Over the previous four decades, numerous technological advancements have been implemented in the perfusion techniques, and cardiopulmonary bypass (CPB) has been improved, establishing cardiac surgery using CPB as a safe routine procedure with a low incidence of mortality. However, during CPB, contact of blood with nonphysiologic surfaces induces several pathophysiologic responses: activation of complement, intrinsic coagulation system with the release of bradykinin (vasodilation), activation of leukocytes and platelets, and the release of various inflammatory cytokines. Postoperatively, this can result in “post pump syndrome”, that is, a nonspecific whole body inflammatory response, which in severe cases can escalate to a systemic inflammatory response syndrome (SIRS), adult respiratory distress syndrome (ARDS), sepsis, or even multiorgan failure (MOF). 1,2
Open heart surgery induces the activation of intrinsic coagulation system and consecutive release of vasoactive bradykinin. Furthermore, it has been shown that blood contacting artificial surfaces during extracorporeal circulation procedures activates blood monocytes. Activated monocytes are known to be responsible for the synthesis of various cytokines. Increased levels of proinflammatory cytokines have generally been associated with negative outcomes after cardiac surgery. Therefore, measurement of specific markers in these blood cells presents a good supplement for the evaluation of the hemocompatibility of biomaterials. Important mediators of the inflammatory response are tumor necrosis factor-alpha (TNF-alpha), interleukin-1 β (IL-1 β), IL-6, and IL-8. They induce a series of biologic effects upon several blood and vascular cells. 3 IL-1 β and TNF-alpha, besides their local action, may be distributed throughout the circulatory system and can trigger other cytokines, such as IL-6 (Colony Stimulating Factors, Interferons) release, 4 initiating the systemic inflammatory response.
To reduce the systemic inflammatory reaction, the medical device industries have developed different coating materials for improving the hemocompatibility of the unphysiologic surfaces.
During the previous few decades, heparin coated circuits for CPB were developed, and their biocompatibility has been improved. Heparin coated circuits can reduce complement activation and the subsequent release of cytokines. 5–7 Recently, oxygenators with poly(2-methoxyethylacrylate) (PMEA) coatings were developed to improve the hemocompatibility of the artificial surfaces. PMEA is a synthetic polymer, which is applied with a quick and easy coating procedure, eliminating the need for other potentially hazardous linkers or organic animal components, like heparin, which may have a small potential to cause allergic reactions, thrombocytopenie (HIT), or to be a source for transfer of new viruses, such as BSE.
We performed an experimental study using an in vitro heart-lung machine (HLM) model to determine whether PMEA coated extracorporeal circuits reduce the release of inflammatory mediators, activation of platelets, and the complement, coagulation, and contact phase system during CPB, in comparison with heparin coated and uncoated circuits.
Materials and Methods
Experimental Conditions and Heart-Lung Machine Model
The experiments were carried out within an in vitro heart-lung machine (HLM) model. Fresh human whole blood (not older than 30 minutes) from healthy volunteers was recirculated in a closed loop cardiopulmonary bypass (CPB) system with oxygenation and simulation of arterial counterpressure. For each HLM run, 500 ml blood from one single donor was used. The length of the tubing was defined in a manner identical to surgical conditions. The test series included 15 membrane oxygenators of the same construction series (CAPIOX SX 18; Terumo, Tokio, Japan). These 15 membrane oxygenators were divided into three groups: uncoated group (NC) (n = 5), PMEA coated group (x coating, XC) (n = 5), and heparin coated group (hepaface, HC) (n = 5). The coating was limited to the oxygenators because these possess 95% of the total surface area; the remaining 5% of tube material (PVC) was neglected and not coated.
Before priming, the oxygenators and tubing were rinsed with 1,000 ml ringer lactate for 30 minutes. The priming volume consisted of 78.3 ml 5% glucose solution (Delta-Pharma GmbH, Pfullingen, Germany), 206 ml ringer lactate (Fresenius, Bad Homburg, Germany), and 15.7 ml 8.4% NaHCO3 (Braun Melsungen AG, Melsungen, Germany). The priming volume was not discarded before addition of blood according to clinical procedures.
500 ml volume of fresh human blood was next circulated in this closed system, using a roller pump (Sarns Inc., Ann Arbor, MI), for 90 minutes. A precisely calculated amount of heparin (Liquemin, Hoffmann-La Roche, Basel, Switzerland) was added to achieve a final heparin concentration in the whole machine filling volume of 3 U/ml. A constant blood flow of 3 L/min and a mean arterial pressure of 60 mm Hg were maintained, while a hypothermic regulator (type Q 102, Haake, Berlin, Germany) held the temperature at 28°C at the arterial exit of the oxygenator.
