Elevations of tissue-type plasminogen activator and differential expression of uroldnase-type plasminogen activator in diseased aorta P a u l a IC S h i r e m a n , M D , W a l t e r J. M c C a r t h y , M D , W i l l i a m H . Pea rce , M D ,
V e r a P . Sh ive ly , M S , M a r i a C i p o l l o n e , BS, and H a u C. K w a a n , M D , Chicago, Ill.
Purpose: Elevations o f plasmin have been implicated in the pathogenesis o f abdominal aort ic aneurysms (AAA) because o f its abil i ty to digest extracellular matr ix proteins. Plasminogen activators regulate the conversion o f plasminogen to plasmin. Tissue-type plasminogen activator ( tPA) is more impor tan t in modula t ion o f fibrinolysis, and uroki- nase-type plasminogen activator (uPA) is p redominan t in tissue remodel ing. The purpose o f this s tudy was to determine the levels o f plasminogen activators in diseased aor ta because they may be responsible for the increased plasmin levels previously described in AAA. Methods: Levels o f t P A and u_PA in AAA, occlusive, and normal (organ donor) aor ta were s tudied in tissue explant supernatants. Supernatant tPA and uPA levels were measured wi th an enzyme-l inked immunosorben t assay. Nor the rn analysis was used to quant i ta te uP_& messenger R N A (mRNA) levels in aort ic tissue. Results: Levels o f tPA in the supernatants were similar in occlusive (20 - 4 n g / m l ) and AAA (23 -+ 8) aorta, bu t threefold higher than in normal aor ta (7 - 5; p < 0.005 for normal vs occlusive and p < 0.001 for normal vs AAA). In contrast , uPA supernatant levels were differentially expressed, wi th the highest level existing in AAA (9.7 + 2.7 n g / m l ) , fol lowed by occlusive (4.9 - 3.5), and the lowest levels in normal aor ta (1.2 -+ 0.7; p < 0.05 for normal vs occlusive, p < 0.001 for normal vs AAA, and p < 0.005 for occlusive vs AAA). Inh ib i t ion o f p ro te in or R N A synthesis by addi t ion o f cyclohexamide or act inomycin D, respectively, revealed no significant difference between treated and control supernatants , suggest ing tha t the increases were caused by pro te in release rather than active synthesis. Levels o f uPA m R N A followed the same t r end as the supernatant uPA levels (AAA 1.07 -+ 0.54, occlusive 0.54 -+ 0.08, and normal aorta 0.01 -+ 0.01). Conclusions: Levels o f t P A were similar in aneurysmal and occlusive aorta, bu t exhibi ted a threefold increase over normal aorta, suggest ing tha t the elevations o f tPA are associated with the arteriosclerosis present in bo th aneurysmal and occlusive disease. Differences in uPA levels were significant between all three groups, wi th the highest levels in AAA and the lowest levels in normal specimens. Nor the rn analysis o f uPA m R N A followed the same t rend, suggest ing tha t the increase in u_PA may be regulated at the level o f t ranscr ipt ion. As uPA plays an impor tan t role in tissue remodel ing, our findings may also reflect the relative tissue repair activities in these three types o f specimens and may explain the previously repor ted increased levels o f plasmin seen in AAA. (J Vasc Surg 1997; 25:157-64. )
From the Division of Vascular Surgery, Department of Surgery; and the Division of Hematology and Oncology, Department of Medicine (Dr. Kwaan); Northwestern University School of Medicine and the Veterans Administration Lakeside Medical Center.
Presented at the Joint Annual Meeting of The Society for Vascular Surgery and the International Society for Cardiovascular Sur-
gery, North American Chapter, Chicago, IU., June 9-12, 1996. Reprint requests: Walter J. McCarthy, MD, Division of Vascular
national Society for Cardiovascular Surgery, North American Chapter.
