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(Circulation. 1997;95:191-196.)
© 1997 American Heart Association, Inc.

Articles

Adhesion Molecule P-Selectin and Vascular Cell Adhesion Molecule–1 in Enhanced Heart Allograft Arteriosclerosis in the Rat

Petri K. Koskinen, MD, PhD; Karl B. Lemstrom, MD, PhD

the Transplantation Laboratory, University of Helsinki and Helsinki University Central Hospital, Helsinki, Finland.

Correspondence to Dr Petri Koskinen, Transplantation Laboratory, University of Helsinki, PO Box 21 (Haartmaninkatu 3), FIN-00014, Helsinki, Finland. E-mail petri.koskinen@helsinki.fi.

	Abstract

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Background The increase of P-selectin on endothelial cells (EC) overlying human atherosclerotic plaques in classic atherosclerosis was recently established. We have previously shown that vascular cell adhesion molecule–1 (VCAM-1) is extensively expressed on EC of occluded arteries during accelerated transplant arteriosclerosis. In the present study, with the use of rat heart allografts under different doses of cyclosporine A (CsA), we investigated whether the expression of P-selectin is increased during chronic rejection and possibly coexpressed with VCAM-1 on EC.

Methods and Results Rat cardiac allografts from DA donors to WF recipients were used. Without immunosuppression, these allografts show an irreversible rejection 7 days after transplantation. In the acute rejection model, syngeneic and allogeneic grafts were harvested 5 days after transplantation. In the chronic rejection model, allograft recipients received triple-drug immunosuppression, including azathioprine, methylprednisolone, and CsA in different doses. The grafts were removed 3 months after transplantation. During acute rejection, a significant expression of P-selectin (P<.01) and VCAM-1 (P<.05) on microvascular endothelia, but not on arteries, was noticed. During intense chronic rejection (5 mg/kg CsA), arterial EC expressed P-selectin (P<.01) and VCAM-1 (P<.05) extensively. The expression of tumor necrosis factor–, a cytokine inducing both P-selectin and VCAM-1 expression, was upregulated in vascular medial cells (P<.05), in intimal cells (P<.01), and in interstitial mononuclear cells (P<.05). Linear regression analysis revealed a significant correlation between arterial P-selectin (P<.01) and VCAM-1 (P<.01) expression and the intensity of intimal thickening. Also, a significant correlation and coexpression of P-selectin and VCAM-1 in epicardial arteries was demonstrated (P<.05).

Conclusions The early expression of P-selectin on microvascular EC during acute rejection may be the basis of cell adhesion and infiltration into the site of inflammation. During chronic rejection, the intensity of arterial intimal thickening was significantly correlated with the intensity of P-selectin expression on EC, in addition to that of previously reported VCAM-1 expression. Thus, P-selectin may have a crucial role in the pathogenesis of chronic rejection in the vascular wall, augmenting the immune-mediated injury against the allograft.

Key Words: transplantation • arteriosclerosis • adhesion molecules

	Introduction

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Adhesion of leukocytes to vascular endothelium is an early step in graft rejection, leading to the migration of inflammatory cells into underlying tissues.¹ EC contribute to adhesion by expressing several inducible cell surface molecules that bind various inflammatory cells. Together with MHC molecules, the adhesion molecules have an important role in T-cell activation.² For inflammatory cell adhesion to activated EC, at least three separate receptor/ligand pairs are involved: ICAM-1/LFA-1, VCAM-1/VLA-4, and E-selectin/sialyl Lewis X, and/or related carbohydrates on leukocytes.

Current models propose that members of the selectin gene family (E-, P-, and L-selectin) mediate the initial adhesive interactions, including leukocyte rolling,³ and that subsequent firm adhesion and diapedesis require activation-dependent engagement of integrins with their endothelial ligands and CD31 (PECAM-1),⁴ respectively.

