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Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:1432-1443

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:1432-1443.)
© 1995 American Heart Association, Inc.

Articles

Plasminogen Activator System in Human Coronary Atherosclerosis

P.N. Raghunath; John E. Tomaszewski; Stephen T. Brady; Robert J. Caron; S. Steve Okada; Elliot S. Barnathan

From the Cardiovascular Division, Department of Medicine, and the Department of Pathology and Laboratory Medicine (J.E.T., R.J.C.), University of Pennsylvania School of Medicine, Philadelphia, Pa.


*    Abstract
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Abstract Altered coronary artery expression of plasminogen activator (PA) system components may predispose to thrombosis and modulate the vascular response to injury. By immunohistochemistry, we studied the expression of PAs (tPA and uPA), their major physiological inhibitor (PAI-1), and a receptor for uPA (uPAR) in human coronary arteries with either pure fibrointimal proliferation (n=15) or developed atherosclerotic plaques (n=10). Overall, the degree of staining showed the following rank order: PAI-1>tPA>uPAR>uPA. A similar pattern was seen in two normal coronary arteries. There were no significant differences in the extent of staining in any vascular compartment between atherosclerotic arteries and those with only fibrointimal proliferation. However, the ratio of intimal to medial expression of tPA (P=.001) and uPAR (P=.004) was significantly increased in atherosclerotic arteries, with a similar trend for uPA (P=.069) but not for PAI-1 (P=.73). Four of 10 atherosclerotic arteries had higher uPAR expression in the intima than in the media, whereas none of the 15 arteries with only fibrointimal proliferation had this pattern (P<.01). Dual labeling studies demonstrated colocalization of all four PA system components in endothelial cells, smooth muscle cells, and macrophages, with a predominance of PAI-1. Thus, coronary arteries with a wide range of vascular pathology express an abundance of antifibrinolytic potential with enhanced local expression of profibrinolytic proteins, mainly within atherosclerotic plaques.


Key Words: endothelium • plasminogen activator inhibitor • urokinase receptor • smooth muscle cells • atherosclerosis


*    Introduction
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 *Introduction
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The PA system is an important protective mechanism against stable thrombus formation. The central reaction of this system is the activation of plasminogen to plasmin by PAs. Two forms of PA have been identified, namely, tPA1 and uPA. Cultured ECs secrete both types and normal ECs in situ have been reported to express tPA but little or no uPA,1 but other investigators have noted variable expression of PAs cultured from vessels of different size.2 3 4 Ljungner and Bergqvist5 demonstrated diminished PA activity in atherosclerotic compared with normal blood vessels, although neither the mechanism of this decreased activity nor the cellular distribution was reported. During the development of atherosclerotic lesions, changes in vessel wall architecture and composition occur that ultimately predispose to plaque rupture and thrombosis.6 The precise role of the fibrinolytic system in the development of atherosclerosis remains unclear, but overexpression of PA inhibitors could predispose to stable thrombus formation. Increased protease expression could facilitate early atherogenesis by enhancing macrophage and SMC migration and could later weaken atherosclerotic plaques, predisposing to rupture and thrombosis.

Fibrinolysis can also be regulated by the expression of PA inhibitors and receptors. The major physiological inhibitor of both PAs in humans is PAI-1, which was first identified in bovine aortic ECs7 but is now known to be expressed in a wide variety of normal and malignant cell types, including vascular SMCs.8 Increased PAI-1 levels have been observed in patients with CAD,9 10 angina pectoris,11 and in young12 and older13 survivors of myocardial infarction, suggesting that PAI-1 may represent a risk factor for acute coronary thrombosis. The discovery of a receptor for uPA14 and its subsequent cloning15 have added a new dimension to the regulation of cell-associated PA activity. ECs and SMCs in culture express uPAR,16 17 but whether it is expressed at all or whether its expression is modulated by atherosclerosis in coronary arteries in vivo is unclear. Recent experiments in cholesterol-fed rabbits have suggested a significant upregulation of uPAR in early atherosclerotic lesions.18 In this report, uPAR expression was variably noted in macrophages and SMCs from atherosclerotic samples removed from diseased peripheral arteries.

