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Circulation. 1997;96:1783-1789

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(Circulation. 1997;96:1783-1789.)
© 1997 American Heart Association, Inc.

Articles

Plasminogen Activator Inhibitor-1 in Neointima of Vein Grafts

Its Role in Reduced Fibrinolytic Potential and Graft Failure

Petteri Kauhanen, MD; Vappu Sirén, PhD; Olli Carpén, MD, PhD; Antti Vaheri, MD, PhD; Mauri Lepäntalo, MD, PhD; ; Riitta Lassila, MD, PhD

From the Wihuri Research Institute, Helsinki (P.K., R.L.); the Department of Virology, Haartman Institute, University of Helsinki (V.S., A.V.); the Department of Pathology, Haartman Institute, University of Helsinki (O.C.); and the Division of Vascular Surgery, Department of Surgery, Helsinki University Central Hospital (M.L.), Finland.

Correspondence to Riitta Lassila, Wihuri Research Institute, Kalliolinnantie 4, FIN-00140, Helsinki, Finland.


*    Abstract
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 *Abstract
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 down arrowMethods
 down arrowResults
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Background Intimal smooth muscle cell proliferation is an underlying pathogenetic mechanism for neointimal hyperplasia and consequent vein graft failure. This study characterizes the expression of tissue-type plasminogen activator (TPA), urokinase-type plasminogen activator (UPA), and plasminogen activator inhibitor-1 (PAI-1) in hyperplastic vein grafts and normal venous tissue.

Methods and Results Failing graft and control vein specimens from 14 donors were homogenized, and TPA and PAI-1 were quantified with ELISA. The amount of PAI-1 was seven times higher (4.2±2.1 versus 0.6±0.6 ng/mg protein, P<.005), but the TPA antigen content was markedly lower (3.1±2.1 versus 8.1±3.7 ng/mg protein, P<.005) in the stenosed grafts compared with the control veins. Strong immunohistochemical PAI-1 reactivity and in situ hybridization signals for PAI-1 and UPA mRNA were associated with the smooth muscle cells of the thickened intima of the grafts. Functional assays of the graft specimens showed an increased UPA/TPA ratio and a decreased total fibrinolytic activity in comparison with normal veins.

Conclusions Upregulation of PAI-1 mRNA expression and markedly increased amounts of PAI-1 antigen were detected in the vein grafts after the development of neointima. Furthermore, augmented UPA activity was found in the graft wall, but TPA was clearly depleted. Altogether, our findings imply decreased fibrinolytic potential in the stenosed graft, which may contribute to the graft occlusion.


Key Words: bypass • fibrinolysis • peripheral vascular disease • plasminogen activator • veins


*    Introduction
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 *Introduction
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An occlusion of the saphenous vein, which is the most important conduit used for both aortocoronary and peripheral bypass grafts, seriously compromises the long-term results of cardiovascular surgery.1 2 The current understanding of the fate of a graft is mainly extrapolated from the response-to-injury hypothesis of atherosclerosis.3 Therefore, the mechanisms responsible for the remodeling of the graft wall, resulting in intimal thickening and subsequent graft failure, are only partially understood.

After denudation of arterial endothelium, UPA and TPA have been shown to be involved in SMC proliferation and migration.4 5 Nevertheless, only limited interest has been paid to the fibrinolytic capacity in the graft,6 7 even though altered fibrinolytic capacity is known to be associated with deep venous thrombosis8 9 and early graft occlusion.10 11 Immunohistochemical studies of normal peripheral arteries and veins have shown TPA staining in both ECs and SMCs.12 13 14 After a bypass, however, when an autologous vein is inserted into the arterial circulation, the venous endothelium will be exposed to increased intraluminal pressure and shear stress.15 16 17 Consequently, the incapacity of ECs to adapt to arterialized flow conditions may lead to endothelial dysfunction and decreased expression of TPA.18 In most circumstances, however, reduced fibrinolytic potential is due to upregulation of PAIs, PAI-1 being the most important PAI in the vascular system. PAI-1 is produced primarily by SMCs, but human ECs can also secrete PAI-1 under stimulation and when cocultured with human SMCs.19 Moreover, an enhanced expression of PAI-1 mRNA has been detected in the intima of the thrombotic veins20 as well as in the proliferated SMCs of atherosclerotic arteries.20 21 22 23 The aim of the present investigation was to study the expression and function of the fibrinolytic system in arterialized veins. We compared the fibrinolytic capacity of failing vein grafts obtained during graft revisions with that of normal saphenous veins. Our findings indicate that the graft failure during the first 2 years after surgery is strongly associated with an upregulation of PAI-1 and reduced total fibrinolytic potential of the graft.


