Potential humoral mediators of remote ischemic preconditioning in patients undergoing surgical coronary revascularization

Remote ischemic preconditioning (RIPC) by repeated brief cycles of limb ischemia/reperfusion may reduce myocardial ischemia/reperfusion injury and improve patients‘ prognosis after elective coronary artery bypass graft (CABG) surgery. The signal transducer and activator of transcription (STAT)5 activation in left ventricular myocardium is associated with RIPC´s cardioprotection. Cytokines and growth hormones typically activate STATs and could therefore act as humoral transfer factors of RIPC´s cardioprotection. We here determined arterial plasma concentrations of 25 different cytokines, growth hormones, and other factors which have previously been associated with cardioprotection, before (baseline)/after RIPC or placebo (n = 23/23), respectively, and before/after ischemic cardioplegic arrest in CABG patients. RIPC-induced protection was reflected by a 35% reduction of serum troponin I release. With the exception of interleukin-1α, none of the humoral factors changed in their concentrations after RIPC or placebo, respectively. Interleukin-1α, when normalized to baseline, increased after RIPC (280 ± 56%) but not with placebo (97 ± 15%). The interleukin-1α concentration remained increased until after ischemic cardioplegic arrest and was also higher than with placebo in absolute concentrations (25 ± 6 versus 16 ± 3 pg/mL). Only interleukin-1α possibly fulfills the criteria which would be expected from a substance to be released in response to RIPC and to protect the myocardium during ischemic cardioplegic arrest.

Concentration of humoral factors. The concentrations of the analyzed humoral factors were not significantly different between RIPC and placebo at baseline, with the exception of prolactin, which was lower with RIPC than with placebo ( Table 2). To normalize for interindividual differences, the concentrations of all factors were also normalized to their baseline.
The IL-1α concentration, when normalized to baseline, increased after the RIPC procedure and remained increased until after ischemic cardioplegic arrest, whereas it was unchanged with placebo. In absolute concentrations, interleukin-1α increased after ischemic cardioplegic arrest over that at baseline and before ischemic cardioplegic arrest with RIPC, whereas it did not change over time with placebo (Table 2 and Fig. 2).
The concentrations of GDF-11 and IL-8 increased after ischemic cardioplegic arrest and were greater with RIPC than with placebo, but after normalization to baseline these changes were no longer significant ( Table 2). The concentrations of pentraxin-3 and survivin increased after ischemic cardioplegic arrest and were lower with RIPC than with placebo, but again after normalization to baseline these changes were no longer significant ( Table 2).
Exclusively after normalization to baseline, the GHRH concentration was lower with RIPC than with placebo throughout the remaining protocol. The normalized concentrations of IL-6 and prolactin were greater with RIPC than with placebo after ischemic cardioplegic arrest. The normalized concentration of IL-17 was greater with RIPC than with placebo before and after ischemic cardioplegic arrest (Table 2).

