Plasminogen Activator Expression in Human Atherosclerotic Lesions
Abstract The plasminogen activator (PA) system may participate in the pathogenesis of atherosclerosis by modulating the turnover of intimal fibrin and extracellular matrix deposits and by contributing to intimal cell migration. We present an analysis of tissue-type PA (tPA) and urokinase-type PA (uPA) expression at three levels: mRNA by in situ hybridization, antigen by immunohistochemistry, and enzymatic activity by histoenzymology and zymography. For PA colocalization with cellular or matrix components, we used double immunofluorescence labeling in conjunction with confocal microscopy. In normal arteries, tPA antigen and mRNA were detected in endothelial cells and smooth muscle cells (SMCs). In atherosclerotic arteries, tPA antigen and mRNA were increased in intimal SMCs and in macrophage-derived foam cells of fibrofatty lesions. Part of the tPA was detected in the extracellular space and colocalized with fibrin deposits. uPA antigen and mRNA were detected in association with the intimal macrophages and SMCs. A particularly high uPA expression was noted on macrophages localized on the rims of the necrotic core. Moreover, using a novel histoenzymological assay as well as classic zymography, we revealed uPA-dependent lytic activity in the advanced lesions, whereas in normal arteries, only tPA-dependent activity was detected, mainly over the vasa vasorum. Also, strong tPA and uPA staining was detected in neomicrovessels of the plaques, suggesting that PAs may play a role in plaque angiogenesis. Our results suggest a local dynamic process of PA-dependent proteolysis in lesion areas that is associated with macrophages and SMCs. A better comprehension of these proteolytic mechanisms in advanced atherosclerotic plaques may provide the basis for therapeutic approaches for plaque stabilization.
- tissue-type plasminogen activator
- urokinase-type plasminogen activator
- smooth muscle cells
The PA system is an enzyme cascade system that generates localized proteolysis in a highly regulated fashion. The zymogen plasminogen is activated to plasmin, a trypsin-like protease, by the action of PAs, of which there are two types, tPA and uPA.1 PAI-1 and PAI-2, as well as specialized cell surface binding proteins such as the uPA and plasminogen receptors, ensure that proteolytic activity is confined in space and limited in time.
The PA system may participate in the development of atherosclerotic lesions by a variety of mechanisms: the extent of PA expression and consequently of plasmin generation not only affects the rate of fibrin degradation on or within advanced plaques but also may influence, directly or by activation of metalloproteinases, the turnover of ECM deposits. Furthermore, plasmin can activate cytokines, such as transforming growth factor-β and basic fibroblast growth factor, that are known to participate in atherogenic processes.2 3 4
Fibrinolytic dysfunctions, in particular elevated plasma levels of PAI-1, have been associated with thromboembolic disease and may represent a risk factor for acute coronary thrombosis5 or postoperative deep vein thrombosis in elective hip surgery.6 An increased expression of PAI-1 antigen and mRNA occurs in atherosclerotic lesions in SMCs and in macrophages located at the periphery of the necrotic core.7 8 In pathological situations, PA expression may also be modified. Thus, in a rat model of injured carotid artery, an increase of both uPA and tPA activity and mRNA was detected in extracts of the media.9 Atherosclerotic lesion areas contain fibrin as well as fibrin degradation products, suggesting a continuous formation of fibrin coupled with a fibrinolytic process in the arterial intima.10
In the present study, we investigated in human atherosclerotic arteries the localization of the activity, antigen, and mRNA of both tPA and uPA. Cell type–specific antibodies were used to identify the producer cells. Our results demonstrate that in atherosclerotic lesions, both PAs are expressed by macrophages and SMCs.
