Reperfusion therapies in coronary heart disease, such as thrombolytic therapy and percutaneous transluminal coronary angioplasty, have made it possible to reduce the mortality rate dramatically (1-3). There is experimental evidence that reperfusion itself after sustained a ischemic condition augments myocardial injury (4). The phenomenon is termed "reperfusion injury." After myocardial ischemia-reperfusion, there is a marked accumulation of neutrophils, and these cells have been implicated in myocardial injury (5,6). It has been reported in a number of experimental studies that neutrophil depletion (7,8) or inhibition of neutrophil activation (9,10) significantly attenuates myocardial-reperfusion injury.
In normal conditions, the circulating neutrophils have continuous random contacts with the vascular wall. However, once vascular endothelium is activated by the ischemia-reperfusion stimulus, neutrophils begin to roll along the luminal surface of the endothelium (11,12). Subsequently the rolling neutrophils are activated by chemoattractants immobilized to proteoglycans on the endothelium (13), and neutrophil adhesion is strengthened by interactions between β2 integrin (LFA-1, Mac-1) and intercellular adhesion molecule-1 (ICAM-1). Then the neutrophils transmigrate to myocardial tissue, produce active oxygen, release proteolytic enzymes and inflammatory cytokines, and consequently induce tissue injury (14,15). Accordingly, inhibition of neutrophil accumulation to myocardium or regulation of activated neutrophils has been expected to become a useful therapy for reperfusion injury. Neutrophil rolling is principally mediated by the selectin family of adhesion molecules, such as P- (16,17), L- (18,19), and E-selectin (20,21). In vitro studies have demonstrated that L-selectin is constitutively expressed on the surface of neutrophils, monocytes, and certain subsets of lymphocytes and rapidly shed after activation (22,23). E-selectin is expressed after de novo synthesis after activation of endothelial cells by inflammatory cytokines [e.g., tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β)] (24,25). P-selectin is stored in α granules of platelets and Weibel-Palade bodies of endothelial cells (26,27) and is rapidly translocated to the surface of these cell types in response to inflammatory stimuli such as thrombin, histamine, or oxidants (28,29).
More recently, treatments with monoclonal antibodies against P- (30-32), L- (33,34), and E-selectin (35) or analogs of sialyl Lewis X (sLeX) ligands of the selectin family (36,37) significantly inhibited neutrophil accumulation to myocardial tissue after ischemia-reperfusion and protected the heart against myocardial injury and endothelium dysfunction. Furthermore, it was reported that inhibition of firm adhesion of neutrophils to endothelium by monoclonal antibodies against either CD18 (38,39) or ICAM-1 (40-42) also significantly attenuates myocardial ischemia-reperfusion injury.
The polysaccharide fucoidin has been demonstrated to block leukocyte adhesion to P- (43,44) or L-selectin (45,46) in vitro. Intravital microscopy studies have shown that fucoidin inhibited P- or L-selectin-mediated leukocyte rolling in mesenteric postcapillary venules (12,47). Moreover, fucoidin attenuated leukocyte-dependent central nervous system damage in rabbit bacterial meningitis (48). However, the effect of fucoidin on the myocardial-injury model is undetermined.
In our study, therefore, we investigated whether polysaccharide fucoidin protects the myocardium against ischemia-reperfusion injury in rat.
Intravital microscopic experiment of leukocyte rolling
Animal preparation for the intravital microscopic study was carried out according to the method described by Suematsu et al. (49). In brief, male Wistar rats (Charles River Japan Inc., Kanagawa, Japan) weighing 250-350 g fasted for 24 h and were anesthetized with sodium pentobarbital (50 mg/kg, i.m.). The trachea was cannulated to facilitate spontaneous breathing. A midline abdominal incision was made, and a segment of the ileum was exteriorized from the peritoneal cavity and placed on a transparent pedestal to allow microscopic observation of the mesenteric microcirculation. To protect against mast-cell degranulation caused by surgical preparation, disodium cromoglycate (5 mg/kg; Biomol Research Laboratory, Plymouth Meeting, PA, U.S.A.) was administered intravenously before exteriorization of the mesentery (50). The exposed tissue was superfused with warmed bicarbonate-buffered saline (37°C, pH 7.4) bubbled with 5% CO2/95% N2. Observation of the mesenteric microcirculation was made by using an inverted microscope (Diaphot-TMD, Nikon, Tokyo, Japan) with a ×40 objective lens connected with a videocamera (C2400; Hamamatsu Photonics). The microscopic image was televised (TM-21; Victor, Tokyo, Japan) and recorded on videotape (BR-S810; Victor). Single unbranched venules (length, 100 μm) with diameters ranging between 20 and 40 μm were selected to analyze rolling of leukocytes on the vessel wall.
