Leukocyte infiltration into tissues is essential to inflammation, and matrix metalloproteinases (MMPs) help support the extravasation and infiltration of leukocytes. MMPs belong to a family of more than 20 zinc-dependent endopeptidases, including collagenases, stromelysins, gelatinases, matrilysins, and membrane-type MMPs (1). In nature, they degrade the basement membrane and extracellular matrix to facilitate embryo development, morphogenesis, and angiogenesis. They also play a critical role in wound healing, inflammatory diseases, and tumor metastasis. MMP-9, also called gelatinase B, is mainly produced by inflammatory cells, such as neutrophils, monocytes/macrophages, and eosinophils (1). During an acute inflammatory response, neutrophils are chemo-attracted to the inflammatory site. MMP-9 is degranulated to degrade type IV collagen, the major constituent of basement membrane, and to facilitate neutrophil extravasation. Significant expression of MMP-9 in inflammatory diseases, such as rheumatoid arthritis (2), asthma (3), sepsis (4), and acute respiratory distress syndrome (ARDS) (5), has been demonstrated. Moreover, increased levels of plasma MMP-9 are detected in acute coronary syndrome (6) and myocardial ischemia-reperfusion (7) and may be predictive for ventricular remodeling after myocardial infarction (8).
There is increasing evidence that cardiopulmonary bypass (CPB) causes a systemic inflammatory response syndrome (9), including activation of neutrophils (10). Activated neutrophils may release a number of reactive oxygen species and proteolytic enzymes, including MMPs and neutrophil elastase (11). Increased circulating MMP-9 levels have been observed in patients undergoing cardiac surgery with CPB (12,13). Nonetheless, the possible sources of increased plasma MMP-9 during CPB have not been examined. We thus hypothesized that an increase of plasma MMP-9 may be caused by activation of neutrophils during cardiac surgery with CPB. The aim of the present study was to investigate the MMP-9 production and expression of circulating neutrophils in patients undergoing cardiac surgery with CPB. To distinguish the effect of major surgery on MMP-9 production, we also examined those patients receiving off-pump cardiac surgery. In addition, the MMP-9 activity, as reflected by zymography, and correlation with tissue inhibitors of metalloproteinase (TIMPs) were also determined.
This study was approved by the ethics committee, and written informed consent was obtained from 21 patients undergoing elective coronary artery bypass grafting (CABG) surgery. CPB was applied in 16 patients, and off-pump technique was used for 5 patients. Patients with rheumatoid arthritis, asthma, chronic bronchitis, cancer, autoimmune disease, or receiving steroid or nonsteroidal antiinflammatory drug therapy were excluded from the study. Aspirin was discontinued in all patients 5 days before the operation.
Patients were premedicated with midazolam for arterial catheterization. Anesthesia was induced with thiopental and maintained with isoflurane in oxygen, fentanyl, and pancuronium. Sixteen patients underwent routine median sternotomy and standard hypothermic CPB (Sarns 8000; Terumo, Ann Arbor, MI) with an extracorporeal membrane oxygenator (Capiox®SX 18; Terumo). In the control group, five patients underwent off-pump CABG with a stabilization device (Guidant, Santa Clara, CA). Porcine heparin was administered to achieve anticoagulation before CPB (activated coagulation time, >480 s by heparin 300 U/kg) or before application of a stabilization device in an off-pump technique (activated coagulation time, 250–350 s by heparin 200 U/kg) and was neutralized with protamine sulfate after grafting. Antifibrinolytic drugs, such as aprotinin, were not used in these patients. Total CPB time and number of bypass grafts were recorded.
Blood was obtained from an indwelling arterial catheter at 7 sequential time points as follows: before the induction of anesthesia, before the incision, before the initiation of CPB, and 30 min, 2 h, 4 h, and 6 h after beginning CPB. In patients having off-pump CABG, the sampling time points were as follows: before the induction, before the incision, before the initiation of the stabilization device, and 30 min, 2 h, 4 h, and 6 h after the initiation of the stabilization device. All blood samples were collected into ethylenediamine tetraacetic acid-containing tubes.
