This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Allaire, E. Articles by Clowes, A. W. Search for Related Content PubMed PubMed Citation Articles by Allaire, E. Articles by Clowes, A. W.

(Circulation. 1998;98:249-255.)
© 1998 American Heart Association, Inc.

Basic Science Reports

Prevention of Aneurysm Development and Rupture by Local Overexpression of Plasminogen Activator Inhibitor-1

Eric Allaire, MD; David Hasenstab, BS; Richard D. Kenagy, PhD; Barry Starcher, PhD; Monika M. Clowes, BA; ; Alexander W. Clowes, MD

From the Department of Surgery, University of Washington, Seattle (D.H., R.D.K., M.M.C., A.W.C.), and the University of Texas Health Center at Tyler, Department of Biochemistry (B.S.). Dr Allaire is currently at the Service de Chirurgie Vasculaire, Hôpital H. Mondor, 51 Avenue du Maréchal de Lattre de Tassigny, 94010 Créteil, France.

Correspondence to Alexander W. Clowes, MD, Department of Surgery, University of Washington School of Medicine, 1959 NE Pacific St, HSB Box 356410, Seattle, WA 98195-6410.

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background—Arterial aneurysms exhibit a loss of elastin and an increase in the plasminogen activators urokinase plasminogen activator (u-PA) and tissue plasminogen activator (t-PA). Because u-PA, t-PA, and plasmin have a limited proteolytic activity against elastin, the role of plasminogen activators in the aneurysmal disease is unclear. To investigate this question, we overexpressed plasminogen activator inhibitor-1 (PAI-1), an inhibitor of t-PA and u-PA, in a rat model of aortic aneurysm.

Methods and Results—Guinea pig–to-rat aortic xenografts were seeded with syngeneic Fischer 344 rat smooth muscle cells retrovirally transduced with the rat PAI-1 gene (LPSN group) or the vector alone (LXSN group). Some grafts were not seeded with cells (NO group). Western blots showed increased PAI-1 in grafts from the LPSN group compared with LXSN and NO groups. All grafts in the NO group (n=8) and 40% in the LXSN group ruptured between days 4 and 14. At 4 weeks in the LXSN group, the remaining unruptured grafts (n=6) were aneurysmal (diameter increase 100%), whereas in the LPSN group (n=6) none of the grafts had ruptured or were aneurysmal. Elastin was preserved in the LPSN group. t-PA, the major PA expressed in the model, was decreased in the LPSN group compared with the other groups, as determined by zymography. Quantitative zymography showed decreased levels of two matrix metalloproteinases (MMPs), a 28-kD caseinase, and activated MMP-9 in the LPSN group.

Conclusions—The blockade of plasminogen activators prevents formation of aneurysms and arterial rupture by inhibiting MMP activation.

Key Words: plasminogen activators • genes • metalloproteinases • aneurysm • transplantation

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Arterial aneurysm development and rupture are thought to be the consequence of elastin and collagen degradation by proteases derived mainly from inflammatory cells infiltrating the media and the adventitia.^{1 2 3 4 5} Serine proteases (t-PA, u-PA, and plasmin)^{6 7 8} as well as MMPs (MMP-1, MMP-2, MMP-3, and MMP-9)^{9 10 11 12 13 14 15} have been detected in aneurysm walls at higher concentrations than in normal or stenotic atherosclerotic arteries.

The role of the PA/plasmin pathway in the formation of aneurysms has not been defined. Infusion of plasmin in rat arteries does not cause aneurysms, whereas infusion of pancreatic elastase does.¹⁶ Because PA and plasmin have a limited proteolytic activity against components of the extracellular matrix, their involvement in aneurysm formation and rupture might be indirect. Both t-PA and u-PA convert plasminogen into plasmin, which can activate proMMP-3.^{17 18} In turn, MMP-3 can activate proMMP-1 and proMMP-9.^{18 19 20} In addition, u-PA and plasmin can activate proMMP-9.^{21 22} Therefore, the PA/plasmin system could act as a trigger to activate the MMP pathway of arterial extracellular matrix degradation in aneurysms.

We developed a model of arterial aneurysm formation by taking advantage of the immunogenicity of guinea pig aortic extracellular matrix grafted into rats.^{23 24} During the rejection of the xenograft, monocyte/macrophages and T lymphocytes penetrate into the graft media where immunoglobulins are deposited. Elastin in the media is degraded, and the graft dilates and becomes an aneurysm by 1 month after engraftment.²⁴ When rat recipients are immunized with guinea pig aortic extracellular matrix before transplantation (preimmunization), the rate of rejection is increased and the xenograft ruptures.²⁵ During aneurysm formation and rupture in this model, the gelatinolytic and elastinolytic MMP-9 is upregulated. In this respect, this xenograft model reproduces the main features of the human disease (arterial dilation and rupture, elastin degradation, inflammatory cell infiltration in the media, and upregulation of MMPs). In a study using this model, we showed that the inhibition of MMPs by local overexpression of TIMP-1 blocks aneurysm formation and rupture and preserves elastin while blocking MMP-9 activity.²⁵ This result suggested to us that MMPs degrade arterial extracellular matrix and cause aneurysm formation and rupture in the xenograft model.

In the present study, we investigated PA activity during aneurysm formation in the xenograft model. To block PA activity, we overexpressed PAI-1 by seeding syngeneic rat SMCs retrovirally transduced with rat PAI-1 cDNA. We studied the impact of PAI-1 overexpression on MMP activity and aneurysm formation and rupture.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Animal Model
Male Fischer 344 rats (250 and 80 g) and male Hartley guinea pigs (400 g) from Simonsen Laboratories Inc (Gilroy, Calif) were housed and cared for in the Animal Facility of the University of Washington School of Medicine, Seattle. All animal experimentation complied with the National Institutes of Heath guidelines (NIH publication 86-23). All protocols were approved by the Animal Care Committee of the University Washington School of Medicine.

Orthotopic engraftments of 10-mm-long segments of decellularized guinea pig aorta (xenograft) or Fischer 344 rat aorta (isograft) into Fischer 344 rats were performed as previously described.²³ For decellularization, the grafts were incubated in 0.1% SDS to obtain arterial extracellular matrix without cells.²⁴ The xenografts dilate and exhibit a progressive loss of elastin, whereas the rat isografts do not dilate after implantation and elastin in their media is preserved. Therefore, we compared decellularized xenografts and isografts to investigate PA and PAI activities during aneurysm formation. To increase the rejection process and obtain xenograft rupture, 80-g Fischer 344 rats were immunized by repeated injections of homogenized decellularized guinea pig arterial extracellular matrix with no adjuvant before transplantation (preimmunization). Preimmunized rats were used as recipients for all graft cell seeding experiments.