0741-5214/97/$5.00 + 0 24/6/77981
157
JOURNAL OF VASCULAR SURGERY 158 Shireman et aL January 1997
Tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) convert the proenzyme plasminogen into its active form, plasmin. Plasmin is important in tissue remodeling and in fibrinolysis. Jean-Claude et al.1 described ele- vated levels of plasmin in abdominal aortic aneu- rysms (AAA) as compared with controls, suggesting that plasmin may be important in the pathogenesis of aneurysm formation. Plasmin plays a key role in the degradation of extraccllular matrix (ECM) by di- rectly digesting matrix proteins, as well as by activat- ing the proteolytic matrix metalloproteinases (MMPs).2
Proteolytic activity in the aortic wall varies with disease state. Vine and Powell 3 demonstrated an in- creased gelatinase activity in homogenates from an- eurysmal and occlusive atherosclerotic aorta. In addi- tion, collagenase and stromelysin were detected in aneurysmal aorta, but rarely in occlusive specimens, and never in normal samples.-Plasmin, a nonspeciflc protease, activates these enzymes as well as directly degrading many ECM proteins. Both tPA and uPA arc present in the aneurysmal wall, and both of these enzymes convert plasminogen to plasmin. Reilly et al.4 studied the level of plasminogen activators in protein extracts derived from aneurysmal, occlusive, and normal aorta. Immunoassay demonstrated dif- ferential expression of tPA, with the highest level in AAA, followed by normal aorta, and the lowest level in occlusive specimens. They were unable to quanti- tate uPA antigen by immunoassay in any of the specimens. A more sensitive method--fibrin autog- raphy--dcmonstrated activity attributable to both tPA and uPA, with the largest amount offibrinolysis occurring because of free tPA. Expression of uPA and tPA mRNA was comparable in aneurysmal and occlusive specimens.
Increased proteolytic activity has been demon- strated in the wall of AAA, and this increase may at least partially be explained by increased plasmin lev- els. Regulation of plasminogen activation is an im- portant area of investigation because of plasmin's involvement in both fibrinolysis and tissue remodel- ing. To date, quantitation of uPA protein levels has not been reported in diseased aorta. The purpose of this study was to compare levels of tPA and uPA in aneurysmal, occlusive, and normal aorta.
M E T H O D S
The following studies were performed with the approval of the Institutional Review Board of North- western University, and all patients gave informed consent. Infrarenal aorta (aneurysmal and occlusive)
was obtained at the time of bypass grafting. Normal thoracic and abdominal aorta was procured from organ donors through the Regional Organ Bank of Illinois.
Tissue explant supernatants. In the operating room, specimens were placed on ice in holding me- dia (Dulbecco's Modified Eagle's Medium, Ham's F-12, 16% fetal bovine serum, penicillin/streptomy- cin, and amphotericin B) and processed within 1 to 2 hours. Using sterile technique, the tissue was washed with phosphate buffered saline solution (PBS) to remove residual thrombus, minced into 1 × 1 mm squares, weighed, and placed into 4 ml Roswell Park Memorial Institute (RPMI) media with penicillin/ streptomycin per 1 g of tissue. The specimens were incubated at 37 ° C with 5% CO 2 for 48 hours. The supematant was removed, centrifuged, aliquoted, and stored at - 8 0 ° C until assayed. Time course experi- ments were performed by placing similar amounts of tissue from the same patient into separate flasks at 37 ° C and collecting the supernatants at 0, 4, 8, 24, 48, and 72 hours. Protein and RNA synthesis were inhibited respectively by cyclohexamide (25 txg/ml) and actinomycin D (5 Ixg/ml). The inhibitors were dissolved in alcohol and added to the RPMI media at time zero and were in contact with the tissue for 72 hours. Control flasks contained tissue from the same patient placed in the RPMI media-alcohol mixture without the respective inhibitors.
Quantitation of tPA and uPA. Total tPA and uPA antigen was quantitated using TintElize tPA and TintElize uPA (Biopool International, Ventura, Calif.), respectively. These enzyme-linked immu- nosorbent assays recognize all forms oftPA and uPA. Manufacturer's instructions for nonplasma samples were followed, and interassay variability was approx- imately 5%.
