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J Thorac Cardiovasc Surg 1995;110:924-933
© 1995 Mosby, Inc.

SURGERY FOR CONGENITAL HEART DISEASE

P-SELECTIN EXPRESSION IN MYOCARDIUM OF CHILDREN UNDERGOING CARDIOPULMONARY BYPASS

Stephanie A. Burns, MDa, Brian J. DeGuzman, MDb, Jane W. Newburger, MDa, John E. Mayer, Jr., MDc, Ellis J. Neufeld, MD, PhDd, David M. Briscoe, MDc


Boston, Mass.

Supported by the Charles Hood Foundation and a Charles Janeway Award of the Children's Hospital, Boston, Mass. (D. M. B.).

Received for publication Dec. 20, 1994. Accepted for publication March 3, 1995. Address for reprints: David M. Briscoe, MD, Division of Nephrology, Children's Hospital, 300 Longwood Ave., Boston, MA 02115.

Abstract

Cardiopulmonary bypass is a planned support technique that results in a period of myocardial ischemia and reperfusion. In addition, it is associated with an inflammatory response likely involving endothelial cell activation. In previous studies, we showed that E-selectin and intercellular adhesion molecule-1 (ICAM-1) messenger ribonucleic acid (mRNA) are increased in human myocardium after cardiopulmonary bypass. We have now examined the expression of P-selectin mRNA by ribonuclease protection in paired atrial biopsy specimens from 12 patients before and after cardiopulmonary bypass. By means of immunocytochemistry, we have also examined the endothelial cell surface expression of P-selectin protein, as well as that of E-selectin and ICAM-1 in three additional patients. Patient ages ranged from 1 day to 8.5 years (median 12 months), and cardiopulmonary bypass times ranged from 46 to 196 minutes (median 144 minutes). By ribonuclease protection, there was marked variability in the expression of P-selectin in biopsy specimens before bypass. However, when compared with prebypass levels, P-selectin mRNA decreased modestly in 10 of 12 patients after bypass (median decrease 1.5-fold, p = 0.016). As seen with immunocytochemistry, P-selectin protein was distributed diffusely through the vascular bed on large vessels and small vessels before bypass but was virtually absent on capillaries in specimens taken after bypass. E-selectin, which was absent in prebypass biopsy specimens, was induced in one of the three specimens after bypass, but no change in ICAM-1 protein expression above baseline was noted. We also find that cultured human endothelial cells treated with tumor necrosis factor-{alpha}in doses which induce ICAM-1 mRNA simultaneously decrease their expression of P-selectin mRNA as compared with untreated cells. These observations suggest that endothelial P-selectin is transcriptionally downregulated after cardiopulmonary bypass at times when E-selectin and ICAM-1 are induced. Furthermore, we find that E-selectin and ICAM-1 are expressed at times and at sites where P-selectin is absent. Although it is possible that P-selectin may have been induced and lost at early times before reperfusion, these data suggest that endothelial P-selectin plays a limited role in the inflammatory response that ensues after cardiopulmonary bypass. (J THORACCARDIOVASCSURG1995; 110:924-33)

Cardiopulmonary bypass is the essential and common support technique used in most cardiovascular operations in pediatric patients. During cardiopulmonary bypass, the myocardial and pulmonary circulations may be interrupted for periods of 1 hour or more resulting in profound local ischemia. Reperfusion of ischemic tissue results in cytokine production with resultant endothelial activation and leukocyte infiltration. Go Go 1-3 In addition, cardiopulmonary bypass is a potent inflammatory event involving the activation of various humoral cascades Go 4 and commonly affects multiple organs. Usually this multiorgan system dysfunction is transient and short-lived, but, in certain instances, it can be prolonged and severe. Given the necessity of cardiopulmonary bypass for reparative cardiac operations and its potential for morbidity, we have chosen to use it as a human model of inflammatory injury. Our studies focus on mechanisms whereby the endothelium might contribute to this inflammatory response.

