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Cardiovascular Research 1997 35(2):241-249; doi:10.1016/S0008-6363(97)00088-6
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Copyright © 1997, European Society of Cardiology

Arterial expression of the plasminogen activator system early after cardiac transplantation

Michael R Garvina, Marino Labinaza, Klaus Pelsa, Virginia M Walleyb, Henry F Mizgalac and Edward R O'Briena,*

aDepartment of Medicine (Cardiology), Vascular Biology Laboratory, University of Ottawa Heart Institute, 1053 Carling Avenue, Ottawa, Ont. K1Y 4E9, Canada
bDepartment of Pathology and Laboratory Medicine, University of Ottawa, Ottawa, Ont., Canada
cDepartment of Medicine, University of British Columbia, Vancouver, BC V6T 1W5, Canada

* Corresponding author. Tel.: +1 (613) 761-5427; fax: +1 (613) 761-4690; e-mail: eobrien@ohi-net.heartinst.on.ca

Received 31 December 1996; accepted 10 March 1997


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objectives: Recent studies suggest that alterations in tissue thrombolysis as well as the inward migration of cells may be specific events that contribute to coronary artery narrowing after cardiac transplantation. Plasminogen activators and inhibitors play a central role in governing not only tissue thrombolysis, but also vascular cell migration. The purpose of this study was to examine arterial wall expression of the plasminogen activation system in coronary arteries during graft vascular disease initiation and progression. Methods: Using in situ hybridization and immunocytochemistry, the expression patterns of uPA and PAI-1 in coronary arteries from cardiac allografts were compared to those of young individuals without disease. Results: Both PAI-1 and uPA were over-expressed early after transplantation and as late as 27 months post grafting. Over-expression of these molecules preceded morphological evidence of graft vascular disease. Of special note was the adventitial expression of uPA and PAI-1 in microvessels and myofibroblasts. In contrast, the expression of uPA and PAI-1 in normal coronary arteries was confined to endothelial cells of the central lumen, as well as low levels of expression in intimal and medial smooth muscle cells. Conclusions: Despite morphologic similarities between normal and transplant coronary arteries, differences were noted in the vascular expression pattern of uPA and PAI-1. The exact role of these molecules in graft vascular disease requires further study; however, it is intriguing to consider that a local imbalance in the plasminogen system may contribute to arterial wall thrombosis and/or excessive cell migration and the genesis of complex vascular lesions.

KEYWORDS Urokinase; Plasminogen; Plasminogen activator inhibitor-1; Human, coronary artery


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Advances in clinical management and immunosuppressive therapies have increased the survival rate of cardiac transplant recipients to over 90% at 1 year [1]. Unfortunately, graft vascular disease (GVD) remains the major cause of mortality, affecting between 5% and 15% of transplant patients each year following surgery [2, 3]. At 1 year after heart transplantation, all hearts have some histological evidence of GVD [4]. The diffuse and concentric involvement of epicardial and intramural transplant coronary arteries is very different from the largely focal lesions seen with atherosclerosis. The fact that GVD is not a systemic disease and spares the recipients' own arteries suggests that immunological mechanisms may be involved; however, the prevalence of GVD does not relate closely with the number of allograft rejection episodes [5].

The pathogenesis of GVD is incompletely understood, however, it has been suggested that medial smooth muscle cells migrate through gaps in the internal elastic lamina to expand the neointima [6–8]. Recent developments have uncovered much information about the factors that govern vascular cell migration [9]. For example, the plasminogen activator (PA) system is a tightly controlled system of serine proteases and protease inhibitors that are not only involved in local fibrinolysis but also vascular cell migration [10]. There are 2 types of plasminogen activators, tissue type (tPA) and urokinase type (uPA), that were so named because of their original identification in tissue extracts and urine, respectively. Both enzymes convert plasminogen to plasmin, although the enzymatic activity of tPA largely depends on its binding to fibrin. Plasminogen activator inhibitor-1 (PAI-1) is the main inhibitor of both proteases in humans and acts by forming 1:1 complexes with each enzyme. As illustrated by several biological events (e.g., embryonic development, ovulation, wound healing, tumor invasion, angiogenesis and clot dissolution) it is the balance between activators and inhibitors, rather than absolute levels of any one of the components, that determines local enzymatic activities and the remodeling of extracellular matrix [11–14].

