 h, followed by a 1-h period of breathing a hypoxic gas mixture (F(i)O(2) ~ 0.15). The CON group breathed a normoxic gas mixture (F(i)O(2) ~ 0.21) for the same duration (2 h). Blood samples were taken just before, after 60 min, and immediately after the 2-h exposure period. Thereafter, samples were taken at 3, 5, 8, 24, 32, and 48 h after the exposure. During the breathing interventions, subjects remained in supine position. There were significant increases in absolute [EPO] within groups at 8 and 32 h in the CON and at 32 h only in the IHH group. No significant differences in absolute [EPO] were observed between groups following the intervention. Relative (∆[EPO]) levels were significantly lower in the IHH than in the CON group, 5 and 8 h following exposure. The tested protocol of consecutive hyperoxic-hypoxic gas mixture breathing did not induce [EPO] synthesis stimulation. Moreover, the transient attenuation in ∆[EPO] in the IHH group was most likely due to a hyperoxic suppression. Hence, our findings provide further evidence against the "normobaric O(2) paradox" theory.
It remains uncertain as how the reduction in systemic oxygen transport limits high-intensity exercise tolerance. 11 participants (5 males; age 35 ± 10 years; peak 3.5 ± 0.4 L min−1) performed cycle ergometry to the limit of tolerance: (1) a ramp test to determine ventilatory threshold (VT) and peak ; (2) three to four constant-load tests in order to model the linear P–t
−1 relationship for estimation of intercept (critical power; CP) and slope (AWC). All tests were performed in a random order under moderate hypoxia (FiO2 = 0.15) and normoxia. The linearity of the P–t
−1 relationship was retained under hypoxia, with a systematic reduction in CP (220 ± 25 W vs. 190 ± 28 W; P < 0.01) but no significant difference in AWC (11.7 ± 5.5 kJ vs. 12.1 ± 4.4 kJ; P > 0.05). However, large individual variations in the change of the latter were observed (−36 to +66%). A significant relationship was found between the % change in CP (r = 0.80, P < 0.01) and both peak (CP: r = −0.65, P < 0.05) and VT values recorded under normoxia (CP: r = −0.65, P < 0.05). The present study demonstrates the aerobic nature of the intercept of the P–t
−1 relationship, i.e. CP. However, the extreme within-individual changes in AWC do not support the original assumption that AWC reflects a finite energy store. Lower hypoxia-induced decrements in CP were observed in aerobically fitter participants. This study also demonstrates the greater ability these participants have to exercise at supra-CP but close to CP workloads under moderate hypoxia.
To determine and compare the erythropoietic response and exercise performance of adolescent cross-country skiers, as a result of "living high-training high" (HH) and "living high-training low" (HL).
Nine female and six male adolescent cross-country skiers volunteered to participate in separate trials. In the first trial (HH), the skiers lived and trained for 21 days at 1550-2050 m, while in the second trial (HL) they trained near sea level (450-500 m) but resided at 1550 m. All participants underwent maximal cycle ergometer tests for the determination of VO2max and cardiorespiratory parameters via an open circuit system at sea level before ascent to altitude, and 1-2 days after descent from altitude. Blood samples were drawn prior to and immediately after maximal cycle exercise testing, at sea level prior to ascent, on days 1 (D1) and 21 (D21) at altitude (1740 m), and 1-2 days post-altitude, for the determination of serum erythropoietin (EPO) concentration, haemoglobin (Hb), hematocrit (Ht), and red blood cell (RBC) volume.
The results showed that both boys and girls cross-country skiers, significantly improved their sea level VO2max after 21 days of living at moderate altitude and training near sea level.
The present study demonstrates that living at moderate altitude, 1550-2050 m and training low, near sea level (450-500 m) significantly increases VO2max and RBC mass for both boys and girls. Results indicate that applying the training concept "living high - training low" in adolescent athletes may improve their endurance performance.
The regimen of aerobic training at sea level with recovery at high altitude has been used by athletes to improve performance. However, little is known about the effects of hypoxia when combined with sprint interval training on performance. The aim of the present study was to determine the effect of a "living high-sprint training low" strategy on hemoglobin, hematocrit and erythropoietin levels in rats. We also wanted to test whether the addition of a hypoxic stress to the program of daily treadmill running at high speeds induces expressional adaptations in skeletal muscle and affects performance. The protein content of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), cytochrome C, pyruvate dehydrogenase kinase (PDK1), heat shock protein 70 (HSP70), manganese superoxide dismutase (MnSOD) and citrate synthase activity were determined in different muscle fiber types in our animals (red and white gastrocnemius muscle). We also determined the maximal aerobic velocity (MAV) before and after the training period. A total of 24 male Wistar rats (3 month old) were randomly divided into four experimental groups: the normoxic control group (n = 6), the normoxic trained group (n = 6), the hypoxic control group (12 h pO(2) 12%/12 h pO(2) 21%) (n = 6) and the hypoxic trained group (12 h pO(2) 12%/12 h pO(2) 21%). Living in normobaric hypoxia condition for 21 days significantly increased hemoglobin, hematocrit and erythropoietin levels in both the rest and the trained groups. The trained animals (normoxia and hypoxia) significantly increased their maximal aerobic velocity. No changes were found in the skeletal muscle in PGC-1α, cytochrome C, PDK1, HSP70, MnSOD protein content and in the citrate synthase activity in any experimental group. Regardless of whether it is combined with sprint interval training or not, after 21 days of living at high altitude we found a significant increase in the hematological values determined in our study. However, contrary to our starting hypothesis, the combination of normobaric hypoxia and sprint training did not improve MAV in our animals.
