The impact of remote ischaemic preconditioning on the human plasma proteome

© 2014 Dr. Joanne Michele HepponstallPublications included in thesis:Hepponstall, M., Ignjatovic, V., Binos, S., Monagle, P., Jones, B., Cheung, M. H. H., d’Udekem, Y. & Konstantinov, E. (2012). Remote ischemic preconditioning (RIPC) modifies plasma proteome in humans. PLoS ONE, 7(11), e48284. DOI: 10.1371/journal.pone.0048284Pepe, S., Liaw, N. Y., Hepponstall, M., Sheeran, F. L., Yong, M. S., d'Udekem, Y., Cheung, M. M. & Konstantinov, I. E. (2013). Effect of remote ischemic preconditioning on phosphorylated protein signalling in children undergoing tetralogy of Fallot repair: a randomised controlled trial. Journal of the American Heart Association, 2(3), e000095. DOI: 10.1161/JAHA.113.000095Remote Ischemic Preconditioning (RIPC) is an intervention involving intermittent periods of ischaemia and reperfusion of peripheral tissue to provide multi-organ protection, including the heart, from ischaemia-reperfusion (IR) injury. RIPC has been shown to improve clinical outcomes in a variety of settings. It appears that humoral factors that are released into the blood in response to the RIPC stimulus. Previous studies have demonstrated a global genomic response to RIPC in healthy adult volunteers. The focus of the studies described in this thesis was to examine the proteomic changes in plasma in response to RIPC in healthy adult volunteers and children undergoing repair of Tetralogy of Fallot (ToF) with cardiopulmonary bypass (CPB). The latter was in the setting of a randomised controlled trial (RCT). RIPC was induced by cyclic inflations of a standard blood pressure cuff applied to a limb, to a pressure exceeding systolic in order to completely interrupt blood flow. Complete interruption of blood flow was confirmed by pulse oximetry. The protocol involved four cycles of five minutes of ischaemia alternating with five minutes of reperfusion. In the preclinical study, the proteomic change in plasma originating from the limb subjected to the RIPC and the proteomic response to RIPC at both 15 min and 24 h post RIPC was studied. Forty eight proteins were found to be differentially expressed with up-regulation dominating during the cycles of reperfusion and down-regulation evident at 15 min and 24 h post RIPC stimulus. These results suggested that in response to brief episodes of ischaemia and reperfusion in an upper limb, proteomic changes in the plasma are induced. This may result in a protective state systemically, that significantly modifies both the early and late proteomic response to IR injury. We then extended this idea into the clinical setting to determine if a similar proteomic response could be detected in children with ToF undergoing repair with CPB. A double blind RCT was performed to investigate whether RIPC modifies clinical markers of IR injury and cardiopulmonary function after CPB in children undergoing ToF repair. Although there was no clinical evidence that RIPC promotes recovery of cardiac function, reduces post-operative complications or inotrope requirement in the first 24 h post CPB, the proteomic response to the RIPC was noticeable. We presented the results characterising the proteomic changes in response to CPB that are most apparent at 6 h and 12 h post CPB and return to baseline within 24 h. CPB induced significant changes to the global proteomic response in plasma of children undergoing cardiac surgery for repair of ToF. We further investigated the impact of the RIPC on modifying the global plasma proteomic response during the peri-operative period in plasma from children undergoing ToF repair with CPB. There were no differences between the control group and the RIPC group at baseline or at the end of CPB. At 6h post CPB, there were 48 peptides that were found to be differentially expressed which related to six proteins. There was a return to the baseline within 24 h of CPB. In conclusion, the findings of this thesis provided evidence for the first time that there were proteomic changes in human plasma in response to RIPC, supporting the hypothesis that humoral factors are released into the blood to render a protective state against IR injury. Together, findings from the preclinical study and the RCT indicated that the observed proteomic changes were consistent in healthy adults and children undergoing heart surgery

Remote Ischemic Preconditioning (RIPC) Modifies the Plasma Proteome in Children Undergoing Repair of Tetralogy of Fallot: A Randomized Controlled Trial