Recently, the potential role of the plasminogen activator system in the artery wall has been extended beyond the topics of atherogenesis and restenosis to include the problem of GVD. Lebarrere and colleagues have noted that the loss of tPA protein in arterioles of endomyocardial biopsy specimens of transplanted hearts is associated with an increase in fibrin deposition in the myocardial microcirculation [15, 16]. Although decreased tPA expression (or enhanced PAI-1 expression) was predictive of transplant graft failure and GVD in these hearts, these investigators did not examine the expression of the PA system in epicardial coronary arteries.

Therefore, in this study we hypothesize that alterations in the expression of uPA and PAI-1 exist locally in transplant coronary arteries and contribute to altered tissue thrombolysis and/or vascular cell migration, leading to lesion formation and eventually GVD. Our results indicate that both uPA and PAI-1 mRNA and protein expression are upregulated in coronary transplant allografts early after transplantation and that this pattern of altered gene expression precedes morphological alterations in the artery wall.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Tissue specimens
To determine the expression of uPA and PAI-1 in normal coronary arteries, such as those from hearts that might be used for cardiac transplantation, post-mortem coronary arteries from young adults who died as a result of trauma were collected from the coroner's service at the Vancouver Hospital and Health Sciences Center in Vancouver, Canada [17]. The left anterior descending (LAD) coronary artery of 7 males (median age 31 years, range 18–38 years) were examined, as the LAD is prone to develop atherosclerosis [18]. In order to ensure the viability of mRNA in this tissue only specimens that had been fixed less than 6 h after death were studied. Specimens were immersion fixed in 10% neutral buffered formalin and embedded in paraffin.

To examine the expression of the plasminogen activation system in coronary allografts early after transplantation, we obtained post-mortem tissue from cardiac transplants performed at the University of Ottawa Heart Institute. From the more than 225 cardiac transplants performed at the institute, we were able to identify 7 cases where post-mortem coronary tissue was collected from transplant hearts. Tissue from 1 arterial subsegment of these grafts was preserved for pathological examination (e.g., 3 LAD and 4 RCA). Five of these patients died due to complications in the early peri-operative period (Table 1). The remaining 2 patients died 3 and 27 months post-transplantation. The median age was 54 years (range 34–65 years). All patients died in hospital and underwent autopsy shortly thereafter. Tissue specimens were fixed in 10% neutral buffered formalin and embedded in paraffin. All specimens have previously been shown to contain viable protein and mRNA [19].


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  		Table 1 Cardiac tansplantation clincial data

 
2.2 In situ hybridization
In situ hybridization was performed as described previously [19]. A 380 base pair human PAI-1 cDNA fragment (kindly provided by Dr. W.E. Holmes, Genentech) was subcloned into pSP72 (Promega, Madison, WI), and linearized with EcoR V. An antisense cRNA probe was created using SP6 RNA polymerase. For transcription of the sense probe, the plasmid was linearized with Xho1 and incubated with T7 RNA polymerase. A 660 base pair fragment of the human uPA cDNA (kindly provided by Dr. F. Blasi) that was subcloned into the Hind III/Pst I sites of pSP64 (Promega, Madison, WI), was linearized with Pst 1 before an antisense cRNA probe was generated using SP6 RNA polymerase [20].

Tissue sections were hybridized overnight at 55°C with 35S-UTP labeled probes at an activity of 400,000 cpm/ul. The following day, slides were washed in a 2xbuffer of sodium chloride/sodium citrate (SSC) followed by a high stringency solution of 0.1xSSC at 55°C for 2 h. After dipping in NTB-2 emulsion, slides were exposed for 32 days at 4°C before being developed with Kodak D-19 developer and fixer at 15°C. Harris hematoxylin was used as a nuclear counterstain. Slides were assessed using dark field microscopy. Human glioblastoma was used as positive control tissue for the expression of uPA and PAI-1 as these tumors are known to express these molecules [21]. The location and identity of individual cells that expressed uPA or PAI-1 mRNA were assessed on adjacent slides immunolabeled with specific cell identity markers.