We previously established that VCAM-1 is extensively expressed on EC in enhanced rat heart allograft arteriosclerosis.⁵ In the present study, we investigate the expression of another adhesion molecule, P-selectin, previously called PADGEM or GMP-140. P-selectin is a member of the selectin family and consists of multiple domains, including a lectin domain, an epidermal growth factor domain, nine consensus repeats related to complement binding proteins, a transmembrane domain, and a short cytoplasmic region.⁶ P-selectin is colocalized with vWF in the Weibel-Palade bodies of EC⁷ and is rapidly mobilized from these granules to the cell surface on stimulus.⁸ Several different ligands on monocytes and neutrophils have been identified to bind P-selectin: the Lewis X antigen,⁹ sialyl-Le-x,¹⁰ sulfated glycans,¹¹ and sulfatides.¹²

The increase of P-selectin in endothelium overlying human atherosclerotic plaques in classic atherosclerosis has recently been described.¹³ We demonstrate with the use of rat heart allografts under different dose regimens of CsA that P-selectin is also increased in accelerated transplant arteriosclerosis and is coexpressed with VCAM-1 in EC of occluded arteries of rat heart allografts.

	Methods

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Experimental Design
Rat cardiac allografts from DA donors to WF recipients, with a strong genetic disparity in the MHC and non-MHC loci, were used. These cardiac allografts show an irreversible rejection 7 days after transplantation.¹⁴ In the acute rejection model, syngeneic and allogeneic grafts were harvested 5 days after transplantation, whereas in the chronic rejection model, allograft recipients received triple-drug immunosuppression, including azathioprine (2 mg·kg^-1·d^-1), methylprednisolone (0.5 mg·kg^-1·d^-1), and CsA (5, 10, or 20 mg·kg^-1·d^-1), and the grafts were removed 3 months after transplantation.

Experimental Animals
Inbred DA (AG-B4, RT1^a) and WF (AG-B2, RT1^u) rat strains were used as donors and recipients, respectively. The animals were purchased from Laboratory Animal Centre, University of Helsinki. They were 2 to 3 months old and weighed 200 to 300 g. The rats were fed regular rat food (Altromin, Standard Diet, Chr. Petersen A/S) and tap water ad libitum. All animals were maintained on a 12-hour light/dark cycle. The animals received humane care in compliance with the "Principles of Laboratory Animal Care" and "Guide for the Care and Use of Laboratory Animals" prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (NIH publication No. 86-23, revised 1985).

Immunosuppressive Regimen
In the acute rejection model, transplant recipients were not immunosuppressed, whereas in the chronic rejection model, they received oral triple-drug immunosuppression for the entire observation time. Perioperatively, the rats received CsA (15 mg/kg SC in the neck as a single dose, Sandimmun; Sandoz Pharma) . For the injection, 50 mg/mL CsA infusion substance was dissolved in Intralipid (200 mg/mL; KabiVitrum) to a final concentration of 3 mg/mL. Thereafter, CsA (100 mg/mL Sandimmun mixture, Sandoz) at a dosage of 5, 10, or 20 mg·kg^-1·d^-1 PO was given with regular rat food. Methylprednisolone (0.5 mg·kg^-1·d^-1, Solu-Medrol 40 mg/mL; Upjohn) and azathioprine (2 mg·kg^-1·d^-1, Imuran; Wellcome) were administered in drinking water.

Heterotopic Cardiac Transplants
Intra-abdominal heterotopic cardiac allografts were transplanted according to a modified technique of Ono and Lindsey,¹⁵ and some of the cardiac transplantation samples used in this study were originally procured in experimental sets described previously.^{5 14} Briefly, the donor rats were anesthetized with ether. After perfusion of 200 IU of heparin in 1 mL of ice-cold PBS into the inferior vena cava, it was ligated with 6-0 silk. The superior vena cavae and pulmonary veins were ligated en bloc with 6-0 silk, and the pulmonary artery and aorta were transected 2 to 3 mm above their origin in the heart. Recipient animals were anesthetized with chloral hydrate (240 mg/kg IP) and were administered 0.25 mg/kg buprenorphine SC (Temgesic; Reckitt & Colman) for postoperative pain relief. A midline incision was made, the great abdominal vessels were dissected free from the surroundings, the graft was implanted in the abdominal cavity, and the aorta and pulmonary artery were anastomized with abdominal aorta and inferior vena cava in a running end-to-side fashion using 9-0 nylon sutures, respectively. Total ischemic time varied from 45 to 60 minutes, during which time the graft was kept in an ice bath of +4°C PBS for 15 minutes. Hearts were cooled throughout the procedure with frequent changes of saline-cooled gauze. The grafts started beating vigorously after perfusion was established. The function of the grafts was evaluated by abdominal palpation, and all the grafts were beating at graft removal. When removed, the grafts were immediately washed with ice-cold PBS, sectioned into four or five cross sections, and processed for histology and immunohistochemistry.