Northern blot analysis. Total RNA was ex- tracted from snap-frozen tissue using the method of Chomczynski and Sacchi. 5 RNA was quantified by measuring absorbance at 260 nm, and approximately 10 ~g of cellular RNA was size fractionated on a 1% formaldehyde agarose gel. The samples were electro- phoresed with ethidium bromide to verify the integ- rity of the ribosomal RNA. The RNA was transferred to a nylon membrane and hybridized overnight. A Prime-It II Kit (Stratagene, LaJolla, Calif.) was used with [32p] deoxyadenosine-triphosphate to synthe- size radiolabeled probes. A 1.1 kb uPA probe was donated by Abbott Laboratories (Abbott Park, Ill.) and a 1.0 kb glyceraldehyde-3-phosphate dehydro- genase (GAPDH) probe was kindly provided by Dr. Kathy Green (Northwestern University, Chicago).
JOURNAL OF VASCULAR SURGERY Volume 25, Number 1 Shireman et al. 159
The signal was quantitated using a Fuji Bio-imager BAS 2000 (Tokyo, Japan) and reported as pulses minus background divided by the area. Tissue cul- ture RNA from microvascular endothelial cells, kindly provided by Dr. T o m Lawley (Centers for Disease Control, Atlanta) and RKO, a colon cancer cell line, generously donated by Dr. Douglas Boyd (M.D. Anderson Cancer Center , H o u s t o n ) , were used as positive controls. The 2.5 kb uPA band was divided by the G A P D H signal to normalize for differences in RNA loading. Before the second hybridizat ion, the membrane was stripped in boil- ing 0.1 × SSC, 0.1% SDS to remove the bound probe.
Statistics. Analysis o f variance (ANOVA) and unpaired and paired Student 's t tests were per- formed, and results for these tests were reported as significant at the p < 0.05 level. Results are reported as the average + standard deviation.
R E S U L T S
Tissue explant superna tan ts , tPA and uPA were assayed in 48 hour supernatants o f 19 patients (7 aneurysms, 4 occlusive, and 8 normal). The aver- age patient age was 66 + 10 years for aneurysmal aorta, 50 + 13 years for occhisive aorta, and48 + 13 years for normal aorta.
Aneurysmal aorta had an average of 23 + 8 ng t P A / m l of supernatant, occlusive specimens had 20 + 4, and normal aorta averaged 7 + 5. ANOVA among all three groups revealed p = 0.0003. Fisch- er's least square differences was used as an adjunct test and revealed a p < 0.001 for normal versus aneurysmal aorta and a p < 0.005 for normal versus occlusive aorta. There was no significant difference between aneurysmal and occlusive aortic tPA levels.
uPA (ng /ml ) in the supernatants averaged 9.7 -+ 2.7 in aneurysmal aorta, 4.9 + 3.5 in occlusive aorta, and 1.2 + 0.7 in normal aorta. ANOVA among all three groups revealed a p < 0.0001. Fischer's least square differences revealed a p < 0.0001 for aneu- rysm versus normal aorta, a p < 0.005 for aneurysm versus occlusive aorta, and p = 0.02 for normal versus occlusive aorta. Supernatant uPA was differen- tially expressed in all three aortic types, with aneurys- mal specimens exhibiting the highest level, followed by occlusive and normal specimens. In contrast, there was no significant difference in supernatant tPA levels in aneurysmal and occlusive aorta, but the diseased specimens exhibited a threefold higher level than normal aorta.
Time point experiments were conducted to see how tPA and uPA levels changed over time in the
tPA nghnl
3o
25
2 0
15
lO.
5.
0.
/
J ~ N o r m a l
0 10
A 20 30 40 50
Time in hours 60 70 80
12
10
8
uPA 6 ng/m!
4
2
0
B
Y / j ~ . A A A
,Normal J
10 20 30 40 50 60 70 80 Time in hours
Fig. 1. Supernatant time course experiments. A, Levels of tPA in aneurysmal supernatants increased over time, while levels of tPA in normal specimens initially increased and slightly declined after 8 hours. B, Levels of uPA in both normal and aneurysmal supernatants increased over time, with aneurysmal specimens exhibiting a steady increase throughout entire time period, and normal specimens in- creasing initially and then remaining relatively constant after 8 hours.
supernatants. A total o f 7 specimens, 3 aneurysms and 4 normals, were assayed and the average patient age was 68 +_ 2 years for aneurysmal aorta and 44 + 18 years for normal aorta. Levels o f tPA (Fig. 1, A) in both normal and aneurysmal supernatants rapidly increased until the 8 hour point. After 8 hours, tPA in the aneurysms continued to slowly rise while tPA levels in the normals slightly decreased.
