#

Granulocyte CD18 expression

After surgery 24 hours 7 days

control reconstruction

Reconstruction + postcond

PLASMA MYELOPEROXIDASE (MPO) CONCENTRATION

Fig. 6. Changes in plasma myeloperoxidase following operation (# p<0.05 vs before surgery;

EXPRESSION OF GRANULOCYTE ADHESION MOLECULES

Before surgery After surgery 24 hours 7 days

AU

500

100

control Before surgery

200

300

400

Fig. 7. The graphs show the changes in expression of granulocyte adhesion during the examined perioperative period. (AU= arbitary unit) (# p<0.05 vs before surgery; \* p<0.05 vs

7 days

\* p<0.05 vs non-conditioned group)

AU

200

400

600

800

1000

1200

1400

1600

control

Granulocyte CD11a expression

#

#

\*

#

After surgery 24 hours

0,2

0,4

0,6

0,8

1,0

1,2

nmol/ml

non-conditioned group)

kontroll Before surgery In the last 3 years the literature of ischaemic postconditioning exponentially increased in the experimental cardiology. The beneficial effects of the manoeuver has been confirmed in various models, including human results as well, and the cellular and biochemical background is intensively examined. Until now this is the first study to evaluate the effect of ischaemic postconditioning on peripheral tissues in abdominal aortic surgery.

Our results demonstrated that after a prolonged ischaemia, postconditioning can reduce free radical production, TNF-alpha expression and leukocyte activation in the early phase of reperfusion in an animal model of abdominal aortic surgery. In this model we also confirmed that PostC could induce antiapoptotic signaling pathways in the skeletal muscle and in far organs as well.

In our human model we could confirm that ischaemic PostC could decrease in some points the revascularization surgery evoked oxidative stress and inflammatory response.

We have demonstrated that the protective effect of postconditioning is a complex process, involving many cell types, the generation of oxidants, cytokines, and inflammatory pathways, has not only one target, but acts on a diverse site. This complexity, the powerful protective effect and the simplicity in the surgery (lasts for a few minutes) can make the manoeuver really a powerful tool of surgeons.

#### **22. Mechanisms involved in postconditioning**

The mechanisms of protection by PostC were initially attributed mainly to improved endothelial function and to the events reducing the detrimental effects of lethal reperfusion injury, such as reduced edema, reduced oxidative stress, reduced mitochondrial calcium accumulation, reduced endothelium damage and reduced inflammation. However, subsequent studies suggest that protection is mediated through the recruitment of signal transduction pathways as in the case of ischaemic preconditioning. Therefore, a distinction in passive and active mechanisms can be proposed. Of course an intricate cross-talk among these events/mechanisms exists, thus this distinction can be useful for a better understanding of the phenomenon, but we must not forget that a single event/mechanism may not be effective if it occurs alone. (*Figure 8*)

#### **23. Passive mechanisms**

Among passive mechanisms we can consider those strictly related with hydrostatic force – hereafter named as 'Mechanical mechanisms' – and those related with reduced endothelial adhesion of leucocytes and subsequent reduction of inflammatory process that we call 'Cellular mechanisms'.

Ischaemic Postconditioning

also been observed52.

cardioprotection53.

**27. Triggers of postconditioning** 

protection remains to be elucidated.

**26. Active mechanisms (intracellular mechanisms)** 

Reduces Reperfusion Injury After Aortic Revascularization Surgery 251

proinflammatory genes. PostC delays the onset and reduces the maintenance of postischaemic inflammation47. As stated before, whether this is a cause or an effect of PostC

Studies have identified a signalling pathway that is recruited at the time of reperfusion and which is similar in ischaemic preconditioning and PostC. This pathway includes the survival kinases phosphatidylinositol 3-kinase (PI3K)-Akt and Erk1/2, the major components of the reperfusion-injury salvage kinase pathway, termed the RISK- pathway, which may influence the mPTP, a non-specific pore of the mitochondrial membrane whose opening in the first few minutes of myocardial reperfusion promotes cell death48. Delayed washout of endogenously produced adenosine and activation of the adenosine receptor also

Thus delayed washout of adenosine in the setting of PostC might recruit RISK at the time of reperfusion through the activation of adenosine responsive G-protein-coupled receptors. It seems that adenosine receptors are repopulated during PostC manoeuvres. While in murine hearts adenosine A2a and A3 subtypes 50 have been seen to be involved, in rabbit hearts PostC seems to depend on A2b subtype 51. An important role of the redox environment has

Therefore, similar to preconditioning, PostC has been proposed to be triggered by receptor stimulation, mediated by one or more complex and interrelated signal transduction pathways, and, ultimately, achieved via phosphorylation of one or more end-effectors of