*    Methods
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 *Methods
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Tissue Specimens
This study was approved by an institutional ethical committee of the Department of Surgery, Helsinki University Central Hospital, and informed consent was obtained from the study subjects. The material consisted of vein graft stenoses and control veins harvested from arteriosclerotic patients, who after femorodistal in situ vein bypass participated in a surveillance program for identification of failing grafts. Surveillance included repeated measurements of ankle/brachial systolic pressure index and duplex scanning of the entire grafts. If stenosis was indicated by these examinations, angiography was performed to identify the lesion before an operation in which the stenosed segment was opened longitudinally and widened with a vein patch. The grafts had served as arterial conduits for an average of 13 months (range, 2 to 46 months). All the grafts were extensively stenosed because of the thickened intima but were patent and without signs of intraluminal thrombi. A longitudinal slice from the edge of the arteriotomy in the stenosed portion of the graft and a specimen from the control vein were carefully collected. The removed tissue samples were immediately snap-frozen in liquid nitrogen, stored at -70°C, and analyzed within 3 months. Antigens for TPA and PAI-1 were quantified from 12 specimen pairs. Because a majority of the graft specimens were relatively small, the functional analyses were selectively performed as follows: radial fibrinolysis (n=7), UPA activity assay (n=6, only 3 controls), and zymography (n=4). Graft and control sections for immunohistochemistry (n=6) and in situ hybridization (n=4) were embedded in optimal-cutting-temperature compound (Tissue-Tek, O.C.T. compound, Miles Scientific), frozen in liquid nitrogen, and stored at -70°C until they were cut.

Tissue Homogenization
Before they were minced, the samples for antigen analyses were thawed in 1.5 mL of PET buffer (PBS–EDTA–Tween 20 buffer from TintElize PAI-1 kit, Biopool) to which aprotinin (0.2 trypsin inhibition units/mL, Sigma Chemical Co) was added. After thawing, the samples were washed three times (5 minutes each) with 1.5 mL of PET buffer on ice. Samples for zymography, radial fibrinolysis, and UPA activity were processed without aprotinin. Then the samples were weighed and cut into small pieces. To complete the homogenization, the tissue was sonicated (Branson Sonifier Cell Disruptor B 15; continuous level 3, 4x30 seconds) in 0.2 mL of PET buffer on ice. After high-speed centrifugation (15 minutes, 1500g), the supernatant was separated and stored at -70°C until analyzed. To optimize the yield of TPA, UPA, and PAI-1, some experimental tests with normal venous tissue were performed. These preliminary experiments showed that all PAI-1 and {approx}80% of the TPA could be extracted by the described mincing procedure. In addition, we observed that the use of a reducing agent (dithiothreitol 2 mmol/L) did not improve the recovery of the proteins of interest, and thus the reductant treatment was omitted. The total protein amount of the supernatant was measured with BSA (Sigma) used as a reference.

Quantitative Assays of TPA and PAI-1
The content of TPA and PAI-1 antigens in the tissue extracts was determined with commercially available ELISA kits (TintElize tPA, TintElize PAI-1, Biopool) according to the manufacturer's instructions. The concentrations of TPA and PAI-1 in the extract were related to the total protein amount.