Preparation of Vascular Tissue
All samples were obtained during cardiovascular surgery, immediately embedded in optimal cutting temperature compound (OCT compound, Miles Scientific), frozen in cold methyl butane (Merck), and stored at −70°C. The aorta samples for this study were small pieces cut from the ascending aorta before the proximal anastomosis of an aortocoronary bypass was constructed. Three specimens had advanced atherosclerotic lesions with a necrotic core and a fibrous cap, four showed a lipid-rich thickened intima that was invaded by numerous macrophages, and three showed a thickened intima with no or minimal macrophage invasion. Ten carotid specimens showing advanced atherosclerosis were obtained after endarterectomy surgery. Segments of normal internal mammary or popliteal arteries were used as control tissues. The tissue samples were cryosectioned at 7 μm and thaw-mounted onto poly-l-lysine–coated slides for in situ hybridization and on gelatin-coated slides for immunohistochemistry. For in situ hybridization, the sections were air-dried, fixed in 4% paraformaldehyde (Sigma Chemical Co) in PBS composed of (in g/L) NaCl 8, KCl 0.2, Na2HPO4 · 2H2O 1.44, and KH2PO4 0.2, pH 7.4, dehydrated, and stored at −20°C with desiccant. Slides for immunohistochemistry were air-dried overnight and fixed in cold acetone.
In addition to the MAbs listed in the Table⇓, two polyclonal goat anti–human melanoma tPA antibodies (American Diagnostica, No. 387, and Biopool) and a rabbit anti–human uPA antibody (American Diagnostica, No. 389) were used at a concentration of 20 μg/mL for immunohistochemical purposes or to block PA activity for zymographic analysis.
An isotype-matched MAb that did not react with human tissue or serum proteins (Ms IgG1, Coulter Immunology), mouse ascites fluid, and nonimmune serum from goat (Sigma) were used as controls.
A 1974-bp fragment from the human tPA cDNA and a 600-bp fragment from the human uPA cDNA were subcloned in the Bluescript M13+ vector (Stratagene Inc) and labeled by run-on transcription with 35S-labeled UTP (specific activity, 1300 Ci/mmol; New England Nuclear). Sense and antisense probes were prepared by linearizing the constructs with the appropriate restriction endonucleases and the use of either T7 or T3 RNA polymerase (Boehringer Mannheim).
For bright-field immunohistochemical staining, either the alkaline phosphatase/anti–alkaline phosphatase double bridge (Dako-APAAP Kit) or the avidin–biotinylated peroxidase complex (Vectastain ABC Kit, Vector Laboratories) technique was applied. Incubation with the first antibody was done for 60 minutes at room temperature. All further steps were performed according to the manufacturer’s instructions.
Immunofluorescence and Confocal Microscopy
To establish the PA distribution in different cell types of normal and atherosclerotic tissues, we used a double immunofluorescence labeling approach. PAs were identified by specific polyclonal antibodies raised in rabbits against recombinant tPA or human uPA (both from American Diagnostica). As cell-specific markers, we used MAbs as listed in the Table⇑. After the proper blocking treatments, the frozen tissue sections were incubated overnight at 4°C with cocktails containing a PA-specific polyclonal antibody and a cell type–specific MAb. As secondary antibodies, a mixture of horse anti-mouse IgG–Texas Red and goat anti-rabbit IgG–FITC was used. The sections were studied with a Bio-Rad MRC 600 confocal laser scanning unit attached to Nikon Diaphot inverted microscope (Bio-Rad Microscience Ltd). The light source was a krypton/argon laser (Ion Laser Technology) with principal lines at 488, 568, and 674 nm. For simultaneous visualization of FITC and Texas Red staining, the K1 and K2 filter blocks were used.