Erythropoietin (EPO) and soluble EPO receptors (sEPOR) have been proposed to play a central role in the ventilatory acclimatisation to continuous hypoxia in mice.
In this study, we demonstrated for the first time in humans (n = 9) that sEPOR is downregulated upon daytime exposure to 4 days of intermittent hypoxia (IH; 6 h·day ⁻¹ , cycles of 2 min of hypoxia followed by 2 min of reoxygenation; peak end-tidal oxygen tension ( P ET,O 2 ) 88 Torr, nadir P ET,O 2 45 Torr), thereby allowing EPO concentration to rise. We also determined the strength of the association between these haematological adaptations and alterations in the acute hypoxic ventilatory response (AHVR).
We observed a nadir in sEPOR on day 2 (-70%), concomitant with the peak in EPO concentration (+50%). Following exposure to IH, tidal volume ( V T ) increased, respiratory frequency remained unchanged, and minute ventilation ( V ′ E ) was increased. There was a negative correlation between EPO and sEPOR (r = -0.261; p = 0.05), and between sEPOR and V T (r = -0.331; p = 0.02). EPO was positively correlated with V ′ E (r = 0.458; p = 0.001).
In conclusion, the downregulation of sEPOR by IH modulates the subsequent EPO response. Furthermore, the alterations in AHVR and breathing pattern following IH appear to be mediated, at least in part, by the increase in EPO.
Spleen contraction occurs in humans during exercise, apnea, and simulated altitude, resulting in ejection of stored red blood cells into circulation. The mechanisms responsible for initiating the contraction are not fully known: hypoxia is likely involved, but other, unknown factors may also contribute. To reveal the initiating factors, we studied its occurrence in two different situations involving similar reductions in arterial oxygen saturation (SaO2). We hypothesized that similar spleen responses would result if the level of hypoxia is the main factor involved.
Five female and four male healthy volunteers performed two different trials on separate days: (1) 20 min of normobaric hypoxic breathing (14.2% oxygen); and II) 2 min of apnea after a deep inspiration of air. Both trials started and ended with 10 min of sitting eupneic rest. Spleen diameter was intermittently measured via ultrasonic imaging in three dimensions to calculate volume. SaO2 and heart rate (HR) were recorded continuously with a pulse oximeter.
Exposures resulted in similar nadir SaO2: 87% after normobaric hypoxia and 89% after apnea. During normobaric hypoxia, spleen volume was reduced by 16% and during apnea by 34%. HR increased by 7% during normobaric hypoxia, but fell by 25% during apnea.
Both normobaric hypoxia and apnea induced spleen contraction, but despite similar levels of SaO2 apnea evoked a significantly stronger response, possibly due to hypercapnia, faster desaturation, or the apneic stimulus in itself. Spleen contraction may facilitate adaptation to altitude and to apneic diving by elevating blood gas storage capacity.
The aim of this study was to follow up whether the modification of pro-antioxidant status by 8-day oral application of N-acetylcysteine (NAC) in healthy men affects the haematological response, whether there is a direct relationship between antioxidant defences and erythropoietin (EPO) secretion and whether NAC intake enhances exercise performance. Fifteen healthy men were randomly assigned to one of two groups: control or NAC (1,200 mg d(-1) for 8 days prior to and 600 mg on the day of exercise trial). To measure the ergogenic effectiveness of NAC, subjects performed incremental cycle exercise until exhaustion. NAC administration significantly influenced the resting and post-exercise level of glutathione (+31%) as well as the resting activity of glutathione enzymes (glutathione reductase, -22%; glutathione peroxidase, -18%). The oxidative damage markers, i.e., protein carbonylation and lipid peroxidation products (thiobarbituric acid reactive substance) were reduced by NAC by more than 30%. NAC noticeably affected the plasma level of EPO (+26%), haemoglobin (+9%), haematocrit (+9%) and erythrocytes (-6%) at rest and after exercise. The mean corpuscular volume and the mean corpuscular haemoglobin increased by more than 12%. Plasma total thiols increased by 17% and directly correlated with EPO level (r = 0.528, P < 0.05). NAC treatment, contrary to expectations, did not significantly affect exercise performance. Our study has shown that 8-day NAC intake at a daily dose of 1,200 mg favours a pro-antioxidant status and affects haematological indices but does not enhance exercise performance.
