© 2000 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Expression of Secretory Group IIA Phospholipase A2 in Relation to the Presence of Microbial Agents, Macrophage Infiltrates, and Transcripts of Proinflammatory Cytokines in Human Aortic Tissues
Presented as a preliminary report at the 71st Scientific Sessions of the American Heart Association, Dallas, Tex, November 8–11, 1998.
From the Institut für Klinische Chemie und Laboratoriumsmedizin (M.M., A.R.-S., R.E., W.J.), the Institut für Informatik und Biometrie (R.K.), and the Institut für Rechtsmedizin (E.M.), Technische Universität Dresden, Medizinische Fakultät "Carl Gustav Carus," Dresden, Germany.
Correspondence to Mario Menschikowski, PhD, Institut für Klinische Chemie und Laboratoriumsmedizin, Technische Universität Dresden, Medizinische Fakultät "Carl Gustav Carus," Fetscherstrasse 74, D-01307 Dresden, Germany. E-mail menschik{at}rcs.urz.tu-dresden.de
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Abstract—Recent seroepidemiological and immunohistochemical studies have demonstrated an association between microbial infections and atherosclerosis. However, the mechanisms underlying this association are widely unknown. In the present study, arterial specimens obtained at autopsy after sudden death were analyzed concerning (1) the presence of Chlamydia pneumoniae, cytomegalovirus, herpes simplex virus, and Helicobacter pylori; (2) the expression of secretory group IIA phospholipase A2 (sPLA2-IIA) and of proinflammatory cytokines; and (3) the stage of atherosclerosis. Genomic DNA of microbial pathogens was determined by the polymerase chain reaction technique. The expression of sPLA2-IIA was studied immunohistochemically by using monoclonal antibodies against human sPLA2-IIA. Transcripts specific for sPLA2-IIA, interleukin-1ß, tumor necrosis factor-
, and interferon-
were identified by reverse
transcription–polymerase chain reaction. In 18 of 102 analyzed
specimens, DNA of microbial pathogens was found. Thirteen sections were
positive for C pneumoniae, whereas 2 specimens were
positive either for cytomegalovirus or for herpes simplex virus. One
section contained genomic DNA of all 3 pathogens
simultaneously. None of the analyzed tissues
exhibited nucleic acids specific for H pylori. In
addition to macrophage infiltrates, the presence of microbial
DNA was closely associated with the occurrence of transcripts specific
for proinflammatory cytokines and sPLA2-IIA.
Pathogens as well as sPLA2-IIA and cytokines were
found to be present not only in advanced but also in early stages
of atherosclerosis. In tissues negative for
sPLA2-IIA and cytokine expression, none of the
pathogens could be identified. Because macrophages exposed to
phospholipase A2–treated lipoproteins are transformed into
foam cells in vitro, the results of this study suggest an alternative
mechanism by which microbial infections may act in a proatherogenic
fashion in vessel walls.
Key Words: infections • secretory phospholipase A2 • inflammation • atherosclerosis
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From previous histopathologic studies, it is known that atherosclerotic lesions share many features with those of inflamed tissues. The causes of inflammatory reactions in the vessel wall, however, are still under discussion. Several hypotheses have been proposed, including the theory of infection as a cause of coronary artery diseases, which was first described by Osler1 at the beginning of this century. Although there is now a growing body of evidence suggesting an association between infections and atherosclerosis, the mechanism by which microbial pathogens may act in a proatherogenic manner is still unresolved.
In a previous study, we identified the expression of a secretory phospholipase A2 (sPLA2), which is identical to group IIA isozyme (sPLA2-IIA) according to the new group type classification of phospholipases A2 proposed by Dennis2 and Murakami et al,3 in human atherosclerotic plaques but not in normal arteries.4 From in vitro studies, it is known that phospholipase A2 can hydrolyze the phospholipids of lipoproteins, resulting in varied physicochemical properties of modified particles.5 6 These alterations apparently lead to an enhanced degradation of modified lipoproteins by macrophages, transforming them into foam cells, a hallmark of early atherosclerotic lesions.7 8 These data led to the hypothesis that sPLA2 could be a new factor in the pathogenesis of atherosclerosis.9 In recent years, further studies have been published involving the analysis of sPLA2 expression in human vascular tissues.10 11 12 13 14 Bobryshev et al10 performed a study on carotid arteries and aortas and found sPLA2-specific immunostaining in atherosclerotic plaques but not in areas of the adjacent normal-appearing arterial wall. Another 2 studies have been published by Hurt-Camejo et al11 and Elinder et al,12 respectively, showing sPLA2 immunoreactivities not only in atherosclerotic arteries but also in nonatherosclerotic arteries.
