American Journal of Pathology, Vol. 167, No. 4, October 2005
Copyright © American Society for Investigative Pathology

Vascular Biology, Atherosclerosis and Endothelium Biology

C-Reactive Protein in Atherosclerotic Lesions
Its Origin and Pathophysiological SignificanceHuijun Sun,*† Tomonari Koike,*
Tomonaga Ichikawa,* Kinta Hatakeyama,‡
Masashi Shiomi,§ Bo Zhang,¶ Shuji Kitajima,储
Masatoshi Morimoto,储 Teruo Watanabe,**Yujiro Asada,‡ Yuqing E. Chen,†† andJianglin Fan*
From the Department of Pathology,* Cardiovascular Disease
Laboratory, Institute of Basic Medical Sciences, University ofTsukuba, Tsukuba, Japan; the First Department of Pathology,‡
Faculty of Medicine, University of Miyazaki, Miyazaki, Japan;
Institute for Experimental Animals,§ Kobe University School of
Medicine, Kobe, Japan; the Department of Cardiology,¶ Fukuoka
University School of Medicine, Fukuoka, Japan; Analytical
Research Center for Experimental Sciences 储 and President
Office,** Saga University, Saga, Japan; Cardiovascular ResearchInstitute,†† Morehouse School of Medicine, Atlanta, Georgia; andthe Department of Pharmacology,† Dalian Medical University,
Dalian, China

C-reactive protein (CRP) is frequently deposited in thelesions of the arterial intima; however, the origin andpathological significance of CRP in these lesions are
not completely understood. In this study, we measured CRP levels in the plasma of hypercholesterolemic rabbits and investigated CRP expression at
both the mRNA and protein levels using rabbit andhuman atherosclerotic specimens. CRP levels weresignificantly elevated in both cholesterol-fed and Watanabe heritable hyperlipidemic rabbits, and CRP levels were clearly correlated with aortic atherosclerotic
lesion size. Immunohistochemical staining coupledwith Western blotting analysis revealed that CRP-immunoreactive proteins were found at all stages ofatherosclerosis from the early to advanced lesions.
CRP was present extracellularly and co-localized withapolipoprotein B but was rarely associated with thecytoplasm of macrophages and foam cells. Real-timereverse transcriptase-polymerase chain reaction analysis revealed that CRP mRNA in atherosclerotic lesions was barely detectable, and isolated macrophages did not express CRP mRNA, suggesting that
CRP proteins found in the lesions were essentiallyderived from the circulation rather than synthesized
de novo by vascular cells. These results suggest that
there is a link between plasma CRP and the degree ofatherosclerosis and that inhibition of plasma CRP
may represent a therapeutic modality for the treatment of cardiovascular disease. (Am J Pathol 2005,
167:1139 –1148)

C-reactive protein (CRP) is a classical plasma protein
marker that is markedly elevated in the acute phase of
inflammation, infection, and tissue damage and thus has
been broadly used for monitoring and differential diagnosis.1,2 The major functions of CRP include its ability tobind to various ligands exposed on damaged tissue orbacteria (opsonization) for the enhancement of phagocytosis and activation of the complement pathway, thereby
enabling it to exert both anti- and proinflammatory functions.2,3 CRP is mainly expressed by hepatocytes, and its
synthesis is regulated at the posttranscriptional level bycytokines.4 Ample data from both clinical and experimental studies have shown that a high level of plasma CRP isa risk factor as well as marker for cardiovascular diseases,5–9 although some recent studies failed to prove therisk of CRP compared to other risk factors.10 Regardlessof this controversy, emerging evidence indicates thathigh levels of CRP may be potentially atherogenic;3,11
therefore, it is essential to clarify the functional roles of
CRP in the arterial wall. Although it has been reported fora long time that CRP is present in human atherosclerotic
lesions,12,13 it is still not unequivocal whether CRP in thearterial wall is completely derived from the circulation or
Supported in part by grants-in-aid for scientific research from Ministry of
Education, Culture, Sports and Technology, Japan (KAKENHI.16390089
and 16659099 to J.F.), the Takeda Science Foundation, the Ono Medical
Research Foundation, and the Novartis Foundation for the Promotion of
Science (to J.F.); and a grant from the Center for Tsukuba Advanced
Research Alliance at the University of Tsukuba (to J.F.).
H.S. and T.K. contributed equally to this work.
Accepted for publication June 27, 2005.
Address reprint requests to Jianglin Fan M.D., Ph.D., Department of
Pathology, Institute of Basic Medical Sciences, University of Tsukuba,
Tsukuba, 305-8575 Japan. E-mail: j-lfan@md.tsukuba.ac.jp.

1139

1140
Sun et al
AJP October 2005, Vol. 167, No. 4

is locally synthesized by the arterial cells.14,15 Several
studies even suggested that CRP may possibly be produced by macrophages16 and smooth muscle cells(SMCs).17 The hypothesis of arterial wall-synthesized
CRP is so intriguing and attractive that it led to a numberof investigations attempting to determine the potential
pathophysiological significance of the local production of
CRP in terms of its atherogenicity in vitro. For example,
CRP has been shown to modulate endothelial cell functions (NO and ET-1 production)18 –21 and increase SMC
migration and mediate the uptake of oxLDL by macrophages.22,23 CRP itself is a chemoattractant factor formonocytes24 and induces the production of MCP-1 andthe expression of adhesion molecules on endothelial
cells in vitro.25,26 If this hypothesis really represents thesituation in vivo, CRP, along with hypercholesterolemia,
may play a pivotal role in the initiation and progression ofatherosclerosis, and therapeutic strategies to block CRP
functions may be useful for treating atherosclerosis. Unfortunately, this hypothesis has not been tested or verified
in vivo using appropriate experimental animals. Several
recent studies have argued that biological effects of CRP
on vascular cells reported previously were possible artifacts caused by the presence of sodium azide in thecommercial CRP.27–29 Furthermore, it has not been determined whether the level of CRP is associated with thedegree of the lesions of atherosclerosis and whether CRP
is involved in the initiation and progression of the lesions
or with plaque rupture. These issues may be difficult toresolve by merely using human advanced lesions or invitro models. Finally, there have been no experimental
animal studies examining the relationship between CRP
levels and degree of atherosclerosis. To address theseissues, we performed the current study using rabbit atherosclerosis models as well as specimens of human coronary arterial plaques. Rabbits are excellent models foratherosclerosis because they are sensitive to cholesterol
diet and rapidly develop atherosclerosis.30 In addition,
rabbit CRP has 70% homology with human CRP1 andrabbit CRP levels are highly inducible and responsive
during the inflammatory reaction.31 We used both cholesterol-fed and Watanabe heritable hyperlipidemic(WHHL) rabbits because these two kinds of rabbit models exhibited different atherogenic lipoprotein profiles(remnant-rich hypercholesterolemia in cholesterol-fed
rabbits versus low-density lipoprotein (LDL)-rich hypercholesterolemia in WHHL rabbits). Using these models,
we were particularly interested in clarifying: 1) whether