Rolling of leukocytes was measured according to the method described by Kubes and Kanwar (50). Rolling leukocytes were defined as those white blood cells that moved at a velocity less than that of red blood cells in the same stream of blood. The flux of rolling leukocytes (FWBC) was determined by counting the number of rolling leukocytes per min passing a reference point in the microvessel. FWBC was expressed as the number of leukocytes per second. Leukocyte rolling velocity (VWBC) was determined by measuring the time required to roll 100 μm of the length of the venule. The number of rolling leukocytes per 100-μm venule length was calculated as FWBC/VWBC.
Histamine was superfused for 90 min. Fucoidin infusion into the jugular vein was started from 60 min before histamine (0.1 mM) superfusion and stopped 30 min after histamine superfusion. Because it was reported that an intravenous bolus injection of 10 mg/kg fucoidin inhibits leukocyte rolling in mesenteric venules (48), the same dose (27 μg/kg/min × 370 min = 10 mg/kg) of fucoidin (Sigma, St. Louis, MO, U.S.A.) was infused constantly during the period of 6-h reperfusion in the myocardial ischemia and reperfusion model, as follows. Therefore in the intravital microscopic study, we examined whether fucoidin inhibits leukocyte rolling at a dose of 27 μg/kg/min by using Harvard Apparatus syringe pump (model 975; 13 μl/min).
Surgical preparation of myocardial ischemia-reperfusion model
Induction of myocardial ischemia-reperfusion injury in rats was carried out according to the method described by Selye et al. (51). In brief, male Wistar rats weighing 200-300 g were anesthetized with sodium pentobarbital (50 mg/kg, i.p.). An intratracheal cannula was inserted through a midline incision, and the rats were artificially ventilated with room air (5 ml/100 g/stroke, 60 cycle/min) with a small-animal respirator (model SN-480-7; Shinano Manufacturing Co., Ltd., Tokyo, Japan). A left lateral thoracotomy was performed, and the heart was exposed. A 4-0 silk ligature was placed around the left anterior descending coronary artery (LAD) 1-2 mm from its origin, and the end of the silk ligature was passed through a short length of polyethylene tubing. After the completion of surgical procedure, the rats were allowed to stabilize for ≥15 min, and the LAD was occluded by pulling the snare, which was then fixed by clamping the tube with a hemostat. Heart rate (HR) and ST-segment elevation were monitored by standard lead II of the electrocardiogram (ECG) and recorded on a pen recorder (RM6200; Nihon Kohden, Tokyo, Japan). A polyethylene catheter was inserted through the left carotid artery for the measurement of mean arterial blood pressure (MABP) via a pressure transducer. The pressure-rate index (PRI), an index of myocardial oxygen demand, was calculated as the product of MABP and HR divided by 1,000. After 30 min of coronary occlusion, the LAD ligature was released, and 30 min-6 h after reperfusion, the heart was removed for postmortem analysis. Sham-operated rats were subjected to the same surgical procedures without LAD occlusion.
Fucoidin administration started 10 min before reperfusion and stopped at 2, 4, or 6 h after reperfusion.
Peripheral blood (50 μl) was collected from the tail artery before the infusion of saline or fucoidin infusion and 6 h after infusion. The collected blood was diluted by Turk's stain solution, and circulating leukocytes were counted by using a hemocytometer (Fuchs Rosenthal, Erma, Tokyo, Japan).
Measurement of myocardial-infarct size
Myocardial-infarct size was measured according to the method described by Boor and Reynolds (52). Six hours after reperfusion, the LAD was reoccluded, and 1 ml of 0.5% (wt/vol) Evans blue solution was perfused retrogradely from abdominal aorta into the coronary circulation to distinguish the area at risk from the area of myocardium that was perfused by the nonoccluded coronary arteries. The heart was rapidly excised and washed with phosphate-buffered saline (PBS). The heart was embedded in 0.2% (wt/vol) agarose and then cut transversely into five sections from apex to base. The heart slices were incubated in 1% (wt/vol) triphenyl tetrazolium chloride (TTC) solution (Nacalai tesque Co. Ltd., Kyoto, Japan) in 0.1 M TrIS/HCl buffer solution (pH 8.0) at room temperature. The viable area in the area at risk was stained red by TTC, whereas the necrotic area was not stained by TTC or Evans blue. Each heart slice was weighed, and computerized planimetry of the area at risk and necrotic area was determined by using a computerized image analyzer (IBAS; Carl Zeiss Co., Ltd., Tokyo, Japan).