The physiological and/or
pathophysiological relevance of
sPLA2 expression in arterial tissues
remains to be elucidated. In vitro studies indicated a substrate
preference of sPLA2 for Gram-negative bacterial
membranes, such as from Escherichia coli, in the presence of
bactericidal/permeability-increasing protein.15
Recently, bactericidal activity of sPLA2 against
Gram-positive bacteria has been observed in human tears.16
Based on these data, which suggest microbicidal properties of
sPLA2, the question arises of whether the
expression of sPLA2 in atherosclerotic lesions
could represent a part of the host defense mechanism against
microbial pathogens. This assumption was supported by further in vitro
and in vivo data showing that (1) the synthesis and secretion of
proinflammatory cytokines, including interleukin (IL)-1ß,
tumor necrosis factor- (TNF-), and interferon- (IFN-), are
induced after exposure to bacterial endotoxins17 18 ;
(2) cytokines such as IL-1ß, IL-6, and TNF- are able to
stimulate the synthesis and secretion of sPLA2 in
smooth muscle cells19 20 ; and (3) proinflammatory
cytokines are expressed in atherosclerotic
plaques.21
The objective of the present study was to investigate the expression of sPLA2-IIA in relation to the stage of atherosclerosis and the presence of microbial agents and signs of inflammatory reactions in human vascular walls. For this purpose, abdominal and thoracic aortic segments obtained at autopsy after sudden death were analyzed by immunohistochemistry, polymerase chain reaction (PCR), and reverse transcription (RT)-PCR methods.
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Tissues
Human arterial samples (n=102) obtained at autopsy from 51 persons (38 males and 13 females ranging in age from 1 to 94 years, mean±SEM 41.7±17.1 years) were taken from 2 cm above the aortic valve and 3 cm below the renal ostia, respectively. To exclude positive results concerning the expression of proinflammatory cytokines and sPLA2-IIA mediated by acute-phase reaction, the tissues were taken from subjects whose death was sudden and who did not show signs of severe acute diseases. Most common causes of death were accidents, homicides, or suicides. The time between death and autopsy, during which the subjects were kept at 4°C, ranged from a few hours to 6 days.
Histopathology and Immunohistochemistry
Immediately after autopsy, the removed aortic segments
were washed in PBS (pH 7.2), fixed in neutral-buffered formalin, and
embedded in paraffin. Subsequently, the samples were coded, and all
further investigations were performed with observers blinded to the
details of the study protocol. To determine the stage of
atherosclerosis, sections were stained by
hematoxylin-eosin staining. Goldner’s trichrome staining was
applied to visualize extracellular components. The immunohistochemical
investigations were performed as described elsewhere4 by
applying the biotin-streptavidin-alkaline phosphatase system (Super
Sensitive Multilink, Biogenex). Before incubation with specific
antibodies, deparaffinized and rehydrated sections were treated with
0.5 mg/mL Pronase (DAKO Diagnostica) for 15 minutes at
24°C. To detect antigens, serial sections were incubated with
monoclonal antibodies recognizing (1) human
sPLA2 (BIOMOL), (2) -actin from smooth muscle
cells (DAKO Diagnostica), and (3) CD68 from
macrophages (clone KP-1, DAKO Diagnostica). The
monoclonal anti-sPLA2 antibody, prepared
originally against the sPLA2 from human sperm,
cross-reacts immunologically with the sPLA2
enzyme expressed in atherosclerotic plaques, as previously
demonstrated.4 The immunoreactivities were visualized by
Fast Red TR as chromogen containing levamisole to block
endogenous alkaline phosphatase activity (Biogenex). In
each case, nonimmune mouse serum instead of specific antibodies was
used as a negative control. Prostate tissue processed in the same way
as aortic tissues after autopsy was used as a positive control because
of the known sPLA2-positive immunoreactivities in
glandular epithelial cells of this tissue.22 The score of
sPLA2-IIA immunostaining was
assessed in the whole area of each section by using the following
grades: 3+, intense immunoreactivity in numerous cells; 2+, positive
immunoreactivity in a moderate number of cells (10 to 100 cells); 1+,
weak immunoreactivity in a few cells; and 0, no immunoreactivity.
