Articles |
From the National Public Health Institute, Oulu (J.J., H-M.S, A.B., M.L., P.S.); the Department of Surgery, Oulu University Hospital and University of Oulu (J.S., T.J.); the Department of Internal Medicine, University of Helsinki, Helsinki (A-M.T.); Hospital Infection Control, Oulu University Hospital (H.S.); the Department of Internal Medicine, Division of Cardiology, Oulu University Hospital and University of Oulu (J.A.); and the Department of Internal Medicine, Central Hospital of Kainuu, Kajaani, Finland (J.J.).
Correspondence to Tatu Juvonen, MD, PhD, Research Asst Professor, Department of Cardiothoracic Surgery, The Mount Sinai Medical Center, One Gustave L. Levy Place, Box 1028, New York, NY 10029.
| Abstract |
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(TNF-
), and interferon-
(IFN-
) were measured in 50
AAA patients (40 men, 10 women), 42 patients with coronary
heart disease (CHD) (23 men, 19 women), and 38 controls whose angiogram
was normal (17 men, 21 women). No differences in cytokine
concentrations were found between the CHD patients and the controls.
AAA disease was found to be associated with significantly higher IL-
1ß and IL-6 concentrations in both male patients (median
concentrations of 19.40 pmol/L and 6.45 pmol/L, respectively) and
female patients (19.26 pmol/L and 7.99 pmol/L) than in either the CHD
patients or the controls (P<.005). TNF-
levels were
slightly higher in the AAA patients (1.64 pmol/L in the males and 1.59
pmol/L in the females) than in the other groups (P <.05).
IFN-
levels were elevated significantly in the female AAA patients
(3.75 pmol/L) compared with levels found in the other female
(P<.05) or male (P<.01) patient groups. The
measured cytokine concentrations were not related to the size
of the aneurysm or the maximal thickness of the thrombus within
the aneurysm. IFN-
concentration showed a significant
positive correlation to the aneurysm expansion
(R=.37, P<.02) and negative correlation to the
concentration of aminoterminal propeptide of type III procollagen
during 6-month follow up (R=-.42, P<.005). The
results show that circulating levels of inflammatory cytokines
are elevated in patients with AAA disease, suggesting that the
production of these cytokines is increased in these
patients compared with CHD patients and controls. Elevated INF-
concentrations seem to predict an increased rate of expansion in AAA.
Key Words: cytokines abdominal aortic aneurysm inflammation
| Introduction |
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Abdominal aortic aneurysms (AAA) are often associated with atherosclerosis. There are, however, certain pathogenetic, epidemiological, and genetic differences between the two diseases.9 10 The basic phenomena in the pathogenesis of AAA are degradation of the extracellular matrix components (elastin and collagens) and loss of the structural integrity of the aortic wall.9 10 The AAA disease typically involves tissue inflammation as seen by the presence of inflammatory leukocytes and various cytokines, which are considered to participate in immunopathogenesis of AAA leading to the destruction of aortic matrix.9 10 11 12 13 14 15 16 Because of the potentially acute and lethal course of the disease, it would be a significant benefit to be able to screen unsuspected AAA and certain patients with known AAA for whom elective surgery is most appropriate.10 In certain diseases, circulating levels of individual cytokines have been found to correlate with the presence or severity of clinical symptoms,1 2 17 18 but these kinds of associations have not been studied in AAA disease.
The aim of the present study was to measure circulating levels of
inflammatory cytokines (IL-1ß, TNF-
, IL-6, and IFN-
) in
AAA patients to asses if the levels of circulating cytokines
show any relationship with clinical parameters or with
collagen metabolism.19 AAA has a strong male
preponderance relative to atherosclerosis, but the
reasons for this are unknown, and, consequently, we analyzed
the cytokine findings separately in male and female patients in
order to evaluate the potential immunologic basis for this
difference.
| Methods |
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The CHD patients and controls had been referred to the Cardiovascular Laboratory at Oulu University Hospital for coronary angiography during the same period of time because of chest pain. All of these patients underwent left-sided cardiac catheterization, including selective coronary arteriography in multiple views performed by Judkin's technique. The angiographic findings were interpreted by two independent cardiologists. A stenosis causing a >50% reduction in the luminar diameter was regarded as significant. The CHD group consisted of a consecutive cohort of 42 patients, 19 females and 23 males, with significant coronary artery disease. Their median symptoms were calculated to be NYHA (New York Heart Association) class III. Seventeen of them had three vessel disease, 10 two vessel disease, and 15 one vessel disease. The 38 randomly selected patients (21 females and 17 males) who comprised the control group had no significant coronary artery stenosis or atherosclerotic lesions in coronary angiography and were comparable with the AAA patients in terms of sex and age distribution. Patients with recent myocardial infarction, cardiomyopathy, congestive heart failure, or any infectious, inflammatory, or neoplastic disease were excluded. The protocol was approved by the Ethical Committee of the Faculty of Medicine at the University of Oulu.