Table 1.

plasma CRP levels are correlated with aortic atherosclerosis; 2) whether CRP deposition patterns are different inearly-stage lesions from the advanced lesions; and 3)
whether vascular wall cells, especially macrophages,
can synthesize CRP. In addition, we investigated the CRP
deposition and expression in unstable and ruptured coronary arteries from human myocardial infarction patients.

Materials and Methods
Atherosclerotic Specimens
In this study, we used both rabbit and human specimens
to examine CRP deposition and the relationship of CRP
with other cellular components in atherosclerotic lesions.
For rabbit experiments, we used two kinds of rabbits withatherosclerosis: 45 cholesterol-diet fed rabbits32 (fed adiet containing 0.3% cholesterol and 3% soybean oil for
16 weeks) and 31 WHHL rabbits33 (fed a chow diet, aged
from 8 to 12 months) and 62 normolipidemic rabbits (feda chow diet, aged from 4 to 10 months) as a control
group. All rabbits were raised in a specific pathogen
free-equivalent barrier facility at either the University ofTsukuba or Saga University and given food and water ad
libitum. Human coronary arteries and aortas were obtained from patients who died of myocardial infarction
within 6 hours after death. Autopsy was performed at the
Department of Pathology, Faculty of Medicine, Universityof Miyazaki. All specimens were fixed in formalin forhistological examinations or snap-frozen in liquid nitrogen and stored at ⫺80°C for protein isolation and RNA
extraction. All protocols for animal studies and use ofhuman specimens were performed according to theguidelines and with the approval of the ethics review
committee of the University of Tsukuba.

Histology and Immunohistochemical Staining
All specimens were embedded in paraffin and cut into
3-␮m-thick serial sections. The specimens were
mounted on slides and deparaffinized with gradedconcentrations of xylene and ethanol, and then stainedwith hematoxylin and eosin (H&E). For immunohistochemical staining, all slides were immersed in 3%
H2O2 in methanol for 45 minutes at room temperature
to block endogenous peroxidase activity. To stain human macrophages and complement 3 (C3), the slides

Antibodies Used for the Current Study

Antibodies

Working dilution

Species

Manufacturers

hCRP
rCRP
KP1
RAM11
HHF35
apoB
C3

⫻500
⫻200
⫻50
⫻400
⫻400
⫻200
⫻100

Mouse
Chicken
Mouse
Mouse
Mouse
Goat
Mouse

Sigma, St. Louis, MO
Immunology Consultants Laboratory Inc., Newberg, OR
DakoCytomation Denmark A/S, Glostrup, Denmark
Dako Japan Inc., Tokyo, Japan
Enzo Biochemicals, NY
Rockland Inc., Gilbertsville, PA
Santa Cruz Biotechnology Inc., Santa Cruz, CA

hCRP, human C-reactive protein; rCRP, rabbit C-reactive protein; KP1, human CD68 macrophage; RAM11, rabbit monocyte/macrophages; HHF35,
smooth muscle ␣-actin; C3, recombinant protein corresponding to amino acids 541 to 840 of C3 precursor of human origin.

CRP and Atherosclerosis
1141
AJP October 2005, Vol. 167, No. 4

Table 2.

Primers Used for Real-Time RT-PCR

Genes

Primers

RabbitForward TCAAAGCCTTCACTGTGTGC
CRP
Reverse AGGTGAGTTGGATCCACAGGGAPDH Forward GCTGAACGGGAAACTCACTG
Reverse CCAGCATCGAAGGTAGAGGA
Human
Forward GTGTTTCCCAAAGAGTCGGATACT
CRP
Reverse CCACGGGTCGAGGACAGTTGAPDH Forward CAATGACCCCTTCATTGACCTC
Reverse AGCATCGCCCCACTTGATTGAPDH, glyceraldehyde-3-phosphate dehydrogenase.

were predigested with 0.4% proteinase K for 5 minutes
at 37°C. The sections were then incubated with 10%
normal goat serum for 60 minutes to reduce nonspecific background staining and reacted with each appropriately diluted antibody (Ab) at 4°C overnight. The
panel of Abs used for examining rabbit and human
CRP, apoB, C3, and macrophages and SMCs in thelesions is listed in Table 1. CRP antibodies were solid
phase absorbed and then immunoaffinity purified of
CRP proteins. Affinity purified CRP antibodies give asingle band by immunoelectrophoresis and gel diffusion against fluids containing CRP. The specificity of
CRP antibodies has been confirmed by Western blots.
This antibody does not cross-react with serum amyloid
P component, haptoglobin, ␣-1-acid glycoprotein and
IgG. It can detect both native and denatured-reduced
CRP proteins. After rinsing with phosphate-bufferedsaline (pH 7.4), the sections were incubated with peroxidase-conjugated secondary Abs at room temperature for 60 minutes and then the staining was visualized
by reaction with 3-amino-9-ethylcarbazole (Nichirei,
Tokyo) as described previously.34 For negative controls, primary Abs were either omitted or replaced bynonspecific IgG.