Histology
At least two midsections of the allografts were fixed in 10% phosphate-buffered formalin for 24 hours, routinely processed, and embedded in paraffin. Four-micron-thick cross sections of cardiac allografts were stained with Mayer's hematoxylin and eosin for general evaluation, with Masson's trichrome for fibrosis, and with Weigert van Gieson's stain for elastin. Slides were examined with the use of light microscopy by two observers in a blind review, and the score assigned was determined by consensus of the observers. Rejection diagnosing and grading were based on the recommendations of the Working Formulation of the International Society for Heart and Lung Transplantation.¹⁶ Epicardial and intramyocardial arterioles were evaluated for histological changes attributable to chronic rejection. The changes in intimal thickness were scored as mild (score 1, <25% occlusion of the lumen) when the intima was readily discernible and as moderate (score 2, 25% to 50% occlusion) or severe (score 3, >50% occlusion) when the lumen was encroached on.

Immunostaining
One of the midsections of hearts was embedded in OCT (Tissue-Tek, Miles), snap-frozen in liquid nitrogen, and stored at -70°C. Serial frozen sections (4 to 6 µm) were cut, air dried onto Silane-coated slides, and fixed in acetone for 20 min at -20°C, and stored at -20°C until use. Before immunostaining, the slides were refixed with chloroform and then air dried. After incubation with 1.5% nonimmune horse serum (Vector Laboratories) for monoclonals or with 1.5% nonimmune goat serum for polyclonals (Vector Laboratories), frozen sections of cardiac allografts were incubated with VCAM-1 at a dilution of 24 µg/mL (5F10 [a mouse IgG_2a monoclonal antibody]; a generous gift from Dr Roy Lobb, Biogen, Cambridge, Mass) at room temperature for 30 min, or P-selectin at a dilution of 5 µg/mL (CD62 [affinity-purified rabbit anti-mouse/rat/human polyclonal antibody]; Pharmingen), or TNF- at a dilution of 5 µg/mL (CY-051 [polyclonal rabbit anti-rat TNF-]; Innogenetics) at +4°C for 12 hours. The primary antibodies were diluted in PBS with 1% BSA and the appropriate 3% nonimmune serum. With intervening washes in Tris-buffered saline, the following steps were performed. The specimens were incubated with bionylated horse anti-mouse/rat absorbed antibodies or bionylated goat anti-rabbit/rat absorbed antibodies in PBS at RT for 30 min; avidin-bionylated horseradish complex (Vectastain Elite ABC Kit, Vector Laboratories) in PBS at RT for 30 min; and the reaction was revealed by chromogen AEC (Sigma Chemical Co) containing 0.1% hydrogen peroxidase, yielding a brown-red reaction product. The specimens were counterstained with hematoxylin, and coverslips were aquamounted (Aquamount; BDH Ltd).

Double Staining
To demonstrate adhesion molecule expression on EC, double staining was applied on representative frozen sections. After staining for VCAM-1 or P-selectin using the peroxidase ABC method described above (yielding a brown-red peroxidase reaction product), cardiac frozen sections were washed in Tris-buffered saline, and avidin-biotin complex from the first step was blocked by incubating the sections with an excess of avidin and biotin (Avidin/Biotin Blocking Kit, Vector Laboratories). After application of vWF (Dako A/S) at RT for 30 min, the sections were incubated with bionylated goat anti-rabbit/rat absorbed antibodies at RT for 30 min, followed by incubation with alkaline phosphatase avidin-biotin complex (Vectastain ABC Kit, Vector Laboratories) and visualized with the use of a Vector blue, alkaline phosphate substrate kit (Vector Laboratories) that produced a blue reaction. Sections were counterstained with hematoxylin.

