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Ann Thorac Surg 2005;80:611-617
© 2005 The Society of Thoracic Surgeons

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Original article: Cardiovascular

Sivelestat Reduces Inflammatory Mediators and Preserves Neutrophil Deformability During Simulated Extracorporeal Circulation

Departments of Cardiovascular Surgery and Cardiology, Institute of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Japan

Accepted for publication February 9, 2005.

^_* Address reprint requests to Dr Hiramatsu, Department of Cardiovascular Surgery, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8575, Japan (Email: yuji3{at}md.tsukuba.ac.jp).

	Abstract

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BACKGROUND: Neutrophil is a major focus in efforts to ameliorate the systemic inflammatory response associated with cardiopulmonary bypass. Neutrophil elastase is a powerful proteolytic enzyme, and plays a pivotal role in the development of the inflammatory response. This study assesses the inhibitory effects of sivelestat, a highly specific neutrophil elastase inhibitor, on elastase levels, cytokine production, and the functional changes of neutrophils in a simulated extracorporeal circulation model.

METHODS: Simulated recirculation was established by recirculating heparinized (3.75 U/mL) human blood for 120 minutes in an oxygenator and a roller pump circuit with and without 100 µmol/L of sivelestat (n = 7 for each group). Neutrophil elastase and interleukin-8 were measured with an enzyme immunoassay. Neutrophil deformability was evaluated by simulated microcapillaries. The neutrophil F-actin and the expression of CD11b and L-selectin were measured by flow cytometry.

RESULTS: Sivelestat reduced both neutrophil elastase levels (p = 0.0006) and interleukin-8 production (p < 0.0001) at 120 minutes of recirculation. Sivelestat also significantly preserved neutrophil deformability (p = 0.017) and reduced F-actin expression (p = 0.0037). The drug did not modulate the changes of CD11b or L-selectin.

CONCLUSIONS: This study suggests that specific elastase inhibition with sivelestat could be a feasible therapeutic strategy for patients undergoing cardiopulmonary bypass to attenuate neutrophil-derived inflammatory response and organ injuries.

	Introduction

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Cardiopulmonary bypass (CPB) induces a systemic inflammatory response that contributes to the morbidity of open heart surgery [1, 2]. One of the most important initiating events of this phenomenon is neutrophil activation and sequestration. Neutrophil sequestration in microvessels is due to a loss of deformability and the changes of adhesive qualities between neutrophils and endothelial cells [3]. Neutrophils also secrete toxic oxygen species and proteolytic enzymes, including neutrophil elastase. Neutrophil elastase is an extremely cytotoxic enzyme in plasma and interstitial fluid. It degrades connective tissue components such as elastin, proteoglycan, fibronectin, and collagen, and potentially causes severe tissue injury and subsequent multiple organ dysfunction [1, 4].

Sivelestat sodium hydrate, sodium N-{2-[4-(2,2-dimethylpropionyloxy) phenylsulfonylamino] benzoyl} aminoacetate tetrahydrate (Elaspol, ONO-5046-Na [C₂₀H₂₁N₂NaO₇S-Na-4H₂O, molecular weight, 528.51]; Ono Pharmaceutical Co., Osaka, Japan), is a synthetic, specific, low-molecular weight neutrophil elastase inhibitor. The inhibitory activity (Ki value) of sivelestat against human neutrophil elastase is 46 nmol/L. Its 50% inhibitory concentration values (IC₅₀) against elastin and neutrophil elastase activity in human plasma are 1.7 µmol/L and 22.8 µmol/L, respectively [5]. The drug is intravenously active, and competitively inhibits the activity of neutrophil elastase in humans, hamsters, and dogs, but does not affect other proteases such as plasmin, thrombin, kallikrein, cathepsin B, or collagenase I [5]. The eliminating phase of the drug is biphasic, and the half-life of the first and second phases are constant at about 2 and 3 hours, respectively [6]. Intrinsic macromolecular antiproteases, which inhibit neutrophil elastase in an ordinary state, are immediately inactivated by superoxide radicals and blocked from close contact with neutrophils during the onset of an inflammatory state [4, 7]. A low-molecular weight antiprotease, such as sivelestat, achieves close contact with neutrophils without rapid inactivation by superoxide and therefore is advantageous [8]. The efficacy of sivelestat on postperfusion lung, ischemia-reperfusion and endothelial cell injuries have been demonstrated in several investigations [9–11]. The use of the drug in humans has been approved in Japan for cases of acute lung injury [12], although there have been conflicting conclusions regarding its benefits [13]. However, no report exists regarding its effect on the inflammatory state associated with CPB. Because ONO-6818, a prototypical neutrophil elastase inhibitor, has demonstrated inhibitory effects on elastase, interleukin-8 (IL-8) and the terminal complement complex levels during simulated CPB [14], we aimed to assess the impact of sivelestat during CPB with an eye toward future clinical applications for controlling the neutrophil-derived inflammatory response. We hypothesized sivelestat reduces IL-8 production, and alters elastase levels, adhesion molecule expression, and the deformability of neutrophils via the attenuation of chemoattractants during simulated extracorporeal circulation.