Interactions between circulating leukocytes and endothelium have been shown to be critical in inflammatory processes such as ischemia-reperfusion injury. A substantial body of evidence suggests that injury which follows ischemia results at least in part from the local accumulation of neutrophils during reperfusion. Go Go 5-7 This process involves a series of events mediated by the activation of complementary members of several families of adhesion molecules expressed on the cell surface of both the leukocyte and the endothelial cell. The selectin family of molecules, including E-selectin (ELAM-1, CD62E) Go Go 8,9 and P-selectin (GMP-140, PADGEM, CD62P), Go Go 10,11 expressed by endothelial cells mediate low-affinity rolling events by means of interactions with sialylated oligosaccharides, whereas the integrin family of molecules, including LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18), mediate higher-affinity binding by means of interactions with endothelial intercellular adhesion molecule (ICAM)-1 or ICAM-2. Go Go 12-16

In a previous study, we showed that mRNA for E-selectin and ICAM-1 are induced in cardiac and skeletal muscle after cardiac operations and cardiopulmonary bypass in children. Go 17 We speculated that these molecules, which bind neutrophils, may have a role in the pathogenesis of the inflammatory response after cardiopulmonary bypass. However, it is likely that other endothelial adhesion molecules also have a role. P-selectin, originally described in the alpha-granules of platelets, is also expressed by endothelial cells where it is stored preformed in Weibel-Palade bodies. Go 18 After stimulation of endothelial cells, P-selectin is rapidly mobilized to the cell surface where it may bind neutrophils, monocytes, and lymphocytes. Go 11 In vitro, P-selectin has been shown to be functional in binding neutrophils within minutes of an initiating stimulus. Go 11 Thus, as an early participant in the adhesive process, P-selectin is an attractive target for potential anti-adhesive therapeutic strategies in ischemia-reperfusion injury. However, in vivo studies using anti-P-selectin antibodies in animal models have been somewhat controversial. In some models of ischemia-reperfusion anti-P-selectin antibodies ameliorate local inflammation, Go 19 whereas in others these antibodies have no local effect but rather ameliorate remote injury. Go 20 In this human study, we have compared the expression of myocardial P-selectin mRNA in paired biopsy samples before and after cardiopulmonary bypass. In addition, we have compared the immunocytochemical protein expression of endothelial P-selectin with E-selectin and ICAM-1 to determine site-specific expression of these molecules.

PATIENTS AND METHODS

Tissue collection.
Tissue was collected in a manner similar to that previously described. Go 17 In brief, samples of atrium, skeletal muscle, and skin were obtained from pediatric patients undergoing cardiac operations with cardiopulmonary bypass for congenital heart disease just before onset of cardiopulmonary bypass and after reperfusion at the conclusion of the surgical procedure. Specimens were immediately snap frozen in liquid nitrogen and stored at -70°C until use. The protocol for tissue collection was approved by the Committee on Clinical Investigation at Children's Hospital, and informed consent was obtained for each patient.

RNA preparation.
Total RNA was extracted with the guanidinium-thiocyanate method of Chomczynski and Sacci Go 21 with RNAzol B (Cinna/Biotecx, Friendswood, Tex.). Quantification of total RNA resuspended in 0.5% sodium dodecylsulfate was done with spectrophotometry.

Antibodies.
Monoclonal antibodies used for immunocytochemistry were SO49 (mouse anti-human P-selectin) Go 22 and RR1/1 (mouse anti-human ICAM-1), Go 14 supplied by T. A. Springer (Boston, Mass.), and H4/18 (mouse anti-human E-selectin), Go 8 a gift of M. P. Bevilacqua, Amgen Inc. (Thousand Oaks, Calif.). Antibody K16/16 (nonspecific mouse immunoglobulin G) provided by D. Menderick (Brigham and Women's Hospital, Boston, Mass.) and antihuman CD31 (Dako Corp., Carpenteria, Calif.) served as negative and positive controls, respectively. With the exception of CD31, all antibodies were used as ascites fluids. Antibodies SO49 and RR1/1 were titrated at dilutions from 1:1000 to 1:100,000 so that changes in staining intensity were easily quantified. Antibody H4/18 was used at a dilution of 1:1000, which optimized staining of positive control appendicitis tissue. Anti-CD31 antibody was used at a dilution of 1:100.

