Atherosclerosis and Lipoproteins |
From the Kinsmen Laboratory of Neurological Research, Department of Psychiatry, University of British Columbia, Vancouver.
Correspondence to Dr Patrick L. McGeer, Kinsmen Laboratory of Neurological Research, University of British Columbia, Vancouver, BC V6T 1Z3. E-mail mcgeerpl{at}interchange.ubc.ca
| Abstract |
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Key Words: CD59 CD46 C4 binding protein classical pathway inflammation
| Introduction |
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There are many regulators that are designed to control unwanted complement activation. These include C1 inhibitor (C1-INH), C4 binding protein (C4BP), decay accelerating factor (DAF), membrane cofactor protein (MCP, CD46), and protectin (CD59).15 C1-INH arrests the complement cascade at the stage at which C1q binds to a target, causing dissociation of the C1 complex. It is a suicide inhibitor that attaches covalently to the exposed esterase sites of C1r and C1s. C4BP inhibits C3 convertase by binding to the active fragment C4b. DAF is a glycosylphosphatidylinositol linked surface protein that accelerates the decay of C4b2a. MCP accelerates cleavage of C3b, primarily in the alternative pathway,16 but it may also have inhibitory activity in the classical pathway.17 CD59 is another glycoprotein Ilinked cell surface protein that binds to the C7 and C8 components of the MAC as it assembles on the surface of host cells. Thus, it prevents C9 attachment and full insertion of the MAC into the membrane. It is also known as protectin because it protects host cells against bystander lysis.18 In the present study, we describe the production of all of these inhibitors by normal artery and plaque tissue. We compare their upregulation in plaques with that of their main complement protein targets to assess their ability to defend against the self-damaging effects of complement activation.
| Methods |
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The techniques used for RNA extraction and preparation of reverse transcription (RT)polymerase chain reaction (PCR) products, as well as for protein extraction, Western blotting, and immunohistochemistry, were as previously reported in detail.11 18 19 23
Briefly, total RNA was extracted from
500 mg of tissue
samples by the acid guanidinium thiocyanatephenolchloroform method.
Single-strand cDNA synthesis was performed on 5-µg samples of total
RNA extract. The resultant cDNAs (1-µL aliquots) were amplified by
using appropriate primers and standard buffer. The primers used, along
with GenBank accession numbers and the endonucleases used for
identifying the RT-PCR products, are shown in
Table 1
. The thermal profile consisted of a denaturation
step of 94°C for 1 minute, an annealing step of 30 seconds at 61°C
for C1-INH, 56°C for DAF, 60°C for C4BP and MCP, and 57°C for
CD59, and an extension step of 72°C for 1 minute, except for 3
minutes in the first cycle. All samples were initially denatured for a
total of 9 minutes (94°C). Preliminary experiments were carried out
with each set of primers to determine the range of cDNA concentration
and PCR cycle amplification number that would give reliable comparative
values for each cDNA product. Cyclophilin mRNA was chosen as the
internal standard because of the consistency of its level
from tissue to tissue and its postmortem stability. A linear
relationship was found between the log of PCR product intensity and
cycle number of cyclophilin (between 20 and 29 cycles), C1-INH and CD59
(between 24 and 30 cycles), DAF (between 27 and 33 cycles), C4BP
(between 27 and 37 cycles), MCP (between 29 and 33 cycles), and
complement components (between 25 and 37 cycles), after which plateaus
were reached. A linear relationship was found between the amount of
cDNA product obtained and the original cDNA added within the range
corresponding to 0.01 to 0.5 µg total RNA. Accordingly, standard
conditions were followed in which cDNA (1 µL) corresponding to 0.1
µg total RNA was added, and the cyclophilin product was amplified
for 27 cycles. In parallel experiments, the C1-INH and CD59
products were amplified for 29 cycles; the DAF, C4BP, and MCP
products, for 30 cycles; and the complement products, for 35
cycles.
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Each PCR reaction product was electrophoresed through a 6% polyacrylamide gel, and the product was visualized by incubation for 10 minutes in a solution containing 10 ng/mL ethidium bromide. Resulting gel bands were imaged by using a GDS 7600 image analyzer (Ultra Violet Product). The relative intensities of the bands, expressed as optical density units, were quantitatively analyzed by using NIH image software 1.62. Each mRNA analysis was made in parallel with a cyclophilin mRNA analysis to provide an internal standard. Values normalized to cyclophilin, as well as uncorrected data, were analyzed. Cyclophilin values were almost constant from sample to sample. Most were within 1% of each other, with the range <4%, so that the normalization corrections were very small. Polaroid photographs of the gels were taken. The PCR products were of the expected size, and endonuclease digestion in each case gave fragments of the predicted size as previously reported.11 18 19 In all experiments, the presence of possible contaminants was checked by control reactions in which amplification was carried out for up to 40 cycles on samples in which we omitted from the RT-PCR reaction mixture either (1) the reverse transcriptase or (2) a template cDNA. No product was obtained under these conditions.