Zymography
The separation of plasminogen activators from the tissue extracts was performed by 8% PAGE in the presence of SDS under nonreducing conditions.24 After electrophoresis, plasminogen activators were characterized by zymography as previously described.25 Before zymography, SDS was removed by washing the gel for 3 hours with PBS, pH 7.4, containing 2.5% Triton X-100 (BDH Ltd). Subsequently, an agarose minigel to which casein and plasminogen (1.7 µg/mL) had been added was placed over the polyacrylamide gel before incubation for 48 to 96 hours at 37°C in a humidified atmosphere. Activity standards of UPA (Calbiochem) and TPA (American Diagnostica) as well as low-molecular-weight marker proteins (Pharmacia) were used for estimation of the molecular weights of the lysis bands. To further characterize the plasminogen activator distribution, an anticatalytic MAb against UPA (10 µg/mL, catalogue No. 394, American Diagnostica) was added over the polyacrylamide gel before the incubation with the casein-agarose gel.

Radial Fibrinolysis Assay
Fibrin/agarose plates were prepared according to Booth.26 Briefly, 2 mL of 1% fibrinogen and 8 mL of 1% agarose in Tris-buffered saline (pH 7.8) to which bovine {alpha}-thrombin (60 mU/mL, Dade, Baxter Healthcare Co) was added were mixed at 44°C. The mixture was poured onto polystyrene Petri dishes (9 cm in diameter) and allowed to coagulate for 5 minutes at 37°C. The wells (3 mm in diameter) were cut and removed by suction, loaded with 7 µL of the sample, incubated for 72 hours at 37°C, and subsequently photographed. In addition, TPA standards (Actilyse, Boehringer Ingelheim, specific activity 460 000±72 000 IU/mg) at several concentrations were used to approximate the level of activity. For quantitative assessments, the protein content of the tissue homogenates was standardized to 1.5 mg/mL, and the diameter of the lysis zones was measured.

Immunocapture Assay for UPA
For assessment of UPA activity, microtiter wells of polystyrene immunoplates (type 269620, Nunc) were coated overnight at 37°C with 50 µL of a solution of goat IgG antibodies to human urokinase (10 µg/mL) (American Diagnostica). After they were rinsed, the wells were incubated with 50 µL of tissue extract for 2 hours at 23°C. To assay the bound enzyme activity, 40 µL of plasminogen solution was incubated in the wells. Subsequently, plasmin generation was assessed colorimetrically. This solid-phase assay for UPA has been described in detail elsewhere.27

Immunohistochemistry
MAb against human PAI-1 (subclass IgG1, No. 3785, American Diagnostica) and a control isotype-matched MAb X63 (ATCC) were used at a concentration of 5 µg/mL. Before staining, the tissues were cryosectioned at 5 µm and placed on gelatin-coated slides, where they were allowed to thaw slowly. Then they were fixed with cold acetone and air-dried. For immunohistochemical staining, the avidin-biotin complex/horseradish peroxidase method (Dakopatts A/S) was used. The whole staining procedure was done according to the manufacturer's instructions. After immunoreaction, the slides were rinsed with water and counterstained with hematoxylin-eosin.

Riboprobes and Labeling
The pHUK-8 UPA clone28 was obtained from ATCC, and PAI-1 cDNA represented a BamH1 fragment.29 It was a kind gift from Dr Leif Lund, Finsen Laboratory, Copenhagen, Denmark. The templates for oligonucleotide probes with either SP6 or T7 promoter sequence on the 5' end were produced by polymerase chain reaction. Probes were 226 bases for PAI-1 (positions, 134 to 360 bases)30 and 240 bases for UPA (positions, 1421 to 1661 bases)28 and were labeled with digoxigenin-uridine triphosphate by in vitro transcription with SP6 and T7 RNA polymerases according to the manufacturer's instructions (Boehringer Mannheim). Positive controls included cells known to produce PAI-1 and UPA. Negative controls included the use of sense RNA probes.