Endothelial cell culture.
Human umbilical vein endothelial cells Go 23 were grown in medium containing 10% fetal calf serum, 1% penicillin-streptomycin, L-glutamine, and endothelial cell growth factor and were used up to passage six. Endothelial cells were treated with human recombinant tumor necrosis factor (TNF)-{alpha} (Boehringer-Mannheim, Indianapolis, Ind.) at a concentration of 100 U/ml for 0, 0.5, 1, 2, 6, and 24 hours, and total RNA was extracted as described previously.

Ribonuclease protection assays.
Complementary deoxyribonucleic acid templates of human P-selectin and {gamma}-actin were chosen to be of different lengths to allow multiplex ribonuclease (RNAse) protection analysis on a given sample. The full-length P-selectin probe was 366 base pairs with the protected sequence 303 base pairs in length, from base pair 329 to 632 in the full length cDNA. Go 10 The selected fragment of P-selectin cDNA (a gift of Dr. R. McEver, Oklahoma City, Okla.) was amplified by polymerase chain reaction with convenient restriction sites in the primers and cloned into the BamH1 and EcoR1 sites of the plasmid vector, pBluescript II KS (Stratagene, La Jolla, Calif.). The P-selectin template was linearized with BamHI. All samples were simultaneously hybridized with {gamma}-actin probe Go Go 24,25 which served as an internal control.

All riboprobes were synthesized by runoff transcription with the appropriate RNA polymerase: T3 for P-selectin and ICAM-1 antisense probes, and SP6 for {gamma}-actin antisense probe as described. Go 26 For each patient specimen pair (before and after bypass), equal amounts of RNA were analyzed in parallel. Ten micrograms of transfer RNA was used as a negative control in each experiment. Extracts from cultured human umbilical vein endothelial cells were analyzed in a similar manner.

Signals were quantified with the ImageQuant software (Molecular Dynamics, Sunnyvale, Calif.) after 24-hour exposures to PhosphorImager screens (Molecular Dynamics) and were controlled for background by subtraction of the tRNA signal. Each P-selectin band was normalized for {gamma}-actin internal control according to the formula (P-selectin – P-selectin background)/({gamma}-actin – {gamma}-actin background). Paired postbypass values were compared with prebypass values, and statistical analysis was performed with the Student t test.

Immunocytochemistry.
Four-micrometer cryostat sections were air dried for 30 minutes, fixed in acetone for 5 minutes, then air dried again. Specimens were incubated in primary antibody then in secondary antibody, peroxidase-conjugated goat anti-mouse immunoglobulin G (Jackson ImmunoResearch, West Grove, Pa.), each for 1 hour at room temperature, as described. Go 27 The slides were washed in phosphate-buffered saline solution/0.2% gelatin between each incubation. Next, the slides were incubated in sodium acetate 0.1 mol/L (pH 5.2), in 3-amino 9-ethyl carbazole (Sigma Chemical Co., St. Louis, Mo.) 12 mg/ml, and in 2% N,N-dimethylformamide (Sigma) each for 5 minutes, rinsed in deionized water, and counterstained with Gills hematoxylin stain. Finally, the slides were mounted in glycerol gelatin (Sigma).

A scoring system was designed to assess the immunocytochemical staining of specimens. Scores for adhesion molecules were based on the qualitative and quantitative staining patterns for each molecule compared with positive control staining of endothelial-specific anti-CD31 antibody. The scores ranged from 0 to 4+: 0 = no visible stain; 1 = few vessels stained faintly; 2 = moderate intensity staining of few vessels; 3 = moderate intensity staining of many vessels; and 4 = intense staining of many vessels. The slides were scored by three independent observers (S. A. B., B. J. D., and D. M. B.).

RESULTS

Patients.
The patient diagnoses and surgical procedures were a mix reflecting the heterogeneity of congenital heart disease seen at this center and are summarized in GoTable I. Patient ages were 1 day to 8.5 years (median 12 months). Total cardiopulmonary support time was 46 to 196 minutes (median 144 minutes), and the aortic crossclamp was applied for 0 to 123 minutes (median 56 minutes). In all patients, cardiopulmonary bypass was performed under hypothermic conditions. Seven patients had a period of deep hypothermic circulatory arrest (range 3 to 61 minutes, median 49 minutes) during which perfusion of all tissues was interrupted. Specimens from 12 patients were used for RNA analysis, and specimens from three other patients were used for immunocytochemistry.