Localized expression and regulation of PA activity have been postulated to be important in a wide variety of normal and pathological conditions. However, expression of PAI-1 in the vasculature of patients with different degrees of atherosclerosis has only recently been explored. Simpson et al19 measured PAI-1 activity and antigen in human necropsy samples and found evidence of PAI-1 in ECs, smooth muscle, liver, and spleen. Lupu et al20 reported increased synthesis of PAI-1 by cellular components of the atherosclerotic plaque and suggested that extracellular accumulation of PAI-1 may contribute to the thrombotic complications associated with plaque rupture. Schneiderman et al21 demonstrated increased levels of PAI-1 mRNA in severely atherosclerotic vessels compared with normal or only mildly affected arteries, although their studies were performed mainly on aortas rather than coronary vessels. Bjorkerud22 characterized the expression of the overall fibrinolytic capacity of cultured vascular SMCs from normal media and atherosclerotic intima and suggested that impaired fibrinolytic capacity existed in SMCs from the latter source. By using immunohistochemistry, Brody et al23 demonstrated alterations in tPA expression in occluded vein grafts. Recently, Herbert et al24 have provided evidence that tPA is mitogenic for SMCs and that PAI-1 inhibits tPA-mediated SMC proliferation.

To date, the relative distributions of PAs, PA receptors, and PA inhibitors in the same arterial wall have not been systematically studied in coronary arteries. For this report, we evaluated the relative expression of uPA, tPA, uPAR, and PAI-1 in coronary arteries with pure FIP and in those with developed ASP, in parallel with histogenetic markers to assign likely cells of origin. We demonstrate that each of these antigens is expressed in three of the major cell types of the vessel wall, namely, ECs, SMCs, and macrophages, with PAI-1 expression predominating in general. Furthermore, we demonstrate upregulation of profibrinolytic proteins in the atherosclerotic plaque.


*    Methods
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Patient Population
Epicardial coronary artery specimens were obtained from native hearts of patients with end-stage congestive heart failure who were undergoing heart transplantation. Hematoxylin and eosin–stained sections of coronary arteries were prospectively scored for inclusion in the study as having either FIP or ASP. The FIP group had 15 arteries from 12 patients (age range, 24 to 66 years); the ASP group had 10 arteries from 7 patients (age range, 49 to 63 years). Not surprisingly, the FIP group was significantly younger on average (mean age ±SD, 44±14 versus 57±5 years; P=.03) and included more women (42% versus 14%, P=NS). Twelve of 15 FIP arteries were obtained from patients with angiographically "normal" arteries and no clinical CAD. Six of 10 ASP arteries were obtained from patients with end-stage clinical CAD. The remaining 4 were obtained from patients with idiopathic dilated cardiomyopathy and no "significant" coronary stenoses by angiography, although all 4 were from patients with one or more risk factors for CAD. All 10 arteries from the ASP group had macroscopically visible evidence of atherosclerosis. Primary analyses were performed on these 25 arteries. Excluding the 3 FIP arteries from patients with clinical CAD and the 4 ASP arteries from patients without clinical CAD did not significantly change the results of any of the analyses. Two additional epicardial coronary arteries from unused normal heart donor tissue were studied. All studies were done with the approval of the Institutional Committee on Studies Involving Human Beings.

Fixation and Processing
Specimens were obtained and rapidly placed in 100% ethanol for 12 to 24 hours, processed, and embedded in paraffin. This fixative was chosen because for this panel of antibodies, 100% ethanol has the best antigen-preserving properties compared with those of 10% neutral buffered formalin, acetone, methanol, and Bouin's fixative. Sections from each paraffin block were stained with hematoxylin and eosin to illustrate the cell morphology of each tissue specimen. Serial sections of 5 µm were cut on ProbeOn Plus slides and used for staining with the different monoclonal antibodies to components of the PA system and to histogenetic markers. All 27 arteries were analyzed for the relative distribution of the antigens of interest and histogenetic markers.

Immunohistochemistry
For immunolabeling procedures, we used a modified streptavidin-biotin immunoperoxidase method25 with capillary-action technology and the MicroProbe System (Fisher Scientific). Isotype-matched, nonimmune mouse immunoglobulin was used as an irrelevant control antibody. In brief, sections were deparaffinized, and endogenous peroxidase activity was quenched with an 8% (vol/vol) H2O2–methanol solution for 4 minutes, washed with 1x automation buffer (Biomeda Corp), and treated with 1.5% (vol/vol) normal horse serum for 20 minutes at 37°C to block nonspecific antibody binding. The slides were incubated with the relevant primary antibodies at 5 µg/mL for 60 minutes at 37°C. The slides were then washed sequentially in 1x automation buffer and 1x automation buffer containing 1% BSA (Sigma Chemical Co) for 5 minutes each. Biotinylated anti-mouse serum (Vector Labs) at a 1:200 dilution was used as the secondary antibody, incubated for 30 minutes at 37°C, and then washed again with sequential 1x automation buffer and 1x automation buffer containing 1% BSA. Binding of monoclonal antibody was demonstrated by the streptavidin-biotin system (Dako Corp) at a 1:50 combined dilution. Sections were incubated for 30 minutes at 37°C, washed once again sequentially, and then developed with 0.05% (vol/vol) 3,3'-diaminobenzidine solution (Sigma) and 0.03% (vol/vol) H2O2 for 5 minutes. After a final wash, all immunoperoxidase preparations were counterstained with aqueous hematoxylin, dehydrated in ethanol and xylene, and coverslipped with Permount (Baxter).

