Skip Navigation

Cardiovascular Research 1997 36(1):28-36; doi:10.1016/S0008-6363(97)00144-2
© 1997 by European Society of Cardiology
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Padró, T.
Right arrow Articles by Kienast, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Padró, T.
Right arrow Articles by Kienast, J.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 1997, European Society of Cardiology

Comparative analysis of plasminogen activator inhibitor-1 expression in different types of atherosclerotic lesions in coronary arteries from human heart explants

Teresa Padróa, Martin Steinsa, Chang-Xun Lia, Rolf M Mestersa, Dieter Hammelb, Hans H Scheldb and Joachim Kienasta,*

aDepartment of Internal Medicine, Division of Hematology/Oncology, University of Münster, Albert-Schweitzer Str. 33, D-48129 Münster, Germany
bDepartment of Cardiovascular Surgery, University of Münster, Münster, Germany

* Corresponding author. Tel. (+49-251) 8347595; fax. (+49-251) 8347595.

Received 23 December 1996; accepted 7 May 1997


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objectives: Clinical manifestations of coronary heart disease result primarily from the progressive development of atherosclerotic plaques and subsequent thrombus formation; processes which may be accelerated by an enhanced expression of plasminogen activator inhibitor (PAI-1) in the vessel wall. In the present study, content and expression of PAI-1 were comparatively analyzed in human coronary arteries in relation to the presence and severity of atherosclerotic lesions. Methods: Segments of coronary arteries obtained from heart explants (n = 15) were classified by the presence and types of atherosclerotic lesions. Antigen and activity levels of PAI-1 were determined in protein extracts of intimal and medial layers. In situ hybridization and immunohistochemical analyses were performed on serial sections of representative tissue specimens. Results: Total PAI-1 antigen consistently increased from macroscopically normal areas (MNAs) to early lesions (ELs) and to maximal levels in fibrous (FPs) and calcified (CPs) plaques. No PAI activity was detected, although PAI-1 in its free form was present in all vascular specimens. Both free PAI-1 and PAI-1 complexed with plasminogen activators were significantly increased in extracts of advanced lesions. However, there was a 2–3 fold molar excess of free versus complexed PAI-1 in FPs and CPs. These findings suggest the presence of relevant amounts of PAI-1 in its substrate rather than in its inhibitor conformation in areas of advanced lesions. Compared with MNAs, PAI-1 mRNA was strongly expressed within the thickened intima of ELs. The highest PAI-1 expression was observed in FPs and CPs, being mainly localized in areas surrounding the necrotic cores in co-localization with infiltrating macrophages. Conclusions: PAI-1 content is consistently increased in relation to the severity of the lesions in atherosclerotic coronary arteries. The concomitant elevation of PAI-1 mRNA suggests that the PAI-1 increase is regulated by local synthesis in the areas of atherosclerotic lesions.

KEYWORDS Atherosclerosis; Plasminogen activator inhibitor-1; Gene expression; Human, coronary artery


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Thrombus formation has been implicated as a major event responsible for the transformation of stable into active atherosclerotic lesions, leading to clinical manifestations in coronary heart disease [1, 2]. Thrombotic vessel occlusion has been demonstrated in the majority of individuals with myocardial infarction [3, 4]. Fibrin has also been found as a significant component in the intimal and subintimal layers of the arterial vessel wall with developing atherosclerotic lesions [5]. Increasing evidence suggests a possible pathogenic role for intramural fibrin deposition. Fibrin might, indeed, influence atherogenesis through several mechanisms, e.g. by modulating endothelial cell permeability [6], by providing a scaffold for smooth muscle cell migration and proliferation [7], and through binding of atherogenic lipoproteins or thrombin [reviewed in [8]].

Fibrin is mainly degraded by the plasminogen activator/plasmin (PA/plm) system [9]. A reduced local activity of the PA/plm system may predispose to the deposition of intramural and intraluminal fibrin in atherosclerotic coronary arteries. The activity of the PA/plm system is highly regulated by the level of plasminogen activator inhibitor-1 (PAI-1), the major physiological inhibitor of tissue-type (TPA)- and urokinase-type (UPA)- plasminogen activators [9]. PAI-1, a serine proteinase inhibitor of the serpin group, may occur in three interconvertible conformations. Besides its active inhibitory form that spontaneously converts into the latent form, a third conformation as a non-inhibitory substrate has been identified [10, 11]. The reaction of PAs with PAI-1 in its substrate form leads to the generation of a degraded PAI-1 without inhibition of the proteinase and without formation of a stable covalent complex in contrast to the active PAI-1 [10]. In addition to PAs, other serine proteinases, like thrombin and elastases, are known to cleave and inactivate PAI-1 in its substrate form [12, 13]. The association rate between thrombin and PAI-1 is highly enhanced in the presence of cofactors as the glycoprotein vitronectin or the glycosaminoglycan heparin [reviewed in [12]].

Clinical studies have demonstrated elevated plasma levels of PAI-1 in patients with coronary artery disease [14–17]and in individuals at high risk of future myocardial infarction [18, 19]. Evidence for a role of intravascular PAI-1 in atherogenesis is provided by experimental animal studies showing increased PAI-1 expression in aortic neointimal cells in response to sustained mechanical injury [20, 21], particularly when accompanied by hypercholesterolemia [21]. In human arteries, increased levels of PAI-1 mRNA [22, 23]and antigen [24–26]have been found in severely atherosclerotic vessels compared with normal or only mildly affected arteries. The enhanced expression of PAI-1 has been localized to plaques and to thickened intima, mostly in association with macrophages and smooth muscle cells [22–29].