Levels o f uPA (Fig. 1, B) in both normal and aneurysmal supernatants continued to rise over the entire 72 hour period. Aneurysms exhibited a steady increase in uPA while normal specimens remained relatively constant after 8 hours.
To determine whether the increase over time was a result o f active synthesis or o f cell death with release of the plasminogen activators, cyclohexamide, or ac- tinomycin D was added to the media to inhibit pro- tein or RNA synthesis, respectively. Inhibitor studies were performed on 7 specimens, which included 4 aneurysms and 3 normal aortas. There was no signif-
JOURNAL OF VASCULAR SURGERY 160 Shireman et al. January 1997
E N N O O A A H
uPA 2.5 kb--)
(--28S
18S
GAPDH 1.2 kb--)
Fig. 2. Northern blot analysis ofuPA. 2.5 kb uPA mRNA band migrates between 28S and 18S ribosomal bands (arrows). Constitutively produced GAPDH was used to correct for RNA loading. E and H on either end represent endothelial cell and colon cancer tissue culture RNA used as positive controls. N denotes normal, O, occlusive, and A, aneurysmal aorta. Notice absence ofuPA bands on lanes containing RNA from normal tissues but strong GAPDH signal, suggesting that amount of uPA in these specimens was below level of detection for Northern analysis.
icant difference in tPA or uPA levels using a paired Student's t test in control, cyclohexamide, or actino- mycin D supernatants (tPA: control, 14 -+ 9; cyclo- hexamide, 11 _+ 8; and actinomycin D, 11 +_ 8; uPA: control, 4.2 _+ 3.1; cyclohexamide, 4.2 -+ 3.2; and actinomycin D, 4.3 +_ 3.1). These results suggest that tPA and uPA are released into the supernatants and are not actively synthesized. Previous studies measuring plasminogen activator inhibitor type 1 (PAId ) in the inhibitor supernatants revealed a de- crease o f PAI-1 to 33% of control levels, indicating that active synthesis had occurred for PAI-1 and demonstrating that both protein and RNA synthesis hadbeen inhibited by the addition of cyclohexamide and actinomycin D. 6 Time point and inhibitor exper- iments were not performed on occlusive samples because o f specimen size limitations.
N o r t h e r n analysis. Six specimens--2 aneu- rysms, 2 occlusive, and 2 normal--were analyzed. The average patient age was 75 +- 8 years for aneu- rysms, 55 --- 6 years for occlusive, and 40 + 13 years for normal aorta. The 2.5 kb uPA band was mea- sured on a bio-imager and normalized to the GAPDH signal, revealing 1.07 -+ 0.54 for aneu- rysms, 0.54 _+ 0.08 for occlusive, and 0.01 _+ 0.01 for normal aorta. An ANOVA was performed which
revealed a p = 0.1. Although the sample sizes were small, the averagc values for the three groups fol- lowed the same trend as the uPA protein levels in the supernatants, with aneurysmal specimens exhibiting the highest levels, followed by occlusive and then normal aorta. This suggests that the elevations o f uPA may be controlled at the level of transcription. On the Northern blot (Fig. 2) there were no clear uPA bands on the lanes with RNA from normal tissue but strong GAPDH signals were present, sug- gesting that the amount of uPA in these specimens was below the level of detection for Northern analy- sis. A 1.1 kb tPA probe obtained from the American Type Culture Collection (#67587, Rockville, Md., U.S. patent #04766075) was also hybridized to this membrane, which was positive on the endothelial cell tissue culture lane but exhibited no clear bands on the tissue RNA lanes (data not shown). Perhaps a more sensitive method of quantitation, such as re- verse transcriptase polymerase chain reaction, would be more useful in detecting tPA mRNA levels in aortic tissue.