Ligands, such as adenosine 54 and bradykinine 55 what accumulate during PostC manoeuvres may initiate the cascade that lead to PostC protection. It has been recently reported that inhibition of opioid receptors with opioid antagonists administered 5 min. before reperfusion in the absence or presence of PostC, reversed the infarct sparing effect of PostC in an in vivo rat model56. The activation of protein kinase C and G (PKC and PKG) and opening of mitochondrial KATP channels after PostC (see below) would be consistent with the involvement of BK and endogenous opioids. Nitric oxide and ROS may be included among the triggers. Nitric oxide is demonstrated to act both as a trigger and as a mediator of the preconditioning response in a variety of species. The role of endogenous NO in classic ischaemic preconditioning was controversial. Cohen and Downey's group suggested that exogenously administered NO could trigger the preconditioned state through a free radical-mediated process not shared by endogenous NO. Very recently these authors questioned whether their observation was due to a bias in the experimental model. These authors are now on the opinion that endogenous NO participates in triggering in vivo preconditioning57. Among the autocaids released by the ischaemic heart there is BK that may induce nitric oxide release (*Figure 3*). It has been suggested that the mechanism whereby NO protects myocardium includes the activation of guanylate-cyclase58. As an inducer of the protection, nitric oxide may also directly open the mitochondrial KATP

seems to be required for PostC protection49, by activating the survival pathway.

Fig. 8. A schematic figure on the mechanisms of ischaemic postconditioning.

#### **24. Mechanical mechanisms**

With regard to mechanical or haemodynamic mechanisms, it has been suggested that the stuttering of reperfusion and pressure during PostC manoeuvres may limit the hydrostatic forces in a very important moment, thus limiting early edema and consequent damages. In experiments performed in isolated heart models, the effect of the PostC on the infarct area has been studied perfusing the hearts either with constant pressure or with constant flow. It has been compared the role of these two types in perfusion in affecting the infarct area during PostC. In the constant pressure model the infarct area was less reduced by PostC than it was with the model of the constant flow reperfusion after PostC46. Considering that during the short period of restoration of flow in the PostC manoeuvres the capillary pressure increases less in the constant flow model, than in the constant pressure model (i.e. at the beginning of reperfusion in the constant flow model there is smaller hydrostatic pressure and so smaller transcapillary pressure), it was argued that in the constant-flow model a reduced edema and consequent reduced damages may explain the increased effectiveness of PostC. In other words, in the constant flow model the effectiveness of PostC is greater than in the constant pressure model supports the idea that the reduction of hydrostatic forces during PostC manoeuvres may play an important role in determining the protective effects.

#### **25. Cellular mechanisms**

Among the cellular mechanisms we consider acute inflammatory response. It occurs through the release of cytokines, activation of vascular endothelial cells and leukocytes with expression of cell surface adhesion molecules, and up-regulation of a program of

NO.

**MECHANICAL CELLULAR MOLECULAR** 

**(-)**

Fig. 8. A schematic figure on the mechanisms of ischaemic postconditioning.

With regard to mechanical or haemodynamic mechanisms, it has been suggested that the stuttering of reperfusion and pressure during PostC manoeuvres may limit the hydrostatic forces in a very important moment, thus limiting early edema and consequent damages. In experiments performed in isolated heart models, the effect of the PostC on the infarct area has been studied perfusing the hearts either with constant pressure or with constant flow. It has been compared the role of these two types in perfusion in affecting the infarct area during PostC. In the constant pressure model the infarct area was less reduced by PostC than it was with the model of the constant flow reperfusion after PostC46. Considering that during the short period of restoration of flow in the PostC manoeuvres the capillary pressure increases less in the constant flow model, than in the constant pressure model (i.e. at the beginning of reperfusion in the constant flow model there is smaller hydrostatic pressure and so smaller transcapillary pressure), it was argued that in the constant-flow model a reduced edema and consequent reduced damages may explain the increased effectiveness of PostC. In other words, in the constant flow model the effectiveness of PostC is greater than in the constant pressure model supports the idea that the reduction of hydrostatic forces during PostC manoeuvres may play an important role in determining the

**CELLULAR PROTECTION** 

KATP mPTP **APOPTOSIS**↓

Ca2+ ↓

**(-)**

( . O2) <sup>↓</sup> IC

**(-)**

pH↑

Citokines

( . O2)

↑PI3 K ↑ P-Akt ↑ ERK 1/2 ↓ P38, JNK ↑ Bcl2/BAX **(-)**

**NECROSIS**↓

J.Vinten-Johansen et al. Basic Res Cardiol 2005

Among the cellular mechanisms we consider acute inflammatory response. It occurs through the release of cytokines, activation of vascular endothelial cells and leukocytes with expression of cell surface adhesion molecules, and up-regulation of a program of

**24. Mechanical mechanisms** 

**EDEMA CELLULAR SWELLING** 

**(-)** 

**H2O** 

endothelium

K+

MITOCHONDRIUM

adenosine

GPCR

**(-) (-)**

**PMN ( . O2)** 

( . O2)

protective effects.