Western Blot Analysis
To examine the expression of CRP proteins in theatherosclerotic lesions, aortic specimens were divided
into lesions and nonlesions based on the gross observation. The aortic lesions were further separated into
the intima, media, and adventitia under a dissecting
microscope. In addition, alveolar macrophages, thioglycollate-elicited peritoneal macrophages, and liver
were used. One hundred mg of each tissue were homogenized in ice-cold suspension buffer (0.02 mol/L
Tris-HCl, pH 7.5) supplemented with phenylmethyl sulfonyl fluoride (1 ␮g/ml), aprotinin (1 ␮g/ml), and ethylenediamine tetraacetic acid (1 mmol/L). Ten-␮g aliquots of the crude proteins from each sample wereseparated by 4 to 20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gradient gel electrophoresis for Western blotting and probed with a mousemonoclonal Ab against human CRP.35

Figure 1. A: Increased plasma CRP levels in cholesterol-fed and WHHL
rabbits. The values are expressed as mean ⫾ SE. **P ⬍ 0.01 versus normal
rabbits or cholesterol-fed rabbits versus WHHL rabbits. Average levels ofplasma total cholesterol are 30 ⫾ 9 mg/dl in normal rabbits, 860 ⫾ 53 mg/dl
in cholesterol-fed rabbits, and 459 ⫾ 21 mg/dl in WHHL rabbits. Total en facelesion area of the aorta is 14 ⫾ 3.3% in cholesterol-fed rabbits and 42.3 ⫾
5.9% in WHHL rabbits. B and C: Correlations of plasma CRP and aortic
atherosclerosis gross size (B) and plasma cholesterol levels (C).

Northern Blot and Real-Time Reverse
Transcriptase-Polymerase Chain Reaction(RT-PCR)
Total RNA was isolated from various tissues of bothnormal and cholesterol-fed and WHHL rabbits using

1142
Sun et al
AJP October 2005, Vol. 167, No. 4

Figure 2. Demonstration of CRP deposition in atherosclerotic lesions of cholesterol-fed rabbits. Two representative lesions were selected from cholesterol-fed
rabbits and stained with H&E or mAbs against rabbit CRP, macrophages (M␾ ), SMCs, and apoB. A: Early-stage lesion, which is composed of a single layer ofmacrophage-derived foam cells. B: Advanced lesion, which is covered by a fibrotic cap. CRP is co-localized with apoB staining. See supplemental data for sections
stained with CRP Abs shown in higher magnification at http://ajp.amjpathol.org.

Trizol reagent (Invitrogen, Carlsbad, CA). Thioglycollate-elicited peritoneal and alveolar macrophages
were collected as described.35 Human specimens(aorta and liver) were obtained at autopsy. Ten ␮g of
RNA was then subjected to electrophoresis in a 1.2%
agarose gel and transferred to a Nytran nylon membrane. The membrane was hybridized in turn with the32
P-labeled human CRP cDNA probe. The membrane
was rehybridized with human ␤-actin cDNA probe as
an internal standard. Total RNA was reverse-transcribed into cDNA by using Invitrogen reverse transcription reagents. Expression of CRP and a housekeeping gene (GAPDH) in the liver, aortic lesions,nonlesion regions of aortas, and macrophages by realtime RT-PCR (DNA Engine Option; MJ Research, Tokyo, Japan) were performed using DyNAmo SYBRGreen qPCR kits (Finnzymes) according to the manufacturers’ instructions.35 The primers for rabbit andhuman CRP and GAPDH are listed in Table 2.

Measurement of Plasma CRP Levels in Rabbits
by Enzyme-Linked Immunosorbent Assay(ELISA)To determine whether CRP was elevated in cholesterolfed or WHHL rabbits, levels of rabbit CRP in the plasma
were quantified using two rabbit high-sensitive CRPELISA kits from Kamiya Biomedical (Seattle, WA) andShibayagi Co. (Gunma, Japan), and compared with normal rabbits. All measurements were performed independently by two researchers using different ELISA kits. The
plasma specimens were mixed thoroughly before
assays.

In Vitro Investigation of CRP Expression
To examine the effects of cytokines on the regulation of
CRP expression in hepatocytes and possibly in mac-rophages, we used the Huh7 cell line (derived fromhuman hepatoma) and U937 monocytic cells. Huh7
cells were cultured in serum-free Dulbecco’s modified
Eagle’s medium and incubated with various concentrations of interleukin (IL)-1␤, IL-6, and tumor necrosis
factor-␣ for 48 hours. U937 cells were cultured in RPMI
1640 medium and treated with phorbol 12-myristate
13-acetate (100 ng/ml) for 3 days before incubation
with cytokines.36 After that, these cells were collected
for the extraction of total RNA using Trizol agent and
CRP expression was evaluated using real-time RT-PCR
as described above.

Statistical Analysis
All values are expressed as mean ⫾ SE. Plasma CRP,
total cholesterol, and aortic en face lesion area werecompared with Student’s t-test, Welch’s t-test, or MannWhitney’s U-test for nonparametric analysis. Correlations
were analyzed using Spearman’s correlation coefficientby rank test.