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  		Table I. Patient characterisitics.
 
P-selectin mRNA in human myocardium.
P-selectin mRNA was detected in all samples examined before and after bypass and was readily quantified by RNAse protection gels (Fig. 1). Marked variability in the expression of P-selectin was found in the prebypass samples among the 12 paired specimens examined. However, P-selectin mRNA expression decreased by a modest amount in 10 of 12 paired postbypass specimens (Fig. 1, A). Overall, the median decrease in message was 1.5-fold (range 1.02 to 29.0 fold; Fig. 1, A). In one pair of samples, postbypass P-selectin mRNA increased slightly above prebypass levels, and, in another pair, P-selectin rose twofold. The change in P-selectin mRNA was not significantly associated with the total support time, circulatory arrest time, crossclamp time, patient age, or immediate postoperative clinical course (data not shown). Thus, it appears that P-selectin message, which is constitutively expressed in human myocardium, may be modestly decreased after cardiopulmonary bypass.




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  		Fig. 1. P-selectin mRNA levels in atrium from patients before and after cardiopulmonary bypass. A, Phosphor-Imager quantitation of ribonuclease protection assays for patients 1 through 12. The overall median decrease is 1.5-fold; p = 0.016 with paired t test. B, Representative autoradiograph of RNAse protection experiment with pairs of atrial tissue. The expected P-selectin protected band (303 nucleotides) and {gamma}-actin control band (140 nucleotides) are shown with bold arrows. The small arrow above P-selectin indicates a nonspecific band which was also present in the tRNA negative control lane.

 
P-selectin protein expression by immunocytochemistry.
Because P-selectin is expressed on other cell types in addition to endothelial cells, we next wished to confirm that the changes in P-selectin mRNA expression noted previously, were reflective of changes in endothelial cell P-selectin protein expression. In addition, because we have previously reported that E-selectin and ICAM-1 mRNA are increased after bypass, Go 17 we wished to evaluate whether all three proteins are expressed at different times and at different sites within the myocardium after bypass. Paired samples of atrium before and after bypass from three additional subjects were studied with immunocytochemistry for the expression of P-selectin, E-selectin, and ICAM-1. The results are summarized in GoTable II. Before bypass, P-selectin, and ICAM-1 were expressed constitutively throughout the vascular bed on all microvessels including capillaries and larger vessels (arterioles and venules). In contrast, E-selectin was not detectable in any of the tissues before the initiation of cardiopulmonary bypass. After bypass, the most notable finding was a decrease in P-selectin expression as compared with prebypass levels. In all specimens examined, the loss of P-selectin staining was most marked on capillaries (Fig. 2, C and D), including postcapillary venules. However, the pattern and intensity of P-selectin staining of larger vessels including arterioles and venules was not appreciably different between prebypass and postbypass specimens. P-selectin did not change in intensity in skeletal muscle after cardiopulmonary bypass but may have slightly increased in some of the postbypass skin specimens. Endothelial ICAM-1 was expressed diffusely throughout the microvascular bed and was not notably different in distribution or intensity between prebypass and postbypass specimens (see GoTable II and Fig. 2, A and B). E-selectin was expressed on an isolated vessel in atrium and skin after bypass in one of the three patients examined.


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  		Table II. Scores for immunocytochemical expression of endothelial cell P-selectin, E-selectin, and ICAM-1.
 






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  		Fig. 2. Immunocytochemical staining of atrial tissue before (Pre) and after (Post) cardiopulmonary bypass from one patient. A (original magnification x 400): prebypass atrium stained for ICAM-1 shows diffuse, constitutive expression of ICAM-1. B (x 400): postbypass atrium stained for ICAM-1 showing a similar staining pattern as that in A. C (x 400){alpha} prebypass atrium showing endothelial P-selectin on a moderate number of small and larger vessels. D (x 400): postbypass atrium showing a significant loss of staining of capillary P-selectin as compared with C. Note that staining of larger vessels did not appreciably change. E (x 1000): higher power of the prebypass specimen in C showing endothelial cell expression of P-selectin.