The extent of immunohistochemical staining was graded by two observers who were blinded to the clinical data for each patient. Grading was performed in a semiquantitative manner by light microscopic observation of the prepared slides. We chose to numerically analyze only the extent of staining to minimize the impact of differences in epitope preservation and antibody affinity on the degree of staining. We used a scale of 0 to 4+, with "0" indicating no staining (background); "1+," <2% of cells stained in that particular compartment; "2+," 3% to 10%; "3+," 11% to 50%; and "4+," >50%. The scored compartments were the luminal endothelium, intima (cells between the endothelium and the internal elastic laminae), media, adventitia, and small vessels. Cell assignments were made by analyzing the histogenetic markers listed below.

Antibodies
Monoclonal antibodies of subclass IgG1 for No. 394 (anti-uPA), No. 373 (anti-tPA), and No. 3785 (anti–PAI-1) and of subclass IgG2A for No. 3936 (anti-uPAR) were used at a final concentration of 5 µg/mL (all were gifts from R. Hart, American Diagnostica Inc). The anti-uPA antibody recognizes all known forms of uPA, including pro-urokinase, two-chain uPA, and receptor-bound uPA. The anti-tPA antibody reacts equally well with single- and two-chain tPA. The anti–PAI-1 antibody reacts with active and latent PAI-1 and with tPA/PAI-1 complexes but does not cross-react with PAI-2 or PAI-3. The anti-uPAR antibody competes for and binds to the uPA binding site on uPAR and detects free uPAR better than occupied uPAR. A series of monoclonal and polyclonal antibodies was used for immunolocalization of histogenetic markers. Both HHF-35 (1:150 dilution; final concentration, 0.05 µg/mL; Enzo Diagnostics) and IA4 (1:800 dilution; final concentration, 0.12 µg/mL; Dako Corp) recognize {alpha}–smooth muscle actin. ECs were identified by staining for vWF by using a polyclonal goat anti-human antibody at 1:3750 dilution (Dako Corp). To confirm the endothelial nature of luminal cells, we also used a monoclonal antibody to CD31 (JC/70A, Dako Corp) at 1:20 dilution (final concentration, 13 µg/mL). Cells of monocyte-macrophage lineage were identified by using a monoclonal antibody to CD68 (KP1) at 1:1000 dilution (final concentration, 0.36 µg/mL; Dako Corp). Slides from which primary antibodies were omitted and that were stained with type- and class-matched irrelevant immunoglobulin at a final concentration of 5 µg/mL served as negative controls for each antibody.

Validation of Antibodies
Immunohistochemical localization of PA system components has been generally hampered by the fixation-sensitive nature of the antigenic epitopes and the lack of well-characterized antibodies with known sensitivities and specificities. A systematic approach was initially made to find known negative and positive cell lines for several antigens of the PA system.26 Cell pellets from various cell lines were fixed in different candidate fixatives, processed, and embedded in paraffin by methods that were identical to those used for tissue. The immunostaining protocol with the various monoclonal antibodies was then optimized. Antigen preservation for this panel of antibodies was optimal when tissues were fixed in 100% ethanol. Monoclonal antibody concentrations <=5 µg/mL in general had the least nonspecific staining with maximal sensitivity. The TCCSUP cell line (transitional cell carcinoma, bladder, primary grade IV, human) was >80% positive (ie, >80% of the cell were positively stained) with all monoclonal antibodies to the PA system and negative with the isotype control antibodies at 5 µg/mL. The following cell lines were found to be negative (<10% staining) for the indicated antigen: HOS (osteosarcoma, human) for uPA and uPAR; HeLa-S3 (epithelioid-carcinoma, cervix, human) for uPA and uPAR; CaCo-2 (adenocarcinoma, colon, human) for tPA; and SW-948 (colon adenocarcinoma, human) for PAI-1 as previously reported.26

