There is now substantial evidence that EPO protects organs and tissues against a number of noxious stimuli including ischemia (6
). In 2004, we discovered that the administration of EPO (300 IU/kg) on resuscitation reduces the organ injury/dysfunction associated with severe trauma hemorrhage by antiapoptotic and antiinflammatory mechanisms (19
). Although EPO also improved survival in critically ill patients with trauma, this came at the expense of a significant increase in thrombotic events (11
). The recent discovery of a nonerythropoietic, tissue-protective peptide mimicking the 3D structure of EPO (pHBSP) allowed us to test the hypothesis that the tissue- protective effects of EPO can be obtained in trauma hemorrhage in the absence of significant side effects. We report here that a single injection of pHBSP given intravenously to rats after the onset of resuscitation attenuated the renal dysfunction, liver injury, pancreatic injury and neuromuscular injury caused by severe HR in the anesthetized rat. There is evidence that EPO attenuates the degree of lung inflammation and injury caused by hyperoxic injury (20
), ischemia-reperfusion (21
) and acute necrotizing pancreatitis (22
). We report here that pHBSP attenuated the degree of inflammatory cell infiltration, alveolar septal thickening and congestion in the lungs of rats subjected to HR. Thus, similar to EPO, pHBSP exerts tissue-protective and antiinflammatory effects in vivo
What, then, is the mechanism(s) by which pHBSP exerts these beneficial effects? Clearly, pHBSP affected neither the metabolic acidosis nor the hemodynamic abnormalities caused by HR. There is some evidence that the beneficial effects of EPO are secondary to the activation of the survival kinase Akt (23
). Akt is a member of the phosphoinositide 3-kinase signal transduction enzyme family, which regulates cellular activation, inflammatory responses, chemotaxis and apoptosis (25
). When phosphorylated by its upstream regulator, phosphoinositide -dependent kinase, Akt modulates cell survival and growth (25
). We report here that HR results in a significant reduction in the phosphorylation of Akt in both the liver and kidney. A reduction in the activation of this important survival pathway will make organs more susceptive to injury and inflammation (26
). Most notably, pHBSP restored the degree of Akt phosphorylation to the level seen in sham- operated animals even when the EPO-mimetic was given as late as 60 min into resuscitation. Both EPO and pHBSP also enhance the phosphorylation of Akt in cardiomyocytes subjected to TNF-α (28
GSK-3β is a serine-threonine kinase that originally was recognized as a kinase that phosphorylates glycogen synthase. In contrast to most other kinases, GSK-3β is active in a resting cell state; however, it is inactivated by phosphorylation of Ser9.
GSK-3β is regulated by multiple signaling pathways including the Akt pathway, which inactivates it by causing Ser9
). Consistent with decline in the phosphorylation/activation of Akt reported here, HR also caused a significant decline in the phosphorylation of GSK-3β on Ser9.
This indicates an excessive activation of GSK-3β which would drive both inflammation (31
) and tissue-injury (32
). Similar to the above-reported effects on Akt phosphorylation, pHBSP restored the degree of Ser9
phosphorylation on GSK-3β to the levels seen in sham-operated animals even when the EPO-mimetic was given as late as 60 min into resuscitation. An increase in Ser9
phosphorylation results in inhibition of this kinase and inhibitors of GSK-3β exert potent antiinflammatory (31
) and antiischemic effects in a number of organs (32
). Interestingly, inhibition of GSK-3β also mediates the cardioprotective effects of EPO (32
Downstream of GSK-3β, several studies have now reported an association between GSK-3β and NF-κB activity in vitro
) and in vivo
). NF-κB is a transcriptional factor that plays an important role in regulating the transcription of a number of genes, especially those involved in producing mediators involved in local and systemic inflammation, such as cytokines, chemokines, cell adhesion molecules, apoptotic factors and other mediators (39
). Treatment of TNF-α stimulated hepatocytes with a specific GSK-3β inhibitor resulted in a decrease of the NF-κB–dependent gene transcription (40
). This study also indicated four potential phosphorylation sites for GSK-3β on the NF-κB subunit p65. Most notably, pretreatment with a number of chemically distinct inhibitors of GSK-3β attenuates organ injury and dysfunction caused by hemorrhage and resuscitation and endotoxemia (31
). This protective effect was associated with inhibition of the activation of NF-κB and NF-κB–dependent proinflammatory genes, along with a reduced phosphorylation of Ser536
on the NF-κB p65 subunit. In addition, GSK-3β also may inhibit the activation of NF-κB by phosphorylating and degrading IκBα, which is required to prevent NF-κB translocation (37
). We report here that HR results in a significant increase in the activation of NF-κB (measured here as nuclear translocation of p65) which was attenuated when pHBSP was given 60 min into resuscitation. All of the above findings support the view that pHBSP restores the activation of Akt, resulting in inhibition of GSK-3β (after phosphorylation on Ser9
) and inhibition of the activation of NF-κB.
In addition to inhibiting the activation of GSK-3β, activation of Akt results in the phosphorylation of eNOS on Ser1177
which, in turn, causes activation of eNOS resulting in an enhanced formation of nitric oxide (NO). In our study, HR did not affect eNOS phosphorylation on Ser1177
. Administration at 60 min into resuscitation of pHBSP, however, caused a pronounced increase in eNOS phosphorylation and, hence, activity. In conditions associated with ischemia-reperfusion injury, activation of eNOS is beneficial as the enhanced formation of NO causes local vasodilation, inhibits adhesion of platelets and neutrophils and regulates angiogenesis (41
). There is good evidence that agents which release NO or enhance the formation of endogenous NO attenuate organ injury/dysfunction in HS (42
). Agents that also inhibit the formation of NO from inducible NOS (iNOS) also attenuate the multiple organ failure associated with HS (43
). Inhibition of eNOS activity also attenuates the cardioprotective effects of EPO (44
). Recently, Su et al
) have demonstrated that CD131, when activated by EPO, is able to activate Akt and, in turn, increases the interaction between CD131 and eNOS. This increase in interaction results in the activation of eNOS and production of NO (45
). Thus, activation of eNOS (possibly secondary to activation of Akt) may contribute to the beneficial effects of pHBSP reported here.
