Journal of Vascular Surgery
Volume 45, Issue 6 , Pages 1109-1113, June 2007

Elevated tissue plasminogen activator in patients with screening-detected abdominal aortic aneurysm

  • Anders Wanhainen, MD, PhD

      Affiliations

    • Department of Surgical Sciences, Section of Vascular Surgery, Uppsala University Hospital, Uppsala, Sweden
    • Corresponding Author InformationCorrespondence: Dr Anders Wanhainen, Department of Surgical Sciences, Section of Vascular Surgery, Uppsala University Hospital, SE-371 85 Uppsala, Sweden.
    • ,
  • Torbjörn K. Nilsson, MD, PhD

      Affiliations

    • Department of Clinical Chemistry, Örebro University Hospital, Uppsala, Sweden
    • ,
  • David Bergqvist, MD, PhD

      Affiliations

    • Department of Surgical Sciences, Section of Vascular Surgery, Uppsala University Hospital, Uppsala, Sweden
    • ,
  • Kurt Boman, MD, PhD

      Affiliations

    • Department of Medicine, Skellefteå County Hospital, Uppsala, Sweden
    • Institution of Public Health and Clinical Medicine, Umeå University Hospital, Uppsala, Sweden.
    • ,
  • Martin Björck, MD, PhD

      Affiliations

    • Department of Surgical Sciences, Section of Vascular Surgery, Uppsala University Hospital, Uppsala, Sweden

Received 25 September 2006; accepted 4 February 2007.

Article Outline

Objective

A population-based case-control study with historical and current data was conducted in a population with a high prevalence of disease to explore the hypothesis that the fibrinolytic system may be involved in the early pathogenesis of abdominal aortic aneurysm (AAA).

Methods

Forty-two patients found to have AAA at population-based screening were compared with 100 controls matched for age and sex. Mass concentration of tissue plasminogen activator (tPA mass) and tissue plasminogen activator/plasminogen activator inhibitor-1 complex (tPA/PAI-1 complex mass) were analyzed in blood samples obtained at the screening (current), and in blood samples obtained from a study conducted 12 years previously on the same population (historical).

Results

Current tPA mass levels were significantly higher in AAA patients compared with controls (13.6 vs 11.4 μg/L, P = .016). A similar trend was observed in historical tPA mass levels (9.8 vs 8.2 μg/L, P = .062). Current and historical mass concentrations of tPA/PAI complex in AAA patients were similar to those in controls. Current tPA mass levels retained the associations with AAA in a logistic regression model after adjustment for history of atherosclerosis (odds ratio [OR], 1.1 per μg/L, P = .039) and current smoking (OR 1.1 per μg/L, P = .039). When family history of AAA was added in a logistic regression model, the OR for current tPA mass was 1.1 per μg/L (P = .056) and 1.1 per μg/L (P = .070) when treated hypertension was added.

Conclusion

The finding of elevated tPA mass, in contrast to tPA/PAI-1 complex, in plasma among patients with screening-detected AAA supports the hypothesis that the fibrinolytic system may be important in the early pathogenesis of AAA.

Clinical Relevance

Early detection by screening and repair is the only option to reduce mortality from abdominal aortic aneurysm (AAA) in the population. As a consequence, AAA screening programs have been launched in many countries. Most AAAs detected by screening are small, however, and the current treatment of these is limited to watchful waiting. Other therapeutic or preventive strategies are needed, necessitating a profound knowledge of the pathogenesis of AAA.

 

One of the most important features of arterial wall in aneurysmal disease is the degradation of the connective tissue,1 and an increased proteolytic activity has been demonstrated in the wall of abdominal aortic aneurysm (AAA).2, 3, 4 The fibrinolytic system is a proteolytic enzymatic system involved not only in the dissolution of fibrin in blood vessels but also in the remodeling of the extracellular matrix (ECM).5 Fibrinolysis is initiated by plasmin, which plays an important role in the degradation of ECM by directly digesting matrix proteins and by activating proteolytic matrix metalloproteinases (MMP).6, 7, 8 Studies have found elevated levels of plasmin in AAA tissue compared with controls9 and demonstrated a correlation between plasmin-antiplasmin complex and the expansion rate of small AAA,10 suggesting plasmin to be important in the pathogenesis of aneurysm formation and progression.

Plasmin is regulated by tissue plasminogen activator (tPA), urokinase-type plasminogen activator, and plasminogen activator inhibitor-1 (PAI-1). In the presence of fibrin, tPA converts the proenzyme plasminogen within the thrombus into plasmin, its active form.11, 12 PAI-1 regulates plasminogen activation by inhibiting free tPA and forming an enzymatically inactive tPA/PAI-1 complex, which results in a loss of plasminogen activation potential and thereby a decreased level of proteolytic and fibrinolytic activity.13 Fig 1 displays an overview of the fibrinolytic system.