**25. Cellular mechanisms** 

proinflammatory genes. PostC delays the onset and reduces the maintenance of postischaemic inflammation47. As stated before, whether this is a cause or an effect of PostC protection remains to be elucidated.

#### **26. Active mechanisms (intracellular mechanisms)**

Studies have identified a signalling pathway that is recruited at the time of reperfusion and which is similar in ischaemic preconditioning and PostC. This pathway includes the survival kinases phosphatidylinositol 3-kinase (PI3K)-Akt and Erk1/2, the major components of the reperfusion-injury salvage kinase pathway, termed the RISK- pathway, which may influence the mPTP, a non-specific pore of the mitochondrial membrane whose opening in the first few minutes of myocardial reperfusion promotes cell death48. Delayed washout of endogenously produced adenosine and activation of the adenosine receptor also seems to be required for PostC protection49, by activating the survival pathway.

Thus delayed washout of adenosine in the setting of PostC might recruit RISK at the time of reperfusion through the activation of adenosine responsive G-protein-coupled receptors. It seems that adenosine receptors are repopulated during PostC manoeuvres. While in murine hearts adenosine A2a and A3 subtypes 50 have been seen to be involved, in rabbit hearts PostC seems to depend on A2b subtype 51. An important role of the redox environment has also been observed52.

Therefore, similar to preconditioning, PostC has been proposed to be triggered by receptor stimulation, mediated by one or more complex and interrelated signal transduction pathways, and, ultimately, achieved via phosphorylation of one or more end-effectors of cardioprotection53.

#### **27. Triggers of postconditioning**

Ligands, such as adenosine 54 and bradykinine 55 what accumulate during PostC manoeuvres may initiate the cascade that lead to PostC protection. It has been recently reported that inhibition of opioid receptors with opioid antagonists administered 5 min. before reperfusion in the absence or presence of PostC, reversed the infarct sparing effect of PostC in an in vivo rat model56. The activation of protein kinase C and G (PKC and PKG) and opening of mitochondrial KATP channels after PostC (see below) would be consistent with the involvement of BK and endogenous opioids. Nitric oxide and ROS may be included among the triggers. Nitric oxide is demonstrated to act both as a trigger and as a mediator of the preconditioning response in a variety of species. The role of endogenous NO in classic ischaemic preconditioning was controversial. Cohen and Downey's group suggested that exogenously administered NO could trigger the preconditioned state through a free radical-mediated process not shared by endogenous NO. Very recently these authors questioned whether their observation was due to a bias in the experimental model. These authors are now on the opinion that endogenous NO participates in triggering in vivo preconditioning57. Among the autocaids released by the ischaemic heart there is BK that may induce nitric oxide release (*Figure 3*). It has been suggested that the mechanism whereby NO protects myocardium includes the activation of guanylate-cyclase58. As an inducer of the protection, nitric oxide may also directly open the mitochondrial KATP

Ischaemic Postconditioning

effect of PostC 81.

**28. Mediators of postconditioning** 

Reduces Reperfusion Injury After Aortic Revascularization Surgery 253

We considered ROS among triggers as they are necessary during PostC manoeuvres. Nevertheless, PostC activated the RISK pathway, with increased expression of the phosphorylated form of endothelial nitric oxide synthase (eNOS) as one of the results77. Thus it is likely that after NOS activation the cGMP is produced and PKG is activated; then mitochondrial ATP-dependent potassium (mKATP) channels are opened and ROS produced. Therefore cGMP, PKG, mKATP and ROS may be considered as mediators of PostC protection, which are likely to be upstream to PKC activation. We demonstrated that cGMP production is increased during reperfusion of postconditioned hearts78. Moreover we showed that in these hearts mKATP and PKC must also be active (i.e. they should not be blocked) during late reperfusion79. Regarding the role of mKATP channels a couple of papers indicate that the mKATP channel is important for PostC80. In these studies two different mKATP channel blockers (glibenclamide and 5-hydroxidecanoate) abolished the protective