2.3 Immunocytochemistry
Specimens were immunolabeled using previously described techniques [22]. Briefly, 5 µm sections were deparaffinized in xylene and decreasing grades of ethanol. Slides were then washed in 3% hydrogen peroxide to inhibit endogenous peroxidase activity. After rinsing in PBS (pH 7.4), a primary antibody was applied at room temperature for 60 min. Biotinylated horse anti-mouse or rabbit anti-goat antibodies were applied for 30 min followed by an avidin-biotin-peroxidase complex (ABC Elite Kit, Vector Laboratories, Burlingame, CA) for 30 min. Slides were then placed in 0.05M Tris containing the peroxidase enzyme substrate, 3,3'-diaminobenzidine (DAB) for 10 min at 37°C to yield a brown reaction product. All specimens were counterstained with Harris hematoxylin.

Tissue sections expressing either uPA or PAI-1 mRNA (as detected by in situ hybridization) were immunolabeled with a goat polyclonal antibody to human PAI-1 (American Diagnostica #395G Greenwich, CT) at a dilution of 1:200 and with a monoclonal antibody to uPA (Amercian Diagnostica #396) at a dilution of 1:1000. The following antibodies were used as cell identification markers: anti-smooth muscle cell {alpha}-actin (Boeringer Mannheim, Mannheim, Germany, at a dilution of 1:100) as a marker for smooth muscle cells, anti-CD-68 (DAKO, Carpinteria, CA, at a dilution of 1:50, after a 5 min digestion with proteus XXIV, Sigma, St. Louis, MO, at room temperature) as a marker of macrophage/monocytes, and anti-VWF (DAKO, at a dilution of 1:300 after a 5 min pretreatment with trypsin at room temperature) to label endothelial cells. For each set of slides a positive control specimen was included to confirm appropriate immunolabeling. Normal human aorta was used to assess smooth muscle cell {alpha}-actin and VWF immunolabeling of smooth muscle and endothelial cells, respectively; human tonsil was used to confirm CD-68 immunolabeling of macrophage/monocytes. All slides were examined by 2 independent investigators who were blinded to all clinical data regarding these specimens.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Histomorphology
All coronary specimens from young individuals displayed benign intimal hyperplasia with a modest accumulation of smooth muscle cells that immunolabeled with an antibody to smooth muscle cell {alpha}-actin (Fig. 1a,b). By light microscopy, the IEL of these normal arteries appeared to be intact. The adventitia was composed of a thin layer of extracellular matrix interspersed with a sparse population of cells, some of which immunolabeled for smooth muscle cell {alpha}-actin. A few macrophage/monocytes were detected in the adventitia of these arteries using an anti-CD-68 antibody. The lumenal endothelium of these arteries was intact and immunolabeled with the anti-VWF antibody.


Figure 1
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  		Fig. 1 Normal coronary. (a) Left anterior descending coronary artery of a 38-year-old male who died as a result of trauma (Movat pentachrome stain; x105). There is minimal thickening of the intima (I), a sparse adventitia (A) and no obstruction of the central lumen (L). The multi-layered IEL (small arrow) is mostly intact and the EEL (large arrow) is continuous. (b) Immunolabeling with anti-smooth muscle cell {alpha}-actin antibody yields a brown color reaction product (hematoxylin nuclear counterstain; x210). (c) uPA protein is primarily expressed by lumenal endothelial cells, as well as some intimal and medial smooth muscle cells (brown color reaction product; hematoxylin nuclear counterstain; x420). (d) In situ hybridization with an 35S-UTP labeled riboprobe demonstrating expression of PAI-1 mRNA by lumenal endothelial cells (darkfield microscopy; x420). (e) The distribution of PAI-1 protein (brown color reaction product) is similar to that of PAI-1 mRNA (hematoxylin nuclear counterstain; x420).