Results
Correlation between Plasma CRP andCholesterol and Aortic Atherosclerosis
In normal rabbits, the range of plasma CRP levels was
narrow with an average of 3.15 ⫾ 0.8 mg/L. It seems
that rabbit CRP levels are close to the mean values ofhealthy middle-aged humans (2.82 mg/L) reported in
the literature.37 Rabbits with hypercholesterolemia, either induced by cholesterol diet feeding or caused by
LDL receptor deficiency, had significantly higher
plasma CRP levels than normal rabbits: 2.5-fold higher
in cholesterol-fed rabbits and 28-fold higher in WHHL
rabbits on average (Figure 1A). We also examined therelationship between plasma CRP and aortic athero-

CRP and Atherosclerosis
1143
AJP October 2005, Vol. 167, No. 4

Figure 3. Demonstration of CRP deposition and apoB in the early-stage lesions of WHHL rabbits. Three representative lesions with different features were selected
and stained with H&E or mAbs against CRP, apoB, macrophages (M␾ ), and SMCs. CRP is deposited on the surface (A) or under foam cells (B) and co-localizedwith apoB and does not overlap with macrophage-rich area (C). See supplemental data for sections stained with CRP Abs shown in higher magnification at
http://ajp.amjpathol.org.

sclerosis en face size or plasma total cholesterol levels
in hyperlipidemic rabbits. As shown in Figure 1B andC, the CRP level in plasma was well correlated with thelesion size in the aorta, and weakly associated with theplasma total cholesterol level.

Demonstration of CRP in Atherosclerotic
Lesions of Rabbits
Because a large range of lesions were obtained from a
sufficient number of rabbits in this study, we were able toselect both early stage lesions (referred to as foamcell-rich lesions) as well as relatively advanced lesions(including fibrous plaques and complicated lesions). Immunohistochemical staining revealed that in the earlystage lesions, where a few layers of macrophages were
present, CRP-immunoreactive proteins were found either
on the surface or beneath the foam cells (Figure 2A). In
advanced lesions, CRP was primarily localized beneaththe fibrous cap and closely associated with apoB deposition (Figure 2B). In either case, CRP did not apparently
overlap with the cytoplasm of macrophages and foamcells (Figure 4). CRP tended to be localized around theextracellular matrix and was seldom, if ever, associatedwith macrophages or SMCs (also see supplemental datafor high magnification at http://ajp.amjpathol.org).
Atherosclerotic lesions of WHHL rabbits were moreadvanced than those of cholesterol-fed rabbits because
these rabbits have higher levels of LDL-cholesterol rather
than the levels of remnant lipoproteins present in cholesterol-fed rabbits. In WHHL rabbits, various kinds of atherosclerotic lesions were seen, from foam cell-rich lesions to complicated lesions in which calcification andnecrotic cores were prominent. Similar to those of cholesterol-fed rabbits, CRP-immunoreactive proteins weredetected on the surface of the lesions, beneath foamcells, or around the lipid or necrotic cores (Figures 3 and
4). CRP was co-localized with apoB but seemed not to be

Figure 4. Demonstration of CRP deposition and apoB in advanced lesions of WHHL rabbits. Two representative lesions with a necrotic core (A) or calcification(B) were selected and both lesions show CRP deposition overlapping with apoB. See supplemental data for sections stained with CRP Abs shown in higher
magnification at http://ajp.amjpathol.org.

1144
Sun et al
AJP October 2005, Vol. 167, No. 4

associated with the cytoplasm of macrophages and foamcells. In these lesions, CRP was not deposited in any
particular areas such as the cap or core. We also performed Western blot analysis. For this purpose, we separated the lesion from nonlesion areas (based on grossobservation) and furthermore, we isolated the intima, media, and adventitia of the aorta from WHHL rabbits undera dissecting microscope. We were especially interested
in examining whether macrophages could express CRP
per se, and therefore we used macrophages in parallel.
As shown in Figure 5, CRP was detected in the lesional
intima but not in the media or adventitia. Isolated macrophages contained smaller amounts of CRP but the immunoreactive bands from macrophages showed faster
mobility on sodium dodecyl sulfate-polyacrylamide gelelectrophoresis, presumably they were degraded CRP
proteins or taken up by macrophages.

Demonstration of CRP in Human Coronary
Plaques
Next, we investigated CRP deposition in human coronary
plaques. CRP was detected in all types of the lesions,
intimal thickening, unstable plaques, and ruptured plaques,
and especially in fragile or ruptured caps. In the vicinity ofthese lesions, strong CRP staining was seen around orconcentrated on the surface, but did not overlap with thecytoplasm of macrophages (Figure 6). In these CRP-positive areas, C3 was also detected (Figure 6B), which wasconsistent with a previous report.38 In stable plaques, CRP
was also detected, either on the surface or in the deep areaof thickened intima (Figure 7). The immunohistochemical
findings were further confirmed by Western blot analysis
and lesions of aortic atherosclerosis contained substantial
amounts of CRP proteins regardless of the lesion characteristics (Figure 8).

CRP mRNA Expression
The fact that CRP-immunoreactive proteins were invariably present in atherosclerotic lesions raised the questionof whether CRP was exclusively derived from the circulation or/and produced locally. To address this issue, we
initially screened the CRP mRNA expression of multiple
tissues from both chow- and cholesterol-fed rabbits using

Figure 5. Immunoblotting analysis of tissue CRP from WHHL rabbits. Aortic
lesions along with isolated alveolar and peritoneal macrophages were analyzed for the presence of CRP protein as described in Materials and Methods.
Liver and plasma were used as positive controls. Note that CRP bands inmacrophages are smaller than other CRP bands.