Macrophage infiltrates were expressed as follows: 3+, massive
infiltration of macrophages; 2+, moderate number of cells (10
to 100 cells); 1+, only a few cells; and 0, no cells. The stages of
atherosclerosis were classified according to the
recommendations made by Stary et al.23
Identification of Nucleic Acids Specific for C
pneumoniae, H pylori, CMV, and HSV
Nucleic acids from paraffin-embedded aortic sections (10x5
µm-thick sections) were prepared by using the DNA isolation kit
Puregene (Gentra Systems). For amplification of genomic DNA specific
for Chlamydia pneumoniae, Helicobacter pylori,
cytomegalovirus (CMV), and herpes simplex virus (HSV), primer pairs
were applied as described24 25 26 27 (Table 1
). The buffers and reagents used in the
PCR were those supplied in the Gene Amp Kit (Perkin-Elmer, ABI). The
conditions of PCR were as follows: an initial denaturing step for 2
minutes at 94°C; 35 cycles at 94°C for 30 seconds, 62°C for 30
seconds, and 72°C for 1 minute; and, finally, an extension step at
72°C for 10 minutes (with the exception of H pylori, for
which 62°C was applied in the extension step). The products of
amplification were analyzed on ethidium bromide–stained 2%
agarose gels under UV light. In cases of negative results, amplified
samples were reamplified by a second PCR under the same condition as
applied in the first PCR. Tissues that showed negative results after
the second amplification were defined as pathogen-free specimens.
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To verify the efficiency of DNA isolation and amplification, genomic DNA specific for pyruvate dehydrogenase was determined in parallel with the microbial pathogens in every aortic specimen. PCR reagents without templates and with nucleic acids isolated from C pneumoniae, H pylori, CMV, and HSV were used as negative and positive controls, respectively. The C pneumoniae prototype strain TW-183 and H pylori were kindly provided by Dr Pressler (Friedrich-Schiller-University, Jena, Germany). CMV and HSV of type 1 were obtained from Dr Wischmann (Technical University, Dresden, Germany). Isolated DNA from HSV of type 2 was purchased from Advanced Biotechnology. At the beginning of the present study, investigations on DNA from HSV of type 1 and type 2 proved the specificity of applied primers to identify both types of HSV.
Identification of mRNA Specific for sPLA2-IIA, IL-1ß,
TNF-, and IFN-
To evaluate the stability of sPLA2-IIA
mRNA in situ, 2 aortic segments obtained in surgery were
analyzed after storage in humidified chambers at 4°C or
24°C for 1, 2, 4, 5, and 6 days before fixing. A segment of each
biopsy was immediately fixed after the removal of tissues for use as a
control. After they were embedded in paraffin, 5-µm-thick and
10x5-µm-thick serial sections were prepared for immunohistochemical
investigations and for extraction of RNA, respectively.
Total RNA was prepared after deparaffinization and rehydration from
10x5-µm-thick autopsy sections by acid guanidinium
thiocyanate–phenol–chloroform extraction as described by Chomczynski
and Sacchi.37 Isolated RNA was converted to cDNA by using
the GeneAmp RNA-PCR Kit (Perkin-Elmer). A portion of the RT reaction
products was then amplified for identification of
sPLA2-IIA and TNF- mRNA by using nested PCR
and identification of IL-1ß and IFN- mRNA by using conventional
PCR. The applied primer pairs, which are respectively designed to span
splicing sites between 2 exons, are summarized in Table 1
. For the analysis of sPLA2-IIA
mRNA, oligonucleotides were synthesized according to
the published nucleotide sequence of human placental
sPLA2-IIA cDNA.28 In the nested PCR,
extrinsic and intrinsic primer pairs were applied in a final
concentration of 0.1 µmol/L and 0.8 µmol/L, whereas in
the conventional PCR, the final concentration averaged 0.8
µmol/L. The conditions for amplification were as follows: 15 cycles
at 94°C for 30 seconds and at 60°C for 50 seconds, followed by 40
cycles at 94°C for 30 seconds and at 72°C for 1 minute. The buffers
and reagents used were the same as supplied in the GeneAmp Kit
(Perkin-Elmer). After amplification, products were analyzed
by electrophoresis on agarose gels. As control, GAPDH mRNA was
determined in every autopsy sample.
