P-selectin and intercellular adhesion molecule-1 (ICAM-1) mediate early interaction and adhesion of neutrophils to coronary endothelial cells and myocytes after myocardial ischemia and reperfusion. In the present study, we examined the physiological consequences of genetic deletions of ICAM-1 and P-selectin in mice. In wild-type mice, after 1 h of ischemia followed by reperfusion, neutrophil influx into the area of ischemia was increased by 3 h with a peak at 24 h and a decline by 72 h. ICAM-1/P-selectin-deficient mice showed a significant reduction in neutrophils by immunohistochemistry or by myeloperoxidase activity at 24 h but no significant difference at 3 h. Infarct size (area of necrosis/area at risk) assessed 24 h after reperfusion was not different between wild-type and deficient mice after 30 min and 1 h of occlusion. Mice with a deficiency in both ICAM-1 and P-selectin have impaired neutrophil trafficking without a difference in infarct size due to myocardial ischemia-reperfusion.
- cell adhesion molecules
- intercellular adhesion molecule 1
polymorphonuclear neutrophils (PMNs) play an important role in the inflammatory response in experimental models of ischemia-reperfusion injury in dogs and other large mammals (15, 17). Inhibition of PMN activation or depletion of PMNs has been shown to reduce myocardial injury after ischemia and reperfusion in these models (31,34). Although larger mammals, such as dogs and primates, have been extensively studied in models of myocardial ischemia and reperfusion, these species are not easily amenable to genetic manipulations, such as the overexpression or targeted disruption of specific genes, which can be routinely performed in the mouse. Because murine models of cardiovascular disease provide a unique opportunity to understand the functional significance of specific genes, we developed a mouse model to study myocardial ischemia and reperfusion injury (19). The process of leukocyte extravasation entails sequential steps of rolling, firm adhesion, and transmigration, involving interactions of molecules in the selectin, immunoglobulin, and integrin gene families. The initial step is the transition from rapid flow of neutrophils to neutrophil rolling, which is mediated by members of the selectin family present on leukocytes (L-selectin) and endothelial cells (P-selectin and E-selectin). P-selectin is stored in the Weibel-Palade bodies, rapidly increases on endothelial cells after exposure to reactive oxygen, and may play a more important role than E-selectin in neutrophil extravasation with cardiac ischemia-reperfusion injury.
Neutrophil activation by chemotactic factors leads to firm adhesion that is dependent primarily on the leukocyte β2(CD11/CD18) integrins and endothelial intercellular adhesion molecule-1 (ICAM-1). ICAM-1 appears to be constitutively expressed at low levels on the endothelial cell surface and can be markedly upregulated by cytokine stimulation. ICAM-1 mRNA is rapidly induced within the first hour of reperfusion, followed by cardiac myocyte ICAM-1 protein expression within 6 h of reperfusion (14). The rapid induction of ICAM-1 occurs in viable myocytes immediately adjacent to nonviable myocardial cells, which coincides with the region of maximal neutrophil infiltration. Neutrophil adhesion to myocytes in vitro has been shown to be mediated by the interaction between ICAM-1 on myocytes and neutrophil CD11b (4).
Genetically altered mice have been generated with targeted mutations leading to a deficiency in ICAM-1 (35), P-selectin (18), and both ICAM-1 and P-selectin (2). As might be predicted by the multiple-step paradigm of sequential rolling and firm adhesion, mice with a deficiency in both ICAM-1 and P-selectin show a greater defect in neutrophil migration in a peritoneal model (2) than mice with a deficiency of either ICAM-1 or P-selectin alone.
In this paper, we characterize the time course and localization of neutrophil trafficking with ischemia-reperfusion injury in the mouse. In addition, we examine the impact of a combined deficiency of ICAM-1/P-selectin on neutrophil trafficking and infarct size.
MATERIALS AND METHODS
Mouse Model of Myocardial Ischemia-Reperfusion Injury
Male C57/BL6 mice 8–12 wk of age (20–30 g body wt,n = 105 control mice) were obtained from Harlan Sprague Dawley (Houston, TX). Mice deficient in both ICAM-1 and P-selectin were developed by targeted homologous recombination as previously reported (2) and backcrossed with C57/BL6 mice for six generations. Mice were confirmed to be homozygous for the combined mutations by Southern blot.