 
Five of the 7 transplant coronary artery specimens were from patients who had died peri-operatively or less than 4 days following surgery, possibly due to hyperacute rejection, although an exact cause was not determined (referred to as Transplant Failure in Table 1). Of these, 4 showed benign intimal hyperplasia due to a modest accumulation of intimal smooth muscle cells. For example, the intimal width of these specimens was comparable to that of the media and appeared histologically indistinguishable from post mortem coronary arteries of normal young individuals. Similarly, the adventitia consisted of a modest layer of dense connective tissue that contained both smooth muscle cell {alpha}-actin immunopositive and immunonegative cells (Fig. 2a,b). In contrast to the minimally diseased specimens of the trauma victims, macrophage/monocytes were abundant in the adventitia of these 4 coronary arteries, and therefore support the possibility of hyperacute rejection. The fifth early death was patient #3 who had more advanced disease than expected with approximately 60% of the area within the IEL obliterated due to the eccentric accumulation of pultaceous material underneath a fibrous cap. As this allograft survived <24 h, it is likely that the coronary artery disease in this specimen was due to atherosclerosis and not GVD. The remaining 2 transplant coronary arteries had advanced disease that was probably due to GVD. For example, patient #6 died within 3 months of transplantation but had significant lumenal narrowing (Fig. 3a). The intima of the LAD from patient #6 included both smooth muscle cell {alpha}-actin immunopositive cells as well as CD-68 immunopositive macrophage/monocytes (Fig. 3b). Patient #7 died 27 months post cardiac transplantation and also had marked lumenal narrowing due to an abundant collection of extracellular matrix, lipid, and CD-68 immunopositive cells. Using light microscopy, both the external elastic lamina (EEL) and IEL of the arteries from the early death group appeared to be largely intact. However, the IEL and EEL of the arteries of the 2 patients who died months post-transplantation were either severely fragmented or no longer present.


Figure 2
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  		Fig. 2 Early post cardiac transplantation. (a) Coronary artery from the transplant heart of patient #4 who died less than 24 h after cardiac transplantation, possibly due to hyperacute rejection. There is minimal thickening of the intima (I), a sparse adventitia and preservation of the arterial lumen (L). White arrow denotes the IEL that is fragmented in some areas. The black arrow indicates the continuous EEL. (Movat stain; x105). (b) Immunolabeling with an antibody to smooth muscle cell {alpha}-actin (brown reaction product; x420). (c) Lumenal endothelial cells of an early specimen expressing uPA protein as denoted by the brown colour reaction (x420). (d) An adventitial microvessel from a human coronary artery 1 day post-transplantation. Note immunolabeling of pericytes with a human anti-smooth muscle cell {alpha}-actin antibody (x1050). (e) and (f) The same microvessel immunolabeled with antibodies to uPA and PAI-1, respectively (hematoxylin nuclear counterstain; x1050).

 

Figure 3
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  		Fig. 3 Late post cardiac transplantation. (a) A hematoxylin-phloxine-saffron stained section of a coronary artery 3 months post cardiac transplantation (x42). The center of this eccentric lesion consists of loose necrosis and calcification. Arrow denotes tissue area shown in subsequent panels at higher magnification. A Movat pentachrome stain of an adjacent slide revealed that the IEL and EEL of this artery was severely disrupted (not shown). (b) Immunolabeling with an antibody to smooth muscle cell {alpha}-actin demonstrates that many but not all intimal cells are smooth muscle cells (brown reaction products; hematoxylin nuclear counterstain; x205). An adjacent section was immunolabeled with an anti-CD-68 antibody and revealed the presence of a few intimal macrophage/monocytes (not shown). (c) Adjacent section hybridized with an 35S-UTP labeled sense probe to PAI-1 demonstrates background signal (darkfield microscopy; x49). (d) Hybridization with the 35S-UTP radiolabeled antisense probe to PAI-1 demonstrates abundant expression of PAI-1 mRNA by lumenal endothelial cells, smooth muscle cells and likely intimal macrophage/monocytes (darkfield microscopy; x205). (e) and (f) Immunolabeling with antibodies to PAI-1 and uPA, respectively (hematoxylin nuclear counterstain; x205). Expression of uPA protein and mRNA (not shown) was less abundant than that of PAI-1.