Statistical Analysis
The prevalence of the expression of sPLA2
in relation to the stage of atherosclerosis in aortic
tissue and data from aortic specimens with and without the
analyzed microbial agents were analyzed by using the
2 test and, if the expected frequencies were
low, by the Fisher exact test. The confidence intervals for odds ratios
were determined by using the Cornfield method. The Student t
test was used to compare the average age of individuals positive and
negative for C pneumoniae, CMV, or HSV.
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Identification of Microbial Agents in Autopsy Aortas
By use of PCR, 18 of the 102 tissue samples investigated showed the presence of DNA fragments of C pneumoniae, CMV, or HSV (Table 2
). In 5 individuals,
nucleic acids of C pneumoniae were found in both
sections of the aorta, whereas in 8 individuals, microorganisms were
found to occur in only 1 location, ie, either in the abdominal or the
thoracic aorta. Samples with early signs of atherosclerotic changes
(types I and II) or preatheromas (type III) were found to
be up to 8.5% positive for analyzed microorganisms, whereas
the tissues with advanced atherosclerotic lesions (types IV to VI)
yielded up to 36% microbial agents. Tissue samples without
atherosclerotic lesions were free of analyzed microorganisms
(Table 3). Genomic DNA of H
pylori was not observed in any of the tissues investigated.
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Identification of sPLA2-IIA Expression and of
Transcripts Specific for Proinflammatory Cytokines in
Autopsy Aortas
Preliminary investigation of 2 arterial tissue samples
obtained during surgery showed that sPLA2-IIA and
GAPDH mRNA were detectable by means of RT-PCR up to 4 days after
removal of the tissue, provided the samples were stored at 4°C before
fixing. After the day 5, there was a reduction of mRNA, and on day 6,
no mRNA specific for sPLA2-IIA and GAPDH could be
determined (Figure 1A). Compared with
tissue samples stored at 4°C, the tissue samples stored at 24°C
before fixing exhibited an accelerated decomposition of the
sPLA2-IIA and GAPDH mRNA (Figure 1A). In
contrast to mRNA, however, the immunohistochemical identification of
sPLA2-IIA was not significantly impaired by
unfixed storage up to 6 days after the intraoperative release of
tissue. Furthermore, no differences in the intensity of
sPLA2-IIA immunostainings were
observed in aortas fixed in Bouin’s solution, ethanol, or formalin
(not shown).
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All the autopsy aortic samples in which DNA sequences of pathogens were
identified exhibited immunostaining and transcripts
specific for sPLA2-IIA. The distributions and
intensities of the sPLA2-IIA
immunostaining and macrophage infiltrates in
the individual vessel layers of the aorta are shown in Table 2.
It can be seen that along with the intima, the adventitia also
exhibited sPLA2-IIA
immunostainings. In 5 samples, the media was
specifically stained for sPLA2-IIA. Transcripts
of IL-1ß, TNF-, or IFN- were identified in 11 of the 18
pathogen-positive tissues. In addition, macrophage infiltration
was found in the intima and, with the exception of 1 sample, in the
adventitia as well (Table 2).
Together with the tissues in which pathogens were detected, a further
61 samples in which no DNA of the investigated microorganisms occurred
revealed sPLA2-IIA–specific
immunostaining. Of these samples, 52
simultaneously exhibited
sPLA2-IIA–specific transcripts. Of the 52
samples, in which sPLA2-IIA expression was
determined both on the protein level and on the mRNA level, 16 samples
yielded transcripts of proinflammatory cytokines. All samples
without sPLA2-IIA expression at the protein and
the mRNA levels (n=23) contained none of the investigated pathogens and
were also free of transcripts of IL-1ß, TNF-, and IFN-. Some of
the results of these PCR and RT-PCR investigations are shown in Figure 1B.