Activation of p38 MAPK promotes cellular stress responses such as proliferation, differentiation and production of proinflammatory cytokines and occurs in response to ischemia and hemorrhage in a number of organs (46
). HR results in the activation of p38 MAPK (in male animals), while inhibition of the activity of this p38 MAPK in hemorrhagic shock attenuates renal, cardiac and lung injury (48
). We report here that HR results in a moderate activation of p38 MAPK (even when measured at 4 h after onset of resuscitation) in the kidney and a more marked activation in the liver, the latter of which was attenuated by pHBSP when given either at 30 or 60 min into resuscitation. Our data are consistent with the hypothesis that prevention of the activation of p38 MAPK contributes to the observed beneficial effects of pHBSP, at least, in the liver. It is well documented that activation of p38 peaks 1 h after hemorrhage in the kidney and thereafter gradually declines. Thus, it is possible that a more marked activation of p38 MAPK occurs early after hemorrhage which may have been attenuated by pHBSP.
Like ischemia, severe HR results in the activation of ERK1/2 and drugs that prevent the activation of ERK1/2 in HS exert beneficial effects (51
). We report here that the activation of both ERK1/2 caused by HR is attenuated by pHBSP. Like trauma hemorrhage, traumatic brain injury results in a significant activation of ERK1/2 which drives brain edema. Interestingly, EPO attenuates brain edema in this model by preventing the activation of ERK1/2 (52
). Similarly, the beneficial effects of EPO in a rat model of spinal cord injury have been attributed to the ability of EPO to reduce the phosphorylation of ERK (53
In conclusion, we have discovered that the nonhematopoietic EPO-analogue pHBSP attenuates the multiple organ injury and dysfunction when given during resuscitation of rats subjected to severe hemorrhage. It should be noted that pHBSP was still effective when given as late as 60 min after the onset of resuscitation. Surprisingly, some of the effects (on outcome or signaling) observed with pHBSP were more pronounced when the peptide was given at 60 min, rather than 30 min, into the resuscitation period. We have reported previously that the efficacy of pHBSP increased when given as late as 6 hours after the onset of reperfusion of the previously ischemic kidney (15
). We do not fully understand this phenomenon, but it is possible that the receptor targeted by pHBSP (and EPO) is upregulated in response to injury, leading to a more pronounced response after binding of the ligand. In addition, pharmacokinetic studies have confirmed that pHBSP has a very short plasma half life of ~2 min in the rat (15
) suggesting that an agent present within the circulation for only a short time after i.v. dosing elicits protective effects equivalent to EPO or CEPO with plasma half lives of 4–6 hours. Whatever the mechanism, this finding is likely to be of therapeutic relevance, as it allows a late intervention in HS and other conditions associated with ischemia-reperfusion. This could be of great importance when aiming to extend the “golden hour” after a major insult.
The acute model used here necessitates the use of heparin to prevent the formation of clots in the catheters used to extract the blood as well as the hemorrhaged blood. It has been known for some time that heparin does have potential therapeutic applications beyond anticoagulation (54
) and these should be considered when evaluating data from this model. However, the model used here demonstrates significant multiple organ failure (MOF) compared with animals subjected to sham operation, suggesting that the dose of heparin used here to prevent coagulation does not produce any observable beneficial effects.
To gain a better insight into the signaling pathways involved in the tissue- protective and/or antiinflammatory effects of pHBSP, we have investigated the effects of pHBSP on signaling pathways known to play a role in tissue injury/survival and/or inflammation. As the time-course in the activation of these signaling cascades may vary between tissues, we have carried out these mechanistic studies in both liver and kidney, two organs that were markedly protected by pHBSP, in the hope to find common signaling events that contribute to the observed beneficial effects of pHBSP in vivo
. In both liver and kidney, HR resulted in a significant inactivation of the survival kinase Akt (measured as reduction in the phosphorylation on Ser473
). Treatment of rats with pHBSP restored the phosphorylation and, hence, activation of Akt, which, in turn, resulted in inhibition of GSK-3β (secondary to phosphorylation on Ser9
) and inhibition of the activation of NF-κB. There is now very good evidence that therapeutic strategies which enhance the activation of Akt and reduce the activation of GSK-3β enhance the resistance of organs to noxious stimuli (including ischemia) and reduce inflammation via inhibition of NF-κB (37
). Activation of Akt by pHBSP also resulted in activation of eNOS (measured as phosphorylation on Ser1177
) which should result in an enhanced formation of NO in the microcirculation. In addition, pHBSP attenuated the HR-induced activation of p38 MAPK and ERK1/2, both of which are known to contribute to the development of organ injury/inflammation in HS (49
). We propose that all of the above signaling events initiated by pHBSP contribute to the beneficial effects of this nonhematopoietic EPO analogue in HS. As the beneficial effects of EPO in patients with trauma are limited by side effects owing to excessive erythropoiesis, we speculate that nonhematopoietic analogues of EPO such as pHBSP may be useful to mimic the tissue-protective effects of EPO without causing the well documented side effects.