The studies on human arteries, however, have been mainly performed on aortic, carotid, or peripheral tissue specimens. To date, only two recent reports have dealt with the immunolocalization of PAI-1 in human coronary arteries [30, 31]. However, PAI-1 protein levels and PAI-1 mRNA expression have not been systematically studied. Thus, the present investigation was undertaken to analyze comparatively the content and activity state of intramural PAI-1 as well as the distribution of PAI-1 mRNA and antigen in human coronary arteries in relation to the presence and severity of atherosclerotic lesions.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Preparation of vascular tissues
Epicardial coronary artery specimens were obtained from heart explants of 15 patients undergoing heart transplantation for end-stage congestive heart failure as consequence of ischemic cardiac disease (n = 11) or dilatative cardiomyopathy (n = 4).

Samples were obtained within a few minutes of excision and processed immediately. Periadventitial tissue and adherent blood were removed and the luminal surface examined. Coronary artery segments were classified by macroscopic criteria for the degree of atherosclerotic involvement in four categories: (1) macroscopically normal areas (MNAs): segments without macroscopic evidence of atherosclerosis; (2) early lesions (ELs): segments with white or yellowish mild intimal thickening; (3) fibrous plaques (FPs): advanced lesions with deep intimal thickening of the vessel wall without macroscopically detectable calcification; (4) calcified plaques (CPs): advanced lesions containing macroscopically detectable calcification. Samples with macroscopic evidence of thrombosis or ulceration were excluded. To confirm the validity of the macroscopic classification, representative sections of the different segments were examined by routine histochemical staining with hematoxylin–eosin and for cell identification by immunohistochemistry. Coronary artery segments for in situ hybridization, immunohistochemistry and routine histochemical analysis were fixed in neutral buffered 4% paraformaldehyde and embedded in paraffin before serially sectioned into 8 µm thick slices and mounted on STAR-FROST adhesive slides (Engelbrecht). In the artery segments used for protein extraction, the adventitia layer was carefully dissected under a magnifier, and the intima-media specimens were immediately snap-frozen in liquid nitrogen and stored at –80°C.

2.2 Tissue extraction
Intima-media specimens of coronary arteries were extracted as described previously [24]. Briefly, individual tissue specimens were ground under liquid nitrogen until reduced to a fine powder, which was resuspended in ice-cold acid acetate buffer (20 mg wet tissue/ml) consisting of 75 mmol/l acetic acid, 225 mmol/l NaCl, 75 mmol/l KCl, 10 mmol/l EDTA, 100 mmol/l arginine, 0.25% (v/v) Triton X-100 (final pH 4.2). Samples were homogenized for 1 min at 4°C. Tissue homogenates were then centrifuged for 10 min at 3000xg, 4°C. The supernatants were filtered (Sartorius filter, pore size 1.2 µm) and stored at –80°C until assayed. In order to control for the efficiency of the extraction procedure, the protein concentration of each tissue extract was determined by the bicinchoninic acid (BCA) method [32], using a commercial assay (Pierce Chemical Co.). Since no significant differences in protein content of the extracts were observed between groups (Kruskal–Wallis analysis, P = 0.306), the results are presented per 100 mg wet tissue weight. The efficacy of the acid acetate buffer (as used) to extract PAI-1 in the free, active and PA-complexed forms was compared with the efficacies of the acetate buffer neutralized at pH 7 and the acetate buffer at acidic pH but in the presence of the aspartic proteinase inhibitor pepstatin A (5 µmol/l, Calbiochem) [33]. For these experiments, tissue powders from ten different coronary artery segments with the various types of atherosclerotic lesions were pooled, mixed, and divided into 9 equal parts. Three parts were extracted with acid acetate buffer, three parts with the neutralized acetate buffer and three parts with the pepstatin A containing acid acetate buffer.

2.3 Measurement of total PAI-1, free PAI-1, PAI-1/TPA and PAI-1/UPA complexes
The concentration of total PAI-1 antigen in the tissue extracts was determined by a commercial ELISA (Tintelize, Biopool). In this assay, the capturing antibody is a mouse anti-PAI-1 monoclonal antibody (MA-7D4B7) and the detecting antibody a goat polyclonal anti-human PAI-1 IgG. According to the manufacturer, the assay recognizes free (active and latent) and complexed PAI-1 to equal extent. For measurement of free PAI-1 levels a commercial ELISA (Imulyse, Biopool) employing two murine monoclonal antibodies MA-7D4 and MA-7F5 as coat and conjugate respectively, was used. This assay detects the various forms of human PAI-1 with the following efficiencies: latent 100%, active 83%, complexed 10%, and degraded 21% [34]. PAI-1/TPA complex was measured by a two-site ELISA (Asserachrom, Stago), using a monoclonal mouse anti-TPA as capture antibody and a monoclonal anti-PAI-1 as detecting antibody. The working range of the ELISA for the measurement of PAI-1/TPA was 0.04 to 1.5 ng/ml (expressed as TPA equivalent). The two-site ELISA for measurement of PAI-1/UPA complex utilized a mouse monoclonal anti-human UPA as capture antibody (MUK-1, Biopool) and a mouse monoclonal anti-human PAI-1 biotin conjugate as detecting antibody (PAI-1 ELISA kit, Monozyme). As standard, PAI-1/UPA complex was prepared by incubation of 10 nmol/l two-chain active UPA (purified from human urine; Calbiochem) with a 50-fold molar excess of active PAI-1 (recombinant, human; Calbiochem) at room temperature (RT) for 60 min. The absence of free UPA in the incubates was controlled spectrophotometrically [35]. For the standard curve, the PAI-1/UPA preparation was used in a range of 0.05 to 2 ng/ml. The assay did not recognize free UPA or PAI-1 even at 200 fold higher concentrations than the highest concentration of the standard curve. The intraassay and interassay variations were below 10%.