Graft diameter and length were measured after engraftment and at harvest. A diameter increase of >100% was defined as graft aneurysm degeneration. This definition corresponds to the generally accepted clinical criterion for significant aortic aneurysm dilation.

Seeding of PAI-1–Transduced Cells
The preparation of replication-deficient retrovirus and the isolation and transfection of the Fischer 344 rat SMCs has been described elsewhere.²⁶ The rat PAI-1 gene (a generous gift from Dr Thomas Gelehrter²⁷ ) was slightly modified by the addition of a perfect Kozak sequence at the 5' end and then cloned into the multiple cloning site of the retroviral vector LXSN. The LXSN vector and packaging cell lines were a generous gift from Dr A.D. Miller, Fred Hutchinson Cancer Research Center, Seattle, Wash.²⁸

Transduced cells were seeded into the graft lumen just after transplantation through a PE 10 catheter introduced through a transverse arteriotomy. Cells (8x10⁶) suspended in 5% BSA were used for each graft. The aortotomy was closed with a 10/0 Ethicon suture, and flow was reestablished.

Elastin Assay
Desmosine, a cross-linking amino acid present only in elastin, was quantified as a measure of elastin content in the tissue. The samples (5 mm long) were freeze-dried, weighed, and hydrolyzed in 6N HCl at 105°C for 24 hours. The samples were evaporated to dryness and redissolved in 500 µL of distilled water, and the radioimmunoassay for desmosine was performed in triplicate on 5-µL samples as described previously.²⁹

Western Blots
For Western analysis, pools of 3 grafts in each group were cut with razor blades, crushed in a mortar, and extracted in iced buffer containing 75 mmol/L potassium acetate, 0.3 mol/L NaCl, 0.1 mol/L L-arginine, and 10 mmol/L EDTA in 0.25% Triton X-100, pH 4.2, for 15 minutes³⁰ and centrifuged at 10 000 rpm for 10 minutes at 4°C. Protein content in the supernatant was measured against a BSA standard. Samples (20 µg) were subjected to 10% SDS-PAGE and transferred onto a nylon membrane (Bio-Rad). Membranes were blocked with 3% BSA and incubated with either a rabbit anti–rat PAI-1 antibody (1 µg/mL) (American Diagnostica Inc), goat anti–human t-PA antibody (15 µg/mL) (American Diagnostica Inc), or sheep anti–human MMP-3 serum (dilution 1:1000, a gift from Dr H. Nagase, University of Kansas Medical Center, Kansas City, Kan). Secondary antibodies were anti-rabbit or anti-sheep IgG conjugated with alkaline phosphatase (dilution, 1/7500) with nitro blue tetrazolium and BCIP as a substrate (Promega). Controls included incubation with a sheep or goat serum. In addition, Western blots of the same extracts incubated with an anti–u-PA antibody (American Diagnostica Inc) did not show bands at the same molecular weight level as Western blots with anti–PAI-1 and anti–MMP-3 antibodies.

Zymography for PAs and Reverse Zymography for PAIs
Pools of 3 grafts were extracted as described for Western blots. Equal protein amounts in each group were subjected to 10% SDS-PAGE. The gel was rinsed in 2.5% Triton X-100 and incubated in 1xPBS for 20 minutes each. The gel was applied to an agar gel containing milk casein and human glu-type plasminogen (American Diagnostica Inc) in 1% agarose.³¹ After incubation at 37°C in a moist chamber, gels were photographed with dark-field illumination. Areas of casein lysis appeared as dark areas in a white background. t-PA and u-PA were identified by incorporating selective inhibitors into the gel (anti–human t-PA No. 387 from American Diagnostica Inc and 1 mmol/L amiloride from Sigma Chemical Co, respectively).³¹

For reverse zymography to detect PAIs, the preparation of the underlay was the same as for zymography, except that 250 mU/mL u-PA was added to the underlay. Areas of PAI activity appeared as white on a black background.³¹

Gelatin and Casein Zymography of MMPs
Pools of 3 grafts were extracted with 0.05 mol/L Tris, pH 7.5, containing 0.01 mol/L CaCl₂, 2 mol/L guanidine, and 0.2% Triton X-100. Extracts were dialyzed against 0.05 mol/L Tris/0.2% Triton X-100, pH 7.5, for 48 hours at 4°C. Equal amounts of extracted proteins were subjected to 10% SDS-PAGE in gels containing 0.1% gelatin or 0.1% casein. Gels were then incubated in 2.5% Triton X-100 for 30 minutes, further incubated for 18 hours at 37°C in 50 mmol/L Tris/10 mmol/L CaCl₂, pH 8.2, and finally stained with 0.008% Coomassie blue R (Sigma).³² Various proteinase inhibitors (30 mmol/L EDTA [Sigma]), recombinant TIMP-1 (a gift from Dr H. Nagase), or 1 mmol/L PMSF (Sigma) was added to the incubating buffer to assess the class of proteinase. Activation with 1 mmol/L APMA was used to identify MMP activities.³³ On treatment of extracts with 1 mmol/L APMA, there was a shift of the high-molecular-weight gelatinolytic bands of MMP-9 to a lower-molecular-weight (80-kD) band, suggesting that the latter is activated MMP-9 (data not shown). For quantification, gels containing the extracts from the groups of grafts to be compared were scanned with ScanJet 3C/T, Hewlett Packard, using Adobe Photoshop 3.0. MMP-9–related gelatinolytic bands (100, 90, and 80 kD) and MMP-3–related caseinolytic bands (48 and 28 kD) were quantified with Image Quant (v3.3, Molecular Dynamics). For MMP-9, results were plotted against a standard curve generated on the same gel with purified human MMP-9 (a gift from Dr H. Welgus, Washington University, St Louis, Mo).