We previously reported the hitherto highest prevalence of AAA in a general population.14 In this population, we found that some traditional risk factors for atherosclerosis were associated with AAA, but others were not, indicating complex etiologic mechanisms.15 One of these may be the interaction between the fibrinolytic system and the pathogenesis of AAA. In this field, several scientific questions remain to be answered, such as the temporal associations between different components of the fibrinolytic system and the development of AAA.

The aim of this investigation was to explore, in a population with a high prevalence of disease and with current as well as historical blood samples, the hypothesis that the fibrinolytic system may be involved in the pathogenesis of AAA.

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Patients and methods 

Between 1984 and 1994, all 60-year-old men and women living in the northern Sweden municipality of Norsjö were invited to a health survey at their local health center. As part the Västerbotten Intervention Program (VIP), they donated blood to be stored at the Northern Sweden Medical Research Bank.16 In 1999, all men and women between 65 and 75 years of age and living in Norsjö were invited to a health examination that included an ultrasonography measurement of the abdominal aortic diameter and a questionnaire on medical history. Fasting blood samples were also drawn and stored. Of 555 invited, 504 subjects (91%) participated, consisting of 248 men and 256 women with a median age of 70 years.

In a previous report on traditional risk factors for atherosclerosis in the same population,15 an AAA was defined as a mean diameter ≥30 mm by both ultrasonography (US) and computed tomography (CT); that is, US diameter + CT diameter/2. However, because few previous investigations have used CT measurements to define the disease, comparisons with other investigations were impeded. In the present study, we decided to use a more accepted definition, where an AAA was defined as a maximum infrarenal aortic diameter ≥30 mm by means of US alone.14 This change in definition was made before blood was retrieved from the blood bank.

The present study was designed as a case–control study. Frozen plasma samples were retrieved for the analysis of historical data for all AAA cases and controls who were identified in the 1999 AAA screening study and who had participated in the VIP study between 1984 and 1994. Thus, two sets of data were used in the comparison between AAA cases and controls: (1) from the VIP study when the participants were at the age of 60 years (historical data), and (2) from the AAA screening when the same subjects were between 65 and 75 years old (current data). The mean time between assessments of historical and current data was 11.6 years (median, 12 years). The study design is displayed in Fig 2. All participants gave their informed consent, and the study was approved by the Ethics Committee of Umeå University.

  • View full-size image.
  • Fig 2. 

    Overview of the study design. VIP, Västerbotten Intervention Program; tPA-PAI; tissue plasminogen activator; PAI, plasminogen activator inhibitor; AAA, abdominal aortic aneurysm.

Venous blood was taken in ethylenediaminetetraacetic acid (EDTA) tubes. The plasma samples were snap-frozen within 1 hour to −20°C and stored at −80°C within a week. All EDTA plasma analyses were thawed and analyzed simultaneously at the same occasion. The tPA and tPA/PAI-1 complex mass concentrations were measured with enzyme linked immunosorbent assay (ELISA) kits (TintElize tPA and TintElize tPA/PAI-1 complex, respectively; Biopool AB, Umeå, Sweden). The coefficients of variation of these two assays were about 10%.

History of disease and smoking were assessed at the AAA screening in 1999. Subjects were classified as current smokers, former smokers, or nonsmokers. History of atherosclerotic disease was defined as any combination of self-reported coronary heart disease, cerebrovascular disease, or peripheral artery occlusive disease. Family history of AAA was defined as having a first degree relative (ie, parents, siblings, or offspring) with a known AAA.

The independent samples t test was used for comparison between cases and controls. Bivariate logistic regression models were used to estimate the odds ratio (OR) for hemostatic factors in relation to AAA after adjustment for history of atherosclerosis, current smoking, family history of AAA, serum creatinine level, and treated hypertension, with “AAA” or “no AAA,” as a dichotomous dependent variable. Statistical evaluations of the data and random selection of controls from different groups matched for sex and age were done with SPSS 14.0 software (SPSS, Chicago, Ill).

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Results 

The screening identified AAAs (n = 42) in 35 men (83%) and seven women (mean age, 71.4 years). From the AAA screening study population, 100 controls matched for age and sex were assigned. The mean aortic diameter was 38 mm (95% confidence interval [CI], 34 to 42 mm; range, 30 to 81 mm) in the AAA group and 24 mm (95% CI, 23 to 25 mm; range, 17 to 29 mm) in the control group. Table I summarizes baseline characteristics of the study population.