2.4 Assay of PAI activity
PAI activity was evaluated spectrophotometrically as described by Verheijen et al. [36].

2.5 Preparation of PAI-1 cRNA probes
A 1977- bp cDNA probe for human-PAI-1 inserted in the pGEM3Z vector (Promega), kindly provided by Dr. P. Quax (Gaubius Laboratory TNO, Leiden, The Netherlands), was used. Antisense and sense cRNA probes were transcribed in vitro from linearized plasmids using digoxigenin-labeled UTP (Boehringer Mannheim) and SP6- or T7-RNA polymerase (Boehringer Mannheim), respectively. Correct synthesis of the cRNA probes was controlled by agarose-gel electrophoresis. RNA probes were cut into 400- up to 500-bp fragments by limited alkaline hydrolysis in 40 mmol/l NaHCO3/60 mmol/l Na2CO3, pH 10.2.

2.6 In situ hybridization
In situ hybridization (ISH) was performed as described by Wilcox et al. [37]and modified as follows: Vascular sections were deparaffinized and pretreated sequentially with 4% (w/v) paraformaldehyde (10 min at 4°C), 2.5 µg/ml proteinase K in 100 mmol/l Tris-HCl, 50 mmol/l EDTA, 2 mmol/l CaCl2, pH 7.5 (10 min at RT), and 0.25% (v/v) acetic acid anhydride in 0.1 mol/l triethanolamine pH 8.0 (10 min at RT). Prehybridization was performed for 1 h at 42°C in buffer containing 50% (v/v) formamide, 10% (w/v) dextran sulfate, 2xstandard saline–citrate buffer (SSC), 1xDenhardt's solution, 0.01% (w/v) sodium dodecyl sulfate, 10 mmol/l dithiotreitol, 5 mmol/l EDTA, 250 µg/ml salmon sperm DNA. Hybridization was performed overnight at 42°C in heat-denatured prehybridization buffer supplemented with 2.5 ng/µl of digoxigenin-labeled cRNA probe and yeast t-RNA (250 µg/ml). Subsequently, sections were washed twice in 2xSSC/50% (v/v) formamide (20 min at 42°C) and in 2xSSC (30 min at RT). Immunological detection of digoxigenin-hybridized sections was performed according to Höltke et al. [38], using alkaline phosphatase-conjugated F(ab')2 fragments of anti-digoxigenin antibody (1:1000, for 2.5 h at RT). The NBT-BCIP substrate (DIG nucleic acid detection kit, Boehringer Mannheim) supplemented with 0.1% (w/v) levamisole was employed for revelation of phosphatase activity (overnight incubation at RT, in darkness). Sections were counterstained with 0.1% (w/v) erythrocin solution.

2.7 Immunohistochemistry
Serial sections adjacent to those used for in situ hybridization were processed for immunohistochemical detection of PAI-1 antigen and identification of macrophages, smooth muscle and endothelial cells. The following murine monoclonal antibodies were used: anti-human PAI-1 (Mab 3785, American Diagnostica; 20 µg/ml), anti-human macrophage (DAKO-CD68, DAKO; working dilution 1:50), anti-human {alpha}-smooth muscle actin (clone 1A4, DAKO; working dilution 1:25), anti-human von Willebrand factor (DAKO-vWF, DAKO; working dilution 1:25). For negative controls mouse IgG (Sigma; 20 µg/ml) was used in substitution of the first antibody. Immunohistolocalization was performed by the peroxidase conjugated biotin–avidin technique as described previously [24].

2.8 Statistics
Data are presented as individual data plots or as medians and interquartile ranges. Statistical significance of overall differences between groups was analyzed by the Kruskal–Wallis one-way analysis. If the test was significant, pairwise comparisons were done by the multiple-comparisons' criterion [39]. The Wilcoxon matched-pairs signed rank test was used to compare levels of free and complexed PAI-1 in each group. A P value of 0.05 or less was considered significant.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
For the determination of PAI-1 (total, free, complexed, and active) in intima-media extracts, coronary arteries with macroscopically normal areas (MNAs) and with each of the different types of atherosclerotic lesions (ELs, FPs, and CPs) were available from seven heart explants of patients with end-stage clinical ischemic cardiac disease. Representative segments of each category were isolated and extracted separately. However, depending on the size of the tissue samples, representative artery segments of the same category and from the same individual explant were pooled for tissue homogenization and extraction. For in situ hybridization and immunohistolocalization of PAI-1, coronary segments with MNAs (n = 5), ELs (n = 6) and FPs or CPs (n = 5) were obtained from eight additional heart explants. Four of 5 MNAs specimens, two of 6 ELs and one of 5 FPs were retrieved from patients with dilative cardiomyopathy (n = 4) as the cause for the failing heart. The remaining specimens were obtained from patients with end-stage ischemic cardiac disease (n = 4).