Statistical Analysis
Comparison between 2 groups of grafts was done with a Mann-Whitney U test. Comparison between 3 groups was done with Kruskall-Wallis one-way ANOVA. If the one-way ANOVA showed significant differences, pairwise comparisons were done with a Dunn's test. A value of P<0.05 was accepted as significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
t-PA Activity and Graft Degeneration
Xenografts become aneurysmal by 4 weeks after implantation.^{24 25} To establish a relationship between aneurysm formation and PA activity in the xenograft model, we extracted pools of decellularized isografts and xenografts harvested 1 and 2 weeks after implantation. Inflammatory cell penetration into the media is evident at these times in xenografts but not isografts.²⁴

By zymography, graft extracts exhibited a plasminogen-dependent, caseinolytic 72-kD band that was more intense in xenograft than in isograft extracts (Figure 1). This caseinolytic band was inhibited by a blocking anti–t-PA antibody but not by amiloride. A 55-kD activity appeared after a longer time of incubation (>48 hours) and was inhibited by amiloride, not by the anti–t-PA blocking antibody, which demonstrated that it was u-PA. Western blots with anti–t-PA antibody showed a 72-kD band more intense in xenografts than in isografts 1 and 2 weeks after engraftment in naive rats (data not shown). No immunoreactive u-PA could be detected on Western blots. On reverse zymography, a 50-kD band was visible in graft extracts. This band, which corresponds to the molecular weight of PAI-1, was intense in isografts and low or absent in xenografts (Figure 1).

View larger version (27K): [in this window] [in a new window]   Figure 1. PAs and PAIs in aneurysm model. Top, Zymogram demonstrating t-PA (Mr 72 kD) activity in normal rat aorta, rat aortic isografts (iso), and guinea pig xenografts (xeno) at 1 and 2 weeks after implantation; bottom, reverse zymogram demonstrating PAI-1 (Mr 55 kD).

Xenografts implanted in preimmunized animals showed similar patterns of PA activity at day 3, but no PAI-1 activity could be detected on reverse zymograms (data not shown).

Graft Diameter and Rupture in Preimmunized Rats
All nonseeded grafts placed in preimmunized rats ruptured between 4 and 14 days. Four of 10 LXSN cell–seeded grafts (40%) ruptured within 4 weeks. The remaining 6 grafts were aneurysmal at 4 weeks (Figure 2). None of the 6 LPSN cell–seeded grafts ruptured, and none were aneurysmal at 4 weeks.

View larger version (16K): [in this window] [in a new window]   Figure 2. Cumulative rupture rates and changes in graft diameter at 4 weeks. Guinea pig aortic xenografts were seeded with control (LXSN) and PAI-1–expressing (LPSN) SMCs and implanted into abdominal aortas of rats previously immunized with guinea pig aortic extracts. Horizontal line corresponds to increase in diameter of 100%. **P<0.01, Mann-Whitney U test.

Elastin Network and Desmosine Content in Preimmunized Rats
Histological cross sections of xenografts harvested 4 weeks after implantation show that elastin was completely deleted in the LXSN cell–seeded group, whereas the internal elastic lamina and the lamellae in the media were preserved in the LPSN cell–seeded grafts (Figure 3). Desmosine content at 4 weeks was lower in the LXSN cell–than in the LPSN cell–seeded grafts (Figure 4). Taken together, these results indicate that elastin structure and content were preserved in the LPSN cell–seeded grafts but not the LXSN cell–seeded grafts. Cell nuclear staining showed similar amounts of inflammatory cells in the media of LXSN cell–and LPSN cell–seeded grafts.

View larger version (53K): [in this window] [in a new window]   Figure 3. Elastin in media of seeded xenografts at 4 weeks. Elastin in media of nonruptured LXSN cell–seeded graft (left) and LPSN cell–seeded graft (right) at 4 weeks after implantation in preimmunized rats. Elastin stains as dark fibers in media (arrow, internal elastic lamina). Magnification x40.

View larger version (14K): [in this window] [in a new window]   Figure 4. Desmosine content in unruptured grafts at 4 weeks. Desmosine content (pmol/mg dry wt) was measured in control (LXSN) and PAI-1–expressing (LPSN) cell–seeded grafts (n=6 in each group). *P<0.05, Mann-Whitney U test.

PAI-1 Protein Overexpression in Preimmunized Rats
Extracts of LXSN cell–and LPSN cell–seeded grafts at 3 days after implantation in preimmunized rats showed equal amounts of the 50-kD band corresponding to rat PAI-1 on Western blots. On reverse zymograms, PAI-1 activity could not be detected in nonseeded, LXSN cell–seeded, or LPSN cell–seeded grafts implanted in preimmunized rats (data not shown). However, bands between 104 and 160 kD were more abundant in the LPSN cell–seeded than in the LXSN cell–seeded grafts (Figure 5); these bands might be complexes formed between PAI-1 and PAs or vitronectin³⁴ in the LPSN cell–seeded grafts. Western blots performed under reducing conditions (2 mmol/L DDT) showed, in addition to the 50-kD band, sharp 29- and 27-kD bands in the LPSN cell–seeded but not in the LXSN cell–seeded grafts, which might correspond to partially degraded forms of PAI-1.

View larger version (43K): [in this window] [in a new window]   Figure 5. PAI-1 in grafts. Extracts of unseeded grafts (no seeding) and grafts seeded with control (LXSN) or PAI-1–expressing (LPSN) SMCs were analyzed for PAI-1 protein by Western blotting. A, PAI (Mr 50 kD) and PAI complexes (Mr 104 to 160 kD); B, PAI-1 degradation products (Mr 27 and 29 kD). Lanes were loaded with equal amounts of protein.

PA Activity in the Seeded Grafts at Day 3 in Preimmunized Rats
PA activities were analyzed after a 3-day implantation in preimmunized rats. At this early time point, no graft had ruptured, and therefore comparisons between groups could be made. The major PA activity was a 72-kD band inhibited by anti–t-PA antibody and not by amiloride, which hence was t-PA. In the LPSN cell–seeded grafts, the t-PA activity was much reduced compared with LXSN cell–seeded grafts and nonseeded grafts (Figure 6).

View larger version (42K): [in this window] [in a new window]   Figure 6. t-PA activity in seeded grafts. Zymograms of rat urine and extracts from unseeded grafts (no seeding) and grafts seeded with control (LXSN) or PAI-1–expressing (LPSN) SMCs. To identify t-PA and u-PA activity, gels were treated with a blocking antibody to t-PA or amiloride, respectively.

MMP Activity in the Seeded Grafts at Day 3 in Preimmunized Rats
On gelatin zymograms, extracts from grafts implanted for 3 days in preimmunized rats displayed bands of gelatinolysis between 100 and 80 kD and between 72 and 68 kD. These bands were inhibited by 30 mmol/L EDTA and by TIMP-1 but not by 1 mmol/L PMSF and therefore were MMP activities likely to correspond to MMP-9 (M_r 100 to 80 kD) and MMP-2 (M_r 72 to 68 kD) (Figure 7A). In LXSN cell–seeded grafts, bands were shifted toward lower molecular weights (M_r 90 and 80 kD), whereas in LPSN cell–seeded grafts, bands were at the highest molecular weights (100 and 90 kD) (Figure 7B), suggesting a lesser degree of MMP-9 activation in the LPSN cell–seeded grafts. No difference in MMP-2 zymographic pattern was evident between the 2 groups.

