Reducing Ischaemia/Reperfusion-Induced Organ Injury
Reducing Ischaemia/Reperfusion-Induced Organ Injury
Preconditioning results in an early phase and a late phase of protection to a subsequent severe prolonged ischaemic insult. The early phase starts immediately after the conditioning stimuli and lasts up to 2 h, and the late phase starts 12–24 h after the stimuli and lasts up to a few days. The generation of small amounts of free radicals during the preconditioning period is essential for both early and late phase of protection, but unlike the early phase, the late phase is associated with the expression of several prosurvival genes, including glucose transporter-1 and -4 (GLUT-1 and GLUT-4), heat shock protein 70, and vascular endothelial growth factor, triggered by the increased levels of hypoxia-inducing factor 1α (HIF-1α) after the ischaemic preconditioning stimuli.
The role of HIF-1α in cell survival during ischaemia–reperfusion has been reviewed. HIF-1α is an important transcription factor mediating cellular responses to hypoxia. In normoxic conditions, HIF-1α is hydroxylated by prolyl hydrolases (PHDs); this process facilitates its binding to von Hippel–Lindau protein leading to its subsequent removal by polyubiquitylation and proteasomal degradation. In hypoxic conditions, PHD activity is inhibited allowing HIF-1α levels to increase and cause the expression of hypoxia response genes in the cells. Natarajan and colleagues showed that silencing the PHD gene in mice 24 h before ischaemia resulted in a reduction in PHD enzyme levels and a time-dependent increase in cellular HIF-1α levels. This produced a reduction in the area of myocardium infarction and better recovery of left ventricular function after reperfusion after a period of myocardial ischaemia. The role of HIF-1α in delayed preconditioning is also confirmed by Adluri and colleagues, in a study in mice, where they showed that transgenic PHD knocked out mice (PDH-1−/− KO) had higher levels of HIF-1α, reduced area of myocardial infarction, reduced myocardial apoptosis, increased β-catenin, endothelial nitric oxide synthase enzyme activity, and antiapoptotic BcL-2 levels compared with the wild-type control mice after an ischaemia/reperfusion protocol.
During ischaemic preconditioning, in the early phase, ischaemic tissues release agonists, including adenosine, bradykinin, and opioids; these activate their respective G-protein-coupled receptors (GPCRs), which eventually phosphorylate and activate matrix metalloproteinase. These in turn phosphorylate and activate growth factor receptors, for example, epidermal growth factor receptors (EGFR) and signalling cascades culminating in cytoprotection.
It is well established that μ-, κ, and δ-opioid agonists, when bound to pertussis toxin-sensitive heterotrimeric G protein receptors, mediate their cell signalling mechanisms by phosphorylating and stimulating the extracellular-signal-regulated protein kinases ERK 1 and 2. In the context of preconditioning, opioids also mediate their cytoprotective effect via this pathway. Ma and colleagues demonstrated, in cultured rat cortical neurones, that preconditioning produced an up-regulation and activation of δ-opioid GPCRs resulting in an increase in phosphorylation of ERK followed by downstream activation of PKC. The latter induced an increase in levels of BcL-2 and a decrease in the release of cytochrome C, the net effect was a decrease in cell death during reperfusion after a period of severe hypoxia. The other released autacoids, adenosine and bradykinins, have also been shown to mediate their preconditioning effects via the ERK pathway. These autacoids, acting via the GPCRs, growth receptors, and MEK/ERK1/2, modulate the cellular apoptotic process by increasing and decreasing the expression of antiapoptotic and proapoptotic BcL-2 family of proteins, respectively, shifting the balance towards a reduction in cell apoptosis much like in cancer cells. The increase in expression of antiapoptotic Bcl-2 proteins can result from an increase in transcription or translation of mRNAs. The important role the Bcl-2 family of proteins plays in apoptosis came to light after studies on cancer pathways. These proteins control the permeabilization of the mitochondrial outer membrane; for example, activation of cytosolic Bcl-2 protein bid (t-bid) results in its translocation to the outer mitochondrial membrane where it anchors proapoptotic Bax allowing the latter to polymerize and form membrane pores. The antiapoptotic Bcl-2 proteins, for example, Bcl-2, Bcl-XL, and Mcl-1, by binding to the activators the BH-3 only proteins, for example, t-bid and Bim, prevent the binding and activation of the proapoptotic Bcl-2 proteins.