In Situ Hybridization
In situ hybridization was performed as described in Boehringer Mannheim's application manual with minor modifications. Frozen 5-µm tissue sections were mounted on Silane-coated slides and fixed for 10 minutes in 4% paraformaldehyde. The sections were washed in PBS before dehydration in ethanol and stored at -70°C until use. Then they were treated with 0.2N HCl for 5 minutes before the permeabilization with proteinase K (5 µg/mL, Sigma) for 15 minutes at 37°C, after which they were rinsed with PBS and 2xSSC (1xSSC being 150 mmol/L NaCl and 15 mmol/L sodium citrate). Solutions were treated with diethylpyrocarbonate (Sigma D5758) before hybridization.

The sections were prehybridized for 2 hours at 37°C in a hybridization mixture (50% formamide, 2xSSC, 500 µg/mL herring sperm DNA, 500 µg/mL Escherichia coli tRNA, and 1xDenhart's solution). After dehydration, the probes (15 ng per section) were added into the hybridization mixture (containing 5% dextran sulfate), which was heated at 80°C for 7 minutes and cooled before addition to the tissue sections.

Hybridization was performed overnight at 42°C in a humidified chamber. Nonspecifically bound probe was removed by two washes with 50% formamide in 2xSSC. After the sections were rinsed for several times with SSC and treated with a buffer containing 0.1 mol/L Tris-HCl (pH 7.5), 0.15 mol/L NaCl, and 0.05% Tween-20 (AP-1), the sections were incubated for 30 minutes at 37°C in solution containing 1% BSA and 0.05% Triton X-100. Alkaline phosphatase–labeled anti-digoxigenin antibody (Boehringer Mannheim) was then added (1:1000 dilution in AP-1) before incubation for 60 minutes at 37°C. After several rinses with the AP-1 and a buffer containing 0.1 mol/L Tris-HCl (pH 9.5), 0.1 mol/L NaCl, and 50 mmol/L MgCl2 (AP-3), the antibody was visualized with nitro blue tetrazolium, bromochloro-indolyl phosphate (Boehringer Mannheim), and levamisol (Sigma) in the AP-3 buffer. Sections were incubated with the substrate in the dark for 2 hours at room temperature before they were counterstained with eosin and detected with light microscopy.

Statistical Analysis
The results are reported as mean±SD. The paired comparisons between grafts and controls were performed with a nonparametric Wilcoxon signed rank test. The graft subclasses were compared by ANOVA. Correlation analyses were done by the nonparametric Spearman rank test.


*    Results
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 *Results
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Total Protein of Vessel Wall
In macroscopic examination, marked thickening of the vessel wall was observed in all the grafts, but the control vessels showed no evidence of hyperplasia. After tissue extraction, the mean protein content of the graft samples was 5.5±2.7 mg/mL versus 2.6±1.1 mg/mL of the control samples, respectively. However, on a weight basis, the proportion of the extracted protein from the two tissues did not differ, being 2.0±1.1% in the graft group and 1.8±0.8% in the control group. To assess the effect of the specimen size (grafts, 75±50 mg; controls, 43±34 mg) on the extractable protein content of the supernatant, we computed a correlation analysis, which showed a strong negative correlation (grafts, r=-.76, P<.05; controls, r=-.60, P<.05) between the weight of the tissue specimens and the proportion (percentage) of the extractable protein.