Discussion
Except for IL-1α, none of the analyzed humoral factors in our study appeared to fulfill the criteria for a transfer factor of cardioprotection by RIC (increase in the factor's concentration after the RIC procedure and before myocardial ischemia as well as association with reduced myocardial ischemia/reperfusion injury), and we thus add another mostly negative study to the so far elusive search for RIC´s transfer factor 17 . Our study was unique in that it was conducted in patients undergoing CABG surgery, where the RIPC procedure indeed induced cardioprotection. However, none of the humoral factors differed in absolute concentration between RIPC and placebo before ischemic cardioplegic arrest. The concentrations of some factors (GDF-11, GHRH, IL-1α, IL-6, IL-8 and IL-17) were greater with RIPC than with placebo after ischemic cardioplegic arrest. For these factors, however, it is unclear whether this difference is truly related to myocardial ischemia/reperfusion injury and protection from it. Cardiopulmonary bypass inflicts a systemic inflammatory injury to the entire body and induces damage to various parenchymal organs 78 . RIC, in turn, is also a systemic response and provides protection to a number of parenchymal organs 79,80 . Therefore, the observed differences in the concentrations of the above humoral factors may originate from other organs than the heart.
The IL-1α concentration, when normalized to baseline, was increased after the RIPC procedure and it remained increased until after ischemic cardioplegic arrest whereas it was not changed throughout the placebo protocol. In absolute concentrations, IL-1α was also greater with RIPC than with placebo after ischemic cardioplegic arrest. IL-1α is a member of the IL-1 cytokine family and involved in inflammatory processes. IL-1α is released from macrophages, monocytes, endothelial and epithelial cells 81,82 but also from cardiomyocytes 83 in response to cell injury. In mice with myocardial infarction, IL-1α was released into the systemic circulation, whereas IL-1α in the myocardial tissue did not change 83 . In isolated perfused rat hearts, IL-1α blockade after reperfusion reduced infarct size 84 , suggesting that intracellular IL-1α contributes to ischemia/reperfusion injury. However, exogenous IL-1α preconditioning 85 and pretreatment 86 in isolated perfused rat hearts improved ventricular systolic pressure and reduced infarct size, suggesting that circulating, extracellular IL-1α induces cardioprotection. A causal role of IL-1α as humoral mediator and trigger for intracellular signaling in RIC remains to be established. Whereas IL-1β is known to activate STATs 54 , the exact role of IL-1α in STAT activation is not clear so far. IL-1α could indirectly activate STATs by induction of IL-6 53 . Except for IL-1α, which has not been associated with RIC before, we could not confirm any of the previously reported humoral factors to be associated with cardioprotection by RIC. There are limitations of our current study: 1) Given our small patient cohort and the high number of analyzed humoral factors, the risk of a type I error with respect to IL-1α is high, in particular since its increase after the RIPC procedure was only evident with normalized data. Our exploratory study is hypothesis generating, so replication in a larger cohort of patients is mandatory. 2) We used plasma samples from a consecutive patient cohort with co-morbidities and co-medications, some of which may potentially interfere with the protection by ischemic conditioning maneuvers [87][88][89] , but also with the release of humoral factors. Patients undergoing RIPC were younger and had lower preoperative risk scores than those undergoing the placebo procedure, and these differences may have contributed to the more pronounced decrease in TnI release than that in the larger cohort reported before 3 . 3) We analyzed the plasma concentrations only at four defined time points, i.e. before/5 min after the RIPC/placebo protocol and before/10 min after ischemic cardioplegic arrest, not considering for the potentially different kinetics of each humoral factor. In particular, the time from the end of the RIPC/placebo procedure to ischemic cardioplegic arrest was a bit longer in patients with RIPC than with placebo, and we may have missed a transient increase or decrease in humoral factors with RIPC.

Methods
Ethics Statement. The study conforms to the principles of the Declaration of Helsinki. With approval by the local ethics committee (Institutional Review Board, University of Duisburg-Essen, no. 08-3683) and patients' written informed consent, arterial blood samples were harvested from a small cohort of consecutive patients (n = 23 RIPC/23 placebo) who underwent elective isolated first-time CABG surgery 3 . These patients were enrolled between February 2012 and April 2013 and within the framework of a larger, randomized, prospective, double-blind, placebo-controlled trial (ClinicalTrials.gov NCT01406678, date of registration: December 1, 2009). The inclusion and exclusion criteria for the trial as well as its results have been reported 3 . Study procedure. Anesthesia was induced with sufentanil (1 µg/kg), etomidate (0.3 mg/kg) and rocuronium (0.6 mg/kg) and maintained with isoflurane (0.6-1.0% end-tidal). The RIPC protocol consisted of 3 cycles of 5 min left upper arm ischemia/5 min reperfusion, and data were compared to placebo (cuff left deflated for 30 min). CABG was performed using median sternotomy, mild systemic hypothermia (>32 °C) and antegrade cold crystalloid Bretschneider (Köhler Chemie GmbH, Bensheim, Germany) cardioplegia, with additional topical cooling and single aortic cross-clamping for all distal anastomoses 3 .
Arterial blood samples and plasma preparation. Arterial blood samples were taken before (baseline) and 5 min after the end of the RIPC/placebo procedure as well as before and 10 min after the ischemic cardioplegic arrest. These time points were chosen to detect changes induced by the RIPC protocol per se and the interaction of RIPC with ischemic cardioplegic arrest. At each time point, 25 mL arterial blood was withdrawn and sampled in vials containing lithium-heparin (Sarstedt, Nümbrecht, Germany). The arterial blood was then immediately centrifuged at 4 °C with 800 g for 15 min, plasma was separated, stored at −80 °C for later use and again centrifuged for 10 min at 4500 g before use. Additionally, 5 mL of arterial blood was withdrawn in separate vials (Sarstedt, Nümbrecht, Germany) to analyze the serum concentration of prolactin.      105 and GHRP 106 (ELISA Cloud-Immunoassay, Houston, USA) biotin-conjugated antibodies against the respective protein were added to the microplate, and the antibodies on the plate and the biotin-labeled antibodies then competed for each other. An avidin-conjugated horseradish peroxidase-conjugated secondary antibody was supplemented. For the detection of thymosin-β4 107 (Immundiagnostik, Bensheim, Germany) an antibody against thymosin-β4 was added to the microplate, which was precoated with the immobilized antigen to thymosin-β4. The antigen of the sample and the immobilized antigen then competed for each other. A horseradish peroxidase-conjugated secondary antibody was supplemented.