After immediate centrifugation at 1500g for 10 min at room temperature, polymorphonuclear neutrophils (PMNs) were isolated from buffy coats by Ficoll-Hypaque (Amersham Pharmacia, Little Chalfont, Bucks, UK) density gradient separation. With the use of a red blood cell lysing solution, the contaminating red cells were lysed, and the PMNs were washed three times. Neutrophil purity determined by flow cytometry exceeded 95%, and cell viability was more than 98%, as determined by trypan blue exclusion. The plasma was frozen at −70°C for later testing.
Enzyme-linked immunosorbent assays (ELISA) were performed to determine the plasma concentrations of MMP-9 and TIMP-1 in each sample. Plasma MMP-9 levels were quantified using the Human MMP-9 (Total) Immunoassay Kit, and TIMP-1 levels were quantified using the DuoSet ELISA Development System (R&D, Minneapolis, MN).
Intracellular MMP-9 was analyzed in neutrophils using a modification of published protocols (14). PMNs (106 per condition) were resuspended in 100 μL of phosphate-buffered saline (PBS). After incubation with fluorescein isothiocyanate-conjugated mouse anti-human CD16b antibody (Chemicon, Temecula, CA) at room temperature in the dark, the PMNs were washed twice with PBS. PMNs were permeabilized and fixed using IntraPrep™ Permeabilization Reagent (Immunotech, Westbrook, ME) and then stained intracellularly with mouse anti-human MMP-9 monoclonal antibody (Chemicon). After being washed twice, the PMNs were resuspended in PBS containing rPE-conjugated F (ab’)2 goat antimouse immunoglobulin (Ig)G antibody (Serotec, Kidlington, Oxford, UK). PMNs were then analyzed using dual-color flow cytometry (FACScan cytometer; Becton Dickinson, San Jose, CA). Each data point was represented by the mean fluorescence intensity of 30,000 gated events.
Total RNA was extracted from neutrophils (1 × 106 cells) using TRIzol reagent (Gibco BRL, Grand Island, NY) and was reversely transcribed using SuperScript II RNase H Reverse Transcriptase (Gibco BRL). Polymerase chain reaction (PCR) was performed, as previously described (15). Briefly, a minimum of three different complimentary DNA concentrations served as the templates for amplification through 25–30 cycles of denaturation (30 s at 95°C), primer annealing (30 s at 55°C), and DNA extension (30 s at 72°C) in a Gene Amp PCR System 2400 (Perkin-Elmer Corp, Narwalk, NJ). Amplification of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used to produce an internal quality standard. The primer sequences were as follows: MMP-9 sense 5′-CCA AAC TGG ATG ACG ATG TCT-3′, antisense 5′-CGT GGA GAG TCG AAA TCT CTG-3′; GAPDH sense 5′-GGG GAG CCA AAA GGG TCA TCA TCT-3′, and anti-sense 5′-GAG GGG CCA TCC ACA GTC TTC T-3′. Amplified products were electrophoresed on 2% agarose gels and then stained with ethidium bromide; a 100-bp DNA ladder (Gibco BRL) was used as a molecular weight marker. The experimental condition and number of PCR cycles were predetermined to ensure the amount of MMP-9 and housekeeping-gene (GAPDH) fragments were in the linear range of amplification. GAPDH was used as the standard to control variations in RNA isolation. The MMP-9 bands were quantified using Photo-Capt (Vilber Lourmat, Marne LuVallee Cedex, France) and represented as percentages of the preinduction values.
Gelatin zymography was performed to determine the proteolytic activities of plasma MMP-9. Each sample, containing 10 μg of total protein, was electrophoresed on a sodium dodecyl sulfate-polyacrylamide gel (10%) containing 0.1% gelatin as the substrate at 130 V for 4–5 h. After electrophoresis, the gel was washed in 2.5% Triton X-100 for 1 h at room temperature and then incubated overnight at 37°C with an incubation buffer (1 M of Tris, pH value of 7.6, 1 M of NaCl, and 1 M of CaCl2; 30% Brij-35). After incubation, the gel was stained with 0.2% (wt/vol) solution of Coomassie blue and then destained in a solution of 10% acetic acid and 30% methanol. The gelatinolytic activity of each MMP-9 sample was qualitatively evaluated as a clear band against the blue-stained gelatin background. The bands were quantified using Photo-Capt (Vilber Lourmat) and were represented as percentages of the preinduction values.