Table 4 shows the frequency of
sPLA2-IIA expression as a function of the stage
of atherosclerosis in the autopsy tissues. Because no
significant differences concerning the stage-dependent enzyme
expression between thoracic and abdominal sections of the aorta were
apparent by multivariate analysis (not shown),
the data of both segments were considered together. In Table 5, the results of the
univariate analysis are summarized. A significant
association could be established between the presence of C
pneumoniae, CMV, or HSV DNA sequences and the expression of
proinflammatory cytokines and sPLA2-IIA
transcripts. Furthermore, CD68-positive cell infiltrates and
sPLA2-IIA immunostainings were
more frequently detectable in the adventitia of aortas with microbial
agents compared with specimens free of agents.
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Immunohistochemical Examinations of Autopsy Aortas
Immunostainings specific for
sPLA2-IIA and macrophage infiltrations
were found in all 3 layers of pathogen-positive vessel walls. In Figure 2, an abdominal aorta is shown that
contained DNA of C pneumoniae (Figure 1B, lane 11);
it exhibited atheromatous lesions of type V. Strong
positive sPLA2-IIA
immunostainings were detectable in the lipid core,
especially at the base of lipid core adjacent to the media (Figure 2A). By use of serial sections (Figures 2B through 2D),
the positive immunostainings were shown to correspond
to CD68-positive macrophages. In addition to
sPLA2-IIA–positive signals in lipid cores,
further immunostainings were evident in the media near
the adventitia (Figure 2B). The
sPLA2-IIA–positive cells exhibited a
spindle-shaped morphology and were positive for -actin (Figures 2B and 2D).
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Figure 3 shows serial sections from an
abdominal aorta with advanced atherosclerotic plaques of type VI in
which DNA of C pneumoniae, CMV, and HSV could
simultaneously be identified (Figure 1B, lane 1). The
plaque was characterized by 2 different lipid cores with a fibrotic cap
overlay (Figure 3A). Within the fibrotic caps, especially
between the lipid cores, heavy cell infiltrations were apparent, of
which individual cells were identifiable as CD68-positive
macrophages (Figure 3B). Furthermore, small blood
vessels were visible, suggesting neovascularization. In addition to
sPLA2-IIA immunostainings
associated with macrophages, further stainings were observed in
regions corresponding to -actin–positive round cells (Figures 3C through 3F).
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A further property of specimens exhibiting nucleic acids of pathogens
consisted in strong sPLA2-IIA
immunostainings of the adventitia, which were
accompanied by infiltrations of numerous CD68-positive
macrophages. Figure 4A through 4C
shows the adventitia of a thoracic aorta that was positive for CMV DNA,
IL-1ß, and TNF- mRNA (Figure 1B, lane 8) and that exhibited
atheromatous lesions of type IV. In addition to smooth
muscle cells and CD68-positive macrophages,
sPLA2-IIA–positive immunoreactions were
associated with connective tissue cells and extracellular matrix
structures of the adventitia. Compared with pathogen-positive specimens
with atherosclerotic changes, the thoracic aorta shown in Figure 4D through 4F did not exhibit nucleic acids of analyzed
microbial pathogens (Figure 1, lane 2) and was free of
microscopically observable atherosclerotic lesions. In this specimen
obtained at autopsy from a 23-year-old man, no
sPLA2-IIA immunostainings were
evident in either the intima (not shown) or the media/adventitia
(Figure 4D). These data were consistent with results
obtained by RT-PCR analysis, demonstrating a complete absence
of mRNA specific for sPLA2-IIA (Figure 1B, lane 2). Comparable data indicating the complete absence of
sPLA2-IIA expressions were observed in an
additional 4 nonatherosclerotic aortas (Table 3).
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Two aortic specimens, although entirely free of atherosclerotic
lesions, exhibited transcripts specific for
sPLA2-IIA. Figure 5
shows an aorta of a 7-year-old child who died after carbon monoxide
poisoning. Strong sPLA2-IIA–positive
immunostainings were detectable in the media
corresponding to -actin–positive smooth muscle cells. By use of
RT-PCR, sPLA2–specific mRNA and transcripts
specific for TNF- and IFN- were identifiable in this tissue (not
shown). In addition, the adventitia was characterized by infiltration
of numerous CD68-positive macrophages, which was not the case
in the other sPLA2-IIA–free nonatherosclerotic
specimens. Comparable findings were made in a further sample taken from
a thoracic aorta of a 10-year-old boy who died after an accident (Table 3).