TPA and PAI-1 Antigen Assays
Quantitative analysis of the extracted fibrinolytic proteins revealed that the fibrinolytic capacity of the graft tissue was significantly reduced. The amount of PAI-1 per total protein of the tissue extract was seven times higher in the stenosed grafts than in the control veins (4.2±2.1 versus 0.6±0.6 ng/mg protein, P<.005) (Fig 1ADown). In contrast, TPA antigen content was markedly decreased in the graft versus the control tissue (3.1±2.1 versus 8.1±3.7 ng/mg protein, P<.005) (Fig 1BDown). To evaluate the internal compatibility of the TPA and PAI-1 measurements, correlation analyses were performed. The amount of TPA and PAI-1 correlated negatively in each specimen (r=-.65, P<.002). The protein-related TPA (r=.18) and PAI-1 (r=-.19) did not associate with the actual protein content of the tissue extract. Thus, the incomplete homogenization of the larger samples did not distort the TPA and PAI-1 analyses.



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  		Figure 1. PAI-1 (A) and TPA (B) in control vein and bypass graft tissues. Antigen concentrations are expressed in ng/mg protein in extract. Mean of group is indicated by horizontal lines. Statistical significance was tested by nonparametric Wilcoxon signed rank test.

To study the influence of time from operation on the fibrinolytic proteins of the graft tissue, the specimens were divided into three subclasses. Although the observed differences in PAI-1 and TPA contents between the grafts of different ages were not statistically significant, this analysis showed that the amount of extractable PAI-1 of the graft tissue was most prominent in veins obtained <6 months after their interposition into the arterial circulation. The lowest TPA content, on the other hand, was detected in the grafts that had been arterialized for 7 to 24 months (Fig 2Down).



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  		Figure 2. Influence of time elapsed from operation on expression of proteins of fibrinolytic system. Antigen concentrations (mean±SD) are expressed in ng/mg protein in extract. A, PAI-1 antigen. B, TPA antigen. Subclasses comprise the following specimens: I, <6 months after graft insertion (n=5); II, 7 to 24 months after graft insertion (n=5); and III, >24 months after graft insertion (n=2); controls, 12 normal vein specimens. Graft subclasses do not differ statistically from each other, but all are different from controls (PAI-1, P<.05; TPA, P<.12).

Radial Fibrinolysis Assay
To study the total activity of the extracted plasminogen activators, samples of graft and control vein homogenates were added to wells cut into plates containing fibrin, the natural substrate of in vivo fibrinolysis. As the control experiments with the added TPA demonstrate (Fig 3ADown), the fibrinogen solution was contaminated with plasminogen.26 These experiments proved that the fibrinolytic potential of the graft tissue on the fibrin plates was consistently decreased compared with that of the control veins (Fig 3Down). Thus, with a standardized protein content of the sample, the mean diameter of the lysis area was 5.9±3.0 mm in the grafts and 9.1±1.9 mm in the controls (P<.05). The fibrinolytic activity of all graft and control specimens was markedly lower than that of the lowest TPA standard, which represented specific activity of 1.5 IU/mg.



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  		Figure 3. Total fibrinolytic activity of tissue extracts. Plates were photographed after incubation for 72 hours at 37°C. A, TPA standards at concentrations of 100, 50, 10, and 5 ng/mL (starting from top left), respectively. B, Two graft-control pairs; controls are on left and grafts are on right side of plate. Actual TPA and PAI-1 concentrations in the upper panels were 34.5 and 1.1 ng/mL for the controls and 2.9 and 10.5 ng/mL for the grafts, respectively; in the bottom panels, the actual TPA and PAI-1 concentrations were 33.9 and 5.2 ng/mL for the controls and 5.5 and 20.5 ng/mL for the grafts, respectively.

Zymography
To characterize the plasminogen activators present, tissue homogenates were analyzed by zymography (Fig 4Down). In the analysis of the control samples (lanes 1 and 3), a strong lysis was evident at the position of the TPA standard (70 kD). In some control specimens (lane 1), faint lysis was also present near the UPA (50-kD) standard. However, this UPA activity was relatively weak compared with the TPA activity of the same specimens. The graft samples essentially differed from one another and from the controls. One specimen, obtained 5 months after the bypass, presented a strong lysis at the position of the UPA standard, but TPA activity was absent (lane 2). Two graft samples obtained 6 months after the bypass demonstrated both TPA and UPA activity. However, the oldest specimen, obtained 14 months after the bypass (lane 4), showed a rather weak lysis close to the UPA standard (after 96 hours), in contrast with strong TPA activity indicating a variable UPA/TPA ratio during the progression of arterialization.