Specificity Controls of Immunostaining
Controls were performed using the same immunoglobulin concentration of species and isotype-matched antibodies: mouse monoclonal IgG₁ antibody (catalog No. X931; Dako) and rabbit polyclonal immunoglobulin fraction (catalog No. X936; Dako) for monoclonal and polyclonal antibodies, respectively. Additional control for the specificity of TNF- staining involved the use of a working dilution of the polyclonal antibody after overnight incubation with a 20-molar excess of recombinant mouse TNF- (Genzyme). None of these control stainings showed any immunoreactivity.

Quantification of Immunohistochemistry
The immunohistochemical analysis was done in a blind review by two observers. The score assigned was determined by consensus of the observers. The intensity of the staining was scored from 0 to 3 as follows: 0, no visible staining; 1, few cells with faint staining; 2, moderate intensity with multifocal staining; and 3, intense diffuse staining of the cells analyzed.

Statistical Analysis
All data are expressed as mean±SEM. A nonparametric test was chosen because of the small sample sizes and inability to determine whether the samples were normally distributed.¹⁷ Total variation between the groups was analyzed with the nonparametric Kruskal-Wallis one-way analysis (Z corrected for ties) by rank (StatView 512+, BrainPower). The rank sums obtained with the Kruskal-Wallis test were used for the Dunn test at the significance level of 5% or 1% (Medstat; Astra Group) to determine which of the groups differed significantly from the others. In addition, linear regression analysis was applied to evaluate the possible relation of adhesion molecule expression to intimal thickening.¹⁸ Values of P<.05 were considered to be statistically significant.

	Results

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P-Selectin and VCAM-1 Expression on Normal Heart and Syngeneic Vascular Endothelia
In nontransplanted native rat hearts, neither P-selectin nor VCAM-1 expression was observed on heart vasculature. In syngeneic grafts, EC of small capillaries and postcapillary venules stained faintly positive for P-selectin and showed trace positivity for VCAM-1. Arteries did not express P-selectin or VCAM-1. (Table 1).

View this table: [in this window] [in a new window]   Table 1. Expression of P-Selectin and VCAM-1 on Endothelial Cells of Heart Vasculature

P-Selectin and VCAM-1 Expression During Acute Heart Allograft Rejection
During acute rejection, a clear induction of P-selectin on endothelium of postcapillary venules was recorded (Table 1). Also, significant upregulation of VCAM-1 expression on microvascular endothelia occurred compared with syngeneic grafts (Table 1). During acute rejection, P-selectin was not expressed in arteries. Arterial VCAM-1 expression was trace to mild.

P-Selectin and VCAM-1 Expression During Chronic Heart Allograft Rejection
To modulate the severity of heart allograft arteriosclerosis (ie, chronic rejection), dosages ranging from 5 to 20 mg·kg^-1·d^-1 of CsA in triple background were used for immunosuppression. We have previously shown at this dosage range an inverse correlation between mean CsA blood level and mean intimal thickness of epicardial arteries and intramyocardial arterioles of rat heart allografts.⁵

P-selectin and VCAM-1 expression in arteries of accelerated type of arteriosclerosis (5 mg/kg CsA) was striking (Table 1 and Fig 1). Double staining with vWF confirmed the endothelial origin of these cells (Fig 2). When the intensity of arteriosclerosis was decreased through an increase in the intensity of immunosuppression (CsA dosage range, 10 to 20 mg/kg), arterial adhesion molecule expression gradually vanished (Table 1). Unlike during acute rejection, no significant differences in microvascular P-selectin or VCAM-1 expression were observed during chronic rejection.

View larger version (67K): [in this window] [in a new window]   Figure 1. Photomicrograph demonstrating P-selectin (A) and VCAM-1 (B) expression in the luminal part of intimal cells of severely occluded arteries under 5 mg·kg-1·d-1 immunosuppression of CsA. Original magnification x400.

