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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2002;22:400.)
© 2002 American Heart Association, Inc.

Vascular Biology

Increased Plasmin and Serine Proteinase Activity During Flow-Induced Intimal Atrophy in Baboon PTFE Grafts

Richard D. Kenagy; Jens W. Fischer; Mark G. Davies; Scott A. Berceli; Suzanne M. Hawkins; Thomas N. Wight; Alexander W. Clowes

From the Division of Vascular Surgery, Department of Surgery (R.D.K., M.G.D., S.A.B., S.M.H., A.W.C.) and Pathology (J.W.F., T.N.W.), University of Washington, Seattle.

Address correspondence to Richard Kenagy, PhD, Department of Surgery, Box 356410, University of Washington, School of Medicine, 1959 NE Pacific St, Seattle, WA 98195-6410. E-mail rkenagy{at}u.washington.edu

	Abstract

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High blood flow causes intimal atrophy and loss of extracellular matrix in PTFE aortoiliac grafts. We have investigated whether matrix-degrading proteinases are altered in this baboon model of atrophy using zymography, western analysis, and a versican degradation assay. After four days of high flow, urokinase was increased and plasminogen activator inhibitor-1 was decreased in the intima. Plasminogen was increased after seven days. Pro-matrix metalloproteinase (MMP)-2, activated MMP-2, and proMMP-9 levels were modestly increased by high flow at 7 days, whereas MMP-3 and tissue inhibitor of metalloproteinases-1 were not altered. Extracts of 4-day high-flow intimas degraded more ³⁵S-methionine–labeled versican than low-flow intimal extracts, and this activity was inhibited by AEBSF, a serine proteinase inhibitor, and a plasmin antibody. In contrast, this activity was not inhibited by the MMP inhibitor, BB-94 (Batimastat). These data suggest that serine proteinases, including plasmin, may be largely responsible for extracellular matrix degradation in this primate model of flow-induced intimal atrophy.

Key Words: intimal atrophy • flow • plasminogen • urokinase • proteoglycan • smooth muscle cells

	Introduction

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In response to changes in blood flow and shear stress, arteries adjust lumen size acutely by changes in vasomotor tone and chronically by remodeling the structure of the intima and media.^1,2 In rigid prosthetic grafts or stented arteries, which cannot dilate or contract, blood flow alters the size of the lumen by inducing growth or regression of the neointima.^3,4 PTFE grafts used to replace segments of iliac artery in baboons under high-flow conditions (created by placement of a femoral arteriovenous fistula) develop less neointima than those under normal flow.⁵ When normal flow is returned to a graft that has been allowed to heal under high-flow conditions, message levels of platelet-derived growth factor (PDGF)-A increase, intimal smooth muscle cells (SMCs) begin to proliferate, and the neointima thickens.⁶ The SMCs also synthesize extracellular matrix so that the volume of matrix per cell is similar between high- and normal-flow conditions (approximately 65% of intimal volume).⁷ Conversely, increasing the blood flow to a graft that has healed under normal flow induces atrophy of the neointimal layer within 2 months.⁸ Because atrophy involves the loss of extracellular matrix as well as cell death, we have investigated the regulation of matrix-degrading proteinases. We demonstrate a large increase of serine proteases, specifically urokinase and plasmin.

	Methods

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Sixteen male baboons (Papio cynocephalus anubis) received bilateral PTFE aortoiliac bypass grafts as described.⁸ After eight weeks, a unilateral arteriovenous fistula was created downstream between the common femoral artery and vein. Midstream graft blood velocities, measured with duplex ultrasonography as previously described,⁷ were increased acutely 2.6-fold. Animals were euthanized 4, 7, or 14 days later. The grafts of one 4-day animal were occluded with thrombosis and not used further. Graft intimas were either fixed in buffered 10% formalin or frozen for extraction. Final numbers of intimal extracts used for this study were 5, 5, and 4 for days 4, 7, and 14, respectively.

Zymography and Western Analysis
Frozen portions of the neointima were pulverized, extracted (0.05 mol/L Tris, pH 7.5, 0.01 mol/L CaCl₂, 2.0 mol/L guanidine HCl, and 0.2% Triton X-100), dialyzed (0.1 mol/L Tris, 5 mmol/L EDTA, 0.2% Triton X-100, pH 7.4), and 10 µg of protein (bicinchoninic acid method with a bovine albumin standard; Pierce) were loaded per lane for SDS-PAGE (10%) and Western blotting. Gelatin, casein, and casein/plasminogen zymography was performed as described.^9,10 Mouse monoclonal antibodies used were: plasminogen (1 µg/mL; NeoMarkers), 2-antiplasmin (2 µg/mL; American Diagnostica), and plasminogen activator inhibitor type 1 (PAI-1; 1 µg/mL; American Diagnostica). Rabbit antibodies to tissue inhibitor of metalloproteinases (TIMP)-1 and TIMP-2 were kindly provided by Dr. Howard Welgus, Parke Davis, Ann Arbor, Mich. Blots were developed using chemiluminescent reagents (Pharmacia). Western blots and zymograms were quantified as described.⁹