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  		Figure 4. Zymography of vein graft and control vein specimens (lanes 1 through 6). Lane 1, control vein (patient 1); lane 2, stenosed graft 5 months after insertion of graft (patient 1); lane 3, control vein (patient 2); lane 4, stenosed graft 14 months after insertion of graft (patient 2); lane 5, TPA standard (70 kD); and lane 6, UPA standard (50 kD). Photograph was taken after incubation for 48 hours at 37°C. After 96 hours, older graft specimen (lane 4) developed a weak lysis also at position of UPA standard (not shown).

Immunocapture Assay for UPA
To quantify the impact of UPA in the total fibrinolytic capacity of the graft, UPA activities (IU/g protein) of six graft and three control samples were assessed by the immunocapture assay. According to these analyses, both the grafts and the controls contained detectable amounts of active UPA, but the mean UPA activity between the grafts and the controls (5.3±3.5 versus 6.5±4.3 IU/g protein) did not differ, suggesting an indistinct distribution of UPA in vascular tissues.

Immunohistochemistry
To determine the localization of PAI-1 antigen, cryosections of the graft and normal vein tissues were studied by use of a specific MAb. Immunohistochemical analysis of PAI-1 showed that this protein was abundantly present in SMCs of the thickened intima (Fig 5ADown). The staining of PAI-1 was most intense in the cells near the vessel lumen, and the intensity decreased toward the adventitial layer. Interestingly, ECs did not demonstrate PAI-1 antigen. No reactivity was seen in vessels stained with a control MAb (Fig 5BDown). Control vein specimens from the same individuals demonstrated only very weak and scattered PAI-1 reactivity (Fig 5CDown).



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  		Figure 5. Immunohistochemical PAI-1 staining of human vein graft and control vein specimens. A, Neointima of stenosed vein graft 39 months after grafting immunostained with an MAb against PAI-1. In ELISA, content of PAI-1 of this specimen was 3.4 ng/mg protein. B, Same graft specimen immunostained with a control isotype-matched MAb X63 (negative control). C, Control vein immunostained with an MAb against PAI-1. In ELISA, content of PAI-1 of this specimen was 1.4 ng/mg protein. Magnification x160 (A through C).

In Situ Hybridization
To localize the expression of PAI-1 and UPA genes, in situ hybridization was performed on cryosections of the vessel wall specimens (Fig 6Down). In the grafts, a strong hybridization signal for PAI-1 mRNA was present in the deeper layers of the thickened intima (Fig 6ADown), which was in good agreement with the aforementioned immunohistochemistry results. However, the superficial layers beneath the endothelium, which showed the presence of PAI-1 antigen in immunohistochemistry, and ECs themselves did not present hybridization signals. Furthermore, UPA transcripts could be detected in SMCs of hyperplastic neointima (Fig 6CDown), although the difference between the grafts and the control vein specimens was not as obvious as when PAI-1 was studied. Hybridization signals for both PAI-1 and UPA of control sections were faint and dispersedly located (not shown).



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  		Figure 6. In situ hybridization of PAI-1 mRNA in graft harvested 39 months after insertion into arterial circulation. In ELISA, content of PAI-1 of this specimen was 3.4 ng/mg protein. Hybridization signals are seen as dark brown areas (arrows). A, Hybridization with digoxigenin-labeled antisense PAI-1 RNA probe. Magnification x400. B, Hybridization with digoxigenin-labeled negative control sense PAI-1 RNA probe. Magnification x400. C, Hybridization with digoxigenin-labeled antisense UPA RNA probe. Magnification x200. D, Hybridization with digoxigenin-labeled negative control sense UPA RNA probe. Magnification x200.