Serum troponin I.
After adding the respective substrate, the enzyme-substrate reaction resulted in a blue product. The color intensity was proportional to the concentration of the protein. The reaction was stopped, and the color changed to yellow. The color intensity was measured at 450 nm using a spectrophotometer (Microplate Reader 680, BIORAD, München, Germany).
For the detection of IL-1β 97 , IL-6 108 , IL-8 108 , IL-10 98 and TNF-α 108 (R&D systems, Abingdon, UK) an enzyme-linked polyclonal antibody and a substrate solution were supplemented. After adding an amplifier enzyme the enzyme-substrate reaction resulted in a violet product. The color intensity was proportional to the enzyme activity, which was related to the concentration of bound proteins. The reaction was stopped, and the color intensity was measured at 490 nm using a spectrophotometer (Microplate Reader 680, BIORAD, München, Germany).
The prolactin concentration was measured in the central laboratory of the University Duisburg-Essen Medical School. The detection range of prolactin assay was 0.3 μg/L to 200 μg/L. The serum concentration of prolactin was measured using a two-side sandwich chemiluminescence immunoassay with an acridinium ester-conjugated antibody against prolactin and a secondary antibody covalently coupled to paramagnetic particles (ADVIAR Centaur XP, Siemens, Tarrytown, USA) 109 .
The concentrations of the respective proteins were quantified by comparison to a standard curve.
Statistics. Data are expressed as mean ± standard error of the mean (SEM). Statistics were performed using SigmaStat software (SigmaStat 2.03, SPSS Inc., Chicago, IL, USA). Patient demographics and intraoperative characteristics were compared using unpaired Student's t-test (continuous data) and 2-tailed Fisher's exact test (categorical data). Serum TnI of patients was analyzed by 2-way (group, time) ANOVA for repeated measures. The AUC for the serum TnI over 72 h was compared between RIPC and placebo by unpaired Student's t-test. Plasma Figure 2. Plasma concentration of interleukin-1α. The plasma concentration of interleukin-1α (IL-1α) before (baseline) and after remote ischemic preconditioning (RIPC; n = 23, black bars) or the placebo protocol (n = 23, white bars) and before and after ischemic cardioplegic arrest, respectively, in patients undergoing coronary artery bypass graft surgery. The plasma concentration of IL-1α was increased after ischemic cardioplegic arrest with RIPC and was greater with RIPC than with placebo (a). After normalization to baseline, the IL-1α plasma concentration was greater with RIPC than with placebo throughout the remaining protocol (b). *p < 0.05 versus baseline, # p < 0.05 versus before ischemic cardioplegic arrest, + p < 0.05 versus RIPC using 2-way ANOVA for repeated measures, followed by Fisher's post hoc tests. concentrations of all humoral factors were analyzed by 2-way (group, time) ANOVA for repeated measures. When a significant difference was detected, ANOVA was followed by Fisher's post hoc tests. Differences were considered significant at the level of p < 0.05.