View larger version (38K): [in this window] [in a new window]   Figure 7. Gelatinase activities in graft extracts. Extracts of grafts seeded with control (LXSN) or PAI-1–expressing (LPSN) cells were made 3 days after implantation and analyzed by gelatin zymography (A), and relative amounts (B) of MMP-9 bands (100, 90, and 80 kD as percent of total activity) were determined. The 80-kD band is the activated form of MMP-9. No changes in MMP-2 activity were detected (72 and 68-kD bands). *P<0.05, Mann-Whitney U test.

A 28-kD lytic band appeared on casein zymograms and was more intense in the LXSN cell–than in the LPSN cell–seeded grafts (Figure 8A and 8B). This lytic activity was inhibited by 30 mmol/L EDTA and by 2 µg/mL TIMP-1 in the incubating buffer but not by 1 mmol/L PMSF and therefore is an MMP activity. The 28-kD band was not detected on Western blots with an anti–human MMP-3 antibody. However, a 55-kD band corresponding to proMMP-3 was present by Western analysis in the LPSN cell–but not in the LXSN cell–seeded grafts (data not shown). Furthermore, the fact that the 28-kD band was unaffected by incubation with 1 mmol/L APMA³³ ruled out the possibility that this 28-kD band could be MMP-7 (matrilysin). These results suggest that LPSN cell seeding prevented the conversion of proMMP-3 into its activated 28-kD form.

View larger version (35K): [in this window] [in a new window]   Figure 8. MMP caseinolytic activities in graft extracts. Extracts of grafts seeded with control (LXSN) or PAI-1–expressing (LPSN) cells were analyzed for proteolytic activity by casein zymography (A). To identify MMPs, gels were treated with either EDTA or recombinant TIMP-1. Quantification of 28-kD activity is shown in B. Lanes were loaded equally with pools of 3 grafts extracted 3 days after implantation. *P<0.05, Mann-Whitney U test.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The serine proteases t-PA, u-PA, and plasmin compose a system involved in fibrinolysis and in focal degradation of fibrin and extracellular matrix during angiogenesis, cancer metastasis, and arthritis.^{35 36} In arteries mechanically injured in vivo, t-PA is upregulated during SMC migration from the media to the intima, a process in the arterial response to injury.³¹ Treatment with heparin, which decreases smooth muscle migration after arterial injury, inhibits the expression of t-PA.³⁷ In arterial explants, the migration of SMCs is inhibited by an anti–t-PA antibody.³⁸ These results suggest that the lysis of arterial extracellular matrix could involve t-PA. In atherosclerotic plaques and arterial aneurysms, inflammation is thought to be associated with extracellular matrix lysis leading to thrombosis and arterial enlargement and either fibrous cap or arterial wall rupture.^{39 40 41 42} Plasmin, u-PA, and t-PA have been detected in these arterial lesions,^{43 44} as well as PAI-1.⁴⁵ In a model in mice, u-PA appears to be necessary for aneurysm formation.⁴⁶ Because plasminogen is synthesized in the liver, its activation to plasmin in focal areas of the arterial wall is thought to be mediated by PAs secreted by SMCs, endothelial cells, and inflammatory cells. In particular, mononuclear phagocytes have been shown to infiltrate the adventitia and the media of arterial aneurysms.^{4 5} In this report, we show in a xenograft model of arterial aneurysm formation that t-PA activity is upregulated. We show that PAI-1 overexpression preserves elastin in the media while decreasing t-PA activity, preserving elastin in the media, and preventing aneurysm formation. A similar approach using PAI-2 overexpression in vivo has been shown to inhibit metastasis, a phenomenon in which extracellular matrix lysis is necessary for the movement of transformed cells in tissues.⁴⁷

PAIs form inhibitory complexes with their C-terminus end and the catalytic domain of PAs.^{48 49} In addition, inactive t-PA–PAI-1 complexes compete with active t-PA to occupy binding sites on fibrin.⁵⁰ By these two mechanisms, the balance between PAs and PAIs might modulate PA activity in tissues. PAI-1 is secreted by endothelial cells,⁵¹ SMCs, and macrophages.⁵² PAI-1 content in atherosclerotic and aneurysmal arteries might be increased by the influx of platelets and mononuclear phagocytes.⁵² However, despite its presence in inflammatory arterial lesions, the concentration of PAI-1 might be inadequate for full PA blockade. In addition, PAI-1 could be inactivated by oxidants generated by inflammatory cells and by thrombin.^{49 53} Our results comparing xenografts with isografts shows that the degradation of medial elastin is accompanied by an upregulation of t-PA activity and a parallel decrease in PAI-1 activity in graft extracts.

Plasmin degrades extracellular matrix glycoproteins,⁵⁴ fibronectin, laminin, and vitronectin,³⁵ but the PA/plasmin system has no direct proteolytic activity against elastin and collagens.⁵⁵ These matrix proteins are substrates for MMPs, such as MMP-9, MMP-2, and MMP-12.^{56 57 58 59 60 61} We recently showed in the xenograft model that the inhibition of MMPs by overexpression of an inhibitor of MMPs, TIMP-1, also results in elastin preservation and prevents aneurysm formation and rupture.²⁵ These findings underline the importance of MMPs in arterial extracellular matrix degradation. Because most MMPs are secreted as zymogens,⁶² MMP activity in the extracellular space is dependent on the activation of the zymogen. In vitro studies suggest that the PA/plasmin system might be a major pathway of MMP activation.^{21 22} In particular, plasmin can convert proMMP-3 (stromelysin 1) into active MMP-3.¹⁸ Therefore, the PA/plasmin system could trigger extracellular matrix degradation by activating the MMP pathway of proteolysis. ProMMP-3 has an apparent molecular weight of 57 kD and can be cleaved into lower-molecular-weight fragments (45, 28, and 24 kD) that have similar proteolytic activities for proteoglycans, type I gelatin, type IV collagen, fibronectin, and laminin.^{63 64} PAI-1 overexpression resulted in a decrease in 28-kD caseinolytic activity. These activities can be inhibited by EDTA and TIMP-1 and are likely to be activated forms of MMP-3. Moreover, Western blot analysis showed a band of 54-kD proMMP-3 in grafts seeded with LPSN cells but not with LXSN cells. Taken together, these results suggest that PAI-1 overexpression partially prevented MMP-3 activation and resulted in a decrease in MMP-3 active forms. Therefore, the PA/plasmin system could trigger extracellular matrix degradation by activating the MMP pathway of proteolysis.