 
3.2 Urokinase plasminogen activator expression
Over-expression of uPA was more commonly found in coronary arteries of transplant hearts compared to those of normal subjects (Table 2). None of the 7 normal coronary artery specimens expressed uPA mRNA as detected by in situ hybridization. Lumenal endothelial cells, as well as some intimal and medial smooth muscle cells (but not adventitial cells) of these normal arteries showed moderate levels of immunolabeling for uPA protein (Fig. 1c). In contrast, all 7 transplant coronary arteries showed moderate levels of uPA mRNA and protein expression in lumenal endothelial cells as well as intimal and medial smooth muscle cells (Fig. 2c, Fig. 3f). In the adventitia of these transplant coronary arteries, endothelial cells and pericytes of microvessels, as well as myofibroblasts over-expressed uPA mRNA and protein (Fig. 2d–e).


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  		Table 2 Expression of uPA and PAI-1 in coronary arteries

 
3.3 Plasminogen activator inhibitor type I expression
The expression of PAI-1 was more abundant in coronary allografts compared to normal coronary arteries. PAI-1 mRNA expression in normal coronary arteries was limited to lumenal endothelial cells in 2 of 7 specimens (Fig. 1d, Table 2). However, modest levels of PAI-1 protein were immunodetected in lumenal endothelial cells, as well as some intimal and medial cells of all normal coronary arteries (Fig. 1e, Table 2). For coronary arteries of allografts that failed early after transplantation, modest levels of PAI-1 protein were found in lumenal endothelial cells and intimal smooth muscle cells. In addition, adventitial pericytes and endothelial cells of these arteries expressed PAI-1 mRNA and protein (Fig. 2f, Table 2). The arteries of patients #6 and #7 who died at later time points and likely had GVD, had intimal smooth muscle cells and macrophage/monocytes that demonstrated very abundant levels of PAI-1 mRNA and protein expression (Fig. 3c–e). In contrast to normal coronary arteries, PAI-1 mRNA or protein was not detected in the media of transplant arteries (Table 2).


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
Although descriptive, the data presented in this study add insight into the incompletely understood pathogenesis of GVD. The main conclusion of this endeavor is that both uPA and PAI-1 are over-expressed in transplant coronary arteries compared to normal coronary arteries. In normal coronary arteries modest levels of uPA and PAI-1 protein expression were found in lumenal endothelial cells, as well as in some intimal and medial smooth muscle cells. These results in non-diseased human coronary arteries are similar to those reported in 2 recent publications [23, 24]. In contrast, all 7 coronary arteries from transplant hearts, including 4 specimens without significant lumenal narrowing, demonstrated over-expression of uPA and PAI-1 mRNA and protein. uPA and PAI-1 were not only expressed in lumenal endothelial cells and the intima of transplant coronary arteries, but also in the adventitia. Adventitial endothelial cells and pericytes, as well as myofibroblasts over-expressed both uPA and PAI-1 protein. The expression of uPA and PAI-1 was observed both early (24 h) and late (27 months) post-transplantation despite that fact that the early specimens appeared histologically similar to the normal coronary arteries. Finally, it is of interest to note the absence of PAI-1 mRNA expression in the media of transplant but not normal coronary arteries.

There are two major reasons to consider why alterations in the expression of the PA system might occur early after allografting. First, it is possible that the activity of the PA system is altered in acute graft rejection. Second, disruptions in the balance of the PA system may play a role in the initiation of graft vascular disease. Unfortunately, in this study we are unable to determine which comes first. Possibly, animal transplantation experiments would be helpful in determining the acute and chronic effects of allografting on the PA system. However, if we accept that GVD is an immune/inflammatory response that involves the recruitment of inflammatory cells, expression of intercellular adhesion molecules, and the release of soluble factors that eventually lead to smooth muscle cell migration, proliferation and the deposition of extracellular matrix, then it is of interest to try to understand the expression pattern of the PA system in these processes [25, 26].