Versican Degradation
Human SMCs were labeled with 40 µCi ³⁵S-methionine/mL for 24 hours, and versican was isolated from the conditioned medium by diethylaminoethyl cellulose and Sepharose CL-2B chromatography as described.^11–13 Ten micrograms of intimal extract protein (in 25 µL of 100 mmol/L Tris, 10 mmol/L EDTA, 0.2% Triton X-100, pH 7.4) was added to 30,000 dpm of ³⁵S-versican with or without various proteinase inhibitors (in 25 µL of 50 mmol/L Tris, 25 mmol/L CaCl₂, pH 7.4). The anti-plasminogen antibody that blocks activity (2D1.2.2; a gift from Lindsey Miles, La Jolla, Calif) and the control immunoglobulin G1 were used at 450 µg/mL. The samples were incubated at 37°C for 20 to 24 hours and quick frozen. Chondroitin sulfate (5 µg) was added to samples with 4 volumes of 1.3% potassium acetate in 95% ethanol and left at -70°C for 3 hours. Samples were centrifuged, and the precipitate was treated with chondroitin ABC lyase (ICN Biomedicals; 0.03 U/mL in 50 mmol/L Tris, 3 mmol/L Na acetate, 0.1 mg/mL bovine serum albumin, pH 8, for 3 hours at 37°C) and run on a 4% to 12% gradient SDS-polyacryamide gel. Dried gels were exposed to photographic film or PhosphorImager and quantitation done using ImageQuant (Molecular Dynamics).

Statistical Analysis
Data are expressed as the mean±SEM of the indicated number of intimal extracts from individual animals. Statistical differences between high- and normal-flow groups were tested with the Wilcoxon signed rank test (SPSS 8.0 for Windows).

	Results

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Matrix Degrading Proteinases
To determine if matrix proteases in intimal extracts were affected by flow, we investigated the expression of the plasminogen activators and matrix metalloproteinases (MMP), which have been shown to play major roles in tissue involution and remodeling.^14–16 Urokinase (48-kd band on casein zymography; inset Figure 1A) was increased by high flow at day 4 (Figure 1A) and at 14 days, although the latter was not statistically significant. Plasminogen was observed as 85-kd and 76-kd bands (inset Figure 1B), which are consistent with Glu- and Lys-plasminogen.¹⁷ There was no significant effect of flow on the proportion of these two species (data not shown), and the total of Glu- plus Lys-plasminogen was increased at day 7 by high flow (Figure 1B).

View larger version (14K): [in this window] [in a new window]   Figure 1. The effect of increased blood flow on proteinases. A, uPA expressed as the ratio of high-to-normal flow values obtained by scanning zymograms (inset shows intimal urokinase from 4-day grafts with normal- [N] and high- [H] flow). * P<0.05 high versus normal flow; n=4 to 5. B, plasminogen expressed as the ratio of high-to-normal flow values obtained by scanning the 85-kd and 76-kd bands on Western blots (inset shows the 85-kd and 76-kd plasminogen bands in 7-day grafts). P<0.05 high versus normal flow; n=5. C, MMP-2 (closed bars, sum of pro- and active MMP-2) and MMP-9 (open bars, sum of pro- and active MMP-9) expressed as a ratio of high-to-normal flow values obtained by scanning gelatin zymograms (inset shows intimal proMMP-2 at 68 kd, active MMP-2 at 60 kd, proMMP-9 at 100 kd, and active MMP-9 at 87 kd from 7-day high- and normal-flow intimas). *P<0.05 high versus normal flow; n=4 to 5. D, MMP-3 expressed as the ratio of high-to-normal flow values obtained by scanning zymograms (inset shows the 50-kd MMP-3 band and the 76-kd and 85-kd plasminogen bands from 4-day high- and normal-flow graft intimas). P>0.1 high versus normal flow for MMP-3.

Gelatin zymography demonstrated the presence of MMP-2 (68-kd proform and 60-kd activated form¹⁸) and MMP-9 (100-kd proform and 89-kd activated form¹⁸) in the graft intima (inset Figure 1C). High flow increased proMMP-2 and proMMP9 at day 7 by 23% and 44%, respectively (Figure 1C). Although activated MMP-2 was also slightly increased at day 7 (1.21±0.08 high-to-normal flow ratio, P=0.043, n=5), active MMP-9 was not (0.66±0.22, 1.31±0.22, and 1.00±0.22 high-to-normal flow ratio for days 4, 7, and 14, respectively; P>0.20, n=4 to 5). MMP-3, which was observed as a 50-kd band in casein zymograms (Figure 1D) and non-reducing Western blots (data not shown), was not significantly changed by flow. The caseinolytic bands between 70 and 100 kd are plasmin, based on inhibition by AEBSF (data not shown), molecular weight, and response to high flow.