It is interesting that many of the RISK elements (e.g. PI3K/Akt and MEK1/2-ERK) involved in the signaling pathway in preconditioning and protection against reperfusion injury have recently been documented also in PostC82. Some differences, however, may exist between pre- and PostC (see also Table 1 and Table 2). Darling et al. 83 showed an increase of phospho-ERK, but not of PI3K/Akt in PostC, while Yang et al.84 showed that ERK is involved in PostC, but not in preconditioning. These findings may explain a certain degree of additive protection between ischaemic preconditioning and PostC, as observed by Yang et al.85. Yet in contrast with Yang et al.86, Cao et al.87 reported that ERK is present in preconditioning trigger pathway. The reasons for the differences are not clear. Different species and/or protocols may play a role88. Different methods of tissue sampling also be may play a role89. Besides protein kinase C, the possible roles for tyrosine kinase, and members of the MAPK family other than ERK1/2 in PostC has been suggested90. (*Figure 9*) Focal disorganization of gap junction distribution and down-regulation of connexin 43 (Cx43) are typical features of myocardial remodelling 91 and Cx43 – especially Cx43 localized in mitochondria – has been indicated as one key element of the signal transduction cascade of the protection by preconditioning. However, Cx43 does not seem to be important for infarct size reduction by PostC 92. These results, together with the above reported differences on kinase activation by pre- and PostC, suggest a certain degree of differences

Mitochondrial PTPs opening represents a fundamental step of reperfusion injury. Among the potential mechanisms responsible for mPTP opening during reperfusion, Ca2+-overload has received particular attention. In particular, mitochondrial Ca2+-overload occurring during ischaemia must bring mitochondria closer to the threshold at which mPTP opening takes place, favouring the occurrence of mPTP opening during reperfusion, a phenomenon described as mitochondrial priming93. Additionally, reduced mitochondrial Ca2+ overload during ischaemia has been pointed out as a potentially important mechanism of ischaemic

between the protective pathways activated by these two procedures.

**29. End-effectors of postconditioning** 

and pharmacological preconditioning94.

channels59. Therefore, nitric oxide acting on mitochondria may play a relevant role in protection both through activation of these channels and via modulation of respiratory chain; both mechanisms favor ROS signalling, which can trigger protection60. A relevant role of nitric oxide may also be attributed to the endothelial protection brought by this molecule 61 or to its role as antioxidant under certain conditions 62.

The one-electron-reduction product of nitric oxide, HNO/NO– (nitrosyl hydride/nitroxyl anion), has been scarcely studied in an I/R scenario. In our laboratory low doses of Angeli's salt, a donor of HNO/NO–, have been seen to induce early/classical preconditioning against myocardial damages63. Intriguingly, the protective effects of HNO/NO– generated by Angeli's salt were more potent than the protective effects induced by equimolar concentration of the pure nitric oxide donor diethylamine/nitric oxide (DEA/NO). While the HNO/NO– donor seems deleterious in reperfusion64, there is evidence that NO may also be involved in the cardioprotection by ischaemic PostC. When the nitric oxide synthase (NOS) inhibitor N-omega-nitro-L-arginine methyl esther (LNAME) was given 5 min. before start of reperfusion of *in vivo* rabbit hearts, the infarct limiting effect was abolished65. We have shown that nitric oxide participates in PostC, but NOS inhibitors given for the entire reperfusion period only blunted the protective effect of PostC66 . Paradoxically, the same inhibitor, given only during PostC manoeuvres completely blocked the protective effects67. At the moment, we do not have an explanation for this apparent paradox. In a previous study, we argued that nitric oxide may be produced in post-conditioned heart both by NOS and by non-enzymatic mechanisms. Nitric oxide can then activate the guanyl cyclase to produce cyclic guanosine monophosphate (cGMP), which mediates protection 68 (see also below). The infusion of a NOS inhibitor only during PostC manoeuvres may alter the equilibrium between ROS and nitric oxide thus leading to the production of the wrong kind of radical which does not trigger the protective pathway. It can be argued that in the absence of this protection the stronger limitation of nitric oxide production by NOS may be protective during reperfusion. In fact, data have demonstrated that NOS inhibitors can attenuate I/R damage69. Also, the different doses of nitric oxide inhibitors applied and the different basal levels of nitric oxide endogenously produced may explain these disparities.

The beneficial and deleterious effects of nitric oxide and nitrite in pathophysiological conditions and contradictory results about the effects of nitric oxide during reperfusion have been reviewed by Bolli in 200170, Wink et al. in 200371, Pagliaro in 200372 and Schulz et al. in 2004 73. ROS could also be included among the triggers of PostC. In fact, ROS scavengers such as N-acetylcysteine and 2-mercapto-propionylglycine given during PostC manoeuvres prevent the protective effects74. It is possible that the low pH during the PostC cycles prevents mPTP opening, while the intermittent oxygen bursts allow mitochondria to make enough ROS in a moment in which other enzymes, able to produce massive quantity of ROS, are not yet re-activated. Then mitochondrial ROS may activate PKC and put the heart into a protected state. The importance of the role of acidosis in the triggering of PostC protection has been recently confirmed by two independent laboratories75. Acidosis may also prevent mPTP opening in the early reperfusion (see below). Recently, it has been reported that redox signaling and a low pH at the time of myocardial reperfusion are also required to mediate the cardioprotection triggered by ischaemic preconditioning76.