Northern blotting. As shown in Figure 9A, the liver was the
only organ that expressed detectable mRNA by Northernblot hybridization. There was no expression of CRP
mRNA in either atherosclerotic lesions or isolated macrophages. This result suggests that the liver is the exclusive
organ for CRP production, while the arterial wall andmacrophages do not express CRP mRNA or the levels of
CRP mRNA expressed by the arterial wall and macrophages are too low to be detected. To further explore thispossibility, we applied a real-time RT-PCR analysis,
which is considered to be more sensitive than Northernblotting analysis and more specific than traditional quantitative RT-PCR.39 In these experiments, we analyzed the
CRP expression of the liver (n ⫽ 22 rabbits), lesions (n ⫽16 rabbits), nonlesion regions of the aorta (n ⫽ 12 rabbits), and macrophages (n ⫽ 28 rabbits). Using the liver
expression of CRP as a standard, we calculated the CRP
expression level using the 2⫺⌬⌬CT method by Livak andSchmittgen.40 We found that isolated macrophages did
not express CRP mRNA (90% of sample values wereactually 0). The average relative CRP expression levels(arbitrary units) of lesions or nonlesions were less than
1/100th of the hepatic expression of CRP (Figure 9B),
suggesting that lesional CRP expression was indeed extremely low and not comparable with the hepatic expression. This conclusion was further strengthened by examining human aortic atherosclerotic lesions. Analysis ofeight freshly isolated human aortic lesions showed nomRNA expression (5 sample values were 0 and 3 were⬍0.02) by real-time RT-PCR (average value of livers of
the same patients: 121 ⫾ 48 arbitrary units). We also
compared CRP mRNA expression using human Huh7
hepatoma cells and U937 macrophages. Huh7 hepatocytes expressed a much lower level of CRP under basal
conditions (compared to normal human liver tissue) but
the level was discernibly up-regulated by incubation with
IL-1␤, IL-6, and tumor necrosis factor-␣ (data not shown).
U937 macrophages did not express CRP mRNA in either
normal state or when stimulated by various cytokines atdifferent concentrations (all sample values were 0).
Therefore, these results demonstrate that macrophages
from both rabbits and humans did not express CRP
mRNA.

Discussion
In this study, we demonstrated for the first time that the
CRP level was significantly elevated in hypercholesterolemic rabbits compared to normal rabbits. Although it iscurrently unknown whether the elevation of CRP in
plasma is a cause or consequence or both of atherosclerosis, it is clear that elevated CRP levels in plasma werecorrelated with the severity of atherosclerosis and hypercholesterolemia in rabbits. It was reported that CRP canselectively bind to apoB-containing atherogenic lipoproteins, such as ␤-VLDL and LDL, in hypercholesterolemic
rabbits as well as in humans41 and thus increased levelsof apoB-containing particles may facilitate the formationof CRP-lipoprotein complexes in plasma. Formation of
CRP-apoB-containing lipoprotein complexes does not af-

CRP and Atherosclerosis
1145
AJP October 2005, Vol. 167, No. 4

Figure 6. CRP was closely associated with unstable vulnerable plaque (A) or ruptured plaques (B) in human coronary arteries. Human coronary arteries, obtained
from patients who died of MI, were used for the detection of CRP immunoreactive proteins. Macrophage-rich areas (stained by CRP, M␾, SMCs, or C3), boxed
area, were shown in higher magnification (B). Note that CRP is concentrated on the surface of macrophages of both lesions. See supplemental data for sections
stained with CRP Abs shown in higher magnification at http://ajp.amjpathol.org.

fect the clearance rate of 125I-labeled rabbit CRP;42
therefore, we speculate that the elevated CRP level inplasma is mainly caused by enhanced hepatic production rather than by reduced catabolism of CRP in rabbits.

Atherosclerosis is an inflammatory process7 and many
inflammatory cells, especially macrophages and foamcells can produce a variety of cytokines that may stimulate the hepatic expression of the CRP gene and up-

Figure 7. Detection of CRP deposition in stable plaques of human coronary arteries. Two macrophage-rich lesions were selected and both show CRP deposition.
CRP was either located on the top (A) or the bottom (B) of the lesions. See supplemental data for sections stained with CRP Abs shown in higher magnification
at http://ajp.amjpathol.org.

1146
Sun et al
AJP October 2005, Vol. 167, No. 4

Figure 8. Demonstration of CRP protein in human aorta by Western blotting.
The samples were from eight autopsy cases and all of them contained CRP
proteins detected by Western blotting. The lesions were divided grossly: DIC,
diffuse intimal thickening and plaques.

regulate CRP production in the liver. The pathophysiological significance of the elevated CRP in hyperlipidemic
rabbits is still unknown; however, it may reflect an inflammatory state related to atherosclerosis or amount or/and
activity of circulating proinflammatory cytokines or both. It
seems unlikely that the increased CRP level in plasma
was caused by de novo extrahepatic synthesis in atherosclerotic lesions because we failed to detect significant
CRP mRNA expression in the lesions or other tissues byeither Northern blot hybridization or real-time RT-PCR.
In accordance with previous reports,12–14,24 our study
clearly demonstrated that CRP protein was invariably
present in atherosclerotic lesions, regardless of lesion
type (early versus advanced) or nature (foam cell-richversus extracellular matrix-rich) and was closely co-localized with apoB, suggesting that CRP may interact with
apoB-containing particles in the intima, thereby facilitating their binding to extracellular matrix, increasing retention time and oxidation in the intima, and enhancing foamcell formation.43,44 As reviewed in the Introduction, thesepremises or assumptions regarding the proatherogenic
effects of CRP, however, should be considered cau-

Figure 9. Northern blot (A) and real-time RT-PCR (B) analysis of CRP
expression in rabbit tissues. A: The liver was the only organ that expresses
detectable CRP by Northern blotting. B: Real-time RT-PCR analysis showedthat compared to the liver, aortic lesions expressed very small amounts(⬃1/100th of the liver) of CRP mRNA whereas macrophages did not produce
detectable CRP mRNA.