Animals were anesthetized by an intraperitoneal injection of pentobarbital sodium (4 mg/ml), 10 ml/kg weight of mouse. Midline sternotomy followed by sternal retraction was performed to permit the visualization and ligation of the left anterior descending artery (LAD) under a microscope (Stemi 2000-C, Carl Zeiss) as previously described by Michael et al. (19). Reperfusion of the previously occluded coronary bed was confirmed by visual inspection and surface electrocardiogram changes. The chest was then closed and the animal allowed to recover under a heating lamp.
Tissues were fixed in 10% paraformaldehyde or Z-fix (Anatech, Battle Creek, MI) for at least 3 h, dehydrated with ethanol (50–100%), cleared with xylene, and embedded in paraffin. The sections (5 μm thick) were rehydrated with deionized water in preparation for immunohistochemistry and were incubated with the primary antibody in a humid chamber overnight at 4°C or for 2 h at room temperature. After the sections were washed in phosphate-buffered saline, the secondary biotinylated antibody (Vector Laboratories, Burlingame, CA) was incubated for 30 min at room temperature. The avidin-biotin complex was incubated for 45 min and washed for 3 min. In a humid chamber, a drop of 3,3′-diaminobenzidine solution was used as the substrate for the detection of neutrophils and myosin, and 3-amino-9-ethyl-carbazole was used as a substrate for the myoglobin.
Immunohistochemical staining for neutrophils was performed using the rat monoclonal antibody anti-mouse neutrophils AMU-0021 (Biosource, Camarillo, CA) as a primary antibody and anti-rat IgG biotinylated monoclonal antibody as a secondary antibody (Vectastain, ABC Kit Peroxidase Rat IgG PK-4004, Vector Laboratories).
Immunohistochemical staining for the infarcted area was performed using rabbit anti-human myoglobin monoclonal antibody (Dako) as a primary antibody and anti-rabbit IgG biotinylated monoclonal antibody as a secondary antibody (Vectastain, ABC Kit Elite Rabbit IgG PK-6101, Vector Laboratories, Burlingame, CA). Immunohistochemical identification of myocytes undergoing necrosis was performed with myosin monoclonal antibody (23). Animals were injected with 3–48 S2 mouse antibody to cardiac myosin heavy chains intravenously (100 mg) 1 h before surgery. The animals were euthanized 24 h postreperfusion, and the hearts were fixed with Z-fix for immunohistochemical staining.
Assessment of Leukocyte Trafficking
Assessment of peripheral white blood cell count.
Blood was drawn from the tail vein and added (10 μl) to Isoton II diluent (Coulter, Miami, FL); then erythrocytes were lysed (Manual Lyse, Stephens Scientific, Riverdale, NJ). The total number of leukocytes was determined (Coulter Counter ZM, Coulter Electronics, Hiateach, FL). Blood smears were prepared with Neat stain (Midlantic Biomedical, Paulsboro, NJ) and analyzed under a Nikon phase contrast microscope. Blood was drawn before surgery or after 30 min of ischemia in five wild-type mice and five ICAM-1/P-selectin knockout mice at each time point.
Neutrophil counting in myocardium.
Neutrophils were counted manually using an Axioskop microscope (Carl Zeiss) at a power of ×100–400. Immunohistochemical staining of neutrophils was confirmed by morphological assessment in serial sections stained with hematoxylin and eosin.
Three transverse sections of the heart [the base (above the occlusion), the middle section (at the level of the LAD occlusion), and the apex (below the occlusion)] were analyzed by immunohistochemistry to quantify neutrophil infiltration in the myocardium and to define the localization of neutrophils in the necrotic zone. The area of the left ventricle (LV) in each section was determined by using an image analysis software program (Image Tools, Houston, TX). Neutrophil numbers in each section were expressed per millimeter squared of section.
Assay for myeloperoxidase activity.
For preparation of the extracts, the tissues were minced and homogenized in 500 ml of phosphate buffer (0.002 M, pH = 7.4) and placed in aliquots. The samples were spun 15 min at 14,000 rpm. The supernatant was discarded, and pellets were dissolved in 300 ml of 5 mM citrate buffer (pH 5.0, 0.5% hexadecyltrimethylammonium bromide). The samples were sonicated until homogenized, freeze-thawed three times (liquid nitrogen/37°C water bath), sonicated again, and spun 15 min at 14,000 rpm. The supernatant was collected and kept in a new tube. A dilution of 1:5 or 1:10 was required for our samples.