View larger version (61K): [in this window] [in a new window]   Figure 2. Double-staining procedure indicating that the P-selectin– (A) and VCAM-1– (B) expressing cells are vWF-positive EC. Original magnification x1000.

The expression of TNF-, a cytokine inducing both P-selectin and VCAM-1 on EC, was significantly upregulated in vascular medial cells, in intimal cells, and in interstitial mononuclear cells of the group with accelerated arteriosclerosis (5 mg/kg) (Table 2 and Fig 3).

View this table: [in this window] [in a new window]   Table 2. TNF- Expression

View larger version (132K): [in this window] [in a new window]   Figure 3. Photomicrograph demonstrating TNF- expression in vascular medial cells and in the abluminal part of intimal cells of severely occluded arteries under 5 mg·kg-1·d-1 immunosuppression of CsA. Original magnification x400.

Linear regression analysis revealed a significant correlation between the intensity of arterial endothelial P-selectin and VCAM-1 expression and the intensity of intimal thickening (Fig 4). Also, through linear regression analysis, a significant correlation of endothelial P-selectin and VCAM-1 expression was demonstrated in epicardial arteries of cardiac allografts (Fig 5).

View larger version (27K): [in this window] [in a new window]   Figure 4. Correlation between the intensity of endothelial P-selectin expression and the severity of intimal thickening in epicardial arteries (A) and in intramyocardial smaller arterioles (B) and between the intensity of endothelial VCAM-1 expression and the severity of intimal thickening in epicardial arteries (C) and in intramyocardial smaller arterioles (D). Linear regression analysis was applied. See text for definitions of scores.

View larger version (16K): [in this window] [in a new window]   Figure 5. Correlation and coexpression of endothelial P-selectin and VCAM-1 in epicardial arteries of cardiac allografts as analyzed with linear regression analysis.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Selectins mediate the binding of leukocytes to endothelium at sites of tissue injury and inflammation. Early studies found that activated neutrophils that have lost L-selectin expression do not infiltrate inflammatory sites in vivo.¹⁹ L-selectin ligands are broadly expressed by a variety of tissues because leukocytes can bind to endothelial cells in a number of vascular beds as well as to myelin sheets of the central nervous system.¹⁹ L-selectin binding is completely dependent on the activation of endothelial cells with proinflammatory mediators that induce a ligand on vascular endothelium. E-selectin mediates the adhesion of neutrophils to activated vascular endothelium²⁰ and may function as a tissue-specific homing receptor for T-cell subsets.²¹ P-selectin mediates the adhesion of myeloid cells to activated endothelium and the adhesion of platelets to monocytes and neutrophils.^{22 23} P-selectin also mediates the binding of activated B cells and a subset of T cells to stimulated endothelium in vitro. In vivo, P-selectin plays a central role in neutrophil accumulation within thrombi, which is important for fibrin deposition.²⁴

Based on the central role of the selectins for the initial contact formation between leukocytes and endothelial cells, it is obvious that the expression and activity of the selectins need to be carefully regulated. L-selectin is constitutively expressed on leukocytes, can be rapidly upregulated in its avidity,²⁵ and is then immediately shed through proteolysis from the cell surface.²⁶ In contrast, the two endothelial selectins are absent from the surface of unstimulated EC. On human EC, two different regulation mechanisms for the endothelial selectins were described. P-selectin is intracellularly stored and is transported to the cell surface within minutes after stimulation by various proinflammatory reagents such as histamine or thrombin.²² E-selectin is transcriptionally induced by cytokines such as TNF- or interleukin-1ß, leading to maximal expression levels at the cell surface 3 to 4 hours after stimulation.²⁷

In our acute rejection model, P-selectin was expressed on the endothelia of small capillaries and venules, underlying the importance of the postcapillary microvascular bed as the site of inflammatory cell transmigration into inflamed allografts.²⁸ During acute rejection, almost no P-selectin expression was observed in arteries. Given the fact that some P-selectin expression of small capillaries and venules of syngeneic graft also occurred, this most likely reflects the response of endothelium to the reperfusion injury. Previous studies have shown rapid expression of P-selectin in the venules of pulmonary vascular endothelium of rats subjected to infusion of cobra venom factor²⁹ and in the myocardial venules of cats subjected to ischemia and reperfusion.³⁰