 
Analysis of P-selectin mRNA in cultured human endothelial cells.
Because we find that P-selectin mRNA is decreased in vivo in endothelial cells, we next wished to determine the effect of cytokines on P-selectin in cultured human endothelial cells. TNF-{alpha} reported to be present in ischemic tissue activates endothelial cells in vitro and is reported to increase the cell surface expression of endothelial P-selectin. Go Go Go Go 2,3,28-30 We performed RNAse protection assays on RNA extracted from cultured human endothelial cells after treatment with TNF-{alpha} and examined the expression of P-selectin and ICAM-1 mRNA. We find that P-selectin mRNA, present in untreated human endothelial cells, was somewhat decreased by 6 hours and was markedly decreased at 24 hours after treatment with TNF-{alpha} (Fig. 3). In contrast, ICAM-1 message increased as early as 1 hour after stimulation, peaked at 6 hours after stimulation, and, although still increased above basal levels, had decreased by 24 hours. (Fig. 3). These in vitro findings confirm that human endothelial P-selectin may be transcriptionally downregulated after activation of endothelial cells with TNF-{alpha} in doses which stimulate ICAM-1 mRNA.




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  		Fig. 3. Time course of P-selectin and ICAM-1 mRNA in human umbilical vein endothelial cells treated with TNF-{alpha}. Endothelial cells were treated with human recombinant TNF-{alpha} (100 U/ml) and total RNA extracted. Ribonuclease protection assays were quantified with PhosphorImager screens. A shows the results of two experiements. B shows the autoradiograph of RNAse protection assay from experiement 2. Each time point was performed in duplicate. (Note: the single solid triangle in experiment 2 indicates P-selectin mRNA level in cells exposed to TNF-{alpha} briefly ("TNF wash"), and time 0 hr P-selectin and ICAM-1 represent cells exposed to medium only).

 
DISCUSSION

The use of cardiopulmonary bypass for cardiac operations in pediatric patients is common, and in most cases is essential for successful cardiac operations. In addition to the widespread "total body inflammation" associated with cardiopulmonary bypass, Go 4 the myocardium may be subjected to periods of ischemia resulting in detrimental events including local inflammation and tissue injury. Although the resultant ischemia-reperfusion inflammatory injury is usually mild and transient, it may be severe and thereby increase the morbidity and mortality after the surgical procedure. In this study, we have used this human model involving planned periods of ischemia and reperfusion to examine patterns of endothelial adhesion molecule expression. We find that P-selectin mRNA and protein are decreased after cardiopulmonary bypass, likely a result of a diminution in endothelial cell P-selectin. We also find that endothelial E-selectin and ICAM-1 may be expressed at times and at sites where P-selectin expression is decreased or absent.

An important question raised by this study is whether endothelial P-selectin has a role in the inflammatory process induced by ischemia-reperfusion in human beings. P-selectin is rapidly transported from endothelial cell storage granules to the cell surface where it binds neutrophils to mediate adhesion events and "rolling" in a shear rate-dependent manner.Go Go 31,32 The rapidity of P-selectin redistribution and loss from endothelial cell surfaces (within minutes) led to the prediction that functional P-selectin events occur early and transiently. Go 33 Indeed, our finding of decreased P-selectin expression at least 1 hour after injury is consistent with these studies. Although we did not examine myocardial biopsy specimens at early times, it is possible that endothelial P-selectin may have been expressed and lost before the onset of reperfusion. Nevertheless, because neutrophil recruitment and associated inflammation occur during the period of reperfusion, our data suggest that there may be a limited role for P-selectin in subsequent neutrophil recruitment. This suggestion has been tested by Seekamp and associates Go 20 in a rabbit limb ischemia model in which anti-P-selectin antibodies failed to ameliorate local reperfusion injury (1 hour ischemia, 1.5 hours reperfusion).