Serial dilutions of neutrophil extract were prepared (for the calibration curve) in 10 mM citrate (pH 5.0), and 75 ml of these dilutions were added to wells of a 96-well, flat-bottomed tissue culture plate (Ninc, GIBCO-BRL, Mississauga, Ontario, Canada). The substrate solution consisted of 3 mM 3,3′,5,5′-tetramethylbenzidine dissolved in 5 mM citrate buffer (pH 5.0, DMSO) and 8.8 mM H2O2 freshly prepared from a 30% stock H2O2 (Fisher Scientific, Montreal, Quebec, Canada). The reaction was started by adding 75 μl of substrate solution; after 2-min protection from light, the reaction was stopped by the addition of 150 μl cold 4 N H2SO4. Absorbance was read at 450 nm. Neutrophils extracted from bone marrow were used to obtain a calibration curve of the absorbance versus the number of neutrophils.
Assessment of Area at Risk and Infarct Size
The aorta was cannulated with a 22-gauge Luer stub, and 1% Evans blue was perfused into the aorta and coronary arteries with distribution throughout the ventricular wall proximal to the coronary artery ligature. After this procedure, the heart was sectioned transversely into four sections, with one section being made at the site of the ligature. Sections of the ventricle were then incubated in 1.5% triphenyltetrazolium chloride (TTC). After TTC staining, viable myocardium is brick red and the infarct appears pale white. The sections were weighed. The apical side of each slice was imaged, and the area of infarction for each slice was determined by computerized planimetry using an image analysis software program (Image Tools, Houston, TX).
The size of infarction was determined by the following equations. Weight of infarction = (A 1 × Wt1) + (A 2 × Wt2) + (A 3 × Wt3) + (A 4 × Wt4), where A is percent area of infarction by planimetry and Wt is the weight of each section. Percentage of infarcted LV = (weight of infarction/weight of LV) × 100. Area at risk (AAR) as a percentage of LV = (weight of LV − weight of LV stained blue)/weight of LV.
In the first protocol, for the characterization of leukocyte trafficking in C57/BL6 control wild-type mice: 1 h of ischemia was followed by 3, 24, 72 h, and 7 days of reperfusion.
In the second protocol, for the characterization of leukocyte trafficking in ICAM-1/P-selectin-double knockout mice, 1 h of ischemia was followed by 3 and 24 h of reperfusion.
Finally, for the characterization of infarct size, infarct size was assessed in mice deficient in both ICAM-1 and P-selectin after either 30 min or 1 h of ischemia followed by 24 h of reperfusion.
All data are expressed as means ± SE. One-way ANOVA was used to compare neutrophil accumulation at different time points. Statistical comparisons between each transgenic or antibody treatment group and the control group were made with Wilcoxon's two-sample rank-sum test (Mann-Whitney test) with P values corrected by the Bonferroni method. Values of P < 0.05 were considered significant.
Characterization of Leukocyte Trafficking in C57/BL6 Mice
Immunohistochemical staining of the heart at various time points after 1 h of LAD occlusion showed that at each time point studied, there were very few neutrophils in sections from the base that were not in the AAR. In contrast to the base of the heart, a marked increase in neutrophils was observed in sections from the middle and apex, which contained ischemic myocardium in the AAR. The number of neutrophils in the area of ischemia (middle level and apex) at 3 h after reperfusion increased by ∼5- to 10-fold compared with the base level (Fig. 1). By 24 h of reperfusion, the influx of neutrophils was increased by 10- to 20-fold compared with the nonischemic base level. By 72 h, the number of neutrophils was markedly diminished. The number of neutrophils in the middle level at 3 h of reperfusion was significantly higher than at 7 days of reperfusion (P < 0.05).
An intense, local, acute inflammatory reaction was seen to develop over the first 3 h of reperfusion which was prominent at the junction between the region of irreversibly injured myocardial cells and intact cells. After 1 h of ischemia followed by 3 or 24 h of reperfusion, neutrophils were localized in the infarcted tissue, identified by myoglobin-negative and myosin-positive staining, as shown (Fig. 2).
Characterization of Leukocyte Trafficking in ICAM-1/P-Selectin-Double Knockout Mice
Assessment of peripheral white blood cell count.