Fluctuations in cellular oxygenation causing intermittent hypoxia and oxidative stress affect the regulation of hypoxia-inducible factor (HIF-1) and the nuclear factor erythroid 2-related factor 2 (Nrf2). HIF-1 is primarily induced in hypoxia, whereas Nrf2 is induced in response to oxidative stress. Whereas HIF-1 regulates the expression of genes important for the adaptation of cells to hypoxia, Nrf2 induces antioxidative enzymes such as thioredoxin 1 (Trx1), exerting a cytoprotective role. Here, we investigated the regulation and cross talk of HIF-1 alpha and Nrf2 in intermittent hypoxia in lung adenocarcinoma A549 cells expressing high levels of the NADPH oxidase subunit NOX1. Whereas continuous hypoxia induced only HIF-1 alpha, intermittent hypoxia induced both HIF-1 alpha and Nrf2, including its target Trx1. NOX1 was determined to be crucial for enhanced ROS production in intermittent hypoxia that in turn mediated induction of Nrf2 and Trx1. The regulation of Nrf2 and Trx1 by NOX1 was confirmed by both inhibition of endogenous NOX1 and overexpression of recombinant NOX1 protein. By using a proteasomal inhibitor, NOX1 was demonstrated to activate Nrf2 by protein stabilization. Subsequently, Nrf2-dependent Trx1 induction turned out to enhance HIF-1 alpha signaling in intermittent hypoxia.
In vitro studies have indicated that reactive oxygen species modifying cellular redox status are involved in hypoxia-induced erythropoietin (EPO) production. However, the effects of redox balance on hypoxia-induced EPO production in vivo are still not fully understood. To investigate the effect of the change in cellular redox status on EPO generation, we determined whether glutathione (GSH) depletion has a significant influence on hypoxia-induced EPO production in rats. For the inhibition of GSH synthesis, DL-buthionine-[S,R]-sulfoximine (BSO) was employed by intraperitoneal injection. Twenty male rats were assigned to one of four experimental groups: (1) normoxic placebo, (2) normoxic BSO, (3) hypoxic placebo, and (4) hypoxic BSO. Hypoxic groups were exposed to a simulated normobaric hypoxic condition (4,500 m above sea level) for 12 hours. BSO treatment resulted in a significant depletion of total GSH levels in kidney and plasma in both conditions. However, the hypoxia-induced elevation in serum EPO concentration was not completely affected by the inhibition of GSH synthesis. These data demonstrate that GSH depletion in the kidney is not involved in the increase in serum EPO concentration in response to systemic hypoxia. It is also conceivable that the cellular redox changes could not function as a primary regulator of hypoxia-induced renal erythropoietin formation in vivo.
Hypoxia-inducible factor (HIF) is an important transcriptional regulator of cell metabolism and the adaptation to cellular stress caused by oxygen deficiency (hypoxia). Phagocytic cells have an essential role in innate immune defence against pathogens and this is a battle that takes place mainly in the hypoxic microenvironments of infected tissues. It has now become clear that HIF promotes the bactericidal activities of phagocytic cells and supports the innate immune functions of dendritic cells, mast cells and epithelial cells. In response to microbial pathogens, HIF expression is upregulated through pathways involving the key immune response regulator nuclear factor-kappaB, highlighting an interdependence of the innate immune and hypoxic responses to infection and tissue damage. In turn, HIF-driven innate immune responses have important consequences for both the pathogen and the host, such that the tissue microenvironment fundamentally influences susceptibility to infectious disease.
Human erythropoiesis is a complex multistep developmental process that begins at the level of pluripotent hematopoietic stem cells (HSCs) at bone marrow microenvironment (HSCs niche) and terminates with the production of erythrocytes (RBCs). This review covers the basic and contemporary aspects of erythropoiesis. These include the: (a) cell-lineage restricted pathways of differentiation originated from HSCs and going downward toward the blood cell development; (b) model systems employed to study erythropoiesis in culture (erythroleukemia cell lines and embryonic stem cells) and in vivo (knockout animals: avian, mice, zebrafish, and xenopus); (c) key regulators of erythropoiesis (iron, hypoxia, stress, and growth factors); (d) signaling pathways operating at hematopoietic stem cell niche for homeostatic regulation of self renewal (SCF/c-kit receptor, Wnt, Notch, and Hox) and for erythroid differentiation (HIF and EpoR). Furthermore, this review presents the mechanisms through which transcriptional factors (GATA-1, FOG-1, TAL-1/SCL/MO2/Ldb1/E2A, EKLF, Gfi-1b, and BCL11A) and miRNAs regulate gene pattern expression during erythroid differentiation. New insights regarding the transcriptional regulation of alpha- and beta-globin gene clusters were also presented. Emphasis was also given on (i) the developmental program of erythropoiesis, which consists of commitment to terminal erythroid maturation and hemoglobin production, (two closely coordinated events of erythropoieis) and (ii) the capacity of human embryonic and umbilical cord blood (UCB) stem cells to differentiate and produce RBCs in culture with highly selective media. These most recent developments will eventually permit customized red blood cell production needed for transfusion.