Demographic data including age, sex, body weight, body height, total CPB time, and number of artery grafts were summarized as mean ± sem. Generalized estimating equations (GEEs) were introduced as a method of dealing with correlated data in applied science (16). We used the GEE to assess the group-, time-, and group-by-time effects, as well as to adjust the correlations arising from repeated measurements with the SAS 8.2 (SAS, Cary, NC). A P value <0.05 was considered indicative of significant differences.
Demographic data are summarized in Table 1. As shown in Figure 1, the plasma levels of MMP-9 in patients with CPB increased significantly 2–6 h after the start of CPB. Peak level of MMP-9 occurred 4 h after beginning CPB (312 ± 35 ng/mL; P < 0.01; compared with 74 ± 8 ng/mL before the induction). In differentiation of CPB from operation per se as the cause of an increase of MMP-9 levels, we found no significant change in plasma MMP-9 levels in patients undergoing off-pump CABG. Using GEE (Table 2), there was an interaction effect on group-by-time (P = 0.033), indicating significant increased levels of MMP-9 over time in patients with CPB, comparing with that in patients without it. Besides, there was no significant difference in preinduction MMP-9 levels between groups (P = 0.190), nor was there MMP-9 alteration in patients who underwent off-pump CABG on time effect (P = 0.631).
Using flow cytometry, intracellular MMP-9 protein in isolated neutrophils (Fig. 2) was detected by double staining of neutrophils and MMP-9, presented as mean fluorescence intensity. The peak mean fluorescence intensity value occurred 2 h after the start of CPB (189 ± 36; P < 0.01; compared with 32 ± 5 before the induction). The effect of CPB on transcriptional expression of MMP-9 messenger (m)RNA in neutrophils was further studied. As shown in Figure 3, the mRNA expression of MMP-9 increased gradually to the peak value 4 h after beginning CPB (up to 182% of the preinduction value; P < 0.01). In clarification of MMP-9 enzyme activity, gelatin zymography (Fig. 4) revealed significant increases with peak value occurring 2 h after the start of CPB (up to 184% of the preinduction value; P < 0.01).
To investigate the balance between MMP-9 and TIMP-1, the plasma levels of TIMP-1 were examined (Fig. 5). Plasma TIMP-1 levels in the patients with CPB decreased initially to the smallest value 30 min after beginning CPB (175 ± 18 ng/mL; P < 0.01; compared with 272 ± 25 ng/mL before the induction) and then increased gradually for 6 h after the start of CPB (466 ± 43 ng/mL; P < 0.01; compared with 272 ± 25 ng/mL before the induction), whereas those in patients without CPB increased gradually but not significantly (Fig. 5). Furthermore, the MMP-9/TIMP-1 ratios increased gradually and exceeded 1 at 2 and 4 h (1.19 and 1.39, respectively) after beginning CPB (Fig. 6).
MMP-9 degrades basement membrane and extracellular matrix and facilitates extravasation of leukocytes and tumor cells (1). Increased MMP-9 levels have been detected in many inflammatory diseases, tumor invasion, and cardiovascular diseases. In this timecourse study, we found that plasma MMP-9 levels increased significantly after beginning CPB, whereas the MMP-9 levels did not increase in patients with off-pump cardiac surgery. Moreover, we clearly demonstrated that both mRNA expression and production of MMP-9 increased in isolated neutrophils after CPB.