Let us focus on the over-expression of the PA system in the adventitia of transplant coronary arteries. Fibrin deposition and the formation of organized mural thrombi are likely dependent on the net effect of the PA system [27]. Therefore, analogous to earlier observations on altered fibrinolytic activity in atherosclerotic vessels, dysregulation of intramural thrombosis in, for example, adventitial microvessels may be important for the initiation and progression of GVD [28]. This concept parallels the recent observations of Lebarrere et al. who report a loss of tPA expression and an increase in PAI-1 expression in arterioles of endomyocardial biopsies from transplant hearts with graft failure and GVD [15]. In the present study we examined epicardial coronary arteries of transplant hearts and found that PAI-1 was over-expressed in the lumenal endothelial cells of adventitial microvessels. Therefore, a relative increase in PAI-1 may facilitate vascular thrombosis not only in endomyocardial arterioles, but also in adventitial microvessels of transplant coronary arteries. The consequences of adventitial microvessel thrombosis may be grave. In experimental models, loss of adventitial microvessels results not only in neointimal formation, but also inadequate arterial remodeling [29, 30]. Similarly, the sequelae of enhanced intimal expression of PAI-1 are not known, however, it would not be unreasonable to expect an increase in intramural coagulation and lesion progression under these circumstances.

Another potentially important role for the PA system in GVD may relate to the facilitation of vascular cell migration [31–33]. uPA, a potent promoter of tissue proteolysis, was more abundantly expressed in transplant compared to normal coronary arteries – especially in the adventitia [34]. Moreover, there was an absence of PAI-1 mRNA, the main inhibitor of uPA, in the media of transplant but not normal coronary arteries. Data from several animal studies suggest that vascular proteolysis occurs between days 2–7 post-injury and is required for cells to migrate. For example, in the rat carotid artery model, intimal smooth muscle cell migration with over-expression of uPA and the uPA receptor is seen within the first 5 days after balloon injury [31]. As well, we have recently demonstrated in a porcine model of coronary artery restenosis that adventitial uPA expression is dramatically upregulated on days 3 and 7 after balloon injury when adventitial cell proliferation is present and migration appears to begin [30, 35]. Therefore, this leaves the possibility that alterations in uPA and PAI-1 expression after cardiac transplantation may facilitate the inward migration of adventitial cells after cardiac transplantation and promote neointimal formation.

This study is not without limitations. First, the number of specimens examined in this study was limited by the availability of tissue from coronary allografts early after grafting. Second, upregulation of the plasminogen activation system has been associated with sepsis or may be due to a pre-terminal systemic response [36]. Although some of the transplant recipients from this study were extremely sick immediately prior to transplantation, only 2 patients had systemic infections documented during the peri-operative period or immediately prior to death. Alternatively, these transplant patients received cyclosporine – an immunosuppressive agent that is known to upregulate the expression of PAI-1 [37]. Therefore, the possibility that cyclosporine played a role in impairing fibrinolysis and causing microvessel thrombosis cannot be excluded. Third, is it possible that post-mortem autolysis resulted in excessive mRNA and protein degradation in the normal coronary arteries compared to the transplant coronary arteries, thereby creating false negative observations? Again, this is unlikely, as others have reported low levels of expression of the PA system in normal arteries and we have been successful in reporting the expression of other housekeeping genes in this tissue [19, 23, 24].

In summary, this study is the first to demonstrate the over-expression of PAI-1 and uPA in transplant human coronary arteries. From this data, we are unable to determine if alterations of the PA system in transplant coronary arteries are directly involved in the pathogenesis of GVD, however, we speculate that diminished local fibrinolysis and/or enhanced vascular cell migration, 2 events that are mediated by PAI-1 and uPA, may play an important role in the development of GVD. Further studies of the potential role of the PA system in accelerated vascular disease are ongoing.

Time for primary review 31 days.


   Acknowledgements
 
This work was supported by a Grant-in-Aid from the Heart and Stroke Foundation of Ontario (Grant# NA-2915). We thank the cardiac transplant service at the University of Ottawa Heart Institute and the Coroner's Service at the Vancouver Hospital for their cooperation in collecting coronary artery specimens, and Val Duffin for secretarial assistance with this manuscript.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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