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Discussion |
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There is increasing evidence that inflammation and immunologic mechanisms play a major role in atherogenesis, but exactly what stimuli are responsible for the initiation of these reactions remains to be determined. In the present study, aortic specimens obtained at autopsy after sudden death were analyzed for the presence of microbial agents and the expression of sPLA2-IIA and proinflammatory cytokines. The data show that 14% of the analyzed specimens exhibited nucleic acid fragments specific for C pneumoniae, whereas 3% were positive either for CMV or for HSV. Recently, Maass et al38 published a study demonstrating that 15% to 26% of analyzed specimens obtained from different vascular regions were positive for C pneumoniae DNA by using a nested PCR protocol. In aortic wall samples, the prevalence of C pneumoniae averaged 18%, a value that closely parallels the frequency observed in the present study. Compared with frequencies observed in other studies, the frequencies observed in the present study and in the study of Maass et al38 are relatively low. This could be due to differences in the incidence of C pneumoniae infections, dependent on the respective geographic regions in which the studies were carried out. Another reason might be that many surgical samples were examined for the presence of C pneumoniae, from which it can be expected that the samples will show a preponderance of atherosclerotic lesions. If this is taken into account and the prevalence of C pneumoniae in aortas with advanced atherosclerotic lesions (types IV to VI) is determined, the data in the present study yield a value of 31%. Indications of the occurrence of H pylori in aortic tissues could not be found in the present study, which is consistent with results published by Blasi et al.39
To determine whether the presence of bacteria or viruses is accompanied
by inflammatory reactions, markers of inflammatory activities such as
infiltrates of CD68-positive macrophages and expressions of
proinflammatory cytokines and sPLA2-IIA
were investigated. In the present study, it was determined that all
of the tissue samples in which genomic DNA of pathogens was found had
strong macrophage infiltrates and
sPLA2-IIA immunostainings in the
intima and in the adventitia. Furthermore, in 11 of these specimens,
transcripts specific for IL-1ß, TNF-, or IFN- could be
identified, which is significantly more frequent (P<0.001)
than in specimens free of C pneumoniae, CMV, or HSV DNA
sequences. These results are in accord with in vitro data showing that
the infection of mouse macrophages or human alveolar
macrophages with C pneumoniae resulted, along with
the generation of reactive oxygen species, in a dose- and
time-dependent release of TNF- and IL-1ß into the culture
supernatant.40 41 Similar results concerning the
production of TNF- and IL-1ß were obtained in human
peripheral blood mononuclear cells after infection with
C pneumoniae.42 In CMV-infected
mononuclear cells, an elevated level of IL-1ß activity was
determined.43 The reason that cytokine-specific
transcripts were not found simultaneously in all
pathogen-containing and sPLA2-IIA–positive
tissues may, on the one hand, be due to the concentration of mRNA being
too low. On the other hand, it is also possible that along with the
cytokines IL-1ß, TNF-, and IFN- investigated in the
tissues examined, other proinflammatory mediators, such as IL-6, that
can also induce sPLA2-IIA expression were
present.44
sPLA2-IIA is known to exhibit a highly alkaline pI, leading to a strongly positively charged protein under physiological conditions.45 46 For this reason, it can be assumed that the enzyme will interact with negatively charged proteoglycans and, for that reason, bind itself, independent of the location of synthesis, to cell surfaces and extracellular matrix structures and be able to remain there for extended periods of time, as recently analyzed by Romano et al.13 Increased sPLA2-IIA activities in the vessel wall may act in a proatherogenic manner in different ways. Besides the direct cytotoxicity due to the hydrolysis of membrane phospholipids, through which the maintenance of plaque structures can be disturbed, and besides the generation of proinflammatory lipid mediators, including eicosanoids and platelet-activating factor, a series of indirect effects may be associated with the expression of sPLA2-IIA. Lysophosphatidylcholine, which is produced in the hydrolysis of phospholipids, was shown to exhibit cytolytic properties,47 to induce the synthesis of heparin-binding epidermal growth factors through macrophages,48 to express vascular cell adhesion molecules in endothelial cells,49 and to be chemotactic for human monocytes50 and T lymphocytes.51 Furthermore, phospholipids of lipoproteins retained in the subendothelial space can be hydrolyzed by the enzyme identifiable at the same sites. It has been shown in vitro that the exposure of macrophages to LDL and HDL treated with phospholipase A2 results in a considerable cholesterol accumulation in the cells, transforming macrophages into foamlike cells.7 8
In addition to the tissue sections containing nucleic acids of
pathogens, sPLA2-IIA
immunostaining could be found in tissues in which there
was no indication of the presence of C pneumoniae, CMV,
or HSV. The mechanisms that induced the sPLA2-IIA
transcription in these tissues are still unexplained. A concomitant
expression of proinflammatory cytokines in some of these aortas
may suggest that other stimuli were present, possibly even other
microorganisms. In this connection, it is noteworthy that in 5
pathogen-free, but sPLA2-IIA–positive sections,
IFN- mRNA was detectable. These data may suggest, at least in
IFN-–positive tissues, that activated T lymphocytes were
present, underscoring the occurrence of immunologic reactions in
these specimens.
