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The Reperfusion Injury Salvage Kinase Pathway: A Common Target for Both Ischemic Preconditioning and Postconditioning
The Reperfusion Injury Salvage Kinase Pathway: A Common Target for Both Ischemic Preconditioning and Postconditioning Derek J. Hausenloy, Andrew Tsang, and Derek M. YellonT
Novel treatment approaches, as potential adjunctive therapy to current reperfusion strategies (such as thrombolysis, primary coronary angioplasty, and cardiac surgery), are required to provide further cardioprotection in the setting of an acute myocardial infarction to effect further reductions in morbidity and mortality. In this regard, the activation of prosurvival kinases, such as Akt and Erk1/2 (which we have termed the reperfusion injury salvage kinase [RISK] pathway), at the time of reperfusion, has been demonstrated to confer powerful cardioprotection against myocardial ischemia–reperfusion injury. In this review, we present evidence suggesting that the cardioprotective phenomena of ischemic preconditioning and the recently described ischemic postconditioning exert their cardioprotective effects through the recruitment of the RISK pathway, at the time of reperfusion, and that the protection in these settings is mediated through the inhibition of mitochondrial permeability transition pore (mPTP) opening at this time. Therefore, the pharmacologic manipulation of the RISK pathway at the time of reperfusion may enable one to harness the powerful cardioprotective benefits of both ischemic preconditioning and postconditioning, and provide a novel approach to cardioprotection. (Trends Cardiovasc Med 2005;15:69–75) D 2005, Elsevier Inc.
Protecting the Heart Against Ischemia–Reperfusion Injury
Coronary artery disease is currently the leading cause of mortality and morbidity in the western world. The serious and
Derek J. Hausenloy, Andrew Tsang, and Derek M. Yellon are at the Hatter Institute and Centre for Cardiology, University College London Hospital, WC1E 6DB London, UK. T Address correspondence to: Derek M. Yellon, PhD, DSc, Hon FRCP, The Hatter Institute and Centre for Cardiology, University College London Hospital, Grafton Way, WC1E 6DB London, UK. Tel.: (+ 44) 207-3809776; fax: (+ 44) 207-388-5095; e-mail: [email protected]. D 2005, Elsevier Inc. All rights reserved. 1050-1738/05/$-see front matter
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often fatal consequence of coronary artery disease is an acute myocardial infarction, which results from the acute occlusion of one of the major coronary arteries. In this scenario, the best hope of limiting the size of the myocardial infarct is the timely restoration of coronary blood flow, by either thrombolysis or primary percutaneous coronary artery angioplasty. Despite these current reperfusion strategies, the morbidity and mortality associated with an acute myocardial infarction remains significant, necessitating the development of novel cardioprotective strategies, which can be used as adjunctive therapy to the current reperfusion strategies. One of the most powerful mechanisms for protecting the myocardium before the
acute coronary artery occlusion occurs is to ischemically precondition the myocardium, a powerful cardioprotective phenomenon first described in 1986 by Murry et al. (1986) in which transient nonlethal episodes of myocardial ischemia confer protection against the myocardial infarction induced by a subsequent sustained episode of lethal myocardial ischemia. This approach however depends crucially on intervening before the ischemic event, which is difficult, given the unpredictable onset of an acute coronary artery occlusion. An alternative, more amenable, approach to cardioprotection is to protect the myocardium after the acute coronary artery occlusion has occurred. In this way, the cardioprotective strategy can be applied at the time of reperfusion to protect the myocardium against the myocyte death induced by the process of reperfusion itself—a phenomenon termed lethal reperfusion-induced injury (Braunwald and Kloner 1985). In this regard, our previous studies have implicated the prosurvival kinases, phosphatidylinositol-3-OH kinase (PI3K)-Akt and the p42/p44 extracellular signal-regulated kinases (Erk1/2), which we have termed the reperfusion injury salvage kinase (RISK) pathway as an important target for cardioprotection (Yellon and Baxter 1999, Hausenloy and Yellon 2004), at the time of reperfusion. We have demonstrated that the pharmacologic activation of the RISK pathway at the onset of reperfusion protects the heart against ischemia– reperfusion injury (Yellon and Baxter 1999, Hausenloy and Yellon 2004). Previous studies have demonstrated that adenovirally transfecting mural hearts (Fujio et al. 2000) or rat hearts (Miao et al. 2000) with constitutively active Akt gene constructs confers protection against ischemia–reperfusion injury. Interestingly, our recent data have demonstrated that the recruitment of this protective RISK pathway, at the time of reperfusion, contributes to the protection of both ischemic preconditioning (IPC) (Hausenloy et al. 2005) and the newly described phenomenon of ischemic postconditioning (Tsang et al. 2004). This suggests that the pharmacologic upregulation of the RISK pathway at the time of reperfusion may enable one to harness the cardioprotective benefits of both IPC and ischemic postconditioning.
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The RISK Pathway
In view of the studies demonstrating that apoptosis may contribute to the myocyte death induced by lethal reperfusion injury (Gottlieb et al. 1994, Freude et al. 2000), and the fact that the prosurvival kinases PI3K-Akt and Erk1/2 exert antiapoptotic protective effects, our laboratory (Yellon and Baxter 1999) first formulated and tested the hypothesis that the pharmacologic activation of these prosurvival kinases, at the time of reperfusion, protects the myocardium against lethal reperfusion-induced injury. We and others have demonstrated that the activation of these kinase cascades (the RISK pathway), with various growth factors including insulin (Jonassen et al. 2001) and urocortin (Schulman et al. 2002), and other agents such as atorvastatin (Bell and Yellon 2003a), bradykinin (Bell and Yellon 2003b, Yang et al. 2004a, 2004b), and opioid receptor agonists (Gross et al. 2004), induces cardioprotection when given during the early reperfusion phase (Yellon and Baxter 1999, Hausenloy and Yellon 2004). Despite their initial premise as mediators of anti-apoptotic protection, studies have revealed that the pharmacologic activation of the RISK pathway at reperfusion protects against both apoptotic and necrotic cell death, as evidenced by a reduction in infarct size (Yellon and Baxter 1999, Hausenloy and Yellon 2004).