Blood samples were taken from the blood bag before circulation (control) and from the arterial exit of the oxygenator at several time-points after starting circulation. Samples were taken into tubes containing various anticoagulant and preservative solutions required for the different assays. The first 2 ml of blood on each sampling point were discarded to avoid artificial activation.
The heparin concentration in each plasma sample was determined (anti-Xa activity, Chromogenix AB, Molndal, Sweden) and neutralized with protamine chloride (Hoffmann-La Roche) in a ratio of 1:1 (some contact system assays are influenced by high heparin levels). All plasma samples were shock frozen in liquid nitrogen and stored at −80°C.
Whole blood samples were assayed on a blood cell counter to determine hematocrit and blood cell counts.
Blood samples for radioimmunoassay of bradykinin were handled and measured as previously described. 8–9 PMN-Elastase-α1-PI complexes and terminal complement complex (SC5b-9) were determined using ELISA kits supplied by Merck AG (Darmstadt, Germany) and Quidel (San Diego, CA), respectively. The ELISAs for β-thromboglobulin were from Boehringer (Mannheim, Germany). Chromogenic peptide substrate assay for FXII was performed with a kit supplied by Unicorn Diagnostics Ltd. (London, UK). All assays were performed in duplicate according to the manufacturer's instructions.
All values derived from diluted samples were corrected for hematocrit. The initial values, which differed according to the individual donor's blood, were normalized to 100%, and subsequent samples were calculated as a percentage of the initial value. Statistical analysis was performed by the statistical software package SPSS (SPSS Software Inc., Chicago, IL) using the method of univariate analysis of variance. Values of p ≤ 0.05 were considered significant, and p ≤ 0.01 was considered highly significant.
Blood samples were taken before circulation and at 45 and 90 minutes after starting the circulation. Samples were immediately incubated on ice for 10 minutes. Afterwards, monocytes were isolated by using the monocyte isolation kit (Dynal Biotech, Oslo, Norway) according to the manufacturer's protocol. Samples were aliquoted, shock frozen with liquid nitrogen, and stored at −80°C until further analyses.
Monocyte mRNA was harvested from monocyte solution by using the mRNA Isolation Kit (Dynal Biotech, Oslo, Norway).
Monocyte mRNA from all probes of one volunteer was always reverse transcribed in the same run with their respective control samples. The oligo(dT)15-primed first strand cDNA synthesis was carried out with Superscript II reverse transcriptase (Invitrogen, Kahrsruhe, Germany) in a total volume of 20 μl according to the manufacturer's prescription.
Semiquantitative real time RT-PCR.
Primer design and optimization in regard to primer dimers, self priming formation, miss priming and amplicon length was done with the Primer 3 (Whitehead Institute Center for Genome Research, Cambridge, MA) 10 and Primer Premier 5 (PREMIER Biosoft International, Palo Alto, CA) primer design software. All primers were optimized to an equal annealing temperature of 60°C, a similar GC-contend between 57% and 63.2% and supplied by MWG Biotech (Ebersberg, Germany).
The product size was verified by electrophoresis through 1.5% agarose gels with ethidiumbromid staining and by melt curve analysis. The sequences of the PCR primers are listed in Table 1.
PCR was performed with standard protocols using SYBR Green as fluorescent detection dye in a real time iCycler (Biorad, Hercules, CA). All PCR reactions had a final volume of 15 μl comprised out of two times SYBR Green PCR kit, 360 μM forward and reverse primer and 2 ng of reverse transcribed RNA. The whole reaction was pipetted on ice into a 96 well plate (Abgene, Rochester, NY), and the well contents were collected by brief centrifugation of the plate. Cycling was started with an activation step at 95°C for 15 minutes, and the amplification program was repeated 45 times (denaturation: 95°C for 20 seconds; annealing: 60°C for 30 seconds; extension: 72°C for 45 seconds) with fluorescence measurement at 72°C. For characterization of the generated amplicons and to control the contamination by unspecific byproducts, a melting curve analysis was applied between 50–100°C at 0.5°C intervals.
Standard curves for six twofold dilution steps between 4 ng and 0.125 ng of reverse transcribed RNA samples were run for all primer pairs in quinticates to determine the PCR efficacy under the experimental conditions for the different target genes and the housekeeping gene.
All PCR reactions for a given sample were pipetted in three replicates to control and adjust for the variability of the PCR amplification. After the exclusion of outliers, the average of the replicates was used for statistical data processing.
Statistical procedure of real time RT-PCR.