*    Discussion
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 *Discussion
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The expression of plasminogen activators (TPA, UPA) and their principal inhibitor, PAI-1, has been widely investigated in vitro by use of vascular ECs and SMCs in cell cultures.19 31 32 Clinically, increased plasma levels of PAI-1 have been detected in connection with thrombotic and inflammatory disease states and several surgical settings,8 including the postoperative period of patent venous bypass.33 Moreover, studies with transgenic mice having overexpressed or underexpressed fibrinolytic proteins have shown that a deficiency of TPA does not affect the intimal changes after vascular trauma, whereas a deficiency of UPA delays the neointima formation.34 Interestingly, the rate of neointimal cell accumulation was accelerated in mice with a deficiency of PAI-1. Because vein graft occlusion caused by underlying neointimal hyperplasia remains an unresolved clinical problem, the present study was designed to elucidate the role of the fibrinolytic system in a developing vein graft failure in humans.

In the present study, we have shown that patent but hyperplastic infrainguinal vein grafts synthesize PAI-1 and UPA. According to the quantitative assessments of the graft extracts by ELISA, PAI-1 content was markedly and consistently increased, whereas TPA was depleted. Because the endothelial and intimal fibrinolytic system is less active in normal arteries than in veins,20 35 it is plausible that our findings reflect the progression of the arterialization process, ie, the adaptation of venous intima to arterial circulation. The total fibrinolytic capacity of the tissue extracts was assessed with radial fibrinolysis, a traditional technique to study fibrinolysis.26 Even after standardization of the protein concentration and other experimental conditions, this method remains only semiquantitative. After all, our results remained consistent and showed that the total fibrinolytic activity was decreased in all graft specimens. Actually, similar observations were reported as early as in the 1970s, when Yao et al6 studied the fibrinolytic activity in thrombotic and patent vein grafts and detected no activity near thrombi and only minimal activity in the endothelium of patent grafts. Also, reduced fibrinolytic activity has been reported in a segment of the feline caval vein interposed in the aorta.7 In our radial fibrinolysis assay, it was impossible to ascertain whether the reduced fibrinolytic capacity of grafts was due to a decreased activity of plasminogen activators or upregulation of PAI-1. It was also impossible to characterize the separate roles of TPA and UPA in the total activity. Therefore, to identify the plasminogen activators present, zymography was performed on some of our samples. In zymography, the graft specimens were found to be TPA-deficient, which paralleled our ELISA results. In addition, zymography showed marked UPA activity during the early adaptation, which was absent in some control specimens. These results are in agreement with the observations by Carmeliet,34 who reported that UPA- rather than TPA-mediated plasmin proteolysis contributes to the intimal healing and restenosis after vascular trauma in transgenic "knockout" mice. Inconsistently, however, our immunocapture assay for UPA showed an even amount of UPA activity in the grafts and the controls. A different contribution of active PAI-1 to the assay is a possible explanation for the disagreement between these two functional UPA determinations (zymography and the immunocapture assay).

Using in situ hybridization and immunohistochemical stainings, we could localize the enhanced PAI-1 and UPA expression to the SMCs of neointima. An increased expression of PAI-1 mRNA has previously been found in the intima around the thrombus in occluded saphenous veins.20 This observation was similar to the early results of Yao et al6 obtained a decade before the discovery of PAI-1. Nevertheless, because we wanted to study the pathophysiology of neointimal hyperplasia, all graft specimens included in the present study were obtained from patent grafts without signs of thrombosis. Patency of the grafts was verified by preoperative duplex scanning and arteriogram, by intraoperative macroscopic inspection, and by postoperative microscopy (cryosections from six grafts). Recently, several research groups have reported localization and increased synthesis of plasminogen activators13 35 and PAI-121 22 36 in atherosclerotic arteries, suggesting active proteolysis. Although the progression of atherosclerosis and the vein graft failure due to neointimal hyperplasia show some pathophysiological similarities, it is important to note that in many respects, they are distinct entities.