tiously, and need to be verified in the future. Despite this,
CRP deposition rarely overlapped with the cytoplasm ofmacrophages and foam cells in our studies of both rabbit
and human specimens. Our findings showed that although some macrophages could be positively stainedby CRP antibodies, this was not a frequent and universal
phenomenon and may occur only when a particular ligand is exposed on these cells. Three methods of analysis (immunohistochemical staining, Western blotting,and real-time RT-PCR) provided consistent observations,and therefore, these results rebut the contention thatmacrophages16,17,38 are the major sources of CRP in thearterial wall or that macrophages are responsible for theincreased plasma CRP levels. This notion is also consistent with a recent report using CRP transgenic mice
showing that no CRP mRNA was detected in apoE knockout mouse lesions.45 Our results are not in agreementwith the report by Yasojima and colleagues,38 whoshowed that human atherosclerotic plaques (10 humanpostmortem samples) contained higher (7.2-fold)amounts of CRP mRNA than liver and proposed thatmacrophages and SMCs are the main sources of plasma
CRP. The discrepancy between our study and previous
studies regarding the macrophage-produced CRP in thelesions is clearly due to the differences in the samples
and methods applied. In sharp contrast to the previous
reports, we used a large number and variety of atherosclerotic samples from two kinds of rabbits along withhuman coronary arteries and aortas as well as macrophages and livers. More importantly, we analyzed CRP
mRNA expression using the most sensitive method available, real-time RT-PCR, whereas the previous studies
performed only conventional semiquantitative RT-PCR,38
which has been criticized as being unreliable.39 It shouldbe emphasized that none of the previous studies that
concluded that CRP was expressed by vascular macrophages or SMCs hitherto were performed using real-time
RT-PCR. In addition, our refutation was also supported bystudies on cultured human Hu7h hepatocytes and U937
macrophages. It is noteworthy that CRP-immunoreactive
proteins were also detected in unstable plaques or
ruptured plaques, particularly on the fibrotic cap. CRP
proteins were concentrated on the surface of macrophages and foam cells in these areas and co-localizedwith C3. Whether CRP in this particular region is directly
involved in or triggers the plaque rupture remains to be
determined.
It should be pointed out that there are several differences between human CRP and rabbit CRP in terms of
their structures and functional properties. For example,
native rabbit CRP has higher affinity ability to apoB-containing lipoproteins and formed a complex whereas human CRP can only bind to apoB-containing lipoproteinswhen they are aggregated. Human CRP can activate
human complement pathway whereas it is completely
unknown whether rabbit CRP can activate rabbit complement pathway (M. Pepys, personal communication). It
remains unclear whether these differences (rabbit versus
human, bound CRP versus free CRP, native versus aggregated CRP) may affect their physiological functions
such as in atherosclerosis. Perhaps a precise elucidation

CRP and Atherosclerosis
1147
AJP October 2005, Vol. 167, No. 4

of CRP pathological roles in atherogenesis awaits thegeneration of appropriate transgenic animals that can
express high levels of inducible CRP in plasma or highlevels of CRP specifically in lesions in the future. Recently, four studies using transgenic mice expressing
either human45– 47 or rabbit48 (K. Reifenberg, personalcommunication) CRP did not lead to a consistent conclusion regarding the atherogenic effect of CRP. One study
showed that increased CRP moderately accelerated atherosclerosis in male apoE knockout mice45 whereas
other studies failed to demonstrate this effect. In mice,endogenous plasma CRP is not inducible and never increases more than 2 to 3 mg/L49 and also apoE knockout
mice have some defects in their complement system thatcannot be activated by transgenic CRP.48 In this regard,
rabbits are apparently superior to mice because rabbit
CRP levels are highly inducible and responsive during
the inflammatory reaction.31 Our separate study also
showed that, as in human studies,6,50 administration of
statin in rabbits could effectively normalize plasma CRP
levels and reduce the CRP levels in both liver and aortic
lesions (J.F., unpublished data). This result suggests thatthe rabbit may be an alternative animal model to evaluate
therapeutic effects of decreasing the CRP level.51 Further
studies will be required to clarify whether decreasing
CRP alone without changing the plasma cholesterol levelcan be beneficial for the treatment of atherosclerosis.
Recently, our laboratory has successfully created a lineof human CRP transgenic rabbits (S.K. and J.F., unpublished data). In view of the multiple and complicatedbiological functions of CRP in many cardiovascular diseases and other inflammatory processes, CRP transgenic rabbits may be extremely valuable for elucidating
both the pathophysiological functional roles and mechanisms of this protein in atherosclerosis, and for testing theefficacy of CRP-lowering drugs in the future.
In conclusion, our studies reported here demonstratethat there is a close relationship between the CRP plasma
level and atherosclerosis in hypercholesterolemic rabbits. CRP immunoproteins were frequently present in thelesions of atherosclerosis regardless of the severity anddegree of the lesions but this CRP was basically derived
from the circulation rather than synthesized by vascular
cells such as macrophages. In human coronary plaques,
CRP was also frequently present in the ruptured fibrotic
caps. In future studies, we are expecting to investigate
whether elevated CRP levels are directly atherogenic orwhether CRP is involved in the progression of atherosclerosis in vivo.

Acknowledgment
We thank T. Hachisu (Shibayagi Co.) for providing rabbit
CRP ELISA kits and analyzing rabbit plasma CRP.

References
1. Pepys MB, Baltz ML: Acute phase proteins with special reference toC-reactive protein and related proteins (pentaxins) and serum amyloid A protein. Adv Immunol 1983, 34:141–212