	Material and Methods

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Simulated Extracorporeal Circulation
Simulated extracorporeal circulation [14, 15] involved a membrane oxygenator (model 60EC, surface area 0.6 m²; MERA, Tokyo, Japan), a soft reservoir (MERA), silastic tubing, polycarbonate connectors, and a roller pump (model MS-033; MERA). Each circuit was primed with 250 mL of fresh undiluted human blood. Blood was obtained from healthy, fasting volunteers, who abstained from all medications for at least 2 weeks before donation. One donor was used for each individual bypass. Written informed consent was obtained from donors and the protocol was approved by the Institutional Review Board of the University of Tsukuba.

In the control group (n = 7), blood was drawn into a reservoir containing heparin (3.75 U/mL) and dextrose (2.25 mg/mL). In the sivelestat group (n = 7), the reservoir contained heparin (3.75 U/mL), dextrose (2.25 mg/mL), and sivelestat (final concentration of 100 µmol/L). This initial dose regimen of sivelestat was determined based on its half-life (2 to 3 hours), IC₅₀ values (22.8 µmol/L against human elastase activity) and a preliminary in vitro study which indicated that sivelestat inhibits the activity of neutrophil elastase in a dose-dependent fashion at 1 to 300 µmol/L but does not affect other proteases even at 500 µmol/L [5]. Sivelestat was provided by Ono Pharmaceutical Co. Blood was recirculated for 120 minutes at a fixed flow rate of 400 mL/min with the blood temperature maintained at 37°C by immersion in a water bath. The oxygenator was ventilated with 95% oxygen – 5% carbon dioxide at a flow rate of 1.0 L/min. Preliminary experiments confirmed that the pH of the circulating blood was maintained at 7.3 to 7.5, and the activated clotting time was over 500 seconds at all times. The circuit pressure was not measured.

Blood samples were obtained for analysis from each donor before any anticoagulant was introduced (Donor sample), then from the reservoir before recirculation (0 minutes), and at 30, 60, and 120 minutes of recirculation. Additionally, a standing control sample was collected from the reservoir and incubated for 120 minutes at 37°C. Blood samples for analysis were obtained with either 3.8% acid-citrate-dextrose (for microchannel analysis, F-actin, CD11b, and L-selectin, 9:1 by volume) or 1.0% ethylenediaminetetraacetic acid disodium (EDTA-2Na, for neutrophil elastase, and IL-8, 9:1 by volume). Blood collected with 1.0% EDTA-2Na was centrifuged immediately for 15 minutes at 2,000 g at 4°C. The plasma was then stored at –80°C for subsequent measurements. Samples for cell counts were collected in EDTA-2Na tubes.

Blood Cell Counts
Blood cell counts were performed with a counter (T-660; Coulter, Hialeah, FL), and differential white cell counts were performed on Wright’s stained blood smears by an independent observer. Neutrophil counts were expressed as a percentage of the Donor value.

Neutrophil Elastase and Interleukin-8 Assay
Neutrophil elastase combined with ₁-protease inhibitor in plasma (Merck Diagnostica, Darmstadt, Germany), and plasma IL-8 levels (Pierce Endogen, Rockford, IL) were measured by enzyme-linked immunosorbent assay kits.

Adhesion Molecules Assay
Changes in the surface expression of L-selectin and CD11b of neutrophils were measured as markers of neutrophil activation using flow cytometry, as previously described [16]. One hundred microliters of whole blood samples were incubated for 30 minutes with 2 mg/mL fluorescein isothiocyanate (FITC)-conjugated anti-human CD62L antibody (Isotype: IgG1, kappa; BD Biosciences Pharmingen, San Diego, CA) and 1 mg/mL R-phycoerythrin (RPE)-conjugated mouse monoclonal anti-human CD11b antibody (Isotype: IgG1, kappa; DAKO, Copenhagen, Denmark) at 4°C. Identical samples were incubated with FITC-conjugated monoclonal mouse IgG1, kappa antibody (DAKO) and RPE-conjugated monoclonal mouse IgG1, kappa antibody (DAKO) as negative controls. The erythrocytes were lysed for 60 seconds with Immuno-lyse, and leukocytes were fixed with Immuno-fix (Coulter Clone, Hialeah, FL). Neutrophils were identified by the typical forward- and side-scatter pattern, and the expression of L-selectin and CD11b was measured as a mean fluorescent intensity of 5,000 cells. The changes of L-selectin and CD11b were expressed as the percentage changes compared to the Donor value.