The Role of Akt and Erk1/2 in IPC: At the Time of Reperfusion
The aforementioned studies implicate PI3K-Akt and Erk1/2 as potential signaling components of the IPC signal. In contrast, we were interested in investigating the role of PI3K-Akt and Erk1/2, and the potential protection their activation may provide at the time of reperfusion, in the setting of IPC. Our recent work on IPC has focused on the reperfusion phase, which follows the index ischemic period, and has revealed that an IPC stimulus applied before the index ischemic event is able to protect the heart by modifying crucial events which occur during the postischemic reperfusion phase, such as the opening of the mPTP, which is a critical determinant of cell death (Hausenloy and Yellon 2003). Therefore, IPC appears to protect the heart by inhibiting the opening of the mPTP at the time of reperfusion (Hausenloy et al. 2002, 2004). Along the same train of thought, we have been investigating whether IPC may protect the heart by activating the RISK pathway at the time of reperfusion. In this regard, we have recently demonstrated, with the use of the isolated perfused rat heart, that a standard IPC stimulus results in the phosphory-
The Role of Akt and Erk1/2 in IPC: Before the Index Ischemic Period
The initial studies investigating the role of these prosurvival kinases, PI3K-Akt and Erk1/2, in the setting of IPC, did not examine these kinases in the context of the RISK pathway per se, that is, their protective role at the time of reperfusion was not examined. Instead, these initial studies investigated the role of these kinases before the index ischemic period, as potential signaling components, conveying the preconditioning signal from the cell membrane to the inner components of the cell such as the mitochondria. Tong et al. (2000) were the first to demonstrate a role for the PI3K-Akt pathway as a potential signaling component of the IPC signaling pathway. With the use of the isolated perfused rat heart, they demonstrated that a standard IPC stimulus induced the phosphorylation of
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both Akt and its downstream target GSK3h, immediately before the index ischemic period, and that the phosphorylation of these kinases was essential for IPC-induced protection (Tong et al. 2000, 2002). Downey’s laboratory (Oldenburg et al. 2004, Krieg et al. 2004a, 2004b) has subsequently elucidated the IPC signaling pathway from the cell membrane to the mitochondria via several different components including the PI3K-Akt pathway (see Figure 1). Whether Erk1/2 also acts as a potential signaling component in the setting of IPC is unclear, with some studies demonstrating that a standard IPC stimulus results in its phosphorylation before the index ischemic period (Fryer et al. 2001, Mocanu et al. 2002), although only some of these studies have actually demonstrated Erk1/2 phosphorylation contributing to IPC-induced protection (Fryer et al. 2001). Furthermore, other studies reported no change in Erk1/2 phosphorylation in response to an IPC stimulus (Behrends et al. 2000). Recent studies suggest that the mitochondrial reactive oxygen species (ROS) release induced by preconditioning may activate Erk1/2 (Samavati et al. 2002), thereby placing Erk1/2 downstream of the mitochondria in the scheme depicted in Figure 1.
Figure 1. The role of Akt and Erk1/2 as potential signaling components during the preconditioning phase. Activation of the G-protein-coupled receptor (GPCR), in response to an IPC stimulus, leads to the trans-activation of the epidermal growth factor receptor (EGRF) via the matrix metalloproteinase (MP), which then activates the PI3K-Akt pathway in a Srckinase-dependent manner. Signaling through the PI3K-Akt pathway results in the phosphorylation and activation of eNOS which then activates guanylate cyclase (GC) via NO. Guanylate cyclase then activates protein kinase G (PKG) via cyclic guanine-5-monophosphate (cGMP). PKG then phosphorylates and opens the mitochondrial KATP channel (mKATP), which results in the mitochondrial release of ROS, generated from the electron transport chain (ETC), which then activates the downstream mediators of preconditioning including Erk1/2. Adapted with permission from a scheme by Downey’s laboratory (Krieg et al. 2002).
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(A)
After IPC/ 15 min stabilization ischemia
35 min 15 min ischemia reperfusion
The RISK Pathway in Ischemic Postconditioning
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Figure 2. Representative Western blots showing that IPC results in a biphasic response in the phosphorylation of both (A) Akt and (B) Erk1/2, with an initial phase of phosphorylation, occurring before the index ischemic period, and a second phase of phosphorylation occurring at the time of reperfusion (n = 6 per group; *P b .05). Adapted with permission from Hausenloy et al. (2005).
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the authors found that delaying the administration of the PI3K inhibitor by 15 min into reperfusion also abrogated IPC-induced protection, suggesting the requirement for Akt phosphorylation for the first 30 min of reperfusion (Solenkova et al. 2005). Control
IPC
Akt phosphorylation at reperfusion Erk1/2 phosphorylation at reperfusion Infarct-risk volume ratio (%)
lation of both the Akt and Erk1/2 components of the RISK pathway at the time of reperfusion (Hausenloy et al. 2005). Interestingly, the IPC stimulus appears to induce a biphasic response in kinase phosphorylation, demonstrating initial kinase phosphorylation before the index ischemic period, and a second phase of kinase phosphorylation at the time of reperfusion (Figure 2) (Hausenloy et al. 2005). Importantly, we show that pharmacologically inhibiting the IPC-induced phosphorylation of these kinases, at the time of reperfusion, completely abrogates the IPC-induced limitation in infarct size, suggesting that both the Akt and Erk1/2 components of the RISK pathway are essential for IPC to manifest its protection (see Figure 3) (Hausenloy et al. 2005). With the use of the isolated rabbit heart model of ischemia–reperfusion injury, Downey’s laboratory has recently demonstrated that the presence of wortmannin (a PI3K inhibitor) during the reperfusion phase is also able to abrogate IPC-induced protection, supporting a role for Akt phosphorylation during the reperfusion phase in mediating protection in this setting (Solenkova et al. 2005). Interestingly, in that study
Vinten-Johansen and colleagues (Zhao et al. 2003) have recently described an apparently new cardioprotective phenomenon, which they have termed ischemic postconditioning, in which they demonstrate that brief intermittent episodes of alternating myocardial ischemia and reperfusion, when applied at the onset of reperfusion after an episode of myocardial ischemia, result in a significant reduction in infarct size, in the canine heart model of ischemia–reperfusion. Subsequent studies have demonstrated that ischemic postconditioning also reduces other markers of reperfusion-induced injury, such as apoptotic cell death, endothelial dysfunction, oxidative stress production, and neutrophil accumulation (Zhao et al. 2003, Kin et al. 2004b). This protective phenomenon is essentially a form of modified reperfusion, which previous studies from the 1980s have established to be cardioprotective (Okamoto et al. 1986). Heusch (2004) recently described the phenomenon of postconditioning as ban old wine in a new bottle.Q Whatever the case, the concept of ischemic postconditioning has clearly elicited renewed interest in the reperfusion phase as a potential target for cardioprotection. In our recent studies (Tsang et al. 2004) we have examined the role of the
60 55 50 45 40 35 30 25 20 15 10 5 0
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Figure 3. Isolated perfused rat hearts were subjected to 35 min of regional ischemia followed by either 15 min of reperfusion (after which samples were taken from the region at risk for Western blot analysis) or 120 min of reperfusion (after which the infarct-risk ratio was determined by tetrazolium staining). Representative Western blots show that IPC results in the phosphorylation of Akt and Erk1/2 after 15 min of reperfusion. Either inhibiting Akt or Erk1/2 phosphorylation, with the known PI3K inhibitor, LY294002 (LY), or MEK1/2 inhibitor, PD98059 (PD), respectively, for the first 15 min of reperfusion abrogates the reduction in infarct-risk volume ratio induced by IPC. This suggests that IPC reduces infarct size by phosphorylating Akt and Erk1/2 at the time of reperfusion (n = 6 per group; *P b .05). Adapted with permission from Hausenloy et al. (2005).
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Control
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Figure 4. Isolated perfused rat hearts were subjected to 35 min of regional ischemia followed by either 15 min of reperfusion (after which samples were taken from the region at risk for Western blot analysis) or 120 min of reperfusion (after which the infarct-risk ratio was determined by tetrazolium staining). Representative Western blots show that ischemic postconditioning phosphorylates Akt and p70S6K at the time of reperfusion. Inhibiting Akt phosphorylation, with the use of the known PI3K inhibitor, LY294002 (LY), for the first 15 min of reperfusion, abrogates the reduction in infarct-risk volume ratio induced by ischemic postconditioning (Postcond). This suggests that ischemic postconditioning reduces infarct size by phosphorylating Akt and p70S6K at the time of reperfusion (n = 6 per group; *P b .05).