The MMP-9 pattern of activation was also modified by PAI-1 overexpression. Higher-molecular-weight gelatinolytic activities were detected in the LPSN cell–seeded grafts compared with LXSN cell–seeded grafts. u-PA and plasmin have been shown to activate MMP-9.^{21 22} PAI-1 overexpression in xenografts might also lower MMP-9 activation by lowering MMP-3, because MMP-3 can activate MMP-9.⁶⁵ Because MMP-9 has elastinolytic properties as well as gelatinolytic activities,⁵⁸ a decrease in MMP-9 activation might account in part for limited elastinolysis on PAI-1 overexpression in xenografts (Figure 9).

View larger version (31K): [in this window] [in a new window]   Figure 9. Interplay between plasminogen and MMP activation cascades in arterial extracellular matrix degradation. Plasminogen activators participate in MMP activation. MMP activity appears to be main effector of extracellular matrix degradation leading to arterial aneurysm formation and rupture in xenograft model. Aneurysm degeneration and rupture can be prevented by PAI-1 or TIMP-1 expressed locally by seeded SMCs.

Our data show for the first time that the blocking of t-PA activity by PAI-1 overexpression results in the prevention of aneurysm degeneration and rupture. These results together with results from TIMP-1 overexpression experiments²⁵ strongly support the idea that MMPs are a major pathway of elastin degradation in the xenograft model of aneurysm. Pharmacological strategies to block aneurysm formation and rupture might include the direct blocking of MMPs as well as the prevention of MMP activation by blocking the PA/plasmin system.

	Selected Abbreviations and Acronyms

 

APMA = 4-aminophenylmercuric acetate MMP = matrix metalloproteinase PA = plasminogen activator PAI = plasminogen activator inhibitor t-PA = tissue plasminogen activator TIMP = tissue inhibitor of matrix metalloproteinases u-PA = urokinase plasminogen activator

	Acknowledgments

 
These studies were supported by grants HL-52459 and HL-18645 from the National Institutes of Health and a grant from F. Hoffmann-LaRoche, Inc. Dr Allaire was supported by a grant from Laboratoire Lafon. The retroviral vector LXSN and the cell lines PE501, PA317, and NIH 3T3 TK- were generously provided by Dr A.D. Miller, Fred Hutchinson Cancer Research Center, Seattle, Wash. We thank Dr T. Gelehrter for providing us with the rat PAI-1 cDNA.

Received November 11, 1997; revision received February 3, 1998; accepted February 4, 1998.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Dobrin PB, Baker WH, Gley WC. Elastolytic and collagenolytic studies of arteries: implications for the mechanical properties of aneurysms. Arch Surg. 1984;119:405–409.[Abstract]
   
2. Anidjar S, Kieffer E. Pathogenesis of acquired aneurysms of the abdominal aorta. Ann Vasc Surg. 1992;6:298–305.[Medline] [Order article via Infotrieve]
   
3. MacSweeney ST, Powell JT, Greenhalgh RM. Pathogenesis of abdominal aortic aneurysm. Br J Surg. 1994;81:935–941.[Medline] [Order article via Infotrieve]
   
4. Brophy CM, Reilly JM, Smith GJ, Tilson MD. The role of inflammation in nonspecific abdominal aortic aneurysm disease. Ann Vasc Surg. 1991;5:229–233.[Medline] [Order article via Infotrieve]
   
5. Koch AE, Haines GK, Rizzo RJ, Radosevich JA, Pope RM, Robinson PG, Pearce WH. Human abdominal aortic aneurysms: immunopathologic analysis suggesting an immune-mediated response. Am J Pathol. 1990;137:1199–1213.[Abstract]
   
6. Jean Claude J, Newman KM, Li H, Gregory AK, Tilson MD. Possible key role for plasmin in the pathogenesis of abdominal aortic aneurysms. Surgery. 1994;116:472–478.[Medline] [Order article via Infotrieve]
   
7. Reilly JM, Sicard GA, Lucore CL. Abnormal expression of plasminogen activators in aortic aneurysmal and occlusive disease. J Vasc Surg. 1994;19:865–872.[Medline] [Order article via Infotrieve]
   
8. Schneiderman J, Bordin GM, Engelberg I, Adar R, Seiffert D, Thinnes T, Bernstein EF, Dilley RB, Loskutoff DJ. Expression of fibrinolytic genes in atherosclerotic abdominal aortic aneurysm wall: a possible mechanism for aneurysm expansion. J Clin Invest. 1995;96:639–645.
   
9. Vine N, Powell JT. Metalloproteinases in degenerative aortic disease. Clin Sci. 1991;81:233–239.[Medline] [Order article via Infotrieve]
   
10. Newman KM, Ogata Y, Malon AM, Irizarry E, Gandhi RH, Nagase H, Tilson MD. Identification of matrix metalloproteinases 3 (stromelysin-1) and 9 (gelatinase B) in abdominal aortic aneurysm. Arterioscler Thromb. 1994;14:1315–1320.[Abstract/Free Full Text]
    
11. Thompson RW, Holmes DR, Mertens RA, Liao S, Botney MD, Mecham RP, Welgus HG, Parks WC. Production and localization of 92-kilodalton gelatinase in abdominal aortic aneurysms: an elastolytic metalloproteinase expressed by aneurysm-infiltrating macrophages. J Clin Invest. 1995;96:318–326.
    