Although erythropoietin (Epo) is produced primarily by the kidneys in response to hypoxia, the precise cell type(s) and mechanisms by which these cells regulate production are poorly understood. In the experiments we report, the kinetics of renal Epo production in response to acute hypoxia and the intrarenal localization of cellular Epo synthesis were studied at the level of Epo mRNA. Erythropoietin mRNA expression was determined by Northern blot analysis of rat kidney RNAs using a probe derived from the mouse Epo gene. Renal Epo mRNA content increased as early as 1 hour after initiation of hypoxia and continued to accumulate during 4 hours of stimulation. Discontinuation of the hypoxic stimulus resulted in rapid decay of mRNA levels. Kidney and plasma Epo levels measured by radioimmunoassay paralleled, with respective lag times, the changes in renal Epo mRNA content, suggesting that Epo production in response to acute hypoxia represents de novo synthesis and is regulated by changes in Epo mRNA. Northern blot analysis of RNAs extracted from separated glomerular and tubular tissue fractions revealed Epo mRNA in the tubular fraction, whereas glomerular tissue did not contain Epo mRNA. Thus, the site of cellular Epo synthesis is located in the renal tubule or its interstitium and not in the glomerular tuft.
All organisms can sense O(2) concentration and respond to hypoxia with adaptive changes in gene expression. The large body size of mammals necessitates the development of multiple complex physiological systems to ensure adequate O(2) delivery to all cells under normal conditions. The transcriptional regulator hypoxia-inducible factor 1 (HIF-1) is an essential mediator of O(2) homeostasis. HIF-1 is required for the establishment of key physiological systems during development and their subsequent utilization in fetal and postnatal life. HIF-1 also appears to play a key role in the pathophysiology of cancer, cardiovascular disease, and chronic lung disease, which represent the major causes of mortality among industrialized societies. Genetic or pharmacological modulation of HIF-1 activity in vivo may represent a novel therapeutic approach to these disorders.
A systematic search was conducted to determine the characteristics of perioperative anemia, its association with clinical outcomes, and the effects of patient blood management interventions on these outcomes in patients undergoing major orthopedic surgery. In patients undergoing total hip or knee arthroplasty and hip fracture surgery, preoperative anemia was highly prevalent, ranging from 24 +/- 9% to 44 +/- 9%, respectively. Postoperative anemia was even more prevalent (51% and 87 +/- 10%, respectively). Perioperative anemia was associated with a blood transfusion rate of 45 +/- 25% and 44 +/- 15%, postoperative infections, poorer physical functioning and recovery, and increased length of hospital stay and mortality. Treatment of preoperative anemia with iron, with or without erythropoietin, and perioperative cell salvage decreased the need for blood transfusion and may contribute to improved patient outcomes. High-impact prospective studies are necessary to confirm these findings and establish firm clinical guidelines.
Objective.
—To assess the efficacy of oral iron therapy in the recovery of patients' hemoglobin levels after major surgery.Design.
—Randomized controlled trial.Setting.
—Private orthopedic practice confined to one large community hospital.Patients.
—One hundred seventy consecutive elderly patients undergoing hip surgery; 75 failed to meet entry hematologic or medical criteria; 95 were randomized, with 16 withdrawn because of complications.Intervention.
—Thirty-seven patients received ferrous sulfate orally four times a day for the duration of their hospitalization. Forty-two patients who received no iron supplement served as the control group.Main Outcome Measures.
—Changes in hemoglobin levels and reticulocyte counts over the 2- to 3-week follow-up period.Results.
—There was no significant difference in mean hemoglobin levels between the treatment and control groups (95% confidence interval [Cl] for difference of —6.6 to 5.4 g/L). Corrected reticulocyte fractions increased equally in both groups (95% Cl for difference of —9 × 103 to 2 × 10-3. The study was designed to detect a difference in mean hemoglobin levels of 8.5 g/L or greater or a difference in mean reticulocyte fraction of 10 × 10-3 between the two groups with a power of 0.80 at the .05 (two-sided) level of significance.Conclusion.
—The administration of oral iron supplements to elderly, healthy orthopedic patients postoperatively did not hasten the recovery of hemoglobin levels, provided adequate tissue iron stores were present.(JAMA. 1992;267:525-527)
Hypoxia plays critical roles in the pathobiology of heart disease, cancer, stroke, and chronic lung disease, which are responsible for 60% of deaths in the United States. This review summarizes advances in our un derstanding of how cells sense and respond to changes in oxygen availability and the physiologic or pathologic consequences of these responses in the context of chronic diseases. The role of hypoxia in inflammatory disorders was recently reviewed in the Journal 1 and is therefore not discussed here. M e ch a nis m s of Signa l T r a nsduc t ion i n H y p ox i a Humans have evolved complex circulatory, respiratory, and neuroendocrine systems to ensure that oxygen levels are precisely maintained, since an excess or deficiency may result in the death of cells, tissue, or the organism. As discussed below, oxygen homeostasis represents an organizing principle for understanding evolution, devel opment, physiology, and disease. Historically, oxygen sensing was thought to be limited to specialized cells, such as the glomus cells of the carotid body, which depo larize within milliseconds in response to hypoxemia by means of incompletely understood mechanisms. 2 We now recognize that all nucleated cells in the body sense and respond to hypoxia. Under conditions of reduced oxygen availability, hypoxia-inducible factor 1 (HIF-1) regulates the expression of genes that mediate adaptive responses. 3-6 In hypoxic cells, the transcription of several hundred messenger RNAs (mRNAs) is increased, and the expression of an equal number of mRNAs is decreased. The changes are dependent on HIF-1 in both cases, but HIF-1 binding is detected only at genes with increased expression. HIF-1 decreases mRNA expression indirectly by regulating transcriptional repressors and microRNAs. 3-6 HIF-1 was first identified in human cells as a regulator of erythropoietin, the hor