RISK pathway in the setting of ischemic postconditioning. We have demonstrated that, in the isolated perfused rat heart, ischemic postconditioning also protects the heart by activating the RISK pathway, at the time of reperfusion. We found that a standard ischemic postconditioning stimulus, comprising six 10-s episodes of alternating myocardial ischemia and reperfusion, results in the phosphorylation of the Akt component of the RISK pathway at the time of reperfusion (Tsang et al. 2004). Importantly, we show that pharmacologic inhibition of the ischemic postconditioning-induced phosphorylation of Akt, at the time of reperfusion, completely abrogates the postconditioning-induced limitation in infarct size, suggesting that the Akt component of the RISK pathway is essential for protection (see Figure 4) (Tsang et al. 2004). Subsequent studies by Downey’s group have demonstrated that inhibiting the Erk1/2 component of the RISK pathway at the time of reperfusion also abrogates the protection induced by ischemic postconditioning (Yang et al. 2004a, 2004b). These findings suggest that the Akt
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and Erk1/2 components of the RISK pathway are also required to mediate the protection induced by ischemic postconditioning.
The RISK Pathway: Potential Activating Mechanisms
The mechanism through which the cardioprotective maneuvers, IPC and ischemic postconditioning, actually induce the activation of the RISK pathway at the time of reperfusion is unclear (see Figure 5). With respect to IPC, the Akt and Erk1/2 components of the RISK pathway may be activated at the time of reperfusion via two possible mechanisms. (a) The initial activation of Akt and Erk1/2, which occurs in response to the IPC stimulus, before the index ischemia, may serve to prime the kinase pathways or else enable the cellular redistribution of these kinases, such that at the time of reperfusion the stimulus for kinase phosphorylation is potentiated in IPC-treated hearts. (b) There may be an intermediary factor, such as adenosine, ROS, or protein kinase C (PKC), which acts as the mediator between the IPC stimulus and the activation of the RISK pathway at the time of reperfusion (see Figure 5). With regard to ischemic postconditioning, the recent finding that the
Ischemic preconditioning
delayed washout of endogenously produced adenosine and the fact that adenosine receptor activation are required for protection (Kin et al. 2004a, Philipp et al. 2004) may provide the explanation through which ischemic postconditioning results in the activation of the RISK pathway, that is, the delayed washout of adenosine in the setting of ischemic postconditioning may recruit the RISK pathway at the time of reperfusion through the activation of G-proteincoupled receptors (see Figure 5).
The RISK Pathway: Potential Downstream Effectors
After an episode of sustained myocardial ischemia, any cardioprotective strategy applied at the time of reperfusion must provide protection against the known mediators of lethal reperfusion injury, which include cellular and mitochondrial calcium overload, a burst of oxidative stress, endothelial dysfunction, reduced nitric oxide (NO) production, and so on. The pharmacologic activation of the RISK pathway at the time of reperfusion, by growth factors or other agents, has been demonstrated to induce cardioprotection through the phosphorylation of various downstream effectors such as p70S6K (Jonassen et al. 2001), endothelial NO synthase (eNOS) (Bell and Yellon 2003a, 2003b), Bcl-2-associated death
Ischemic postconditioning
Pharmacological agents (eg statin, EPO, insulin)
Activation of The RISK pathway Preconditioning protocol
Ischemic Reperfusion phase period
Inhibition of mPTP opening at reperfusion
Cardio-Protection
Figure 5. Hypothetical scheme demonstrating that both IPC and ischemic postconditioning result in the activation of the RISK pathway at the time of reperfusion. A typical IPC protocol, followed by the index ischemic period and subsequent reperfusion phase, is depicted in the figure. Pharmacologic agents such as growth factors erythropoietin (EPO) and insulin as well as other agents such as statins may be given at the time of reperfusion to pharmacologically activate the RISK pathway and induce the same protective effects as IPC and postconditioning. Recruitment of the RISK pathway at the time of reperfusion results in cardioprotection through the inhibition of mPTP opening. We speculate that the RISK pathway inhibits mPTP opening via several mechanisms, including (1) the phosphorylation and inhibition of the pro-apoptotic factors BAD and Bcl-2-associated X protein (BAX); (2) the phosphorylation and activation of eNOS; (3) the phosphorylation and inhibition of GSK3h; and (4) the phosphorylation and mitochondrial translocation of PKC q. Potential mechanisms through which the RISK pathway may be activated by IPC and postconditioning include adenosine and PKC.
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promoter (BAD) (Jonassen et al. 2001), and GSK3h (Gross et al. 2004). In terms of the nonpharmacologic activation of the RISK pathway at the time of reperfusion (Figure 5), we have demonstrated that IPC induces the phosphorylation of p70S6K (Hausenloy et al. 2005), and that ischemic postconditioning induces the phosphorylation of p70S6K and eNOS (Tsang et al. 2004). Many of these protective signaling pathways appear to converge on the mitochondria, particularly the mPTP, which is believed to open during the first few minutes of reperfusion, in response to mitochondrial calcium overload, oxidative stress, and ATP depletion (Hausenloy and Yellon 2003). Our recent data suggest that the activation of the RISK pathway may actually confer its protection through the inhibition of mPTP opening (Davidson et al. 2004). The evidence in support of a central role for the mPTP in this scenario include (see Figure 5) (a) the fact that both IPC (Hausenloy et al. 2002, 2004) and ischemic postconditioning (Argaud et al. 2005) have been demonstrated to protect the heart through the inhibition of mPTP opening; (b) the recruitment of the RISK pathway induces many protective mechanisms which may be expected to act in concert to inhibit the opening of the mPTP. These include (a) the phosphorylation and inactivation of various pro-apoptotic factors such as BAD and Bcl-2-associated X protein (Tsuruta et al. 2002), which are believed to exert their apoptotic actions via the opening of the mPTP; (b) the phosphorylation and activation of eNOS, which would be expected to inhibit mPTP opening through its release of NO (Brookes et al. 2000); (c) the phosphorylation and inhibition of GSK3h, which has been demonstrated to mediate inhibition of mPTP opening (Juhaszova et al. 2004); (d) the Erk1/2 component of the RISK pathway has been demonstrated to form functional complexes with mitochondrial PKCq (Baines et al. 2002), the second of which has been demonstrated to confer cardioprotection through the inhibition of mPTP opening (Baines et al. 2003).
Recruiting the RISK–mPTP Pathway at Reperfusion: A Universal Cardioprotective Strategy
The Akt and Erk1/2 components of the RISK pathway together with the subseTCM Vol. 15, No. 2, 2005
quent inhibition of mPTP opening appear therefore to provide a common pathway for cardioprotection, and their powerful protective actions can be recruited with either pharmacologic agents or cardioprotective maneuvers such as IPC and ischemic postconditioning (see Figure 5). Importantly, this common pathway can be recruited at the time of reperfusion, which makes it an attractive strategy for cardioprotection in the clinical settings of reperfusion.