12. McMillan WD, Patterson BK, Keen RR, Shively VP, Cipollone M, Pearce WH. In situ localization and quantification of mRNA for 92-kD type IV collagenase and its inhibitor in aneurysmal, occlusive, and normal aorta. Arterioscler Thromb Vasc Biol. 1995;15:1139–1144.[Abstract/Free Full Text]
    
13. Irizarry E, Newman KM, Gandhi RH, Nackman GB, Halpern V, Wishner S, Scholes JV. Demonstration of interstitial collagenase in abdominal aortic aneurysm disease. J Surg Res. 1993;54:571–574.[Medline] [Order article via Infotrieve]
    
14. Newman KM, Jean Claude J, Li H, Scholes JV, Ogata Y, Nagase H. Cellular localization of matrix metalloproteinases in the abdominal aortic aneurysm wall. J Vasc Surg. 1994;20:814–820.[Medline] [Order article via Infotrieve]
    
15. Deleted in proof.
    
16. Anidjar S, Salzmann JL, Gentric D, Lagneau P, Camilleri JP, Michel JB. Elastase-induced experimental aneurysm in rat. Circulation. 1990;82:973–981.[Abstract/Free Full Text]
    
17. Nagase H, Enghild JJ, Suzuki K, Salvesen G. Stepwise activation mechanisms of the precursor of matrix metalloproteinase 3 (stromelysin) by proteinases and (4-aminophenyl)mercuric acetate. Biochemistry. 1990;29:5783–5789.[Medline] [Order article via Infotrieve]
    
18. He CS, Wilhelm SM, Pentland AP, Marmer BL, Grant GA, Eisen AZ, Goldberg GI. Tissue cooperation in a proteolytic cascade activating human interstitial collagenase. Proc Natl Acad Sci U S A. 1989;86:2632–2636.[Abstract/Free Full Text]
    
19. Ogata Y, Itoh Y, Nagase H. Steps involved in activation of the pro-matrix metalloproteinase 9 (progelatinase B)-tissue inhibitor of metalloproteinases-1 complex by 4-aminophenylmercuric acetate and proteinases. J Biol Chem. 1995;270:18506–18511.[Abstract/Free Full Text]
    
20. Shapiro SD, Fliszar CJ, Broekelmann TJ, Mecham RP, Senior RM, Welgus HG. Activation of the 92-kDa gelatinase by stromelysin and 4-aminophenylmercuric acetate: differential processing and stabilization of the carboxyl-terminal domain by tissue inhibitor of metalloproteinases (TIMP). J Biol Chem. 1995;270:6351–6356.[Abstract/Free Full Text]
    
21. Baramova EN, Bajou K, Remacle JM, L'Hoir C, Krell HW, Weidle UH, Noel A, Foidart JM. Involvement of PA/plasmin system in the processing of pro-MMP-9 and in the second step of pro-MMP-2 activation. FEBS Lett. 1997;405:157–162.[Medline] [Order article via Infotrieve]
    
22. Mazzieri R, Masiero L, Zanetta L, Monea S, Onisto M, Garbisa S, Mignatti P. Control of type IV collagenase activity by components of the urokinase-plasmin system: a regulatory mechanism with cell-bound reactants. EMBO J. 1997;16:2319–2332.[Medline] [Order article via Infotrieve]
    
23. Allaire E, Guettier C, Bruneval P, Plissonnier D, Michel JB. Cell-free arterial grafts: morphologic characteristics of aortic isografts, allografts, and xenografts in rats. J Vasc Surg. 1994;19:446–456.[Medline] [Order article via Infotrieve]
    
24. Allaire E, Bruneval P, Mandet C, Becquemin JP, Michel JB. The immunogenicity of the arterial extracellular matrix in arterial xenografts. Surgery. 1997;122:73–81.[Medline] [Order article via Infotrieve]
    
25. Allaire E, Forough R, Wang T, Clowes MM, Clowes AW. Tissue metalloproteinase inhibitor-1 (TIMP-1) overexpression prevents arterial rupture and dilation in a xenograft model. FASEB J. 1996;10:A1134. Abstract.
    
26. Lynch CM, Clowes MM, Osborne WR, Clowes AW, Miller AD. Long-term expression of human adenosine deaminase in vascular smooth muscle cells of rats: a model for gene therapy. Proc Natl Acad Sci U S A. 1992;89:1138–1142.[Abstract/Free Full Text]
    
27. Zeheb R, Gelehrter TD. Cloning and sequencing of cDNA for the rat plasminogen activator inhibitor-1. Gene. 1988;73:459–468.[Medline] [Order article via Infotrieve]
    
28. Miller AD, Rosman GJ. Improved retroviral vectors for gene transfer and expression. Biotechniques. 1989;7:980–989.[Medline] [Order article via Infotrieve]
    
29. Starcher B, Conrad M. A role for neutrophil elastase in the progression of solar elastosis. Connect Tissue Res. 1995;31:133–140.[Medline] [Order article via Infotrieve]
    
30. Camiolo SM, Siuta MR, Madeja JM. Improved medium for extraction of plasminogen activator from tissue. Prep Biochem. 1982;12:297–305.[Medline] [Order article via Infotrieve]
    
31. Clowes AW, Clowes MM, Au YPT, Reidy MA. Smooth muscle cells express urokinase during mitogenesis and tissue-type plasminogen activator during migration in injured rat carotid artery. Circ Res. 1990;67:61–67.[Abstract/Free Full Text]
    
32. Zehr BD, Savin TJ, Hall RE. A one-step, low background Coomassie staining procedure for polyacrylamide gels. Anal Biochem. 1989;182:157–159.[Medline] [Order article via Infotrieve]
    
33. Abramson SR, Conner GE, Nagase H, Neuhaus I, Woessner JFJ. Characterization of rat uterine matrilysin and its cDNA: relationship to human pump-1 and activation of procollagenases. J Biol Chem. 1995;270:16016–16022.[Abstract/Free Full Text]
    
34. Eitzman DT, Fay WP, Lawrence DA, Francis Chmura AM, Shore JD, Olson ST, Ginsburg D. Peptide-mediated inactivation of recombinant and platelet plasminogen activator inhibitor-1 in vitro. J Clin Invest. 1995;95:2416–2420.
    
35. Plow EF, Herrer T, Redlitz A, Miles LA, Hoover-Plow JL. The cell biology of the plasminogen system. FASEB J. 1995;9:939–945.[Abstract]
    
36. Ossowski L. Invasion of connective tissue by human carcinoma cell lines: requirement for urokinase, urokinase receptor, and interstitial collagenase. Cancer Res. 1992;52:6754–6760.[Abstract/Free Full Text]
    
37. Clowes AW, Clowes MM, Kirkman TR, Jackson CL, Au YPT, Kenagy RD. Heparin inhibits the expression of tissue-type plasminogen activator by smooth muscle cells in injured rat carotid artery. Circ Res. 1992;70:1128–1136.[Abstract/Free Full Text]
    
38. Kenagy RD, Vergel S, Mattsson E, Bendeck M, Reidy MA, Clowes AW. The role of plasminogen, plasminogen activators, and matrix metalloproteinases in primate arterial smooth muscle cell migration. Arterioscler Thromb Vasc Biol. 1996;16:1373–1382.[Abstract/Free Full Text]
    
39. Libby P. Molecular bases of the acute coronary syndromes. Circulation. 1995;91:2844–2850.[Free Full Text]
    
40. Galis ZS, Sukhova GK, Lark MW, Libby P. Increased expression of matrix metalloproteinases and matrix degrading activity in vulnerable regions of human atherosclerotic plaques. J Clin Invest. 1994;94:2493–2503.
    