Dual Labeling
To confirm and colocalize expression of the PA system components in the endothelium, SMCs, and macrophages, a sequential double immunoenzymatic staining procedure for simultaneous localization of both antigens in the same tissue was used. After the sections were deparaffinized and endogenous peroxidase activity was blocked, they were treated with 1.5N HCl at room temperature for 15 minutes (except for vWF) and blocked with 5% (vol/vol) normal horse serum for 20 minutes at room temperature. After they were stained for PA system component antigens at 10 µg/mL using the peroxidase ABC method described above, the sections were then washed in 1x automation buffer for 5 minutes. Once again blocking was done initially with 5% (vol/vol) fetal bovine serum followed by 5% (vol/vol) normal horse or goat serum for 15 minutes at room temperature. Primary antibodies for each histogenetic marker were then applied at 37°C for 45 minutes. Sections were then incubated with the respective biotinylated secondary antibody for 45 minutes at 37°C. An alkaline phosphatase–avidin-biotin complex (Vecstatin ABC kit AK-5000, Vector) was used as a reporter system, and this second reaction was visualized with the alkaline phosphatase substrate Vector Blue (alkaline phosphatase substrate kit III SK-5300, Vector) that produced a blue reaction product. Stained sections were dehydrated and coverslipped. Doubly stained cells showed mixtures of brown (PA components) and blue (SMA, CD68, or vWF) tones.

Statistics
For comparisons between groups (eg, FIP versus ASP), a two-tailed unpaired Student's t test was used (STATVIEW). For comparisons between different compartments of the same artery, a two-tailed paired t test was used. In all cases P<.05 was considered significant.


*    Results
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 up arrowIntroduction
 up arrowMethods
 *Results
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We first analyzed the localization and extent of expression of PAI-1, uPA, tPA, and uPAR in serial sections of epicardial coronary arteries with varying degrees of FIP or with ASP. Fig 1Down shows an example of serial arterial sections with relatively mild FIP on the left and ASP on the right. The media had the most extensive staining for all components of the PA system analyzed. The relative intensities of staining were PAI-1>tPA>uPAR>uPA for FIP arteries. Histogenetic marker analysis of arteries with only FIP (not shown) demonstrated an intact endothelium with an intima composed mainly of SMCs but devoid of macrophages. Staining for each PA system antigen was generally less intense in intimal compared with medial SMCs. Large-vessel ECs were positive for PAI-1 and tPA, with variable weak or absent staining for uPA and uPAR. The relative intensities of staining overall for atherosclerotic arteries followed a similar pattern, with PAI-1>tPA>uPAR>uPA. Of note is that in arteries with eccentric lesions and a more "normal" thinner side, the staining intensity pattern on this side was similar to that in FIP arteries with more homogeneous expression. Much more local heterogeneity was evident in areas involved with ASP. Relatively hypocellular central areas often stained positively for PAI-1 (Fig 1BDown). Shoulder areas, however, sometimes appeared to have lower PAI-1 expression, and adjacent areas near the fibrous cap were noted to have an increased profibrinolytic component (eg, uPA, uPAR, and tPA) as well as PAI-1 (Fig 1HDown, 1FDown, 1DDown, and 1BDown, respectively). In the preserved media and areas of myointimal proliferation, PAI-1 staining correlated with {alpha}–smooth muscle actin staining (Fig 2ADown and 2BDown), although abundant PAI-1 staining was often noted in macrophage-rich areas (Fig 2DDown) as well. The endothelium (Fig 2FDown), denoted by CD31-positive staining, was also positive for PAI-1. Double staining with PAI-1 (brown) and histogenetic markers (blue) demonstrated PAI-1 expression in these three cell types in atherosclerotic arteries (Fig 2CDown, 2EDown, and 2GDown).



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  		Figure 1. Localization of PA system components in human coronary artery: FIP vs ASP. Sections were fixed in 100% ethanol, embedded, sectioned, stained with monoclonal antibodies (5 µg/mL) to the following antigens, and detected with a streptavidin-biotinylated peroxidase complex: PAI-1 (A and B), tPA (C and D), uPAR (E and F), and uPA (G and H). Parallel sections stained with histogenetic markers (not shown) revealed the absence of macrophages in the artery with FIP (A, C, E, and G) but a macrophage-rich area in the plaque noted in the artery to the right (B, D, F, and H) (not shown). Parallel sections with isotype control antibodies (not shown) had no detectable staining (magnification x12.5).