Erythropoiesis is affected during deep saturation dives. The mechanism should be related to a downregulation of serum Erythropoietin (s-EPO) concentration or to a toxic effect of the hyperbaric hyperoxia. We evaluated s-EPO and other haematological parameters in 6 scuba divers before, during and after a 14-days guinness saturation dive (8-10 m). Athletes were breathing air at 1.8-2 ATA, under the control of a team of physicians. Serum parameters were measured before diving (T0) and: 7 days (T1), 14 days (T2) after the beginning of the dive and 2 h (T3) and 24 h (T4) after resurfacing. Hgb, and many other haematological parameters did not change whereas Ht, s-EPO, the ratio between s-EPO predicted and that observed and reticulocytes (absolute, percent) declined progressively from T0 to T3. At T4 a significant rise in s-EPO was observed. Hgb did not vary but erythropoiesis seemed to be affected as s-EPO and reticulocyte counts showed. All these changes were statistically significant. The experiment, conducted in realistic conditions of dive length, oxygen concentration and pressure, allows us to formulate some hypotheses about the role of prolonged hyperbarism on erythropoiesis. The s-EPO rise, 24 h after resurfacing, is clearly documented and related to the "Normobaric Oxygen Paradox". This evidence suggests interesting hypotheses for new clinical applications such as modulation of s-EPO production and Hgb content triggered by appropriate O2 administration in pre-surgical patients or in some anemic disease.
Based on a report of a marked increase in the erythropoietin concentration ([EPO]) a few hours after the cessation of a single 2-h session of O(2) breathing, short periods of O(2) administration have been advocated as a therapy for anaemia. Accordingly, the purpose of the present study was to evaluate this theory by investigating the effect of 10 daily short-term exposures to normobaric O(2) over a 2-week period on the plasma [EPO] in healthy individuals.
Twenty men were assigned to either an experimental (NBO(2)) or to a control (AIR) group. The NBO(2) group breathed 100% normobaric O(2) for 2 h every weekday over a 2-week period. The AIR group breathed air within the same time protocol. Blood samples were collected at the pre-, mid- and post-intervention periods to determine [EPO].
[EPO] of the NBO(2) group was significantly lower than that of the AIR group during the mid- and post-periods (P < 0·001). [EPO] of the NBO(2) group showed a slight, albeit statistically nonsignificant, decrease during the mid (∼11%)- and post (∼16%)-periods.
Daily short-term exposures to normobaric hyperoxia do not increase the [EPO] in healthy individuals. The increased O(2) tension suppresses [EPO]. Hence, administration of pure O(2) to enhance erythropoiesis is not warranted.
The maintenance of oxygen homeostasis is critical for survival, and the master regulator of this process in metazoan species is hypoxia-inducible factor 1 (HIF-1), which controls both O(2) delivery and utilization. Under conditions of reduced O(2) availability, HIF-1 activates the transcription of genes, whose protein products mediate a switch from oxidative to glycolytic metabolism. HIF-1 is activated in cancer cells as a result of intratumoral hypoxia and/or genetic alterations. In cancer cells, metabolism is reprogrammed to favor glycolysis even under aerobic conditions. Pyruvate kinase M2 (PKM2) has been implicated in cancer growth and metabolism, although the mechanism by which it exerts these effects is unclear. Recent studies indicate that PKM2 interacts with HIF-1α physically and functionally to stimulate the binding of HIF-1 at target genes, the recruitment of coactivators, histone acetylation, and gene transcription. Interaction with HIF-1α is facilitated by hydroxylation of PKM2 at proline-403 and -408 by PHD3. Knockdown of PHD3 decreases glucose transporter 1, lactate dehydrogenase A, and pyruvate dehydrogenase kinase 1 expression; decreases glucose uptake and lactate production; and increases O(2) consumption. The effect of PKM2/PHD3 is not limited to genes encoding metabolic enzymes because VEGF is similarly regulated. These results provide a mechanism by which PKM2 promotes metabolic reprogramming and suggest that it plays a broader role in cancer progression than has previously been appreciated.
Previous studies in healthy subjects have shown an increase in erythropoietin (EPO) production after administration of N-acetyl-cysteine (NAC). These authors hypothesized that NAC increases intracellular reduced glutathione, decreasing reactive oxygen species and enabling EPO production. We investigated if EPO production could be stimulated with a single dose of NAC, after 90 min of pure oxygen breathing. Thirty-eight healthy volunteers were randomized into either the control (C) group or the NAC group, which received 600 mg NAC PO dissolved in a glass of orange juice, 60 min before breathing 15 L/min of 100% normobaric oxygen. Orange juice was administered to both groups. Blood samples for EPO measurement were taken at T0, before the orange juice administration, and T1, T2, T3 and T4, respectively, 8, 24, 32 and 48 h after the orange juice. The EPO concentrations of the NAC group decreased significantly at T1, followed by a significant increase compared to baseline, which was obvious until T4. The EPO concentrations of the C group did not show any significant variations. In this study, a significant increase of EPO production was observed after a short-term hyperoxic stimulus only when preceded with the administration of a single dose of NAC.