The RISK–mPTP Pathway: Clinical Implications
Recruiting the RISK pathway and inhibiting mPTP opening, in the clinical settings of reperfusion such as primary percutaneous coronary intervention and thrombolysis for an acute myocardial infarction, requires the presence of the intervention at the point of reperfusion. In this regard, previous clinical studies had suggested cardioprotection with GIK (glucose-insulin-potassium) therapy, an intervention which would be expected to recruit the RISK pathway, as a potential novel approach to cardioprotection (Fath-Ordoubadi and Beatt 1997). However, in a recent large clinical study, the administration of GIK therapy as an adjunct to current reperfusion strategies, to patients presenting with an acute MI, did not reduce mortality (Mehta et al. 2005). However, there was a substantial delay in the administration of GIK therapy (median of 4.7 h from onset of pain to randomization with a further 20% of patients having a delay of 8–12 h) which may account for this lack of cardioprotection. In addition, the lack of cardioprotection observed in this study may be due to the higher levels of glucose in the patient group treated with GIK therapy compared with the control group (Mehta et al. 2005). Other newer agents which have been demonstrated to activate components of the RISK pathway, such as the novel antidiabetic agent glucagon-like peptide-1 (Nikolaidis et al. 2004) and erythropoietin, may provide protection against ischemia–reperfusion injury in the clinical setting. A more novel approach would be to target several components of the cardioprotective pathway at reperfusion, by giving individual pharmacologic agents which separately inhibit mPTP
opening, upregulate the RISK pathway, or protect against the calcium overload or oxidative stress which accompanies reperfusion injury.
Conclusion
The Akt and Erk1/2 components of the RISK pathway appear to act as a point of convergence for the apparent diverse and unrelated cardioprotective phenomena of IPC and ischemic postconditioning. Importantly, the recruitment of the RISK pathway at the time of reperfusion exerts powerful protection against ischemic– reperfusion injury, through various cellular protective mechanisms, including the inhibition of mPTP opening, a known mediator of cell death. The RISK pathway therefore provides a novel target for protecting the heart against lethal reperfusion injury, in the clinical settings of reperfusion, which include thrombolysis, primary coronary artery angioplasty, and cardiac surgery. Components of the RISK pathway may be upregulated by administering pharmacologic agents at the time of reperfusion, thereby delivering the powerful protective benefits associated with IPC and postconditioning.
Acknowledgment
The authors would like to thank the British Heart Foundation for the support provided for the work mentioned in this review.
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Yang XM, Krieg T, Cui L, et al.: 2004a. NECA and bradykinin at reperfusion reduce infarction in rabbit hearts by signaling through PI3K, ERK, and NO. J Mol Cell Cardiol 36:411– 421.
Murry CE, Jennings RB, Reimer KA: 1986. Preconditioning with ischemia: a delay of
Yang XM, Proctor JB, Cui L, et al.: 2004b. Multiple, brief coronary occlusions during
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early reperfusion protect rabbit hearts by targeting cell signaling pathways. J Am Coll Cardiol 44:1103–1110. Yellon DM, Baxter GF: 1999. Reperfusion injury revisited: is there a role for growth factor signaling in limiting lethal reperfusion injury? Trends Cardiovasc Med 9:245–249.
Zhao ZQ, Corvera JS, Halkos ME, et al.: 2003. Inhibition of myocardial injury by ischemic postconditioning during reperfusion: comparison with ischemic preconditioning. Am J Physiol 285:H579–H588. PII S1050-1738(05)00025-3
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Regulation of Hematopoietic Stem Cells by the Niche Fumio Arai*, Atsushi Hirao, and Toshio Suda*
The quiescent state in the cell cycle is thought to be indispensable for the maintenance of hematopoietic stem cells (HSCs). Interaction of HSCs with their particular microenvironments, known as niches, is critical for maintaining the stem cell properties of HSCs, including cell adhesion, survival, and cell division. Hematopoietic stem cells balance quiescence and cell division in the stem cell niche and also maintain the potential for long-term hematopoiesis. We have recently reported that HSCs expressing the receptor tyrosine kinase Tie2 are in the G0 phase and antiapoptotic, and comprise a side-population (SP) of HSCs, which contacts osteoblasts (OBs), the source of the angiopoietin-1 (Ang-1) ligand for Tie2 in the bone marrow (BM) niche. Tie2/Ang-1 signaling occurs in interactions between HSCs and niche cells. The interaction of Tie2 with Ang-1 in vitro induces tight adhesion of HSCs to stromal cells and is sufficient to maintain the long-term blood-repopulating (LTR) activity of HSCs in vivo by preventing cell division. In addition, Ang-1 enhances the ability of HSCs to become quiescent and induces their adhesion to the bone surface in vivo, resulting in protection of the HSC compartment from stresses suppressing hematopoiesis. These data suggest that the Tie2/Ang-1 signaling pathway plays a critical role in the maintenance of HSCs in the adult BM niche. Ang-1 produced by OBs activates Tie2 on HSCs and promotes tight adhesion of HSCs to the niche, resulting in quiescence and enhanced survival of HSCs. (Trends Cardiovasc Med 2005;15:75–79) D 2005, Elsevier Inc.
Fumio Arai, Atsushi Hirao and Toshio Suda are at the Department of Cell Differentiation, The Sakaguchi Laboratory of Developmental Biology, School of Medicine, Keio University, Tokyo 160-8582, Japan. * Address Correspondence to: Fumio Arai, PhD, and Toshio Suda, MD, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. Tel.: (+81) 3-5363-3475; fax: (+81) 3-5363-3474; email: farai @sc.itc.keio.ac.jp. D 2005, Elsevier Inc. All rights reserved. 1050-1738/05/$-see front matter
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The fate of stem cells is controlled by a particular microenvironment known as the stem cell niche. The concept of the stem cell niche was first proposed for the human hematopoietic system in the 1970s (Schofield 1978). A similar concept has also been proposed for the epidermis, intestinal epithelium, nervous system, and gonads (Fuchs et al. 2004). A unique feature of hematopoietic stem cells (HSCs) is that they migrate
toward the stem cell niche during their ontogeny. Definitive hematopoiesis develops from the para-aortic splanchnopleural mesoderm (P-Sp) region as early as E7.5 (Cumano et al. 1996). By E10.5, HSCs originate in the aorta– gonad–mesonephros (AGM) region (Medvinsky and Dzierzak 1996). Hematopoietic stem cells from this region colonize the fetal liver (FL) and then move to the spleen and bone marrow (BM). Embryonic HSCs are closely associated with and often adhere to endothelial cells on the ventral surface of the aorta. In the adult BM, it was reported that a subpopulation of OB is a definitive regulatory component of the HSC niche (Calvi et al. 2003, Zhang et al. 2003). Zhang et al. (2003) showed that N-cadherin-positive spindle-shaped OBs are niche cells, and N-cadherin is asymmetrically localized between HSCs and OBs in the adult BM niche. An increase in the number of OBs correlates with an increase in the number of longterm repopulating (LTR) HSCs, suggesting that OBs are key components of the stem cell niche in vivo (Calvi et al. 2003, Zhang et al. 2003). We hypothesized that cell-cycle regulation by the niche is critical for the fate of HSCs.