Neutrophils are activated initially in the inflammatory response. Degranulation of the inflammatory mediators from neutrophils, such as reactive oxygen species and proinflammatory cytokines (11), could cause pulmonary dysfunction after cardiac surgery with CPB by augmenting both neutrophil-pulmonary endothelial adhesion and change of alveolar-endothelial permeability (17). Neutrophils are the major source of MMP-9 in glycogen-induced peritonitis (18) and pancreatitis-associated lung injury (19). In this study, we demonstrated that intracellular MMP-9 protein and mRNA expression of neutrophils increased after beginning CPB, consistent with the concept that neutrophils may contribute to the increase of plasma MMP-9 during cardiac surgery with CPB.
The MMP family is classified by substrates, such as collagenases, gelatinases, or matrilysins. Some of them are synthesized in specific tissues. For examples, MMP-1 is a predominant form of interstitial collagenase in many tissues, whereas MMP-2 (gelatinase A) expression is limited to myocardial and stromal cells. MMP-7, one of matrilysins, is found in the glandular epithelial cells of the gastrointestinal tract and endometrium (1). Most MMPs are synthesized and secreted from cells as latent proenzymes (zymogens). In neutrophils, pro-MMP-9 is stored in tertiary granules (11). Once neutrophils are activated, pro-MMP-9 is degranulated into the extracellular space and then activated by proteinases (including plasmin, trypsin, or neutrophil elastinase), by nonproteolytic compounds such as SH reactive agents, or by heat treatment (11). The α2-macroglobulins can serve to eliminate MMP-9 by binding its exposed bait sequence (20), resulting in conformational change in the macroglobulin molecule and entrapment of the proteinases. At termination of CPB in cardiac surgeries, Mayers et al. (21) found pro-MMP-9 activity increased both in heart tissues and plasma, whereas pro-MMP-2 activity increased in the heart tissues but not in plasma. In this study, we found that the enzyme activity of MMP-9 increased significantly in the beginning of CPB and declined after the end of CPB, indicating increased MMP-9 activation during CPB.
TIMP-1, the inhibitor of MMP-9, is secreted by various types of cells to inhibit catalytic activity of MMP-9 in vivo through the formation of a tight, noncovalent complex (1). In our study, the plasma TIMP-1 levels were downregulated in the beginning of CPB and then increased gradually later for six hours after the start of CPB. It suggested that the upregulation of TIMP-1 was related to the increased activity and production of MMP-9 in patients with CPB. Furthermore, the imbalance between MMPs and TIMPs has been proposed to play a role in the pathogenesis of ARDS. In bronchoalveolar lavage fluid, Ricou et al. (22) demonstrated increased MMP-9 levels in the early phase of ARDS, whereas the MMP-9/TIMP-1 ratio remained increased (>1) in the late phase of the disease. Postoperative pulmonary dysfunction is recognized as the result of CPB-associated systemic inflammatory response in patients undergoing cardiac surgery (17). There are no studies concerning the correlation between MMP-9 and clinical outcomes after cardiac surgery. However, in animal studies of myocardial infarction, reduced infarct size (23), less ventricular dilation (24), and less frequent cardiac rupture (25) are found in MMP-9 knockout mice. In our study, we clearly demonstrated that neutrophil-mediated MMP-9 expression and secretion as well as MMP-9/TIMP-1 ratio increased during cardiac surgery with CPB. Further investigation is required to verify the significance of our finding on the pathogenesis of acute lung injury after CPB.
The increasing popularity of off-pump CABG surgery reflects an attempt to avoid the postoperative morbidity associated with CPB (26). There is growing evidence that the inflammatory mediators, including cytokines (tumor necrosis factor-α, interleukin [IL]-6, IL-8, and IL-10) (27) and neutrophil activation (28), diminish in patients undergoing off-pump CABG surgery. Similarly, we observed no significant changes in plasma MMP-9 and TIMP-1 levels in patients having off-pump CABG surgery. However, more patients are required to determine the clinical significance of MMP-9 and TIMP-1 between off-pump CABG and CPB.
In summary, we demonstrated increases of MMP-9 mRNA expression and production of neutrophils during cardiac surgery with CPB. The increased MMP-9 activity and MMP-9/TIMP-1 ratios may both play an important role in the systemic inflammatory response after CPB.
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© 2005 International Anesthesia Research Society
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