In Situ Hybridization
The procedure was performed according to the method of Holland.11 The slides were brought to room temperature at least 2 hours before use, acid-treated in 0.2 mol/L HCl for 20 minutes, washed, and fixed in 4% (wt/vol) paraformaldehyde for 10 minutes. This was followed by two wash steps in PBS and 10 minutes of incubation in 0.25% acetic anhydride in 0.1 mol/L Tris HCl, pH 8. After three wash steps in PBS, the slides were dehydrated and air-dried. The cRNA probe (in 50% formamide [BRL], 0.3 mol/L NaCl, 1× Denhardt’s solution, 0.02 mol/L Tris-HCl, pH 8.0, 5 mmol/L EDTA, 5% dextrane sulfate, 50 mmol/L dithiothreitol, and 500 μg/mL yeast total RNA [Boehringer Mannheim]) was used at 106 cpm per slide. Sections were covered with siliconized coverslips and hybridized at 50°C overnight (12 hours) in a chamber humidified with 50% formamide, 0.3 mol/L NaCl, and 1× Denhardt’s solution. To remove coverslips, sections were immersed in 4×SSC (1×SSC is 0.15 mol/L NaCl and 0.015 mol/L trisodium citrate, pH 7.0) at 37°C and then washed in 4×SSC at 37°C. After an RNase A (Boehringer Mannheim) treatment (20 mg/mL) for 30 minutes at 37°C, the slides were washed in 0.5 mol/L NaCl, 10 mmol/L Tris HCl, pH 7.5, and 1 mmol/L EDTA, followed by 1 hour of incubation at 50°C in 1×SSC and 2×1 hour at 50°C in 0.1×SSC. All solutions of the posthybridization wash steps contained 2 mmol/L dithiothreitol. The graded alcohol series for the final dehydration contained 300 mmol/L ammonium acetate. The sections were air-dried and covered with an autoradiographic emulsion (NTB-2, Kodak) according to the manufacturer’s instructions, stored in black airtight boxes at 4°C, and developed after 10 to 20 days. The slides were viewed with a Nikon Optiphot 2 microscope equipped with a mercury UV lamp and epipolarization filters.
Localization and Quantification of PA Proteolytic Activity
We developed a novel histoenzymological method using a synthetic peptide coupled to 4-methoxy-β-naphthylamide (4MβNA) as a substrate to localize and quantify the proteolytic activity of uPA. Similar methods have already been developed for other proteases, such as elastase and cathepsin B.12 To demonstrate urokinase activity, unfixed cryostat sections were prepared as described above. The incubation medium (1 mL 0.1 mol/L Tris-HCl buffer, pH 7.2, 30.5 mg NaCl [final concentration 0.5 mol/L], 10 μL 2-hydroxy-5-nitrobenzaldehyde [5′ nitrosalicyl aldehyde; 17 mg dissolved in 200 μL ether and 800 μL dimethylformamide, final concentration 1 mmol/L], and 1 mg of H-Gly-Arg-4MβNA [Bachem] as a specific substrate13 ) was applied to the section, onto which a coverslip was placed. The development of yellow fluorescence as an indicator of protease activity was monitored with the CLSM equipment. A semiquantitative evaluation of the fluorescent reaction product was done with pseudocolor bands. Samples incubated in substrate-free medium were used as controls.
The zymography technique, which is not confined in space and does not provide quantifiable results, was used simply to confirm the histoenzymological data. The protocol is based on the method of Todd.14 Briefly, cryosections of vascular tissue were overlaid with 100 μL of a mixture consisting of 2% casein, 0.8% agarose, and 40 μg/mL Glu-plasminogen at 50°C. The slides were incubated at 37°C in a humidified chamber for 1 to 3 hours. Controls were performed with a plasminogen-free overlay or a mixture to which specific neutralizing antibodies such as goat anti–human melanoma tPA IgG or specific rabbit anti–human uPA IgG (both from American Diagnostica) were added. Photographs were taken under dark-field illumination.
PAs in Normal Arterial Tissue
A panel of monoclonal and polyclonal antibodies specific for human tPA and directed against different epitopes was used to determine the localization of this protein in normal arterial tissue. In apparently healthy human mammary arteries, tPA antigen was detected both in endothelial cells and in SMCs (Fig 1a⇓) identified by anti–von Willebrand factor (Fig 1c⇓ through 1e) or anti–α-actin (not shown) antibodies, respectively. In situ hybridization analysis revealed that the same cells were also positive for tPA mRNA (Fig 1f⇓). In the adventitia, expression of tPA antigen (Fig 1b⇓) and mRNA (Fig 1g⇓) was restricted to SMCs and endothelial cells of small arteries and venules, whereas no tPA was detected in cells of the adventitial connective tissue. Controls for immunostaining using isotype-matched MAbs, mouse ascites fluid, and nonimmune goat serum (not shown) or for in situ hybridization using sense cRNA (Fig 1h⇓) were consistently negative.