3.1 Histological characterization of the atherosclerotic lesions
Histological examination of the coronary artery specimens confirmed that very little or no intimal thickening existed in segments with MNAs. The endothelium was intact in these segments as detected by immunostaining for von Willebrand factor. Occasional cells staining positive for {alpha}-actin, but no infiltrating CD68-positive macrophages were detected in the intima. Coronary artery segments with early atherosclerotic lesions exhibited the characteristic thickening of the tunica intima. {alpha}-Actin immunostaining was found throughout the subendothelial intima indicating that a significant proportion of the cells in the intimal layer were SMCs. Scattered CD68-positive macrophages were localized immediately beneath the luminal endothelium, which was found always intact in segments with ELs. Fibrous and calcified plaques consistently contained a well-defined core region of amorphous material. Lesions with evidence of haematomas or thrombotic deposits were excluded from the analyses. Luminal endothelium was frequently absent in this type of lesions, and immunohistochemical staining identified SMCs and macrophages as the principal cellular elements. The latter cells mainly concentrated at the adjacent sides of the core regions.

3.2 PAI-1 levels in coronary artery extracts
The amount of extractable total PAI-1 antigen consistently increased from macroscopically normal areas to early lesions to maximal levels in fibrous and calcified plaques (Fig. 1; Kruskal–Wallis analysis: P<0.001). This trend was observed for each individual heart explant and regardless of whether the results were expressed as total PAI-1 antigen per mg of wet tissue extracted (as presented) or per amount of extractable protein (not shown). The difference in PAI-1 content between MNAs and advanced lesions was highly significant as was the difference between ELs and CPs (P values by multiple group comparisons' criterion: MNAs versus FPs=0.025; MNAs versus CPs=0.001; ELs versus CPs=0.025).


Figure 1
View larger version (21K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 Total PAI-1 antigen in intima-media extracts of coronary artery segments with different types of atherosclerotic lesions. Results are expressed in fmol per 100 mg wet tissue (wt) extracted. Coronary arteries from n = 7 individual heart explants were analyzed. Representative segments of each type of lesions were available from all but one explant, where a CP for tissue extraction was not obtainable. Data points represent individual values in each group and those linked by lines originate from the same heart explant. Significance testing for overall group differences was performed by the Kruskal–Wallis test (P<0.001). The multiple-comparisons' criterion was employed to identify differences between groups. The * denotes a significant increase versus the MNA group (FP: P = 0.025, CP: P = 0.001) and the {blacktriangleup} versus the EL group (CP: P = 0.025).

 
To determine the levels of free and PA-complexed PAI-1, tissue extracts were analyzed by different immunoassays. A similar, though less marked trend as for total PAI-1 was found for free PAI-1 antigen (Fig. 2A) measured by an ELISA which recognizes specifically latent and active PAI-1 [34]. Significant overall group differences were confirmed by Kruskal–Wallis analysis (P = 0.004). Subsequent multiple group comparisons revealed a significant increase in extractable free PAI-1 in areas with CPs compared with MNAs (P = 0.01). Levels of PAI-1 complexed with PAs were expressed as the sums of PAI-1/TPA and PAI-1/UPA concentrations. As shown in Fig. 2B, the content of PAI-1/PA complex was slightly but significantly increased in atherosclerotic segments of coronary arteries (Kruskal–Wallis test: P = 0.005). The median value for CPs (193.5 fmol/100 mg tissue) was significantly higher than for MNAs (104.5 fmol/100 mg tissue; P = 0.005) and for ELs (106.0 fmol/100 mg tissue; P = 0.05). Separate data for PAI-1/TPA and PAI-1/UPA complex in the coronary extracts are shown in Table 1. Free and complexed PAI-1 were found in equimolar concentrations in extracts of MNAs and ELs, but the median values of free PAI-1 content in advanced lesions were 2- to 3-fold higher than the median values for PAI-1/PA complex (FPs: 450 versus 149 fmol/ 100 mg tissue, P = 0.01; CPs: 463 versus 193 fmol/100 mg tissue, P = 0.02).


Figure 2
View larger version (23K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 Levels of free PAI-1 (A) and PAI-1 complexed with PAs (B) in intima-media extracts of coronary arteries with different types of atherosclerotic lesions. Points linked by lines represent data from the same heart explant. Overall group differences were tested for statistical significance by Kruskal–Wallis analysis (free PAI-1: P = 0.004; PAI-1/PA complex: P = 0.005). Subsequent pairwise group comparisons using the multiple comparisons' criterion revealed: (A) a significant difference between CPs and MNAs (P = 0.01 denoted by *), (B) a significant difference between CPs and MNAs (P = 0.005; denoted by *) and between CPs and ELs (P = 0.05; denoted by {blacktriangleup}).

 

View this table:
[in this window]
[in a new window]

  		Table 1 Concentrations of PAI-1/TPA and PAI-1/UPA complex in intima-media extracts of coronary artery segments by types of atherosclerotic lesions

 
Using a spectrophotometric assay, we did not detect PAI activity in any of the coronary artery extracts analyzed. Transformation of active into latent PAI-1 during the extraction procedure was excluded in previous experiments [24]. PAI-1 activity was neither detected when coronary artery tissues were extracted with neutralized acetate buffer nor when pepstatin A was added to the acid acetate buffer. The extraction of coronary tissue at neutral pH or at acidic pH in the presence of pepstatin A did not affect the relative levels of free and complexed PAI-1 in the extracts (Table 2). According to these results, an underestimation of active PAI-1 due to lysosomal degradation (encouraged by the acidic pH of the buffer) during the extraction procedure is unlikely to have occurred.


View this table:
[in this window]
[in a new window]

  		Table 2 Efficiency of three different buffers for extraction of free PAI-1 and PA-complexed PAI-1 from coronary artery tissue

 
3.3 PAI-1 localization in coronary artery sections
Representative specimens of normal areas and of atherosclerotic lesions were serially sectioned and processed in parallel for in situ hybridization and immunohistochemical analyses.