DISCUSSION
Understanding the regulation of fibrinolysis and tissue remodeling, two important functions of plas-
JOURNAL OF VASCULAR SURGERY Volume 25, Number 1 S h i r e m a n et al. 161
min, may provide future therapeutic methods for the treatment of atherosclerotic and aneurysmal disease. Evidence is accumulating to support a significant role for uPA in ECM degradation and tissue remodeling. Migrating endothelial cells exhibit an increased uPA activity that decreases to background levels on cessa- tion of migration. 7 Activation of uPA on the cellular surface is localized by the uPA receptor. The receptor is rapidly polarized to the leading front o f migrating cells where it binds uPA, allowing localized activa- tion of plasminogen and degradation of the ECM. 8 Various cancers, which can be thought of as unregu- lated forms of tissue remodeling, have been shown to exhibit an increased expression ofuPA. Ovarian can- cer cells in tissue culture have been shown to secrete 17 to 38 times higher levels of uPA than normal ovarian cell lines, 9 and this elevated level of uPA has been associated with malignant transformation of the ovarian surface epithelium. 1° Additionally, increases in uPA in breast cancer have been associated with a poor prognosis, n presumably as a result o f its inva- sire effect.
Diseased aorta has been shown to contain inflam- matory cell infiltrates, with aneurysms exhibiting the largest amount followed by occlusive atherosclerotic aorta and a relative absence of inflammation in nor- mal aorta. 12 In situ hybridization studies of AAA localized uPA mRNA to these infiltrates as well as to scattered inflammatory cells with a morphology re- sembling macrophages in the thickened intima and media. In contrast, normal aorta and areas of AAA devoid of inflammation were negative for uPA mRNA. ~3 Aneurysms are heterogeneous, with areas of arteriosclerosis exhibiting increased wall thickness and areas o f relative thinning, which presumably are the site of potential rupture. Many cell types, includ- ing endothelial cells, 14 vascular smooth muscle cells, as and macrophages, 16 synthesize uPA, and the expression o f uPA is regulated by various cytokines such as interleukin-1 and tumor necrosis factor-a, s
In addition to tissue remodeling, plasmin plays an important role in fibrinolysis. Fibrin clots are de- graded by plasmin, resulting in dissolution of throm- bus and sometimes vessel recanalization. Plasmino- gen is normally incorporated into thrombus where it can be converted to plasmin by tPA, and fibrin must be present for the activation o f plasmin by tPA. s PAI-1 regulates this process by complexing with both tPA and uPA, thereby resulting in a loss of plasminogen activation potential. High plasma PAI-1 levels have been associated with thrombotic complications such as deep venous thrombosis ~7 and myocardial infarction is by promoting an overall de-
crease of fibrinolytic activity. Peripheral arterial dis- ease has been associated with both elevated plasma tPA antigen levels and increased PAI-1 activity. The tPA antigen is increased because of the inactive tPA- PAI-1 complexes and results in an overall decrease in fibrinolytic activity. 19
The blood vessel wall is the site of origin of plasma tPA. In AAA, tPA mRNA has been localized by in situ hybridization to macrophage-like cells in the inflammatory infiltrate, and it has been shown within the lumenal endothelial cells in normal aor- ta. 13 111Indium labeled monoclonal antibodies against tPA injected into patients before AAA repair demonstrated increased tPA accumulation in the an- eurysms on scintigrams. In this same study, tissue specimens collected at the time of Surgery exhibited tPA within the aneurysmal wall and the lumenal thrombus. 2° In vitro studies have shown that tPA stimulates vascular smooth muscle cell proliferation 2~ and migration, is
Using tissue explant supematants, we have dem- onstrated elevated levels of both plasminogen activa- tors in diseased aorta. In the present study, levels of tPA are threefold higher in diseased versus normal aorta. There was no significant difference between occlusive and aneurysmal specimens. In contrast to these findings, a previous report from Reilly et al. 4 measuring tPA in diseased aorta found significant differences between all three aortic types, with aneu- rysms exhibiting the highest level, followed by nor- mal aortas and the lowest level in occlusive aortas. This study used protein extracts, and tPA levels were reported as ng o f an t igen/mg of protein in the cx- tract. We also performed protein extract experiments but found no significant differences in tPA levels between the aortic types using this method 4,6 (data not shown). Technical difficulties arose from measur- ing total protein in the extracts. We used two differ- ent methods but observed variance within the same samples to be as high as 40%, and there were marked differences in total protein levels between the two methods. Dialyzing the extracts before measurement o f total protein did not improve the variance. We believed that total protein was not a consistent de- nominator for comparison of plasminogen activator levels and used the method of tissue explant superna- tants instead.