MEK/ERK1/2 is not the only pathway mediating preconditioning. Research have demonstrated that the autacoids released during preconditioning acting on their respective GPCRs also mediate their effect via the phosphatidylinositol 3-kinase (PI3-K) pathway. They showed that inhibition of PI3-K with wortmannin resulted in the reduction in the protective effect of preconditioning. It is well known that one of the downstream targets of PI3-K is the oncogene product of the retrovirus AKT8, AKT/protein kinase B (PKB), a serine/threonine kinase. Phosphorylation of PI3-K, after, for example, activation of growth factor receptors, produce phosphoinositides which bind to pleckstrin homology (PH) domain of AKT/PKB and phosphoinisitide-dependent protein kinase (PDK) leading to the translocation of AKT/PKB to the plasma membrane where they are phosphorylated and activated by PDK. This PI3-K/AKT/PKB pathway is also involved in preconditioning. Kunuthur and colleagues showed, in a transgenic mice model, that ischaemic preconditioning produced phosphorylation and activation of Akt1 and Akt2 receptors and this resulted in the inhibition of downstream GSK-3β, similar to the inhibition of this enzyme produced by stimulation of insulin receptors. Human δ-opioid receptors mediate the inhibition of GSK-3β activity via a complex signalling pathway involving Src-dependent transphosphorylation of platelet-derived growth factor receptor β, insulin growth factor-1 receptor, PI3-Kα activation of AKT, and AMP-activated protein kinase. This pathway is also involved in cytoprotection after preconditioning. Indeed, prosurvival kinase pathways have been shown to converge onto and inhibit GSK-3β by phosphorylating the N-terminal serine residue (ser9). GSK-3β is involved in the regulation of many cell signalling events; it is active in the basal state and it phosphorylates serine or threonine amino acids on substrates and its effects are terminated by phosphatases. GSK-3β is present in the mitochondria and it is associated with components of the mPTP. It is the convergence point of the cytoprotective pathways and it controls the opening of the mPTP. Inhibition of GSK-3β by upstream cytoprotective signalling pathways involving PI3-K/AKT/PKB probably results in the release of inhibition of downstream signalling pathways, allowing the latter to prevent the opening of mPTP. It has also been suggested that the mammalian target of rapamycin (mTOR) is the downstream pathway of GSK-3β mediating ischaemic and pharmacological preconditioning. mTOR is a member of the PI3-K kinase enzymes and one of its known functions is to control proteins which modulate the translation of mRNAs.
The PI3-K/AKT/PKB pathway not only mediates the reduction in ischaemia/reperfusion injury, after preconditioning, by inhibiting GSK-3β-mediated opening of mPTPs but also by inhibiting apoptosis mediated by the Bcl-2 protein-induced mitochondrial outer membrane permeabilization. The effect of PI3-K/AKT/PKB on the Bcl-2 family of proteins can be explained by the cross-talk between PI3-K/AKT/PKB and MEK/ERK1/2 pathways. Kunuthur and colleagues showed that, after ischaemic preconditioning, phosphorylation and activation of ERK 1/2 pathway occurred in the wild-type AKT1 mice, but this was significantly reduced in the Akt1 mice.
Molecular Mechanisms of Ischaemic Preconditioning
Preconditioning results in an early phase and a late phase of protection to a subsequent severe prolonged ischaemic insult. The early phase starts immediately after the conditioning stimuli and lasts up to 2 h, and the late phase starts 12–24 h after the stimuli and lasts up to a few days. The generation of small amounts of free radicals during the preconditioning period is essential for both early and late phase of protection, but unlike the early phase, the late phase is associated with the expression of several prosurvival genes, including glucose transporter-1 and -4 (GLUT-1 and GLUT-4), heat shock protein 70, and vascular endothelial growth factor, triggered by the increased levels of hypoxia-inducing factor 1α (HIF-1α) after the ischaemic preconditioning stimuli.
The role of HIF-1α in cell survival during ischaemia–reperfusion has been reviewed. HIF-1α is an important transcription factor mediating cellular responses to hypoxia. In normoxic conditions, HIF-1α is hydroxylated by prolyl hydrolases (PHDs); this process facilitates its binding to von Hippel–Lindau protein leading to its subsequent removal by polyubiquitylation and proteasomal degradation. In hypoxic conditions, PHD activity is inhibited allowing HIF-1α levels to increase and cause the expression of hypoxia response genes in the cells. Natarajan and colleagues showed that silencing the PHD gene in mice 24 h before ischaemia resulted in a reduction in PHD enzyme levels and a time-dependent increase in cellular HIF-1α levels. This produced a reduction in the area of myocardium infarction and better recovery of left ventricular function after reperfusion after a period of myocardial ischaemia. The role of HIF-1α in delayed preconditioning is also confirmed by Adluri and colleagues, in a study in mice, where they showed that transgenic PHD knocked out mice (PDH-1−/− KO) had higher levels of HIF-1α, reduced area of myocardial infarction, reduced myocardial apoptosis, increased β-catenin, endothelial nitric oxide synthase enzyme activity, and antiapoptotic BcL-2 levels compared with the wild-type control mice after an ischaemia/reperfusion protocol.
During ischaemic preconditioning, in the early phase, ischaemic tissues release agonists, including adenosine, bradykinin, and opioids; these activate their respective G-protein-coupled receptors (GPCRs), which eventually phosphorylate and activate matrix metalloproteinase. These in turn phosphorylate and activate growth factor receptors, for example, epidermal growth factor receptors (EGFR) and signalling cascades culminating in cytoprotection.