Western blots were performed on extracts of the soluble fraction of homogenates of normal or plaque tissue as previously described.11 Briefly, tissue samples were homogenized in 5 times (vol/wt) extraction buffer (0.02 mol/L Tris-HCl, pH 7.5, and 0.1% Triton X) containing the protease inhibitors phenylmethylsulfonyl fluoride (10 µg/mL) and aprotinin (10 µg/mL) and 1 mmol/L EDTA. Homogenates were centrifuged at 18 000g at 4°C for 30 minutes. The protein content of the supernatants was determined by Lowrys method. The samples were diluted in SDS sample buffer (60 mmol/L Tris, pH 6.8, 2.5% SDS, and 5% 2-mercaptoethanol) to a final protein content of 5 mg/mL and were boiled for 3 minutes. Samples containing 70 µg of protein were loaded onto 7.5% polyacrylamide minigels. Life Technologies high-range prestained standards were used as molecular weight markers. The separated proteins were transferred onto nitrocellulose membranes, stained with Ponceau staining solution (Sigma), and scanned densitometrically. Membranes were blocked in 5% skim milk for 6 hours. The immunoblots were next treated for 6 hours at room temperature with a primary antibody, followed by treatment for 2 hours with a secondary antibody labeled with horseradish peroxidase. Immunoreactivity was visualized by incubation with Supersignal CL-HRP chemiluminescent substrate (Pierce Chemical Co). After draining, the membranes were covered in clear plastic wrapping and exposed to x-ray film (Hyper film ECL, Amersham Life Science) for 0.3 to 2 minutes, depending on the strength of the signal. Protein loading differences were corrected for by densitometric quantification of Ponceau staining. The primary antibodies were goat antiC1-INH (Quidel, 1: 3000), rat anti-CD59 (Serotec, 1:500), goat anti-C1r (ICN, 1:2000), goat anti-C1s (Quidel, 1:2500), rabbit anti-C4BP (Calbiochem, 1:2000), goat anti-C4 (Chemicon, 1:10 000), mouse anti-C4d (Quidel, 1:5000), goat anti-C7 (Quidel, 1:3000), goat anti-C8 (Calbiochem, 1:5000), and mouse monoclonal antifraction Bb (Quidel, 1:500). All secondary antibodies were from Sigma (1:8000).
Immunohistochemistry was carried out on paraffin-embedded tissue, as previously described,24 25 with the same antibodies used for Western blotting. Sections were cut at 10 mm, deparaffinized in toluene, and processed in the free-floating state. The C1-INH antibody was used at a concentration of 1:15 000, and the CD59 and fraction Bb antibodies were used at a concentration of 1:500. Secondary antibodies were from Vector (1:1000), as was the avidin-biotinylated horseradish peroxidase (Vector ABC-Elite) labeling reagent. Color was developed with 0.01% diaminobenzidine (Sigma) enhanced with nickel ammonium sulfate.
The data on relative levels of the mRNAs were first analyzed by ANOVAs, with subsequent Student t tests being used to determine the probability value for differences between plaque and normal tissue. The plaque/normal tissue ratio was calculated for each pair and analyzed by ANOVA, followed by Student t tests for the difference from 1. The probability values were corrected by the method of Holm26 for multiple assays.
| Results |
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Table 2
gives quantitative values for the mRNAs. The
housekeeping gene cyclophilin was almost constant for all tissues,
indicating the stability of the mRNAs. Moreover, the values for normal
tissue from case to case and for plaques from case to case varied
little for any of the other mRNAs. This indicates that such confounding
factors, such as cause of death and postmortem delay, are not
significant contributors to the values under study.
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Table 2
also gives the average ratio of plaque to normal
artery mRNA levels for the 11 matched pairs. For the regulators, they
were as follows: C1-INH 1.04, C4BP 0.97, MCP 0.94, and CD59 1.06. None
of these ratios was significantly different from 1. However, C1r, C1s,
C4, C7, and C8 all showed significant increases
(P<0.001), with ratios of
2.35, 4.96, 1.34, 2.61, and 3.25, respectively.
Figure 2
shows typical Western blots comparing protein
extracts from a normal artery and a paired nearby plaque. This is
illustrated for C1-INH compared with its 2 substrates C1r and C1s, C4BP
compared with C4 and C4d, and CD59 compared with C7 and C8.
Figure 2
also compares Western blots of fraction Bb of
factor B in normal human serum compared with the same serum
activated by IgG, a normal artery, and a nearby plaque. The
figure illustrates that no discernible differences between normal
arterial and plaque tissue can be seen for the regulators
C1-INH, C4BP, CD59, and fraction Bb, with the last being a specific
marker of alternative pathway activation. In contrast, compared with
its companion normal artery, plaque tissue shows strong upregulation
for C1r and C1s, for C4 and C4d, and for C7 and C8. C4d is a specific
marker of classical pathway activation. These results are
consistent with the mRNA data of
Table 2
. Additionally, serum activated by IgG, but
not normal serum, gave a strong band for fraction Bb, indicating that
aggregated IgG, when added to serum, will activate the
alternative pathway and that no apparent activation of the alternative
pathway is occurring in plaque tissue.