In the current study, uPA was differentially ex- pressed using tissue explant supernatants in the three aortic types. Reilly et al. 4 were unable to quantitate uPA levels in their protein extract experiments. They diluted all of their samples to contain 1 m g / m l of total protein before assay o f u P A levels, which prob-
JOURNAL OF VASCULAR SURGERY 162 Sh i reman et al. January 1997
ably led to concentrations of uPA that were below the level of detection in their immunoassay. They did, however, detect uPA activity in the extracts with fibrin autography, which is a more sensitive assay for detecting uPA. We did not standardize the protein levels in our protein extracts before assay and were able to detect uPA antigen, but found no statistically significant differences between the three aortic types (data not shown). Once again, difficulties with mea- suring total protein in the extracts led us to use the method of tissue explant supematants.
To further characterize tissue explant superna- tants, we performed time course and inhibitor stud- ies. Levels of both plasminogen activators increased over time in the aneurysmal supernatants and in the uPA normal supernatants, but levels o f tPA increased initially and then slightly decreased after 8 hours in the normal samples. Inhibition of protein and RNA synthesis by the addition of cyclohexamide and acti- nomycin D revealed no significant differences in ei- ther of the plasminogen activator levels, suggesting that the proteins were being released into the super- natants and not being actively synthesized.
Protein levels of uPA were elevated, which led us to perform Northern analysis to quantitate mRNA levels. Although not statistically significant, the dif- ferences in the uPA transcripts followed the same trend as the protein data, with aneurysms expressing the highest levels, followed by occlusive aorta, and normal aorta exhibiting the lowest values. In fact, normal aorta revealed essentially no signal above background, suggesting that the level o fuPA expres- sion is below the level of detection for Northern analysis and that more sensitive methods, such as reverse transcriptase competitive polymerase chain reaction, should be used. Hybridization with tPA revealed message on the positive control lanes, but no signal above background levels on the aortic tis- sue lanes. Reilly et al. 4 also performed Northern analysis on aneurysmal, occlusive, and normal aorta. They obtained their normal specimens from autop- sies, and the RNA extracted from these specimens was degraded and did not allow comparison with diseased aorta. Comparison of uPA and tPA mRNA in aneurysmal and occlusiv~e aorta revealed variability of plasminogen activator expression, but no signifi- cant differences. Similar to our study, sample num- bers were small (n = 2 for each group), making meaningful statistical analysis difficult.
The differential expression ofuPA, with the high- est levels appearing in AAA, mal~e it an attractive molecule to explain the previously described eleva- tions of plasmin seen in aneurysms.1 Localization o f
uPA to the inflammatory infiltrate by in situ hybrid- ization 13 correlates with earlier observations of in- creased inflammation in AAA as compared with oc- clusive aorta 12 and may be responsible for the differential expression of uPA observed in this study. Elevations of tPA were also observed in this study, but levels in aneurysmal and occlusive disease were similar. Previous reports have described elevations of PAI-1 in diseased as compared with normal aorta, and this elevation was associated with the arterioscle- rosis present in both aneurysmal and occlusive dis- ease. 6,22 Possible explanations for this association of increased tPA with arteriosclerosis include thrombus in the diseased aortic wall, which may be responsible for releasing incorporated tPA. It has also been shown, in tissue culture models, that thrombin, which is incorporated into the thrombus, increases tPA release from vascular smooth muscle cells 23 and endothelial cells. 24