Proteinase Inhibitors
We also investigated some inhibitors of the plasminogen activators and the MMPs. PAI-1 was observed as doublets at 44 kd and 57 kd and variably observed as a doublet at 102 kd (inset Figure 2A; the 68-kd band is nonspecific). These represent intact PAI-1 (57 kd), PAI-1 clipped by plasminogen activators (44 kd),^19,20 and a plasminogen activator–PAI-1 complex (100 kd).²¹ The 44-kd and 57-kd doublets were decreased by high flow by 50% at 4 and 14 days (Figure 2A). This trend was also apparent for the 102 kd doublet (0.47±0.17, 2.81±0.98, and 0.36±0.02 for the high-to-normal flow ratio for days 4, 7, and 14, respectively; P=0.138, 0.138, and 0.068, respectively; n=4 to 5). The inhibitor of MMPs, TIMP-1, was mostly present as 4 to 5 bands between 120 kd and 230 kd with much less of the 28 kd free form (inset, Figure 2B). Neither the complexes nor the free TIMP-1 (Figure 2B) were significantly altered by high flow. No specific staining for TIMP-2 was observed (data not shown). Finally, the plasmin inhibitor 2-antiplasmin was observed as three bands between 50 kd and 68 kd (inset, Figure 2C); the 68-kd form represents intact 2-antiplasmin, and the others are partially cleaved forms.²² There was no statistically significant difference seen between normal- and high-flow samples in the total amount of all forms of 2-antiplasmin (Figure 2C) or in the amount of cleaved forms (data not presented).

View larger version (15K): [in this window] [in a new window]   Figure 2. Proteinase inhibitors expressed as the high-to-normal flow ratio of values obtained by quantitation of Western blots. A, PAI-1 (sum of 44-kd and 55-kd doublets). Inset shows intimal PAI-1 in 4-day graft intimal extracts. P<0.05 high versus normal flow for day 4 (n=5) and P=0.068 for day 14 (n=4). B, High molecular weight TIMP-1 immunoreactive bands (five bands from 110 to 230 kd, open bars) and free (28 kd) TIMP-1 (closed bars). Inset shows free TIMP-1 and TIMP-1 complexes in a 7-day normal-flow intimal extract. P>0.1; n=4 to 5. C, 2-Antiplasmin (sum of the three bands; P>0.2; n=4 to 5). Inset shows 2-antiplasmin in a 7-day high-flow intimal extract.

³⁵S-Versican Degradation In Vitro
Neointimal extracts were examined for proteolytic activity by incubation with ³⁵S-versican, because versican is a major vascular proteoglycan present in abundance in the graft intima. Versican core protein prepared from human arterial SMC cultures runs on SDS-polyacrylamide electrophoresis as two major bands, which we have identified as the V0 and V1 isoforms.²³ Extracts of 4-day high-flow intimas consistently degraded the V0 and V1 core proteins to a greater degree (Figure 3A and inset) than did the normal-flow intimal extracts. Extracts of 7-day high-flow intimas demonstrated variable activity compared with normal-flow extracts (two of four extracts had increased activity). Extracts of 14-day high-flow intimas consistently demonstrated higher activity than normal-flow extracts, but the results were not statistically significant. The versican-degrading activity in the day 4 high-flow extracts was completely inhibited by the serine proteinase inhibitor AEBSF, while EDTA had some inhibitory effect (Figure 3B). However, BB-94 at concentrations known to inhibit all MMPs and RG101625 (a MMP inhibitor specific for MMP-7) did not prevent versican degradation (Figure 3B). In addition, TIMP-1 and TIMP-2 at 750 nmol/L had little or no effect (data not presented). The aspartate and cysteine proteinase inhibitors, pepstatin A and E-64, also had no effect (Figure 3B). Because the serine proteinase zymogen plasminogen was detected by Western analysis in extracts, a plasmin-blocking antibody was tested and found to significantly inhibit the versican-degrading activity (Figure 3C).