It is well established that μ-, κ, and δ-opioid agonists, when bound to pertussis toxin-sensitive heterotrimeric G protein receptors, mediate their cell signalling mechanisms by phosphorylating and stimulating the extracellular-signal-regulated protein kinases ERK 1 and 2. In the context of preconditioning, opioids also mediate their cytoprotective effect via this pathway. Ma and colleagues demonstrated, in cultured rat cortical neurones, that preconditioning produced an up-regulation and activation of δ-opioid GPCRs resulting in an increase in phosphorylation of ERK followed by downstream activation of PKC. The latter induced an increase in levels of BcL-2 and a decrease in the release of cytochrome C, the net effect was a decrease in cell death during reperfusion after a period of severe hypoxia. The other released autacoids, adenosine and bradykinins, have also been shown to mediate their preconditioning effects via the ERK pathway. These autacoids, acting via the GPCRs, growth receptors, and MEK/ERK1/2, modulate the cellular apoptotic process by increasing and decreasing the expression of antiapoptotic and proapoptotic BcL-2 family of proteins, respectively, shifting the balance towards a reduction in cell apoptosis much like in cancer cells. The increase in expression of antiapoptotic Bcl-2 proteins can result from an increase in transcription or translation of mRNAs. The important role the Bcl-2 family of proteins plays in apoptosis came to light after studies on cancer pathways. These proteins control the permeabilization of the mitochondrial outer membrane; for example, activation of cytosolic Bcl-2 protein bid (t-bid) results in its translocation to the outer mitochondrial membrane where it anchors proapoptotic Bax allowing the latter to polymerize and form membrane pores. The antiapoptotic Bcl-2 proteins, for example, Bcl-2, Bcl-XL, and Mcl-1, by binding to the activators the BH-3 only proteins, for example, t-bid and Bim, prevent the binding and activation of the proapoptotic Bcl-2 proteins.
MEK/ERK1/2 is not the only pathway mediating preconditioning. Research have demonstrated that the autacoids released during preconditioning acting on their respective GPCRs also mediate their effect via the phosphatidylinositol 3-kinase (PI3-K) pathway. They showed that inhibition of PI3-K with wortmannin resulted in the reduction in the protective effect of preconditioning. It is well known that one of the downstream targets of PI3-K is the oncogene product of the retrovirus AKT8, AKT/protein kinase B (PKB), a serine/threonine kinase. Phosphorylation of PI3-K, after, for example, activation of growth factor receptors, produce phosphoinositides which bind to pleckstrin homology (PH) domain of AKT/PKB and phosphoinisitide-dependent protein kinase (PDK) leading to the translocation of AKT/PKB to the plasma membrane where they are phosphorylated and activated by PDK. This PI3-K/AKT/PKB pathway is also involved in preconditioning. Kunuthur and colleagues showed, in a transgenic mice model, that ischaemic preconditioning produced phosphorylation and activation of Akt1 and Akt2 receptors and this resulted in the inhibition of downstream GSK-3β, similar to the inhibition of this enzyme produced by stimulation of insulin receptors. Human δ-opioid receptors mediate the inhibition of GSK-3β activity via a complex signalling pathway involving Src-dependent transphosphorylation of platelet-derived growth factor receptor β, insulin growth factor-1 receptor, PI3-Kα activation of AKT, and AMP-activated protein kinase. This pathway is also involved in cytoprotection after preconditioning. Indeed, prosurvival kinase pathways have been shown to converge onto and inhibit GSK-3β by phosphorylating the N-terminal serine residue (ser9). GSK-3β is involved in the regulation of many cell signalling events; it is active in the basal state and it phosphorylates serine or threonine amino acids on substrates and its effects are terminated by phosphatases. GSK-3β is present in the mitochondria and it is associated with components of the mPTP. It is the convergence point of the cytoprotective pathways and it controls the opening of the mPTP. Inhibition of GSK-3β by upstream cytoprotective signalling pathways involving PI3-K/AKT/PKB probably results in the release of inhibition of downstream signalling pathways, allowing the latter to prevent the opening of mPTP. It has also been suggested that the mammalian target of rapamycin (mTOR) is the downstream pathway of GSK-3β mediating ischaemic and pharmacological preconditioning. mTOR is a member of the PI3-K kinase enzymes and one of its known functions is to control proteins which modulate the translation of mRNAs.
The PI3-K/AKT/PKB pathway not only mediates the reduction in ischaemia/reperfusion injury, after preconditioning, by inhibiting GSK-3β-mediated opening of mPTPs but also by inhibiting apoptosis mediated by the Bcl-2 protein-induced mitochondrial outer membrane permeabilization. The effect of PI3-K/AKT/PKB on the Bcl-2 family of proteins can be explained by the cross-talk between PI3-K/AKT/PKB and MEK/ERK1/2 pathways. Kunuthur and colleagues showed that, after ischaemic preconditioning, phosphorylation and activation of ERK 1/2 pathway occurred in the wild-type AKT1 mice, but this was significantly reduced in the Akt1 mice.