We have previously shown that occluded arteries of heart allografts under low-dose CsA expressed VCAM-1 on the endothelium. The use of higher CsA doses significantly reduced the expression of endothelial VCAM-1. In other words, the thickness of the intimal lesion of arteries and the intensity of VCAM-1 expression were positively correlated.⁵ We now report that the expression of P-selectin is also strongly correlated to development of chronic heart allograft rejection. Both VCAM-1 and P-selectin were expressed in the thickened intimas of occluded epicardial arteries, and a similar positive correlation to the intensity of P-selectin expression and increasing intimal thickening was observed, as was the case with VCAM-1. This upregulation of endothelial P-selectin and VCAM-1 coincided with the elevated expression of TNF- in vascular medial and intimal layers, as well as in interstitial mononuclear infiltrates. This is expected because TNF- is one of the potent inducers of both VCAM-1 and P-selectin.^{31 32}

The enhanced chronic allograft vasculopathy that we previously observed during cytomegalovirus infection was related particularly to subendothelial and perivascular/adventitial inflammation consisting of clusters of MNC in the subendothelial space or periphery of the vascular wall.³³ The induction of VCAM-1 and P-selectin on EC of the accelerated type of chronic rejection in this model offers a pathogenetic mechanism by which these inflammatory cells could adhere and penetrate into the vessel wall. After penetration, the MNC, along with EC, would produce a variety of cytokines and growth factors, control the migration of smooth muscle cells from media to the intima, and induce smooth muscle cells to replicate. Thus, VCAM-1 and P-selectin expression during enhanced chronic rejection is strongly suggestive for a continuous and ongoing immune activation within the vessel wall.

Cell-surface expression of P-selectin is generally short lived (minutes), which makes it a good candidate for mediating early leukocyte/EC interactions. In vivo studies have also demonstrated that the expression of P-selectin may have a role at later time points as well. Levels of P-selectin mRNA are increased in mice after treatment with cytokines such as TNF- or lipopolysaccharide.³² These findings support the pathogenetic mechanisms suggested in the present study. P-selectin has also been shown to contribute predominantly to monocyte attachment to venules in human rheumatoid synovium.¹⁹ In addition, neutralization of P-selectin protects cat heart and endothelium during myocardial ischemia and reperfusion injury.³⁰

In conclusion, the early expression of P-selectin on microvascular endothelia during acute rejection may be basis of marked cell deposition and infiltration into the site of inflammation. During chronic rejection, the intensity of arterial intimal thickening was significantly correlated with the intensity of P-selectin on EC, in addition to that of previously reported VCAM-1 expression and generation of inflammation in the vascular wall (vasculitis).³³ Thus, P-selectin may have a crucial role in the development of chronic rejection by attracting inflammatory cells into the vascular wall and augmenting the immune-mediated injury against the allograft.

	Selected Abbreviations and Acronyms

 

AEC = 3-amino-9-ethylcarbazole CsA = cyclosporine A EC = endothelial cells GMP-140 = granule membrane protein–140 ICAM-1 = intercellular adhesion molecule–1 MNC = mononuclear inflammatory cells PADGEM = platelet activation-dependent granule-external membrane protein PECAM-1 = platelet/endothelial cell adhesion molecule TNF- = tumor necrosis factor– VCAM-1 = vascular cell adhesion molecule–1 vWF = von Willebrand factor

	Acknowledgments

 
This study was supported by grants from Finnish Foundation for Cardiovascular Research, Paavo Nurmi Foundation, Technology Development Center, Helsinki, and Farmos Pharmaceuticals, Turku, Finland. We are grateful to Maria Sandberg, RN, Lab; E. Wasenius, RN, Lab; and T. Lahtinen, RN, Lab; for excellent technical assistance.

Received June 10, 1996; revision received July 30, 1996; accepted August 19, 1996.

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