Identification of the Quiescent Hematopoietic Stem Cell in Adult BM
A stem cell niche includes three compartments: localized niche cells (supporting cells), the extracellular matrix (ECM), and soluble factors derived from niche cells (Lin 2002). Key features of stem cells in a niche are that they are quiescent and adhere to surrounding cells. Indeed, it has been reported that HSCs are relatively quiescent when compared with transiently amplifying progenitor cells (Cheshier et al. 1999). To mark HSCs in the niche, we identified a marker for quiescent HSCs. To do so, we used the myelosuppressive model of treatment with 5-FU, a drug that induces apoptosis in cycling cells, and analyzed SP cells in HSCs. Side population is a cell fraction weakly labeled with the DNA dye Hoechst 33342 (Goodell et al. 1996). It is known that HSCs are enriched in side population. Moreover, SP cells have been found in hematopoietic tissues of mice, pigs, monkeys, and humans, as well as in nonhemato-
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Novel treatment approaches, as potential adjunctive therapy to current reperfusion strategies (such as thrombolysis, primary coronary angioplasty, and cardiac surgery), are required to provide further cardioprotection in the setting of an acute myocardial infarction to effect further reductions in morbidity and mortality. In this regard, the activation of prosurvival kinases, such as Akt and Erk1/2 (which we have termed the reperfusion injury salvage kinase [RISK] pathway), at the time of reperfusion, has been demonstrated to confer powerful cardioprotection against myocardial ischemia–reperfusion injury. In this review, we present evidence suggesting that the cardioprotective phenomena of ischemic preconditioning and the recently described ischemic postconditioning exert their cardioprotective effects through the recruitment of the RISK pathway, at the time of reperfusion, and that the protection in these settings is mediated through the inhibition of mitochondrial permeability transition pore (mPTP) opening at this time. Therefore, the pharmacologic manipulation of the RISK pathway at the time of reperfusion may enable one to harness the powerful cardioprotective benefits of both ischemic preconditioning and postconditioning, and provide a novel approach to cardioprotection. (Trends Cardiovasc Med 2005;15:69–75) D 2005, Elsevier Inc.
Protecting the Heart Against Ischemia–Reperfusion Injury
Coronary artery disease is currently the leading cause of mortality and morbidity in the western world. The serious and
Derek J. Hausenloy, Andrew Tsang, and Derek M. Yellon are at the Hatter Institute and Centre for Cardiology, University College London Hospital, WC1E 6DB London, UK. T Address correspondence to: Derek M. Yellon, PhD, DSc, Hon FRCP, The Hatter Institute and Centre for Cardiology, University College London Hospital, Grafton Way, WC1E 6DB London, UK. Tel.: (+ 44) 207-3809776; fax: (+ 44) 207-388-5095; e-mail: [email protected]. D 2005, Elsevier Inc. All rights reserved. 1050-1738/05/$-see front matter
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often fatal consequence of coronary artery disease is an acute myocardial infarction, which results from the acute occlusion of one of the major coronary arteries. In this scenario, the best hope of limiting the size of the myocardial infarct is the timely restoration of coronary blood flow, by either thrombolysis or primary percutaneous coronary artery angioplasty. Despite these current reperfusion strategies, the morbidity and mortality associated with an acute myocardial infarction remains significant, necessitating the development of novel cardioprotective strategies, which can be used as adjunctive therapy to the current reperfusion strategies. One of the most powerful mechanisms for protecting the myocardium before the
acute coronary artery occlusion occurs is to ischemically precondition the myocardium, a powerful cardioprotective phenomenon first described in 1986 by Murry et al. (1986) in which transient nonlethal episodes of myocardial ischemia confer protection against the myocardial infarction induced by a subsequent sustained episode of lethal myocardial ischemia. This approach however depends crucially on intervening before the ischemic event, which is difficult, given the unpredictable onset of an acute coronary artery occlusion. An alternative, more amenable, approach to cardioprotection is to protect the myocardium after the acute coronary artery occlusion has occurred. In this way, the cardioprotective strategy can be applied at the time of reperfusion to protect the myocardium against the myocyte death induced by the process of reperfusion itself—a phenomenon termed lethal reperfusion-induced injury (Braunwald and Kloner 1985). In this regard, our previous studies have implicated the prosurvival kinases, phosphatidylinositol-3-OH kinase (PI3K)-Akt and the p42/p44 extracellular signal-regulated kinases (Erk1/2), which we have termed the reperfusion injury salvage kinase (RISK) pathway as an important target for cardioprotection (Yellon and Baxter 1999, Hausenloy and Yellon 2004), at the time of reperfusion. We have demonstrated that the pharmacologic activation of the RISK pathway at the onset of reperfusion protects the heart against ischemia– reperfusion injury (Yellon and Baxter 1999, Hausenloy and Yellon 2004). Previous studies have demonstrated that adenovirally transfecting mural hearts (Fujio et al. 2000) or rat hearts (Miao et al. 2000) with constitutively active Akt gene constructs confers protection against ischemia–reperfusion injury. Interestingly, our recent data have demonstrated that the recruitment of this protective RISK pathway, at the time of reperfusion, contributes to the protection of both ischemic preconditioning (IPC) (Hausenloy et al. 2005) and the newly described phenomenon of ischemic postconditioning (Tsang et al. 2004). This suggests that the pharmacologic upregulation of the RISK pathway at the time of reperfusion may enable one to harness the cardioprotective benefits of both IPC and ischemic postconditioning.
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The RISK Pathway
In view of the studies demonstrating that apoptosis may contribute to the myocyte death induced by lethal reperfusion injury (Gottlieb et al. 1994, Freude et al. 2000), and the fact that the prosurvival kinases PI3K-Akt and Erk1/2 exert antiapoptotic protective effects, our laboratory (Yellon and Baxter 1999) first formulated and tested the hypothesis that the pharmacologic activation of these prosurvival kinases, at the time of reperfusion, protects the myocardium against lethal reperfusion-induced injury. We and others have demonstrated that the activation of these kinase cascades (the RISK pathway), with various growth factors including insulin (Jonassen et al. 2001) and urocortin (Schulman et al. 2002), and other agents such as atorvastatin (Bell and Yellon 2003a), bradykinin (Bell and Yellon 2003b, Yang et al. 2004a, 2004b), and opioid receptor agonists (Gross et al. 2004), induces cardioprotection when given during the early reperfusion phase (Yellon and Baxter 1999, Hausenloy and Yellon 2004). Despite their initial premise as mediators of anti-apoptotic protection, studies have revealed that the pharmacologic activation of the RISK pathway at reperfusion protects against both apoptotic and necrotic cell death, as evidenced by a reduction in infarct size (Yellon and Baxter 1999, Hausenloy and Yellon 2004).
