Triggers And Signal Transduction

In addition to exogenous growth factors, cytokines and circulating progenitor cells that offer protection to ischemia, the endogenous responses of these systems may be involved in the natural history of ischemia-reperfusion injury. More traditional mediators however have also been extensively studied.

When compared to early preconditioning, the protection offered by delayed preconditioning seems to have a broader spectrum of protective mechanisms (eg. also anti-stunning), to be longer-lasting (3 to 4 days) and to depend upon de novo protein synthesis [98] and mobilization of transcription factors. Figure 4B depicts the various phases, which could be schematically recognized in the processes leading to delayed preconditioning. Comparison between panels A and B in figure 4 illustrates the differences between early and late preconditioning.

5.1. Receptor Activation

Biochemical triggers of condition include adenosine, opioids and bradykinin. Of these, adenosine is best described. Adenosine's involvement in the protective mechanism has been implicated using adenosine receptor agonists and antagonists [99]. Adenosine is a trigger for preconditioning -early and late-and postconditioning, albeit that it appears that adenosine's protective effects during preconditioning are mediated by the A1 and A3 receptors and its postconditioning effects by A2 and A3 receptors [64].

5.2. Dual Role for NO and NOS

Nitric oxide (NO) has a direct protective effect on the heart, as demonstrated by Bolli and colleagues [100]. While in preconditioning NO appears to play a role primarily in the late phase of protection [100], blockade of NOS impaired postconditioning as well [67, 101]. NOS has been demonstrated to be a protective protein, through the vaso-active activity of its products [102]. The heart is equipped with three different isoforms of NOS. The inducible NOS, iNOS is expressed by the cardiomyocyte and is highly inducible by stress. eNOS is constitutively being expressed in the endothelial cells and responsice to shear stress. Finally, n(euronal)NOS is found in neurons in the heart. Possibly the best known action of eNOS is the generation of NO for maintaining vasodilatation of the coronary vessels. However, eNOS is also triggered by preconditioning and postconditioning stimuli. The protective effect of NO during postconditioning appears to be mediated through increasing intracellular cGMP concentrations. The thus stimulated production of NO plays a role as end-protector through a not yet completely understood mechanism [64]. Activation of eNOS through the akt pathway is a prominent effect of VEGF-A [103], HGF [104], G-CSF [57] and of PDGF-BB [105], so ischemic protection by these growth factors may be mediated by induction of NO release.

5.3. Survival Kinases

Three kinases, the PI3-kinase, Akt and MEK/Erk-1 have been called the reperfusion injury salvage kinases (RISK), because of their important role in protection. These kinases are activated by various stimuli, both in relation to preconditioning and postconditioning. The kinases form an important, but intermediary signal. They couple to more final processes, such as mPTP opening, anti-apoptotic signaling and Katp channels [64]. Interestingly, ischemic protection by IGF-1, FGF-1, FGF-2, HGF and TGF(1 effect appears to be mediated through a MEK/ERK/GATA-4 pathway. Others, such as VEGF-A [106] and PDGF-BB [105] exert a similar effect on reperfused myocardium, but signal through the PI3kinase/akt pathway.

5.4. Transcription Factors

A set of triggers and pathways that require activation of transcription factors and subsequent gene transcription, has more prominently been described for late preconditioning. Delayed preconditioning seems associated with the activation of protein kinase C [107, 108], mitogen-activated protein kinases [109] and the protein tyrosine kinases, such Src PTKs [110]. Downstream these kinases activate transcription factors, such as heat shock transcription factor (HSF), JAK-STAT or NF-kappaB [111] that are implicated in the delayed cardioprotection. JAK-STAT activation results in a number of phosphorylated stats, of which stat1 and stat3 seem to be important for protection against ischemia reperfusion injury [112]. HIF1a and VEGF are downstream targets of stat3 and may therefore mediate this effect [113]. No data are available on the relation of HSF and growth factors or growth factor signaling in the heart or with respect to ischemia reperfusion injury. NF-kB activation is responsible for the upregulation of iNOS and cyclo-oxygenase-2 (COX-2) [114]. The relationship between vaso-active products of COX-2, but also the expression of COX-2 itself, with VEGF is intricate and reciprocal in pathologic conditions that favor neovascularization such as tumor growth and diabetic retinopathy [115]. It appears that the combination of multiple transcription regulatory proteins, acting in concert, is required for the activation of cardiopro-tective genes [116].

5.5. Heat Shock Proteins

The heat shock proteins comprise a group of highly conserved proteins and are divided into specific families depending on their molecular size. Most studies on HSP and cardioprotection have focused on HSP72 since it is the major inducible HSP in heart and other tissues. The involvement of HSPs in the delayed phase of preconditioning is still unclear. It is well known that brief periods of ischemia/reperfusion induce HSP-overexpression [117]. In addition, genetically modified mice, overexpressing specific members of the heat shock families such as HSP72, HSP60/10 or HSP32 (HO-1), display decreased vulnerability to various stressors like ischemia/reperfusion or heat [118]. However, other studies using a less severe ischemic preconditioning protocol did not confirm the association of presence of protection and enhanced synthesis of HSP72 [119]. Therefore, HSP72-when present- could merely be a marker of ischemic stress. Interestingly, HSP32 is HIF-1 a dependent, and attenuates proinflammatory chemokine production by microvascular endothelium in vitro and in vivo [120]. HSP32-overexpression ameliorates postischemic myocardial damage and is involved in wound healing after an infarction [118].

5.6. Mechanical Stimulation

Of all conditioning stimuli, the protective mechanism of mechanical stimulation is least understood. A common denominator of mechanical stimulation is increased myocardial stretch. An important role for stretch in preconditioning is supported by publications reporting the inhibition of the protection by the stretch activated channel (SAC) blocker gadolinium [91, 92]. Stretch induced preconditioning is mediated by common factors such as PKC and KATP-channels. Stretch-induced preconditioning was also shown to be blocked by an adenosine receptor blocker [92].

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