Because the number of neutrophils and other leukocyte types in the peripheral circulation may influence leukocyte trafficking during myocardial reperfusion injury, we assessed the peripheral blood leukocytes before and at different time points (0, 3, and 24 h) after myocardial reperfusion. As shown in Table1, the total number of leukocytes in P-selectin/ICAM-1-deficient mice was 2.6 times higher than in wild-type mice (P < 0.01). Although neutrophils, monocytes, and eosinophils tended to be increased, only lymphocytes were significantly increased. Immediately after reperfusion (time 0) and also at 3 and 24 h postreperfusion, the total peripheral white blood cell count remained significantly higher in ICAM-1/P-selectin knockout mice than in wild-type mice, with a 4.1- and 2.6-fold increase in neutrophils at 0 and 3 h (P < 0.01).
Assessment of leukocyte trafficking in myocardium.
There were no significant differences in the number of neutrophils in the myocardium that were not in the ischemic area between the control and transgenic animals at any time point. The number of neutrophils increased significantly between 3 and 24 h in control mice but not in the ICAM-1/P-selectin-double knockout mice. After 24 h of reperfusion, the ICAM-1/P-selectin-deficient mice had significantly fewer neutrophils in the middle level than did the control mice (Fig.3).
Assessment of myeloperoxidase activity.
We measured myeloperoxidase activity of the nonischemic and ischemic areas of the myocardium as another means to assess leukocyte accumulation in ischemic tissue. In the nonischemic myocardium (area not at risk), myeloperoxidase activity was very low in all groups and there was no significant difference between the groups, indicating that few neutrophils infiltrated in the nonischemic myocardium. Wild-type mice exhibited a marked increase in myeloperoxidase activity in the ischemic region at 24 h of reperfusion compared with 3 h of reperfusion (Fig. 4). Although mice deficient in ICAM-1/P-selectin had a significant reduction in myeloperoxidase activity at 24 h compared with wild-type mice, there was no significant difference at 3 h. For both control and mutant mice, leukocyte trafficking was higher after 24 h of reperfusion than after 3 h of reperfusion.
Assessment of area at risk and infarct size
The coronary artery occlusion created a large AAR and infarct, shown by delineation using Evans blue dye and TTC staining, respectively, in the transverse heart sections with the area of infarction appearing pale; AAR, red; and the area not at risk, blue.
The infarction as a percentage of the LV and of the AAR after 1 h of ischemia followed by 24 h of reperfusion was 21 ± 3 and 37 ± 5%, respectively, in C57/BL6 control wild-type mice (n = 12) and 23 ± 3 and 40 ± 5% in ICAM-1/P-selectin-double knockout mice (n = 12). The AAR as a percentage of LV after 1 h of ischemia followed by 24 h of reperfusion was 56 ± 3% in C57/BL6 control wild-type mice (n = 12) and 56 ± 4% in ICAM-1/P-selectin-double knockout mice (n = 12).
The infarction as a percentage of the LV and of the AAR after 30 min of ischemia followed by 24 h of reperfusion was 14 ± 3 and 21 ± 4%, respectively, in C57/BL6 control wild-type mice (n = 16) and 17 ± 3 and 26 ± 5% in ICAM-1/P-selectin-double knockout mice (n = 8). The AAR as a percentage of LV after 30 min of ischemia followed by 24 h of reperfusion was 66 ± 3% in C57/BL6 control wild-type mice (n = 16) and 67 ± 4% in ICAM-1/P-selectin-double knockout mice (n = 8) (Fig.5).
Although murine models of cardiovascular diseases present a unique opportunity to better define the role of specific genes in both normal cardiac function and diseases, murine models have not yet been as extensively characterized as disease models in larger animals. One of the objectives, therefore, was to characterize neutrophil trafficking with ischemia-reperfusion in the mouse.
Assessment of neutrophils by both immunohistochemistry and myeloperoxidase activity showed an increase within 3 h of reperfusion, with a peak at 24 h and decline by 72 h with further reduction at 1 wk. At 3 h of reperfusion, increased neutrophils were observed only in the myocardium at risk that had been ischemic. The most intense inflammation was noted at the junction between the region of irreversibly injured myocardial cells and the intact cells, in a manner similar to that observed in a canine model of ischemia-reperfusion injury (8, 14). The border zone of ischemia-reperfusion injury was determined by the presence of contraction-band necrosis, absence of myoglobin, and staining with antimyosin monoclonal antibody. Thus the temporal sequence and spatial location of leukocyte influx after ischemia-reperfusion in the mouse are similar to those in other mammalian species.