The Role of Akt and Erk1/2 in IPC: At the Time of Reperfusion
The aforementioned studies implicate PI3K-Akt and Erk1/2 as potential signaling components of the IPC signal. In contrast, we were interested in investigating the role of PI3K-Akt and Erk1/2, and the potential protection their activation may provide at the time of reperfusion, in the setting of IPC. Our recent work on IPC has focused on the reperfusion phase, which follows the index ischemic period, and has revealed that an IPC stimulus applied before the index ischemic event is able to protect the heart by modifying crucial events which occur during the postischemic reperfusion phase, such as the opening of the mPTP, which is a critical determinant of cell death (Hausenloy and Yellon 2003). Therefore, IPC appears to protect the heart by inhibiting the opening of the mPTP at the time of reperfusion (Hausenloy et al. 2002, 2004). Along the same train of thought, we have been investigating whether IPC may protect the heart by activating the RISK pathway at the time of reperfusion. In this regard, we have recently demonstrated, with the use of the isolated perfused rat heart, that a standard IPC stimulus results in the phosphory-
The Role of Akt and Erk1/2 in IPC: Before the Index Ischemic Period
The initial studies investigating the role of these prosurvival kinases, PI3K-Akt and Erk1/2, in the setting of IPC, did not examine these kinases in the context of the RISK pathway per se, that is, their protective role at the time of reperfusion was not examined. Instead, these initial studies investigated the role of these kinases before the index ischemic period, as potential signaling components, conveying the preconditioning signal from the cell membrane to the inner components of the cell such as the mitochondria. Tong et al. (2000) were the first to demonstrate a role for the PI3K-Akt pathway as a potential signaling component of the IPC signaling pathway. With the use of the isolated perfused rat heart, they demonstrated that a standard IPC stimulus induced the phosphorylation of
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both Akt and its downstream target GSK3h, immediately before the index ischemic period, and that the phosphorylation of these kinases was essential for IPC-induced protection (Tong et al. 2000, 2002). Downey’s laboratory (Oldenburg et al. 2004, Krieg et al. 2004a, 2004b) has subsequently elucidated the IPC signaling pathway from the cell membrane to the mitochondria via several different components including the PI3K-Akt pathway (see Figure 1). Whether Erk1/2 also acts as a potential signaling component in the setting of IPC is unclear, with some studies demonstrating that a standard IPC stimulus results in its phosphorylation before the index ischemic period (Fryer et al. 2001, Mocanu et al. 2002), although only some of these studies have actually demonstrated Erk1/2 phosphorylation contributing to IPC-induced protection (Fryer et al. 2001). Furthermore, other studies reported no change in Erk1/2 phosphorylation in response to an IPC stimulus (Behrends et al. 2000). Recent studies suggest that the mitochondrial reactive oxygen species (ROS) release induced by preconditioning may activate Erk1/2 (Samavati et al. 2002), thereby placing Erk1/2 downstream of the mitochondria in the scheme depicted in Figure 1.
Figure 1. The role of Akt and Erk1/2 as potential signaling components during the preconditioning phase. Activation of the G-protein-coupled receptor (GPCR), in response to an IPC stimulus, leads to the trans-activation of the epidermal growth factor receptor (EGRF) via the matrix metalloproteinase (MP), which then activates the PI3K-Akt pathway in a Srckinase-dependent manner. Signaling through the PI3K-Akt pathway results in the phosphorylation and activation of eNOS which then activates guanylate cyclase (GC) via NO. Guanylate cyclase then activates protein kinase G (PKG) via cyclic guanine-5-monophosphate (cGMP). PKG then phosphorylates and opens the mitochondrial KATP channel (mKATP), which results in the mitochondrial release of ROS, generated from the electron transport chain (ETC), which then activates the downstream mediators of preconditioning including Erk1/2. Adapted with permission from a scheme by Downey’s laboratory (Krieg et al. 2002).
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(A)
After IPC/ 15 min stabilization ischemia
35 min 15 min ischemia reperfusion
The RISK Pathway in Ischemic Postconditioning
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Figure 2. Representative Western blots showing that IPC results in a biphasic response in the phosphorylation of both (A) Akt and (B) Erk1/2, with an initial phase of phosphorylation, occurring before the index ischemic period, and a second phase of phosphorylation occurring at the time of reperfusion (n = 6 per group; *P b .05). Adapted with permission from Hausenloy et al. (2005).
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the authors found that delaying the administration of the PI3K inhibitor by 15 min into reperfusion also abrogated IPC-induced protection, suggesting the requirement for Akt phosphorylation for the first 30 min of reperfusion (Solenkova et al. 2005). Control
IPC
Akt phosphorylation at reperfusion Erk1/2 phosphorylation at reperfusion Infarct-risk volume ratio (%)
lation of both the Akt and Erk1/2 components of the RISK pathway at the time of reperfusion (Hausenloy et al. 2005). Interestingly, the IPC stimulus appears to induce a biphasic response in kinase phosphorylation, demonstrating initial kinase phosphorylation before the index ischemic period, and a second phase of kinase phosphorylation at the time of reperfusion (Figure 2) (Hausenloy et al. 2005). Importantly, we show that pharmacologically inhibiting the IPC-induced phosphorylation of these kinases, at the time of reperfusion, completely abrogates the IPC-induced limitation in infarct size, suggesting that both the Akt and Erk1/2 components of the RISK pathway are essential for IPC to manifest its protection (see Figure 3) (Hausenloy et al. 2005). With the use of the isolated rabbit heart model of ischemia–reperfusion injury, Downey’s laboratory has recently demonstrated that the presence of wortmannin (a PI3K inhibitor) during the reperfusion phase is also able to abrogate IPC-induced protection, supporting a role for Akt phosphorylation during the reperfusion phase in mediating protection in this setting (Solenkova et al. 2005). Interestingly, in that study
Vinten-Johansen and colleagues (Zhao et al. 2003) have recently described an apparently new cardioprotective phenomenon, which they have termed ischemic postconditioning, in which they demonstrate that brief intermittent episodes of alternating myocardial ischemia and reperfusion, when applied at the onset of reperfusion after an episode of myocardial ischemia, result in a significant reduction in infarct size, in the canine heart model of ischemia–reperfusion. Subsequent studies have demonstrated that ischemic postconditioning also reduces other markers of reperfusion-induced injury, such as apoptotic cell death, endothelial dysfunction, oxidative stress production, and neutrophil accumulation (Zhao et al. 2003, Kin et al. 2004b). This protective phenomenon is essentially a form of modified reperfusion, which previous studies from the 1980s have established to be cardioprotective (Okamoto et al. 1986). Heusch (2004) recently described the phenomenon of postconditioning as ban old wine in a new bottle.Q Whatever the case, the concept of ischemic postconditioning has clearly elicited renewed interest in the reperfusion phase as a potential target for cardioprotection. In our recent studies (Tsang et al. 2004) we have examined the role of the
60 55 50 45 40 35 30 25 20 15 10 5 0
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IPC+PD IPC+LY at reperfusion at reperfusion
Figure 3. Isolated perfused rat hearts were subjected to 35 min of regional ischemia followed by either 15 min of reperfusion (after which samples were taken from the region at risk for Western blot analysis) or 120 min of reperfusion (after which the infarct-risk ratio was determined by tetrazolium staining). Representative Western blots show that IPC results in the phosphorylation of Akt and Erk1/2 after 15 min of reperfusion. Either inhibiting Akt or Erk1/2 phosphorylation, with the known PI3K inhibitor, LY294002 (LY), or MEK1/2 inhibitor, PD98059 (PD), respectively, for the first 15 min of reperfusion abrogates the reduction in infarct-risk volume ratio induced by IPC. This suggests that IPC reduces infarct size by phosphorylating Akt and Erk1/2 at the time of reperfusion (n = 6 per group; *P b .05). Adapted with permission from Hausenloy et al. (2005).
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Control
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Figure 4. Isolated perfused rat hearts were subjected to 35 min of regional ischemia followed by either 15 min of reperfusion (after which samples were taken from the region at risk for Western blot analysis) or 120 min of reperfusion (after which the infarct-risk ratio was determined by tetrazolium staining). Representative Western blots show that ischemic postconditioning phosphorylates Akt and p70S6K at the time of reperfusion. Inhibiting Akt phosphorylation, with the use of the known PI3K inhibitor, LY294002 (LY), for the first 15 min of reperfusion, abrogates the reduction in infarct-risk volume ratio induced by ischemic postconditioning (Postcond). This suggests that ischemic postconditioning reduces infarct size by phosphorylating Akt and p70S6K at the time of reperfusion (n = 6 per group; *P b .05).