Table I. Baseline characteristics of the study population
AAA patients (n = 42)Controls (n = 100)P
Age (mean years)71.471.4.95
Men83%83%.96
Aortic diameter (mean mm)3824<.001
Self-reported atherosclerotic disease43%25%.046
Current smokers18%1%<.001
Family history of AAA23%5%.002
Diabetes mellitus10%9%.87
Treated hypertension45%26%.034
Serum creatinine (mean μmol/L)8887.95

AAA, Abdominal aortic aneurysm.

Current tPA mass levels were significantly higher among AAA patients compared with controls (13.6 vs 11.4 μg/L, P = .016; Table II). The difference observed in historical tPA mass levels did not reach significance (9.8 vs 8.2 μg/L, P = .062; Table I). Current and historical levels of tPA/PAI-1 complex mass in AAA patients were similar to those in controls (Table I). There was a significant increase in tPA mass levels within the AAA group (P = .002) and within the control group (P < .001) over time, but no change was observed in tPA-PAI complex mass (Table III).

Table II. Laboratory variables in patients with screening-detected AAA and controls matched for age and sex
Variable⁎AAA patients (n = 42)Controls (n = 100)P
Historical†
tPA/PAI-1 complex (μg/L)4.6±2.34.9±3.0.622
tPA mass (μg/L)9.8±3.58.2±4.0.062
Current†
tPA/PAI-1 complex (μg/L)5.3±2.24.7±2.4.128
tPA mass (μg/L)13.6±4.711.4±4.3.016

AAA, Abdominal aortic aneurysm; tPA/PAI-1 complex, tissue plasminogen activator-plasminogen activator inhibitor complex; tPA mass, tissue plasminogen activator mass concentration.

⁎Variables presented as means ± standard deviation.

†Current data obtained at AAA screening (at the age of 65-75 years, average 71.4 years), and historical data 5-15 years (average 11.6 years) prior to screening (at the age of 60 years).

Table III. Differences between current⁎ and historical† variables (Δ data‡) in patients with screening-detected AAA and controls matched for age and sex.
AAA patients (n = 42)Controls (n = 100)
ΔDataPΔDataP
tPA/PAI-1 complex (ug/L)+ 0.3.623− 0.2.558
tPA mass (ug/L)+ 3.8.002+ 3.1<.001

AAA, Abdominal aortic aneurysm; tPA/PAI-1 complex, tissue plasminogen activator-plasminogen activator inhibitor-1 complex; tPA mass, tissue plasminogen activator mass concentration.

⁎Current data obtained at AAA screening at the age of 65 to 75 years (average, 71.4 years).

†Historical data obtained 5 to 15 years (average, 11.6 years) before screening at the age of 60 years.

‡Differences between current and historical data within each group, using paired tests.

Current tPA mass levels retained the associations with AAA in a logistic regression model after adjustment for history of atherosclerosis (OR 1.1 per μg/L, P = .039), current smoking (OR 1.1 per μg/L, P = .039) and serum creatinine levels (OR 1.1 per μg/L, P = .022). The OR was 1.1 per μg/L (P = .056) when family history of AAA was added in the model and 1.1 per μg/L (P = .070) when treated hypertension was added.

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Discussion 

The present study demonstrated a significant elevation of tPA mass concentration in plasma in AAA patients compared with controls. Although a novel finding, this observation is consistent with previous histologic studies.17, 18, 19 Reilly et al17 found high levels of tPA in aneurysmal aortas compared with normal aortas and occlusive aortas,17 whereas Shireman et al18 found a similar threefold elevation of tPA in aneurysmal and occlusive aortas compared with normal aorta.18 In AAA, tPA messenger RNA was localized to macrophage-like cells in the inflammatory infiltrate in AAA,20 smooth muscle cells (SMCs),21 and the luminal thrombus.19, 22

Lindholt et al23 found a positive correlation between plasma levels of tPA and the annual AAA expansion rate as well as with serum cotinine, suggesting that smoking may interact with this pathway.23 As reported previously,15 current smoking together with duration of smoking, rather than pack-years, was the most important smoking variable associated with AAA. In the present study, the elevation of tPA mass concentration in plasma among AAA patients was independent of current smoking, as well as of reported history of atherosclerosis. However, self-reported data, in particular smoking habits are, prone to recall bias.