Transmigration of neutrophils from blood vessels requires the sequential interactions of neutrophil and endothelial cell adhesion molecules. A number of mice with targeted mutations in specific endothelial and leukocyte cell adhesion molecules have been developed to help define the function of these molecules. Mice deficient in both ICAM-1 and P-selectin were previously observed to have greater defects in neutrophil extravasation than mice deficient in either ICAM-1 or P-selectin alone (2). We decided to examine neutrophil trafficking and infarct size in mice with a combined deficiency in ICAM-1 and P-selectin, based on the hypothesis that the combined mutations would lead to a greater reduction in neutrophil extravasation and, possibly, infarct size than either mutation alone. These mice had a significant reduction in neutrophil influx into the zone of ischemic myocardial injury compared with wild-type mice. At 24 h, the ICAM-1/P-selectin-deficient mice had a 40% reduction, compared with wild-type mice, in neutrophils in the myocardium distal to the LAD ligation as assessed by myeloperoxidase activity. Although the reduction in total number of neutrophils in the heart seen at 24 h was only 40% in mice deficient in ICAM-1/P-selectin, this reflects a larger reduction in neutrophil trafficking into the heart because peripheral neutrophil counts after reperfusion are two- to fourfold higher in the ICAM-1/P-selectin knockout mice than observed in wild-type mice. In addition, the lack of a significant reduction at 3 h postreperfusion may be at least partially due to the 2.6-fold increase noted in peripheral neutrophils at 3 h postreperfusion.
Even though neutrophil extravasation was reduced, these experiments clearly show that the absence of both ICAM-1 and P-selectin is not sufficient to prevent neutrophil trafficking in the heart, as a substantial number of neutrophils were present at both 3 and 24 h postischemia. Therefore, ICAM-1 and P-selectin are not absolute requirements for neutrophil adherence/transmigration after ischemia-reperfusion injury in the heart. Previous studies have shown that the molecular mechanisms for leukocyte extravasation exhibit both tissue and stimulus specificity. Acute neutrophil influx into the peritoneal cavity with chemical peritonitis is completely abolished in ICAM-1/P-selectin-deficient mice, whereas neutrophil influx into the lung with Streptococcus pneumoniae is not affected (2). Present studies clearly demonstrate that neutrophils may use pathways independent of both ICAM-1 and P-selectin for adhesion/migration in the heart after ischemia-reperfusion injury.
Although P-selectin appears to play a predominant role in mediating neutrophil rolling (16), mice deficient in both E- and P-selectin have much greater defects in rolling, emigration, and susceptibility to infection (1, 6) than mice deficient in P-selectin alone. P-selectin is essential for the early increase in leukocyte rolling seen within the first hour of ischemic injury to skeletal muscle (12), whereas at 3 h, rolling and adhesion were independent of P-selectin. With only surgical manipulation of the cremaster muscle, early adhesion was also dependent on P-selectin, but at later time points (60–120 min), leukocyte rolling was dependent on L-selectin and independent of P-selectin (16). With tumor necrosis factor-α stimulation of the cremaster muscle, rolling was independent of both P- and E-selectin but dependent on L-selectin (16).
Other cell adhesion molecules besides ICAM-1 may serve to facilitate firm adhesion and transendothelial migration of neutrophils. Mice with a partial deficiency of CD18 were found to have a more substantial reduction in neutrophil infiltration after 2 h of reperfusion than mice deficient in ICAM-1, suggesting that a mechanism dependent on CD18, but independent of ICAM-1, may be involved (25). Both CD11a/CD18 lymphocyte function-associated antigen-1 (LFA-1) and CD11b/CD18 macrophage antigen-1 (Mac-1) can bind to ICAM-2, and CD11b/CD18 can also bind to fibrinogen, fibronectin, and other ligands. Rat neutrophils use the β1 integrin very late antigen 4 (VLA-4) to mediate migration to arthritic joint and dermal inflammation (10). VLA-4 can mediate adhesion of neutrophils to stimulated endothelium in the presence of anti-selectin therapy (29). VLA-4 can also mediate neutrophil adhesion to cardiac myocytes by adhesion to fibronectin instead of vascular cell adhesion molecule-1 (VCAM-1) (30) and impair myocyte contraction and relaxation (28). The integrin α9β1 may also contribute to both adhesion and migration of neutrophils (37). At least some of the differences in tissue-specific leukocyte recruitment may be due to differences in cell adhesion molecule expression in different microvascular beds (9). Baseline expression and changes in expression after cytokine stimulation in endothelial cell adhesion molecules vary widely in different tissues. Both heart and skeletal muscle have low levels of P-selectin, which increase 5- to 10-fold within 5 min of histamine stimulation. Compared with other tissues examined, the mouse heart has very high baseline levels of E-selectin, ICAM-1, and VCAM-1, with marked increases observed after stimulation with lipopolysaccharide (5, 9, 13, 22, 26). Levels of ICAM-1 are approximately fivefold higher in the heart than in skeletal muscle, and levels of VCAM-1 in the heart are threefold higher than in skeletal muscle at baseline and increase to levels sevenfold higher than skeletal muscle after stimulation with lipopolysaccharide (26).