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  		Figure 2. PAI-1 compared with histogenetic marker expression in atherosclerotic coronary arteries. Serial sections were stained with antibodies to PAI-1 (A) and to histogenetic markers to identify SMCs ({alpha}–smooth muscle actin; B), macrophages (CD68; D), and ECs (CD31; F) (magnification x8 for A, B, D, and F). In another atherosclerotic artery (C, E, and G) double immunohistochemical labeling was performed with PAI-1 (shown in each in brown). Demonstrated in blue are SMCs ({alpha}–smooth muscle actin; C), macrophages (CD68; E) or ECs (vWF; G) (magnification x100 for C and x125 for E and G).

We next performed dual-labeling studies, with the first label to localize the PA component and the second to identify histogenetic markers in an artery with a relatively normal side and an eccentric atherosclerotic plaque on the other (Fig 3Down). Macrophages (blue, first column) were observed immediately beneath the endothelium and expressed each PA system component. Similarly, SMCs (blue, second column) and ECs (blue, third column) expressed each component, with uPA the least expressed overall. The intima of the atherosclerotic side demonstrated increased expression of each component compared with the intima of the normal side.



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  		Figure 3. Double immunohistochemical analysis of an early atherosclerotic plaque in a coronary artery. Antigens and corresponding cell types identified with ABC–alkaline phosphatase and Vector Blue are listed at the top, whereas antigens detected with ABC-peroxidase and DAB substrate (brown) are noted at left. In the bottom row, the control section had no primary antibodies added (No 1° Ab) but was otherwise processed in an identical fashion. A hematoxylin and eosin–stained (H&E) section is shown at both low and high power (magnifications x10 and x100, respectively). The top right side of the artery contains an early atherosclerotic plaque (as noted by macrophage infiltration), whereas the lower half represents the thinner, more "normal" side.

To confirm that this distribution of PA system components was not specific to failing hearts, we studied two arteries from unused portions of normal donor hearts. Fig 4Down depicts a small, normal, epicardial coronary artery with only a few SMCs and no macrophages in the intima (Fig 4DDown and 4FDown). PAI-1 staining was similarly high in the intima and media (Fig 4ADown), whereas tPA, uPAR, and uPA were all detected in the media and to a lower and variable extent in the intima (Fig 4CDown, 4EDown, and 4GDown). Control antibodies showed no staining (Fig 4HDown and 4IDown). We also examined an epicardial coronary artery with FIP (Fig 5Down). The intimal thickening consisted mainly of SMCs (Fig 5EDown), although subendothelial macrophages were rarely detected (Fig 5HDown). Staining was seen for each PA system component in intimal and medial SMCs and ECs.



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  		Figure 4. PA system component expression in normal coronary artery. Serial sections of a branch of an epicardial coronary artery from a normal heart were stained with antibodies to the following proteins: (A) PAI-1; (B) CD31, an EC marker; (C) tPA; (D) {alpha}–smooth muscle actin, an SMC marker; (E) uPAR; (F) CD68, a macrophage marker; (G) uPA; (H) IgG1 control antibody; (I) IgG2A control antibody; and (J) hematoxylin and eosin stain. Black arrows (J) denote location of the internal elastic laminae (magnification x250).



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  		Figure 5. PA system component expression in an epicardial coronary artery with FIP from a normal heart. Representative sections were stained with antibodies to the following antigens: (A) PAI-1; (B) CD31, an EC marker; (C) uPA; (D) tPA; (E) {alpha}–smooth muscle actin, an SMC marker; (F), control IgG1; (G) uPAR; and (H) CD68, a macrophage marker (note the single subendothelial cell macrophage). Section in panel I was stained with hematoxylin and eosin. Black arrows depict location of the internal elastic laminae (magnification x250).

Quantitative analyses that compare arteries with FIP and those with ASP are shown in Fig 6Down. There were no statistically significant differences for any of the PA system components between groups. There was a trend toward increased PAI-1 expression in the endothelium from ASP vessels (3.2 versus 2.5, P=.11) and decreased expression in the media (3.0 versus 3.5, P=.10). Vascular media had the most extensive staining for all four PA system components studied. PAI-1 staining was the most prominent in all arterial compartments in both groups. The only significant differences between the FIP and ASP groups occurred in the adventitia for PAI-1 and tPA, although the extent of staining was low for all components. tPA was found in many but not all periadventitial small vessels, consistent with prior reports.4



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  		Figure 6. Quantitation of PA system antigen expression by vascular compartments: FIP vs ASP. For each compartment, there were no significant differences in mean extent of staining for PAI-1, tPA, uPAR, or uPA comparing the FIP group (n=15) to the ASP group (n=10) using a two-tailed, unpaired, Student's t test except for PAI-1 and tPA in the adventitia (P<.05). However, staining for PAI-1, uPAR, tPA, and uPA was significantly lower in the intima compared with that in the media of FIP arteries; for ASP arteries, staining extent was not significantly different in the intima/plaque area compared with the media for tPA or uPAR, suggesting upregulation of these components in the intima.