The purpose of the present study was to evaluate the 'normobaric oxygen paradox' theory by investigating the effect of a 2-h normobaric O(2) exposure on the concentration of plasma erythropoietin (EPO).
Ten healthy males were studied twice in a single-blinded counterbalanced crossover study protocol. On one occasion they breathed air (NOR) and on the other 100% normobaric O(2) (HYPER). Blood samples were collected Pre, Mid and Post exposure; and thereafter, 3, 5, 8, 24, 32, 48, 72 and 96 h, and 1 and 2 weeks after the exposure to determine EPO concentration.
The concentration of plasma erythropoietin increased markedly 8 and 32 h after the NOR exposure (approx. 58% and approx. 52%, respectively, P ≤ 0.05) as a consequence of its natural diurnal variation. Conversely, the O(2) breathing was followed by approx. 36% decrement of EPO 3 h after the exposure (P ≤ 0.05). Moreover, EPO concentration was significantly lower in HYPER than in the NOR condition 3, 5 and 8 h after the breathing intervention (P ≤ 0.05).
In contrast to the 'normobaric oxygen paradox' theory, the present results indicate that a short period of normobaric O(2) breathing does not increase the EPO concentration in aerobically fit healthy males. Increased O(2) tension suppresses the EPO concentration 3 and 5 h after the exposure; thereafter EPO seems to change in a manner consistent with natural diurnal variation.
The current practice of mechanical ventilation comprises the use of the least inspiratory O2 fraction associated with an arterial O2 tension of 55 to 80 mm Hg or an arterial hemoglobin O2 saturation of 88% to 95%. Early goal-directed therapy for septic shock, however, attempts to balance O2 delivery and demand by optimizing cardiac function and hemoglobin concentration, without making use of hyperoxia. Clearly, it has been well-established for more than a century that long-term exposure to pure O2 results in pulmonary and, under hyperbaric conditions, central nervous O2 toxicity. Nevertheless, several arguments support the use of ventilation with 100% O2 as a supportive measure during the first 12 to 24 hrs of septic shock. In contrast to patients without lung disease undergoing anesthesia, ventilation with 100% O2 does not worsen intrapulmonary shunt under conditions of hyperinflammation, particularly when low tidal volume-high positive end-expiratory pressure ventilation is used. In healthy volunteers and experimental animals, exposure to hyperoxia may cause pulmonary inflammation, enhanced oxidative stress, and tissue apoptosis. This, however, requires long-term exposure or injurious tidal volumes. In contrast, within the timeframe of a perioperative administration, direct O2 toxicity only plays a negligible role. Pure O2 ventilation induces peripheral vasoconstriction and thus may counteract shock-induced hypotension and reduce vasopressor requirements. Furthermore, in experimental animals, a redistribution of cardiac output toward the kidney and the hepato-splanchnic organs was observed. Hyperoxia not only reverses the anesthesia-related impairment of the host defense but also is an antibiotic. In fact, perioperative hyperoxia significantly reduced wound infections, and this effect was directly related to the tissue O2 tension. Therefore, we advocate mechanical ventilation with 100% O2 during the first 12 to 24 hrs of septic shock. However, controlled clinical trials are mandatory to test the safety and efficacy of this approach.
The "normobaric oxygen paradox" is a dual mechanism by which oxygen regulates the expression of the Hypoxia Inducible Factor 1 alpha (HIF-1α). The HIF-1α-depending gene regulation is responsible for many different genetic expressions including EPO and VEGF that are usually expressed in parallel. First, VEGF under-expression could decrease tumor angiogenesis leading to a decrease in tumor growth or even apoptosis of cancer cells. Second, induction of EPO-expression can provide cytoprotection. Altogether, this could be deleterious for cancer cells while helping non-malignant cells (at least neural and cardiac) cells to be protected from the side effects of chemotherapy. Eventually, HIF induction could boost immune response by inflammatory cells, increasing their antitumor activity.
Rhodiola rosea is commonly used in China and Tibet folk medicine for the treatment of high altitude sickness, anoxia and mountain malhypoxia.
Salidroside (SDS) is an active ingredient of Rhodiola rosea. This study attempted to examine the potential erythropoiesis-stimulating and anti-oxidative effect of SDS in TF-1 erythroblasts.
The erythropoiesis-promoting effect was determined by treating human TF-1 cells, one of the popular in vitro models for studying erythropoiesis, with SDS in the presence and absence of erythropoietin (EPO) through the measurement of the expression of a series of erythroid markers such as glycophorin A (GPA), transferrin receptor (CD71) and hemoglobin (Hb). The potential protective effect of SDS against H(2)O(2)-induced apoptosis and its underlying mechanism in TF-1 erythroblasts were examined by flow cytometry and Western blot analysis.
SDS promotes erythropoiesis in the EPO-treated cells and it also reduces the number of apoptotic cells in TF-1 erythroblasts after H(2)O(2) treatment probably through the up-regulation of protective proteins thioredoxin-1 (Trx1) and glutathione peroxidase-1 (GPx1).