#### **28. Mediators of postconditioning**

252 Vascular Surgery

channels59. Therefore, nitric oxide acting on mitochondria may play a relevant role in protection both through activation of these channels and via modulation of respiratory chain; both mechanisms favor ROS signalling, which can trigger protection60. A relevant role of nitric oxide may also be attributed to the endothelial protection brought by this molecule

The one-electron-reduction product of nitric oxide, HNO/NO– (nitrosyl hydride/nitroxyl anion), has been scarcely studied in an I/R scenario. In our laboratory low doses of Angeli's salt, a donor of HNO/NO–, have been seen to induce early/classical preconditioning against myocardial damages63. Intriguingly, the protective effects of HNO/NO– generated by Angeli's salt were more potent than the protective effects induced by equimolar concentration of the pure nitric oxide donor diethylamine/nitric oxide (DEA/NO). While the HNO/NO– donor seems deleterious in reperfusion64, there is evidence that NO may also be involved in the cardioprotection by ischaemic PostC. When the nitric oxide synthase (NOS) inhibitor N-omega-nitro-L-arginine methyl esther (LNAME) was given 5 min. before start of reperfusion of *in vivo* rabbit hearts, the infarct limiting effect was abolished65. We have shown that nitric oxide participates in PostC, but NOS inhibitors given for the entire reperfusion period only blunted the protective effect of PostC66 . Paradoxically, the same inhibitor, given only during PostC manoeuvres completely blocked the protective effects67. At the moment, we do not have an explanation for this apparent paradox. In a previous study, we argued that nitric oxide may be produced in post-conditioned heart both by NOS and by non-enzymatic mechanisms. Nitric oxide can then activate the guanyl cyclase to produce cyclic guanosine monophosphate (cGMP), which mediates protection 68 (see also below). The infusion of a NOS inhibitor only during PostC manoeuvres may alter the equilibrium between ROS and nitric oxide thus leading to the production of the wrong kind of radical which does not trigger the protective pathway. It can be argued that in the absence of this protection the stronger limitation of nitric oxide production by NOS may be protective during reperfusion. In fact, data have demonstrated that NOS inhibitors can attenuate I/R damage69. Also, the different doses of nitric oxide inhibitors applied and the different

basal levels of nitric oxide endogenously produced may explain these disparities.

ischaemic preconditioning76.

The beneficial and deleterious effects of nitric oxide and nitrite in pathophysiological conditions and contradictory results about the effects of nitric oxide during reperfusion have been reviewed by Bolli in 200170, Wink et al. in 200371, Pagliaro in 200372 and Schulz et al. in 2004 73. ROS could also be included among the triggers of PostC. In fact, ROS scavengers such as N-acetylcysteine and 2-mercapto-propionylglycine given during PostC manoeuvres prevent the protective effects74. It is possible that the low pH during the PostC cycles prevents mPTP opening, while the intermittent oxygen bursts allow mitochondria to make enough ROS in a moment in which other enzymes, able to produce massive quantity of ROS, are not yet re-activated. Then mitochondrial ROS may activate PKC and put the heart into a protected state. The importance of the role of acidosis in the triggering of PostC protection has been recently confirmed by two independent laboratories75. Acidosis may also prevent mPTP opening in the early reperfusion (see below). Recently, it has been reported that redox signaling and a low pH at the time of myocardial reperfusion are also required to mediate the cardioprotection triggered by

61 or to its role as antioxidant under certain conditions 62.

We considered ROS among triggers as they are necessary during PostC manoeuvres. Nevertheless, PostC activated the RISK pathway, with increased expression of the phosphorylated form of endothelial nitric oxide synthase (eNOS) as one of the results77. Thus it is likely that after NOS activation the cGMP is produced and PKG is activated; then mitochondrial ATP-dependent potassium (mKATP) channels are opened and ROS produced. Therefore cGMP, PKG, mKATP and ROS may be considered as mediators of PostC protection, which are likely to be upstream to PKC activation. We demonstrated that cGMP production is increased during reperfusion of postconditioned hearts78. Moreover we showed that in these hearts mKATP and PKC must also be active (i.e. they should not be blocked) during late reperfusion79. Regarding the role of mKATP channels a couple of papers indicate that the mKATP channel is important for PostC80. In these studies two different mKATP channel blockers (glibenclamide and 5-hydroxidecanoate) abolished the protective effect of PostC 81.