Although we observed a reduction in neutrophil emigration in ICAM-1/P-selectin-double knockout mice, we found no difference in infarct size compared with wild-type controls with either 30 min or 1 h of ischemia. Previous studies in the dog have shown there may be a relatively brief “window of opportunity” to reduce reperfusion injury by neutrophil inhibition. In the dog, interventions have not shown benefit after coronary artery occlusion of 3 h or more (3, 11). In the rat, no benefit was noted with coronary artery occlusion of 1 h with monoclonal antibody to ICAM-1, whereas infarct size was reduced with 30 min of ischemia followed by reperfusion. The shorter time interval in rodents may be related to the rate of myocardial contractions, i.e., the heart beats in the mouse at 500–600 beats/min (7); in the rat, at 300 beats/min (21); and in the dog, at 134 beats/min (38). In the mouse, infarcts as a percentage of the AAR were smaller after 30 min than after 60 min of ischemia, and we believe that 30 min was an appropriate time point to examine potential benefit.
Other studies have also found no difference in infarct size in mice deficient in either ICAM-1 or P-selectin alone with 60 min of ischemia followed by 2 h of reperfusion (2, 24), but the studies did show a reduction in infarct size with 30 min of ischemia followed by 120 min of reperfusion. In mice with a combined deficiency of ICAM-1 and P-selectin, we found no difference in infarct size with 30 min of ischemia followed by 24 h of reperfusion. One potential explanation of why a deficiency in ICAM-1 or P-selectin led to a reduction in infarct size measured at 2 h but not observed at 24 h in mice deficient in both ICAM-1 and P-selectin is that a deficiency of ICAM-1 or P-selectin delays myocyte death in ischemia-reperfusion injury, which delays loss of NADH and of dehydrogenases from irreversibly damaged myocytes that determine demarcation of the infarct zone by TTC staining, but does not ultimately reduce infarct size. Simpson et al. (32, 33) previously showed that infusion of a prostacyclin analog for the first 2 h of reperfusion resulted in smaller infarct size at 6 h but not at 72 h after reperfusion, and Miki et al. (20) has shown that assessment of infarct size by TTC in the rabbit after 30 min of ischemia followed by 3 h of reperfusion did not correlate to infarct size assessed at 72 h by conventional histology.
The severity of ischemia and the extent of collateral blood flow are other important variables that may have influenced our results. In some cases, antibodies to CD18 have not shown benefit in reducing infarct size when collateral blood flow is ≤0.1 ml · min−1 · g−1 (27,36). One of the limitations of the mouse model is the difficulty in assessment of flow in the myocardium. We were not able to assess collateral blood flow and recognize that collateral flow may differ in the mouse and could be an important variable.
In summary, mice deficient in ICAM-1 and P-selectin have reduced neutrophil trafficking after ischemic injury and reperfusion, but do not have a reduction in infarct size. Future studies should help clarify the role of other leukocyte and endothelial cell adhesion molecules in regulating neutrophil adhesion and extravasation in the heart, particularly in the early period after ischemia when neutrophils may have the most prominent effect on potentiating myocyte injury, and whether the absence of adhesion molecules and changes in leukocyte infiltration alter healing of the myocardium.
The authors acknowledge Kerrie Jara for editorial assistance.
This work was supported in part by National Heart, Lung, and Blood Institute Grant HL-42550 (M. L. Entman, C. M. Ballantyne) and by an American Heart Association Established Investigator Award (C. M. Ballantyne).
Address for reprint requests and other correspondence: C. M. Ballantyne, Baylor College of Medicine, 6565 Fannin, M.S. A-601, Houston, TX 77030 (E-mail:).
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- Copyright © 2001 the American Physiological Society