The finding of a correlation between the prevalence of sPLA2-IIA expressions and the stage of atherosclerosis provided further evidence that the induction of the enzyme synthesis and secretion in the aortic wall may be of importance not only for the progress but also for the initiation of atherosclerotic processes. The immunohistochemical investigations demonstrated that sPLA2-IIA–positive immunoreactivities were present, especially in regions of lipid cores (primarily at the base of lipid cores adjacent to the media) and in areas of extracellular matrix structures. When the sPLA2-IIA and CD68-specific immunostainings are compared, the enzyme has never been identified in intima free of considerable numbers of macrophages. On the other hand, the presence of macrophages did not correlate strongly with sPLA2-IIA expression, in view of the fact that macrophage infiltrations with and without evidence of sPLA2-IIA were detectable, suggesting a different activation state of macrophages in atherosclerotic lesions, which is in accordance with data published by van der Wal.52 Furthermore, a number of tissues have been found to exhibit adventitia, including the media half directed toward it, and to a lesser extent intima, which were positive for sPLA2-IIA. At the same time, massive infiltrations of CD68-positive macrophages were to be found in the adventitia of these tissues. Because sPLA2-IIA–specific immunostainings could be attributed, among other cellular and extracellular structures, to macrophages in the adventitia, the question arises as to whether inflammatory cells may initiate the induction of the sPLA2-IIA gene after they have entered the arterial wall not only from the vascular region but also from the adventitia via the vasa vasorum.
In addition to CD68-positive macrophages, -actin–positive
round cells associated with areas of plaque neovascularization and
strong cell infiltrates were identified as
sPLA2-IIA positive. The nature of these cells
remains to be elucidated. Other -actin– and
sPLA2-IIA–positive cells frequently
exhibited a typical stellate-shaped appearance. Recently, Tjurmin et
al53 have shown that stellate cells of myxomatous tissue
represent a heterogeneous cell population, sharing
features of macrophages, smooth muscle cells, and
antigen-presenting dendritic cells. The latter cell type is
characterized by immunoreactivity with antibodies recognizing
-actin, HLA-DR, and CD1a.53 Bobryshev et
al10 have argued that in addition to macrophages,
at least in part, vascular dendritic (CD1a-positive) cells may be
responsible for the sPLA2-IIA expression in
atherosclerotic plaques. These data suggest that in addition to
CD68-positive macrophages, smooth muscle cells, and adventitial
fibroblasts, stellate cells may be responsible for the expression of
sPLA2-IIA identified in atherosclerotic lesions.
Interestingly, Tjurmin et al53 hypothesized that in
atherosclerotic lesions, stellate cells might be involved in local
immune responses, in which they act as antigen-presenting
cells.