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Figure 3
illustrates immunohistochemical results for C1-INH,
CD59, and fraction Bb staining. Overall, they show a similar intensity
of staining in plaque compared with normal artery for both C1-INH and
CD59, although the staining in plaque tissue is more widespread. The
distribution is somewhat different. C1-INH staining is more prominent
in the intimal and adventitial layers than in the medial layer
(Figure 3A
). In plaque tissue, C1-INH staining is widespread
in the proliferating fibrous zone and appears to be intracellular and
extracellular
(Figure 3B
). CD59 staining is intense in the
endothelial layer, as previously
described27
(Figures 3C
and 3D
), with weaker staining in the other layers.
In fibrous plaque tissue, the antibody to CD59 weakly stains some
elongated fibromyocytes and some round cells resembling leukocytes
(Figure 3D
). Overall, the immunohistochemical findings for
C1-INH and CD59 are consistent with the mRNA and Western blot
data, showing a failure to upregulate the inhibitors in
inflamed plaque tissue. Fraction Bb staining was very faint
(Figure 3E
), with no structures being
identifiable.
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| Discussion |
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We have previously shown that all components of the classical complement pathway are produced within arterial tissue and that they are all significantly upregulated in plaques.11 Moreover, we have shown that CRP, a known complement activator, is also produced by arterial tissue and is more upregulated than any of the complement proteins. Thus, the mechanism for a self-sustaining complement attack generated within plaque tissue itself is clearly present.
In several previous publications, it has been shown that CRP and the MAC colocalize on damaged cells in the proliferating zone of plaque tissue.4 10 11 The fact that the relevant mRNAs are also colocalized to these same cells indicates that the process is internal to plaque tissue. Although the plaque deposits build up over time and are, thus, the end result of a long-standing pathological process, the half-life of mRNAs is usually minutes to hours. Therefore, the mRNA levels give a dynamic picture of what is continuously occurring in plaque tissue.
The reason for the proliferation of cells in the deep intimal layers of evolving plaque tissue is unclear. It is proposed that oxidized LDL is a stimulant for such proliferation.30 This may be an initial stimulus, but it is possible that continuing complement attack is also acting as a stimulus, thus enhancing the plaque enlargement process. Macrophage attack occurs on opsonized targets, and such an attack is easily discerned within the proliferating plaque shoulder area. The process leads to 2 unfortunate outcomes. The first is dissolution of the fibrous coat by macrophage collagenase activity, releasing material that causes platelet aggregation and thrombotic activity.31 The second is continued growth of the plaque, resulting in arterial stenosis.
The inflammatory reaction within plaques is somewhat analogous to the situation in Alzheimer brain tissue. Amyloid deposits stimulate macrophages, which provide a stressful environment for neurons. The neurons produce upregulated levels of CRP32 and all of the complement proteins.24 However, they do not produce upregulated levels of C1-INH and CD59,18 and fraction Bb is not detected.23 The consequence is full activation of the classical complement pathway. Extracellular deposits are opsonized, but the MAC localizes on damaged neurites, providing evidence of autodestruction.24 However, unlike myocytes, neurons cannot proliferate, so loss of vital tissue occurs rather than an expansion of unwanted tissue.
The reaction in plaques is also somewhat analogous to the situation in infarcted myocardium. Heart tissue is also capable of synthesizing all of the complement proteins, and there is substantial upregulation, coupled with complement activation in infarcted areas.20 The MAC is observed attacking damaged myocardial fibers, not only in acutely infarcted areas33 but also in the region of old infarcts.20 CRP colocalizes with the MAC in infarcted tissue.34
Other activators of the classical complement
pathway may be involved. These include mannan-binding protein, an
activator of the closely related lectin
pathway,35 36
enzymatically modified
LDL,37 and even IgG. But
these are all inhibited, including the lectin
pathway,35 36 by
the regulators described in the present study. Of course, there may
be other less specific inhibitors, such as
2-macroglobulin and
1-antitrypsin, which play a role in
inhibiting the complement cascade, but it cannot be a decisive role
because robust activation is clearly taking place in plaque
tissue.
In summary, the data reported in the present study suggest that complement activation is at the core of the inflammatory process that characterizes atherosclerosis. The inflammation is silent because arteries lack pain fibers. Therapeutic intervention to reduce the inflammatory process may be an effective addition to reducing the levels of cholesterol and other lipids. Once the inflammatory process in a plaque is initiated, arrest may not be achieved by simple lowering of circulating lipid levels. Controlling the autodestructive local process by inhibiting complement activation may be essential.
| Acknowledgments |
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Received April 11, 2001; accepted April 25, 2001.
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