It is interesting that many of the RISK elements (e.g. PI3K/Akt and MEK1/2-ERK) involved in the signaling pathway in preconditioning and protection against reperfusion injury have recently been documented also in PostC82. Some differences, however, may exist between pre- and PostC (see also Table 1 and Table 2). Darling et al. 83 showed an increase of phospho-ERK, but not of PI3K/Akt in PostC, while Yang et al.84 showed that ERK is involved in PostC, but not in preconditioning. These findings may explain a certain degree of additive protection between ischaemic preconditioning and PostC, as observed by Yang et al.85. Yet in contrast with Yang et al.86, Cao et al.87 reported that ERK is present in preconditioning trigger pathway. The reasons for the differences are not clear. Different species and/or protocols may play a role88. Different methods of tissue sampling also be may play a role89. Besides protein kinase C, the possible roles for tyrosine kinase, and members of the MAPK family other than ERK1/2 in PostC has been suggested90. (*Figure 9*)

Focal disorganization of gap junction distribution and down-regulation of connexin 43 (Cx43) are typical features of myocardial remodelling 91 and Cx43 – especially Cx43 localized in mitochondria – has been indicated as one key element of the signal transduction cascade of the protection by preconditioning. However, Cx43 does not seem to be important for infarct size reduction by PostC 92. These results, together with the above reported differences on kinase activation by pre- and PostC, suggest a certain degree of differences between the protective pathways activated by these two procedures.

#### **29. End-effectors of postconditioning**

Mitochondrial PTPs opening represents a fundamental step of reperfusion injury. Among the potential mechanisms responsible for mPTP opening during reperfusion, Ca2+-overload has received particular attention. In particular, mitochondrial Ca2+-overload occurring during ischaemia must bring mitochondria closer to the threshold at which mPTP opening takes place, favouring the occurrence of mPTP opening during reperfusion, a phenomenon described as mitochondrial priming93. Additionally, reduced mitochondrial Ca2+ overload during ischaemia has been pointed out as a potentially important mechanism of ischaemic and pharmacological preconditioning94.

Ischaemic Postconditioning

clarify this issue.

**31. Conclusion** 

Reduces Reperfusion Injury After Aortic Revascularization Surgery 255

It has been already established that preconditioning triggering, that is the period that precedes the index ischaemia, is redox sensitive. This was demonstrated by both avoiding preconditioning with ROS scavengers and inducing preconditioning with ROS generators given before the index ischaemia 99. Also, several metabolites, including acetylcholine, BK, opioids and phenylephrine, trigger preconditioning-like protection via a mKATP-ROS dependent mechanism100. As stated in the case of reperfusion injury, ROS are also implicated in the sequel of myocardial reperfusion injury101. These studies supported the paradigm that ROS may be protective in pre-ischaemic phase, but are deleterious in the post-ischaemic phase. Thus the main idea was that ROS play an essential, though double-edged, role in cardioprotection: they may participate reperfusion injury or may play a role as signaling elements of protection in pre-ischaemic phase102. The importance of ROS signalling (as opposed to excess ROS in the development of injury) has been examined closely in great detail in recent years103. Intriguingly, and in contrast to the above-described theory of ROS as an obligatory part of reperfusion induced damage, some studies suggest the possibility that some ROS species at low concentrations could protect ischaemic hearts104. Yet, from the above reported mechanisms of PostC, it appears that also ischaemic PostC is a cardioprotective phenomenon that requires the intervention of redox signaling to be protective105. Moreover, as mentioned, very recently it has been shown that redox signalling is also required at the time of myocardial reperfusion to mediate the cardioprotection elicited by ischaemic preconditioning106. Therefore, the role of ROS in reperfusion may be reconsidered as they are not only deleterious. This fact may help to understand the variability in the results of studies aimed at proving a role of ROS in reperfusion injury. For instance, negative results came from trials in which free radical scavengers such as recombinant human superoxide dismutase or vitamin E were administered to patients with coronary artery disease or risk factors for cardiovascular events107. In addition to the dual role of ROS (beneficial versus deleterious), among the reasons why these scavengers did not show any consistent benefit in these human studies may be: (1) the type of ROS generated (e.g. superoxide dismutase only removes the superoxide and not the hydroxyl radical); (2) the site of ROS generation (e.g. most scavengers scarcely enter into the cells) and (3) the rate of reaction between two ROS and/or scavengers. The importance of the rate of reaction can be understood if we consider that, despite a five times lower concentration of nitric oxide with respect to superoxide dismutase, 50% or more of the available superoxide will react with nitric oxide to form ONOO– instead of reacting with superoxide dismutase108. Notwithstanding the evidence of a protective role of ROS signalling in reperfusion, we were unable to reproduce cardioprotection with ROS generation by purine/xanthine oxidase given at reperfusion109. Since ROS scavengers (N-acetyl-L-cysteine or 2-mercapto-propionylglycine), given at the beginning of reperfusion, abolished both IP- and PostC-induced protection110 it is likely that the type, the concentration and/or the compartmentalization of ROS may play a pivotal role in triggering protection at reperfusion time. We are performing studies in the attempt to