The CTs obtained for the different amplicons were statistically processed by the software package REST, applying pair wise fixed reallocation randomization test, which has particularly been designed for the evaluation of semiquantitative real time PCR results. 11
For graphic presentation of results and statistical analysis by paired Student's t-test and analysis of variance (ANOVA), the mean normalized expression (MNE) of the target genes were calculated by using the average CT value from the three replicates for the target (CTtarget, mean) and the average for the three replicates for the appropriate reference (CTreference, mean). For the statistical comparison of the group of coating at the different time-points, a significant ANOVA was followed by a pair wise fixed reallocation randomization test for post hoc analysis. To control for the variability of the PCR amplification step, the standard error for the MNE (SEMNE) was calculated by using the standard error for the target replicates (SE CTtarget, mean) and the reference replicates (SE CTreference, mean). 12 The coefficient of variation (CV) was thereafter determined.
During the recirculation period, no increase in heparin concentration could be found in the samples for the heparin coated (HC) oxygenators. Moderate falls in heparin concentration during the circulation period were seen with the NC and the XC oxygenators. The results indicate no significant differences by comparing NC with XC and NC with HC, but significant differences at 60 and 90 minutes were shown by comparing XC with HC (60 min, p = 0,014; 90 min, p = 0,047) (Figure 1).
The concentration of white blood cells showed a slight decrease during the 90 minutes of the circulation period. The decrease in the NC group was more pronounced than in the other groups and shows low significant differences after 30 minutes to the XC (p = 0.0424) and the HC group (p = 0.0311); after 60 minutes there were only significant differences to the HC group (p = 0.0307) left (data not shown).
Platelets and β-Thromboglobulin
The number of platelets decreased immediately as soon as the circulation period started in all groups (Figure 2). In the group with the noncoated oxygenators, the platelet count dropped dramatically within the first 20 minutes and then increased continuously after 30, 60, and 90 minutes. During the whole circulation period, the platelet levels were significantly higher in groups XC and HC than were found in group NC. At 90 minutes, there was no significant difference between the platelet counts for groups NC, XC, and HC. However, the levels of β-thromboglobulin indicated a continuous increase during the entire investigation period in all groups. The increase in the group with the uncoated oxygenators was greatly pronounced, indicating high activation of platelets (Figure 3).
During the 90 minutes of recirculation, the PMN-elastase concentrations rose continuously. In the first 30 minutes, there was a gradual increase in all three groups, followed by a sharper increase by 60–90 minutes. At 90 minutes, concentrations in uncoated oxygenators were at 1259.78 ± 319.74%, in the XC group at 1175.77 ± 484.56%, and the group of oxygenators with heparin-coated surfaces at 684.28 ± 238.96% of the original concentration (Figure 4).
During the whole circulation period, the SC5b-9 level continuously increased in all groups. The only significant difference between the groups was observed after 30 minutes, by comparing the x coated group (164% of the initial value) with the heparin coated group (132% of the initial value) (p = 0.03). None of the other time-points showed any significant differences between the three groups investigated (data not shown).
After 5 minutes the levels of bradykinin increased significantly in all three groups (p = 0.01). The highest level in the uncoated and heparin coated groups was observed at 5 minutes, whereas the peak in the PMEA-coated group was after 10 minutes, and it showed the lowest values in all time points (Figure 5).
Changes in gene expression
Interleukin-1 β and interleukin-6.
IL-1 β (Figure 6) showed a continuous upregulation in all three groups, being lowest in the PMEA coated group. Groups NC and HC showed significant changes in gene expression in IL-1 β as well as in IL-6 after 45 minutes (NC: IL-1 βp = 0.002, IL-6 p = 0.02; HC: IL-1 βp = 0.006, IL-6 p = 0.02) and after 90 minutes (NC: IL-1 β and IL-6 p = 0.002; HC: IL-1 βp = 0.007, IL-6 p = 0.001).
The expression curves of IL-8 indicated a low upregulation of all groups after 45 minutes, followed by a significant upregulation at the end of the HLM procedure in group NC by the factor 71.244 (p = 0.005), in group XC by the factor 41.239 (p = 0.014) (Table 2), and in the group HC by the factor 126.497 (p = 0.001) (Figure 8). XC group showed moderate increase during the entire period.
TNF-alpha. During the whole investigation period, no significant changes in gene expression were observed in the XC group, whereas the NC and the HC group showed significant differences at both time-points. An upregulation after 45 minutes was indicated in the NC group by the factor 8.486 (p = 0.009) as well as in the HC group by the factor 9.078 (p = 0.023). Furthermore, NC and HC showed an upregulation after 90 minutes by the factors NC = 8.845 (p = 0.009) and HC = 10.727 (p = 0.006) (Table 2).
In summary, gene expression of cytokines interleukin-1 β, interleukin-6, interleukin-8, and TNF-alpha was upregulated in all groups. In the group comparison, a remarkable trend to elevated cytokine genes expression was detected in the un-coated group of oxygenators in contrast to PMEA coated group.
Improvement of hemocompatibility of artificial surfaces is a viable approach to reduce complications caused by extracorporeal circulation. 13 Recently a new polymer PMEA was developed for surface coatings of blood contacting materials. 14 Tanaka et al. 14 first used PMEA as a soluble polymer for quick and easy surface coating.