When staining extent within individual arteries was compared, several interesting patterns emerged. The extent of PAI-1 staining was significantly lower in the intima than in the media of arteries with only FIP (P=.007), although a similar trend was seen in ASP arteries (P=.08). The extent of uPA staining was also lower in the intima than in the media for both FIP (0.33 versus 1.66, P<.001) and ASP (0.8 versus 1.9, P=.003) arteries. tPA antigen was lower in the intima than in the media, but only in FIP arteries (P<.001). In a similar manner, uPAR antigen was lower in the intima of FIP arteries (P<.0001) but not in ASP arteries (P=.52), suggesting that uPAR and tPA are upregulated in the ASP.

As an additional index of the difference between intimal and medial staining in FIP and ASP groups, ratios of intimal to medial staining were compared. All ratios were <1 on average. However, 4 of 10 ASP vessels had more uPAR expression in the intima and plaque area than in the media (ie, ratio >1.0), whereas ratios >1 were not seen in any of the 15 FIP arteries examined (P<.01 by {chi}2 analysis). As shown in the TableDown, the ratio of PAI-1 staining was not significantly different between the FIP and ASP groups, whereas for each of the other antigens, ASP arteries had higher ratios than those in FIP arteries. These data imply that ASP vessels may have increased intimal expression, decreased medial expression, or some combination thereof for tPA and uPAR. Nevertheless, PAI-1 expression remained high in both compartments.


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  		Table 1. Comparison of Staining Extent in the Intima Compared With That in the Media: FIP vs ASP


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
Localized expression of PA activity has been postulated to be important in a wide variety of normal and pathological conditions. Disturbances of the PA system have been reported to be associated with atherosclerotic and thrombotic vascular diseases.5 20 21 27 28 In this study, we have reported the immunohistochemical localization of two PAs, the major inhibitor of both (PAI-1), and a cellular receptor for uPA in normal human epicardial coronary arteries and those with either FIP or ASP. The origin of cells expressing these proteins has been further characterized by histogenetic markers in serial sections and double-label experiments. We consistently showed that the degree of staining was in the order PAI-1>tPA>uPAR>uPA in all arteries examined. Staining for all antibodies was most extensive in the vascular media. There were no significant differences in staining extent for any of the markers between arteries with FIP and those with ASP when the same compartments in the two groups were compared, although there was a trend toward increased PAI-1 expression in the endothelium of ASP arteries and increased profibrinolytic proteins in atherosclerotic versus nonatherosclerotic intimas. However, several interesting patterns emerged when staining extent was compared within individual arteries. The extent of staining for profibrinolytic antigens was significantly lower in the intima than the media of FIP arteries. However, this difference was generally absent in atherosclerotic arteries, mainly due to increased expression in the plaque area. For tPA and uPAR, the ratios of intimal to medial expression were significantly increased in atherosclerotic arteries, with a similar trend for uPA but not for PAI-1. These data suggest upregulation of profibrinolytic proteins in discrete vascular compartments amidst a rather extensive background of PAI-1 expression in atherosclerotic coronary arteries. Thus, heterogeneous expression may enable different net fibrinolytic profiles in different areas within the same vessel, thereby retarding or accelerating matrix degradation and cell migration.

The precise role of cell-associated plasminogen activation as protection against or in the acceleration of intimal thickening or overt atherosclerosis is still uncertain. Some investigators have suggested that plasmin generation in the vessel wall activates latent transforming growth factor–ß, which in turn suppresses SMC proliferation29 and possibly migration.30 Overexpression of apo(a) in mice increases their susceptibility to diet-induced atherosclerosis, purportedly by decreasing cell-associated plasminogen activation in the vessel wall.31 PAI-1 expression in the vessel wall would have a similar potential and thus may be proatherogenic. Our finding of widespread PAI-1 expression in the media and intima of diseased vessels supports this concept, although PAI-1 was readily found in normal vessels as well. The susceptibility of mice deficient in PA system components to diet-induced atherosclerosis is currently being tested. However, PAI-1–deficient mice were not protected from intimal thickening after vascular injury, whereas uPA-deficient mice had less SMC accumulation in the intima, suggesting that PA expression by SMCs may facilitate intimal thickening after vascular injury.32 This finding accords with data that have been generated in the rat carotid injury model, which suggest a role for PAs in cell migration.33 34 These data in turn suggest that injury models may be more dependent on concentrated PA expression for enhanced cell migration and invasion, and in that context, PAI-1 could inhibit intimal thickening. In native atherosclerosis, which occurs over decades, dependence on the PA system for facilitated cell migration may be episodic or less pronounced. We have demonstrated a relative increase in profibrinolytic components in discrete areas of atherosclerotic plaques within an overall background of relatively widespread though nonuniform PAI-1 expression. Focal concentrations of profibrinolytic activity in atherosclerotic plaques may facilitate cellular migration, extracellular matrix degradation, and weakening of fibrous caps, perhaps thereby increasing the likelihood of plaque rupture. None of the vessels that we studied had ulcerated lesions or dissections and thus may not have been representative of recently ruptured vessels.