To evaluate the temporal relationship between the arterialization process and altered synthesis of fibrinolytic proteins, PAI-1 and TPA contents were compared in grafts of different ages. We observed that PAI-1 production was strongly increased in the graft samples obtained during the first 6 months after the revascularization. Subsequently, the content of PAI-1 was lower, but still above the levels in control veins, in the grafts that had been exposed longer to the arterial circulation. Thus, the lowest TPA levels were detected in the graft specimens obtained 7 to 24 months after the primary reconstruction. The long-term observations described in the present study differ markedly from the findings of Diamond et al,31 who reported that when human ECs were cultured for 86 hours under arterial flow conditions, they produced more than twice as much TPA as under venous flow conditions. We therefore suggest that venous ECs have only a limited capacity to produce TPA during adaptation to arterial circulation. A similar decline in TPA expression has been reported after experimental angioplasty injury37 and cardiac transplantation.38 After a few days, however, SMCs begin to produce both UPA and TPA, especially during migration from media to intima,5 and the fibrinolytic potential partially recovers.37 Although the described changes in the fibrinolytic system may be necessary to maintain the patency of the graft, it is also possible that this new balance of plasminogen activators (ie, upregulation of UPA) has mitogenetic effects on SMCs.34 This process can be modulated by several growth factors, such as transforming growth factor-ß or platelet-derived growth factor, which are both detectable in vein grafts before the development of neointima.39

Up to the present, the use of autologous saphenous vein grafts has been a mainstay of the surgical treatment of coronary artery disease and critical leg ischemia.1 40 Despite its extensive use, the saphenous vein is far from a perfect conduit for the purposes of cardiovascular surgery, and it is widely accepted that the internal mammary artery is superior to the autologous vein as a graft in coronary artery bypass. To explain this phenomenon, it has been reported that the glycosaminoglycan composition of veins with an abundance of dermatan sulfate differs markedly from that of arteries, in which heparan sulfate predominates, and this difference may favor atherogenesis in arterialized veins.41 In addition, arteries seem to be resistant to SMC proliferation caused by prolonged exposure to pulsatile stretch,42 and endothelial nitric oxide production and prostacyclin release are also greater in arteries than in veins.43 Moreover, it has been shown that products from aggregated platelets relax the mammary artery but weakly contract the saphenous vein.44 In most cases, however, the saphenous vein is still the best and only possible biological conduit for arterial reconstructions. Therefore, to improve the results of surgery, further efforts should be focused on the pathophysiology of vein graft failure.

In summary, the hyperplastic intimal layer of vein graft produced increased amounts of PAI-1 and UPA. Simultaneously, the natural TPA production of the graft tissue was markedly depleted, resulting in a change in the distribution of plasminogen activators. It is evident that this decreased and altered fibrinolytic activity of the graft tissue is associated with graft failure. It remains unclear, however, whether the pronounced upregulation of PAI-1 is a cause or a consequence of neointimal hyperplasia and whether it is the prevention of fibrinolytic proteins by pharmacological agents, by specific antibodies, or by gene therapy that will be beneficial during the evolution of vein graft disease.


*    Selected Abbreviations and Acronyms
 
EC = endothelial cell
MAb = monoclonal antibody
PAI = plasminogen activator inhibitor
SMC = smooth muscle cell
TPA = tissue-type plasminogen activator
UPA = urokinase-type plasminogen activator


*    Acknowledgments
 
This work was supported by grants from the Paavo Nurmi Foundation, the Aarne Koskelo Foundation, the Medical Research Council of the Academy of Finland, and the Finnish Society of Angiology. The authors wish to thank Satu Cankar, Tuula Halmesvaara, and Tuula Järvenpää for their excellent technical assistance. Dr Vesa Manninen is acknowledged for his constant encouragement.

Received December 31, 1996; revision received April 8, 1997; accepted May 1, 1997.


*    References
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 *References
 

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