F-actin Content Assay
A 50-µL sample was fixed with formaldehyde and the cells were permeabilized using IntraPrep (Immunotech Coulter, Marseilles, France). Neutrophils were stained for 30 minutes at 37°C with 1 U of BODIPY FL phallacidin (Molecular Probes Inc., Eugene, OR). Cells were washed with phosphate-buffered saline solution (PBS), and the F-actin content was measured using a flow cytometer as previously described [16]. The change in the F-actin content was expressed as the percentage change from the Donor value.

Neutrophil Deformability Assay
The transit time of whole blood through the microchannel array was measured as a surrogate marker of neutrophil deformability. A microchannel array flow analyzer (MC-FAN, KH-2; Hitachi Haramachi Electronics, Hitachi, Japan) was used for the analysis, and the detailed procedures have been previously described [15]. The microchannel array (Bloody-3S; Hitachi Haramachi Electronics) was made with a silicon substrate to closely resemble the size of capillaries (2,600 channels [width, 6 µm; depth, 4.5 µm; length, 10 µm]). Whole blood samples, diluted with PBS (1:1 by volume), were made to flow through the microchannels under a pressure difference of 10 cm H₂O. The transit time for each 100-µL sample was determined to assess the blood filterability. Results were expressed as a percentage of the transit time of the Donor samples.

Statistics
One-way analysis of variance as compared with the Donor value was used for within group comparison. Comparison of two groups as a function of time was performed by two-way analysis of variance (ANOVA) with repeated measures. Data were further compared by the use of post-hoc tests with Bonferroni correction. All values are expressed as mean ± standard error of the mean.

	Results

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Changes in measured blood and plasma constituents and the microchannel transit time observed during the experiments are shown in Table 1. The hematocrit value (data not shown) did not change significantly in either group throughout the recirculation. Neutrophil counts decreased to approximately 88% to 90% of the Donor value in both the control (p = 0.011, versus Donor) and the sivelestat (p = 0.0023, versus Donor) groups by 120 minutes of recirculation.

View larger version (13K): [in this window] [in a new window]   Fig 1. Changes in plasma neutrophil elastase levels before and during recirculation. Values are expressed as the mean ± standard error of the mean. • = control group; = sivelestat group; p < 0.05, p < 0.01 by two-way analysis of variance with Bonferroni correction between the sivelestat group and the control group; *p < 0.05, ***p < 0.001 by one-way analysis of variance as compared with the donor value. (SC = standing control.)

View larger version (10K): [in this window] [in a new window]   Fig 2. Changes in plasma interleukin-8 levels before and during recirculation. Values are expressed as the mean ± standard error of the mean. • = control group; = sivelestat group; p < 0.01 by two-way analysis of variance with Bonferroni correction between the sivelestat group and the control group; ***p < 0.001 by one-way analysis of variance as compared with the donor value. (SC = standing control.)

The F-actin content of the neutrophils increased after 30 minutes of recirculation in the control group. The sivelestat group did not show any significant increases during recirculation. There were significant differences between the two groups over time (Fig 3; p = 0.0037).

View larger version (12K): [in this window] [in a new window]   Fig 3. The expression of neutrophil F-actin before and during recirculation. Data points are standardized as a percentage of the donor value for each time point. Values are expressed as the mean ± standard error of the mean. • = control group; = sivelestat group; p < 0.05, p < 0.01 by two-way analysis of variance with Bonferroni correction between the sivelestat group and the control group; *p < 0.05, **p < 0.01, ***p < 0.001 by one-way analysis of variance as compared with the donor value. (SC = standing control.)

View larger version (11K): [in this window] [in a new window]   Fig 4. Changes in the microchannel transit time before and during recirculation. Data points are standardized as a percentage of the donor value for each time point. Values are expressed as the mean ± standard error of the mean. • = control group; = sivelestat group; p < 0.01 by two-way analysis of variance with Bonferroni correction between the sivelestat group and the control group; *p < 0.05, ***p < 0.001 by one-way analysis of variance as compared with the donor value. (SC = standing control.)

	Comment

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Plasma neutrophil elastase levels were reduced by the sivelestat administration, though the drug is reported to inhibit the activity of secreted neutrophil elastase competitively without direct inhibition on its release [5]. A similar result was observed in our previous study with ONO-6818 [14]. It could be that further amplification of neutrophil degranulation is attenuated by the reduction of IL-8 and possibly other chemoattractant levels through the inhibition of neutrophil elastase activity. A recent study demonstrating sivelestat directly inhibits the production and secretion of intracellular neutrophil elastase, as well as the extracellular elastase activity in neutrophil-mediated endothelial cell injury, supports this data [22].