Measurement of Cytokines
The sera for the cytokine assays were frozen and stored
at -20°C until tested. Serum TNF-
concentrations were measured
with a solid phase radioimmunoassay (RIA),20 the detection
limit of which was 0.41 pmol/L. Serum IL-1ß was measured by a
RIA modified from that of Cannon et al,21 in which values
<1.18 pmol/L were considered normal, and serum IL-6 was also
determined by RIA,22 the detection limit for which was
0.58 pmol/L. Serum IFN-
was measured by EIA (Bender
Medsystems, Vienna, Austria); the detection limit of the test, as
indicated by the manufacturer, was 0.06 pmol/L.
Analysis of Collagen Metabolism
Collagen biosynthesis was analyzed by measuring a
collagen turnover, the aminoterminal propeptide of type III procollagen
(PIIINP), with an equilibrium-type RIA based on human antigen (Orion
Diagnostica, Oulunsalo, Finland) with use of duplicate 200
µL aliquots of serum as previously described by Satta et
al.19 Concentration of PIIINP is expressed as
µg/L because its molecular weight slightly varies.
Statistical Methods
The statistical analyses were performed with SPSS
statistical software (Windows 6.0), with comparisons among the three
groups made by Kruskal-Wallis analysis of variance and
differences between two groups analyzed with the Mann-Whitney
U-test. Pearson correlation coefficients were used to test associations
between continuous variables. Categorical variables were
compared by
2 test.
| Results |
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As shown in Fig 1
, cytokine
concentrations were comparable between the CHD patients and the
controls, but IL-1ß, IL-6, and TNF-
concentrations were
significantly higher in the sera of male AAA patients (median
concentrations 19.40, 6.45, and 1.64 pmol/L respectively) than
in controls (2.06, 0.39 and 1.35 pmol/L) or in males with CHD
(2.23, 2.31, and 1.35 pmol/L). No difference in IFN-
concentrations was found between the male patient groups. Serum
concentrations of IL-1ß, IL-6, and TNF-
were also significantly
higher in the female AAA patients (19.26, 7.99 and 1.59 pmol/L,
respectively) than in either the control (2.06, 1.54, and 1.29
pmol/L) or the CHD female patients (2.21, 1.73, and 1.35
pmol/L). IFN-
levels were significantly higher in the AAA
females (median 3.75 pmol/L) than in the other female groups
(1.89 pmol/L in the controls and 2.61 pmol/L in CHD,
P<.05) or in the male patient groups (P<.05).
No correlation was found between the serum levels of any
cytokine and either age, diameter of aneurysm, or
thickness of thrombus. Cytokine levels in either the 14
patients with a small AAA (maximal diameter <45 mm) or the seven
patients with no ultrasonographically detectable thrombus were not
significantly different from cytokine levels for the rest of
the patients (P>.1).
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At the time of diagnosis, median maximal diameter of aneurysms in the AAA male and female patients was 48 mm (range 33 to 66 mm; SD 0.78, which showed a significant correlation to the median thickness of the thrombus 10.0 mm (range 0 to 35.0 mm; SD 9.69; R=.45, P<.001). Median PIIINP concentration (3.15 µg/L, range 1.80-5.30, SD 0.78), presence of hypertension, and COPD had no significant correlation to the aneurysm size. The thickness of thrombus correlated to PIIINP concentration (R=.73, P<.001).
As shown in Table 2
, the average rate of
aneurysm expansion was 4 to 5 mm within the first 12
months of follow-up. The aneurysm growth was not found to be
influenced by hypertension, COPD, or smoking. The aneurysm
expansion in 6 months follow-up showed a positive correlation to the
IFN-
concentration measured at the time of AAA diagnosis
(R=.35, P<.02) and to low PIIINP concentration
(R=.49, P<.0001). A correlation coefficient
between IFN-
and PIIINP concentrations was -.42
(P<.005). No correlation was found with the
aneurysm expansion and IL-1ß, IL-6, or TNF-
(P>.1).
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| Discussion |
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, have been detected locally in
atherosclerotic and AAA tissues.13 14 15 16 In this study we
were able to demonstrate significantly higher levels of circulating
IL-1ß, IL-6, and TNF-
in the serum of AAA patients than in either
CHD patients or angiographically normal controls. IL-1ß levels were
especially high and provided a good demarcation between AAA patients
and the other patient groups. The measured cytokine levels were
the same in the AAA patients with small and large aneurysms
(maximal diameter > or <45 mm) and in patients with or
without detectable thrombus, suggesting that the
cytokine-mediated inflammatory process is present as early
as it is possible to diagnose the AAA disease and is not restricted to
an advanced or "end stage" of the disease.
The presence of elevated cytokines (IL-1ß, IL-6, and TNF-
)
was associated with AAA disease but not with
atherosclerosis because CHD patients had
cytokine levels that did not differ from those of the controls
with no angiographically verified lesions. However, AAA patients often
have advanced atherosclerosis, and, therefore, we
cannot exclude the possibility that the extent of
atherosclerosis in the AAA patients was greater than
that present in those with CHD. If this were true, it would be at
least partly responsible for our results. Recently, Uno et
al23 published data supporting this possibility,
demonstrating elevated levels of circulating cytokines in
patients suffering from end stage occlusive
arteriosclerosis, with signs of poor
peripheral circulation such as cold, colored, or ulcerated
extremities. We must also consider that in the general population, the
incidence of unsuspected AAA has been estimated to be 1 to 2% and in
screening studies of patients more than 50 years of age, 1 to
8%.10 24 Our CHD and control groups were not screened in
this sense, and some cases with unsuspected AAA in these groups could
possibly have affected on our results. However, because the control
patients were quite young, the risk for unsuspected AAA is low.