2. Black S, Kushner I, Samols D: C-reactive protein. J Biol Chem 2004,
279:48487– 48490
3. Pepys MB, Hirschfield GM: C-reactive protein: a critical update. J Clin
Invest 2003, 111:1805–1812
4. Taylor AW, Ku NO, Mortensen RF: Regulation of cytokine-induced
human C-reactive protein production by transforming growth factorbeta. J Immunol 1990, 145:2507–2513
5. Ridker PM, Rifai N, Rose L, Buring JE, Cook NR: Comparison of
C-reactive protein and low-density lipoprotein cholesterol levels in theprediction of first cardiovascular events. N Engl J Med 2002,
347:1557–1565
6. Ridker PM, Rifai N, Pfeffer MA, Sacks F, Braunwald E: Long-term
effects of pravastatin on plasma concentration of C-reactive protein.
The Cholesterol and Recurrent Events (CARE) Investigators. Circulation 1999, 100:230 –235
7. Libby P, Ridker PM: Inflammation and atherosclerosis: role of C-reactive protein in risk assessment. Am J Med 2004, 116(Suppl
6A):9S–16S
8. Nissen SE, Tuzcu EM, Schoenhagen P, Crowe T, Sasiela WJ, Tsai J,
Orazem J, Magorien RD, O’Shaughnessy C, Ganz P: Statin therapy,
LDL cholesterol, C-reactive protein, and coronary artery disease.
N Engl J Med 2005, 352:29 –38
9. Ridker PM, Cannon CP, Morrow D, Rifai N, Rose LM, McCabe CH,
Pfeffer MA, Braunwald E: C-reactive protein levels and outcomes after
statin therapy. N Engl J Med 2005, 352:20 –28
10. Danesh J, Wheeler JG, Hirschfield GM, Eda S, Eiriksdottir G, Rumley
A, Lowe GD, Pepys MB, Gudnason V: C-reactive protein and othercirculating markers of inflammation in the prediction of coronary heartdisease. N Engl J Med 2004, 350:1387–1397
11. Labarrere CA, Zaloga GP: C-reactive protein: from innocent bystander to pivotal mediator of atherosclerosis. Am J Med 2004,
117:499 –507
12. Hatanaka K, Li XA, Masuda K, Yutani C, Yamamoto A: Immunohistochemical localization of C-reactive protein-binding sites in humanatherosclerotic aortic lesions by a modified streptavidin-biotin-staining method. Pathol Int 1995, 45:635– 641
13. Reynolds GD, Vance RP: C-reactive protein immunohistochemical
localization in normal and atherosclerotic human aortas. Arch Pathol
Lab Med 1987, 111:265–269
14. Zhang YX, Cliff WJ, Schoefl GI, Higgins G: Coronary C-reactive
protein distribution: its relation to development of atherosclerosis.
Atherosclerosis 1999, 145:375–379
15. Ishikawa T, Imamura T, Hatakeyama K, Date H, Nagoshi T, Kawamoto
R, Matsuyama A, Asada Y, Eto T: Possible contribution of C-reactive
protein within coronary plaque to increasing its own plasma levels
across coronary circulation. Am J Cardiol 2004, 93:611– 614
16. Dong Q, Wright JR: Expression of C-reactive protein by alveolar
macrophages. J Immunol 1996, 156:4815– 4820
17. Kobayashi S, Inoue N, Ohashi Y, Terashima M, Matsui K, Mori T, Fujita
H, Awano K, Kobayashi K, Azumi H, Ejiri J, Hirata K, Kawashima S,
Hayashi Y, Yokozaki H, Itoh H, Yokoyama M: Interaction of oxidative
stress and inflammatory response in coronary plaque instability: important role of C-reactive protein. Arterioscler Thromb Vasc Biol 2003,
23:1398 –1404
18. Verma S, Wang CH, Li SH, Dumont AS, Fedak PW, Badiwala MV,
Dhillon B, Weisel RD, Li RK, Mickle DA, Stewart DJ: A self-fulfilling
prophecy: C-reactive protein attenuates nitric oxide production andinhibits angiogenesis. Circulation 2002, 106:913–919
19. Wang CH, Li SH, Weisel RD, Fedak PW, Dumont AS, Szmitko P, Li RK,
Mickle DA, Verma S: C-reactive protein upregulates angiotensin type
1 receptors in vascular smooth muscle. Circulation 2003,
107:1783–1790
20. Venugopal SK, Devaraj S, Yuhanna I, Shaul P, Jialal I: Demonstrationthat C-reactive protein decreases eNOS expression and bioactivity inhuman aortic endothelial cells. Circulation 2002, 106:1439 –1441
21. Venugopal SK, Devaraj S, Jialal I: C-reactive protein decreases prostacyclin release from human aortic endothelial cells. Circulation 2003,
108:1676 –1678
22. Zwaka TP, Hombach V, Torzewski J: C-reactive protein-mediated low
density lipoprotein uptake by macrophages: implications for atherosclerosis. Circulation 2001, 103:1194 –1197
23. Chang MK, Binder CJ, Torzewski M, Witztum JL: C-reactive protein
binds to both oxidized LDL and apoptotic cells through recognition of