The functional responses of neutrophils including shape change, adherence, and transendothelial migration are induced after chemotactic stimulation. All these responses are initiated by the binding of the agonist to its receptor, and the subsequent interaction of the agonist-receptor complex with a GTP-binding protein. This event further leads to a transient rise in cytosolic calcium levels [20, 23]. Because cyclic adenosine monophosphate, an intracellular messenger, modulates F-actin expression via the regulation of cytosolic calcium levels as demonstrated in a previous study [24], it seems appropriate to endorse the fact that sivelestat attenuates F-actin expression if the drug reduces the levels of chemotactic agonists such as IL-8.

During neutrophil activation, rapid changes occur in the amount of polymerized actin and in the structure of the microfilament network inside the cells with a rapid increase of F-actin assembly from the G-actin pool. The decrease in the neutrophil deformability is mediated by this rapid assembly of F-actin [25]. Therefore, in the present study, it is probable that the attenuation of the changes in the neutrophil cytoskeleton with F-actin assembly is responsible for the preservation of cell deformability in the sivelestat group. Although platelet microaggregates and conjugates may affect the results of the transit time data [15], sivelestat is not supposed to act upon platelets and there were no differences in platelet counts or aggregation response to adenosine diphosphate as measured by an aggregometer (PAC-4S; NBS Hematracer, Tokyo, Japan) between the two groups (data not shown). Likewise, there were no differences in hematocrit between groups. Because the loss of neutrophil deformability would contribute to cell sequestration in vivo [26], our results from the cytoskeleton and deformability tests suggest that sivelestat may contribute to the control of neutrophil sequestration in organ capillaries, even if the drug does not alter the adhesion molecules [27].

The fact that the adhesion molecules are not modulated by the drug but the cytoskeleton is not easily explained, but this may have to with the in vitro nature of the model used in the experiment. The fact that no neutrophil-endothelial cell interaction occurs may be responsible for this perceived inconsistency. A recent ex vivo study suggested this possibility when it demonstrated that the expression of E-selectin, a key molecule in the neutrophil-endothelial cell interaction, was increased by the adhesive reaction between neutrophils and endothelial cells after supplementation of neutrophil elastase, and such responses were inhibited by a synthetic neutrophil elastase inhibitor, ZD 8321 [28].

Another limitation of this study is the fact that the in vitro CPB model used does not consider the effects of hemodilution, hypothermia, or ischemia-reperfusion. Moreover, the model does not involve the role of tissue factor because of the absence of a wound. Therefore, the results of this study cannot be immediately applied clinically or directly compared with the results of in vivo studies. However, a similar simulated extracorporeal circulation model has been used extensively to evaluate inhibitors [14, 24]. The system prevents the loss of cells or markers from the circuit, and also prevents new cells being recruited. This is a screening method and is not intended to be a substitute for animal CPB studies. In addition, we did not study the effects of complement activation. The results of previous studies [10, 14] suggest that the neutrophil elastase activity may have close interactions with components of the complement cascade.

In summary, the results of this study provide incremental knowledge regarding the beneficial effects of specific neutrophil elastase inhibition by sivelestat on the attenuation of the inflammatory mediators and preservation of the neutrophil cytoskeleton and deformability during simulated CPB. Although we cannot elucidate the mechanism, sivelestat has a clear advantage over a similar synthetic inhibitor, ONO-6818, regarding its effect on cytoskeleton and cell deformability maintenance. Although limiting initial neutrophil activation is, in principle, a more appropriate and efficacious strategy, inhibition of the extracellular elastase activity may be a more practical approach given our current state understanding. Further investigations in an in vivo primate CPB are necessary to completely clarify the efficacy of sivelestat, one of the most promising neutrophil elastase inhibitors, on the attenuation of the neutrophil-derived inflammatory response and subsequent organ injury.

	The Society of Thoracic Surgeons Policy Action Center

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The Society of Thoracic Surgeons (STS) is pleased to announce a new member benefit—the STS Policy Action Center, a website that allows STS members to participate in change in Washington, DC. This easy, interactive, hassle-free site allows members to:

• Personally contact legislators with one’s input on key issues relevant to cardiothoracic surgery

• Write and send an editorial opinion to one’s local media

• E-mail senators and representatives about upcoming medical liability reform legislation

• Track congressional campaigns in one’s district—and become involved

• Research the proposed policies that help—or hurt— one’s practice

• Take action on behalf of cardiothoracic surgery

This website is now available at www.sts.org/takeaction.

	Acknowledgments

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The authors wish to thank Dr Joseph H. Gorman III for reviewing the manuscript; Avi Landau for language assistance; and Dr Shiro Hinotsu for statistical assistance. This work was supported by a University of Tsukuba Institutional Research Grant in 2003.

	References

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Personal Folders Download to citation manager Author home page(s): Yuji Hiramatsu Yuzuru Sakakibara Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Matsuzaki, K. Articles by Sakakibara, Y. Search for Related Content PubMed PubMed Citation Articles by Matsuzaki, K. Articles by Sakakibara, Y.