A potential confounding factor in this study is the differences in the ages o f the three groups (normal, 48 + 13 years; occlusive, 50 +_ 13 years; and AAA, 66 -+ 10 years). Most of the previous work dealing with levels of tPA and age have been performed on serum samples, with the majority of the authors showing an age-related increase, 2s-29 whereas an- other report reveals an age-related decrease. 3° A lit- erature search failed to find any data regarding serum uPA levels and age. The current report studied levels of the plasminogen activators in the aortic wall, and no age-related differences were noted; however, the number of samples was small. The age of the normal and occlusive groups was very similar and yet there was a statistically significant difference between the tPA levels o f these two groups. On the other hand, no significant difference in the tPA levels of the oc- clusive and AAA groups was detected, even though the AAA group, on the average, was older than the occlusive group. We are not aware of any previous studies that compared arterial wall levels of the plas- minogen activators and age. Other researchers have studied tissue levels of the plasminogen system and found no correlation with age of tPA levels in breast cancer. 31 Increased levels of uPA, however, were associated with increased age in pulmonary adeno- carcinoma. 32
Finally, antigen levels o f tPA and uPA were mea- sured in the tissue explant supernatant studies rather than the activity o f these proteins as a result o f the instability of these molecules in the tissue culture media. PAL1 is thought to be secreted in an active, unstable form with a half-life of 75 minutes at 37 ° C 33 and in this system, is present in approxi-
JOURNAL OF VASCULAR SURGERY Volume 25, Number 1 Shireman et al. 163
mately 30-fold to 200-fo ld higher levels than the p lasminogen activators. 6 Both uPA and tPA are se-
creted in a p roenzyme form that is activated by plas- m in cleavage. Active uPA and t_PA in tissue culture media are rapidly inhibi ted by b ind ing to the more
a b u n d a n t PAI-134; therefore, there would be essen-
tially no active componen t s of the p lasminogen sys-
t em at the 48 hou r t ime po in t chosen in this study. We conclude that tPA levels in atherosclerotic
aorta are threefold higher than in normal specimens and that uPA is differentially expressed in aneurys-
mal, occlusive, and normal aorta. As uPA plays an impor tan t role in tissue remodel ing , our findings
may also reflect the relative tissue repair activities in these three types of specimens and may explain the previously described elevations of plasmin observed
in AAA. 1
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Submitted June 17, 1996; accepted Sep. 13, 1996.
T H E V O N L I E B I G F O U N D A T I O N A W A R D F O R V A S C U L A R S U R G E R Y R E S E A R C H , 1 9 9 7 - 1 9 9 8
3 rd Annua l von Liebig F o u n d a t i o n A w a r d for Ear ly-Career Academic S u r g e o n s - - $10 ,000 Award . Eligibility requirements: • The applicants are expected to be in the first 5-year period of their academic career. • Manuscripts accompanied by a signed letter from the author 's Division or Depar tment
head verifying the author 's length of appointment and attesting that the author per- formed all the essential parts o f the experimental work reported.
• The work must be original and unpublished and cannot be a summary or retrospective review o f previous work.
• A full curriculum vitae must be attached which includes a bibliography o f prior publica- tions.
Genera l r equ i rements for the award: • The research may be experimetnal or clinical in nature dealing with some fundamental
or clinical aspect o f vascular surgery. Clinical research papers are especially encouraged. • Research performed by an individual on staff at an institution in the United States,
Canada or Mexico. • Must be an original, unpublished work (nor submitted elsewhere for publication),
except to the ACS Surgical Forum. • Submitted in English (6 copies of the typed manuscript and 6 copies of glossy prints o f
illustrations), complying with "Instructions to Authors" o f the Journal of Vascular Surgery and including an abstract o f 2 50 words or less.
• A cover sheet indicating the manuscript is to be considered for: "The 3rd Annual von Lieberg Foundation Award for Early Career Academic Surgeons"
The manuscripts submitted will be reviewed by a select committee of vascular surgeons. The ECAS award will be presented at the annual meeting of the New England Society for Vascular Surgery. The yon Liebig Foundation reserves the right to withhold the grant of the award at the sole discretion of the Award Committee whose judgment with respect thereto shall be final and conclusive.
Further inquiries may be directed to the same address. Manuscripts must be postmarked no later than May 1, 1997.
Jean A. Goggins, PhD, Award Commit tee Secretary, The von Liebig Foundation, 281 Broad Ave S., Naples, FL 34102; (941) 262-3868.
