Articles |
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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Key Words: endothelium plasminogen activator inhibitor urokinase receptor smooth muscle cells atherosclerosis
| Introduction |
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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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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) H2O2methanol
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 (antiPAI-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 antiPAI-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
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 phosphataseavidin-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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smooth muscle actin staining (Fig 2A
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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 3
).
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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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 4
depicts a small,
normal, epicardial coronary artery with only a few SMCs and no
macrophages in the intima (Fig 4D
and 4F
). PAI-1 staining was
similarly high in the intima and media (Fig 4A
), whereas tPA, uPAR, and
uPA were all detected in the media and to a lower and variable
extent in the intima (Fig 4C
, 4E
, and 4G
). Control antibodies showed no
staining (Fig 4H
and 4I
). We also examined an epicardial
coronary artery with FIP (Fig 5
).
The intimal thickening consisted mainly of SMCs (Fig 5E
), although
subendothelial macrophages were rarely
detected (Fig 5H
). Staining was seen for each PA system component in
intimal and medial SMCs and ECs.
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Quantitative analyses that compare arteries with FIP and those
with ASP are shown in Fig 6
. 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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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
2 analysis). As shown in the
Table
, 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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| Discussion |
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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 factorß, 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-1deficient 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 mRNAexpressing 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 |
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| Acknowledgments |
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| Footnotes |
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Received December 29, 1994; accepted June 22, 1995.
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