41. Nikkari ST, O'Brien KD, Ferguson M, Hatsukami T, Welgus HG, Alpers CE, Clowes AW. Interstitial collagenase (MMP-1) expression in human carotid atherosclerosis. Circulation. 1995;92:1393–1398.[Abstract/Free Full Text]
    
42. Nikkari ST, Geary RL, Hatsukami T, Ferguson M, Forough R, Alpers CE, Clowes AW. Expression of collagenase, and tissue inhibitor of metalloproteinase-1 in restenosis after carotid endarterectomy. Am J Pathol. 1996;148:777–783.[Abstract]
    
43. Raghunath PN, Tomaszewski JE, Brady ST, Caron RJ, Okada SS, Barnathan ES. Plasminogen activator system in human coronary atherosclerosis. Arterioscler Thromb Vasc Biol. 1995;15:1432–1443.[Abstract/Free Full Text]
    
44. Lupu F, Heim DA, Bachmann F, Hurni M, Kakkar VV, Kruithof EK. Plasminogen activator expression in human atherosclerotic lesions. Arterioscler Thromb Vasc Biol. 1995;15:1444–1455.[Abstract/Free Full Text]
    
45. Schneiderman J, Sawdey MS, Keeton MR, Bordin GM, Bernstein EF, Dilley RB, Loskutoff DJ. Increased type 1 plasminogen activator inhibitor gene expression in atherosclerotic human arteries. Proc Natl Acad Sci U S A. 1992;89:6998–7002.[Abstract/Free Full Text]
    
46. Carmeliet P, Moons L, Lijnen R, Baes M, Lemaître V, Tipping P, Drew A, Eeckhout Y, Lupu F, Collen D. Urokinase-generated plasmin activates matrix metalloproteinases during aneurysm formation. Nat Genet. 1997;17:439–444.[Medline] [Order article via Infotrieve]
    
47. Mueller BM, Yu YB, Laug WE. Overexpression of plasminogen activator inhibitor 2 in human melanoma cells inhibits spontaneous metastasis in scid/scid mice. Proc Natl Acad Sci U S A. 1995;92:205–209.[Abstract/Free Full Text]
    
48. Bjorquist P, Brohlin M, Ehnebom J, Ericsson M, Kristiansen C, Pohl G, Deinum J. Plasminogen activator inhibitor type-1 interacts exclusively with the proteinase domain of tissue plasminogen activator. Biochim Biophys Acta. 1994;1209:191–202.[Medline] [Order article via Infotrieve]
    
49. Loskutoff DJ, Sawdey M, Mimuro J. Type 1 plasminogen activator inhibitor. Prog Hemost Thromb. 1989;9:87–115.[Medline] [Order article via Infotrieve]
    
50. Wagner OF, de Vries C, Hohmann C, Veerman H, Pannekoek H. Interaction between plasminogen activator inhibitor type 1 (PAI-1) bound to fibrin and either tissue-type plasminogen activator (t-PA) or urokinase-type plasminogen activator (u-PA): binding of t-PA/PAI-1 complexes to fibrin mediated by both the finger and the kringle-2 domain of t-PA. J Clin Invest. 1989;84:647–655.
    
51. Hekman CM, Loskutoff DJ. Endothelial cells produce a latent inhibitor of plasminogen activators that can be activated by denaturants. J Biol Chem. 1985;260:11581–11587.[Abstract/Free Full Text]
    
52. Simpson AJ, Booth NA, Moore NR, Bennett B. Distribution of plasminogen activator inhibitor (PAI-1) in tissues. J Clin Pathol. 1991;44:139–143.[Abstract/Free Full Text]
    
53. Lawrance DA, Loskutoff DJ. Inactivation of plasminogen activator inhibitor by oxidants. Biochemistry. 1986;25:6351–6355.[Medline] [Order article via Infotrieve]
    
54. Chapman HA, Reilly JJ, Kobzik L. Role of plasminogen activator in the degradation of extracellular matrix protein by live human alveolar macrophages. Am Rev Respir Dis. 1988;137:412–419.[Medline] [Order article via Infotrieve]
    
55. Montgomery AM, De Clerck YA, Langley KE, Reisfeld RA, Mueller BM. Melanoma-mediated dissolution of extracellular matrix: contribution of urokinase-dependent and metalloproteinase-dependent proteolytic pathways. Cancer Res. 1993;53:693–700.[Abstract/Free Full Text]
    
56. Murphy G, Cockett MI, Ward RV, Docherty AJ. Matrix metalloproteinase degradation of elastin, type IV collagen and proteoglycan: a quantitative comparison of the activities of 95 kDa and 72 kDa gelatinases, stromelysins-1 and -2 and punctuated metalloproteinase (PUMP). Biochem J. 1991;277:277–279.
    
57. Senior RM, Griffin GL, Fliszar CJ, Shapiro SD, Goldberg GI, Welgus HG. Human 92- and 72-kilodalton type IV collagenases are elastases. J Biol Chem. 1995;266:7870–7875.[Abstract/Free Full Text]
    
58. Katsuda S, Okada Y, Imai K, Nakanishi I. Matrix metalloproteinase-9 (92-kd gelatinase/type IV collagenase equals gelatinase B) can degrade arterial elastin. Am J Pathol. 1994;145:1208–1218.[Abstract]
    
59. Okada Y, Morodomi T, Enghild JJ, Suzuki K, Yasui A, Nakanishi I, Salvesen G, Nagase H. Matrix metalloproteinase 2 from human rheumatoid synovial fibroblasts: purification and activation of the precursor and enzymic properties. Eur J Biochem. 1990;194:721–730.[Medline] [Order article via Infotrieve]
    
60. Mecham RP, Broekelmann TJ, Fliszar CJ, Shapiro SD, Welgus HG, Senior RM. Elastin degradation by matrix metalloproteinases: cleavage site specificity and mechanisms of elastolysis. J Biol Chem. 1997;272:18071–18076.[Abstract/Free Full Text]
    
61. Shipley JM, Doyle GAR, Fliszar CJ, Ye QZ, Johnson LL, Shapiro SD, Welgus HG, Senior RM. The structural basis for the elastinolytic activity of the 92-kDa and 72 kDa gelatinases. J Biol Chem. 1996;8:4335–4341.
    