Other studies in animal models have shown that anti-P-selectin antibodies may decrease the reperfusion inflammatory response. Go Go 34,35 Several possibilities exist for the observed differences between these animal studies and the current study. First, human cardiopulmonary bypass may not be a pure ischemia-reperfusion phenomenon. It involves exposure of the circulating leukocytes to foreign surfaces in the bypass circuit, which may activate neutrophils and enhance their ability to localize within microvascular tissue beds. Second, the degree of ischemia suffered by the heart during bypass procedures may be much less with use of protective hypothermia and myocardial protective infusions of cardioplegic solution, than that induced by vessel clamping in experimental ischemia-reperfusion models. Third, P-selectin function may vary with changes in the shear flow rate, evident in various vascular beds under different experimental conditions. Go Go Go 31,32,36 Fourth, interspecies differences may exist in the expression of P-selectin. For instance, we find that TNF-{alpha}downregulates P-selectin transcription in cultured human endothelial cells at times when it has been reported to induce P-selectin mRNA in mouse endothelial cells. Go 29

Cell surface expression of P-selectin may be regulated at several levels, including transcriptional control of synthesis of the protein or regulation of the expressed cell surface protein. P-selectin protein may be lost into the circulation, reinternalized and degraded, or reinternalized and returned to intracellular storage granules. Our analysis suggests that transcriptional control of P-selectin is unlikely. We observed that total P-selectin mRNA levels from extracts of atrium decrease only slightly at times when microvascular protein appears to decrease substantially. In addition, our findings that P-selectin mRNA decreases in activated cultured endothelial cells at times when cell surface expression is present (6 hours) Go 30 suggests that the predominant regulatory mechanism is not likely to be at the level of de novo synthesis. Regulation of P-selectin may be at the level of the protein itself because rapid reinternalization of P-selectin is seen in activated endothelial cells. Go 11 The fate of the reinternalized P-selectin is controversial. Green and associates Go 37 have shown that loss of P-selectin from the surface of transfected PC 12 and CHO cells is most likely due to internalization followed by rapid lysosomal degradation of P-selectin protein. Subramanian, Koedam, and Wagner Go 38 have suggested that a substantial portion of P-selectin surface protein which is reinternalized reappears in Weibel-Palade bodies and is thus "recycled". Go 38 Recycling of P-selectin would not account for our observation of the absolute loss of P-selectin staining from capillaries at early times. Furthermore, Silber and associates Go 39 noted that P-selectin protein which fell to low amounts by 24 hours did not return to baseline constitutive levels until 72 hours, suggesting that P-selectin is degraded and later synthesized de novo rather than recycled. Go 39

In this study, we find that ICAM-1 protein is constitutively expressed on endothelium in both prebypass and postbypass specimens. In addition, we find that E-selectin, which was not present on endothelial cells in prebypass specimens, was expressed on an isolated vessel in one postbypass atrial specimen. These immunohistochemical findings are consistent with our previous report in which we found that ICAM-1 and E-selectin mRNA had increased in most postbypass specimens. Go 17 The earliest time course for maximal E-selectin and ICAM-1 protein expression in vivo is 2 hours and 9 hours, respectively, after an inflammatory stimulus. Go 27 Thus, it is likely that the postbypass specimens used in this study were obtained too early to detect any significant amounts of cell surface E-selectin and ICAM-1 protein expression.

In summary, we find that both endothelial P-selectin mRNA and protein expression are decreased in human myocardium after cardiopulmonary bypass. Although P-selectin may have been expressed at early times (minutes) during the period of ischemia and had decreased by the time of postbypass biopsy and analysis, our observations suggest endothelial P-selectin has a limited role in subsequent myocardial tissue injury. Moreover, the expression of E-selectin and ICAM-1 suggests a role for these molecules in the inflammatory process after bypass. Future studies to further elucidate the mechanisms underlying cardiopulmonary bypass-related tissue injury will ultimately lead to new therapeutic strategies to improve the clinical outcome of children undergoing cardiac operations.

Acknowledgments

We thank R. S. Cotran, MD, for his critical review of this manuscript and Amy Walsh, RN, for her assistance with patient information and samples.

Footnotes

From the Divisions of Nephrologyc and Hematology,d Department of Medicine, the Departments of Cardiologya and Cardiac Surgery,c Children's Hospital, and the Division of Cardiac Surgery,b Brigham and Women's Hospital, and Harvard Medical School, Boston, Mass. Back

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