Postconditioning has the advantage of being a way to influence and modify reperfusion injury after it has occurred. This may open a therapeutic alternative in situations of

**30. Cytoprotection by pre- and postconditioning is redox-sensitive** 

### **ACTIVATION OF REPERFUSION INJURY SURVIVAL KINASES**

opening J Vinten-Johansen, D Yellon, L Opie. Circulation.2005.Oct..2085- 2088

Fig. 9. Possible contexts in the activation of reperfusion injury survival kinases in ischaemic postconditioning

mPTP

SURVIVAL

Neonatal rat cardiomyocytes subjected to 3 hrs of hypoxia and 6 hrs of re-oxygenation, "hypoxic PostC" with alternating exposure to three cycles of 5 min. hypoxic and normoxic conditions preceding re-oxygenation reduced intracellular and mitochondrial Ca2+ loading compared to non-postconditioned cardiomyocytes. This was associated with a reduction in cardiomyocyte death assessed by propidium iodide and lactate dehydrogenase release95. However, the signalling pathways and physiological consequences of this lower intracellular Ca2+ by PostC are not known at present, especially in vivo. For instance, it cannot be excluded that reduced mitochondrial Ca2+ overload could actually be a consequence of a more preserved Ca2+ handling by the sarcoplasmic reticulum in postconditioned cardiomyocytes rather than a cause of protection. It has been reported that PostC reduces calcium-induced opening of the mPTP in mitochondria isolated from the myocardial area at risk96. PostC was also associated with a reduction in infarct size after both acute and long-term (72 hrs) reperfusion. Bopassa et al.97 demonstrated in isolated perfused rat hearts that maintenance of mPTP closure was associated with PI3K activation, which is consistent with the activation of survival kinase pathways described above, but the functional involvement of these pathways and regulation of the mPTP in vivo is not yet clear. It seems that in the PostC scenario the inhibition of GSK3 contributes to the prevention of mPTP opening98. Taken together it would appear that the trigger pathway for PostC involves the following sequence of events: occupation of surface receptors (adenosine and NOS and non-enzymatic processes to make nitric oxide, activation of cGMP-dependent kinase (PKG), opening of mKATP, production of ROS and finally activation of PKC and MAPKs as well as inhibition of GSK3 which put the heart into a protected state. The protect state may include a central role of the prevention of mPTP opening by acidosis in the early phase and by the aforementioned mechanisms in the late reperfusion (*Figures 2 and 3*).

**ACTIVATION OF REPERFUSION INJURY SURVIVAL KINASES** 

**POSTCONDITIONING**

passive active

**Intermittent RISK**

Neutrophils

↓ ROS ↓

**reperfusion**

↓ mito[Ca2+]

J Vinten-Johansen, D Yellon, L Opie. Circulation.2005.Oct..2085- 2088

Fig. 9. Possible contexts in the activation of reperfusion injury survival kinases in ischaemic

mPTP opening

eNO

PI3K MEK

Akt ERK1/2

GSK

CELL SURVIVAL

Neonatal rat cardiomyocytes subjected to 3 hrs of hypoxia and 6 hrs of re-oxygenation, "hypoxic PostC" with alternating exposure to three cycles of 5 min. hypoxic and normoxic conditions preceding re-oxygenation reduced intracellular and mitochondrial Ca2+ loading compared to non-postconditioned cardiomyocytes. This was associated with a reduction in cardiomyocyte death assessed by propidium iodide and lactate dehydrogenase release95. However, the signalling pathways and physiological consequences of this lower intracellular Ca2+ by PostC are not known at present, especially in vivo. For instance, it cannot be excluded that reduced mitochondrial Ca2+ overload could actually be a consequence of a more preserved Ca2+ handling by the sarcoplasmic reticulum in postconditioned cardiomyocytes rather than a cause of protection. It has been reported that PostC reduces calcium-induced opening of the mPTP in mitochondria isolated from the myocardial area at risk96. PostC was also associated with a reduction in infarct size after both acute and long-term (72 hrs) reperfusion. Bopassa et al.97 demonstrated in isolated perfused rat hearts that maintenance of mPTP closure was associated with PI3K activation, which is consistent with the activation of survival kinase pathways described above, but the functional involvement of these pathways and regulation of the mPTP in vivo is not yet clear. It seems that in the PostC scenario the inhibition of GSK3 contributes to the prevention of mPTP opening98. Taken together it would appear that the trigger pathway for PostC involves the following sequence of events: occupation of surface receptors (adenosine and NOS and non-enzymatic processes to make nitric oxide, activation of cGMP-dependent kinase (PKG), opening of mKATP, production of ROS and finally activation of PKC and MAPKs as well as inhibition of GSK3 which put the heart into a protected state. The protect state may include a central role of the prevention of mPTP opening by acidosis in the early phase and by the aforementioned mechanisms in the late reperfusion (*Figures 2 and 3*).