62. Birkedal-Hansen H, Moore WGI, Bodden MK, Windsor LJ, Birkedal-Hansen B, DeCarlo A, Engler JA. Matrix metalloproteinases: a review. Crit Rev Oral Biol Med. 1993;4:197–250.[Abstract/Free Full Text]
    
63. Okada Y, Nagase H, Harris EDJ. A metalloproteinase from human rheumatoid synovial fibroblasts that digests connective tissue matrix components: purification and characterization. J Biol Chem. 1986;261:14245–14255.[Abstract/Free Full Text]
    
64. Okada Y, Harris EDJ, Nagase H. The precursor of a metalloendopeptidase from human rheumatoid synovial fibroblasts: purification and mechanisms of activation by endopeptidases and 4-aminophenylmercuric acetate. Biochem J. 1988;254:731–741.[Medline] [Order article via Infotrieve]
    
65. Okada Y, Gonoji Y, Naka K, Tomita K, Nakanishi I, Iwata K, Yamashita K, Hayakawa T. Matrix metalloproteinase 9 (92-kDa gelatinase/type IV collagenase) from HT 1080 human fibrosarcoma cells: purification and activation of the precursor and enzymic properties. J Biol Chem. 1992;267:21712–21719.Although an increase in plasminogen activators has been detected in human aortic aneurysms, the role of plasmin in aneurysm development and rupture is unclear. We overexpressed PAI-1 in a rat model of arterial aneurysm by seeding rat smooth muscle cells retrovirally transfected with PAI-1 cDNA. As a result, the activity of t-PA, the major plasminogen activator in this system, was blocked, and aneurysm development and rupture were prevented. Our data suggest that the protective effect of PAI-1 overexpression on matrix degradation is mediated in part by decreased MMP activation.[Abstract/Free Full Text]
    

This article has been cited by other articles:

				J. P. Schwartz, M. Bakhos, A. Patel, S. Botkin, and S. Neragi-Miandoab Repair of aortic arch and the impact of cross-clamping time, New York Heart Association stage, circulatory arrest time, and age on operative outcome Interactive CardioVascular and Thoracic Surgery, June 1, 2008; 7(3): 425 - 429. [Abstract] [Full Text] [PDF]

				H Senzaki The pathophysiology of coronary artery aneurysms in Kawasaki disease: role of matrix metalloproteinases Arch. Dis. Child., October 1, 2006; 91(10): 847 - 851. [Abstract] [Full Text] [PDF]

				E. Choke, M. M. Thompson, J. Dawson, W. R. W. Wilson, S. Sayed, I. M. Loftus, and G. W. Cockerill Abdominal Aortic Aneurysm Rupture Is Associated With Increased Medial Neovascularization and Overexpression of Proangiogenic Cytokines Arterioscler. Thromb. Vasc. Biol., September 1, 2006; 26(9): 2077 - 2082. [Abstract] [Full Text] [PDF]

				Z. Touat, V. Ollivier, J. Dai, M.-G. Huisse, A. Bezeaud, U. Sebbag, T. Palombi, P. Rossignol, O. Meilhac, M.-C. Guillin, et al. Renewal of Mural Thrombus Releases Plasma Markers and Is Involved in Aortic Abdominal Aneurysm Evolution Am. J. Pathol., March 1, 2006; 168(3): 1022 - 1030. [Abstract] [Full Text] [PDF]

				J. Dai, F. Losy, A.-M. Guinault, C. Pages, I. Anegon, P. Desgranges, J.-P. Becquemin, and E. Allaire Overexpression of Transforming Growth Factor-{beta}1 Stabilizes Already-Formed Aortic Aneurysms: A First Approach to Induction of Functional Healing by Endovascular Gene Therapy Circulation, August 16, 2005; 112(7): 1008 - 1015. [Abstract] [Full Text] [PDF]

				A. Garcia-Touchard, T. D. Henry, G. Sangiorgi, L. G. Spagnoli, A. Mauriello, C. Conover, and R. S. Schwartz Extracellular Proteases in Atherosclerosis and Restenosis Arterioscler. Thromb. Vasc. Biol., June 1, 2005; 25(6): 1119 - 1127. [Abstract] [Full Text] [PDF]

				Yasmin, S. Wallace, C. M. McEniery, Z. Dakham, P. Pusalkar, K. Maki-Petaja, M. J. Ashby, J. R. Cockcroft, and I. B. Wilkinson Matrix Metalloproteinase-9 (MMP-9), MMP-2, and Serum Elastase Activity Are Associated With Systolic Hypertension and Arterial Stiffness Arterioscler. Thromb. Vasc. Biol., February 1, 2005; 25(2): 372 - 378. [Abstract] [Full Text] [PDF]

				E. Ribourtout and J. Raymond Gene Therapy and Endovascular Treatment of Intracranial Aneurysms Stroke, March 1, 2004; 35(3): 786 - 793. [Abstract] [Full Text] [PDF]

				O. D Defawe, A. Colige, C. A Lambert, C. Munaut, P. Delvenne, C. M Lapiere, R. Limet, B. V Nusgens, and N. Sakalihasan TIMP-2 and PAI-1 mRNA levels are lower in aneurysmal as compared to athero-occlusive abdominal aortas Cardiovasc Res, October 15, 2003; 60(1): 205 - 213. [Abstract] [Full Text] [PDF]

				M. J. Mulligan-Kehoe, H. K. Kleinman, M. Drinane, R. J. Wagner, C. Wieland, and R. J. Powell A Truncated Plasminogen Activator Inhibitor-1 Protein Blocks the Availability of Heparin-binding Vascular Endothelial Growth Factor A Isoforms J. Biol. Chem., December 6, 2002; 277(50): 49077 - 49089. [Abstract] [Full Text] [PDF]

				I. Loftus and M. Thompson The role of matrix metalloproteinases in vascular disease Vascular Medicine, May 1, 2002; 7(2): 117 - 133. [Abstract] [PDF]

				F. J. Miller Jr, W. J. Sharp, X. Fang, L. W. Oberley, T. D. Oberley, and N. L. Weintraub Oxidative Stress in Human Abdominal Aortic Aneurysms: A Potential Mediator of Aneurysmal Remodeling Arterioscler. Thromb. Vasc. Biol., April 1, 2002; 22(4): 560 - 565. [Abstract] [Full Text] [PDF]