The luminal endothelium in MNAs and ELs stained positive for PAI-1 mRNA (Fig. 3A and 3D). In the medial layer, in situ hybridization analysis revealed a widespread distribution of positive signals for PAI-1 mRNA, but the staining was markedly enhanced in ELs (Fig. 3A and 3D). In addition, PAI-1 was strongly expressed within the thickened intima of ELs (Fig. 3D) in cell-rich areas staining positive for smooth muscle {alpha}-actin (not shown). In agreement with the in situ hybridization results, immunohistochemical analysis revealed a positive reactivity for PAI-1 antigen at the luminal surface. Positive signals were also fairly evenly distributed throughout the intimal and medial layers of the vessel wall (MNAs: Fig. 3C; ELs: Fig. 3F).


Figure 3
View larger version (132K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 Localization of PAI-1 mRNA and antigen in serial sections of macroscopically normal areas (panels A through C), early lesions (panels D through F), and advanced lesions (panels G through I) of human coronary arteries. In situ hybridization with digoxigenin-labeled antisense (A,D,G) and sense (B,E,H) human cRNA probes and immunostaining with a monoclonal antibody for human PAI-1 (C,F,I) were performed as described in Section 2. Note the positive staining for PAI-1 mRNA (blue dots) overlying the luminal endothelial surface and the spotted pattern of blue signals throughout the medial layer in MNAs and ELs. In addition, PAI-1 mRNA was strongly expressed in the thickened intima of ELs (panels A and D). Immunostaining for PAI-1 antigen in MNAs and ELs (brown signals, panels C,F) matched the areas of PAI-1mRNA expression. Advanced atherosclerotic lesions exhibited abundant PAI-1mRNA expression in areas adjacent to the necrotic core regions (panel G). Immunostaining with anti-human PAI-1 (panel I) disclosed some cell associated staining throughout the cap (shown with arrows). However, the strongest signal for PAI-1 antigen was associated with amorphous material in the necrotic core. The use of sense PAI-1 cRNA probes for hybridization (B,E,H) and nonimmune mouse IgG in the immunohistochemical analyses (not shown) did not exhibit any labeling. The arrowheads in A–C point to the internal elastic lamina. I, tunica intima; M, tunica media. Original magnification x325 (A,B,C), x162 (D,E,F), x81 (G,H,I).

 
The findings reported on advanced atherosclerotic lesions apply to fibrous and to calcified plaques (Fig. 3, G through I). In these areas, PAI-1 mRNA was highly expressed compared with MNAs and ELs. PAI-1 mRNA specific signals were observed throughout the fibrous cap and in the base of the lesions in co-localization with {alpha}-positive staining for SMCs (not shown). However, PAI-1 mRNA signals were particularly intense in regions adjacent to the necrotic cores (Fig. 3G), in cell-rich areas which also stained intensively for CD68-positive macrophages (not shown). Some PAI-1 antigen staining was detected in the cap at sides of mRNA expression, but the strongest PAI-1 antigen signal was detected in the core of the lesions, in areas mainly containing amorphous material (Fig. 3I).

Controls for in situ hybridization using a sense cRNA probe (Fig. 3B, 3E and 3H) and for immunostaining using nonimmune murine IgG in substitution for first antibodies (not shown) were consistently negative.


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
Clinical manifestations of coronary heart disease result primarily from the progressive development of atherosclerotic plaques and subsequent thrombus formation. Expression of PAI-1 in the arterial vessel wall has been postulated to play an important role in atherogenesis and mural thrombosis. Up to now, only two studies have dealt with PAI-1 expression in human coronary arteries [30, 31]. Using immunohistochemistry, both studies detected PAI-1 immunoreactivity in normal and diseased vessel walls.

The present investigation, based on quantitative protein determinations in extracts of coronary arteries, unambiguously demonstrates a consistent increase of PAI-1 content (total, free and PA-complexed) from macroscopically normal areas to early lesions and to peak values in advanced atherosclerotic plaques. These results are in line with those reported before by our group and others on human aortic tissue [24–26]. Furthermore, in the intima and media of atherosclerotic human coronary arteries Raghunath et al. [31]detected a widespread staining for PAI-1, with a trend towards more intense staining in the endothelium of areas with plaques. However, our study is the first that quantitates PAI-1 protein comparatively in various types of atherosclerotic lesions isolated from coronary arteries obtained from individual heart explants.

In the extracts of macroscopically normal areas, free and PA-complexed forms of PAI-1 were in equimolar concentrations and almost quantitatively accounted for the total level of PAI-1 antigen. Despite the presence of free PAI-1, we were unable to detect PAI activity in the intima-media extracts of MNAs by a spectrophotometric assay. These results suggest that free PAI-1 is mainly in its latent form in these areas. Following activation of latent PAI-1 within the coronary vessel wall, e.g. by negatively charged phospholipids [40, 41], active PAI-1 becomes rapidly inactivated by complex formation with PAs.

In extracts of early and advanced atherosclerotic lesions, the sum of free PAI-1 and PAI-1/PA contents did not exceed 65% of the total PAI-1 antigen concentration. This may indicate the presence of substantial amounts of degraded PAI-1 in these areas, which was not recognized by the immunoassay for free PAI-1 [34]. In addition, we have found a 2–3 fold molar excess of free versus PA-complexed PAI-1, but no PAI activity in the extracts of advanced atherosclerotic lesions. Taking these results together, we hypothesize that in atherosclerotic lesions of human coronary arteries a relevant amount of PAI-1 is in its substrate rather than in its inhibitor conformation for PAs [10]. The excess of total PAI-1 antigen compared with the sum of free and PA-complexed PAI-1 could be explained by an enhanced cleavage and degradation of PAI-1 in its substrate form. The potential enzymes that cleave PAI-1 could be thrombin with vitronectin as cofactor, since both components are localized in atherosclerotic lesions [42, 43], or elastase released by leucocytes derived from microthrombi incorporated into the vessel wall [44].