This study showed that PMEA and heparin coatings (covalent heparin binding) were advantageous over uncoated surfaces with respect to platelet preservation. Correspondingly, concentration of β-thromboglobulin, as an indicator of platelet activation, was significantly higher in the uncoated oxygenators. Li et al. 15 reported changes in platelet count during CPB, measured by analyzing platelet adhesion to the surfaces of CPB circuits treated with surface modifying additives (SMA) using gamma scintigraphy. Their data indicated that some circulating platelets adhered to the surface 5–10 minutes after beginning of CPB and that some of these adhered platelets were released back into the circulation during CPB. The present findings show that the level of circulating platelets continuously decreased in all three groups until 20 minutes after beginning the circulation procedure. The decrease in the uncoated group was significantly stronger than in the heparin or PMEA coated oxygenators. The present data suggest that, in the uncoated system, adhered platelets were released back into the circulation 30 minutes after the initiation of CPB, whereas the authors could not observe this effect in the heparin and PMEA coated systems. The released platelets, especially of the group with uncoated oxygenators, were highly activated, as shown by significantly elevated levels of β-thromboglobulin (Figure 3). The SMA surface's microscopic structure of alternating hydrophobic and hydrophilic regions carries a net neutral charge and might reduce platelet and leukocyte deposition. 15 The PMEA coating may function by a similar mechanism. 16,17
Activation of PMN neutrophils results in release of PMN-elastase. In the present experiments, only small differences between the three groups were detected.
One of the major humoral defense mechanisms of human blood is the activation of the complement system for elimination of pathogenic invaders. Contact of blood with artificial surfaces does lead to the activation of the complement cascade. Both pathways, classical and alternative, could be initiated during CPB. Activation by alternative pathway could result from conformational changes of C3 complement component upon binding to the foreign surfaces. Classical pathway activation is possible when immunoglobulins bound to the surface undergo conformational changes, or as a reaction to endotoxins. In the present experiments, the authors could observe moderate activation of complement system without significant differences between the groups at the end of circulation period. 18,19 As soon as the blood comes in contact with the negatively-charged surface, F XII, which occurs in an inactive complex with prekallikrein, F XI, and high molecular weight kininogen (HMWK), splits into alpha and beta factor XIIa fragments. These fragments then initiate the entire contact activation system. β-factor XIIa converts the zymogen prekallikrein into its active form, kallikrein, which splits off the vasodilator bradykinin from HMWK. 20 No apparent changes in the F XII value were noted in this study, and there were no significant differences between the three groups. Possibly, this outcome could be related to the low sensitivity of the method of F XII determination (chromogenic substrate assay). However, bradykinin measurement with a highly sensitive radioimmunoassay (RIA) showed significant differences between the three groups investigated (Figure 5). The group with uncoated oxygenation systems showed a tremendous release of bradykinin after 5 minutes circulation. The group with PMEA showed the lowest activation. The heparin coated circuits demonstrated almost as much release of bradykinin as the uncoated control. This finding is in agreement with previously published data. 21 It can be postulated that the low bradykinin levels in the case of PMEA coated circuits result from the minimization of the interaction of the PMEA coated surface with F XII, F XI, and HMWK because of the absence of surface charges.
Cytokines are a group of low molecular weight polypeptides, which are responsible for the intercellular communication. They have a central role in inflammatory reactions, particularly acting on the heart, lung, liver, coagulation system, and central nervous system, subsequently causing damaging effects. 22–25 Various reports have been published concerning CPB caused enhancement of inflammatory cytokine production. 26,27 These results show that the coating of blood contacting surfaces results in a diminished gene expression of IL-1 β, IL-6, IL-8, and TNF-alpha, indicating an improved hemocompatibility. (Figures 6–9) Ninomiya et al. 28 could not show significant differences in gene expression of IL-6 when comparing PMEA-coated with uncoated oxygenators in a clinical study, whereas Gunaydin et al. 29 showed clear benefits of PMEA coating upon the reduction of IL-6 release.
This study, using an in vitro heart-lung machine model with fresh human blood, showed that heparin and PMEA coated oxygenation systems caused significantly less activation of platelets, bradykinin release, and expression of genes encoding inflammatory cytokines. Therefore, the authors assume that PMEA coated materials for CPB may ameliorate the postperfusion syndromes arising from ECC procedures, particularly unspecific inflammation, hyperfibrinolysis, and blood loss. It is important to note that the activation of cellular and protein systems in the human body is regulated by a complex network of activators and inhibitors acting upon the principal of positive or negative feedback, which is the unfulfilled condition in the in vitro situation. Therefore, such data should be interpreted only as a trend observed and should be confirmed in well designed clinical trials.
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