Immunohistochemical localization of P-selectin expression
Thirty min or 6 h after reperfusion, hearts were perfused with 20 ml of saline and then 20 ml of 4% (wt/vol) paraformaldehyde (PFA) in 0.1 M sodium phosphate buffer solution (pH 7.4) retrogradely from the abdominal aorta. The hearts were removed, postfixed in 4% (wt/vol) PFA for 24 h at 4°C, and then embedded in O.T.C. compound (Tissue-Tek; Miles Inc., Elkhart, IN, U.S.A.) in dry ice. Sections (10μm) were cut vertically along the apex-base axis by using a cryostat and placed on gelatin-coated slides. Immunohistochemical procedures were performed by using the avidin-biotin immunoperoxidase technique (Vectastain ABC Kit; Vector Laboratories, Burlingame, CA, U.S.A.). The sections were sequentially incubated at room temperature each for 30 min with normal horse serum, monoclonal antibody (mAb) to P-selectin (NPL44-10; Takara, Kyoto, Japan), 0.3% (vol/vol) H2O2 in methanol, the biotinylated antimouse antibody, avidin-biotinylated peroxidase complex, and a peroxidase substrate, 0.02% (wt/vol) 3,3′-diaminobenzidine tetrahydrochloride. By using Western blotting under nonreducing conditions of rat platelet lysates, we confirmed that NPL44-10 (Takara), mAb to P-selectin, bound to a band identified as having the same apparent molecular weight as P-selectin (data not shown). The sections were counterstained with Mayer's hematoxylin. Immunohistochemical localization of P-selectin was examined by using microscopy (BH-2; Olympus, Tokyo, Japan).
Measurement of tissue myeloperoxidase (MPO) activity
Myocardial MPO activity was measured according to the method described by Mullane et al. (53). The area at risk was delineated 0, 30, and 60 min and 2, 4, and 6 h after reperfusion, as described previously. Myocardial tissue weighing 150-300 mg was taken from the area at risk, frozen in liquid nitrogen, and stored at −80°C until MPO assay. The frozen myocardial tissue was pulverized, weighed, and suspended in 1 ml of 50 mM potassium phosphate buffer solution (pH 6.0) containing 0.5% (wt/vol) hexadecyltrimethylammonium bromide (Sigma). The samples were blended for 90 s (30 s × 3) in a homogenizer (HG30; Hitachi Koki, Tokyo, Japan). The homogenates were sonicated for 10 s, freeze-thawed 3 times with liquid nitrogen, and were again sonicated for a further 10 s. Specimens were ultracentrifuged at 45,000 g for 15 min, and then the volume of supernatant was measured. The supernatant (10 μl) was mixed with 290 μl of 50 mM potassium phosphate buffer solution (pH 6.0) containing 0.167 mg/ml o-dianisidine hydrochloride (Sigma) and 0.0005% (vol/vol) hydrogen peroxide. The change in absorbance at 460 nm was measured every 30 s for 4 min by using microplate reader (Thermo max; Molecular Devices Corp., Menlo Park, CA, U.S.A.). One unit of MPO activity was defined as that degrading 1 μmol peroxide/min at 25°C, and the results was expressed as units of MPO/100 mg tissue.
All values are expressed as mean ± SEM. Comparisons of the time course of MPO activity after reperfusion were subjected to analysis of variance followed by Dunnett's multiple comparison test. Data between the saline group and the fucoidin-treated group were compared by using the unpaired Student's t test. Values of p < 0.05 were considered to be statistically significant.
Effect of fucoidin on rolling of leukocytes in the mesenteric venule
To test whether fucoidin inhibits rolling of leukocytes, the effect of fucoidin was investigated by using intravital microscopy in rat mesenteric venules. Fucoidin (27 μg/kg/min) markedly inhibited histamine-induced rolling of neutrophil in rat mesenteric venules (Fig. 1). Accordingly, fucoidin in the following experiments was administered at 27 μg/kg/min. When fucoidin infusion was stopped, the inhibitory effect of fucoidin on the neutrophil rolling disappeared within 15 min.
Effect of fucoidin on myocardial infarct size
To test the effect of fucoidin on the degree of actual myocardial salvage of ischemic tissue 6 h after reperfusion, we measured the amount of necrotic cardiac tissue expressed as a percentage of either the area at risk or of left ventricular mass. There was no difference in the area at risk expressed as a percentage of the left ventricular mass between the vehicle- and the fucoidin-treated groups. However, the infarct size expressed as a percentage of the area at risk and that expressed as a percentage of the total left ventricular mass were significantly smaller (p < 0.01) in rats treated with fucoidin (32.5 ± 3.2% and 15.5 ± 1.3%, respectively) than those in the vehicle control group (57.0 ± 3.3% and 28.0 ± 1.9%, respectively), which showed that fucoidin significantly attenuated the myocardial damage induced by ischemia and reperfusion (Fig. 2).