To understand the possible role of sPLA2-IIA in the pathogenesis of atherosclerosis, the determination of whether this enzyme is expressed in normal arteries is of major importance. In our previous study, sPLA2-IIA–positive immunostainings were visible in arterial and aortic specimens exhibiting atherosclerotic plaques but not in nonatherosclerotic aortas or nonaffected parts of the arterial wall.4 Whereas Bobryshev et al10 found these results to be confirmed in their studies, Hurt-Camejo et al11 and Elinder et al12 described sPLA2-IIA immunoreactivities both in normal nonatherosclerotic arteries and in those with atherosclerotic lesions. In the present study, 7 specimens were classified as nonatherosclerotic. Five of them showed neither sPLA2-IIA–specific immunostainings nor traces of sPLA2-IIA mRNA. Because of the significantly faster decomposition of sPLA2-IIA mRNA in removed aortic tissues after storage at 24°C compared with those at 4°C before fixing, the present study only considered autopsy samples taken from subjects who were delivered to the facility within a few hours of death and were kept at 4°C until autopsy. Furthermore, only those samples were evaluated in which GAPDH mRNA could be detected as a control. This applied also to the 5 sections classified as nonatherosclerotic. Finally, the preliminary investigations showed that when samples were stored at 4°C, sPLA2-IIA levels remained immunohistochemically unchanged up to 6 days post mortem, so that false negatives concerning the sPLA2-IIA immunostaining in the 5 nonatherosclerotic aortas and in further aortic specimens with early stages of atherosclerosis (n=18) can be ruled out.
In 2 nonatherosclerotic thoracic aortas, strong positive
sPLA2-IIA immunostainings were
evident. These findings were confirmed in both tissue samples by the
detection of sPLA2-IIA–specific mRNA. The
investigations of proinflammatory cytokines have shown,
however, that both specimens simultaneously exhibited
transcripts specific for TNF- and IFN- or IL-1ß and IFN-,
which was not the case in the other
sPLA2-IIA–free nonatherosclerotic specimens.
Such results point to inflammatory reactions as the possible cause of
the sPLA2-IIA expressions observed in both
samples, although the reason for the inflammation in these tissues is
presently unexplained. Therefore, the different results concerning
whether sPLA2-IIA is expressed in normal
arterial sections4,10–13 are possibly
explained with reference to whether inflammatory reactions were ongoing
before the removal of tissues.
In summary, the present study has shown that the presence of
microbial agents is closely associated with the expression of
sPLA2-IIA and macrophage infiltrates in
human aortic specimens. This finding strongly suggests that (1) the
induction of the sPLA2-IIA gene in cells of the
aortic wall can be considered a further indicator of ongoing
inflammatory reactions in these tissues and (2) the expression of
sPLA2-IIA may be a constituent of the host
defense mechanism in vessel walls. Furthermore, because
macrophages exposed to phospholipase
A2–modified lipoproteins are transformed into
foam cells in vitro, the present study provides evidence for a
potential mechanism by which microbial infections may act in a
proatherogenic manner. This mechanism is in accordance with the
response-to-injury hypothesis of atherogenesis, which proposes that
atherosclerotic lesions represent a specialized form of a
protective inflammatory response to various forms of insults to the
vessel wall.54 In the progression of this response, a set
of proinflammatory cytokines including IL-1ß, TNF-, and
IFN- may be released by inflammatory cell infiltrates, which in turn
induce the synthesis and secretion of sPLA2-IIA.
However, when infectious agents persist in the arterial
wall, the expression of sPLA2-IIA, possibly
triggered as a protective response to microbial invasions, may become
excessive. In addition to a series of
pathophysiological reactions propagating
inflammatory processes in the arterial wall, chronically
increased sPLA2-IIA activities may be associated
with the hydrolysis of lipoprotein phospholipids. Thereby, the process
of foam-cell formation can be forced, especially when high
concentrations of lipoproteins are simultaneously
present in the subendothelium of the vessel wall.
Interestingly, a recently published study involving mice with LDL
receptor deficiency provided evidence that the chlamydial
proatherogenic effects are dependent on elevated serum
cholesterol levels.55 Although native
lipoproteins proved to be a poor substrate for
sPLA2-IIA, an increased susceptibility of
lipoproteins to phospholipid hydrolysis after minimal oxidation, which
is especially likely to occur under inflammatory conditions in vivo,
could recently be shown.56 This mechanism, if proven in
vivo, may inaugurate an alternative strategy for prevention and therapy
of coronary artery disease by applying specific
sPLA2-IIA inhibitors.
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Acknowledgments |
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This study was supported by Deutsche Forschungsgemeinschaft (DFG, Ja 565/2-3). We thank Dr Ockert and Dr Dressler for providing aortic specimens obtained in vascular surgery and autopsy. The expert technical assistance of Helga Kunze and Margot Vogel is greatly appreciated.
Received June 16, 1999; accepted September 13, 1999.
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