Although PAI-1 may suppress excessive PA activity in the vessel wall, PAI-1 can also inactivate circulating PAs, especially when they are expressed on the EC surface, perhaps increasing the likelihood of formation of local thrombi. A deficiency of uPA, tPA, or PAI-1 in mice results in normal vascular development, thus excluding an absolutely essential role for any of these proteins during development.35 However, uPA- and tPA-deficient mice, although viable, have marked fibrin deposition and a shortened lifespan, suggesting that these proteins do play a role in the resolution of fibrin thrombi.

Numerous in vitro studies have demonstrated regulated expression of components of the PA system in cultured ECs and SMCs.36 37 Several studies have demonstrated tPA expression in ECs of different-size vessels, with uPA expression found mainly in ECs only after some stimulus, such as inflammation.38 Levin and del Zoppo4 recently reported tPA localization in smaller-vessel ECs in the brains of normal baboons. Keeton et al39 demonstrated low-level PAI-1 mRNA expression in the media of normal murine renal arteries, with dramatic upregulation of PAI-1 mRNA in ECs after endotoxemia. Few studies to date have systematically examined localized protein expression of PAs, PA inhibitors, and PA receptors in normal or diseased human vascular tissue. Furthermore, most studies, which have often focused on the expression of one component, have examined normal or severely diseased, often aneurysmal, atherosclerotic aortas or peripheral arteries.18 20 21 27 40 Few studies have examined the effects of atherosclerosis on the PA system in coronary arteries. Yorimitsu et al41 recently reported detection of PAI-1 antigen, mainly in ECs and SMCs and along collagen fibers in the intimas, in the coronary arteries of three patients dying of acute myocardial infarction.

Several studies have reported fibrinolytic activity in atherosclerotic arteries in general but with conflicting results of both decreased and increased PA activity. Earlier studies were based on overall assessment of tissue-related fibrinolytic activity that used a semiquantitative fibrin slide technique. Bjorkerud22 characterized the expression of overall fibrinolytic capacity in cultured vascular SMCs from normal media and atherosclerotic intima and suggested that impaired fibrinolytic capacity existed in SMCs from the latter source. Reilly et al40 recently compared the expression of uPA and tPA in aortas that were either aneurysmal or obstructed as a result of atherosclerosis. These investigators found above-normal levels of tPA in tissue extracts of aneurysmal aortas and below-normal levels in obstructed aortas. uPA was not detectable with this antigen assay but was found by immunohistochemistry in mononuclear inflammatory cells. We also found more extensive staining with anti-tPA antibodies in all arteries. tPA tended to be more abundant in the intimas of atherosclerotic arteries, with the intima-to-media ratio of tPA being significantly higher in atherosclerotic arteries. Given the recent discovery that tPA can be mitogenic for vascular SMCs,24 upregulation of tPA in the intima may participate in sustaining the SMC proliferation that is often found in atherosclerotic plaques. Because tPA mitogenicity requires an intact catalytic site, local PAI-1 levels may be critical in determining whether the released tPA in fact affects proliferation at all.

Three recent studies of PAI-1 expression in normal and atherosclerotic vessels have suggested an association between increased PAI-1 expression and the progression of human atherosclerotic disease. Schneiderman et al21 demonstrated increased levels of PAI-1 mRNA in severely atherosclerotic aorta vessels compared with normal or mildly affected arteries. More recently Lupu et al20 identified PAI-1 mRNA–expressing cells in atherosclerotic aorta as ECs, SMCs, and macrophages. Chomiki et al27 suggested that increased PAI-1 mRNA expression in atherosclerotic lesions could be due to the abundant neovascularization in the lesion as well as an increased expression in SMCs. In our study, the same extent of PAI-1 antigen expression was detected in FIP and ASP arteries. In addition, using the same antibodies and fixation technique, we were able to demonstrate a similar expression of PAI-1 in normal coronary arteries from a healthy donor heart.