Immunohistochemical localization of P-selectin expression after reperfusion after 30-min ischemia in myocardium
The typical immunohistochemical results of coronary endothelial P-selectin expression are shown in Fig. 3. There was little detectable immunoreactivity of P-selectin on the endothelial surface of venules in sham-operated rat hearts (Fig. 3A). On the other hand, in the myocardium 30 min after reperfusion, P-selectin immunoreactivity was markedly observed on the endothelial surface (Fig. 3C) and persisted 6 h after reperfusion (Fig. 3D).
MPO activity in the ischemia-reperfusion rat hearts
MPO activity as an indicator of neutrophil infiltration into the myocardium of the area at risk increased with time after reperfusion and significantly increased 2 h after reperfusion (p < 0.05). The MPO activity 6 h after reperfusion increased to 0.28 ± 0.05 units/100 mg tissue, which was 100 times higher than that of sham-operated rat heart (Fig. 4).
Treatment with fucoidin (27 μg/kg/min) from 10 min before to 6 h after reperfusion significantly decreased the MPO activity to 0.10 ± 0.03 units/100 mg tissue, which was one third of that of the vehicle control group (Fig. 5).
Although the saline-infused rats had a slight tendency toward leukocytosis (4.38 × 108 cells/ml in preinfusion, 5.25 × 108 cells/ml in postinfusion) by fucoidin infusion for 6 h, the number of leukocytes in peripheral blood significantly increased (p < 0.01) from 3.81 × 108 cells/ml before infusion to 7.19 × 108 cells/ml after the fucoidin infusion.
Effects of various periods of fucoidin infusion on MPO activity were compared to find the minimal effective period of fucoidin infusion. Reduction of neutrophil infiltration could not be detected in rats treated by fucoidin for only 2 h after reperfusion (0.30 ± 0.06 vs. 0.24 ± 0.02 units/100 mg tissue). However, when administration of fucoidin lasted for 4 h, neutrophil infiltration at 6 h was significantly reduced to 0.11 ± 0.03 units/100 mg tissue (Fig. 6).
Effect of fucoidin on cardiac electrophysiology and hemodynamic parameters
Several minutes after LAD occlusion, the ST segment of the ECG was significantly elevated in the saline- and fucoidin-treated groups. There was no significant difference in ST-segment elevation 30 min after occlusion between both groups (data not shown). These results indicated that the ischemic insult was similar in both groups. After reperfusion, the ST-segment elevation returned to nearly preischemic values. Moreover, the PRI also showed no significant difference between both groups during ischemia and reperfusion (Fig. 7), suggesting that administration of fucoidin did not affect myocardial oxygen demand.
One of the pivotal risk factors inducing myocardial ischemia-reperfusion injury seems to be activated neutrophils that infiltrate myocardial tissue. Activated neutrophils produce active oxygen and release proteolytic enzymes, resulting in tissue injury. Neutrophil adhesion to endothelial cells and extravasation are part of a sequential process (54). That is, once myocardial ischemia-reperfusion occurs, (a) rolling of neutrophils is initiated by the selectin family, such as E- or P-selectins expressed on the surface of stimulated endothelium or L-selectin constitutively expressed on the neutrophils, (b) neutrophils rolling on the vessel wall are activated by immobilized chemokines on the surface of endothelial cells, (c) they adhere firmly to the endothelium by β2-integrin interaction with ICAM-1, and (d) they migrate to surrounding tissue. Therefore inhibition of neutrophil adhesion to vessel walls has been regarded as one of the effective therapies for myocardial ischemia-reperfusion injury.
In this study, we took note of the neutrophil rolling that is the first step in the adherent processes. Polysaccharide fucoidin has been known to bind to P- (43,44) or L-selectin (45,46) and to inhibit leukocyte rolling in venules (12,54). Accordingly, we studied the protective effect of fucoidin against myocardial ischemic reperfusion injury in vivo. Because of the short half-life of fucoidin in peripheral blood (47,48) fucoidin was intravenously infused. Indeed, our intravital microscopy study in rat mesenteric venule also showed that the inhibitory effect of fucoidin on neutrophil rolling disappeared within 15 min after stopping fucoidin infusion.