1148
Sun et al
AJP October 2005, Vol. 167, No. 4

a common ligand: phosphorylcholine of oxidized phospholipids. Proc
Natl Acad Sci USA 2002, 99:13043–13048
24. Torzewski M, Rist C, Mortensen RF, Zwaka TP, Bienek M, Waltenberger J, Koenig W, Schmitz G, Hombach V, Torzewski J: C-reactive
protein in the arterial intima: role of C-reactive protein receptordependent monocyte recruitment in atherogenesis. Arterioscler
Thromb Vasc Biol 2000, 20:2094 –2099
25. Pasceri V, Willerson JT, Yeh ET: Direct proinflammatory effect of
C-reactive protein on human endothelial cells. Circulation 2000,
102:2165–2168
26. Pasceri V, Cheng JS, Willerson JT, Yeh ET, Chang J: Modulation of
C-reactive protein-mediated monocyte chemoattractant protein-1 induction in human endothelial cells by anti-atherosclerosis drugs.
Circulation 2001, 103:2531–2534
27. Lafuente N, Azcutia V, Matesanz N, Cercas E, Rodriguez-Manas L,
Sanchez-Ferrer CF, Peiro C: Evidence for sodium azide as an artifact
mediating the modulation of inducible nitric oxide synthase by C-reactive protein. J Cardiovasc Pharmacol 2005, 45:193–196
28. Swafford Jr AN, Bratz IN, Knudson JD, Rogers PA, Timmerman JM,
Tune JD, Dick GM: C-reactive protein does not relax vascular smoothmuscle: effects mediated by sodium azide in commercially available
preparations. Am J Physiol 2005, 288:H1786 –H1795
29. van den Berg CW, Taylor KE, Lang D: C-reactive protein-induced invitro vasorelaxation is an artefact caused by the presence of sodium
azide in commercial preparations. Arterioscler Thromb Vasc Biol
2004, 24:168 –171
30. Fan J, Watanabe T: Transgenic rabbits as therapeutic proteinbioreactors and human disease models. Pharmacol Ther 2003,
99:261–282
31. Kushner I, Feldmann G: Control of the acute phase response. Demonstration of C-reactive protein synthesis and secretion by hepatocytes during acute inflammation in the rabbit. J Exp Med 1978,
148:466 – 477
32. Fan J, Shimoyamada H, Sun H, Marcovina S, Honda K, Watanabe T:
Transgenic rabbits expressing human apolipoprotein(a) develop
more extensive atherosclerotic lesions in response to a cholesterolrich diet. Arterioscler Thromb Vasc Biol 2001, 21:88 –94
33. Sun H, Unoki H, Wang X, Liang J, Ichikawa T, Arai Y, Shiomi M,
Marcovina S, Watanabe T, Fan J: Lipoprotein(a) enhances advanced
atherosclerosis and vascular calcification in WHHL transgenic
rabbits expressing human apolipoprotein(a). J Biol Chem 2002,
277:47486 – 47492
34. Ichikawa T, Unoki H, Sun H, Shimoyamada H, Marcovina S, Shikama
H, Watanabe T, Fan J: Lipoprotein(a) promotes smooth muscle cellproliferation and dedifferentiation in atherosclerotic lesions of human
apo(a) transgenic rabbits. Am J Pathol 2002, 160:227–236
35. Wang X, Liang J, Koike T, Sun H, Ichikawa T, Kitajima S, Morimoto M,
Shikama H, Watanabe T, Sasaguri Y, Fan J: Overexpression of humanmatrix metalloproteinase-12 enhances the development of inflammatory arthritis in transgenic rabbits. Am J Pathol 2004, 165:1375–1383
36. Wu L, Fan J, Matsumoto S, Watanabe T: Induction and regulation of
matrix metalloproteinase-12 by cytokines and CD40 signaling inmonocyte/macrophages. Biochem Biophys Res Commun 2000,
269:808 – 815
37. Ockene IS, Matthews CE, Rifai N, Ridker PM, Reed G, Stanek E:

Variability and classification accuracy of serial high-sensitivity C-reactive protein measurements in healthy adults. Clin Chem 2001,
47:444 – 450
38. Yasojima K, Schwab C, McGeer EG, McGeer PL: Generation of
C-reactive protein and complement components in atherosclerotic
plaques. Am J Pathol 2001, 158:1039 –1051
39. Bustin SA: Absolute quantification of mRNA using real-time reverse
transcription polymerase chain reaction assays. J Mol Endocrinol
2000, 25:169 –193
40. Livak KJ, Schmittgen TD: Analysis of relative gene expression datausing real-time quantitative PCR and the 2(-delta delta C(T)) method.
Methods 2001, 25:402– 408
41. Rowe IF, Soutar AK, Trayner IM, Baltz ML, de Beer FC, Walker L,
Bowyer D, Herbert J, Feinstein A, Pepys MB: Rabbit and rat C-reactive proteins bind apolipoprotein B-containing lipoproteins. J Exp
Med 1984, 159:604 – 616
42. Rowe I, Baltz M, Soutar A, Pepys M: In vivo turnover studies of
C-reactive protein. Clin Exp Immunol 1984, 58:245–252
43. Torzewski J, Torzewski M, Bowyer DE, Frohlich M, Koenig W, Waltenberger J, Fitzsimmons C, Hombach V: C-reactive protein frequently
colocalizes with the terminal complement complex in the intima ofearly atherosclerotic lesions of human coronary arteries. Arterioscler
Thromb Vasc Biol 1998, 18:1386 –1392
44. Bhakdi S, Torzewski M, Klouche M, Hemmes M: Complement andatherogenesis: binding of CRP to degraded, nonoxidized LDL enhances complement activation. Arterioscler Thromb Vasc Biol 1999,
19:2348 –2354
45. Paul A, Ko KW, Li L, Yechoor V, McCrory MA, Szalai AJ, Chan L:
C-reactive protein accelerates the progression of atherosclerosis inapolipoprotein E-deficient mice. Circulation 2004, 109:647– 655
46. Hirschfield GM, Gallimore JR, Kahan MC, Hutchinson WL, Sabin CA,
Benson GM, Dhillon AP, Tennent GA, Pepys MB: Transgenic humanC-reactive protein is not proatherogenic in apolipoprotein E-deficient
mice, Proc Natl Acad Sci USA 2005, 102:8309 – 8314
47. Trion A, de Maat MP, Jukema JW, van der Laarse A, Maas MC,
Offerman EH, Havekes LM, Szalai AJ, Princen HM, Emeis JJ: No
effect of C-reactive protein on early atherosclerosis development in
apolipoprotein E*3-leiden/human C-reactive protein transgenic mice,
Arterioscler Thromb Vasc Biol 2005, 25:1635–1640
48. Reifenberg K, Lehr HA, Baskal D, Wiese E, Schaefer SC, Black S,
Samols D, Torzewski M, Lackner KJ, Husmann M, Blettner M, Bhakdi
S: Role of C-reactive protein in atherogenesis: can the apolipoprotein
E knockout mouse provide the answer? Arterioscler Thromb Vasc Biol
2005, 25:1641–1646
49. Pepys MB: Isolation of serum amyloid P-component (protein SAP) in
the mouse. Immunology 1979, 37:637– 641
50. Plenge JK, Hernandez TL, Weil KM, Poirier P, Grunwald GK, Marcovina SM, Eckel RH: Simvastatin lowers C-reactive protein within 14
days: an effect independent of low-density lipoprotein cholesterol
reduction. Circulation 2002, 106:1447–1452
51. Ivashchenko Y, Kramer F, Schafer S, Bucher A, Veit K, Hombach V,
Busch A, Ritzeler O, Dedio J, Torzewski J: Protein kinase C pathway
is involved in transcriptional regulation of C-reactive protein synthesis
in human hepatocytes. Arterioscler Thromb Vasc Biol 2005,
25:186 –192