The lack of PAI activity in the extracts of coronary atherosclerotic lesions is consistent with the excess of extractable PA activity reported in atherosclerotic coronary specimens [45, 46]. It is interesting to note that an opposite fibrinolytic profile exists in the human aortic vessel wall, where excess of PAI activity is present in the intimal and medial layers [24]. The present data corroborate our previous suggestion that quantitative differences in the modulation of intramural PAs by PAI-1 exist between coronary and aortic vessel walls.

Our data on PAI-1 mRNA distribution in atherosclerotic coronary arteries are consistent with the previous reports in aorta, carotid, and peripheral arteries [22, 23, 26–28]. They confirm the presence of PAI-1 mRNA in the luminal endothelium and throughout the medial layer both in normal and atherosclerotic segments. Furthermore, they corroborate the detection of widespread PAI-1 mRNA within the thickened intima of ELs in co-localization with intimal smooth muscle cells. In advanced lesions, intense hybridization signals for PAI-1 were observed in the cap and in areas adjacent to the necrotic cores, mainly in association with infiltrates of macrophage-like cells. The increased PAI-1 expression in the cap of the fibrous plaques in coronary arteries is in accordance with the findings in aorta reported by previous studies. However, in the cap of advanced aortic atherosclerotic lesions PAI-1 expression was mainly related to SMCs [22, 27]. In contrast to MNAs and ELs where PAI-1 antigen was co-localized with sides of mRNA expression, in advanced lesions there was a discrepancy in the distribution pattern between synthesis and localization of PAI-1. The accumulation of PAI-1 antigen in the necrotic core suggests that after secretion from cells, e.g. macrophages, PAI-1 becomes locally concentrated by binding to extracellular matrix proteins, most likely fibrin and vitronectin, which also have been detected in these areas [8, 43].

Mural TPA and UPA may affect atherogenesis through a variety of mechanisms that include fibrin digestion, regulation of growth factor activities [47], and extracellular matrix degradation [48]. Local content and conformation of PAI-1 in the vessel wall are of great interest because of its function in regulating the activities of mural PAs. A local increase of PAI-1 in the inhibitory conformation may promote coronary atherogenesis by predisposing to local thrombosis and fibrin deposition. On the other hand PAI-1 as an inhibitor may contribute to plaque stabilization. The conformational change of PAI-1 towards its substrate form may represent a regulatory mechanism as well. Since PAI-1 in its substrate conformation is susceptible for proteolytic degradation, it could increase the local concentration of free PAs and subsequently promote plasmin generation, as has been demonstrated in studies in vitro [49]. This could represent a protective mechanism by increasing fibrin degradation, but in turn an enhanced plasmin formation in advanced atherosclerotic plaques could lead to extracellular matrix breakdown and therefore might contribute to plaque destabilization.

In conclusion, our results demonstrate a consistent increase of PAI-1 content in relation to the presence and severity of atherosclerotic lesions in human coronary arteries. The concomitant elevation of PAI-1 mRNA suggests that the quantitative alterations in PAI-1 content are regulated by local synthesis in the areas of lesion. Further studies are necessary to elucidate the role of the different forms of PAI-1 in atherogenesis.

Time for primary review 21 days.


   Acknowledgements
 
This work was supported by an institutional grant from the "Interdisciplinary Clinical Research Center" at the University of Münster.