Histopathologic localization of uPAR in human atherosclerosis has not been previously reported, although it has been widely studied in breast cancer,42 metastatic melanoma,43 colon cancer,44 and ovarian cancer.45 It has been suggested that cell-associated plasminogen activation enhances migration through tissue barriers. In atherosclerosis, migration of ECs (in areas of neovascularization), monocytes, and SMCs may occur, in addition to the migration of lymphocytes and fibroblasts. In vitro studies have demonstrated uPAR expression in human vascular ECs,46 which may be upregulated during EC migration.47 uPAR has been considered as an "activation" antigen on monocytes/macrophages, because its expression can be increased dramatically by various cytokines.48 In addition, uPAR can polarize to the leading edge of migrating monocytes.49 50 uPARs may also be important for cell adhesion, possibly by binding to vitronectin in the extracellular matrix.51 Cultured vascular SMCs also express uPAR,17 and in a fashion similar to that of monocytes, polarization has also been found in migrating SMCs in wounded cultures.52 These studies document the in vivo expression of uPAR protein in ECs, SMCs, and macrophages in normal arteries and in those with varying degrees of atherosclerosis. As with tPA, atherosclerotic arteries tended to have increased intimal uPAR expression, and the ratio of intima-to-media protein expression was increased the most for uPAR.

The present study has several limitations. First, comparisons of the extent of antigen expression between antibodies must be performed with the realization that epitope preservation may differ for each antigen and that the affinity of each antibody for the antigen is likely to be different also. Therefore, antigen levels cannot be directly compared from staining intensity alone. We attempted to account for this problem by relying on staining extent in our scoring system. It is still possible that significant differences in total antigen could exist in arteries with FIP and those with ASP despite similar staining scores. This may be particularly true for arterial sections with higher intimal volumes, as is often the case with atherosclerotic vessels. Second, our studies have not identified with certainty the cell of origin of the proteins, as proteins could be deposited or concentrated in particular areas but synthesized in different cells. Third, grouping of the arteries into those with FIP and those with ASP was based solely on their pathological appearance and not the patients' clinical histories. This resulted in slightly inhomogeneous clinical populations. However, when we repeated our analyses by comparing only FIP arteries from patients with no clinical CAD and ASP arteries from patients with severe end-stage CAD, the results were unchanged.

In summary, we have demonstrated that in normal and diseased human coronary arteries, three identifiable cell types, namely, ECs, SMCs, and macrophages, express uPA, uPAR, tPA, and PAI-1 in vivo. PAI-1 expression appears to predominate over that of both PAs in the endothelium, intima, and media of coronary arteries with a wide range of vascular pathology and could predispose to coronary thrombosis. We also detected moderately enhanced expression of profibrinolytic proteins in particular regions of atherosclerotic plaques compared with nonatherosclerotic intimas. Whether this increased regional expression of profibrinolytic proteins in fact facilitates cellular migration or plaque rupture remains to be determined.


*    Selected Abbreviations and Acronyms
 
ASP = complex atherosclerotic plaque
BSA = bovine serum albumin
CAD = coronary artery disease
EC(s) = endothelial cell(s)
FIP = fibrointimal proliferation without atherosclerotic plaque
PA = plasminogen activator
PAI-1 = PA inhibitor type 1
SMC(s) = smooth muscle cell(s)
tPA = tissue-type PA
uPA = urokinase-type PA
uPAR = urokinase-type PA receptor
vWF = von Willebrand factor


*    Acknowledgments
 
Dr Raghunath is the recipient of a fellowship from the Southeastern Pennsylvania Affiliate of the American Heart Association. Dr Okada is the recipient of a National Institutes of Health (NIH) Clinician Investigator Development Award. This work was also supported by grants from the NIH (HL47839, F32-HL0862, K08-HL02870) and American Diagnostica Inc. The authors wish to acknowledge the contribution of all members of the heart transplant team at the University of Pennsylvania for their assistance with expeditious tissue procurement.


*    Footnotes
 
Reprint requests to Elliot S. Barnathan, MD, University of Pennsylvania School of Medicine, 524 Johnson Pavilion, 3610 Hamilton Walk, Philadelphia, PA 19104-6060.

Received December 29, 1994; accepted June 22, 1995.


*    References
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