RISK pathway in the setting of ischemic postconditioning. We have demonstrated that, in the isolated perfused rat heart, ischemic postconditioning also protects the heart by activating the RISK pathway, at the time of reperfusion. We found that a standard ischemic postconditioning stimulus, comprising six 10-s episodes of alternating myocardial ischemia and reperfusion, results in the phosphorylation of the Akt component of the RISK pathway at the time of reperfusion (Tsang et al. 2004). Importantly, we show that pharmacologic inhibition of the ischemic postconditioning-induced phosphorylation of Akt, at the time of reperfusion, completely abrogates the postconditioning-induced limitation in infarct size, suggesting that the Akt component of the RISK pathway is essential for protection (see Figure 4) (Tsang et al. 2004). Subsequent studies by Downey’s group have demonstrated that inhibiting the Erk1/2 component of the RISK pathway at the time of reperfusion also abrogates the protection induced by ischemic postconditioning (Yang et al. 2004a, 2004b). These findings suggest that the Akt
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and Erk1/2 components of the RISK pathway are also required to mediate the protection induced by ischemic postconditioning.
The RISK Pathway: Potential Activating Mechanisms
The mechanism through which the cardioprotective maneuvers, IPC and ischemic postconditioning, actually induce the activation of the RISK pathway at the time of reperfusion is unclear (see Figure 5). With respect to IPC, the Akt and Erk1/2 components of the RISK pathway may be activated at the time of reperfusion via two possible mechanisms. (a) The initial activation of Akt and Erk1/2, which occurs in response to the IPC stimulus, before the index ischemia, may serve to prime the kinase pathways or else enable the cellular redistribution of these kinases, such that at the time of reperfusion the stimulus for kinase phosphorylation is potentiated in IPC-treated hearts. (b) There may be an intermediary factor, such as adenosine, ROS, or protein kinase C (PKC), which acts as the mediator between the IPC stimulus and the activation of the RISK pathway at the time of reperfusion (see Figure 5). With regard to ischemic postconditioning, the recent finding that the
Ischemic preconditioning
delayed washout of endogenously produced adenosine and the fact that adenosine receptor activation are required for protection (Kin et al. 2004a, Philipp et al. 2004) may provide the explanation through which ischemic postconditioning results in the activation of the RISK pathway, that is, the delayed washout of adenosine in the setting of ischemic postconditioning may recruit the RISK pathway at the time of reperfusion through the activation of G-proteincoupled receptors (see Figure 5).
The RISK Pathway: Potential Downstream Effectors
After an episode of sustained myocardial ischemia, any cardioprotective strategy applied at the time of reperfusion must provide protection against the known mediators of lethal reperfusion injury, which include cellular and mitochondrial calcium overload, a burst of oxidative stress, endothelial dysfunction, reduced nitric oxide (NO) production, and so on. The pharmacologic activation of the RISK pathway at the time of reperfusion, by growth factors or other agents, has been demonstrated to induce cardioprotection through the phosphorylation of various downstream effectors such as p70S6K (Jonassen et al. 2001), endothelial NO synthase (eNOS) (Bell and Yellon 2003a, 2003b), Bcl-2-associated death
Ischemic postconditioning
Pharmacological agents (eg statin, EPO, insulin)
Activation of The RISK pathway Preconditioning protocol
Ischemic Reperfusion phase period
Inhibition of mPTP opening at reperfusion
Cardio-Protection
Figure 5. Hypothetical scheme demonstrating that both IPC and ischemic postconditioning result in the activation of the RISK pathway at the time of reperfusion. A typical IPC protocol, followed by the index ischemic period and subsequent reperfusion phase, is depicted in the figure. Pharmacologic agents such as growth factors erythropoietin (EPO) and insulin as well as other agents such as statins may be given at the time of reperfusion to pharmacologically activate the RISK pathway and induce the same protective effects as IPC and postconditioning. Recruitment of the RISK pathway at the time of reperfusion results in cardioprotection through the inhibition of mPTP opening. We speculate that the RISK pathway inhibits mPTP opening via several mechanisms, including (1) the phosphorylation and inhibition of the pro-apoptotic factors BAD and Bcl-2-associated X protein (BAX); (2) the phosphorylation and activation of eNOS; (3) the phosphorylation and inhibition of GSK3h; and (4) the phosphorylation and mitochondrial translocation of PKC q. Potential mechanisms through which the RISK pathway may be activated by IPC and postconditioning include adenosine and PKC.
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promoter (BAD) (Jonassen et al. 2001), and GSK3h (Gross et al. 2004). In terms of the nonpharmacologic activation of the RISK pathway at the time of reperfusion (Figure 5), we have demonstrated that IPC induces the phosphorylation of p70S6K (Hausenloy et al. 2005), and that ischemic postconditioning induces the phosphorylation of p70S6K and eNOS (Tsang et al. 2004). Many of these protective signaling pathways appear to converge on the mitochondria, particularly the mPTP, which is believed to open during the first few minutes of reperfusion, in response to mitochondrial calcium overload, oxidative stress, and ATP depletion (Hausenloy and Yellon 2003). Our recent data suggest that the activation of the RISK pathway may actually confer its protection through the inhibition of mPTP opening (Davidson et al. 2004). The evidence in support of a central role for the mPTP in this scenario include (see Figure 5) (a) the fact that both IPC (Hausenloy et al. 2002, 2004) and ischemic postconditioning (Argaud et al. 2005) have been demonstrated to protect the heart through the inhibition of mPTP opening; (b) the recruitment of the RISK pathway induces many protective mechanisms which may be expected to act in concert to inhibit the opening of the mPTP. These include (a) the phosphorylation and inactivation of various pro-apoptotic factors such as BAD and Bcl-2-associated X protein (Tsuruta et al. 2002), which are believed to exert their apoptotic actions via the opening of the mPTP; (b) the phosphorylation and activation of eNOS, which would be expected to inhibit mPTP opening through its release of NO (Brookes et al. 2000); (c) the phosphorylation and inhibition of GSK3h, which has been demonstrated to mediate inhibition of mPTP opening (Juhaszova et al. 2004); (d) the Erk1/2 component of the RISK pathway has been demonstrated to form functional complexes with mitochondrial PKCq (Baines et al. 2002), the second of which has been demonstrated to confer cardioprotection through the inhibition of mPTP opening (Baines et al. 2003).
Recruiting the RISK–mPTP Pathway at Reperfusion: A Universal Cardioprotective Strategy
The Akt and Erk1/2 components of the RISK pathway together with the subseTCM Vol. 15, No. 2, 2005
quent inhibition of mPTP opening appear therefore to provide a common pathway for cardioprotection, and their powerful protective actions can be recruited with either pharmacologic agents or cardioprotective maneuvers such as IPC and ischemic postconditioning (see Figure 5). Importantly, this common pathway can be recruited at the time of reperfusion, which makes it an attractive strategy for cardioprotection in the clinical settings of reperfusion.