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

  1. Fuster V., Badimón L., Badimón J.J., Chesebro J.H. The pathogenesis of coronary artery disease and the acute coronary syndromes (part one). N Engl J Med (1992) 326:242–250.[Web of Science][Medline]
  2. Fuster V., Badimón L., Badimón J.J., Chesebro J.H. The pathogenesis of coronary artery disease and the acute coronary syndromes (part two). N Engl J Med (1992) 326:310–318.[Web of Science][Medline]
  3. Davies M.J., Thomas A. Thrombosis and acute coronary-artery lesions in sudden cardiac ischemic death. N Engl J Med (1984) 310:1137–1140.[Abstract]
  4. DeWood M.A., Spores J., Notske R., Mouser L.T., Burroughs R., Golden M.N., Lang H.T. Prevalance of total coronary occlusion during the early hours of transmural myocardial infarction. N Engl J Med (1980) 303:897–902.[Abstract]
  5. Smith E.B., Keen A., Grant A., Stirk C. Fate of fibrinogen in human arterial intima. Arteriosclerosis (1990) 10:263–275.[Abstract/Free Full Text]
  6. Kadish J.L., Butterfield C.E., Folkman J. The effects of fibrin on cultured vascular endothelial cells. Tissue Cell (1979) 11:99–108.[CrossRef][Web of Science][Medline]
  7. Naito M., Funaki C., Hayashi T., Yamada K., Asai K., Yoshimine N., Kuzuya F. Substrate-bound fibrinogen, fibrin and other cell attachement-promoting proteins as a scaffold for cultured vascular smooth muscle cells. Atherosclerosis (1992) 96:227–234.[CrossRef][Web of Science][Medline]
  8. Smith E.B., Thompson W.D. Fibrin as a factor in atherogenesis. Thromb Res (1994) 73:1–19.[CrossRef][Web of Science][Medline]
  9. Collen D., Lijnen H.R. Basic and clinical aspects of fibrinolysis and thrombolysis. Blood (1991) 78:3114–3124.[Free Full Text]
  10. Declerck P.J., de Mol M., Vaughan D.E., Collen D. Identification of a conformational distinct form of plasminogen activator inhibitor-1, acting as a non-inhibitory susbstrate for tissue-type plasminogen activator. J Biol Chem (1992) 267:11693–11696.[Abstract/Free Full Text]
  11. Urano T., Strandberg L., Johansson L.B.A., Ny T. A substrate-like form of plasminogen-activator-inhibitor type 1. Conversions between different forms by sodium dodecyl sulphate. Eur J Biochem (1992) 209:985–992.[Web of Science][Medline]
  12. van Meijer M., Pannekoek H. Structure of plasminogen activator inhibitor 1 (PAI-1) and its function in fibrinolysis: an update. Fibrinolysis (1995) 9:263–276.[CrossRef][Web of Science]
  13. Wu K., Urano T., Ihara H., Takada Y., Fujie M., Shikimori M., Hashimoto K., Takada A. The cleavage and inactivation of plasminogen activator inhibitor type 1 by neutrophil elastase: the evaluation of its physiologic relevance in fibrinolysis. Blood (1995) 86:1056–1061.[Abstract/Free Full Text]
  14. Páramo J.A., Colucci M., Collen D. Plasminogen activator inhibitor in the blood of patients with coronary artery disease. Br Med J (1985) 291:73–574.
  15. Aznar J., Estelles A., Torno G., Sapena P., Torno V., Blanch S., España F. Plasminogen activator inhibitor activity and other fibrinolytic variables in patients with coronary artery disease. Br Heart J (1988) 58:535–541.
  16. Olofsson B.O., Dahlen G., Nilsson T.K. Evidence for increassed levels of plasminogen activator inhibitor and tissue plasminogen activator in plasma of patients with angiographically verified coronary artery disease. Eur Heart J (1990) 10:77–82.[Web of Science]
  17. Juhan-Vague I., Alessi M.C. Plasminogen activator inhibitor 1 and athero-thrombosis. Thromb Haemost (1993) 70:138–143.[Web of Science][Medline]
  18. Hamsten A., de Faire U., Walldius G., Dahlen G., Szamosi A., Landou C., Blombäck M., Wiman B. Plasminogen activator inhibitor in plasma: risk factor for recurrent myocardial infarction. Lancet (1987) 2:3–9.[CrossRef][Web of Science][Medline]
  19. Gram J., Jespersen J. A selective depression of tissue plasminogen activator (t-PA) activity in euglobulins characterizes a risk group among survivors of acute myocardial infarction. Thromb Haemost (1987) 57:137–139.[Web of Science][Medline]
  20. Sawa H., Lundgren C., Sobel B.E., Fujii S. Increassed intramural expression of plasminogen activator inhibitor type 1 after balloon injury. A potential progenitor of restenosis. J Am Coll Cardiol (1994) 24:1742–1748.[Abstract]
  21. Sawa H., Sobel B.E., Fujii S. Potentiation by hypercholesterolemia of the induction of aortic intramural synthesis of plasminogen activator inhibitor type 1 by endothelial injury. Circ Res (1993) 73:671–680.[Abstract/Free Full Text]
  22. Schneiderman J., Sawdey M.S., Keeton M.R., Bordin G.M., Bernstein E.F., Dilley R.B., Loskutoff D.J. Increased type 1 plasminogen activator inhibitor gene expression in atherosclerotic human arteries. Proc Natl Acad Sci USA (1992) 89:6998–7002.[Abstract/Free Full Text]
  23. Arnman V., Nilsson A., Stemme S., Risberg B., Rymo L. Expression of plasminogen activator inhibitor-1 mRNA in healthy, atherosclerotic and thrombotic human arteries and veins. Thromb Res (1994) 76:487–499.[CrossRef][Web of Science][Medline]
  24. Padró T., Emeis J.J., Steins M., Schmid K.W., Kienast J. Quantification of plasminogen activators and their inhibitors in the aortic vessel wall in relation to the presence and severity of atherosclerotic disease. Arterioscler Thromb Vasc Biol. (1995) 15:893–902.[Abstract/Free Full Text]
  25. Robbie L.A., Booth N.A., Brown P.A.J., Bennett B. Inhibitors of fibrinolysis are elevated in atherosclerotic plaque. Arterioscler Thromb Vasc Biol (1996) 16:539–545.[Abstract/Free Full Text]