Immunocytochemical analysis for uPA revealed that this protein was present in luminal endothelial cells and in medial SMCs (Fig 1i⇑). The distribution of uPA in the media was discontinuous, which is in contrast to the more homogeneous distribution of tPA. In the adventitia, a positive reaction for uPA was detected in association with SMCs and endothelial cells of small arteries and venules (Fig 1j⇑).
PAs in Atherosclerotic Tissue
Successive sections of human aortas with fibrous lesions were analyzed for tPA mRNA expression by in situ hybridization. The cell types present in the lesion areas were identified by use of specific MAbs. A particularly strong tPA mRNA signal was detected over the thickened intima (Fig 2a⇓ and 2b⇓) compared with the subjacent media. The silver grains were associated with cells positive for the macrophage-specific marker (Fig 2c⇓) or with α-actin–positive SMCs (Fig 2d⇓). Medial SMCs appear to express lower tPA levels.
tPA antigen was detected both in the thickened intima and in the media (Fig 3⇓). Double immunofluorescence staining for tPA (Fig 3a⇓) and von Willebrand factor (Fig 3b⇓) revealed the colocalization of the two antigens over luminal endothelial cells (Fig 3c⇓). Serial frozen sections stained for tPA (Fig 3d⇓) or α-actin (Fig 3e⇓) suggest that SMCs located both in the intima and in the media contain tPA. Simultaneous immunofluorescence staining on the same section of tPA (Fig 3f⇓) and α-actin (Fig 3g⇓) revealed a high degree of coincident labeling (Fig 3h⇓). In fibrofatty lesions of human aortas and carotid arteries, tPA antigen was detected also in foam cells positive for macrophage-specific markers, as was revealed by immunolabeling for the two antigens on successive sections (Fig 3i⇓ and 3j⇓) and double immunofluorescence labeling on the same section (Fig 3k⇓ through 3m). By means of confocal imaging, integrating Z-serial optical sections (0.5 μm thick) through a double-labeled tissue cryosection (20 μm), it was possible to follow the track of tPA-immunostained microvessels in the thickness of the section. By use of this method, a very strong tPA labeling was observed in neomicrovessels located in the fibrous cap of the plaque and identified by von Willebrand factor staining (Fig 3n⇓).
tPA localization was mainly cell-associated, but in advanced atherosclerotic plaques, we could detect some extracellular tPA, mainly in association with fibrin deposits (Fig 3o⇑).
In atherosclerotic aortas, uPA mRNA and antigen were detected in the thickened intima and in the media (Fig 4a⇓ and 4b⇓). Immunohistochemical analysis using cell type–specific antibodies revealed that uPA is expressed both in macrophage-rich intimal areas (Fig 4c⇓) and in SMC-rich areas (Fig 4d⇓). uPA antigen was detected in association with von Willebrand–positive microvessels located within the cap of the plaque (Fig 4g⇓ and 4h⇓). Particularly strong uPA-specific mRNA signals were seen over macrophages located in the cap of the necrotic core (Fig 5a⇓ and 5b⇓). To colocalize uPA antigen and activity with macrophages within the atherosclerotic plaque, serial frozen sections were analyzed both by immunocytochemistry and/or in situ histoenzymology and zymography. The double immunofluorescence labeling for uPA and macrophage marker confirmed the colocalization of uPA antigen and macrophages within the shoulders of the plaque surrounding or located within the plaque core (Fig 5c⇓ through 5e). The histoenzymological localization of uPA activity by the method described above showed the fluorescent reaction product in large amounts in the areas of the plaque shoulders and core. Image analysis by pseudocolor banding demonstrated high-intensity fluorescent staining (Fig 5g⇓, magenta) in the areas stained by uPA- and macrophage-specific antibodies, revealing the presence of active uPA associated with macrophages (Fig 5f⇓ and 5g⇓). The fluorescent precipitate was found in low amounts or was absent over these areas when the substrate was omitted (Fig 5h⇓) or in the presence of uPA-neutralizing antibodies (not shown), proving the specificity of the method. However, some unspecific fluorescent staining was found over calcified regions.