postconditioning

#### **30. Cytoprotection by pre- and postconditioning is redox-sensitive**

It has been already established that preconditioning triggering, that is the period that precedes the index ischaemia, is redox sensitive. This was demonstrated by both avoiding preconditioning with ROS scavengers and inducing preconditioning with ROS generators given before the index ischaemia 99. Also, several metabolites, including acetylcholine, BK, opioids and phenylephrine, trigger preconditioning-like protection via a mKATP-ROS dependent mechanism100. As stated in the case of reperfusion injury, ROS are also implicated in the sequel of myocardial reperfusion injury101. These studies supported the paradigm that ROS may be protective in pre-ischaemic phase, but are deleterious in the post-ischaemic phase. Thus the main idea was that ROS play an essential, though double-edged, role in cardioprotection: they may participate reperfusion injury or may play a role as signaling elements of protection in pre-ischaemic phase102. The importance of ROS signalling (as opposed to excess ROS in the development of injury) has been examined closely in great detail in recent years103. Intriguingly, and in contrast to the above-described theory of ROS as an obligatory part of reperfusion induced damage, some studies suggest the possibility that some ROS species at low concentrations could protect ischaemic hearts104. Yet, from the above reported mechanisms of PostC, it appears that also ischaemic PostC is a cardioprotective phenomenon that requires the intervention of redox signaling to be protective105. Moreover, as mentioned, very recently it has been shown that redox signalling is also required at the time of myocardial reperfusion to mediate the cardioprotection elicited by ischaemic preconditioning106. Therefore, the role of ROS in reperfusion may be reconsidered as they are not only deleterious. This fact may help to understand the variability in the results of studies aimed at proving a role of ROS in reperfusion injury. For instance, negative results came from trials in which free radical scavengers such as recombinant human superoxide dismutase or vitamin E were administered to patients with coronary artery disease or risk factors for cardiovascular events107. In addition to the dual role of ROS (beneficial versus deleterious), among the reasons why these scavengers did not show any consistent benefit in these human studies may be: (1) the type of ROS generated (e.g. superoxide dismutase only removes the superoxide and not the hydroxyl radical); (2) the site of ROS generation (e.g. most scavengers scarcely enter into the cells) and (3) the rate of reaction between two ROS and/or scavengers. The importance of the rate of reaction can be understood if we consider that, despite a five times lower concentration of nitric oxide with respect to superoxide dismutase, 50% or more of the available superoxide will react with nitric oxide to form ONOO– instead of reacting with superoxide dismutase108. Notwithstanding the evidence of a protective role of ROS signalling in reperfusion, we were unable to reproduce cardioprotection with ROS generation by purine/xanthine oxidase given at reperfusion109.

Since ROS scavengers (N-acetyl-L-cysteine or 2-mercapto-propionylglycine), given at the beginning of reperfusion, abolished both IP- and PostC-induced protection110 it is likely that the type, the concentration and/or the compartmentalization of ROS may play a pivotal role in triggering protection at reperfusion time. We are performing studies in the attempt to clarify this issue.

#### **31. Conclusion**

Postconditioning has the advantage of being a way to influence and modify reperfusion injury after it has occurred. This may open a therapeutic alternative in situations of

Ischaemic Postconditioning

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We think, that many more examinations are needed to describe and understand in details the mechanism of ischaemic PostC. We are sure, that this manoeuver is easy to perform, quick, and does not any expensive instruments, so it may have a place in the therapeutic arsenal of vascular surgeons.

#### **32. References**


256 Vascular Surgery

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### *Edited by Dai Yamanouchi*

This book aims to provide a brief overview of conventional open vascular surgery, endovascular surgery and pre- and post-operative management of vascular patients. The collections of contributions from outstanding vascular surgeons and scientists from around the world present detailed and precious information about the important topics of the current vascular surgery practice and research. I hope this book will be used worldwide by young vascular surgeons and medical students enhancing their knowledge and stimulating the advancement of this field.

Vascular Surgery

*Edited by Dai Yamanouchi*

ISBN 978-953-51-0328-8

ISBN 978-953-51-6913-0

Vascular Surgery

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