Our study provides evidence to explain the ethnopharmacological role of SDS and Rhodiola rosea in Chinese medicine. Our findings also support the use of SDS as an erythropoiesis-adjuvant agent to correct anemia and malhypoxia.
Metazoan life is dependent upon the utilization of O 2 for essential metabolic processes and oxygen homeostasis is an organizing principle for understanding metazoan evolution, ontology, physiology, and pathology. Hypoxia‐inducible factor 1 (HIF‐1) is a transcription factor that is expressed by all metazoan species and functions as a master regulator of oxygen homeostasis. Recent studies have elucidated complex mechanisms by which HIF‐1 activity is regulated and by which HIF‐1 regulates gene expression, with profound consequences for prenatal development, postnatal physiology, and disease pathogenesis. Copyright © 2009 John Wiley & Sons, Inc.
This article is categorized under: Physiology > Organismal Responses to Environment
Hypoxia is known to play important role in cancer biology. In sarcomas, hypoxia-induced protein biomarkers such as Hypoxia Inducible Factor-1alpha (HIF-1alpha), vascular endothelial growth factor (VEGF) and Erythropoietin (Epo) have been previously reported in only a few studies. Moreover, the biologic significance and relationship to tumorigenesis of these hypoxia-induced biomarkers is not well understood in the context of sarcoma. The HIF negative regulator, Prolyl Hydroxylase Domain protein 2 (PHD2) has not been evaluated in sarcomas. We examined the expression of PHD2, HIF-1alpha, and several other hypoxia induced biomarkers in a series of clinically characterized, retroperitoneal sarcomas with immunohistochemical methods. Expression of these proteins was analyzed and correlated with clinical outcome. Increased HIF-1alpha expression was associated with shorter overall and disease free survival. PHD2 expression was detected in the majority of sarcoma cases, with increased expression correlating with high tumor grade but not with survival. Though changes in PHD2 expression alone did not correlate with overall and disease free survival, reduced/absent PHD2 expression in the presence of HIF-1alpha expression was associated with shorter overall and disease-free survival than that of other HIF-1alpha/PHD2 expression profiles. These observations suggest that regulation and expression of both PHD2 and HIF-1alpha are important to the biology of sarcomas, and that loss of PHD2 function has an additional adverse effect in the prognosis of sarcomas in tumors expressing HIF-1alpha. The biologic and therapeutic implications of HIF-1alpha and PHD2 expression in retroperitoneal sarcomas warrant further investigation.
A new type of competitive human GST inhibitors has been developed via the bioisostere and structure activity profile strategies; we report their discovery, preparation, inhibitory activity, and synergetic effect in combination with chemotherapy drugs against breast cancer cells.
We have previously shown that high-dose erythropoietin (EPO) treatment improves hippocampal plasticity and cognitive performance in rodents and in patients suffering from neuropsychiatric diseases. It was therefore of interest to explore whether upregulation of endogenous EPO in brain by hypoxia inducible factor (HIF) stabilization would increase hippocampal memory similar to exogenous EPO. HIFs are transcription factors involved in the cellular response to low oxygen, including upregulation of transcripts like vascular endothelial growth factor (VEGF) and EPO. Under normal oxygen, prolylhydroxylases decrease HIF-alpha stability. This is banned by prolylhydroxylase inhibitors, which prevent oxygen dependent degradation and thus prolong HIF-alpha half life. In an experimental set-up identical to the one yielding strong cognitive effects with EPO, healthy male 28-day-old mice received FG-4497, a HIF prolylhydroxylase inhibitor, or placebo intraperitoneally every other day for 3 weeks. Behavioral testing and hematocrit determinations were conducted in independent cohorts at 1, 3, or 4 weeks after treatment completion. Increased EPO and VEGF mRNA expression in hippocampus or primary hippocampal neurons 6h after the application of FG-4497 confirmed its ability to stabilize HIF and upregulate HIF dependent transcription in brain. At 3 and 4 weeks after the last injection, respectively, FG-4497 treated mice compared to placebo mice had improved hippocampal memory in fear conditioning without change in hematocrit. In contrast, no improvement in memory was detected at 1 week, when the hematocrit was increased, indicating that cognitive improvement and hematocrit are not directly related. FG-4497 application for 3 weeks leads to delayed but lasting enhancement of hippocampal memory, making HIF stabilization an attractive target for pharmacological manipulation of cognition.
One of the abiding mysteries of all multi-cellular organisms is the requirement for controlled death —apoptosis — of unwanted cells. It has been estimated that without apoptosis an 80 year old person would have two tons of bone marrow and lymph nodes and an intestine 16 kilometers long.1 Progress in defining pathways of apoptosis has revealed complex interconnections between various cell death programs that may affect the treatment of a wide range of diseases.2–10 This article reviews advances in our understanding of mechanisms of cell death and highlights current and potential therapies based upon these concepts.