<div><p>Background</p><p>Remote ischemic preconditioning (RIPC) has been applied in paediatric cardiac surgery. We have demonstrated that RIPC induces a proteomic response in plasma of healthy volunteers. We tested the hypothesis that RIPC modifies the proteomic response in children undergoing Tetralogy of Fallot (TOF) repair.</p><p>Methods and Results</p><p>Children (n=40) were randomized to RIPC and control groups. Blood was sampled at baseline, after cardiopulmonary bypass (CPB) and 6, 12 and 24h post-CPB. Plasma was analysed by liquid chromatography mass spectrometry (LC-MS) in an untargeted approach. Peptides demonstrating differential expression (p<0.01) were subjected to tandem LC-MS/MS and protein identification. Corresponding proteins were identified using the NCBI protein database. There was no difference in age (7.3±3.5vs6.8±3.6 months)(p=0.89), weight (7.7±1.8vs7.5±1.9 kg)(p=0.71), CPB time (104±7vs94±7 min)(p=0.98) or aortic cross-clamp time (83±22vs75±20 min)(p=0.36). No peptides were differentially expressed at baseline or immediately after CPB. There were 48 peptides with higher expression in the RIPC group 6h post-CPB. This was no longer evident at 12 or 24h, with one peptide down-regulated in the RIPC group. The proteins identified were: inter-alpha globulin inhibitor (42.0±11.8 vs 820.8±181.1, p=0.006), fibrinogen preproprotein (59.3±11.2 vs 1192.6±278.3, p=0.007), complement-C3 precursor (391.2±160.9 vs 5385.1±689.4, p=0.0005), complement C4B (151.5±17.8 vs 4587.8±799.2, p=0.003), apolipoprotein B100 (53.4±8.3 vs 1364.5±278.2, p=0.005) and urinary proteinase inhibitor (358.6±74.9 vs 5758.1±1343.1, p=0.009). These proteins are involved in metabolism, haemostasis, immunity and inflammation.</p><p>Conclusions</p><p>We provided the first comprehensive analysis of RIPC-induced proteomic changes in children undergoing surgery. The proteomic changes peak 6h post-CPB and return to baseline within 24h of surgery.</p><p>Trial Registration</p><p>ACTR.org.au <a href="http://www.anzctr.org.au/trial_view.aspx/ACTRN12610000496011" target="_blank">ACTRN12610000496011</a></p></div

Complement C3 concentration for the control and RIPC groups at the time points in the study.

<p>Individual samples were assessed in this ELISA. There was significantly higher levels of Complement C3 in the RIPC group at 6 h after CPB (p<0.05).</p

Time points of the sample collection in panel A.

<p>Pooling of the plasma samples at each time point in panel <b>B.</b> Plasma from all 20 patients of one group was pooled into 5 samples (each representing plasma of 4 patients).</p

Integrated peak area of the peptides in the 5 pooled samples in each group.

<p>Expression of each peptide is depicted as a line with the lines in red each representing the significantly higher expressed peptides in the RIPC group compared to the control group. There were 48 up-regulated peptides in the RIPC group compared to the control group. The histogram on the left indicates the false discovery rate.</p

Significantly changed proteins 2D DIGE/MS.

*<p>Proteins that were also found to change significantly using LC-MS.</p><p>□The protein score indicates the confidence with which the proteins identified match the NCBInr human protein database. Only scores greater than 40 were considered to match with sufficient confidence. Average ratio indicates the degree of difference in the abundance between two protein spot groups. Values below zero indicate a down-regulation, whereas, values greater than zero indicate up-regulation.</p

Two complementary proteomic methods used to assess RIPC induced changes in the plasma proteome.

<p>2D DIGE- 2Dimensional Difference in gel electrophoresis, LC– Liquid chromatography, -MS-mass spectrometry.</p

Protein data for the 6 proteins that were up-regulated in RIPC group as compared to the control group at 6 hours after CPB.

<p>Protein data for the 6 proteins that were up-regulated in RIPC group as compared to the control group at 6 hours after CPB.</p

Venn diagram detailing the number of differentially expressed peptides in RIPC as compared to control samples at each time point.

<p><b>A.</b> Baseline. <b>B.</b> End CPB. <b>C.</b> 6 hours post CPB sample. <b>D.</b> 12 hours post CPB. <b>E.</b> 24 h post CPB. <b>*—</b>down-regulated, †<b>—</b>up-regulated, ‡<b>—</b>significantly differentially expressed.</p

CONSORT diagram describing flow of the participants though the enrolment, allocation, follow up and analysis phases of the trial.

<p>CONSORT diagram describing flow of the participants though the enrolment, allocation, follow up and analysis phases of the trial.</p