Although circulating immunoreactive cytokines reflect
production of these cytokines at sites of inflammation,
they are not necessarily biologically active because immunoassays
measure cytokine immunoreactivity regardless of its
inactivation in specific circulating soluble receptors,
beta-2-microglobulin, or other serum proteins.17 18 Matrix
metalloproteinases (MMPs) have been suggested as mediators of the
degradation of the structural proteins in the aneurysmal aortic
wall,25 26 27 28 29 although this hypothesis has been questioned
quite recently.30 It must be emphasized that
aneurysmal degeneration has been shown to involve additional
proteinases, such as plasminogen
activators.31 32 Based on previous
publications, IL-1ß and TNF-
are linked to AAA pathogenesis by
inducing MMP expression and reducing the expression of tissue
inhibitors of proteinases (TIMP).13 14 15 16
Together with IL-6, TNF-
plays a role in neovascularization, and
IL-6 seems to be at least partly responsible for the accumulation of
immunoglobulins in the aneurysmal wall.33 34 35 36 37 38 39
Therefore, high concentrations of inflammatory cytokines in the
AAA tissue may lead to increased degradation of the extracellular
matrix, weakening of the vessel wall, and eventual dilatation of the
aorta. Interestingly, it has been discovered recently that a
metalloproteinase-like enzyme also mediates the posttranslational
processing step required for conversion of membrane-bound pro TNF-
(26kDa) to its 17 kDa soluble secreted form and that TNF-
release is
suppressed by MMP inhibitors.40 41 42
Inflammatory cytokines are also known to participate in the
progression of atherosclerosis in several
ways,7 8 17 39 and IL-1ß treatment has been shown to
induce coronary intimal lesions in pigs in vivo.43
On this basis, it is tempting to speculate that
atherosclerosis may not be merely associated with AAA
but that the inflammatory cytokines produced by AAA tissue can
accelerate its progression in AAA patients.
According to our results, the expansion rate of the aneurysm
could not be predicted by elevated concentrations of the
proinflammatory cytokines (IL-1ß, IL-6, or TNF-
). Instead,
elevated IFN-
levels seemed to predict aneurysm enlargement
during the first 6 months follow-up correlating to low PIIINP
concentration. IFN-
, which is mostly produced by T cells,
activates macrophages, stimulates the
production of TNF-
and IL-1ß, and has a specific effect on
collagen metabolism by inhibiting the synthesis of type I
and type III collagen alone or synergistically with
TNF-
.17 44 45 Increased IFN-
production and
positive correlation with both IFN-
mRNA expressions and degree of
adventitial inflammation have been demonstrated in AAA
tissue.16 46 In accordance with our results, an inverse
correlation between IFN-
and PIIINP has been reported in patients
with pulmonary fibrosis.47 Our previous study
provided evidence that collagen turnover is increased in AAA
patients.19 However, the actual concentration of collagen
in relation to aneurysm dilatation and rupture risk is debated
continually.10 Most theories of aneurysm rupture
have suggested inadequate collagen synthesis as the primary pathologic
factor.48 In the present study, the enhanced
aneurysm expansion was associated with lowered PIIINP
concentrations, indicating that collagen metabolism can be
depressed in some stage of weakening of the aortic wall because of AAA
disease. A positive correlation between IFN-
concentration and
aneurysm expansion suggests that collagen synthesis and repair
processes are interrupted in the AAA wall. According to this study, the
INF-
levels were higher in female AAA patients than in other patient
groups. Whether this is actually related to gender differences in AAA
pathogenesis requires further study with larger patient groups.
Actively secreted cytokines associated with
AAA13 14 15 16 are indicative of chronic inflammation in the AAA
patients, and secreted IFN-
suggests involvement of
activated T cells. The primary reason for the inflammation
remains obscure, but our recent demonstration of Chlamydia
pneumoniae in AAA tissue49 suggests that C.
pneumoniae infection could be a causative agent. The fact that
chlamydia can cause persistent infection, accelerate the
production of inflammatory cytokines,50
produce protease in HL cells,51 and provoke
aneurysm formation in acute vascular infections52
makes C. pneumoniae a candidate for triggering AAA
formation.
In conclusion, several cytokines are implicated in AAA and atherosclerosis processes, having multiple and complex effects on the pathogenic mechanisms involved in these diseases. An understanding of these mechanisms may thus help us to approach the problem of developing new treatments. In fact, many potential pharmacological strategies for the management of small AAAs have been presented.10 Future research will show whether the circulating concentrations of cytokines can be used to monitor the effect of these therapies.
| Selected Abbreviations and Acronyms |
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| Acknowledgments |
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Received July 2, 1996; accepted April 7, 1997.
| References |
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