View larger version (31K): [in this window] [in a new window]   Figure 3. Degradation of 35S-versican by intimal extracts. A, The effect of time after fistula placement on degradation of 35S-versican by intimal extracts. Data are presented as % of intact versican (V0 plus V1) remaining after exposure to high-flow (solid bars) or normal-flow (open bars) extracts. * P<0.05 high versus normal flow. The number of paired extracts from individual animals at each time point is 5, 4, and 3 for days 4, 7, and 14, respectively. Inset, autoradiogram of degradation products of 4-day high- and normal-flow extracts. B, The effect of proteinase inhibitors on the versican-degrading activity in 4-day extracts. In this autoradiogram, undegraded versican (V0 and V1) is shown in the first lane, and the last lane shows the effect of purified MMP-7. C, An autoradiogram showing the effect of an activity-blocking antibody against plasmin (2B1.2.2 450 µg/mL) compared with a nonimmune immunoglobulin G on the degradation of versican by 4-day intimal extracts.

	Discussion

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We have presented evidence consistent with the idea that increased proteolytic activity caused by increased blood flow contributes to PTFE graft intimal atrophy. Our results suggest that serine proteinases are a major factor in matrix degradation in this model of intimal atrophy. Plasmin and urokinase plasminogen activator (uPA) are increased more than 3-fold over normal-flow levels, whereas MMP-2 and MMP-9 are increased less than 50%. The ability of a plasmin-blocking antibody to inhibit versican degradation by intimal extracts further supports the role of this protease in graft intimal atrophy. Despite the fact that MMP-1,²⁴ MMP-2,^24,25 MMP-3,²⁴ MMP-7¹³ (Figure 3), and MMP-9²⁵ can degrade versican and MMPs are involved in arterial remodeling in response to changes in blood flow,^26,27 BB-94 had no effect on versican degradation by the intimal extracts. Although this result suggests that active MMPs are not available in the intima, this may be an artifact of the extraction process during which active enzyme may be brought together with inhibitors. It is also possible that MMPs not inhibited by BB-94 or that MMPs with specificity for substrates other than versican may be involved in matrix removal.

Although non-hepatic tissue, such as kidney, testis, intestine, and brain,^28–30 can produce plasminogen mRNA, there is no evidence for the synthesis of plasminogen by vascular SMCs. Therefore, our data suggest that high blood flow increases the transport of plasminogen across the endothelial barrier and/or increases retention of plasminogen in the intima. Numerous studies have demonstrated increased endothelial cell permeability resulting from increased shear stress supporting the former possibility. Data supporting the latter possibility are that apoptosis of SMCs is increased 2- to 3-fold by high blood flow in this model (S.A. Berceli, M.G. Davies, R.D. Kenagy, A.W. Clowes, unpublished data, 2001) and that dead and dying cells possess increased proteolytic capacity. Endothelial cells committed to apoptosis after growth factor removal have increased MMP-1, activated MMP-2, and uPA compared with starving, but viable, endothelial cells (B. Levkau, R.D. Kenagy, unpublished data, 1999). Apoptotic U937 and epithelial cells express increased uPA and plasminogen binding^31,32 mediated by the uPA receptor and plasminogen receptors.³³ Therefore, dying SMCs might bind plasminogen from the plasma and mediate the removal of extracellular matrix.

An unusual aspect of this study is the biphasic time course of uPA, PAI-1, and versican degradation. Although the changes are internally consistent, increased uPA and decreased PAI-1 with increased degradation of versican, this result is not easily explained. Because macrophages can express uPA at high levels,³⁴ one possibility is that increased flow may induce an early influx of monocytes. A loss of macrophages might then be followed later by increased expression of SMC urokinase. However, the number of monocyte/macrophages in the intima was <0.1% of intimal cells and was not changed by high flow (S.A. Berceli et al, unpublished data, 2001). Clearly more study is required to understand the mechanisms regulating protease induction in this model.

Our data suggest that by increasing plasmin activity the restenotic process that leads to failure in many vascular grafts may be reversed. Consistent with this idea is the observation that plasmin is required for the positive arterial remodeling observed after arterial injury.^15,35 In addition, arterial lesion development mediated by hyperlipidemia is accelerated by the absence of plasminogen,³⁶ indicating a protective role in atherogenesis. However, in the normolipemic context, plasmin is required for the intimal hyperplasia observed after arterial injury³⁷ and it has been demonstrated to play a role in aneurysmal expansion.^37,38 It is clear that plasmin is a multifunctional protease. Whether the regulation of this enzyme can be of value therapeutically will depend on the context in which its activity is modified.

	Acknowledgments

 
Supported by grants from the National Institutes of Health, US Public Health Service (HL30946, RR00166, HL07828, and HL18645); PTFE grafts were provided by W.L. Gore & Associates; Dr Davies was a recipient of an National Institutes of Health Cardiovascular Training Grant Fellowship (HL07828). The authors thank Holly Lea and Selina Vergel for technical assistance, Sue Perigo-Potter for preparing the ³⁵S-methionine–labeled SMC versican, and Lindsey Miles for the blocking antibody to plasmin.

Received November 21, 2001; accepted January 4, 2002.

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