The RISK–mPTP Pathway: Clinical Implications
Recruiting the RISK pathway and inhibiting mPTP opening, in the clinical settings of reperfusion such as primary percutaneous coronary intervention and thrombolysis for an acute myocardial infarction, requires the presence of the intervention at the point of reperfusion. In this regard, previous clinical studies had suggested cardioprotection with GIK (glucose-insulin-potassium) therapy, an intervention which would be expected to recruit the RISK pathway, as a potential novel approach to cardioprotection (Fath-Ordoubadi and Beatt 1997). However, in a recent large clinical study, the administration of GIK therapy as an adjunct to current reperfusion strategies, to patients presenting with an acute MI, did not reduce mortality (Mehta et al. 2005). However, there was a substantial delay in the administration of GIK therapy (median of 4.7 h from onset of pain to randomization with a further 20% of patients having a delay of 8–12 h) which may account for this lack of cardioprotection. In addition, the lack of cardioprotection observed in this study may be due to the higher levels of glucose in the patient group treated with GIK therapy compared with the control group (Mehta et al. 2005). Other newer agents which have been demonstrated to activate components of the RISK pathway, such as the novel antidiabetic agent glucagon-like peptide-1 (Nikolaidis et al. 2004) and erythropoietin, may provide protection against ischemia–reperfusion injury in the clinical setting. A more novel approach would be to target several components of the cardioprotective pathway at reperfusion, by giving individual pharmacologic agents which separately inhibit mPTP
opening, upregulate the RISK pathway, or protect against the calcium overload or oxidative stress which accompanies reperfusion injury.
Conclusion
The Akt and Erk1/2 components of the RISK pathway appear to act as a point of convergence for the apparent diverse and unrelated cardioprotective phenomena of IPC and ischemic postconditioning. Importantly, the recruitment of the RISK pathway at the time of reperfusion exerts powerful protection against ischemic– reperfusion injury, through various cellular protective mechanisms, including the inhibition of mPTP opening, a known mediator of cell death. The RISK pathway therefore provides a novel target for protecting the heart against lethal reperfusion injury, in the clinical settings of reperfusion, which include thrombolysis, primary coronary artery angioplasty, and cardiac surgery. Components of the RISK pathway may be upregulated by administering pharmacologic agents at the time of reperfusion, thereby delivering the powerful protective benefits associated with IPC and postconditioning.
Acknowledgment
The authors would like to thank the British Heart Foundation for the support provided for the work mentioned in this review.
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Zhao ZQ, Corvera JS, Halkos ME, et al.: 2003. Inhibition of myocardial injury by ischemic postconditioning during reperfusion: comparison with ischemic preconditioning. Am J Physiol 285:H579–H588. PII S1050-1738(05)00025-3
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Regulation of Hematopoietic Stem Cells by the Niche Fumio Arai*, Atsushi Hirao, and Toshio Suda*
The quiescent state in the cell cycle is thought to be indispensable for the maintenance of hematopoietic stem cells (HSCs). Interaction of HSCs with their particular microenvironments, known as niches, is critical for maintaining the stem cell properties of HSCs, including cell adhesion, survival, and cell division. Hematopoietic stem cells balance quiescence and cell division in the stem cell niche and also maintain the potential for long-term hematopoiesis. We have recently reported that HSCs expressing the receptor tyrosine kinase Tie2 are in the G0 phase and antiapoptotic, and comprise a side-population (SP) of HSCs, which contacts osteoblasts (OBs), the source of the angiopoietin-1 (Ang-1) ligand for Tie2 in the bone marrow (BM) niche. Tie2/Ang-1 signaling occurs in interactions between HSCs and niche cells. The interaction of Tie2 with Ang-1 in vitro induces tight adhesion of HSCs to stromal cells and is sufficient to maintain the long-term blood-repopulating (LTR) activity of HSCs in vivo by preventing cell division. In addition, Ang-1 enhances the ability of HSCs to become quiescent and induces their adhesion to the bone surface in vivo, resulting in protection of the HSC compartment from stresses suppressing hematopoiesis. These data suggest that the Tie2/Ang-1 signaling pathway plays a critical role in the maintenance of HSCs in the adult BM niche. Ang-1 produced by OBs activates Tie2 on HSCs and promotes tight adhesion of HSCs to the niche, resulting in quiescence and enhanced survival of HSCs. (Trends Cardiovasc Med 2005;15:75–79) D 2005, Elsevier Inc.
Fumio Arai, Atsushi Hirao and Toshio Suda are at the Department of Cell Differentiation, The Sakaguchi Laboratory of Developmental Biology, School of Medicine, Keio University, Tokyo 160-8582, Japan. * Address Correspondence to: Fumio Arai, PhD, and Toshio Suda, MD, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. Tel.: (+81) 3-5363-3475; fax: (+81) 3-5363-3474; email: farai @sc.itc.keio.ac.jp. D 2005, Elsevier Inc. All rights reserved. 1050-1738/05/$-see front matter
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The fate of stem cells is controlled by a particular microenvironment known as the stem cell niche. The concept of the stem cell niche was first proposed for the human hematopoietic system in the 1970s (Schofield 1978). A similar concept has also been proposed for the epidermis, intestinal epithelium, nervous system, and gonads (Fuchs et al. 2004). A unique feature of hematopoietic stem cells (HSCs) is that they migrate
toward the stem cell niche during their ontogeny. Definitive hematopoiesis develops from the para-aortic splanchnopleural mesoderm (P-Sp) region as early as E7.5 (Cumano et al. 1996). By E10.5, HSCs originate in the aorta– gonad–mesonephros (AGM) region (Medvinsky and Dzierzak 1996). Hematopoietic stem cells from this region colonize the fetal liver (FL) and then move to the spleen and bone marrow (BM). Embryonic HSCs are closely associated with and often adhere to endothelial cells on the ventral surface of the aorta. In the adult BM, it was reported that a subpopulation of OB is a definitive regulatory component of the HSC niche (Calvi et al. 2003, Zhang et al. 2003). Zhang et al. (2003) showed that N-cadherin-positive spindle-shaped OBs are niche cells, and N-cadherin is asymmetrically localized between HSCs and OBs in the adult BM niche. An increase in the number of OBs correlates with an increase in the number of longterm repopulating (LTR) HSCs, suggesting that OBs are key components of the stem cell niche in vivo (Calvi et al. 2003, Zhang et al. 2003). We hypothesized that cell-cycle regulation by the niche is critical for the fate of HSCs.
Identification of the Quiescent Hematopoietic Stem Cell in Adult BM
A stem cell niche includes three compartments: localized niche cells (supporting cells), the extracellular matrix (ECM), and soluble factors derived from niche cells (Lin 2002). Key features of stem cells in a niche are that they are quiescent and adhere to surrounding cells. Indeed, it has been reported that HSCs are relatively quiescent when compared with transiently amplifying progenitor cells (Cheshier et al. 1999). To mark HSCs in the niche, we identified a marker for quiescent HSCs. To do so, we used the myelosuppressive model of treatment with 5-FU, a drug that induces apoptosis in cycling cells, and analyzed SP cells in HSCs. Side population is a cell fraction weakly labeled with the DNA dye Hoechst 33342 (Goodell et al. 1996). It is known that HSCs are enriched in side population. Moreover, SP cells have been found in hematopoietic tissues of mice, pigs, monkeys, and humans, as well as in nonhemato-
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