The localization of PA activity in association with macrophages was established by tissue zymography also. Zymographs performed in the presence of plasminogen showed PA-dependent lytic activity over cell-rich areas of atherosclerotic lesions (Fig 6⇑). Polyclonal antibodies to uPA abolished the lytic zone (Fig 6b⇑), whereas anti-tPA had no effect (Fig 6a⇑). No caseinolysis was detected in the absence of plasminogen (not shown). On zymographs it is very difficult to determine the cell type expressing proteolytic activity. However, by studying the time course of caseinolysis, one can observe that the lytic process started in the macrophage-rich areas (Fig 6a⇑ and 6d⇑). In normal tissue, only tPA-dependent activity was detected, mainly in association with blood vessels of the adventitia (not shown).
Proteolytic events participate in many aspects of atherogenesis and its major complications, myocardial infarction and stroke. They are involved in (1) intimal migration of macrophages and SMCs; (2) activation of basic fibroblast growth factor and transforming growth factor-β, which play a role in atherogenesis2 3 ; (3) turnover of intimal fibrin deposits; (4) degradation of ECM proteins, leading to weakening of the fibrous cap of the plaque; and (5) lysis of occlusive thrombi in stenosed coronary arteries, which is the cause of myocardial infarction.
Previously an increase of PAI-1 expression was reported in atherosclerotic areas.7 8 The aim of the present work was to determine to what extent and by which cells PAs are expressed in normal and atherosclerotic arterial tissue. In normal human arteries, we found that endothelial cells and SMCs express not only tPA antigen and protein but also uPA antigen. The presence of tPA immunoreactivity in all three layers of rat aorta was recently reported by Padro et al.15 Signals for uPA mRNA were weak over SMCs and undetectable over endothelial cells. The absence of a signal over endothelial cells may be due to insufficient sensitivity of the in situ hybridization technique. However, it is also possible that intimal uPA is derived from the blood circulation and becomes bound to the endothelial uPA receptor.16
The role of the various factors of the PA system in the normal blood vessel wall is at present only partially understood. Endothelial cells in vivo contain tPA,17 which is present to some extent in storage granules18 and can be released at high levels after a thrombogenic stimulus.19 The presence of tPA in and of uPA on endothelial cells may contribute to the antithrombotic properties of the blood vessel wall. The role of the PA system in the arterial media is less obvious. SMCs located in the media showed a strong staining for tPA mRNA and antigen, suggesting a constant tPA production. It has not yet been established whether SMCs, like endothelial cells, contain tPA storage vesicles. Also, uPA was detected in the media. By zymographic analysis we detected tPA-dependent activity associated with the vasa vasorum, but we detected no PA activity over the media, which is consistent with the presence of a high local concentration of PAI-1. These observations do not exclude PA-dependent proteolysis in the media, which could take place at the level of cell surfaces. Such a mechanism could provide for a regulated, steady turnover of ECM, which would contribute to the plasticity of the vascular tissue.
In atherosclerotic tissue, both tPA and uPA were detected. These proteins were expressed by SMCs in the intima and in the adjacent media. Particularly strong tPA and uPA antigen and mRNA signals were detected in macrophage-rich lesion areas, in macrophage-derived foam cells, and in macrophages located in the cap of the necrotic core. Recently, increased fibrinolytic activity was reported in extracts of the intima of atheromatous arteries.20 In accordance with these findings, we observed increased uPA-mediated activity over the macrophage-rich area of an advanced lesion. The increased expression of tPA and of uPA in intimal SMCs may contribute to the migration of these cells into and within the intima. Indeed, in rats, balloon catheter injury of the carotid artery resulted within days in an increased expression of uPA and tPA in intimal SMCs.9 Inhibition of the PA system by orally administered tranexamic acid led to a significant reduction of the number of cells that had migrated into the intima.21 In this report, we show an increase of tPA mRNA expression in SMCs located in active lesions, possibly mediated by platelet-derived growth factor,21 which suggests that, in humans, tPA is produced by SMCs and may facilitate their migration into an atherosclerotic intima.