  26. Shireman P.K., McCarthy W.J., Pearce W.H., Patterson B.K., Shively V.P., Cipollone M., Tamarina N., Verrusio E.N., Kwaan H.C. Elevated levels of plasminogen activator inhibitor type 1 in atherosclerotic aorta. J Vasc Surg (1996) 23:810–818.[CrossRef][Web of Science][Medline]
  27. Lupu F., Bergonzelli G.E., Heim D.A., Cousin E., Genton C.Y., Bachmann F., Kruithof E.K.O. Localization and production of plasminogen activator inhibitor -1 in human healthy and atherosclerotic arteries. Arterioscler Thromb (1993) 13:1090–1100.[Abstract/Free Full Text]
  28. Chomiki N., Henry M., Alessi M.C., Anfosso F., Juhan-Vague I. Plasminogen activator inhibitor-1 expression in human liver and healthy or atherosclerotic vessel walls. Thromb Haemost (1994) 72:44–53.[Web of Science][Medline]
  29. Falkenberg M., Tjärnström J., Örtenwall P., Olausson M., Risberg B. Localization of fibrinolytic activators and inhibitors in normal and atherosclerotic vessels. Thromb Haemost (1996) 75:933–938.[Web of Science][Medline]
  30. Yorimitsu K., Saito T., Toyozaki T., Ishide T., Ohnuma N., Inagaki Y. Immunohistochemical localization of plasminogen activator inhibitor-1 in human coronary atherosclerotic lesions involved in acute myocardial infarction. Heart Vessels (1993) 8:160–162.[CrossRef][Web of Science][Medline]
  31. Raghunath P.N., Tomaszewski J.E., Brady S.T., Caron R.J., Okada S.S., Barnathan E.S. Plasminogen activator system in human coronary atherosclerosis. Arterioscler Thromb Vasc Biol (1995) 15:1432–1443.[Abstract/Free Full Text]
  32. Smith P.K., Krohn R.I., Hermanson G.T., Mallia A.K., Gartner F.H., Provenzano M.D., Fujimoto E.K., Goeke N.M., Olson B.J., Kleuk D.C. Measurement of protein using bicinchoninic acid. Biochem (1985) 150:76–85.
  33. Strack P.R., Waxman L., Fagan J.M. ATP-stimulated degradation of oxidatively modified superoxide dismutase by cathepsin D in cardiac tissue extracts. Biochem Biophys Res Commun (1996) 219:348–353.[CrossRef][Web of Science][Medline]
  34. Declerck P.J., Verstreken M., Collen D. Measurement of different forms of plasminogen activator inhibitor 1 (PAI-1) using various monoclonal antibody-based enzyme-linked immunosorbent assays. Fibrinolysis (1990) 4(Supp 2):132–133.[CrossRef][Web of Science]
  35. Nauland U., Rijken D.C. Activation of thrombin-inactivated single-chain urokinase-type plasminogen activator by dipeptidyl peptidase I. Eur J Biochem (1994) 223:497–501.[Web of Science][Medline]
  36. Verheijen J.H., Chang G.T.G., Kluft C. Evidence for the occurrence of a fast-acting inhibitor for tissue-type plasminogen activator in human plasma. Thromb Haemost (1984) 51:392–395.[Web of Science][Medline]
  37. Wilcox J.N., Smith K.M., Williams L.T., Schwartz S.M., Gordon D. Platelet-derived growth factor mRNA detection in human atherosclerotic plaques by in situ hybridization. J Clin Invest (1988) 82:1134–1143.[Web of Science][Medline]
  38. Höltke H.J., Kessler C. Non-radioactive labeling of RNA transcripts in vitro with the hapten digoxigenin (DIG); hybridization and ELISA based detection. Nucl Acid Res (1990) 18:5843–5851.[Abstract/Free Full Text]
  39. Siegel S, Castellan JN, Nonparametric statistics for the behavioral sciences, Singapore: McGraw-Hill Book Co; 1988:190-223.
  40. Lambers J.W.J., Cammenga M., Konig B., Pannekoek H., van Mourik J.A. Activation of human endothelial cell-type plasminogen activator inhibitor (PAI-1) by negatively charged phospholipids. J Biol Chem (1987) 262:17492–17496.[Abstract/Free Full Text]
  41. Woolf N, Thrombosis and atherosclerosis. In: Bloom AL, Thomas DP, eds. Haemostasis and Thrombosis. Edinburgh: Churchill Livingstone, 1981:527-553.
  42. Smith E.B. Fibrin deposition and fibrin degradation products in atherosclerotic plaques. Thromb Res (1994) 75:521–538.[CrossRef][Web of Science][Medline]
  43. Niculescu F., Rus H.G., Porotiu D., Ghiurca V., Vlaicu R. Immunoelectron-microscopic localization of S-protein/vitronectin in human atherosclerotic wall. Atherosclerosis (1989) 78:197–203.[CrossRef][Web of Science][Medline]
  44. Edward F.P. Leukocyte elastase release during blood coagulation. J Clin Invest. (1982) 69:564–572.[Web of Science][Medline]
  45. Underwood M., De Bono D.P. Increased fibrinolytic activity in the intima of atheromatous coronary arteries — protection at a price. Cardiovasc Res (1993) 27:882–885.[Abstract/Free Full Text]
  46. Padró T, Steins M, Hammel D, Kienast J, Atherosclerosis-related alterations of the plasminogen activator/plasmin system in vascular wall: studies in human coronary arteries, Thromb Haemost 1993;69:971 (Abstract).
  47. Falcone D.J., McCaffrey T.A., Haimovitz-Friedman A., Vergilio J.A., Nicholson A.C. Macrophage and foam cell release matrix-bound growth factors. Role of plasminogen activation. J Biol Chem (1993) 268:11951–11958.[Abstract/Free Full Text]
  48. Vassalli J.D., Sappino A.P., Belin D. The plasminogen activator/plasmin system. J Clin Invest (1991) 88:1067–1072.[Web of Science][Medline]
  49. Ehrlich H.J., Klein Gebbink R., Preissner K.T., Keijer J., Esmon N., Mertens K., Pannekoek H. Thrombin neutralizes plasminogen activator inhibitor 1 (PAI-1) that is complexed with vitronectin in the endothelial cell matrix. J Cell Biol (1991) 115:1773–1781.[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?



This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Padró, T.
Right arrow Articles by Kienast, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Padró, T.
Right arrow Articles by Kienast, J.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?