Fucoidin significantly reduced the myocardial infarct size 6 h after reperfusion. The mechanism of this protective effect may not be caused by reducing the degree of the ischemia, because there was no difference in the area at risk as a percentage of the left ventricle of the saline- and fucoidin-treated groups, and ischemia-induced elevation of the ST segement was similar in both groups. In addition, there was no difference in the PRI of the groups. These results rule out the possibility that the cardioprotective effect of fucoidin was related to alterations in myocardial oxygen demand. Because fucoidin also markedly reduced MPO activity in the area at risk, the cardioprotective effect of fucoidin seemed to be caused by its inhibition of neutrophil infiltration into the myocardial tissue. In addition, to attenuate neutrophil infiltration 6 h after reperfusion, fucoidin infusion had to last for ≥4 h after reperfusion.
Immunohistochemical P-selectin expression was highly localized in the venules of myocardial tissue after ischemia-reperfusion. In addition, fucoidin inhibited histamine-induced leukocyte rolling in the mesenteric venules. These results taken together suggest that fucoidin binds to P-selectin expressed on the endothelial cells, inhibits neutrophil rolling, and results in reducing the neutrophil infiltration. Recently it was reported that mAb to P-selectin significantly attenuated ischemia- reperfusion-induced neutrophil rolling in feline (55) and rat (56) mesenteric venules and protected against myocardial ischemia-reperfusion injury in feline (30,32) and canine experimental models (31). Furthermore, we found that chimera-protein of P-selectin and human immunoglobulin-G1 (IgG1) Fc region dramatically inhibited the myocardial ischemia-reperfusion injury in rats (unpublished data). These reports and unpublished data support the hypothesis described. In this model, the expression of P-selectin in myocardial tissue up-regulated within 30 min after reperfusion, whereas there was no significant increase in neutrophil infiltration and P-selectin expression sustained until 6 h after reperfusion. Weyrich et al. (55) showed in a feline myocardial ischemia-reperfusion model that P-selectin is maximally expressed within 20 min after reperfusion. Another ischemia-reperfusion model of rats has shown that P-selectin expression in the myocardium was detected until 10 h after reperfusion (56). Because active oxygen-induced P-selectin expression on endothelial cells is sustained for several hours (29), mediators that upregulated P-selectin expression in this model might be active oxygen derived from ischemia-reperfusion-stimulated endothelium.
Because fucoidin also binds to L-selectin (45,46), it is likely that fucoidin also blocked L-selectin-mediated adhesion. Actually, in the feline model of 90-min myocardial ischemia followed by 4.5-h reperfusion, mAb to L-selectin inhibited neutrophil infiltration and significantly attenuated myocardial necrosis (33,34). Although we do not know whether mAbs to L-selectin protect against myocardial injury in the rat ischemia-reperfusion model, the cardioprotective effect of fucoidin could have been partly caused by inhibition of L-selectin-mediated neutrophil adhesion.
On the other hand, it was recently reported that fucoidin inhibited the migration of neutrophils into the highly expressed sLeX intestinal epithelial cell (57) and that ischemia-reperfusion induced the increase of sLeX expression on myocardial cells (56). From these two reports, it is likely that fucoidin could also inhibit neutrophil migration into highly sLeX-expressing myocardial tissue in addition to the inhibitory effect on neutrophil rolling and adhesion.
Fucoidin infusion increased the number of circulating leukocytes to ∼2 times that before infusion in the rat, as previously reported (48). The mechanism involved in the increase of circulating leukocytes by fucoidin remains undetermined. However, this event excluded the possibility that fucoidin-induced attenuation of neutrophil infiltration into the myocardium could be attributed to the decrease in circulating neutrophils.
In conclusion, we demonstrated for the first time that fucoidin attenuates myocardial-infarct size and neutrophil accumulation in the rat myocardial ischemia- reperfusion injury model. One of inhibitory mechanisms of fucoidin on neutrophil accumulation seemed to be blockade of P-selectin-mediated neutrophil rolling on the vessel wall. We cannot rule out the possibility that cardioprotective effects of fucoidin are the result of mechanisms other than the P-selectin-mediated effect, such as inhibition of L-selectin-mediated neutrophil adhesion.
Acknowledgment: We thank Dr. M. Suematsu (Department of Biochemistry, School of Medicine, Keio University) for his excellent technical advice about intravital microscopic study and Dr. F. Satoh, the Director of the Suntory Institute for Biomedical Research, and Dr. T. Nakanishi, the general manager of Pharmaceutical Research Laboratory II, for their support and encouragement throughout this study.
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