Macrophages constitute a regular component of an atherosclerotic lesion and locally produce relatively high amounts of tPA, uPA, and PAI-1, resulting in detectable uPA-mediated lytic activity. Most likely, macrophage-associated PAs contribute to their migration into and within the lesion areas. The simultaneous expression of the PAIs and the uPA receptor would confine PA activity at the cell surface.22 The mechanism of tPA expression by the intimal macrophages is not clear. Resting monocytes do not normally produce tPA, but under certain conditions, eg, after stimulation with lipopolysaccharide or interleukin-4,23 they are able to do so. Which of the cytokines or growth factors in lesion areas are responsible for the induction of tPA production by macrophages remains to be established.
Plaque disruption with subsequent platelet aggregation and thrombosis is the most important mechanism by which atherosclerosis leads to acute cardiovascular disease, such as myocardial infarction and sudden cardiac death.24 25 Plaques that are disrupted and cause thrombotic disease contain higher concentrations of lipids and macrophages than nondisrupted plaques.26 27 The detection of uPA within the advanced, macrophage-rich atherosclerotic plaques gives new insights into the mechanisms governing the proteolytic weakening of the plaque.28 The key enzymes that regulate the extracellular proteolysis are uPA and plasmin. Both bind to specific surface receptors and thus focus the proteolytic activity on the cell surface.29 30 31 Plasmin may activate the metalloproteinases released by macrophages, such as collagenases, gelatinases, and stromelysin, with subsequent degradation of collagen, elastin, and proteoglycans of the fibrous cap.32 33 34 Recently, Galis et al35 showed that plaque shoulders and regions of foam cell accumulation display locally increased expression of 92-kD gelatinase, stromelysin, and interstitial collagenase, suggesting that focal overexpression of activated matrix metalloproteinases may promote destabilization and complication of atherosclerotic plaques. Macrophage foam cells are frequently found at the rupture site, where the cap is usually thin, probably because of preceding tissue destruction causing progressive plaque weakening.36 Ultimately, the cap may disintegrate or rupture whether it is stressed or not. Macrophages may be “bad,” producing proteolytic enzymes34 37 and other tissue-destroying substances, but they may also constitute an important defense mechanism, attempting to clear lipids from the plaque. Transforming growth factor-β is an interesting example of the complexities of local regulation and participates in a double-negative feedback regulation. Local activation of the PA system leads to enhanced ECM degradation but also to activation of transforming growth factor-β. This growth factor is a powerful inducer of PAI-1 biosynthesis in endothelial cells and SMCs38 and thereby induces a decrease of PA activity. In addition, it is a powerful inducer of the biosynthesis of ECM components.
The overexpression of plasminogen activators in the atherosclerotic plaques, especially in the advanced ones, may have important functions in their destabilization and rupture and provide novel targets for therapeutic interventions.
Selected Abbreviations and Acronyms
|CLSM||=||confocal laser scanning microscope|
|PAI||=||plasminogen activator inhibitor|
|SMC||=||smooth muscle cell|
|SSC||=||standard saline-citrate buffer|
|tPA||=||tissue-type plasminogen activator|
|uPA||=||urokinase-type plasminogen activator|
This work was supported by stipends from the Roche Foundation (Dr Heim), the Sandoz Foundation, the Foundation for Research on Atherosclerosis and Thrombosis (Dr Bachmann), the Swiss National Fund for Scientific Research (grant 31.26302.89), and in part by the British Heart Foundation grant PG 94188 (Drs Lupu and Kakkar). We thank Elisabeth Cousin for preparing the plasmids used for in situ hybridization and Patricia Harley for her expert technical assistance in the immunofluorescence work.
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