In addition, those not reporting a history of cardiovascular disease may have atherosclerosis that is not clinically manifest. An interesting trend in the present study was the higher tPA mass levels in blood obtained in mean 11.6 years before screening. The fact that this trend was present long before the detection of small AAA by screening, suggests that plasmin-mediated proteolysis may be of pathophysiologic importance in the early development of AAA.

An elevation of PAI-1 in diseased aortas was associated with the arteriosclerosis present in both aneurysmal and occlusive disease.24, 25 A higher concentration of tPA in SMCs obtained from AAA compared with SMCs from normal aorta was not followed by the corresponding upregulation of PAI-1, causing an imbalance favoring proteolysis.21 Defawe et al26 showed that two physiologic inhibitors of proteases, of which one was PAI-1, were expressed to a lesser degree in AAA than in occlusive aortas, “suggesting a significant role for protease inhibitors during the divergent evolution of the initial atherosclerotic plaque towards either aneurysmal or occlusive aortic disease.”26 Thus, the significantly higher mass concentration of tPA among AAA patients, but the lack of an increase in tPA-PAI complex in plasma, compared with controls matched for age and sex observed in the present study may be explained by a lower expression of PAI-1.

A limitation of this study is the small number of patients, making it susceptible to type II statistical error. Large number of patients with AAA and historical blood samples are not readily available, however. Despite the small numbers, the significantly increased levels of tPA mass concentration in current blood samples and the trend (P = .06) towards higher levels in the historical blood samples that was found among patients with screening-detected AAA compared with matched controls support the hypothesis that proteolysis through an increased activity (or a decreased inhibition) of the fibrinolytic system may be an early event in the pathogenesis of AAA.

With multiple factors analyzed there is also a risk of mass significance, or type I error. Only two fibrinolytic factors were analyzed in the present study, but in a previous study, 12 atherosclerotic risk factors were analyzed in the same population,15 and we also analyzed antibodies against three bacterial proteins in a report to be published. The small number of patients studied made it impossible to perform a multivariate analysis including all the 17 measured variables, but we tried to minimize the risk of confounding by performing a bivariate logistic regression for the four strongest risk factors for AAA identified in the previous publication.15 The associations between current tPA mass levels and AAA was lost (P = .056) only when family history of AAA was entered into the model.

Another limitation is that not all the components of the fibrinolytic system were analyzed, such as urokinase-type plasminogen activator mass concentration and PAI-1 as well as other inhibitors, to get the whole picture of the fibrinolytic balance over time. Some of these factors are, however, undetectable in most plasma samples or are unstable, necessitating other storage medium than used in the present study. PAI-1 is an extremely unstable molecule, especially when measured by activity-based assays. We therefore did not measure this variable in the study.

Determination of tPA mass concentration and tPA/PAI-1 have, however, been found to be stable both against degradation and freeze/thaw events. Because tPA and its complex does not bind to albumin and thus is not affected by serum albumin levels, albumin does not need to be measured.

Another important aspect to consider is that components of the fibrinolytic system measured in the systemic circulation might differ from the situation within vascular beds of different organs.27 On January 1, 1999, the total population of Norsjö was 4804, of whom 555 (11.6%) were 65 to 75 years old. Unfortunately, no information was available on the original cohort that entered the VIP study between 1984 and 1994. It is essential to analyze and report subjects lost to follow-up in cohort studies. The present study is therefore not a true cohort study, although the historical data were collected prospectively. Considering this limitation it was, however, possible to analyze changes over time at an individual level. Plasma levels of tPA mass concentration increased over time (ie, with age) among both cases and controls, emphasizing the importance of the study design using controls matched for age and sex. The age-dependency of tPA was described previously.28

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Conclusions 

To our knowledge, the present study has demonstrated for the first time a significant and independent elevation of tPA mass concentration, in contrast to tPA/PAI-1 complex, in plasma among AAA patients compared with controls. This finding supports the hypothesis that the fibrinolytic system may be important in the development of AAA.

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Author contributions 


Conception and design: AW, TN, DB, KB, MB

Analysis and interpretation: AW, TN, DB, KB, MB

Data collection: AW

Writing the article: AW

Critical revision of the article: TN, DB, KB, MB

Final approval of the article: AW, TN, DB, KB, MB

Statistical analysis: AW

Obtained funding: Not applicable

Overall responsibility: AW

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We thank professor Göran Hallmans and his staff at the Northern Sweden Medical Research Bank, Umeå University Hospital, for professional storing of the blood samples.

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References 

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 Competition of interest: none.

PII: S0741-5214(07)00252-2

doi:10.1016/j.jvs.2007.02.001

Journal of Vascular Surgery
Volume 45, Issue 6 , Pages 1109-1113, June 2007

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