Ischaemic preconditioning has been shown to protect against ischaemia-reperfusion injury in both animal models and human studies [1, 3, 15, 18], however, the signalling mechanisms responsible remain unclear. To date, relatively little data describing the genomic response to ischaemic preconditioning in humans has been reported. Therefore, to identify the genomic response induced by ischaemic preconditioning, we analysed gene expression patterns in a cohort of total knee arthroplasty patients using the Affymetrix Human U113 2.0 microarray system.
While such a cohort of patients is unlikely to develop serious complications of ischaemia-reperfusion, this study provided a model for investigating the local and systemic effects of ischaemic preconditioning, as standard practice for total knee arthroplasty in our institution already involves the application of a tourniquet for the duration of the operation. In this study, ischaemic preconditioning was induced by three five-minute cycles of tourniquet insufflation on the operative lower limb interrupted by five-minute cycles of reperfusion; this preconditioning protocol has previously been shown to be effective in other clinical studies [15, 16].
We investigated the mechanism of local IPC by comparing the gene expression profile of muscle biopsies taken from the operative leg of control and IPC-treated patients using microarray analysis. IPC was found to induce a gene expression profile which was indicative of a protective genomic response in muscle biopsies taken from IPC-treated patients. A comparison of the gene expression profiles of the control and IPC groups indicated that the effect of ischaemic preconditioning was correlated with increased expression of genes involved in immediate early response, defence against oxidative stress, pro-survival functions, and a decrease in gene expression associated with cell death.
IPC triggers the expression of early response genes
In the present study, increased expression of immediate early response genes was shown to be associated with the protective response induced by IPC. This was exemplified by an upregulation in the expression of egr1, ier2, c-fos, c-jun and myc. Immediate early response genes are a group of genes that are activated transiently and rapidly in response to a wide variety of cellular stimuli. Furthermore, a number of these genes have previously been reported to be involved in the adaptation to ischaemia and in the IPC mechanism [19, 20]. In a rat model of IPC, increased expression of c-fos and myc was found to be associated with cardioprotection as evidenced by improved ventricular function and reduced infarct size . More recently, increased expression of egr1 was associated with a predicted cardioprotective phenotype induced by intraoperative ischaemia-reperfusion . The high incidence of early response gene expression indicates that the induction of these genes may be an important element of the protective response induced by IPC.
IPC induces stress response and prosurvival gene expression
The cytoprotective abilities of anti-oxidant proteins induced by IPC are well documented in in vitro and animal models [19, 22, 23]. In the present study, microarray analysis revealed increased expression of anti-oxidant genes in IPC-treated patients following one hour of ischaemia, including catalase and glutathione S-transferase theta 1. Increased ROS generation occurs in ischaemic tissue upon reperfusion. An important element of the cellular defence against ROS is the induction of antioxidant enzymes and detoxifying enzymes such as catalase and glutathione S-transferase. Catalase functions in the decomposition of hydrogen peroxide to water and oxygen while glutathione S-transferases catalyze the conjugation of reduced glutathione to a variety of electrophilic and hydrophobic compounds. Nuclear factor-erythroid 2-related factor 2 (Nrf2) is a transcription factor and an important regulator of the cells response to oxidative stress . It regulates the expression of a network of cytoprotective enzymes and has recently been shown to be involved in the ischaemic preconditioning mechanism [25, 26]. Pathway analysis revealed induction of a number genes involved in Nrf2 signalling in IPC-treated patients, including catalase, glutathione S-transferase, sequestosome 1, jun and fos. Nrft2 signalling has recently been shown to protect against ischaemia-reperfusion injury in both a kidney cell line and in liver biopsies [25, 26]. Results of our study give further support to the idea that Nrf2 signalling is an important protective signalling pathway activated by IPC.
Analysis of microarray data demonstrated increased expression of genes with pro-survival or chaperone functions in IPC patients. Increased expression of heat shock protein 22 kDa protein 8, BCL2/adenovirus E1B 19 kDa interacting protein 1, and BCL6 co-repressor and DnaJ (Hsp40) homolog, subfamily B, member 6 was observed in IPC-treated patients. Studies have shown that heat shock proteins play a key role in the protection provided by IPC, in particular HSP70 and HSP27 [27–29]. The induction of pro-survival gene expression was also associated with a reduction in pro-apoptotic gene expression (caspase 7 and 8) suggesting that IPC may modulate both cell survival and cell death pathways.
The systemic effect of IPC
Ischaemic preconditioning, induced by transient ischaemia of a limb, has been shown to protect remote organs against the effects of ischaemia-reperfusion injury [15, 18, 30]. In a study of children undergoing cardiopulmonary bypass surgery, patients that received remote IPC (via transient ischaemia of the leg) had less cardiac and pulmonary insult . Similarly, in adult patients, decreased serum troponin levels were detected after cardiopulmonary bypass surgery in those patients that received remote IPC via transient ischaemia of the upper arm . It has also been proposed that remote preconditioning may protect against ischaemia-reperfusion injury through a potent suppression of inflammatory signals. Evidence to support this has been demonstrated in healthy volunteers where ischaemic preconditioning of the upper arm has been shown to provide remote protection in the form of reduced inflammatory cell activation and reduced endothelial dysfunction in the contralateral arm , and to suppress pro-inflammatory gene expression in circulating leukocytes .
In this study, we investigated the effect of ischaemic preconditioning on the systemic inflammatory response to ischaemia-reperfusion in our cohort of total knee arthroplasty patients (n = 20). While the patients in this cohort were unlikely to suffer serious complications of ischaemia-reperfusion, a statistically significant increase in the circulating levels of IL-6 was observed in both groups at 24 hours post-reperfusion indicating a post-operative systemic inflammatory response occurred in both patient groups. While skeletal muscle is relatively resistant to ischaemic-reperfusion injury, studies have shown that tourniquet-induced ischaemia-reperfusion leads to systemic activation of PMNs and T cells [16, 30]. In the present study, no significant difference in the mean levels of circulating cytokines was observed between patient groups. However, IPC patients had a tendency for a reduction in IL-6 and ESR at 24 hours post-reperfusion indicating that IPC may attenuate the post-operative inflammatory response in these patients. Other studies have shown that a local IPC stimulus, induced via transient ischaemia of the lower limb, can modulate the systemic inflammatory response following ischaemic-reperfusion in a rat model of limb ischaemic-reperfusion and in patients undergoing cruciate ligament reconstruction [16, 30]. While these studies, and the current study, have shown that local IPC exerts distant anti-inflammatory effects, it is important to note that local and remote IPC are two separate forms of preconditioning and that the signalling mechanisms underlying both forms are not entirely similar.