Perhaps the most widely used classification of mammalian cell death consists of two types: apoptosis and necrosis.3,4,11 Autophagy, which has recently been proposed as a third distinct mode of cell death, is a process by which cells generate energy and metabolites by digesting organelles or macromolecules.12–15. Normally, autophagy allows a starving cell, or a cell deprived of growth factors to survive.12–15 Ultimately, however, cells deprived of nutrients for extended periods will digest all available substrates and die an ‘autophagy-associated cell death’. Distinctions between apoptosis, necrosis, and autophagy entail differences in mode-specific or selective morphologic, biochemical, and molecular attributes (Fig. 1).3,4,11
Figure 1
Schematic diagram showing 3 possible pathways of cell death
An important concept embodied in part by these attributes is “programmed” cell death. Cell death is “programmed” if it is genetically controlled. The two fundamental types of programmed cell death are apoptosis and autophagy-associated cell death.3,12 The recognition that cell death can occur by genetically controlled processes has enabled advances in unraveling the mechanisms of many diseases. As a result, we now have improved knowledge of the initiation of cell death programs and the relevant signaling pathways. This information has facilitated development of pharmacologic agents that initiate or inhibit programmed cell death.6–8,16 Moreover, there is now evidence that necrosis, traditionally considered an accidental form of cell death, can, in certain instances, be initiated or modulated under programmed control mechanisms.17–21
Anemia is frequent in cancer patients and its incidence increases with chemotherapy. The probability of requiring transfusions also increases with chemotherapy. Anemia negatively impacts survival and accentuates fatigue in cancer patients. Cancer promotes inflammatory cytokine production, which suppresses erythropoiesis and erythropoietin (EPO) production. Erythropoiesis-stimulating agents (ESAs) improve erythropoiesis and reduce transfusion needs in anemic cancer patients receiving chemotherapy. However, meta-analyses have shown an increased risk of thromboembolic (TE) events with ESA use during chemotherapy, but not increased on-study mortality or reduced overall survival. Three reasons have been proposed to explain why ESAs might have adverse effects in anemic cancer patients: tumor progression due to stimulation of tumor cell EPO receptors; increased risk of TE; and reduced survival. However, erythropoietin is not an oncogene, nor is the EPO receptor. It has also been demonstrated that erythropoietin does not stimulate tumor proliferation. Increased TE risk associated with ESAs is probably a consequence of increased blood viscosity due to excessive RBC mass elevation with concomitant plasma volume contraction, nitric oxide scavenging, and endothelial cell activation. Increased ESA dosing may also impact survival negatively because EPO contracts the plasma volume and stimulates inflammatory cytokine production independently of increasing erythropoiesis. Furthermore, transfusions themselves are associated with an increase in TE and plasma volume contraction, and these events are potentiated when ESAs are given with transfusions. An update on the management of anemia in oncology, the potential adverse events of ESAs, the benefits and risks of transfusions, and QoL are discussed in this paper.
Therapeutic red blood cell (RBC) transfusion is widely utilized in the management of anaemia. Critically ill intensive care unit (ICU) patients in particular, as well as medical and haematology-oncology patients, are among the largest groups of users of RBC products. While anaemia is common in these patients, its treatment and management, including appropriate thresholds for RBC transfusion, remain controversial. We review here the function of RBCs in oxygen transport and physiology, with a view to their role in supporting and maintaining systemic tissue oxygenation. Adaptive and physiological compensatory mechanisms in the setting of anaemia are discussed, along with the limits of compensation. Finally, data from clinical studies will be examined in search of evidence for, or against, a clinically relevant transfusion trigger.
Ischemic postconditioning (IPost) and erythropoietin (EPO) have been shown to attenuate myocardial reperfusion injury using similar signaling pathways. The aim of this study was to examine whether EPO is as effective as IPost in decreasing postischemic myocardial injury in both Langendorff-isolated-heart and in vivo ischemia-reperfusion rat models. Rat hearts were subjected to 25 min ischemia, followed by 30 min or 2 h of reperfusion in the isolated-heart study. Rats underwent 45 min ischemia, followed by 24 h of reperfusion in the in vivo study. In both studies, the control group (n=12; ischemia-reperfusion only) was compared with IPost (n=16; 3 cycles of 10 s reperfusion/10 s ischemia) and EPO (n=12; 1,000 IU/kg) at the onset of reperfusion. The following resulted. First, in the isolated hearts, IPost or EPO significantly improved postischemic recovery of left ventricular developed pressure. EPO induced better left ventricular developed pressure than IPost at 30 min of reperfusion (73.18+/-10.23 vs. 48.11+/-7.92 mmHg, P<0.05). After 2 h of reperfusion, the infarct size was significantly lower in EPO-treated hearts compared with IPost and control hearts (14.36+/-0.60%, 19.11+/-0.84%, and 36.21+/-4.20% of the left ventricle, respectively; P<0.05). GSK-3beta phosphorylation, at 30 min of reperfusion, was significantly higher with EPO compared with IPost hearts. Phosphatidylinositol 3-kinase and ERK1/2 inhibitors abolished both EPO- and IPost-mediated cardioprotection. Second, in vivo, IPost and EPO induced an infarct size reduction compared with control (40.5+/-3.6% and 28.9+/-3.1%, respectively, vs. 53.7+/-4.3% of the area at risk; P<0.05). Again, EPO decreased significantly more infarct size and transmurality than IPost (P<0.05). In conclusion, with the use of our protocols, EPO showed better protective effects than IPost against reperfusion injury through higher phosphorylation of GSK-3beta.